Scientific Team: University of Novi Sad, School of Sciences, Department of Chemistry, Trg Dositeja Obradovica 3, Novi Sad, Serbia and Montenegro

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1 Partner No: 006 Scientific Team: University of Novi Sad, School of Sciences, Department of Chemistry, Trg Dositeja Obradovica 3, Novi Sad, Serbia and Montenegro Team leader: Prof. Mirjana Vojinovic-Miloradov, PhD Participating scientists: Prof. Radmila Kovacevic, PhD; Prof. Mira Popovic, Ph.D. Danica Buzarov MSc, Jasna Adamov MSc, Nebojsa Andric MSc, Jan Sudji MSc, Stanka Jovetic BSc, Sonja Zoric BSc Contractual link to other partners: None Subcontractor: UBEG.FMG.IH Team leader: Dr Ivan Matic MSc, senior scientist, Participating scientists Prof. Slobodan Vujasinovic PhD, Miroslav Tanaskovic PhD, Aleksandar Lozajic, MSc, Branislav Sekulović BSc, Zvonko Vincic BSc, Contractual link to other partners: Subcontractor to Partner No. 006 Subcontractor: PUBHEAL.CED Team leader: Dr Danica Djarmati, MSc, scientific associate Participating scientists: Marija Ignjatovic BSc, Svetlana Labus-Blagojevic BSc Contractual link to other partners: Subcontractor to Partner No. 006 Objectives: 1) Participation in laboratory intercalibration of POP analysis 2) The aim is to resulting from transformer oil spills at Zastava car factory in Kragujevac, and other places in Serbia and Monte Negro. Chemical analyses of contaminated media, together with development of mathematical models of stream-aquifer determine the level of contamination of soil, water and sediment in the zone potentially impacted by the PCB contamination interactions, groundwater flow, transport of contaminants would help identifying the hotspots and quantifying the impacts, and associated risks to exposed receptors. 3) Within WP 4, POPs will be monitored in the atmospheric samples of several sites, which are close to the ground argumentative contaminated with POPs in eastern and western part of former Yugoslavia to establish real data about level of contamination this very important part of human ecosphere. 4) Postulation of mathematical model of POPs intake into human organism via food chain. 5) Cytochromes P450 and oxidative stress in PCB toxicity s 6) Sensitive biomarkers in ecological risk assessment of PCBs. Work plan: Work Package 1 Intercalibration of some POPs analysis in environmental samples Applicability of monitoring and other environmental data depends on their quality measured in terms of reliability, which depends on precision (reproducibility) and accuracy (true values). The aim of quality assurance (QA) is to ascertain that the established results have the necessary accuracy and veracity. Though some institutions from these regions possess good experiences and performances in consecutive international intercalibration exercises, the many institutions possess very restricted experiences. Some of them didn't enable laboratories for this very complex and sensitive analysis so that is very important task of this projects training the young people in the more experienced laboratory. Together, engaged scientists and experts from these areas will help in the qualifying people in these laboratories. The check their knowledge will be carried out on the second stage of intercalibration, so that in case of the success they can in one's own areas continue with very important monitoring of this chemical compounds in the life neighborhoods. Unless are succeeding, continue in the further refinement one's own knowledge to the successful mastering this technique. Person-months required: 2 Work Package 3 The level and hydrogeological fate of some POPs in several Serbia and Monte Negro areas as a consequence of war damages The work shall include following activities: detailed analysis of available site data, identification of data gaps in existing data, definition of necessary minimum of additional field investigative activities, sampling of soil, water and sediment, GC analysing of samples followed by external quality control, creation of conceptual hydrogeological model of investigated area, implementation of surface and groundwater level monitoring campaign, development of 43

2 numerical models of groundwater flow and stream-aquifer interactions and, if sufficient impute data is available, development of models for prediction of contaminant fate and transport in groundwater. Person-months required: 21 Work Package 4 The level and atmospheric transportation of some POPs in Croatian, Bosnian & Herzegovina, and Serbia and Monte Negro as consequence of war damages Collection of air samples from determinated hot spots Kragujevac, Pancevo, Novi Sad for further POPs analyses. Person-months required: 1 Work Package 5 Investigation of intake and some ecotoxicological consequences of exposure of living organisms to POPs in some Serbia and Monte Negro and Croatian areas as consequence of war damages Due to their lipophilic and processes of bioaccumulation and biomagnification POPs build up in living organisms plants, animals and humans. Man is at the top of trophic pyramid, so the human organism bioaccumulates significant amounts of organochlorine contaminants, which produce adverse and cumulative effects. In risk analysis, one of the most important steps is assessment of exposure to toxic chemicals. Exposure factors of members of food chains depend on meteorological conditions, physiological characteristics of living organisms, as well as of nature of contaminants. Determine mathematical relationship between members of the food chain. Determine average daily intake of foods per unit of mass of humans. Analyse dependence of intake of organochlorine into human organisms on season, age and gender. Oxidative metabolism of PCBs and other organochlorine compounds involves induction of cytochrome P450 enzymes yielding either activated or inactivated metabolites. Also, it provokes oxidative stress leading to the disbalance in the antioxidative defense system. The extent of adverse effects can be assessed via model system of lipid peroxidation. Also, 8-hydroxyguanine in human urine samples will be used as biomarkers of oxidative stress. Biomarkers provide an integrated response to all the toxicological and pharmacological interactions of the environmental pollutants to which organism are exposed. For evaluation of the environmental impacts of PCB contamination, it is planned to use several laboratory models to assess the values of specific biomarkers of action of PCBs as chemical stressors. Person-months required: 12 Deliverable title Delivery date The participation in laboratory intercomparison exercise 6 3 (phase I) The participation in laboratory intercomparison exercise 18 4 (phase II) 9 Report on background data on hydrogeological conditions and PCB 12 Concentrations in soil, water, and sediment. 10 Design for additional investigative activities and implementation of 14 surface water and groundwater monitoring campaign. 11 Report on the results of additional environmental media sampling and 16 Additional hydrogeological and hydrological investigative activities 12 Hydrogeological model of the zone of spill-site and potentially impacted 20 groundwater resources 13 Mathematical model of groundwater flow and transport of contaminants Final Report with input for risk assessment procedures. Estimate of 35 potential health risk with recommendations for further actions 20 Mathematical model of human intake of POPs well suited for ecological and social state of the investigated region Results of investigation the correlation between the level of environmental contamination with investigated POPs and biological response of living organisms

3 Research activities during the first reporting period: Work Package 1 Intercalibration of some POPs analysis in environmental samples UNS.FS.IC and PUBHEAL.CED were involved in the laboratory intercomparison and intercalibration exercise on analysis of POPs standard solution and reference soil sample. Analytical laboratory of UNS.FS.IC has achieved satisfactory results and acceptable ranges of precision and accuracy. However, analytical laboratory of PUBHEAL.CED hasn t passed intercalibraton test and evaluation. All further activities in determination of POPs in investigated samples of soil and sediment were performed by the UNS.FS.IC laboratory. Analysis of POPs content in soil from Kragujevac obtained by PUBHEAL.CED were repeated by UNS.FS.IC and stated in this report. The corresponding deliverables 1, 2, and 3 were reached, milestone 1 and expected results were partially achieved. Deliverable 1: Harmonisation of sampling procedures and analytical methods Deliverable 2: Adoption of Principles of Good Laboratory Practice Deliverable 3: The organization of laboratoru intercomparison (phase 1) Milestone 1: Internationally acceptable results from laboratory measurements, laboratory and field experiments The plan for the second year of research: In the second phase of intercalibration PUBHEAL.CED will try to achieve expected accuracy. Work Package 3 The level and hydrogeological fate of some POPs in several FR Yugoslavian areas as a consequence of war damages Zastava car factory in Kragujevac (industrial town in Central Serbia) was targeted twice during the conflict 1999, and spillage of the great amount of PCB inside building of the factory in the thick populated city area occurred. The following activities were performed: detailed analysis of the available data on PCB in Kragujevac; identification of the lacking data; determining of the necessary minimum of the additional investigations; sampling of soil, mud and river sediments; GC sample analysis of PCBs combined with bioassay analysis (based on biomarkers of response); creating of conceptual hydrogeological model of the investigated area; creation of the numerical model of the ground water flow, model of interaction between surface water and aquifer and if input data appear to be reliable - design of pollutant transport model. Investigative works have been performed at the location of destroyed trafo station TS 305 within the military facility, very close to the "Zastava" car factory and in the surroundings of Kragujevac, including rivers Zdraljica, Lepenica and Morava, and lakes of Sumarice and Bubanj A A-1 B-4 B-3 B-2 B-5 C-1 Sewage collector B-1 B C D a) b) Fig 1. Sampling location near TS 305 (a) and paint shop of ZASTAVA car factory (b) Sampling zone around TS 305 (12x11m) was divided in quadrants and soil samples were taken from the various depths. 45

4 Table 1. Content of PCBs in soil samples determined by GC/ECD (ppb) PCB Concentration of PCBs in different sampling sites congener A-1 (0.4 m) B-1 (0.2 m) B-5 (1.0 m) C-1 (0.2 m) B-2 (0.6 m) B-3 (0.2 m) B-4 (0.4 m) PCB 28 < 5 < 5 < 5 < 5 < 5 < 5 < 5 PCB PCB PCB PCB PCB TOTAL Sample Table 2. Content of PCBs in soil samples from the Kragujevac area (2003) P1, P2, 4.5 m P3, 4 m P4, 4 m P5, 3 m P6, 3 m S2, PCB28 <5 <5 <5 <5 <5 <5 <5 <5 < 5 <5 <5 9 < 5 < 5 PCB52 <5 <5 <5 <5 <5 <5 <5 <5 < 5 <5 < < 5 PCB101 <5 <5 <5 <5 <5 <5 <5 <5 6 <5 < PCB153 <5 <5 <5 <5 <5 <5 <5 <5 15 <5 < PCB138 <5 <5 <5 <5 <5 <5 <5 <5 20 <5 < PCB180 <5 <5 <5 <5 <5 <5 <5 <5 10 <5 < TOTAL (ppm) - -* * *BA parallel biological assay done S2, 5.5 m S3, 1m S3, 3 m JB, 0.4 m Z1, 0.4 m Z1, 0.2 m Z2, m Fig. 2. Effects of different soil extract on EROD and PROD activities in liver microsomes of immature female rat. Columns represent means ± SE from 5 animals per group. Legend in figure represent total amount of soil extract received for 3 days. Basal EROD and PROD levels were 35.98±3.41 and 2.165± pmol/min/mg prot respectively. Significance: *p<0.05 vs. controls, using ANOVA followed by Duncan s test The results of in vivo bioassay show that all samples significantly induced EROD activities in immature female rats in a dose-dependent manner. The samples A-1 and B-1 were the most potent, which is in accordance with their high PCB contamination, as determined by GC/ECD. 46

5 Dose response curve for soil samples P-2 and P-6 and sediment samples Z-1 and Z-2 demonstrated the presence of EROD-inducing compounds (Fig. 4.). Results demonstrated that EROD-inducing potencies of these samples are Z-1(0.4) > Z-2(0.2) >P-6>P-2. Fig. 3. Micro-EROD bioanalysis of PCB 126 and Pyralene in primary rat hepatocytes culture. The EC 25PCB126 value was calculated as the concentration giving 25% of the maximal induction caused by 10-8 M of PCB126.. Significance: p*<0.05 vs control Fig. 4. Micro-EROD bioanalysis of soil sample extracts P-2 and P-6 and sediment samples (Z-(0.4)1 and Z-1(0.2)) in primary rat hepatocytes culture. For explanations see legend in Fig. 3 Although GC/MS analyses did not show presence of certain PCB congeners in extract of investigated soil samples, induction of EROD activity in both P-2 and P-6 sample indicate to a presence of some non-analyzed, EROD-inducing compounds in these samples. Soil samples from the vicinity of the Paint shop were subjected to TOC analysis. Obtained results are in accordance with the results of GC: samples with high TOC percentage are those in which PCBs residues were detected (Table 3). Table 3. Content of TOC in soil samples from the Kragujevac region (% in dry soil) P1 P2 4.5m P3 4m P4 4m P5 P6 S2 S2 5.5m S3 1m S3 JB 0.4m Z1 0.4m Z1 0.2m Z m M M2 <0.1 < * Results obtained by two described methods differ: content obtained in apparatus are usually twice smaller. It is probable that the investigated soil samples contained large content of highly volatile hydrocarbons and thus the greater differences between results given in Table 3. 47

6 The corresponding deliverable 9 was completely achieved, deliverables 10 and 11, milestone 3 and expected results were partially achieved. Deliverable 9: Report on background data on hydrogeological conditions and PCB concentrations in soil, water, and sediment. Deliverable 10: Design for additional investigative activities and implementation of surface water and groundwater monitoring campaign. Deliverable 11: Report on the results of additional environmental media sampling and additional hydrogeological and hydrological investigative activities Milestone 3: Data collection and POPs monitoring in soil and water environment of Serbia and Montenegro endangered with war wastes The plan for the second year of research: Improvement of the used laboratory method of combined GCbioassay analysis for PCBs. Conceptual hydrogeological model of the zone of spill-site and potentially impacted groundwater resources based on the collected data. Risk assessment. Work Package 4 The level and atmospheric transportation of some POPs in Croatian, Bosnian & Herzegovina, and Serbia and Monte Negro areas as consequence of war damages UNS.FS.IC. has, together with UTHR.DEE.APL, taken part in organization of sampling of air from the selected localities of FRY: three sites in ZASTAVA factory in Kragujevac hot spot (total of 18 samples), three sites in Pancevo: Oil refinery, chemical plant and urban area-meteorological station (total of 18 samples). The corresponding deliverable 18, milestone 4 and expected results were partially achieved. Deliverable 18: Local significant sources of contamination the atmosphere with investigated POPs will be established so that remedial action could be taken on the most dangerous places. Milestone 4: Real danger of POPs sources in atmosphere and the health of local residents of highly contaminated area. The plan for the second year of research: Collection of atmosphere samples from Novi Sad. Work Package WP5 Investigation of intake and some ecotoxicological consequences of exposure of living organisms to POPs in some FR Yugoslavian and Croatian areas as consequence of war damages The following activities were performed: POPs (PCBs, organochlorine insecticides) analysis of bioindicator organisms from Kragujevac (12 samples of frog Rana ridibunda, September 2002), Determination of POPs in samples of food of plant and animal origin collected from the region of Novi Sad in 2002/2003; Determination of the average daily intake of various types of food used in human diet of the population of Novi Sad a questionnaire on dietary habits was answered by 409 persons of both genders and different age (2-65 years) Meat Milk Fruits and vegetables Eggs Grains Fig. 4. Consumption of different types of food by 4 population groups in Novi Sad 13 Children (1-10) Children (11-15) Adults (16-55) Elderly (>55) 48

7 Table 4. Content of non-dioxin like PCB congeners detected in livers of frogs from the Lake Sumarice PCB congener Range (ng/g liver) PCB PCB 52 BDL PCB 101 BDL PCB PCB PCB BDL below detection limit Table 5. Content of organochlorine compounds detected in livers of frogs from the Lake Sumarice Organochlorine contaminant Range (ng/g liver) Average content (ng/g liver) Lindan ,4`-DDE ,4`-DDT Table 6. Content of organochlorine pesticides in investigated types of foodstuffs (µg/kg) No. Type of foodstuff p,p -DDE p,p -DDT TOTAL 1. Vegetables 0.01 BDL Fruits BDL Grains Beef Pork Chicken Fish Eggs 2.69 BDL Milk Table 7. Content of organochlorine pesticides in fish from the Danube (near Novi Sad) Contaminant in fish (µg/kg) Lindan α-hch β-hch δ-hch p,p -DDE p,p -DDT TOTAL It was shown that content of OCI is the highest in the food of animal origin, due to the high lipid content in which organochlorines are readily stored. In all meat samples the highest concentration of pesticides were detected, especially in pork samples. Since pork comprises approximately 63% of total meat diet, or 9.5% of the total diet of the population of the investigated region, content of toxic organochlorine insecticides represent a serious threat to their health. Significant content of OCI in plants indicates more recent, probably atmospheric pollution. No samples exceeded the maximal residue limits for organochlorines set by FAO and national legislative. Oxidative metabolism of PCBs. It was investigated the extent of adverse effects of PCBs (Ascarel and Pyralene) via model system of lipid peroxidation and OH radical production, which cause oxidative stress. It was shown that in mixture with laurel poppies Ascarel and Pyralene showed no prooxidative properties and that they not stimulate the production of OH radicals. Due to the structural similarities of pyralene and ascarel, the obtained results confirm that the increase of the concentration of these substances leads to the decrease of lipid peroxidation intensity to control values (or even less), which could be explained by their negative synergism with radicals produced during lipid peroxidation of liposomes. Sensitive biomarkers in ecological risk assessment of PCBs. Micro-EROD bioassay based on biomarker of response indicating aryl hydrocarbon receptor activity was evaluated and standardized using primary rat hepatocyte culture. Micro-EROD bioassay was used to test the possible presence of dioxin-like compounds in samples analysed under WP3. 49

8 The corresponding deliverables 20 and 21, milestone 5 and expected results were partially achieved. Deliverable 20: Mathematical model of human intake of POPs well suited for ecological and social state of the investigated region. Deliverable 21: Results of investigation the correlation between the level of environmental contamination with investigated POPs and biological response of living organisms. Milestone 5: Obtained results weill be help in better understanding the processes and consequences of POPs intake into various living organisms (including also human beings) in this part of the Balkans. The plan for the second year of research: Determination of POPs levels in bioindicator organisms in the area of Kragujevac. Assessment of the exposure of fishermen s families in Zadar to PCBs. Development of the mathematical model human dietary exposure to POPs, which will help in better understanding the processes of intake of POPs by humans in the polluted regions. Understanding of the response of various living organisms to the POPs exposure and possible health consequences (including human beings). Significant difficulties or delays experienced during the first reporting period: Analytical laboratory of PUBHEAL.CED failed to pass intercalibraton test and evaluation, so their results could not be taken into consideration. Publications A) Refereed journals J. Adamov, M. Vojinović-Miloradov, D. Buzarov, S. Jovetić, J. Sudji, E. Popović, (2003) DISTRIBUTION OF PCBS AT DIFFERENT LEVELS OF AQUATIC ECOSYSTEM IN THE DANUBE RIVER IN VOJVODINA, Fresenius Environmental Bulletin, 12, D. Buzarov, M. Vojinović-Miloradov, J. Adamov, P. Jurlina (2003) DISTRIBUTION OF PCBs IN TRICOMPONENT SYSTEMS: WATER-SEDIMENT-BIOORGANISMS IN OPEN AND CLOSED AQUATIC ECOSYSTEMS, Fresenius Environmental Bulletin, 12, T. Vukavić, M. Vojinović-Miloradov, S. Pavkov, S. Đilas (2003) PESTICIDE RESIDUES AND POLYCHLORINATED BIPHENYLS IN HUMAN COLOSTRUM SEASONAL VARIATIONS IN YUGOSLAVIA A DECADE LATER, Fresenius Environmental Bulletin, 12, B) Refereed Proceedings M. Vojinović-Miloradov, R. Kovačević, D. Djarmati, I. Matić, D. Buzarov, J. Adamov, S. Jovetić, N. Andrić, J. Sudji, COMPARATIVE RESULTS OF THE RESIDUAL LEVELS OF PCBs OBTAINED BY GC/ECD AND BIOASSAY IN SOIL SAMPLES FROM KRAGUJEVAC HOT SPOT AFTER THE WARFARE IN FORMER YUGOSLAVIA, Organohalogen compounds, Vol. 60, Analytical, Editor: Gary Hunt, TRC Environmental, USA, rd Symposium on Halogenated Environmental Organic Pollutants and POPs - Dioxin, 2003, Boston, MAS. USA, August M. Vojinović-Miloradov, S. Jovetić, D. Buzarov, J. Adamov, J. Sudji, E. Popović, S. Krajinović, THE RESIDUES OF PCBs AND ORGANOCHLORINE HYDROCARBONS IN THE FROG LIVER FROM THE KRAGUJEVAC HOT SPOT, Organohalogen compounds, Vol. 62, Environmental levels, Emerging POPs, Site Assessment, Editor: Gary Hunt, TRC Environmental, USA, rd Symposium on Halogenated Environmental Organic Pollutants and POPs - Dioxin, 2003, Boston, MAS. USA, August C) Lectures J. Adamov, M. Vojinović-Miloradov, D. Buzarov, S. Jovetić, MONITORING OF ORGANOCHLORINE INSECTICIDES IN FOODSTUFFS. 6 th International Symposium Interdisciplinary Regional Research, Abstract (on CD), Novi Sad, October J. Adamov, M. Vojinović-Miloradov, S. Jovetić, D. Buzarov, J. Sudji, MATHEMATICAL MODEL FOR THE EXPOSURE OF VEGETATION TO PERSISTENT ORGANIC POLLUTANTS. II Regional Symposium Chemistry and the Environment, Abstract, Kruševac, 2003,

9 S. Jovetić, M. Vojinović-Miloradov, D. Buzarov, J. Adamov, J. Sudji, E. Popović, FROG LIVERS AS BIOINDICATORS OF AQUATIC AND TERRESTRIAL PCB CONTAMINATION OF THE KRAGUJEVAC AREA. II Regional Symposium Chemistry and the Environment, Abstract, Kruševac, 2003, B. Kaurinović, M. Popović, T. Cebović, M. Vojinović-Miloradov, EFFECTS OF ASCAREL AND PYRALENE IN COMBINATION WITH VARIOUS EXTRACTS OF LAUREL POPPIES (Laurus nobilis L.) ON LIPID PEROXIDATION IN LIPOSOMES. II Regional Symposium Chemistry and the Environment, Abstract, Kruševac, 2003, R. Kovačević, N. Andrić, S. Zorić, I. Gut, D. Đarmati, M. Vojinović-Miloradov, BIOANALYSIS AS A COMPLEMENTARY SCREENING TOOLS FOR INSTRUMENTAL ANALYSIS IN XENOBIOTIC MONITORING. II Regional Symposium Chemistry and the Environment, Abstract, Kruševac, 2003, I. Matić, S. Vujasinović, A. Lozajić, M. Tanasković, CONCEPTUAL HYDROGEOLOGICAL AND MATHEMATICAL MODEL OF BEHAVIOR AND FATE OF PCBS IN SOIL OF THE LOCALITY OF KRAGUJEVAC. II Regional Symposium Chemistry and the Environment, Abstract, Kruševac, 2003, M. Popović, B. Kaurinović, T. Cebović, M. Vojinović-Miloradov, EFFECTS OF ASCAREL AND PYRALENE IN COMBINATION WITH VARIOUS EXTRACTS OF LAUREL POPPIES (Laurus nobilis L.) ON OXYGEN RADICALS IN LIPOSOMES. II Regional Symposium Chemistry and the Environment, Abstract, Kruševac, 2003, M. Vojinović-Miloradov, R. Kovačević, D. Buzarov, I. Matić, D. Djarmati, J. Sudji, PCBs GENERATED BY WARFARE DETERMINED BY GC/ECD AND BIOASSAY METHODS IN SOIL SAMPLES FROM KRAGUJEVAC AREA. II Regional Symposium Chemistry and the Environment, Abstract, Kruševac, 2003,