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.

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.

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.

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.

Skin and liver diseases induced in flounder (Platichthys flesus) after long-term exposure to contaminated sediments in large-scale mesocosms.

Disease development in flounder (Platichthys flesus) was studied over a period of 3 years in three large mesocosms (40 m x 40 m x 3 m). Two of the mesocosms contained clean sand and the third, sharing a common water circulation with one of the clean-sand mesocosms, was stocked with contaminated dredged spoil. In this way, one of the clean-sand mesocosms was indirectly polluted via the water phase, and analysis of contaminant concentrations in sediments and flounder tissues showed that it had a status intermediate between the other two. Random samples of the flounder populations from the indirectly polluted and reference mesocosms were examined every 2 months for epidermal diseases (lymphocystis, skin ulcers, fin rot) and then released. In addition, every 6 months, random samples of fish from all three mesocosms were sacrificed for histological and chemical investigation. With regard to the development of epidermal disease, the results showed little difference between the reference mesocosm and the indirectly polluted mesocosm, with the exception that lymphocystis was significantly elevated in the indirectly polluted mesocosm. Although pollution may be a risk factor in the etiology of this disease, such a relationship would probably be obscured under field conditions due to variation arising from other factors. Histopathological analysis of the livers revealed in total four cases of hepatocellular adenoma (1.5% of sampled population) in fish from the polluted mesocosms, the first occurring after 2.5 years of exposure in fish from the indirectly polluted mesocosm. Furthermore, several other liver lesions, including foci of cellular alteration and hydropic vacuolated lesions, developed during the course of the experiment before tumor formation was apparent. Prevalences of these conditions were very much lower in the reference mesocosm than in the two polluted mesocosms. Densities of melanomacrophage centers in the liver showed a similar trend. The findings clearly indicate that long-term exposure to chemically contaminated dredged spoil can induce liver neoplasia and other liver lesions in flounder at contaminant levels comparable to those found in the natural environment.

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.

Low inducibility of CYP1A activity by polychlorinated biphenyls (PCBs) in flounder (Platichthys flesus): characterization of the Ah receptor and the role of CYP1A inhibition.

Several studies have reported a low inducibility of hepatic cytochrome P4501A (CYP1A) activity in European flounder (Platichthys flesus) following exposure to mixtures of polychlorinated biphenyls (PCBs). Here we report on mechanistic studies toward understanding this low CYP1A inducibility of flounder, involving molecular characterization of the Ah receptor (AhR) pathway as well as inhibition of the CYP1A catalytic activity by PCB congeners. Hepatic cytosolic AhR levels in flounder were determined using hydroxylapatite, protamine sulfate adsorption analysis, or velocity sedimentation on sucrose gradients. AhR levels in flounder (approximately 2-7 fmol/mg protein) were much lower than observed generally in rodents (approximately 50-300 fmol/mg protein). Molecular characterization of the flounder AhR was provided by first-strand cDNA synthesis and amplification of flounder hepatic poly(A)+ RNA using RT-PCR. A 690-bp product was found, similar in size to a Fundulus AhR cDNA. The specificity of the 690-bp band was established by Southern blotting and hybridization with a degenerate AhR oligonucleotide. The deduced amino acid sequence of the flounder AhR fragment was 59-60% identical to mammalian AhR sequences. Although the AhR is present in flounder cytosol, we were unable to demonstrate detectable amounts of inducible TCDD-AhR-DRE complex in gel-retardation assays. High induction levels of CYP1A protein and associated EROD activity have been previously found in flounder following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In contrast, the induction of CYP1A catalytic activity by PCB mixtures remains unexpectedly low. Therefore, we further characterized the inhibitory potential of PCB congeners on CYP1A activity in flounder and compared this with inhibitory effects of PCB congeners on rat CYP1A activity. Analysis in vitro demonstrated that 3,3',4,4'-tetraCB, 3,3',4,4',5-pentaCB, 2,2',4,4',5,5'-hexaCB, 3,3',4,4',5,5'-hexaCB, and the commercial PCB mixture Clophen A50 are potent competitive inhibitors of hepatic microsomal CYP1A catalytic activity in flounder and rat. The K(m) for ethoxyresorufin (0.095 microM) in flounder is strikingly close to Ki's found for the tested PCBs. This emphasizes the possible involvement of PCB congeners in inhibition of EROD activity in PHAH exposed fish. Finally, our data indicate that flounder CYP1A is more efficient in metabolizing ethoxyresorufin than that of rat CYP1A.

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.

Fish as biomarkers in immunotoxicology.

This paper presents an overview on the state of the art in the development and application of biomarkers for immunotoxicology in fish. There are several reasons for developing this field: many fish diseases are related to environmental quality, various environmental pollutants have immunotoxic potential and many fish diseases have an immunological component. As in immunotoxicology in general, this aspect, in fish, has received ample attention in the recent past. Much benefit has been obtained from progress in related fields of science, such as fish immunology and rodent immunotoxicology. Meanwhile there is a broad spectrum of potential biomarkers for immunotoxicology in fish, from which macrophage parameters seem to be most widely used. The application of others, such as lymphoid cell parameters is still limited, probably due to practical problems such as lack of experience with conduct, validation and interpretation. Specific problems include the paucity of background data in the case of epidemiological field studies and the important role of other (non-chemical) stress factors in the immune response, and hence the lack of specificity of potential biomarkers.

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

Low hepatic 7-ethoxyresorufin-O-deethylase (EROD) activity and minor alterations in retinoid and thyroid hormone levels in flounder (Platichthys flesus) exposed to the polychlorinated biphenyl (PCB) mixture, Clophen A50.

The effect of the polychlorinated biphenyl (PCB) mixture Clophen A50 on hepatic cytochrome P4501A1 dependent EROD (7-ethoxyresorufin-O-deethylase) activity, plasma thyroid hormone levels and plasma, kidney and liver retinoid concentrations of the euryhaline flatfish flounder (Platichthys flesus) was determined 2 and 10 days after i.p. (intraperitoneal) injection with 20, 100 and 500 mg Clophen A50/kg body weight. No effect of Clophen A50 on total cytochrome P450 content in flounder liver was observed at both time points. A six-fold, dose-dependent, significant increase in EROD activity was found at exposure day 10 in flounder receiving 100 or 500 mg Clophen A50/kg body weight. Plasma retinol concentrations were not altered at both time points after Clophen A50 administration, whereas renal retinol levels showed a minor dose-related increase at day 2 and day 10 of exposure. Significant alterations in hepatic retinoid concentrations were observed, which were not dependent on the dose of PCB administered. In addition Clophen A50 administration did not result in a dose-related alteration of total T4 concentrations in plasma. Total T3 concentrations in plasma were only significantly increased at day 2 after exposure, whereas free T4 concentrations were increased at both time points after Clophen A50 administration. These data indicate that with regard to the parameters investigated and in contrast to other fish species studied, the flounder is not a sensitive species to PCB exposure.

Analysis and occurrence of estrogenic hormones and their glucuronides in surface water and waste water in The Netherlands.

An analytical procedure was developed that enables routine analysis of four estrogenic hormones in concentrations below 1 ng/l in surface water and waste water. The recovery was 88-98% with a limit of detection of 0.1-2.4 ng/l depending on the compound and the matrix measured. This method was used to determine the occurrence of 17 beta-estradiol, 17 alpha-estradiol, estrone and 17 alpha-ethinylestradiol in the aquatic environment in The Netherlands. The data show that estrogenic hormones can be detected at low concentrations (up to 6 ng/l) at some locations in surface water. In selected effluents of waste water treatment plants estrone and 17 beta-estradiol were detected in concentrations in the ng/l range. Concentrations of 17 alpha-estradiol and the contraceptive 17 alpha-ethinylestradiol were in most of these samples below the limit of detection. Hormone glucuronides were not detected in most surface water and effluents.

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.

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.