In Search of Mercury Lost from Sediments in a Previously Contaminated Coastal Area, Harboøre Tange, Denmark.

Concentrations of mercury in sediment and benthic invertebrate fauna of Nissum Broad, North-western Jutland, Denmark were investigated. The western coast of Nissum Broad is Harboøre Tange, along which heavy mercury contamination - caused by discharge from production of mercury containing seed dressers in the 1950 and 1960s - was documented in the 1980s. Recent investigations showed marked decreases in mercury contamination in the near shore sediments along Harboøre Tange since the 1980s and the present investigation was initiated to learn if the loss of mercury from Harboøre Tange had led to an increased mercury contamination in the neighbouring marine area, Nissum Broad. Mercury concentrations in the surface sediment correlated with the content of organic matter and the slope of the regression is a good indicator for the degree of mercury contamination. Average mercury concentrations in the upper 5 cm of the sediments ranged between 0.9 and 71 ng g- 1 dry weight (dw) with only 1 station exceeding the Background Assessment Concentration of 70 ng g- 1 dw. Average mercury concentrations in blue mussels Mytilus edulis (169-260 ng g- 1 dw) and periwinkles Littorina littorea (66-203 ng g- 1 dw) exceeded those in uncontaminated areas and the Environmental Quality Standard of approximately 100 ng g- 1 dw. Present sediment mercury concentrations in Nissum Broad are approximately half of what they were in the 1980s, rendering it unlikely that mercury lost from Harboøre Tange has been deposited there. Sediment and organism concentrations did not show any correlation.

Investigation of the in vivo estrogenicity of the UV-filters 4-methylbenzylidene camphor and octyl methoxy cinnamate in rainbow trout (Oncorhynchus mykiss).

The estrogenic activity of the chemical UV-filters, 4-methylbenzylidene camphor (4-MBC) and octyl methoxy cinnamate (OMC) was investigated in an in vivo rainbow trout (Oncorhynchus mykiss) assay. Plasma vitellogenin concentrations were quantified by means of an Enzyme Linked Immuno Sorbent Assay (ELISA) in juvenile rainbow trout before and after intraperitoneal injection of the test compounds. Injection of 4-MBC on day 0, 3, 6 and 10 in the exposure period caused dose and time dependent increases in the concentration of plasma vitellogenin. Significant elevation of vitellogenin concentrations in plasma was demonstrated from 151 mg 4-MBC kg-1 injection-1. Logistic regression analysis relating the percentage of responding fish to the injected dose of 4-MBC resulted in ED10, ED50 and ED90 values of 37, 115 and 194 mg kg-1 injection-1, respectively, after 14 days of exposure (4 injections). Injections with OMC (up to 202 mg kg-1 injection-1) did not result in a statistically significant response in groups of exposed fish, although some individual fish showed elevated concentrations of vitellogenin in plasma. The results confirm that 4-MBC is estrogenic in fish in vivo.

Mercury levels in humpback whales, and other Southern Ocean marine megafauna.

Mercury is a known potent neurotoxin. The biogeochemical cycle of mercury in the remote Antarctic region is still poorly understood, with Polar climate change contributing added complexity. Longitudinal biomonitoring of mercury accumulation in Antarctic marine megafauna can contribute top-down insight into the bio-physical drivers of wildlife exposure. The bioaccumulative nature of organic mercury renders high trophic predators at the greatest risk of elevated exposure. Humpback whales represent secondary consumers of the Antarctic sea-ice ecosystem and an ideal biomonitoring species for persistent and bioaccumulative compounds due to their extended life-spans. This study provides the first results of mercury accumulation in humpback whales, and places findings within the context of mercury accumulation in both prey, as well as six other species of Antarctic marine megafauna. Combined, these findings contribute new baseline information regarding mercury exposure to Antarctic wildlife, and highlights methodological prerequisites for routine mercury biomonitoring in wildlife via non-lethally biopsied superficial tissues.

Effect of size on concentrations and cadmium inducibility of metallothionein in the shore crab Carcinus maenas.

Metallothionein (MT) plays an important role in protecting organisms from the adverse effects of Cd, Hg, Zn and Cu. Investigations on mammals show variations in metallothionein concentrations and inducibility with age. This has never been investigated in invertebrates, and we determined the concentrations and inducibility of metallothionein in gills and midgut gland of different size classes of shore crabs from uncontaminated areas. Metallothionein concentrations in gills and midgut gland ranged between 30 and 40 µg g-1 dry weight with no differences among the different size classes. Concentrations of cadmium, copper and zinc in the midgut gland increased with increasing size of the crabs when the concentrations were expressed on a dry weight basis; water content in the midgut gland increased with the size and only the cadmium concentration increased with size when concentrations were expressed on wet weight basis. There was an inverse relationship between metallothionein and both copper and cadmium concentrations. Smaller crabs exposed to 1 mg Cd L-1 accumulated higher concentrations of cadmium in midgut gland and gills than larger ones and metallothionein concentrations in the midgut gland were higher in the smaller crabs. However, the increase in metallothionein concentration per accumulated unit of cadmium showed a linear increase with the size of the crabs. The ratio [Cd]midgut/[Cd]gills decreased with the size of the crabs. The overall conclusion is that baseline metallothionein concentrations do not change with age in shore crabs, but that the inducibility of metallothionein upon cadmium challenge does.

Exposure to methylmercury and inorganic mercury in the food does not lead to trophic magnification in the sea star Asterias rubens.

Methylmercury accumulated at the top of aquatic food chains constitutes a toxicological risk to humans and other top predators. Biomagnification of methylmercury takes place among vertebrates at the higher trophic levels, but this process is less elucidated in benthic invertebrates at the lower trophic levels. Therefore, we investigated the accumulation from food and elimination of methylmercury and inorganic mercury in the benthic sea star Asterias rubens (L.) - a representative of trophic level ~3 - in laboratory experiments. Sea stars fed over 49 days with contaminated mussels (Mytilus edulis) accumulate methylmercury and inorganic mercury to the highest concentrations in the digestive glands, the pyloric caeca, less in stomach, gonad, tube feet, aboral body wall and not to detectable levels in the coelomic fluid. Concerning whole body contents, steady states were reached for both methylmercury and inorganic mercury during the 7-week feeding period and the sea stars reached approximately ½ and » of the concentrations in the mussel food for the two mercury forms, respectively. Half-lives for the elimination of the two mercury forms varied between 45 and 173 days in a 140-d elimination period following the feeding period; inorganic mercury was eliminated faster than methylmercury. Examination of total mercury concentrations in field-collected sea stars confirmed this lack of trophic magnification in relation to the major food items, soft parts of molluscs. We suggest that mercury is not trophically magnified in sea stars 1) because they eliminate methylmercury faster than larger fish and decapod crustaceans and 2) maybe more importantly, because inorganic mercury with its faster elimination constitutes a larger fraction of the total mercury in the food at the lower trophic levels - as opposed to methylmercury which dominates at the higher trophic levels.

Mercury (Hg2+) interferes with physiological adaptations to freezing in the arctic earthworm Enchytraeus albidus.

Freezing temperatures is an important stressor in the arctic regions and has a significant influence on the population dynamics and geographic distribution of terrestrial invertebrates. Toxic metals in the environment can interfere with protective cold-acclimation responses of organisms. It is therefore important to evaluate the combined effects of cold stress and environmental contaminants. Here, we aimed to investigate the effects of Hg (HgCl2) on various physiological aspects of freeze-tolerance in the earthworm (Enchytraeus albidus). We measured the levels of the cryoprotectant glucose, the glycogen content (source of glucose molecules for cryoprotection and fuel for metabolism), and changes in the composition of membrane phospholipid fatty acids (PLFA) as an indicator of lipid peroxidation. Freezing at -6 °C had no effect on survival in uncontaminated soil, however, survival of freezing in Hg contaminated soil was clearly reduced, especially at extended exposure times. Thus, the LC50 value in frozen soil decreased from 8.3 mg Hg kg-1 (when exposed for 17 days) to only 4.2 mg Hg kg-1 after 36 days' exposure indicating that combined effects of Hg and freezing became larger at prolonged exposure times. Hg caused a depletion of glycogen reserves (almost 50% at 12 mg kg-1 dry soil), but despite this effect worms were able to maintain a constant cryoprotectant level (about 0.12 mg glucose mg-1 dry weight) at all Hg concentrations. Hg had clear negative effects on the proportion of unsaturated PLFAs, which could be an indication of lipid peroxidation. Since a high proportion of unsaturated fatty acids in the membrane is important for invertebrate freeze-tolerance, our results suggest that the negative effect of Hg on freeze-tolerance in E. albidus is related to degraded membrane functionality at low temperature.

Elevated mercury concentrations in biota despite reduced sediment concentrations in a contaminated coastal area, Harboøre Tange, Denmark.

Metals sequestered in coastal sediments are normally considered to be stable, but this investigation shows - somewhat surprisingly - that mercury concentrations in a previously contaminated area, Harboøre Tange, Denmark, have decreased since the 1980s. Mercury concentrations were determined in sediment and benthic biota and present values were compared to values in the 1980s and values from areas without known; history of mercury contamination. Concentrations in both the upper 20 cm of the sediments and; biota are considerably lower now compared to latest monitoring (1980s). Sediment. concentrations at most locations have decreased from the 100-300 ng Hg g-1 dry weight (dw) level to levels below the Background Concentration (BC) of 50 ng Hg g-1 dw defined by Oslo-Paris Convention for the Protection of the Marine Environment of the North-East Atlantic; some stations are at the 2-10 ng Hg g-1 dw level characteristic of Danish coastal sediments with no known history of mercury contamination. Concentrations of mercury in the benthic biota along Harboøre Tange have also decreased since the 1980s but despite the lowered mercury concentrations in the sediments, concentrations in most samples of benthic invertebrate fauna still exceed those in uncontaminated coastal areas and also the Environmental Quality Standard (EQS) of 20 ng Hg g-1 wet weight (≈100 ng Hg g-1 dry weight) defined by the European Union's Water Framework Directive. Concentration ranges in selected organisms are: (Harboøre Tange l980s/Harboøre Tange now/uncontaminated areas - given in ng Hg g-1 dw): Periwinkles Littorina littorea 9000/150-450/55-77, blue mussels Mytilus edulis up to 9000/300-500/40-170, cockles Cerastoderma edule up to 8000/400-1200/200, brown shrimp Crangon crangon 700-2200/150-450/47, eelgrass Zostera marina up to 330/25-70/12. The present results - together with a literature review - show that a simple and straight forward relationship between the concentrations of mercury in sediment and benthic organisms does not necessarily exist.

Investigation of the potential endocrine effect of nitrate in zebrafish Danio rerio and brown trout Salmo trutta.

Nitrate has the potential to affect steroid production. Nitrate concentrations in streams in agricultural areas may exceed concentrations showing effects in laboratory studies. The effects of nitrate and/or nitrite on endocrine relevant endpoints were tested in zebrafish and brown trout. Zebrafish were exposed in two experiments to nitrate (8.8 to 89 mg NO3-/L) and nitrite (3.6 to 19 mg NO2-/L) during the period of sexual differentiation and sex ratios were determined. Vitellogenin concentrations were determined in the second experiment. The sex ratio was unaffected by the exposure to nitrate and nitrite. Vitellogenin concentrations were slightly elevated in males (but not females) in all of the groups exposed to nitrate. Juvenile brown trout were exposed to 5.7, 14, and 31 mg NO3-/L for 8 days and vitellogenin levels in liver were determined. Vitellogenin concentrations in the females were not affected by exposure, but in the males, there was an overall statistically significant effect of exposure to nitrate with the group exposed to 5.7 mg NO3-/L showing a trend of higher vitellogenin concentrations than the control group; levels in the males of the groups exposed to 14 and 31 mg NO3-/L were not statistically different from those of the control group. In conclusion, some marginal effect of nitrate in male fish on endocrine activity was observed but the present results for zebrafish, using environmentally relevant concentrations, do not define nitrate and nitrite as endocrine disrupting chemicals according to the generally accepted WHO/IPCS definition because no adverse effects (altered sex ratios) were demonstrated.

Retention and distribution of methylmercury administered in the food in marine invertebrates: Effect of dietary selenium.

Methylmercury is transported along aquatic food chains from the lower trophic levels and selenium modulates the biokinetics of mercury in organisms in complex ways. We investigated the retention of orally administered methylmercury in various marine invertebrates and the effect of selenium hereon. Shrimps (Palaemon adpersus and P. elegans), blue mussels (Mytilus edulis), shore crabs (Carcinus maenas) and sea stars (Asterias rubens) eliminated methylmercury slowly (t½â€¯= ½ to >1 year) and the copepod (Acartia tonsa) faster (t½â€¯âˆ¼â€¯12-24 h). Orally administered selenite augmented elimination of methylmercury in the copepod (in one of two experiments) and blue mussels, but not in shrimps, crabs and sea stars. Selenium generally alters the distribution of the body burden of mercury, leaving more mercury in muscle and less mercury in digestive glands or rest of the body - also in the species where total body retention is not affected.

Vitellogenin concentrations in feral Danish brown trout have decreased: An effect of improved sewage treatment in rural areas?

Feminization of male and juvenile fish because of exposure to estrogens or estrogenic chemicals in effluents from central wastewater treatment plants (WWTPs) is a worldwide issue of concern. Intersex and induction of the female yolk protein, vitellogenin, in male and juvenile fish are robust biomarkers for estrogenic exposure, and feminized fish have been observed downstream of WWTP outlets in many countries. Danish central WWTPs reduce effluent estrogenicity effectively by advanced sewage treatment, and feminizations have not been observed downstream of central WWTP outlets. However, between 2000 and 2004, investigations of Danish streams not receiving sewage from central WWTPs revealed a high variation in vitellogenin concentrations of male juvenile brown trout (Salmo trutta); some individuals had high concentrations, probably as a result of estrogenic point sources, and the plasma concentration was >50 ng mL-1 in 79% of the juvenile males. The streams were reinvestigated in 2010 to 2016, and the average male level had decreased to a hitherto unseen baseline level; in 2010 only 0.7% (one individual) of the males had a vitellogenin concentration >50 ng mL-1 , which could indicate that the estrogenicity of the streams decreased after 2004. We examined possible estrogenic sources in streams unaffected by central WWTP effluents, and found that the reduced vitellogenin levels are most likely explained by a national effort to improve on-site wastewater treatment in scattered houses not connected to central WWTPs. Environ Toxicol Chem 2018;37:839-845. © 2017 SETAC.

Two common mild analgesics have no effect on general endocrine mediated endpoints in zebrafish (Danio rerio).

Mild analgesics such as acetylsalicylic acid (ASA) and acetaminophen (APAP) exert their pain-relieving effect in humans by inhibition of prostaglandin synthesis. Prostaglandins play key roles in developmental and reproductive processes in vertebrates, and in recent years, it has been suggested that weak analgesics might also act as endocrine disrupters. In a set of experiments we investigated if ASA and APAP affect well-established endocrine endpoints in zebrafish (Danio rerio), which is a commonly used model organism in the investigation of endocrine disrupting chemicals. Zebrafish were exposed to APAP (0.22, 2.3, and 30mgL-1) or ASA (0.2, 0.5, 1.7, and 8.2mgL-1) from hatch to sexual maturity in a test design resembling the OECD Fish Sexual Development Test. No effects on sex ratio and vitellogenin levels were observed. Adult zebrafish were exposed to high concentrations (mgL-1) of ASA or APAP for eight or 14days. ASA reduced the levels of prostaglandin E2, but had no effect on the concentration of 11-ketotestosterone and vitellogenin. Overall, ASA decrease prostaglandin E2 concentrations, but well-established endpoints for endocrine disruption in zebrafish are generally not affected by aquatic exposure neither during development nor adulthood. According to the WHO/IPCS definition of an endocrine disrupter, the present results do not define APAP and ASA as endocrine disrupters.

Recommended approaches to the scientific evaluation of ecotoxicological hazards and risks of endocrine-active substances.

A SETAC Pellston Workshop® "Environmental Hazard and Risk Assessment Approaches for Endocrine-Active Substances (EHRA)" was held in February 2016 in Pensacola, Florida, USA. The primary objective of the workshop was to provide advice, based on current scientific understanding, to regulators and policy makers; the aim being to make considered, informed decisions on whether to select an ecotoxicological hazard- or a risk-based approach for regulating a given endocrine-disrupting substance (EDS) under review. The workshop additionally considered recent developments in the identification of EDS. Case studies were undertaken on 6 endocrine-active substances (EAS-not necessarily proven EDS, but substances known to interact directly with the endocrine system) that are representative of a range of perturbations of the endocrine system and considered to be data rich in relevant information at multiple biological levels of organization for 1 or more ecologically relevant taxa. The substances selected were 17α-ethinylestradiol, perchlorate, propiconazole, 17ß-trenbolone, tributyltin, and vinclozolin. The 6 case studies were not comprehensive safety evaluations but provided foundations for clarifying key issues and procedures that should be considered when assessing the ecotoxicological hazards and risks of EAS and EDS. The workshop also highlighted areas of scientific uncertainty, and made specific recommendations for research and methods-development to resolve some of the identified issues. The present paper provides broad guidance for scientists in regulatory authorities, industry, and academia on issues likely to arise during the ecotoxicological hazard and risk assessment of EAS and EDS. The primary conclusion of this paper, and of the SETAC Pellston Workshop on which it is based, is that if data on environmental exposure, effects on sensitive species and life-stages, delayed effects, and effects at low concentrations are robust, initiating environmental risk assessment of EDS is scientifically sound and sufficiently reliable and protective of the environment. In the absence of such data, assessment on the basis of hazard is scientifically justified until such time as relevant new information is available. Integr Environ Assess Manag 2017;13:267-279. © 2017 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals, Inc. on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Uncertainties in biological responses that influence hazard and risk approaches to the regulation of endocrine active substances.

Endocrine-disrupting substances (EDS) may have certain biological effects including delayed effects, multigenerational effects, and may display nonmonotonic dose-response (NMDR) relationships that require careful consideration when determining environmental hazards. Endocrine disrupting substances can have specific and profound effects when exposure occurs during sensitive windows of the life cycle (development, reproduction). This creates the potential for delayed effects that manifest when exposure has ceased, possibly in a different life stage. This potential underscores the need for testing in appropriate (sensitive) life stages and full life cycle designs. Such tests are available in the Organisation for Economic Co-operation and Development (OECD) tool box and should be used to derive endpoints that can be considered protective of all life stages. Similarly, the potential for effects to be manifest in subsequent generations (multigenerational effects) has also been raised as a potential issue in the derivation of appropriate endpoints for EDS. However, multigenerational studies showing increasing sensitivity of successive generations are uncommon. Indeed this is reflected in the design of new higher tier tests to assess endocrine active substances (EAS) that move to extended one-generation designs and away from multi-generational studies. The occurrence of NMDRs is also considered a limiting factor for reliable risk assessment of EDS. Evidence to date indicates NMDRs are more prevalent in in vitro and mechanistic data, not often translating to adverse apical endpoints that would be used in risk assessment. A series of steps to evaluate NMDRs in the context of endocrine hazard and risk assessment procedures is presented. If careful consideration of delayed, multigenerational effects and NMDRs is made, it is feasible to assess environmental endocrine hazards and derive robust apical endpoints for risk assessment procedures ensuring a high level of environmental protection. Integr Environ Assess Manag 2017;13:293-301. © 2016 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals, Inc. on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Severe malformations of eelpout (Zoarces viviparus) fry are induced by maternal estrogenic exposure during early embryogenesis.

Pregnant eelpout were exposed via the water to known endocrine disrupting compounds (EDCs) to clarify if EDCs could be causing the increased eelpout fry malformation frequencies observed in coastal areas receiving high anthropogenic input. The presence of a teratogenic window for estrogen-induced malformations was also investigated by starting the exposure at different times during eelpout pregnancy. Both 17α-ethinylestradiol (EE2) (17.8 ng/L) and pyrene (0.5 µg/L) significantly increased fry malformation frequency whereas 4-t-octylphenol (4-t-OP) up to 14.3 µg/L did not. Vitellogenin was significantly induced by EE2 (5.7 and 17.8 ng/L) but not by 4-t-OP and pyrene. A critical period for estrogen-induced fry malformations was identified and closed between 14 and 22 days post fertilization (dpf). Exposure to 17ß-estradiol (E2) between 0 and 14 dpf caused severe malformations and severity increased the closer exposure start was to fertilization, whereas malformations were absent by exposure starting later than 14 dpf. Data on ovarian fluid volume and larval length supported the suggested teratogenic window. Larval mortality also increased when exposure started right after fertilization.

Manufacturing doubt about endocrine disrupter science--A rebuttal of industry-sponsored critical comments on the UNEP/WHO report "State of the Science of Endocrine Disrupting Chemicals 2012".

We present a detailed response to the critique of "State of the Science of Endocrine Disrupting Chemicals 2012" (UNEP/WHO, 2013) by financial stakeholders, authored by Lamb et al. (2014). Lamb et al.'s claim that UNEP/WHO (2013) does not provide a balanced perspective on endocrine disruption is based on incomplete and misleading quoting of the report through omission of qualifying statements and inaccurate description of study objectives, results and conclusions. Lamb et al. define extremely narrow standards for synthesizing evidence which are then used to dismiss the UNEP/WHO 2013 report as flawed. We show that Lamb et al. misuse conceptual frameworks for assessing causality, especially the Bradford-Hill criteria, by ignoring the fundamental problems that exist with inferring causality from empirical observations. We conclude that Lamb et al.'s attempt of deconstructing the UNEP/WHO (2013) report is not particularly erudite and that their critique is not intended to be convincing to the scientific community, but to confuse the scientific data. Consequently, it promotes misinterpretation of the UNEP/WHO (2013) report by non-specialists, bureaucrats, politicians and other decision makers not intimately familiar with the topic of endocrine disruption and therefore susceptible to false generalizations of bias and subjectivity.

Endocrine-disrupting effect of the ultraviolet filter benzophenone-3 in zebrafish, Danio rerio.

The chemical ultraviolet (UV) filter benzophenone-3 (BP-3) is suspected to be an endocrine disruptor based on results from in vitro and in vivo testing. However, studies including endpoints of endocrine adversity are lacking. The present study investigated the potential endocrine-disrupting effects of BP-3 in zebrafish (Danio rerio) in the Fish Sexual Development Test (Organisation for Economic Co-operation and Development TG 234) and a 12-d adult male zebrafish study. In TG 234, exposure from 0 d to 60 d posthatch caused a monotone dose-dependent skewing of the phenotypic sex ratio toward fewer males and more female zebrafish (no observed effect concentration [NOEC]: 191 µg/L, lowest observed effect concentration [LOEC]: 388 µg/L). Besides, gonad maturation was affected in both female fish (NOEC 191 µg/L, LOEC 388 µg/L) and male fish (NOEC 388 µg/L, LOEC 470 µg/L). Exposure to BP-3 did not affect the vitellogenin concentration in TG 234. After 12 d exposure of adult male zebrafish, a slight yet significant increase in the vitellogenin concentration was observed at 268 µg/L but not at 63 µg/L and 437 µg/L BP-3. Skewing of the sex ratio is a marker of an endocrine-mediated mechanism as well as a marker of adversity, and therefore the conclusion of the present study is that BP-3 is an endocrine-disrupting chemical in accordance with the World Health Organization's definition.

A path forward in the debate over health impacts of endocrine disrupting chemicals.

Several recent publications reflect debate on the issue of "endocrine disrupting chemicals" (EDCs), indicating that two seemingly mutually exclusive perspectives are being articulated separately and independently. Considering this, a group of scientists with expertise in basic science, medicine and risk assessment reviewed the various aspects of the debate to identify the most significant areas of dispute and to propose a path forward. We identified four areas of debate. The first is about the definitions for terms such as "endocrine disrupting chemical", "adverse effects", and "endocrine system". The second is focused on elements of hormone action including "potency", "endpoints", "timing", "dose" and "thresholds". The third addresses the information needed to establish sufficient evidence of harm. Finally, the fourth focuses on the need to develop and the characteristics of transparent, systematic methods to review the EDC literature. Herein we identify areas of general consensus and propose resolutions for these four areas that would allow the field to move beyond the current and, in our opinion, ineffective debate.

17ß-estradiol causes abnormal development in embryos of the viviparous eelpout.

Elevated frequencies of malformations among the offspring of Baltic eelpout (Zoarces viviparus) have been observed in aquatic environments receiving high anthropogenic input suggesting that manmade chemicals could be the causative agent. However, causal links between exposure to chemicals and abnormal development have never been confirmed in laboratory experiments. The purpose of this study was to investigate if exposure to 17ß-estradiol (E2) causes abnormal development in larvae of the viviparous eelpout. Wild female eelpout were collected immediately after fertilization and exposed to E2 concentrations ranging from 5.7 to 133 ng L(-1) for 6 weeks in a flow through test system. The experiment shows that E2 concentrations of 53.6 and 133 ng L(-1) cause severe abnormal development among eelpout embryos. Reduced amount of ovarian fluid and increased weight of the ovarian sac indicate disturbance of ovarian function. Female plasma concentrations of E2 and vitellogenin increase in a monotonic concentration-response relationship with significant induction in the low concentration range. Our findings support the plausibility that the abnormal development among eelpout embryos encountered in monitoring programs may actually be caused by exposure to chemicals in the environment.

Evaluation of yolk protein levels as estrogenic biomarker in bivalves; comparison of the alkali-labile phosphate method (ALP) and a species-specific immunoassay (ELISA).

Altered concentration of the vertebrate yolk protein precursor vitellogenin is a recognized biomarker for endocrine disruption in fish, and within recent years yolk protein alteration has also been associated with endocrine disruption in bivalves. Species-specific, direct and sensitive methods for quantification of vitellogenin in fish have been available for years whereas bivalve yolk protein levels have been estimated indirectly by alkali-labile phosphate (ALP) liberated from high molecular weight proteins because the sequence and biochemical structure of most bivalve yolk proteins are unknown. By applying a species-specific enzyme-linked immunosorbent assay (ELISA) for accurate determination of yolk protein level the impact of 17ß-estradiol (57, 164 and 512 ng/L) on the freshwater bivalve Unio tumidus was investigated and compared with ALP estimations. Seven weeks of exposure during the pre-spawning and spawning period had no consistent effect on yolk protein concentration in hemolymph, and ALP levels in hemolymph also remained unchanged in both males and females. Further, basal male and female ALP levels were indistinguishable whereas the ELISA demonstrated that yolk protein levels of females exceeded male levels at the time of sampling, although male basal levels were high compared to fish. Altogether it is shown that individual ALP levels do not reflect yolk protein levels and hence hemolymph ALP levels cannot serve as biomarker for estrogenic exposure during the pre-spawning and spawning period in U. tumidus. The necessity of sensitive and validated biomarkers for reliable interpretation of data and the utility of ALP and yolk protein levels as biomarkers in bivalves are discussed.