September 2019 NATIONAL RESEARCH AND DEVELOPMENT INSTITUTE

FOR INDUSTRIAL ECOLOGY

NO. 1

EXCELLENCE IN RESEARCH AND ENVIRONMENTAL SERVICES

Romanian Journal of
Ecology & Environmental Chemistry

Pollution control and monitoring

Sustainable environmental technologies

Pollution assessment and management systems

 ROMANIAN JOURNAL OF ECOLOGY &
ENVIRONMENTAL CHEMISTRY

BUCHAREST, ROMANIA

SCIENTIFIC COMMITTEE

Gheorghe BATRINESCU Jean Louis MARTY

INCD ECOIND, Romania University of Perpignan via Domitia, France
Asher BRENNER Grigore MIHAESCU
Ben-Gurion University of the Negev (BGU), Israel University of Bucharest, Romania
Elena BUCUR Gheorghe NECHIFOR
INCD ECOIND, Romania Polytechnic University of Bucharest, Romania
Costel BUMBAC Mihai NITA-LAZAR
INCD ECOIND, Romania INCD ECOIND, Romania
Alina BUTU Ines NITOI
National R&D Institute for Biological Sciences, Romania INCD ECOIND, Romania
Mariana Carmen CHIFIRIUC Cristina ORBECI
University of Bucharest, Romania Polytechnic University of Bucharest, Romania
Lucian CONSTANTIN Luoana Florentina PASCU
INCD ECOIND, Romania INCD ECOIND, Romania
Liliana CRUCERU Jana PETRE
INCD ECOIND, Romania INCD ECOIND, Romania
Andrei Florin DANET Ovidiu POPA
University of Bucharest, Romania University of Agronomic Sciences and Veterinary Medicine
Laurentiu DINU of Bucharest, Romania
INCD ECOIND, Romania Elefteria PSILLAKIS
Timothy EHLINGER Technical University of Crete, Greece
University of Wisconsin – Milwaukee, U.S.A. Elena Cristina RADA
Toma GALAON University of Trento, Italy / Insubria University, Varese, Italy
INCD ECOIND, Romania Jaroslav SLOBODNIK
Liviu-Daniel GALATCHI Environmental Institute of Kos, Slovakia
Ovidius University of Constanta, Romania Jacobus (Koos) F van STADEN
Lucian Puiu GEORGESCU INCD EMC, Romania
“Dunarea de Jos” University of Galati Andrea-Cristina STAICU
Stefania GHEORGHE University of Bucharest, Romania
INCD ECOIND, Romania Miruna STAN
Eugen GHEORGIU University of Bucharest, Romania
International Centre of Biodynamics, Romania Michaela Dina STANESCU
Helmut KROISS Society of Chemistry, Bucharest, Romania
Vienna University of Technology, Institute for Water Daniela Simina STEFAN
Quality and Resource Management, Austria Polytechnic University of Bucharest, Romania
Lidia KIM Ivana TEODOROVIC
INCD ECOIND, Romania University of Novi Sad, Serbia
Stanisław KOTER Giuliano TEVI
Nicolaus Copernicus University in Toruń, Poland University of Ecology Bucharest, Romania
Carol Blaziu LEHR Gabriela Geanina VASILE
INCD ECOIND, Romania INCD ECOIND, Romania
Irina LUCACIU Caner ZANBAK
INCD ECOIND, Romania TCMA, Turkey
Tudor LUPASCU
Institute for Chemistry, Academy of Science Moldova
ROMANIAN JOURNAL OF ECOLOGY & ENVIRONMENTAL CHEMISTRY

Editors-in-Chief: dr. chem. Luoana Florentina PASCU

dr. eng. Carol Blaziu LEHR
Deputy Editor-in-Chief: dr. ecol. Catalina STOICA
Technical Editor: eng. Gheorghe-Cristian SERBANESCU

The issue is annually published.

Copyright ROMANIAN JOURNAL OF ECOLOGY & ENVIRONMENTAL CHEMISTRY

Address: 71-73 Drumul Podu Dambovitei, Bucharest 060652, ROMANIA,

Tel: +4.021.410.03.77 / Fax: +4.021.410.05.75

www.incdecoind.ro

ISSN 2668-5418, ISSN-L 2668-5418

Romanian Journal of Ecology &Environmental Chemistry, vol. 1, issue 1, 2019

Edited by EIKON
INTRODUCTORY WORDS

“The concerns regarding the sustainability of our influence on the environment are steadily
increasing even there is great progress in scientific understanding of human interference with nature. Also
technology development has made great progress in solving environmental problems. Environmental
Chemistry is a key discipline in both areas in science as well as in technology. In order to meet the
challenges coming from the UN Sustainable Development Goals we urgently need an improved insight
into the close relationship between human development and the natural environment which is becoming a
primary task of Ecology in the future. In fact, ecology is a very complex scientific area where many
scientists of different disciplines must work together in order to improve our understanding on how we will
have to change our behaviour in order to achieve the SDGs. Industry is playing a dominant role in two
ways: industry is one of the main users of natural resources and key player in materials transformation to
products and waste material which are released to the environment water, air and soil. At the same time
industry is still a major source of jobs and is markedly contributing to increasing our wellbeing, health
and life expectance.
The 17 goals are all interrelated and wherever quality criteria play an important role
Environmental Chemistry is one of the primary disciplines to quantify the actual flow of materials
through our society and allowing to measure the progress in the future. The new Journal has the goal to
enhance the international scientific knowledge transfer as well as to promote innovations contributing to
achieve the SDGs with a focus on industrial development based on sound science and proven technological
experience which both are also an important basis for political decisions.”
Em. O. prof. dr. Helmut KROISS
Vienna University of Technology

“I am pleased to hear about the creation of „Romanian Journal of Ecology & Environmental
Chemistry”. Unfortunately, the present development of our civilization is inevitably associated with the
production of goods (often trashy or useless). A side effect of such development is the devastation of the
natural environment including the chemical pollution, especially dangerous for living organisms. Thus the
setup of the new journal devoted to chemical issues of environmental protection should be considered a
highly desirable undertaking. I wish the Editorial Team of the „Romanian Journal of Ecology &
Environmental Chemistry” many significant articles helpful in solving ecological problems”.
Prof. dr. Stanislaw KOTER
Nicolaus Copernicus University in Toruń, Poland

“I am glad and honored to express my appreciation and support to INCD-ECOIND

Bucharest Romania, for launching the Romanian Journal of Ecology & Environmental Chemistry.
Following closely the annual conferences organized by INCD-ECOIND Bucharest, I have been
exposed to the advances in industrial ecology and environmental chemistry, presented by the participants.
The scientific and practical achievements of the Romanian scientists, demonstrate an impressive progress
in environmental science, and deserve international exposure in order to distribute the knowledge gained.
We all share the deep care for our planet, to prevent pollution and disease, and to preserve biodiversity.
The new Journal will serve as a significant means to disseminate national and international
accomplishments in these aspects, and thus will deeply contribute to science, ecology, and human welfare.”
Prof. Asher BRENNER
Ben-Gurion University of the Negev, Faculty of Engineering Sciences, Unit of Environmental
Engineering, Beer-Sheva, ISRAEL
 “The goal of this new journal is the promotion of research results both in the field of industrial
ecology and environmental chemistry. So it is largely open on very acute actual society problems such as
pollution control and monitoring, water use, energy consumption, and management of resources and
development of sustainable environmental technologies. In the field of analytical chemistry, it will be
certainly a catalyst both
- For the generalization of difficult analytical tasks: more analytes in difficult matrix with high
sensitivity
- For simplification of common analytical tasks: faster, continuous, more automatic and low costly
methods.
We hope it will be a good interface between research and transfer technology in order to give to the
industry very performing analytical devices used over long periods”.
Prof Jean Louis MARTY
University of Perpignan, France

“Environmental issues, including global warming, water, air and soil pollution, are major topics
for discussion both globally and locally. Throughout time there has been a stable balance in nature. There
is a harmony between biotic and abiotic factors in the environment. Since the 1950s as a result of
advances in industry, agriculture, and everyday life, increasing amounts of waste have accumulated in the
environment. Gases, liquids and solid waste, including domestic and waste water, pollute environment.
Where biodegradable chemical substances, from the use of polluting technologies for the production
of material goods, irrational use of pesticides and mineral fertilizers in the agrarian sector, household
waste etc. penetrate into the environment, natural balance is lost and this imbalance can have disastrous
effects on life on Earth.
Results of studies of processes and mechanisms of transformation, migration, chemical and
biochemical decomposition, immobilization, oxido-reduction, adsorption, etc. of organic and inorganic
pollutants entering environmental objects will be published in the new Journal of Ecology and
Environmental Chemistry founded by INCO-ECOIND and will form the basis for monitoring,
assessment and control of pollution of the environment, development of efficient chemical, biochemical and
biological technologies for environmental treatment. The journal will have a positive impact on the
ecological education of the population, and especially on the younger generation.”
Acad., Prof. Dr. Habilitat, Tudor LUPASCU
Institute for Chemistry, Academy of Science Moldova
 CONTENT

pag.

National R&D Institute for Industrial Ecology – ECOIND: “42 years of excellence in
environmental research and services”………………………………………………................... 1

Elena Bucur, Valeriu Danciulescu, Gheorghita Tanase, Carol Blaziu Lehr, Andrei
Vasile, Odour pollution assessment through indirect methods based on the monitoring of
technological parameters – case study………………………………………………………….. 4

Mirela Alina Constantin, Lucian Alexandru Constantin, Ines Nitoi, Ionut Cristea, Ellea
Boulac, Photo-degradation of 5-fluorouracil. Comparative study on the efficiency of
UV/H2O2, UV/TiO2, UV/H2O2/TiO2 processes………………………………………………… 11

Toma Galaon, Alina Banciu, Florentina Laura Chiriac, Mihai Nita-Lazar,

Biodegradation of antibiotics: the balance between good and bad…………………………...... 16

Stefania Gheorghe, Irina Lucaciu, Iuliana Paun, Gabriela Geanina Vasile, Jana Petre,
Vasile Ion Iancu, Catalina Stoica, Daniel Mitru, Mihai Nita-Lazar, Anthropogenic
pollutants: 10 years of progress in ecotoxicological studies and aquatic risk
assessment………………………………………………………………………………………. 26

Ines Nitoi, Lucian Alexandru Constantin, Ionut Cristea, Mirela Alina Constantin,
Advanced oxidation processes (AOPs) alternative methods for degradation of toxic pollutants
from wastewater………………………………………………………………………………… 39

Jana Petre, Vasile Ion Iancu, Toma Galaon, Marius Simion, Marcela Niculescu,
Gabriela Geanina Vasile, Luoana Florentina Pascu, Carol Blaziu Lehr, Liliana Cruceru,
Simona Calinescu, Gabriel-Lucian Radu, Occurrence of pharmaceutical and disinfectants
in the dissolved water phase of the Danube River and three major tributaries from
Romania………………………………………………………………………………………… 55

Bogdan Stanescu, Adriana Cuciureanu, Soil management in the urban environment: today
and future……………………………………………………………………………………….. 66

Catalina Stoica, Elena Stanescu, Iuliana Paun, Alina Banciu, Stefania Gheorghe, Irina
Lucaciu, Gabriela Geanina Vasile, Mihai Nita-Lazar, Danube Delta: monitoring and
ecological status. A link between the past and the future ……………………………………… 72
 National R&D Institute for Industrial Ecology – ECOIND: “42 years of excellence in
environmental research and services”
The institute was founded in 1977 under the
name of ICPEAR (Institute of Research and
Design for Wastewater Treatment) with
headquarter located in Bucharest, one subsidiary
in Timisoara and later in 1982 another
subsidiary was established at Ramnicu Valcea,
having as activity domain: technological
development and design of installations for
treatment of wastewater generated by chemical
and petrochemical industries.
Due to its unicity, obtained results,
multidisciplinary approaches and adequate
response to the environmental issues of
economic units, in 1999 ICPEAR was
transformed through accreditation into National
Research and Development Institute for
Industrial Ecology – NR&DI ECOIND (GD no.
261/1999 and Regulation of Organization and
Operation approved by GD no. 961/2005).
From its establishment (1977) until present days,
the institute continuously evolved from a
research institute dedicated to development of
technologies and design of installations for
treatment of wastewater generated by chemical
and petrochemical industries to a national
research and development institute, with o
global approach of environmental aspects in the
field of industrial ecology, well known at
national level and with a growing presence
within international partnerships.
ECOIND was reaccredited as national institute
in 2001, 2008 and 2012 by the Ministry of
Education and Research and by National
Agency for Scientific Research respectively.
Nowadays, ECOIND is operation under
coordination of Ministry of Research and
Innovation (GD 13/2017) and represents the
only institute at national level with a global
approach of environmental issues in the field
of industrial ecology.

Maintenance as national leader, recognised at international level in the

Vision field of environmental research and services, through quality and
performance

To respond through R&DI works to national / international tendencies

in the field of industrial ecology and to the needs of economic units on
Mission
the road to knowledge based sustainable development

Professional standards for environmental research and services

Capacity de perform and innovate
Fundamental Integrity and ethic in research
Values Commitment, responsibility, guarantees to the client
Sustainable management of resources
Respect to the law and environment
Respect to other research institutes, work, partners, clients, colleagues

With a specific focus on research – development Management System, is implemented within

– innovation mainly on applied research in the
field of environmental protection, industrial
pollution assessment and control, institute’s
activity is carried in accordance with a Quality –
Environmental Management Integrated
System, SR EN ISO 9001:2015 and SR EN ISO
14001:2015 (Certificate Q –
5364/17/26.05.2017 and E-5364/17/26.05.2017).
An Occupational Health and Safety
the institute, in accordance with SR ISO 45001:
2018.
Analytical tests laboratories within the
Department for Pollution Control (Laboratory
for Water, Soil, Waste Pollution Control,
Laboratory for Air Pollution Control, Biotests
Laboratory – Microbiological Analysis) and
Laboratories for Environmental Analyses within
Timisoara and Ramnicu Valcea subsidiaries are

Romanian Journal of Ecology & Environmental Chemistry ● No.1 ● 2019

www.incdecoind.ro
1
accredited by RENAR in accordance with SR
EN ISO/CEI 17025:2005 (Certificate of
accreditation no. LI 941/19.11.2018).
Laboratory for Air Pollution Control is
complying with the requirements of SR CEN/TS
15675:2009 for measurement of pollutants
emissions in air. ECOIND is diversifying testing
and analyses domains in that way to totally
respond to the needs of economic area in the
field of integrated prevention and control of
environmental media. The institute is certified /
accredited for:
- development of environmental documentation
(Certificate of registration position 79 in
National Register of environmental studies
elaborators issued by Ministry of Environment)
- development of technical documentation for
obtaining water management authorizations
(Certificate of Attestation no. 248/17.12.2018
issued by Ministry of Water and Forests);
- sampling and monitoring of drinking water
quality (Certificate no. 533/22.04.2019 issued by
Ministry of Health, General Direction for
Medical Assistance and Public Health).
Development of ECOIND infrastructure was
mainly done within two projects co-financed
by European Regional Development Fund via
Sectoral Operational Programme “Increase of
Economic Competitiveness”:
- SOP-IEC 115/2600 Development of
ECOIND research infrastructure for the
extension and diversification of researches in
the field of industrial ecology – INFRAECO
(Contract No.: 08 / 01-03-2009),
http://erris.gov.ro/Research-Center---
INFRAECO
The present institute headquarter
together with all needed infrastructure was
developed within the project in order to assure
conditions for research works at European level.
Moreover, the institute endowment was
completed with modern equipment, fact that
facilitated the diversification of environmental
research and services domains in the same time
with the modernization of existing laboratories
and development of new ones.
- SOP-IEC 1881/48693 Environment and
industry advanced interdisciplinary research
centre – MEDIND (Contract No.: 657/07-08-
2014), http://erris.gov.ro/Research-Center-
MEDIND
Romanian Journal of Ecology & Environmental Chemistry ● No.1 ● 2019
www.incdecoind.ro
Project objective was to modernise and
extend ECOIND infrastructure through creation
of a new Advanced Interdisciplinary Research
Centre in the aim to develop new research
directions, that covers activities related to
industrial ecology in the fields of: molecular
biology, genetics, electronic microscopy,
biological analyses and eco-toxicological
studies, assessment of air quality within
buildings and smell level via dynamic
olfactometry, complete characterisation of
industrial waste and sludge. These domains are
less studied at national level but in accordance
with environmental research tendencies at
European and international level.
Beside the extension of existing infrastructure, 5
new advanced research laboratories were created
in order to respond to research tendencies at
European and international level in the domain
of environmental pollution assessment and
control. Capitalization of research results and
existing infrastructure is realised through an
ongoing project co-financed through European
Regional Development Fund through
Operational Programme Competitiveness,
Priority Axis 1 – Research, technological
development and innovation to support
economic competiveness and business
development, Action 1.2.3. – Partnerships for
knowledge transfer:
- POC-A1-A.1.2.3-G-2015 - “Promotion,
identification and development of
partnerships for knowledge transfer in the
field of industrial ecology – Partner
ECOIND”-ID/SMIS 2014+:P_40_300/105581
(Contract No.:55/05.09.2016),
www.partenerecoind.incdecoind.ro
The project has as objective the improvement of
knowledge, technological and R&D personnel
transfer in the field of industrial ecology
between ECOIND and enterprises from
economic sector, through promotion,
identification and foundation of partnerships in
the aim to achieve joint research-development-
innovation objective and increase value added at
the level of environmental products/processes/
services and technologies.
It is an ongoing project and the main envisaged
results are: 45 contracts with economic
environment, 20 scientific publications, 3 patent
requests.
2
ECOIND carried various research projects
within both national programs such as National
Research Development Innovation Programme
II and III (PED, PCE, PTE, PCCF), NUCLEU,
ADER 2020 and international programmes such
as H2020-WATER-2015, HUNGARY/
BULGARIA – ROMANIA Cross-Border
Cooperation Programme 2007-2013, CORINT
EUREKA EUROSTARS, CORINT EUREKA,
FP7, Norwegian Cooperation, LIFE+, PHARE,
Sectoral Operational Programme Development
of Human Resources. Those projects led to
elaboration of new and/or optimised
technologies for wastewater treatment,
technologies for treatment of underground water
sources intended for human consumption,
development of new methods and techniques for
pollution assessment and control, modelling of
phenomenon and processes, as well as new
methodologies assessment of environmental
pollution and risks.
Recognition of ECOIND results led to numerous
medals and diplomas obtained within
international invention events and to
appreciation from the scientific community in
the environmental research field.

We consider that the new scientific journal „ROMANIAN JOURNAL OF ECOLOGY &
ENVIRONMENTAL CHEMISTRY”, next to International Symposium “The Environment and
The Industry” now at the 22 edition and institutional repository “Library of National Research and
Development Institute for Industrial Ecology ECOLIB” developed since 2017, will contribute to
promotion of the obtained results both at national and international level, in the field of industrial
ecology and environmental chemistry.

No matter how many barriers we’ll have to overcome our motto will remain the same:
“EXCELLENCE IN ENVIRONMENTAL RESEARCH AND SERVICES”

ECOIND Team

Romanian Journal of Ecology & Environmental Chemistry ● No.1 ● 2019

www.incdecoind.ro
3
 E. Bucur et. al.: Odour pollution assessment through indirect methods based on the monitoring of technological
parameters - case study

Odour pollution assessment through indirect methods based on the monitoring

of technological parameters - case study

ELENA BUCUR1, VALERIU DANCIULESCU1, GHEORGHITA TANASE 1, CAROL BLAZIU

LEHR1, ANDREI VASILE 1*
1NationalResearch and Development Institute for Industrial Ecology ECOIND Bucharest, 71-73 Drumul Podul Dambovitei, 060652,
Bucharest, Romania
*corresponding author (e-mail): poluare.aer@incdecoind.ro

The odours in the ambient air, through the discomfort that they induce, represent an actual problem
for the communities located nearby companies with technological processes that emit in the air
different strong and unpleasant odorous substances.
The standard method for odour assessment involves measuring the concentration through dynamic
olfactometry according with SR EN 13725:2003, a very complex method that requires specialized
working staff and expensive equipment.
The paper presents an indirect method for odour assessment in the ambient air, based on
monitoring the process and meteorological data - Predictive Emission Monitoring Systems (PEMS)
and it is applied to a livestock farm. Using the multiple regression analysis of the monitoring data
for the most important specific technological and meteorological parameters it can be developed a
mathematical model that could be used for the calculation of odour concentration in air, without
the necessity of direct measurement, after the initial step. For the case study presented in the paper,
the distance between the slurry lagoon was identified as a significant statistical parameter that can
determine in a proportion of 72% the concentration of odour in the ambient air nearby the farm;
the margin of error for odour concentration assessment, according to the model validation tests, is
± 8%, acceptable value for an estimation method by mathematical modelling.

Keywords: odour, dynamic olfactometry, multiple regression method, intensive livestock farming

Odour can be defined as sensations resulting
from the interaction of volatile chemical species
inhaled through nose, making contact with the
olfactory area and registering in the brain [1]; in
this context, the standardized method for odour
determination, the method based on dynamic
olfactometry [2-4] uses the human nose,
respectively a group of odour assessor connected
to an olfactometer – a performant system that
dilutes, present the samples to the human
assessors, and does all the calculations using a
data processing software. An automatic
alternative based on electrochemical cells is
represented by the electronical nose, used
mainly in comparative studies [5]. Even though
the direct methods are preferred, in the case
when these analyses are very complex, involves
very high costs or impracticable conditions,
indirect methods can be used, methods based on
statistical analysis of the parameters values that
are correlated with the level of odour emission.
In this regard, the use of statistical correlation
and regression methods have been applied in
various activity, including environmental
protection. [6-10].
The paper presents an indirect method for odour
assessment based on the monitoring of process,
meteorological and geographical parameters -
Predictive Emission Monitoring Systems [11] –
applied to a livestock farm. Using the multiple
regression analysis applied to specific
monitoring parameters data it is intended to
obtain a mathematical model that can be used
for the odour concentration in air estimation,
without direct measurement, except the initial
phase.

EXPERIMENTAL PART
Location. The case study was conducted at a requires a 1500 m sanitary protection area. The
livestock farm operating at maximum capacity, farm is in a hilly area, in the immediate vicinity
which, according to Order no. 994/2018 [12] of a locality, characterized by winds in the
Romanian Journal of Ecology & Environmental Chemistry ● No.1 ● 2019
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4
 E. Bucur et. al.: Odour pollution assessment through indirect methods based on the monitoring of technological
parameters - case study

direction of NV, as shown by the graphical expression of the wind rose (Figure 1).

Fig. 1. Wind rose of the area

The very small distance, sometimes under 100 m
from houses, determined over the years some
problems due to the air pollution with NH3,
H2S, and odours largely due to the activities
carried out on the farm but also to the non-
compliance with the sanitary protection area.
Technological processes carried out on the farm
meet the requirements of the BAT Conclusions
[13], both in terms of the application of the best
technologies and also regarding the methods for
reducing pollutant emissions in the air. Thus,
animal feeding and shelter ventilation systems
are automated, the ventilation rate being set
according to the temperature and humidity
parameters of the shelter to ensure optimal
animal development conditions. The slurry is
automatically transferred to pre-sealed lagoons
that prevent soil and ground infiltration and are
covered with sealed geomembranes where they
are enzymatically treated to reduce
pollutant/odour emissions in the air.
Materials and Methods. The evaluation method
is based on the statistical correlation and
multiple regression analysis applied to the data
series obtained by monitoring the odour
concentration, the main specific odorous
pollutants (NH3 and H2S) and the technological /
geographic parameters and involves the
following steps: i) identification the main
sources of odour emission and their specific
parameters, ii) monitoring of the identified
parameters and of the odour concentration,
iii) correlation and multiple regression analysis
applied to the data series obtained by
Romanian Journal of Ecology & Environmental Chemistry ● No.1 ● 2019
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monitoring, iv) obtaining the equation of the
mathematical model and its validation by direct
measurements and by the mathematical
modelling of the odour dispersion in the air.
Based on the analysis of technological processes
from a livestock farm and the main BAT
techniques [13] to reduce the odour level but
also on the specific conditions of the farm in
which the case study was conducted, three
potential sources of odour pollution and strong
smelling compounds have been identified:
ventilation openings for animal shelters (NH3,
odour), the slurry transfer system (NH3, H2S,
odour) and the lagoons for storage / treatment of
slurry (NH3, H2S, smell) to which are added the
sources of diffuse emissions. In these conditions
the parameters that can characterize the
emissions of odour and strong smelling specific
compounds, NH3 and H2S, which were taken
into account for the mathematical model
development are: i) ventilation rate, expressed in
% from the maximum capacity (vent-cap), ii)
distance to shelters, m (dist), iii) distance to
lagoons, m (dist), iv) wind direction, expressed
in degrees N (dir-vant), v) wind speed, m/s (vit-
vant), vi) atmospheric temperature, 0C (temp),
(vii) atmospheric humidity, % (RH) and viii) the
concentration of NH3 and H2S in air (in the case
of odour calculation) in μg/m3 or mg/m3. For
concentration of NH3 and H2S determination,
Horiba automatic analysers were used, with
detection limits of 5 ppbv for NH3 and 2 ppbv
for H2S. The odour analyzis was done following
the dynamic olfactometry method according to
5
 E. Bucur et. al.: Odour pollution assessment through indirect methods based on the monitoring of technological
parameters - case study

SR EN 13725:2003 using a Odournet T08
dynamic olfactometer, developed by Odournet
GMBH, Germany. Meteorological parameters
were monitored using a MetPack Wind Sonic
Portable Weather Station. For the statistical
interpretation of the data - Pearson correlation
and multiple regression analysis - the SPSS 20.0
(Statistical Package for Social Sciences, version
20.0) software was used. The correlation
analysis provides information regarding the
intensity and direction of the statistical link
between two or more variables without
necessarily involving a causal link between
them. In the case study Pearson correlation was
used, whose coefficient (r) can take values in the
range [-1, + 1]; the closer is the value of the r
coefficient to ± 1, the better is the correlation
between the two analyzed parameters. To assess
the statistical significance of the correlation
coefficient and the regression coefficients, the p
value - the probability that the value of the
correlation coefficient is equal to zero (the null
hypothesis) - is used; if the probability is less
than the significance level (ex. p<0.05 for a
confidence level of 95%), then the correlation
coefficient is statistically significant. Statistical
correlation is always the first step in predictive
studies preceding regression analysis.
The regression represents the process by which
it can estimate the value of the dependent
variable based on the values of the predictor
variables and the mathematical relationship
expressing their interdependence. Such an
estimate is, in fact, a prediction. In this case
study, the multiple regression was used, which
basically relies on the same fundamental
concepts as simple linear regression, but must
deal with the specific problems of a multivariate
model. The multiple regression equation (Ec. 1)
is similar to the simple regression equation,
applied to several predictors:
(1)
in which:
x1…xk = values of the k predictor
variables
a = ordinate to origin
b1…bk = regression coefficients
corresponding to the k predictor
variables
Y = estimate values for the criterion variable
(dependent)

RESULTS AND DISCUSSION

Starting from the actual conditions on the site, 9
points were identified outside the farm enclosure
at different distances from animal shelters and
lagoons for slurry manure handling and storage,
around the site; at each point measurements of
the concentration were made for NH3, H2S,
odour and weather parameters, and the results of
the measurements are found in Table 1.
Analyzing the results of the measurements and
compared with the limit values for the
concentration of H2S and NH3 in the air [14] it
can see that in all 9 points the concentration
exceed the limit values for short-term
measurements (0.015 mg/m3 for H2S and 0.300
mg/m3 for NH3); it should be noted that these
points are located inside the sanitary protection
area (at distances of 80 - 440 m from the farm),
demonstrating once again the necessity of regard
the sanitary protection zones imposed by Order
no. 994/2018 [12].
In this case the 1500 m sanitary protection area
is not respected, with housing reaching in some
cases 100 m from animal shelters. With regard
to odour concentrations, for which limit values
are not set in the ambient air, it finds values that
fall within the normal range for these types of
activities, taking into account the small distance
from the emission sources.

Table 1. Values of parameters monitored in the livestock farm

Point Location H2S, NH3, Odour, Temp, RH, vent- Dist, dist-lag, dir-vant, vit-vant,
µg/m3 mg/m3 oue/m3 0C % cap, % m m grade m/s
0 1 2 3 4 5 6 7 8 9 10 11
1 Farm gate 35.40 1.30 16.00 17.00 56.00 50.00 310.00 600.00 104.00 0.60
Close to lagoon and
2 69.50 3.90 31.00 21.00 44.00 50.00 290.00 .00 150.00 0.70
slurry distribution area
25m from the pt 2. to
3 38.60 5.60 47.00 22.00 41.00 64.00 240.00 90.00 163.00 1.70
farm
4 25m from pt 1. to N 39.50 1.51 19.00 23.00 41.00 64.00 370.00 660.00 220.00 1.90
5 On the road (house) 33.20 10.20 4.40 23.00 41.00 70.00 160.00 600.00 211.00 2.19

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 E. Bucur et. al.: Odour pollution assessment through indirect methods based on the monitoring of technological
parameters - case study

0 1 2 3 4 5 6 7 8 9 10 11
6 On the road (pension) 35.60 3.98 8.00 23.00 41.00 95.00 440.00 810.00 213.00 1.35
In the farm, close to the
7 36.30 13.06 18.00 23.00 42.00 100.00 80.00 520.00 171.00 0.70
monitoring system
CMA (30 min) 15 0.300
Average 41.15 5.65 20.48 21.71 43.71 70.42 270.00 468.57 176.00 1.305
Standard deviation 4.789 1.672 5.470 .837 2.089 7.55 46.291 114.775 15.87 .245
Median 36.30 3.98 18.00 23.00 41.00 64.00 290.00 600.00 171.00 1.35
Minimum value 33.20 1.30 4.40 17.00 41.00 50.00 80.00 .00 104.00 0.60
Maximum value 69.50 13.06 47.00 23.00 56.00 100.00 440.00 810.00 220.00 2.19
8 30m from pt. 2 46.5 3.21 29 18 54 35 220 200 110 0.9
On the road, close to the
9 42.4 10.1 24 20 46 80 120 410 165 1.2
office

Statistical interpretation of data

First step in the statistical interpretation of data
was the Pearson correlation analysis, whose
results are found in Table 2 (the Pearson
correlation coefficients, r) reveals for the
interest indicators, odour, NH3 and H2S a series
of very good and good correlations with some of
the monitored parameters, as follows:
 in the case of the odour concentration a
very good inverse correlation with the
distance from the lagoon (rodour = - 0.847) and
moderate, direct correlation with the H2S
concentration (rodour = 0.425) is observed;
 in the case of H2S concentration a very
good inverse correlation with the distance to
the lagoon (rH2S = - 0.718), and also moderate
correlations with the ventilation capacity of
the shelters (rH2S = - 0.468) and odour
concentration (rodour = 0.425);
 for the concentration of NH3 we can
observe a very good inverse correlation with
the distance from the animal shelters (r = -
0.865), a good correlation with the ventilation
capacity of the shelters (rH2S = 0.618) and a
moderate one with the atmospheric
temperature and humidity.

Table 2. The values of the Pearson correlation coefficients, r

odour dist dist-lag dir-vant vit-vant temp RH vent-cap H2S NH3
odour 1.000
dist -.075 1.000
dist-lag -.847 .270 1.000
dir-vant -.376 .155 .432 1.000
vit-vant -.123 .068 .216 .754 1.000
temp -.107 -.154 .131 .891 .586 1.000
RH -.080 .116 .061 -.835 -.605 -.974 1.000
vent-cap -.379 -.209 .493 .506 .042 .640 -.510 1.000
H2S .425 .118 -.718 -.257 -.394 -.116 -.015 -.468 1.000
NH3 -.169 -.865 -.014 .213 .065 .504 -.430 .618 -.239 1.000

Colour legend:
Colour
Correlation significance very week week moderate good very good

Under these conditions, the results of the
Pearson correlation analysis provide the first
indices for predictor variables with significance
for the value of the dependent variable (odour
concentration). Once a correlation between the
variables is established, the statistical regression
method is used to establish the mathematical
relationship. In our case, multiple regression, the
Forward method, was applied to the monitoring
data series obtained in the first 7 measurement
points (Table 1), including the concentrations of
NH3 and H2S; points 8 and 9 were used to
validate the mathematical model.
The results of multiple regression (Table 3) for
odour concentration as a dependent variable
eliminate as statistically insignificant all
variables from the equation, except the distance
from the lagoon, the only statistically significant

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 E. Bucur et. al.: Odour pollution assessment through indirect methods based on the monitoring of technological
parameters - case study

variable (Sig. = 0.016).

Table 3. The results of multiple regression analysis for odour concentration.

Model Summaryb
Model R R Adjusted R Std. Error of Change Statistics
Square Square the Estimate R Square F Change df1 df2 Sig. F
Change Change
1 .847a .718 .662 8.41942 .718 12.728 1 5 .016
a. Predictors: (Constant), dist-lag
b. Dependent Variable: odour
Coefficientsa
Model Unstandardized Coefficients Standardized T Sig. Collinearity Statistics
Coefficients
B Std. Error Beta Tolerance VIF
(Constant) 39.408 6.185 6.371 .001
1
dist-lag -.040 .011 -.847 -3.568 .016 1.000 1.000
a. Dependent Variable: odour
Excluded Variablesa
Model Beta In t Sig. Partial Collinearity Statistics
Correlation Tolerance VIF Minimum
Tolerance
dist .166b .631 .562 .301 .927 1.078 .927
b
dir-vant -.013 -.044 .967 -.022 .814 1.229 .814
vit-vant .064b .236 .825 .117 .953 1.049 .953
b
temp .003 .013 .990 .007 .983 1.017 .983
1
RH -.028b -.105 .922 -.052 .996 1.004 .996
vent-cap .051b .168 .875 .084 .757 1.321 .757
b
H2S -.378 -1.141 .317 -.496 .484 2.064 .484
NH3 -.181b -.725 .509 -.341 1.000 1.000 1.000
a. Dependent Variable: odour
b. Predictors in the Model: (Constant), dist-lag

The value of R2 = 0.718 indicates that this
variable determines 71.8% of the odour
concentration in the analyzed area. We mention
that these results apply only to the conditions
specific to the period in which the measurements
and data sets were used.
Applying the same procedure in the case of H2S,
similar results have been obtained,
demonstrating that odour and H2S emissions are
from the same source, i.e. the lagoon in which
the slurry effluent is discharged, the only
parameter with statistical significance. In this
case, however, this parameter characterizes the
H2S emission only in a proportion of 51.6%. In
the case of NH3 concentration, the results of the
multiple regression analysis reveal two
statistically significant parameters, the distance
from the animal shelters and the ventilation rate,
which together account for 94.8% of the
emission.
The equations of the mathematical models for
the three indicators and statistically significant
parameters can be found in Table 4.

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 E. Bucur et. al.: Odour pollution assessment through indirect methods based on the monitoring of technological
parameters - case study
Table 4. Equations of mathematical models for estimating odour concentration, NH3 and H2S
Indicator Statistically significant indicators
Odour - distance from the lagoon, m (dist-lag)
H2S - distance from the lagoon, m (dist-lag)
NH3 - ventilation rate, % from maximum
capacity (vent-cap)
- distance from the shelters, m (dist)
In order to validate the equations of the
mathematical models, the data obtained in points
8 and 9 were used; the error margin for odour
concentration assessment according to model
validation tests is -8%, for NH3 -6%, and for
H2S -15%, acceptable for an estimation method
by mathematical modelling. Based on the
mathematical equations developed for H2S, NH3
and odour, the concentrations of these pollutants
can be calculated at any point on the site starting
from the statistically significant parameters,
respectively the distance from the lagoon for
H2S and odour and the distance from the shelter
and ventilation rate for NH3. Thus, the minimum
distances from the animal shelters for NH3 and
from lagoon in the case of H2S and odour were
calculated that should be respected in order not
to exceed the limit values in the legislation; for
H2S the estimated minimum distance from the
lagoon is 1340 m and for NH3, for a maximum
ventilation rate of 100%, the distance from
shelters must be at least 580 m; in the case of
odour, at an estimated distance of 960 m from
the lagoon, the odour concentration would be
below the theoretical threshold of odour
perception (the odour concentration perceived
by 50% of the members of a panel constituted
according to SR EN 13725:2003) of 1 ou/m3.
Considering the value of the Maximum
Allowable Concentration for H2S (15 μg/m3) it
can be concluded that at a distance of at least
1340 m from the lagoon the concentration of
H2S is below the limit value, i.e. if the sanitary
protection zone is respected the concentration of
H2S in sensitive areas would fall within the limit
values imposed by environmental legislation in
force [14]. The same applies to the odour and
NH3 concentration.
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Equation of the mathematical model
x = distance from the lagoon, m
Y = estimated value for the odour concentration, ou e/m3
x = distance from the lagoon, m
Y = estimated value for the concentration of H2S, µg/m3
x1 = distance from the shelter, m
x2 = ventilation rate, %
Y = estimated value for the concentration NH3, mg/m3
CONCLUSION
Starting from the results obtained in the case
study, we find once again a high level of
chemical pollution (NH3 and H2S) and odour
generated by the livestock farm activities, but
also the need to comply with the requirements of
the regulations in force regarding the sanitary
protection areas related to these farms, to ensure
comfort and reduce the effect on the health of
the population.
The indirect method of assessing the
concentration of odour, NH3 and H2S based on
the analysis of the data series from the
monitoring of the technological/geographic
parameters through the multiple regression
method is applicable to this type of activity; the
method allows the estimation of odour, NH3 and
H2S concentration at any point on the site using
mathematical relationships resulting from
multiple regression analysis with an assessment
error margin of -8% for odour, -6% for NH3 and
-15% for H2S, acceptable values for an
estimation method by mathematical modelling.
Using these relationships, the minimum values
of the influence parameters on H2S, NH3 and
odour emission (lagoon distance, shelter
distance at an 100% ventilation rate) can be
calculated for which the concentration of these
indicators in air reaches the maximum
concentration value admitted.
For the case study, the following results were
obtained:
 for H2S the minimum distance to the
lagoon: 1340 m;
 in the case of NH3, for the maximum
ventilation rate (100%) distance to:
minimum 580m;
 in case of odour at a distance of 960 m
from the lagoon, the odour concentration
9
 E. Bucur et. al.: Odour pollution assessment through indirect methods based on the monitoring of technological
parameters - case study

is below 1 ou/m3 - the theoretical sanitary protection areas these concentrations

threshold of odour perception;
We can appreciate that, for the case studied,
outside the 1500 m of the sanitary protection
area, the estimated odour concentrations, NH3
and H2S are below the limit values; inside the
exceed the limit values for sensitive areas [14] in
the case of the settlements that do not respect the
minimum distance from the farm imposed by the
legislation.

ACKNOWLEDGEMENTS. The authors acknowledge the financial support offered by The

National Research Nucleu Program through Project 20N/2019; the authors would like to thank all
those who contributed to this study, too.

REFERENCES
1. GREGORY, L., KENDALL, D., BARNARD, N., J Air Pollut Control Assoc, 19(2), 1969, p.
91;
2. SR EN 13725:2003 Air quality. Determination of odour concentration by dynamic
olfactometry;
3. VASILE, A., DANCIULESCU, V., TANASE, G., KIM, L., DEDIU, V., Rev. Chim.
(Bucharest), 68(8), 2017, p. 1749;
4. JIANG, J., COFFEY, P., TOOHEY, B., J Air Waste Manag Assoc, 56, 2006, p. 675;
5. HUDON, G., GUY, C., HERMIA, J., J Air Waste Manag Assoc, 50, 2000, p. 1750;
6. MORRISON, S., FORDYCE, F.M., SCOTT, E.M., Environ Geochem Health, 36, 2014, p.
319.
7. YOO, J.M., LEE, Y.R., KIM, D., JEONG, M.J., STOCKWELL, W.R., KUNDU, P.K., OH,
S.M., SHIN, D.B., LEE, S.J., Atmos Environ, 82, 2014, p. 226.
8. KLIUCININKAS, L., KRUGLY, E., STASIULAITIENE, I., RADZIUNIENE, I.,
PRASAUSKAS, T., JONUSAS, A., KAUNELIENE, V., MARTUZEVICIUS, D., Atmos
Environ, 97, 2014, p. 83.
9. BUCUR E., DANET, A.F., LEHR, C.B., LEHR, E., NITA-LAZAR, M., J Air Waste Manag
Assoc., 67(4), 2017, p. 391.
10. DANCIULESCU, V., BUCUR, E., PASCU, L.F., VASILE, A., BRATU, M., J Environ Prot
Ecol., 16(3), 2015, p. 815
11. http://eippcb.jrc.ec.europa.eu/reference/ - JRC Reference Report on Monitoring of
Emissions to Air and Water from IED installations, 2017.
12. Ordinul nr. 994/2018 pentru modificarea si completarea Normelor de igiena si sanatate
publica privind mediul de viata al populatiei, aprobate prin Ordinul ministerului sanatatii nr.
119/2014.
13. Commission Implementing Decision (EU) 2017/302 of 15 February 2017 establishing best
available techniques (BAT) conclusions, under Directive 2010/75/EU of the European
Parliament and of the Council, for the intensive rearing of poultry or pigs.
14. STAS 12574-87. Aer din zonele protejate. Conditii de calitate (in Romanian).

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 M.A. Constantin et. al.: Photo-degradation of 5 – Fluorouracil. Comparative Study on the Efficiencies of UV/H 2O2,
UV/TiO2, UV/H2O2/TiO2 Processes

Photo-degradation of 5 – Fluorouracil. Comparative Study on the Efficiencies of UV/H2O2,

UV/TiO2, UV/H2O2/TiO2 Processes

MIRELA ALINA CONSTANTIN1*, LUCIAN ALEXANDRU CONSTANTIN1, INES NITOI1,

IONUT CRISTEA1, ELLEA BOULAC2
1 National Research and Development Institute for Industrial Ecology ECOIND Bucharest, 71-73 Drumul Podul Dambovitei,
060652, Bucharest, Romania
2IUT University Toulouse III Paul Sabatier, 118 route de Narbonne 31062, Toulouse, France

* Corresponding author (e-mail): alina.constantin@incdecoind.ro

Degradation experiments on 5-Fluorouracil synthetic solutions were performed using three types of
advanced oxidation systems: UV/H2O2, UV/TiO2 and UV/H2O2/TiO2. Optimum parameters for all
three systems were established and 5-Fluorouracil degradation efficiencies were calculated, for all
systems being more than 97%. The results showed that the combined UV/H2O2/TiO2 system is
offering shortest irradiation time, the possibility to recover and reuse the photo catalyst as well as
the possibility to use solar radiation. Obtained results proved also that advanced oxidation
processes represent a viable option for degradation of hazardous pollutants that cannot be removed
properly via conventional wastewater treatment processes.
Keywords: 5-Fluorouracil, TiO2, H2O2, degradation efficiency

5-Fluorouracil (5FU) is one of the most common drugs used in cancer treatment worldwide [1].

H
N O

NH
F
O
Fig. 1. 5-Fluorouracil (5FU) structure

Due to its non-selective action it affects not only
cancerous but healthy cells, presenting an
carcinogenic, genotoxic, mutagenic and
teratogenic potential [2]. Moreover about 15%
of the administered 5FU is leaving the human
body with no transformation ending within the
sewerage network [3]. Recent studies
demonstrated that this type of pollutant cannot
be removed within the wastewater treatment
plants [4]. Even if cytostatic residues
concentrations within the sewerage system of
hospitals are low,varying from micrograms to
hundreds of micrograms, its removal through
conventional wastewater treatment processes is
not possible asking for application of more
performant processes [5], [6]. Therefore, 5FU
degradation experiments were performed in
order to determine the optimum parameters for
the following systems: UV/H2O2, UV/TiO2 and
UV/H2O2/TiO2.

EXPERIMENTAL PART
Degradation experiments were performed using
a Heraeus UV reactor with the following
characteristics: UV lamp - TQ150-Z3 type,
cooling jacket, reaction vessel from quartz,
volume = 400 cm3, equipped with a magnetic
recirculation pump. Determination of 5FU
concentration was carried out using an Agilent
7890A gas chromatograph coupled with an
Agilent 240MS ionic trap spectrometer [7].
Synthetic solutions were prepared using 5FU
supplied from Sigma-Aldrich.
All samples were bubbled with air (50 L/h) prior
to irradiation in order to avoid electrons – holes
recombination.
All experiments were at least duplicated in order
to assure their repeatability.

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 M.A. Constantin et. al.: Photo-degradation of 5 – Fluorouracil. Comparative Study on the Efficiencies of UV/H 2O2,
UV/TiO2, UV/H2O2/TiO2 Processes

Fig. 2. Experimental installation

RESULTS AND DISCUSSIONS

UV/H2O2 system
From the main parameters that are influencing
the degradation processes the following were
investigated: H2O2 dose and irradiation time.
Effect of H2O2 dose
In order to set up optimum H2O2 dose, 5FU
degradation experiments were performed for an
initial 5FU concentration [5FU]0 = 13.35 mg/L
= 1.03 x 10-4 M for an irradiation time of 30
minutes and H2O2 doses within the interval
[5FU]0/[H2O2] = 1:3 – 1:28.
Obtained experimental results are presented
within Table 1 and Figure 3.

Table 1. 5FU degradation efficiency in UV/H2O2 system

[5FU]0 = 13.35 mg/L=1.03 x 10-4 M; irradiation time = 30 min
[H2O2]/[5FU]0 [5FU] [5FU] -ln([5FU]/ k Eficienta
mg/L M [5FU]0) s-1 %
3 1.65 1.27 x 10-5 2.09 1.16 x 10-3 87.64%
11 1.32 1.02 x 10-5 2.31 1.29 x 10-3 90.11%
-6 -3
17 1.11 8.54 x 10 2.49 1.34 x 10 91.69%
-5 -3
22 1.45 1.12 x 10 2.22 1.23 x 10 89.14%
28 1.53 1.18 x 10-5 2.17 1.20 x 10-3 88.54%

Fig. 3. H2O2 dose effect upon the 5FU degradation in UV/H2O2 system
[5FU]0 = 13.35 mg/L=1.03 x 10-4 M; irradiation time = 30 min

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 M.A. Constantin et. al.: Photo-degradation of 5 – Fluorouracil. Comparative Study on the Efficiencies of UV/H 2O2,
UV/TiO2, UV/H2O2/TiO2 Processes

Results showed that optimum H2O2 dose
correspond to [5FU]0/[H2O2] = 1:17 which
assures a degradation efficiency of 91.69% after
30 minutes of irradiation. Optimum dose was
used in all the subsequent experiments for
UV/H2O2 system.
Effect of irradiation time
In order to establish the optimum irradiation
time 5FU degradation experiments were
performed for an initial 5FU concentration
[5FU]0 = 13.35 mg/L = 1.03 x 10-4 M for H2O2
dose corresponding to [5FU]0/[H2O2] = 1:17,
and irradiation time up to 45 minutes.
Experimental results showed that the increase of
irradiation time has a positive impact upon 5FU
degradation efficiency. Therefore, the optimum
irradiation time was set at 45 minutes, which
assured the substrate degradation with a 97.08%
efficiency and a residual 5FU concentration of
0.39 mg/L.

Fig. 4. 5FU degradation kinetic in UV/H2O2 system

[5FU]0 = 13.35 mg/L=1.03 x 10-4 M; [5FU]0/[H2O2] = 1:17

UV/TiO2 system
Previous reported researches on 5FU
degradation in UV/TiO2 system [7] led to the
following optimal parameters: pH = 7; photo
catalyst dose 100 mg/L; irradiation time 180
minutes. These conditions assured 5FU
degradation with an efficiency of more than
99%. Moreover, the processes obey Langmuir –
Hinshelwood model proving that degradation
process occurs mainly on TiO2 particle surface.
UV/H2O2/TiO2 system
From the main parameters that are influencing
the degradation processes the following were
investigated: H2O2 dose and irradiation time. All
experiments were performed using a synthetic
solution with initial concentration [5FU]0 =
13.35 mg/L = 1.03 x 10-4 M; photo catalyst
concentration [TiO2] = 100 mg/L at pH = 7
(optimum conditions for UV/TiO2 system).
Effect of H2O2 dose
The oxygen presence plays an important role
due to its electron scavenger properties fact that
is affecting electrons – holes recombination
process. It was demonstrated that oxygen can
have both a positive or negative impact upon the
pollutant degradation [8].
In order to investigate these parameters; molar
ratios [5FU]0/[H2O2] were varied within the
domain 1:0.5 – 1:5. Obtained experimental
results are presented within Table 2 and Figure
5.

Table 2. 5FU degradation efficiency in UV/H2O2/TiO2 system

[5FU]0 = 13.35 mg/L=1.03 x 10-4 M; [TiO2] =100 mg/L; irradiation time = 30 min
[H2O2]/[5FU]0 [5FU] [5FU] -ln([5FU]/ k Efficiency
mg/L M [5FU]0) s-1 %
0,5 2,77 2.13 x 10-5 1,57 0,80 x 10-3 79,25%
1 1,73 1,33 x 10-5 2,04 1,13 x 10-3 87,04%
3 1,29 9,92 x 10-6 2,34 1,29 x 10-3 90,34%
5 1,13 8,69 x 10-6 2,47 1,37 x 10-3 91,54%

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 M.A. Constantin et. al.: Photo-degradation of 5 – Fluorouracil. Comparative Study on the Efficiencies of UV/H 2O2,
UV/TiO2, UV/H2O2/TiO2 Processes

Fig. 5. 5FU degradation kinetic in UV/H2O2/TiO2 system

[5FU]0 = 13.35 mg/L=1.03 x 10-4 M; [TiO2] =100 mg/L; irradiation time = 30 min

Obtained results proved that optimum H2O2 dose
corresponds to the [5FU]0/[H2O2] ratio of 1:5.
Effect of irradiation time
For the optimum H2O2 dose, the influence of
irradiation time upon 5FU degradation
efficiency was investigated, the results being
presented within Figure 6.
The increase of irradiation time has a positive
effect upon 5FU degradation efficiency.
Optimum irradiation time was set at 45 minutes,
assuring the substrate degradation with a
97.37% efficiency and a residual 5FU
concentration of 0.139 mg/L.

Fig. 6. 5FU degradation kinetic in UV/H2O2/TiO2 system

5FU]0 = 13.35 mg/L=1.03 x 10-4 M; [TiO2] =100 mg/L; [5FU]0/[H2O2] = 1:5

CONCLUSIONS
Optimum parameters for 5FU degradation using
UV/H2O2, UV/TiO2, UV/H2O2/TiO2 were
established and degradation efficiencies for all
three systems were calculated.
A synthesis of these data is presented within
Table 3.
UV/H2O2/TiO2 system presents the advantage
of using solar radiation simultaneously with the
shortest irradiation time that leads to energy
costs reduction and also to reduction of
implementation (compact installations) and
operating costs. It also offers the possibility to
recover the photo catalyst and reuse it in further
treatment cycles.

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 M.A. Constantin et. al.: Photo-degradation of 5 – Fluorouracil. Comparative Study on the Efficiencies of UV/H 2O2,
UV/TiO2, UV/H2O2/TiO2 Processes

Table 3. 5FU degradation efficiency using various systems

System Efficiency
UV/H2O2: [5FU]0/[H2O2] = 1:17
97.08%
Irradiation time = 45 min
UV/TiO2: [TiO2] = 100 mg/L
pH = 7 99.85%
Irradiation time = 180 min
UV/H2O2/TiO2: [5FU]0/[H2O2] = 1:5
[TiO2] = 100 mg/L
97.37%
pH = 7
Irradiation time = 45 min

ACKNOWLEDGEMENT. The work was funded by Ministry of Research and Innovation of

Romania through Programme Nucleu Project PN 18 05 03 01.

REFERENCES
1. LIN, H., LIN, A., Water Res., 48, 2014, p. 559.
2. KUMMERER, K., HAIB, A., SCHUSTER, A., HEIN, A., EBERT, I., Environ. Sci. Pollut.
Res. Int, 23, no. 15, 2014, p. 14791.
3. ZHANG, J., CHANG, V., GIANNIS, A., WANG, J., Sci. Total Environ., 445, 2013, p. 281.
4. WANG, X.-H., LIN, A.-C., Environ. Pollut., 186, 2014, p. 203.
5. CONSTANTIN, L., NITOI, I., CRISTEA, I., OANCEA, P., ORBECI, C., NECHIFOR
A.C., Rev. Chim. (Bucharest), 66, no. 5, 2015, p. 597.
6. NITOI, I., OANCEA, P., CRISTEA, I., CONSTANTIN, L., NECHIFOR, G., J. Photochem.
Photobio. A, 298, 2015, p. 17-23.
7. CONSTANTIN, L., NITOI, I., CRISTEA, I., OANCEA, P., Rev. Chim. (Bucharest), 67,
no. 8, 2016, p. 1447.
8. MOZIA, S., Sep. Purif. Technol., 73, 2010, p. 71

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 T. Galaon et. al.: Biodegradation of antibiotics: the balance between good and bad

Biodegradation of antibiotics: the balance between good and bad

TOMA GALAON, ALINA BANCIU, FLORENTINA LAURA CHIRIAC AND MIHAI NITA-
LAZAR*

National Research and Development Institute for Industrial Ecology ECOIND Bucharest, 71-73 Drumul Podul Dambovitei, 060652,
Bucharest, Romania
*corresponding author (e-mail): mihai.nita@incdecoind.ro

The worldwide population rise corroborated with the raise of the health-care standards have
generated an escalation of the antibiotic production and uncontrolled usage. The subsequent effects
of this escalation have led to an increase of the antibiotic resistance rates, Romania is in the top of
the EU countries regarding the antibiotic resistance rates, and to a continuous presence in the
environment, including the aquatic environment. Unfortunately, the present design of the classical
WWTPs is not optimized for the efficient removal of antibiotics since these compounds may have
highly soluble and polar molecular structures. Instead, antibiotics removal using microorganisms
could be an ecofriendly solution to this environmental issue, as long as their antibiotic degradation
structures are not more toxic than the antibiotic itself.
In the present review, we focus on the environmental presence and biodegradation of the most
commonly used antibiotics as well as on their biodegradation, based on bacterial model, monitored
by mass-spectrometric methods.

Keywords: biodegradation, antibiotics, bacteria, degradation pathway, antibiotic resistance

The penicillin discovery by Fleming in 1928
was a turning point in the medicine and
pharmacology fields, but the quest of finding
chemical compounds with microbial growth
inhibition activity started long time before.
In 1871 J. B. Sanderson and his coworkers
reported a mold strain, Penicillium, with
bacterial growth inhibition effect, but the actual
effect was effectively proved by E. Duchesne in
1897. First reports on penicillin were in 1938,
but soon after that, 1939-1940, S. Waksman and
R. Dubos reported over 20 potential antibiotics.
Since then there is a continuous race between
antibiotic resistance and synthesis of new
chemical compound with antibacterial effect.

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 T. Galaon et. al.: Biodegradation of antibiotics: the balance between good and bad

Fig. 1. Timeline of antibiotic resistance and new antibiotic synthesis [1]

At the present, a growing number of chemical
compounds with more and more specific
antimicrobial activity, antibiotics, have been
continuously synthetized for human, animal and
plants health. There are many classes of
antibiotics regardless of their natural, synthetic
and semi-synthetic origin [2] with a very well
characterized antibacterial effect (Table I) [3]
and subsequently there are largely used due to
their benefic effects, up to 2,000,000 tons of
antibiotics (AB) consumption per year,
according to Centers for Disease Control and
Prevention in 2015.
Antibiotics structural characterization
From a structural point of view antibiotics
can be divided into several types of
chemical structures. Logically, this
classification is co-related with their
mechanism of action (bactericidal,
bacteriostatic, bacteriolytic) and their
spectrum of activity (types of controlled
bacteria). The most important classes of
antibiotics are summarized in Table 1. A
brief description of some of these classes of
antibiotics is presented in the following
paragraphs together with their structural
characteristics which are given in Figure 2.
Beta-lactams are one of the most used
classes of antibiotics containing a beta-
lactam ring which consists of a 3-carbon
and 1-nitrogen atom ring and a ketone
group.
These antibiotics interfere with proteins
essential for synthesis of bacterial cell wall
[4]. Some of the most prescribed
representatives are Penicillins (Penicillin G,
Penicillin V, Oxacillin, Ampicillin,
Amoxicillin), Cephalosporines,
Monobactams (Aztreonam), and
Carbapenems (Imipenem, Meropenem,
Ertapenem).
Macrolides are large molecules containing
a 14 to 16 membered macrolide ring
(macrocyclic lactose ring). They inhibit
protein synthesis by bacteria leading
sometimes to cell death. Some of the most

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 T. Galaon et. al.: Biodegradation of antibiotics: the balance between good and bad
important Erythromycin, Azithromycin and
Clarithromycin [5].
Tetracyclines are antibiotics with 4
condensed hydrocarbon rings (6 carbon
atoms). Tetracycline was discovered in
1945 by Benjamin Duggar from a soil
bacterium of the genus Streptomyces.
Tetracyclines may be obtained by
biosynthesis (1st generation), semi-synthesis
(2nd generation), or by chemical synthesis
(3rd generation). Significant representatives
of the 1st generation are Tetracycline,
Chlortetracycline and Oxytetracycline,
while for the 2nd generation drugs like
Doxycycline, Lymecycline are important.
All tetracyclines target the ribosome from
bacteria disrupting addition of amino acids
to polypeptide chains during protein
synthesis inside ribosome, preventing in this
mode bacterial growth. Tetracyclines have a
very broad spectrum of activity with
bacteriostatic effect for aerobic and
anaerobic bacteria both gram-positive and
gram-negative. Over time, many bacteria
have become resistant to Tetracyclines
reducing in this mode their activity
spectrum [6].
Quinolones interfere with bacteria DNA
replication and transcription. These
molecules contain condensed aromatic rings
with a carboxylic acid group attached and in
many cases a fluorine atom. Significant
representatives of this class of antibiotics
are Ciprofloxacin, Norfloxacin,
Levofloxacin, Ofloxacin, Nalidixic acid and
so on. Additional rings and different
chemical moieties extend the spectrum of
antimicrobial activity. Also, these structural
Table 1. Commonly used classes of antibiotics
Antibiotic class
Aminoglycosides
Beta-Lactams
Glycopeptides
Lincosamides
Macrolides
Nitrofurans
Amphenicols
Phosphonates
Polyether ionophores
Quinolones and fluoroquinolones
Rifamycin
Sulfonamides
Tetracyclines
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modifications of the quinolones have
improved bioavailability of the drugs
increasing also activity spectrum and drug
potency. Quinolones are prescribed for the
treatment of urinary, respiratory tract and
systemic infections caused by both gram-
positive and gram-negative bacteria [7]. All
tetracyclines target the ribosome from
bacteria disrupting addition of amino acids
to polypeptide chains during protein
synthesis inside ribosome, preventing in this
mode bacterial growth. Tetracyclines have a
very broad spectrum of activity with
bacteriostatic effect for aerobic and
anaerobic bacteria both gram-positive and
gram-negative. Over time, many bacteria
have become resistant to Tetracyclines
reducing in this mode their activity
spectrum [6].
Quinolones interfere with bacteria DNA
replication and transcription. These
molecules contain condensed aromatic rings
with a carboxylic acid group attached and in
many cases a fluorine atom. Significant
representatives of this class of antibiotics
are Ciprofloxacin, Norfloxacin,
Levofloxacin, Ofloxacin, Nalidixic acid and
so on. Additional rings and different
chemical moieties extend the spectrum of
antimicrobial activity. Also, these structural
modifications of the quinolones have
improved bioavailability of the drugs
increasing also activity spectrum and drug
potency. Quinolones are prescribed for the
treatment of urinary, respiratory tract and
systemic infections caused by both gram-
positive and gram-negative bacteria [7].
Type Target
Bactericidal Inhibit protein synthesis
Bactericidal Inhibit cell wall synthesis
Bactericidal Inhibit cell wall synthesis
Bactericidal Inhibit protein synthesis
Bacteriostatic Inhibit protein synthesis
Bactericidal Inhibit nucleic acid synthesis
Bacteriostatic Inhibit protein synthesis
Bacteriostatic Inhibit cell wall synthesis
Bacteriostatic Disrupt cellular permeability
Bactericidal Inhibit DNA replication
Bactericidal Inhibit nucleic acid synthesis
Bacteriostatic Inhibit folic acid synthesis
Bacteriostatic Inhibit protein synthesis
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 T. Galaon et. al.: Biodegradation of antibiotics: the balance between good and bad

Beta-lactams (Amoxicillin) Aminoglycosides (Streptomycin)

Sulfonamides (Sulfamethoxazole) Tetracyclines (Tetracycline)

Macrolides (Erythromycin) Quinolones (Ciprofloxacin)

Fig. 2. Structural representation of some of the most important classes of antibiotics.

Aminoglycosides are another important gram-negative bacteria such as

class of antibiotics which act by inhibiting Pseudomonas, Acinetobacter and
the synthesis of proteins by bacteria leading Enterobacter. Aminoglycosides are
generally to cell death [8]. Important generally used to treat complicated intra-
representatives of aminoglycosides are abdominal, urinary tract infections, and
Streptomycin, Neomycin, Kanamycin, respiratory tract infections.
Tobramycin and Gentamycin. Another important group of antibiotics is
Their structure is usually made of 3-amino represented by Sulfonamides which were
sugar (glycoside) rings which are linked the first class of commercially available
together by glyosidic bonds. antibiotics. These drugs inhibit both gram-
Aminoglycosides are active against aerobic positive and gram-negative bacteria such as

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 T. Galaon et. al.: Biodegradation of antibiotics: the balance between good and bad
E. coli, Salmonella, Chlamydia trachomatis
[9]. Main members of this class are:
Sulfamethoxazole, Sulfadiazine,
Sulfamethazine, Sulfadimethoxine,
Sulfanilamide, Sulfoxazole, etc. From
structural point of view, Sulfonamides have
a common sulfonamide group grafted on
different hydrocarbon structures (aliphatic
or aromatic in nature). These antibiotics are
given to treat urinary tract infections,
septicemia, meningitis and so on.
Antibiotic resistance genes acquisition and
dissemination
In addition to the human consumption of
antibiotics, they are widely used in agriculture
enhancing the livestock growth, bee-keeping,
and fish farming [10]. Unfortunately, the
antibiotic low degree of metabolization and its
high environmental reminiscence combined with
a high dissipation property has become a major
threat to public health by compromising
bacterial infection treatments. In spite of
banning the usage of antibiotics in agriculture
and livestock industries in EU from 2006, a
large number of world population from China
and India keep using them in agriculture and
livestock industries [11].
Moreover, the excessive use of antibiotics in
human and animal medicine [12] over amplify
the antibiotic presence in the environment and
more dangerously this has triggered the selection
of antibiotic resistant bacteria (ARB) and their
antibiotic resistance genes (ARGs) [13]. The use
of antibiotics in hospitals did not mean that
humans are their only target and, unfortunately,
the antibiotics have had also an impact on others
not-targeted organisms [14, 15] inducing
resistance in all organisms after a longtime
exposure [16]. Significant quantities (30–90%)
of consumed antibiotics may be eliminated
unchanged or as active metabolites through
urinary and/or fecal excretion [17, 18]. In spite
of the fact, that hospitals from European
countries discharge only 5–20%
pharmaceuticals, the antibodies concentrations
in hospital effluents have been 100-fold higher
than those of municipal effluents [19, 20]. The
antibiotics could be detected in municipal
sewage water, WWTP biosolids, effluents, soil,
surface waters, groundwaters, sediments, biota
and drinking water [21, 22, 23].
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In all those areas, especially wastewaters and
WWTP, there have been a mixt of antibiotic
chemical structures as well as an exchange
between human and environmental bacteria
could generate environmental stress and
antibiotic resistance [24, 25]. The antibiotic
resistance has been accentuated by the
persistence of low molecular weight (< 1000 D)
antibiotics which are very soluble in water
bodies such as b-lactams, aminoglycosides,
lincosamides, macrolides, nitrofurans,
amphenicols, quinolones, fluoroquinolones,
rifamycins, sulfonamides and tetracyclines [15,
26]. The wastewater treatment has not been
sufficient to completely eliminate
pharmaceutical compounds, including ABs [17]
and the lower remaining amount released to the
environment boost the antibiotic resistance
process.
Long-term exposure at lower concentration of
antibiotics than their minimal inhibitory
concentration (MIC) increased the bacterial
DNA mutation rate of genes, the horizontal gene
transfer (HGT) in the bacterial population and
modulate genes expression, altogether inducing
an antibiotic resistance [27, 28, 29] as well as
further increasing the minimal inhibitory
concentration (MIC) of antibiotics [30].
Antibiotic resistance mechanisms
The ARGs allowed (micro)organisms to develop
resistance by avoiding a specific drug-target
interaction such as DNA, RNA or protein,
rejecting of antibiotics from the cell through
efflux pump (Fig. 3a) and antibiotics
degradation (Fig. 3b) by biotic and abiotic
processes [3].
The functionality of ARGs is extended also to
the survival fight between bacterian strains
which naturally produce antibiotics to eliminate
the competition for the same resources. The
structural characteristics of cell walls (Gram
positive vs Gram negative bacteria –extra outer
membrane- or exopolysaccharide and
extracellular DNA) as well as the bacterial
aggregation in biofilms [31] prevent antibiotic to
reach their targets increasing the level of
resistance [32, 33]. Moreover, the biofilm is
suitable environment for resistance genes
acquisition and spreading among bacterial
populations [34].
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 T. Galaon et. al.: Biodegradation of antibiotics: the balance between good and bad
Fig. 3. Defense mechanisms [35]
Efflux pumps
One of the most efficient bacterial defense
mechanisms relies on the efflux pump through
which bacteria expel ingested chemical
compounds, including the antibiotics (Fig. 3a).
These bacterial efflux pumps may expel specific
substrates or they may have a very wide
specificity, transporting multiple substrates.
Moreover, the bacterial efflux pump has a high
mechanistic homology with the multiple drug
resistance (MDR) mechanisms of Eukariotic
cells. In this respect, a study showed that LmrA,
an ATP-based ABC transporter, present in
Lactococcus lacis is involved in antibiotic
resistance mechanisms by pumping out
amphiphilic compounds from inside the
bacterium and it is homologue with P-
glycoprotein, which is also an ATP-based ABC
transporter involved in the resistance of tumor
cells to chemotherapy [36]. Due to the high
homology between bacterial LmrA and human
P-glycoprotein, LmrA genes were inserted into
fibroblast cells, which generated resistance
effects comparable to P-glycoprotein. Also, P-
glycoprotein-specific inhibitors retain their
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inhibitory properties on LmrA [37]. The fact
that these genes of different origins are
interchangeable shows that the mechanism of
resistance induced by efflux pumps has been
conserved from bacteria to humans.
Physical-chemical degradation of
antibiotics
The abiotic degradation of antibiotics relies
on adsorption, hydrolysis, photolysis,
oxidation and reduction reactions [17, 38,
39]. The antibiotic susceptibility to abiotic
degradation depends on their chemical
structure and up to 70% of administered
antibiotics could be found in the
environment unchanged or degraded to
different degradation products which in
some cases present also antibiotic activity
[40].
Some of these compounds may exert higher
toxicity on the ecosystem than the
antibiotics themselves [41]. The antibiotics
degradation degree and rate depend on their
chemical structure: in some cases, they are
degraded by certain abiotic factors, and
may be unaffected by others.
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 T. Galaon et. al.: Biodegradation of antibiotics: the balance between good and bad
Tetracyclines have smaller structure than
cephalexin and degrade faster by UV-light,
and photolysis [42]. Overall tetracyclines,
sulphonamides, tylosin, nitrofuran or
(fluoro)quinolones antibiotics are sensitive
to UV light, but fluoroquinolones are
resistant to hydrolysis [43]. Oxygen and
ozone are widely spread abiotic factors and
they are involved in the sulfamethoxazole
and oxytetracycline antibiotics degradation
by oxidation [44, 45]. Metal ions such
mercury, copper, zinc, cadmium, and cobalt
are involved in beta-lactam antibiotics
degradation, like Cephalosporin and
Penicillin, by breakage of the β-lactam ring
[46].
Enzymatic degradation of antibiotics
The enzymes play an important part in the
mechanism of antibacterial resistance by
modifying or degrading the antibiotics (Fig. 3b).
Studies showed a correlation between ARG
expression, antibiotic degrading enzyme
production and antibiotic resistant bacteria. In
addition, some studies point to the idea that
ARGs involved in the expression of
aminoglycoside-modifying and β-lactamase
enzymes were present in the environment long
before the use manmade antibiotics in a
therapeutic scope [47, 48, 49] showed that β-
lactamase family shared a common origin with
enzymes involved in cell wall biosynthesis.
In spite of using the antibiotics and their
presence in the environment, bacteria resistant
to β-lactams are thriving and there are a
constant presence in more and more countries
[50, 51, 52].
The same emerging resistance pattern it is also
observed for bacteria producing carbapenemase
[53, 54, 55]. Unfortunately, bacteria producing
carbapenemase, cephalosporinase or oxacillinase
exchange their ARG among them or with other
bacteria from the environment, subsequently
spreading the resistance to antibiotics to other
bacterial communities and other distant
geographical areas [56, 57]. Fluoroquinolone
class of antibiotics is one of the most prescribed
group of antibiotics worldwide. They are used
for treating infections generated by aerobic
gram-positive and gram-negative bacteria.
Among fluoroquinolones, some of the most
important representatives are Ciprofloxacin,
Norfloxacin, Levofloxacin, Ofloxacin and
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others. The main mechanism of action is by
inhibiting DNA replication. For example,
Ciprofloxacin and Ofloxacin inhibit DNA
topoisomerases of bacteria, affecting thus
nucleic acid synthesis. In a biodegradability
study [58] the authors presented several possible
bacteria biodegradation pathways (LC-MS/MS
identification) of Ciprofloxacin generated by a
mixed culture of Gammaproteobacteria,
Bacteroidia, and Betaproteobacteria. Authors
identified several biodegradation products of
Ciprofloxacin generated by deamination (C-N
bond cleavage), hydroxylation, defluorination
(C-F bond cleavage), dealkylation (C-C bond
cleavage) reactions. The structural changes of
Ciprofloxacin are promoted by bacteria
enzymatic degradation. Approximately 47% of
the Ciprofloxacin amount was biodegraded by
bacteria after 7 days and more than 89% the
antibiotic was dissipated after 28 days of
incubation.
The Sulfonamide class of antibiotic drugs are
another important group of antibiotics with high
volume of consumption worldwide. Among its
representatives, some of the most important are
represented by Sulfamethoxazole (SMX),
Sulfadiazine (SDZ), Sulfamethazine (SMZ),
Sulfadimethoxine (SDM), Sulfanilamide (SAD)
and others. In a comprehensive review, Chen et
al., describe the recent advances in
understanding Sulfonamide antibiotics
biodegradation by microorganisms present in
natural and engineered active sludge systems
[9]. The authors describe biodegradation
pathway of Sulfonamides which is mainly
generated by hydroxylation and acetylation
reactions at the aniline or the aminated
heteroaromatic groups of the sulfonamide
structure. Other chemical changes arise by C-N,
C-S and C-C bond cleavage, along with
oxidation. As with respect to the main bacterial
population able to degrade these types of
compounds, the authors report Proteobacteria
and Acidobacteria, Firmicutes and
Bacteroidetes for Sulfamethazine (SMZ)
biodegradation [59]. In the case of
(Sulfanilamide) SAD biodegradation was
promoted by Firmicutes, Bacteroidetes, Bacillus
and Chryseobacterium genus groups [60].
Concerning activated sludge bacteria species
which are able to biodegrade many of the
sulfonamide antibiotics, it is worthwhile to
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 T. Galaon et. al.: Biodegradation of antibiotics: the balance between good and bad

mention Rhodopirellula baltica, Methylibium are not completely processed / biodegradable

petroleiphilum, Micrococcus luteus, Delftia
acidovorans, Oligotropha carboxidovorans,
Acinetobacter, and Pseudomonas [61]. The
biodegradation rate generated by some of these
microorganisms is quite high and can reach even
100% in the case of Sulfamethoxazole.
CONCLUSIONS
Antibiotics are chemical compounds of natural
or chemical synthesis origin which exhibit a
microorganisms killing or growth inhibition
effects. They have a variable range of bacterial
specificity effect from one group of bacterial
strain to many groups of bacterial strains of
Gram positive and Gram negative bacteria. The
beneficial effect of using antibiotics have
expanded to many applications from agriculture
to livestock growth and human health.
The reverse of the medal consists in producing
of more and more amounts of antibiotics which
and subsequently large quantities of antibiotics
will reach the environment.
Uncontrolled usage of antibiotics enhanced their
pollution effect and more important enhancing
antibiotic resistance features. In a natural way,
bacteria have an innate or acquired resistance to
the antibiotics, but the misusage of antibiotics
enhances the antibiotic features.
A solution to prevent the full bacterial antibiotic
resistance has been to continuously synthetize
new chemical compounds which are obtained by
changing or adding different chemical moieties.
The new molecules exert the same or higher
antimicrobial effect and are efficient concerning
bacteria inhibition until the latter develop
adaptation mechanisms. After that we could
focus on the balance between good effects
versus bad effects and their solution of
producing new chemical structure with
antibacterial effect.

ACKNOWLEDGEMENTS. The authors acknowledge the financial support offered by The

National Research Program “Nucleu” through Project codes PN 19 04 01 01 and PN 19 04 02 01.

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 S. Gheorghe et. al.: Anthropogenic pollutants: 10 years of progress in ecotoxicological studies and aquatic risk assessment

Anthropogenic pollutants: 10 years of progress in ecotoxicological studies and aquatic risk

assessment
STEFANIA GHEORGHE1, #, IRINA LUCACIU1, IULIANA PAUN1, GABRIELA GEANINA
VASILE1, JANA PETRE1, VASILE ION IANCU1, CATALINA STOICA1, DANIEL MITRU1,2,
MIHAI NITA-LAZAR1
1 National Research and Development Institute for Industrial Ecology ECOIND Bucharest, 71-73 Drumul Podul Dambovitei,
060652, Bucharest, Romania
2 The Polytechnic University of Bucharest - Faculty of Chemistry Engineering
# corresponding author (e-mail): stefania.gheorghe@incdecoind.ro

The market expansion due to globalization generated more anthropogenic contaminants, enhancing
their negative impact on the environment. In order to prevent and balanced their negative effect,
more rapid, sensitive and eco-friendly ecotoxicological studies as risk assessments were developed.
Each country developed national programs to ensure the research infrastructure for
implementation and development of new testing methodologies, generating new experimental data
transferable to governmental authorities, economic agents and academia.
In this review, we intend to highlighted the progress of Romanian researchers in the last 10 years in
the field of ecotoxicology and aquatic risk assessment. The studies focused on aquatic systems as a
non-targeted system to the anthropogenic pollutants impact.
The implemented methodologies according with international requirements for chemical control
like REACH Regulation, Detergents Regulation, Biocide Products Regulation or the Aquatic Risk
Assessment Guidelines were presented.
The ecotoxicological effects of various contaminants such as detergents, pharmaceuticals,
pesticides, biocides or heavy metals and environmental polluted samples (surface water, sediment
or sludge) were provided.
Overall, the studies presented in our studies have a nationally novelty through the variety of test
substances, the complexity of laboratory experiments and data processing. The research work has a
continue applicability in industry and for control authorities. New accredited testing services that
providing useful information about the chemicals and their impact on the environmental were
introduced. In addition, the ecotoxicology studies and their challenges were a great source for
professional training like PhD and master studies and for impact published papers.

Keywords: chemicals, ecotoxicity, aquatic, risk, bio-indicator, biomarker

The interest of scientists to identify, solve and
prevent the environmental problems has become
a priority in the context of population growth,
waters contamination, global warming and
ecosystems damage or their extinction [1].
Moreover, the human well-being led to chemical
market expansion that have generated new
anthropogenic contaminants in larger amounts,
thus enhancing their negative impact on the
environment. The ecotoxicology of
anthropogenic contaminants became one of the
most published life science topic, due to vast
and complex research sources.
Ecotoxicology field is a complex scientific
discipline base on principles of toxicology,
biology, chemistry and ecology, being focused
on the research of chemical compounds effects
on living organisms, other than man. The
ecotoxicology is separated in two section:
pollutant dynamics – that evaluate the mobility,
migration, transformation, degradation in
different environmental area; and bio toxicology
- that examine the effects of toxic compounds on
sensitive biological systems correlated with field
data [1].
This review intends to highlight the progress of
Romanian research, especially from National
Research and Development Institute for

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 S. Gheorghe et. al.: Anthropogenic pollutants: 10 years of progress in ecotoxicological studies and aquatic risk assessment

Industrial Ecology - ECOIND Bucharest, in the
last 10 years in the field of anthropogenic
pollutants ecotoxicology and their aquatic risk
assessment.
Trend of ecotoxicity studies
The ecotoxicity studies were mainly aimed the
environmental protection through the
compliance of international norms by chemical
industry. The studies have novelty through the
variety of the test substances available on the
national market (such as pesticides, surfactants,
pharmaceutical compounds, biocides), the
complex laboratory experiments (acute, sub-
acute or chronic exposure tests using different
organisms and high performance chemical
detection new methods) and data analyses (such
as risk characterization, REACH classification,
threshold approach system or natural water
classification after Persoone 2003).
The research studies performed in the last 10
years match the international rising trend of
scientific researches in Thomson ISI Web of
Knowledge – Web of Science Platform (Fig. 1).
An exponential increase of published studies in
the field of chemicals ecotoxicology was
observed.
The same trend is maintained also for biotests
studies or risk assessments studies (Fig. 2). The
search of “ecotoxicity” topic in Wiley Online
Library revealed more than 2300 papers
published in the same period. The high interest
for environmental ecotoxicity studies have been
also maintained in 2019. At the start of 2019,
more than 8500 studies were obtained based on
a “ecotoxicology” topic search in Science Direct
Platform. The scientists interest is focused booth
on fundamental and applicative researches. The
fallowed issues are covered: to testing new
toxicants, to develop new eco-friendly biotests,
to find new acceptable environmentally
technologies applied in chemical industry,
agriculture and households and to elaborate new
environmental legislation / guidelines /
regulations.
Concerning the studied ecosystems, the most
papers were published for freshwater toxicity,
followed by marine and terrestrial studies. The
most used biological systems were crustaceans
and fish followed by insects, mollusks, algae,
bacteria and plants. The mono-species tests were
the most used in research studies during 2010 -
2016 followed by multispecies test and two-
species test and in finally community-cosm [1].
This was happening because single species tests
were in the routine of laboratories, easy to be
reproduced and more economic.
The use of bio-indicators / biomarkers in
ecotoxicology is a top subject in fundamental
researches in order to prevent, control and solve
different environmental pollution issues. A
search of the topic “environmental biomarkers”
or “environmental bio-indicators” on Wiley
Online Platform reveled more than 45 000
published documents in the last 10 years, their
number maintaining constant for each year, that
suggest a great interest for this subject.

Fig. 1. Trend of ecotoxicological studies published Fig. 2. Trend of aquatic risk assessment studies
on Web of Science Platform published on Web of Science Platform

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 S. Gheorghe et. al.: Anthropogenic pollutants: 10 years of progress in ecotoxicological studies and aquatic risk assessment
Legal support of ecotoxicity research in
Romania
The ecotoxicology research represents an
important tool of environmental risk assessment
studies, hazard characterization of new
chemicals or the highlighting of some defense /
action mechanisms. Most of these studies have
been carried out in a few universities from
Romania and national research institutes such as
National Research and Development Institute
for Industrial Ecology -ECOIND Bucharest
(http://www.incdecoind.ro).
The Biotests and Biological Analysis Laboratory
of ECOIND Bucharest is the only accredited
laboratory from Romania for ecotoxicity testing
(aquatic toxicity and biodegradation) for
chemical substances / preparations (including
commercial detergents), wastewater, surface
water, groundwater and leachates. The
laboratory has a modern infrastructure with
specific equipment’s for acclimatization and
testing with living organisms obtained following
several successive stages of modernization in
accordance with the scientific progress.
Modern ecotoxicological methodologies were
developed and they are presently in use for the
aquatic risk evaluation of different classes
ofanthropogenic chemical compounds / must be fully biodegradable. In addition, it
industrial chemical preparations / dangerous
chemicals and contaminated environmental
samples.
Legislative framework
Most of the studies were based on industry
research considering the international norms /
guidelines and the requirements for the
integration of Romania into the European
Union. Below are presented some legal norms
that assure the control of chemicals market and
prevent the harmful of environment and humans.
The REACH legislation (Registration,
Evaluation, and Authorization of Chemicals)
and their amendments is in force in Romania
until June 2008. During 2009 – 2018 great
efforts were made to implement and comply the
requirements of this program, by chemicals
producers and also by ecotoxicology assessment
laboratories.
Like other countries, Romania tried to assure a
high level of human health and environment
protection, as well as the free circulation of the
qualitative and environment friendly chemicals
on the national and international market and the
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increase of innovation and competitively, as
priorities of environmental and social politics
(https://osha.europa.eu/ro).
Regulation (EC) no 1272/2008 on
Classification, Labelling and Packaging (CLP)
of substances and mixtures is based on the
United Nations’ Globally Harmonized System
(GHS) and its purpose is to ensure a high level
of protection of health and the environment, as
well as the free movement of substances,
mixtures and articles. It requires manufacturers,
importers or downstream users of substances or
mixtures to classify, label and package their
hazardous chemicals appropriately before
placing them on the market. The hazard classes
in CLP cover physical, health, environmental
and additional hazards
(https://echa.europa.eu/ro).
Special norms are established for detergents,
biocides or pharmaceutical compounds, as well
as for the risk assessment.
Detergents Regulation (EC) No 648/2004
establishes common rules to enable detergents
and surfactants to be sold and used across the
EU, while providing a high degree of protection
to the environment and human health. It
stipulates that surfactants used in detergents
regulates how products should be labelled with
ingredient and dosage information in order to
protect human health (e.g. skin allergies) and
avoid overuse of detergents. This Regulation
was updated by Regulation (EU) No 259/2012
which imposes a ban on inorganic phosphates in
domestic laundry and dishwasher detergents.
Also, the Regulation (EC) no. 66/2010 for EU
Ecolabel establish the ecolabel criteria for the
product with a reduced environmental impact
during their life cycle based on relevant
scientific information (especialy biodegrdability
and toxicity) - https://eur-lex.europa.eu [2,3].
The Biocide Regulation 528/2012 covers the
authorization process and placing of biocide
products on market, the exact amount of biocide
production. According to guidance the biocides
risk assessment needs to be evaluated
compartments and in multispecies experimental
settings.
Part of chemical mixtures enriches the aquatic
environment as anthropogenic pollutants, all
marketed chemicals must have a toxicological
profile for both humans and animals. For
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 S. Gheorghe et. al.: Anthropogenic pollutants: 10 years of progress in ecotoxicological studies and aquatic risk assessment
ecotoxicological assessment and aquatic risk
assessment, laboratory tests on fish, aquatic
invertebrates (Daphnia magna), green algae /
aquatic plants are mostly recommended; in
addition, studies of bio-concentration and
behavior in the aquatic environment (water and
sediment) - degradation, biodegradation,
absorption, etc. are required.
Today, Romania should respond to all European
reglementation concerning the chemicals testing
according with the OECD Guidelines. The
OECD methods on chemical testing summing
up more than 150 standards periodically
upgraded according to scientific progress, used
by authorities, industry or testing laboratories
for chemicals ecotoxicological assessment.
Moreover, our interest section “Effects on Biotic
Systems” include more than 40 biotests with
different species (http://www.oecd.org).
The European chemicals safety guidelines
promote the reduction of animal (especially
vertebrate) testing by introducing the mandatory
data sharing requirement and by encouraging the
use of alternative testing methods [4].
Microbiotests resulted based on OECD test or
ISO / EPA standards as alternative tests more
easy to use, economic and reproducible, ready to
use for establish the harmful effects of
chemicals/contaminated environmental samples
on small organisms (invertebrates, algae,
ciliates, rotifers, plants and bacteria) [5, 6, 7].
The aquatic risk assessment is in force since
2003 based on “Technical Guidance Document
on Risk Assessment: Commission Directive
93/67/EEC on Risk Assessment for New Notified
Substances” publication.
Furthermore, the European Medicines
Evaluation Agency (EMEA) and the US Food
and Drug Administration (FDA), developed and
implemented various environmental risk
assessment guidelines for the ecosystems safety
[8, 9,10]. The documents establish the steps for
hazard characterization through estimation of
PEC (Predicted Environmental Concentration)
and PNEC (Predicted Non-Effect Concentration
on living organisms) values. The value of PEC/
PNEC ratio is widely accepted as endpoint in
aquatic risk assessment model taking into
account the unfavorable scenarios [11].
Supplementary, the EU Water Framework
Directive 2000/60/EC (WFD), suggests that the
hazardous chemicals need to be monitored in
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European continental waters in order to protect
the water resources quality. The scientific
community as well as the EU composed a list of
priority substances including harmful chemicals
such as: pesticides, metals, volatile organic
compound, dioxins, polychlorinated biphenyl
compound and recently pharmaceuticals
substances, which is regularly upgraded under
Water Framework Directive (WFD
2000/60/EC). The Water Directive promote an
integrate approach concerning the evaluation of
natural receptors through collection and
correlation of interdisciplinary data (physical
and chemical parameters, as well as biological,
ecological, hydro-geomorphological and socio-
economic data).
Methodologies and testing strategies
Romania developed national programs to ensure
the research infrastructure for implementation
and development of new testing methodologies
to generate new experimental data transferable
to governmental authorities, economic agents
and academia.
In the field of ecotoxicology were developed
adequate procedures in order to evaluate and
control the effect of anthropogenic
contaminants, more rapid, sensitive and eco-
friendly bioassays as well as risk assessments
studies [11, 12]. For the ecotoxicity bioassay
were needed different species easy to be
acclimatized in laboratory conditions, low-cost,
sensitive to the toxicants, internationally
recognition, representative for the exposed
biological system, indigenous species and
related to possible changes in the affected
ecosystems. Given this, our experimental studies
were conducted on various species to cover the
entire food chain (table 1). Two species of
freshwater fish with economic interest for
Romania (Cyprinus carpio and Carassius
auratus gibelio), two species of crustaceans
(planktonic Daphnia magna and benthic
Heterocypris incongruens), one species of green
algae (Selenastrum capricornutum renamed
Pseudokirchneriella subcapita), one rotifer
(Brachiounus calyciflorus), one protozoan
(Tetrahymena thermophile), a variety of bacteria
species, an aquatic plants (Spirodela polyrhiza)
and three terrestrial plants (Lepidium sativum,
Sinapis alba, Sorghum saccharatum) were
selected to be used in ecotoxicity tests. The
mortality / immobilization, growth inhibitions or
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 S. Gheorghe et. al.: Anthropogenic pollutants: 10 years of progress in ecotoxicological studies and aquatic risk assessment

biochemical changes (gene expression) were the
most used effect endpoints in the laboratory
bioassays. The acute tests (15 min to 96 h period
of test) were frequently used to obtain a rapid
dose – response (L(E)C50). The long term
effects were underlined through chronic fish test
(30-90 days) or ciliates tests (24h). The acute
effects are the most reproducible and can be
calculated with high reliability. Static or semi-
static systems were performed for the acute
exposure of testing organisms to the
contaminants. The bioaccumulation tests were
also used for quantified the metal accumulation
(bio concentration / bioaccumulation factor
(BCF) factors) in the fish or plants tissues in
controlled laboratory conditions or directly in
surface water / sediment.
In addition, the enzymatic activities and /or
histology modifications were assessed to
evaluate the effects of toxicants on fish.

Table 1. Biological tests used in INCD ECOIND, Bioassay-Biological Analysis Laboratory

Type of Test period /
Species Test Matrices Endpoint effect
test incubation
Freshwater fish
OECD 203 Chemical / Mortality, LC50
acute 96h, 21-22°C
SR EN ISO 7346-1 water samples Clinical signs
Growth instant rate, Mortality rate,
Chemical / Biomass mean, Production, used 30-90 days,
In house procedure chronic
water samples food rate and biochemical indicators 21-22°C
Cyprinus carpio / – hepatic enzymes activity, MATC
Carassius auratus gibelio
Enzymatic activity of antioxidant
Chemical / acute or > 96h, 21-
In house procedure enzyme
water samples chronic 22°C
Histopatological modification
OECD 305 Metals acute or Bioconcentration factor (BCF) > 96h, 21-
(Laboratory tests) chronic 22°C
Aquatic plants
ISO 20227, Spirodela Chemical /
Spirodela polyrhiza acute Growth inhibition, EC50 72h, 25°C
Duckweed Toxkit water samples
Planktonic Crustacean
OECD 202, Chemical /
Daphnia magna acute Mortality / immobilization LC50 24-48h, 20°C
Daphtoxkit F water samples
Benthic Crustacean
Chemical /
ISO 14370,
Heterocypris incongruens water samples / chronic Mortality / Growth inhibition, LC50 6 days, 25°C
Ostracodtoxkit F
sediments
Green Algae
Selenastrum capricornutum / OECD 201, Chemical / acute / Growth rate inhibition / biomass 72h,
Pseudokirchneriella subcapita Algaltoxkit F water samples chronic inhibition, EC50 21-25°C
Rotifers
ASTM Standard Guide Chemical /
Brachiounus calyciflorus acute Mortality, EC50 24h, 25°C
E1440-9, Rotoxkıt F water samples
Protozoan
Chemical /
Tetrahymena thermophila Protoxkıt F chronic Reproduction inhibition, EC50 24h, 30°C
water samples
Bacteria
Marine luminescent bacteria - DIN EN ISO 11348-3 Chemical / 15 / 30 min,
acute Luminescence inhibition, IC50
Aliivibrio fischeri BioFix Lumi water samples 20°C

Microbacterium
sp.,Brevundimonas diminuta,
Citrobacter freundii,
Comamonas testosterroni,
MARA test (Microbial
Enterococcus casseliflavus, Chemical /
Array for toxicity Risk acute Microbial growth inhibition, MTC 18 h, 30°C
Delftia acidovorans, Kurthia water samples
Assessment)
gibsonii, Sthaphilococcus
warnerii, Pseudomonas
aurantiaca,
Serratia rubidae, Pichia
anomalia
Geno toxicity test Chemical / Effects on genes expression
Escherichia coli acute 18h, 37°C
SOS Chromotest water samples (β-galactosidase)
Terrestrial plants
Lepidium sativum, Chemical, soil,
SR EN ISO 11269, Germination and root growth
Sinapis alba, Sorghum sludge, waste, acute 72 h, 25°C
Phytotoxkit inhibition, EC50
saccharatum sediment, water
Note: LC50 / EC50 / IC50 – median lethal / effect / inhibitory concentration; MATC - Maximum Acceptable Toxicant Concentration in aquatic
systems, MTC – microbial toxic concentration

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 S. Gheorghe et. al.: Anthropogenic pollutants: 10 years of progress in ecotoxicological studies and aquatic risk assessment

Table 2 Analytical methods for environmental pollutants detection

Environmental pollutants Analytical methods Matrices
High-performance liquid chromatography with diode array detection
(HPLC-DAD) / Agilent 1200 (Agilent Technologies, Germany);
High performance liquid chromatography (HPLC) with multi-waved Water
Pesticides detection (MWD) 1100 (Agilent Technologies, USA); [17, 18, 19,
Liquid chromatographic tandem mass spectrometry method (LC-MS/MS) 20]
1260 / 6410 Agilent (triple quadrupole MS equipped with an electrospray
ionization /ESI source);
High performance liquid chromatography (HPLC) with multiwaved
detector (MWD) 1100 (Agilent Technologies, USA)
Water
Pharmaceuticals Liquid chromatographic tandem mass spectrometry method (LC-MS/MS)
[21, 22]
1260 / 6410 Agilent (triple quadrupole MS equipped with an electrospray
ionization /ESI source)
Inductively coupled plasma atomic emission spectrometry (ICP-EOS)
Water / soil /
technique, Optima 5300 DV Perkin Elmer
biota / fish
Metals High performance microwave digestion system, Milestone Ethos
organs
Atomic absorption spectrometry (AAS) with and without enrichment
[23, 24, 25]
Could-vapor atomic absorption spectrometry (CV-AAS)
Disulfide blue method Water
Surfactants
Orange II method [26]

Our data accumulated over the time, allowed us concentration - UTC) [28]. The smallest EC50
to conclude that the use of microbiotests (with between algae and crustacean will be used for
algae, crustacean and bacteria) have had a good acute LC50 biotests with fish.
reproducibility, relevant and specific results. If no mortality was observed, the test was
Otherwise, because the vertebrate represents the stopped and LC50 greater than (>) the UTC
final target of aquatic pollution, the toxicity tests value was reported. If the mortality was
with fish have an important role in risk observed the test continue at low concentrations
assessment, therefore this can be used in using a dilution factor until no mortality was
combination with microbiotests. observed.
The multi - species tests can offer relevant data
concerning the chemicals risk that may lead to
an economical and efficient assessment of acute
and chronic toxicity [13, 16]. In the Fig. 3 the
acute testing plan of chemicals under REACH
Regulation applied in the ecotoxicity studies was
presented.
Firstly, the data about the chemical / chemical
product/ environment sample were collected to
have a predicted vision about their toxicity in
water. Considering the Klimisch score to
evaluate the quality of experimental data [27]
the need for future information and what test
should be performed were established. If the
data are not sufficient for risk assessment, new
tests are needed.
Secondly, tests using algae, Daphnia and
bacteria were performed to obtain de lowest
EC50 (effective concentration with inhibition /
mortality/ immobilization effect for 50% of Fig. 3. Acute toxicity laboratory testing plan
tested organisms set as upper threshold under REACH [13]
The results led to predict the maximum allowable environmental risk coefficients [10, 11].
surface water concentrations, predicted non effect By regularly consulting of the stage of
concentrations and also to estimate the knowledge’s in the research field, our
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 S. Gheorghe et. al.: Anthropogenic pollutants: 10 years of progress in ecotoxicological studies and aquatic risk assessment
ecotoxicity topics are fitted in the international
development requirements. A statistical analysis
of the frequency uses of fish in toxicity tests
during the last 10 years revealed a 60% decrease
in 2018 compared to 2009 due to the
implementation of new testing strategies and the
use of invertebrates in the first stage of toxicity
assessment (Figure 4). Because the fish, daphnia
Fig. 4. The fish toxicity test trend in the period Fig. 5. Frequency of biotests use in period 2009-
2009-2018
In this context the next paper sections will
present the most important results obtained in
the least 10 years of ecotoxicological research.
The studies were focused on ecotoxicological
characterization of chemicals or environmental
samples using bio-indicators or biomarkers.
Generally, the aquatic risk assessment
representing de the end point of ecotoxicological
studies.
RESULTS AND DISCUSSIONS
Aquatic toxicity - Mono-species experiments
The single species toxicity tests were
appropriate for analyzing toxicological effects
on individual characteristics of species such as
mortality or growth, but with limited
significance for the consequences on the natural
ecosystem.
Before 2008, our laboratory performed LC50
individual species tests using fresh water bio
indicators such as Cyprinus carpio and Daphnia
magna to evaluate the acute or chronic toxicities
of various chemicals (pesticides, metals or other
inorganic or organic compounds) [11, 29].
Currently, this tests are frequently request by
producers or control authorities for chemical
products authorization (such as biocide,
industrial products or industrial wastes) or
environmental samples toxicity control.
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and algae methods are accredited and required
by the economic media, their frequency in the
last period was higher compared to the other
species (such as bacteria and other small
organisms) (Figure 5). Also, ecotoxicity and risk
assessment studies have included test batteries
that specifically targeted these three species,
their use being internationally regulated.
2018
The ecotoxicity studies with single species tests
were performed in case of: i) chronic tests with
fish (Cyprinus carpio) to highlight the chemical
bioconcentration or sublethal effects using
enzymatic activity or protein expression; ii)
efficiency tests with algae (Selenastrum
capricornutum / Pseudokirchneriella subcapita)
developed for the algaecide efficiency test; ii) or
in research studies when the species are selected
to be bio-indicator of natural ecosystem.
To assess the toxic hazard of contaminated
sediment samples, meiobenthic ostracods
Heterocypris incongruens were used as bio-
indicator for a six days’ chronic test.
The percentage of organism’s growth-inhibition
was assessed. The research study involved the
spatial and temporal monitoring of some
physical, chemical and biological indicators both
from water and sediment and subsequently a
correlation with the laboratory ostracods toxicity
test was made. The toxicity tests presented a
realistic view of field effects. The effects on
benthic organisms were directly influenced by a
mixed content of contaminants from sediment,
especially in those Danube areas where the
anthropogenic activities were more intense
(Ivancea and Murighiol). This fact was
correlated with the benthic diversity from this
area [6]. Others specific studies were performed
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 S. Gheorghe et. al.: Anthropogenic pollutants: 10 years of progress in ecotoxicological studies and aquatic risk assessment
to evaluate the subacute effects of chemicals
using the biomarkers expressed in living
organism such as: antioxidant defense enzymes,
hepatic enzymes or protein expression.
Chemicals such as: benzenthonium chloride
(quaternary ammonium compound - cationic
surfactant), cocamidopropyl betaine
(zwitterionic compound amphoteric), atrazine
and mevinphos (organophosphorus pesticides),
trichlorfon and monolinurone (organochlorine
pesticides) were tested. The level of oxidative
stress biomarkers (superoxide dismutase – SOD,
catalase - CAT, glutathione peroxidase - GPx,
glutathione reductase - GR, glutathione S
transferase - GST, glucose -6- phosphate
dehidrogenase – G6PDH) and of lipid
peroxidation - LPO were evaluated. After 96h of
exposure, the toxicity of surfactants (1 mg/l) is
selective at organs levels. The liver and gills
were the most affected by toxic action. The
enzymatic biomarkers assessment highlighted
deficit in reactive oxygen species balance
followed by oxidative stress in fish organs [30,
31]. Also, it was proved that acute intoxication
of fish with atrazine (10 mg/l) and mevinphos
(0.1 mg/l) inhibit the enzymatic activity of
MnSOD and CuZnSOD, the detoxifying
function of SOD enzyme being discredited,
resulting in high death rate during the test [32].
Also, trichlorphon (1 mg/l) and monolinuron
(1.5 mg/l) could induce an easy change in
antioxidant enzymatic activity in fish liver [33].
The hepatic enzymes activity of glutamic
oxaloacetic transaminase (GOT) and glutamic
pyruvic transaminase (GPT) were used as
biomarkers of acute toxicity of some industrial
products (fluids used in metalic processing). The
tests suggested that the products do not have
significant effect on detoxification system and
the mixtures could be considerate ecological
products [34].
The protein expression in algae extracts were
used to evaluate the toxicity of two pesticides
(bentazon and 4-chloraniline).
After seven days of test, a differentiated
expression of cellular protein was showed
according to chemical structure of contaminant,
concentration and exposure time. New
adaptation or defense proteins were identified in
the protein profile comparative with the control.
The results are in the publication process.
Aquatic toxicity - Two-species experiments
Romanian Journal of Ecology & Environmental Chemistry ● No.1 ● 2019
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In the context of new interest of the European
Commission concerning the improvement of the
environmental performance of products and the
increase the demand for more sustainable goods
and production technologies by 2030, our
laboratory developed ecotoxicity test suitable for
each class of chemicals including the biocide.
The use of two species from the same trophic
level test is exceptionally applied to showed the
sensibility difference. A study of biocides lethal
and sub lethal effects was performed using two
representative crustacean Daphnia magna
(pelagic system) and Heterocypris incongruens
(benthic system). The tests showed a significant
difference of sensitivity between species. The
biocides induced a very toxic or toxic effect on
Daphnia magna. On the contrary, the observed
ostracods lethal effects were less than 50%
mortality and no significant growth inhibitions
were observed [35].
Aquatic toxicity - Three-species experiments
The phytotoxic effects of some contaminated
environmental matrices such as metal /
petroleum products polluted soil or sewage
sludge were test for three days in the same
condition using three higher plants Sorghum
saccharatum, Lepidium sativum and Sinapis
alba. The inhibition of germination and roots
growth inhibition were monitored. The
anthropogenic pollutants from the contaminated
samples determined different sensitivity
responses. The tests could be use as routine
toxicity screening tests to provide rapid
informations for soil contamination [36], waste
water or sludge reuse [37, 38], bioremediation
and environmental risk assessment.
Aquatic toxicity - Multi-species experiments
The biotests battery is based on acute sensitivity
responses of a various representative species of
trophic chain in order to predict the natural
system behavior to the chemical contamination
distress. The multi-species tests include species
of algae, crustacean, fish, and also rotifers,
plants or bacteria. Thereby, a series of specific
laboratory studies were performed to assess the
toxicological effects of cationic and amphoteric
surfactants [39], pharmaceutical compounds
(analgesics, antibiotics, nervous stimulators)
[16], pesticides [11] and other organic
compounds complexed with metals [40]. The
final results allowed us: i) to reveled a
differentiated response for each trophic chain
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 S. Gheorghe et. al.: Anthropogenic pollutants: 10 years of progress in ecotoxicological studies and aquatic risk assessment
level; ii) to establish a specific effect
concentration (LC50/ EC50/ IC50/NOEC); iii)
to classified the toxicity hazard according to
international norms for chemical classification;
iv) to select the unfavorable case (the most
sensitive organism); v) to evaluate the PNEC
values taking in consideration de environmental
dilution factors; vi) to calculate the risk
coefficients considering the real contamination
of natural water.
The organism’s sensitivity depended on
chemical class, exposure time or tested
concentration.
Generally, the chemicals designed for a specific
target organism (such as antibiotics / biocide /
some surfactants with biocide action for bacteria
or pesticide for plant photosynthesis inhibition)
generated toxicity on this species or related to
them. A statistical analysis of acute toxicity
results obtained in period of 2008 - 2011
revealed that 60 % of results showed that
Daphnia magna is the most sensitive to
chemical exposure (respectively pesticides,
anilines, nitroderivates, metals and surfactants
and other dangerous organic compounds) [11].
Fish and bacteria were affected in 15% from
tests. Algae responded only in case of some
cationic surfactants or biocide with algaecide
action.
The biotests battery was also applied to evaluate
the complementary toxicity generated by
surfactant biodegradation in laboratory
condition. Using a classification system of
effluent toxicity [36] based on acute toxicity
data of at least three different aquatic species
(fish, algae and daphnia), was concluded that
some cationic surfactant (such as
benzenthonium chloride) persists in the
biodegradation effluents, being classified as
toxic acute for aquatic system especially for
bacteria and algae [26, 39].
The performance of some waste water or
drinking water treatment technologies for the
natural or anthropogenic pollutants removal,
were evaluated through the effluents toxicity
testing. For example, flotation processes that use
caffeic acid complexed with Cr(III) and Pb(II)
were monitored for effluents toxicity using a
multi species battery. The experiment showed
that pelagic crustacean and luminescent bacteria
were the most sensitive. On the fish, algae and
gram positive and negative bacteria no toxic
Romanian Journal of Ecology & Environmental Chemistry ● No.1 ● 2019
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effects were observed [40].
Danube River and Danube Delta water and
sediment were tested and diagnosed for their
toxicity using a battery tests with producers
(algae - Selenastrum capricornutum, plants -
Sorghum saccharatum, Lepidium sativum,
Sinapis alba) and consumers (rotifers –
Brachionus calyciflorus, crustaceeans –
Daphnia magna, Heterocypris incongruens).
The water toxicity was classified according to
the hazard classification system for natural
water. The effect percentages (EP) or
LC/EC50% of each type of organisms is
transformed in toxicity units (TU) using
formula: TU = 100/EP (LC/EC50%.) The
current classification system is based on acute
toxic / hazard being divided in five hazard
classes [41]. The largest number of water toxic
responses was observed in case of algae,
followed by crustaceans and rotifers. In case of
sediment, the order was ostracods followed by
plants [15].
Aquatic toxicity - Bioconcentration /
bioacumulation
Bioconcentration studies represented a novelty
for our laboratory due to the test complexity and
also because of analytical challenges. Some non-
clinical studies, similar with the pollution of
some rivers (Danube River or Olt River), were
performed for bioconcentration capacity of
heavy metals (Cu, Zn, Ni, Mn or Hg) in
common carp (Cyprinus carpio). It was found
that the metals are bioacumulated in organs and
tissues as followed: liver > kidneys > gills>
gonads > red muscles > white muscles > skin>
brain = heart > intestines. Differences in metal
concentration between individuals or between
receptor organ (e.g. liver versus kidneys or gills)
were highlighted, suggesting the specificity in
metals metabolism [23, 42]. Interesting results
were obtained using field samples (organs of
Cyprinus carpio and Carrasius gibelio) as well
bivalves (Dreissena sp. and Anodonta sp.)
collected from Olt River. The total Hg content
was above the limit allowed by the World
Health Organization (0.5 mg/kg) in muscle and
spleen samples harvested from carp specimens.
In benthic invertebrate specimens, the Hg
content ranged from 0.19 to 0.52 mg/kg dry
matter in internal organs, while Hg value in
shells was below the method quantification limit
[23].
34
 S. Gheorghe et. al.: Anthropogenic pollutants: 10 years of progress in ecotoxicological studies and aquatic risk assessment
More results and final conclusion of metals
bioconcentration studies are in publication
process. Other studies used lichens Parrmelia
spp. as biosensors in the monitoring of
environmental pollution with heavy metals (Cd,
Zn, Pb). The lichens have classified the polluted
sites by the characteristics of bioaccumulation of
metals in lichens. Based on the corticols lichens
specificity a correlation index of air quality in
the interest geographical areas was calculated.
Also, the capacity to accumulate metal in the
lichen communities has been used to identify the
level of air pollution due road traffic. Based on
the degree of heavy metal accumulation such as
Cd, Cr, Cu, Fe, Mn, Pb, Ni and Zn in the
Parmelia spp. lichens, a correlation has been
made, with the road traffic. According to the
metal bioaccumulation degree in lichens tissue,
it has been established that the sources such as
traffic from the outskirts of cities, from the
perimeter of gas stations and of county roads
continuously spread products which contain
these elements, into the atmosphere [43].
The aquatic plants such as Lemna minor L. has
been reported to accumulate toxic metals and is
therefore used as an experimental model to
Fig. 6. Chemical aquatic risk assessment
Thereby, risk characterization researches were
initiated for some hazardous chemicals (4-
chloroaniline, 1-chloro-4-nitrobenzene and 4-
chloro-2-nitrotoluene), pesticides (azinphos-
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investigate the accumulation of heavy metals.
The study shows that the Lemna minor L.,
accumulates considerable amounts of Zn, Cu
and Ni without any symptoms of toxicity [44].
Risk assessment studies
The ecosystem health is usually based on risk
assessment considering the chemical
concentration in the environment (PEC) and the
impact on organisms (PNEC). A ratio of
PEC/PNEC < 1 indicated no aquatic risk and no
future assessments is deemed necessary.
Our studies on chemical risk assessment applied
the international methodology adjusted to our
laboratory conditions and indigenous organisms.
After 2009, the final results of our research
studies was to obtain an risk study. The
integrated chemicals risk studies involved data
of chemical detection in laboratory and field
condition, toxicity experimental data of
chemical on living organisms, biodegradability,
environmental contaminated samples collection
(toxicity testing and chemical detection), sub
lethal effects evaluation, selection of the most
sensitive organisms and risk level establishment
(Fig. 6).
methyl and bentazone), pharmaceuticals
(diclophenac, acetaminophen, ketoprophen,
indomethacin, naproxen, ibuprophen,
carbamazepine, caffeine, ciprofloxacine and
35
 S. Gheorghe et. al.: Anthropogenic pollutants: 10 years of progress in ecotoxicological studies and aquatic risk assessment

trimethoprim), surfactants (benzenthonium The research studies results were quantified as

chloride and cocamidopropyl betaine) and heavy
metals. The studies showed the risk level of each
chemical compound base on field data
concentration respectively Romanian surface
waters (Danube River, Danube Delta, Arges
River, Mures River, Ciorogarla River and
Ghimbasel stream) comparatively with other
international rivers [11, 16, 29]. The results
showed different levels of risk in accordance to
detected environmental concentration, to acute
and chronic toxicities and the environmental
compartment (water or sediment). High level of
risk for water compartment for ciprofloxacin,
benzenthonium-chloride, azinphos methyl and
arsenic were showed [16, 29]. Moderate risk for
diclofenac and ibuprofen was estimated [16].
CONCLUSIONS
The research national program of Romanian
Research and Innovation Ministry supported in
the last 10 years’ various studies on the toxic
effects and aquatic risk generated by the
anthropogenic chemicals. The Biotests -
Biological Analyses Laboratory of National
Research and Development Institute for
Industrial Ecology ECOIND Bucharest
developed and implemented ecotoxicological
methodologies for different classes of chemical
compounds / industrial chemical mixtures /
dangerous chemicals and contaminated
environmental samples and characterization of
aquatic risk.
The results were used at socio-economic level
(creation of new services, partnerships /
collaborations with SMEs / universities,
development personal training, external
visibility) and in environmental protection
(providing useful information to manufacturers /
importers and control authorities) or for in the
establishment of new research partnerships.
follows: seven experimental studies and
methodology for toxic effect evaluation on
aquatic organisms for different classes of
chemicals and aquatic risk assessment; two
experimental studies for characterization and
hazard classification of contaminated
environmental samples; seven toxicity tests that
could be offered as services; eight new detection
methods; three PhD thesis; more than 30 ISI
published papers; more than 150 citations; three
chapters book; more than 30 conferences /
symposiums participation; about new 100
customers. Also, beside of the NUCLEU
projects (2009-2018), other seven national
research project and a European cooperation in
science and technology - COST action was
developed. After 10 years of experience the
most demanding issues in ecotoxicology studies
were: 1) the identification of relevant and
reproducible data sources and 2) the access to
authority’s databases such as consumptions and
discharges of pollutant type. In addition, there
are still gaps in understanding of regulations and
in communication between environmental
authority’s, testing laboratory, producer /
importer and not least the consumer. Given that,
great efforts are being made to collect and sort
information on testing / interpreting and
reporting results so that the final result
correspond to the ultimate standards.
Our future interests are to expand the research at
molecular level in order to understand the
pathways involved in acute and chronic toxicity
and predict the mortality, to implement new
OECD tests (such as early stage assay) and to
use alternative tests (chemical exposure of fish
cell line and bioaccumulation). We intend to
develop biomarkers to assess the toxicity effect
of a large range of pollutants on various
biological models.

ACKNOWLEDGEMENTS. These researches were performed by National Research and

Development Institute for Industrial Ecology Bucharest with the financial support of the Romanian
Research and Innovation Ministry in the period of 2009 – 2018 (NUCLEU Program, CEEX
Program, Inovation Program, POS CCE Program, POC Program).

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 S. Gheorghe et. al.: Anthropogenic pollutants: 10 years of progress in ecotoxicological studies and aquatic risk assessment

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 I. Nitoi et. al.: Advanced Oxidation Processes (AOPs) alternative methods for degradation of toxic pollutants from wastewater

Advanced Oxidation Processes (AOPs) alternative methods for degradation of toxic pollutants
from wastewater

INES NITOI, LUCIAN ALEXANDRU CONSTANTIN, IONUT CRISTEA, MIRELA ALINA

CONSTANTIN

National Research and Development Institute for Industrial Ecology ECOIND Bucharest, 71-73 Drumul Podul Dambovitei, 060652,
Bucharest, Romania
*Corresponding author (e-mail): ines.nitoi@incdecoind.ro

The degradation of some toxic pollutants like chlorobenzenes, nitrobenzene and 4-chloroaniline in
various advanced oxidation system such as: UV/ H2O2, UV-VIS/Fe-TiO2 and UV-VIS/TiO2/ H2O2
were studied. The influence of working conditions (pH0, H2O2 dose, photocatalyst dose, pollutant
initial concentration and irradiation time) on pollutant degradation rate constant and efficiency
were investigated. For any studied advanced oxidation systems, pollutant degradation followed a
pseudo first order kinetics. The degradation pathway of chlorinated and nitro aromatic pollutants
includes initial •OH radicals attack to aromatic ring with hydroxylated intermediates formation,
followed by their step by step oxidation up to carboxylic acids. Inorganic ions like Cl-, NO3-, NH4+
are also formed as mineralization products.

Keywords: chlorobenzene, nitrobenzene, 4-chloroaniline, photocatalyst, H2O2, kinetics and

pathway degradation

Water is essential for life and although
approximately 70% of the terrestrial area is
covered with water, only a small fraction (2.5%)
is freshwater compatible with terrestrial life [1].
This low stock of available freshwater is being
accompanied by an increase of human
consumption as consequence of demographic
growth, industry demand and improved living
conditions. It is even estimated that in some
world regions, the water use has risen twice
faster than the human population. In fact, half of
the European countries are already facing water
stress and it is estimated that within the next 50
years more than 40% of the population
worldwide will face water stress or scarcity [1-
3]. All this together represents a solemn
inducement to achieve sustainable management
options of the water resources [1]. The human
being plays a major role on the issue of
pollutants released in wastewater, which
includes heavy metals, antibiotics, pesticides,
polyaromatic hydrocarbons, halogenated
compounds, and endocrine disruptors, most of
them known as emerging pollutants [4, 5].
As conventional methods applied in wastewater
treatment are limited and their associated
operational and capital costs are high, the issue
of emerging pollutants removal from
environment is not completely solved [6].
The results achieved in latest years showed that
Romanian Journal of Ecology & Environmental Chemistry ● No.1 ● 2019
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Advanced Oxidation Processes (AOPs) based on
photochemical and photocatalytic approaches
seem to be very promising as viable alternatives
for appliance on wastewater treatments of
different sources.
These approaches allow the transformation of
pollutants into less toxic substances and/or with
structural features that are more biodegradable
[7] and are based on the combination of
oxidizing agents with an appropriated catalyst
and/or light [7, 8].
These processes may be of particular interest for
the effluent’s treatment containing highly toxic
compounds like emerging pollutants, and for
which the biological processes might not be
appropriate [7].
AOPs involves specific chemical reactions that
generate highly reactive chemical oxidizing
species which are capable to oxidize and
mineralize almost any organic molecule, even
the most recalcitrant molecules to CO2 and
inorganic ions [7–10]. These processes generally
involve use of light. The degradation by
photochemical reactions of organic pollutants
present in water media can be achieved, but the
degradation degree is dependent on their
chemical structure [11]. The AOPs efficiency
tends to be maximized when the use of an
appropriate catalyst is considered. Although
there are different oxidative systems, the
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 I. Nitoi et. al.: Advanced Oxidation Processes (AOPs) alternative methods for degradation of toxic pollutants from wastewater
production of powerful oxidizing agents, such as
hydroxyl radical (HO•), is the main objective of
most AOPs, since these oxidizing agents react
very fast [9]. Also, their nonselective attack to
organic compounds is a useful attribute for
wastewater treatment and a potential solution of
pollution issues. These oxidizing species can act
at various levels such as hydrogen abstraction
from aliphatic carbon, electron transfer or by
addition to double bonds and aromatic rings
[7,10,12]. This type of reactions generates
organic radicals as intermediates, which then
undergo additional reactions, ending in final
products [13]. The hydroxyl radicals can react
with a vast range of organic compounds at rates
often nearby the diffusion-controlled limit
[7,14,15].
The versatility of the AOPs is likewise enhanced
by the fact that exist different ways to produce
hydroxyl radicals, facilitating the compliance
with specific treatment requirements in
accordance with effluent’s characteristics [9,10].
The AOPs as a photochemical process can occur
according two main ways: homogeneous (when
both reactants and pollutants are in the same
phase) and heterogeneous systems (when
reactants and catalysts are in different phases. In
general, photocatalytic process can be conducted
under visible-light irradiation either with or
without the presence of UV radiation [7]. The
region of the electromagnetic spectrum used to
the photochemical process is dependent on the
reactant/catalyst absorption features [10].
Methods based on ultraviolet light (UV), and
H2O2 use the photolysis of this oxidizing agent
to produce hydroxyl radicals [9,10].
Additionally, other applied methods like
heterogeneous photocatalysis, are based on the
use of a wide-band-gap metal oxide
semiconductor without or with addition of H2O2
to retardation of electrons-holes recombination
process which assures enhance of catalyst
photoactivity, respectively, and irradiation with
UV–VIS light. Both processes are of interest due
to fact that the homogenous system has
advantages of hydrogen peroxide which is easy
to handle and environmentally safe, since in the
heterogeneous system sunlight can be used,
especially for doped semiconductors, which
brings a set of economic benefits.
There are several types of AOPs used in
WWTPs, each one of them can be effective in
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certain types of wastewater and operational
conditions. The AOPs which will be reviewed in
the following subsections are hydrogen peroxide
assisted photocatalysis (UV/H2O2) and
photocatalytic process in the following systems:
UV/0.5Fe-TiO2 and UV/TiO2/H2O2. Target
studied pollutants have been halogenated
aromatic compounds like chlorobenzenes (CB)
and 4-chloraniline(4CLA) and nitro aromatic
derivative, respectively nitrobenzene(NB), all of
them included in the emerging compounds lists.
EXPERIMENTAL PART
The pollutants photodegradation was carried out
in a Laboratory-UV-Reactor system (Heraeus)
with medium-pressure mercury lamps which
emit in the range of 200-280 nm (UVC) (TQ150
lamp), and respectively in the range 300-500nm
(UV-VIS) (TQ150-Z2).
The lamp equipped with a quartz cooling jacket
was immersed in the center of the reactor
containing the pollutant solution. The photon’s
flow of the emitted radiations was determined
by ferrioxalate actinometry and values I0 = (5.3-
6) x10-6 einstein s-1 were found.
Solutions with CB concentrations between
0.1x10-3 - 1.8x10-3M and of H2O2 between
0.3x10-2 – 9x10-2 M, which correspond to molar
ratio [H2O2]/[CB] in the range 5/1-50/1, were
used in peroxide assisted photolysis
experiments. In the case of photocalysis
experiments in UV-VIS/metal doped TiO2,
solutions with (0.37-8.45) x 10-4 M NB content
were photo-oxidized in the following working
conditions: pH= 4-10; photo catalyst dose = 50-
500 mg/L; irradiation time = 30 – 240 minutes.
Experiments performed in UV/TiO2/H2O2system
were conducted with various concentration of
H2O2 between 1 x 10-4 – 1 x 10-2 M, at fixed
pH=4, TiO2 dose 150 mg/L and 1.42 x 10-3 M 4-
CLA concentration. Prior to irradiation, the
required powder photocatalyst dose was added
to samples, and the solutions were bubbled with
air (50L/hour) for 30 min in the dark for both
photocatalytic systems studied. The sample’s pH
was controlled using a Consort C532 pH-meter.
The solution within the reactor was
homogenized continuously using a magnetic
circulation pump.
The concentration of CB and NB were
determined via Gas Chromatography couple
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 I. Nitoi et. al.: Advanced Oxidation Processes (AOPs) alternative methods for degradation of toxic pollutants from wastewater
with flame ionization detector (GC-FID) using
an Agilent 6890N equipment. The concentration
of 4-CLAwas analyzed by GC-MS using an
Agilent 7890A Gas Chromatograph with 5%
phenyl methyl siloxane capillary column
coupled with an Agilent 240 Ion Trap Mass
Spectrometer.
GC-MS screening method was applied for
qualitative identification of 4-CLA degradation
intermediates, by coupling Agilent 6890N with
DB-5MS capillary column to mass spectrometer
5975 BinterXL with electron ionization (70 eV).
Total Organic Carbon concentration was
determined using an MULTI N/C 3000 Analyzer
(Analytik Jena).
Mineralization products (Cl-, NH4+, NO3-) were
determined using aDionex ICS-3000 Ion
Chromatograph, with separate anions and
cations columns and suppressed conductivity
detectors.
All experiments were duplicated to assure data
quality.
The following reagents were used:
chlorobenzene (Merck) of 99wt. % investigate
purity;nitrobenzene (Merck) of 99wt. %
purity;4- chloroaniline (Aldrich) of 99wt.%
purity;TiO2 (Merck) powder with 80 wt.%
anatase; metal - doped (0.5-5wt% Fe, Ni, Co)
TiO2 powders synthesizedby alkoxide route of
sol-gel method (Institute of Physical
Chemistry);hydrogen peroxide (Merck) reagent-
grade 35wt. % solution; Na2SO3 (Merck)
98wt.% purity used for the removal of
unconsumed H2O2 from irradiated samples;
disodium ethylenediaminetetracetate (Sigma
Aldrich) of 98 wt.% purity, isopropyl alcohol
(J.T.Baker) of 99.7wt% purity and 1,4-
benzoquinone (Sigma Aldrich) of 99.5 wt.%
purity utilised as holes (h+), hydroxyl radical
(•OH) and superoxide radical (O2•-) scavengers;
NaOH (Merck) 99wt. % purity and H2SO4
1N(Merck)were used for pH correction; n-
hexane (UniSolv) GC grade used for pollutants
and intermediates extraction from water
samples; anhydrous Na2SO4 (Merck) 99wt. %
purity used for removal of water traces from n-
hexane extracts; KI (Merck) 99wt. % purity and
Na2S2O30.1N (Merck) used for iodometric
determination of H2O2.
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RESULTS AND DISCUSSION
H2O2 assisted photolysis of CB
This process is effected by irradiating the
pollutant solution containing H2O2 with UV
light having wavelengths < 280nm, that causes
the homolytic cleavage of H2O2 [16].
H2O2+hν→2•OH (1)
In aqueous solution, the cage effect of water
molecules lowers the primary quantum yield to
0.5:
2AB +hν→ 2(A•-------- B•) → A• + B•
↓
AB
Since H2O2 itself is attacked by OH radicals
2(H2O2+•OH) →2H2O+2HO2• (2)
2HO2•→H2O2+O2 (3)
the overall quantum yield becomes one. The
major drawback of the process is due to the
small molar extinction coefficient of H2O2
which is only 18.6M−1 cm−1 at 254nm, only a
relative small fraction of incident light is
therefore exploited [17].
Various studies have been carried out to
the effectiveness of AOP
technologies toward the degradation of a wide
variety of halo aromatic compounds [18-21].
These studies revealed that in a UV/H2O2
system, degradation process rates were strongly
affected by the operating conditions like
H2O2/pollutant molar ratio, intensity of UV
radiation, reactor geometry, and irradiation time
[22,23]. Some authors which investigated
degradation of chlorobenzene in UV/H2O2
system showed that depending upon the
irradiation time, various intermediates were
formed such as chlorophenol, different isomers
of chlorobiphenyl and dichlorobiphenyl [24].
They observed the pH decrease during the
irradiation and attributed it to the release of
chlorine as HCl in the solution. The
investigation of formed intermediates was also
carried out by other authors [25-27], which
reported low molecular weight oxygenated
compounds as oxidation products. At longer
enough irradiation times (hours), the subsequent
degradation of these compounds may lead up to
mineralization [24]. It was reinvestigated the
photodegradation of CB in UV/ H2O2 system in
order to bring new kinetic information which
support degradation mechanism of CB reported
in literature [24,27,28].
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 I. Nitoi et. al.: Advanced Oxidation Processes (AOPs) alternative methods for degradation of toxic pollutants from wastewater

Kinetics of CB degradation in UV/ H2O2 system Using the slopes of these straight-lines the rate
and H2O2 concentration influence constants kCB and kCl- were calculated. In Figure
The photodegradation kinetic of CB in the 2 there is presented the evolution of the rate
presence of H2O2, at pH=7, under UV irradiation constants versus the hydrogen peroxide
verified a pseudo-first-order reaction rate with concentration.
respect to the pollutant and chloride ion, one of It is observed that values of kCl-are always lower
the mineralization final product [28]. Figure 1 than values of kCB, suggesting that the Cl-
presents the kinetics monitored at pH=7, for a liberation does not take place at the same time
sample with an initial CB concentration of with the attack of •OH radicals on CB
1.8x10-3M at various H2O2 concentrations molecules. In a first stage chlorinated organic
corresponding to a molar ratio of [H2O2]/[CB] intermediates are formed, which are further
between 0/1-50/1. releasing chlorine as Cl- ion. In the range of low
H2O2 concentration the both rate constants
Fig. 1. The pseudo-first-order kinetics at various increase linearly with H2O2concentration. On the
H2O2 concentration of contrary, at high H2O2 concentration, these rate
a) CB degradation and b) Cl- formation constants decrease due to the fact that •OH radi-
cals are scavenged by the excess of H2O2.
0 Between these two ranges kCB and kCl- reach
their maximum values 15x10-4 s-1 and 9x10-4 s-1,
[H2O2]/[CB]
-1 respectively, for a short interval of H2O2
0/1 concentration (5.4x10-2 - 7.2x10-2 M)
-2
0/1 R2 = 0.9808
corresponding to a [H2O2]/[CB] molar ratio 30/1 –
5/1 R2 = 0.9768
40/1. The extrapolation of the linear part leads to
ln[CB t]/[CB 0]

5/1
10/1 R2 = 0.9982
-3 20/1 R2 = 0.9798 10/1 rate constants values near to those experimentally
25/1 R2 = 0.9982
50/1 obtained in the absence of photosensitizer,
30/1 R2 = 0.9871
-4 via CB direct photolysis.
40/1 R2 = 0.9911 20/1
50/1 R2 = 0.9592
Therefore, this behavior can be described by the
25/1
-5 40/1 kinetic equation: kCB = k0 + k∙OH [H2O2], in
a)
30/1
which kCB is the rate constant of CB degradation
-6 determined for various initial H2O2
0 300 600 900 1200 1500 1800 2100 2400 2700 3000 3300 3600
Time, s
concentrations, k0is the rate constant of CB
photolysis and k∙OH the second-order rate
constant of CB oxidation by OH radicals. In Fig.
3.5 [H2O2]/[CB]
30/1
2 the first linear part represents in fact the plot of
0/1 R2 = 0.9811
2
5/1 R = 0.9989
40/1 before mentioned kinetic equation. A similar
3
b)
10/1 R2 = 0.9955 25/1
equation is valid also for Cl- formation. Some
2.5
2
20/1 R = 0.9981 20/1 other researchers [24] using similar molar ratio
2
25/1 R = 0.9975 [H2O2]/[CB] than in our experiments found that
-ln(1-Cl t]/[Cl f])

the straight-line obtained obeys equation: kCB =

-

2 30/1 R2 = 0.9978 50/1

2
40/1 R = 0.9976 k∙OH [H2O2], which shows that only H2O2
-

10/1
1.5 50/1 R2 = 0.99 mediated degradation takes place in their
irradiated system. However, a similar behavior
1 5/1
of the system to that we reported was presented
0/1 by the same authors in their earlier work, but it
0.5
was not explained. We consider that the
0
contribution of direct photolysis to CB degrada-
0 300 600 900 1200 1500 1800 2100 2400 2700 3000 3300 3600 tion is due to the manipulation of the samples at
Time, s the room light before the irradiation in the photo
reactor.

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 I. Nitoi et. al.: Advanced Oxidation Processes (AOPs) alternative methods for degradation of toxic pollutants from wastewater
Fig.2. The influence of H2O2 concentration on the rate constants kCB and kCl-.
Reaction mechanism
Many scientists studied the photodegradation of
CB in various experimental conditions, like
photolysis, UV/H2O2, UV/O3, UV/TiO2 but only
some of them analyzed the systems in order to
obtain information on the reaction mechanism
[24,27,29]. We tried to correlate these data with
our results to obtain a general plausible reaction
mechanism. This includesH2O2photolysis under
UVC irradiation and generation of •OH radicals
which by addition to the double bound, or by
hydrogen abstraction attacks the CB leading to
various intermediates [30], the majority being
hydroxylated aromatic compounds. Using GS-
MS analysis, some authors identified
chlorophenol, chlorobiphenyl and
dichlorobiphenyl [24]. Other authors studding
the CB degradation in UV/TiO2 systemrevealed
that o- and p-chlorophenol are further oxidized to
hydroquinone and catechol [29]. Using flash
photolysis/HPLC technique for the degradation
of chlorophenol in the presence of H2O2, have
been put in to evidence the same hydroxylated
compounds [27]. It has also been shown that
increase of H2O2 concentration changes the
product distribution, catechol concentration drop
in the favor of hydroquinone and 1,2,4-three-
hydroxybenzene. The formation of these
unchlorinated intermediates agrees with our
results which show the total liberation of the
organic bound chlorine as HCl, during
irradiation. On the other hand, our finding that
kCB>kCl- demonstrates that Cl- ions liberation
does not take place simultaneously with the
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attack of •OH radicals on CB, but in a further
stage, oxidizing the chlorophenols to
hydroquinone and three hydroxibenzene.
According to our studies, formed intermediates
compete with CB for •OH radicals consumption
as experimental results are emphasizing by the
rate constant values. The rate constants values
decrease with an increase in the initial CB
concentration, at constant [H2O2]/[CB] molar
ratio. The before cited authors [27] reported that
hydroquinone is oxidized to benzoquinone
which is cleaved in muconic acid and then under
•OH radical attack leads to maleic, fumaric and
oxalic acids. In the favor of this presumption we
have a qualitative argument: by the appearance
of a reddish-brown color, characteristic to
benzoquinone, which under prolonged exposure
time bleaches due to carboxylic acids formation.
The presence of many stable intermediates is
justified by the low removal yield of total
organic carbon (TOC) of about 14%, determined
after irradiation, in our experiments [28].
Photocatalytic pollutant degradation
These processes use a semiconductor metal
oxide as catalyst and oxygen as oxidizing agent
[31]. Many catalysts have been so far tested,
although only TiO2 in the anatase form seems to
havethe most interesting attributes such as high
stability, good performance and low cost [32,
33]. The initiating step in the photocatalytic
process is the absorption of the radiation with
the formation of electron-hole pairs:
TiO2+hν→e−+h+ (4)
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 I. Nitoi et. al.: Advanced Oxidation Processes (AOPs) alternative methods for degradation of toxic pollutants from wastewater

The considerable reducing power of formed other oxidizing agents. There is another
electrons allows them to reduce some metals and advantage related to the use of this type of
dissolved oxygen with the formation of the oxidant when solar energy is the photon source.
superoxide radical ion O2•− whereas remaining This is related to increase of the photocatalytic
holes are capable of oxidizing adsorbed H2O or reaction rate which assures proportionally
OH − to reactive •OH radicals: decrease of photo reactor dimensions and
TiO2(h+) + H2Oad→TiO2+•OHad+H+ (5) dramatically decrease of overall costs.
TiO2(h+) + OH−ad →TiO2+•OHad (6) Hydrogen peroxide is the obvious candidate. It
These reactions are of great importance in can increase the efficiency of the process at high
oxidative degradation processes due to the high irradiance and it has been tested with a large
concentration of H2O and OH− adsorbed on the number of compounds. Also, it is a very
catalyst particle surface. commonly used chemical and, therefore, very
cheap. Being an electron acceptor, hydrogen
Some adsorbed substrate can be directly peroxide reacts with conduction band electrons
oxidized by electron transfer: to generate hydroxyl radicals, which are
TiO2(h+) +RXad→TiO2+RXad•+ (7) required for the photo mineralization of organic
Unfortunately, a significant part of electron-hole pollutants.
pairs recombine thus reducing the quantum yield
of photocatalytic process. In the most part of the H2O2+e-→•OH+HO- (8)
works devoted to the photocatalysis the possible The following reactions (eqs. 9,10) can also
exploitation of the wavelengths of the solar produce •OH (eq.10, which requires λ < 300 nm,
spectrum is stressed. However, this is only does not take place with solar radiation).
partially true since the overlapping between the
absorption spectrum of TiO2 and that of the sun H2O2 +O2•- → •OH + HO- + O2 (9)
at ground is rather poor [33]. Intensive H2O2+hν → 2 •OH (10)
researches are carried out worldwide to obtain The effect of this electron acceptor deserves
modified (doped) TiO2 with broader absorption some further comment. In some cases, the
spectrum and characterized by higher quantum addition was found to be beneficial, increasing
yield. the degradation rate. The effect depends on the
Another strategy for improving the H2O2 concentration, generally showing an
photocatalytic activity consists of inhibiting e- optimum range of concentration. At higher
/h+ recombination by addition of other concentrations the improvement starts to
(irreversible) electron acceptors to the reaction. decrease. Whereas this beneficial effect can
Outstanding enhancement of the rate of easily be explained in terms of prevention of
degradation of various organic contaminants electron/hole recombination and additional •OH
through the use of inorganic peroxides has been production through reactions 8,9 and 10,
demonstrated [34,35]. Beside increasing of the inhibition could be explained in terms of TiO2
number of trapped e- in the e-/h+ pairs and, surface modification by H2O2 adsorption,
consequently, avoid their recombination, scavenging of photo produced holes (eq.11) and
peroxides addition could have also several reaction with hydroxyl radicals (eq.12).
different effects: • generation of more •OH and
other oxidizing species; • increase the oxidation H2O2+ 2h+ → O2+ 2H+ (11)
rate of intermediate compounds; • avoiding H2O2+•OH →H2O+ HO2•- (12)
problems caused by a low O2 concentration. The inhibition of adsorption not only depends on
It must be mentioned here that in highly toxic the characteristics of the pollutant, but also on
wastewater where degradation of organic the H2O2/pollutant concentration ratio.
pollutants is the major concern, the addition of
an inorganic anion to enhance the organic Metal doped TiO2 assisted photocatalytic
degradation rate may often be justified. degradation of NB under UV-VIS irradiation
For better results, these additives should fulfill Because of the high energy band gap (3.2 eV),
the following criteria: dissociate into harmless titania can be activated only by ultraviolet (UV)
by-products and lead to the formation of •OH or light [36]. In addition, low photo quantum

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 I. Nitoi et. al.: Advanced Oxidation Processes (AOPs) alternative methods for degradation of toxic pollutants from wastewater

efficiency and high recombination of electron-
hole pairs restrict the titania application. In order
to improve optical absorption and photocatalytic
activity of TiO2 many attempts have been made
[37]. One feasible approach consists of doping
oxide semiconductor with metal [38-40].
The degradation of NB from aqueous solution
under UV-VIS irradiation using heavy metal
doped titania was investigated. Dopant type (Fe,
Ni, Co) and their concentration were assessed in
relation with pollutant degradation kinetic. Also
catalyst dose, pH and initial NB concentration
influence on degradation efficiency were
evaluated in order to set up the optimal working
conditions which assure pollutant’s advanced
degradation.
Photocatalytic activity of metal doped TiO2
The trapping of electrons can inhibit
electron/hole recombination during irradiation,
thereby increasing the lifetime of charge
carriers. Doping of TiO2 with metal ion
introduces a new energy level (dopant impurity
level) by the dispersion of metal nanoparticles in
titania matrix which acts as electron trap, and
also assures enhancement of photocatalytic
activity [41]. Since, charges recombination rate
depends on dopant type and its concentration,
the photocatalytic activity of undoped and Fe,
Co, Ni doped TiO2 in degradation of NB from
aqueous solution (2.52x10-4 M) was investigated
[42]. The dopant concentrations, reported to
TiO2 were 0.5; 1; 2; 5wt.%. All experiments
were performed at pH=7, with 100 mg/L
catalyst dose and irradiation time between 30-
240 min. The efficiency of pollutant degradation
was assessed by determination of NB
concentration vs. irradiation time in treated
samples. Kinetic curves obtained in various
working conditions, were linearized by a first
order reaction kinetic. From the slope of each
equation pseudo-first order rate constants, kNB
were calculated. Pollutant degradation yields
registered at 120min. irradiation time and kNB
values for undoped and doped TiO2 are
presented in Table 1.

Table 1. NB photocatalytic degradation rate constants and efficiencies of undoped TiO2

and Fe, Co, Ni - doped TiO2 with various dopant content
Catalyst Dopant type [M], wt.% kNBI x 104, s-1 *NB, %
Undoped TiO2 - - 1.08 54.14
Doped TiO2 Fe 0.5 2.54 84.91
1 2.09 74.77
2 1.76 71.77
5 1.69 67.97
Co 0.5 1.48 66.07
1 2.24 81.03
2 1.61 67.93
5 1.12 55.51
Ni 0.5 1.81 72.25
1 2.19 78.49
2 1.35 61.48
5 1.24 58.95
*irradiation time =120 min

Dopant presence in the catalyst matrix assures
enhancement of TiO2 photocatalytic activity, the
values of pollutant degradation rate constants are
higher than those obtained in the presence of
undoped titania. At the same dopant content, the
rate constants vary as function of dopant type,
photocatalytic activity generally decreasing in
the following order Fe - doped TiO2 > Co -
doped TiO2 > Ni - doped TiO2. Various dopants
induced different modification on catalyst
photoactivity depending on their ionic metal
oxidation state and electronic configuration.
According with our previous studies, metals are
presented in the oxidation state M3+, whose ionic
radii are closer to Ti4+ionic radius in comparison
with M2+. This data is confirmed by small
differences between unit cell volume registered
for undoped titania and 0.5-2wt. % metal doped
TiO2 [35]. Among dopants, Fe3+ ion which
presents only 5 electrons on d orbitals is
probably more accessible to electron trapping
than Co3+ or Ni3+ ions (with 6 and 7 electrons)

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 I. Nitoi et. al.: Advanced Oxidation Processes (AOPs) alternative methods for degradation of toxic pollutants from wastewater
which have less available half-occupied d
orbitals. The enhanced photocatalytic activity of
Fe - doped titania compared with Co or Ni-
doped titania is revealed by the NB degradation
efficiencies obtained at 120 min. irradiation and
2wt. % dopant content, which are 71.77; 67.93
and 61.48%, respectively.
Beside dopant type, other parameter which
modifies the photocatalytic activity is dopant
content. As shown in table1, each dopant
presents an optimum concentration level which
assures the higher NB degradation efficiency.
According to experimental results the optimum
concentration for Fe dopant is 0.5wt.% and
1wt.% in the case of Co and Ni dopants,
corresponding kNB values being 2.5x10-4 and
2.2x10-4 s-1. Dopant level increase up to
optimum concentration positively affected the
photocatalytic activity. In the case of Co and Ni
dopants, enhancement of metal content from 0.5
to 1wt.% assures increasing of kNB with 45 and
21%, respectively. An opposite behaviour is
observed when metal level exceeds optimum
content. kNB values are decreasing with 18; 24;
and 38% for Fe, Co, and Ni, respectively, in the
case of twice higher content than optimum
concentration. In accordance with our results,
other authors reported the improved
photocatalytic activity of 0.5wt.% Fe-doped
TiO2, which showed drastically increases of
charge-carrier lifetime to minutes and even
hours beside undoped titania (average lifetime of
an electron/hole pair is about 30ns) [43-45].
0.40
0.35
0.30
Absorbance
0.25
0.20
0.15
0.10
0.05
0.00
200 300
Fig. 3. UV-VIS absorption spectra of heavy metal doped and undoped TiO2
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Doping of TiO2 with metals not only enhanced
the separating efficiency of photo-induced
electrons and holes, but also increases the visible
light absorption due to shortening of band gap.
The absorbance spectra for the three catalysts
doped with (experimentally set) optimum metal
concentrations, were measured at room
temperature with a Helios UV/VIS
spectrophotometer and comparisons have been
made with undoped titania, as shown in Figure
3.
The undoped TiO2 spectrum shows an
absorption onset at 390 nm, which is in good
agreement with the intrinsic energy band gap of
titania at 3.2eV. The absorption spectra for all
doped catalysts are shifted to larger wavelength
(red shift). The higher absorbance in the range
of 400-600nm (visible light region) is observed
for (0.5wt.%Fe) – TiO2 catalyst, which confirms
its superior photocatalytic activity on NB
degradation in aqueous solution under UV-VIS
irradiation.
The influence of photocatalyst loading
In order to determine the effect of catalyst
loading, a series of experiments were carried out
on NB solution with 2.52x10-4 M content and
pH=7, by varying the (0.5wt.%Fe) - TiO2photo
catalyst dose between 50-500mg/L. From
determined pollutant concentration at various
irradiation times (60- 240min.) the NB
degradation efficiency and rate constant to each
applied catalyst dose was calculated.
TiO2Ni
TiO2Fe
TiO2Co
TiO2
400 500 600 700
nm
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The results, presented in table 2, show that
degradation efficiency increases to almost 99%
by increasing the catalyst dose from 50 to 250
mg/L. Increase of catalyst loading above
250mg/L, leads to degradation rate decrease.
Thus, 79% degradation efficiency was observed
at 500mg/L dose. The enhancement of
degradation efficiency with catalyst loading was
due to the increases in the surface area of the
catalyst available for NB adsorption and
degradation.
But higher values than optimum catalyst dose
(250mg/L) also increases solution opacity
leading to decreases in the penetration depth of
light into suspension and decreasing the
degradation rate [46, 47]. In all subsequent
Table 2. The influence of photocatalyst loading on NB degradation efficiency and rate constant
Photocatalyst dose, mg/L
50
100
250
500
* irradiation time = 240min
The pH of the solution has complex effects on
the photocatalytic oxidation reaction, depending
on pollutant type and zero-point charge (pHZPC)
of catalyst which in the case of Fe-doped TiO2 is
around 7. When doped catalyst is dispersed in
water, the first step is hydration of catalyst
surface followed by protonation (pH < pHZPC) or
deprotonation (pH >pHZPC) depending on
solution pH. Thus, the photocatalytic
degradation of the ionisable organic compounds
will be strongly affected by the pH due to their
Table 3. Effect of pH on NB degradation efficiency and rate constant
pH kNBx104, s-1
4 1,98
7 3,05
10 1.47
* irradiation time = 240min
Kinetics of NB degradation
Langmuir Hinshelwood (L-H) model is the most
commonly expression used to describe the
kinetics of the heterogeneous catalytic
processes. The Langmuir adsorption model has
been applied to organic compounds in aqueous
suspension according to the eq.13:
Romanian Journal of Ecology & Environmental Chemistry ● No.1 ● 2019
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experiments the optimum catalyst loading of
250mg/L was applied.
The influence of initial pH
The effect of pH on NB photocatalytic
degradation was evaluated over the range 4–10,
into solution with 2.52x10-4 M pollutant content,
at optimum photocatalyst dose (0.5wt.%Fe -
TiO2 = 250mg/L) for irradiation time between
60-240 min. The NB degradation efficiencies
and rate constants for several initial pH
conditions are presented in Table 3. The
degradation efficiency increases from 94.4 to
99% as the pH value is incremented from 4 to 7
and then decreases under 90% with the
increased value of pH from 7 to 10.
kNBx104, s-1 *NB, %
1,35 85,71
2,50 97,22
3,05 98,80
1,08 78,96
interaction with charged catalyst. Less or
non-polar compounds as it is the case of NB will
be better adsorbed on uncharged catalyst surface
and their degradation rate achieve maximum
value near the catalyst ZPC [48]. Therefore, the
acidic or alkaline pH does not favour the
adsorption of NB on the Fe-TiO2 particles.
According to results presented in Table 3, the
best pH value for NB photocatalytic degradation
under mentioned condition is 7, similar data
being reported by other authors [49].
NB*, %
94.44
98.80
88,04
dC k r K ads C 0
r  
0 dt 1  K C
ads 0 (13)
In which r0 is the NB degradation initial rate, C0
is initial NB concentration, Kads the equilibrium
constant of NB adsorption on catalyst particles
and kr the reaction rate constant [50,51]. In order
to determine the kr and Kads experiments on
0.5% Fe - TiO2 at optimum catalyst dose and
47
 I. Nitoi et. al.: Advanced Oxidation Processes (AOPs) alternative methods for degradation of toxic pollutants from wastewater

pH=7, for different initial NB concentrations only the experimental points during the first 30
between (0.37-8.45) x 10-4 M were performed. min. irradiation. The plot of 1/r0 = f (1/C0)
The r0 values were independently obtained from showed in Figure 5 attests that Langmuir-
kinetic curves by the linear feet, using the Hinshelwood model describes NB
pseudo-first order kinetics of NB degradation at photodegradation which occurs at catalyst
various initial pollutant concentrations are surface. From the intersection of this straight
presented in Figure 4. line with the ordinate results 1/kr and the
Eq. (13) was linearized by its reciprocal calculated value of the reaction rate is kr=1.23 x
expression: 10-7 Ms-1. The adsorption equilibrium constant
1

1

1 Kads=2022 M-1 was calculated from the slope.
r k k K C
0 r r ads 0 (14)

-4
[NB]0=0.37x10 M
6

-4
5 [NB]0=0.67x10 M
ln([NB]0/[NB]t

4 -4
[NB]0=2.52x10 M

2 -4
[NB]0=8.45x10 M

0
0 50 100 150 200 250
t, min

Fig. 4. The pseudo-first order kinetic of NB degradation in different initial concentrations.

1.2x10
8 kr=0.12 x 10-6Ms-1
Kads= 2022 M-1
8
1.0x10

7
8.0x10
1/r0, M-1s

7
6.0x10

7
4.0x10

7
2.0x10

0.0
0 1x10
4 4
2x10
4
3x10

1/c0, M-1

Fig. 5. Linearized eq. of Langmuir-Hinshelwood for NB degradation on 0.5%Fe-TiO2

Reactive species involved in photocatalytic (irradiation area=78cm2) [52]. Disodium

degradation of NB on iron doped TiO2 ethylenediaminetetracetate (EDTA-2Na, 5mM),
Trapping experiments were performed in order isopropyl alcohol (IPR, 5mM) and 1,4-
to identify the main oxidizing species formed benzoquinone (BQ, 5mM) were utilised as holes
during the photocatalytic degradation of NB (h+), hydroxyl radical (•OH) and superoxide
(2.51 x 10-4 M) on Fe doped TiO2film catalyst radical (O2•-) scavengers, respectively. As is

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48
 I. Nitoi et. al.: Advanced Oxidation Processes (AOPs) alternative methods for degradation of toxic pollutants from wastewater

presented in Figure 6 the presence of EDTA-
2Na and BQ do not affected NB degradation,
while i-PR drastically decreased the pollutant
conversion efficiency. This suggests that
hydroxyl radical (•OH) are the main active
species involved in NB degradation. Taking into
consideration trapping experimental results but
also the presence of oxygen vacancy defects
(VO) and Fe species on the catalyst surface we
try to propose a possible mechanism of
photocatalytic NB degradation on Fe doped
TiO2 under UV-VIS irradiation. It is well known
that delay/retardation of charges recombination
has a positive effect on photocatalytic activity of
TiO2.
One method applied in this respect is trapping of
separated charges like electron reduction of
oxygen.
In the case of oxygen-deficient TiO2 catalysts,
like ours Fe doped TiO2, the reduction of
adsorbed oxygen is inhibited due to VO defects
states localize at 0.78-1.1eV below the
conduction band (CB) minimum of TiO2
increases the reaction barrier for the single
electron reduction of oxygen towards superoxide
radical formation. This disadvantage is
overcome by the presence of Fe species on the
surface of catalyst which served as electron-
trapping centers for the multi-electron reduction
of adsorbed oxygen. Consequently, upon UV-
VIS irradiation electrons in the VB of catalyst
are first transferred to the Fe species (redox
potential of Fe3+/Fe2+=0.771V), which act as co-
catalysts, and subsequently are consumed via the
multi-electron oxygen reduction process. The
holes left in the VB may react with water and
generate •OH radicals that are further involved
in pollutant oxidation. The efficient charge
carriers’ separation achieved by Fe doped TiO2
assures an enhanced catalyst activity on
pollutant degradation under UV-VIS irradiation,
due to the synergistic effect of the presence on
the catalyst surface of Fe species and oxygen
vacancies. This is in accordance with results
reported by other authors for Fe species-grafted
on TiO2 catalyst applied in the β-naphtol
degradation under visible light irradiation [53].

1.0

0.9

0.8

0.7
[NB]t/[NB]0

0.6

0.5 i-PR

0.4

0.3
BQ
0.2
EDTA 2Na
0.1
No scavenger
0.0
0 60 120 180 240
Time, min

Fig. 6. Photocatalytic degradation of NB on 0.5Fe-TiO2 under UV-VIS irradiation

with and without addition of oxidizing species scavengers

TiO2/H2O2 assisted photocatalytic degradation reactive radical intermediates [54-56]. We

of CLA under UV-VIS irradiation studied photo oxidation of 4-CLA in
UV/TiO2/H2O2usinga wide range of H2O2
The use of inorganic oxidants like hydrogen dosage. Degradation pathway of 4-CLA was
peroxide, persulphate, chlorate and bromate in proposed according to GC-MS identified
UV/TiO2 system could increase the degradation organic intermediates [57].
efficiency either by inhibiting electron-hole pair Influence of H2O2 addition
recombination through scavenging conduction- H2O2, which is an electron acceptor, was added
band electrons at the surface of photocatalyst, or into the solution with 4-CLA content in order to
offering additional oxygen atom as an electron improve pollutant degradation efficiency by
acceptor to form the superoxide radical ion or enhancing hydroxyl radical concentration and

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 I. Nitoi et. al.: Advanced Oxidation Processes (AOPs) alternative methods for degradation of toxic pollutants from wastewater

also inhibiting electron/hole pair recombination. Increase of H2O2concentration between 1 x 10-4

Thus, experiments were conducted with various
concentration of H2O2 between 1 x 10-4 - 1 x 10-2
M, at fixed pH = 4, TiO2 dose 150 mg/L and
pollutant concentration 1.42 x 10-3 M. The
pseudo-first order kinetic equations of pollutant
degradation and organic chlorine mineralization
without electron acceptor addition, and in the
presence of various H2O2concentrations were
plotted. From the slope of kinetic equations, the
rate constants were calculated and values
obtained are presented in table 4.
– 1 x 10-3 M has a positive effect on pollutant
degradation; values of kI4-CLA and kIClare
increasing too. Further addition of H2O2 does
not modify pollutant photocatalytic degradation
rate, because, when electron acceptor is over
dosed (1 x 10-2 M in the present study), the
excess H2O2molecules scavenge the valuable
hydroxyl radicals and generate a much weaker
hydroperoxyl radicals. These ones can further
react with the remaining strong hydroxyl
radicals to form ineffective oxygen and water.

Table 4. The influence of H2O2 concentration on thepseudo-first-order rate constants

of 4-CLA degradation and organic chlorine mineralization
[H2O2] x 104, M kI4-CLA x 104, s-1 kIC l x 104, s-1
- 5.50 0.88
1 8.71 1.52
10 11.94 1.97
100 11.32 1.96

In addition, the pollutant photocatalytic
oxidation could be inhibited when the excess
H2O2 reacts with oxidative holes on catalyst
surface, as equations11,12 show [54, 58]. Based
on presented experimental results it was found
that optimum H2O2 concentration is 1 x 10-3M,
which assures after 180 minutes irradiation,
99.99 % pollutant degradation and 88 % organic
chlorine mineralization.
The identification of intermediates and 4-CLA
degradation mechanism
Several organic intermediates like aniline (A), 4-
aminophenol (4-AP), 4-chlorophenol (4-CP), 4-
chloronitrobenzene (4-CNB), 4,4’-
dichloroazobenzene (DCAB) were identified
during 4-CLA photocatalytic degradation in
UV/TiO2/H2O2 system, by GS-MS method.
In order to obtain information on subsequent
intermediates degradation we compared the
specific peak area (directly related with
compound concentration) of each GC-MS
identified byproduct presented in total
chromatogram at different irradiation time and
the following aspects have been observed:
- increase of irradiation time leads to 4-CLA
and some of its degradation intermediates (4-
CP, 4-AP, DCAB) concentrations decrease,
emphasizing that these are subsequently
involved in secondary reactions;
- an opposite behavior is registered in the case
of A and 4-CNB which are accumulated in
the system because these are hardly oxidized
in the tested working conditions;
- the evolution of DCAB and 4-CNB
concentration confirmed that the second one
is the degradation product of halogenated
diazobenzene derivate;
- after 60 min of irradiation, 4-AP
concentration was almost neglectable
compared with 4-CP, suggesting that
halogenated derivate is hardly degraded; in
addition, 4-CP concentration is almost
constant between 60 and 120 min showing
that its generation rate is higher than its
subsequently degradation.
Based on previously presented results it was
proposed a reaction sequence for the generation
of the main intermediates identified during the
photocatalytic degradation of 4-CLA as it is
presented in Figure 7.

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 I. Nitoi et. al.: Advanced Oxidation Processes (AOPs) alternative methods for degradation of toxic pollutants from wastewater

2
. NH Cl

Cl NH NH Cl

OH
Cl N N Cl (DCAB)
OH

2 Cl NO2 (4-CNB)
NH2

OH -NO2- carboxylic

OH   acids
OH OH
OH Cl O O C2 C6
Cl -
(4-CP) (benzoquinone)
Cl 
HOH OH
(4-CLA) -HN4+
-HCl
NH2 OH
CO2 + H2O
-Cl (4-AP)

OH

NH2

(A)
Fig. 7. Proposed photocatalytic degradation pathways of 4-CLA

The proposed mechanism presents four possible
pollutant degradation routes:
- HO• radicals attack on target pollutants with
replacement of the amino group and 4-CP
formation;
- hydrogen abstraction with anilinyl-radical
formation, that is subsequently stabilized by
dimerization to DCAB; The attack of HO•
radical on diazogroup of DCAB leads to 4-
CNB which is further transformed to 4-CP as
other researchers also reported [59, 60];
- Cl- release with A formation by a several
steps heterolytic mechanism consisting of
hydrogen and chloride ions liberation, as
HCl, followed by second hydrogen atom
elimination with nitrene formation. This one
is subsequently rearranged into seven
members’ carbene, which is converted to
aniline [60,61].
- A is oxidized to 4-AP by HO• radicals
addition to the aromatic ring, in accordance
with other studies [62]. Since the nature of
HO• radical is highly photolytic electrophilic,
it preferentially attacks the position with
highest electron density of aromatic ring.
In the case of aniline, the hydroxylation take
place in para positions with respect to amino
group, with lower steric hindrance compared
with ortho position; photohydrolisys of 4-CLA
with 4-AP formation, is a difficult reaction due
to HO• radicals and chlorine have similar
electronegativity which inhibiting substitution of
halogen atom by hydroxyl radicals;
The secondary intermediates (4-CP, 4-AP) are
hydroxilated [63,64] and further oxidized to
benzoquinone [65]. This one is subsequently
converted to other degradation products like
muconic, maleic, fumaric and oxalic acids by
ring cleavage under HO• radical attack [66]. The
final degradation products include Cl-, NH4+,
NO3- resulted in various steps of proposed
degradation routes, as is presented in Fig. 9. The
formation of non-chlorinated intermediates like
A, 4-AP agrees with experimental results which
show Cl- releasing as HCl, during irradiation.
CONCLUSIONS
H2O2 assisted photolysis and photocatalysis in
various systems like UV-VIS/Fe-TiO2 or UV-
VIS/TiO2/ H2O2 are efficient methods for
degradation of chloro and nitroaromatic
pollutants. In proper working conditions
pollutants advanced degradation (η > 99.6%) is
obtained:

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 I. Nitoi et. al.: Advanced Oxidation Processes (AOPs) alternative methods for degradation of toxic pollutants from wastewater

•
 UV/H2O2 system: [CB]0 = 1.8x10-3M, pH = OH radicals attack to double bound of aromatic
7, [H2O2] = 5.4x10-2M, tirr = 60 min; ring with hydroxylated intermediates formation,
 UV-VIS/Fe-TiO2 system: [NB]0 = 0.47x10- followed by their oxidation to benzoquinone,
4 which can be subsequently converted into
M, pH = 4, [Fe-TiO2] = 250mg/L, tirr = 240
min; carboxylic acids through ring cleavage.
 UV-VIS/TiO2/H2O2 system: [4CLA]0 = Inorganic ions like Cl-, NO3-, NH4+ are also
1.4x10-3M, pH = 4, [TiO2] = 200mg/L, formed as mineralization products. Results
[H2O2] = 1x10-3M, tirr = 180 min; obtained recommended studied advanced
degradation processes as performant alternative
The degradation pathway for any of studied treatment methods of wastewater with toxic
advanced degradation systems includes initial pollutants content.

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64. DUNNIVANT, F.M., SCHWARZENBACH, R.P., MACALADY, D.L., Environ. Sci.
Technol., 26, 1992, p. 2133.
65. BANDARA, J., MIELCZARSKI, J.A., LOPEZ, A., KIWI, J., Appl. Catal. B, 34, 2001, p.
321.
66. WINARO, E.K., GETOFF, N., J. Biosci., 57, 2002, p. 512.

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 J. Petre et. al.: Occurrence of Pharmaceuticals and Disinfectants in the Dissolved Water Phase of the Danube River
and Three Major Tributaries from Romania

Occurrence of Pharmaceuticals and Disinfectants in the Dissolved Water Phase of the Danube
River and Three Major Tributaries from Romania

JANA PETRE1*, VASILE-ION IANCU1,2, TOMA GALAON1, MARIUS SIMION1, MARCELA

NICULESCU1, GABRIELA-GEANINA VASILE1, LUOANA FLORENTINA PASCU1, CAROL
BLAZIU LEHR1, LILIANA CRUCERU1, SIMONA CALINESCU, GABRIEL-LUCIAN RADU2

1National Research and Development Institute for Industrial Ecology ECOIND Bucharest, 71-73 Drumul Podul Dambovitei, 060652,
Bucharest, Romania
2University Politehnica Bucharest, Faculty of Applied Chemistry and Materials Science, 1-7 Polizu Str., 011061, Bucharest,

Romania
*correspondence author (e-mail): janapetre@gmail.com

The presence of pharmaceuticals in the aquatic ecosystem has received great attention from the
scientific community in the last decades, due to their potential impact on living organisms. This
paper presents a short review of the results of investigations performed by INCD-ECOIND
concerning the occurrence of 32 pharmaceutical compounds belonging to important therapeutic
classes and 2 disinfectants along the Danube River and its tributaries. Grab water samples were
collected from multiple points along the River (10 sites) and from 2 locations for each of the
tributaries Jiu, Olt and Argeș, upstream and downstream of large cities. All samples were
quantified via solid phase extraction, followed by liquid chromatography-tandem mass
spectrometry (LC-MS/MS). Results of these studies show that various pharmaceutical compounds
are present in the dissolved water phase of the Danube River and its tributaries in low to moderate
amounts with variation due to season and location.

Keywords: pharmaceuticals, disinfectants, surface waters, Danube River, tributaries

In recent years, pharmaceutical compounds have
been recognized as a new class of contaminants
for water resources, with adverse effects on
aquatic and terrestrial life.
The presence and concentration levels of
pharmaceuticals in natural waters require
investigation in order to assess their potential to
disturb the equilibrium of the aquatic
environment. Thus, a continuous exposure of
bacterial fauna, even to very low concentrations
of antibiotics, can lead to the persistence of
resistant bacterial strains [1]. Also, the exposure
of aquatic organisms to endocrine disrupting
chemicals, for instance estrogen hormones can
affect the reproduction and development of these
living organisms [2]. Some studies reported that
bacteria isolated from treated sewage sludge
have shown resistance to several antibiotics
including ampicillin, tetracycline, ciprofloxacin,
trimethoprim and erythromycin [3,4]. Animal
and fish farms, where antibiotics are
administered as feed additives and for
therapeutic purposes, are also sources of drug-
resistant bacteria [5]. Nowadays there are
numerous pathways through which
pharmaceutical compounds reach the aquatic
environment (depicted in Figure 1). Effluents of
the municipal wastewater treatment plants
(WWTPs) are a major source of continuous
introduction of numerous human pharmaceutical
compounds in natural waters. Unused or expired
drugs not properly landfilled are also a potential
source of contamination of surface waters.
Moreover, runoffs from agricultural fields,
livestock from farms and fish ponds may contain
pharmaceutical residues which end up in the
aquatic environment [6].

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 J. Petre et. al.: Occurrence of Pharmaceuticals and Disinfectants in the Dissolved Water Phase of the Danube River
and Three Major Tributaries from Romania

Fig.1. Source of pharmaceuticals in the aquatic environment

A number of pharmaceutical compounds, once
arrived in the aquatic environment, suffer a
decrease in concentration either by dilution,
adsorption on solids, or by biochemical
decomposition, whilst other compounds are
resistant to degradation and may persist, and end
up accumulating in living organisms.
Contamination of natural waters with
pharmaceutical products has a direct impact on
aquatic organisms causing the extinction of
sensible species, and ultimately a decline in
biodiversity. Due to the risks connected with the
ubiquity of various pollutants in natural waters,
the European Union established a watchlist of
substances or groups of substances found to be a
threat to the safety of the aquatic environment.
[7]. This list is updated every two years and the
latest one approved by the European
Commission (EU Decision 2018/840) includes
the following pharmaceuticals: 17α-
ethinylestradiol, 17β-estradiol, estrone,
erythromycin, amoxicillin, clarithromycin,
azithromycin and ciprofloxacin. These new rules
amend the Water Framework Directive (WFD
2000/60/EC) as well as the Directive
(2008/105/EC). The presence of pharmaceutical
compounds in the aquatic environment has
caught the attention of the scientific world due
to its threat on the health of aquatic organisms.
This resulted in an increased number of reports
concerning the concentrations of the
pharmaceutical compounds detected in WWTP
effluent samples as well as in the emissaries.
It is known that the removal efficiency of the
wastewater treatment process can vary and is
influenced by the specific characteristics of the
drugs in question and also by other factors such
as hydraulic retention time, environmental
conditions and age of the activated sludge [8-
10]. It has been noticed that conjugation and
deconjugation processes occur in the treatment
plants and therefore, it is observed an increase in
the initial concentrations of the pharmaceutical
compounds after the treatment process occurs.
It should be emphasized that in the WWTP
effluents the pharmaceuticals and disinfectants
are present in complex mixtures together with
their metabolites and their transformation
products [11].
Pharmaceuticals often detected in some
conventional WWTP effluents are listed in
Table 1, with their reported concentration range
[12].

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 J. Petre et. al.: Occurrence of Pharmaceuticals and Disinfectants in the Dissolved Water Phase of the Danube River
and Three Major Tributaries from Romania

Table 1. Presence of pharmaceuticals and disinfectants in WWTP effluents.

Concentration
Pharmaceuticals Country References
(ng/L)
BQL-34.4 Greece [13]
Sulfamethoxazole
19-198 Spain [14]
BQL-27.4 Greece [13]
Trimethoprim
BQL-108 Spain [14]
Ofloxacin 63-101 Spain [14]
Clarithromycin 172-229 Spain [14]
Norfloxacin BDL-149 Spain [14]
3.89-15.4 Portugal [15]
Triclosan
BQL -111,2 Greece [13]
BDL-592 Spain [14]
Azithromycin
8-190 Czech Republic [16]
Erythromycin 30-350 Czech Republic [16]
BQL-Below Quantification Limit; BDL-Below Detection Limit

Danube River is a large stream receiving high
anthropogenic inputs and whose water is at the
same time an important source of drinking water
for riparian population, in some cases without
being subjected to any treatment process.
Therefore, there is a need of data on the
presence of these contaminants in the Romanian
part of the Danube River basin. The aim of these
works conducted by INCD-ECOIND and
summarized in this short review article, was to
document the occurrence of selected
pharmaceuticals along the Romanian part of the
Danube River and assess the contribution of its
three main tributaries: Jiu, Olt and Argeș.
Presence of pharmaceuticals and disinfectants
in WWTP effluents
Conventional treatment plants, mostly used in
Romania, are not designed to completely
remove organic compounds, such as the
pharmaceuticals with lower levels of
concentration, which can be found in
wastewaters.
In this paper [17] we investigated the occurrence
and removal of nine pharmaceuticals in the
WWTP of Bucharest. The influent and effluent
samples were 24h composites and were
collected using flow proportional samples
during a nine-day period in the autumn of 2012.
The compounds investigated in this study were
acetaminophen (ACE), caffeine (CAF),
trimethoprim (TRI), norfloxacin (NOR),
ciprofloxacin (CIP), azithromycin (AZI),
sulfamethoxazole (SMX), erythromycin (ERY)
and carbamazepine (CBZ). The concentration
ranges of pharmaceuticals found in influent and
effluent samples are presented in Table 2.

Table 2. Concentrations of pharmaceuticals in influent and effluent samples of WWTP of

Influent Effluent
Compound
(µg/L) (µg/L)
ACE 8.96 - 30.16 0.42 - 5.03
CAF 21.78 - 74.54 2.82 - 11.64
CBZ 0.31- 2.34 0.71 - 1.57
CIP <LOD - 4.97 <LOD - 1.83
SMX 0.30 - 0.73 0.13 - 0.61
TRI 0.05 - 0.50 0.19 - 0.49
AZI <LOD - 0.49 <LOD - 0.15
Abbreviations: ACE: acetaminophen, CAF: caffeine, CBZ: carbamazepine, CIP: ciprofloxacin,
SMX: sulfamethoxazole, TRI: trimethoprim, AZI: azithromycin

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 J. Petre et. al.: Occurrence of Pharmaceuticals and Disinfectants in the Dissolved Water Phase of the Danube River
and Three Major Tributaries from Romania

Fig. 2. Mean removal efficiencies (%) of detected pharmaceuticals in Bucharest WWTP

ERY and NOR were not detected in the
analyzed samples, probably due to their limited
usage during the period of investigation.
Excepting CIP and AZI, all compounds were
ubiquitous in WWTP influent and effluent
samples. The most abundant one was caffeine.
This widely used compound was found in
effluents in concentrations ranging from 2.82
µg/L to 11.64 µg/L. In effluent samples CBZ
concentration varied from 0.71 μg/L to1.57
μg/L, similar to 1.18 μg/L found in Källby
WWTP in Sweden [18]. Similar concentrations
of SMX and TRI were observed at WWTPs
effluents in Spain [14], these two
pharmaceuticals being used in the same drug.
Mean removal rates of the compounds
determined in this work, calculated as the
difference between 9 days mean concentrations
of each compound in the influent and effluent
samples are depicted in Figure 2.
ACE and CAF had the highest average
elimination yields of 83.3% and 81.0%,
respectively. Low or no removal rates of CBZ
were also previously reported [19,20]. This
organic compound is known as being very
persistent to biodegradation, even at low
concentrations. An increase in CBZ, TRI and
SMX concentrations in the effluent samples was
observed, a phenomenon also reported in other
studies [20-22].
The fluctuation of the concentrations during the
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sampling period [21] and the deconjugation of
conjugated metabolites during the treatment
process [22] are among the possible
explanations.
Another investigation was carried out in the
effluents of municipal WWTP of the City of
Brăila, having the Danube River as natural
receiver [23]. Treatments in this WWTP include
primary settling and biological processing
(activated sludge). All samples were grab
samples and were collected between the 16th
and the 20th of November 2015.
The studied compounds were diclofenac (DCF),
ketoprofen (KET), piroxicam (PIR), ibuprofen
(IBU), naproxen (NPX), hydrochlorothiazide
(HCT), furosemide (FUR), triclosan (TCS),
triclocarban (TCC), and gemfibrozil (GEM).
The results showed that all effluent samples
contained pharmaceuticals and disinfectants.
Only IBU was not detected in any of the
analysed samples. Maximum concentrations
were determined for PIR, with average
concentration of 103 ng/L. TCS and TCC, the
disinfectants investigated in samples from
effluents were found in average concentrations
of 15.6 ng/L and 14.1 ng/L, respectively.
Occurrence of pharmaceuticals in surface
water of Danube River and tributaries
In several investigations conducted in INCD-
ECOIND since 2012, the presence and fate of 32
58
 J. Petre et. al.: Occurrence of Pharmaceuticals and Disinfectants in the Dissolved Water Phase of the Danube River
and Three Major Tributaries from Romania

pharmaceuticals belonging to various site closed to its confluence with Danube,

therapeutic classes and 2 disinfectants were
investigated in the dissolved water phase of river
water at 16 sampling locations along the
Romanian side of the Danube and its three main
tributaries, Jiu, Olt and Argeș rivers [24-26].
Sampling sites and sample collection
Several sampling campaigns were performed
from February 2014 to October 2015. Samples
were collected from 10 locations along the
Romanian part of the Danube River and 2
locations from each of its 3 tributaries as
follows: (a) Jiu river - upstream and downstream
Craiova city; (b) Olt river - downstream Slatina
and a site closed to its confluence with Danube;
(c) Argeș river - downstream Pitești city and a
according to Figure 3. All samples were grab
samples and were taken from a depth of
approximately 0.5 m to 2 m from the riverbank
with the exception of samples from sites S3, S7,
S11 and S13 which were sampled at 10 m from
the bank, by being gathered from a bridge.
Location data, GPS coordinates and descriptions
are given in Table 3. Investigated
pharmaceuticals in these studies are:
analgesics/anti-inflammatory drugs, antibiotics,
diuretics, anti-epileptics, lipid regulators,
nervous stimulant, disinfectants and estrogen
hormones, due to their extensive prescription or
usage. The individual compounds are presented
in Figure 4.

Fig. 3. Map of the sampling sites in the Danube River basin. S = sampling site

Table 3. Geographic location with GPS coordinates and description of sampling sites.
Sampling point River GPS coordinates
S1- Bazias Danube 44°47'32.61"N; 21°23'20.07"E
S2- Gura Vaii, upstream Drobeta Turnu Magurele Danube 44°40'7.40"N; 22°33'10.74"E
S3- Calafat Danube 43°57'50.94"N; 22°54'15.72"E
S4- Filiasi, upstream Craiova city Jiu 44°34'8.32"N; 23°27'18.14"E
S5- Podari, downstream Craiova city Jiu 44°15'18.48"N; 23°47'25.08"E
S6- Rast, upstream of the confluence with Jiu river Danube 43°51'24.84"N; 23°17'18.79"E
S7 -Bechet, downstream of the confluence with Jiu river Danube 43°45'11.32"N; 23°56'30.69"E
S8 -Downstream Slatina city Olt 44°23'29.63"N; 24°21'4.84"E
S9 -Izbiceni, upstream of the confluence with Danube Olt 43°48'41.71"N ; 24°42'27.71"E
S10 -Islaz, upstream of the confluence with Olt river Danube 43°42'22.72"N; 24°44'4.01"E
S11- Drobeta, downstream of the confluence with Olt river Danube 43°43'2.69"N; 24°48'56.65"E
S12 -Downstream Pitesti city Arges 44°28'45.25"N; 25°40'47.87"E
S13- Giurgiu, upstream of the confluence with Arges river Danube 43°52'37.50"N; 25°58'49.92"E
S14- upstream of the confluence with Danube river Arges 44° 6'38.09"N; 26°38'15.33"E
S15 -Oltenita, downstream of the confluence with Arges river Danube 44° 3'51.89"N; 26°38'45.49"E
S16 -Calarasi Danube 44° 8'15.69"N; 27°20'8.26"E

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 J. Petre et. al.: Occurrence of Pharmaceuticals and Disinfectants in the Dissolved Water Phase of the Danube River
and Three Major Tributaries from Romania

Fig. 4. Investigated pharmaceuticals and disinfectants

Sample treatment and analysis of the monitored, corresponding to the transition

pharmaceuticals
Analytical protocol was based on previously
optimized methods reported elsewhere [24-26].
Sample treatment was based on solid phase
extraction with an enrichment factor of 500, and
using internal labeled compounds. The
chromatographic separations of compounds
were performed on an Agilent 1260 series LC
system (Agilent, Waldbronn, Germany)
consisting of binary pump, thermostated
autosampler and thermostated column
compartment coupled with an Agilent 6410B
triple-quadrupole mass analyzer fitted with an
electrospray ionization source. Data acquisition
and analysis were performed using Mass Hunter
software, revision B.05.00. Target analytes
detection was done in Multiple Reaction
Monitoring (MRM) mode with dual polarity (±).
Full scan MS mode coupled with different
fragmentor voltage values was used to obtain
product ion rich MS spectra for all analytes.
For each compound, two signals were
between the precursor ion and the two most
abundant product ions. The most abundant one
was used for quantification while the other one
was used for confirmation. Agilent Mass Hunter
software was used for data acquisition and
quantitative determinations.
RESULTS AND DISCUSSIONS
By using the optimized analytical methods, the
undertaken studies showed that all investigated
samples contained pharmaceutical compounds.
During a first investigation, the samples were
collected during the months of February, April
and June 2014 and the following compounds
were analyzed: diclofenac (DCF), ketoprofen
(KET), piroxicam (PIR), ibuprofen (IBU),
naproxen (NPX), hydrochlorotiazide (HCT),
furosemide (FUR), triclosan (TCS), triclocarban
(TCC) and gemfibrozil (GEM) [24]. The
obtained results and detected percentages are
presented in Table 4.

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 J. Petre et. al.: Occurrence of Pharmaceuticals and Disinfectants in the Dissolved Water Phase of the Danube River
and Three Major Tributaries from Romania

Table 4. Concentration ranges and frequency of detected pharmaceuticals.

Range (ng/L) Median ± IQRa (ng/L) Detection freq. (%)
Analyte
Feb. Apr. June Feb. Apr. June Feb. Apr. June

DCF 0.8 - 7.2 1.3 - 7.5 0.9 - 5.9 1.9 ± 1.4 3.3 ± 2.6 2.1 ± 1.6 50 100 56

KET 0.9 - 3.6 0.8 - 8.4 2.2 - 12.9 1.8 ± 0.6 2.5 ± 3.8 6.4 ± 4.1 44 87 94

TCS 1.7 - 9.1 0.7 - 7.8 1.9 - 18.4 4.4 ± 4.1 2.7 ± 3.2 2.1 ± 2.0 62 100 50

TCC 0.8 - 6.3 0.8 - 54 0.6 - 4.1 2.4 ± 1.7 3.6 ± 1.8 1.5 ± 1.2 100 100 44

PIR - 0.7 - 1.6 - - 1.2 ± 0.5 - - 37 -

IBU - - 2.0 - 27.2 - - 11.9 ± 5.5 - - 100

NPX - - 2.4 - 106 - - 44.8 ± 19.2 - - 100

a
IQR-interquartile range

Only 7 compounds were determined in the detection frequency in April, in median

analyzed water samples, namely: DCF, KET,
TCS, TCC, PIR, IBU and NPX in relatively low
concentrations with median values between 1.2
and 44.8 ng/L, and their recovery rates varied
between 81.5 % and 106.0 %. Increasing levels
of concentrations of the seven compounds were
observed in the samples collected from
tributaries Jiu, Olt and Argeș, downstream of the
big cities, as opposed to upstream values,
highlighting the contribution of the municipal
effluents.
Detected levels of DCF, KET, NPX, IBU in the
Danube and tributaries were comparable to those
reported for Danube River and Argeș tributary
[27].
DCF, a highly prescribed anti-inflammatory
drug with proved mutagenic effects included in
the priority substances list, was found in all 3
sampling campaigns in concentrations ranging
from 0.8 ng/L to 7.5 ng/L, and detection
frequencies between 50% and 100%. Triclosan
and triclocarban, highly used disinfectants in
personal care products such as soap or
toothpaste, were both found with a 100%
concentration of 2.7 ng/L and 3.6 ng/L,
respectively.
TCS was detected in the surface waters of Spain
in higher concentration than in this investigation
(ND-223 ng/L) [28] and up to 95 ng/L in UK
[29]. TCC levels detected in Central Spain
Rivers being up to 20 ng/L were similar to those
determined in this study [28].
A total of 18 pharmaceuticals belonging to
different therapeutic classes were studied in a
sampling campaign performed in February 2014
[25]: an analgesic (ACE), an anti-epileptic drug
(CBZ), a nervous stimulant (CAF) and 15
antibiotics: TRI, SMX, CIP, norfloxacin (NOR),
azithromycin (AZI), erythromycin (ERY),
roxithromycin (ROX), ampicillin (AMP),
cephalexin (CEF), amoxicillin (AMX),
penicillin G (PEN G), penicillin V (PEN V),
oxacillin (OXA), ofloxacin (OFL),
clarithromycin (CLA). CAF, CBZ, TRI, CIP,
SMX, CLA and CEF were the pharmaceuticals
found in investigated samples. Their ranges of
concentration, median and frequencies are
presented in Table 5.

Table 5. Range and median of pharmaceutical concentrations and detection frequency of individual
pharmaceuticals from surface water samples
Compound Range (ng/L) Median ± IQR (ng/L)a Frequency (%)b
Caffeine 28.3 - 128.0 76.4 ± 35.6 100
Carbamazepine 5.4 - 15.4 9.65 ± 6.1 100
Sulfamethoxazole 3.2 - 15.7 7.9 ± 2.4 87
Clarithromycin 1.2 - 23.2 3.0 ± 2.7 75
a
Trimethoprim 1.3 - 11.1 2.6 ± 1.2 69 IQR-interquartile range
b
Percentage of particular
Cephalexin 5.6 -17.8 9.9 ± 4.6 50
pharmaceuticals in 16
Ciprofloxacin 3.6 - 4.8 3.7 ± 0.6 19 sampling sites

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 J. Petre et. al.: Occurrence of Pharmaceuticals and Disinfectants in the Dissolved Water Phase of the Danube River
and Three Major Tributaries from Romania

Fig. 5. Concentrations of pharmaceuticals detected in Danube River tributaries

Figure 5 shows the scatter plots of individual
pharmaceutical concentration versus sampling
points on Jiu, Olt and Argeș rivers. As depicted
in the figure below, the most contaminated
samples were collected from tributary rivers
downstream of big cities like Craiova (Jiu
River), Slatina (Olt River) and Bucharest (Argeș
River).
CAF and CBZ were also detected in this
investigation in all surface water samples in
concentrations which varied from 28.3 to 128
ng/L and from 5.4 to 15.1ng/L, respectively.
Taking into account their ubiquitous occurrence
in WWTP effluents, CAF and CBZ are
considered markers of surface water pollution
[30].
The CBZ anticonvulsant, found in the surface
waters of Romanian Danube River basin, had
similar concentrations to those reported for
surface waters in Serbia (6-130 ng/L) [31]. The
high frequency detection of CBZ in river waters
is explained by its low removal rate in WWTPs
and by its high resistance in the aquatic
environment [31].
SMX and TRI, usually used in combination
pharmaceutical formulations, are among the
antibiotics most frequently detected in surface
waters, in concentrations of 69% and 87%
respectively. The fairly regular occurrence of
SMX and TRI in the collected sample is
consistent with the compounds occurrence in the
upper Tennessee River basin [32] and Douro
River estuary (Portugal) [33]. Their high degree
of persistence in natural waters is explained by
their low values of log Kow of 0.91(TRI) and
0.89 (SMX) [34]. Similar concentrations of TRI
and SMX were found in the surface waters of
Canal Danube-Tisa-Danube from Serbia [35]
and in the surface waters of the Henares-Jarama-
Tajo River system (Madrid, Spain) [36].
Fluctuations of pharmaceutical concentrations in
the Danube River waters were observed
downstream of the Argeș river discharges, the
most contaminated tributary. TRI, SMX, CLA
and CEF, which were not detected upstream of
the Argeș river discharge, occurred downstream
of the confluence in concentrations ranging from
1.3 ng/L to 15.9 ng/L. Estrogen hormones levels
in surface water of Danube River and the three
tributaries were tested during a sampling
campaign carried out in October 2015 using a
newly developed LC-ESI (-)-MS/MS method
with automated SPE extraction [26]. The
investigated compounds were: Equilin (Eq),
Estrone (E1), 17α-Estradiol (αE2), 17β-Estradiol
(βE2), 17α-Ethinylestradiol (EE2), Estriole (E3).

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 J. Petre et. al.: Occurrence of Pharmaceuticals and Disinfectants in the Dissolved Water Phase of the Danube River
and Three Major Tributaries from Romania

E3, Eq and α-E2 are compounds that were not CONCLUSIONS

detected in any of the analyzed samples, while
E1, β-E2 and EE2 were determined in all the
samples at concentrations ranging from 0.5 ng/L
to a maximum of 3.7 ng/L. In the graph
presented in Fig. 6, it can be observed that the
Estrone steroid was found in higher levels of
concentration than its quantification limit (1.0
ng/L) in samples taken along the Danube River,
with concentrations ranging from 1.2 to 1.6
ng/L. An explanation of the low level of βE2 in
the tested river water can be the result of the
easy transformation of β-E2 into estrone in the
environment through oxidation [37]. Higher
values of estrone, up to 3.8 ng/L, were
determined in tributaries downstream of major
cities like Craiova and Bucharest when
compared to upstream values (Fig. 7). This
easily explains the contribution of their WWTPs
to surface water pollution. Estrogen hormones
levels in Danube water and its tributaries are
generally comparable in magnitude order with
those reported in other studies within Europe,
with levels ranging from <0.3 to 4.9 ng/L
[38,39].
Nowadays, in the absence of solutions that could
effectively prevent the pollution of the natural
waters of our country, it is important to monitor
the level of contamination from
pharmaceuticals, especially from antibiotics and
endocrine disrupting substances. The studies
show that some pharmaceuticals and
disinfectants are not completely eliminated in
the municipal WWTPs and therefore they enter
the aquatic environment. A nervous stimulant
and a psychiatric drug, caffeine and
carbamazepine respectively, were found in all
surface water samples. Occurrences with a
frequency higher than 50% were observed in
collected samples for sulfamethoxazole,
trimethoprim, clarithromycin and cephalexin.
The development and implementation of more
efficient wastewater treatment processes, fit to
remove organic contaminants such as the
pharmaceutical compounds from effluents
discharges in natural emissaries, should be the
key to reducing the spread of these contaminants
in the Romanian aquatic environment.

Fig. 6. Estrogen hormones levels (ng/L) detected along the Danube River

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 J. Petre et. al.: Occurrence of Pharmaceuticals and Disinfectants in the Dissolved Water Phase of the Danube River
and Three Major Tributaries from Romania

Fig. 7. Estrogen hormones levels (ng/L) detected in Jiu, Olt and Argeș tributaries

ACKNOWLEDGEMENTS. The work has been funded by The Program Nucleu 13N/2009, Project
code: PN 09-13 01 09.

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COVACI, A., GONZÁLEZ-MARIÑO, I., HAPESHI, E., KASPRZYK-HORDERN, B.,
KINYUA, J., LAI, F.Y., LETZEL, T. LOPARDO, L., MEYER, M.R., O'BRIEN, J.,
RAMIN, P., ROUSIS, N.I., RYDEVIK, A., RYU, Y., SANTOS, M.M., SENTA, I.,
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and Three Major Tributaries from Romania

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 B. Stanescu et. al.: Soil management in the urban environment: today and future

Soil management in the urban environment: today and future

BOGDAN STANESCU, ADRIANA CUCIUREANU

National Research and Development Institute for Industrial Ecology ECOIND Bucharest, 71-73 Drumul Podul Dambovitei, 060652,
Bucharest, Romania
*correspondence author (e-mail): stanescubogdan2@gmail.com

The present article presents the expertise realized by the Department of Environmental Monitoring
Pollution Evaluation within the INCD ECOIND, in the evaluation of the quality of urban soils in
the municipality of Bucharest and the main big cities in Romania. The current data available at the
level of the 27 member states of the European Union show that annually over 100,000 hectares of
land are introduced into the urban environment, a direct consequence of the development of cities.
There are a number of legislative obstacles to strategic soil protection measures. Moreover, at the
level of the local authorities there is a conflict regarding the measures of soil protection in the long
term, on the one hand, and, the accelerated economic development in the short term, on the other.
European environmental experts consider that the urban development, absolutely necessary for the
economic growth, requires an adequate management of the natural resources in order for the
development to be done on a sustainable basis, respectively to follow a series of strategic
objectives. In our country, at least in the last decade, we find on a large scale the conversion of
industrial areas into commercial or residential areas. The footprint of industrial activities can be
found even after long periods of time present by identifying the remnant of soil pollution or in those
areas known as historically polluted (for example the town of Copsa Mica).
The conclusions stemming from the assessment of pollution in urban areas over large areas, in
correlation with the potential sources of pollution, underline the need to monitor the quality of soils
in the urban environment, but also to apply a performance management in order to protect this
natural resource in the long term.

Keywords: assessment, urban soil, pollution, management

The activities presented in this article aimed at
the level of the main cities in Romania: Iasi,
Timisoara, Cluj Napoca, Constanta, Craiova,
Galati, Brasov, Ploiesti and Oradea to highlight
the quality of the soils in the urban environment.
Urban ecology studies show that soil pollution is
especially important because it is directly related
to public health. The increase of the population
density in the urban environment is felt by the
influence of the anthropic activities and on the
quality of the soils (mainly the industrial
activities). It is found that urban soils differ
greatly from natural ones, respectively from
those not found under the direct influence of the
anthropic factors in the big cities.
Urban soils accumulate different types of
pollutants from different sources of pollution.
The development of cities in Romania has
undergone major changes in the last decades,
whether we are talking about the rehabilitation
of the existing infrastructure or the construction
of new infrastructure, or the restriction of
industrial areas and, very important, their
conversion into commercial or residential areas.
Soil is at the same time a very important natural
resource that must be kept under sustainable
conditions for future generations.
Figure 1 shows the cities in Romania that were
investigated within the project for evaluating the
quality of urban soils.

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 B. Stanescu et. al.: Soil management in the urban environment: today and future

Fig.1. City soil investigations on physical map of Romania

It is known that the urban population is zones [9].

constantly growing. Currently, estimates show
50 per cent of the world's population is
concentrated in the urban environment and the
trend is increasing by up to 70 per cent in 2050
[1]. It is found that urban areas are growing
continuously, and the growth rate is faster than
any other land use, respectively land intended
for urbanization [2]. Therefore, the importance
of green spaces in the urban environment with
their ecosystem functions and services is
beginning to become more and more important
to the sustainability of the environment in urban
areas [3]. Manageable soil properties including
the changes caused by additions in soil
conditions [4] and vary across the landscapes [5,
6, 7]. Soil natural capital needs to be quantified
in such a way that it can be integrated into
environmental policy and land management
decision-making to inform the provision of
ecosystem services [8]. Figure 2 presents
graphically a series of problems, ranked
according to their importance, in direct
connection with the quality of the soils in urban
The role of soil in the urban environment is an
essential role in the development of the human
society. Public awareness is focused on soil as a
basis for space for housing, industrial and
commercial purposes as well as infrastructure,
recreation areas and food production.
The main soil services in the urban environment
is shown in figure 3.
It is important to mention that the processes
related to the water cycle in the urban
environment are deteriorated, especially the
buffer function represented by the soil cover in
relation to the precipitation, respectively the
main meteoric waters are drained and can
produce floods and only a small part it infiltrates
the soil, in the unsaturated area, without
ensuring recharge of the groundwater.
In the current stage there is a lack of information
of the population regarding the importance of
the soil and of its functions in the urban
environment, therefore the importance of the
soil is greatly diminished from the attention of
the general public.

Fig.2. Problems hierarchy regarding soil quality in urban zones

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 B. Stanescu et. al.: Soil management in the urban environment: today and future

Fig. 3. Soil services in the urban environment

Soil management is a priority to protect this influences, industrial pollution sources in each
resource so important, especially in the urban city studied, land use and functionallity, etc);
environment where the anthropic pressures are - GPS localizations of the samples and maps of
very high. It is absolutely mandatory to take into the experimental field for each city.
account the relationships between soil and
human health, as well as between soil and B. Laboratory activities:
climatic conditions, respectively the adaptation - relevant quality indicators determined by
to climate changes of the last decades. modern analytical techniques (ICP-MS);
- adequate test methods, standardized and
EXPERIMENTAL PART validation of the results using Certified
The location of study areas on the maps are References Materials.
presented in Figure 1. Activites was conducted Figure 4 shows schematically the activities
in two main directions: carried out for investigating the quality of urban
A. Field activities: soils.
- dimensioning the number of samples to soil to It is noted that the in terrain documentation
be sampled with adequate equipment for activities occupy the first place, especially
sampling; important, because the office documentation
- obtaining relevant information and details cannot supplement the updated information’s
(microrelief, hydrographic network, climatic obtained on the site.

Fig.4. Scheme for conducting soil survey in urban areas

The involvement of local public authorities with
competences in ensuring the quality of the
environmental conditions in urban areas is
defining and necessary. That is why the final
report of the obtained results is directed to them,
it is a processed information by specialists on
environmental assessment who make a series of
recommendations and monitoring proposals for
future stages, practically a contribution and a
support for the realization of management on
soil resources in the urban environment.

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 B. Stanescu et. al.: Soil management in the urban environment: today and future
RESULTS AND DISCUSSION
The most important aspects related to the
chemical pollution of the soils in the analyzed
cities, especially heavy metal pollution, reveal a
series of details that highlight the specific mark
of each analyzed city. For example, in Iasi city,
32 soil samples were collected and analyzed
from the top soil layer, the first 10 cm. of the 11
quality indicators analyzed, from the heavy
metals category were analyzed: total chromium,
lead, copper and zinc. All the results obtained
were compared with the reference values
contained in Order 756 of November 3, 1997
[10] for approving the Regulation regarding the
assessment of environmental pollution.
For the city of Iasi, there is a significant,
historical pollution, the remanent from the
periods of intense activity of some large
economic units with industrial profile that
worked in the city of Iasi, such as “Tepro”
(metallurgical profile), the “Heavy Equipment
Facility” and “Terom SA”; thus the recorded
pollution is located in the industrial area of the
city of Iasi with exceeding the intervention
threshold at the indicator "zinc".
Another example, for the city of Timisoara,
which does not have an industry concentrated in
a single area, soil pollution was reported only
punctually (1 sample out of 29 analyzed),
without a define a correlation with the industrial
sources of pollution.
In the city of Cluj-Napoca the experimental field
consisted of 21 soil samples scattered
throughout the city, and the analytical results
obtained revealed only exceedances of the alert
thresholds for lead, cadmium and total
chromium in different point areas (in the city of
Cluj Napoca in one the area with a filling land
used for the arrangement of a commercial
outdoor green space).
In the city of Constanta heavy metal pollution
was identified in a degraded urban area where
household waste was stored. A similar situation
was identified in the city of Ploiesti. There are
local problems identified as important issues,
which are still partially solved, the existence of
these degraded areas where uncontrolled waste
is stored. Unfortunately, these areas are not
always inventoryed and, therefore, there is no
prompt intervention to restore the quality
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conditions acceptable to the soil.
What is appreciated is the fact that local
authorities want to stop this phenomenon and
eliminate it in the near future.
A special study case belongs to the city of Copsa
Mica, a small city, but with a very polluting
industry that has been active for over 8 decades.
There were identified a number of negative
aspects regarding the quality of the soils, which
consist in the frequent exceedance of the
intervention threshold.
An extremely unfavorable situation was
recorded in an area located near DN14 B, at
approx. 700m southbound from the dumps of
the company Sometra SA, which is the main
potential source of heavy metal pollution. As a
general conclusion for the soil environment
component: the soil is still affected by the
remaining pollution, of historical character,
induced by the industrial activities carried out in
the area for long periods of time, which had a
significant impact on the environment (Figure
5). In Craiova city the heavy metal pollution
induced by the values of the copper quality
indicator was identified near the industrial area
represented by the industrial platform
Electroputere and SC MAT SA Craiova. Also,
the values that exceeded the alert threshold of
copper lead and zinc quality indicators, were
identified both in Craiova, as well as in the cities
of Galati and Brasov, the latest metionated being
also cities with a well represented industrial
profile. The city of Oradea was shown with a
good representation of the compliance of the
quality conditions of the soil over in the urban
environment, which is a noticeable and a
positive fact. The capital of Romania, Bucharest
has highlighted both the existence of degraded
urban areas, marked by the uncontrolled wastes
directly on soil, but also important exceedances
of heavy metals in the soil in areas where
metallurgical activities, highly polluting, such as
“Laromet industrial platform”, in the northern
part of the city. Bucharest has major
environmental problems related to road traffic,
which brings a major contribution of
pollution.Also, particulate matter pollution of
the city atmosphere is a major source of soil
pollution by direct deposits on the ground or
through precipitation.
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 B. Stanescu et. al.: Soil management in the urban environment: today and future

Fig .5. Pollution in Copsa Mica city related to actual potential source

CONCLUSIONS other European countries that already took

Results obtained and the concrete situation in
the investigated urban areas led to the following
conclusions:
- to sensitize the general public on the
importance of keeping good soil conditions in
the cities of Romania;
- to actively involve the responsible factors,
respectively the Local Public Authorities, to
initiate soil monitoring programs in the urban
environment in order to follow in time and with
a large spatial distribution the evolution of the
soil quality and to intervene promptly if
problems that may affect soil quality are
reported. Figure 6 shows a proposed model for
approaching urban soil management for the best
monitoring and protection of this important
natural resource, soil. From the experience of
important steps in urban soil management, it was
found that degraded areas, through an adequate
strategy, were limited by implementing
measures for high soil quality protection.
In the modern acceptance, soil protection is
regarded as a source of maintaining the health of
the environment, through its role to ensure the
urban microclimate, to temper the thermal
contrasts and to attenuate the circulation of fine
powders. There is also an economic approach,
because the costs of discharging the fluvial
water through the drainage networks of the
urban infrastructure can be greatly diminished if
the soil in the urban environment allows the
inflection, as close to the values specific to the
natural environment.

Fig. 6. Proposed model for Soil Management in urban areas

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 B. Stanescu et. al.: Soil management in the urban environment: today and future

ACKNOWLEDGEMENTS. This work was carried out through the “Program Nucleu”, financed
by the Ministry of Research and Innovation of Romania, project codes: PN 16 25 02 10 and
PN 16 25 02 06.

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4. MCBRATNEY, A., FIELD, D.J., KOCH, A., Geoderma, 213, 2014, p. 203–213.
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7. SPARLING, G.P., SCHIPPER, L.A., BETTJEMAN, W., HILL, R., Agric. Ecosyst.
Environ., 104, 2004, p. 523–534.
8. BRISTOW, K.L., MARCHANT, S.M., DEURER, M., CLOTHIER, B.E., Proc. 19th World
Congress of Soil Science, Brisbane, Australia, 2010.
9. HUBER, S., VERNIK, T., SIEBIELEC, G., VRSCAJ, B., Soil in the City. Urban Soil
Management Strategy, Book, 2012,
10. *** Order no. 756 of November 3, 1997 for approving the Regulation regarding the
assessment of environmental pollution

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 C. Stoica et. al.: Danube Delta: monitoring and ecological status. A link between the past and the future

Danube Delta: monitoring and ecological status. A link between the past and the future

CATALINA STOICA*1, ELENA STANESCU2, IULIANA PAUN1, ALINA BANCIU1,

STEFANIA GHEORGHE1, IRINA LUCACIU1, GABRIELA VASILE1, MIHAI NITA-LAZAR1
1NationalResearch and Development Institute for Industrial Ecology ECOIND Bucharest, 71-73 Drumul Podul Dambovitei, 060652,
Bucharest, Romania
2Research Centre for Ecological Services “Dan Manoleli” (CESEC), University of Bucharest, 1-3 Aleea Portocalelor, Bucharest,

Romania
*corresponding author (e-mail): catalina.stoica@incdecoind.ro

The paper aims a review of the water quality monitoring and evaluation studies conducted at
National R&D Institute for Industrial Ecology over the last ten years, at the level of aquatic
ecosystem complexes in the Danube Delta.
The paper will include a broad compendium of data including: a data bank of abiotic variables
which control the structure and composition of biotic communities; studies of ecological status
assessment; methodology development for assessing the bioaccumulation of chemical pollutants
(metals) in the biota; ecotoxicological study of surface water and sediment on aquatic organisms
(algae, crustaceans, rotifers); studies on the benthic invertebrate species as biological vectors for
pathogens; technical / scientific support for 5 national / international project proposals; 19 papers
published in international / national journals; 22 papers presented at international / national
scientific conferences.

Keywords: ecological status assessment, Danube Delta, ecotoxicity, bioaccumulation

It is well-known that the aquatic ecosystems of the aquatic environment quality to

(including coastal waters, rivers, lakes) provide
numerous ecosystem services, including
aquaculture, fisheries, transportation, electricity
generation, water supply for agriculture
(irrigation), raw water source for drinking and
human consumption, exploitation of natural
resources (oil, gas) as well as cultural and
recreational services [1]. However, the constant
increase of the anthropogenic pressure,
especially in the last years, have diminished the
capacity of aquatic ecosystems to sustain
ecological communities. The Water Framework
Directive (2000/60 / EC) (WFD) adopted a
global sets of measurement to solve the
environmental issues and to achieve
environmental sustainability in a holistic,
integrative way by taking into account the social
requirements, too. The WFD preceded the US
Clean Water Act in 1979, and a clear
correspondence between the two
reglementations regarding the objectives,
implementation and ecological approach was
noted. The WFD purpose was to establish a
framework for the implementation of sustainable
water management strategies for the long-term
protection of water resources. The main
objectives were the protection and improvement
achievement of "good ecological status" for all
water bodies by 2015 [2] or at the latest by 2027
[3]. However, after 18 years of implementation,
WFD continues to be a major challenge
[4,5,6,7]. Member States of the European Union
have spent considerable resources and time
developing the instruments and obtaining the
necessary data for the preparation of River Basin
Management Plans (RBMP). In addition, WFD
has imposed the change of environmental
management objectives from the simplistic
approach that includes pollution control, to the
holistic approach, considering the system
integrity as a whole.
Thus, the “backbone” of the ecosystems
biomonitoring have been based on the response
of biotic communities to stress factors, rather
than the variability of the physical-chemical
parameters. Nowadays, there is a need to
develop rapid, cost-effective tools such as
genomic instruments which represent a
promising alternative to meet the environmental
protection and monitoring goals. Many studies
published in the last three years focused on
comparability between conventional methods
based on morphological identification of
biological models and alternative methods of

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 C. Stoica et. al.: Danube Delta: monitoring and ecological status. A link between the past and the future
DNA metabarcoding. The development of DNA
sequencing technologies has been a promising
alternative for biodiversity monitoring,
especially as these techniques can allow rapid
taxonomic identification, high accuracy, cost-
effective analysis allowing multiple samples to
be analyzed simultaneously by contrast with
conventional methods, higher costs and time
analysis as well as the vast and specific
knowledge on groups of organisms
(identification and quantification of
phytoplankton, phytobenthos and benthic
invertebrates) [8, 9]. The use of specific DNA
sequences to identify species can overcome the
problems mentioned above. In other words,
these alternative methods have the ability to
fundamentally change the assessment of the
ecological status of freshwater systems
worldwide.
In the present paper, the main results of Danube
and Danube Delta systems water quality
obtained after more than ten years monitoring
period were presented.
EXPERIMENTAL PART
Study area. The monitoring program was
developed at spatial (Danube-Danube Delta
ecological system) and temporal level (monthly
data over ten years’ period).
The main sampling sites were selected on the
Pontic Ecoregion along Danube: S1) Isaccea
(km 103) (N 45ᵒ17’051”; E 28ᵒ26’956”, (RO14
typological category) and Danube Delta (RO15
typological category): S2) Tulcea Upstream (km
82) (N 45ᵒ11’757”; E 28ᵒ47’372”), S3) Tulcea
Downstream (km 70) (N 45ᵒ11’492”; E
28ᵒ48’733”); S4) Nufaru (km 101) (N
45ᵒ09’111”; E 28ᵒ55’099”), S5) Baltenii de Sus
(km 97) (N 45ᵒ06’752”; E 28ᵒ59’212”, S6)
Mahmudia (km 90) (N 45ᵒ04’685”;
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E 29ᵒ04’749”), S7) Murighiol (km 64) (N
45ᵒ02’426”; E 29ᵒ11’090”), S8) Uzlina (km 70)
(N 45ᵒ04’452”; E 29ᵒ13’384”), S9) Ivancea (km
16) (N 44ᵒ58’219”; E 29ᵒ27’829”), S10) Sfantu
Gheorghe Port (N 44ᵒ53’467”; E 29ᵒ35’663”),
S11) Black Sea Confluence (km 0) (N
44ᵒ53’098”; E 29ᵒ36’655”). The geographical
localization of the sampling sites was performed
using GPS type system map 60CSx-Garmin
(Figure 1).
Data bank. Physico-chemical quality elements
such as pH, conductivity, dissolved oxygen
(DO), total dissolved solides (TDS), chemical
oxygen demand (COD), biochemical oxygen
demand (BOD), heavy metals (Fe, Mn, Zn, Cd,
Cr, Cu, Ni, Pb, Hg, As), amonium (N-NH4),
nitrates (N-NO3), nitrites (N-NO2), total nitrogen
(TN), phosphates (P-PO4), total phosphorus
(TP), chlorides, sulphates, sulphides, anionic
surfactants, calcium, magnesium, sodium,
petroleum products, polycyclic aromatic
hydrocarbons (PAH) (12 compounds), phenolic
compounds, organochlorine pesticides (α-HCH,
ß-HCH, -HCH, -HCH, heptaclor, aldrin,
dieldrin, endrin, DDT/DDD/DDE), triazine
pesticides (atrazin, simazin), ureic pesticides
(diuron, isoproturon, monolinuron) were
characterized in water samples according to
specific EN ISO standard methods. Alongside
chemical indicators detected in water samples,
numerous parameters such as As, Cd, Cr, Co,
Cu, Hg, Ni, Pb, Zn, Fe, Mn, PAH (12
compounds), organochlorine pesticides (α-HCH,
ß-HCH, -HCH, -HCH, heptaclor, aldrin,
dieldrin, endrin, DDT/DDD/DDE),
polychlorinated biphenyls (PCBs) and benzene,
toluene, ethylbenzene and xylene (BTEX) were
characterized in sediment samples.
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 C. Stoica et. al.: Danube Delta: monitoring and ecological status. A link between the past and the future

S1

S2 S3

S4
S5
S6 S8
S7

S9
S10
S11
0
Fig.1. The geographical localization of sampling sites along Danube: (Isaccea (S1) and Danube
Delta: Tulcea Upstream (S2), Tulcea Downstream (S3), Nufaru (S4), Baltenii de Sus (S5),
Mahmudia (S6), Murighiol (S7), Uzlina (S8), Ivancea (S9), Sf. Gheorghe Port (S10), Black Sea
Confluence (S11)

Biological quality elements such as values were compared with the reference area
phytoplankton and bacterioplankton (total and values (with little or no anthropic impact or
faecal coliforms, streptococci) as well as benthic historical data) and the subsequent classification
invertebrates and bacteriobenthos were detected in ecological states (very good, good, moderate,
both in water and sediment samples. weak, bad).
Ecological status assessment. Abiotic Ecotoxicological study. The toxicity of water
(temperature, oxygenation, mineralization, and sediment samples was evaluated using
acidification, nutrients, pollution with priority / various aquatic organisms such as: algae
priority hazardous substances) and biotic (Selenastrum capricornutum), crustaceans
characteristics (diversity, species presence, (Daphnia magna, Heterocypris incongruens)
numerical abundance / biomass) were and rotifers (Brachionus calyciflorus).
considered for assessing the ecological status of The ecotoxicological study performed along
Danube and Danube Delta aquatic ecosystems Danube Delta freshwater samples was done
following the WFD and RBMP requirements. during 2013. The methods used and the tests
Briefly, the ecological status assessment of the conditions are described in Table 1.
studied freshwater systems based on benthic The Danube Delta freshwater toxicity was
invertebrates’ fauna involved the organisms classified according to the hazard classification
sampling considering the standard methods, the system for natural water described by Persoone
drawing up of the taxonomic groups list, the et al. (2003) [11]: Class I – no acute hazard, no
morphological identification and the abundance test did not reveal toxic effect; Class II – slight
estimation. The resulting data were used for acute hazard, 20%≤ effect percentage <50%, in
various indices calculation (such as at least one test; Class III – acute hazard, 50% ≤
Ephemeroptera-Plecoptera-Trichoptera index, effect percentage <100%, in at least one test;
Shannon-Wiener diversity index, family number Class IV – high acute hazard, effect percentage
index, Oligochaeta-Chironomidae index, = 100%, in at least one test; Class V – very high
functional groups index, water flow preference acute hazard, effect percentage =100%, in all
index and multimetric index). The indexes tests.
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 C. Stoica et. al.: Danube Delta: monitoring and ecological status. A link between the past and the future

Methodology for assessing the metals
bioaccumulation in benthic invertebrates. The
bioaccumulation of numerous metals (such as:
As, Se, Sb, Cd, Cr, Cu, Co, Fe, Mn, Mg, Ni, Pb
and Zn) was quantified from two bivales species
and one gasteropod species collected during
summer and autumn 2013 from S7 and S8. The
applied methodology was detailed in Gheorghe
et al. (2017) [12].
Benthic invertebrates as biological vectors for
pathogens. S6, S7, S8 and S10 sampling sites
were selected along Danube Delta for the
identification of potential vectors of pahogens in
the benthic communities’ structure. The samples
were collected in May and October of 2015.
Benthic invertebrates assemblages (live
specimens) were sampled using Van Veen
dredge (sampling depth of 10 cm).
The samples for microbiological parameters
analysis were collected in 1 L glass bottles (for
water) and 400 g (for sediment). The recipients
were properly sterilized at 160˚C. Benthic
macroinvertebrates were separated by taxonomic
groups using Motic stereomicroscope and
identified by specific determination keys. The
identification and characterization of
microorganisms was carried out on the
OmniLog automated system (Biolog Inc., USA).

Table 1. Bioassays used to assess the water and sediment toxicity in Danube Delta (according with
Gheorghe et al. (2016) [10]
Method / Tested Examined Test duration/ Sample
Test type Treatment
Microbiotest organisms behavior temperature matrix
OECD 201 Selenastrum Acute / Growth 72h, Undiluted
Water
AlgaltoxKit FTM capricornutum Chronic inhibition 21-25oC
OECD 202 Acute
Daphnia magna Mortality 24-48h, 20oC Water Undiluted
DaphtoxKit FTM
ASTM Standard Guide
24 h,
E1440-91 Brachionus calyciflorus Acute Mortality Water Undiluted
25oC
RotoxKit FTM
Mortality
ISO 14371 Heterocypris 6 days,
Chronic Growth Sediment Untreated
OstracodtoxKit FTM incongruens 25oC
inhibition

RESULTS AND DISCUSSIONS

Data bank. In the last ten years, National R&D
Institute for Industrial Ecology developed
various studies aiming to characterize the water
quality of Danube Delta systems under an
extensive and intensive monitoring program. A
large database comprising the monthly values of
abiotic variables and the dynamics of biotic
communities was established.
The data collected from numerous physico-
chemical parameters of the water samples,
collected along Sfantu Gheorghe Branch,
showed a high organic load (expressed as COD
and BOD) mainly at S7 during 2003-2013 and a
high nutrients concentration (1.08 mg/L to 7.72
mg/L of total nitrogen and 0.03 mg/L to 0.48
mg/L of total phosphorus) [37].
The increased concentration of COD, BOD and
nutrients were due to the conversion of flood
zones into agricultural lands associated with the
extensive use of fertilizers which occurred in the
second half of the 19th century. In the same
period of time, the industrialization and the
development of socio-economical systems [13]
led to the accumulation of a broad range of
chemical compounds, an effect which was found
worldwide in hydrographic basins, including
Danube and Danube Delta systems [14]. For
instance, at the level of ecosystem complexes
represented by Mahmudia meander, in the last
10 years, there was an over the limit (imposed
by the Romanian / European legislation)
presence of specific organic pollutants such as:
polycyclic aromatic hydrocarbons (benz (a)
anthracene, benzo (b) fluorantren, fluorantren,
phenanthrene), γ - HCH (lindan), heptachlor,
DDT / DDE / DDD, polychlorinated biphenyl
compounds (PCBs) and petroleum hydrocarbons
as well as heavy metals (Hg) [15, 16, 17, 18, 19,
20].
In spite of an improvement of water quality
observed in recent years, the Danube and the
Danube Delta (Sfantu Gheorghe Branch)
continue to be exposed to numerous point and
nonpoint pollution sources [21].
The changes that occurred in the structure, the
hydrological regime and the hydrochemistry at

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 C. Stoica et. al.: Danube Delta: monitoring and ecological status. A link between the past and the future

the level of the ecosystem complexes
represented by the Danube and the Danube
Delta (Sfantu Gheorghe Branch), were followed
by profound changes in the taxonomic
composition and distribution of the biocenoses.
The densities of both coliform bacteria and
Enterococcous in water samples were high
during the summer and autumn sampling
campaigns. Moreover, various antibiotic
resistant bacteria were identified [22]. However,
annual averages of faecal bacteria densities
decreased in recent years in Danube Delta, due
to the reduction of nutrient concentration (N, P)
[23, 24]. The qualitative analysis of
phytoplankton community along the Danube
Delta sampling sites over ten-years study,
emphasized the presence of species belonging to
the classes Baccilariophyceae, Chlorophyceae,
Euglenophyceae and Cyanophycae. In all
sampling sites, the oligo-betamezosaprobe,
betamezosaprobe, beta-alfamezosaprobe and
alfamezosaprobe species dominated. The
taxonomic diversity of phytoplankton included
64 species of algae, but the most frequent were
Asterionella formosa, Cymatopleura solea,
Melosira varians, Navicula gracilis, Cymbella
ventricosa, Phacus longicauda, Scenedesmus
quadricauda, Pediastrum duplex, Amphipleura
pellucida, Fragilaria crotonensis, Nitzschia
sigmoidea [25]. The distribution of
phytoplankton species was conditioned by the
availability of light, dissolved oxygen, nutrients,
but also by the dynamics of the hydrological
regime. The phytoplankton densities increased
from Isaccea (S1) (32.750 ind/L, average
numerical density calculated during 2012-2013)
located on the Danube downstream to the Sfantu
Gheorghe Branch sites, due to the intake of
phytoplankton from lakes and canals. It is the
case of S8 (Uzlina) sampling site which
recorded the highest numerical densities during
2003-2013 (40.145 ind/L).
The benthic invertebrate fauna was composed of
species belonging to Gasteropoda, Bivalvia,
Oligochaeta, Insecta, Crustacea taxonomic
groups, with greater abundance and diversity in
the stations located on Sfantu Gheorghe Branch,
compared to those on the Danube likewise
phytoplankton communties, but lower compared
to those reported by other studies conducted on
Danube [26, 27, 28, 29, 30, 31, 32, 33]. This
reduction of the taxonomic richness from
upstream to downstream was considered to be
due to the heterogeneity and size of the particles
/ sediment granulation, alongside the increase of
control factors pressure [34].
The heavy metal profile in the Danube Delta
sediment had a specific configuration. The
concentration of heavy metals (Zn, Hg, Cu)
increased until 1989, due to the increase of
industrialization in Central and Eastern Europe
[35], including in Romania. However, during the
last 5 decades a decrease of metals concentration
in the Danube Delta was recorded (Table 2).
Although, the concentration of metals in the
sediment diminished in the last 10 years
between 2 to 5 folds, however, some of them
have been adsorbed on the surface of the debris
or humic acids. The neglecting of plans
regarding intensive agriculture in the Danube
Delta, contributed to the reduction of Cd
concentration as this compound is part of
fertilizers.

Table 2. Variation of heavy metals (mg / kg s.u) in the sediments of the Danube Delta during the
last five decades
1950 2003-2009
Quality element 2012-2013
U.M Winkels et al., 1998 [35] Vosniakos et al., 2010 [18]
Cu 38 14.8-194 4.65-45.9
Pb 36 7.5-51.3 4.76-41.3
Zn mg/kg d.m 90 29.8-218 17.7-93.1
Cr 50 7.5-61.9 7.62-32.5
Ni 56 19-111 10.8-49.8
Cd <0.5 <0.5-1.5 BDL*
*BLD-below detection limit

Decreased Pb may also be associated with time, the phasing out of gasoline with Pb is a
changes in land use, as it also includes complementary method of reducing this
fungicides and other pesticides. At the same contaminant. The increase of metals

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 C. Stoica et. al.: Danube Delta: monitoring and ecological status. A link between the past and the future
concentration along Danube Delta sampling
points was due to the nonpoint contamination
from non-ferrous deposits abandoned after the
transitions in the Romanian economy [36]. In
addition, PAH concentrations increased almost
seven-fold compared to concentrations
determined in 1950 [35], confirming the
negative impact of navigation development (data
not shown).
Moreover, the organochlorine pesticides
concentration decreased after 1990, with
changes in the political sector, but still persist in
the sediments of the Danube Delta. A visible
decrease between 2012-2013 and 2003-2011,
with the exception of DDT and its degradation
products was observed [15, 16, 17, 18, 25, 37].
However, there was a positive correlation
between the concentrations of the
organochlorine pesticides and the benthic
indices: Saprobic index (IS), Oligochaeta-
Chironomidae (OCH/O) and Hilsenhoff biotic
index (IBH) at S6 (Mahmudia), S7 (Murighiol),
S9 (Ivancea) and S10 (Sf. Gheorghe) in terms of
increase values of the indices due to the
increased values of organochlorine pesticides
(data not shown).
As a result, the structure and composition of the
benthic communities changed, by reducing the
number of taxonomic groups, the replacing of
sensitive sepcies with the tolerant ones, often
invasive as well as the replacing of shredders or
collectors with detritivores (data not shown).
Studies conducted after 1990 have shown a
pronounced simplification of biocenoses
integrated into aquatic systems [38,39,25],
preceding a period of restoration of the structure
and composition of the benthic invertebrate
fauna [40].
Overall, a general tendency of benthic fauna
recovery in terms of taxonomic richness was
observed during the monitoring period at the
level of ecosystem complexes in the Danube and
Danube Delta (data not shown).
A large database was created based on the
monitoring of Danube Delta water quality over
ten years’ period (2003-2013) and subsequently,
it was possible to perform a comprehensive
study of Danube Delta ecological status
assessment. The macroinvertebrates’ community
composition was used as they are closely related
to habitat and substrate heterogeneity.
Moreover, ‘biological quality elements’ were
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more affected by anthropic and environmental
pressure factors rather than ‘chemical quality
elements’ [41].
Ecological status assessment.
Characterization of the state variables (both
chemical and biological quality elements) that
influence the composition and structure of
macroinvertebrates organisms, as well as the
dynamics of these biological elements, allowed
the assessment of the ecological status of the
Danube and Danube Delta. Therefore, taking
into account the "worst case" principle and the
results obtained during 2012-2013 from both
biological and chemical quality elements, the
assessment induced „good” ecological status for
Danube system (typological category RO14) and
"moderate" ecological status for Danube Delta
(typological category RO15) (data not shown).
The assessment of freshwater systems ecological
status was completed by an in-vitro
ecotoxicological study.
Ecotoxicological study. Primary producers
(algae, Selenastrum capricornutum) and
consumers (planktonic crustaceans, Daphnia
magna and rotifers, Brachyonus calyciflourus as
well as benthic crustaceans (Heterocypris
incongruens) were used to evaluate the Danube
Delta water and sediment quality according to
hazard classification system of natural water
[11]. The water samples showed no acute toxic
effect (Class I) or slightly acute toxic effect
(Class II). The sediment samples were more
toxic than water samples due to the pollutants
accumulation (e.g metals, PAH, organochlorine
pesticides). Their main toxicity classes were
from Class II – slightly acute toxic effect and
Class III – acute toxic effect up to Class IV –
high acute toxicity. The largest number of water
toxic responses was observed in case of
Selenastrum capricornutum algae, followed by
Daphnia magna crustaceans and Brachyonus
calyciflourus rotifers. In case of sediment, the
highest number of toxic responses were obtained
from Heterocypris incrongruens ostracods [10].
In addition to the development of the database
comprising a large number of water quality
indicators, the study of freshwater systems
ecological status assessment as well as an
ecotoxicological study, a methodology for the
evaluation of metals bioaccumulation in biota
was implemented.
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 C. Stoica et. al.: Danube Delta: monitoring and ecological status. A link between the past and the future
Methodology for assessing the metals
bioaccumulation in benthic invertebrates. The
researches that founded the above mentioned
methodology was carried out in 2013 (July and
September) along Danube Delta (S7, S8 and
S10) regarding the accumulation of chemical
pollutants (metals) in Mollusks (two Bivalve
species: Anodonta sp., Unio sp. and one
Gasteropode: Viviparus sp.) [42].
The results pointed out that the metals involved
in the metabolic processes (Fe, Mn, Zn, Cu and
Mg) have a greater accumulation capacity than
the toxic ones (Pb and Cd). This fact was also
explained by the presence of these metals in
high concentrations, reported in previous studies
[43]. Moreover, Vasile et al. (2006, 2005) [20,
44] observed that Pb was either mobile or bound
to manganese oxides and organic matter in
sediment samples. The mobile Pb concentration
was higher in the sediment at S7 due to the
increase of the total concentration of metals in
the sediment, compared to the concentration of
metals detected at S8, where a decrease
concentration in the mobile form for Cu, Zn and
Pb was detected in 2004, compared to 2003.
Furthermore, Vosniakos et al. (2010) [18]
showed that the mobile fraction (1.2-3.1 mg /
kg) of Cu represents 41%, and Ni showed a
relatively low mobility compared to its total
concentration.
Gheorghe et al (in tech) highlithed that the
bioaccumulation level fluctuated considering the
species, metals type, and sampling sites. A
greater difference of bioaccumulative metals
impact (Ni and Zn) at S7 compared to S8 was
observed. The study of metal bioaccumulation
capacity in the mollusks shell from Danube
Delta aquatic system underlined that metals
involved in metabolic processes (such as Fe,
Mn, Zn, Cu, and Mg) had a greater storage
capacity than the toxic ones (such as Pb and Cd)
[12]. The selectivity of the metal
bioaccumulation was represented as follows:
Fe> Mn> Zn> Cu> Pb> Co> Cd, in case of
Viviparus sp. shell, while the shell of Anodonta
sp. had a higher accumulation capacity of metals
(Cu, As, Cr, Zn) compared to Unio sp. (data not
shown). Overall, bioaccumulation factors were
subunitary, which indicated a slowly
bioaccumulation process along Danube Delta.
Since, the macroinvertebrates represent the key
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component of aquatic ecosystems, the group was
largely examined in our over ten-years studies.
A number of functions performed by benthic
invertebrates’ fauna have been mainly related to
trophic relations (accumulating, transforming
and transferring the stored energy from organic
detritus decomposition and nutrient transfer to
the higher trophic levels, information circulation
and systems self-regulation [45,46, 47, 48]. The
advantage of using benthic invertebrates’ fauna
in the freswater quality evaluation studies was
due to their ability to maintain in their structure
the effects of environmental pressure [49, 40].
Moreover, the invertebrates have relatively long
life and widespread distribution, limited
mobility and they cannot avoid adverse
conditions when exposed to environmental
issues (such as eutrophication, acidification, low
oxygen concentrations, hydromorphological
changes or other problems combined with
habitat loss (e.g. embankments, etc.).
Thus, the benthic communities were considered
suitable for either the bioaccumulation of
specific toxic pollutants in their structure, or
characterization and assessment of ecological
status of aquatic ecosystems quality, including
the identification of various benthic taxonomic
groups as vector for pathogens.
Benthic invertebrates as biological vectors for
pathogens. The identification of potential
pathogen vectors of the benthic communities’
structure was carried out in S6, S7, S8 and S10
sampling sites along Danube Delta. The samples
were collected in May and October of 2015.
The results pointed out that alongside the
structure of benthic invertebrate fauna (potential
vectors for pathogens) from the ecological
system of the Danube Delta, a wide range of
stable endogenous bacteria were identified.
Since the frequency of occurrence reported in
2014 was greater than 50% (Figure 2) [40],
several benthic invertebrates’ groups were used
as sentinels to assess the prevalence of
pathogens microorganisms, such as: filters
(Dreissena polymorpha, Anodonta cygnea, Unio
pictorum), scrapers (Viviparus acerosus,
Esperiana esperi, Esperiana acicularis,
Lithoglyphus naticoides, Theodoxus danubialis
and Chironomidae) and detritivores
(Oligochaeta, Ceratopogonidae).
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Fig. 2. Frequency of occurrence of benthic invertebrates

Although 15 pathogen species were detected in
all sampling sites subjected to untreated
wastewater discharges, mainly linked with
animal and human fecal waste, Citrobacter
freundii and Raoultella plancticola /
ornithinolytica were the most common bacteria
species, especially associated with Diptera
larvae and Mollusks species.
CONCLUSIONS
The paper presented a summary of all the studies
carried out in the National R&D Institute for
Industrial Ecology for more than 10 years at the
level of aquatic ecosystems complexes of
Danube and Danube Delta, considering the
anthropogenic pressure after the industrialization
period. The studies aimed to highlight the
ecological integrity of aquatic systems
considering both chemical and biological
components monitoring. The main issues that
Danube and Danube Delta still facing are: high
organic load (expressed COD and BOD),
pathogenic microbial load as well as, the
increase of antibiotic bacterial resistance, high
concentrations of heavy metals (copper, nickel,
zinc, mercury), organochlorine pesticides
(lindane, DDT/DDE/DDDs), PAHs and PCBs,
mosly detected in sediment. A reduction of
sensitive benthic invertebrates’ species and
replacing with the tolerant ones was observed.
Moreover, benthic invertebrates’ fauna was
strongly related with pathogens and can be
further used as alternative monitoring systems
for waterborne contamination. In addition, since
the Danube Delta water body was framed in
2013 as „moderate” ecological status according
with WFD, alternative methods needs to be
tested in order to meet the good ecological status
by 2027.
Future recommendations. Since the good
ecological status for all water bodies by 2015
was not achieved, alternative methods needs to
be tested to meet this criterion by 2027. Several
steps further were made at international level
[50] by means of DNA metabarcoding
identification of prokaryotic and eukaryotic
organisms, yet certain "methodological
limitations" [51] have been identified, namely:
a) biomass estimation [52]; b) detection of rare
species [53]; c) incomplete reference libraries
[54, 4].
Considering those issues, our future studies will
focus on the application of alternative methods
at national level, which may contribute to the
identification of functional diversity based on
gene expression (transcription), and thus, to
meet a WFD target that could not be adequately
covered based on conventional morphological
identification methods.

ACKNOWLEDGEMENTS. This work was financially supported by the National Core Program
within several contracts: 20N/2019, Project no. PN 19- 04 02 01, 13N/2009, Projects no. 09-13 02
03, PN 09-13 02 14, 12N/2006, Project no. PN 06-12 01 13.

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