Endocrine Disruption In Fish Research Articles

Salicylate Disrupts Interrenal Steroidogenesis and Brain Glucocorticoid Receptor Expression in Rainbow Trout

Varying levels of pharmaceuticals, including salicylate, ibuprofen, and acetaminophen, have been reported in the aquatic environment, but few studies have actually addressed the impact of these drugs on aquatic organisms. We tested the hypothesis that these pharmaceuticals are endocrine disruptors in fish by examining their impact on interrenal corticosteroidogenesis in rainbow trout. Indeed, acute adrenocorticotrophic hormone (ACTH)-mediated cortisol production in trout interrenal cells in vitro was significantly depressed (20-40%) by these pharmaceutical drugs. Furthermore, we investigated whether this interrenal dysfunction involved inhibition of the steroidogenic capacity in rainbow trout. To this end, we fed trout salicylate-laced feed (100 mg/kg body weight) for 3 days and assessed the transcript levels of key proteins involved in corticosteroidogenesis, including steroidogenic acute regulatory protein (StAR), peripheral-type benzodiazepine receptor (PBR), cytochrome P450 cholesterol side chain cleavage (P450scc), and 11beta-hydroxylase. Salicylate treatment did not affect the resting plasma cortisol or glucose levels, whereas the acute ACTH-stimulated cortisol production was significantly depressed in the interrenal tissue. This disruption of steroidogenesis by salicylate corresponded to a significant drop in the gene expression of StAR and PBR, but not P450scc or 11beta-hydroxylase, compared to the sham-treated fish. Also, brain glucocorticoid receptor (GR) protein content and not GR mRNA level was significantly reduced by salicylate. Taken together, salicylate is a corticosteroid disruptor in trout and the targets include the key rate-limiting step in interrenal steroidogenesis and brain glucocorticoid signaling.

Contamination of headwater streams in the United Kingdom by oestrogenic hormones from livestock farms

Most studies of hormonal activity in rivers have focused on inputs from sewage treatment works (STW), and their consequences for endocrine disruption in fish. It is possible that livestock is contributing to this hormonal activity in rivers. This study represents a search for evidence of steroid hormone contamination in streams associated with livestock farms. The majority of the 10 sites selected were streams running through dairy farms, although some examples of beef, sheep and pigs were included. Passive water samplers (Polar Organic Chemical Integrative Samplers – POCIS) were deployed up- (control) and down-stream of the farms for 3 to 10 weeks (mean = 39 days) during the period November 2004 to January 2005. At one site, water samples were also taken automatically during rainfall events. All samples were solvent-extracted. Total oestrogenic activity in concentrates of the extracts was analysed using the Yeast Estrogen Screen (YES) calibrated against 17β-oestradiol (E2), while oestrone (E1), E2 and 17α-ethinylestradiol (EE2) were analysed by liquid chromatography–mass spectrometry (LC–MS/MS). Stream water from the entirety of only one rainfall event was sampled directly, but this revealed background activity (E2 equivalents) of 0–0.3 ng/l, rising to a transient peak of 9.4 ng/l. Average oestrogenic activity at this site as estimated from the POCIS samplers was 1.8–2.7 ng E2 equiv./l. Estimated average oestrogenic activity across all sites (with one exception) lay in the range 0– 26.5 ng E2 equiv./l (mean = 2.0 ng/l; S.D. = 5.1), based on the POCIS samples. The outlier was 292 ng/l, and this could not be specifically linked with livestock rearing. 92% of monitoring stations (at least one on each farm) contained some oestrogenic activity, and activity was higher at downstream sites in 50% of cases. Although no EE2 was detected analytically in any stream, E1 and E2 were almost ubiquitous, with E2 equivalents ranging from 0.04 to 3.6 ng/l across all sites. Furthermore, steroid concentrations downstream of livestock were higher than upstream in 60% of cases, more markedly so than for the YES data. In several cases, activity upstream was greater than downstream, and this tended to be associated with higher activity than could be accounted for by the hormone analyses. Both the YES and chemical analytical data suggest that fish in headwater streams on or near some livestock farms may be at risk of endocrine disruption.

Vitellogenin gene expression as a biomarker of endocrine disruption in the invertebrate, Mytilus edulis

Vitellogenin levels are often used as a biomarker of endocrine disruption in fish. For invertebrates, there is a general lack of knowledge regarding endocrine regulation and, consequently, there are few direct biomarkers of endocrine disruption in such species. This study focuses on the marine mussel Mytilus edulis, which is often employed in biomonitoring studies. A partial vitellogenin mRNA has been isolated and gene expression quantified using real-time PCR.

Screening and Testing for Endocrine Disruption in Fish—Biomarkers As “Signposts,” Not “Traffic Lights,” in Risk Assessment

Biomarkers are currently best used as mechanistic “signposts” rather than as “traffic lights” in the environmental risk assessment of endocrine-disrupting chemicals (EDCs). In field studies, biomarkers of exposure [e.g., vitellogenin (VTG) induction in male fish] are powerful tools for tracking single substances and mixtures of concern. Biomarkers also provide linkage between field and laboratory data, thereby playing an important role in directing the need for and design of fish chronic tests for EDCs. It is the adverse effect end points (e.g., altered development, growth, and/or reproduction) from such tests that are most valuable for calculating adverseNOEC (no observed effect oncentration) or adverseEC10 (effective concentration for a 10% response) and subsequently deriving predicted no effect concentrations (PNECs). With current uncertainties, biomarkerNOEC or biomarkerEC10 data should not be used in isolation to derive PNECs. In the future, however, there may be scope to increasingly use biomarker data in environmental decision making, if plausible linkages can be made across levels of organization such that adverse outcomes might be envisaged relative to biomarker responses. For biomarkers to fulfil their potential, they should be mechanistically relevant and reproducible (as measured by interlaboratory comparisons of the same protocol). VTG is a good example of such a biomarker in that it provides an insight to the mode of action (estrogenicity) that is vital to fish reproductive health. Interlaboratory reproducibility data for VTG are also encouraging; recent comparisons (using the same immunoassay protocol) have provided coefficients of variation (CVs) of 38–55% (comparable to published CVs of 19–58% for fish survival and growth end points used in regulatory test guidelines). While concern over environmental xenoestrogens has led to the evaluation of reproductive biomarkers in fish, it must be remembered that many substances act via diverse mechanisms of action such that the environmental risk assessment for EDCs is a broad and complex issue. Also, biomarkers such as secondary sexual characteristics, gonadosomatic indices, plasma steroids, and gonadal histology have significant potential for guiding interspecies assessments of EDCs and designing fish chronic tests. To strengthen the utility of EDC biomarkers in fish, we need to establish a historical control database (also considering natural variability) to help differentiate between statistically detectable versus biologically significant responses. In conclusion, as research continues to develop a range of useful EDC biomarkers, environmental decision-making needs to move forward, and it is proposed that the “biomarkers as signposts” approach is a pragmatic way forward in the current risk assessment of EDCs.

Assessment of Feminization of Male Fish in English Rivers by the Environment Agency of England and Wales

In recent years there has been considerable concern over the ability of substances discharged into the environment to disrupt the normal endocrine function of wildlife. In particular, the apparent widespread feminization of male fish in rivers has received significant attention from regulators in the United Kingdom, the United States, Europe, and Japan. The U.K. and European epidemiological data sets have demonstrated that the occurrence of feminized fish is associated with effluent discharges and that the incidence and severity is positively correlated with the proportion of treated sewage effluent in receiving waters. Although weakly estrogenic substances may contribute to the overall effect, studies have concluded that steroid estrogens are the principal and most potent estrogenic components of domestic sewage. Extensive laboratory data sets confirm that steroid estrogens are capable of eliciting the effects observed in wild fish at concentrations that have been measured in effluents and in the environment. Based on evaluation of the available information, the Environment Agency (England and Wales) has concluded that the weight of evidence for endocrine disruption in fish is sufficient to develop a risk management strategy for estrogenically active effluents that discharge to the aquatic environment.

Detailed Endocrine Assessments of Wild Fish in the Northern River Basins, Alberta, in Comparison to EEM Monitored Endpoints

Abstract During the Northern River Basins Study (NRBS), several issues were highlighted regarding the health of resident fish populations. Based on a basin-wide study of gonad morphology and circulating sex steroid levels, there was preliminary evidence of potential endocrine disruption in fish within the basins. The main concern with this was the potential loss of reproductive capability and possible declining populations. The Northern Rivers Ecosystem Initiative was established in response to the NRBS and an endocrine study was conducted to evaluate in more detail reproductive endocrine function of fish within the identified areas. A suite of endocrine endpoints was used and compared to overall fish health assessments using the Environmental Effects Monitoring (EEM) Programs protocol in areas exposed to sewage and pulp and paper mill effluents. As these northern rivers are nutrient-limited, fish responded to effluent addition (increased nutrients) by showing signs of increased growth and development relative to the upstream reference fish. Some signs of altered reproductive function were observed downstream of the one mill site, however additional responses were observed downstream of a municipal sewage treatment plant discharge from the neighbouring community. The reproductive responses were minimal compared to earlier studies at other older pulp and paper facilities. Follow-up studies at the Grande Prairie site documented changes in the fish responses to the effluents in terms of enrichment. The sewage treatment plant had installed tertiary treatment prior to the latest studies and reductions in the release of nutrients may be the cause. It is not known at the present time whether the pronounced eutrophication response that is still present in these fish may mask other endocrine alterations, as these effluents have endocrine-active compounds in them. The results also emphasize the need for EEM-like monitoring downstream of municipal sewage discharges.

The human lipid regulator, gemfibrozil bioconcentrates and reduces testosterone in the goldfish, Carassius auratus

Human and veterinarian pharmaceuticals have been detected in the aquatic environment for a number of years, but the potential for biological effects in exposed aquatic organism is only now being reported. The lipid regulator, gemfibrozil (GEM) is detected at μg/L concentrations in domestic wastewater and ng/L concentrations in surface waters. We investigated the uptake of GEM in goldfish ( Carassius auratus) over a 96 h time period by measuring GEM in blood plasma using LC–MS/MS. Results indicated that GEM can be taken up from water through the gills. In goldfish exposed to GEM by a single intraperitoneal injection, concentrations of GEM in the blood plasma declined rapidly over 96 h post-injection, with a half-life estimated at approximately 19 h. Exposure of goldfish to waterborne GEM at an environmentally relevant concentration over 14 days resulted in a plasma bioconcentration factor of 113. In goldfish exposed to aqueous concentrations of GEM for 96 h or 14 days, plasma testosterone (T) was reduced by over 50% in fish from all treatments. As a possible mechanistic explanation for the observed reduction in T, levels of steroid acute regulatory (StAR) protein transcript in goldfish testes were assessed by RT–PCR. StAR protein is involved in the transport of cholesterol from the outer to the inner mitochondrial membrane for transformation by the first enzyme in steroidogenesis. After exposure to GEM for 96 h, a 50% decrease in StAR mRNA levels was observed in goldfish. Gonadal StAR mRNA levels were not affected in the 14 days exposure, indicating that the observed decreases in plasma testosterone were not solely due to impaired delivery of cholesterol to the inner mitochondrial membrane. Our results demonstrate that exposure to environmental levels of GEM leads to bioconcentration of the drug in plasma and the potential for endocrine disruption in fish.

Effects of alkylphenols on redox status in first spawning Atlantic cod ( Gadus morhua)

Offshore oil production releases large amounts of lipophilic compounds in produced water and into the ocean. The discharge of produced water from the Norwegian petroleum sector has increased from 26 million m 3 in 1993 to 120 million m 3 in 2001, and it continues to increase. Produced water contains significant amounts of alkylphenols, which have been reported to be estrogenic, causing endocrine disruption in fish. In year 2000, approximately 44 tons of alkylphenols were released on the Norwegian continental shelf in connection with discharge of produced water. Except from being estrogenic, relatively little is known about the effects of alkylphenols when released in the marine environment. Our objective was to study how alkylphenols affect the redox status in first spawning Atlantic cod ( Gadus morhua) of both sexes. Model compounds tested included 4- tert-butylphenol (C 4), 4-n-pentylphenol (C 5), 4-n-hexylphenol (C 6) and 4-n-heptylphenol (C 7), all found in produced water. First spawning Atlantic cod were force-fed a mixture of these four alkylphenols, ranging between 0.02 and 80 ppm or 5 ppm 17β-estradiol (E 2), for 1 or 4 weeks. Increased hepatic total glutathione concentration in response to alkylphenol exposure was detected in female fish compared to control group after 1-week exposure, an effect not seen after 4 weeks. Furthermore, hepatic total glutathione concentration was sex dependent, where male fish sampled after 4 weeks had higher levels of glutathione than female fish. Increased glutathione reductase catalytic activities in both male and female fish were seen after exposure to 0.02 ppm alkylphenol mixture in 4 weeks. The glutathione S-transferase activity was only affected in male fish exposed to 0.02 ppm alkylphenols, and glucose-6-phosphate dehydrogenase activity increased in female fish exposed to 0.02 ppm alkylphenol mixture for 1 week. The increase of hepatic total glutathione content as well as the effects on glutathione reductase activities suggests that alkylphenol exposure affects the redox status in Atlantic cod.

Effects of alkylphenols on redox status in first spawning Atlantic cod (Gadus morhua)

Offshore oil production releases large amounts of lipophilic compounds in produced water and into the ocean. The discharge of produced water from the Norwegian petroleum sector has increased from 26 million m3 in 1993 to 120 million m3 in 2001, and it continues to increase. Produced water contains significant amounts of alkylphenols, which have been reported to be estrogenic, causing endocrine disruption in fish. In year 2000, approximately 44 tons of alkylphenols were released on the Norwegian continental shelf in connection with discharge of produced water. Except from being estrogenic, relatively little is known about the effects of alkylphenols when released in the marine environment. Our objective was to study how alkylphenols affect the redox status in first spawning Atlantic cod (Gadus morhua) of both sexes. Model compounds tested included 4-tert-butylphenol (C4), 4-n-pentylphenol (C5), 4-n-hexylphenol (C6) and 4-n-heptylphenol (C7), all found in produced water. First spawning Atlantic cod were force-fed a mixture of these four alkylphenols, ranging between 0.02 and 80 ppm or 5 ppm 17β-estradiol (E2), for 1 or 4 weeks. Increased hepatic total glutathione concentration in response to alkylphenol exposure was detected in female fish compared to control group after 1-week exposure, an effect not seen after 4 weeks. Furthermore, hepatic total glutathione concentration was sex dependent, where male fish sampled after 4 weeks had higher levels of glutathione than female fish. Increased glutathione reductase catalytic activities in both male and female fish were seen after exposure to 0.02 ppm alkylphenol mixture in 4 weeks. The glutathione S-transferase activity was only affected in male fish exposed to 0.02 ppm alkylphenols, and glucose-6-phosphate dehydrogenase activity increased in female fish exposed to 0.02 ppm alkylphenol mixture for 1 week. The increase of hepatic total glutathione content as well as the effects on glutathione reductase activities suggests that alkylphenol exposure affects the redox status in Atlantic cod.

Histopathology as a tool for the evaluation of endocrine disruption in zebrafish (Danio rerio)

The importance of histology as a tool in the evaluation of endocrine disruption in fish depends on the choice and interpretation of appropriate endpoints, as is illustrated by the analysis of the effects of exposure to the estrogen 17beta-estradiol (E2) and the nonaromatizable androgen 17-methyldihydrotestosterone (MDHT). The E2 led to the disappearance of vitellogenic oocytes in the ovary and an increased area of relatively large, eosinophilic cells in the testis, which were identified as spermatogonia under high-power magnification; this was a relative increase, as was shown by histomorphometry, because of a decreased size of spermatogenic cysts and a relative decrease of spermatocyte cysts. The E2 also induced an accumulation of acidophilic fluid in vessels and interstitial spaces, confirmed by immunohistochemistry as vitellogenin, and basophilia in the liver also associated with the production of vitellogenin. The MDHT induced activation of Sertoli cells in the testis and a decreased presence of vitellogenic oocytes and a reduced growth of previtellogenic oocytes in the ovary. These observations indicate the advantages of examining multiple organ systems on whole-body sections and the application of adequate magnifications. Inclusion of additional techniques such as morphometry and immunohistochemistry is valuable to further uncover insidious effects of endocrine disruptors.

Endocrine disruption in wild freshwater fish

Endocrine disruption has been reported in freshwater fish populations around the world. This phenomenon ranges from subtle changes in the physiology and sexual behavior of fish to permanently altered sexual differentiation and impairment of fertility. Despite widespread reports of endocrine disruption in fish (and this is well characterized at the individual level), few studies have demonstrated population-level consequences as a result of exposure to endocrine-disrupting chemicals (EDCs). An exception to this is in Lake Ontario Lake trout where precipitous declines in the population have been linked with periods of high exposure to organochlorine chemicals (known EDCs). Recently, it has been established that roach (Rutilus rutilus) exposed to treated sewage effluent (that contains complex mixtures of EDCs) in UK rivers, have a reduced reproductive capacity. This, in turn, may have population-level consequences. Evidence for a link between exposure to effluents from kraft mill (BKME) and sewage treatment works (STWs) and altered reproductive function in freshwater fish is compelling. In most cases, however, a causal link between a specific chemical and a physiological effect has not been established. Indeed, identifying specific chemical(s) responsible for adverse effects observed in the wild is difficult, given that tens of thousands of man-made chemicals enter the aquatic environment and that mixtures of chemicals can have combination (e.g., additive) effects. Some EDCs are known to act at a number of different body targets to affect a variety of physiological processes, further complicating the identification of the causative agent(s). Endocrine disruption appears to be particularly widespread in freshwater fish populations. There is little evidence, however, to suggest fish are more susceptible to EDCs relative to other wildlife. Notwithstanding this, there are some features of the endocrine physiology of fish that may be particularly susceptible to the effects of EDCs, including the processes of sex-determination and smoltification (in salmonids). Furthermore, their aquatic existence means that fish can be bathed constantly in a solution containing pollutants. In addition, uptake of chemicals readily occurs via the gills and skin, as well as via the diet (the major exposure route for most EDCs in terrestrial animals). The exposure of fish early life stages to the cocktail of EDCs present in some aquatic environments may be of particular concern, given that this is an especially vulnerable period in their development. The challenge, from the point of view of ecological risk assessment, is to determine effects of EDCs on freshwater fish populations and freshwater ecosystems. In order to meet this challenge, high-quality data are required on the population biology of freshwater fish, on the effects of EDCs on their various life history characteristics, and comprehensive and appropriate population models. Basic information on the population biology of most species of wild freshwater fish is, however, extremely limited, and needs significant improvement for use in deriving a sound understanding of how EDCs affect fish population sustainability. Notwithstanding this, we need to start to undertake possible/probable predictions of population level effects of EDCs using data derived from the effects found in individual fish. Furthermore, information on the geographical extent of endocrine disruption in freshwater fish is vital for understanding the impact of EDCs in fish populations. This can be derived using published statistical associations between endocrine disruption in individual fish and pollutant concentration in receiving waters. Simplistic population models, based on the effects of EDCs on the reproductive success of individual fish can also used to model the likely population responses to EDCs. Wherever there is sufficient evidence for endocrine disruption in freshwater fish and the need for remediation has been established, then there is a need to focus on how these problems can be alleviated. Where industrial chemicals are identified as causative agents, a practical program of tighter regulation for their discharge and/or a switch to alternative chemicals (which do not act as EDCs) is needed. There are recent examples where such strategies have been adopted, and these have been successful in reducing the impacts of EDCs from point source discharges on freshwater fish. Where EDCs are of natural origin (e.g., sex steroid hormones from human and animal waste), however, remediation is a more difficult task. Regulation of the release of these chemicals can probably be achieved only by improvements in treatment processes and/or the implementation of systems that specifically remove and degrade them before their discharge into the aquatic environment.

A model of fish preference and mortality under hypoxic water in the coastal environment

Eutrophication and associated occurrence of hypoxic condition could cause significant damage to marine ecosystems, resulting in considerable economic losses to fisheries and aquaculture and is a major source of stress that fish often have to contend with in order to survive. This problem is likely to be exacerbated in the coming years, since the wastewater treatment facilities is unlikely to catch up with increasing human activities. Moreover, large-scale reclamation projects in coastal areas have recently been increased, and these activities certainly have adverse impacts on water quality and fisheries resources. Coastal construction has a significant role in the development of hypoxic water by changing the current and mixing pattern of water. Changes in species composition and decreases in species richness and diversity have been well documented in hypoxic systems. Hypoxia could cause endocrine disruption in fish and eliminate populations of sensitive species. Shallow coastal areas are of great importance for the special nursery of fish and shellfish and land reclamation in these areas cause strong damage to fisheries. Although the tolerance of aquatic life to hypoxia is known, there is no information about the mortality of fish caused by hypoxia because fish can swim around it and no modeling study has yet been carried out. Criteria that influence the movement of fish are: amounts of food, water temperature and depth, dissolved oxygen concentration and nature of seabed. However, among these, water temperature and dissolved oxygen are the most crucial parameters that affect survival, movement and growth of fish. In this paper, a model of fish preference and mortality for environmental conditions was developed and applied to the Hakata Bay where hypoxic water occurs every summer. For the purpose of this study, a field survey of fish behavior under hypoxic water was conducted by releasing marbled sale (Pleuronectes yokohamae) in the inner bay. Moreover, a series of preference tests for DO, salinity and temperature in the laboratory were conducted in order to decide preference parameters of fish. Using the results of both field and laboratory studies, a sub-module of fish preference and mortality was coded within an integrated hydrothermal and eutrophication model (CHEM) to predict the behavior and mortality of marbled sale when hypoxia would occur in the bay. The model could reasonably simulate the behavior of the fish under hypoxia. An assessment of the impact of the ongoing land reclamation project of about 401 ha in the Wajiro tidal flat zones at the head of the bay on the fisheries resources was also conducted using the model. The results showed that the artificial land lowered the mortality rate of fish under hypoxic condition in the bay during the summer period.

Sequence analysis of choriogenin H gene of medaka (Oryzias latipes) and mRNA expression

Zona radiata proteins of medaka (Oryzias latipes) consist of two major subunit groups, ZI-1,2 and ZI-3. The precursor of ZI-1,2, which is a glycoprotein with a molecular weight of 74,000 to 76,000 Da, is called choriogenin H, and the precursor of ZI-3, with a molecular weight of 49,000 Da, is called choriogenin L. The precursor proteins are synthesized in the liver in response to estrogen in sexually mature female medaka. However, they are also induced in the male medaka when fish are exposed to estrogenic chemicals. Therefore, choriogenin is known as a possible sensitive biomarker for endocrine disruption in fish. In this study, the choriogenin H cDNA sequence was reanalyzed and the genomic DNA sequence was newly analyzed. This was done for the selection of proper reverse transcription-polymerase chain reaction (RT-PCR) primers to measure the choriogenin mRNA induction by estrogenic chemicals. In the results, the full cDNA sequence was found to be 2,109 bp long and the size of the open reading frame (ORF) was found to be a total of 1,998 bp encoding 666 predicted amino acids, which was found to be different from previously reported cDNA sequence of medaka choriogenin. In the choriogenin H cDNA, a repetitive domain and a nonrepetitive domain were shown. Regarding the repetitive domain, seven complete repeats of the 45 bp of 5'-ccc cag tac cca tca aag cct cag ccc cct cag aat cct cag gtc-3' encoding PQYPSKPQPPQNPQV were found. Medaka choriogenin H gene was found to possess seven exons and six introns, and the total length was 2,643 bp long. The seven repetitive nucleotide residues described above existed in exon 1, which was found to be 1,033 bp long. Based on the full sequence information, proper primers were synthesized for RT-PCR to detect choriogenin H mRNA induction in male and juvenile medaka by 17alpha-ethinylestradiol, and this type of measurement system was found to be effective as a simple tool for the screening of endocrine-disrupting chemicals.

Choriogenin mRNA induction in male medaka, Oryzias latipes as a biomarker of endocrine disruption

The fish egg envelope has been known to consist of two distinct layers. The outer layer is thin and is formed around the oocytes during the later stage of previtellogenic development. The inner layer, called zona radiata, is thicker than outer layer and occupies most of the egg envelope. Zona radiata proteins of medaka ( Oryzias latipes) consist of two major subunit groups, ZI-1,2 and ZI-3. The precursors of ZI-1,2 and ZI-3 have been named choriogenin H and choriogenin L, respectively. They are synthesized in the liver in response to estrogen, and then released into the blood stream and incorporated into the zona radiata in sexually matured female medaka. However, choriogenin is also induced in male medaka when the fish are exposed to estrogenic chemicals. Choriogenin can be suggested as a possible biomarker of endocrine disruption in fish, however, relatively little has been known about chorogenin gene expression by estrogenic chemicals. In this study, full sequence of choriogenin L DNA was identified, and measurement of choriogenin mRNA induction was established in medaka by use of RT-PCR technique. Also estrogenic effects of industrial chemicals were evaluated by this tool. When mature male medaka was treated with bisphenol A, nonylphenol, and 17α-ethinylestradiol for 6 days, respectively, the expression levels of choriogenin L and H mRNA were elevated in treated groups. The induction of choriogenin subunits expression by these chemicals showed a dose-dependent pattern and choriogenin L was found to be more sensitive than choriogenin H.

Skewed embryonic sex ratios in a viviparous fish: a result of endocrine disruption?

Sex ratios in catches of wild adult fish are often affected by sex related differences in growth, behaviour and survival. Therefore, such sex ratios may not reflect the relative number of males and females that are recruited to the population. However, this can be circumvented by studying unborn fish embryos. We have found skewed sex ratios among embryos from the viviparous eelpout ( Zoarces viviparus) which could indicate exposure to endocrine disrupting chemicals. The sex ratio was close to 50/50 at two presumably clean sites from Kattegat/Skagerrak and two from the Baltic Sea. On the Swedish Baltic coast near a large pulp mill the relative number of female embryos was significantly lower (42%; P=0.006) but approached 50% further south in the discharge gradient. Treatment of females during early pregnancy with methyltestosterone inhibited oocyte development in all embryos, and instead testis-like tissue was formed. Thus, masculinization found in the field could be caused by exposure to androgen mimics or substances interfering with steroid-synthesis, activity or excretion. This may arise from exposure to the pulp mill effluents, since endocrine disruption in fish has been reported earlier near North American mills. Irrespectively of the cause, reduced numbers of female offspring may have negative impacts on the recruitment capacity of a population.

Endocrine Disruption in Fish. David E. Kime

Endocrine Disruption in Fish. David E. Kime

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 nonexist-ing. 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 contami nated 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.

Gamete viability as an indicator of reproductive endocrine disruption in fish

Xenobiotic pollutants may disrupt reproductive endocrine function by acting at the hypothalamus, pituitary, gonad or liver. Disruption at any of these sites may result in either changes in the rate of gonadal development or in the viability of the gametes. Production of high quality gametes is dependent upon the correct hormonal milieu throughout their development, and any disruption of hormonal balance may result in abnormal sperm or eggs. Such effects may be apparent at levels of pollutant lower than that which causes arrest of gametogenesis or decreased gonadosomatic index. In this paper the use of sperm motility, morphology and fertilising ability in males, and egg numbers, size and hatching success in females as monitors of endocrine disruption will be discussed. Data are presented to show that exposure of adult zebra danios ( Danio rerio) to 5 ng l −1 ethynylestradiol leads to arrest in development of the eggs produced at the early blastula stage and induces vitellogenesis in exposed males.

Feminisation of young males of the common carp, Cyprinus carpio, exposed to 4- tert-pentylphenol during sexual differentiation

In recent years, a hierarchy of techniques has become available for detecting chemicals which may cause endocrine disruption in the aquatic environment. The molecular structure of a chemical provides a first indication about estrogenic activity, i.e. their likelihood of interfering with the female hormone receptor. In vitro competitive binding assays for this receptor and specific cell cultures are also used to demonstrate an estrogenic response, but this does not adequately indicate whether the substance will cause adverse reproductive effects in an entire organism. An elevated level of vitellogenin, a typical female lipoprotein in the plasma of male fish is an in vivo estrogen-mediated response. However, its direct relationship to reproductive developmental effects is as yet unclear. The present study aims at investigating this relationship for assessing endocrine disruption in fish exposed to an estrogenic substance during relevant life stages. A monosex population of male carp, Cyprinus carpio, was exposed to 4-tert-pentylphenol (TPP) and to 17 β-estradiol as a positive control during the period of sexual differentiation, starting at 50 days post hatch. The fish were sampled every 10 days for the histological examination of the development of the testes, i.e. the formation of the reproductive tract, the multiplication and subsequent meiosis of the primordial germ cells, and gametogenesis in the early gonad. At the end of the experiment, blood was extracted for the quantification of vitellogenin by radioimmunoassays in the plasma. The average number of primordial germ cells (PGCs) per gonadal section was significantly reduced ( P<0.001) at all sampling periods in the testes of carp exposed to 17 β-estradiol (positive control). The PGCs had commenced oogenesis after 50 days exposure and developed into oocytes as in a `normal' ovary. The number of PGCs in the testes of the carp exposed to TPP was reduced in a dose dependent manner. Spermatogenesis was severely inhibited in the testes of the TPP-exposed carp, where, few oocytes were ever observed. 20 days exposure to 17 β-estradiol were sufficient to induce the formation of an oviduct instead of a male vas deferens, whereas this occurred after 30 days in TPP-exposed carp. A dose effect relationship with TPP was established and the EC 50 calculated (63 μg TPP l −1). Vitellogenin levels were significantly elevated in the plasma of the carp exposed to both 17 β-estradiol concentrations, but only at the highest 4-tert-pentylphenol concentration (256 μg TPP l −1, P<0.05). At the end of the exposure, the no observed effect concentration for the oviduct formation was <36 μg TPP l −1 and that for increased levels of vitellogenin was between 90 and 256 μg TPP l −1.

Vitellogenin – a biomarker for endocrine disruptors

This article describes the use of vitellogenin as a biomarker for endocrine disruption in fish. The lowest effective concentrations of vitellogenin are reported for oestrogenic compounds like 17β-oestradiol, nonylphenol and bisphenol A. Comparative studies of oestrogens detected in sewage plants from Germany and the UK are discussed. The article points out future directions in the application of vitellogenin ELISA assays and how these assays can provide additional information for a better understanding of the environmental behaviour of pollutants.