Characterizing export of land-based microplastics to the estuary - Part II: Sensitivity analysis of an integrated geospatial microplastic transport modeling assessment of tire and road wear particles.

Integrated models addressing microplastic (MP) generation, terrestrial distribution, and freshwater transport are useful tools characterizing the export of MP to marine waters. In Part I of this study, a baseline watershed-scale MP mass balance model was developed for tire and road wear particles (TRWP) in the Seine watershed. In Part II, uncertainty and sensitivity analysis (SA) methods were used to identify the parameters that determine the transport of these particles to the estuary. Local differential, local range and global first-order variance-based SA identified similar key parameters. The global SA (1000 Monte Carlo simulations) indicated that most of the variance in TRWP exported to the estuary can be apportioned to TRWP diameter (76%), TRWP density (5.6%), the fraction of TRWP directed to combined sewers with treatment (3.9%), and the fraction of TRWP distributed to runoff (versus roadside soil; 2.2%). The export fraction was relatively insensitive to heteroaggregation processes and the rainfall intensity threshold for road surface washoff. The fraction of TRWP exported to estuary in the probabilistic assessment was centered on the baseline estimate of 2%. This fraction ranged from 1.4 to 4.9% (central tendency defined as 25th to 75th percentile) and 0.97% to 13% (plausible upper bound defined as 10th to 90th percentiles). This study emphasizes the importance of in situ characterization of TRWP diameter and density, and confirms the baseline mass balance presented in Part I, which indicated an appreciable potential for capture of TRWP in freshwater sediment.

Characterizing export of land-based microplastics to the estuary - Part I: Application of integrated geospatial microplastic transport models to assess tire and road wear particles in the Seine watershed.

Human and ecological exposure to micro- and nanoplastic materials (abbreviated as MP, < 5 mm) occurs in both aquatic and terrestrial environments. Recent reviews prioritize the need for assessments linking spatially distributed MP releases with terrestrial and freshwater transport processes, thereby providing a better understanding of the factors affecting MP distribution to the sea. Tire and road wear particles (TRWP) have an estimated generation rate of 1 kg tread inhabitant-1 year-1 in Europe, but the fate of this MP source in watersheds has not been systematically assessed. An integrated temporally and geospatially resolved watershed-scale MP modeling methodology was applied to TRWP fate and transport in the Seine (France) watershed. The mass balance considers TRWP generation and terrestrial transport to soil, air, and roadways, as well as freshwater transport processes including particle heteroaggregation, degradation and sedimentation within subcatchments. The per capita TRWP mass release estimate in the Seine watershed was 1.8 kg inhabitant-1 yr-1. The model estimates indicated that 18% of this release was transported to freshwater and 2% was exported to the estuary, which demonstrated the potential for appreciable capture, degradation, and retention of TRWP prior to export. The modeled pseudo-steady state sediment concentrations were consistent with measurements from the Seine watershed supporting the plausibility of the predicted trapping efficiency of approximately 90%. The approach supported the efficient completion of local and global sensitivity analyses presented in Part II of this study, and can be adapted to the assessment of other MPs.

Below the surface: Twenty-five years of seafloor litter monitoring in coastal seas of North West Europe (1992-2017).

Marine litter presents a global problem, with increasing quantities documented in recent decades. The distribution and abundance of marine litter on the seafloor off the United Kingdom's (UK) coasts were quantified during 39 independent scientific surveys conducted between 1992 and 2017. Widespread distribution of litter items, especially plastics, were found on the seabed of the North Sea, English Channel, Celtic Sea and Irish Sea. High variation in abundance of litter items, ranging from 0 to 1835 pieces km-2 of seafloor, was observed. Plastic tems such as bags, bottles and fishing related debris were commonly observed across all areas. Over the entire 25-year period (1992-2017), 63% of the 2461 trawls contained at least one plastic litter item. There was no significant temporal trend in the percentage of trawls containing any or total plastic litter items across the long-term datasets. Statistically significant trends, however, were observed in specific plastic litter categories only. These trends were all positive except for a negative trend in plastic bags in the Greater North Sea - suggesting that behavioural and legislative changes could reduce the problem of marine litter within decades.

Microplastics en route: Field measurements in the Dutch river delta and Amsterdam canals, wastewater treatment plants, North Sea sediments and biota.

Environmental contamination by plastic particles, also known as 'microplastics', brings synthetic materials that are non-degradable and biologically incompatible into contact with ecosystems. In this paper we present concentration data for this emerging contaminant in wastewater treatment plants (WWTPs) and freshwater and marine systems, reflecting the routes via which these particles can travel and the ecosystems they potentially impact along their path. Raw sewage influents, effluents and sewage sludge from seven municipal WWTPs in the Netherlands contained mean particle concentrations of 68-910L-1, 51-81L-1 and 510-760kg-1 wet weight (ww), respectively (particle sizes between 10 and 5000µm). Even after treatment, wastewater constitutes a source of microplastic pollution of surface waters, and via biosolids applications in farming and forestry, plastic retained in sewage sludge can be transferred to terrestrial environments. The WWTPs investigated here had a mean microplastics retention efficiency of 72% (s.d. 61%) in the sewage sludge. In the receiving waters of treated and untreated wastewaters, we detected high microplastic levels in riverine suspended particulate matter (1400-4900kg-1 dry weight (dw)) from the Rhine and Meuse rivers. Amsterdam canal water sampled at different urban locations contained microplastic concentrations (48-187L-1), similar to those observed in wastewater that is emitted from sewage treatment facilities in the area. At least partial settling of the particles occurs in freshwater as well, as indicated by microplastics in urban canal sediments (<68 to 10,500particleskg-1dw). Microplastics in suspension in the water column have the potential to be discharged into the sea with other riverine suspended particulates. We report microplastic concentrations from 100 up to 3600particleskg-1 dry sediment collected at 15 locations along the Dutch North Sea coast. The high microplastic enrichment in marine sediments compared to most literature data for seawater at the surface supports the hypothesis of a seabed sink for these materials. Marine species are heavily exposed to plastic particles. Body residues between 10 and 100particlesg-1dw were measured in benthic macroinvertebrate species inhabiting the Dutch North Sea coast: filter-feeding mussels and oysters (species for human consumption) as well as other consumers in the marine food chain.

PAH metabolites in fish bile: From the Seine estuary to Iceland.

Polycyclic aromatic hydrocarbons (PAH) are environmental contaminants that pose significant risk to health of fish. The International Workshop on Integrated Assessment of Contaminant Impacts on the North Sea (ICON) provided the framework to investigate biomarker responses as well as contaminant concentrations side by side in marine ecosystems. Concentrations of the main PAH metabolites 1-hydroxypyrene, 1-hydroxyphenanthren and 3-hydroxybenzo(a)pyrene were determined in bile by HPLC with fluorescence detection. Fish species under investigation were dab (Limanda limanda), flounder (Platichthys flesus) and haddock (Melanogrammus aeglefinus). A contamination gradient was demonstrated from the low contaminated waters of Iceland and off-shore regions of the North Sea towards higher concentrations in coastal areas. Concentrations of PAH metabolites differed primarily according to sampling region and secondarily to species.

Integrated chemical and biological analysis to explain estrogenic potency in bile extracts of red mullet (Mullus barbatus).

A biological screening was performed to establish the total exposure to estrogenic compounds of red mullet (Mullus barbatus) collected at several sites along the Spanish Mediterranean coast by testing male fish bile extracts using the in vitro ER-LUC reporter gene assay. In addition, major metabolites were identified and measurements of OH-PAHs (1-naphthol, 9-phenantrol, 9-fluorenol, 1-pyrenol, 1OH-BaP and 3OH-BaP) and alkylphenols (4-n-nonylphenol (4-n-NP) and 4-tertoctylphenol (4-tert-OP)) in the same fish bile extracts were taken by gas chromatography-mass spectrometry in electron ionization mode (GC-EI-MS). Relative in vitro estrogenic potencies of the chemically quantified compounds were also tested. The highest biliary concentrations of 1-pyrenol, 9-fluorenol and 4-n-NP were found in fish from Barcelona and from the Mar Menor coastal lagoon. However, these concentrations can be considered relatively low compared to values reported in red mullet from other polluted waters in the Mediterranean Sea. The contribution of 1-pyrenol, 4-n-NP and 4-tert-OP to the total estrogenic potency measured in male fish bile was found to be negligible, indicating the presence of other estrogenic compounds in red mullet bile. Estrogenic potency in bile from male fish was markedly elevated in Mar Menor lagoon (234.8±5.7 pg E2EQ/µl), and further research will be necessary to explain whether the presence of natural and synthetic-hormones in the lagoon contributed to this finding. Values of approximately 15-16E2EQ pg/mg bile can be regarded as preliminary baseline levels of bile estrogenicity in male red mullet from the western Mediterranean Sea.

Health status of red mullets from polluted areas of the Spanish Mediterranean coast, with special reference to Portmán (SE Spain).

A suite of general physiological indicators and hepatic biomarker responses were determined in red mullet (Mullus barbatus) from priority pollution areas of W Mediterranean Sea, including the highly metal polluted area of Portmán (Cartagena, SE Spain). Concentrations of metals and persistent organic pollutants in fish muscle tissue and sediment samples were also analysed. Our results showed that fish from Portmán accumulated the highest concentrations of mercury, lead and arsenic and also of some polycyclic aromatic hydrocarbons and polychlorinated biphenyls congeners. In addition, they had significantly lower condition factor, muscle lipid content and gonadosomatic index, as well as the lowest levels of DNA integrity and the highest ethoxyresorufin-O-deethylase (EROD) activity in liver of the areas investigated. Contaminant body burden in fish only partly corresponded to chemical characteristics of the sediments in the areas in which they were collected. Our findings indicate that red mullets from Portmán had suboptimal health status that warrant further study.

Using biological effects tools to define Good Environmental Status under the European Union Marine Strategy Framework Directive.

The use of biological effects tools offer enormous potential to meet the challenges outlined by the European Union Marine Strategy Framework Directive (MSFD) whereby Member States are required to develop a robust set of tools for defining 11 qualitative descriptors of Good Environmental Status (GES), such as demonstrating that "Concentrations of contaminants are at levels not giving rise to pollution effects" (GES Descriptor 8). This paper discusses the combined approach of monitoring chemical contaminant levels, along side biological effect measurements relating to the effect of pollutants, for undertaking assessments of GES across European marine regions. We outline the minimum standards that biological effects tools should meet if they are to be used for defining GES in relation to Descriptor 8 and describe the current international initiatives underway to develop assessment criteria for these biological effects techniques.

Histopathological effects of chronic aqueous exposure to bis(tri-n-butyltin)oxide (TBTO) to environmentally relevant concentrations reveal thymus atrophy in European flounder (Platichthys flesus).

Although the use of tributyltin in antifouling paints has been banned, this compound is still a serious pollutant of the marine environment. This paper describes a unique study in which European flounder (Platichthys flesus) were chronically (8 months) exposed to bis(tri-n-butyltin)oxide (TBTO) in the water under controlled laboratory conditions. Residue levels in selected tissues (liver, muscle) and general health status indices were measured and the effects on several organs (gills, liver, mesonephros, ovary/testis, spleen, and gastrointestinal tract) were examined histopathologically. Additionally, morphometric analysis of the thymus was performed. The major finding is that exposure of flounder to 5 microg TBTO/l over a period of 8 months, resulting in body burdens comparable to high field levels, induced significant reduction of thymus volume, possibly affecting immunocompetence of the animals. Chronic exposure of European flounder to tributyltin is therefore likely to affect the general health status of this species in heavily polluted aquatic environments.

Evaluation of three-year monitoring with biomarkers in fish following the Prestige oil spill (N Spain).

A previous study using a suite of hepatic enzymatic biomarker in two demersal fish species (Lepidorhombus boscii and Callionymus lyra) indicated exposure of the fish to the hydrocarbons in the oil spilled by the Prestige five months after the accident. The main objective of this follow-up study is to determine whether the same biomarkers in both fish species show any significant variations in responses over the years following the Prestige oil spill. Detoxification and antioxidant enzyme measurements--of ethoxyresorufin-O-deethylase (EROD), glutathione-S-transferase (GST), glutathione reductase (GR) and catalase (CAT)--were taken from immature specimens. The results show significant lowering of biomarker activity two and three-years after the oil spill, indicating a decreasing level of exposure of the fish to residual hydrocarbons associated with the spillage (p < 0.01) and a recovery to baseline levels existing before the accident. Overall, spatial biomarker patterns over time are in agreement with the oil slick trajectories and the spatial distribution of tar aggregates found on the bottom shelf after the accident. The results also indicate that the Prestige oil spill had an impact on sublethal responses in fish not only in inshore areas, but also in offshore areas along the middle/outer northern Iberian shelf. In both species, EROD activity was found to be the most discriminating biomarker.

Toxicity of analytically cleaned pentabromodiphenylether after prolonged exposure in estuarine European flounder (Platichthys flesus), and partial life-cycle exposure in fresh water zebrafish (Danio rerio).

Residues of polybrominated diphenylethers (PBDEs), extensively applied as flame retardants, are widely spread in the aquatic environment and biota. The present study investigates effects of the environmentally relevant lower brominated diphenylethers in two fish species in vivo under controlled laboratory conditions. Euryhaline flounder (Platichthys flesus) and freshwater zebrafish (Danio rerio) were exposed to a range of concentrations of a commercial pentabromodiphenylether mixture, DE-71. Chemical analysis of exposed fish showed a pattern of PBDE congeners that was very similar to that in wild fish. The resulting range included environmentally relevant, as well as higher levels. Animals were investigated histopathologically with emphasis on endocrine and reproductive organs. In zebrafish, hatching of embryos and larval development were assessed. Biochemical parameters were investigated in flounder as markers for suggested dioxin-like activity (ethoxyresorufin-O-deethylase=EROD), and activation of endogenous estrogen synthesis (gonad aromatase activity). Thyroid hormones were analyzed in plasma in both species. Benchmark analysis using internal PBDE concentrations showed a mild dose-dependent decrease of hepatic EROD and ovarian aromatase activities, and plasma thyroxin levels in flounder, and an increase of plasma thyroid hormone levels in zebrafish. These trends did not result in statistically significant differences from control fish, and major histopathological changes were not observed. Reproduction in zebrafish appeared to be the most sensitive parameter with statistically significantly reduced larval survival and non-significant indications for decreased egg production at internal levels that were more than 55 times the highest environmental recordings. The present results indicate limited risk for endocrine or reproductive effects of current environmental PBDE contamination in fish.

Long-term exposure of European flounder (Platichthys flesus) to the flame-retardants tetrabromobisphenol A (TBBPA) and hexabromocyclododecane (HBCD).

Tetrabromobisphenol A (TBBPA) and hexabromocyclododecane (HBCD) are widely used flame retardants that have increasingly been found as contaminants in the aquatic environment. In the present study, European flounder (Platichthys flesus) were chronically exposed to TBBPA; (105 days) and HBCD (78 days), in a wide range including environmentally relevant concentrations. TBBPA was administered via the water, whereas HBCD was administered in food and sediment, or in sediment alone. Chemical analysis of muscle showed an average increase in internal concentrations of approximately two orders of magnitude for both compounds tested. Animals exposed to HBCD via sediment alone (8000 microg/g total organic carbon, TOC) showed a proportional increase of alpha-HBCD in muscle compared to animals exposed via food and sediment. In both studies, exposure to the test compounds did not affect general health and toxicity parameters (behavior, survival, growth rate, relative liver and gonad weight). Hepatic microsomal enzyme activities (TBBPA: EROD; HBCD: EROD, PROD, and BROD) were not induced by any of the tested chemicals. Aromatase activity in male gonads showed a mild increase with rising TBBPA levels. There were no morphological and immunohistochemical indications for increased production of the yolk precursor protein vitellogenin (VTG) in animals exposed to TBBPA and HBCD; immunochemical analysis of plasma VTG levels showed no dose response in animals exposed to TBBPA. In animals exposed to TBBPA, levels of the thyroid hormone thyroxin (T(4)) increased with internal concentrations of the test compound, possibly indicating competition of TBBPA for plasma protein binding. Triiodothyronin (T(3)) levels were not affected and histology showed no signs of altered thyroid gland activity. Other organs investigated (liver, gills, kidney, skin, and gonads) revealed no histological changes related to TBBPA or HBCD exposure. Overall, the present results indicate limited endocrine effects of these widely used flame retardants in a test species representative of European estuaries at environmentally relevant exposure levels and at internal levels up to 4300 ng TBBPA/g wet weight, and 446 microg HBCD/g lipid weight in flounder muscle.

Effects of natural and synthetic estrogens and various environmental contaminants on vitellogenesis in fish primary hepatocytes: comparison of bream (Abramis brama) and carp (Cyprinus carpio).

Interaction of environmental estrogens with the estrogen receptor (ER) has been shown in various fish species. Our objective was to compare the sensitivity of bream (Abramis brama) to (xeno-)estrogens with that of the carp (Cyprinus carpio), by measuring the effects of 17beta-estradiol (E2), estrone (E1), ethynylestradiol (EE2), bisphenol A (BPA), nonylphenol (NP), methoxychlor (MXCL), and halogenated aromatic hydrocarbons (HAHs) such as polychlorinated biphenyls (PCB126, PCB118), 2,3,7,8-tetrachlorodibenzo-dioxin (TCDD), and 2,3,4,7,8-pentachlorodibenzofuran (PCDF) on vitellogenesis in primary hepatocytes. Comparing the EC50 values in bream hepatocytes: EE2 (0.1-0.2 microM) < E1 (0.6-0.2 microM) < E2 (1.9 microM) with those of carp hepatocytes EE2 (0.03-0.06 microM) < E2 (0.3 microM) approximately E1 (0.2-0.3 microM) we found differences in sensitivity and ranking of the estrogenic potency of E2 and E1, indicating interspecies differences. Exposure to BPA, NP, MXCL, and HAHs did not or only weakly induce vitellogenesis. Bream hepatocytes coexposed to E2 and TCDD, PCB126 or PCDF showed a concentration-dependent inhibition of E2-induced vitellogenesis. IC50 (concentration of a compound that elicits 50% inhibition of E2-induced vitellogenesis) values determined in bream were: TCDD (0.02-0.09 nM) < PCB126 (0.35-0.1 nM) < PCDF (2.0-0.1) and in carp were: TCDD (0.01 nM) < PCB126 (0.4 nM). PCB118 showed no (anti-)estrogenic response. IC50 values and benchmark-concentration for TCDD and PCB126 in bream and carp hepatocytes were in the same range, indicating similar sensitivity to these compounds. Due to their anti-estrogenic capacity with benchmark-concentrations in the pM range TCDD, PCDF, and PCB126 may form a potential hazard for the reproductive success of fish species by inhibition of vitellogenesis.

Environmental impact and recovery at two dumping sites for dredged material in the North Sea.

The environmental impact and recovery associated with the long and uninterrupted disposal of large volumes of moderately contaminated dredged material from the port of Rotterdam was studied at nearby dumping sites in the North Sea. Observations were made on sediment contamination, ecotoxicity, biomarker responses and benthic community changes shortly after dumping at the 'North' site had ceased and at the start of disposal at the new dumping site 'Northwest'. During the period of dumping, very few benthic invertebrates were found at the North site. Concentrations of cadmium, mercury, polychlorinated biphenyls (PCBs), polyaromatic hydrocarbons (PAHs) and tributyltin (TBT) in the fine sediment fraction (<63 microm) from this site were 2-3 times higher than at the reference site. In four different bioassays with marine invertebrates the sediments showed no acute toxic effects. In tissue (pyloric caeca) of resident starfish Asterias rubens, residual levels of mercury, zinc, PCBs and dioxin-like activity were never more than twice those at the reference site. Four different biomarkers (DNA integrity, cytochrome P450 content, benzo[a]pyrene hydroxylase activity and acetylcholinesterase inhibition) were used on the starfish tissues, but no significant differences were found between North and the reference site. Minor pathological effects were observed in resident dab Limanda limanda. One year after dumping had ceased at the North site, a significant increase in the species richness and abundance of benthic invertebrates and a concomitant decrease in the fine sediment fraction of the seabed were observed. After 8.2 million m3 of moderately contaminated dredged material had been dumped at the new dumping site Northwest, the species richness and abundance of benthic invertebrates declined over an area extending about 1-2 km eastwards. This correlated with a shift in sediment texture from sand to silt. The contamination of the fine sediment fraction at the Northwest location doubled. It is concluded that marine benthic resources at and around the dumping sites have been adversely affected by physical disturbance (burial, smothering). However, no causal link could be established with sediment-associated contaminants from the dredged spoils.

Aquatic toxicology: opportunities for enhancement through histopathology.

This paper briefly reviews the application of histopathology as aninstrument or endpoint in toxicity studies in fish. For long this has been applied rather occasionally in (regulatory) toxicology, and was mainly of interest in fundamental studies and limited carcinogenicity experiments. However, nowadays there are various incentives that ask for the application of pathology, such as field monitoring of pollution effects, the wish for optimal use and lower species of laboratory animals, the availability of modern histology techniques, and insight and interest in mechanistic data. This is timely illustrated by the current broad interest in endocrine disrupting pollutants-a threat mainly in the aquatic environment-where histopathological organ and tissue changes in intact sentinel fish species provide pivotal diagnostic and mechanistic features.

Organic contaminants and trace metals in flounder liver and sediment from the Amsterdam and Rotterdam harbours and off the Dutch coast.

Organic contaminants [polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs), polybrominated diphenylethers (PBDEs), nonylphenols], organotin compounds and trace metals (cadmium, chromium, mercury and zinc) were determined in flounder (Platichthys flesus) liver and sediment from the Amsterdam harbour (North Sea Canal) and Rotterdam harbour (Euromonding) and off the Dutch coast between the Amsterdam and Rotterdam harbour mouths in order to assess the level of contamination in these harbours and to study contamination gradients.

Toxicity of PCB-126 in European flounder (Platichthys flesus) with emphasis on histopathology and cytochrome P4501A induction in several organ systems.

A series of experiments was set up to elucidate the effects of pollution on marine and estuarine fish health, since the European flounder (Platichthys flesus) has shown a relatively high prevalence of (pre)neoplastic liver lesions and lymphocystis virus disease in Dutch coastal and estuarine waters. The hypothesis of a causal relationship between pollution and the above-mentioned diseases was supported by results from semi-field experiments. Therefore several laboratory experiments were carried out to substantiate causality further and to identify the xenobiotics that may play a major role in the field. The present study focuses on polychlorinated biphenyls (PCBs). European flounders (Platichthys flesus) were orally exposed to a single dose of 0, 0.5, 5 or 50 mg PCB-126/kg body weight under controlled laboratory conditions. The effects on liver, gills, gastrointestinal tract, gonads, spleen and mesonephros were examined histologically after 16 days. Induction and localization of cytochrome P4501A (CYP1A) immunoreactivity, and effects on hepatocyte proliferation were visualized immunohistochemically. Effects on thymus size were examined by morphometric analysis of serial sections. Three out of five animals of the highest dose group showed haemorrhages in the fins and tail after 16 days. All animals showed reduced activity in the later stages of the experiment, and some animals of the highest dose group discontinued feeding 14 days after exposure. Strong and exposure-related induction of CYP1A immunoreactivity was noted in hepatocytes, endothelium in all organs examined, and epithelium of the digestive tract and mesonephros at PCB-126 levels of 0.5, 5 and 50 mg/kg. In addition, the strong induction of CYP1A immunoreactivity in a distinct population of haematopoietic cells in the mesonephros and in circulating blood is remarkable, and has not been described previously in other fish species. Furthermore, a morphometrically determined significant reduction in relative thymus size was noted in animals exposed to 50 mg PCB-126/kg. Although the functional implications for the immune system of this reduction need to be further investigated, an impact on the specific resistance against infectious diseases as observed in the field, e.g. viral lymphocystis disease, is not implausible. In addition, a significant increase in absolute liver weight, in hepatosomatic index, and in number of proliferating hepatocytes [measured as immunoreactivity against proliferating cell nuclear antigen (PCNA)] was noted in animals of the highest dose group. From these findings we suppose that PCB-126 (and related chemicals) may play a role in the promotion of tumour development in the liver of European flounders as observed in the field. The results of the present experiment show relatively stronger effects than effects previously reported from experiments with TCDD, suggesting that the TEF of 0.005 assigned to PCB-126 from early life stage mortality experiments in rainbow trout (Oncorhynchus mykiss), underestimates the toxic potential of PCB-126.

Toxicology of environmental chemicals in the flounder (Platichthys flesus) with emphasis on the immune system: field, semi-field (mesocosm) and laboratory studies.

European flounder (Platichthys flesus) has shown an increased prevalence of liver tumors and lymphocystis disease (a viral infection) that correlated with pollution in field research in Dutch coastal and estuarine waters. Semi-field or mesocosm experiments confirmed the supposed causality. Although these types of research are highly relevant for the feral population, laboratory experiments are necessary to establish causal relationships between specific chemical pollutants and disease. Therefore, the effects on flounder of some of the potentially causative chemicals such as benzo[a]pyrene (BaP), dimethyl-benz[a]anthracene (DMBA), 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD), 3,3',4,4',5 pentachlorobiphenyl (PCB-126), and bis(tri-n-butyltin)oxide (TBTO) were examined in several laboratory experiments. These effects were evaluated using general toxicological parameters and histopathology. For immune function assessment, attempts to develop an infection model with the lymphocystis virus were made, but appeared unsuccessful and immune function tests are not fully operational at the moment. Flounder has been successfully maintained and exposed to toxic substances in captivity in our laboratory. Short-term aqueous exposure to high levels of BaP or DMBA did not induce marked effects under our experimental conditions. Results of oral exposure of flounder to low levels of TCDD, PCB-126 or harbor sludge extract show significant induction of cytochrome P4501A (CYP1A) in hepatocytes. Oral exposure to high levels of TCDD or PCB-126 also significantly induced CYP1A immunoreactivity in epithelium in mesonephros and digestive tract and in endothelium in several organs. Remarkable was the induction of CYP1A in a distinct population of mononuclear cells in the mesonephros. Moreover, oral exposure to TCDD resulted in an increased mitotic activity and an increase of the hepatosomatic index in the 20 and 500 microgram TCDD/kg group respectively. Therefore, exposure to TCDD and related substances may promote the development of liver tumors in the field. Exposure to PCB-126 also significantly reduced the relative thymus volume, but other results indicate that flounder is relatively insensitive to this type of chemicals. Short-term aqueous exposure of flounder to TBTO, in concentrations that were in the same order of magnitude as upper TBT levels measured in the field, caused mortality after 7-12 days associated with gill lesions, and induced reduction of the non-specific resistance and decrease of the relative thymus volume. From these results we therefore conclude that TBTO might play a causal role in, for instance, increased prevalence of lymphocystis virus infections in the field

Health effects of endocrine-disrupting chemicals on wildlife, with special reference to the European situation.

Many wildlife species may be exposed to biologically active concentrations of endocrine-disrupting chemicals. There is strong evidence obtained from laboratory studies showing the potential of several environmental chemicals to cause endocrine disruption at environmentally realistic exposure levels. In wildlife populations, associations have been reported between reproductive and developmental effects and endocrine-disrupting chemicals. In the aquatic environment, effects have been observed in mammals, birds, reptiles, fish, and mollusks from Europe, North America, and other areas. The observed abnormalities vary from subtle changes to permanent alterations, including disturbed sex differentiation with feminized or masculinized sex organs, changed sexual behavior, and altered immune function. For most reported effects in wildlife, however, the evidence for a causal link with endocrine disruption is weak or nonexisting. Crucial in establishing causal evidence for chemical-induced wildlife effects appeared semifield or laboratory studies using the wildlife species of concern. Impaired reproduction and development causally linked to endocrine-disrupting chemicals are well documented in a number of species and have resulted in local or regional population changes. These include: Masculinization (imposex) in female marine snails by tributyltin, a biocide used in antifouling paints, is probably the clearest case of endocrine disruption caused by an environmental chemical. The dogwhelk is particularly sensitive, and imposex has resulted in decline or extinction of local populations worldwide, including coastal areas all over Europe and the open North Sea. DDE-induced egg-shell thinning in birds has caused severe population declines in a number of raptor species in Europe and North America. Endocrine-disrupting chemicals have adversely affected a variety of fish species. In the vicinity of certain sources (e.g., effluents of water treatment plants) and in the most contaminated areas is this exposure causally linked with the effects on reproductive organs that could have implications for fish populations. However, there is also a more widespread occurrence of endocrine disruption in fish in the U.K., where estrogenic effects have been demonstrated in freshwater systems, in estuaries, and in coastal areas. In mammals, the best evidence comes from the-field studies on Baltic gray and ringed seals, and from the Dutch semifield studies on harbor seals, where both reproduction and immune functions have been impaired by PCBs in the food chain. Reproduction effects resulted in population declines, whereas impaired immune function has likely contributed to the mass mortalities due to morbillivirus infections. Distorted sex organ development and function in alligators has been related to a major pesticide spill into a lake in Florida, U.S.A. The observed estrogenic/antiandrogenic effects in this reptile have been causally linked in experimental studies with alligator eggs to the DDT complex. Although most observed effects currently reported concern heavily polluted areas, endocrine disruption is a potential global problem. This is exemplified by the widespread occurrence of imposex in marine snails and the recent findings of high levels of persistent potential endocrine-disrupting chemicals in several marine mammalian species inhabiting oceanic waters.

Development of a stably transfected estrogen receptor-mediated luciferase reporter gene assay in the human T47D breast cancer cell line.

Development of an estrogen receptor-mediated, chemical-activated luciferase reporter gene-expression (ER-CALUX) assay was attempted by stable transfection of luciferase reporter genes in a number of cell lines. Stable transfection of the chimeric Gal4 estrogen receptor and luciferase gene constructs in MCF-7 breast cancer and Hepa.1c1c7 mouse hepatoma cell lines, as well as transfection of a newly constructed luciferase reporter gene pEREtata-Luc in the ECC-1 human endometrial cell line, resulted in constitutive, non-estradiol-inducible clones. Stable transfection of pEREtata-Luc in the T47D breast cancer cell line, however, resulted in an extremely sensitive, highly responsive cell line. Following a 24-h exposure to estradiol (E2), stably transfected T47D.Luc cells demonstrated a detection limit of 0.5 pM, an EC50 of 6 pM, and a maximum induction of 100-fold relative to solvent controls. No clear reduction in responsiveness has been found over extended culture periods (50 passages). Anti-estrogens ICI 182,780, TCDD, and tamoxifen inhibited the estradiol-mediated luciferase induction. Genistein, nonylphenol, and o,p'DDT were the most potent (pseudo-)estrogens tested in this system (EC50 100, 260, and 660 nM, respectively). Determination of interactive effects of the (pseudo-)estrogens nonylphenol, o,p'DDT, chlordane, endosulfan, dieldrin, and methoxychlor revealed that, in combination with 3 pM E2, (pseudo-)estrogens were additive. Slightly more than additive effects (less than 2-fold) were found for combinations of dieldrin and endosulfan tested in the range of 3 to 6 microM. At these concentrations, the combination of endosulfan and chlordane demonstrated additive interaction. The ER-CALUX assay with T47D cells can provide a sensitive, responsive, and rapid in vitro system to detect and measure substances with potential (anti-)estrogenic activity.