Mikrobiologija Pijace Vode

Monitoring and
control of drinking
water quality
Inventory and evaluation of monitoring
technologies for key-parameters

Techneau
October 2008

2008 TECHNEAU
TECHNEAU is an Integrated Project Funded by the European Commission under the Sixth Framework
Programme, Sustainable Development, Global Change and Ecosystems Thematic Priority Area
(contractnumber 018320). All rights reserved. No part of this book may be reproduced, stored in a database
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Techneau
October 2008

Monitoring and
control of drinking
water quality
Inventory and evaluation of monitoring
technologies for key-parameters

This report is:

PU = Public

Colofon
Title
Monitoring and control of drinking water quality Inventory and
evaluation of monitoring technologies for key-parameters

Author(s)
Margreet Mons (Ed.), all WA3 partners

Quality Assurance
All WA 3 partners

Deliverable number
D 3.1.3

Monitoring and control of drinking water quality
TECHNEAU - 4 - October 2008

Contents
Contents 4
1 Introduction 9
2 Method 10
3 Microbiological parameters 14
3.1 E. coli and coliform bacteria 14
3.1.1 Monitoring technology nr 1: Lactose TTC Tergitol Method (ISO 9308-1) 14
3.1.2 Monitoring technology nr 2: Colilert-18/Quanti-Tray (IDExx, UK National Standard
Method W 18) 16
3.1.3 Monitoring technology nr 3: Membrane Lauryl Sulphate Agar (MLSA) (NEN 6553) 18
3.1.4 Monitoring technology nr 4: Membrane Lauryl Suphate Broth (MLSB) (UK National
Standard Method W 2) 20
3.1.5 Monitoring technology nr 5: Chromocult Coliform Agar (Merck) 22
3.2 Intestinal enterococci 25
3.2.1 Monitoring technology nr 1: ISO method 7899-1 25
3.3 Clostridium perfringens 29
3.3.1 Monitoring technology nr. 1: Guideline according to Council Directive 98/83/EC -
membrane filtration and cultivation on m-CP agar 29
3.3.2 Monitoring technology nr. 2: Membrane filtration and cultivation on TSC agar,
subsequent confirmation tests (draft of ISO 6461 CD part 2) 31
3.3.3 Monitoring technology nr 3: Membrane filtration and cultivation on fluorogenic TSC
agar 34
3.3.4 Confirmation technology nr. 1: C. perfringens Detection System (Biotecon
Diagnostics) 36
3.3.5 Confirmation technology nr. 2: API 32 A (Biomerieux) 36
3.4 Heterotrophic Plate Counts 38
3.4.1 Monitoring technology nr 1: ISO 6222 38
3.4.2 Monitoring technology nr 2: Plating on R2A medium 40
3.5 Enteroviruses 43
3.5.1 Monitoring technology nr 1: Cell culture methods 43
3.5.2 Monitoring technology nr 2: RT-PCR 44
3.6 Giardia/Cryptosporidium 46
3.6.1 Monitoring technology nr 1: Method 1622 47
3.6.2 Monitoring technology nr 2: UK method 48
3.6.3 Monitoring Technology nr. 3: method 1623 48
3.6.4 Monitoring technology nr 4: ISO 15553 48
3.6.5 Monitoring technology nr 5: cross flow ultrafiltration 48
3.7 Thermotolerant Campylobacter species 52
3.7.1 Monitoring technology nr 1: Detection and enumeration of thermotolerant
Campylobacter species (ISO 17995:2005) 52
3.8 Legionella and Legionella pneumophila 55
3.8.1 Monitoring technology nr 1: ISO method 11731:1998 55
3.8.2 Monitoring technology nr 2: Q-PCR for Legionella pneumophila 56


3.9 Pseudomonas aeruginosa 58
3.9.1 Monitoring technology nr. 1: Filtration and cultivation according to EN ISO 16266 58
3.9.2 Monitoring technology nr. 2: VIT-Pseudomonas aeruginosa 60
3.9.3 Confirmation technology nr. 1: API-Test 20E 62
3.10 Aeromonas 63
3.10.1 M onitoring technology nr 1: EPA Method 1605 63
3.10.2 Monitoring technology nr 2: MALDI 65
3.10.3 Monitoring technology nr 3: PCR 67
3.11 Bacteriophages 70
3.11.3 Monitoring technology nr 3: ISO method 10705-4 (modified) 74
3.12 Aerobic spore forming bacteria 77
3.12.1 Monitoring technology nr 1: HPC counts 77
3.12.2 Monitoring technology nr 2: Membrane filtration 79
3.12.3 Monitoring technology nr: 3. Assays, based on cell constituents 80
3.12.4 Monitoring technology nr 4: PCR 84
3.13 Biofilm formation rate (BFR) 87
3.13.1 Monitoring technology nr 1: ATP measurement of biofilm 87
3.14 Total cell counts 90
3.14.1 Monitoring technology nr 1: Direct total microbial count (based on fluorescence
microscopy) 90
3.14.2 Monitoring technology nr 2: Direct total microbial count (based on flow cytometry) 92
3.15 Cultivation free viability analysis 95
3.15.1 Monitoring technology nr 1: Cultivation free viability analysis (based on flow
cytometry or fluorescence microscopy) 95
3.15.2 Monitoring technology nr 2: Analysis of bacterial ATP 98
4 Chemical parameters 100
4.1 Metals: Antimony, arsenic, boron, cadmium, chromium, copper, lead, mercury,
nickel, selenium, sodium, calcium, magnesium, aluminum, iron, manganese 100
4.1.1 Monitoring technology nr 1: AAS (Atomic adsorption spectroscopy) 101
4.1.2 Monitoring technology nr 2: AFS (Atomic fluorescence spectroscopy) 102
4.1.3 Monitoring technology nr 3: ICP-OES: Inductively-coupled plasma with optical
emission spectroscopy 103
4.1.4 Monitoring technology nr 4: ICP-MS: Inductively-coupled plasma with mass
spectrometry 104
4.2 Benzene 105
4.2.1 Monitoring technology nr 1: Liquid-liquid extraction, GC-FID or GC-MS 105
4.2.2 Monitoring technology nr 2: Headspace, GC-FID or GC-MS 106
4.2.3 Monitoring technology nr 3: Purge&trap, GC-FID or GC-MS 107
4.2.4 Monitoring technology nr 4: Solid-phase micro extraction (SPME), GC-FID or GC-MS108
4.3 Benzo(a)pyrene and other PAHs 110
4.3.1 Monitoring technology nr 1: Liquid-liquid extraction, HPLC/FLD 110
4.3.2 Monitoring technology nr 2: Liquid-liquid extraction, GC/MS 111
4.4 Bromate 113
4.4.1 Monitoring technology nr 1: Ion chromatography with conductivity detection
(IC/CD) 113
4.4.2 Monitoring technology nr 2: Ion chromatography with UV detection (IC/UV) 114
4.4.3 Monitoring technology nr 3: Ion chromatography with fluorescence detection
(IC/FLD) 115


4.4.4 Monitoring technology nr 4: Ion chromatography with inductively coupled plasma
mass spectrometry detection (IC/ICP-MS) 116
4.5 Cyanides 118
4.5.1 Monitoring technology nr 1: Photometric method (batch mode) 118
4.5.2 Monitoring technology nr 2: Continuous flow analysis 119
4.6 1,2-Dichloroethane 121
4.6.1 Monitoring technology nr 1: Liquid-liquid extraction, GC-ECD(-ECD) 122
4.6.2 Monitoring technology nr 2: Headspace, GC-ECD-(ECD) 123
4.6.3 Monitoring technology nr 3: purge&trap, GC-MS 124
4.7 Fluoride 125
4.7.1 Monitoring technology nr 1: Ion-selective electrode (ISE) 125
(IC/CD) 126
4.8 Nitrite 127
4.8.1 Monitoring technology nr 1: Ion Chromatography 127
4.8.2 Monitoring technology nr 2: Wet chemical analysis Colorimetric method 128
4.8.3 Monitoring technology nr 3: Spectrometric method, derivative spectroscopy 130
4.9 Nitrate 132
4.9.1 Monitoring technology nr 1: Wet Chemical Analysis 132
4.9.2 Monitoring technology nr 2: Electrochemical method 134
4.9.3 Monitoring technology nr 3: Spectrometric method, single wavelength 135
4.9.4 Monitoring technology nr 4: Spectrometric method, derivative spectroscopy 137
4.10 Polycyclic aromatic hydrocarbons 139
4.11 Pesticides 139
4.12 Tetra- and trichloroethene 140
4.13 Disinfection byproducts (trihalomethanes) 143
4.14 Radioactivity 146
4.14.1 Semiconductor Detectors 146
4.14.2 Liquid Scintillation Counting 147
4.14.3 Inductively Coupled Plasma Mass Spectrometry 148
4.15 Endocrine disruption chemicals 150
4.15.1 Introduction 151
4.15.2 Analysis of water using bioassay 151
4.15.3 Analysis using bioassays 152
4.15.4 General evaluation of the bioassays 153
4.16 Genotoxicity 157
4.16.1 Introduction 157
4.16.2 Mechanisms of genotoxicity 157
4.16.3 Tests to detect genotoxicity 158
4.16.4 General review 158
4.16.5 Sensitivity and specificity 160
4.16.6 Robustness 161
4.16.7 Time to result 161
4.16.8 Ease of use and instrumentation 161


4.17 Acute toxicity 162
4.17.1 Standard toxicity tests 162
4.17.2 Monitoring technology nr 2: Daphnia toximeter 164
4.17.3 Monitoring technology nr 3: Fish toximeter 166
4.17.4 Monitoring technology nr 4: Combined Fish and Daphnia toximeter 170
4.17.5 Monitoring technology nr 5: Algae toximeter 172
4.17.6 Monitoring technology nr 6:Luminiscent bacteria 174
4.17.7 Monitoring technology nr 7: Mussel monitor 175
4.18 Algae toxins 178
4.18.1 Monitoring technology nr 1-4: LC-DAD, LC-MS/MS, ELISA, PPIA 179
4.19 Pesticides, pharmaceuticals, industrial chemicals and other organic micropollutants183
4.19.1 Monitoring technology nr 1: Gas Chromatography-Mass Spectrometry 183
4.19.2 Monitoring technology nr 2: High-Performance Liquid Chromatography-UltraViolet
Diode Array Detection 184
4.19.3 Monitoring technology nr 3: High-Performance Liquid Chromatography-Mass
Spectrometry 185
4.20 pH 187
4.21 Monitoring technology nr 1: pH indicator 187
4.21.1 Monitoring technology nr 2: pH meter 189
4.22 Chloride/nitrate/sulphate 191
(IC/CD) 191
4.23 Conductivity 192
4.23.1 Monitoring technology nr 1: Conductimeter 192
4.24 Calcium & magnesium 194
4.25 Sulphate 194
4.26 Aluminium 194
4.27 Ammonium 195
4.27.1 Monitoring technology nr 1: Ion Selective Electrode 195
4.27.2 Monitoring technology nr 2: Ion Chromatography 196
4.27.3 Monitoring technology nr 3: Photometric test kit 197
4.27.4 Monitoring technology nr 4: Automatic Analyser 198
4.28 Iron 200
4.29 Manganese 200
4.30 Taste & Odor 201
4.30.1 Monitoring technology nr 1: Sensory panel 202
4.30.2 Monitoring technology nr 2: GC- MS 203
4.30.3 Monitoring technology nr 3: Electronic Nose & Tongue 204
4.31 Colour 207
4.31.1 Monitoring technology nr 1: Visual Comparison method 207
4.31.2 Monitoring technology nr 2: Colorimetric analysis 209
4.31.3 Monitoring technology nr 3: Online spectrophotometric measurement 210
4.32 Turbidity 213
4.32.1 Monitoring technology nr 1: Analysis using a laboratory colorimeter 213
4.32.2 Monitoring technology nr 2: Online turbidity meter 214
4.32.3 Monitoring technology nr 3: Online spectrophotometric measurement 216
4.33 AOC 218
4.33.1 Monitoring technology nr. 1: The original van der Kooij assay 218


4.33.2 Monitoring technology nr. 2: The Werner and Hambsch assay 220
4.33.3 Monitoring technology nr. 3: The Stanfield and Jago ATP-based assay 222
4.33.4 Monitoring technology nr. 4: The LeChevallier assay 224
4.33.5 Monitoring technology nr.5: The Eawag-AOC assay 226
4.34 DOC/TOC 228
4.34.1 Monitoring technology nr 1: High temperature combustion 229
4.34.2 Monitoring technology nr 2: Persulfate oxidation 230
4.34.3 Monitoring technology nr 3: UV - oxidation / conductivity 232
4.34.4 Monitoring technology nr 4: UV-spectroscopy 233
4.35 UV absorbing organic constituents 235
4.35.1 Monitoring technology nr 1: Single Wavelength absorption measurement 235
4.35.2 Monitoring technology nr 2: Full spectral analysis 236
4.36 Particle counts 239
4.36.1 Monitoring technology nr 1: Particle counting systems 239
4.37 Oxygen 243
4.37.1 Monitoring technology nr 1: Titration 243
4.37.2 Monitoring technology nr 2: Electrochemical Measurement 244
4.37.3 Monitoring technology nr 3: Optical Measurement 246
Annex I. Individual evaluation forms for endocrine disrupting effects 249
Annex II. Individual evaluation form for genotoxic effects 281


1 Introduction
Monitoring and control technologies are indispensable for the production of
safe drinking water. They allow for the surveillance of source water quality
and the detection of biological and chemical threats, thus defining the
boundary conditions for the subsequent treatment and providing early-
warning in case of unexpected contaminations. They are mandatory for the
permanent control of the treatment process and the efficacy of each single
treatment step, and they safeguard the high quality of finished water.
Furthermore, appropriate analytical techniques are indispensable for the
detection of changes in water quality during distribution and for monitoring
drinking water quality at consumers tap. Reliable monitoring technologies
contribute to a large extent to the consumers trust in a high drinking water
quality.

Following the overall objective of the TECHNEAU project, the major
objective of WA 3 is to provide a set of analytical techniques and methods
that ensure the provision of safe high quality drinking water that has the trust
of the consumers. In WP 3.1 existing monitoring technologies are evaluated
according to their suitability for application in controlling water quality in the
whole drinking water production process. This evaluation includes not only
basic analytical techniques, but also new and innovative monitoring
technologies like effect-related DNA-arrays or electronic nose technology.

A first report resulting from WP 3.1 dealt with selection of key-parameters to
assess water quality. It described the different locations and purposes in
which monitoring and control technologies need to be applied. The respective
biological and chemical water quality parameters that provide essential
information for water suppliers (so-called 'key-parameters') are identified and
listed.
The current report is a follow-up and describes the results of a survey on
monitoring technologies for the selected key-parameters. The existing
monitoring technologies are identified and evaluated based on information
on e.g. ease-of-use, maintenance requirements, cists, and technical
specifications. Also the suitability of the techniques for use in small-scale-
systems (3S) is evaluated.

This report can be used as reference when deciding on the analytical chemical
and biological techniques to be used for monitoring water quality from source
to tap.


2 Method
All evaluations of the monitoring technologies have been made by the
participants in WA3. Although there is a large expertise among the WA3
partners, it should be kept in mind that these evaluations are based on
personal experience and judgement and should not be taken as absolute.
Evaluations focus on the methods for the selected key-parameters, not on the
suitability or accuracy of instruments from different suppliers.

The basis for this report is the table that was prepared in the TECHNEAU
report Monitoring and control of drinking water quality - Selection of key-
parameters (Mons et al., 2007, see www.techneau.org ). This table is presented
in table 1.
To prepare evaluations in a uniform format, and make sure that evaluations
from different partners include information on similar aspects, a standard
evaluation form was prepared. The basic evaluation form is shown in Fig 1.
As most expertise within the WA3 partners lies in the field of chemical
and/or microbiological techniques, the process parameters (including
inhibitors) were left out of this evaluation. Also for radioactivity there was no
good option for a partner to create an evaluation. This problem was solved by
including an evaluation retrieved from the literature.

Fig. 1 Evaluation form


Table 1. Total list of parameters and where they are/can be used
Parameter

C
a
t
c
h
m
e
n
t

S
o
u
r
c
e

w
a
t
e
r

S
W

S
o
u
r
c
e

w
a
t
e
r

G
W

T
r
e
a
t
m
e
n
t

1

F
i
n
i
s
h
e
d

w
a
t
e
r

D
i
s
t
r
i
b
u
t
i
o
n

i
n
g
r
e
s
s

D
i
s
t
r
i
b
u
t
i
o
n

t
i
m
e

r
e
l
a
t
e
d

C
u
s
t
o
m
e
r
s

t
a
p

Microbiological parameters
E. coli
Enterococci
Clostridium perfringens
Total coliforms
Colony count/HPC
Enteric viruses
Giardia/Cryptosporidium
Campylobacter
Legionella
Pseudomonas aeruginosa
Aeromonas
F-specific RNA phages
Aerobic spore-forming
bacteria

Biofilm formation
Total cell counts
Cultivation-free viability
analysis

Chemical parameters
antimony
arsenic
benzene
benzo(a)pyrene
boron
bromate
cadmium
copper
chromium
cyanides
1,2-dichloroethane
fluoride
lead
mercury
nickel
nitrite
nitrate
PAHs
pesticides
selenium
tetra- & trichloroethene
disinfection byproducts
2

radioactivity
EDCs
genotoxicity
acute toxicity
algae toxins


Parameter

C
a
t
c
h
m
e
n
t

S
o
u
r
c
e

w
a
t
e
r

S
W

S
o
u
r
c
e

w
a
t
e
r

G
W

T
r
e
a
t
m
e
n
t

1

F
i
n
i
s
h
e
d

w
a
t
e
r

D
i
s
t
r
i
b
u
t
i
o
n

i
n
g
r
e
s
s

D
i
s
t
r
i
b
u
t
i
o
n

t
i
m
e

r
e
l
a
t
e
d

C
u
s
t
o
m
e
r
s

t
a
p

pharmaceuticals
industrial chemicals
organic micropollutants
3

pH
chloride
alkalinity
saturation index
4

sodium
conductivity
calcium
magnesium
sulphate
aluminum
ammonium
iron
manganese
taste
odour
colour
turbidity
AOC/BDOC
DOC/TOC
UV absorption
particle counts
oxygen
inhibitors

Process parameters
5

head loss
filter velocity
residence time
ozone dose, contact time (Ct)
ozone concentration
residual ozone
UV dose
oxidant dose
residual oxidant conc.
disinfectant dose
residual disinfectant conc.
inhibitors
sediments (e.g. iron oxides)
flow rate
transmembrane pressure
pressure drop
particle size distribution
membrane (bio)fouling

1. Various parameters are suitable/preferable for different treatment steps. For details see TECHNEAU
report "Monitoring and control of drinking water quality. Selection of key-parameters".


2 disinfection by-products: chlorination by-products, ozonation by-products, UV/AOP by-products
3 general group, consisting of e.g. pharmaceuticals, industrial pollutants etc
4 saturation in dex is only a calculation and will not be further evaluated in this report
5 process parameters will not be evaluated in this report


3 Microbiological parameters
3.1 E. coli and coliform bacteria

Prepared by: TZW

Required technical specifications:
For the detection and enumeration of E. coli and coliform bacteria in drinking
water a detection limit of one bacterial cell / 100 mL water sample is
required. Methods suitable for analyses of drinking waters with higher
bacterial numbers or source waters have to provide a high selectivity to avoid
interference with background flora.

Monitoring technologies:
1. Lactose TTC Tergitol Method (ISO 9308-1)
2. Colilert-18/Quanty-Tray (IDExx, UK National Standard Method W 18)
3. Membrane Lauryl Sulphate Agar (MLSA) (NEN 6553)
4. Membrane Lauryl Suphate Broth (MLSB) (UK National Standard Method
W 2)
5. Chromocult Coliform Agar (Merck)

3.1.1 Monitoring technology nr 1: Lactose TTC Tergitol Method (ISO 9308-1)

Description:
The European Drinking Water directive (EU DWD) defines Lactose TTC
Tergitol Method according to ISO 9308-1 as reference method for the
detection and enumeration of E. coli and coliform bacteria.

Method and mode of action
E. coli and coliform bacteria are detected and enumerated by membrane
filtration and subsequent culture on the differential agar medium Lactose
TTC Tergitol as described in ISO 9308-1. Lactose is degraded to acid by E. coli
and coliform bacteria which is indicated by a colour change of the medium.
Tergitol 7 (sodium heptadecylsulfate) and TTC (triphenyl-
tetrazoliumchloride) inhibit the growth of gram positive non-target
organisms. TTC is also part of the differential system. The reduction of TTC
by lactose negative bacteria produces dark red colonies, whereas lactose
positive E. coli and coliform bacteria reduce TTC only weakly resulting in
yellow-orange colonies.

Procedure and Evaluation
100 mL water sample is filtered through a membrane filter. The membrane
filter is transferred to Lactose TTC Tergitol Agar and incubated at 36 2C for
21 3 hours. Lactose positive bacteria produce yellow-orange colonies and
under the membrane yellow-orange halos. The count of these typical colonies
is considered to be presumptive coliform bacteria count. For confirmation of
E. coli and coliform bacteria count further subculture of typical colonies on a
non selective agar ( e.g. Tryptic Soy Agar) for oxidase test and in Tryptophane


Broth for indole production is required. Colonies that are oxidase negative are
considered to be coliform bacteria. Coliform bacteria that form indole from
tryptophane at 44 0.5C within 21 3 hours are considered to be E. coli.
Results are obtained after 1 day (negative results) or 2 days.

Equipment and consumables
As described in ISO 9308-1 (e.g. autoclave, incubator, water bath, filtration
unit, scale, pH-meter, gas burner, glassware, Petri dishes, membrane filter,
Lactose TTC Agar with Tergitol 7, Tryptic Soy Agar, Tryptophane Broth,
Kovacs-Indole and oxidase reagent etc.).

References
Anon. (2000) ISO 9308-1: Water quality - Detection and enumeration of
Escherichia coli and total coliform bacteria - Part 1: Membrane filtration
method (ISO 9308-1:2000). Geneva, Switzerland: International Organisation
for Standardisation.

Evaluation:
The membrane filtration method ISO 9308-1 for the detection and
enumeration of E. coli and coliform bacteria is suitable for disinfected waters
and other drinking waters with low bacterial numbers. Due to the low
selectivity of Lactose TTC Tergitol Agar background growth can interfere
with the reliable enumeration of E. coli and coliform bacteria. It is therefore
not recommended for drinking waters with high bacterial numbers or for
source waters. For these waters alternative methods e.g. Colilert-18/Quanti-
Tray (see monitoring technology nr 2) or MLSA (see monitoring technology
nr 3) are more suitable. The costs for consumables are low, but experienced
laboratory staff is required for test performance and evaluation. Time to
result is 1-2 days.


Monitoring technology nr 1: Lactose TTC Tergitol Agar (ISO 9308-1)
Criteria 1 2 3 4 5 Comments
Technical specifications
sensitivity (A)
source water
drinking water

x

x

Heavy background growth can
occur , only for waters with low
bacterial numbers (e.g.
disinfected drinking water)
robustness (A)
operational robustness
selectivity

x

x

Growth of non target organisms
can occur
time to result

1-2 days
Operational specifications
ease-of-use (B) x
maintenance requirements (C) x

Costs
instrumentation (C) x
operational costs (C)
consumables
maintenance

x
x

Recommendation for use in SSS (D) x

Overall conclusion Inexpensive method, but requires experienced laboratory
staff. Due to low selectivity, it is only recommended for
very clean water samples
(A): 1 = very low 2 = low 3 = average 4 = high 5 = very high
(B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good
(C): 1 = very high 2 = high 3 = average 4 = low 5 = very low
(D): 2 = no 3 = yes 4 = strong
SSS: small-scale systems

3.1.2 Monitoring technology nr 2: Colilert-18/Quanti-Tray (IDExx, UK National
Standard Method W 18)

Description:
Colilert-18/Quanti-Tray has been approved as alternative method for the
detection and enumeration of E. coli and coliform bacteria according to the
EU DWD in a number of EU countries e.g. Germany, Italy, Czech Republic
and Hungary. Colilert-18/Quanti-Tray is included in American and UK
Standard Methods.

Colilert-18/Quanti-Tray is a Most Probable Number test and allows
simultaneous detection of E. coli and coliform bacteria. The method is based
on an enzyme substrate reaction. The major carbon sources are ONPG (o-
nitrophenyl- -D-galactopyranoside) and MUG (4-methyl-umbelliferyl--D-
glucuronide), which are metabolised by the enzymes -galactosidase


(coliform bacteria) and -glucuronidase (E. coli). When ONPG is metabolised
by coliform bacteria the medium changes from colourless to yellow. The
degradation of MUG by E. coli creates fluorescence. Most non-coliforms do
not have these enzymes and are unable to grow and interfere. Furthermore,
Colilert uses Defined Substrate Technology (DST) to inhibit growth of
non-target organisms.

Procedure and evaluation
100 mL water sample is transferred to a sterile bottle with antifoam reagent.
Colilert reagent is added and sample is mixed until reagent is completely
dissolved. Sample/reagent is filled in a sterile Quanti-Tray. The tray is
sealed and incubated for 18 - 22 h at 36C. Positive wells are counted (yellow
= coliform bacteria, yellow and fluorescent (UV light) = E. coli) and the
numbers of E. coli and coliform bacteria are determined from the MPN tables.

As described e.g. in the UK National Standard Method W 18 (e.g. incubator,
water bath, gas burner, Quanti-Tray heat sealer, long wavelength UV light
source and viewer, Quanti-Trays and reference comparator, Colilert
reagent, antifoam reagent, sterile glassware).

References
IDExx Laboratories, Milton Court, Churchfield Road, Chalfont St Peter,
Buckinghamshire, SL9 9EW.

Health Protection Agency (2004). Enumeration of coliforms and Escherichia
coli by Idexx (Colilert 18) Quanti-Tray
TM
. National Standard Method W 18
Issue 2.
http://www.hpastandardmethods.org.uk/pdf_sops.asp.

Chromogenic Substrate Coliform Test (9223) (1997) in: Standard Methods for
the Examination of Water and Wastewater, 21st Edition (2005), American
Public Health Association, 1015 Fifteenth Street, NW, Washington, DC 20005.

Evaluation:
The Colilert-18/Quanti-Tray method is applicable to the enumeration of E.
coli and coliform bacteria in drinking water, source water and environmental
water. Due to the presence of -galactosidase in some species which are
unable to produce acid from lactose using Colilert-18 Quanti-Tray can result
in higher coliform numbers compared to other culture-based tests (e.g.
Lactose TTC Tergitol Method or MLSA). The method is easy to perform and
does not require further identification tests. It can be performed by less
experienced laboratory staff and is suitable for small water supplies, but
higher costs for consumables incur. Test results are available after 1 day.


Monitoring technology nr 2: Colilert-18/Quanti-Tray (IDExx, UK National
Standard Method W 18)
sensitivity (A)
source water
drinking water

x
x

No interference in waters with
higher bacterial numbers
robustness (A)
selectivity

x
x

time to result

1 day
ease-of-use (B) x

Costs
consumables
maintenance

x

x


Overall conclusion Fast and easy to use method. More expensive compared to
other culture-based tests due to higher costs for
consumables. Is suitable for drinking water and source
water.

3.1.3 Monitoring technology nr 3: Membrane Lauryl Sulphate Agar (MLSA) (NEN 6553)

Description:
Membrane Lauryl Sulphate Agar (MLSA) has been approved as alternative
method for the detection and enumeration of E. coli and coliform bacteria
according to the EU DWD in the Netherlands.

E. coli and coliform bacteria are simultaneously detected and enumerated by
membrane filtration and subsequent culture on the differential agar medium
MLSA which contains lactose as major carbon source. E. coli and coliform
bacteria degrade lactose to acid which is indicated by a change of the colony
colour to yellow. Laurylsulphate inhibits the growth of non-target organisms.
Yellow oxidase negative colonies are considered as coliform bacteria and
yellow oxidase negative colonies which produce indole from tryptophane are
considered as E. coli.


100 mL water sample is filtered through a membrane filter. The membrane
filter is transferred to MLSA and incubated at 25 1 C for 5 1 hours
followed by incubation at 36 2 C for 14 2 hours. Typical yellow lactose-
positive colonies are transferred to a non-selective agar and incubated at 36
2 C for 21 3 hours to test for oxidase activity. Parallel the same colonies are
transferred to tryptophane broth and incubated at 44 0.5 C for 21 3 hours
to test for indole production. Yellow colonies which are oxidase negative are
considered total coliforms. Those being oxidase negative and indole positive
are considered E. coli. Results are obtained after 1 day (negative results) or 2
days.

As described in NEN 6553 (e.g. autoclave, incubator, water bath, filtration
unit, scale, pH-meter, gas burner, glassware, Petri dishes, membrane filter,
MLSA, Tryptone Soy Agar, Tryptophane Broth, Kovacs-Indole and oxidase
reagent etc.).

References
Anon. 1981: NEN 6553. Bacteriological analysis of drinking water.
Quantification of coliform bacteria using membrane filtration. [In Dutch] NNI,
Delft, The Netherlands.

Evaluation:
The membrane filtration method NEN 6553 for the detection and
enumeration of E. coli and coliform bacteria is suitable for drinking waters
with various contamination levels and for source water. MLSA is more
selective for target organisms compared to Lactose TTC Tergitol Agar (ISO
9308-1). However, it is not recommended for highly contaminated surface
water. The costs for consumables are low, but experienced laboratory staff is
required for test performance and evaluation. Late sample delivery can cause
inconvenience for the laboratory workflow due to the two-stage incubation
procedure. Time to result is 1-2 days.


Monitoring technology nr 3: Membrane Lauryl Sulphate Agar (MLSA) (NEN
6553)
sensitivity (A)
source water
drinking water

x

x

robustness (A)
selectivity

x
x

More selective than Lactose TTC
Tergitol Agar
time to result

1-2 days
ease-of-use (B) x

Costs
consumables
maintenance

x
x


staff. Late sample delivery can cause inconvenience due to
the two-stage incubation procedure. Is suitable for water
samples with various contamination levels.

3.1.4 Monitoring technology nr 4: Membrane Lauryl Suphate Broth (MLSB) (UK
National Standard Method W 2)

Description:
Membrane Lauryl Sulphate Broth (MLSB) has been approved as alternative
method for the detection and enumeration of E. coli and coliform bacteria
according to the EU DWD in the UK. MLSB is included in the UK National
Standard Methods.

E. coli and coliform bacteria are detected separately and enumerated by
membrane filtration and subsequent culture on an absorbent pad saturated
with MLSB as differential medium.
MLSB contains lactose as major carbon source, which is degraded to acid by
E. coli and coliform bacteria, indicated by a change of the colony colour to
yellow. Laurylsulphate inhibits the growth of non-target organisms. Yellow


colonies are to be confirmed as E. coli and coliform bacteria by further
confirmatory tests (acid from lactose, oxidase activity, indole production).

100 mL of water sample is filtered through a membrane filter (2 filters for
each sample). The 2 membrane filters are transferred to pads soaked with
MLSB and incubated (at 30 1 C for 4 1 hours followed by incubation at
37C 1 C for 14 1 hours for coliform bacteria and at 30 1 C for 4 1
hours followed by incubation at 44 2 C for 14 1 hours for E. coli). Yellow
colonies are counted on each pad as presumptive coliform bacteria (37 C)
and presumptive E. coli (44 C). Yellow colonies from both membrane filters
are transferred to Lactose Peptone Water (LPW), MacConkey Agar (MA) and
Nutrient Agar (NA) for incubation at 37 C for 20 4 hours and to LPW and
Trypton Water (TP) for incubation at 44 C for 20 4 hours. Colonies grown
on NA are checked for oxidase activity and indole test is done in TP. Coliform
bacteria produce red colonies on MA, are oxidase negative and produce acid
from lactose in LPW at 37 C. E. coli produces acid from lactose at 44 C, is
oxidase negative and indole positive. Results are obtained after 1 day
(negative results) or 2-3 days.

As described in the UK National Standard Method W 2 (e.g. autoclave,
incubator, water bath, filtration unit, scale, pH-meter, gas burner, glassware,
Petri dishes, membrane filter, Membrane Lauryl Sulphate Broth, Nutrient
Agar, McConkey Agar, Kovacs-Indole and oxidase reagent etc.).

References
Health Protection Agency (2007). Enumeration of coliform bacteria and
Escherichia coli by membrane filtration. National Standard Method W 2 Issue
4. http://www.hpastandardmethods.org.uk/pdf_sops.asp.

Evaluation:
The membrane filtration method NSM W 2 for the detection and enumeration
of E. coli and coliform bacteria is suitable for drinking waters with various
contamination levels and for source water. MLSB is like MLSA more selective
for target organism compared to Lactose TTC Tergitol Agar (ISO 9308-1).
However, it is also not recommended for highly contaminated surface water.
The costs for consumables are low, but experienced laboratory staff is
required for test performance and evaluation. Compared to MLSA the MLSB
method is more involved and labour-intensive. Late sample delivery can
cause inconvenience for the laboratory workflow due to the two-stage
incubation procedure. Time to result is 1-3 days.


Monitoring technology nr 4: Membrane Lauryl Suphate Broth (MLSB) (UK
National Standard Method W 2)
sensitivity (A)
source water
drinking water

x

x

robustness (A)
selectivity

x

x

More selective than Lactose TTC
Tergitol Agar
time to result

1-3 days
ease-of-use (B) x

Costs
consumables
maintenance

x
x


staff. Late sample delivery can cause inconvenience due to
the two-stage incubation procedure. Is suitable for water
samples with various contamination levels.

3.1.5 Monitoring technology nr 5: Chromocult Coliform Agar (Merck)

Description:
Chromocult Coliform Agar (Merck) is a selective agar for the simultaneous
detection of total coliforms and E. coli in drinking water and processed food
samples. The approval of this method by US-EPA is pending. Furthermore, it
has been taken into consideration to develope an ISO standard.

E. coli and coliform bacteria are detected and enumerated by membrane
filtration and subsequent culture on CCA as selective and differential agar
medium. CCA contains chromogenic substrates which change colour to
salmon-red when degraded by -galactosidase positive coliform colonies and
to dark blue-violet when degraded by -galactosidase and -glucuronidase
positive E. coli colonies. Non-target organisms are largely inhibited by
addition of Tergitol 7 and E. coli/Coliform Selective-Supplement. In case of
growth colonies of non-target organisms appear colourless or light-blue.


100 mL of water sample is filtered through a membrane filter. The membrane
filter is placed on CCA and incubated at 35-37 C for 24 hours. Salmon to red
-galactosidase positive colonies are counted as non E. coli coliforms. No
further confirmatory tests are required for non E. coli coliforms. Dark-blue to
violet -galactosidase and -glucuronidase positive colonies are counted as E.
coli and are confirmed by testing for indole production. Results are available
after 1 day.

Usual laboratory equipment and in addition: autoclave, incubator, water
bath, filtration unit, scale, pH-meter, gas burner, glassware, Petri dishes,
membrane filter, Chromocult Coliform Agar (see Merck description Cat.
No. 1.10426.0100/500).

References
Ossmer, R., Schmidt, W., Mende, U. (1999). Chromocult Coliform Agar -
Influence of Membrane Filter Quality on Performance. - xVII Congresso de la
Sociedad, Granada.

Finney, M., Smullen, J., Foster, H.A., Brokx, S., Storey, D.M. (2003). Evaluation
of Chromocult coliform agar for the detection and enumeration of
Enterobacteriaceae from faecal samples from healthy subjects. Journal of
Microbiological Methods 54: 353-358.

Evaluation:
Chromocult Coliform Agar should be suitable for the enumeration of E. coli
and coliform bacteria in drinking water, source water and environmental
water. Due to the presence of -galactosidase in some species which are
unable to produce acid from lactose CCA can like Colilert-18/Quanti-Tray
result in higher coliform numbers compared to other culture-based tests. No
further cultivation step is required for confirmation, hence the method is
faster and can be performed by less experienced laboratory staff than. Test
results are obtained after 1 day. Validation data are not yet available


Monitoring technology nr 5: Chromocult Coliform Agar (Merck)
sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

time to result

1 day
ease-of-use (B) x

Costs
consumables
maintenance

x
x


Overall conclusion Fast method, no further cultivation step is required for
confirmation. Suitable for drinking water and source water


3.2 Intestinal enterococci

Prepared by: Kiwa Water Research

The concentration range that is normally encountered in water samples is:
0 cfu per 100 ml water sample in drinking water and groundwater
0 to 1000 cfu per 100 ml in surface water

The required resolution of the method is:
1 cfu in 100 ml water sample

1. ISO method 7899-1: MPN cultivation in liquid MUD/SF medium.
2. ISO method 7899-2: membrane filtration and cultivation on agar medium
containing azide and 2,3,5-triphenyltetrazoliumchloride.

3.2.1 Monitoring technology nr 1: ISO method 7899-1

Description:
- The diluted sample is inoculated in a row of microtitre plate wells
containing dehydrated MUD/SF culture medium. The microtiter plates are
examined under ultraviolet light at 366 nm in the dark after an incubation
period of between 36 h and 72 h at 44C 0.5 C. The presence of enterococci
is indicated by the fluorescence resulting from the hydrolysis of 4-
methylumbelliferyl--D-glucoside (MUD). The results are given as Most
Probable Number (MPN) per 100 ml.

- To perform ISO method 7899-1 an apparatus for sterilization by dry heat or
steam, a thermostatic incubator, a membrane filtration apparatus, a tunnel
drier or vertical laminar air flow cabinet, UV observation chamber (Woods
lamp 366 nm), pre-set 8-channel multi-pipette and sterile microtiter plates are
required.

- The method is one of the two ISO-approved methods for the enumeration of
intestinal enterococci in water.

- Reference: ISO 7899-1.

Evaluation:
- In general, an accurate method to determine the number of enterococci in
surface water samples, but enumeration is based on MPN, which might be
less reliable than the colony count method described below.
- The detection limit of the method is 15 bacteria per 100 ml, which is too high
for use with drinking water.
- The use of microtiter plates, liquid media and multi-pipette makes the
method more robust than the colony count method described below.


- Incubation time is rather long, because results are obtained after 1.5 to 3
days. However, a fast standard method is not available, although methods
based on DNA-detection have been published and might come available in
the near future.
-It is known that in some water samples (especially sea-water) other micro-
organisms might give false-positive results. However, the other ISO-method
described below has a similar limitation because with that method agar plates
can be overgrown with other micro-organisms, preventing reliable
enumeration of intestinal enterococci colony types.
- A disadvantage compared to monitor technology 2 is that the method does
not contain confirmation steps.

Monitoring technology nr 1: ISO-method 7899-1

sensitivity (A)
source water
drinking water

x

x
Detection limit: 15 bacteria in 100
ml water
robustness (A)
selectivity

x

x

time to result

x Total incubation time: 36 to 72 h
ease-of-use (B) x

Costs
consumables
maintenance
x


Overall conclusion Acceptable method to determine intestinal enterococci in
surface waters. Not applicable to drinking water, because
the detection limit of the method is too high (15 bacteria per
100 ml). Some water samples might give false-positive
results. Quantification is based on MPN. Positive results are
not confirmed by additional reactions. Because of the
cultivation step this standard method is rather time
consuming; a faster standard method is preferred, but not
yet available.



Description:
- A specified volume of water sample is filtered through a membrane filter
with a pore size (0.45 m) sufficient to retain the bacteria. The filter is placed
on a solid selective medium containing sodium azide (to suppress growth of
Gram-negative bacteria) and 2,3,5-triphenyltetrazolium chloride, a colourless
dye, that is reduced to red formazan by intestinal enterococci. Typical
colonies are raised, with a red, maroon or pink colour, either in the centre of
the colony or throughout.

If typical colonies are observed, a confirmation step is necessary, by transfer
of the membrane, with all the colonies, onto bile-aesculin-azide agar,
preheated at 44 C. Intestinal enterococci hydrolyse aesculin on this medium
in 2 h. The end-product, 6,7-dihydroxycoumarin, combines with iron(III) ions
to give a tan-coloured to black compound which diffuses into the medium

Confirmed colonies are expressed as colony forming units (cfu) per 100 ml.

steam, two thermostatic incubators (37C and 44C), a membrane filtration
apparatus, sterile membrane filters (0.45 m) and a water bath (100C) (to
dissolve the agar medium) are required.

- The method is one of the two ISO-approved methods for the enumeration of
intestinal enterococci in water.


Evaluation:
- In general, an accurate method to determine the number of enterococci in
water.
- The detection limit of the method is 1 cfu per 100 ml, which makes the
method suitable for determining intestinal enterococci in drinking water.
- The use of selective agar media and membrane filtration and the
interpretation of colony characteristics make the method slightly less robust
than the first monitoring technology described above.
- Incubation time is rather long, because results are obtained after 2 days.
However, a fast standard method is not available, although methods based on
DNA-detection have been published and might come available in the near
future.
-It is known that agar plates can sometimes be overgrown with other micro-
organisms, preventing reliable enumeration of intestinal enterococci colony
types (especially in source surface water). However, the other ISO-method
described above has a similar limitation because with that method some
water samples might give false-positive results.
- An advantage compared to monitor technology 1 is that the method
contains a confirmation step, making the chance of false-positive results
lower.


- Because this method is suitable for both source and drinking water, the
method is preferred over monitoring technology nr 1, which was described
above.


sensitivity (A)
source water
drinking water

x
x
Detection limit: 1 bacterium in
100 ml water
robustness (A)
selectivity

x

x

time to result

x Total incubation time: 46 h
ease-of-use (B) x

Costs
consumables
maintenance
x


Overall conclusion Acceptable and ease-to-use method to determine intestinal
enterococci. Applicable to both source and drinking water.
Surface source water samples might give overgrowth of
non-target bacteria on the agar plates. Because of the
cultivation step this standard method is rather time
consuming; a faster standard method is preferred, but not
yet available.


3.3 Clostridium perfringens

Prepared by: vermicon AG

C. perfringens is a rod-shaped, gram-positive, endo-spore forming and non-
motile bacterium of the genus Clostridium. It is strictly anaerobic, but can
survive a short exposure to oxygen. The micro-organism can be detected in
anaerobic zones of soil, in water, foodstuff and in the intestine of humans and
animals.
C. perfringens is selected out of the genus Clostridium to serve as
microbiological parameter, because it is the most important species out of the
sulphite reducing Clostridia and it is normally present in human and animal
faeces.

The limit value for C. perfringens (including spores) according to the Drinking
Water Directive (DWD) is 0 cells/100 ml drinking water. The analysis of this
parameter is only necessary, if the water originates from surface water or is
influenced by surface water. When the limit value is exceeded, the competent
authority will initiate exploratory research to assure that there is no danger
for human health, due to the occurrence of a pathogenic micro-organism.
Furthermore if no C. perfringens is detected in 100 ml drinking water, it can be
assumed that no resistant dormant bodies/cysts of a parasitic protozoa are
present in the water.
For the analysis quantitative detection methods have to be applied. Yet, there
is no approved ISO standard procedure available for the detection of
Clostridium perfringens.

Monitoring and confirmation technologies:
1. Guideline according to DWD - membrane filtration and cultivation on
m-CP agar
2. Membrane filtration and cultivation on TSC agar, subsequent
confirmation tests according to draft of ISO 6461 CD part 2
3. Membrane filtration and cultivation on fluorogenic TSC agar (Araujo
et al., 2004)
4. Clostridium perfringens PCR-based-detection system, Biotecon
Diagnostics, Germany
5. API 32A, biochemical screening test, Biomerieux, France

3.3.1 Monitoring technology nr. 1: Guideline according to Council Directive 98/83/EC -
membrane filtration and cultivation on m-CP agar

Description:
Cultivation-based quantitative method for the enumeration of C. perfringens
in water samples. 100 ml water samples is filtrated on a membrane filter.
Membrane filtration is followed by an anaerobic incubation of the membrane
on m-CP selective agar at 44 +/- 1 C for 21 +/- 3 hours. Opaque yellow
colonies that turn pink or red after exposure to ammonium hydroxide vapors
are counted.


Material & method
m-CP medium is a selective, chromogenic medium for the rapid identification
and enumeration of Clostridium perfringens in water samples. The medium is
designed to improve the differentiation of Clostridium perfringens from other
Clostridia species and background flora.
Chromogenic compounds within the m-CP medium cause Clostridium
perfringens colonies to turn yellow (based on their ability to ferment sucrose),
thus differentiating them from other Clostridium species, whilst colonies of
background flora turn purple (based on their ability, unlike C. perfringens, to
hydrolyse indoxyl-b-D-glucoside).
An additional confirmation of the result is proposed by exposing the culture
plate to ammonium hydroxide. This highly specific reaction causes acid
phosphatase-producing C. perfringens yellow colonies to turn into a
distinctive dark pink color.

The addition of D-cycloserine and polymyxine B, and an incubation
temperature of 44 C improves selectivity of m-CP agar by inhibiting the
growth of gram-negative bacteria and Staphylococci.
No further verification steps are necessary according to the directive.

M-CP agar
Basal medium
Membrane filtration manifold
Sterile filter funnels graduated to 100 ml
Vacuum pump with moisture trap or protective filter, or
alternative vacuum force
Incubator: 44 C +/- 1C
Facilities for anaerobic incubation
Petri dishes
Cellulose ester 0,45 m pore size filters

Evaluation
The fastest method for the detection of C. perfringens is monitoring technology
no. 1. This procedure is based on membrane filtration and subsequent
cultivation on m-CP agar. Time to result is 1 day, the handling is quite simple
and it can be performed with standard laboratory equipment on low cost
basis. Nevertheless, this approach is criticized by experts regarding the poor
sensitivity. Due to this fact another method for the detection of C. perfringens
was established in the draft of ISO 6461 CD part 2.


Monitoring technology nr. 1: Guideline according to Council Directive
98/83/EC - membrane filtration and cultivation on m-CP Medium

sensitivity (A)
source water
drinking water

-
x

Not yet analysed
robustness (A)
selectivity

x
x

time to result

24 h
ease-of-use (B) x

Costs x
instrumentation (C) x Standard water laboratory
equipment
consumables
maintenance

x
x


Overall conclusion Very fast, easy & low cost test, but poor reliability

3.3.2 Monitoring technology nr. 2: Membrane filtration and cultivation on TSC agar,
subsequent confirmation tests (draft of ISO 6461 CD part 2)

Description:
Filtration of 100 ml water sample on a membrane filter (maximal pore size
0,45 m, no specification of filter material is given). Subsequent incubation of
the membrane filter under anaerobic conditions on TSC-Agar at 44 +/- 1 C
for 21 +/- 3 hours. All colonies are counted, that show a black or grey to
yellow brown staining of the TSC agar when viewed either from above or
below the filter. Some colonies may exhibit very faint staining of the medium,
but should still be counted. Further confirmatory tests should be performed
for purposes of identification.

Material & method:
The nutrient base provides optimal conditions for the development of
Clostridia. Colonies producing hydrogen sulfide are characterized by
blackening due to the reaction with sulfite and iron salt. In TSC Agar
cycloserine inhibits the accompanying bacterial flora and causes the colonies,


which develop, to remain smaller. It also reduces a diffuse and thus
disturbing blackening around the C. perfringens colonies.
C. perfringens produces black colonies. Further tests should be performed for
purposes of identification.

Confirmatory tests
Subculture each colony on two blood agar plates. Place one plate in an
incubator for aerobic incubation at 36+/-2 C and the other in an incubator
for anaerobic incubation at the same temperature. Examine the plates after 21
+/- 3 hours for the presence or absence of growth and for purity. Perform
confirmatory tests on subcultures that only grow anaerobically. Colonies of C.
perfringens characteristically produce clear zone of haemolysis on blood agar.

- Confirmatory test a: Buffered Nitrate Motility medium:
Testing for motility
Incoculate by stabing into the medium and incubate under anaerobic
conditions at 36 C +/- 2 C for 21 +/- 3 hours. After incubation examine the
medium for growth along the line of the stab. Motility is evident as diffuse
growth out into the medium away from the stab line.
Nitrate reduction
Mix equal volumes of nitrate reagents A and B immediately before use and
test for the presence of nitrate by adding 0,2 ml to 0,5 ml of this mixture to
each of Buffered Nitrate Motility medium. The formation of a red colour
confirms the presence of nitrite produced by the reduction of nitrate. If a red
colour does not develop within 15 minutes add a small amount of zinc dust
and allow to stand for 10 minutes.

If a red colour develops no reduction of nitrite has taken place and the test is
considered negative. If no red colour develops following the addition of zinc
dust this means no nitrate remains and has been completely converted to
nitrogen gas and the test is recorded as positive.

- Confirmatory test b: Lactose gelatine medium :
Inoculate the medium and incubate anaerobically at 36 C +/- 2 C for 21 +/-
3 hours. Examine the tube for a yellow colour indicating the production of
acid. Chill the tubes for 1- 2 hours at 5 +/- 3 C and check for gelatine
liquefaction. The tubes can be examined after 1 hour but any that have not
been solidified should be returned to the refrigerator for a further hour. If the
medium has solidified reincubate at 36 C +/- 2 C for an additional 21-/- 3
hours and again check for liquefaction of gelatine.

C. perfringens produces black or grey to yellow brown colonies on TSCA agar,
is non-motile, reduces nitrit to nitrate, produces acid from lactose and
liquefies gelatine within 44 +/- 4 hours.

Equipment and consumables:


Tryptose sulphite cycloserine agar
Buffered Nitrate-Motility medium
Nitrate reagent A
Nitrate reagent B
Lactose-gelatine medium:
Blood agar: Columbia agar or any other suitable base with 5 %
horse blood
Petri dishes
0,45 m pore size filters

Evaluation
Monitoring technology nr. 2 (ISO 6461 CD part 2) contains different
confirmatory tests. In comparison to monitoring technology no. 1 this
approach is more time-consuming, but a higher reliability and sensitivity is
achieved. This ISO standard is still not approved yet and comprising
evaluation studies are missing.


Monitoring technology nr. 2: Membrane filtration and subsequent cultivation on TSC
agar and subsequent confirmation tests

sensitivity (A)
source water
drinking water

x

not analysed
robustness (A)
selectivity

x
x

time to result

3-4 days
ease-of-use (B) x
x
Costs
equipment
consumables
maintenance

x
x


Overall conclusion Low cost test, time consuming, handling is feasible, not
robust, but reliability is very good

3.3.3 Monitoring technology nr 3: Membrane filtration and cultivation on fluorogenic
TSC agar

Description:
Membrane filtration procedure is performed according to Monitoring
technology no 1 and 2, but confirmatory tests are not necessary. By the
addition of the fluorogenic substrate 4-Methylumbelliferyl-phosphate (MUP)
Clostridium perfringens colonies can be identified by UV light.

Method
D-Cycloserine inhibits the accompanying bacterial flora and causes the
colonies which develop to remain smaller. It also reduces a diffuse and thus
disturbing blackening around the C. perfringens colonies. 4-
Methylumbelliferyl-phosphate (MUP) is a fluorogenic substrate for the
alcaline and acid phosphatase. The acid phosphatase is a highly specific
indicator for C. perfringens. The acid phosphatase splits the fluorogenic
substrate MUP forming 4-methylumbelliferone, which can be identified as it
fluorescence in long wave UV light. Thus a strong suggestion for the presence


of C. perfringens can be obtained. The acid phosphatase splits the fluorogenic
substrate MUP forming 4-methylumbelliferone which can be identified as it
fluorescence in long wave UV light. Thus a strong suggestion for the presence
of C. perfringens can be obtained. (method by VWR, Germany)

Tryptose sulphite cycloserine agar
Additive for the preparation of TSC-Agar (Base), Fluorocult
TSC-Agar supplement.
Petri dishes
Cellulose ester 0,45 m pore size filters

Evaluation
Monitoring technology nr. 3, which is somehow an enhancement of
monitoring technology nr. 2 (ISO 6461 CD part 2), gives also a higher
reliability and sensitivity compared to monitoring technology nr. 1


Monitoring technology nr. 3: Membrane filtration and cultivation on
fluorogenic TSC agar

sensitivity (A)
source water
drinking water

x

not analysed
robustness (A)
selectivity

x
x

time to result

24 h
ease-of-use (B) x
x
Costs
equipment
consumables
maintenance

x
x


Overall conclusion Low cost and fast test, handling is feasible and reliability is
good

3.3.4 Confirmation technology nr. 1: C. perfringens Detection System (Biotecon
Diagnostics)
Oligonucleotides for the specific detection amplification and detection of C.
perfringens DNA by PCR suitable for gel based detection are provided by
Biotecon Diagnostics. The application of this detection system indicates only
the presence of C. perfringens DNA, while no indication for the presence of
active cells is given. Additionally well trained personal is needed for the
performance of this non-quantitative test. Thus this method can only be used
as a confirmatory test.

3.3.5 Confirmation technology nr. 2: API 32 A (Biomerieux)
API-Test 32 A (Vendor: Biomerieux) is a test for the identification of different
anaerobic bacteria. The biochemical test is a confirmatory test for colonies
and identification is possible within 4 hours.
For the application of this test a pure culture is needed. Thus the test can be
applied after the subculture of a colony as an additional test for the
confirmation of the result.


References
1. Araujo M. Sueiro R.A . Gmez Garrido M.J. (2004) Enumeration of
Clostridium perfringens in groundwater samples: comparison of six
culture media. Journal of Microbiological Methods, 57 (2), 175-180
2. Armon P. and Payment P. (1988) A modification of m-CP medium for
enumerating Clostridium perfringens from water samples. Can. J.
Microbiol. 34 78-79
3. Barthel H. Krger W. Mendel B. Suhr R. Die Trinkwasserverordnung
2001 bewhrt oder revisionsbedrftig. Bundesgesundheitsblatt
Gesundheitsforschung
Gesundheitsschutz 2007, 50: 265-275, Springer Medizin Verlag 2007
4. Payment P. and Franco E. (1993) Clostridium perfringens and somatic
coliphages as indicators of the efficiency of drinking water treatment
for viruses and protozoan cycts. Appl. Environ. Microbiol. 59, 2418-
2124
5. Sartory D. P. (2005) Validation, verification and comparison: Adopting
new methods in water microbiology Revised paper. Water SA Vol. 31
No.3 July 2005

Guidelines
1. Council Directive 98/83/EC of November 1998 on the quality of water
intended for human consumption. Official Journal of the European
Communities
2. Enumeration of Clostridium perfringens by membrane filtration.
Issue no: 3.1 Issue date: 03.05.05 Issued by: Standards Unit,
Evaluations and Standards Laboratory on behalf of the Regional Food,
Water and Environmental Microbiologist Forum. www.evaluations-
standards.org.uk


3.4 Heterotrophic Plate Counts

Prepared by: EAWAG

Definition of HPC
Heterotrophic plate counts are defined as the microbial colonies that form on
semi-solid nutrient-rich growth media after a selected time of incubation at a
selected incubation temperature. The colonies can arise from single cells,
pairs, clusters or strings of cells (also called colony forming units, CFU). The
methodological descriptions below have relied heavily on the methods as
described in 9215 Heterotrophic Plate Counts, In: Standard Methods for the
Analysis of Water and Wastewater (Clesceri et al., 1998), and on the
standardised ISO 6222 method (ISO, 1998).

Analysis of HPC is included in the legislation/guidelines of most countries,
as well as the European Drinking Water Directive (DWD - Council Directive
98/83/EC of 3 November 1998). Current drinking water guidelines give HPC
limits that vary typically in the range of 10 300 colony forming units (CFU)
per mL depending on the country, the water and the specific method which is
used. The DWD states as parametric value only no abnormal change.

1. Method according to ISO 6222
2. R2A method and common variations

3.4.1 Monitoring technology nr 1: ISO 6222

Description:
If necessary the water sample is diluted 10-fold in sterile peptone-water. From
the various dilutions, a volume not exceeding 2 mL is transferred into a sterile
Petri-dish. Sterile, warm Yeast Extract Agar (15 20 mL) is added and mixed
with the sample according to the so-called pour-plate method (see Clesceri et
al (1998) for details). Separately prepared samples are subsequently incubated
at respectively 36 C for 44 h 4 h, and 22 C for 68 h 4 h. After incubation,
the colonies that have formed on the plates are counted. Plates with in excess
of 300 colonies should not be considered for the analysis. The result is given
as the number of colony forming units (CFU) per mL.

Equipment:
Autoclave; incubators (22 C, 36 C)

Consumables:
Sterile Petri dishes; Yeast Extract Agar

Status of the technique
The basic HPC method has been in use for nearly 100 years and is generally
accepted in drinking water treatment as standard indicator of the general


microbiological quality of water. Several European countries have adopted
the ISO 6222 method as standard method according to the DWD (e.g.,
TrinkwV, 2001).

Evaluation:
The method has the advantage that it has been used for a long time and a lot
of experience and data exists in the peer reviewed literature. The main
general disadvantage of all HPC methods is that cultivation only detects a
small percentage (typically ca. 1%) of the total microbial concentration in a
water sample, and that the methodology is time and labour consuming
method. Several authors have suggested that the growth medium, incubation
temperature and incubation time is not optimal to achieve the highest plate
count results (Reasoner and Geldreich, 1985; Uhl and Schaule, 2004; Berney et
al., 2008).

References
ISO, 1998. Water Quality-Enumeration of Culturable Micro-organism-Colony
Count by Inoculation in a Nutrient Agar Culture medium, prEN ISO 6222
European Committee for Standardisation, Brussels.
Clesceri, L.S., Greenberg, A.E. and Eaton, A.D. (Eds.) (1998) Standard
Methods for the examination of water and wastewater; 9215 Heterotrophic
Plate Counts. ISBN 0-87553-235-7
Reasoner DJ and Geldreich EE (1985). A new medium for the enumeration
and subculture of bacteria from potable water. Appl. Environ. Microb. 49: 1-7.
Uhl, W. and Schaule, G. (2004) Establishment of HPC (R2A) for regrowth
control in non-chlorinated distribution systems. International Journal of Food
Microbiology, 92: 317 325.
Berney, M., M. Vital, I. Huelshoff, H.-U. Weilenmann, T. Egli, and F.
Hammes. Rapid, cultivation-independent assessment of microbial viability in
drinking water. Accepted for publication in Water Research, July 2008
TrinkwV, 2001. verordnung ber die Qualitt von Wasser fr menschlichen
Gebrauch (Trinkwasserverordnung - TrinkwV 2001). BGB1 I, Nr. 24, issued
May 28th 2001, pp.959-980.
DWD - Council Directive 98/83/EC of 3 November 1998 on the quality of
water intended for human consumption.


Monitoring technology nr 1: ISO 6222


3.4.2 Monitoring technology nr 2: Plating on R2A medium

Description:
Heterotrophic plate counts (HPC) are estimated according to the R2A method
by the spread plating method in which 0.1 mL of the water sample is spread
out on a pre-prepared sterile agar plate containing R2A medium (see
Reasoner and Geldreich, 1985). If the bacteria in the sample are expected in
high concentratiions, a 10-fold dilution series in sterile phosphate buffer or
physiological solution can be used. R2A-agar plates are typically incubated
for 7-10 days at 22 2C before the colony forming units (CFU) are counted
by eye. Results are expressed as the mean number of bacterial CFU per mL of
water sample.

Variations
sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x
x

time to result

44 h (36 C) and 68 h (22 C)
ease-of-use (B) x

Costs
consumables
maintenance

x

x


Overall conclusion The method detects only a small percentage of the natural
microbial community in a water sample. The nutrient rich
medium used in this method contributes to lower HPC
counts than other methods.


Several alternative methods for HPC determination are described and
standardised. For example, Standard Methods (Clesceri et al., 1998) describe
three different methods (spread plate method, pour plate method, membrane
filter method) and four different agars (Plate Count Agar; m-HPC agar; R2A
agar; NWRI agar). Typical variations on the methods include the use of
alternative growth media (e.g. nutrient agar), different incubation
temperatures and different incubation time periods. In some cases, colony
counting with a magnifying glass or with an automated colony counter has
also been promoted. The membrane filter method can be used if the cell
density of the sample is too low for direct spread plating. The membrane
filters have a diameter of 47 mm and a pore size of 0.2 um. The sample up
from 10 mL can be filtered and the membrane filter is placed on one agar
plate (m-HPC agar is prescribed in this instance) (Clesceri et al., 1998). For an
overview, see Bartram et al., 2003.

Equipment:
Incubator; autoclave

Consumables:
Sterile Petri dishes and sterile medium (e.g. R2A agar)

The method is used for research but not typically included in drinking water
directives/legislation.

Evaluation:
The method has similar disadvantages to the ISO 6222 method. However,
several users agree that the R2A displayes higher numbers for HPC bacteria
when drinking water is analysed (Reasoner and Geldreich, 1985; Uhl and
Schaule, 2004; Berney et al., 2008)

References
Bartram, J., Cotruvo, J., Exner, M., Fricker, C. and Glasmacher, A. (2003)
Heterotrophic plate counts and drinking-water safety. London, UK: IWA
Publishing on behalf of the World Health Organization.
Reasoner DJ and Geldreich EE (1985). A new medium for the enumeration
and subculture of bacteria from potable water. Appl. Environ. Microb. 49: 1-7.
Uhl, W. and Schaule, G. (2004) Establishment of HPC (R2A) for regrowth
control in non-chlorinated distribution systems. International Journal of Food
Microbiology, 92: 317 325.


Monitoring technology nr 2: R2A method


sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x
x

time to result

7 10 days
ease-of-use (B) x

Costs
consumables
maintenance

x

x


Overall conclusion The method detects only a small percentage of the natural
microbial community in a water sample. It is generally
giving higher numbers for drinking water HPC bacteria
compared to the ISO 6222 method.


3.5 Enteroviruses

Prepared by: EAWAG

Enteroviruses are widely present in water environments

Levels of contamination are widely unknown, also because molecular
methods, which are increasingly used, are not quantitative.
Concentrations in freshwaters (Guillot, 2006):
1-100 pfu/L in contaminated surface water
1-10 pfu/100 L in less polluted surface water
1-10 pfu/1000 L in treated drinking water

Since the infectious dose is very low (1-10 infectious particles), detection
methods should be very sensitive.

1. Cell culture methods
2. RT-PCR

3.5.1 Monitoring technology nr 1: Cell culture methods

Description:
Concentration:
- Adsorption/elution technique using positively charged filters or
electronegative filters (APHA, 1998)
- Ultrafiltration
- Flocculation (APHA, 1998)
- Tangential flow ultrafiltration (Bigliardi et al., 2004)

Detection:
Cell cultures for most enteric viruses are commercially available (Fout et al.,
1996). Viruses are grown in cell culture monolayers and form visible plaques
or other visible changes to infected cells in the monolayer.

Evaluation:
The cell culture detection method is regarded as the golden standard.
However, it is time consuming (6-15 days) and requires a lot of expertise in
order to obtain reliable results.


Monitoring technology nr 1: Cell culture methods

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

time to result

The method is rather time
consuming: 6 - 15 days
ease-of-use (B) x Requires expertise with
cell cultures

Costs
consumables
maintenance

x

x


Overall conclusion The cell culture detection method is regarded as the golden
standard. However, it is time consuming and requires a lot of
expertise in order to obtain reliable results.

3.5.2 Monitoring technology nr 2: RT-PCR

Description:
Concentration:
As above

Detection
Many studies report the detection of different enteric viruses using RT-PCR
(for a review see Guillot, 2006)). Integrated-RT-PCR procedures have also
been used (Guillot, 2006).

Evaluation:
Primers for all different kinds of enteric viruses are available. The method
generally has a low detection limit and can be performed rapidly. Humic
substances or other compounds present in the water sample can interfere
with the PCR reaction resulting in a false-negative result. Furthermore,
conventional RT-PCR methods are not quantitative and do not detect
infectivity.


Monitoring technology nr 2: RT-PCR


References
APHA, AWWA, et al. (1998). Standard Methods for the Examination of Water
and Wastewater. 20th ed.

Bigliardi, L., Cesari, C., Zoni, R., Sansebastiano, GE. (2004). The concentration
of viruses in water using the tangential flow ultrafiltration. Recovery
effectiveness in experimental conditions. Ann Ig. 16 (1-2): 281-9.

Fout, GS., Schaefer III, FW., Messer, JW., Dahling, DR., Stetler, RE. (1996). ICR
microbial laboratory manual. Washington, D.C., U.S. Environmental
Protection Agency.

Guillot, E., Loret, J.F. (2006). Waterborne Pathogens. London: Global Water
Research Coalition.
sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

time to result

Time to result 4h, rapid
method
ease-of-use (B) x

Costs
consumables
maintenance

x
x

Recommendation for use in SSS (D) x needs skilled personel

Overall conclusion The method can be performed rapidly. Humic substances or
other compounds present in the water sample can interfere
with the PCR reaction resulting in a false-negative result.
Furthermore, conventional RT-PCR methods are not
quantitative and do not detect infectivity.


3.6 Giardia/Cryptosporidium


Giardia is a genus of protozoan parasites potentially found in water and other
media. The recent taxonomy of the genus Giardia includes the following
species and their potential hosts: G. lamblia (also called G. intestinalis or G.
duodenalis; humans and other mammals); G. muris (rodents); G. agilis
(amphibians); G. psittaci and G. ardeae (birds). As with Cryptosporidium, the
parasite is shed with the faeces as environmentally robust cyst that is
transmitted to a new host. Waterborne outbreaks of giardiasis have been
reported for almost 30 years and in the US it is the most commonly identified
pathogen with more than 100 waterborne outbreaks (Craun 1990).

In the last decade most attention on parasitic pathogens in water was focused
on Cryptosporidium because of its higher resistance to chlorine the most
common drinking water disinfectant.

There are no technical specifications required with respect to Cryptosporidium
in the official European drinking water standards. In the EU directive on
drinking water (Council Directive 98/83/EC) the minimum requirement is
that water intended for human consumption shall be wholesome and clean
defined as free from any micro-organisms and parasites and from any
substances which, in numbers or concentrations, constitute a potential danger
to human health. Consequently, the assumption is made that compliance with
the standards of the Directive will satisfy this minimum requirement. One of
the standards directly related to Cryptosporidium is the standard for C.
perfringens (including spores) of 0/100 ml drinking water. Monitoring of this
parameter is not required unless the water originates from or is influenced
by surface water. In the event of non-compliance with this parametric value,
the Member State concerned must investigate the supply to ensure that there
is no potential danger to human health arising from the presence of
pathogenic micro-organisms, e.g. Cryptosporidium. Member States must
include the results of all such investigations in the reports they must submit
under Article 13(2). Note 2, Annex 1, Part C.

Countries with Cryptosporidium monitoring requirements
The only country where a monitoring requirement for Cryptosporidium in
drinking water has been regulated, is the United Kingdom (Anonymous 1999;
Anonymous 2000). This regulation sets a standard of <1 oocyst per 10 liter of
drinking water to be monitored continuously at a rate of 40 liter/h at least 23
hours a day. With respect to the analysis requirements in the UK regulation
there is a Standard Operational Procedure (SOP) prescribed with no
requirement for the assessment of infectivity, species identity and recovery of
the analysis. A recovery of 30-60% is assumed for this SOP. As to the
concentration, however, strict procedures have been documented in the UK-
regulations for confirmation of concentrations of 0.5 oocysts per 10 liter of
drinking water.


A risk-based approach for Cryptosporidium in drinking water is regulated in
the United States and the Netherlands. In the US the Long Term 2 Enhanced
Surface Water Treatment Rule (LT2) (USEPA 2006) source water monitoring
is required to quantify the decree of log reduction (removal or inactivation)
required to protect public health. The required analytical technique for
Cryptosporidium analysis in the source water is similar to the SOP of the UK
regulation in that they prescribe specific approved and validated methods
with ongoing proficiency testing for quality assurance/quality control
(QA/QC; (Clancy and Hargy 2008)) This method 1622 (US;
www.epa.gov/microbes) was originally developed for 10 liter samples but
has been adapted for larger volumes of 50 1000 liter (McCuin and Clancy
2003).
The risk-based approach in the Netherlands on Cryptosporidium requires
source water monitoring to assess treatment requirement followed by
assessment of treatment elimination efficiency (removal and inactivation) and
drinking water exposure. Water suppliers must demonstrate compliance with
an annual infection risk level of <10
-4
. For analytic requirements the
regulation (AMVD, 2005) refers to the SOP used by KWR (formerly known as:
Kiwa Water Research) and RIVM.

1. Method 1622. Cryptosporidium detection method. (USEPA 2006)
published by US Environmental Protection Agency (EPA;
www.epa.gov/microbes)
2. Supplements of the UK Water Supply Regulation (Anonymous,
1999;2000) published by the Drinking Water Inspectorate (DWI;
www.dwi.gov.uk)
3. Method 1623. Cryptosporidium and Giardia detection method. (USEPA
2006) published by US Environmental Protection Agency (EPA;
www.epa.gov/microbes)
4. ISO 15553: Isolation and identification of Cryptosporidium oocysts and
Giardia cysts from water
5. Method 1622/1623 using Cross flow ultrafiltration (Hemoflow-filter)

3.6.1 Monitoring technology nr 1: Method 1622
Summary of the method (USEPA, 2006)
A water sample is filtered and the oocysts and extraneous materials are
retained on the filter.
Although EPA has only validated the method using laboratory filtration of
bulk water samples shipped from the field, field-filtration also may be used.
Materials on the filter are eluted and the eluate is centrifuged to pellet the
oocysts, and the supernatant fluid is aspirated. The ocysts are magnetized by
attachment of magnetic beads conjugated to anti-Cryptosporidium antibodies.
The magnetized oocysts are separated from the extraneous materials using a
magnet, and the extraneous materials are discarded. The magnetic bead
complex is then detached from the oocysts.
The oocysts are stained on well slides with fluorescently labeled monoclonal
antibodies and 4',6-diamidino-2-phenylindole (DAPI). The stained sample is
examined using fluorescence and differential interference contrast (DIC)
microscopy. Qualitative analysis is performed by scanning each slide well for


objects that meet the size, shape, and fluorescence characteristics of
Cryptosporidium oocysts. Quality is assured through reproducible calibration
and testing of the filtration, immunomagnetic separation (IMS), staining, and
microscopy systems.

Since the interlaboratory validation of EPA Method 1622, inter laboratory
validation studies have been performed to demonstrate the equivalency of
modified versions of the method using the following components (USEPA,
2006):
- IDExx Filta-Max filter
- Pall Gelman Envirochek HV filter
- Portable Continuous-Flow Centrifugation (PCFC)
- Waterborne Aqua-Glo G/C Direct FL antibody stain
- Waterborne Crypt-a-Glo and Giardi-a-Glo antibody stains
- BTF EasyStain antibody stain
- BTF EasySeed irradiated oocysts for use in routine QC samples
3.6.2 Monitoring technology nr 2: UK method
This method is very similar to the method 1622 (Clancy and Hargy 2008).
3.6.3 Monitoring Technology nr. 3: method 1623
This method is similar to the method 1622.
3.6.4 Monitoring technology nr 4: ISO 15553
This method is similar to the method 1622.
3.6.5 Monitoring technology nr 5: cross flow ultrafiltration
This method is identical to the method 1622/1623 with respect to the
treatment of the water concentrate and microscopical counting of the
(oo)cysts. The method uses cross flow ultrafiltration (hemo-flow) for
concentration of the particulates (all microbes) in the water and was first
described by Simmons et al. (2001). The method was further developed for on
site sampling (Veenendaal and Brouwer-Hanzens 2007).

Evaluation (based on Medema, 2008):

Recovery
An important drawback of the current concentration methods is that many
factors in the water matrix (suspended solids, algae) and also age/history of
the oocysts can have significant effect on the recovery efficiency.
Ideally, the recovery efficiency is determined for every sample. Since this is
laborious and expensive, recovery efficiency data are usually collected from a
subset of samples. (Warnecke, Weir et al. 2003) describe the use of pre-stained
oocysts that can be discriminated from natural oocysts for seeding of every
sample.

Viability/infectivity
Another drawback of the immunofluorescence detection assay is that it does
not allow differentiation of viable from dead oocysts. DIC microscopy can be
used to determine if the internal morphology is compromised as an indication
of non-viability. DAPI (diaminophenylindole)-staining is used as support-


stain that allows the assessment of the presence of sporozoites in the oocyst,
again as mark of viability. Vital staining (PI (Campbell, Robertson et al. 1992)
or Syto59 (Belosevic and Finch 1997)) can be used in combination with the
IFA test and gives an indication of cell membrane integrity. These dye
exclusion assays provide some information about viability, but should be
used with caution as they can (largely) overestimate viability of oocysts that
have been exposed to stressors such as exposure to UV light (Clancy, Hargy
et al. 1998).
Another assay to assess the viability of Cryptosporidium oocysts is cell culture.

Specificity
The specificity of the immunofluorescence assay is based on the specificity of
the monoclonal antibody-antigen reaction. Although this is highly specific,
non-specific binding is observed in natural samples. Many of the particulates
that react with the monoclonal antibody can be discriminated from oocysts by
a trained observer, but occasionally particles (algae) occur in samples that are
very difficult to discriminate from oocysts. This may lead to false-positive
results. The immunofluorescence method is also not specific to
Cryptosporidium species and genotypes that are infectious to humans, also
species that are infectious to animals are detected. Molecular techniques
(PCR, genotyping) are rapidly evolving and some laboratories are now using
these methods for environmental monitoring (Xiao, Alderisio et al. 2001;
LeChevallier 2004; Xiao, Bern et al. 2004; Heijnen, Wullings et al. 2005).

References
AMVD (2005). Inspection Guidance Document 'Analysis of the
microbiological safety of drinking water'. Ministry of Public Housing,
Spatial Planning and the Environment. [In Dutch].
Anonymous (1999). Water Supply (Water Quality) (Amendment)
Regulations, SI 1524. Stationery Office, London, UK.
Anonymous (2000). Water Supply (Water Quality) Regulations, SI 3184.
Stationery Office, London UK..
Belosevic, G. M. and G. F. Finch (1997). Int. Symp on Waterborne
Cryptosporidium, Newport Beach Ca, USA.
Campbell, I., L. J. Robertson, et al. (1992). "Viability of Cryptosporidium
parvum oocysts - correlation of in vitro exystation with inclusion or
exclusion of fluorgenic vital dyes.." Appl. Environ. Microbiol. 58: 3488-
3493.
Clancy, J. F. and T. M. Hargy (2008). Waterborne: drinking water.
Cryptosporidium and Cryptodiosis. R. Fayer and L. Xiao. Boka Raton
London New York, CRC Press: 305-326.
Clancy, J. L. (2000). "Sydney's 1998 water quality crisis." J. Am. Water Works
Assoc. 92: 55-66.
Clancy, J. L., T. M. Hargy, et al. (1998). "UV light inactivation of
Cryptosporidium oocysts." J. Am. Water Works Assoc. 90(9): 92-102.
Craun, G. F. (1990). Waterborne giardiasis.. Human parasitic diseases. E. A.
Meyer. Amsterdam, the Netherlands, Elsevier Science Publ. 3: 267-293.
Heijnen, L., B. Wullings, et al. (2005). "Genetic analysis of Cryptosporidium
oocysts from surface water." Submitted for publication.


LeChevallier, M. W. (2004). Removal of Cryptosporidium and Giardia by water
treatment processes. Intern. Cryptosporidium and Giardia Conf.,
Amsterdam, the Netherlands.
McCuin, N. E. and J. L. Clancy (2003). "Modification of USEPA method 1622
and 1623 for detection of Cryptosporidium oocysts and Giardia cysts
in water." Appl. Environ. Microbiol. 69: 267-274.
Medema, G.J., Teunis, P.F.M., Blokker, M., Deere, D., Davison, A., Charles, P.
and Loret, J.F. (2008) WHO Guidelines for Drinking Water Quality:
Risk Assessment of Cryptosporidium in drinking water. London UK:
World Health Organization.
Simmons III, Otto D., Mark D. Sobsey, Christopher D. Heaney, Frank W.
Shaefer III and Donna S. Francy(2001). Concentration and Detection
of Cryptosporidium oocysts in Surface Water Samples by Method
1622 Using Filtration and Capsule Filtration. Applied and
Environmental Microbiology vol. 67, No.3, p. 1123-1127
USEPA (2006). "National primary drinking water regulations, long term 2
enhanced surface water treatment rule. Final Rule. Federal Register 40
CFR Parts 9, 141, and 142."
Veenendaal, H. R. and A. J. Brouwer-Hanzens (2007). A method for the
concentration of microbes in large volumes of water. Techneau
Deliverable D3.2.4.
Warnecke, M., C. Weir, et al. (2003). "Evaluation of an internal positive control
for Cryptosporidium and Giardia testing in water samples.." Lett. Appl.
Microbiol. 37(3): 244-248.
Xiao, L., K. Alderisio, et al. (2001). "Identification of species and soources of
Cryptosporidium oocysts in storm waters with a small-subunit rRNA-
based diagnostic and genotyping tool.." Appl. Environ. Microbiol. 66:
5492-5498.
Xiao, L., C. S. Bern, I.M., et al. (2004). Molecular epidemiology of human
cryptosporidiosis. Cryptosporidium: from molecules to disease.. R. C. A.
Thompson, A. Armson and U. M. Ryan. Amsterdam, the Netherlands,
Elsevier.


Monitoring technology nr 1,2,3,4,5: Method 1622, 1623, ISO15553


sensitivity (A)
source water
drinking water

x

x
Recovery of the method has
been improved over time but
still is low and variable (50%)
robustness (A)
selectivity

x

x

The method requires well-
trained analysts (microscopic
counting and oocyst
identification); no differentiation
in species/infectivity
time to result

The method is time consuming
ease-of-use (B) x Once trained, the method is easy
to perform for analysts.
maintenance requirements (C) x Due to the high variability
internal QC is required

Costs
instrumentation (C) x IF Microscopy
consumables
maintenance

x

x


Overall conclusion Available methods to quantify (oo)cysts in water samples
have been optimized to an acceptable level of accuracy and
reproducibility when pre-cautions such as recovery
assessments are implemented in monitoring. Methods to
specify the observed (oo)cysts and to verify the infectivity
though are more comprehensive and only advisable for
research objectives rather than routine monitoring.


3.7 Thermotolerant Campylobacter species

Prepared by: TZW

For the detection and enumeration of thermotolerant Campylobacter species in
drinking water sample volumes of 10 mL, 100 mL and 1000 mL are
recommended to be analysed (ISO 17995:2005).

1. Detection and enumeration of thermotolerant Campylobacter species (ISO
17995:2005)
3.7.1 Monitoring technology nr 1: Detection and enumeration of thermotolerant
Campylobacter species (ISO 17995:2005)

Description:
The International Standard specifies a method for detection and semi
quantitative enumeration of thermotolerant Campylobacter species. The
method can be applied to all kinds of filterable waters. The method can also
be used as a presence/absence test in a specified sample volume. A
quantitative result can be obtained by using a MPN set-up (see ISO 8199).
Thermotolerant Campylobacter species of relevance in human infections
include C. jejuni, C. coli, C. lari and C. upsaliensis.

Thermotolerant Campylobacter species are detected and enumerated by
membrane filtration and subsequent incubation in 2 enrichment broths
(highly and less selective) under microaerobic conditions as described in ISO
17995. Following incubation, inoculum from each broth is streaked onto
selective solid medium (mCCDA) and incubated under microaerobic
conditions. Campylobacter species require enriched substrates for optimal
growth and they prefer an atmosphere containing approx. 5 % oxygen and
approx. 10 % CO2. They are sensitive to toxic oxygen derivates like peroxides,
which can arise in media exposed to light and oxygen. Colonies resembling
Campylobacter species are tested for aerobic growth and, if negative, examined
by microscopy for motility and characteristic morphology. If necessary,
further biochemical tests are performed.

Sample volumes of 10 mL, 100 mL and 1000 mL are filtered through a
membrane filter. For each volume 2 filters are prepared and immediately
transferred to highly (Preston) and less (Bolton) selective enrichment broth.
Both broths are incubated in modified atmosphere at 37 1C for 44 4 hours
leaving caps open. After incubation, approx. 10 L inoculum from each broth
is transferred onto selective mCCDA plates. mCCDA plates are incubated at
modified atmosphere at 41,5 1C and checked for visible growth after 44 4
hours. Suspect colonies from mCCDA are transferred to 2 non-selective agar
plates. Plates are incubated aerobically and in modified atmosphere at 41,5
1C for 21 3 hours. Campylobacter species grow under microaerobic but not


under aerobic conditions. The presence of Campylobacter species is confirmed
by microscopy. Campylobacter species are highly motile, slender rods with
spiral morphology. In doubt, further tests (oxidase, catalase and Gram stain)
are necessary for verification. Campylobacter species are oxidase and catalase
positive and Gram-negative. The results are reported as detected in the
sample volume examined, whether the Campylobacter species are
demonstrated in one or in both enrichment broths. A semi quantitative
estimate of the numbers is made from results with different test volumes.

As described in ISO 17995 (e.g. microscope, autoclave, incubators, equipment
for membrane filtration and microaerobic incubation, glassware, Petri dishes,
usual laboratory equipment, culture media, diluents and reagents).

References
Anon. (2005) ISO 17995: Water quality - Detection and enumeration of
thermotolerant Campylobacter species (ISO 17995:2005). Geneva, Switzerland:
International Organisation for Standardisation.

Anon. (2005) ISO 8199: Water quality - General guide to the enumeration of
microorganisms by culture (ISO 8199:2005). Geneva, Switzerland:
International Organisation for Standardisation.

Evaluation:
The method ISO 17995 for the detection and semi quantitative enumeration of
thermotolerant Campylobacter species is suitable for clean drinking waters and
also for dirty surface waters.
The method is very laborious and costs for consumables are higher compared
to other culture-based methods e. g. for the detection and enumeration of. E.
coli or Enterococci. Furthermore, more expensive laboratory equipment like a
microscope and incubators for microaerobic cultivation is required. Some
Campylobacter species are pathogenic to man and therefore isolation and
identification must be carried out by trained laboratory staff in a properly
equipped laboratory. Time to result is 5 days.


Monitoring technology nr 1: ISO 17995:2005

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

Campylobacter species are very
sensitive to adverse conditions

time to result

5 days
ease-of-use (B) x

Costs
consumables
maintenance

x
x


Overall conclusion Method is laborious and requires a well equipped
microbiological laboratory and experienced and well trained
staff. Method is suitable for all kinds of waters.


3.8 Legionella and Legionella pneumophila


0 - 10
6
cfu per 250 ml in drinking water
0 - 10
6
cfu per 100 ml in water from cooling towers or surface water

The required resolution of the parameter is:
100 cfu in litre drinking water (Dutch legislations)

1. ISO method 11731:1998: Detection and enumeration of Legionella by direct
membrane filtration and cultivation on agar medium;
2. Detection and quantification of Legionella pneumophila with the Quantitative
Polymerase Chain Reaction (Q-PCR)

3.8.1 Monitoring technology nr 1: ISO method 11731:1998

Description:
Bacteria in a water sample are concentrated by membrane filtration or by
centrifugation. To reduce the growth of unwanted bacteria, a portion of the
concentrated sample is subjected to treatment with acid or heat. Treated and
untreated concentrated sample are then inoculated onto plates of agar
medium (semi)-selective for Legionella. Plates are incubated at 37 C for 7
days. After incubation, morphologically characteristic colonies which form on
the selective medium are to be confirmed as Legionella by subculture to
demonstrate their growth requirements for L-cysteine and iron. The culture
and subculture of Legionella will require 10 days before results are available.

To perform the ISO culture method no specific equipment, other than
standard microbiological laboratory equipment, is needed. The ISO method is
the generally accepted method and agar medium is commercially available.

Evaluation:
The culture method is the standard method generally used to enumerate
Legionella in water. It uses semi-selective GVPC or BCYE agar medium to
culture Legionella. In water samples containing a high microbiological
background, the agar medium is likely to be overgrown by other bacteria
then Legionella. The method is therefore less useful for water samples from
cooling towers or surface water.


Monitoring technology nr 1: ISO method 11731:1998

sensitivity (A)
source water
drinking water

x

x

robustness (A)
selectivity

x
x

time to result

10 days
ease-of-use (B) x

Costs
consumables
maintenance

x

x


Overall conclusion Standard accepted method but faster and more specific
method is needed.

3.8.2 Monitoring technology nr 2: Q-PCR for Legionella pneumophila

Description:
The Q-PCR method is a molecular detection technique which amplifies
specific Legionella pneumophila DNA. The method targets the human pathogen
Legionella pneumophila which is responsible of more than 90% of the cases of
Legionellose. The results are given as DNA copies per litre and are available
with 4 hours. The detection and quantification consists of three phases: (1)
concentration of the water sample by membrane filtration, (2) lyses of the
bacteria, DNA extraction and purification of the DNA, and (3) amplification
of the specific DNA fragment PCR and real-time quantification of the
amplified DNA using a probe. An internal control is included in the method
and added after filtration to each sample. The result of this internal control is
used to give insight in the recovery of the DNA isolation and possible
inhibition of the DNA amplification. A Real-time PCR machine is required to
perform the Q-PCR analysis. Laboratory technicians should have experiences
in applying molecular techniques in the laboratory. The method is submitted
to ISO for acceptation. Adaptation of legislation will be necessary for
application of this DNA based method. However, it is expected that the
approval of this method will occur in the near future.


Evaluation:
In general, the method is accurate, specific and robust. The detection results
are available within a few (4) hours, this in comparison with the 7 days
required for the standard culture method. The quantification if based on a
DNA standard. The results are therefore given in DNA copies per litre. For
risk analysis it can be necessary to know if recent disinfections have been
carried out. Heat disinfection can result in uncultivable but PCR detectable
DNA copies. The method is some what sensitive to inhibition of the PCR or
loss of DNA during DNA isolation. However, the result of the internal
control quantitatively shows the overall performance of the analysis per
sample.

In a few years, this method will presumably be used as the standard detection
method for Legionella pneumophila. The Q-PCR results are available within one
day. If necessary, within 24 hours after sampling the samples can additionally
be analyzed with the culture method.

Monitoring technology nr 2: Q-PCR for Legionella pneumophila

sensitivity (A)
source water
drinking water

x

x

robustness (A)
selectivity

x

x

time to result

result within 4 hours
ease-of-use (B) x

Costs
consumables
maintenance

x
x

Recommendation for use in SSS (D) x needs skilled personel

Overall conclusion Fast, reliable, and sensitive detection method for the
detection of Legionella pneumophila in water. The
method detects specific Legionella pneumophila DNA in
water samples, but no differentiation between live and
dead cells is possible


3.9 Pseudomonas aeruginosa

Prepared by: Vermicon AG

P. aeruginosa is a gram-negative, rod-shaped, none spore-forming, aerobic
bacterium with low nutrient requirements. The optimal growth temperature
is 37 C . It is a known biofilm-forming bacterium and therefore used as an
indicator for deficiencies within cleaning processes. Moreover, this
opportunistic pathogenic micro-organism can cause infection of wounds in
immune-suppressed and elderly persons.

Pseudomonas aeruginosa is a bacterium that is naturally found in many types of
drinking water and water used for human consumption. For example, it is a
violation of European regulations to have Pseudomonas aeruginosa present in a
250 ml bottled mineral water. However, no comparable regulation exists in
the United States. Apparently, the Pseudomonas aeruginosa regulation in
Europe originated as a quality control issue and not as a health effects issue.
Nevertheless, during the last decade, a number of papers have been
published that indicate that Pseudomonas aeruginosa in hospitals can be a
health threat, but that it is normally no health risk if it is present in drinking
water (Hardalo, C. and Edberg, S.C, 1997).

The analysis is only necessary in the case of water offered for sale in bottles or
containers. Limit value for P. aeruginosa according to the European Council
Directive 98/83/EC of November 1998 is 0 cells/250 ml. The guidelines for
the detection is EN ISO 16266. For health protection reasons, occasionally
water for human consumption or drinking water is analysed on this
microbiological parameter, too.

References
Hardalo, C. and Edberg, S.C., Pseudomonas aeruginosa: Assessment of Risk
from Drinking Water, Critical Reviews in Microbiology, 23(1):47-75 (1997).

Council Directive 98/83/EC of November 1998 on the quality of water
intended for human consumption. Official Journal of the European
Communities.

Monitoring and confirmation technologies:
1. Filtration and cultivation according to EN ISO 16266
2. VIT-Pseudomonas aeruginosa
3. API-Test, confirmatory test

3.9.1 Monitoring technology nr. 1: Filtration and cultivation according to EN ISO 16266

Method
Filtration of 250 ml water on a cellulose-nitrate membrane filter (0,45 m pore
size and 50 mm diameter), subsequent incubation of the membrane filter


under aerobic conditions on a cetrimid containing selective-agar (CN-Agar) at
36 +/- 2 C for 44 +/- 4 hours.
All colonies that show a blue-green pigment (pyocyanin) are regarded as
confirmed P. aeruginosa colonies.
Subsequently the filter is exposed for a short time to UV-light. All colonies
that show fluorescence and but do not produce pyocyanin are regarded as
suspicious colonies and have to be confirmed by the acetamid utilization test.
Additionally not fluorescent, red-brown pigmented colonies are also
suspicious and have to be confirmed by an oxidase-test, acetamid utilization
and a fluorescein formation test.

Membrane filtration manifold, sterile filter funnels
Cellulosenitrate-membrane filter (0,45 m pore size and 50
mm diameter)
Incubator: 36 +/- 2 C
CN-agar
NB (Nutrient Broth)-agar
Kings B medium
Oxidase-test stripes
acetamide-solution

Evaluation:
The method according to EN ISO 16266 is the only accepted method. But the
procedure for the confirmation of colonies is very time-consuming (up to 5
days).


Monitoring technology nr. 1: Filtration and cultivation according to EN ISO
16266
sensitivity (A)
source water
drinking water

-
x

not applied
robustness (A)
selectivity

x
x

Experience is needed
time to result

Up to 5 days
ease-of-use (B) x

Costs
equipment
consumables
maintenance

x
x


Overall conclusion method is time-consuming when colonies have to be
confirmed

3.9.2 Monitoring technology nr. 2: VIT-Pseudomonas aeruginosa

Description:
For the application of VIT-Pseudomonas aeruginosa a pre-enrichment of the
water sample in Malachit green broth is necessary (48 h, incubation at 37 C).
An aliquot (2 ml) of a suspicious broth (turbid, colour change into yellow) is
centrifuged, fixed and analysed with VIT-Pseudomonas aeruginosa.
Additionally the kit can be used as a confirmatory test for colonies.

Material & Method
5 l of the sample (pre-enrichment or fixed colony) is placed on each well of a
slide. The sample is fixed on the slide and the VIT-solution is added to the
sample. This solution contains specific oligonucleotide probes for P.
aerugionosa, labeled with fluorescent dyes. During an incubation step (90 min,
at 46 C) the probes bind specifically to their matching signatures on the
genetic material (16S rRNA). Following this a stringent washing step removes
all unbound and surplus oligonucleotide probes from the cells (15 min, 46
C). The results are evaluated using a fluorescence microscope, whereby P.


aeruginosa, if present in the sample lights up specifically. The test is not
providing a quantitative result.

- medium (malachitgreen-broth), agar plates (CN-agar)
- mini-centrifuge
- micro-pipettes and tips
- incubator 37 C and 46 C +/- 2 C
- VIT-adapted fluorescence microscope

Evaluation:
By the application of VIT-Pseudomonas the results is available within 2 days,
but the analysis is not quantitative. Special knowledge is not required for the
performance of the test. The application of the test is not officially accepted by
a directive, but comprehensive and numberous studies were performed and
the reliability of the test was confirmed. Moreover, the test can also be
applied as a confirmation test for colonies.

Monitoring technology no. 2: VIT-Pseudomonas aeruginosa

sensitivity (A)
source water
drinking water

-
x

robustness (A)
selectivity

x
x

time to result

2 days
ease-of-use (B) x

Costs
instrumentation (C) x Micro-centrifuge, Fluorescence
microscope
consumables
maintenance

x

x


Overall conclusion Easy-to-use rapid test, can be used in addition as
confirmation test for colonies, not quantitative


3.9.3 Confirmation technology nr. 1: API-Test 20E

API-Test 20 E (Biomerieux) is a screening test for the identification of
Enterobacteriaceae and other gram-negative rods. The biochemical test is a
confirmatory test for colonies and identification is possible within 18-24
hours.

For the application of this test a pure culture is needed. Thus the test can be
applied after the subculture of a colony as an additional test for the
confirmation of the result.

Evaluation:
For the application of the API-test a pure culture is needed and the test can be
used instead of tedious typical confirmatory tests.


3.10 Aeromonas

Prepared by: RTU

The concentration range that should be covered for this parameter is 1
cell/100 ml. There are no current regulations in Europe but indicative values
in The Netherlands (20 CFU/100 ml as a median value over a 1-year period in
the treated drinking water and 200 CFU/100 ml as the 90th-percentile value
of the Aeromonas counts of drinking-water in the distribution system in a 1-
year period) In the US Aeromonas are monitored since 2003. A minimum
reporting level of 0.2 CFU/100 ml is set (source: EPA). EPA Method 1605 is
required.
Sensitivity/resolution required is 1 cell.

1. EPA Method 1605
2. MALDI
3. PCR
3.10.1 M onitoring technology nr 1: EPA Method 1605

Description:
Aeromonas is a common genus of bacteria indigenous to surface waters, and
may be found in non-chlorinated or low-flow parts of chlorinated water
distribution systems. Monitoring their presence indistribution systems is
desirable because some aeromonads may be pathogenic and pose a
potentialhuman health risk. Method 1605 describes a membrane filtration
technique for the detection and enumeration of Aeromonas species. This
method uses a selective medium that partially inhibits thegrowth of non-
target bacterial species while allowing most species of Aeromonas to grow.
Aeromonas is presumptively identified by the production of acid from dextrin
fermentation and the presence of yellowcolonies on ampicillin-dextrin agar
medium with vancomycin (ADA-V). Yellow colonies are counted and
confirmed by testing for the presence of cytochrome c (oxidase test), and the
ability to ferment trehalose and produce indole.
This method is adapted from Havelaar et al. (1987) for the enumeration of
Aeromonas species in finished water by membrane filtration. The method
provides a direct count of Aeromonas species in water based on the growth of
yellow colonies on the surface of the membrane filter using a selective
medium. A water sample is filtered through 0.45-m-pore-size membrane
filter. The filter is placed on ampicillin-dextrin agar with vancomycin (ADA-
V) and incubated at 35C 0.5C for 24 2 hours. This medium uses
ampicillin and vancomycin to inhibit non-Aeromonas species, while allowing
most Aeromonas species to grow. It is a quantitative assay that uses a selective
medium which partially inhibits the growth of non-target bacterial species
while allowing Aeromonas to grow. Aeromonas is presumptively identified by
the production of acid from dextrin fermentation producing yellow colonies.
Presumptively positive colonies are counted and confirmed by testing for the


presence of cytochrome c (oxidase test), and the ability to ferment trehalose,
and produce indole.

The method is quite inexpensive to perform. It requires only the basic
equipment of any laboratory. The prices of the growth media can vary,
depending on the selected method. The price of consumables for further
identification should be added.

A traditional method, widespread for drinking water analysis.

Guidelines
In U.S.A. EPA Method 1605 is used and the minimum reporting level is set to
0.2 CFU/100ml.
The health authorities in the Netherlands in 1985 introduced indicative
maximum values for Aeromonas densities in drinking-water. The values were
based on a national survey of aeromonads in drinking-water in the
Netherlands and have been defined as follows: 20 cfu/100 ml as a median
value over a 1-year period in water leaving the treatment facility; 200 cfu/100
ml as the 90th-percentile value of the Aeromonas counts of drinking-water
collected from the distribution system in a 1-year period (Trouwborst, 1992).

References:
1. Havelaar, A.H., M. During, and J.F.M. Versteegh. 1987. Ampicillin-
dextrin agar medium for the enumeration of Aeromonas species in
water by membrane filtration. Journal of Applied Microbiology.
62:279-287.
2. Trouwborst T (1992). Overheidsbeleid ten aanzien van het voorkomen
van Aeromonas in drinkwater. [Government policy with regard to
occurrence of Aeromonas in drinking-water.] In: van der Kooij D, ed.
Aeromonas in Drinkwater: Voorkomen, Bestrijding en Betekenis.
[Aeromonas in drinking water: occurrence, control and significance.]
Nieuwegein, Kiwa NV: 95104.

Evaluation:
It is a simple, inexpensive method that requires basic routine bacteriology
laboratory facilities. Water samples containing colloidal or suspended
particulate material may clog the membrane filter and prevent filtration or
cause spreading of bacterial colonies which could interfere with identification
of target colonies.
Other ampicillin/vancomycin resistant bacteria that are not aeromonads may
be able to grow on this medium. Some of these bacteria may also produce
yellow colonies if they are able to produce acid byproducts from the
fermentation of dextrin or some other media component, or if they produce a
yellow pigment. Enterococci are reported to produce pinpoint-size yellow
colonies on ADA. Confirmation of presumptive Aeromonas colonies is
necessary to mitigate false positives.


Monitoring technology nr 1: EPA Method 1605

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

time to result

1-2 days
ease-of-use (B) x

Costs
consumables
maintenance

x

x


Overall conclusion Not complicated but laborious method. Robust, does not
require skilled personnel. An additionl identification of
the positive colonies may be necessary.

3.10.2 Monitoring technology nr 2: MALDI

Description:
Matrix-assisted laser desorption/ionization (MALDI) is a soft ionization
technique used in mass spectrometry, allowing the analysis of biomolecules
and large organic molecules, which tend to be fragile and fragment when
ionized by more conventional ionization methods. The ionization is triggered
by a laser beam (normally a nitrogen laser). A matrix is used to protect the
biomolecule from being destroyed by direct laser beam and to facilitate
vaporization and ionization. The type of a mass spectrometer most widely
used with MALDI is the TOF (time-of-flight mass spectrometer), mainly due
to its large mass range. MALDI-MS was used to analyze the whole cells of
both reference strains and unknown Aeromonas isolates obtained from water
distribution systems (Donohue, et al., 2007). A library of over 45 unique m/z
signatures was created from 40 strains that are representative of the 17
recognized species of Aeromonas, as well as 3 reference strains from genus
Vibrio and 2 reference strains from Plesiomonas shigelloides. The library was
used to help speciate 52 isolates of Aeromonas. The environmental isolates


were broken up into 2 blind studies. Group 1 contained isolates that had a
recognizable phenotypic profile and group 2 contained isolates that had an
atypical phenotypic profile. MALDI-MS analysis of the water isolates in
group 1 matched the phenotypic identification in all cases. In group 2, the
MALDI-MS-based determination confirmed the identity of 18 of the 27
isolates. These results demonstrated that MALDI-MS analysis can rapidly and
accurately classify species of the genus Aeromonas.

The equipment is expensive and requires certain skills for operating and
analyzing the obtained data.

This method is still under development as this is a very recent study.

For MALDI-based equipment one of the market leaders is Waters
(www.waters.com).

References:
1. Donohue, M. J., J. M. Best, A. W. Smallwood, M. Kostich, M. Rodgers,
and J. A. Shoemaker. 2007. Differentiation of Aeromonas isolated from
drinking water distribution systems using matrix-assisted laser
desorption/ionization-mass spectrometry. Anal Chem 79:1939-46.

Evaluation:
This could potentially be a powerful tool especially suited for environmental
monitoring and detection of microbial hazards in drinking water, but it can
only be used for confirmation.


Monitoring technology nr 2: MALDI

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

time to result

ease-of-use (B) x

Costs
consumables
maintenance

x

x


Overall conclusion This is a very promising method but not enough
verified to be recommended for routine analysis.

3.10.3 Monitoring technology nr 3: PCR

Description:
The polymerase chain reaction (PCR) is a biochemistry and molecular biology
technique for exponentially amplifying DNA, via enzymatic replication.
Quantitative real time polymerase chain reaction (RT-PCR) is a laboratory
technique used to simultaneously quantify and amplify a specific part of a
given DNA molecule. It is used to determine whether or not a specific
sequence is present in the sample; and if it is present, the number of copies in
the sample. It is the real-time version of quantitative polymerase chain
reaction (Q-PCR), itself a modification of polymerase chain reaction. The
procedure follows the general pattern of polymerase chain reaction, but the
DNA is quantified after each round of amplification; this is the "real-time"
aspect of it. Two common methods of quantification are the use of fluorescent
dyes that intercalate with double-strand DNA, and modified DNA
oligonucleotide probes that fluoresce when hybridized with a complementary
DNA.
There are quite a number of studies which have taken advantage of this
technique for Aeromonas detection. As a result of these studies it is apparent
that Aeromonas are present in source water (Ormen and Ostensvik, 2001),
bottled water (Biscardi, et al., 2002), drinking water (Emekdas, et al., 2006;


Figueras, et al., 2005; Sen and Rodgers, 2004) distribution systems and most
of them possess aerolysin gene. They have been shown to be resistant to the
first generation beta-lactam antibiotics as well (Emekdas, et al., 2006).

The equipment varies depending on whether the conventional or real-time
approach is chosen. The conventional cycler is not expensive but it must be
completed with gel running and imaging system. RT-PCR cycler is
considerably more expensive and also there an elctroforesis and imaging unit
might be needed (to check for eventual unspecific products). Also the running
cost, the price of enzyme, nucleotides, primers, fluorescent dyes and probes
etc. has to be considered.

This is a quite popular method which, when well designed, gives an
opportunity to screen several pathogens (along with Aeromonas) in one run.

PCR equipment can be purchased from Applied Biosystems
(www.appliedbiosystems.com) and Eppendorf (www.eppendorf.com),
among others.

References:
1. Biscardi, D., A. Castaldo, O. Gualillo, and R. de Fusco. 2002. The
occurrence of cytotoxic Aeromonas hydrophila strains in Italian
mineral and thermal waters. Sci Total Environ 292:255-63.

2. Emekdas, G., G. Aslan, S. Tezcan, M. S. Serin, C. Yildiz, H. Ozturhan,
and R. Durmaz. 2006. Detection of the frequency, antimicrobial
susceptibility, and genotypic discrimination of Aeromonas strains
isolated from municipally treated tap water samples by cultivation
and AP-PCR. Int J Food Microbiol 107:310-4.

3. Figueras, M. J., A. Suarez-Franquet, M. R. Chacon, L. Soler, M.
Navarro, C. Alejandre, B. Grasa, A. J. Martinez-Murcia, and J. Guarro.
2005. First record of the rare species Aeromonas culicicola from a
drinking water supply. Appl Environ Microbiol 71:538-41.

4. Ormen, O., and O. Ostensvik. 2001. The occurrence of aerolysin-
positive Aeromonas spp. and their cytotoxicity in Norwegian water
sources. J Appl Microbiol 90:797-802.

5. Sen, K., and M. Rodgers. 2004. Distribution of six virulence factors in
Aeromonas species isolated from US drinking water utilities: a PCR
identification. J Appl Microbiol 97:1077-86.

Evaluation:
This is a rather accepted method which works quite well in drinking water. If
there is enough genetic material in the sample, the results can be achieved in
a couple of hours or less. If there is not enough cells, pre-enrichment might be
necessary which then increases the analysis time. However, the problem


might be the biofilm analysis as this technique is rather sensitive to inhibitors.
The equipment, however, is expensive and skills in operating it are needed.

Monitoring technology nr 3: PCR


sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

time to result

ease-of-use (B) x

Costs
consumables
maintenance

x

x


Overall conclusion A promising method but the equipment is expensive to
purchase and run. Skilled personnel is also required. It
still needs to be developed prior to application,
however, the method has a potential for being used by
larger systems in the future


3.11 Bacteriophages


The concentration range that is normally encountered in water samples is:
0 pfu per 10 ml water sample in drinking water and groundwater
0 to 1000 pfu per ml surface water

The required resolution of the method is:
1 pfu in 10 ml water sample

1. ISO method 10705-1: Enumeration of F-specific RNA bacteriophages.
1a. Method to determine the serotype of isolated F-specific RNA
bacteriophages
2. ISO method 10705-2: Enumeration of somatic coliphages
3. ISO method 10705-4 (modified): Enumeration of bacteriophages that infect
Bacteroides


Description:
- The sample is mixed with a small volume of semisolid nutrient medium. A
culture of host strain Salmonella typhimurium strain WG49 is added and
plated on solid nutrient medium. After this, incubation and reading of agar
plates for visible plaques takes place. Plaques can be confirmed on the same
medium with added RNAse, which inhibits infection of the host strain by F-
specific RNA bacteriophages. The results are expressed as the number of
plaque forming units (pfu) per unit of volume.

steam, a thermostatic incubator, a water bath, a counting apparatus, a
spectrophotometer, a pH-meter and a deep freezer (-20C 5C) are required.

- The method is one of the three ISO-approved methods for the enumeration
of bacteriophages (as indicators for enteric viruses) in water.


Monitoring technology nr 1a: method to determine the serotype of isolated F-
specific RNA bacteriophages
- A duplex RT-PCR using primers that are specific for the replicase gene of F-
specific RNA bacteriophages is performed on isolated F-specific RNA
bacteriophages. The DNA strands of the RT-PCR product are separated by
heat treatment and the product is added to a miniblotter that contains
covalently bound oligonucleotide probes of the replicase gene of each


serotype. After hybridization and washing, bound PCR-product is detected
by chemiluminescence and visualized by exposure to X-ray film.

- To use this method, a PCR apparatus, a miniblotter, a water bath and an X-
ray film are required.

- Reference: Vinj, J. et al. 2004. Molecular detection blot hybridization.
Appl. Environ. Microbiol. 70:5996-6004.

Evaluation:
- In general, an accurate method to determine the number of bacteriophages
(as indicator for enteric viruses) in water samples. The best indication of the
presence of enteric viruses is obtained by using this method in combination
with methods to determine somatic coliphages and bacteriophages that infect
Bacteriodes (Leclerc et al 2000. Bacteriophages as indicators of enteric viruses
and public health risk in groundwaters. J. Appl Microbiol. 88:5-21).
- From literature it is known that F-specific RNA bacteriophages are a better
indicator for the presence of enteric viruses in water than somatic coliphages,
but might be a less reliable indicator than bacteriophages that infect
Bacteriodes.
- Plaques are often vague, making them more difficult to count than plaques
of somatic coliphages.
- ISO method 10705-1 states that confirmation by added RNAse to the
medium should be performed in parallel with the method to enumerate F-
specific RNA bacteriophages. In case of low number (1 to 5) of pfu of F-
specific RNA bacteriophages it is better to take out the plaque and confirm
inhibition of infection by addition of RNAse for each isolated plaque.
- Incubation time is rather long, because results are obtained after 18 to 22
hours. However, a fast standard method is not available, although methods
based on RNA-detection have been published and might come available in
the near future.
- Determination of the serotype of isolated F-specific RNA bacteriophages can
be used to determine the source of fecal contamination, because with few
exceptions serotype II and III have been associated with human waste
affected water. Serotype I and IV are associated with animal waste affected
water.





Description:
culture of host strain Escherichia coli strain C is added and plated on solid
nutrient medium. After this, incubation and reading of agar plates for visible
plaques takes place. The results are expressed as the number of plaque
forming units (pfu) per unit of volume.

sensitivity (A)
source water
drinking water

x
x
Detection limit: 1 bacteriophage in 10
ml water. In combination with
concentration of the water sample via
Hemoflow, detection limits in
drinking water can be 1
bacteriophage in 10 L or even 1
bacteriophage in 2000 L.
robustness (A)
selectivity

x

x

time to result

Total incubation time: 18 to 22 h
ease-of-use (B) x

Costs
consumables
maintenance

x
x


Overall conclusion Most used method together with the method for somatic
coliphages to determine bacteriophages (as indicator for enteric
viruses) in water samples. One method is not better over the other
and it is advised to determine F-specific RNA bacteriophages,
somatic coliphages and bacteriophages that infect Bacteroides.
Serotyping isolated F-specific RNA bacteriophages can be used to
determine the source (human versus animal) of faecal
contamination.


spectrophotometer, a pH-meter and a deep freezer (-20C 5C) are required.



Evaluation:
- From literature it is known that somatic coliphages are slightly less reliable
as indicator for the presence of enteric viruses in water than F-specific RNA
bacteriophages or bacteriophages that infect Bacteriodes.
- Plaques are clear, making them easier to count than plaques of F-specific
RNA bacteriophages.
hours. However, a fast standard method is not available.




3.11.3 Monitoring technology nr 3: ISO method 10705-4 (modified)

Description:
culture of host strain Bacteroides is added and plated on solid nutrient
medium. After this, incubation and reading of agar plates for visible plaques
takes place. The results are expressed as the number of plaque forming units
(pfu) per unit of volume.

-The Bacteroides strains that have been used as host strains are Bacteroides
fragilis RYC2056 (detection of bacteriophages from animal and human
sources; this strain is mentioned in ISO 10705-4), Bacteroides
sensitivity (A)
source water
drinking water

x
x
Detection limit: 1 bacteriophage in
10 ml water. In combination with
concentration of the water sample
via Hemoflow, detection limits in
robustness (A)
selectivity

x

x

time to result

ease-of-use (B) x

Costs
consumables
maintenance

x
x


Overall conclusion Most used method together with the method for F-specific RNA
bacteriophages to determine bacteriophages (as indicator for
enteric viruses) in water samples. One method is not better over
the other and it is advised to determine F-specific RNA
bacteriophages, somatic coliphages and bacteriophages that infect
Bacteroides.


thetaiotaomicron GA17 (detection of bacteriophages from human sources;
Blanch, A. R. et al. 2006, Appl. Environ. Microbiol. 72:5915-5926), Bacteroides
ovatus GB124 (detection of bacteriophages from human sources; Blanch, A. R.
pers. comm.) and Bacteroides thetaiotaomicron HB13 (detection of
bacteriophages from human sources; Blanch, A. R. pers. comm.).

spectrophotometer, an anaerobic cabinet or jars or bags, Hungate glass tubes,
a pH-meter and a deep freezer (-20C 5C) are required.



Evaluation:
- From literature it is known that bacteriophages that infect Bacteroides might
be the most reliable indicator for enteric viruses in water.
- Host strain B. fragilis RYC2056 can be used to determine animal and human
related bacteriophages that infect Bacteroides. Host strains B.
thetaiotaomicron GA17 and HB13 and B. ovatus GB124 can be used to
determine human related bacteriophages that infect Bacteroides. However,
the human related host strains seem to perform only in a specific geographic
area (GA17: Spain; HB13: Spain and Columbia; GB124: Great Britain), making
their interlaboratory use more difficult.
- Bacteroides is an obligate anaerobic bacterium, which is inhibited by
oxygen. All lab procedures should, therefore, be kept to a minimum amount
of time. Incubation in anaerobic jars, bags or cabinet is required.
hours. However, a fast standard method is not available.



sensitivity (A)
source water
drinking water

x
x
Detection limit: 1 bacteriophage in
10 ml water. In combination with
concentration of the water sample
via Hemoflow, detection limits in
robustness (A)
selectivity

x

x

Laboratory procedures should be
performed in a short time and
incubation is under anoxic
conditions
time to result

ease-of-use (B) x Anaerobic incubations required

Costs
consumables
maintenance

x
x


Overall conclusion Because of the required anaerobic conditions, the method is not
used as often as the other two methods. However, one method is
not better over the other and it is advised to determine F-specific
RNA bacteriophages, somatic coliphages and bacteriophages that
infect Bacteroides. The fact that Bacteroides is an obligate
anaerobic bacterium makes the procedure more complicated than
the methods to determine F-specific bacteriophages and somatic
coliphages. An European water survey has shown that source
tracking with human related Bacteroides strains is one of the
most reliable ways to determine human versus animal faecal
contamination.


3.12 Aerobic spore forming bacteria

Prepared by: RTU

Concentration range that should be covered for this parameter is starting
from 1 cell/spore/100 ml. For the highest limit information is not available.
Sensitivity/resolution required (distinguish between source water and
drinking water, if applicable).
1 cell/spore/100 ml

1. HPC counts
2. Membrane filtration
3. Assays based on the cell constituents
4. PCR

3.12.1 Monitoring technology nr 1: HPC counts

Description:
A variety of simple culture-based tests that are intended to recover a wide
range of microorganisms from water are collectively referred to as
heterotrophic aerobic and aerobic spore-forming bacterial counts or HPC
test procedures. There is no universal HPC measurement. Although
standardized methods have been formalized, HPC test methods involve a
wide variety of test conditions that lead to a wide range of quantitative and
qualitative results. Temperatures employed range from around 20 C to 40
C, incubation times from a few hours to seven days or a few weeks, and
nutrient conditions from low to high. The test itself does not specify the
organisms that are detected.
They are of a little sanitary significance, however useful for assessment of the
efficiency of the water treatment where the colony counts should be as low as
possible. Media can be made more selective, e.g. MYP media (Cereus
Selective Agar Base) can be used (Mazoua and Chauveheid, 2005).
The culture method can be adapted to count spores only by exposing samples
to temperatures of 70-80C for 10-20 minutes before culturing. If necessary,
biochemical testing of the colonies, such as API 20 E or API 50 CHB, may
further clarify the identity of the colony (Mazoua and Chauveheid, 2005).
There are even studies where the authors have devised an identification key
for Bacillus species, based on series of culture-based and biochemical tests
(Reva, et al., 2001), however, this is rather of an interest for taxonomical
studies than treatment efficiency monitoring.

The method is very cheap to perform. It requires only the basic equipment of
any laboratory. The prices of the growth media can vary, depending on the
selected method.


According to COUNCIL DIRECTIVE 98/83/EC of 3 November 1998
(UNION, 1998) a colony count at 22C and 37C is required for drinking water
monitoring (method prEN ISO 6222). HPC is accepted and commercially
available method. Many laboratories/companies supply ready growth
medium mixtures, solutions etc.

References:

1. Mazoua, S., and E. Chauveheid. 2005. Aerobic spore-forming bacteria
for assessing quality of drinking water produced from surface water.
Water Res 39:5186-98.
2. Reva, O. N., I. B. Sorokulova, and V. V. Smirnov. 2001. Simplified
technique for identification of the aerobic spore-forming bacteria by
phenotype. Int J Syst Evol Microbiol 51:1361-71.
3. The Council of the European Union. 1998. COUNCIL DIRECTIVE
98/83/EC of 3 November 1998 on the quality of water intended for
human consumption. In EU (ed.), L 330/32 ed. Official Journal of the
European Communities.

Evaluation:
Conventional treatment processes in series (coagulation, sedimentation,
filtration and disinfection) are essential to the processing of river water
because of fluctuating water quality. Where lakes and large watershed
impoundments are utilized, treatment may include only disinfection.
Because fluctuations in raw water quality impact successful treatment, daily
sampling should be done to provide information on storm water runoff
conditions and alert the utility to sewage treatment bypasses released
upstream. The most important sampling site in the treatment train is at the
entry point for finished water (plant effluent) release into the distribution
system. Here the purpose is to verify that treatment process barriers are
preventing the passage of the contaminants. In some cases, this sampling site
may be located at the first customers tap, if contact time for disinfectant
action must be extended beyond that available in the plant contact basin
(Geldreich, 1996).
The measurement of spores of aerobic spore-forming bacteria (ASFB) is
becoming a widely accepted method for validating the effectiveness of
treatments applied in drinking water treatment plants. ASFB have been
shown to be conservative indicators for Cryptosporidium and Giardia. HPC are
provided by simple, inexpensive methods that require basic routine
bacteriology laboratory facilities and can be performed by relatively unskilled
persons. However, HPC will not register the presence of metabolically active
but not dividing organisms and they are not rapid.

1. Geldreich, E. E. 1996. Microbial quality of water supply in distribution
systems. CRC Press LLC, Boca Raton, FL, USA.


Monitoring technology nr 1: HPC

sensitivity (A)
source water
drinking water

x

x

robustness (A)
selectivity

x

x

time to result

x
ease-of-use (B) x

Costs
consumables
maintenance

x

x


Overall conclusion Not complicated but laborious method, not very useful for
identification. Robust, does not require skilled personnel. No
information on stress-injured cells, unable to divide.

3.12.2 Monitoring technology nr 2: Membrane filtration

Description:
Certain volume of water is filtered through 0.2-0.45 nm membrane filter and
then the filter is incubated on a pad, soaked in nutrients. The colonies are
then counted. However, due to the low concentrations of spores, the rate of
the recovery is influenced both by the both the presence of a cake on the filter
and the aggregation of the spores. It has been shown that the addition of a
surfactant Tween 80, recovery can be increased for up to 1000 times(Cartier,
et al., 2007).

See monitoring technology nr 1 above.

Accepted and commercially available method. Many laboratories/companies
supply ready growth medium mixtures, solutions etc.


References:
1. Cartier, C., B. Barbeau, M. Besner, P. Payment, and M. Prevost. 2007.
Optimization of the detection of the spores of aerobic spore-forming
bacteria (ASFB) in environmental conditions. Journal of Water Supply:
Research and Technology - AQUA 56:191-202.

Evaluation:
The same considerations as for HPC method apply (see above).

Monitoring technology nr 2: Membrane filtration

sensitivity (A)
source water
drinking water

x

x

robustness (A)
selectivity

x

x

time to result

x
ease-of-use (B) x

Costs
consumables
maintenance

x

x


Overall conclusion Simple method, not very useful for identification without
additional tests. Robust, does not require skilled personnel. More
potential compared to HPC method. No information on stress-
injured cells, unable to divide.

3.12.3 Monitoring technology nr: 3. Assays, based on cell constituents

Description:
More contemporary methods proposed for Bacillus spp. identification include
the fatty acid profile detection using capillary gas chromatography with
flame ionization detection (Whittaker, et al., 2005) which was applied quite
successfully to a number of pathogens. Several techniques applied for
detection of other groups of pathogens could also potentially be used, such as
pyrolysis in combination with mass spectrometry (Py-MS) (Freeman, et al.,
1990), (Magee, et al., 1991) and matrix-assisted laser desorption/ionization


time-of-flight (MALDI-TOF) in combinations with mass spectrometry (Smole,
et al., 2002) and liquid chromatography (LC) (Zhang, et al., 2004). The latter
approach, LC/MALDI and tandem MS is an instrument of shotgun
proteomics, which refers to the direct analysis of complex protein mixtures to
rapidly generate a global profile of protein complement within a mixture. FT-
IR technique has also been applied for Bacillus detection, however it was
shown to require uniform culture conditions for obtaining well differentiated
spectrum (Filip, et al., 2004). Flow cytometry has been tested for application
of spore and vegetative cell enumeration since the 1980ies(Phillips and
Martin, 1983). Nowadays it is possible to assess spore viability using this
method as well (Laflamme, et al., 2005). The method can be used together
with fluorescent dyes and antibodies.

Pyrolysis is the chemical decomposition of organic materials by heating in the
absence of oxygen or any other reagents. A particular piece of equipment
performing this heating can be coupled to GC-MS for detection of
components. Some gas chromatographs are connected to a mass spectrometer
which acts as the detector. The combination is known as GC-MS. In most
modern GC-MS systems, computer software is used to draw and integrate
peaks, and match MS spectra to library spectra. In general, substances that
vaporize below ca. 300 C (and therefore are stable up to that temperature)
can be measured quantitatively.

Fourier transform (FT) spectroscopy is a measurement technique whereby
spectra are collected based on measurements of the temporal coherence of a
radiative source, using time-domain measurements of the electromagnetic
radiation or other type of radiation. It can be applied to a variety of types of
spectroscopy including infrared spectroscopy (IR). FT-IR spectroscopy
involves the observation of vibrations of molecules that are excited by an
infrared beam. Molecules are able to absorb the energy of distinct light quanta
and start a rocking or rotation movement. The FT-IR spectrum uses only
vibrations that lead to a change in the dipole moment. An infrared spectrum
represents a fingerprint which is characteristic for any chemical substance.
The composition of biological material and, thus, of its FT-IR spectrum, is
exceedingly complex, representing a characteristic fingerprint. In principle, a
reference spectrum library is assembled based on well-characterized strains
and species. The FT-IR spectrum of any unidentified isolate is then measured
under the same conditions as those used for the reference spectra and is
compared to spectra in the reference spectrum library. If the library contains
an identical or a very similar spectrum, identification is possible. The success
of the method is, therefore, directly dependent on the complexity of the
reference spectrum library.

Flow cytometry is a technique for counting, examining and sorting
microscopic particles suspended in a stream of fluid. It allows simultaneous
multiparametric analysis of the physical and/or chemical characteristics of
single cells flowing through an optical and/or electronic detection apparatus.
A flow cytometer has 5 main components:


a flow cell - liquid stream (sheath fluid) carries and aligns the cells so that
they pass single file through the light beam for sensing.
a light source - commonly used are lamps (mercury, xenon); high power
water-cooled lasers (argon, krypton, dye laser); low power air-cooled lasers
(argon (488nm), red-HeNe (633nm), green-HeNe, HeCd (UV)); diode lasers
(blue, green, red, violet).
a detector and Analogue to Digital Conversion (ADC) system - generating
FSC and SSC as well as fluorescence signals.
an amplification system - linear or logarithmic.
a computer for analysis of the signals.
A beam of light of a single wavelength is directed onto a hydro-dynamically
focused stream of fluid. A number of detectors are aimed at the point where
the stream passes through the light beam; one in line with the light beam
(Forward Scatter or FSC) and several perpendicular to it (Side Scatter (SSC)
and one or more fluorescent detectors). Each suspended particle passing
through the beam scatters the light in some way, and fluorescent chemicals
found in the particle or attached to the particle may be excited into emitting
light at a lower frequency than the light source. This combination of scattered
and fluorescent light is picked up by the detectors, and by analyzing
fluctuations in brightness at each detector (one for each fluorescent emission
peak) it is then possible to extrapolate various types of information about the
physical and chemical structure of each individual particle. FSC correlates
with the cell volume and SSC depends on the inner complexity of the particle
(i.e. shape of the nucleus, the amount and type of cytoplasmic granules or the
membrane roughness).

The methods and the equipment is well known for other applications but as
detection methods for Bacilli or other spore-forming aerobes these are still
under development, perhaps, except flow cytometry which is both more
documented and closest to be applied for bacteria and spore
detection/enumeration in water.

Pyrolysis equipment can be obtained from PerkinElmer Life
(www.perkinelmer.com), Analytical Sciences and Shimadzu Scientific
Instruments Inc (www.shimadzu.com). A FTIR apparatus can be purchased
from e.g. Varian (www.varian.com). BeckmanCoulter
(www.beckmancoulter.com) and BD Biosciences (www.bdbiosciences.com)
provide reliable flow cytometers.

References:
1. Filip, Z., S. Herrmann, and J. Kubat. 2004. FT-IR spectroscopic
characteristics of differently cultivated Bacillus subtilis. Microbiol Res
159:257-62.
2. Freeman, R., M. Goodfellow, F. K. Gould, S. J. Hudson, and N. F.
Lightfoot. 1990. Pyrolysis-mass spectrometry (Py-MS) for the rapid
epidemiological typing of clinically significant bacterial pathogens. J
Med Microbiol 32:283-6.
3. Laflamme, C., J. Ho, M. Veillette, M. C. de Latremoille, D. Verreault,
A. Meriaux, and C. Duchaine. 2005. Flow cytometry analysis of


germinating Bacillus spores, using membrane potential dye. Arch
Microbiol 183:107-12.
4. Magee, J. T., J. M. Hindmarch, and C. D. Nicol. 1991. Typing of
Streptococcus pyogenes by pyrolysis mass spectrometry. J Med
Microbiol 35:304-6.
5. Phillips, A. P., and K. L. Martin. 1983. Immunofluorescence analysis of
bacillus spores and vegetative cells by flow cytometry. Cytometry
4:123-31.
6. Verberkmoes, N. C., W. J. Hervey, M. Shah, M. Land, L. Hauser, F. W.
Larimer, G. J. Van Berkel, and D. E. Goeringer. 2005. Evaluation of
"shotgun" proteomics for identification of biological threat agents in
complex environmental matrixes: experimental simulations. Anal
Chem 77:923-32.
8. Whittaker, P., F. S. Fry, S. K. Curtis, S. F. Al-Khaldi, M. M. Mossoba,
M. P. Yurawecz, and V. C. Dunkel. 2005. Use of fatty acid profiles to
identify food-borne bacterial pathogens and aerobic endospore-
forming bacilli. J Agric Food Chem 53:3735-42.
9. Wilkes, J. G., K. L. Glover, M. Holcomb, F. Rafii, X. Cao, J. B.
Sutherland, S. A. McCarthy, S. Letarte, and M. J. Bertrand. 2002.
Defining and using microbial spectral databases. J Am Soc Mass
Spectrom 13:875-87.

Evaluation:
It should be noted that some of these methods will often require pre-
enrichment step/cultivation and therefore are not really rapid methods. All
of these methods require costly equipment. Furthermore, the fingerprints
from mass spectral patterns from microbial isolates are affected by variations
in instrumental condition, by sample environment and by sample handling
factors (Wilkes, et al., 2002). A recent study evaluated LC/MALDI and
tandem MS this approach for E. coli detection, however the conclusion was
that low concentrations of a pathogen in a mixed sample were not detected
and the size of the database also had severe effect on the identification
(Verberkmoes, et al., 2005). Although a promising technology, it must be
significantly improved and therefore is not described in detail in this report.
From the described methods flow cytometry should be considered closest to
the practical application as it (i) will not require cultivation and (ii) has a
potential to provide the information whether the cell is viable or not.


Monitoring technology nr 3: Assays based on cell constituents

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x
x

time to result

x
ease-of-use (B) x

Costs
consumables
maintenance

x


Overall conclusion Promising methods but still to far from the application.
Flow cytometry could be interesting for larger systems.
The equipment is expensive to purchase and maintain.
Skilled personnel in performing the analysis and
interpreting data is also required.

3.12.4 Monitoring technology nr 4: PCR

Description:
The polymerase chain reaction (PCR) is a biochemistry and molecular biology
technique for exponentially amplifying DNA, via enzymatic replication. PCR-
based methods have been used for the detection of Bacilli, more specifically,
ARDRA-PCR (amplified ribosomal DNA restriction analysis) which was
capable to identify most environmentally important species of Bacillus and
distinguish these from 15 other Eubacteria species (Wu, et al., 2006). This
method implies first using restriction enzymes and then specific primers to
amplify the genome fragments and then identify a certain pattern, specific for
e.g Bacilli.

This technique requires PCR thermocycler, which nowadays supports 96
reactions simultaneously. If there is enough genetic material in the sample,
the results can be achieved in a couple of hours or less. Thermocycler is not


very expensive, however this technique requires consumables such as
restriction enzymes, polymerization enzyme(s), nucleotides, buffers, and gel
imaging systems, if the cycler is conventional. Real-Time PCR does not
always require gel imaging but the cycler is more expensive and the
operation more difficult.

PCR is a promising technique which could be tested for aerobic spore-
forming bacteria analysis through the water treatment steps. The equipment,
however, is expensive and skills in operating it are needed.

PCR equipment can be purchased from Applied Biosystems
(www.appliedbiosystems.com) and Eppendorf (www.eppendorf.com),
among others.

References:
1. Wu, X. Y., M. J. Walker, M. Hornitzky, and J. Chin. 2006. Development
of a group-specific PCR combined with ARDRA for the identification
of Bacillus species of environmental significance. J Microbiol Methods
64:107-19.

Evaluation:
This method has very good potential but the ability to distinguish aerobic
spore-formers from other Eubacteria species than tested so far must be
investigated. Another important question is the sensitivity of the reaction,
enzymes, in particular, to substances found in the treated waters. It is known
that certain metal ions inhibit polymerases thus it is likely that sample
preparation, clean-up, amplification facilitators might be necessary.


Monitoring technology nr 4: PCR

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

time to result

x
ease-of-use (B) x

Costs
consumables
maintenance

x

x


purchase and run. Skilled personnel is also required. It
still needs to be developed prior to application, however,
the method has a potential for being used by larger
systems in the future


3.13 Biofilm formation rate (BFR)

Prepared by: NTNU

- Concentration range that should be covered: 1ng adenosine triphosphate
(ATP)/l to >1000 ngATP/l mg/l
- sensitivity/resolution required: An ATP signal of 2000-3000 Relative Light
Units (ca. 0.01 nM ATP) is considered the lower detection limit above which
statistically accurate measurements were possible (standard deviation < 5%).

1. ATP measurement of biofilm

3.13.1 Monitoring technology nr 1: ATP measurement of biofilm

Description:
The methodology was developed for at-line monitoring of biofilm formation
in drinking water systems [1] and the biofilm formation rate was determined
with a biofilm monitor containing cylinders. Several monitoring systems have
been reported [2][Fehler! Verweisquelle konnte nicht gefunden werden.].
The water to be investigated is flowing through the monitor at a selected rate,
e.g. 0.2 m/s. Biofilm formation is determined as a function of time by
collecting sacrificial supports from the monitor at regular intervals and
determining the active biomass concentration on the surface of these
supports. ATP is used as active biomass parameter. For the ratio between cell
carbon and ATP, generally a value of 250 is used [3]. Attached biomass is
released from the surface of the support by e.g. sonication, and cells in
suspension are permeabilised to extract nucleotides before measurement. The
concentration of ATP is determined in the biomass suspension and the ATP
concentration of the biofilm is calculated. The BFR (pg ATP cm
-2
d
-1
) of the
water can be calculated as the slope of the linear increase of the biofilm
concentration with time.

1. The monitoring system
2. Ultrasonic cleaning bath for transfer of biofilm from supports into
suspension.
3. Luminometer for light measurement (ATP analysis)
4. ATP analysis reagents: Nucleotide releasing reagent; Enzymatic
reaction reagents; ATP standard

Status of the technique:
Several monitoring systems have been used, e.g. LUMAC Biocounter,
Propella reactor, Rotating Annular Reactor.
ATP is proposed by European experts as a parameter of active biomass of
biofilms in drinking water systems [2].


References

1 Van der Kooij, D.,Veenendaal, H.R., Baars-Lorist, C., Klift, D.W. and Drost,
Y. C. (1995) Water Res. 29 1665-1662
2 Miettinen and Schaule (2003) Handbook for analytical methods and
operational criteria for biofilm reactors. SAFER WP2. EVKT-CT-2002-00108
May 2003
3 Karl, D. M. (1980) Cellular nucleotide measurements and applications in
microbial ecology. Microbol. Rev. 44 739-796
4 Van der Kooij, D., Vrouvenwelder, J. S. and Veenendaal, H. R. (2003).
Elucidation and control of biofilm formation processes in water treatment and
distribution using the unified biofilm approach. Water Sci. Technol. 47 83-90

Vendors
ATP-assay reagents: Promega; Celsis
Luminometers: Turner Biosystems; Celsis; Perkin Elmer

Evaluation:
The measurement of BFR requires several days/weeks of exposure of
sacrificial supports to water. ATP measurement is a more rapid and sensitive
technique for detection of active biomass on the supports, than enumeration
of culturable heterotrophic bacteria.

The ATP method can be used for measuring biofilm/biofouling biomass in
different parts of the water supply system, e.g. in drinking water distribution
[2] and on membranes used in drinking water treatment [4].

BFR ATP measurement is applied for monitoring biofilm formation both in
research projects and by water works.

The consumables of ATP analysis are relatively expensive.

Alternative methods for at-line/in-line biofilm monitoring of active biomass
such as (advanced) microscopy, combined with staining of biofilm
components can provide measures (i.e. thickness, volume, components) of
undisturbed biofilm on sacrificial supports. Such techniques are more time
consuming than ATP measurement and require expensive instrumentation
and skilled personnel. Biofilm enzymatic activity measurements can
potentially be used for at-line/in-line biofilm activity monitoring. These
methods are not providing direct measures of biomass.

The assimilable organic carbon (AOC) test is commonly used to assess the
concentration of growth promoting organic compounds in water. This
method gives a measure of the potential for biofilm growth, but is not
providing a direct quantitative measure of biofilm formation.


Monitoring technology nr 1:ATP measurement of biofilm

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x
x

time to result

x
x
Biofilm development: several d
ATP measurement: rapid
ease-of-use (B) x

Costs
consumables
maintenance

x

x

Recommendation for use in SSS (D)
x
Overall conclusion Reliable method, but expensive consumables ,
method o.k for active biomass measurement ,but not for
measurement of total biofilm components , i.e. all ( live
and dead) cells; EPS


3.14 Total cell counts

Prepared by: EAWAG

- There are no current drinking water guidelines or legislation covering this
parameter.
- The concentration range that should be covered is 10
2
10
9
bacterial cells/ml
- The sensitivity/resolution required is:
source water: 10
2
bacterial cells/ml
drinking water: 10
2
bacterial cells/ml

1. Direct total microbial cell count (based on fluorescence microscopy)
2. Direct total microbial cell count (based on flow cytometry)

3.14.1 Monitoring technology nr 1: Direct total microbial count (based on fluorescence
microscopy)

Description
The method consists of (1) fixing the water sample with gluteraldehyde (5%
w/v) for storage; (2) staining the fixed sample with acridine orange (0.1 %
w/v); (3) filtering a known volume (typically 1 mL) of the sample onto a non-
fluorescing polycarbonate membrane filter; (4) visualization and enumeration
of the stained cells on the filter with an epifluorescence microscope; and (5)
calculation of the total cell number based on the microscopy field size, the
total filter area and the total volume of water that was filtered (Hobbie et al.
(1977), Clesceri et al (1998).

Variations on the method:
Sample preparation: Alternative fixation agents can be used, e.g., formaldehyde
(2%), or in case of immediate sample processing, fixation can be omitted
entirely.
Staining: Alternative fluorescent dyes can be used, e.g., DAPI and SYBR
Green I (depending on the availability of relevant hardware in the fluorescent
microscope set-up).
Enumeration: Automated microscopy detection and enumeration hardware
and software is available and used by several research groups.

Equipment and consumables (basic method)

Instruments:
Epifluorescence microscope with the appropriate filter sets (see Standard
Methods 9216 (Clesceri et al., 1998))
Filtration unit (see Standard Methods 9216)


Consumables:
Membrane filters, Syringes, Test tubes, Phosphate buffer, Fixative, Acridine
orange, Immersion oil

This is a generally accepted method for total cell counting. The method
requires a skilled operator with experience in distinguishing bacterial cells
from background particles.

References
Methods for the examination of water and wastewater; 9216 Direct Total
Microbial Count. ISBN 0-87553-235-7

Hobbie, J.E., Daley, R. J. und Jasper, S. (1977). Use of Nuclepore filters for
counting bacteria by fluorescence microscopy. Appl. Environ. Microbiol., Vol.
33:1225-1228.

Evaluation
The concept to quantify bacteria in solution after filtration is based on
unspecific staining of their nucleic acids with a chemical fluorochrome and
enumeration by epifluorescence microscopy. Depending on the kind of
enumeration (by eye or digitally) it can be more or less time consuming.


Monitoring technology nr 1: Direct total microbial count (based on
fluorescence microscopy)

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x
x

time to result

< 20 min
ease-of-use (B) x

Costs
consumables
maintenance

x
x


Overall conclusion High initial investment costs for the purchase of a
fluorescence microscope. Some automation methods for
counting are also available to speed up analysis and
reduce labour requirements.

3.14.2 Monitoring technology nr 2: Direct total microbial count (based on flow
cytometry)

Description
The method consists of staining the bacterial cells in a water sample with a
fluorochrome (e.g. SYBR Green I or DAPI) and enumeration of bacterial
numbers by flow cytometry (Hammes et al., 2008; Lebaron et al., 1998). The
fluorescent dye is selected based on the availability of the relevant light
source and filters in the flow cytometer set-up (see also Lebaron et al., 1998).
The stain concentration and staining time are selected based on the
approximate concentration of bacteria in the sample and the specific features
of the fluorochrome. For example, for a cell concentration smaller than 1 x 10
6
cells/mL (typical for drinking water), a concentration final concentration of
10,000x diluted SYBR Green I is used with a staining time of 10 minutes
(Hammes et al., 2008). In case of high cell concentrations, enumeration with
flow cytometry might require pre-dilution. For the latter, cell-free (0.1 um
filtered) phosphate buffer or cell-free (0.1 um flitered) drinking water can be


used. Dilution should be performed immediately before the flow cytometric
measurement. For enumeration, some brands of flow cytometers are
equipped with volumetric counting hardware, while other brands use the
addition of a commercially available standard bead solution for counting.


Instruments:
Conventional flow cytometer equipped with a specific light source (e.g. 200
mW blue laser (488 nm)).

Consumables:
Test tubes, fluorochrome (e.g. SYBR Green I), cell-free phosphate buffer

Accepted method, currently in preparation for Standard Methods

Reference
Lebaron, P., N. Parthuisot, and P. Catala. 1998. Comparison of blue nucleic
acid dyes for flow cytometric enumeration of bacteria in aquatic systems.
Appl Environ Microbiol 64:1725-30.

Hammes, F., M. Berney, W. Yingying, M. Vital, O. Kster, and T. Egli (2008)
Flow-cytometric total bacterial cell counts as a descriptive microbiological
parameter for drinking water treatment processes. Water Research, 42(1-2),
269-77.

Evaluation
Flow cytometry is well adapted to enumerate high particle numbers in a short
time period.


Monitoring technology nr 2: Direct total microbial count (based on flow
cytometry)

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x
x

time to result

x < 15 min

ease-of-use (B) x

Costs
consumables
maintenance

x
x


Overall conclusion High initial investment costs for the purchase of a flow
cytometer. Otherwise the method is fast, accurate and
easy to use.


3.15 Cultivation free viability analysis

Prepared by: Eawag

Parameter: Cultivation free viability analysis

- There are no current drinking water guidelines or legislation covering this
parameter.
- The concentration range that should be covered: 10
3
10
9
bacterial cells/ml
- The sensitivity/resolution that is required
source water: 10
3
bacterial cells/ml
drinking water: 10
3
bacterial cells/ml

1. Cultivation free viability analysis (based on flow cytometry or fluorescence
microscopy)
2. Analysis of bacterial adenosine tri-phosphate (ATP)

3.15.1 Monitoring technology nr 1: Cultivation free viability analysis (based on flow
cytometry or fluorescence microscopy)

Note: The flow cytometric technology described herein consists of a
combination of several different staining methods. The different methods
target different aspects of bacterial viability. While individual stains can be
useful for specific applications, the use of several stains in concert is
suggested as the best-available approach to general viability analysis in an
unknown water sample. The method below is described for flow cytometry,
but can be used in combination with fluorescence microscopy as well (see
section 3.14 above)

Description
Water samples: It is essential that fresh water samples without pre-treatment
or fixation be used in the analysis. Any treatments may affect viability and
thus alter the outcome of the analysis.
Staining: The water samples are stained with four different fluorochromes
namely SYBR Green I (total cell count, see above), propidium iodide
(membrane integrity), DiBAC4(3) (membrane potential) and CFDA (enzyme
activity)) (Berney et al., 2008; Hammes et al., 2008; Hoefel et al., 2003). The
specific concentrations of the fluorochromes and the staining times are based
on the features of the individual stains and on the concentration of cells in the
water sample (Berney et al., 2008). For example, for a standard drinking water
sample (c.a. 1 x 10
5
cells/mL), the following stain combinations are used (2
uL/200 uL):
- SYBR Green I: 100x diluted in DMSO; 10 minutes incubation
- Propidium Iodide: 0.3 mM in SYBR Green I solution (above), 15 minutes
incubation
- DiBAC4(3): 0.1 mM in DMSO, 20 minutes


- CFDA: 10 mM in DMSO, 30 minutes at 30 C
Flow cytometry: In case of high cell concentrations, enumeration with flow
cytometry might require pre-dilution. For the latter, cell-free (0.1 um filtered)
phosphate buffer or cell-free (0.1 um flitered) drinking water can be used.
Dilution should be performed immediately before the flow cytometric
measurement. Correct instrument settings for the flow cytometer have to be
established for the specific instrument being used, in combination with a set
of relevant control samples (Berney et al., 2008).

Variations on the method: The list of stains mentioned here is not exhaustive,
since many alternative stains exist (Porter et al., 1995; Davey and Kell, 1996;
Joux and Lebaron, 2000).

Instruments:
Flow cytometer equipped with a blue laser (488 nm) and the appropriate filter
sets

Consumables:
Test tubes, fluorochromes (e.g. SYBR green I, propidium iodide, DiBAC4(3),
CFDA), sterile filtered (0.1 um) bottled mineral water or phosphate buffer for
dilution.

Method widely used for scientific purposes (pure cultures, bacterioplankton)
but not in drinking water routine analysis. Currently under development in
the Techneau project (Deliverable 3.3.7 and 3.3.8)

References
Porter, J., J. Diaper, C. Edwards, and R. Pickup. 1995. Direct measurements of
natural planktonic bacterial community viability by flow cytometry. Appl
Environ Microbiol 61:2783-2786.
Davey, H. M., and D. B. Kell. 1996. Flow cytometry and cell sorting of
heterogeneous microbial populations: the importance of single-cell analyses.
Microbiol Rev 60:641-96.
Joux, F., and P. Lebaron. 2000. Use of fluorescent probes to assess
physiological functions of bacteria at single-cell level. Microbes Infect. 2:1523-
35.
Hoefel, D., W. L. Grooby, P. T. Monis, S. Andrews, and C. P. Saint. 2003.
Enumeration of water-borne bacteria using viability assays and flow
cytometry: a comparison to culture-based techniques. J. Microbiol. Methods
55:585-97.

Evaluation


This method is currently under development (validation) and has a huge
potential for routine analysis and monitoring of microbial activity in drinking
water (during treatment and in the distribution system) because it is fast and
very reproducible. A combination of flow cytometric analysis with
conventional heterotrophic plate counts (HPC) and adenosine tri-phosphate
(ATP) analysis is encouraged (Berney et al., 2008). At the moment the method
requires trained personnel. Alternatively the analysis can be done on a
fluorescence microscope.

Monitoring technology nr 1: Cultivation free viability analysis (based on flow
cytometry)

sensitivity (A)
source water
drinking water

x

x

robustness (A)
selectivity

x

x

time to result

30 min
ease-of-use (B) x Requires expertise with
flow cytometry operation
and interpretation

Costs
consumables
maintenance

x
x


Overall conclusion Viability-free analysis is a different way to study/assess
the entire microbial community in drinking water. It can
be used to assess the efficacy of disinfection methods.
Some expertise in operation and interpretation is
required.


3.15.2 Monitoring technology nr 2: Analysis of bacterial ATP

Description
General: The ATP method is a bulk water microbiological paramter that
measures the concentration of ATP extracted from planktonic cells. It is based
on the principle that a lysis buffer extracts the ATP from the cells, and that
this ATP then reacts with the substrate-enzyme complex luciferin-luciferase
to produce light that can be measured in a luminometer. The resulting
relative light units (RLU) can then be related to a given ATP concentration by
means of a pre-established standard curve with pure ATP. Several ATP kits
are commercially available from different manufactures.
Water samples: It is essential that fresh water samples without pre-treatment
or fixation are used in the analysis. Any treatment may affect viability and
thus alter the outcome of the analysis.
Total ATP: The total ATP in the bulk water sample is measured directly.
Typically a protocol would be provided with each kit.
Free ATP: The free ATP is measured in the water following a 0.1um filtration
step.
Bacterial ATP: Calculated as the difference between total ATP and free ATP.
It is absolutely essential that sterile equipment is used for all the steps
involved in the measurement of ATP.

Instruments:
Standard luminometer (e.g. Glomax, Turner Biosystems)

Consumables:
ATP reagent (e.g. Promega); Sterile eppendorf tubes andpipettes; sterile 5 mL
syringes and sterile 0.1 um syringe filters.

There are no legal guidelines for ATP analysis, but the method has been used
for several decades, and have been suggested as a useful parameter for
drinking water analysis (Siebel et al., 2008; Delahaye et al., 2003).

References
Methods for the examination of water and wastewater; 9211 Bioluminescence
Test. ISBN 0-87553-235-7
Delahaye, E., Welte, B., Levi, Y., Leblon, G., and Montiel, A.: An ATP-based
method for monitoring the microbiological drinking water quality in a
distribution network. Water Res., 37(15), 3689-3696, 2003.
Hammes, F., M. Berney, W. Yingying, M. Vital, O. Kster, and T. Egli (2008)
Flow-cytometric total bacterial cell counts as a descriptive microbiological
parameter for drinking water treatment processes. Water Research, 42(1-2),
269-77.

Evaluation


The method has been used for several decades, but the use in drinking water
has been limited due to a lack of sensitivity of the assay (Clesceri et al., 1998).
Sample concentration and optimization of the measurement protocol can
overcome these shortcomings. A major problem is the absence of accurate
conversion factor to relate ATP concentrations to bacterial concentrations
(Berney et al., 2008). It is also essential to differentiate between bacterial ATP
and free ATP (Hammes et al., 2008), which make the analysis more expensive
and time consuming.

Monitoring technology nr 2: Analysis of bacterial ATP

sensitivity (A)
source water
drinking water

x

x

robustness (A)
selectivity

x

x

time to result

10 min
ease-of-use (B) x The method is extremely
easy to perform

Costs
consumables
maintenance

x

x


Overall conclusion The method is fast and easy to perform, but some skill is
required to measure low concentrations of ATP in water
samples.


4 Chemical parameters
4.1 Metals: Antimony, arsenic, boron, cadmium, chromium, copper, lead,
mercury, nickel, selenium, sodium, calcium, magnesium, aluminum, iron,
manganese

Prepared by: TZW

1. AAS: Atomic adsorption spectroscopy
a) Graphite furnace AAS
b) Hydride generation AAS
c) Cold vapour AAS
d) Flame AAS
2. AFS: Atom fluorescence spectroscopy
3. ICP-OES: Inductively-coupled plasma with optical emission spectroscopy
4. ICP-MS: Inductively-coupled plasma with mass spectrometry

The following table summarizes information about the suitability of each
technique for analyzing metals with the sensitivity required by the DWD.
Other technologies like ion chromatography or voltametric methods are
applicable to single methods, however, most often suffer from limited
sensitivity and/or selectivity and thus will not be considered here.

Parameter

P
a
r
a
m
e
t
r
i
c

v
a
l
u
e

D
W
D

[
m
g
/
L
]

S
e
n
s
i
t
i
v
i
t
y

r
e
q
u
i
r
e
d

[
g
/
L
]

G
r
a
p
h
i
t
e

f
u
r
n
a
c
e

A
A
S

H
y
d
r
i
d
e

G
e
n
e
r
a
t
i
o
n

A
A
S

C
o
l
d

v
a
p
o
u
r

A
A
S

C
o
l
d

v
a
p
o
u
r

A
F
S

F
l
a
m
e

A
A
S

I
C
P
-
O
E
S

I
C
P
-
M
S

Antimony 0,005 1,25 x x
Arsenic 0,010 1,0 x x
Boron 1,0 100 x x
Cadmium 0,005 0,5 x x
Chromium 0,050 5,0 x x
Copper 2,0 200 x x x
Lead 0,010 1,0 x x
Mercury 0,001 0,20 x x
Nickel 0,020 2,0 x x
Selenium 0,010 1,0 x x
Sodium 200 20000 x x x
Calcium - - x x x
Magnesium - - x x x
Aluminium 0,50 50 x x x
Iron 0,20 20 x x x
Manganese 0,050 5,0 x x x


4.1.1 Monitoring technology nr 1: AAS (Atomic adsorption spectroscopy)

Description:
The water sample is acidified to pH < 2 with nitric acid. Then an aliquot is
injected into the AAS instrument. For concentrations < 100 g/l the graphite
furnace technique (GF-AAS) is recommended, whereas for concentrations >
100 g/L the flame AAS is preferred. For the hydride forming metals
(antimony, arsenic, selenium) the hydride generation technique (HG-AAS)
could be used to improve method performance. For a sensitive determination
of mercury by AAS, the cold vapour technique (CV-AAS) must be used.

Evaluation:

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x
x

time to result

x
ease-of-use (B) x

Costs
Operational costs (C)
consumables
maintenance

x
x

Recommendation for use in SSS (D) x cheap instrument which can be
used for multiple parameters

Overall conclusion Cheap method with a limited linear range; rather time
consuming if several elements have to be analysed as it is a
sequential technique.


4.1.2 Monitoring technology nr 2: AFS (Atomic fluorescence spectroscopy)

Description:
Special detection technology based on the cold vapour technique for the
sensitive analysis of mercury. The water sample is acidified to pH < 2 with
nitric acid. For preservation hydrobromic acid or potassium dichromate are
added. Then an aliquot is injected into the AFS instrument.

Evaluation:

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x
x

time to result

x
ease-of-use (B) x

Costs
Operational costs (C)
consumables
maintenance

x
x


Overall conclusion Method of choice for mercury analysis.


4.1.3 Monitoring technology nr 3: ICP-OES: Inductively-coupled plasma with optical
emission spectroscopy

Description:
injected into the AFS instrument.

Evaluation:

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x
x

Selectivity depends on the
optical resolution of the
instrument.
time to result

x
ease-of-use (B) x

Costs
consumables
maintenance

x
x


Overall conclusion Multi-element method for determination of elements in the
mg/L range; Large linear range but not suitable for trace
analysis.


4.1.4 Monitoring technology nr 4: ICP-MS: Inductively-coupled plasma with mass
spectrometry

Description:
injected into the AFS instrument.

Evaluation:

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x
x

Selectivity can be improved by
using a reaction or collision cell.
time to result

x
ease-of-use (B) x

Costs
consumables
maintenance

x
x


Overall conclusion Fast and sensitive multi-element method with a large linear
range.


4.2 Benzene

Prepared by: TZW

- concentration range
0.25 to 10 g/L (drinking water)

- sensitivity required
0.25 g/L (drinking water)

1. Liquid-liquid extraction, gas chromatography with flame ionization
detection or mass spectrometric detection (GC-FID or GC-MS)
2. Headspace, GC-FID or GC-MS
3. Purge&trap, GC-FID or GC-MS
4. Solid-phase micro extraction (SPME), GC-FID or GC-MS
4.2.1 Monitoring technology nr 1: Liquid-liquid extraction, GC-FID or GC-MS

Description:
100 mL water sample are extracted with 1 mL of n-pentane or n-hexane. After
separation of the organic layer, an aliquot is injected into the gas
chromatograph. Detection of benzene is possible with both, a flame ionization
detector (FID) or a mass spectrometer (MS). Both detectors exhibit
comparable sensitivity but a MS detector offers better selectivity.


Evaluation:

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x
(FID)

x
x
(MS)

time to result

x ca. 1 h
ease-of-use (B) x

Costs
(FID)
x
(MS)

consumables
maintenance

x
x


Overall conclusion Good and reliable technology that is rather easy to use (but
needs trained personal).

4.2.2 Monitoring technology nr 2: Headspace, GC-FID or GC-MS

Description:
30 mL water sample (depending on system used) are filled in a vial that is set
on an elevated temperature (e.g. 60 C). Then, an aliquot of the headspace is
automatically transferred by a syringe into the gas chromatograph. Detection
of benzene is possible with both, a flame ionization detector (FID) or a mass
spectrometer (MS). Both detectors exhibit comparable sensitivity but a MS
detector offers better selectivity.


Evaluation:

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x
(FID)

x

x
(MS)

time to result

x ca. 0.5 h
ease-of-use (B) x

Costs
(FID)
x
(MS)

consumables
maintenance

x
x



4.2.3 Monitoring technology nr 3: Purge&trap, GC-FID or GC-MS

Description:
30 mL water sample are purged in a nitrogen stream. The volatile water
ingredients are stripped and trapped onto a trap (which most often is some
activated carbon material). By heating the trap material the target compounds
are transferred into the gas chromatograph. Detection of benzene is possible
with both, a flame ionization detector (FID) or a mass spectrometer (MS).
Both detectors exhibit comparable sensitivity but a MS detector offers better
selectivity.


Evaluation:

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x
x
(FID)

x
(MS)

time to result

x ca. 0.5 h
ease-of-use (B) x

Costs
(FID)
x
(MS)

consumables
maintenance

x
x


4.2.4 Monitoring technology nr 4: Solid-phase micro extraction (SPME), GC-FID or GC-
MS

Description:
30 mL water sample are extracted with a fiber coated with polymeric
material. The target analytes adsorb onto the polymeric material. The fiber is
transferred into the injector of the gas chromatograph. When the injector is
heated, the target compounds desorb from the fiber and thus are transferred
onto the chromatographic column. Detection of benzene is possible with both,
a flame ionization detector (FID) or a mass spectrometer (MS). Both detectors
exhibit comparable sensitivity but a MS detector offers better selectivity.


Evaluation:

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x
x
(FID)

x
(MS)

time to result

x ca. 0.5 h
ease-of-use (B) x

Costs
(FID)
x
(MS)

consumables
maintenance

x
x




4.3 Benzo(a)pyrene and other PAHs

Prepared by: TZW

0.0025 to 0.5 g/L per single compound (drinking water)
For contaminated sites higher concentrations can be expected.

0.0025 g/L per single compound (drinking water)

1. Liquid-liquid extraction, Liquid chromatography with fluorescence
detection (HPLC/FLD)
2. Liquid-liquid extraction, GC/MS

4.3.1 Monitoring technology nr 1: Liquid-liquid extraction, HPLC/FLD

Description:
1 L (or 2 L, depending on the sensitivity of the detection system) water
sample are extracted with 45 mL of cyclohexane. After separation of the
organic layer, the volume is carefully reduced to ca. 200 L. Then 50 L
dimethylformamide (DMF) are added as keeper and the volume is further
reduced. An aliquot of the organic extract is injected into the liquid
chromatograph. Detection of the PAH is done with fluorescence detection at
the respective wavelength combinations.


Evaluation:

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

time to result

x
ease-of-use (B) x

Costs
consumables
maintenance

x
x


Overall conclusion Sensitive and selective analytical method. Use for drinking
water analysis is recommended. In contaminated waters,
problems with interferences might occur.

4.3.2 Monitoring technology nr 2: Liquid-liquid extraction, GC/MS

Description:
1 L (or 2 L, depending on the sensitivity of the detection system) water
sample are extracted with 25 mL of cyclohexane. After separation of the
organic layer, the volume is carefully reduced to ca. 500 L. Then, an aliquot
is injected into the gas chromatograph. Detection of the PAH is done with
mass spectrometric detection.


Evaluation:

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

time to result

x
ease-of-use (B) x

Costs
consumables
maintenance

x
x


Overall conclusion Sensitive analytical method with a large range of linearity
and high robustness. Method can especially be
recommended for analysis of contaminated waters.


4.4 Bromate

Prepared by: TZW

1 to 10 g/L (drinking water)

1 g/L (drinking water)
This parameter is only relevant for drinking waters if ozonation is used.

1. Ion chromatography with conductivity detection (IC/CD)
2. Ion chromatography with UV detection (IC/UV)
3. Ion chromatography with fluorescence detection (IC/FLD)
4. Ion chromatography with inductively coupled plasma mass spectrometry
detection (IC/ICP-MS)

(IC/CD)

Description:
Depending on the sensitivity of the detection system, an aliquot of the sample
can be directly injected into the ion chromatographic system. To improve
sensitivity, on-line pre-concentration is possible. Details of both methods
(direct injection and on-line pre-concentration are described in EN ISO 15061).


Evaluation:

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

time to result

x
ease-of-use (B) x

Costs
consumables
maintenance

x

x


Overall conclusion Standardized method with good sensitivity and selectivity.

4.4.2 Monitoring technology nr 2: Ion chromatography with UV detection (IC/UV)

Description:
An aliquot of the sample is directly injected into the ion chromatographic
system. After separation of the ions, post-column derivatisation takes place.
Different methods with different agents are available (e.g. tribromide method,
o-dianisidine method, methylene blue method, chloropromazine method).
During all reactions, colored compounds are formed which are then detected
on-line by UV photometric measurements.


Evaluation:

sensitivity (A)
source water
drinking water

x

x

robustness (A)
selectivity

x

x

time to result

x
ease-of-use (B) x

Costs
consumables
maintenance

x
x


Overall conclusion Method is very sensitive but lacks of limited selectivity due
to possible interferences with other compounds reacting
with the derivatization agent.

4.4.3 Monitoring technology nr 3: Ion chromatography with fluorescence detection
(IC/FLD)

Description:
An aliquot of the sample is directly injected into the ion chromatographic
system. After separation of the ions, post-column derivatisation takes place.
Different methods with different agents are available. During all reactions,
compounds which are fluorescence-active are formed which are then detected
on-line by fluorescence measurements.


Evaluation:

sensitivity (A)
source water
drinking water

x

x

robustness (A)
selectivity

x

x

time to result

x
ease-of-use (B) x

Costs
consumables
maintenance

x
x


Overall conclusion Method is very sensitive but lacks of limited selectivity due
to possible interferences with other compounds reacting
with the derivatization agent.

4.4.4 Monitoring technology nr 4: Ion chromatography with inductively coupled plasma mass
spectrometry detection (IC/ICP-MS)

Description:
An aliquot of the sample can be directly injected into the ion chromatographic
system. After ion chromatographic separation, the effluent is coupled with a
suppressor unit to the nebulizer of the ICP-MS. Detection of bromate is
preferably done via the
79
Br mass.


Evaluation:

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x
x

time to result

x
ease-of-use (B) x

Costs
consumables
maintenance

x

x


Overall conclusion Excellent method that combines high selectivity with
outstanding sensitivity, if ICP-MS is available.


4.5 Cyanides

Prepared by: TZW

0.01 to 0.5 mg/L

0.01 mg/L

1. Photometric method (batch mode)
2. Continuous flow analysis

4.5.1 Monitoring technology nr 1: Photometric method (batch mode)

Description:
Cyanide is released from its complexes by addition of special chemicals.
Hydrogen cyanide is formed which is transferred into caustic soda where the
cyanide is photometrically determined after addition of a barbituric
acid/pyridine mixture. Several systems from different manufacturers are
commercially available.


Evaluation:

sensitivity (A)
source water
drinking water

x
x
Sensitivity of the method
strongly depends on the size of
the cuvette used for the
photometric detection.
robustness (A)
selectivity

x

x

time to result

x
ease-of-use (B) x

Costs
consumables
maintenance

x
x


Overall conclusion Low cost and easy to use method for fast analysis of
cyanide.

4.5.2 Monitoring technology nr 2: Continuous flow analysis

Description:
First, the water sample is on-line digested by UV light. After distillation of the
cyanide it and addition of a barbituric acid/pyridine mixture, a photometric
on-line measurement takes place. Commercial systems from different
manufacturers are on the market.


Evaluation:

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x
x

time to result

x
ease-of-use (B) x

Costs
consumables
maintenance

x
x


Overall conclusion Sensitive and robust method which requires advanced
instrumentation.


4.6 1,2-Dichloroethane

Prepared by: TZW

- concentration range: 0.3 3 g/L (drinking water)

- sensitivity required: 0.3 g/L (drinking water); according to DWD, accuracy and
precision of the method used should be below 20% of the parametric value

1. Liquid-liquid extraction, GC-ECD(-ECD)
2. Headspace, GC-ECD-(ECD)
3. purge&trap, GC-MS


4.6.1 Monitoring technology nr 1: Liquid-liquid extraction, GC-ECD(-ECD)

Description:
EN ISO 10301: Liquid-liquid extraction of 100 mL water sample with 1 mL n-
pentane; analysis of extract with GC-ECD or GC-ECD-ECD (using two GC
columns with different polarity)

Evaluation:

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

time to result

x
ease-of-use (B) x

Costs
consumables
maintenance

x
x


Overall conclusion Reliable standard method. Sensitivity for 1,2-dichloroethane,
however, is low and the requirements of the DWD can
hardly be fulfilled. For increasing selectivity, two GC
columns should be used.


4.6.2 Monitoring technology nr 2: Headspace, GC-ECD-(ECD)

Description:
Headspace analysis of ca. 30 mL water sample; analysis of extract with GC-
ECD or GC-ECD-ECD (using two GC columns with different polarity)

Evaluation:

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

time to result

x
ease-of-use (B) x

Costs
consumables
maintenance

x
x


Overall conclusion Reliable method. Sensitivity for 1,2-dichloroethane,
however, is low and the requirements of the DWD can
hardly be fulfilled. For increasing selectivity, two GC


4.6.3 Monitoring technology nr 3: purge&trap, GC-MS

Description:
Purge&trap analysis (dynamic headspace) of ca. 30 mL water sample;
analysis of extract with GC-MS (GC-ECD or GC-ECD-ECD analysis is also
possible).

Evaluation:

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x
x

time to result

x
ease-of-use (B) x

Costs
consumables
maintenance

x
x


Overall conclusion Reliable and sensitive method. Method of choice for analysis
of drinking water samples.


4.7 Fluoride

Prepared by: TZW

0.05 to 0.5 g/L

0.05 g/L

1. Ion-selective electrode (ISE)

4.7.1 Monitoring technology nr 1: Ion-selective electrode (ISE)

Description:
A buffer solution (TISAB) is added to the water sample to adjust pH and salt
content. Then fluoride is measured by using an ion-selective electrode (which
is available from different manufacturers).

Evaluation:

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

time to result

x
ease-of-use (B) x

Costs
consumables
maintenance

x

x


Overall conclusion Fast and low-cost method for selective analysis of fluoride.


(IC/CD)

Description:
An aliquot of the water sample is directly injected into the ion
chromatographic system.

Evaluation:

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

The separation power of the
column is crucial for a good
selectivity.
time to result

x
ease-of-use (B) x

Costs
consumables
maintenance

x

x


Overall conclusion Fast and reliable method which can also be used for analysis
of other ions.


4.8 Nitrite

Prepared by: S::can

Concentration ranges that can be encountered are the following:
source water (ground or surface water)
0 - 50 mg/L (measured as NO2)
drinking water
0 - 0.50 mg/L (measured as NO2)

Required sensitivity of the measurement: 0.05 mg/L

1. Ion chromatography
2. Wet chemical analysis / colorimetric method
3. Spectrometric method, derivative spectroscopy

4.8.1 Monitoring technology nr 1: Ion Chromatography

Description:
In ion chromatography, a pre-treated sample is processed in the laboratory.
The sample in injected in chromatograph, and the components present are
separated on the column. Based on retention time the desired component is
identified.

Equipment: ion chromatograph, including chromatographic column, HPLC
pump.
Consumables: solvents, filters, columns
Lab technique available for many years. Highly reliable and accurate. Very
expensive. Available from Waters, Metrohm, Thermo Electron, Dionex and
others.

Evaluation:
- Highly accurate technique. Only suitable for off-line, batch wise laboratory
analysis. Useful for regulatory measurements.
- Most accurate technique, but also by far the most expensive (investment in
equipment).
- This method fulfils the needs in terms of measuring range and accuracy.


Monitoring technology nr 1: Ion chromatography

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

time to result

x
ease-of-use (B) x calibration requires some
training

Costs
instrumentation (C) x price indication: > 15.000 Euro
consumables
maintenance

x

x


Overall conclusion Suitable method for reference / regulatory measurements in
the laboratory

4.8.2 Monitoring technology nr 2: Wet chemical analysis Colorimetric method

Description:
Nitrite is determined through formation of a reddish purple dye produced at
pH 2. The application range of the method for spectrometric measurements is
10 - 1000 g/L nitrite. Higher NO2 concentrations can be determined after
dilution of the sample. The analysis has to be performed immediately after
sampling to ensure reliable NO2 concentrations.

- Equipment needed: photometer, test-tube with reagent (pr-mixed, easy to
handle), filter to remove solids / turbidity from the sample before starting
test
- Consumables: test tubes (approx 2 - 5 Euros per test)
- Photometry is established technique (see Standard Methods for the
examination of water and Waste Water APHE, AWWA, WEF publication).
Available from many vendors, such as WTW, Dr. Lange.


Evaluation:
- Relatively accurate tests that can be used in the lab and in the field.
Relatively easy to use (any lab technician can handle it, but the operator will
need at least some lab training).
- Only off-line analysis possible.
- This method fulfils the needs in terms of range and accuracy.

Monitoring technology nr 2: Wet chemical analysis colorimetric method

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x
x

time to result

x 15 minutes
ease-of-use (B) x

Costs
consumables
maintenance

x

x


Overall conclusion Suitable method for referencing, calibration, periodical
measurements


4.8.3 Monitoring technology nr 3: Spectrometric method, derivative spectroscopy

Description:
Even though nitrite absorbs strongly in the ultraviolet light, determining
nitrite by measuring the absorbance at one wavelength is not reliable because
natural organic matter and other solutes also absorb UV light. In natural
water and drinking water, the nitrate concentrations are normally so much
higher and its signal can not be distinguished from the nitrite signal when
using a single wavelength. In the nitrite spectrum the absorbance increase
rapidly from 230 to 210 nm, but with a different slope compared to the
absorbance of the nitrate ion. Computing the first or second derivative of the
spectrum allows differentiation between nitrite and nitrate, and both can be
measured independently.

Using an instrument of sufficient optical resolution, it is possible to measure
both nitrate and nitrite simultaneously.

Bromide is known to interfere with this type of measurement at sea water
concentrations. However, as the bromide concentration in sea water is rather
stable, a single calibration will cancel out this effect.

Laboratory spectrophotometers have been used for this type of analysis since
the 1960's. Nowadays, such systems are also available as online, in situ probe.
Full spectrum spectrometers suited for NO2 measurement can be obtained
e.g. from s::can.
The costs for a spectrometer, lies in the range of 8000 - 12000 Euros. Each of
these probes will provide multiple parameters, such as DOC, TOC, UV254,
nitrate and others simultaneously.

Evaluation:
This is the most reliable online technique for nitrite measurement. Calibration
is required most of the time at the start of the installation. Long term stability
of the optical parts ensures long term measurement stability when no fouling
of the optical parts takes place. Some instruments are equipped with
automatic cleaning to counter fouling.


Monitoring technology nr 3: Spectrometric method, derivative spectroscopy

sensitivity (A)
source water
drinking water

x

x

robustness (A)
selectivity

x

x

time to result

x < minute
ease-of-use (B) x

Costs
consumables
maintenance

x
x

Recommendation for use in SSS (D) x Spectrometers capable of
performing this measurement can
monitor a range of parameters
simultaneously. Initial cost per
instrument might be higher, but
are cheaper than multiple cheap
instruments combined. As soon
as 3 - 4 parameters (such as
nitrate, turbidity and DOC) are
needed, these instruments are
competivite.

Overall conclusion Reliable method, but rather expensive for a single
parameter. When multiple parameters provided by the
instrument are of interest, however, price is very
competitive. Operationally very robust and very low
maintenance requirements.


4.9 Nitrate

Prepared by: S::can

drinking water

Required sensitivity of the measurement: 1 mg/L

1. Wet chemical analysis/colorimetric method
2. Electrochemical method
3. Spectrometric method, single wavelength
4. Spectrometric method, derivative spectroscopy
5. Ion chromatography (see for this technique par. 4.22)

4.9.1 Monitoring technology nr 1: Wet Chemical Analysis

Description:
The nitrate present in a sample is reduced to nitrite (NO2) in the presence of
cadmium. The NO2 thus produced is determined by diazotising with
sulfanilamide and coupling to N-(1-naphthyl)-ethylenediamine- Thus a
highly coloured dye is formed, which is measured colorimetrically.

The applicable range is 0.01 - 1.0 mg/L. Samples at higher concentration need
to be diluted before analysis. The method is sensitive to particulate matter
present in the sample, as well as presence of iron and copper.

Alternatively, reaction of nitrate with hydrazine sulphate, followed by
diazotisation as after reduction with cadmium has been used. This can be
used in the range 0.01 - 10 mg/L NO3-N.

This analysis can be performed using standard laboratory equipment.
Alternatively, ready to use cuvettes prefilled with reagent can be obtained. In
this case a micropipette is required to accurately dispense the correct amount
of liquid into the cuvette. After waiting for the prescribed time ( 10 - 15
minutes), the solution is analyses using a colorimeter.
This analysis is well established and described in standard methods, such as
APHA Standard methods for the examination of water and waste water (21st
edition). The cuvette tests are available from various manufacturers such as
WTW, LaMotte, Hach Lange.


The costs for a colorimeter are approx 1000 - 2000 Euros and one cuvette (one
concentration measurement) costs approximately 2 - 3 Euros.

Alternatively, this analysis has also been automated. So-called process-
analysers perform this analysis fully automatic, generally with a frequency of
one measurement per minute. As these instruments perform the classical wet
chemical analysis, they consist of many pumps for dosing chemicals,
collection waste etc. This makes them prone to failures and very intensive to
maintain. These systems have mainly been use in industrial applications and
waste water treatment plants.

Evaluation:
The cuvette tests for nitrate are a reliable and fast method for concentration
determinations. They are suited for use in the field as well as in the
laboratory. The only limitation is the need to perform them manually. The
process analysers that automate this procedure suffer from many issues,
mainly complexity, to make them a good alternative to the techniques for
online measurement described below.

This method is particularly suited for low concentration measurements (0.01
mg/L range) where the other techniques are less accurate.

Monitoring technology nr 1: Wet chemical analysis

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x
x

time to result

x 15 minutes
ease-of-use (B) x for the cuvette tests
maintenance requirements (C) x for the cuvette tests, for the
online system score would be '2'

Costs
instrumentation (C) x colorimeter
consumables
maintenance

x

x


measurements


4.9.2 Monitoring technology nr 2: Electrochemical method

Description:
The NO3 ion electrode is a selective sensor that develops a potential across a
thin, porous, inert membrane that holds in place a water-immiscible liquid
ion exchanger. The electrode responds to NO3 ion activity between about 0.14
and 1400 mg/L as NO3-N). Chloride and bicarbonate ions can interfere when
their concentrations are 5 - 10 times higher than nitrate. Cross sensitivity for
nitrite is also known.

As the measurement is pH sensitive, for reliable measurement either stable
pH is required, or a pH sensor is needed to correct for the actual pH.

Equipment
ion selective electrode for nitrate
control device / data logger
calibration solutions with known nitrate concentrations

Systems using ion selective electrodes have been used for many years. They
function well in case of good maintenance (regular cleaning, membrane
replacement, re-calibration). The method has been included in APHA
Standard methods for the examination of water and waste water (21st
edition).

Selective electrodes for NO3 can be obtained from manufacturers such as
WTW, LaMotte, Hach Lange, Endress + Hauser, Jumo, YSI,.

The costs for an electrode, including controller are 2000 - 3000 Euros. Price
depends on application. Water proof industrial controller is more expensive
than lab equipment.

Evaluation:
In principle a well proven and satisfactory technique. However, the use of
electrodes always suffers from limitations, such as cross sensitivity,
maintenance requirements. For continuous monitoring, therefore, optical
instruments are preferred.


Monitoring technology nr 2: Electrochemical method

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x
x

time to result

x < minute
ease-of-use (B) x
maintenance requirements (C) x frequent calibration etc

Costs
consumables
maintenance

x

x


Overall conclusion Suitable method for online monitoring, however
maintenance intensive

4.9.3 Monitoring technology nr 3: Spectrometric method, single wavelength

Description:
This method is suitable for water with low organic content, ie.
uncontaminated natural waters or drinking water. Measurement of light
absorption at 220 nm allows rapid determination of the NO3 concentration.
NO3 absorps very strongly in this region of the spectrum, so strong even that
it is the main cause of absorption there. However, because only one
wavelength is used, and no distinction can be made between substances, this
method is highly sensitive to other substances as well. A crude correction is
sometimes made for organics by measurement at a second wavelength, 275
nm, where NO3 does not absorb but some organic substance do absorb.
However, even in that case cross sensitivity for nitrite, dissolved organic
matter and chromium are often encountered.

Equipment
Single or dual wavelength spectrometer

Systems using single and dual wavelength spectrometers for nitrate
measurement have been in use now for approximately 20 years. For stable
water quality they can provide good data. They are currently being replaced


by multiwavelength instrumentation (see part 4 of this datasheet). The
method has been included in APHA Standard methods for the examination of
water and waste water (21st edition).

Single and dual wavelength spectrometers for NO3 can be obtained from
manufacturers such as Hach Lange, Endress + Hauser, YSI.

The costs for a spectrometer lies in the range of 3000 - 4000 Euros.

Evaluation:
In principle a well proven and satisfactory technique. However, the use of
single or dual wavelength optical systems suffers from limitations, mainly
cross sensitivity for organics and turbidity.

Monitoring technology nr 3: Spectrometric method, single wavelength

sensitivity (A)
source water
drinking water

x

x

robustness (A)
selectivity

x

x

time to result

x < minute
ease-of-use (B) x

Costs
consumables
maintenance

x

x

Recommendation for use in SSS (D) x could be used for control of
drinking water nitrate levels in
case of stable source (ground
water)

Overall conclusion Online method that requires low maintenance. Method is
not very selective, therefore false results are often obtained.


4.9.4 Monitoring technology nr 4: Spectrometric method, derivative spectroscopy

Description:
Even though nitrate absorbs strongly in the ultraviolet light, determining
nitrate by measuring the absorbance at one wavelength is not realiable
because natural organic matter and other solutes also absorb UV light. The
spectra of these organics are very different between water sources, therefore
the method described under 3 can be very inaccurate. The UV spectrum of
nitrate is quite different from that of organic matter. In the nitrate spectrum
the absorbance increase rapidly from 230 to 210 nm, whereas the organic
spectrum in natural water this absorbance only increases gradually in this
region. Computing the first derivative of the spectrum will effectively
eliminate the background signal of the organic and only indicate the nitrate
absorption.

The main interference possible in this measurement is nitrite, that has a
similar but not identical spectrum. Using an instrument of sufficient optical
resolution, it is possible to measure both nitrate and nitrite simultaneously.

Bromide is known to interfere with this type of measurement at sea water
concentrations. However, as the bromide concentration in sea water is rather
stable, a single calibration will cancel out this effect.

Equipment
Spectrophotometer.

Laboratory spectrophotometers have been used for this type of analysis since
the 1960's. The method has been included as a proposed method in APHA
Standard methods for the examination of water and waste water (21st
edition).

Nowadays, such systems are also available as online, in situ probe. Full
spectrum spectrometers suited for NO3 measurement can be obtained from
various manufacturers such as s::can, Trios, Stip.

The costs for a spectrometer, lies in the range of 8000 - 20000 Euros. Each of
these probes will provide multiple parameters, such as DOC, TOC, UV254,
nitrate and others simultaneously.

Evaluation:
This is the most reliable online technique for nitrate measurement.
Calibration is required most of the time at the start of the installation. Long
term stability of the optical parts ensures long term measurement stability
when no fouling of the optical parts takes place. Some instruments are
equipped with automatic cleaning to counter fouling.


Monitoring technology nr 4: Spectrometric method, derivative spectroscopy

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

time to result

x < minute
ease-of-use (B) x

Costs
consumables
maintenance

x
x

Recommendation for use in SSS (D) x Spectrometers capable of
performing this measurement can
monitor a range of parameters
simultaneously. Initial cost per
instrument might be higher, but
are cheaper than multiple cheap
instruments combined. As soon
as 3 - 4 parameters (such as
nitrate, turbidity and DOC) are
needed, these instruments are
competitive.

Overall conclusion fast, online method with high selectivity. Equipment is
more expensive than single wavelength.


4.10 Polycyclic aromatic hydrocarbons

Prepared by: TZW

See par. 4.3: Benzo(a)pyrene and other PAHs

4.11 Pesticides

See par. 4.19: Organic microcontaminants


4.12 Tetra- and trichloroethene

Prepared by: TZW

- concentration range: 0.1 100 g/L (drinking water; raw water concentration can
be higher)
- sensitivity required: 0.1 g/L (drinking water)



Description:
hexane; analysis of extract with GC-ECD or GC-ECD-ECD (using two GC
columns with different polarity).
Evaluation:

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

time to result

x
ease-of-use (B) x

Costs
consumables
maintenance

x
x


Overall conclusion Reliable standard method. For increasing selectivity, two GC



Description:

Evaluation:

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

time to result

x
ease-of-use (B) x

Costs
consumables
maintenance

x
x


Overall conclusion Reliable method. For increasing selectivity, two GC columns
should be used.



Description:
possible).

Evaluation:

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x
x

time to result

x
ease-of-use (B) x

Costs
consumables
maintenance

x
x

x

Overall conclusion Reliable and sensitive method.


4.13 Disinfection byproducts (trihalomethanes)

Prepared by: TZW

- concentration range: 0.1 100 g/L (drinking water; depending on the chlorine
doses)
- sensitivity required: 0.1 g/L (drinking water)


Description:
hexane; analysis of extract with GC-ECD or GC-ECD-ECD (using two GC
columns with different polarity)

Evaluation:

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

time to result

x
ease-of-use (B) x

Costs
consumables
maintenance

x
x


Overall conclusion Reliable standard method. For increasing selectivity, two GC



Description:

Evaluation:

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

time to result

x
ease-of-use (B) x

Costs
consumables
maintenance

x
x


Overall conclusion Reliable method. For increasing selectivity, two GC columns
should be used.



Description:
possible).

Evaluation:

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x
x

time to result

x
ease-of-use (B) x

Costs
consumables
maintenance

x
x

x

Overall conclusion Reliable and sensitive method.


4.14 Radioactivity

Regarding radio-activity there was no partner within WA3 having sufficient
expertise to prepare an evaluation form. To enable readers to have some
information on analytical techniques, information from the literature will be
presented below. The information is retrieved from:
Wisser, S. and Hartkopf, J. (2006) Natural and artificial radioactivity in the
Rhine and its tributaries. In: Knepper (ed) The Handbook of Environmental
Chemistry. Volume 5 Water Pollution, Part L: The Rhine. p255-306.

Measurement systems for radioactive isotopes are primarily based on the in-
teraction between ionizing radiation and matter. Presently, three categories of
radiation detection systems are mainly used for radionuclide determination:
Semiconductor detectors (-/- and -spectrometry)
Scintillation counters (- and /-measurements)
Proportional counters (- and /-measurements)
Counting techniques are still highly applicable for the determination of
radioisotopes with half-lives of less than a few thousand years. However,
inductively coupled plasma mass spectrometry is the current state-of-art in-
strumentation for measuring multiple elements at trace level. Unfortunately,
this outstanding method is presently only applicable for stable isotopes or
long-lived primordial isotopes, such as 232Th and 238U.
The following sections provide details of measurement techniques for the
determination of radionuclides and present a brief summary of the advan-
tages and disadvantages of the respective counting method.

4.14.1 Semiconductor Detectors
The use of solid detection medium is of great advantage for the detection of
high-energy electrons, heavy particles and -rays. In the first place, detector
dimension can be kept smaller due to the high density of semiconductors. In
addition to that, a superior energy resolution can be achieved by using a solid
semiconductor detector. There are different semiconductor materials
available today, such as silicon and germanium. The fundamental informa-
tion carriers are electron-hole pairs created in the crystal lattice by primary or
secondary charged particles along their path to the detector [1]. The move-
ment of electron-hole pairs through the crystal generates the basic electrical
signal from the detector.
Since the intensity of the electric pulse is proportional to the energy of the
penetrating particle, semiconductor detectors contribute full energy informa-
tion. Multi-channel analyzers (MCA) provide the energy resolution and the
primary analog signal is transformed into digital units by use of an analog-
digital-converter. Compared with other counting techniques, semiconductor
detectors possess advantages, such as excellent energy resolution, good
stability, excellent timing characteristics and simplicity of operation [1].

Alpha-Spectrometry
Alpha-spectrometry is an extremely useful and sensitive method for the de-
tection of -emitting radionuclides in a variety of materials. One of the main
reasons for the sensibility of this method is the very low background radia-


tion. High-purity silica surface barrier detectors have become the detectors of
choice for the measurement of -particles. The relatively small size of a silicon
detector might be a limitation, especially when a large surface area is
required. Modern -spectrometers are equipped with vacum chambers to
achieve a high-energy resolution of 20-30 keV. The obtained a-spectrum can
easily be analyses by using simple region of interest (ROI) settings. In
addition, the energy resolution is generally sufncient to separate the energy
peaks of the most important natural and artificial radionuclides present in
waters.
Unfortunately, extended measurement times (2-3 days) are necessary to
achieve low detection limits in the range of several mBq/L.

Gamma-Spectrometry
High-purity germanium (HPGe) detectors are preferably used for complex -
ray spectrometry. This is due to the lower melting point of germanium
compared with silicon, which makes the exclusion of impurities in the re-
fining process much easier [1]. Additionally, the extremely high density of
germanium (5.33 g/cm
3
) leads to a higher adsorption probability for -rays
[1]. Another advantage of gamma-spectrometry is the relatively low self-
absorption of -rays within the sample, which results in a simple sample
preparation. However, the counting efficiency of HPGe detectors is com-
parably low and the naturally occurring -background radiation might be
problematic when trying to achieve low detection limits.
These three effects of radiation interactions with matter are fundamental of -
spectrometry:
- Photo effect (detector-atom absorbs the energy of the incident photon
entirely)
- Compton effect (incident photons are scattered by electrons of the
detector)
- Pair production (positron-electron pair is created by the incident
photon)
The operation of germanium detectors at room temperature conditions is
impossible. This is because of the thermally induced leakage current that
would occur at room temperature. Hence, germanium detectors must be
cooled with liquid nitrogen to reduce the leakage current to the point that the
associated noise does not spoil their excellent energy resolution [1]. Usually,
the temperature is reduced to -196 C by using an insulated vessel in which
liquid nitrogen is kept in thermal connection with the detector.

4.14.2 Liquid Scintillation Counting
The liquid scintillation method is one of the oldest and most useful method
for detection and spectroscopy of ionizing radiations [1]. It is mainly utilized
for the detection of low-energetic -emitting radioisotopes, such as 3H and
14C, but -emitting radioisotopes can also be detected with high efficiencies.
The approach involves dissolving the sample directly into the liquid scintilla-
tor. Problems relating to sample self-adsorption or beta-backscattering from
the detector are completely avoided [1]. The scintillation technique is based
on the phenomenon of fluorescence and detects photons produced by the
scintillator material. These light flashes are caused by exited states of the scin-
tillator molecules after the absorption of energy from radioactive decay. The


photons are converted in electrical pulses by a photo multiplier tube (PMT)
and can be analyzed by electronic equipment. The liquid scintillation cocktail
is composed of a primary and a secondary scintillatorthat are able to trans-
form the decay energy to light flashesand an organic solvent (e.g., toluene)
as carrier substance. Thus, the scintillator molecules can be regarded as the
actual radiation detector in a liquid scintillation counter.
Modern liquid scintillation counters are able to distinguish between the
different types of radiation and even between the different energies of the
same type of radiation. The discrimination between - and -radiation in
water samples is applicable for determining gross-alpha and gross-beta ac-
tivity concentrations. One major disadvantage of liquid scintillation counters
is their relatively poor energy resolution. Furthermore, quench effects may
reduce the counting efficiency and background radiation might negatively
affect the measurement. On the other hand, the counting efficiency for - and
-particles can potentially be close to 100% in unquenched samples [1].

4.14.3 Inductively Coupled Plasma Mass Spectrometry
Inductively coupled plasma mass spectrometry (ICP-MS) provides a rapid
and sensitive technique for determining long-lived radionuclides and stable
isotopes. The principal use of the ICP-MS is that of a mass spectrometerto
detect the mass of elements according to their mass to charge ratio (m/z).
As the analytes enter the plasma, they are stripped off to their ionic form. This
is only possible due to the high temperature of the plasma, which goes up to
approximately 6000-10000 K [2]. There are different types of mass separation
devices, which can be used in a mass spectrometer. The most com-mon ICP
mass spectrometers are quadrupole-based instruments (ICP-QMS), which
allow isotope ratio measurements with a precision for short-term isotope
ration measurements from 0.1% to 0.5% relative standard deviation [3]. The
achievable lowest limit of detection (LLD) of an ICP-MS for the de-
termination of uranium isotopes is about 0.1 ng/L - without further sample
preparation and sample concentration, respectively.
Major advantages of ICP-MS are the small sample sizes, high sample
throughputs, and short measurement times with only few sample preparation
steps [74]. Not only that, ICP-MS offers efficient and sensitive multi-elemental
analysis in trace or ultra-trace levels [3]. However, the instrument itself is
rather costly and it requires high maintenance. High sample matrix
concentration may interfere with the determination of low levels of long-lived
radioisotopes, but solid-phase extraction may overcome these problems [4].


References

1. Knoll GF (2000) Radiation detection and measurement, 3rd edn. Wiley,
New York, p 802
2. Ponce de Lon CA, Montes-Bayn M, Caruso JA (2002) Elemental
speciation by chromatographic separation with inductively coupled plasma
mass spectrometry detection. J Chromatogr 974:1
3. Becker JS, Dietze HJ (2000) Precise and accurate isotope ratio measurements
by ICP-MS. Fresenius J Anal Chem 368:23
5. Unsworth ER, Cook JM, Hill SJ (2001) Determination of uranium and
thorium in natural waters with a high matrix concentration using solid-phase
extraction inductively coupled plasma mass spectrometry. Anal Chim Acta
442:141


4.15 Endocrine disruption chemicals

Prepared by: BDS

There are several types of endocrine systems where chemicals may interact
with. For each type of endocrine interaction there exists several techniques.
Below, an overview is presented. For specific details regarding individual
interactions, analytical techniques and references see Annex I.

Concentration range that should be covered: no limits have been set for
endocrine disrupting compounds in the (aquatic) environment.

Sensitivity/resolution required: not applicable.


Bioassays detecting estrogenic activity

1. ER CALUx

2. MVLN and MELN
3. T47D-KBluc
4. YES and related assays
5. E-Screen

Bioassays detecting androgenic activity

6. AR CALUx

7. MDA-bk2
8. PALM
9. YAS and related assays
10. A-Screen

Bioassays detecting progestagenic activity

11. PR CALUx

12. TM-Luc
13. Yeast based assays

Bioassays detecting glucocorticoid activity

14. GR CALUx

15. TGRM-Luc
16. MDA-kb2

Bioassays detecting thyroid activity

17. TR CALUx

18. T-Screen
19. PC-DR-LUC
20. xL58-TRE-Luc


4.15.1 Introduction

The focus of this document is on bioassays that have been applied and
validated for the analysis of hormone receptor mediated endocrine disrupting
compounds in water, including extraction and clean-up. Although many
different cell lines which are an essential part of the bioassay analysis - have
been developed to detect several types of endocrine activity, only a limited
number of bioassays have been applied in the detection of endocrine activity
in the aquatic environment. Bioassays for the detection of non-estrogenic
responses have almost never been applied for environmental monitoring and
have not been subjected to comparison studies, but are included for
completeness. However, no information is known about their robustness and
sensitivity towards the analysis of water.

4.15.2 Analysis of water using bioassay

Many different cell line based bioassays have been developed, generally
based on mammalian or yeast cells. However, assays utilizing fish or
amphibian cell lines have also been described, also depending on the species
of interest. Differences exist regarding the ease of culture, sensitivity to toxic
compounds and sensitivity towards compounds of interest. Regardless of the
cell line used in the assays, they mainly fall in one of the following categories:
Reporter gene assays. These assay measures hormonal receptor
binding dependent transcriptional activity
Cell proliferation assay. These assays measure the increase in the
amount of cells as a response to specific type of compounds.

Reporter gene assays
The principle behind reporter gene assays is that binding to a specific
receptor results in activation of this receptor. The activated receptors bind to
the corresponding responsive elements (REs), resulting in transcription of the
normal target genes. In reporter gene assays, a DNA sequence has been
added for the production of an easily quantifiable gene product under control
of similar REs. Activated receptors bind to these REs as well, resulting in the
production of the product, e.g. an enzyme. The reporter gene can be stably
transfected (incorporated) into the cells or can be transfected transiently.
Since transient transfections require more work and show more variability
within and between assays they are considered not suitable for water quality
monitoring.

To be able to activate the reporter gene, an activated receptor is needed.
Several reporter gene assays make use of naturally present (i.e. endogenous)
receptors. The advantage of using endogenously present receptors is that the
resulting bioassays are sometimes more sensitive. Using endogenously
present receptors however, increases the risk of non-specific responses since
often other types of receptors are co-expressed. Therefore, reporter gene
assays have been developed utilizing transfected (i.e. artificially introduced)


receptors. These receptors are mostly derived from human cells but can also
be derived from other species.

Beside mammalian cells, yeast cells are frequently utilized for making
bioassays responding to hormones because they are easier to handle and do
not endogenously express hormone receptors. However, bioassays based on
yeast cells are generally much less sensitive than bioassays based on
mammalian cells and have problems identifying antagonistic activity. Beside
general problems regarding sensitivity, the presence of a cell wall in yeast
combined with very active membrane transport mechanisms can results in
different activity for compounds and extracts and false negatives. Therefore,
yeast cells are generally not recommended for areas involving risk
assessment.

Cell proliferation assays
Cell proliferation assays make use of fact that proliferation (i.e. increase in
number) of cells can be induced or inhibited as a response to compounds. By
relating the amount of cell growth to that of cells exposed by the reference
compound, the response can be quantified.

4.15.3 Analysis using bioassays

Method of analysis
Although different in details, most bioassays described in this document
follow similar methods for the analysis. In general, water samples are
extracted using solid phase extraction of liquid-liquid extraction. The solvents
are evaporated and the extract is transferred to a carrier, generally DMSO,
because of its low volatility and good dissolving capabilities.

To analyse the activity in the extract, bioassay cells are seeded into 96 well
plates, usually using medium that is supplemented with hormone-stripped
serum. The next day, when the cells have attached, the cells are exposed by
replacing the cell culture medium by medium that contains the extracts to be
tested. After exposing the cells for a specified amount of time (generally 24
hours), the amount of response produced is quantified by lysing the cells and
the adding the appropriate substrate. When luciferase is used as a reporter,
luciferin is added and the amount of light produced is quantified using a
luminometer. When -galactosidase is produced, chlorophenol red--D-
galactopyranoside (CPRG) or 2-nitrophenyl- -D-galactosidase (ONPG) can
be used to determine the amount of red or yellow colour produced
respectively using a spectrophotometer. The response of bioassays producing
Green Fluorescent Protein can be quantified using a fluorometer. Yeast
bioassays are generally performed similarly mammalian bioassays, but
without the need of the cells attaching to the 96 well plate.

Proliferation assays are seeded and performed like all other mammalian
bioassays, however exposure times are longer (3-7 days). This is due to the
kinetics of induction of cell proliferation. The amount of cells can be
quantified by counting nuclei or by an enzymatic endpoint.


In all assays, the amount of response is a direct measure of amount of
agonistic activity. By also exposing cells to a concentration series of known
agonists, the amount of response can be expressed as reference compounds
equivalents. Beside agonists, also antagonists can be present in the water
extract. By co-exposing the cells to a fixed amount of reference agonist
(generally at the EC50 level), the amount of decrease in activity can be used as
a measure of antagonistic activity.

For the analysis of water samples, all bioassays are exposed to extracts.
Several different methods have been described, usually based on solid phase
extraction or liquid-liquid extraction.

Since most bioassays use mammalian cell lines, the equipment needed for
culturing and performing the bioassay is similar for all bioassays. Cells are
maintained in an incubator to provide optimum temperature, pH and
humidity. Handling of the cells usually takes place in a laminar flow cabinet
to provide sterile conditions. All equipment needed (pipettes, culture flasks,
96 well culture plates) has to be sterile.

Differences exist in the equipment needed to quantify the response, whether
being a luminometer (luciferase reporter), fluorometer (GFP reporter) or
spectrophotometer (-galactosidase reporter).

4.15.4 General evaluation of the bioassays

It is important to realize that monitoring of water quality using bioassays
comprises not only a responsive cell line, but also robust extraction and clean-
up procedures. Since some cell lines have demonstrated to be more sensitive
to matrix interferences, validation of the bioassay for the matrices of interest
is very important. Very recently, an inter-laboratory study focusing on
estrogen responsive bioassays was performed by the GWRC (2008), allowing
for a direct comparison of the responses of the individual bioassays. This
study showed that clear differences exist between bioassays with regard to
the sensitivity and coefficients of variation. Unfortunately, similar studies are
not frequently conducted. While inter-laboratory studies regarding water
analysis for estrogenic activity using bioassays are scarce, similar studies
focussing on other than estrogenic responses are virtually non-existed.
Although most of the information is available for the estrogenic bioassays
only, to some extent conclusions and remarks can be made regarding the
suitability of the other bioassays.

Sensitivity
In general, all mammalian cell lines bioassays evaluated in this document are
more sensitive than GC-MS analysis, but LODs for the specific cell lines are
generally not provided in the original manuscripts. A recent study conducted
by the GWRC (Leusch, 2008) showed that of the five estrogen responsive
bioassays tested, the E-Screen and the ER CALUx were the most sensitive,
being approximately 10 times more sensitive than GC-MS. The ER CALUx
and the E-Screen also showed the lowest coefficient of variation. The YES


bioassay was shown to be at least 10 times less sensitive than mammalian cell
line based bioassays (Leusch, 2008; Murk et al., 2002). This low sensitivity,
combined with a risk of false negative due to very active membrane transport
mechanisms that are present in yeast (ICCVAM, 2003), makes that the YES
(and other yeast based bioassays) are not suited for drinking water quality
assessment. Information on sensitivity regarding bioassays measuring other
receptors mediated processes is unavailable, although the PALM bioassay
metabolizes testosterone, resulting in an underestimation of the amount of
androgenic activity when testosterone is present.

Robustness
Many cell lines have been developed, although not always for the application
of (aquatic) environmental monitoring. Most cell lines have been utilized only
by a very limited number of labs or samples, making it difficult to evaluate
there robustness as a bioassays. Few cell lines have successfully been used by
different labs or have been applied in comparison studies for the use of water
monitoring. However, this seems to be restricted to ER responsive cell lines.
Bioassays that have shown to produce good results when handled by
different laboratories are considered to be robust. When no information is
available, as is the case with some ER and AR assays and all PR, GR and TR
assays, this type of robustness of the assay cannot be assessed.

Another form of robustness that is of importance is the robustness of the cells
that are used in the bioassay. Yeast cells are generally easier to handle than
mammalian cells. However, this also leads to an increased risk of false
negatives. Some mammalian cell lines have been reported to be more
sensitive to matrix effects.

Selectivity
Differences exist between the specificity of the bioassays, mostly as a result of
the reporter construct used. One of the most frequently used reporter
constructs is MMTV-Luc. However, this reporter can be activated by
androgens, progestins and glucocorticoids. Bioassays that utilize this
construct, in combination with cells that endogenously express receptors
other than the receptor of interest, do generally not respond to the
compounds of interest exclusively. For examples the androgenic MDA-kb2
and TARM-Luc cell lines were also shown to respond to glucocorticoids and
progestins respectively, limiting their use in screening for androgenic activity.
Similar problems exist with several bioassays using other receptors. This low
selectivity makes these bioassays less suitable for selective screening,
especially for complex mixtures like (surface) water extracts. The cell
proliferation bioassays E-Screen has also been shown not to respond to
estrogens exclusively. Yeast based assays like the YES generally cannot
differentiate between agonistic and antagonistic activity. Reporter gene
assays utilizing a construct containing only the responsive elements of
interest allow for a more selective analysis of endocrine activity.

Time to result
Most reporter gene bioassays use an exposure time of 24 hours, although
differences exist ranging from 16 to 48 hours. Proliferation assay generally
take more time to produce a quantifiable response (generally 3-7 days) and


are therefore considered not to be suitable for routine monitoring. The
response of yeast cells is in some occasions detectable more quickly, allowing
for exposure times of approximately 2 hours or more, although most yeast
based bioassays use exposure times of 16-48 hours.

Ease of use
All mammalian cell lines require similar equipment and culturing techniques.
Yeast cells are slightly easier to culture than mammalian cells, requiring less
sterile conditions. All cell lines can become contaminated with mycoplasm,
which has to be checked regularly. This places a responsibility on the source
of the cells. Some bioassays like CALUx
and EcoScreen are marketed by

commercial companies. Several cell lines have been used by different users
world wide, indicating their transferability and ease of use. The CALUx cell
lines are offered including training and customer support, improving the
reliability and ease of use of the bioassay.

Maintenance requirements
All mammalian cell lines require more or less the same handling, equipment
etc. which includes culturing the cells once or twice a week. Commercially
available cell lines can include troubleshooting and customer support,
facilitating maintenance.

Instrumentation
Most bioassays make use of a luciferase or GFP reporter gene, requiring a
luminometer or fluorometer respectively. The response of cells producing -
galactosidase can be quantified using a spectrophotometer.

Operational costs
The mammalian cell based bioassays do not vary greatly in the need of
consumables or labour. Therefore, these costs are not assessed further. Several
assays, including commercial ones, include licensing costs.

Recommendation for use in small scale systems
Due to the equipment and techniques needed, in vitro bioassays are not
recommended for use in small scale systems.


Table of evaluation: overall rating of the different monitoring technologies.

Sensitivity (A)

Robustness (A)

Time to
result
Operational
specifications

source
water
drinking
water
operational
robustness
selectivity ease-of-
use (B)
maintenance
requirements
(C)
1. ER CALUx
4 4 5 3 3 4 3
2. MVLN and MELN 2 2 2 2-3 2
3. T47D-KBluc 3 3 3
4. YES and related assays 1 1 4 3 2-3 3 3
5. E-Screen 4 4 4 2 1 3 3
6. AR CALUx
3 3 3 5 3 4 3
7. MDA-bk2 3 3 1 3
8. PALM 3 3 3 3 3
9. YAS and related assays 2 1 3 3 3
10. A-Screen 3 3 2 1
11. PR CALUx
3 3 3 5 3 4 3
12. TM-Luc 3 3 1 3
13. Yeast based reporter
gene assays
2 1 4 3
14. GR CALUx
3 3 3 5 3 4 3
15. TGRM-Luc 3 3 1 3
16. MDA-kb2 3 3 1 3
17. TR CALUx
3 3 4 3
18. T-Screen 1
19. PC-DR-LUC
20. xL58-TRE-Luc


4.16 Genotoxicity

Prepared by: BDS


Gene mutation assays
1. Ames test
2. Vibrio harveyi test

Chromosomal aberration tests
3. Comet assay
4. Micronucleus assay
5. Alkaline elution assay
6. Polymerase inhibition assay

DNA repair assays
7. UMU test
8. SOS Chromotest
9. Vitotox

10. Mutatox
11. GreenScreen

12. GreenScreen HC
13. MCF-7-p53R2

4.16.1 Introduction
A large number of tests have been developed for detecting genotoxicity in
water samples, of which some are commercially available and/or have been
validated for water specifically. The focus of this document is on assays that
could - in principle - be utilized to detect genotoxicity in surface and drinking
water. Ideally, these tests can be performed in 96 well plates or other high-
throughput format, and would take less than two days to perform. Assays
that have been developed recently, but that have not been used (frequently)
for the analysis of genotoxicity in water, have also been included, although
the potential of these novel assays still needs to be assessed.

An overview in general is presented below. Evaluation forms for the specific
tests and references can be found in Annex II.

4.16.2 Mechanisms of genotoxicity
Many different compounds have the ability to elicit a genotoxic effect, i.e.
enter the cell and damage DNA. The resulting genotoxic stress can trigger
appropriate responses to cope with the damage, such as growth arrest, DNA
repair and in extreme circumstances apoptosis (programmed cell death).
However, sometimes the repair mechanisms fail and the exposure to the
compound(s) can result in DNA changes, either small such as gene mutations
and/or large such as chromosomal aberrations.


Mutations are small-scale changes in the nucleotide sequence in the DNA.
Generally, mutations can be subdivided into two types: base-pair
substitutions and frameshift mutations. In base-pair substitutions, one or
more base-pairs are substituted by others, mostly resulting in a changed
amino acid in the related protein. With frameshift mutations, one or more
base-pairs are added or deleted from the DNA sequence. Due to the triplet
nature of gene expression by codons, the insertions and deletions can disrupt
the reading frame, resulting in a completely different translation, and thus in
a completely different protein.

While mutations affect only nucleotides, chromosomal aberrations are large-
scale changes in the DNA that can be structural or numerical. Structural
chromosomal aberrations (caused by so called clastogens) are DNA breaks
and exchanges in the chromosomes, where numerical aberrations (caused by
so called aneugens) change the number of chromosomes in the cell.

4.16.3 Tests to detect genotoxicity
Several methods exist for the detection of genotoxicity. These methods are
based on different principles, enabling detection of specific or nonspecific
types of DNA damage. The existing methods fall into one (or more) of the
following categories:
- Gene mutation assays. These assays belong to the most applied and
well known methods, like the Ames test. Gene mutation assays rely
on the detection of compounds that can induce gene mutations,
utilizing different strains of bacteria, yeast cells or mammalian cells.
These mutations can be forward (from wild type to mutant) or back
(from mutant to wild type). In general, mutations assays rely on the
acquired ability of bacteria or cells to grow on selection medium when
a specific mutation occurs. After exposure, the number of cells or
colonies can be detected and this number is a measure of the
mutagenic potential of a compound or extract.
- Chromosomal aberration tests. Chromosomal aberrations can consist
of DNA breaks and/or unsuccessful separation of chromosomes
during cell replication. Chromosomal aberration tests make use of the
visualization of chromosomal damage as an indication of DNA
damage.
- DNA repair systems. As a response to damaged DNA, several DNA
repair systems can be activated. The activation of these systems can be
linked to a reporter gene, resulting in the production of an easily
detectable enzyme or protein. Most DNA repair assays utilize bacteria
and their SOS repair system, although yeast and mammalian cell line
based assays have been developed recently as well. However, large
differences exist between the prokaryotic and eukaryotic DNA repair
mechanisms, with the latter generally being more complex.
4.16.4 General review
Screening water samples for genotoxicity equals screening of complex
mixtures with unknown compounds and concentrations. In this respect it is
different, and more complex, than the regulatory testing of pure compounds
where generally (very) high concentrations of a single compound are tested,


and where positive findings can be followed by in vivo testing for
confirmation. Therefore, there is a need for assays that are sensitive, accurate,
reproducible and robust for the detection of genotoxins in water. A large
number of (commercially available) tests exist to determine genotoxicity in a
variety of matrices. Most of the assays developed have been applied to the
aquatic matrix and some have been standardized at the international level by
ISO.

Most of the assays to detect genotoxicity utilize bacteria, which have the
advantage of being relatively easy and fast to perform, but have some
limitations related to the use of prokaryotic cells and their repair systems.
Due to the polysaccharide envelope, not present in mammalian cells, in
combination with active transport mechanisms, chemicals cannot easily enter
bacteria. Bacteria also do not have the metabolic capacity of mammals,
although this has partly been solved by utilizing S9 fractions. Most bacterial
mutation assays can only detect specific types of (back) mutations, covering
only a small part of the genome, i.e. the part where the mutation was
originally introduced. Although this has in part been solved by utilizing the
SOS responses of bacteria, a more general indication of DNA damage, the
eukaryotic DNA repair mechanisms are generally more complex. Damage
like chromosomal aberrations cannot be detected using micro organisms.
Existing bacterial tests are known to suffer from detecting a high number of
false positives when testing pure compounds (Kirkland et al., 2005; Tweats et
al., 2007), which can be problematic when testing complex mixtures consisting
of many compound.

To solve the shortcomings of the bacterial assays, several genotoxicity tests
have been developed based on mammalian cells. Depending on the
mammalian cell line used, cells can in part metabolize genotoxic compounds;
otherwise S9 fractions can be utilized. The most used and validated tests, like
the micronucleus test and the Comet assay, allow for the sensitive detection
of clastogens and aneugens, a type of DNA damage that cannot be tested for
using bacterial assays. However, these tests are very labour intensive and
have difficulty detecting types of DNA damage that do not include
fragmentation of DNA. Mammalian mutagenic assays like the TK assays or
HPGRT test are capable of detection mutagenic activity, but are very time
consuming since they can take several weeks to perform.

All studies clearly show that no single test is capable of detecting all relevant
genotoxic activity in water. Therefore, the genotoxic potential of water can
only be evaluated using a battery of tests, using at least two assays aimed at
different endpoints (Corbisier et al., 2001; Heringa, 2005). Tests with real
water samples showed that while most tests are able to detect genotoxins as
pure compounds, the same compounds are frequently not detected when
spiked to surface water (Corbisier et al., 2001). Similar tests revealed that
some water samples were scored as non-genotoxic by most bacterial assays,
while the opposite was found by tests utilizing higher target organisms
(yeast, mammalian cells). These results illustrate that results from bacterial
tests might not reflect the actual risk for higher organisms. The first results
from novel assays utilizing cells from higher organisms, and focussing on a
general measure of DNA damage, look promising. However, these assays


have some draw-backs, like the use of yeast cells and the use of GFP, are not
expected to detect all types of genotoxicity, and have not been validated for
water specifically. Therefore, there remains a need for quick and easy
genotoxicity assays, preferably based on mammalian cells, that are sensitive
enough to detect a broad range of genotoxins in water.

4.16.5 Sensitivity and specificity
Several reviews exist with regard to the sensitivity and specificity of assays to
detect genotoxins. These reviews mainly focus on the genotoxicity of pure
compounds, although some have been directed at genotoxins in water. The
high concentrations that are sometimes necessary to elicit a detectable
response are easily achieved when using pure compounds. However, when
analyzing water samples (or extracts), the sensitivity of the assay is of high
importance.

With regard to the pure compounds, it was shown that the currently applied
in vitro bioassays are able to detect genotoxins correctly (albeit at high
concentrations), but suffer from detecting a high number of false positives
(Kirkland et al., 2005; Tweats et al., 2007). Since for complex mixtures like
(extracts from) surface water the chemical composition is unknown, and no in
vivo studies are performed with positive water samples, it is difficult to assess
the trueness of the applied genotoxicity assay in this respect. However,
studies aimed at the detection of genotoxicity in water showed that there are
great differences between sensitivity of the assays, both regarding the
response to pure compounds (in water) as to water and/or water extracts
(Farr et al., 2007; Reifferscheid and Grummt, 2000). Assays relying on the
induction of bacterial responses, like SOS chromotest, Vitotox and Umu were
shown to be much more sensitive than other bacterial tests like Mutatox and
Ames. Although the Ames test is commonly used for environmental samples,
it is not recommended for rapid water quality analysis due to the relatively
low sensitivity and long analysis time (Corbisier et al., 2001; Wegrzyn and
Czyz, 2003).

Because some compounds are not genotoxic agents themselves, most assays
can also be performed including a metabolisation step, generally by adding
liver S9 fraction. This mixture contains microsomal and cytosolic fractions of
liver cells, incorporation both phase I (transformation) and phase II
(conjugation) enzymes. Although this can give an indication of genotoxicity
of metabolites, differences can exist between batches, species of origin and
induction of enzymes. Most frequently, S9 fractions are derived from rat
livers treated with phenobarbital or Aroclor 1254. However, the balance of
activating enzymes usually is unrepresentative of what will occur in human
livers (Ku et al., 2007). Tests have been described using human derived S9
fractions, which behave differently than rat derived S9 (Hakura et al., 1999).
The use of S9 - as well as the presence of other autofluorescent compounds -
can interfere with the measurement when using GFP based assays, since S9 is
fluorescent and therefore interferes with GFP analysis.


4.16.6 Robustness
Although most assays have been used frequently for the detection of
genotoxins in surface and drinking water, only a limited amount of
interlaboratory studies have been performed. One study, comparing 14
genotoxicity tests on 11 samples, showed that small differences in assay
conditions of a particular test, as well as differences in interpretation of the
generated data, often greatly influence the outcome, even for identical tests
(Farr et al., 2007). Some assays also showed matrix interference when testing
pure compounds that are added to water samples (Corbisier et al., 2001),
stressing the validation of the assay for the matrix of interest.

4.16.7 Time to result
Most bacterial assays use an exposure time of 24 hours, although the Vitotox
assay is somewhat faster with an exposure time of 18 hours. The Ames test,
with exposure times ranging from 2-5 days, takes too long to perform and is
therefore not recommended for routine monitoring. The GreenScreen assay
has an exposure time ranging from 4-18 hours, with longer exposure times
resulting in lower detection limits. However, a similar yeast assay utilizing
another reporter gene can produce responses more quickly, but this assay has
not been tested for water samples. Because the Comet assay can utilize many
different cells, exposure times depend on the source of the cells. Most Comet
assays using in vitro cell lines use an exposure time of 24 hours, but the
analysis of the response also takes time since it utilizes electrophoresis.

4.16.8 Ease of use and instrumentation
Bacterial assays are relatively easy to perform, as bacteria are robust and can
be grown in a simple incubator. However, since some bacterial assays utilize
pathogenic bacteria (e.g. Salmonella typhimurium), a laboratory suited for the
appropriate risk level is required. Regarding eukaryotic cells, yeast cells are
relatively easier to culture than mammalian cells, requiring less sterile
conditions. More complicated tests like the alkaline elution test, Comet assay
and micronucleus test are much more labour intensive.

Many differences exist in the way the assays quantify the final response. The
response of reporter gene assays can easily be determined by measuring
luminescence, fluorescence or colour formation using a luminometer,
fluorometer or spectrophotometer respectively. The Mutatox assay even
comes with a specific Mutatox Analyser, reagents and media. Some assays
produce responses that can even be quantified by simply counting the
amount of colonies. Automated versions have been developed for the alkaline
elution test, Comet assay and micronucleus, but these require more expensive
and specialised equipment. Unlike other tests, the polymerase inhibition
assay requires a PCR machine.


4.17 Acute toxicity

Prepared by: IGB/bbe Moldaenke

For toxicity detection the use of biological systems is inevitable. Here, model
organisms act as reliable indicators for harmful agents, e.g. toxins. Unlike
analytical instruments, they will respond to several toxic compounds without
pre-calibration. The majority of the standard acute toxicity tests are
performed for the examination of waste water, sludge and for the
characterisation of the toxic potential of a chemical.

To detect toxicity in drinking water it is essential to measure the water quality
in real-time, on line, and at different points of the source. For that reason new
biomonitoring technologies were developed that can assess the toxicity of
water samples by analysis of living organism behaviour within a short time
period, whereby significant changes of behaviour trigger an alarm. This was
made possible by advances in digital video processing, signal analysis, and
by fast computers.

Most of the online biomonitoring tests are performed on fish and selected
aquatic invertebrates. The sensitivity of the alarm can often be pre-selected
and adjusted by the user based on the specific application. Though it is
known that different species vary in their sensitivity to different substances,
the so called biological early warning systems (BEWS) are sensitive to a wide
range of harmful agents including synergistic effects of toxic mixtures. The
following biomonitoring technologies are suitable for testing drinking water.
If source water does not interfere the optical recognition systems (e.g. by high
concentrations of humic substances), test are also applicable in this case.

1. Standard toxicity tests
2. Daphnia toximeter
3. Fish toximeter
4. Combined Fish and Daphnia toximeter
6. Algae toximeter
7. Luminiscent bacteria
8. Mussel monitor

4.17.1 Standard toxicity tests

Description:
A variety of methods of toxicity test methods have been standardized in
different countries. Some of these procedures are recognized internationally,
notably the standard methods published by the International Organisation
(ISO), and the guidelines given by the OECD.
For determining the acute toxicity of substances to aquatic organisms, the
following ISO methods are used:


ISO 6341:1996 Water quality - Determination of the inhibition of the
mobility of Daphnia magna Straus (Cladocera, Crustacea)
ISO 7346-1:1996 Water quality - Determination of the acute lethal toxicity
of substances to a freshwater fish [Danio rerio Hamilton-Buchanan
(Teleostei, Cyprinidae) - Part 1: Static method.
(Teleostei, Cyprinidae)] - Part 2: Semi-static method.
(Teleostei, Cyprinidae)] - Part 3: Flow-through method.
ISO 12890:1999 Water quality - Determination of toxicity to embryos and
larvae of freshwater fish. Semi-static method.
DIN 38415-6: 2003 German standard methods for the examination of
water, waste water and sludge Sub-animal testing (group T) - Part 6:
Determination of the effect of nonacute toxicity of waste water on the
development of fish eggs (K 9).
ISO 8692:2004 Water quality Freshwater algal growth inhibition test
with unicellular green algae.

Most of these standard acute toxicity tests use the water flea Daphnia magna,
the fish Danio rerio and fish eggs of Danio rerio; the procedures also allow for
the use of some other fish species. The basic data obtained is the LC50 for
periods of 24, 48, 72 and 96 hours; for Daphnia the usual maximum exposure
period is 48 hours.

The following standard describes the performance testing of on-line
sensors/analysing equipment for water (but it is not an analytical method
itself):
ISO 15839:2003 Water quality - On-line sensors/analysing equipment for
water - Specifications and performance tests.
The standard is applicable to most sensors/analysing equipment, but it is
recognized that for some sensors/analytical equipment certain performance
tests cannot be carried out. This International Standard
defines an on-line sensor/analysing equipment for water quality
measurements;
defines terminology describing the performance characteristics of on-line
sensors/analysing equipment;
specifies the test procedures (for laboratory and field) to be used to
evaluate the performance characteristics of on-line sensors/analysing
equipment.

Evaluation:
These standard toxicity tests are primarily performed for the examination of
waste water, sludge and for the characterisation of the toxic potential of a
chemical. A continuous observation of drinking water quality is not possible.
It needs too long to get the results. For single toxicity tests in suspected cases
of contamination especially in the distribution system or at the consumers tap
test kits can be useful.


The ISO guideline 15839:2003is a good starting point for describing the
performance characteristics of on-line biomonitors.

Monitoring technology nr 1: Standard toxicity tests

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

time to result (B)

x
ease-of-use (B) x

Costs x
consumables
maintenance
x


Overall conclusion The test methods are not useful for continuous online water
monitoring

4.17.2 Monitoring technology nr 2: Daphnia toximeter

Kerren Aqua-Tox-Control Daphnia, bbe Daphnia Toximeter

Description:
The daphnia toximeters directly monitor the activity of the test organism, the
common freshwater water flea (Daphnia magna). Computer software analyzes
the movements respectively the location of the daphnia while they are
exposed to sample stream water. The values are recorded and analysed
continuously and in real time to determine if changes occur in any or all of
the observed parameters.
Both instruments consist of housing, an industrial PC, monitoring chambers,
sample pumps and an automatic food supply. For feeding an algae solution
can be added. Under normal conditions e.g. operation without toxic events,
the tests run continuously, without maintenance, for about seven days.


The Aqua-Tox-Control works with a control and a test chamber. For the
control chamber a supply of guaranteed uncontaminated water is necessary.
The reaction of daphnia to changing light conditions is continuously
monitored by sensors and compared between both chambers. Significant
behavioural deviations trigger an alarm.

The bbe Daphnia Toximeter uses a video recognition technology to observe
behavioural changes of daphnids in one or two chambers. The behavioural
parameters evaluated by the instrument are: average velocity, speed class
distribution, average distance of organisms, the curviness of swimming
patterns as determined by two fractal dimension equations, average altitude
and the recognition rate of Daphnia. The Toxicity Index, a parameter
calculated from individual behaviours, gives an overall status of the daphnia
during testing. If behavioural changes occur in the course of time suddenly or
dramatically, the Toxicity Index will rate the change and will give an alarm
when alarm conditions are met. The contribution of the individual
parameters to the Toxicity Index can be weighted differently thereby
adjusting the sensitivity of the instrument. A pre-filtering system,
dechlorination and a peltier heating/cooling system ensure constant keeping
conditions for the daphnids. The software provides a graphic presentation of
the measured results with live, real-time pictures, offline viewing and an
intuitive user interface.

Both daphnia toximeters are commercially available and applicable for
continuous drinking water monitoring and protection.

Evaluation:
Daphnia Toximeters are sensitive methods to detect hazardous compounds in
water. Based on the Extended Dynamic Daphnia Test (which is not available
anymore), a new sensitive method to detect hazardous compounds in water
was developed. Both small doses (allowable for short-term water ingestion)
and graduated higher concentrations induced toxic reactions lead to alarms in
the toximeter systems. The systems are sensitive to a wide range of toxic
agents. The bbe system was evaluated by using different test pollutants e.g
Sarin, Tabun, Soman and Cyclosarin. Concentrations which are below acute
human toxicity could be discovered in a very short time. In every case alarms
occurred within two hours at concentrations which are considered allowable
for drinking water in exceptional conditions.

Both systems need some experience in keeping daphnids. The adjustable high
sensitivity may lead to false positive alarms in some cases. The systems can be
applied also in chlorinated drinking water.
The bbe system seems to be technologically advanced and more intensively
tested.
The systems are generally priced between $30,000 and $50,000 operating costs
are rather modest, consisting mostly of replacement costs of organisms and
power supply.


Monitoring technology nr 2: Daphnia toximeter

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

time to result (B)

x
ease-of-use (B) x

Costs
consumables
maintenance

x
x


Overall conclusion Sensitive method to detect hazardous compounds in water.


4.17.3 Monitoring technology nr 3: Fish toximeter

Kerren Aqua-Tox-Control, BMI BIO-SENSOR
, IAC 1090 intelligent Aquatic

BioMonitoring System
, CIFEC Truitel TruitoSEM, bbe Fish Toximeter, bbe

ToxProtect

Description:
The measuring technology of all fish toximeters is based on the observation
and evaluation of behavioural changes of actively swimming fish when
exposed to toxic influences.
Different types of sensors and evaluation methods are used.

The Aqua-Tox-Control monitors a group of fish in a swimming tunnel and
registers changes in swimming power and capacity when fish are touching
the grid barrier at the end of the tunnel.

The Bio-Sensor 7008 features eight fish (on independent sensor channels)
monitoring a single water source, noninvasive electronic sensors measure
changes in ventilatory behaviour and certain locomotor activities.


The IAC 1090 detects breathing, coughing and movements of fish by
noncontact sensors mounted in an aquarium. These movements or ventilatory
parameters (ventilation rate, average depth, cough rate, and percentage of
motion) along with water quality parameters are processed and analyzed to
provide an assessment of water toxicity.

The Truitel TruitoSEM also measures changes in the movements of fish by
noncontact sensors in an aquarium. The data processing and alarm
algorithms are not described in detail. Sensitivity can be technically adjusted
or by the selection of different fish species.

The bbe Fish Toximeter uses a video camera to directly monitor the activity of
fish under the influence of a stream of sample water. Tracking all movements
online, the instrument records any changes in behaviour. On the occurrence
of a toxic event the toximeter triggers an alarm at an early stage of exposure.
Toxicity computations and assessments are based on the measurement of the
following behavioural parameters: speed, behaviour (height, turns, circular
motion), determination of size, number of active fish. An integrated
parameter - the so-called toxic index - is calculated continuously.

The bbe ToxProtect is an automated monitoring system to protect especially
drinking water supplys from accidental or malicious contaminations due to
harmful substances. It is mainly designed to detect relatively high
concentration of dangerous substances that occurs suddenly. The ToxProtect
monitors the swimming activity of up to 20 fish by measuring the frequency
of interruption of an array of light barriers. The result is given in interrupts
per minute per fish. Furthermore activity or inactive objects at the surface or
bottom of the tank are evaluated additionally. In order to prevent false
alarms, an alarm verification system is integrated. In the event of values
falling below a given threshold for a certain period of time, the alarm
verification process is activated.

All systems use computational algorithms and statistical approaches to
evaluate behavioural changes and trigger alarms in the case of toxic events.
The systems can optionally be combined with physicochemical sensors to
assist in analysis and be equipped with automated water samplers. Systems
offer the opportunity of remote communications and viewing of activity.

The fish toximeters are equipped with life-guarding systems like flow control,
temperature regulation, automated feeding and optional dechlorination.
These fish toximeters are commercially available and are applicable for a
continuous drinking water monitoring.

Evaluation:
Continuous biological monitoring with the Fish Toximeters enables a rapid
detection of toxic substances in water and provides an online early warning
system. The Fish Toximeters are well suited for the rapid detection of wilful
or negligent damage to water systems. A high toxic response relation between
fish and humans exists, so real alarms based on the fish behaviour responses
have a high hazard probability for human beings too.


Fish are relatively easy to keep and systems are easy to set-up, fish tank, tubes
and connectors are accessible at a low-maintenance level compared to
daphnia toximeters. All systems can be applied also in chlorinated drinking
water.
The systems do not require special technical skills. Adjustments are almost
easy to perform and require no expert knowledge. The sensitivity and
reliability is dependent on the different technologies, data evaluation
techniques and the chosen fish species.

The continuous analysis of live video images in the bbe Fish Toximeter
enables a rapid and accurate determination of the behaviour and health of the
fish. The integrated bbe software recognises significant changes in the
behavioural data obtained from the live observation and recording of the
fishs movement. Toxic events are clearly indicated as "alarms". A statistical
approach enables alarm recognition even under difficult real-world
conditions such as "noisy" or slow drift of the measured behavioural curve(s).
The sensitivity of the device is high but can be adjusted by the user based on
the specific application. As mentioned a correlation exists between sensitivity
and the probability of false positive alarms. Costs for this highly sophisticated
system are relatively high. But beside the suitability for the detection of acute
toxicity it is also conceived as a long-term monitor of water quality during
"strategic" analysis.

The BMI Bio-Sensor and the IAC 1090 use similar technologies to detect
behavioural impairments in fish. Both are designed to detect rapidly and
reliable developing water toxicity threats. That means systems are relatively
sensitive but suitable for the detection of acute toxicity. The flexibility in the
range of applications seems to be less than in the bbe Fish Toximeter, the
price is comparably high.

The Aqua-Tox-Control uses a detection method which is based on a loss of
swimming capacity of fish. That means an alarm signal can only be generated
when a physical impairment of fish already occurred. Sensitivity of the device
will thus be less than that of the previously discussed systems, delay times to
get an alarm signal will be longer. It may be sufficient sensitive for acute
toxicity detection. The costs for the Aqua-Tox-Control are relatively high.

A system especially designed for drinking water acute toxicity monitoring is
the bbe ToxProtect. Continuous water monitoring with the ToxProtect
enables rapid detection of a wide range of toxins. Usage of behavioural
changes as parameters will make the device sufficiently sensitive to detect all
seriously dangerous substances and situations. ToxProtect is a very easy
operation system at a affordable costs. The required maintenance time is
approximately 10 min/day. The special integrated alarm verification
algorithm leads to a high reliability of the system and avoidance of false
alarms. The use of fish gives a close practical comparison in the expected
scenario of contamination with substances that are harmful to humans.


Monitoring technology nr 3: Fish toximeter

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

time to result (B)

x
ease-of-use (B) x

Costs
consumables
maintenance

x
x


Overall conclusion Sensitive method to detect hazardous compounds in water,
most comparable to human toxicity.



Monitoring technology nr 3a: ToxProtect

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

time to result (B)

x
ease-of-use (B) x

Costs
consumables
maintenance

x
x


Overall conclusion Reliable and robust test system to detect high toxic
concentrations, most comparable to human toxicity.


4.17.4 Monitoring technology nr 4: Combined Fish and Daphnia toximeter

Kerren Aqua-Tox-Control, bbe Fish and Daphnia Toximeter

Description:
Both Kerren and bbe have the possibility to combine their instruments for fish
and daphnia.

Kerren offers a combination of two single devices and assures a significant
increase in sensitivity and reliability by evaluating the output parameters of
both systems integratively.

Bbe combines the advanced Fish and Daphnia Toximeter in one device based
on the two chamber Daphnia toximeter. In one chamber young living fish and
in the other daphnids are observed under the influence of a stream of sample
water. The toximeters continuous visual analysis of fish and daphnia
movement enables rapid assessment of fish and daphnia behaviour and
health (for technical description see the single Daphnia Toximeter). For the


integrated evaluation of any change in the behaviour of the fish or daphnia
the continuously calculated "toxicity index" as a dimensionless parameter
proved to be especially usefiul.
The bbe software integrates all data components of the bbe Toximeter. It
provides a graphic presentation of the measured results with live, real-time
pictures, offline viewing and an intuitive user interface.

Both combined Fish and Daphnia toximeters are commercially available and
useful for continuous drinking water protection.

Evaluation:
Continuous water monitoring with combined Fish and Daphnia toximeters
enables rapid detection of toxic substances in water and provides an online
real time warning system. The combined Fish and Daphnia toximeters are
well suited to the detection of wilful or negligent damage to water systems.
Both systems can be applied in chlorinated drinking water too.
Both Fish and Daphnia Toximeters can also be used for long-term monitoring
for the "strategic" evaluation of water quality.
The integrated use of two simultaneous online test systems does not only
improve the quality of analysis statistically, but enhances also the sensitivity
for harmful compounds by combining the different reactiveness of biological
species to different groups of chemical substances.
However the handling of different species and especially of fish larvae makes
the maintenance of such a system more costly and time consuming as single
systems.
The Kerren solution of two combined single systems will probably nearly
double the instrumentation costs.


Monitoring technology nr 4: Combined bbe Fish and Daphnia toximeter

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

time to result (B)

x
ease-of-use (B) x

Costs
consumables
maintenance

x
x


Overall conclusion Sensitive method to detect hazardous compounds in water.


4.17.5 Monitoring technology nr 5: Algae toximeter

bbe Algae Toximeter

Description:
Beside the bbe Algae taximeter, a Korean solution for an Algae test shall be
existent, but no reliable information about this device was recently available.
The bbe Algae Toximeter uses algae from a standardized culture which is
established within the instrument. The cultivation of the algae takes place in a
fermenter which is regulated "turbidostatically" by a second fluorescence
measurement. This regulation ensures that the algae concentration and their
activity are maintained. As species Chlorella vulgaris, Scenedesmus spec. and
others can be used. The instrument compares the effects of toxins on one
portion of algae to another portion kept in uncontaminated clean water (e.g.
carbon filtered tap water). The principle of operation is based on the
determination of the fluorescence spectrum and kinetics of the algae. If the
algal cells are damaged by toxins in the sample water, the light energy will
not be used by the cell and even without an additional background light there
will be a lower fluorescence response to the light impulse. In the case of
significant deviations between the sample water and the reference water, an
alarm is generated by the instrument to alert the operator. In addition to


monitoring toxic substances, the device can determine different amounts of
chlorophyll within different algae classes.

The bbe Algae toximeter is commercially available and partly useful for
continuous drinking water protection.

Evaluation:
The bbe Algae Toximeter continually determines toxic substances in water.
The bbe Toximeter provides a highly sensitive biological system for early
detection of potentially dangerous unknown substances, in a wide variety of
circumstances. These instruments are ideal for detecting water quality
contamination in real-time. The Toximeter provides means to detect (and
alert users at) potentially dangerous circumstances.
The test mechanism of this monitor is very similar to a Biological Oxygen
Demand (BOD) test, as the effect on fluorescence is a measure for electron
transfer. The time required for the conventional 5-day bench test cannot
compete with the 10-minute response time of this instrument.
Maintenance costs and time for this system are low. Instrumentation costs in
the same range as for fish and daphnia toximeters. Algae toximeters are more
suitable for detecting herbicides and a lot of chronic toxic substances, in acute
toxicity measurements values are less comparable to human toxicity.

Monitoring technology nr 5: bbe Algae toximeter

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

time to result (B)

x
ease-of-use (B) x

Costs
consumables
maintenance

x
x


less comparable to human toxicity.



4.17.6 Monitoring technology nr 6:Luminiscent bacteria

Microtox
, Deltatox
, BioTox, AbraTox Kit, LUMIStox; MicroLAN

TOxcontrol Biomonitor

Description:
Most of these tests are 15-60 minute in vitro exposure metabolic inhibition
tests which use luminescent bacteria to assess the acute toxicity of water, soil
or sediment samples.
The tests use strains of naturally occurring luminescent bacteria, Vibrio fischeri
or Photobacterium phosphoreum. Both are nonpathogenic, luminescent bacteria
that are sensitive to a wide range of toxicants. When properly grown,
luminescent bacteria produce light as a by-product of their cellular
respiration. Cell respiration is fundamental to cellular metabolism and all
associated life processes. Bacterial bioluminescence is related directly to cell
respiration, and any inhibition of cellular activity (toxicity) results in a
decreased rate of respiration and a corresponding decrease in the rate of
luminescence.

The described luminescent bacteria tests are commercially available but do
only allow discontinuous measurements.
The TOxcontrol Biomonitor is the single device with this technology working
continuously as a flow-through system.

Evaluation:
These toxicity test systems provide rapid screening and confirmatory results
that are cost-effective and easy to perform. Costs range between 3,000 and
10,000 $ for the discontinuous test sets. TOxcontrol is with 30,000 the most
expensive device but even the only one suitable for an online monitoring of
acute toxicity.
Manual quality differs between tests, initial light measurements served as a
good check of bacterial health and instrument operation, sample handling is
easy, and sample throughput can reach 15-50 samples per hour. Some of the
tests are applicable in laboratory as well as field environments. Operators
dont need scientific backgrounds, based on the observations of the
verification test coordinator, operators with little technical training would
probably be able to follow the instructions to analyze samples successfully.
Detection of hazards is limited to substances inhibiting or disturbing the
bacterial cell respiration, so not all test substances were detected and not all
results allow conclusions to human imperilment.
For single toxicity tests in suspected cases of contamination especially in the
distribution system or at the consumers tap test kits can be useful.


Monitoring technology nr 6: Luminiscent bacteria

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

time to result (B)

x
ease-of-use (B) x

Costs
consumables
maintenance

x
x


Overall conclusion Medium sensitive but selective method to detect hazardous
compounds in water, not continuous.


4.17.7 Monitoring technology nr 7: Mussel monitor

Delta Consult MOSSELMONITOR

(Dreissena polymorpha, Unio pictorum)

Description:
The behaviour of bivalves in response to toxicity in water is seemingly
simple, they will close their shells. That means the sensor in the
MOSSELMONITOR
is the mussel itself.

The measurement of gape, or opening of the shells, and the frequency of this
opening is generally accepted as a measure of stress to the organisms. In the
normal movement pattern of the mussel shells, the shell halves will remain
open approximately 70-80% of the time, for the intake of food and oxygen.
The shells only occasionally close, and re-open after only a short period of
time. Gape behaviour is measured using proximity sensors which detect a
stainless steel proxy attached to the shell of each clam. By comparing the
organisms baseline, or normal, gape behaviours to that of a test water,
information regarding toxicity of a water can be determined.


The various movements, which the mussel can make as a result of differing
types and levels of contamination, are as follows:
Keeping the shell halves closed for a certain (longer) period.
An increased activity level, i.e. the mussel opens and closes more
frequently than normal.
A reduction in the average value of opening over a certain period of time.
No further movement, the shell remains open far more than the normal,
maximum open position. This occurs if the mussel is no longer alive
("gaping").

The monitor uses normally 8 bivalves per system and evaluates its behaviour,
systems with 16 bivalves are optionally available.
An Automated Food Device (AFD) was developed to enable the use in places
where little or no food is present for mussels as e.g. in drinking water
protection.
By entering information about the movements of the shell into a
microprocessor, and by carrying out certain calculations on this data, it is
possible to ascertain the behaviour of the mussel. Currently, a memory is
being used to store the data for evaluation of trends in shell movement
behaviour
The monitor is a stand alone unit; it contains all electronics required for
measurement, data evaluation and communication. For data storage any PC
can be used.

Evaluation:
The flow-through version of the Mosselmonitor is a broad spectrum sensor
that can easily be used as an early warning system for on line monitoring of
drinking water. It can be applied also in chlorinated drinking water. The
Mosselmonitor operates unattended for weeks, whereby zebra mussels are
still alive and responding to chemicals for more than 3 months. So
maintenance costs and time are low. Instrumentation costs are moderate.
Detection limits are sometimes not directly comparable to human toxicity
values.


Monitoring technology nr 7: Mussel Monitor

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

time to result (B)

x
ease-of-use (B) x

Costs
consumables
maintenance

x
x


sometimes not directly comparable to human toxicity.



4.18 Algae toxins

Prepared by: TZW

Actual reports suggest that toxic cyanobacteria blooms are an emerging issue
world wide because of increased source water nutrient pollution causing
eutrophication. A third of the freshwater cyanobacteria genera are capable of
producing toxins. Actual investigation shows that the strategies of early
warning and protection drinking water treatment trains have been updated
continuously because of the increasing number of fresh water toxins
identified. The most important groups of fresh water toxins are the cyclic
peptides like microcystins and alkaloid toxins like cylindrospermopsin.
On the other hand, other less common and studied algal toxins from the large
group of non fresh water species are disseminate world wide. The possibility
exists for these toxins to be detected in freshwaters in future due to increased
salinity of freshwater supplies. This increased salinity is partly the result of
salt intrusion and salt contamination caused by the mining and commercial
industry. Additional the use of sea and brackish waters for drinking water
production is increasing word wide.
Cyanotoxins can occur cell bound and dissolved in water. Cell bound
cyanotoxins can released into the water by natural mechanisms
(metabolization) and induced mechanisms (physical, chemical as well
biological impact during water treatment).

The most common toxins, the microcystins occur in water in a range up to
mg/L. Most of the toxins are cell bound. Ca. 10% of the total toxin content is
found in dissolved form. Therefore the relevant concentration level of toxins
in water can be expected between several g/L to mg/L in some cases.
For analysis of drinking water we propose a sensitivity of 0.1 g/L for each
single toxin (cell bound and dissolved). The analytical technique applied
should be able to separate and to recognize single toxin structures. In some
cases, e.g. if the pollution level of the raw water is very high, the sensitivity of
the analysis of single toxins with modern techniques available is about 1 g/L
up to now.
For analysis of cyanotoxins certified reference standards are used. The
availability of these materials is world wide a problem.

1. LC-DAD
2. LC-MS/MS
3. ELISA-Tests
4. PPIA (protein phosphatase inhibition assay)

Monitoring sequence:
The monitoring sequence must be flexible because the algal bloom as well as
the cell stability and therefore the toxin release can change rapidly.


4.18.1 Monitoring technology nr 1-4: LC-DAD, LC-MS/MS, ELISA, PPIA

Description:
Dissolved toxins will be separated by solid phase extraction from water
phase. Cell bound toxins wil be separated by physical (freezing) and chemical
(solvents) destruction of algal cells. Following to this, analysis with LC-DAD,
LC-MS/MS, ELISA, PPIA can be conducted.

Standard operational procedures (SOP) for trace analysis of microcystins,
anatoxin-a and cylindrospermopsin were developed within the frame of the
joint European Research Project TOxIC [TOxIC, EVK1-CT-2002-00107],
Barriers against Cyanotoxins in Drinking Water.
For detailed information see: TOxIC: Cyanobacterial Monitoring and
Cyanotoxin Analysis, Abo Akademi Turku, ISBN 951-765-259-3 (2005).

In the frame of the TECHNEAU-Project the SOPs for analysis of the so called
new toxins will be designed.

Monitoring technology nr 1: LC-DAD

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x
x

time to result

several days
ease-of-use (B) x qualified staff needed

Costs high
consumables
maintenance

x

x


Overall conclusion Sensitive and selective method, but expensive; faster
methods are needed in case of algal blooms.


Monitoring technology nr 2: LC-MS/MS
sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

time to result

several days
ease-of-use (B) x qualified staff needed

Costs very high
consumables
maintenance

x

x


Overall conclusion Sensitive and selective method, but expensive, faster
methods are needed in case of algal blooms.


Monitoring technology nr 3: ELISA test

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

x

time to result

immediately if there is a critical
situation 1-2 days in general
ease-of-use (B) x
maintenance requirements (C)

x

Costs average
consumables
maintenance

x
x

average


Overall conclusion Comparable not expensive and fast method, but low
selectivity, because of matrix effects and cross activities the
sensitivity of the test kits which is published by the assay
producing companies seems too optimistic, ELISA tests
should used as pre-examination,


Monitoring technology nr 4: algal toxins PPIA

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

x

time to result

immediately if there is a critical
situation 1-2 days in general
ease-of-use (B) x

x

Costs average
consumables
maintenance

x
x

average


Overall conclusion Fast and cheap method with low selectivity and sensitivity,
PPIA test can be used as pre-screening method.

Evaluation:
ELISA tests give a first view concerning the toxin content in the water, if the
level of toxins is higher than 1 g/L. ELISA kits cannot identify single toxins
in general. On the other hand, companies offer test kits on the market with
sensitive detection limits of 0.1 g/L. Because of the cross activities and
matrix effects in the water this value seems to be too optimistic. This can be
true in some cases, but it is not a general rule. PPIA tests seem to be more
sensitive. Nevertheless, also this procedure cannot identify single toxins. Both
methods are influenced by cross-activities.
For drinking water monitoring (as well as raw water monitoring) the
identification and quantification of cyanobacterial toxins should be done by
MS-detection. This requires expensive technical equipment and qualified
staff.
If the water contains cyanobacteria and if pre-screening methods like ELISA
and PPIA indicate a positive signal LC-MS/MS measurements should be
performed.


4.19 Pesticides, pharmaceuticals, industrial chemicals and other organic
micropollutants


Required technical specifications
Concentration ranges that should be covered are the following:
0-10 g/L
drinking water
0-1 g/L, concentration should be less than 0.1 g/L (quality requirement for
pesticides in drinking water)

1. Gas Chromatography-Mass Spectrometry (GC-MS)
2. Liquid Chromatography-Ultraviolet Diode-Array Detection (LC-UV DAD)
3. Liquid Chromatography-Mass Spectrometry (LC-MS)

4.19.1 Monitoring technology nr 1: Gas Chromatography-Mass Spectrometry

GC-MS is a widely-accepted method and commercially available from
Agilent, Thermo Fisher, Waters, LECO, and so on. A large number of CEN
methods for the determination of organic micro-pollutants by GC/MS are
available.

Evaluation:
- Highly accurate and reliable technique, but also relatively expensive.
- Method fulfills the need for the parameter as long as the compounds are
volatile, thermo-stable and not too polar.
- Method can be used for regulatory measurement in a routine setting.
Operator needs certain skills to acquire and process data.


Monitoring technology nr 1: Gas Chromatography-Mass Spectrometry

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x
x

time to result

x 20-60 min
ease-of-use (B) x

Costs
consumables
maintenance

x
x


Overall conclusion Reliable, sensitive method for a wide range of compounds,
but expensive

4.19.2 Monitoring technology nr 2: High-Performance Liquid Chromatography-
UltraViolet Diode Array Detection

HPLC-UV DAD is a widely-accepted method and commercially available
from Agilent, Beckman Coulter, PerkinElmer, Shimadzu Scientific
Instruments, Thermo Fisher Scientific, Varian, Waters Corporation and
others.
A large number of CEN methods for the determination of organic micro-
pollutants by HPLC are available.

Evaluation:
- Highly accurate, sensitive and reliable universal technique. The technique is
less expensive than GC-MS and HPLC-MS.
- Method fulfills the need for the parameter as long as the compound absorbs
UV light.


Monitoring technology nr 2: High-Performance Liquid Chromatography-
Ultraviolet Diode Array Detection

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x
x

time to result

x 30-60 min
ease-of-use (B) x

Costs
consumables
maintenance

x
x


Overall conclusion Reliable, sensitive and universal method


4.19.3 Monitoring technology nr 3: High-Performance Liquid Chromatography-Mass
Spectrometry

HPLC-MS is a commercially available from Agilent, PerkinElmer, Shimadzu
Scientific Instruments, Thermo Fisher Scientific, Bruker Daltonics, Waters
Corporation and others.

Evaluation:
- Highly accurate, sensitive and reliable technique, but expensive.
- Method fulfills the need for the parameter as long as the compound can be
ionized i.e. brought into the gas/damp phase from a liquid phase and can be
ionized either in positive-ion mode by the addition of an proton and in
negative-ion mode by reduction of a proton.
Operator needs training and certain skills to acquire and process data.


Monitoring technology nr 3: High-Performance Liquid Chromatography-
Mass Spectrometry

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x
x

time to result

x
30-60 min
ease-of-use (B) x

Costs
consumables
maintenance

x
x


Overall conclusion Reliable, sensitive and selective method for wide range of
compounds, but expensive. Operator needs certain
skills/training


4.20 pH

Prepared by: AlphaMos

pH is a measure of the acidity or alkalinity of a solution. Aqueous solutions at
25 C with a pH less than 7 are considered acidic, while those with a pH
greater than 7 are considered basic (alkaline). pH values in water are
commonly in the range 0-14, though more extreme values, even negative
values, are possible. Because pH is dependent on ionic activity, a property
which cannot be measured easily or fully predicted theoretically, it is difficult
to determine an accurate value for the pH of a solution. The pH reading of a
solution is usually obtained by comparing unknown solutions to those of
known pH, and there are several ways to do so.

The normal range for pH in surface water systems is 6.5 to 8.5 and for
groundwater systems 6 to 8.5.
The pH of pure water (H20) is 7 at 25C, but when exposed to the carbon
dioxide in the atmosphere this equilibrium results in a pH of approximately
5.2. Because of the association of pH with atmospheric gases and
temperature, it is strongly recommended that the water be tested as soon as
possible. The pH of the water is not a measure of the strength of the acidic or
basic solution and alone does not provide a full picture of the characteristics
or limitations of a water sample.

1. pH indicator
2. pH meter

4.21Monitoring technology nr 1: pH indicator

Description:
A pH indicator is added into the solution under study. The indicator color
varies depending on the pH of the solution.
Using indicators, qualitative determinations can be made with universal
indicators that have broad color variability over a wide pH range and
quantitative determinations can be made using indicators that have strong
color variability over a small pH range.

Evaluation:
Because of the subjective determination of color, pH indicators are susceptible
to imprecise readings. For applications requiring precise measurement of pH,
a pH meter is frequently used.


Monitoring technology nr 1: pH indicator

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

time to result

x x immediat
ease-of-use (B) x

Costs
instrumentation (C)
consumables
maintenance

x


Overall conclusion Quick, cheap, but imprecise



4.21.1 Monitoring technology nr 2: pH meter

Description:
A pH meter is an electronic instrument used to measure the pH (acidity or
basicity) of a liquid. A typical pH meter consists of a special measuring probe
connected to an electronic meter that measures and displays the pH reading.
The pH probe measures pH as the activity of hydrogen ions surrounding a
thin-walled glass bulb at its tip. The probe produces a small voltage (about
0.06 volt per pH unit) that is measured and displayed as pH units by the
meter.
The standard hydrogen electrode (abbreviated SHE), also called normal
hydrogen electrode (NHE), is a redox electrode which forms the basis of the
thermodynamic scale of oxidation-reduction potentials. Hydrogen electrode
is based on the redox half cell: 2H+(aq) + 2e- H2(g) This redox reaction
occurs at platinized platinum electrode.
Quinhydrone electrode is one of several oxidation-reduction electrode's in
which the ratio of the two forms (quinone-quinhydrone), determined by the
hydrogen ion concentration, sets up a potential that can be measured and
converted to a pH value (fails above pH 8).
An ISFET is an ion-sensitive field effect transistor used to measure ion
concentrations in solution; when the ion concentration (such as pH) changes,
the current through the transistor will change accordingly. Here, the solution
is used as the gate electrode. A voltage between substrate and oxide surfaces
arises due to an ions sheath. An ISFET's threshold voltage depends on the pH
of the substance in contact with its ion-sensitive barrier.
The two basic adjustments performed at calibration set the gain and offset.
Calibration with at least two, but preferably three, buffer solution standards
is usually performed every time a pH meter is used. One of the buffers has a
pH of 7.01 (almost neutral pH) and the second buffer solution is selected to
match the pH range in which the measurements are to be taken: usually pH
10.01 for basic solutions and pH 4.01 for acidic solutions. The gain and offset
settings of the meter are adjusted repeatedly as the probe is alternately placed
in the two calibration standards until accurate readings are obtained in both
solutions.
pH meters range from simple and inexpensive pen-like devices to complex
and expensive laboratory instruments with computer interfaces and several
inputs. Pocket pH meter are readily available today for a few tens of Euros
that automatically compensate for temperature. More information can be
found at
http://www.radiometer-analytical.com/
http://www.metrohm.com/
http://us.mt.com/home
http://www.hannainst.com/


Evaluation:
For applications requiring precise measurement of pH, a pH meter is
frequently used.

Monitoring technology nr 2: pH meter

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x
x

time to result

immediate
ease-of-use (B) x

Costs
instrumentation (C) x All prices available
consumables
maintenance

x
x


Overall conclusion Common method easy & reliable



4.22 Chloride/nitrate/sulphate

Prepared by: TZW

- concentration range: 1 to 500 mg/L (chloride), 0.5 to 50 mg/L (nitrate), 1 to 300
mg/L (sulphate)
- sensitivity required: 1 mg/L (chloride), 0.5 mg/L (nitrate), 1 mg/L (sulphate)

For monitoring technologies that are only suited for analysis of nitrate see 4.9.

(IC/CD)

Description:
An aliquot of the water sample is directly injected into the ion
chromatographic system.

Evaluation:
sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

time to result

x
ease-of-use (B) x

Costs
consumables
maintenance

x

x


Overall conclusion Standard multi-method for analysis of common anions.


4.23 Conductivity

Prepared by: AlphaMOS

Conductivity of a substance is defined as 'the ability or power to conduct or
transmit heat, electricity, or sound'. Its units are Siemens per meter [S/m] in
SI and mhos per centimeter [mho/cm] in U.S. customary units. Its symbol is
or .
An electrical current results from the motion of electrically charged particles
in response to forces that act on them from an electrically applied electric
field. In water or fluids a net motion of charged ions can occur. This
phenomenon produces an electric current and is called ionic conduction.
Electrical conductivity is defined as the ratio between the current density (J)
and the electric field intensity () and it is the opposite of the resistivity (,
[*m]): = J/ = 1/
Conductivity is a measurement used to quickly determine the variation or
changes in natural water or wastewaters.
Elevated dissolved solids can cause "mineral tastes" in drinking water.
Corrosion or encrustation of metallic surfaces by waters high in dissolved
solids causes problems with industrial equipment and boilers as well as
domestic plumbing, hot water heaters, toilet flushing mechanisms, faucets,
and washing machines and dishwashers.

Pure water is not a good conductor of electricity. Because the electrical
current is transported by the ions in solution, the conductivity increases as the
concentration of ions increases.
Thus conductivity increases as water dissolved ionic species.
Ultra pure water 0.055 S/cm
Deionised water 1 S/cm
Rainwater 50 S/cm
Drinking water 500 S/cm
Industrial wastewater 5 mS/cm
Mineral water 4 7 mS/cm
Sea water 50 mS/cm
An EC meter is normally used to measure conductivity in a solution.

1. Conductimeter

4.23.1 Monitoring technology nr 1: Conductimeter

Description:
Conductivity is measured by applying an alternating electrical current at an
optimal frequency to two electrodes immersed in a solution and measuring
the resulting voltage. During this process, the cations migrate to the negative
electrode, the anions to the positive electrode and the solution acts as an
electrical conductor.


The conductivity depends on the value of the pH, on the temperature of
measurement and on the amount of CO2 which has been dissolved in the
water to form ions. The conductivity due to these factors is intrinsic
conductivity.
The conductivity is also affected by the concentration of ions already present
in the water such as chloride, sodium and ammonium. This contribution to
the conductivity is extraneous conductivity.
Consumables include conductivity standards. This technology is
commercially available. EC meters range from simple and inexpensive pen-
like devices to complex and expensive laboratory instruments with computer
interfaces and several inputs.
interfaces and several inputs. Information about technical products can be
found at
http://www.radiometer-analytical.com/
http://www.metrohm.com/
http://us.mt.com/home
http://www.hannainst.com/.

Evaluation:
Easy to use & maintain, economical. Different systems (at different prices) can
be used depending on the requested precision and conductivity range.
It is recommended to check the cell constant regularly due to contamination
risk.

Monitoring technology nr 1: Conductimeter
sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x
x

time to result

immediate
ease-of-use (B) x

Costs
consumables
maintenance

x
x


Overall conclusion Reliable method, easy & affordable



4.24 Calcium & magnesium

See par 4.1: Metals

4.25 Sulphate

See par 4.20: chloride, nitrate, sulphate

4.26 Aluminium

See par 4.1: Metals


4.27 Ammonium

Prepared by: S::can

0 - 1 mg/L (drinking water, EU limit is 0.5 mg/L)
0 - 50 mg/L (raw water)
Required resolution of the measurement: 1 mg/L
0.05 mg/L (drinking water)
1 mg/L (raw water)

Monitoring technologies
1. Ion selective electrode
2. Ion chromatography
3. Photometric test kits
4. Automatic Analyser

4.27.1 Monitoring technology nr 1: Ion Selective Electrode

Description:
- the ISE is an electrode that is equipped with a membrane that is permeable
for ammonium ions and not for other ions. The presence of ammonium ions
will result in a change in the potential measured by the electrode because of
diffusion of ammonium ions through the membrane into the electrode. The
potential is indicative for the concentration.
- ISEs for ammonium have been available for many years. Reliability has
been, however, poor due to instability of the measurement and cross
sensitivities. Over the last 2 years a number of manufacturers have
introduced improved instruments with additional electrodes next to the ISE
for ammonium to compensate for cross sensitivities (pH, temperature,
potassium)

Equipment
Equipment needed: electrode, electrolyte (to be changed on a periodical
basis), selective membranes (need to be changed on a periodical basis)

References
Vendors selling this latest generation of ammonium probes: s::can
Messtechnik, Nadler Chemische Analysen Technik, Dr. Lange, Endress and
Hauser and others.

Evaluation:
- When the techniques do what the marketing sheets tell, then this is very
suitable for online monitoring of source water and drinking water. Only own
experience is with s::can and Nadler instruments and they work well (stable
without calibration for up to two months, no membrane replacement needed
until 6 months of operation in Waste Water (more difficult and demanding
matrix for ISE).


- This is the only online method. It's much cheaper than ion chromatography.
Photometric test equipment is cheaper, but not online and costs around 5
Euros per test.
- This method fulfils the needs.

Monitoring technology nr 1: Ion Selective Electrodes

sensitivity (A)
source water
drinking water

x

x

robustness (A)
selectivity

x
x

time to result

x measurement every 5 seconds
training

Costs
instrumentation (C) x price indication: s::can
instrument 3500
consumables
maintenance

x
x



4.27.2 Monitoring technology nr 2: Ion Chromatography
In ion chromatography, a pre-treated sample is processed in the laboratory.
The sample in injected in chromatograph, and the components present are
separated on the column. Based on retention time the desired component is
identified.

- ion chromatograph, including chromatographic column, HPLC pump.
- consumables: solvents, filters, columns.

Available from Waters, Metrohm, Thermo Electron, Dionex and others.
Evaluation:


- Highly accurate technique. Only suitable for off-line, batch wise laboratory
analysis. Useful for regulatory measurements.
- Most accurate technique, but also by far the most expensive (investment in
equipment).
- This method fulfils the needs in terms of measuring range and accuracy.

Monitoring technology nr 2: Ion Chromatrography

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

time to result

training

Costs
instrumentation (C) x price indication: > 15.000 Euro
consumables
maintenance

x
x


Overall conclusion Suitable method for reference/regulatory measurements in
the laboratory
4.27.3 Monitoring technology nr 3: Photometric test kit
In the photometric test kit, a grab sample is taken, to this sample is added a
reagent that forms a coloured substance by reaction with the targeted species.
This coloured substance is then measured using a simple photometer.

- equipment: photometer, test-tube with reagent (pr-mixed, easy to handle),
filter to remove solids / turbidity from the sample before starting test
- consumables: test tubes (approx 2 - 5 Euros per test)

Reference
- Photometry is established technique (see Standard Methods for the
Available from many vendors, such as WTW, Dr. Lange and others.


Evaluation:
- Relatively accurate tests that can be used in the lab and in the field.
Relatively easy to use (any lab technician can handle it, but the operator will
need at least some lab training).
- Only off-line analysis possible.

Monitoring technology nr 3: Photometric Test Kit

sensitivity (A)
source water
drinking water

x

x

robustness (A)
selectivity

x
x

time to result

x reaction before measuring takes
15 minutes
ease-of-use (B) x sampling and filtering of liquids
needed manually

Costs
instrumentation (C) x price indication: WTW
instrument < 2000
consumables
maintenance

x

x


measurements

4.27.4 Monitoring technology nr 4: Automatic Analyser

Descritpion:
In the photometric test kit, a grab sample is taken, to this sample is added a
reagent that forms a coloured substance by reaction with the targeted species.
This coloured substance is then measured using a simple photometer.
Sampling, sample preparation and handling are automated.

Alternatively, ISE is used instead of photometry in such automated analysers.



- equipment: cabinet analyser, comprising of sampling line, filtering unit,
dosing pumps, photometer,
- consumables: reagents for photometric reaction, filters, tubes in analyser

References
Photometry is an established technique (see Standard Metods for the
The automatic analysers have been in use for more than 10 years. They
remain however relatively expensive to buy and highly expensive / time
consuming to operate as their complexity makes them prone to failure.
Exception: Danfoss Evita which uses membrane technology for isolating the
ammonia from the water and small volumes to reduce reagent use and
maintenance. Range and resolution only sufficient for waste water use.
- Available from Applikon, WTW, Nico2000, Kelma and others.

Evaluation:
- Performance is OK, but costs of equipment and maintenance are relatively
high.
- Online analysis is possible, maintenance intensive.

Monitoring technology nr 4: Automatic Analyser

sensitivity (A)
source water
drinking water

x

x

robustness (A)
selectivity

x

x

time to result

x cycle time per measurement at
least 15 minutes
ease-of-use (B) x maintenance troublesome

Costs
instrumentation (C) x price indication: > 10000
consumables
maintenance

x

x


Overall conclusion Expensive equipment, especially because it requires a lot of
maintenance. Not user friendly.


4.28 Iron

See par 4.1: Metals

4.29 Manganese

See par 4.1: Metals


4.30 Taste & Odor

Prepared by: AlphaMos

Water is called the universal solvent. It dissolves a little of anything that
comes in contact with it. Wherever water goes, it gathers minerals, trace
elements, contaminants, etc. Taste & odor are two of the organoleptic
properties of drinking water: properties that can be identified by sensory
perceptions.

Main problems with off-taste or off-odor are:
- Chlorinous, chemical or medical taste / odor problems
Two common causes are either the addition of chlorine to the water by the
supplier or the interaction of that chlorine with organic material in the
plumbing system. Generally this occurs when the water is treated at the water
treatment plant to disinfect it. The addition of chlorine is used to kill off
bacteria and other harmful microorganisms.
- Sulfurous, sewage like, musty, moldy, earthy, grassy or fishy taste /
odor problems
The main cause is due to bacteria or other organisms (algae, fungi, .).
Organic matter can accumulate and bacteria can grow on these organic
deposits. These bacteria can produce a gas that smells like rotten eggs or
sewage.
This is usually a result of decaying organic deposits underground. As water
flows through these areas, hydrogen sulfide gas is picked up, and when this
water reaches the surface or comes out of the faucet, the gas is released into
the air. Hydrogen sulfide gas produces the rotten egg odor, can be corrosive
to plumbing at high concentrations.
The odor of water with as little as 0.5 ppm of hydrogen sulfide concentration
is detectable by most people. Concentrations less than 1 ppm give the water a
"musty" or "swampy" odor. A 1-2 ppm hydrogen sulfide concentration gives
water a "rotten egg" odor and makes the water very corrosive to plumbing.
Generally, hydrogen sulfide levels are less than 10 ppm, but have been
reported as high as 50 to 75 ppm.
The absence of sulfur and chlorine add to the taste of water.
The musty smell is normally a result of organic matter or even some
pesticides in the water supply. Even very low amounts can introduce
unpleasant odors into the water.
- Petroleum, gasoline, turpentine, fuel or solvent like odor problems
This smell can be a result of MTBE (Methyl Tert butyl Ether ) contamination
in the water. The odor threshold is fairly low, below any toxic thresholds. So
even though one can smell it, the MTBE is more than likely not at a level to
cause harmful effects.
- Salty taste problem
A salty taste in water is usually due to the presence of naturally occurring
sodium, magnesium or potassium. For water to taste correctly on the palate,
certain minerals must be present. Good tasting water is a subtle combination
of flavors with an ideal combination and concentration of minerals.
Potassium, magnesium, calcium and even small amounts of sodium give
water its fullness. If not, water, such as distilled water, tastes flat and dull.


- Alkali taste problem
An alkali taste in water is usually due to high level of total dissolved solids.
- Metallic taste problem
A metallic taste in water is due to low pH, corrosive water, high content of
iron (0.004 mg/L), copper (2-5 mg/L), zinc (4-9 mg/L), and manganese. Too
high a mineral concentration and water takes a tinny metallic taste.

1. Sensory panel
2. GC MS (for individual compounds)
3. Electronic tongue & nose

4.30.1 Monitoring technology nr 1: Sensory panel

Description:
The European Standard EN 1622:2006 specifies a qualitative method for
determining any abnormal odour and/or flavour.
The odour and flavour of a water sample may also be assessed qualitatively
by only one selected assessor or a test panel to detect any abnormal odour
and/or flavour.
In European regulations relative to water intended for human consumption,
the only requirement for odour and flavour is acceptable for the consumer
and no abnormal change.
In these cases, no quantitative odour (TON) and/or flavour (TFN) assessment
is required.
The qualitative simplified procedure can be used to check the water on a
routine basis and also in cases of consumer complaints.
Samples shall be taken directly from the consumer tap and shall be assessed
immediately without any dilution.
The selected assessor shall judge whether the test environment is appropriate
(absence of odours likely to mask the sample odours). If not, the selected
assessor should carry out the test in another room.
The selected assessor shall shake thoroughly each flask, remove the stopper,
smell, and then transfer a suitable volume of water in his/her glass, hold in
the mouth for several seconds before discharging it without swallowing.
The selected assessor shall record if there is abnormal odour and/or flavour.
If it is not the case, he/she will note no abnormal odour and/or flavour.
If an abnormal odour and/or flavour is detected, take a fresh sample for
further assessment.

Evaluation:
Reference method


Monitoring technology nr 1: Sensory panel

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x
x

time to result

x
ease-of-use (B) x

Costs
consumables
maintenance

x
x


Overall conclusion reliable method

4.30.2 Monitoring technology nr 2: GC- MS

Description:
Gas chromatography-mass spectrometry (GC/MS) is a method that combines
the features of gas-liquid chromatography and mass spectrometry to identify
different substances within a test sample. As such it can also be used to
analyse individual compounds that cause taste and/or odor problems.

Evaluation:
Time consuming method, high skilled user needed


Monitoring technology nr 2: GC-MS

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x
x

time to result

x
ease-of-use (B) x

Costs
consumables
maintenance

x
x


Overall conclusion Time consuming method


4.30.3 Monitoring technology nr 3: Electronic Nose & Tongue

Description:
Over the last decade, electronic sensing or e-sensing technologies have
undergone important developments from a technical and commercial point of
view. The expression electronic sensing refers to the capability of
reproducing human senses using sensor arrays and pattern recognition
systems. Since 1992, research has been conducted to develop technologies,
commonly referred to as electronic noses, that could detect and recognize
odors and flavors. The stages of the recognition process are similar to human
olfaction and are performant for identification, comparison, quantification
and other applications. However, hedonic evaluation is a specificity of the
human nose given that it is related to subjective opinions. These devices have
undergone much development and are now used to fulfil industrial needs.
Electronic Nose EN & Tongue ET include three major parts: a sample delivery
system, a detection system, a computing system.
The sample delivery system of ET enables the liquid sample to be in contact
with the detection system. The sample delivery system of EN enables the
generation of the headspace (volatile compounds) of a sample, which is the


fraction analysed. The system then injects this headspace into the detection
system.
The detection system, which consists of a sensor set, is the reactive part of
the instrument. When in contact with samples, the sensors react, which means
they experience a change of physical properties. The more commonly used
sensors include metal oxide semiconductors (MOS), conducting polymers
(CP), quartz crystal microbalance, surface acoustic wave (SAW), and field
effect transistors (FET). In recent years, other types of electronic noses have
been developed that utilize mass spectrometry or ultra fast gas
chromatography as a detection system.
A specific response is recorded by the electronic interface transforming the
signal into a digital value. Recorded data are then computed based on
statistical models. The computing system works to combine the responses of
all of the sensors, which represents the input for the data treatment. This part
of the instrument performs global fingerprint analysis and provides results
and representations that can be easily interpreted.
As a first step, an electronic nose or tongue need to be trained with qualified
samples so as to build a database of reference. Then the instrument can
recognize new samples by comparing volatile compounds fingerprint to those
contained in its database. Thus they can perform qualitative or quantitative
analysis.
Information about commercial instruments can be found at:
www.alpha-mos.com
http://www.airsense.com/
www.gsg-analytical.com/

Evaluation:
Simple technique allowing to obtain a correlation with a given panel
Needs to be validated on the application & trained based on either
concentration or panel characterizations.


Monitoring technology nr 3: Electronic Nose & Tongue

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x
x

time to result

x
ease-of-use (B) x

Costs
consumables
maintenance

x
x


Overall conclusion Simple technique allowing to obtain a correlation with a
given panel


4.31 Colour

Prepared by: S::can

Colour in water is caused by contaminations present. Natural water is
normally coloured because of the presence of clay particles, iron particles
and/or humic and fulvic acids. This normally results in a yellow to brown
colour of the water. Alternatively, a green colour is sometimes observed
which is caused by algae. True colour is measured after filtration of the water
through a 0.45 m filter.

Colour is expressed either in absorbance units (m
-1
) or with reference to a
standard (Hazen, Pt-Co or APHA units for example).

In general, expressed in Hazen Units, natural waters are expected to be in the
range of 0 - 200 Hazen Units, and drinking water in the range of 0 - 15 Hazen
units. The EU guideline define colour has to be "acceptable for consumers and
not subject to abnormal change".

1. Visual comparison method
2. Colorimetric analysis
3. On-line spectrophotometric measurement

4.31.1 Monitoring technology nr 1: Visual Comparison method

Description
In this method water colour, after filtration and pH adjustment to pH 7, is
compared to the colour of reference solutions. The reference solutions are
prepared from standardised platinum and cobalt salts. The sample is then
visually compared to the standard solutions. This comparison is performed
manually using a so-called nessler tube. The colour is estimated using this
apparatus by looking through it towards a white surface. The colour is then
expressed in Colour Units (CU), and need to be corrected for sample dilution,
which is necessary if colour is too high.

Required for this measurement are:
nessler tube
filter assembly and 0.45m filters
K2PtCl6 (potassium chloroplatinate)
CoCl2.6H2O (cobaltous chloride)
HCl (hydrochloric acid)
NaOH (sodium hydroxide)

This is a long established and accepted method. However, it is no longer used
much as it has been replaced by automated tests, as described under
technique 2.


Reference
Technique is described in APHA Standard Methods for the examination of
water and waste water (21st edition). Equipment available for example from
Kimble, Fischer Scientific, Taylor Scientific and many other laboratory
equipment resellers.

Costs
Costs per test set-up is in the order of 30 .

Evaluation:
This method is simple and cheap. However, it is laborious, highly subjective
(as it depend on the judgement of the person the makes the comparison with
the standard). The performance of a reproducible measurement with this
technique requires a skilled laboratory technician. The use of current
colorimeters is much simpler and has higher reproducibility. This technique
is only suited for laboratory analysis, not for online purposes.

An experienced technician can perform one colour measurement per 5
minutes (measurement only, excluding preparation of stock solutions etc)


Monitoring technology nr 1: Visual comparison method

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

time to result

x only manual analysis in lab
possible.
ease-of-use (B) x
maintenance requirements (C) x only washing of glassware
needed

Costs
instrumentation (C) x very cheap
consumables
maintenance

x

x

Recommendation for use in SSS (D) x not recommended as it requires
skilled technician

Overall conclusion Primitive method. Cheap but relies on operator and is
subjective. Not suited for online use.

4.31.2 Monitoring technology nr 2: Colorimetric analysis

Description:
As above. But in this case, the analysis is performed using a laboratory
instrument. Used for this is a simple single wavelength spectrophotometer
that measures colour between 450 and 465 nm and correlates this with an
internally stored calibration curve.
This is a long established and accepted method. It is the most commonly used
technique for colour measurement.

Equipment
spectrophotometer
filter assembly and 0.45m filters

Reference


WTW, LaMotte, Hach Lange, Elektro Physik, Thermo Electron.

Costs
Costs of the spectrometer are in the range of 1000 - 3000 Euro, depending on
the number of different tests that can be performed with it. Most can handle
tests for several up to more than 20 different parameters. Each parameter
requires a different cuvette with reagents. Colour is normally performed
without the need for reagents.

Evaluation:
This method is simple and cheap. It is objective and reproducible. It only
provides a colour indication against platinum cobalt standards, so is only
suitable for orange, yellow and brown colours.
This technique is only suited for laboratory analysis, not for online purposes.
An experienced technician can perform one colour measurement per 2
minutes.

Monitoring technology nr 2: Colorimetric Analysis
sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

time to result

possible.
ease-of-use (B) x

Costs
instrumentation (C) x cheap
consumables
maintenance

x

x


Overall conclusion Reliable method, but requires filtration of sample and
manual analysis. Method o.k., but faster method needed.

4.31.3 Monitoring technology nr 3: Online spectrophotometric measurement
Description:


In this method water colour, without filtration or pH adjustment, is measured
at one wavelength. The result is compared to a stored calibration curve made
against platinum- cobalt (HAZEN) reference solutions.
This is a long established and accepted method. It is the online equivalent of
technique 2.

Equipment
spectrometer

Reference
Siegrist, WTW, s::can, Applikon.

Costs
Costs per instrument vary between 2000 and 8000 Euro. More expensive
instruments will provide additional parameters simultaneously, such as
turbidity (Siegrist), UV absorbance at 254 nm or even NO3 and TOC/DOC
(s::can). These instruments can also provide true and apparent colour because
they use multiple wavelengths to compensate for the influence of particles.

Evaluation:
This instrument installation is simple. A simple flow through setup is all that
is necessary. Furthermore, some instruments are submersible and thus can be
installed directly in rivers or reservoirs. The instruments are more expensive
than the laboratory spectrometer, but provide continuous information, often
in combination with additional important process parameters.


Monitoring technology nr 3: Online spectrometric measurement

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

time to result

x continuous
ease-of-use (B) x
maintenance requirements (C) x monthly cleaning recommended

Costs
consumables
maintenance

x
x

Recommendation for use in SSS (D) x suitable

Overall conclusion This is a reliable and low maintenance method. Equipment
is not too expensive, so well suited to online applications.


4.32 Turbidity

Prepared by: S::can

Turbidity is the scattering of light caused by particles, colloids and / or air
bubbles in water. Turbidity is related to the clarity of water, higher turbidity
resulting in less clear water.

Turbidity ranges expected for
surface water: 0 - 1500 FTU
drinking water: 0 - 60 FTU

In the drinking water guidelines, it is stated: turbidity should be "acceptable
for the consumer and not show unacceptable changes".

In general, it is expressed either in FTU (formazine turbidity units) or NTU
(nephelometric turbidity units). Both are based on the same method, but are
obtained by calibration of the turbidity meter with a different reference
solution.

1. Analysis using a laboratory Colorimeter
2. On-line turbidity meter
3. On-line spectrophotometric measurement

4.32.1 Monitoring technology nr 1: Analysis using a laboratory colorimeter

Description:
In this method turbidity is analysed by sampling followed by analysis onsite
(using a handheld analyser) or in a laboratory.
The measurement is based on the following principle: turbidity is caused by
scattering of light by particles in the water. This scattering is measured by
sending a light beam through a sample and measuring the scattering at 90
degree angle of the incident beam. In general, IR light at 830 nm is used for
this measurement, although some instruments used visible light instead.

Equipment
spectrophotometer
glass cuvette fitting spectrophotometer

Reference:
water and waste water (21st edition) and specified in DIN 27027, ISO 7027,
US EPA 180.1. Equipment available for example from WTW, LaMotte, Hach
Lange, Elektro Physik, Thermo Electron.


Costs
Costs of the spectrometer are in the range of 1000 - 3000 Euro, depending on
the number of different tests that can be performed with it. Most can handle
tests for several up to more than 20 different parameters. Each parameter
requires a different cuvette with reagents. Turbidity measurement is normally
performed without the need for reagents.

Evaluation:
This method is simple and cheap. It is objective and reproducible.
This technique is only suited for laboratory analysis, not for online purposes.
An experienced technician can perform one turbidity measurement per
minute.

Monitoring technology nr 1: Analysis using a laboratory colorimeter

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x
x

time to result

possible.
ease-of-use (B) x
maintenance requirements (C) x only cleaning of cuvettes

Costs
consumables
maintenance

x
x



4.32.2 Monitoring technology nr 2: Online turbidity meter

Description:
As above. But in this case, the analysis is performed using on online
instrument. Used for this is a simple single wavelength spectrophotometer
that measures turbidity. Either flow through or submersible instruments can
be used.


This is a long established and accepted method. It is the most commonly used
technique for turbidity measurement.

Equipment
spectrophotometer

Reference
WTW, YSI, Endress + Hauser, SWAN, Hach Lange, Siegrist, etc.

Costs
Costs of the turbidy meter are in the range of 2000 - 4000 Euro. The more
expensive one, such as from Siegrist, provide additional parameters such as
colour and UV254 at the same time.

Evaluation:
This method is simple and affordable. Many run with low maintenance,
although keeping the measurement chamber clean and replacement of optical
parts (e.g. the lamp) can be periodically required.
Cross sensitivity is limited: main reasons for inaccurate readings are: air
bubbles, dirty measuring cell. Further causes for discrepancies in
measurements are the use of difference reference standards and different
design of the measuring cell (if not according to ISO or US EPA norms).
This technique is suited for online purposes.


Monitoring technology nr. 2: Colorimetric Analysis

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x
x

time to result

x continuous online measurement
ease-of-use (B) x

Costs
consumables
maintenance

x
x


Overall conclusion Reliable method, equipment affordable. Suited for online
monitoring, but referencing/ calibration is difficult due to
instable standard solutions.

4.32.3 Monitoring technology nr 3: Online spectrophotometric measurement

Description:
In this method turbidity is measured by monitoring the light absorbance of
the water at multiple wavelengths. The obtained spectrum is fitted with
known absorption curves for turbidity and from this fit the turbidity is
calculated. Because multiple wavelengths are used, the effects of cross
sensitivity are minimised.

Equipment
spectrometer

Reference
This is a relatively new technique, not yet included in standard methods. It is
founded on many scientific papers, but only has been available for online
water quality measurement since 1999. Available from e.g. s::can.


Costs
Costs per instrument vary between 8000 and 12000 Euro. The instruments
will provide additional parameters simultaneously, such as colour, UV
absorbance at 254 nm, NO3 and TOC/DOC.

Evaluation:
This instrument installation and use is simple. A simple flow through setup is
all that is necessary. Furthermore, the instruments are submersible and thus
can be installed directly in rivers or reservoirs. The instruments are more
expensive than the laboratory spectrometer, but provide continuous
information in combination with additional important process parameters.

Monitoring technology nr 3: Online spectrometric measurement

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x
x

time to result

ease-of-use (B) x
maintenance requirements (C) x no replaceable parts, automatic
cleaning

Costs
instrumentation (C) x competitive when used for
multiple parameters at once.
consumables
maintenance

x
x

Recommendation for use in SSS (D) x suitable. espeically because of
low maintenance and multiple
parameters in one instrument

Overall conclusion Reliable method, equipment affordable. Suited for online
monitoring, but referencing/ calibration is difficult due to
instable standard solutions.


4.33 AOC

Prepared by: Eawag


AOC analysis is not covered by current drinking water legislation or
guidelines. However, a suggested AOC value for biologically stable water is
10 g-C/L acetate-carbon equivalents (Van der Kooij, 1982). A concentration
range that should at least be covered by the AOC method is 10 1000 g-C/L


1. The original van der Kooij assay (van der Kooij et al., 1982)
2. The Werner and Hambsch assay (Hambsch et al., 1992)
3. The Stanfield and Jago ATP-based assay (Stanfield and Jago, 1989)
4. The LeChevallier assay (LeChevallier et al., 1993)
5. The Eawag-AOC assay (Hammes and Egli, 2005)

Methods 1 & 4 are incorporated/combined in Standard Methods for the
Analysis of Water and Wastewater (APHA, 1998). Numerous other AOC
methods are described in peer-reviewed literature. For further information,
see for example Page and Dillon (2007) and Volk (2001).
A complete overview is given in Biodegradable organic matter in drinking
water treatment and distribution (Prevost et al., 2005).

References
APHA (1998). Standard Methods for the Examination of Water and
Wastewater. 20th ed.
Page, D. and Dillon, P. (2007) Measurement of the biodegradable fraction of
dissolved organic matter relevant to water reclamation via aquifers.
www.csiro.au, ISSN: 1835-095x.
Volk, C.J. (2001) Biodegradable organic matter measurement and bacterial
regrowth in potable water. Methods in Enzymology, 337: 144 170.
Prevost, M., Laurent ,P. R., Servais, P., Joret J.-C. (2005). Biodegradable
organic matter in drinking water treatment and distribution. ISBN
1583213678.

4.33.1 Monitoring technology nr. 1: The original van der Kooij assay

Description:
This method measures the total growth until stationary phase of two bacterial
pure cultures in a pasteurized water sample at 15 C, using plate counting on
agar as enumeration method. The concept is that the pure culture strains
consume the AOC and produce cells. The test organisms are Pseudomonas
fluorescens P17 and Aquaspirillum NOX. The obtained cell concentration is
converted to an AOC concentration using growth of the pure cultures on
acetate-carbon as a standard. Typical variations on this method include using


a higher incubation temperature (LeChevallier et al., 1993), adding an
inorganic mineral buffer to ensure only carbon limitation (Lehtola et al.,
2001), and using alternative strains (van der Kooij, 2002). The detection limit
of the method is < 10 /L acetate-carbon equivalents.

Equipment:
Incubator, carbon-free glassware and standard equipment for plate counting
Consumables:
Agar used for plate counting

The method has been in use for more than 20 years and is generally accepted
as the established method. It is also included in Standard Methods (APHA,
1998).

Evaluation:
The method has the advantages that it has been used for a long time and a lot
of experience and data exists in the peer reviewed literature. The method is
generally regarded as rather time consuming (< 14 days) and the detection of
all AOC compounds can be limited due to the use of selected pure cultures.

References
Van der Kooij D., Visser A., Hijnen W.A.M. (1982) Determination of easily
assimilable organic carbon in drinking water. J. Am. Water Works Assoc. 74:
540-545.
Van der Kooij, D. Assimilable organic carbon (AOC) in treated water:
determination and significance. In Encyclopedia of Environmental
Microbiology, Bitton, G. (Ed.), John Wiley & Sons, Hoboken, NJ, USA, 2002.
pp 312 327.


Monitoring technology nr 1: The original van der Kooij assay


4.33.2 Monitoring technology nr. 2: The Werner and Hambsch assay

Description:
The method is based on correlation between turbidity and total bacterial cell
number (TCN) after growth of an inoculum in the target water sample. The
sample is filter-sterilized, placed into a cuvette (250 mL), and a sterile nutrient
salt solution containing no carbon is added. The sample is inoculated to about
5 x 10
4
TCN/mL with a suspension of bacteria washed from the sterilizing
filter, and the cuvette is incubated in a specially modified turbidimeter at
approximately 22 C. Turbidity, by applying 12-degrees forward scattering on
a specifically designed instrument, is measured every 30 minutes for 2 to 4
days, until stationary phase is reached. The growth curves are plotted as
logarithm of turbidity versus incubation time. The slope of the curve gives the
growth rate () and is an indicator of substrate quality and the growth factor
indicates the substrate quantity. Moreover, acetate-C-equivalents are
calculated from the turbidity increase with the turbidity yield on acetate-C.
sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

Might not detect all AOC
compounds
time to result

up to 14 days
ease-of-use (B) x All AOC methods require ultra-
clean handling
maintenance requirements (C) x All AOC methods require ultra-
clean handling

Costs
consumables
maintenance

x
x


Overall conclusion The method is established, and does not require complex
instrumentation or expertise to perform. The drawback is
that it is time and labour intensive.


The bottom detection limit is 10 g/L acetate-C-equivalents. Also, DOC-
removal and total cell number increase are analyzed at the start and at the
end of the experiments.


Equipment
Turbidity meter (12 forward scattering), carbon-free glassware. Optional:
DOC analyzer and fluorescence microscope (for TCN determination)

Evaluation:
The method is significantly faster than the original van der Kooij assay (2-4
days). The use of a natural microbial community allows the detection of a
broader range of AOC substrates. The additional advantage of the method is
that it also produces kinetic information (specifically growth rates relating to
AOC quality) and additional values like DOC removal and increase of total
cell number). The disadvantage is a need for specialized equipment
(turbidimeter) and a limited number of samples that can be processed at one
time.

References:
Werner, P. (1984). Untersuchungen zur Substrateigenschaft organischer
Wasserinhaltsstoffe bei der Trinkwasseraufbereitung. Zbl. Bakt. Hyg. I Abt.
Orig. B 180 S. 46-61.
Hambsch, B., Werner, P., Frimmel, F. H. Bakterienvermehrungsmessungen in
aufbereiteten Wssern verschiedener Herkunft. Acta hydrochim. hydrobiol.
20:1, p. 9-14 (1992)
Hambsch B., Werner, P. (1996). The removal of regrowth enhancing organic
matter by slow sand filtration, in: Advances in Slow Sand and Alternative
Biological Filtration, John Wiley & Sons, , p. 21-27.


Monitoring technology nr 2: The Werner and Hambsch assay


4.33.3 Monitoring technology nr. 3: The Stanfield and Jago ATP-based assay

Description:
The concept is similar to the original van der Kooij assay in that the growth
of bacteria on AOC is quantified and related to the consumed AOC.
However, this method differs essentially in that it uses a natural microbial
community instead of pure cultures, and that it utilizes an ATP assay instead
of plating (due to the natural microbial community) (Stanfield and Jago,
1989).

Equipment
Incubator, carbon-free glassware and standard equipment for ATP analysis
(luminometer)
Consumables
ATP reagents
sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x
x

Natural microbial communities
probably consume more AOC
compounds than pure cultures
time to result

2-4 days
clean handling
clean handling

Costs
consumables
maintenance

x

x


Overall conclusion The method is fast compared to the original AOC method,
but the specific instrumentation (turbidometer) limits the
number of samples that can be processed.


The method has bee restricted to a limited number of published applications
(Stanfield and Jago, 1989; Gibbs et al, 1993).

Evaluation:
The method is significantly faster than the original van der Kooij assay (3
days). There is also the notion that the use of a natural microbial community
allows the detection of a broader range of AOC substrates. Potential
disadvantages are that additional conversion factors are required to convert
ATP results to cell concentrations, which is complicated by the fact that
natural microbial communities can have broader differences in cellular ATP
than pure cultures.

Monitoring technology nr 3: The Stanfield and Jago ATP-based assay


sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x
x

time to result

3 days
clean handling
clean handling

Costs
consumables
maintenance

x

x


Overall conclusion The method is rather fast, but has as a main drawback the
additional conversion of ATP concentrations to cell
concentrations.


References:

Stanfield, G. and Jago, P.H. (1989) Application of ATP determinations to
measure the concentration of assimilable organic carbon in water. In: ATP
luminescence, Edited by Stanley et al. Blackwell Scientific, Oxford, England,
99 - 108.

4.33.4 Monitoring technology nr. 4: The LeChevallier assay

Description:
Essentially similar to the original van der Kooij assay, this adaptation
employs a higher incubation temperature (22 C) and higher inoculum
concentration (10
4
cells/mL) to achieve a faster result. In addition, ATP
analysis is used to quantify the growth of the pure cultures (LeChevallier et
al., 1993).

Equipment:
Incubator, carbon-free glassware and standard equipment for ATP analysis
(luminometer)
Consumables:
ATP reagents

The method has been in use for more than 10 years and is also included in
Standard Methods (APHA, 1998).

Evaluation:
The method is significantly faster than the original van der Kooij assay (3
days). Disadvantages are that additional conversion factors are required to
convert ATP results to cell concentrations. This is supposedly less
problematic for pure cultures compared to natural communities.


Monitoring technology nr 4: The LeChevallier assay


References:

APHA, AWWA, et al. (1998). Standard Methods for the Examination of Water
and Wastewater. 20th ed.

LeChevallier, M.W., Shaw, N.E., Kaplan, L.A., Bott, T.L. Development of a
rapid assimilable organic carbon method for water. Appl. Environ. Microb.
1993, 29: 1526 1531.

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

time to result

3 days
clean handling
clean handling

Costs
consumables
maintenance

x

x


Overall conclusion The method is similar to the Van der Kooij assay but faster.
However, it also requires the additional conversion of ATP
concentrations to cell concentrations.


4.33.5 Monitoring technology nr.5: The Eawag-AOC assay

Description:
Essentially similar to the Stanfield and Jago (1989) method, this assay
measures the total growth of a natural microbial community in a filter-
sterilized water sample at 30 C, using fluorescent staining and flow
cytometry (FCM) enumeration method. The obtained cell concentration is
converted to a carbon concentration using a theoretical conversion value (1
ug/L AOC = 1 x 10
7
cells) (Hammes and Egli, 2005; Vital et al., 2007)

Equipment & consumables
Instruments:
Incubator, carbon-free glassware and a flow cytometer equipped with a blue
laser (488 nm) and the appropriate filter sets.

Consumables:
Fluorochromes (e.g. SYBR GREEN I); Sterile syringe filters.

The method has been in use at Eawag since 2005, and is under
development/testing in the TECHNEAU project.

Evaluation:
The method is easy to use and robust. It requires a maximum of 3 days to
perform, and the FCM enumeration allows for large amount of samples to be
processed at little cost of time/labour. The natural microbial community
erases the limitation that might go along with a pure culture. A drawback is
the relative expensive nature of the instrumentation (flow cytometry).


Monitoring technology nr 5: The Eawag-AOC assay


References

Hammes F.A., Egli T. (2005) New method for assimilable organic carbon
determination using flow-cytometric enumeration and a natural microbial
consortium as inoculum. Environ. Sci. Technol. 39: 3289-3294.

Vital, M., Fuchslin, H. P., Hammes, F. & Egli, T. (2007). Growth of Vibrio
cholerae O1 Ogawa Eltor in freshwater. Microbiol 153, 1993-2001.

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x
x

time to result

3 days
clean handling
clean handling

Costs
instrumentation (C) x Initial investment in a flow
cytometer can be high
consumables
maintenance

x

x


Overall conclusion The method is fast and allows processing of a large number
of samples. However, some degree of expertise in flow
cytometry is required, and investment costs are high.


4.34 DOC/TOC

Prepared by: S::can

TOC stands for the total organic carbon present in water. This is composed of
a variety of organic compounds in various oxidation states. TOC is a
convenient method for expression of the total organic content (rather then
parameters such as BOD, COD, AOC, which only provide information on a
specific part of the organics present). TOC is independent of the oxidation
state of the organic molecules and does not measure other organically bound
elements, such as nitrate and hydrogen, nor inorganic that can contribute to
e.g. COD. TOC does not replace the other parameters, such as COD and BOD,
as it produces a different type of information entirely.

DOC stands for Dissolved Organic Carbon. It measures the same components
as TOC, but after filtration of the water through a 0.45 m pore size filter. As
such it represents the total amount of dissolved organic carbon in the water.
DOC concentrations are generally in the same range, but always lower than,
TOC.

Concentration ranges commonly encountered in
source water:
TOC 0.1 - 100 mg/L
drinking water
TOC 0.1 - 25 mg/L

Required sensitivity of the measurement:
source water: 1 mg/L
drinking water: 0.1 mg/L

1. High temperature combustion
2. Persulfate oxidation
3. UV oxidation
4 UV-Spectroscopy


4.34.1 Monitoring technology nr 1: High temperature combustion

Description:
To determine the quantity of organically bound carbon, the organic molecules
are broken down into a molecular form that can be measured quantitatively
(most often to CO2). With this method, a sample is homogenised and diluted.
Then a micro portion is injected in a heated reaction chamber packed with
oxidative catalyst, such as cobalt oxide. The water is vaporised and the
organic carbon is oxidised to CO2 and H2O. The CO2 from the oxidation is
transported in a carrier gas stream and measured by means of an infra red
analyser or titrated.

Common interferences are inorganic carbons, mainly from carbonate and
bicarbonate. This must be removed before analysis by acidification and
purging with purified gas. This however, can result in the loss of volatile
organic substances.

Equipment
- a TOC analyser using combustion techniques
- sampling, sample preparation and injection accessories
- blender
- magnetic stirrer
- filtering apparatus

The required analysers are available as laboratory equipment or as fully
automated analysers for on-site operation.

References
This technique is internationally accepted and certified (APHA Standard
methods for Water and Waste water analysis, 21st edition, DIN EN 1484, ISO
8254 und EPA 415.1)

Equipment is available from e.g.: LAR Process Analysers AG (online),
Analytik Jena (lab), Shimadzu (lab), Thermo Electron (lab), Star instruments
(lab).

Evaluation:
This technique is a reliable and reproducible one, but the apparatus and
sample preparation required mean it is only useful in the laboratory and not
in field application (combustion temperatures of 900 C are used.

Its only benefit over the lower temperature oxidation procedures is that it
does not need strongly oxidising chemicals. However, this technique is less
and less often used because of the extreme conditions required during
combustion.


Monitoring technology nr 1: High temperature combustion

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x
x

time to result

x
ease-of-use (B) x

Costs
consumables
maintenance

x
x


Overall conclusion Reliable method. Only suited for well equipped lab, due to
sample preparation. Suited for regulatory monitoring, not
for process control.

4.34.2 Monitoring technology nr 2: Persulfate oxidation

Description:
To determine the quantity of organically bound carbon, the organic molecules
are broken down into CO2 that can be measured quantitatively. The
oxidation is performed using persulfate which is activated either thermally or
by UV light.
After oxidation, the produced CO2 is purged from the sample, dried and
transferred into a carrier gas. It can then by measured by IR analysis,
coulorimetric titration or conductivity in ultra pure water.

Heated instruments use a persulfate solution of around 95 C.

Common interferences are inorganic carbons, mainly from carbonate and
bicarbonate. This must be removed before analysis by acidification and
purging with purified gas. This however, can result in the loss of volatile
organic substances.


- a TOC analyser using persulfate principle
- sampling, sample preparation and injection accessories
- reagents (various persulfate and peroxydisulfate solutions can be used)
- filtering apparatus

The required analysers are available as laboratory equipment or as fully
automated analysers for on-site operation.

References
methods for Water and Waste water analysis, 21st edition, DIN EN 1484, ISO
8245, ASTM D4839, ASTM D 4779, USEPA 9060, USP 26.

Equipment is available from e.g.: Sievers (lab), Endress and Hauser (online),
Hach Lange (online), Mettler Toledo, WTW (online), WTW (cuvette test,
which requires thermal reactor as well) Thermo Scientific (lab), Applikon
(online), Analytik Jena (lab).

Evaluation:
his technique is a reliable and reproducible one, but the apparatus and
sample preparation required mean it is mainly useful in the laboratory. In
field applications the automated analyser have proven to be very
maintenance intensive, with pumps, reagents and tubings requiring frequent
cleaning and / or replacement. Up-time of these analysers is often between
50 - 90 %.


Monitoring technology nr 2: Persulfate oxidation
sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

for online use, as lab technique it
functions without problems
time to result

x approx 15 minutes for online
instruments
ease-of-use (B) x
maintenance requirements (C) x in case of online operation

Costs
instrumentation (C) x unit price between 5000 and
20000 Euro
consumables
maintenance

x

x

Recommendation for use in SSS (D) x needs small lab

Overall conclusion Reliable mehod in laboratory. Field use less practical due to
complexity of analysers which require frequent
maintenance.

4.34.3 Monitoring technology nr 3: UV - oxidation / conductivity

Description:
Technique uses relatively weak oxidising power to destroy larger organic
molecules. The content of the total organics is measured by the conductivity
of the water. This method is only suitable for ultra pure water, where
conductivity is naturally very low and the amount of organics is very low.

Equipment
Required equipment
- a TOC analyser

Used in semi conductor and other clean water industries. Often used as TOC
indicator for Deionised Water units.

References
Equipment is available from e.g.: Sievers, Mettler Toledo


Evaluation:
This technique is not suitable for source water or drinking water monitoring.

Monitoring technology nr 3: UV-oxidation / conductivity

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

time to result

x
ease-of-use (B) x

Costs
consumables
maintenance

x

x


Overall conclusion Not suited because of background signal in water too high.

4.34.4 Monitoring technology nr 4: UV-spectroscopy

Description:
Organic matter absorbs light in the UV and Visible range. Although not all
substances absorb light strongly, 20 - 50 % of the substances in TOC are
normally visible without need for preconcentration. It is possible to use the
correlation of the absorption spectrum of this fraction to monitor TOC. This
requires analysis of the spectrum, as well as a small number ( 2 - 5) of
reference analyses using classical TOC analysis (method 1 or 2).

This method is a more advanced version of the single wavelength UV254
method (see separate datasheet). Because a full spectral measurement is used,
cross sensitivities are much reduced and TOC and DOC can be measured
with a single instrument.

Common interferences: a change in the composition of the TOC which results
in a significant change in the shape of the absorption spectrum can lead to
loss in accuracy. This can be corrected by calibration.


Equipment
- UV/Vis spectrophotometer

Reference
This technique has been described in numerous scientific papers.

The required equipment is either a normal laboratory spectrophotometer (e.g.
Shimadzu, Hitachi) or an online instrument (s::can, Stip).

Evaluation:
This is a very efficient technique for process control and monitoring. The high
measurement frequency allows online and real time analysis. As it is a
surrogate parameter, its accuracy relies on relatively stable correlations with
the total TOC. This correlation should be verified at the start of any
application, as well as with regular intervals. However, the low maintenance
requirements of the online instruments means that for process control they
are ideal.

Monitoring technology nr 4: UV-spectroscopy

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

time to result

x
ease-of-use (B) x

Costs
instrumentation (C) x starting at 8000 Euro
consumables
maintenance

x
x


Overall conclusion A surrogate method that depends on calibration at the start
of operation. After calibration, is it fast, stable, easy to use
and cost efficient.


4.35 UV absorbing organic constituents

Prepared by: S::can

Some organic commonly found in water and waste water, such as humic
substances and aromatic compounds, strongly absorb ultraviolet radiation.
UV absorption is a useful surrogate parameter to measure selected organic
constituents in water. Strong correlations may exist between UV absorption
and organic carbon components, colour and precursors for trihalomethanes
and other disinfection by-products.
UV absorbance is measured in absorbance units or absorbance units per
meter. The advantage of using the latter, is the fact that it is standardised to
one optical cell length. Because different spectrometers use different cell
lengths, only this makes possible intercomparision of results.

Concentration ranges commonly encountered in
source water:
Abs: 0 100 m
-1
drinking water
Abs: 0 50 m
-1

Required sensitivity of the measurement:
source water & drinking water: 0.1 Abs/m

1. Single wavelength absorption measurement
2. Full spectral analysis

4.35.1 Monitoring technology nr 1: Single Wavelength absorption measurement

Description:
Using a spectrometer UV-absorption is measured at one wavelength. This
wavelength is classically 254 nm, because the lamps used in these instruments
emit light at this wavelength. It is not by definition the best wavelength, but
suffices in many applications.

UV-absorbance at one wavelength generally provides a rough indication of
the organics present in the water. It has however extremely high cross
sensitivity, as it can not distinguish between organic materials and other
substances and it can not distinguish between different organic substances,
turbidity and suspended particles also cause absorption.

A more advanced measurement uses a second wavelength to assess turbidity
and compensates based on this. This improves the relationship between
dissolved substances and UV254 but it is still highly unselective.

Equipment


A single or dual wavelength spectrometer

Reference:
methods for Water and Waste water analysis, 21st edition).

Equipment is available from e.g.: Endress+Hauser, HachLange, s::can, WTW,
Stip, Trios, ABB.

Evaluation:
This technique is simple and can be effective. However, its extremely low
selectivity means it is only suitable for crude process monitoring.

Monitoring technology nr 1: Single Wavelength

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

time to result

x
ease-of-use (B) x

Costs
instrumentation (C) x approx 1000 2000 Euro
consumables
maintenance

x
x


Overall conclusion Simple instrument, very unspecific, only for process control.

4.35.2 Monitoring technology nr 2: Full spectral analysis

Description:
Using a spectrometer the full UV-absorption spectrum of the water is
measured. This allows using of the shape of the spectrum as well as the
absorption values themselves. This combination provides a much better
correlation with organic materials than the single wavelength method
described under 1. The shape information makes it possible to classify TOC,


distinguish between TOC from different sources and can even be used to
measure single substances and perform a highly efficient turbidity
compensation, so that dissolved substances only can be measured without the
need for filtration.

Cross sensitivities: calibration against TOC for accurate readings is often
required. If the composition of the mixture of organic substances changes
after calibration, this can result in loss of accuracy. This will be accompanied
by a shape change in the spectrum, which can be automatically detected and
thus provide warning that the instrument is less accurate.

The shape change is also used as a direct indicator for water quality changes.

Equipment
Required equipment is a full spectrum spectrometer.

Reference
This technique has been described in numerous scientific papers and is
succesfully applied by many waterworks.
Equipment is available from e.g.: s::can, Stip, Trios.

Evaluation:
This technique uses robust equipment to obtain a high amount of information
(TOC, DOC, turbidity, Nitrate, others) from a single measurement that needs
no pre-treatment. Although the TOC and DOC parameters are surrogate
parameters, local calibration means they can be used very efficiently for
process monitoring and control.

The instruments are highly robust, need no consumables and can be installed
in-situ (submersible instruments).


Monitoring technology nr 2: Full spectral analysis

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

time to result

x < 1 minute
ease-of-use (B) x

Costs
instrumentation (C) x unit price between 8000 and
20000 Euro
consumables
maintenance

x
x

none


Overall conclusion Reliable method for process control. Not very cheap, but
provides multiple parameters simultaneously making it
competitive when more than one parameter is required.


4.36 Particle counts

Prepared by: RTU

Concentration range that should be covered is 10-1000 particles/mL.
Sensitivity/resolution is a minimum of 50 particles/mL.

1. Particle counting systems

4.36.1 Monitoring technology nr 1: Particle counting systems
A particle counter is an instrument that detects and counts particles. Optical
particle counters detect particles and measure their size by using the
interaction of particles with a light beam. They can count and size particles
(dust, particulate contamination, or dirt) in air, other gases, or liquids. Liquid
Particle Counters are used to determine the quality of the liquid passing
through them. The size and number of particles can determine if the liquid is
clean enough to be used for the designed application. Liquid particle counters
can be used to test the quality of drinking water. Two methods are commonly
used for detecting and measuring particles in water; light extinction and light
scattering. In both methods, a change in light intensity measured by the
detector is converted to an electrical signal. Light extinction is useful for
particle 1 m and greater in size. In this method, the detector looks directly
into the light source and measures the size of the "shadow" of the particle as it
passes through the beam. To detect particles smaller than 1 m, light
scattering is used. In a scattering sensor, the detector does not look directly
into the light source, but views the beam from the side. It looks at the region
of the beam through which the particles pass. When the particle passes
through the beam, it scatters light, which is measured by the detector.

All particle counting systems have three parts: the sensor, counter, and
sampler. The sensor is the most important part of the system. It contains a
light source, optics, a detector, and a flow channel that directs the sample into
the light beam. With newer instruments, the light source is a laser or laser
diode. The sensor determines the particle sizes that can be measured. Sensors
are available which can measure particles as small as 0.05 m and as large as
2500 m (2.5 mm). The range over which a sensor will measure depends on
its optical design. The counter analyzes the electrical signals from the sensor.
The counter also allows the user to define the operating parameters (for
example, the particle sizes of interest and sample time) and it outputs the
data. Today, nearly all counters contain a microprocessor, providing the user
with lots of versatility in operating particle counter and reporting the results.
The sampler delivers the sample to the sensor at a known, controlled flow
rate. Samplers may also provide other features, such as volume measurement,
flow rate monitoring, stirring, and vacuum degassing (required for some
samples, since bubbles look like particles to the sensor).


Fig.1. An overview on a particle counter

Types of Particle Counting Systems
The sensor, counter, and sampler can be implemented in many different ways
to measure the product or process. Different applications require different
designs of the components. Furthermore, the particle counting system can be
designed for use in the laboratory, in the field, or on-line at the point of
interest. Liquid samples are collected in containers, then brought to the
laboratory for analysis. (There are no laboratory systems for particle counters
for air or gases.) Theses samples are often called "batch" or "grab" samples.
Laboratory systems allow you to analyze a wide variety of samples from
many different sites. As an example, in the pharmaceutical industry, R & D
and Quality Control could use the same instrument on a wide variety of
samples. Portable particle counters, as the name implies, can be taken around
a plant or to different sites to measure the level of contamination directly at
the location of interest. This method eliminates some problems that can be
introduced when taking batch samples (such as dirty sample containers). It
also provides immediate results, which can be used to assess the
contamination level and, if necessary, to take redial action quickly. For critical
processes, dedicated particle counters can be installed to monitor the
contamination level either continuously or on a regular schedule. On-line
systems have all the advantages of the portable systems, with the further
benefit that increases in the particulate level can be detected and responded to
very quickly.
The system requires maintenance expenses and needs to be calibrated one or
twice a year (according to the instructions of manufacturer).

Potable water is an industry that has been using particle counters
sporadically for many years, but has only recently seen a wide acceptance of
their use. In USA the particle counters received increased attention following
the Milwaukee outbreak in 1993 where over 100 people died as a result of
Cryptosporidium infection. In fact, in 1996 US Governement brought an
antitrust case (http://www.usdoj.gov/atr/cases/f0700/0722.htm) against the
merger of Pacific Scientific and Met One, former competitors in particle


counter sales. COUNCIL DIRECTIVE 98/83/EC of 3 November 1998
(UNION, 1998)does not specify particle count for drinking water but only
turbidity.

References
Pacific Scientific (http://www.particle.com/) is perhaps a market leader in
particle counters. Particle Measuring Systems
(http://www.pmeasuring.com/aux/about) and CCsTec
(http://www.ccstec.at/en/index.htm) also provide an extensive range of
counters. Particle Measuring Systems offer a possibility to rent or lease a
counter.

1. The Council of the European Union, 1998. COUNCIL DIRECTIVE
98/83/EC of 3 November 1998 on the quality of water intended for human
consumption. In Official Journal of the European Communities L 330/32,
5.12.98.

Evaluation:
Particle counters extend the sensitivity of particle detection beyond that
achievable with turbidimeters. Water filtration technologies available today
can produce almost particle free water, but current pathogens, like
Cryptosporidium, can avoid disinfection and infect a host with a single
organism. Therefore, single particle detection is critical, and indeed
mandated, for some technologies. The sensitivity of the particle counter can
be utilized to detect the effects on effluent quality due to operational
procedures, chemical dosage and type, and parametric changes. As a result,
simple and affordable means of filtration enhancement can often be evaluated
for their effectiveness before considering complicated and expensive ones.
With increasing requirements to remove pathogens, such as Giardia and
Cryptosporidium, the need to optimize processes to remove particles has
become obvious. Particle counters reveal minute changes in water quality that
can identify opportunities to modify operations or design for particle
removal. Particle counting provides an important tool for assessing source
water quality, finished water quality, unit process performance, and total
treatment efficiency. Plant influent and filter effluent are the most important
sampling locations for assessing plant performance.
Particle counters are best suited to monitoring water with turbidity less than 7
to 10 ntu. Sampling locations with high particle concentrations tend to require
frequent maintenance and cleaning. Particle counts in excess of the
instrument concentration limit will introduce coincidence errors into the
results. Because both particle number and size can be used in optimizing
drinking water treatment, the most useful information available from many
sequential particle count analyses includes the cumulative total number of
particles, the differential number of particles, and percentile statistics.
Increasing concentrations of particles are generally associated with increasing
concentrations of Cryptosporidium and Giardia.
Care must be taken when using the particle counter on raw water because
clogging and biological growth can cause major functional problems at the
instrument level. This limits the applications of particle counters in this
industry. Attempts to place particle counters upstream of the final filter
effluent have proven to be problematic. In fact, several manufacturers


discourage the use of particle counters on settling basins or raw water unless
the operators are willing to support high maintenance costs. Cost of
ownership, maintenance, and the generation of large volumes of data limit
the application of particle counters in the drinking water industry. Particle
counters are initially more expensive to purchase than turbidimeters and
often require additional accessories to run the instrumentation. Maintenance
is another issue; particle counters are difficult instruments to calibrate on-site.
If on-site calibration is performed, the cost is high. Calibration verification
methods have been developed but often are seen as difficult and not highly
accurate. The WTP operator may lack confidence that the results from a
particle counter are indeed accurate. In summary, when particle counters are
properly used on filter effluent water, they can be very effective. Many water
plants have adopted this technology and have optimized their treatment
processes; claiming production results that could not be accomplished using
only traditional turbidimetric techniques. To successfully use particle
counters, operators must adhere to proper setup, calibration, and
maintenance.

Monitoring technology nr 1: Particle counting systems

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

time to result

x
ease-of-use (B) x

Costs
consumables
maintenance

x


purchase and maintain. Skilled personnel is also required.


4.37 Oxygen

Prepared by: S::can

Oxygen dissolved in water can be present between 0 - 20 mg/L. The
concentration is often expressed as saturation in %, between 0 and 200%.
The measuring range is identical for source water and for finished drinking
water.

1. Titration
2. Electrochemical measurement
3. Optical measurement

4.37.1 Monitoring technology nr 1: Titration

Description:
Various titration methods for dissolved oxygen (DO) are described. The most
commonly used one is the iodometric titration. It is the most precise and
reliable titrimetric procedure for DO analysis. It is based on the addition of a
divalent manganse solution, followed by strong alkali, to the sample in a
closed glass bottle. The oxygen rapidly oxidises an equivalent amount of the
manganese, upon which manganese hydroxide precipitates. In the presence
of iodide in acidic solution, the manganese is reduced back to its original
divalent state, liberation iodine equivalent to the original DO content. The
iodine is then titrated with a standard solution of thiosulfate, with the
endpoint of the titration being determined visually. The liberated iodine can
also be analysed using a spectrophotometer, providing reliable results in the
microgram per liter range, providing no interferences from particulate matter,
colour or chemical interferences are present.

For this analysis are required (azide modification):
- manganese sulphate solution
- alkali iodide azide reagent
- sulfuric acid
- starch
- thiosulphate titrant
- potassium bi-iodate solution
- glassware for titration

This technique is a standard method for DO measurement (described in
APHA Standard Methods for the examination of water and waste water (21st
edition)).

Costs
The materials and chemicals required for this analysis can be obtained from
any chemicals supplier and lab instrumentation supplier. Total costs for first


experiment should not exceed 200 Euros. Costs per analysis are in the range
of < 10 Euro.

Evaluation:
This method is well proven and accurate in determining DO content of a
sample. However, as DO is not a constant but very susceptible to change, the
need to perform this analysis in a laboratory is a big drawback. Even with
sample preservation, DO is not very suitable for laboratory analysis.
Furthermore, a skilled technician is required to perform this analysis reliably.
The laborious nature of this test means that it is no longer used widely,
because more rapid and simpler technique with comparable accuracy are
now available (electrochemical methods, see point 2).

Monitoring technology nr 1: Titration

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

time to result

x
ease-of-use (B) x

Costs
consumables
maintenance

x

x

Recommendation for use in SSS (D) x needs a least a simple laboratory

Overall conclusion Suitable method for reference/regulatory measurements in
the laboratory.

4.37.2 Monitoring technology nr 2: Electrochemical Measurement

Description:
Membrane covered electrode minimises the problems of the titration method
with regards to applicability in online and field applications. The sensing
element is an electrode covered with an oxygen permeable membrane. This


serves as a barrier against impurities. Under steady state conditions, the
current in the electrode is directly proportional to the DO concentration.

Two different types are available: galvanic (passive, oxygen is not consumed)
and poloragraphic (active, oxygen is consumed and sample needs to be
refreshed after measurement).

Replacement of membranes, recalibration as well as substantial temperature
effects, that has to be compensated for, on the measurement are the main
drawbacks of this method.

For this analysis are required:
- oxygen sensitive electrode
- data logger
- sodium sulphite for referencing of the instrument
- replacement membranes and electrolyte

This type of instrument can be obtained from a great number of suppliers.
Versions exist for laboratory use only (simply put in sample and ready or real
classical electrodes) to handheld systems to robust instruments for
continuous in-situ measurement.

Costs
Electrodes only are available for 100 - 500 Euro, whereas total equipment can
be obtained for 1000 - 3000 Euro.

References
This technique is a standard method for DO measurement (described in
APHA Standard Methods for the examination of water and waste water (21st
edition)).

Manufacturers are for example: WTW, Jumo, Hach Lange, YSI, Swan, Mettler
Toledo, Thermo Electron, Palintest, Zllig,

Evaluation:
This method is well proven and accurate in determining DO content of a
sample. However, stability of the sensor can be problematic, hence frequent
calibration is required. This limits the robustness of the technique for online
insitu applications. Also, water flow is required for proper functioning of
these electrodes.


Monitoring technology nr 2: Electrochemical measurement

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

time to result

x continuous or in minutes
(polarographic measurement)
ease-of-use (B) x

Costs
consumables
maintenance

x
x

needs replacement membranes
and frequent calibration

Recommendation for use in SSS (D) x needs a least a simple laboratory


4.37.3 Monitoring technology nr 3: Optical Measurement

Description:
The sensing element in optical DO probes consists of a fluorescing compound
suspended stably in a robust optical window or membrane. A light source
at controlled wavelength briefly pulses the optical window. This causes
excitation of the fluorescent material, causing it to emit a specific wavelength
of light. The intensity of this fluorescence or light is determined by the
amount of dissolved oxygen in the water that is in contact with the optical
window. An optical sensor measures the emitted fluorescence, and from the
light intensity the concentration is calculated.

Alternatively, luminescence instead of fluorescence is used for DO
measurement. In this case not the intensity of fluorescence is measured but
the lifetime of the fluorescence. Otherwise the principle and equipment are
similar.

Equipment
- optical DO probe


This technique is relatively new. It is being offered by many manufacturers
already, as ease of use is much better than for electrochemical analysis.
However, it is not yet included in standards.

This type of instrument can be obtained from a great number of suppliers.
Versions exist for field use (dipping probe) or for continuous insitu use
(submersible probes).

Costs
The probes for only use can be obtained for 1500 - 2500 Euro. Price differences
are caused for example by internal temperature and pressure compensation
sensors.

References
Manufacturers are for example: Endress + Hauser, YSI, s::can, Hach Lange,
Zebra Tech Ltd, In-Situ Inc., WTW,

Evaluation:
This method is now well proven and accurate in determining DO content of
water. It is the most simple and easy to use method of the three described
here, and ideally suited for online measurements.

Independent studies, for example by the US Geological Survey (USGS) have
proven that optical DO probes are less prone to fouling and sensor drift than
electrochemical instruments.

The fluorescence lifetime probes usually require membrane replacement only
once a year, whereas the fluorescence probes often need no membrane
replacement during the lifetime of the sensor.


Monitoring technology nr 3: Optical Measurement

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x
x

time to result

x result every few seconds
ease-of-use (B) x

Costs
consumables
maintenance

x
x

cleaning needed, depending on
the water quality.

Recommendation for use in SSS (D) x simple to use, robust. If this
parameter needs to be measured,
this is the best method.

Overall conclusion Reliable, relatively inexpensive measurement. well suited
for online monitoring purposes.


Annex I. Individual evaluation forms for
endocrine disrupting effects

Monitoring technology nr 1: ER CALUx

Description:
The ER CALUx bioassay (Estrogen Receptor mediated Chemically Activated
Luciferase gene expression) is developed by Legler and co-workers in the
Netherlands (Legler et al., 1999). The bioassay utilizes T47D human breast
cancer cells expressing endogenous ER (ER and ER). The cells have been
stably transfected with pERE-tata-Luc, a pGL3 based estrogen responsive
luciferase reporter gene under control of three Estrogen Responsive Elements.
Recently, two bioassays have been developed allowing for the detection of
the activation of specifically ER or ER (Sonneveld et al., 2005). These ER
CALUx and ER CALUx utilize U2-OS human osteoblastic osteocarcoma
cells that do not endogenously estrogen receptors.

Evaluation:
The ER CALUx bioassay has been validated for the detection of low
concentrations of estrogens in both drinking water and surface water. The
bioassay is very sensitive and the response of the bioassay is very
reproducible. The ER CALUx bioassay has been used extensively by research
groups in the Netherlands in several projects regarding the quantification of
estrogenic compounds in the Dutch aquatic environment (e.g Vethaak et al.,
2005; CSIRO, 2007); Vethaak, 2002). In a recent comparison by the GWRC, the
ER CALUx performed best overall, being one of the most sensitive bioassays
and with the lowest average coefficient of variation (Leusch, 2008). Other
mammalian cell line based bioassays were shown to be equally or less
sensitive, have higher coefficients of variation or take longer to perform. The
ER CALUx is marketed by BioDetection Systems B.V., the Netherlands.
Training in analysis and service are provided. ER CALUx analyses are
performed by BDS under ISO 17025.


Monitoring technology nr 1: ER CALUx

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

time to result

x
ease-of-use (B) x Training and technical support
by supplier

Costs
consumables
maintenance

x
x


Overall conclusion Suitable. Among the most sensitive and reproducible
bioassays.


Description: & evaluation

Monitoring technology nr 2: MVLN and MELN

Description:
Several bioassays expressing luciferase have been developed using the MCF-7
human breast cancer cell line that expresses endogenous ER. Of these cell
lines, the MVLN and the MELN have been used most frequently the
determination of estrogens in aqatic matrices. The MVLN (MCF-7-p-Vit-tk-
Luc-Neo) bioassay utilizes cells that have been transfected with a luciferase
gene under control of an ERE derived from the xenopus vitellogenin (Pons et
al., 1990; Demirpence et al., 1993). The time of exposure for the MVLN
bioassay is 48 hours (Gutendorf and Westendorf, 2001). The MELN (MCF-7-
ERE-Glob-Luc-Neo) uses a stably transfected plasmid with the luciferase
gene driven by an ERE in front of the -globulin promoter (Balaguer et al.,
1999).

Evaluation:
Both cell lines have been used for the detection of estrogens in a variety of
aquatic matrices in a variety of studies. However, both cell lines seem to be
less sensitive than other mammalian cell line based bioassays (Kinnberg,
2003; Leusch, 2008). In a recent comparison by the GWRC, the MELN also
showed higher coefficients of variation (Leusch, 2008). The same study also
showed that accurate quantification of the estrogenic activity in water extracts
was problematic for the MELN, possibly due to matrix interference.


Monitoring technology nr 2: MVLN and MELN

sensitivity (A)
source water
drinking water

x
x

Less sensitive than comparable
methods
robustness (A)
selectivity

x
x

x

Matrix interference in water
extracts
time to result

x x 48 hours exposure for MVLN
ease-of-use (B) No information
maintenance requirements (C) No information

Costs
consumables
maintenance

x
x


Overall conclusion Unknown



Monitoring technology nr 3: T47D-KBluc

Description:
The T47D-Kbluc bioassay makes use of human breast cancer cell line T47D
that endogenously expresses ER and ER. These cells have been are stably
transfected with a 3x ERE-tata-lnr-luc-neo construct (Wilson et al., 2004).

Evaluation:
The T47D-Kbluc bioassay has only been described for estrogen analysis in
water in a recent comparison study (Leusch, 2008). In that study, the bioassay
showed lower responses (in fold inductions) and was slightly less sensitive
than the T47D based ER CALUx.

Monitoring technology nr 3: T47D-KBluc

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

No information
time to result

x

Costs
consumables
maintenance

x
x





Monitoring technology nr 4: YES

Description:
The Yeast Estrogen Screen (YES) (Routledge and Sumpter, 1999) utilizes
Saccharomyces cerevisiae yeast cells that have been stably transfected with
human ER and a plasmid containing EREs and the lacZ gene as a reporter
gene coding for the enzyme -galactosidase. The response is mesured by
adding the yellow substrate chlorophenol red--D-galactopyranoside
(CPRG), which changes the colour to red which can be quantified
spectrophotometrically at 540 nm.

Evaluation:
The YES bioassay is by far the most widely used yeast-based reporter gene
assay and has been used extensively to assess estrogenic activity of both
compounds and environmental extracts by research groups world wide.
However, the YES bioassay is less sensitive than comparable mammalian
assays (Murk et al., 2002; Leusch, 2008) and the thick cell wall may impede
active and passive transport of chemicals to the intracellular space, increasing
the risk of false negatives (Legler et al., 2002). Yeast cells are also not always
capable of detecting anti-estrogenic activity (Kinnberg, 2003). Adaptations
have been made to account for this problem (Gaido et al., 1997). However, the
resulting yeast bioassays were shown to be more sensitive to toxic
compounds, growth inhibition and matrix effects (Saito et al., 2002) and are
therefore not suitable for water quality monitoring. Recently, a yeast based
bioassay has been developed expressing Green Fluorescent Protein as a
reporter (Bovee et al., 2004). This bioassay has not been applied to
environmental monitoring and is less sensitive than mammalian cell based
bioassays.


Monitoring technology nr 4: YES

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

Less sensitive, thus more sample
needs to be added -> matrix
effects
time to result

x x Exposure takes 1-3 days
ease-of-use (B) x

Costs
consumables
maintenance

x
x


Overall conclusion Not suitable due to low sensitivity



Monitoring technology nr 5: E-Screen

Description:
The E-Screen bioassay (Soto et al., 1992; Soto et al., 1995) utilizes the estrogen
responsive growth of the MCF-7 cell line. The E-Screen is based on the
premises that factors in the serum inhibit cell growth and that estrogens
(when added as compound or extract) negate this inhibitory effect. Because
the bioassay relies on the quantification of growth, the bioassay takes more
time than reporter gene assays, having times of exposure of 4-6 days. The
amount of cells is determined by counting nuclei (Soto et al., 1992; Soto et al.,
1998) or by using a colorimetric end point (Krner et al., 2001).

Evaluation:
The E-Screen bioassay has been widely used in studies analyzing estrogenic
activity of both pure compounds and extracts. The bioassay is approximately
as sensitive as reporter gene bioassays based on mammalian cells. However,
research has shown that proliferation is not induced by estrogens exclusively,
but could also be effected by a range of mitogens, cytokines, growth factors,
nutrients and hormones other than estrogens (Kinnberg, 2003), leading to
false positives. Because of the relatively long time of exposure, the E-Screen is
more time-consuming compared to other estrogen responsive bioassays,
making it less suitable for routine analysis (Leusch, 2008; Kinnberg, 2003).


Monitoring technology nr 5: E-Screen

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

Does not respond to estrogens
exclusively
time to result

x Exposure takes several days
ease-of-use (B) x

Costs
consumables
maintenance

x
x


Overall conclusion Not suitable due to long exposure time



Monitoring technology nr 6: AR CALUx

Description:
The AR CALUx (Sonneveld et al., 2005) utilizes U2-OS human osteoblastic
osteocarcoma cells that do not endogenously express the androgen receptors.
The cells have been stably transfected to express human AR and luciferase
under control of three HREs coupled to the minimal TATA-promoter.

Evaluation:
The AR CALUx has been shown to responds to androgens exclusively and
has recently been applied for water quality monitoring (Van der Linden et al.,
2008). The AR CALUx is part of a U2-OS based bioassay panel responding to
different nuclear receptor ligands. The cell line is marketed by BioDetection
Systems, the Netherlands.

Monitoring technology nr 6: AR CALUx

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

time to result

x
by supplier

Costs
consumables
maintenance

x
x


Overall conclusion Suitable



Monitoring technology nr 7: MDA-kb2

Description:
The MDA-kb2 bioassay (Wilson et al., 2002) utilizes stably transfected MDA-
MB-453 breast cancer cells. These cells endogenously express high levels of
AR, while ER is only expressed at very low levels and ER and progesterone
receptor (PR) are not detectable at the mRNA level. This cell line does
however also constitutively express the glucocorticoid receptor (GR). The
cells have been transfected with an androgen-responsive luciferase reporter
plasmid driven by the mouse mammary tumour virus promoter (MMTV).

Evaluation:
The MDA-kb2 bioassay is approximately equally sensitive as the AR CALUx.
However, since in addition to AR also GR (and PR) have high affinity for, and
can activate the MMTV promoter, the MDA-kb2 cell line responds to both
androgens and glucocorticoids. The specificity (whether AR or GR) of the
signal can be determined by additional tests, existing of co-administrating a
known androgen receptor antagonist, e.g. hydroxyflutamide. However, since
also antagonists are not exclusively selective, additional testing makes it
difficult to quantify the response. This relatively unspecific response of the
cells is an important drawback when testing complex mixtures like
environmental extracts.



sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

No information
Also respond to glucocorticoids
time to result

x

Costs
consumables
maintenance

x
x


Overall conclusion Not suitable due to low selectivity



Monitoring technology nr 8: PALM

Description:
The PALM (PC3-Androgen-receptor-Luciferase-MMTV) cell line (Trouanne
et al., 2000) is derived from the AR-deficient human prostate adenocarcinoma
PC3 cell line, stably transfected with hAR and an MMTV-driven luciferase
reporter gene. The time of exposure is 30 hours.

Evaluation:
The PALM bioassay has been applied in detecting androgens in
environmental samples (Balaguer et al., 2000). However, the PALM cells are
reported in metabolize testosterone, possibly due to the relatively high
expression of 17b-hydroxysteroid dehydrogenase which is present in wild
type PC-3 cells (Trouanne et al., 2000; Castagnetta et al., 1997). This may be
result in underestimation of the androgenic activity when testing complex
mixtures containing natural androgens like testosterone.

Monitoring technology nr 8: PALM

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x
x

Risk of metabolizing compounds
of interest
time to result

x

Costs
consumables
maintenance

x
x





Monitoring technology nr 9: YAS

Description:
The YAS bioassay is based on the same principle as the YES bioassay. It
utilizes the yeast strain PGKhAR that containes a gene for the human
androgen receptor (hAR), which is constitutively expressed (Sohoni and
Sumpter, 1998). The yeast cells express the lacZ gene, encoding for the
enzyme -galactosidase. The amount of -galactosidase can be quantified by
adding the yellow substrate chlorophenol red--D-galactopyranoside (CPRG)
which is converted by the enzyme to a red end product that can be quantified
spectrophotometrically at 540 nm. A correction for cell concentration is
sometimes including by measuring the cell density at the end of the
experiment at 620 nm (Urbatzka et al., 2007). Similar bioassays have been
developed expressing either also expressing lacZ (Gaido et al., 1997) or other
reporters like luciferase (Michelini et al., 2005) or Green Fluorescent Protein
(Bovee et al., 2007).

Evaluation:
The yeast based YAS bioassay has been applied in the detection of androgens
in environmental monitoring (e.g. Thomas et al., 2002). However, yeast cells
are less sensitive to androgens than mammalian cell line based bioassays,
limiting their use for monitoring androgenic activity in surface water and
drinking water.


Monitoring technology nr 9: YAS

sensitivity (A)
source water
drinking water

x

x

robustness (A)
selectivity

x
x

time to result

x

Costs
consumables
maintenance

x
x





Monitoring technology nr 10: A-Screen

Description:
The A-Screen bioassay utilizes MCF-7-AR1 cells (Szelei et al., 1997), wild type
MCF-7 cells that have been stably transfected with the human androgen
receptor. These cells express approximately five times more AR than wild
type MCF-7 cells. The A-Screen is based on the principles that sex steroid
control proliferation is a combined effect of two different pathways:
proliferative response and inhibition of cell proliferation. Proliferation is
induced by exposing the cells to known concentrations of estrogen.

Evaluation:
The hAR transfected cells were shown to have an inhibited proliferation
when exposed to a combination of estrogens and androgens; this inhibitory
effect was reversed by adding anti-androgens indicating that the inhibitory
effect was indeed caused by androgens. However, since a combination is
needed of estrogens and androgens, measuring complex environmental
extracts containing both estrogens and androgens might result in false
positives and/or negatives. The A-Screen was also shown to respond non-
specifically to the glucocorticoid hydrocortisone. The time of exposure in the
A-Screen (5 days) is comparable to that of the E-Screen, which is relatively
time-consuming compared to other androgen responsive bioassays, making it
less suitable for routine analysis.


Monitoring technology nr 10: A-Screen

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x
Responds to estrogens,
androgens and glucocorticoids.
Unknown performance for
complex environmental mixtures
time to result

x Exposure takes several days

Costs
consumables
maintenance

x
x


Overall conclusion Not suitable due to long exposure time. Selectivity
unknown



Monitoring technology nr 11: PR CALUx

Description:
The PR CALUx (Sonneveld et al., 2005) utilizes U2-OS human osteoblastic
osteocarcoma cells that do not endogenously express the progesterone
receptors. The cells have been stably transfected to express hPR and luciferase
under control of three HREs coupled to the minimal TATA-promoter.

Evaluation:
The PR CALUx was shown to respond exclusively to progestins, making the
bioassay suitable for the specific detection of this group of compounds. The
bioassay has recently been applied for water monitoring (Van der Linden et
al., 2008). No comparative studies have been published for bioassays
responding the progestins. The PR CALUx is part of a U2-OS based bioassay
panel responding to different nuclear receptor ligands.

Monitoring technology nr 11: PR CALUx

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

time to result

x
by supplier

Costs
consumables
maintenance

x
x





Monitoring technology nr 12: TM-Luc

Description:
The TM-luc bioassay utilizes T47D cells that have been stably transfected with
the reporter plasmid MMTV-Luc (Willemsen et al., 2004). The T47D cells
endogenously express PR, which in combination with the presence of PR
ligands leads to the production of luciferase which can easily be quantified
using a luminometer.

Evaluation:
The cell line is sensitive to progestins and has been utilized in the detection of
progestins in a variety of matrices. However, the cell line does not respond to
progestins exclusively, but is shown to respond to certain androgens
(Willemsen et al., 2004). The bioassay is therefore not suitable for the specific
detection of progestins in water and surface water.

Monitoring technology nr 12: TM-Luc

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

No information
Also responds to androgens
time to result

x

Costs
consumables
maintenance

x
x





Monitoring technology nr 13: Yeast based reporter gene assays

Description:
This assay utilizes Saccharomyces cerevisiae strain YPH500, which is transfected
with a human PR expression plasmid and a reporter plasmid carrying the
lacZ gene under control of two PREs (Gaido et al., 1997). When compounds
bind to the PR the enzyme -galactosidase is produced, which can be
quantified by adding the substrate 2-nitrophenyl- -D-galactosidase (ONPG).
The formation of the yellow orthonitrophenol can be quantified
spectrophotometrically at 420 nm. Similar assays have been developed (Wang
et al., 2005; Li et al., 2006), sometimes using GFP as a reporter (Chatterjee et al.,
2008).

Evaluation:
The yeast bioassay provides a rapid analysis of progesterone-like activity in a
variety of matrices. However, due to the limited sensitivity of yeast cell,
combined with the risk of false negatives, the yeast based reporter gene assay
is less suitable for the detection of low concentrations of progestins.

Monitoring technology nr 13: Yeast based reporter gene assay

sensitivity (A)
source water
drinking water

x

x

robustness (A)
selectivity

x

No information

time to result

x

Costs
consumables
maintenance

x
x





Monitoring technology nr 14: GR CALUx

Description:
The GR CALUx (Sonneveld et al., 2005) utilizes U2-OS human osteoblastic
osteocarcoma cells that do not endogenously express significant levels of
glucocorticoid receptors. The cells have been stably transfected to express
human GR and luciferase under control of three HREs coupled to the
minimal TATA-promoter.

Evaluation:
The GR CALUx bioassay is shown to respond specifically to glucocorticoids
and has recently been applied in water quality monitoring (Van der Linden et
al., 2008). The GR CALUx is part of a U2-OS based bioassay panel responding
to different nuclear receptor ligands.

Monitoring technology nr 14: GR CALUx

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

time to result

x
by supplier

Costs
consumables
maintenance

x
x





Monitoring technology nr 15: TGRM-Luc

Description:
The TGRM-Luc bioassay utilizes T47D cells that have been stably transfected
with the reporter plasmid MMTV-Luc (Willemsen et al., 2004). T47D cells
endogenously express a non-functional GR. Therefore, the cell line was
cotransfected with the RS-hGR expression vector coding for the human
glucocorticoid receptor.

Evaluation:
The TGRM-Luc bioassay utilizes T47D cells that endogenously express ER,
PR and GR. Because of the non-specific MMTV-Luc reporter construct used,
the TGRM-Luc bioassay does not respond to glucocorticoids exclusively, but
also to progestins and androgens. It is therefore not suitable as a selective
screening tool for the detection of glucocorticoids in surface water and
drinking water.

Monitoring technology nr 15: TGRM-Luc

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

No information
Also responds to progestins
time to result

x

Costs
consumables
maintenance

x
x






Description:
The MDA-kb2 bioassay (Wilson et al., 2002) utilizes stably transfected MDA-
MB-453 breast cancer cells. These cells endogenously express high levels of
AR and GR, while ER is only expressed at very low levels and ER and
progesterone receptor (PR) are not detectable at the mRNA level. The cells
have been transfected with an MMTV-Luc reporter gene.

Evaluation:
The sensitivity of MDA-kb2 is in the same range as other mammalian cell line
based bioassays. However, since in addition to GR also AR (and PR) have
high affinity for, and can activate the MMTV promoter, the MDA-kb2 cell line
responds to both glucocorticoids and androgens. The specificity (whether AR
or GR) of the signal can be determined by additional tests, existing of co-
administrating a known androgen receptor antagonist, e.g.
hydroxyflutamide. However, since also antagonists are not exclusively
selective, additional testing makes it difficult to quantify the response. This
relatively unspecific response of the cells is an important drawback when
testing complex mixtures like environmental extracts.



sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

No information
Also responds to androgens
time to result

x

Costs
consumables
maintenance

x
x





Monitoring technology nr 17: TR CALUx

Description:
The TR CALUx (Sonneveld et al., unpublished) utilizes U2-OS human
osteoblastic osteocarcoma cells that do not endogenously express the thyroid
receptors. The cells have been stably transfected to express human TR and
luciferase under control of three TREs coupled to the minimal TATA-
promoter.

Evaluation:
The TR CALUx bioassay responds specifically to compounds that bind to
the TR receptor. It is part of a U2-OS based bioassay panel responding to
different nuclear receptor ligands. However, since the bioassay has been
developed very recently, no information is available concerning
environmental monitoring.

Monitoring technology nr 17: TR CALUx

sensitivity (A)
source water
drinking water

No information
No information
robustness (A)
selectivity

x

Not yet established fully
time to result

x
by supplier

Costs
consumables
maintenance

x
x





Monitoring technology nr 18: T-Screen

Description:
The T-Screen bioassay is based on the GH3 rat pituitary somatotroph cell line
(Gutleb et al., 2005). This cell, expressing TR1, TR2 and TR1, shows
thyroid hormone dependent proliferation when seeded at low density.
Because the bioassay relies on the quantification of growth, the bioassay takes
more time than reporter gene assays, having a time of exposure of 4 days. The
amount of cells is determined by fluorometric or spectrophotometric
methods.

Evaluation:
The T-Screen bioassay has been applied for the detection of thyroid-like
compounds in environmental matrices (Gutleb et al., 2005) and is shown to
sensitively respond to thyroid hormones. However, since this bioassay like
E-Screen and A-Screen - relies on proliferation, it takes relatively much time
compared to the other (reporter gene) thyroid hormone bioassays, and is
likely to be prone to non-specific influences on proliferation.

Monitoring technology nr 18: T-Screen

sensitivity (A)
source water
drinking water

No information
No information
robustness (A)
selectivity

No information
No information
time to result

x Time of exposure is 4 days

Costs
consumables
maintenance

x
x





Monitoring technology nr 19: PC-DR-LUC

Description:
The PC-DR-LUC bioassay utilizes the rat pheochromocytoma cell line PC12
(Jugan et al., 2007). These cells, previously engineered to express TR1 of
avian origin, have been stably transfected with a pGL3-DR4 reporter plasmid.
The bioassay was shown to respond to various iodothyronines.

Evaluation:
The bioassay is shown to be sensitive to thyroid hormones and can be applied
for the detection of compounds that bind to the TR1 receptor. However,
since the bioassay has been developed very recently, no information is
available concerning environmental monitoring.

Monitoring technology nr 19: PC-DR-LUC

sensitivity (A)
source water
drinking water

No information
No information
robustness (A)
selectivity

No information
No information
time to result


Costs
consumables
maintenance

x
x





Monitoring technology nr 20: xL58-TRE-Luc

Description:
This bioassay is based on a cell line of xenopus laevis, a water-living amphibian
(Sugiyama et al, 2005). The cells, endogenously expressing TR, are stably
transfected with a plasmid containing the firefly luciferase gene under control
of the SV40 promoter and x. laeves TREs.

Evaluation:
The bioassay is shown to be sensitive to thyroid hormones and can be applied
for the detection of compounds that bind to the TR receptor. It has recently
been applied in environmental monitoring (Murata and Yamauchi, 2008).
Note however, it utilizes not a mammalian cell line but an amphibian cell
line.

Monitoring technology nr 20: xL58-TRE-Luc

sensitivity (A)
source water
drinking water

No information
No information
robustness (A)
selectivity

No information
No information
time to result


Costs
consumables
maintenance

x
x




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Annex II. Individual evaluation form for
genotoxic effects
Monitoring technology nr 1: Ames test

Description:
The Ames test is a bacterial reverse mutation test, utilizing Salmonella
typhimurium bacteria that harbour a mutation which makes them unable to
make histidine (Ames et al., 1975). Therefore, these mutants cannot grow in
medium without histidine. When a genotoxic compound or extract is added,
mutations can occur that reverse the original mutation, enabling the bacteria
to grow on medium without histidine. This genotoxic potential is related to
growth, which can be quantified by determining the number of colonies
formed. Based on the amount of colonies, the mutagenicity of the water is
assessed to be low, moderate, high and extreme for samples for <500, 5002500,
25005000 and >5000 revertants/L respectively. Beside Salmonella also
Escheria coli bacteria can be used for bacterial reverse mutation assays. Several
strains of S. typhimurium are used, allowing for the detection of a variety of
base pair substitutions or frameshift mutations either at AT sites (e.g. TA104)
or at GC sites (TA98 and TA100).

The classical Ames test is performed in Petri dishes and consists of counting
the number of colonies after 2-3 days of exposure. An important
improvement of the Ames test is the Ames II (Flckiger-Isler et al., 2004). This
bioassay, using 96 well plates, utilizes a pH indicator (bromocresol) in the
medium to monitor the pH change caused by growing bacteria. As an end-
point, not the number of colonies is determined but the colour change in the
well is assessed. Hence the assay is also known as the MutaChromo or Ames
fluctuation test.

Evaluation:
The Ames test is a widely recognized and utilized test that allows for the
rapid detection of mutagens. The test is standardized for water quality
monitoring at the international level (ISO 16240). Modified versions have
been described that are more sensitive (Reifferscheid and Grummt, 2000).
Both TA98 and TA100 strains are the most frequently used strains for
monitoring the amount of mutagenicity of surface water, although generally
the TA98 strain is found to be more sensitive than the TA100 strain (Ohe et
al., 2004; Umbuzeiro et al., 2001). Various other strains are used in water
monitoring, overexpressing nitroreductase or O-acetyltransferase. These
strains can aid in the detection of nitroarenes and aromatic amines, since
Salmonella strains are usually very efficient at nitroreduction, possibly
resulting in overestimation of the genotoxic potential (Ohe et al., 2004;
Cobisier et al., 2001).

The Ames test takes relatively long to perform and is therefore difficult to
incorporate into standard monitoring processes. Compared to other bacterial
assays like the Umu and SOS chromo test, the Ames test is less sensitive,
making it less suitable for the screening of possibly low concentrations of


genotoxins in surface and drinking water (Reifferscheid and Grummt, 2000;
Cobisier et al., 2001; Czyz et al., 2002). Like most bacteria based tests, the
Ames test sensitive enough for testing pure compounds at high
concentrations, but shows low specificity, resulting in a relatively large
number of false positives (Kirkland et al., 2002). The Ames II is equally
sensitive and can be performed in 96 well plates, allowing high throughput
screening and automation. The Ames II test has shown to perform well by
different laboratories.

Monitoring technology nr 1: Ames test

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x
x

time to result

x
ease-of-use (B) x Pathogenic bacteria

Costs
consumables
maintenance


Overall conclusion



Monitoring technology nr 2: Vibrio harveyi

Description:
The principle of the Vibrio harveyi test is similar to the Ames test, but in stead
utilizes V. harveyi bacteria that are sensitive to the antibiotic neomycin
(Wegrzyn et al., 2003). Genotoxic agents can make these bacteria resistant to
neomycin due to induced mutations in the neo-gene. This genotoxic potential
is related to growth, which can be quantified by determining the number of
colonies formed. The Vibrio harveyi bacteria are more robust than e.g. S.
typhimurium and E. coli. generally utilized, making it possible to detect
genotoxicity in water samples without extracting the water samples.

Evaluation:
The Vibrio harveyi test has been applied for the detection of genotoxins in a
variety of aquatic matrices. The bacteria in this test are more robust than the
Salmonella bacteria utilized in the Ames and Umu test, which can be an
advantage when testing unextracted water samples (Ohe et al., 2004; Czyz et
al., 2002). Recently, an adapted version of the bioassay has been developed
and applied in water monitoring (Podgrska et al., 2007).

Monitoring technology nr 2: Vibrio harveyi

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

No information
No information
time to result

x

Costs
consumables
maintenance

Unknown


Overall conclusion



Monitoring technology nr 3: Comet assay

Description:
The Comet assay (also known as single cell gel electrophoresis) was
introduced as a microelectrophoretic method to visualize structural DNA
damage in individual cells (stling and Johanson, 1984). This method can be
applied to any mammalian cell type and involves lysis of the cells followed
by gel electrophoresis of the lysate. Chromosome breaks shorten the
chromosome pieces, and smaller pieces move faster through the agarose gel
towards the anode. These shorter pieces thus form a tail, extending the cell
nucleus, giving the cell the appearance of a comet. Quantification of the
clastogenicity can be performed in several ways, determining e.g. the length
of the comet, the percentage of DNA in the tail or the product of the previous
two (called the tail moment). The original method applies neutral pH, so that
only double-strand breaks can be detected. However, the method has been
adapted to detect single-strand breaks and alkali label sites by performing the
method at high pH (pH >13) (Singh et al., 1988). Recent adaptation made it
possible to perform the comet in a 96 well format, which, combined with
image analysis software, enables automation of the method (Kiskinis et al.,
2002).

Evaluation:
The Comet assay is sensitive and relatively fast test that can performed with
any mammalian cell type, both from in vivo and in vitro studies. The assay can
be performed in 96 well plates, allowing automated high throughput
screening (Kiskinis et al., 2002; Witte et al., 2007). In a recent comparison of
genotoxicity assays, the Comet assay was less strongly recommended than
other assays for the analysis of water samples, mainly due to the variability in
the cell to cell response (Corbisier et al., 2001). However, the assay has been
applied frequently for water quality monitoring. If the Comet assay is to be
used routinely, standardization and inter-laboratory calibration will be
required (Frenzilli et al., 2008). In general, the Comet assay is more
complicated to perform than bacterial mutation and reporter gene assays and
requires more expensive equipment, but in return reflects genetic endpoints
that cannot be assessed using bacterial tests.


Monitoring technology nr 3: Comet assay

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

No information
time to result

x

Costs
consumables
maintenance

Unknown


Overall conclusion



Monitoring technology nr 4: Micronucleus assay

Description:
In the micronucleus assay, mammalian cells are made to undergo a division
step. This division step is stopped right after the cell nuclei have divided into
two separate daughter nuclei but before the cells have divided (i.e.
binucleated cells). When chromosomal breaks are present, the chromosome
fragments are not transported to the daughter cells but rather will form a
separate, small nucleus called a micronucleus. The presence of micronuclei in
binucleated cells indicates that the original cell had structural chromosomal
damage, caused by the presence of DNA damaging agents.

Evaluation:
The micronucleus test has been standardized for water quality monitoring at
the international level using amphibian larvae (ISO 21427-1) and V79 cells
(ISO 21427-2). A 96 well format of the assay, utilizing a mouse lymphoma cell
line, has been described by Nesslany and Marzin (1999). The read-out of the
method is mostly performed using a microscope, making the method labour-
intensive. However, recent advances in image analysis and flowcytometric
methods have made automated analysis possible (Hayashi et al., 2007; Diaz et
al., 2007). Exposure times for in vitro analysis of micronuclei vary
considerably, ranging from 3 hours to several days (Diaz et al., 2007; Kirsch-
Volders et al., 2003).


Monitoring technology nr 4: Micronucleus assay

sensitivity (A)
source water
drinking water

No information
No information
robustness (A)
selectivity

No information
No information
time to result

x x Varies between several hours to
several days

Costs
instrumentation (C) x Depends on level of automation
consumables
maintenance


Overall conclusion



Monitoring technology nr 5: Alkaline elution assay

Description:
The alkaline elution assay detects single- and double-strand breaks in DNA
and alkali-labile sites (Kohn and Ewig, 1973). For this assay, cells are exposed
to the compound or extract to be test for 3-10 hours. After exposure, cells are
lysed on a 2.0 m filter after which an alkaline buffer is added and different
subsequent fractions are eluted and collected. The amount of DNA in each
fraction and in the filter residue is quantified using a fluorescent label. The
idea behind the assay is that DNA strand breaks lead to smaller pieces of
DNA (the same principle that applied to the Comet assay), which will go
through the filter more easily. Exposure of cells to DNA damaging chemicals
will lead to a lower amount of DNA left in the residue than normal. By
comparing exposed to non-exposed cells, the difference in amount of DNA is
a measure of clastegenicity (Storer et al., 1996).

Evaluation:
The alkaline elution assay can detect strand breaks but like all other strand
breaks assays - cannot distinguish between direct genotoxic insult and
cytotoxicity-mediated DNA degradation (Elia et al., 1993). The assay as
originally described by Kohn and Ewig (1973) takes approximately 3 days to
perform and is rather labour-intensive. Recently, an automated, high-
throughput version of the assay utilizing rat hepatocytes has been described
(Gaely et al., 2007). This assay utilizes the 96 well plate format and has a
reduced analysis time of 1.5 days. However, the automated assay has not
been applied for water monitoring and custom made equipment is needed.


Monitoring technology nr 5: Alkaline elution assay

sensitivity (A)
source water
drinking water

No information
No information
robustness (A)
selectivity

x

No information
Both genotoxicity and
cytotoxicity
time to result

x x

Costs
instrumentation (C) x Custom made for automated
version
consumables
maintenance


Overall conclusion



Monitoring technology nr 6: Polymerase inhibition assay

Description:
The polymerase inhibition assay is based on the fact that DNA damage blocks
DNA polymerases during the polymerase chain reaction (PCR). DNA
exposed to genotoxicants is therefore less amplified than non-exposed DNA,
which can be measured by intensity analysis (Jenkins et al., 2000). This assay
allows for the detection of general DNA damage, in the form of gene
mutations or chromosomal aberration.

Evaluation:
The polymerase inhibition assay is fast and can be used for high throughput
screening, although samples cannot be processed in parallel. However, it is
not widely used and needs to be validated before it can be used as a full
genotoxicity screen.

Monitoring technology nr 6: Polymerase inhibition assay

sensitivity (A)
source water
drinking water

No information
No information
robustness (A)
selectivity

No information
No information
time to result

x x Depending on the amount of
samples

Costs
instrumentation (C) x PCR needed
consumables
maintenance

No information


Overall conclusion



Monitoring technology nr 7: UMU test

Description:
The UMU test utilizes genetically modified Salmonella typhimurium bacteria
posessing an elevated O-acetyltransferase level. These bacteria are transfected
with a plasmid vector (pACYC184) in order to express an introduced lacZ
gene under control of the umuC gene, one of the SOS functions induced by
genotoxins (Oda et al., 1985). When these bacteria are exposed to genotoxins,
the SOS repair system is activated, including the lacZ gene, leading to the
production of -galactosidase. The amount of -galactosidase can be
quantified by adding the appropriate substrate which is converted to a
coloured product that can be measured spectrophotometrically. Several
adaptations exist of this bioassay, utilizing e.g. more sensitive strains (Oda et
al., 1995), bacteria expressing human CYPs for the detection of promutagens
and procarcinogens (Aryal et al., 1999) or bacteria expressing luciferase
(Schmid et al., 1997) or GFP (Arai et al., 2001) in stead of -galactosidase

Evaluation:
The UMU test is easy and relatively quick to perform and has been used for
extracts of a variety of environmental matrices. The assay is applicable in 96
well plates, making it more suitable for automation. Compounds not
inducing the SOS system will not be detected. The UMU test has been
standardized in Germany by DIN (DIN 38415T3) and at the international
level by ISO (ISO 13829). UMU tests expressing luciferase or GFP are
generally more sensitive than UMU tests expressing -galactosidase (Arai et
al., 2001; Schmid et al., 1997).


Monitoring technology nr 7: UMU test

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x
x

x
Only detects SOS activating
damage
time to result

x
ease-of-use (B) x

Costs
instrumentation (C) x spectrophotometer
consumables
maintenance


Overall conclusion



Monitoring technology nr 8: SOS Chromotest

Description:
The SOS Chromotest is based E. coli bacteria in which the activation of the
bacterial SOS repair system (specifically the sfiA gene) is coupled to the
production of -galactosidase (Quillardet et al., 1982. When these bacteria are
exposed to genotoxins, the SOS repair system is activated, including the lacZ
gene, leading to the production of -galactosidase. The amount of -
galactosidase can be quantified by adding the appropriate substrate which is
converted to a coloured product that can be measured
spectrophotometrically.

Evaluation:
The SOS Chromotest test is easy and relatively quick to perform and can be
obtained as a kit (EBPI, Canada). The assay is applicable in 96 well plates,
making it more suitable for automation. Compounds not inducing the SOS
system will not be detected. The assay has been applied regularly in water
quality monitoring. In a recent study the SOS Chromotest was shown to be
more sensitive than Ames and Mutatox (Guzzella et al., 2004) and Vitotox
(Cobisier et al., 2001).

Monitoring technology nr 8: SOS Chromotest

sensitivity (A)
source water
drinking water

No information
No information
robustness (A)
selectivity

time to result

x

Costs
consumables
maintenance

No information


Overall conclusion



Monitoring technology nr 9: Vitotox

Description:
The Vitotox
assay utilizes S. typhimurium bacteria that have been genetically

modified to express a Luc-gene coupled to the RecN gene of the SOS repair
system (van der Lelie et al., 1997). DNA damage leads to the production of
luciferase which can be quantified by adding the substrate luciferin and
determine the amount of light produced using a luminometer.

Evaluation:
The Vitotox assay is easy and relatively quick to perform and is developed
and marketed by Labsystems/Thermo (USA). The assay is applicable in 96
well plates, making it suitable for automation. Compounds not inducing the
SOS system will not be detected. In a recent comparison study, the Vitotox
assay was shown to perform similarly to the Umu and SOS-lux test (Cobisier
et al., 2001). The Vitotox assay was shown to respond differently to PAHs
than the SOS chromotest (van der Lelie et al., 1997), which can cause different
results between assays when analysing environmental samples.

Monitoring technology nr 9: Vitotox

sensitivity (A)
source water
drinking water

No information
No information
robustness (A)
selectivity

No information
No information
time to result

x

Costs
instrumentation (C) x Required luminometer
consumables
maintenance

No information


Overall conclusion



Monitoring technology nr 10: Mutatox

Description:
The Mutatox assay uses a special (not genetically engineered) dark strain
(M169) of the luminescent bacterium Vibrio fischeri (formerly Photobacterium
phosphoreum) (Kwan et al., 1990). When these bacteria are exposed to
mutagenic chemicals, they can restore the luminescence by activation of the
SOS repair system. Activation of this system leads to the formation of
protease, which brakes down a repressor protein of the lux pathway leading
to luminescence. Exposure times are generally 24 hours. The Vibrio harveyi
bacteria are more robust than e.g. S. typhimurium and E. coli. generally
utilized, making it possible to detect genotoxicity in water samples without
extracting the water samples.

Evaluation:
The Mutatox can be obtained as a complete kit, including analyser, media
and reagents (Microbics, AZUR Environmental, UK). The test has been
applied for the detection of genotoxicity in a variety of environmental
samples. The test has been used by different laboratories world wide and is
relatively quick and easy to perform. The bacteria in this test are more robust
than the Salmonella bacteria utilized in the Ames test when testing
unextracted water samples, which can be an advantage (Ohe et al., 2004).
However, in a recent comparison study, the Mutatox assay was not
recommended for the rapid analysis of genotoxicity of environmental
samples (Corbisier et al., 2001).


Monitoring technology nr 10: Mutatox

sensitivity (A)
source water
drinking water

x
x

robustness (A)
selectivity

x

x

time to result

x

Costs
instrumentation (C)
consumables
maintenance


Overall conclusion



Monitoring technology nr 11: GreenScreen
GC

Description:
The GreenScreen
GC utilizes yeast cells that are genetically modified to

contain a gene for the green fluorescent protein (GFP) under control of a
DNA repair system (RAD54) promoter (Walmsley et al., 1997; Cahill et al.,
2004). This repair system is part of recombinational repair pathway, which
repairs double strand breaks and interstrand crosslinks. However, the system
is also induced by agents causing gene mutations. Exposure to genotoxic
agents leads to activation of the DNA repair system and to the production of
GFP which can be quantified using a fluorometer. By counting the cells after
exposure (through cell density), the assay serves simultaneously as a control
for cytotoxicity. A similar assay has been described utilizing a yeast strain
deleted in several multidrug transporters to enhance the sensitivity of the
assay (Lichtenberg-Frat et al., 2003).

Evaluation:
The GreenScreen
GC can be performed relatively quickly and can be used

for high throughput screening as it can be performed in 96 well plates and
determination of fluorescence can be automated. Because this assay uses
yeast cells, which are just as mammalian cells eukaryotic cells, it is claimed
that this assay performs better at detecting clastogenic agents than assays
based on bacteria. It has been used for environmental monitoring, including
surface waters and effluents and the results from a recent comparison study
are promising (Corbisier et al., 2001). However, the use of S9 - as well as the
presence of autofluorescent compounds - can possibly interfere with the
measurement, as S9 is fluorescent and therefore interferes with GFP analysis.

The GreenScreen
GC is marketed by Gentronix (UK), and an adaptation of

the test is available as GreenScreen
EM (environmental monitoring), which

consists of the assay supplied as a portable kit. The yeast assay described by
Lichtenberg-Frate et al. (2003) has not been utilized for water monitoring, but
showed a more sensitive response regarding model genotoxins like 4-NQO
and MNNG.


GC

sensitivity (A)
source water
drinking water

No information
No information
robustness (A)
selectivity

No information
Responds to fluorescence
time to result

x

Costs
instrumentation (C) x Requires fluorometer
consumables
maintenance


Overall conclusion



HC

Description:
The GreenScreen
HC is based on the same principle as the GreenScreen
GC
but in stead utilizes the human lymphoblastic TK6 cell line; hence the name
HC i.e. Human Cells. These cells produce Green Fluorescent Protein gene
under control of the GADD45 (Growth Arrest and DNA damage) gene
(Hastwell et al., 2006). The assay is performed in 96 well plates and utilizes
incubation times of 24-48 hours and claims better prediction for the human
situation regarding genotoxicity.

Evaluation:
The GreenScreen
HC bioassay is more sensitive than the GreenScreen
GC
test and bacterial based assays, while retaining the specificity of other
mammalian cell line based bioassays (Hastwell et al., 2006; Billtinton et al.,
2008). However, because S9 is fluorescent and can therefore interferes with
GFP analysis, metabolic activation using S9 mixtures - as well as the presence
of autofluorescent compounds - can be problematic. Recently, protocols have
been developed allowing the use of S9. The GreenScreen
HC assay has not

been used for environmental monitoring. The GreenScreen
HC is marketed
by Gentronix (UK). In a recent interlaboratory study, the assay was shown to
perform well at other laboratories (Billinton et al., 2008).


HC
sensitivity (A)
source water
drinking water

No information
No information
robustness (A)
selectivity

No information
No information
time to result

x

Costs
instrumentation (C) Requires fluorometer
consumables
maintenance


Overall conclusion



Monitoring technology nr 13: MCF-7-p53R2

Description:
This bioassay utilizes MCF-7 human breast cancer cells that are stably
transfected to express luciferase under control of the p53R2 gene (Ohno et al.,
2005). These cells express wild-type p53, which regulate the expression of
p53R2. The p53R2 gene is known to be activated by -ray and UV-irradiation
in a p53-dependent manner and to play a central role in cell survival,
repairing damaged DNA. The assay can be performed in 96 well plates and
includes an exposure time of 24 hours, allowing high throughput analysis.

Evaluation:
The MCF-7-p53R2 allows for the detection of general DNA damage, linked to
the expression of p53. The assay is relatively quick and easy to perform and
allows for high throughput screening. However, the test has not been applied
to extracts from environmental matrices.

Monitoring technology nr 13: MCF-7-p53R2

sensitivity (A)
source water
drinking water

No information
No information
robustness (A)
selectivity

No information
No information
time to result

x

Costs
instrumentation (C) x Requires luminometer
consumables
maintenance


Overall conclusion


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Monitoring and

and EcoScreen are marketed by

, IAC 1090 intelligent Aquatic

, CIFEC Truitel TruitoSEM, bbe Fish Toximeter, bbe

, BioTox, AbraTox Kit, LUMIStox; MicroLAN

is the mussel itself.

assay utilizes S. typhimurium bacteria that have been genetically

GC utilizes yeast cells that are genetically modified to

GC can be performed relatively quickly and can be used

GC is marketed by Gentronix (UK), and an adaptation of

EM (environmental monitoring), which

HC is based on the same principle as the GreenScreen

HC bioassay is more sensitive than the GreenScreen

HC assay has not

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