10.1.
Environmental Risk Assessment and Mitigation of Hazardous
Chemical Contamination
Business Plan
LABORATORY FOR MOLECULAR ECOTOXICOLOGY (LME)
LABORATORY FOR BIOGEOCHEMISTRY OF ORGANIC COMPOUNDS (LBOC)
M. AHEL, D. HRŠAK, M. PICER AND T. SMITAL
Vision
The main idea is to form a regional center for risk assessment and mitigation of
hazardous chemical contamination in the marine and terrestrial ecosystems. A
comprehensive assessment of environmental risk associated with the increasing use of
a large variety of toxic chemical contaminants requires an extremely high level of
multidisciplinary expertise, which can be found exclusively in specialized
interdisciplinary-oriented scientific institutions. Moreover, a truly objective judgment
of the environmental issues can be achieved only through fully independent institutes
that are capable to respond to growing public demands for a safe and healthy
environment.
Mission
The mission of the future Center comprises research, postgraduate education
and performing of services in the field of environmental protection, including
monitoring, ecotoxicological assessment and life cycle analysis of harmful
substances, as well as developing strategies for the prevention and mitigation of
hazardous chemical pollution. It also envisages a market-oriented dissemination of the
results of fundamental and applied research to various users, such as governmental
agencies, local authorities and industry in order to promote a scientifically-based
approach to the detection, assessment and mitigation of risks associated with usage
and disposal of hazardous substances.
Objectives
Research:
 Systematic investigation of hazardous chemical contamination in the marine
and freshwater ecosystems as a basis for sustainable development of the
Republic of Croatia
 Development of new highly specific methods for the determination of
xenobiotic compounds in environmental samples
 Investigation of detoxification mechanisms in aquatic organisms and
development of new biomarkers to detect hazardous chemical contamination
(e.g. multixenobiotic resistence MXR)
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Evaluation of the combined use of chemical and biomarker approach in the
ecological risk assessment
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Study of microorganisms as catalysts in biotransformation processes and
cycling of elements in the biosphere
Investigation of metabolic pathways and biotransformation kinetics of
ecotoxicologically relevant chemical contaminants, with the emphasis on
highly toxic, genotoxic and endocrine disrupting substances

Education:
 Participation in postgraduate studies in marine sciences (specialized courses,
execution of Ms and PHD theses)
 Participation in postgraduate studies in environmental protection
(specialized courses, execution of Ms and PHD theses)
 Teaching and providing specialized training courses in environmental
analytical chemistry, ecotoxicology, environmental microbiology,
implementation of environmental directives
Development of new measurement techniques:
 Introduction of the most reliable state-of-the-art methodologies for the
determination the prominent pollutant classes foreseen by the Croatian
regulations and EU Water Directive
 Becoming a center of excellence for the analysis of war-related hazardous
pollution in soil and aquatic systems
 Obtaining ISO certification for the standardized methods for the
determination of the most common xenobiotic pollutants such as PAH and
organo-halogen compounds
 Development of suitable in vitro and in vivo bioassays for the determination
of transport activity of specific proteins included in MXR phenomenon;
 Development of appropriate methods and statistical models for the
determination of additive, antagonistic and synergistic effects of binary (or
even more complex) combination of pollutants
 Introduction, standardisation and intercalibration of in vitro and in vivo
methods for the detection of xenoestrogenic effects of environmentally
relevant pollutants; development of molecular biology techniques (PCR,
DGGE and in situ rRNA hybridization) for the characterisation of
detoxification mechanisms in aquatic organisms and for the detection and
identification of active microorganisms, participating in biotransformation
processes
 Validation of the reference methods for biodegradability and ecotoxicity
testing
Services:
 Monitoring of various pollutants and exposure biomarkers in the framework
of national projects supported by Ministries and governmental agencies
 Expert assistance in accidental situations
 Authorised reference laboratory for the assessment of ecotoxicity,
biodegradability and other biological parameters in wastewaters and natural
waters
 Environmental impact studies and consulting
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Technology development and support:
 Participation in the development of biotechnological processes for the
treatment of xenobiotic substances in wastewater and in situ
biotransformation. (e.g designing a treatment plant for purification of
wastewaters from the herbicide production)
 Development of new procedures for the advanced wastewater treatment
based on membrane technologies (e.g. treatment of landfill leachate
containing biorefractory pharamaceuticals)
Background
The co-workers of the future Center presently work in 2 Laboratories of the Center for
Marine and Environmental Research, Laboratory for biogeochemistry of organic
compounds (LBOC) and Laboratory for molecular ecotoxicology (LME). The
Laboratories consist, in fact, of 4 autonomous research groups with distinct goals and
funding resources. With respect to the proposed new association, the groups have
complementary structures including environmental analytical chemistry,
biogeochemistry, molecular ecotoxicology, environmental microbiology and
biotechnology. All these disciplines form a promising network regarding the high
requirements, which are needed to cope with the complex issues of environmental risk
assessment. Besides the fundamental research, which considered as the only solid
basis for all intended applications, the expertise gathered around the proposal allows
the initiation of activities towards development and support of new technologies for
wastewater treatment and in situ remediation. Besides an adequate scientific
background, each of individual groups has a long experience in applying their
fundamental research to the practical problems in the environment.
The Group for organic pollutant analysis was founded in 1974 with the main task to
initiate gas chromatographic methods for the analysis of organochlorine pollutants in
the marine environment. The members of the group have gradually broadened their
interest to other pollutant classes, such as hydrocarbons, methyl-mercury, volatile
halogenated hydrocarbons, anionic and nonionic surfactants and their metabolites,
triazine herbicides and pharmaceutical chemicals, evolving into two separate groups
(LME-analytical and LBOC-biogeochemical). Research activity of the LMEanalytical group can be divided into two main branches: a) development of methods
for the analysis of organic pollutants in environmental samples and b) application the
developed methods in various field studies aimed at determining exposure levels and
fate of selected pollutants in soil, marine and freshwater environments. Methods for
analysis mineral oil and chlorinated hydrocarbons have been successfully
intercalibrated in numerous domestic and international intercalibration exercises.
Research programme of the LBOC-biogeochemical group comprises systematic
investigation of biogeochemical behaviour and fate of biogenic and anthropogenic
organic compounds in different freshwater and marine environments using highly
specific analytical techniques such as high-resolution gas chromatography, highperformance liquid chromatography and mass spectrometry. Regarding biogenic
compounds, a special attention was paid to the natural compounds which play a major
role in organic carbon cycling (e.g. photosynthetic pigments and carbohydrates),
while for xenobiotic chemicals emphasis was on anionic and nonionic surfactants and
emerging pollutants having pharmaceutical origin. The research programme includes
studies of environmental processes, which govern the fate of organic compounds in
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real systems with a special emphasis on biodegradation. Intensive field studies have
been carried out in the large variety of aquatic environments, ranging from marine
environments to the alluvial aquifers. Frequent sampling campaigns in the marine
environments are mainly focussed on the shelf and estuarine areas influenced by
increased freshwater inputs (e.g. northern Adriatic and Krka River and Zrmanja River
estuaries) with the emphasis on eutrophication-related phenomena.
The members of both groups participated in UNEP MED POL Projects of
Monitoring the Adriatic Sea (Phase I. Phase II and Phase III) and former YugoslavItalian cooperation in monitoring the Adriatic Sea. The Group have taken part in
numerous ecological studies of the various parts of the Adriatic environment andfresh
water environment in Croatia. During the 1991-1995 war in Croatia, and especially
after the “Oluja“-action in 1995, the LME-group has been intensively involved in the
investigation of the ecological consequences of the war damages in the karst area of
Croatia.
The members of analytical/biogeochemical groups published more than 200 scientific
and professional papers dealing with the analytical determination environmental fate
of various organic and organo-metallic pollutants in soil and water, earning more than
1600 citations.The average impact factor per paper published in CC-journals (being
an indicator of the quality of journals) is 1.286 The total number of citations received
by the researchers of our laboratory (period 1978-2001) is about 1400 (including
some cross-citations In terms of scientific productivity in the period 1996-2001, the
number of papers published in CC and SCI-journals per faculty is about 5.
The basic goal of the Laboratory for molecular ecotoxicology (LME) is to improve
Ecological Risk Assessment by discovering and establishing new, better and more
relevant biomarkers as important indicators of environmental quality.
The research group is primarily focused on the identification of detoxification and/or
defense mechanisms that are present in taxonomically various classes of marine and
freshwater organisms. The work comprises explanation of the physiological and
biochemical relationships between the activity of detoxification mechanisms and the
level of environmental pollution with the aim to develop and standardize biomarkers
that would allow the measurement of the activity of these defense mechanisms on the
molecular, and even more important on the sublethal level. This approach offers
important information on biologically relevant exposure levels and effect of
environmental pollutants and provides a scientific basis for both national and
international legislation as well as for rational spending of funds assigned to
environmental management and/or protection.
In the last 5 years LME published 24 papers in SCI quoted journals, with the
average impact factor per paper of 1.264 (Note: average impact factor of journals listed in
Environmental Sciences is  0.737) and average citations per paper of 6.4. Average
number of papers per faculty is about 4. One patent was filed. The LMEecotoxicology research group discovered the presence of a new defense mechanism
inherently present in aquatic organisms. The discovery of this defense system,
hereafter in aquatic organisms named Multixenobiotic Resistance Mechanism
(MXR), had exceptional ecotoxicological significance, especially after investigations
that demonstrated that MXR might be inhibited by specific chemicals, so-called MXR
inhibitors that are also present in aquatic environment. Therefore, MXR-inhibitors
may be considered as a new potentially dangerous class of chemicals. During the last
five-year period LME actively participated in several ecological and biomonitoring
studies and as a consequence of this MXR mechanism was recently classified as a
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promising (“under development”) biomarker within international (US EPA, ICES,
UNEP MED POL) biomonitoring programs.
The main research interest of the research group for Environmental microbiology and
biodegradation (LBOC- environmental microbiology) is in basic principles governing
the biotransformation of complex synthetic organic compounds (xenobiotics) with a
special emphasis on microorganisms as essential factor (catalysts) in
biotransformation processes. The research activities are focused on the enrichment,
isolation, identification and characterization of bacterial communities from various
pristine and polluted environments with the aim to study their roles in the
transformation of complex synthetic organic compounds. This research is a
prerequisite for better understanding the principles of the biogeochemical cycling of
elements in the biosphere and is crucial for opening the possibility to influence the
rate and the extent of xenobiotics transformation. Furthermore, this research provides
a basis for the optimisation of the existing and development of new biotechnological
procedures for the remediation of chemically-polluted sites as well as for improving
the efficiency of traditional treatment of domestic and industrial wastewaters that
regularly contain various xenobiotic compounds.Besides these basic investigations, a
significant part of the research has been carried out in the framework of applied
projects. Currently, an innovative-technological project is in progress with the main
goal for designing the biotechnological procedure for the treatment of wastewater
from atrazine production. Specific goals of these projects are possible improvements
in maximizing atrazine recovery from technological wastewater and to meet the new,
stringent discharge standards (minimize raw water use, wastewater generation and
pollution of natural waters).
All research groups involved in this proposal have had an intensive co-operation
with many internationally recognized laboratories, including those in EU and USA.
The staff actively participated in several international projects, in some of them acting
as co-proposers and scientific co-ordinators. Very recently, the leader of the LMEanalytical group has been accepted as the scientific co-ordinator of a project on
“Assessment of the selected POPs (PCBs, PCDDs/F, OCPs) in the athmosphere and
water ecosystems from the waste materials generated by warfare in area of former
Yugoslavia”, which is being realised within the EU Fifth framework INCO
Copernicus Programme, involving 12 institutions from Croatia (3 institutions),
Slovenia, Bosnia and Herzegovina, Kosovo, FR Yugoslavia (3 institutions), Austria,
Czech Republic and Greece.
Current situation
LME-Ecotoxicology group
Staff:

The Lab staff include 5 researchers (educated in ecology, molecular biology
and ecotoxicology) + 1 technician;
Existing infrastructure:
 Lab space (6 rooms, 136 m2);
 Supraspeed centrifuge Sorvall RC 28S (2 x) + other centriguges;
 Microplate fluorimeter Fluorolite 1000 (Dynatech); Microplate photometer
Anthos HT III (Anthos);
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Incubator, LaminAir, inverse microscope (Olympus) and other cell culture
equipment (Heraeus);
Colony counter and other equipment for Ames microsomal test;
Freezers (-80C, 2 x), liquid nitrogen containers;
"Wet" lab (basins, experimental aquaria, flow of dechlorineted tap water;
Rotary evaporator, sterilizators, autoclav, balances, etc.
Major projects/ revenues
 ”Multixenobiotic Resistance Mechanism as a Biomarker of Environmental
Quality”, 2002-2004) – Project supported by the Ministry of Science and
Technology of The Republic of Croatia - estimated budget ~ 22.000 $
(EUR/year);
 “Projekt Jadran” - Systematic investigation of the Adriatic Sea as a basis for
sustainable development of the Republic of Croatia (a project funded by
Ministry of Science and Technology) ) – estimated budget ~ 11.000 $
(EUR/year);
 ”An integrated environmental monitoring system for Croatian freshwater,
estuarine and coastal marine areas”, (2002-2004) – Bilateral Project
supported by the Research Council of Norway and the Ministry of Science
and Technology of the Republic of Croatia – our budget ~ 28.000 $
(EUR)/year;
 ”Ecological study of gas fields in the northern Adriatic”, (2001.-) – Pilot
project supported by INA (Croatian oil and gas company) – estimated
budget ~ 10.000 $ (EUR)/year.
 ”An integrated environmental monitoring system for Croatian freshwater,
estuarine and coastal marine areas”, (2002-2004) – Bilateral Project
supported by the Research Council of Norway and the Ministry of Science
and Technology of the Republic of Croatia – our budget ~ 28.000 $
(EUR)/year;
 ”Ecological study of gas fields in the northern Adriatic”, (2001.-) – Pilot
project supported by INA (Croatian oil and gas company) – estimated
budget ~ 10.000 $ (EUR)/year.
LME-analytical group
Staff:

The Lab staff include 2 researchers (educated in analytical chemistry) + 1
PhD student;
Existing infrastructure:
 Lab space (2 rooms, 40 m2);
 1 Gas chromatograph equipped with EC-detector
 Rotary evaporator
 Accelerated solvent extraction
Major projects/ revenues
 Persistent organohalogen pollutants in coastal areas of Dalmatia, project
funded by Ministry of Science and Technology of the Republic of Croatia.
(7000 USD/year)
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
“Projekt Jadran” - Systematic investigation of the Adriatic Sea as a basis for
sustainable development of the Republic of Croatia (a project funded by
Ministry of Science and Technology) (10000 USD/year)
 “Assessment of the selected POPs (PCBs, PCDDs/F, OCPs) in the
athmosphere and water ecosystems from the waste materials generated by
warfare in area of former Yugoslavia” - a 3-year project realised within the
EU Fifth framework INCO Copernicus Programme (40 000 USD/year)
LBOC-biogeochemistry group
Staff:

The Lab staff consists of 2 researchers (educated in environmental analytical
chemistry, 2 PhD students + 1 technician)
Existing infrastructure:
 Lab space (5 rooms, 70 m2);
 GC with mass spectrometric detection
 GC chromatography with FID and NPD-detection
 HPLC with serially coupled spectrophotometric and spectroflurimetric
detection
 HPLC/MS/MS system with ESI and APCI
 Spectrofluorimeter
Major projects/ revenues
 Biogeochemistry of organic compounds in natural waters, the basic project
funded by the Ministry of Science and Technology of the Republic of
Croatia (30 000 USD/year).
 “Projekt Jadran” - Systematic investigation of the Adriatic Sea as a basis for
sustainable development of the Republic of Croatia (a project funded by
Ministry of Science and Technology) (25 000 USD/year)
 Aromatic Surfactants and Emerging Chemical Contaminants – Analyses,
Occurrence, and Fate in the Aquatic Environment (ASECCO), a bilateral
Croatian-Swiss project between the Ruđer Bošković Institute and Swiss
Federal Institute for Environmental Science and Technology funded by the
Swiss programme SCOPES (principal investigators Walter Giger and
Marijan Ahel) (cca. 12 000 USD/year)
 Mitigation of environmental consequences of war in Croatia – Risk
assessment of hazardous chemical contamination” – a bilateral CroatianNorwegian project funded by the Norwegian government (principal
investigator Marijan Ahel) (cca 25 000 USD/year)
LBOC-environmental microbiology
Staff:

The Lab staff include 1 researchers (educated in environmental
microbiology) + 2 PhD students;
Existing infrastructure:
 Lab space (5 rooms, 78 m2);
 1 spectrophotometer
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Laboratory equipment for microbiological studies (sterilizers, incubators,
water baths, devices for membrane filtration, shakers, microscopes, counting
apparatus, refrigerators, freezers)
 Ordinary laboratory equipment and specific equipment for biodegradability
studies (centrifuge, pH meter, oxygen measuring instrument, agitators,
different test vessels, activated sludge units, continuous flow units, dosing
pumps, aeration devices, ultrasonic baths, device for filtration etc.)
Major projects/ revenues
 “Microbial communities as catalysts in biotransformation processes,
scientific project funded by Ministry of Science and Technology of the
Republic of Croatia. (16 000 USD/year).
 “Projekt Jadran” - Systematic investigation of the Adriatic Sea as a basis for
sustainable development of the Republic of Croatia (a project funded by
Ministry of Science and Technology) (5 000 USD/year)
 “Assessment of the selected POPs (PCBs, PCDDs/F, OCPs) in the
athmosphere and water ecosystems from the waste materials generated by
warfare in area of former Yugoslavia” - a 3-year project realised within the
EU Fifth framework INCO Copernicus Programme (20000 USD/year)
 Conceptual model for biotechnological treatment of atrazine production
waste water - Development and innovative-technological project funded by
Ministry of Science and Technology and herbicide factory Herbos d.d.,
Sisak ( principal investigator Dubravka Hršak) (50 000 USD/year)
 Authorized Reference Laboratory Activities (ecotoxicity, biodegradability
and other biological parameters for natural water and wastewater quality
control), a contract between the Ruđer Bošković Institute and Croatian
Authority for Waters (11 000 USD/year)
SWOT and PEST analysis
SWOT (Strengths, Weaknesses, Opportunities, Threats):
Strength of the Laboratory is in well-educated and specialized staff, most
probably among the best specialists in Croatia dealing with molecular ecotoxicology,
analysis organic compounds in environmental samples, biogeochemistry and
biodegradation of xenobiotic compounds. At the same time, the weakness is in
relatively unfavorable age structure (scientists staff average cca 55 years) and the fact
that it is very difficult to attract young graduate students willing to work in this
extraordinary demanding laboratory practice, and who, at the same time, satisfy the
high demands of the Ministry of Science and Technology. An additional and very
important strength of the proposed unit is the multidisciplinary environment of the
Department for Marine and Environmental Research and the “Rudjer Boskovic”
Institute. As to the existing infrastructure, for the Croatian circumstances the
laboratories are relatively well-equipped but compared with average European
laboratories of the same kind we still need a significant upgrading. The main
weakness is the lack of adequate laboratory space for all of the activities envisaged,
especially for the trace analyses of organic contaminants. Recent purchase of
sophisticated analytical equipment including hyphenated system HPLC/MS/MS
system and an automatic extractor for solid samples, will allow a significant
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enlargement of the spectrum of possible organic compounds to be analyzed as well as
the sample throughput, safety and reliability analytical methods used. As
consequence, there is an opportunity to make our services more competitive in
attracting more contract analyses on the national and international level as well as to
broaden our collaboration with other research groups. Threats are in taking over too
many duties (contracts, analyses), which cannot be solved by limited laboratory
capacities. An important threat could be from unfair competition of little private
laboratories and even from some public laboratories, which can offer much lower
prices for the same kind of analyses by sacrifying often some of the necessary
analytical quality control thus affecting the reliability of the results. The same applies
for the contract work in the field of classical ecotoxicology, biodegradation
assessment and environmental impact studies.
PEST (Political/legal, Economic, Social and Technological factors)
Besides the overall factors that influence the RTD sector in Croatia, an
important problem that hinders a more intensive and efficient application of scientific
research to the benefit of the society are the high taxes imposed to the revenues
deriving outside the direct support through the Ministry of Science and Technology.
In addition to the high VAT level (22 %), there is an additional fee of 30 % at the
level of the Institute, leaving only 48 % of the income to the laboratories that carry
out the contract. Such a reduced funding is therefore mostly fully spent for
consumables, spare parts and necessary maintenance of the equipment, which is older
than 10 years on average. Very seldom, the money earned through the contract work
suffices to purchase new equipment, which is essential to keep high competitiveness
of the labs on the market. In rare cases, when some funding remains for possible
allocation to salaries of the personnel involved in the contract-work, there are some
legal constraints that limit the allowed salary increase to only 30 % of the salary
deriving from budgetary support. All these facts act very de-stimulating on both
scientists and technical staff, regarding the motivation to perform services and
contract work for various customers. The limitations mentioned above, which are
valid for marked-oriented science in general, are even more stringent for
environmental sciences. Namely, lacking a consequent and strict regulatory system
that would force industries to demand services in environmental issues more
frequently, the main customer of environmental institutes remain the governmental
agencies and local authorities.
Marketing plan
Market segmentation
Possible customers for the services given by this association mostly belong to
governmental and public institutions, while the number of industrial clients and repeat
customers have been very limited over the last 5 years. The main repeat customer is
the State Directorate for Water Protection, to whom we offer our services as
Authorized Reference Laboratory for the assessment of ecotoxicity, biodegradability
and other biological parameters in wastewaters and natural waters. When problems to
be dealt with have national or regional scale, the contracts are realized through the
Ministry of the Environment and Physical Planning, State Directorate for Water
Resources and Croatian Water Management, who are usually asking for trustworthy
super-analyses besides the ones obtained by other public laboratories. In case of
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environmental problems having a local character, county and town authorities
responsible for environmental issues, require help in estimating the environmental
risk associated with a given case of hazardous chemical contamination.
During the last several years the research groups of this unit have been involved in
several studies aimed at assessing environmental impact of various pollutants on soil
and groundwater quality. One of the prominent case studies was the assessment of
polychlorinated biphenyls in the area of the cities of Zadar and Dubrovnik. An
important customer are authorities of the city of Zagreb, in particular those dealing
with groundwater protection and solid waste management issues. We have coordinated a major risk assessment study at the main landfill of the city of Zagreb,
which was used as a scientific basis for ongoing remediation activities.
Although the improvement of the wastewater treatment in our chemical industries is
one of the key prerequisites in their obligation to meet stringent criteria on chemical
emissions into natural waters and waste generation required for EU-exporting
industries, demands for such services are still limited. The expected contributions to
industry are possible improvements in maximizing recovery of chemicals during
manufacture, minimizing usage of raw water and wastewater generation and
emissions of xenobiotic chemicals into the enviroment. Consultancies will be
provided in designing and setting up wastewater treatment facilities for chemical
industries.
Recently, a biotechnology project of LBOC-environmental microbiology group
aimed at designing a treatment plant for purification of wastewaters from the
herbicide production was approved by the factory “Herbos” d.d., Sisak. Another
wastewater treatment project of the LBOC-biogeochemistry group, proposed in cooperation with the Faculty of Food Technology and Biotechnology to pharmaceutical
industries PLIVA, Zagreb, deals with membrane techniques for the advanced
purification of wastewaters from pharmaceutical industry.
In the forthcoming period more attention will be paid to advertisements (web
pages, printed material), contacts with potential customers and presentation of our
possibilities at national and international congresses.
Projecting demand
 Since the Republic of Croatia proclaimed joining the EU as its primary
political goal, one of the important prerequisites of this process is the
adoption of all environmental standards envisaged by EU environmental
legislation. This fact will enormously increase the demand for the
determination of a large variety of organic and inorganic constituents in
environmental samples using ISO-certified methodologies. Along these
lines, the adoption of methods for the determination of the most prominent
pollutants from the EU Water Directive will be the highest priority.
Specifically a high priority will be given to the introduction of highly
specific methods for PCBs and other organohalogen pollutants as well as to
representative classes of endocrine-disrupting compounds.
 According to the comprehensive EU scheme for the detection of hot-spots
and execution of compliance monitoring of hazardous chemical
contamination, there will be necessary to implement a suite of biomarker
methods, which are available or are under development in the LMEecotoxicology group.
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
Moreover, expected growth of investments in Croatian industries will
require an increased number of baseline studies, environmental impact
assessment studies and compliance monitoring studies.
 One of the key areas, which are expected to open the market for the
envisaged activities, is the treatment, of domestic and industrial wastewaters.
Since the classical activated sludge treatment is not sufficiently efficient to
remove biorefractory xenobiotic compounds, the minimization of the
emissions has to be based on advanced treatment technologies.
 On the other hand, most of the solid waste disposal sites in the Republic of
Croatia are open dumps, which require an urgent remediation, primarily with
respect to groundwater protection. Therefore, there will be a high demand
for monitoring activities to assess groundwater quality and possible transport
of pollutants from the dumping sites into groundwater aquifers.
 A highly relevant issue specific for this geographic region is a significant
contamination that has resulted from war activities in Croatia (as well as in
neighboring countries such as Bosnia-Herzegowina and Serbia). Intensive
efforts, including radiological, chemical and biomarker analyses will be
needed to detect the hot-spots of hazardous chemical contamination.
Moreover, the possible remediation activities would require innovative
technologies for the in situ remediation.
Competition analysis
There are only few laboratories in Croatia and in neighboring countries of
Western Balkan, which are capable of reliably measuring low levels of xenobiotic
compounds in environmental samples and to offer a full spectrum of biomarker
parameters foreseen by EU regulations. In fact, highly specialized institutions,
providing a full scale of services necessary for the comprehensive assessment of
hazardous chemical contamination and development of mitigation strategies, do not
exist in the Republic of Croatia, which leaves a significant gap in the execution
environmental policy of the country. The gap is partially filled by combining the
expertise available in various leading institutions. However, the process of combining
expertise into the final assessment is often carried out through the small private
companies that are specialized in environmental management, which provide contract
work for the Institutes or hire the experts on a personal basis. One of the possibilities
to by-pass this problem, which might be worth considering, would be establishing a
small unit for environmental management to provide similar services for the proposed
association. The optimal solution might be expanding such a unit to cover ecological
modeling, data management issues and marketing.
Competence building
Our experience in international co-operation and successful participation in
international inter-comparison studies corroborate our competence in the chemical
analysis of various anthropogenic organic pollutants as well in the determination of
biomarkers. The future activities will be directed towards the establishment of a
regional center of excellence for the determination of hazardous chemical
contaminants and for their ecotoxicological assessment with a special emphasis on the
assessment of war-related chemical contamination. The process of capacity building
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should provide linking the fundamental research with the application-oriented
research and technological development.
Resource Requirements
HR Plan (Human Resources)
As indicated in the description of the current situation, human resources of all
participating research teams need to be improved. One of the main problems is the
unfavorable age profile of the permanent research staff. The LME-ecotoxicology
group is expected to improve the personnel structure by promoting 2 postdoctoral
scientists into the tenured positions (research associate). It is foreseen to increase the
group by employing another PhD student. The improvement of human resources in
the LME-analytical group will be achieved by employing two new PhD research
students in next two years and another two by 2008. The LBOC-biogeochemistry
group will also be enlarged in the next 2 years by employing two younger scientists
(PhD student and/or Ms Sci.), while increasing activities within the LBOCenvironmental microbiology group require 3 PhD students in the next 2 years. All
PhD students will be trained in the participating Laboratories.
Facilities Plan
 Adaptation of a new laboratory space for the trace organic analyses in
environmental samples (50 000 USD)
 Purchase of a high throughput automatic extractor (accelerated solvent
extraction) for solid environmental samples (55 000 USD);
 Purchase of an automated system for sample clean-up (35 000 USD)
 Purchase of a capillary column gas chromatograph equipped with EC
detector, automatic sampler and computerized data processing (40 000
USD).
 Purchase of a capillary column gas chromatograph equipped with MS/MS,
detector including a negative chemical ionization option, automatic sampler,
large volume injection and computer-assisted data processing (100 000
USD).
 Purchase of a HPLC system equipped autosampler and DAD and
spectrofluorimetric detectors (55 000 USD).
 Expanded activities will require adaptation of an dedicated instrumental
room (20 000 USD).
 Purchase of the low temperature cell culture incubator and other equipment
for the introduction of fish cell culture techniques (15 000 USD);
 Purchase of the microplate scanning photofluorimeter (20 000 USD);
 Purchase of DNA, RNA and protein sequencing and analysis equipment (50
000 USD);
 Improvement/building of animal holding facilities (50-70 m2 of additional
lab space is needed for this purpose) (50 000 USD).
 Purchase of the PCR (14 000 USD)
 Purchase of the refrigerated microlitre centrifuge (14 000 USD)
 Purchase of the horizontal gel electrophoresis system equipped with
documentation and analysis system (20 000 USD)
 Purchase of the incubated, refrigerated and illuminated shaker (16 000 USD)
 Purchase of the shaker cabinet (25 000 USD)
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
Purchase of the total organic carbon analyser equipped with autosampler
and solid sampler module (54 000 USD)
 Purchase of the gas chromatograph for headspace analyses (18 000 USD)
Business process improvements
Adaptation of new laboratories and installation of state-of-the-art
instrumentation equipped with automatic sampling devices should allow an increase
in sample throughput and expanding applied research activities with no negative
consequences for the scientific output of individual research groups. Job and task
description will be adapted to individual skills of the personnel. Moreover, an attempt
for better organization of work process will be made. Measurement equipment,
dislocated from laboratory rooms and offices, will be made available on-line to
authorized users through LAN, which should allow fast and flexible data processing
and management.
Organizational plan
The organizational structure will reflect the management needs of the ongoing
research projects. Principal investigator of a given project should have a great deal of
freedom in organizing activities necessary for a successful completion of the project,
including the financial management of the project. A part of MIS (Management
Information System) will be entrusted to Institute administration, while the head of
each Group will take care of proposals, contracts, customers and financial situation.
Quality plan
A strict compliance with good laboratory practice (GLP) and quality assurance (QA)
protocols is the prerequisite for the collection of reliable analytical data. Based on the
frequency of participation and the quality of the data reported, the IAEA has issued a
list of selected laboratories around the Mediterranean for trace organochlorine
compound analysis. Of the 56 laboratories from 14 Mediterranean countries that
participated in the intercalibration runs to determine chlorinated hydrocarbons in
marine samples, six were selected for their excellent analytical performance. Among
them, the LME-analytical group has been on that list.
The first step in assuring the reliability of analytical data is the use of reference
methods and reference materials. A careful estimation of the accuracy and precision
of each individual method must be performed before the application of newly
developed method to the analysis of environmental samples. For the systematic
monitoring studies, it is important to use certified analytical methods.
Accreditation of a given laboratory is highly desirable but a cost-benefit analysis of
possible accreditation process is advisable.
Financial plan
Income and expenditure projections
During the last several years the income of the participating laboratories greatly
varied. In 2002, the revenues from various customers were in the some groups greater
than the funding from Ministry of Science and Technology for material expenditures
(total 75 000 USD) excluding salaries (total 200 000 US$/ year). In the period of the
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next three years it is planned to increase income from local authorities in Zadar and
Dubrovnik by 20% taking into account expanding of activity of analysis PCB
congeners in jeopardized Dalmatian areas. A significant contract was obtained for the
project on “Assessment of the Selected POPs (PCBs, PCDDs/F, OCPs) in the
Atmosphere and Water Ecosystems from the Waste Materials Generated by Warfare
in Area of Former Yugoslavia “ funded by European Commission “The fifth
framework Programme (Call Identifier ICFP501A2PR02 - Copernicus) providing
additional 40 000 EUR yearly. Increased incomes will be spent mainly for purchasing
new instruments, wages for new young scientists, consumables, chemicals and
fieldwork. Another significant project, entitled “Mitigation of environmental
consequences of war in Croatia – Risk assessment of hazardous chemical
contamination”, was obtained by LBOC-biogeochemistry group in the framework of
financial support provided by Norwegian Government (cca 50 000 USD/year).
Collaboration with Norway obtained by LME-ecotoxicology group within the
project ”An integrated environmental monitoring system for Croatian freshwater,
estuarine and coastal marine areas”, (28 000 EUR)/year) represent another significant
funding that will be spent for the development and/or transfer of new biomarkers.
Action plan
Based on a FP5 European Commission Programme, one of the priorities for the period
2002-2005 is the investigation into fate of PCBs in the karstic region of the Middle
and South Dalmatia. A similar project will be extended to the continental parts of
Croatia, including central mountainous region and the areas along the Sava and Drava
Rivers. Feasibility of an in situ remediation of soils contaminated with PCBs
compounds will be also considered.
In the same period an intensive analytical programme to determine hot-spots
associated with hazardous pollution resulting from war activities will be carried out.
This will comprise a programme to develop methods for war-related contaminants.
Simultaneously, the analytical groups will start a focused programme of introducing
certified state-of-the-art methodologies for the determination of organic pollutants
quoted in the recently revised EU Water Directive as a preparation of Croatia for
compliance monitoring along the lines of Croatia’s aspirations to apply for the
membership in EU next spring. A special emphasis will be on persistent organic
pollutants (POPs), especially halogenated hydrocarbons. Compliance and trend
monitoring studies will be offered for the most critical chemical contaminants starting
from 2005. In the first phase the emphasis will be on water, aquatic sediments and
soils, while in the later phase it is envisaged to intensify monitoring of organisms that
are suitable to give an early indication of pollution trend as well of those used for the
human consumption.
In the period 2004-2007 we plan to intensify our participation in FP 6 EU project with
the emphasis on environmental protection and global change.
In 2003, LME-ecotoxicology group will introduce and standardise in vitro methods
using fish cell lines for the determinations of the exposure to environmental pollutants
that are inducers of mixed function oxygenases (MFO) detoxification system present
in aquatic vertebrates. By 2004 in vitro and in vivo methods for the determination the
exposure and effect of estrogen-like chemicals in environmental samples would be
established. During 2005-2007 appropriate state of the art animal holding facilities for
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aquatic invertebrates and vertebrates should be constructed. Ecotoxicological courses
dealing with biomarkers and bio-test available within our research groups would be
established in between 2004 and 2006. Concerning our research and marketing
activities as well as the intensifying of international collaboration and participation in
EU projects we expect that determination of MXR inhibitors as environmentally
hazardous chemicals might be internationally established bio-test by the end of 2006.
In addition, extensive surveys aimed to illustrate the advantages of the of MXR
activity measurement in aquatic organisms as a relevant biomarker of exposure will
be performed in 2004-2008 period. Introduction of DNA, RNA and protein
sequencing and characterisation knowledge and equipment is planed for 2004-2007
period. As a general goal in terms of capacity building during the period covered by
this business plan, we plan the implementation of high throughput gene and protein
expression profiling (microarrays technology) for the aquatic ecotoxicology.
Regarding biodegradability testing, validation of some reference methods for is
planned for the period 2003-2004. Development of molecular techniques for
detection, identification and characterization of active microorganisms as catalists in
biotransformation processes is foreseen for the period 2003-2006. Parallel to this
activity and extending in the period 2006-2010, the LBOC-environmental
microbiology group will perform reserach aimed at investigating growth kinetic of
microorganisms capable of efficiently degrading xenobiotic compounds. The growth
stimulation studies of selected (the most efficient) microbial cultures will be focused
on designing biodegradation (bioremediation) strategies for target xenobiotic
compounds. Based on the ongoing fundamental studies of xenobiotic compound
transformation, it is foreseen in 2004 to start pilot laboratory and field
biotransformation studies to assess of the efficiency of the proposed wastewater
treatment or soil bioremediation procedures.
Expected support from the World Bank
Croatia is actively participating in the development of the Regional Environmental
Reconstruction Plan in the framework of the Stability Pact for the SE Europe. Given
the fact that scientific institutions specialized for environmental risk assessment do
not exist in the region, a support from the World Bank should be primarily seen as a
part of the capacity building programme to increase the capacity of SE countries to
deal with complex environmetal issues, resulting from the transition process as well
as those resulting from the recent war activites. A support is required for improving
our research facilities dedicated for environmental risk assessment and the total
amount is estimated at aproximately 600 000 USD.
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