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7SSG/5035 Monitoring Environmental

Change

Student Number: 0813160

Word Count: 2,262

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Alternatives to Indophenol Blue for

ammonia measurement: modification of
OPA method and application to the Thames

Abstract

A variation of the OPA technique has been shown to give results that are not statistically
different than published methods while being more cost effective. This method has been
used to calculate the concentration of ammonia in a sample taken from the Thames.
Instrument error on the Aquafluor handheld fluorimeter has been quantified at 0.41% and
has been judged to be suitably low for most lab analyses of ammonia. A separate study to
find the dependence of fluorescence on time since the sampling showed a strong
degradation with time, levelling off after 24 hours. The maximum reading was two hours
after sampling and offers an avenue for further research.

Keywords

Ammonia measurement, ortho-phthaldialdehyde, fluorimetry, Thames

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1.0 Introduction

1.1 Ammonia impact and legislation

Ammonia is important in the aquatic environment as it can have significant impact on fish health,
causing both chronic and acute effects in fish and invertebrates. This is widely reported in the
literature (Arthur et al. (1987), Hazel et al. (1971) and Roseboom and Richey (1977), among others)
and has caused both the US Environmental Protection Agency (EPA) and the European Union to
legislate. The EU Freshwater Fish Directive (Directive 2006/44/EC) states that ammonia
concentration should be monitored on a monthly basis on all rivers containing freshwater fish. The
directive indicates that mean values of un-ionised NH3, the fraction of ammonia with the greatest
toxicity to fish (Thurston et al. 1981), must be below 0.004 mg l-1. Also, no more than 5% of values
recorded in a year can be above 0.0206 mg l-1.

Due to the negative impact on fish health, and the associated legal requirement, the accurate
measurement of ammonia is vital. The European directive, as well as the World Health Organisation,
WHO (1990), recommends either the Indophenol Blue (IPB) or Nessler method for the determination
of total ammonia concentration. It is also necessary to record temperature and pH to calculate the
un-ionised NH3 fraction (US EPA 1979) to ensure compliance with legislation.

1.2 Indophenol blue method

The IPB method involves treatment of a sample to form indophenol blue which can then be
measured via colourimetry (Chaykin 1969). It is possibly the most characterised and well used of the
methods, being widely reported with improvements and variations in the literature, as can be seen
in Table 1.

Table 1: Literature review of IPB method

Study Variation / Improvement described

Chaykin (1969) Effect of molecules competing with NH3
Patton and Crouch (1977) Catalysts and substituted phenol
Boo et al. (1976) Effect of metal ion contaminants
Ngo et al. (1982) Interference from amines and thiols
Harfmann and Crocuh (1988) Use of coupling reagents
Kempers and Kok (1989) Less toxic forms of phenol
Von Staden and Taljaard (1997) Accuracy of automatic sampling
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Although widely employed, the accuracy of the IPB has been called into question. A study by Aminot
et al. (1998) collated results of three concentrations of ammonia solution from 132 analytical
laboratories which were using variations on the IPB method. Figure 1 shows the results of this study.

Figure 1: Laboratory results for three levels of ammonia concentration. Taken from Aminot et al.
(1997).

The study showed that for the medium and high concentrations standard deviations between
laboratories were 22% and for the lower concentration 56%. This indicates that the IPB method has
weaknesses in repeatability, especially at low ammonia concentrations. This is supported by Genfa
and Dasgupta (1989) who report the lower detection limit of the IPB method to be 10 μg l-1.

1.3 Alternative Methods

A number of alternative methods exist for ammonia measurement including ion-selective

electrodes, described by Koryta (1986), and cathodic stripping voltametery, described by Harbin and
Van den Berg (1993).

A fluorometric method has been reported (Roth (1971), Genfa and Dasgupta (1989), Kerouel and
Aminot (1997), Holmes et al. (1999)) to have better sensitivity at lower concentrations than IPB.
Fluorimetry is generally preferred over colourimetry as an analytical technique as the measurement
is taken at 900 to the sample and therefore is not affected by the incident light (Turner Designs
2010).
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This method involves the treatment of a sample with ortho-phthaldialdehyde (OPA) in the presence
of a reducing agent to give an OPA-NH3 molecule which fluoresces. The method has been shown to
be up to 500x more kinetically favourable to NH3 than other nitrogen containing compounds,
significantly reducing sources of error (Genfa and Dasgupta 1989).

The reaction is reported to progress as in Eq. 1, modified from Zuman (2003).

Eq. 1

The advantages of the method reported by Holmes et al. (1999) include a large range of effective
concentrations, use of a working reagent (WR) that can be stored for months, lack of complicated
techniques employed and ability to easily account for background fluorescence (BF).

2.0 Methodology

2.1 Decon90 Investigation

The methodology employed by Holmes et al. (1999), protocol B, was followed except for one
modification. Holmes et al. rinse all sample bottles with 20ml of WR before samples are taken. As
the WR is relatively expensive a study was undertaken to see if this could be replaced by rinsing and
soaking overnight with Decon90, a commercially available laboratory cleaning agent.

Eight samples of ammonia and WR were made up to 0.7 mg l-1 concentration following Holmes et al.
and 8 samples were made up using the Decon90 method. These were then tested after being stored
for 2 hours on an AquaFluor fluorimeter. The resulting data was analysed statistically to test if there
was a significant difference in means between sample populations.

2.2 Calibration curve for protocol B and Wandle samples

The Decon90 method was then used to create a calibration curve of fluorescence against
concentration for the 420nm excitation channel. 10 samples were analysed between concentrations
of 0.033 and 0.625 mg l-1. Secondary data obtained from the Environment Agency was used to
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estimate the expected range of results. Each sample was analysed in triplicate and a mean taken to
reduce instrument error.

14 water samples from the river Wandle were obtained from Dr. Mike Chadwick and treated with
WR before analysis. Each sample was again run in triplicate and a mean taken. Three of the samples
were also treated with borate buffer (a component of the WR) and then analysed in triplicate to
calculate the background fluorescence (BF). The BF is due to any other components of a real water
sample which may fluoresce at 420nm which wouldn’t be accounted for by the calibration where
ultrapure de-ionised (DI) water is used.

Eq. 2 shows how the fluorescence due to ammonia is calculated using the samples fluorescence and
the measurement using borate buffer.

𝐹𝑆𝑎𝑚𝑝𝑙𝑒 𝑁𝐻3 = 𝐹𝑆𝑎𝑚𝑝𝑙𝑒 𝑜𝑏𝑠 − 𝐹𝑆𝑎𝑚𝑝𝑙𝑒 𝐵𝐹

Eq. 2

8 of these samples were diluted with DI as high values were expected as analysis reported
elsewhere had shown very high levels of phosphates.

2.3 Calibration Protocol A and Thames Sample

Figure 2.1 shows the method for making the WR, figure 2.2 shows the procedure for making
standards and figure 2.3 shows procedure for samples and BF. Standards and samples should start
the reaction with WR at the same time.

Figure 2.1: Method for WR.

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Figure 2.2: Procedure for standards.

Figure 2.3: Procedure for samples and BF.

Protocol A was employed from Holmes et al. using the Decon modification to create a second
calibration curve and then analyse a sample from the Thames. Figure 3 shows the sampling location.
The sample was taken at 12:10 on 30/11/10 at the RNLI Tower station, upstream from the boats to
ensure no direct contamination of hydrocarbons or similar.
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Figure 3: Marker A shows sampling location. Modified from Googlemaps (2010).

2.4 Sample Degradation

To test the effect of incubation of the samples between taking and treating with WR the sample
from the Thames was used and treated using Protocol B. This was then repeated at intervals with
the original sample (approximately 1l) being stored at room temperature.

3.0 Results

3.1 Decon90 investigation

The two samples were first tested for normality using The Shapiro-Wilk test (Shapiro and Wilk,
1965), then for similarity of variances via Levene’s F-test (Levene 1960). Finally a T-test (Student,
1908) was done to indicate any differences in means, the results are shown in Table 2.

Table 2: Statistical analysis on WR and Decon identical samples.

Test Test Score Significance

Shapiro-Wilk (WR) 0.929 0.509
Shapiro-Wilk (Decon90) 0.907 0.331
F-Test 0.916 0.456
t-Test -1.522 0.150 (two tailed)
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Figure 4 shows boxplots of the two samples.

Figure 4: Identical ammonia concentrations run with Decon90 method and Holmes et al. method.

3.2 Calibration curve Protocol B and Wandle samples

Figure 5 shows the calibration curve created for ammonia stock solutions. Below 0.1 mg l -1 a non-
linear response was seen so these have been discarded and 0.1 mg l -1 set as the lower detection
limit.
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Figure 5: Calibration curve for protocol B.

The acquired samples from the Wandle were analysed and the calculated BF subtracted, the results are
shown in Table 3 and 4.

Table 3: Analysis on undiluted samples

Sample Mean raw Fluorescence due to

fluorescence NH3 (BF subtracted)
2 67.89 52.93
3 71.64 56.68
3- spring 42.28 27.32
5 57.21 42.25
6 58.39 43.43
7 58.65 43.69
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Table 4: Analysis on diluted samples

Sample Mean raw Fluorescence due to

fluorescence NH3 (BF subtracted)
1 39.61 38.11
8 46.56 45.07
9 40.93 39.43
10 49.50 48.00
12 32.24 30.74
B 64.05 62.55
E 45.23 43.73
G 27.77 26.27

3.3 Calibration curve Protocol A and Thames sample

A second calibration curve was performed using Protocol A from Holmes et al. which is designed for
analysing dilute samples of ammonia. Figure 6 shows the resulting graph.

Figure 6: Calibration curve for protocol A.

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The resulting equation from this graph was then used to process the fluorescence of a sample taken from
the Thames and treated with the protocol A method. Table 5 gives the result of this.

Table 5: Thames water sample

Sample 𝑭𝑺𝒂𝒎𝒑𝒍𝒆 𝒐𝒃𝒔 𝑭𝑺𝒂𝒎𝒑𝒍𝒆 𝑩𝑭 𝑭𝑺𝒂𝒎𝒑𝒍𝒆 𝑵𝑯𝟑 Concentration / mg l-1
Lifeboat station 127.9 110.6 17.3 0.052

3.4 Sample Degradation

Figure 7 shows the results for the sample degradation tests.

Figure 7: Fluorescence degradation over time.

4.0 Discussion

Analysis on Holmes et al. versus the Decon method showed that the two samples were normally distributed
(Shapiro-Wilk test) and that they had similar variances (Levene’s test). It was therefore decided to perform a
two-sample t-test which assumed equal variances. This test showed (test score =-1.522 with a two-tailed
significance of 0.150) that the means of the two groups were not statistically different and therefore using
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the Decon method did not alter the results obtained. This provides justification of adopting the alternate
method which significantly reduces the cost of running a sample.

The creation of a calibration curve for protocol B only showed a linear response down to 0.1 mg l-1, which
was lower than reported in the literature. Holmes et al. use protocol B lower than this, however this is at the
lower end of the detection limit (0.1 µM) of the handheld fluorimeter used (Turner Designs 2010). This
model is a relatively inexpensive fluorimeter with variable repeatability and is only capable of measuring
fluorescence on two channels. Reported literature generally uses more accurate fluorimeters capable of
recording a spectrum however none of this type was available for this study. Greater accuracy could have
been achieved in the laboratory procedure by using graduated glass pipettes rather than auto-pipettes as
these can suffer from significant deviation from calibration.

Each sample run on the Fluorimeter was done in triplicate and a mean taken. This was to reduce instrument
error and also quantify this uncertainty. For the most part the instrument showed high repeatability but in
some cases the standard deviation was as high as 1.3% (mean value 0.41%) of the recorded value. This adds
to the uncertainty in the technique but it dwarfed by the size of possible error due to poor standard
preparation or storage of samples before treatment.

The samples obtained from Dr. Mike Chadwick were all below the lower detection limit of 0.1 mg l-1, when
analysed using protocol B. This was unexpected considering the Wandle is known to be a polluted stream
where sewage effluent can contribute up to 80% of discharge (Wandle Trust 2005). Interrogation of the
method used provides possible reasons for this. Holmes et al. recommend that samples are not filtered
before analysis. This is justified as Eaton and Grant (1979) note that ammonia from river samples can be
removed during the filtering process. The samples should also be treated with WR as soon as they are
collected whereas the samples obtained by Dr. Chadwick were not treated for approximately three days.
These two deviations from the published methodology are likely to account for the samples being below the
detection limit.

As no actual samples were within the detection limits a new sample was taken from the Thames using
protocol A and was treated with WR immediately without being filtered. After creating a calibration curve
from standard ammonia solutions it was possible to calculate the concentration of ammonia in the Thames
at the location shown in Figure 2. The calibration curve for protocol A showed linearity across the full range
and had a much higher r2 value, giving more confidence in these results.
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The analysis on sample degradation showed that the fluorescence decreases by around 13% after 24 hours
of storage and then levels off. This would account for the low values recorded for the Wandle samples. To
accurately quantify this source of error a more thorough study should be undertaken as the curve of
degradation of fluorescence was not linear. The fact that fluorescence actually increased after two hours of
storage is interesting in that it could provide a more accurate measurement when small concentrations of
ammonia and therefore low fluorescence readings are a problem. However, before a change to the
methodology is recommended to reflect this, the exact effect responsible for the increase would have to be
quantified.

5.0 Conclusions

This study has shown that replacing the WR wash recommended by Holmes et al. with a Decon90 rinse does
not produce statistically different results. This alternate method can therefore be adopted to reduce the cost
of analysing samples. Both protocols employed by Holmes et al. were tested and it was found that protocol
A gave a linear response over the entire range tested (whereas B was non-linear at low concentration) and
has an r2 value much greater than protocol B. To get more accurate results it is suggested to dilute samples
into the working range of protocol A.

It has become clear that prompt treatment of samples with WR is critical to achieving accurate results.
Samples stored for three days were unable to be analysed due to significant degradation causing an
artificially low fluorescence to be recorded. The peak fluorescence recorded was actually two hours after
collection of the sample. This provides an avenue for further research as this may offer a more accurate
point to take measurements.

The instrument error of the Aquafluor fluorimeter, at 0.41% of reading, has been shown to be small in
comparison to other possible sources of error and therefore acceptable for this level of lab analysis.

Overall the OPA method has proved to be cost effective and offers accuracy and simple operation with very
little equipment required. Further characterisation and improvements in the literature could improve the
technique to replace the widely adopted Indophenol Blue method.
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