First report of intersex roach residing in Irish rivers downstream of several wastewater treatment plants

Tyler C.R., Jobling S.

Tarrant H., Mousakitis G., Wylde S., Tattersall N., Lyons A., Maloney M., Llewellyn N.

Reid A.M., Brougham C.A., Fogarty A.M., Roche J.J.

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)...

Jobling S., Williams R., Johnson A., Taylor A., Gross-Sorokin M., Nolan M., Tyler C.R., van Aerle R., Santos E., Brighty G.

Steroidal estrogens, originating principally from human excretion, are likely to play a major role in causing widespread endocrine disruption in wild populations of the roach (Rutilus rutilus), a common cyprinid fish, in rivers contaminated by treated sewage effluents. Given the extent of this problem, risk assessment models are needed to predict the location and severity of endocrine disruption in river catchments and to identify areas where regulation of sewage discharges to remove these contaminants is necessary. In this study we attempted to correlate the extent of endocrine disruption in roach in British rivers, with their predicted exposure to steroid estrogens derived from the human population. The predictions of steroid estrogen exposure at each river site were determined by combining the modeled concentrations of the individual steroid estrogens [17beta-estradiol (E2), estrone (E1), and 17alpha-ethinylestradiol (EE2)] in each sewage effluent with their predicted dilution in the immediate receiving water. This model was applied to 45 sites on 39 rivers throughout the United Kingdom. Each site studied was then categorized as either high, medium, or low "risk" on the basis of the assumed additive potency of the three steroid estrogens calculated from data derived from published studies in various cyprinid fish species. We sampled 1,438 wild roach from the predicted high-, medium-, and low-risk river sites and examined them for evidence and severity of endocrine disruption. Both the incidence and the severity of intersex in wild roach were significantly correlated with the predicted concentrations of the natural estrogens (E1 and E2) and the synthetic contraceptive pill estrogen (EE2) present. Predicted steroid estrogen exposure was, however, less well correlated with the plasma vitellogenin concentration measured in the same fish. Moreover, we found no correlation between any of the end points measured in the roach and the proportion of industrial effluents entering the rivers we studied. Overall, our results provide further and substantive evidence to support the hypothesis that steroidal estrogens play a major role in causing intersex in wild freshwater fish in rivers in the United Kingdom and clearly show that the location and severity of these endocrine-disrupting effects can be predicted.

Schulte-Oehlmann U., Albanis T., Allera A., Bachmann J., Berntsson P., Beresford N., Carnevali D.C., Ciceri F., Dagnac T., Falandysz J., Galassi S., Hala D., Janer G., Jeannot R., Jobling S., et. al.

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) .

Liney K.E., Jobling S., Shears J.A., Simpson P., Tyler C.R.

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.

Tyler C.R., Spary C., Gibson R., Santos E.M., Shears J., Hill E.M.

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.

Quinn B., Gagné F., Costello M., McKenzie C., Wilson J., Mothersill C.

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.

Wang C., Wang Y., Kiefer F., Yediler A., Wang Z., Kettrup A.

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.

Schultz I.R., Skillman A., Nicolas J., Cyr D.G., Nagler J.J.

The synthetic estrogen 17α-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 17α, 20β-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.

Billiard S.M., Khan R.A.

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.

Jobling S., Tyler C.R.

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.

Nolan M., Jobling S., Brighty G., Sumpter J.P., Tyler C.R.

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.

Baronti C., Curini R., D'Ascenzo G., Di Corcia A., Gentili A., Samperi R.

Sewage treatment plant (STP) effluents with primarily domestic inputs are strongly suspected to be an important source of natural and synthetic estrogens contaminating the aquatic environment. Even a few ng/L of some of these substances can provoke reproductive disturbances in riverine fish. The main purpose of this investigation has been that of ascertaining whether activated sludge STPs (ASSTPs) are able to produce significant amounts of free estrogens. For this purpose, we have monitored monthly estriol (E3), estradiol (E2), estrone (E1) and ethinylestradiol (EE2) in influents and effluents of six Roman ASSTPs for five months. To do this, we have developed an original analytical method involving analyte extraction with a Carbograph 4 cartridge and LC coupled with negative turbo ion spray tandem mass spectrometry in the selected reaction monitoring mode. Analyte recovery ranged between 86 and 91%, and limits of quantification were below 1 ng/L. Over five months, inlet concentrations of E3, E2, E1 and EE...

Sumpter J.P.

There is now unequivocal evidence that a wide variety of chemicals capable of disrupting the endocrine system are present in the aquatic environment. These range from natural (e.g. 17beta-oestradiol) and synthetic (e.g. ethinyl oestradiol) oestrogens through to industrial chemicals that can mimic endogenous hormones (e.g. nonylphenol, some pesticides). Relatively little is known about the fate and behaviour of these chemicals in the aquatic environment, and hence it is unclear which organisms are exposed to the chemicals, and to what degree. This makes predicting possible effects difficult. Nevertheless, enough examples, such as the masculinisation of female molluscs exposed to the anti-fouling agent TBT (tributyltin), and the feminisation of male fish exposed to oestrogenic chemicals in effluent from sewage-treatment works, are documented to demonstrate that adverse affects are occurring. The challenge now is to assess how wide-ranging these adverse effects are, to determine their severity at the population level, and to gauge how serious an issue endocrine disruption is compared to other factors (such as habitat loss) which are also adversely impacting aquatic organisms.

P R., M. S., G. T., R. J., A.K. A.N., M. S., K.K. A., A. A.M., N. K., T.T. R.

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.

Lange A., Paris J.R., Gharbi K., Cézard T., Miyagawa S., Iguchi T., Studholme D.J., Tyler C.R.

Oestrogenic wastewater treatment works (WwTW) effluents discharged into UK rivers have been shown to affect sexual development, including inducing intersex, in wild roach (Rutilus rutilus). This can result in a reduced breeding capability with potential population level impacts. In the absence of a sex probe for roach it has not been possible to confirm whether intersex fish in the wild arise from genetic males or females, or whether sex reversal occurs in the wild, as this condition can be induced experimentally in controlled exposures to WwTW effluents and a steroidal oestrogen. Using restriction site-associated DNA sequencing (RAD-seq), we identified a candidate for a genetic sex marker and validated this marker as a sex probe through PCR analyses of samples from wild roach populations from nonpolluted rivers. We also applied the sex marker to samples from roach exposed experimentally to oestrogen and oestrogenic effluents to confirm suspected phenotypic sex reversal from males to females in some treatments, and also that sex-reversed males are able to breed as females. We then show, unequivocally, that intersex in wild roach populations results from feminisation of males, but find no strong evidence for complete sex reversal in wild roach at river sites contaminated with oestrogens. The discovered marker has utility for studies in roach on chemical effects, wild stock assessments, and reducing the number of fish used where only one sex is required for experimentation. Furthermore, we show that the marker can be applied nondestructively using a fin clip or skin swab, with animal welfare benefits.

De Waal T., Egan J., Read C., Pender J.

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.

Bahamonde P.A., Munkittrick K.R., Martyniuk C.J.

Intersex is defined as the simultaneous presence of male and female gonadal tissue in a gonochoristic (fixed-sex) species. The intersex condition has been documented in both wild and laboratory animals, including fish, amphibians, and reptiles. In aquatic animals, intersex is often viewed as a signature effect of exposure to endocrine disrupting compounds. At least 37 fish species from 17 families have been identified with intersex gonads in 54 field survey studies. However, reports of the occurrence of intersex at reference sites have led to speculation that a baseline level of intersex is "normal". The objective of this critical review was to assess factors potentially associated with baseline levels of intersex in fish and to examine the mechanisms involved in the intersex condition in order to identify priority research areas. Based on current literature, the relationship between intersex and physiological parameters such as plasma sex steroids and vitellogenin is not well characterized or conclusive. Moreover, the literature is not definitive on whether field studies are distinguishing between natural intersex and intersex due to stressors. High throughput transcriptomics will improve understanding of how intersex condition manifests after exposure to aquatic pollution and it is recommended that studies consider both males with and without intersex that inhabit the same polluted site in order to differentiate pathways associated with xenobiotic responses versus molecular pathways associated with intersex. Other experimental design considerations for field studies examining intersex include data collection on life history (e.g. migratory patterns) and improved reference site characterization.

Quinn-Hosey K.M., Roche J.J., Fogarty A.M., Brougham C.A.