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First report of intersex roach residing in Irish rivers downstream of several wastewater treatment plants
Abstract
Endocrine disrupting chemicals (EDCs) are chemicals that have the ability to mimic or disrupt the endocrine system in wildlife. Many of these EDCs are present in treated Irish sewage effluents and landfill leachate and have recently been identified in Irish rivers. The effects of exposure to EDCs were investigated in Irish brown trout (Salmo trutta), sampled from selected Irish rivers downstream of Irish wastewater treatment plants (WWTPs) with control fish sampled upstream of the same WWTP. The effects were also investigated in Irish roach (Rutilus rutilus) sampled downstream of Irish WWTPs. Control roach were sampled from a pristine salmon fishery river (River Deel) located in the west of Ireland. Histopathological and macroscopic analyses of downstream trout showed no adverse reproductive effects, compared to control fish sampled upstream of the WWTP. Statistically significant (p <0.05) populations of wild roach from three sampled Shannon basin rivers were, however, observed to be phenotypically intersex (ovo-testis) when compared to the control river. This, it is hypothesised, is as a direct result of exposure to WWTP effluents. The results reported herein are the first observations of intersex fish in Irish rivers.
... been the case of some marine gastropod molluscs, whereby male characteristics (penis and vas deferens) were superimposed onto females in response to TBT exposure derived from antifouling paints which, in severe cases in some species resulted in sterility and population decline ( Barroso et al., 2000;Bauer et al., 1995Bauer et al., , 1997Bryan et al., 1993Bryan et al., , 1986bBryan et al., , 1987Gibbs et al., 1991aGibbs et al., , 1987Gibbs et al., , 1988Gibbs et al., , 1991bOehlmann et al., 1998). The opposite response, feminisation of male individuals, has been most widely reported in freshwater fish exposed to EDCs derived from sewage treatment works and other sources (Hinck et al., 2009;Jobling et al., 1998;McGee et al., 2012;Pollock et al., 2010;Sanchez et al., 2011;Tetreault et al., 2011;Vajda et al., 2011). However, the physical manifestation of intersex (feminization) may take several forms including production of the egg-yolk precursor protein vitellogenin ( Arukwe et al., 2000;Harries et al., 1997;Kime et al., 1999;Leonardi et al., 2010;Matozzo et al., 2008;Routledge et al., 1998); formation of female-like ducts ( Jobling et al., 2002;Liney et al., 2005;Nolan et al., 2001); and the occurrence of oocytes in testicular tissue (ovotestis) ( Bateman et al., 2004;Chesman and Langston, 2006;Langston et al., 2007;Liney et al., 2005;Matthiessen et al., 2002;Nadzialek et al., 2010;Stentiford and Feist, 2005;Stentiford et al., 2003). ...
... In gonochoristic (dioecious) organisms, intersex is therefore defined as the abnormal condition of being intermediate between male and female sexes. In recent decades the phenomenon of intersex (measured by the manifestations described above) has been used as a biological indicator of the impact of endocrine disrupting chemicals (EDC) in aquatic environments ( Bateman et al., 2004;Chesman and Langston, 2006;Ford et al., 2004Ford et al., , 2007Hashimoto et al., 2000;Jobling et al., 2002;Kirby et al., 2004;McGee et al., 2012;Moore and Stevenson, 1991;Nolan et al., 2001;Pollock et al., 2010;Stentiford and Feist, 2005). Arguably however, it is the simply determined endpoints, such as ovotestis or imposex, that are the most valuable and cost-effective to apply within large-scale assessments over a broad geographical area ( Gibbs and Bryan, 1996;Gibbs et al., 1991aGibbs et al., , 1987Langston et al., 2007;Oehlmann et al., 1996). ...
Estuarine clams Scrobicularia plana were sampled from 108 intertidal locations around the English Channel and adjacent areas. Although S. plana is believed to be a strict gonochorist, 58% of the populations sampled included intersexed individuals (described as male clams exhibiting ovotestis). Over the entire region, on average, 8.6% of male clams exhibited intersex, although proportions of affected males ranged from 0% to 53% depending on location. The severity of intersex was assessed using a simple classification scale, with the majority of individuals showing low levels of impact. Sex ratios were significantly skewed at some sites. There were no significant relationships between incidence or severity of intersex; or with size or parasitism of individual clams. Intersex in S. plana is a useful tool to assess endocrine disruptive effects in estuaries, although mechanisms of impact and causative agents remain uncertain.
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... Since the first report on intersex in fish (Goldschmidt, 1931) there has been a dramatic increase over the past eight decades in reports of intersex in fish. Many of the intersex incidences are correlated with different anthropogenic sources of pollutants, especially those chemicals that have an estrogenic mode of action (Dutney et al., 2017;Hinck et al., 2009;Jobling et al., 2002;McGee et al., 2012;Miller et al., 2012). There is only a limited evidence of anthropogenic impact in causing intersex in marine fishes. ...
The present study reports the first incidence of intersex in Cobia, Rachycentron canadum maintained at sea cages in the Gulf of Mannar, India. During the 10 year study period (2010–2019), at total of 815 broodstock (386 wild captured and 429 captive raised) were maintained under captivity in sea cages in the Gulf of Mannar, India for the purpose of seed production. Of which, 318 male and 252 female broods’ gonads were examined post-mortem following a natural death. Only one male fish was found with a visible gonadal anomaly. Microscopic analysis of gonad revealed the presence of both the characteristic female and male tissues in testes of a single specimen (testis-ova). Only the abnormal nodular portion contained primary oocytes, whereas the central testicular tissue contained spermatocytes and spermatids in the seminiferous lobules. This is the first reported intersex of cobia from the Gulf of Mannar, India. It could be interpreted as low-level rare incidence of natural aberration.
... Synthetic chemicals enter freshwater ecosystems directly from domestic and industrial effluents (Petrovic et al., 2002) and from diffuse agricultural runoff (Schulz, 2004;Wauchope, 1978). Of particular environmental concern are endocrine active chemicals (EACs) which have been reported to cause adverse effects on fish and their populations (Tyler and Jobling, 2008;Kidd et al., 2007;McGee et al., 2012;Geraudie et al., 2017). EACs have been defined in various ways, but put simply they may alter the synthesis, metabolism or function of natural endogenous hormones, causing physiological stress to individuals and subsequent changes in life-cycle processes (Calow, 1991;Beyers et al., 1999). ...
... 4-Nonylphenol was found at these locations 100 and 10 fold higher than levels required to elicit a response in the E-SCREEN and MVLN respectively. Intersex in populations of wild roach from rivers in the Shannon basin of Ireland have also been reported for the first time and it is hypothesised that the incidence of ovo-testis is a direct result of exposure to EDCs in waste water effluents [53]. ...
A diverse range of endocrine disrupting chemicals (EDCs) was examined, using an in vitro test system, for critical events required for the onset of carcinogenesis in vivo. The initiation stage of carcinogenesis is a genotoxic process. 4-Octylphenol (alkylphenol), bisphenol A (plasticiser), coumestrol and genistein (phytoestrogens), 2,4-dichlorophe-noxyacetic acid and toxaphene (pesticides) and ethinylestradiol (synthetic hormone) were investigated for potential mutagencicity, DNA strand breakage, clastogenicity and DNA repair. Significant induction in the percentage of cells containing micronuclei was observed for all the EDCs. Toxaphene and coumestrol were mutagenic in the Ames assay. They also induced significant levels of unscheduled DNA synthesis and DNA strand breakage. Bisphenol A induced low level DNA strand breakage in HepG2 cells in the comet assay. The EDCs, with the exception of toxaphene, in-duced transcriptional activation in the yeast estrogen screen (YES) assay. They were potently oestrogenic in the mam-malian based MVLN (transactivation) and E-SCREEN (proliferation) assays. This report on the transactivational, pro-liferative and genotoxic ability of the EDCs suggests that these chemicals may play a role in the etiology of male and female reproductive cancers.
... To date, the focus of understanding effects of anthropogenic chemicals has mainly been on identifying responses inside aquatic vertebrates. Accordingly, numerous laboratory and field studies have documented the endocrine disrupting effects of EDCs in fish (Crago et al., 2011;Jobling et al., 2003;Lange et al., 2011;McGee et al., 2012;Nash et al., 2004;Tyler and Jobling, 2008), which in severe cases may result in individual fish carrying both male and female reproductive organs Katsu et al., 2007;Kidd et al., 2007). However, negative effects of EDCs have also been documented in natural populations of mollusks (Giusti and Joaquim-Justo, 2013). ...
... Roach are a species that has been widely studied for intersex in Europe, i.e. England ( Jobling et al., 1998Jobling et al., , 2006Minier et al., 2000;Nolan et al., 2001), France ( Maltret-Geraudie et al., 2008;Minier et al., 2000), Sweden ( Andersen et al., 2001), Denmark ( Bjerregaarda et al., 2006) and Ireland ( McGee et al., 2012). This species was specifically studied at a reference site in Normandy (France) that was thought to be free of anthropogenic influences (industrial, agricultural or municipal effluents). ...
During recent decades, survey studies have documented the widespread presence of oocytes in the testes of male Smallmouth Bass Micropterus dolomieu collected from surface waters throughout the United States. There are few published reports of testicular oocytes (TO) in Smallmouth Bass before the 1990s, so it is difficult to know how long this has been occurring. Consequently, this study was conducted to evaluate the prevalence and severity of TO occurrence in whole fish specimens from two archival collections-the Smithsonian Institution's National Museum of Natural History in Suitland, Maryland, and Cornell University's Museum of Vertebrates in Ithaca, New York. Gonads were excised from 167 preserved male Smallmouth Bass that were originally collected between 1875 and 2004, and routine histologic sections were prepared and examined. The severity of TO was determined using a semiquantitative scoring system. Overall, 52.1% of male Smallmouth Bass were found to have TO. Affected fish had been collected in 11 of the 18 represented states, and TO were found in specimens harvested during decades as early as the 1880s and 1900s. Unfortunately, the small number of samples acquired at the earliest time periods precluded analyses of prevalence and severity trends over time. The results of this study demonstrated that the phenomenon of TO in male Smallmouth Bass is at least a century old and confirmed the widespread nature of this finding throughout the species' historic range. Further research efforts should focus on determining the baseline prevalence of TO in laboratory-reared male Smallmouth Bass that have not been exposed to endocrine active substances or the effects of experimental estrogen exposure on such fish.
A group of potential emerging contaminants in two Irish drinking water supplies were investigated. The aim was to investigate the presence of emerging contaminants which are not currently routinely monitored or regulated as part of the European Communities Drinking Water Regulations SI 278 of 2007 but are known or anticipated to occur in drinking water supplies. A monthly sampling and analysis programme was carried out to examine the presence of seven groups of potential emerging contaminants in two Irish drinking water supplies. The seven groups selected were: herbicides, molluscides, endocrine disrupters, perfluorinated chemicals, disinfection by-products, personal care products, and heavy metals. The investigation showed that the majority of the seven selected groups of contaminants were not detected at either drinking water site. Results from the first site (water treatment plant (WTP) 1) showed elevated levels of musk xylenes, a member of the personal care products suite of tests. The specific compound detected was galaxolide, a polycyclic musk found in perfumes, soaps, cosmetics and detergents. Results from the second site (WTP 2) showed elevated levels of oestrone, a member of endocrine disrupter steroid suite of tests. Oestrone is one of three types of oestrogen made by the body and is produced by the ovaries as well as by adipose tissue and the adrenal glands. Results from both sites showed that of the seven groups of contaminants chosen for examination most were not detected. The musk xylene compound galaxolide was detected on one occasion at a level just above the guideline limit and oestrone a component of the endocrine disrupting chemicals steroid suite of tests was found on two occasions throughout the study.
Given the impact of endocrine disrupting compounds (EDCs) on fish, both at individual and population level, it may be expected that these substances impede the achievement of international objectives aiming at restoring and securing the quality of the environment, nature and biodiversity. More specifically, the quality objectives for natural habitats and species as defined under the Habitats Directive (HD), and the targets of ecological and chemical quality of our water ecosystems as defined in the Water Framework Directive
(WFD) are at stake. This report analyses through a study of literature, how endocrine
disruption in fish can impact the achievement of these international targets.
The first chapter includes a brief summary on hormonal disruption in fish, while Chapter 2
provides an overview of the research thus far conducted in Belgium. Chapters 3 and 4 deal with the potential impact of the occurrence of endocrine disruptors on reaching the targets of the Habitats Directive and the Water Framework Directive respectively.
Many field studies focusing on a broad variety of species demonstrate the presence of feminized fish as a result of the release of EDC’s in the aquatic environment, and suggest
that in hormonally disturbed fish, reproductive capacity is reduced.
In Flanders (northern Belgium) measurements of estrogenic effects in surface water were
positive in all assessed sites. Despite the rather poor state of the Flemish water quality, and the clear impact of EDCs on fish stocks and the ecological quality of surface water, only very limited studies on endocrine disruption in Flandrian freshwater fishes have been undertaken. Preliminary studies have shown that substances with estrogenic activity also cause certain effects on fish populations, e.g. male roach shows signs of feminization (presence of oocytes - increased vitellogenin (VTG) concentrations) in 50% of the cases.
Studies aiming at predicting the potential impact of endocrine disruptors on species selected under the HD lack for Flanders. However, some international papers document EDC impact on a limited number of HD species. Species-related differences in distribution, reproduction, migration, and trophic ecology are supposed to greatly influence sensitivity of HD species to endocrine disruptors. The reports showed for some of the HD species an adverse impact of EDCs on individual or population level. It may be expected that these compounds will have a major impact on the status and possibilities for restoration of the Flemish HD species, given the high concentrations of substances with estrogenic activity in Flanders compared with other European countries.
Exploratory research on a broad set of sites has shown significant inverse correlation between the levels of certain substances, some of which known as EDCs, and the indices used in reporting the ecological status of the WFD. Another study on a more limited set of sites showed a significant negative correlation between biomarkers for estrogenicity (measured as estrogenicity of the water and VTG levels in male roach) and indicators for the integrity of aquatic communities (BBI Belgian Biotic Index and IBI Index of Biotic Integrity). The higher the estrogenic activity, the lower the environmental quality seems to be. Given these findings and other research showing that EDCs (and by extension, other toxic substances) generate negative effects on fish population and community level, we can conclude that hormonal disturbance is one of the many anthropogenic pressures with a significant impact on achieving the objectives of the WFD.
The report concludes with recommendations for further research to better understand the
impact of endocrine disruptors on the achievement of the objectives of the HD and the WFD in Flanders.
Feminization of the male roach, Rutilus rutilus, a freshwater, group-spawning fish, is widespread in English rivers; among the causative agents are natural and synthetic
steroidal estrogens and chemicals that mimic estrogens. In feminized male roach, concentrations of the egg-yolk protein vitellogenin
are elevated, sex steroid hormone dynamics are altered, and gonad development is disrupted (most notably, a female reproductive
duct or developing eggs [oocytes] are present in the testis). In some English rivers containing high levels of estrogens,
all male roach sampled have been feminized to varying degrees. In the more severely affected males, individuals produce low-quality
sperm with a reduced capability for fertilization. Laboratory studies have shown that the environmental estrogens responsible
for inducing gonadal feminization in roach can also alter reproductive behavior, disrupting normal breeding dynamics (parentage)
in the zebrafish, another group-spawning fish. Together these findings indicate that feminization of wild roach may result
in adverse population-level effects, but this hypothesis has yet to be fully addressed.
Surveys of U.K. rivers have shown a high incidence of sexual disruption in populations of wild roach (Rutilus rutilus) living downstream from wastewater treatment works (WwTW), and the degree of intersex (gonads containing both male and female structural characteristics) has been correlated with the concentration of effluent in those rivers. In this study, we investigated feminized responses to two estrogenic WwTWs in roach exposed for periods during life stages of germ cell division (early life and the postspawning period). Roach were exposed as embryos from fertilization up to 300 days posthatch (dph; to include the period of gonadal sex differentiation) or as postspawning adult males, and including fish that had received previous estrogen exposure, for either 60 or 120 days when the annual event of germ cell proliferation occurs. Both effluents induced vitellogenin synthesis in both life stages studied, and the magnitude of the vitellogenic responses paralleled the effluent content of steroid estrogens. Feminization of the reproductive ducts occurred in male fish in a concentration-dependent manner when the exposure occurred during early life, but we found no effects on the reproductive ducts in adult males. Depuration studies (maintenance of fish in clean water after exposure to WwTW effluent) confirmed that the feminization of the reproductive duct was permanent. We found no evidence of ovotestis development in fish that had no previous estrogen exposure for any of the treatments. In wild adult roach that had previously received exposure to estrogen and were intersex, the degree of intersex increased during the study period, but this was not related to the immediate effluent exposure, suggesting a previously determined programming of ovotestis formation.
Tens of thousands of man-made chemicals are in regular use and discharged into the environment. Many of them are known to interfere with the hormonal systems in humans and wildlife. Given the complexity of endocrine systems, there are many ways in which endocrine-disrupting chemicals (EDCs) can affect the body's signaling system, and this makes unraveling the mechanisms of action of these chemicals difficult. A major concern is that some of these EDCs appear to be biologically active at extremely low concentrations. There is growing evidence to indicate that the guiding principle of traditional toxicology that "the dose makes the poison" may not always be the case because some EDCs do not induce the classical dose-response relationships. The European Union project COMPRENDO (Comparative Research on Endocrine Disrupters--Phylogenetic Approach and Common Principles focussing on Androgenic/Antiandrogenic Compounds) therefore aims to develop an understanding of potential health problems posed by androgenic and antiandrogenic compounds (AACs) to wildlife and humans by focusing on the commonalities and differences in responses to AACs across the animal kingdom (from invertebrates to vertebrates) .
Japanese medaka were exposed to environmentally-relevant concentrations of environmental estrogens: nonylphenol (NP, 0.5, 0.8 and 1.9 μg l−1), methoxychlor (MXC, 0.2, 0.6 and 2.3 μg l−1) and estradiol (E2, 0.01, 0.12 and 1.66 μg l−1). Exposure occurred throughout the first month following hatch. E2 survival ratios following the exposure period were significantly altered compared to control groups. Following a month period of `growout' in dilution water only, sex ratios were measured and reproductive capabilities assessed. No alteration in sex ratios was observed following treatment with NP or MXC. All three concentrations of E2 were sufficient to produce exclusively female populations. There was no depreciation in reproductive capability in the NP or MXC-treated fish as measured by fecundity, viability of eggs, or hatchability of eggs. E2-treated female fish had a lower fecundity in the highest concentration.
To determine the levels of known endocrine disrupting chemicals such as phthalates and alkylphenols in environmental samples such as leachate water and sediment, suitable isocratic high performance liquid chromatographic (HPLC) methods utilising a narrow bore column were developed. The study was an analytical challenge in terms of developing a method, which would be sensitive enough to detect trace levels of these compounds, while still retaining the advantages of being suitable for relatively inexpensive instrumentation and featuring reasonable throughput. Generally speaking, when the internal diameter of the HPLC column is decreased by a factor of two, the signal of a sample component increases by a factor of four, the square of the change in diameter. However, combining a narrower bore column along with the isocratic method enabled us to see 20-fold increases in peak signal. The detection of these compounds was further improved by using pre-concentrating techniques known as solid-phase extraction (SPE) and accelerated solvent extraction (ASE). Limits of detection in the ng L−1 range were reached for target analytes. Maximum values (µg L−1) of 7.05 dibutyl phthalate (DBP), 7.37 diethylhexyl phthalate (DEHP), 5.56 diisononyl phthalate (DINP), 1.19 diisodecyl phthalate (DIDP) and 6.16 4-nonylphenol (NP) were found in sampled leachates, whilst maximum values (mg kg−1) of 42.3 DBP, 49.8 DEHP, 36.2 DINP, 20.0 DIDP and 1.14 NP were found in sediments. Concentrated leachate contained up to 226 µg L−1 DEHP. The highest levels of NP were found to leach from an unlined landfill, with concentrations of 10.6 µg L−1. The levels, which were quantified in the Irish midlands, are clearly linked to anthropogenic activity and were comparable to levels found in other pan-European studies.
This paper is an illustrative guide to intersex in the roach Rutilus rutilus, based on 150 intersex individuals. Most intersex roach had female germ cells, or oocytes, within a predominantly male gonad (testis), and/or malformed/intersex reproductive ducts. The number, pattern and developmental stage of oocytes within testicular tissue in intersex fish varied greatly. In most intersex fish, a few primary oocytes, or numerous primary and secondary oocytes, were scattered randomly throughout the testicular tissue (multifocal intersex). In other, more severely feminized individuals, large areas of ovarian tissue were separated clearly from testicular tissue (focal intersex). Almost all intersex individuals had a female-like reproductive duct (ovarian cavity). In mild cases of intersex (in which the majority of the germ cells were male) the ovarian cavity was present together with the male sperm duct/vas deferens, whilst in certain severe cases, the sperm duct was absent or vestigial.
In 1993 TBT contamination in areas of aquaculture and small boat activity about Ireland was found to have improved significantly since 1987, but in areas of shipping, contamination remained. Cork Harbour, a sea inlet with heavy shipping traffic, was examined in 1994 to determine the extent of contamination, using imposex and intersex in two prosobranch snails. Both Nucella lapillus (dogwhelk) and Littorina littorea (periwinkle) provided data on the varying levels of TBT contamination throughout Cork Harbour. N. lapillus is particularly sensitive to TBT and has become extinct in some areas of the harbour since 1968. Littorina littorea tolerates lower salinities, is less affected by TBT, and so can be used to monitor in more brackish and heavily contaminated areas. Intersex is used for the first time to map areas of TBT contamination. Shell thickness in the Pacific oyster, Crassostrea gigas, from 1985–1987 and 1994 was also compared. All three indicator species are consistent in terms of the pattern of TBT contamination in Cork Harbour. TBT contamination in the western side is greater than in the eastern side of the harbour, reflecting the inputs. Contamination levels generally decline with increased distance from the harbour.
The pattern of spermatogenesis was studied during an annual reprodutive cycle in adult male brown trout. The part of the testicular volume occupied by the nuclei of germ cells and of Sertoli cells, the lobular lumen and the extra-lobular space were measured. The number of germ cells was estimated by the total volume occupied by each cell divided by the individual volume of each cell. The permanent number of spermatogonia A (spermatogonia isolated or in groups of 2) was about 50 million. Each GA cell produced several spermatogenetic waves during the same reprodutive cycle. Sperm production was about 25×109/g of testis/year, or about 6.8 × 107/g of body weight/year.
Males stocked separately from females, thus presumably not releasing spermatozoa, were killed in December, January and February. Since these fish contained the same amount of spermatozoa it appears that the process of spermiation initiated in December did not continue in January and February. During this period, some spermatozoa were phagocytized by macrophages (spermatophages) in the lobular lumen and by Sertoli cells.
Chronic changes were measured in fish downstream from a pulp and paper mill located in the Humber Arm, Newfoundland, Canada. Cunner (Tautogolabrus adspersus) downstream of the mill were longer and heavier at an earlier age than reference fish. Condition factor was similar among sites. Hepatosomatic indices and ethoxyresorufin-O-deethylase activity were higher in fish near the mill. Gondal condition suggested that gametogenesis might have been delayed in these same fish. Although splenosomatic indices were greater in exposed fish, splenic hemosiderin was not significantly different from reference values. Gill and liver changes in exposed cunner were significant. Gill parasites showed no site difference; however, the prevalence and abundance of gastrointestinal acanthocephalans were significantly greater in fish from impact sites. These results suggest that effluent from this mill may be responsible for observed biological effects; however, further study is needed to determine whether gradients in nutrient loading and untreated sewage discharge may also contribute to fish health in the Humber Arm.
The synthetic estrogen 17 alpha-ethynylestradiol (EE2) is a commonly used oral contraceptive that has been increasingly detected in sewage effluents. This study determined whether EE2 exposure adversely affected reproduction in sexually maturing male rainbow trout (Oncorhynchus mykiss). We exposed male trout to graded water concentrations of EE2 (10, 100, and 1,000 ng/ L) for 62 d leading up to the time of spawning. Semen and blood plasma samples were removed from each fish. Semen was used to fertilize groups of eggs from one nonexposed female. As a measure of fertility, eggs were incubated for 28 d after fertilization to determine the proportion that attained the eyed stage of embryonic development. Additional endpoints also measured included sperm motility, spermatocrit, gonadosomatic and hepatosomatic indices, testis histology, and circulating plasma levels of the sex steroids 17 alpha, 20 beta-dihydroxyprogesterone (17,20-DHP) and 11-ketotestosterone (11-KT). Exposure to 1,000 ng/L of EE2 caused complete mortality of the treatment group by day 57. Exposure to lower EE2 water concentrations (10 and 100 ng/L) caused an increase in sperm density, while a significant reduction in testis mass was observed only in the 100-ng/L exposure group. Most significantly, semen harvested from fish exposed to 10 and 100 ng/L EE2 caused an approximately 50% reduction in the number of eggs attaining the eyed stage of embryonic development. Plasma levels of 17,20-DHP in exposed fish were roughly twice the level of the controls, while levels of 11-KT were significantly reduced in fish exposed to 100 ng/L EE2. These results suggest that sexually maturing male rainbow trout are susceptible to detrimental reproductive effects of short-term exposures to environmentally relevant levels of EE2.
Municipal effluents have been shown to contain a cocktail of endocrine disrupting chemicals (EDCs). The estrogenic effect of these effluents has been demonstrated on both vertebrate and invertebrate species by the feminisation of the exposed males. This effect was investigated on the freshwater zebra mussel (Dreissena polymorpha) after exposure to tertiary treated effluent from a municipal sewage treatment works (STW). Mussels were exposed to the effluent in situ for 112 days during gametogenesis (December to mid-March). Levels of vitellin (Vn)-like proteins (the major protein found in oocytes) were measured indirectly using the alkali-labile phosphate (ALP) technique and confirmed by gel electrophoresis. Significant increases (P < 0.05) in Vn-like proteins were found in both male and female mussels after exposure to the effluent, indicating that endocrine disruption (ED) had occurred. Using High-performance thin layer chromatography (HPTLC) levels of the mussels main steroid, cholesterol were found to more than double after effluent exposure. General physiological (survival, condition, etc.) and histological effects were also investigated. Histological effects observed included a large increase in interstitial tissue between the seminiferous tubules of the gonad in male mussels exposed to effluent. Effluent samples were tested for estrogenic compounds using the toxicity identification and evaluation method (TIE). A complex mixture of compounds with estrogenic activity was found with 17beta-estradiol, 17alpha-ethynlestradiol and bisphenol A accounting for the majority of the effluents estrogenic activity. Results indicate that the zebra mussel is a suitable bioindicator of endocrine disruption in freshwater environments.
Effluents from wastewater treatment works (WwTWs) contain estrogenic substances that induce feminizing effects in fish, including vitellogenin (VTG) synthesis and gonadal intersex. Fish vary in their responsiveness to estrogenic effluents, but the physiological basis for these differences are not known. In this study, uptake of estrogen from two WwTW effluents (measured in hydrolyzed bile) and estrogenic response (VTG induction) were compared in a salmonid (rainbow trout, Onchorhynchus mykiss) and a cyprinid fish (roach, Rutilus rutilus). Immature rainbow trout were more responsive than maturing roach to the estrogenic effluents. The more potent of the two estrogenic effluents (containing between 24.3 and 104.1 ng estradiol-17beta equivalents/L [E2eq/L]) resulted in a 700-fold and 240-fold induction of plasma VTG in male and female trout, respectively, but only a 4-fold induction in roach (and in males only). The less potent effluent (varying between 4.1 and 6.8 ng E2eq/L) induced VTG in the trout only, with a 4-fold and 18-fold induction in males and females, respectively. In fish exposed to tap water, the estrogenicity of the hydrolyzed bile was 0.03+/-0.01 ng E2eq/microL (for both sexes in trout), 0.18+/-0.04 ng E2eq/microL in male roach, and 0.88+/-0.15 ng E2eq/microL in female roach. The higher bile content of estrogen in control roach reflected their more advanced sexual status (and thus higher endogenous estrogen) compared with the immature female trout. In trout maintained in effluents, the bile content of estrogen was 100-fold and 30-fold higher than controls at WwTW A and B, respectively. Bioconcentration factors (BCFs) for estrogenic activity in bile were between 16 344 and 46 134 in trout and between 3543 and 60 192 in roach (no gender differences were apparent). There were strong correlations between VTG induction and the estrogenic activity of bile extracts for both trout and roach. The results confirm that estrogenic contaminants bioconcentrate to a high degree in fish bile and that the level (and nature) of this accumulation may accountfor responsiveness to the endocrine disruptive effects of estrogenic effluents. Immature fish were the more appropriate life stage for quantifying estrogen exposure and uptake in bile, as they contain little circulating endogenous oestrogen compared with sexual maturing fish. The nature of the estrogenic contaminants is detailed in an accompanying paper.
The triolein-containing semipermeable membrane devices (SPMDs) were deployed for 4 weeks in a sewage treatment plant in Beijing, China, to sample and concentrate priority hydrophobic organic pollutants in a sewage treatment process. The chemical analyses and ecotoxicities of the residuals of SPMDs dialysate were examined. The data from the chemical analyses by gas chromatography-mass spectrometry selected ion monitoring mode indicated the lower removal for polychlorinated biphenyls (PCB) congeners and polycyclic aromatic hydrocarbons (PAHs) coincided with the persistence of them in the environment. The acute toxicity examined by bioluminescence test with Vibrio fischeri revealed approximately only 20% decrease in the overall toxicity of the influent after the activate sludge treatment process. The ethoxy resorufin-O-deethylase (EROD) induction with a micro-EROD assay in vitro using H4-IIE rat hepatoma cell cultures demonstrated the presence of persistent organics in influent and sequency effluents. Results obtained suggested that integration of the SPMD technique and chemical analyses and bioassay might be a valuable approach for the risk assessment of hydrophobic organic pollutants in water ecosystem. It revealed the necessity for organic pollutants monitoring and ecotoxicities examining of sewage treatment plants.
Alterations in development and reproduction as a consequence of exposure to endocrine-disrupting chemicals (EDCs) have been demonstrated in many wildlife species. Animals living in, or closely associated with, the aquatic environment are particularly vulnerable to endocrine disruption because thousands of chemicals are actively disposed into rivers, estuaries and seas. Fish have thus been a focus in endocrine disruption studies, and some of the most comprehensive studies on the disruption of sexual development and function are on the roach (Rutilus rutilus). This paper provides a critical analysis of the roach as a sentinel for studies into endocrine disruption. The paper starts by describing what is known on the basic reproductive biology of the roach, information essential for interpreting chemical effect measures on sexual development and function. We then analyze where and how the roach has been applied to improve our understanding of the estrogenic nature of discharges from wastewater treatment works (WWTWs) and describe the phenomenon of feminized male roach in UK rivers. In this paper, the causation of these effects and issues of relative susceptibility and sensitivity of the roach to the effects of estrogenic EDCs are addressed. The paper then describes the ongoing work on the development of genetic and genomic resources for roach and analyses how these are being applied in studies to understand the mechanisms of disruption of sexual development. Finally, the paper addresses the biological significance of sexual disruption and intersex for the individual and discusses the possible implications for wild populations.
Ovotestis (intersex) and raised plasma vitellogenin in male fish are widely employed biomarkers of estrogen contamination in the aquatic environment. In the present study, these biomarkers were used to determine whether Irish rivers contain estrogenic chemicals at levels capable of affecting the reproductive health and success of exposed fish populations. A number of aquatic ecosystems were investigated (the rivers Liffey, Lee, and Bandon and the Killarney lakes). A survey of male wild brown trout (Salmo trutta) was carried out to assess the incidence of endocrine disruption in the feral fish population; no evidence of intersex was found in any of the wild fish sampled. Raised plasma vitellogenin was detected, however, in the wild brown trout downstream of a major municipal wastewater treatment plant on the river Liffey.