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Feminisation of fish

3. Reproductive disturbances in feral and caged fish from sewage effluent affected aquatic environments

3.1 The freshwater environment

3.1.1 Feral fish

3.1.1.1 Effects in male fish

The current concern about estrogens in the aquatic environment and their endocrine disrupting effects on fish has been raised by observations of feminised male fish made in England during the early and mid 90’ies (1;104;105). These observations supported the estrogen hypothesis put forward in 1993 by Skakkebæk and Sharpe which proposed that exposure of males (animals and humans) in a critical period of their life time to female hormones estrogens or to chemicals acting like estrogens (called xenoestrogens) can disrupt the normal development and impair the normal function of the male reproductive system (106).

Roach (Rutilus rutilus) collected from eight rivers throughout England downstream of discharges from sewage treatment plants (STP) were found to have a high frequency of intersex – an abnormal occurrence of hermaphoditism (1). Intersex was found in 16-100 % of male fish compared to 4-18 % intersex in males from control sites. In four sewage effluent receiving rivers the intersex frequency was above 50 % and in two of these, all examined males were showing feminisation. The intersex condition was characterised by a presence of both early and developed stages of egg cells (oocytes), and the presence of the female duct, the ovarian cavity, in the testes of presumptive male fish. The ovarian cavity is the structure within the ovary to which mature eggs are released before being passed on to the oviduct. The severity of the condition varied among individuals from only a few primary oocytes interspersed among otherwise normal testicular tissue to testes where more than 50 % of the tissue was female. In some cases the male sperm duct was further missing. The study showed a correlation between the degree of intersex and the concentration of sewage effluent in the river indicating that compounds in the sewage effluent were responsible for inducing the intersex condition. Observations of concomitantly high levels of the female egg yolk protein vitellogenin (vitellogenin) in sewage effluent exposed male fish indicated exposure to estrogens and/or xenoestrogens which has been verified by cage experiments and chemical analyses (25;104;107) (see section 3.1.2.1 and chapter 4).

Synthesis of vitellogenin by male or immature fish is an internationally used marker for estrogenic exposure. Vitellogenin is normally produced by female fish during their reproductive cycle. It is induced in the liver by the endogenous estradiol production of the female, excreted to the blood stream and incorporated into the developing oocytes in which it is degraded to yolk proteins. These act as nutrition for the developing larvae (108). Males and immature fish normally contain no or very little vitellogenin in plasma possibly due to very low circulating levels of estradiol. If male fish are exposed to (xeno-)estrogens this will, however, induce the synthesis of vitellogenin which in severe cases can reach levels as high as or higher than found in sexually mature females.

A second species of cyprinid fish, the gudgeon (Gobio gocio) has also been demonstrated to show intersex in English rivers (15). The frequency was not as high as demonstrated among roach from the same rivers. This is despite the fact that the gudgeon is a bottom living fish which could be expected to be exposed to compounds both in the water phase and in the sediment. The highest frequency of intersex found in this species was 15 %. This was found at the same river site from which 100 % intersex was seen among roach. However, in line with the high frequency of disruption in roach, the most severe cases of intersex among gudgeon were found among fish from this site and the male fish also had elevated levels of vitellogenin. As for roach, intersex in gudgeon seems to occur to some extent naturally since it was found at a background frequency of 6 % at control sites without sewage effluent discharges. In general, however, this species seems to be less sensitive compared to roach towards endocrine disruption.

The observations of feminised feral fish from English rivers have led to studies of roach populations in both Denmark and France and of populations of other fish species in other European countries and USA.

In Denmark intersex has been found in 7, 11 and 27 % of male roach collected in 1999 from three sewage effluent receiving streams in Aarhus County. The highest frequency of intersex occurred in the stream, Kristrup Landkanal which receives sewage from the city of Randers and which all year round consists of more than 97 % sewage effluent. Among the roach populations from two clean lakes with no known effluent discharge intersex occurred in 4.5 -5 % of male fish (2). A tendency to a higher degree or extent of feminisation was found in intersex individuals from the three sewage effluent affected streams, but only significantly at one site, compared to the extent in intersex fish from the two control sites. In general, a milder degree of feminisation was found in the examined Danish roach populations compared to that observed in English roach. Occurrence of the female ovarian cavity was not observed and only primary growth stages of oocytes were seen from few to a maximum of 200 per testes section.

In accordance with the Danish study, a study of roach from 4 rivers in the north eastern part of France concluded a lower incidence of intersex compared to English roach. An intersex frequency of 0-40 % and 2-60 oocytes per histological testes section were found (13).

In France estrogenic effects have also been observed in a preliminary study of another fish species the chub (Leuciscus cephalus) from one river (7). Male fish from a polluted river stretch of Moselle had higher levels of vitellogenin compared to fish from a control river and 50 % of the males had necrotic sperm cells whereas this was not seen in males from the unpolluted river. This study is, however, a study based on relatively few fish from each site.

Brown trout (Salmo trutta) is another fresh water species which has been studied in Denmark with regard to endocrine disruption (2). In one stream with low sewage effluent load, Voel Brook, an increased level of vitellogenin was found among males. Fortyfour % of these males also had testes with a high degree of vacuolation⁴ and only a presence of the earliest sperm cell stage, spermatogonia. This was also found in 5 % of fish from a clean control site and in approximately 2 % of fish from two other sewage effluent receiving brooks. The high levels of vitellogenin in males from Voel show that the fish have been exposed to estrogens and/or xenoestrogens. Whether the different testes structure compared to most control fish also was a consequence of hormonal exposure or is due to other factors such as a different age structure among fish from Voel or exposure to other chemicals is not known. Vacuolation in sertoli cells in the brown trout can be the result of the reabsorption of sperm cells which have not been spawned in the previous spawning cycle or absorption of degenerated sperm cell stages from the present reproductive cycle (109;110).

In Spain, male carp (Cyprinus carpio) has been examined for feminising effects in two rivers, Anoia and Cardener, with high and low to moderate presence of estrogens, respectively (8;14). Males from both rivers showed presence of vitellogenin in plasma in approximately 30 % of the examined fish. In accordance with the higher estrogenic load in the Anoia river, male carp from this river showed a higher plasmatic vitellogenin concentration than males from Cardener River. Intersex fish with simultaneous presence of testicular and ovarian tissue were further observed among fish from River Anoia. In this case the presence of high vitellogenin levels in the male fish was not consequently indicative of gonadal effects since intersex fish both were found to have vitellogenin levels higher than females and levels not significantly different from control males.

A large national survey in the Netherlands of occurrence and effect of estrogens and xenoestrogens in the aquatic environment has examined the reproductive health of approximately 800 bream (Abramis brama) (6). High levels of vitellogenin were detected in male bream from several freshwater sites including the rivers Rhine and Meuse and in three major river sedimentation areas. The highest vitellogenin concentrations were found among males from a small stream, Drommel, from which the largest percentage of intersex was also recorded. Fortyseven % male fish collected in spring and 33 % of male fish collected in fall had oocytes in the testes. Lower frequencies of intersex (4 % and 9 % respectively) were found at two other sites. Initial results from other smaller Dutch waters, which have not been published yet, have indicated that in one other stream, equally high levels of vitellogenin and intersex in male bream as in the Drommel were found. Generally, estrogenic effects appeared at half the selected locations (6).

The bream population in the river Elbe in Germany has also been examined for endocrine disruption at nine different sites from Hamburg to the Czech border (5). Weak estrogenic effects were seen along the entire length of the river since higher levels of vitellogenin in male bream were found at all sites compared to levels in males from a control lake with no domestic or industrial effluent input. The strongest estrogenic influence, detected as the highest vitellogenin induction (20-100 times the level in controls), was found at locations characterised by high levels of effluent discharge to the river (discharges from STPs with 825,000 and 1,534,000 population equivalents (P.E.s)⁵, respectively). Conversely, no marked vitellogenin induction was  found downstream of a very large STP discharge (2,000,000 PE at Hamburg) probably reflecting the importance of the greater dilution and better treatment of the sewage at this site compared to the above mentioned. Suppression of reproductive function was also assessed by altered steroid hormone status (lowered 17b -estradiol in females and lowered 11-ketotestosterone in males) and lowered gonadosomatic index (GSI)⁶ in both sexes. Low incidences of intersex were found at all sites along the Elbe from 2-6 % compared to 0.5 % fish with ovotestis in the control lake. Mostly, mild degrees of intersex were observed but cases where more than 45 % of the testis was ovarian were also found. In general it was concluded that disruption of the endocrine system of the bream population was observed but only the higher levels of vitellogenin were ascribed to estrogenic exposure whereas other effects might have happened through multiple mechanisms also involving other groups of chemicals such as polyaromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) also known to exist in higher levels in the Elbe. Levels of estrogens were not measured (5).

Outside Europe intersex has been detected in 29 % of shovelnose turgeon (Scaphirhynchus platyorynchus) in USA from a stretch of the Mississippi River which is rich in contaminants (9). Other studies of carp (Cyprinus carpio) and walleye (Stizostedion vitreum) have demonstrated higher vitellogenin levels in males collected near discharges from STPs (11). In both species lower levels of serum testosterone were also detected in male fish. In walleye a delay in spermatogenesis relative to fish from control sites was observed and there were indications that viable sperm was not being produced in males from the STP site. Again another American study has shown that there in a population of chinook salmon (Oncorhynchus tshawytcha) were 84 % of phenotypical⁷ females (having both female external secondary sexual characters and ovaries) which were genotypical⁸ males. These fish were therefore apparently completely sex reversed males (111). A connection to a chemical/compound release has not been found.

3.1.1.2 Effects in female fish

Due to their low endogenous levels of estrogens, male fish are considered to be the most sensitive sex towards reproductive disrupting effects caused by estrogenic compounds in sewage effluent. There does, however, exist some observations indicating that endocrine disruption of female feral fish has also taken place.

New results on English roach from two of the most affected rivers in which males had developed intersex have also demonstrated a higher incidence of oocyte atresia or degeneration in the sewage effluent-exposed females compared to control females. Sex hormone levels were also disturbed since the estradiol concentration was only half that measured in control fish illustrating that the female reproductive system was also affected (60).

Another report on female reproductive effects come from a study in Sweden reported in 2001, which described endocrine disruption in fish from a lake which was receiving leakage water from a nearby refuse dump(112). The situation is therefore not entirely comparable to the exposure cases with STP, but included since strong indications of endocrine disruption were seen. Effects were observed in both males and females but with the most pronounced effects in females. A large percentage (75 %) of the females was found to be sexually immature compared to fish from a clean reference lake. They showed an 80 % reduction in the gonadosomatic index (GSI) which shows a marked reduction in gonadal size. Among the sexually mature females a lower aromatase activity in the brain and altered steroid levels in plasma were detected. Testosterone levels were lower in fish from the leakage water affected lake and a concomitant tendency to a lower level of plasma estradiol was observed. The authors suggested that the reduced level of estradiol might be a result of the lower brain aromatase⁹ activity and that the reduced estradiol production could have lowered the production of vitellogenin and therefore inhibited the development of oocytes in the females. A reduced GSI (23 %) and reduced plasma testosterone levels were also observed among female roach from the same lake. In both perch (Perca fluvalis) and roach reductions in GSI were also seen in male fish with a 36 % reduction in male roach.

Changed steroid levels have also been reported in female walleye from a sewage effluent affected site in the USA (11).

3.1.2 Cage experiments and other controlled sewage effluent exposure studies

A number of caging experiments where fish have been placed in streams in close proximity to the STP outlets and experiments where fish have been exposed to various controlled dilutions of sewage effluent have verified the estrogenic and feminising capacity of sewage effluent from several STPs on male fish. Dose-response relationships among effluent dilution and feminising effects have been demonstrated (4;24;107;113;114) along with consequences of exposure time (24).

3.1.2.1 Cage experiments

A series of cage experiments in England where male rainbow trout (Oncorhynchus mykiss) were placed in cages for three weeks in vicinity of discharges from STPs were the first to demonstrate the estrogenic nature of UK sewage effluent being discharged to English rivers through observations of up to a 10,000-fold induction of vitellogenin in male fish (104;105). The survey of six rivers demonstrated induction of vitellogenin in caged fish in five of these rivers. In one river the estrogenicity was shown to persist as far as 5 km from the discharge point but was in the other rivers (in a three week trial) only observed in immediate vicinity of the STPs (107). Fractionation of the sewage effluent and chemical analysis of the water have subsequently shown that the compounds responsible for the observed feminisation in most cases primarily were natural and synthetic estrogens, 17b -estradiol, oestrone and ethinylestradiol (25). In the case of one river, however, outlets of alkylphenolic chemicals with industrial sewage from wool scouring and other textile industries (34) were assessed to contribute to the majority of the estrogenic activity.

Estrogens from humans are excreted in a hormonally inactive form as steroid glucoronides and sulphates primarily via urine. The steroid conjugation takes place in the liver and is a normal metabolic pathway used to facilitate the excretion of compounds from the body (115). In humans estrogens are primarily excreted as estrogen glucuronide conjugates. Women may excrete around 2.4 mg of estradiol, 4.6 mg of estriol and 7 mg of estrone daily via urine. Smaller quantities are also excreted with faeces (in (116)). (For more information regarding women as a source of estrogens see section 10). The microbial activity in the sewage treatment works and possibly also in the sewage before it reaches the treatment works (117) results, however, in a deconjugation of the steroid thereby restoring the estrogenicity of the steroid components of the sewage effluent (118).

Cage experiments using juvenile rainbow trout have also demonstrated the estrogenic nature of Swedish sewage effluent from a relatively small STP which mainly receives domestic sewage (3). The STP had chemical and biological treatment steps but no anaerobic denitrification step and processed sewage effluent from a smaller number of people (3500). Still, a very high induction of vitellogenin was found in male fish after an exposure period of two weeks in the stream. Both estradiol, estrone, ethinylestradiol, nonylphenol and bisphenol A could be detected in the bile of the fish. Chemical analyses of the stream water showed high levels of all three estrogens but with ethinylestradiol at a 45 times higher concentration (4.5 ng/l) than the one experimentally set as lowest concentration which will induce estrogenic effects in fish (119). Ethinylestradiol was therefore considered to be responsible for most of the estrogenicity of the examined effluent.

Very high levels of EE2 detected in a final STP effluent in Texas were also considered the likely cause of elevated vitellogenin levels, reduced GSI and reduced secondary sexual characteristics in fathead minnows (Pimephales promelas) kept for three weeks in cages in a treatment wetland close to the outlet of effluent (120). EE2 was measured in concentrations as high as 200-900 ng/l. No information were given regarding the treatment steps of the STP from which the effluent was coming.

On the contrary, no effects on the same parameters in a similar study of fathead minnow were observed when caged fish were exposed in situ at riverine sites to representative effluents from central Michigan sewage treatment plants (121). All plants were using at least secondary treatment technologies and at some sites tertiary treatment such as sand or charcoal filters were also applied. Chemical analysis of possible estrogenic compounds in the sewage effluent or surface water was not performed.

Different sensitivities of different fish species have to be taken into consideration when using cage studies to assess or confirm the estrogenic capacity of sewage effluent. In the dutch study mentioned above where a high degree of feminisation was observed in wild bream from the small stream, Drommel, cage experiments in the same stream with either carp or rainbow trout showed high vitellogenin induction in rainbow trout but not in carp when male fish were placed in cages for 21 days (6). The results from the cage experiments with rainbow trout did, however, confirm that the feminisation of feral bream could be ascribed to sewage effluent exposure. This was confirmed by dose-response related vitellogenin induction in rainbow trout (but again not in carp) with a threshold concentration of 25-50 % sewage from two of the sites with feminisation of wild bream.

The Danish studies which have examined the estrogenicity of sewage by caged fish have in general found no or a relatively weak induction of vitellogenin in the sewage exposed fish. A weak vitellogenin induction has been found after 6 weeks among rainbow trout placed near the outlet of sewage from the treatment plant, Ejby Mølle, Odense (122) and in flounder exposed for three weeks to sewage effluent from Avedøre STP (123). In the last mentioned study no induction was, however, not seen in two other species, rainbow trout and eel (Anguilla anguilla). A study in which rainbow trout were placed in cages for 9 weeks in a highly sewage effluent receiving brook, Kristrup Landkanal and in the brook, Egå for 13 weeks also did not cause induction of vitellogenin (124). This is despite the observation of relatively high frequencies of intersex among roach from Kristrup Landkanal. The explanation for this discrepancy might be differences in exposure time for the two species. Further, for the Atlantic croaker (Micropogonias undulatus), it has been demonstrated that the estrogen receptor in the testis is saturated at four times lower concentrations of estrogens and a number of estrogenic compounds compared to the estrogen receptor in the liver, in which the vitellogenin synthesis is induced. Alterations in the gonads such as intersex or sex reversal might therefore be more sensitive indicators of an estrogen effect than vitellogenin.

3.1.2.2 Other controlled experiments with graded concentrations of sewage effluent

In line with the last study just mentioned, a number of controlled studies with graded concentration of sewage effluent have further demonstrated a dose-response relationship between vitellogenin induction or intersex and the percentage of sewage effluent verifying again the estrogenicity of sewage effluent from several European countries and adding proof to the sewage effluent as the source of compounds causing the feminisations of wild fish.

Sewage effluent from a municipal sewage plant which receives domestic sewage from Oslo was estrogenic to rainbow trout at concentration of 2.5, 10 and 20 % sewage when exposing the fish for 3 weeks (4). In the same study effluent from an oil refinery treatment works was estrogenic to rainbow trout. A Swedish study has also demonstrated estrogenicity of 50 % effluent from a pulp mill (125) by vitellogenin induction in zebrafish (Danio rerio). Concerns about endocrine disruption by effluent from pulp mills have mainly been in the direction of masculinising effects, which has been seen in a number of cases (115;126). Both estrogenicity and androgenicity of the effluent were observed in the Swedish study since exposure to the pulp mill effluent besides inducing vitellogenin also resulted in a higher proportion of males. This indicates the presence of both estrogenic and androgenic compounds in the water.

Increasing levels of vitellogenin in male rainbow trout exposed for six months to increasing levels of effluent (10, 20, 30, 40 %) from the Berlin-Ruhleben sewage plant in Germany have also been found(114;127). Ethinylestradiol, 17ß-estradiol and estrone were measured at concentrations of <0.2-3 ng/l, <0.5-1.5 ng/l and 0.5-1 ng/l, respectively in the effluent which, compared to the measured concentration of xenoestrogens i.e. alkylphenols and bisphenol A, were closer to the concentrations known to exert effects on fish (see chapter 6) (127).

In roach, it has further been shown that vitellogenin synthesis induced by sewage effluent besides being dose dependent also depends on the exposure period (24). The percentage of the sewage effluent which was able to induce vitellogenin in mature adult roach fell from 37.9 to 9.4 % sewage effluent by prolonging the exposure period from 1 to 4 months. In the sewage effluent used in this study the levels of estrogens were 1.7-3.4 ng/l ethinylestradiol, 4-88 ng/l 17ß-estradiol and 15-220 ng/l estrone also illustrating the high levels of all three hormones relative to their estrogenic potential (see chapter 6) but also the great fluctuation in hormone levels over time ( 8 months).

Sewage from the same STP could in another study induce a dose-response dependent development of intersex in roach when exposing juvenile fish from 50 to 200 days after hatch. The fish developed the female duct called the ovarian cavity also detected in wild roach (1) at a threshold concentration of 23.6 % sewage. Exposure to 100 % effluent resulted in 100 % occurrence of intersex in males. This feature seemed to be irreversible since depuration of the effluent exposed fish in clean water for 150 days did not result in any alteration in the duct formation. No induction of oocytes was induced in the testes of any male fish in the present study and therefore the causality of the oocytes in the testes in wild roach still needs complete elucidation. Since other studies have demonstrated induction of ovotestes with (xeno-)estrogens (53;128), the lack of oocyte induction in males in the study with juvenile roach might be because the exposure period did not cover the sensitive window/critical period for development of this feature (113).

3.2 The marine environment

Marine organisms have been expected to be less affected by endocrine disruption from estrogenic discharges to the marine environment compared to freshwater fauna due to the usually high degree of dilution of sewage effluent in estuaries and open waters (18). A number of surveys have, however, demonstrated feminisation of some marine species, predominantly in flounder (Platichthys flesus) (16-22). The flounder has been chosen as a suitable marine test monitoring species because it lives in close contact with the sediment in which it buries itself and it lives most of the time in the estuaries migrating only to sea for breeding (19). One disadvantage with this species is, however, that controlled laboratory studies using water exposure have demonstrated the flounder to be less sensitive towards estrogens compared to for instance the rainbow trout (17). Still, elevated levels of vitellogenin has been detected in flounder from several industrialised estuaries in England (especially in Tyne, Tees and Mersey) compared to reference sites in a number of studies conducted over a period of 6 years (16-18;21;22). The routes by which flounder is exposed to environmental estrogens are not fully understood. Exposure through the food chain might contribute to the observed effects in flounder since experiments where flounders were fed mussels held in the Tees estuary for 3 months showed a 10-fold increase in plasma vitellogenin (18). Exposure of male flounder to water and sediment in cages at two sites of the Tees and Tyne for three weeks did not induce vitellogenin production (129). Occasionally high vitellogenin levels have also been detected among males collected offshore. The reason for this is unclear but might rather be due to migration of males from the more polluted estuarine areas than caused by local pollution offshore (16). Similar observations of single very high levels of vitellogenin among male flounder from several near cost areas in Denmark have been made (130). How these results should be interpreted is also very uncertain and a need for more knowledge on seasonal and age related vitellogenin levels in this species is needed.

Intersex has also been detected among male flounder in the English surveys. Depending on the year of sampling, intersex has been found in between 7 and 20 % of male fish from the sites where also high vitellogenin induction was found among males. Other gonad changes which have been reported from flounder are abnormal testes morphology such as disorganised lobules¹⁰, vacuolation, thickening of connective tissue and lack of germ cells pointing toward an inhibited spermatogenesis (22). These changes were detected in a higher proportion (53-67 %, depending on the time of the year) in the fish from the polluted Tyne estuary compared to fish from a reference site (maximum 12 %). A higher proportion of degenerating oocytes were seen among females from Tyne in the same study.

In a Dutch study of offshore, estuarine and inland locations including 1400 flounders, levels of vitellogenin were found to be low among males at most investigated sites (6) except at two sites in the North Sea Canal, an industrialised port zone which receives effluent from sewage treatment works. Here moderately elevated levels of vitellogenin were found among the examined males in a fall but not a spring sampling. None of the captured males had oocytes in their testes. Based on these results it was therefore concluded that estrogenic effects in the marine waters of the Netherlands were few compared to the situation reported from the English and Welch Coast.

Both a higher level of vitellogenin and intersex were found in male flounder from the Tokyo Bay which receives large amounts of industrial and domestic sewage effluent. Oocytes were found in testes of 15 % of the males compared to none in the control population (12).

As mentioned, studies of feminising effects on marine fish species have mainly concentrated on the flounder but a few results on other species have been reported. A small scale study of the eelpout (Zoarches viviparous) from English estuaries has shown that up to 85 % of the males caught at particular stations contained vitellogenin mRNA in the liver compaired to none at a reference site. Gonads were normal among the control individuals whereas intersex was found in up to 17 % of male from polluted estuaries (18).

Two species of sand gobies (Pomatoschistus minutus and P. lozanoi) have recently also been proposed as suitable monitoring organisms for endocrine disruption related to estrogen exposure (18;23). A phenomenon called Morphologically Intermediate Papilla Syndrome (MIPS) has been found in 33-75 % of males from four estuarine sites with known estrogenic contamination compared to a 0-15 % prevalence of the condition among males from the control and other estuarine sites. MIPS is a condition in which the male urogenital papilla (a secondary sexual organ used for depositing sperm) has developed villi at the papilla tips which resembles the villi on the female papilla used for oviposition. This is a condition which has been demonstrated to be inducible by controlled exposure to estradiol (18).

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