Feminisation of male fish in freshwater and marine environments

Feminisation of male fish has now been detected in a number of countries world-wide. These kinds of hormone disruptions have been seen in a range of both freshwater and marine species of fish though most frequently among freshwater species.

The feminisations are believed to be caused by release of natural and synthetic estrogens and estrogenic compounds being released to the aquatic environment via sewage effluent.

Signs of feminisation in male fish are generally a synthesis of the yolk protein vitellogenin, an estrogen marker, and intersex, an abnormal type of hermaphoditism in which males develop egg cells in the testes.

Worst cases of feminisation in regard to both occurrence and degree of the disruption have been seen in England while a lower extent has been found in other countries including Denmark.

Care must be taken in using results from short-term exposure studies as ultimate estimates of risk for wild populations of fish which live their entire life in sewage effluent receiving waters.

The estrogenic components of sewage effluent

A combination of cell based in vitro assays and chemical analyses of sewage water has verified and quantified the estrogenicity of sewage effluent from numerous countries. These have also demonstrated that the natural estrogens, 17ß-estradiol and estrone, and the synthetic estrogen, ethinylestradiol used in contraceptives are likely candidates for some observed disturbances in fish from sewage effluent receiving rivers. In single cases, the estrogenic chemicals, alkylphenols, have also been suggested as possible causative agents.

Sewage effluent and surface water concentrations of estrogens

Concentrations of the three natural estrogens, 17ß-estradiol, estrone and estriol have internationally been detected in sewage effluent at concentrations of < 0.1 – 88 ng/l (typical 1 – 10 ng/l), < 0.1 – 220 ng/l (typical 5 – 20) and < 0.1 – 42 ng/l, respectively. Ethinylestradiol has been found at concentrations of < 0.053 – 62 ng/l (typically below 1 or seldom above 10 ng/l).

Concentrations in surface waters have been found in the ranges 0.05 – 15.5 ng estradiol/l, < 0.1 – 17 ng estrone/l, < 0.1 – 3.4 ng estriol/l and < 0.053 – 30.8 ng ethinylestradiol/l with typical concentrations of less than 5 ng/l for estradiol and estrone and less than 1 ng/l for ethinylestradiol.

Fate of estrogens in the aquatic environment

Ethinylestradiol is more persistent than the natural estrogens both in water and sediment. Average half-lives of 2.8 and 3.0 days in water has been calculated. Ethinylestradiol has been demonstrated to have a ten times as long half-life compared to estradiol. In anaerobic sediment 17ß-estradiol is rapidly converted to estrone, but both estrone and ethinylestradiol show very low degradability in the sediment and might accumulate.

Occurrence and fate of alkylphenols and bisphenol A in sewage effluent and surface water

Some of the more potent estrogenic compounds which might be released with sewage effluent are the alkylphenols, nonylphenol and octylphenol, and bisphenol A. Nonylphenol and octylphenol have in sewage effluent generally been detected in concentrations below 10 µg/l and below 1 µg/l in surface water, though, few example of concentrations above 300 µg/l in sewage effluent and 600 µg/l in surface water have been seen. Bisphenol A is seldom detected above 1 µg/l in either sewage effluent or surface water. Both alkylphenols and bisphenol A have great potential for accumulating in the sediment.

Lowest effect concentrations for feminisation by estrogens, alkylphenols and bisphenol A

Laboratory experiments have for 17ß-estradiol found a lowest effect concentration for induction of vitellogenin on 5 ng/l and for induction of intersex on 10 ng/l. A range of other effects have been seen at concentrations between 10 and 50 ng/l.

Estrone has an equal or slightly lower estrogenic potency compared to estradiol, and lowest effect concentration for vitellogenin and intersex induction in male by estrone is 30 and 10 ng/l, respectively. There is little knowledge on the potency of estriol but in vivo it appears to be 100 times less potent than estradiol.

Ethinylestradiol is more potent than the natural estrogens in regard to inducing feminising effects. Vitellogenin production and intersex in males have been induced by 0,1 ng/l and changed sex ratio by 0,6 ng/l. A range of other testis effects have been seen at concentrations below 10 ng/l.

Nonylphenol, octylphenol and bisphenol A have lower estrogenicity compared to the natural and synthetic estrogens. Induction of vitellogenin has been made by 5 µg/l nonylphenol or octylphenol, and intersex, changed ratio and other effects at nonylphenol concentrations between 30 and 100 µg/l. Male reproductive disorders have been seen with 2 µg/l octylphenol. Bisphenol A has exerted effects at concentrations between 10 and 40 µg/l.

Relationsship between lowest effect concentrations of estrogens/estrogenic compounds and their presence in the environment

Comparing sewage effluent and surface water concentrations of the estrogens, alkylphenols and bisphenol A with the lowest effect concentrations for reproductive disruptions by the individual compounds demonstrate that concentrations of estradiol, estrone, ethinylestradiol, nonylphenol and octylphenol in some cases have been high enough to be suspected of causing feminisations in wild fish populations. Based on the present knowledge on environmental occurrence of and the estrogenic potential of estriol it is not possible to estimate the contribution of this estrogen to observed feminisations. Bisphenol A is generally detected at concentrations below the lowest effect concentrations for inducing reproductive disorders in male fish.

Which compounds are responsible for observed feminisations of fish in Denmark is still uncertain due to a limited knowledge on water concentrations of estrogens.

Several aspects have to be taken into account when assessing the possible implications for the reproductive health of fish of estrogenic compounds in the environment. Estrogens and estrogenic chemicals in sewage effluent will act in an additive manner thereby lowing the concentration of a single compounds which is needed to induce effects. Different species exert different sensitivities. The timing of the exposure relative to critical periods in the fish life-cycle is of great importance to the resulting effects, and intermittent exposures to high concentrations of estrogens seems to give unproportionately large effects.

Effect of feminisation or estrogenic exposure on fertility of male and female fish

Little is known about the importance of the observed signs of feminisation on the fertility and reproductive success of fish. However, observations among intersex roach in England of asynchrony of gamete maturation between males and females due to reduced spermatogenesis as well as reduced sperm volume, density and motility have indicated reduced fertility. The normal development of egg cells in females was also affected. Controlled exposure experiments with the compounds in question have also demonstrated reduced fertilisation success among males and reduced spawning of eggs among females.

Impact of estrogens and estrogenic compounds on the reproductive capacity might also be indirectly via reduced energy sources to reproduction.

Sources of estrogens to sewage effluent

The pool of estrogens which enters the municipal sewage system origins form the natural production of estrogens by humans, from hormone and estrogen replacement therapies and the intake of hormone contraceptives containing ethinylestradiol.

The estrogens are mainly excreted as water-soluble conjungates. Estimation of the excretion of estrogens by humans in Denmark showed that the main part of the estrogens originates from the natural production in humans which contributes to approx. 87 % of the total excretion of estrogens. Excretion from hormone therapy accounts for approx. 12 % while excretion of hormones from contraceptives only accounts for approx. 1 % of the total excreted amount of estrogens.

The estimates of the total humane excretion of the four estrogens per 24 h were: 36 g of estradiol, 69 g of estrone, 340 g of estriol and 3.2 g of ethinylestradiol.

Sources of alkylphenols and bisphenol A to sewage effluent

Alkylphenols are mainly used in production of other products as e.g. alkylphenolethoxylates. Alkylphenolethoxylates are relatively easy degraded to alkylphenols and, therefore, important sources of alkylphenols in sewage systems. No release from production of either alkylphenols and alkylphenolethoxylates in Denmark is expected.

The nonylphenol family represents approx. 85 % of the alkylphenol market and the remaining 15 % are assumed to be octylphenol.

The total release of nonylphenolpolyethoxylates to waste water in Denmark was estimated to be between 37 and 996 tonnes per year.

No production of bisphenol A is expected to take place in Denmark. The release in Denmark is expected to arise solely from processing, use and disposal of bisphenol A containing materials. The release of bisphenol A to waste water in Denmark per year was estimated to 17 kg from PVC processing, 256 kg from PVC use and 492 kg from paper reuse.

Fate of estrogens in sewage treatment works

Conjungated steroids have been found to contribute to approx. 50 % in influent and approx. 70 % in effluent of German STPs. The total amount of estrogens (estrone, estradiol and ethinylestradiol) was 25.5 ng/l and 9.3 ng/l in the influent and effluent, respectively.

Based on laboratory experiments it is expected that the conjugates of estradiol are de-conjungated relatively fast and that estradiol is oxidised into estrone which is further eliminated.

Typical average removal efficiencies of the four estrogens from the water phase of STPs are: 61 ± 38 % of estrone, 87 ± 9 % of estradiol, 96 ± 6 % of estriol and 85 ± 14 % of ethinylestradiol. Analyses of glucuronates were not included in these studies. The removal of the estrogens may occur by degradation as well as sorption to sludge particles.

Fate of alkylphenols and bisphenol A in sewage treatment works

The resulting products of the degradation of alkylphenolethoxylates under aerobic conditions in STPs are mono- and diethoxylates, the more hydrophobic carboxylates and alkylphenols. The final product alkylphenol seems to be degradable under aerobic conditions. The transformation under anaerobic condition results in production of mono- and diethoxylates and finally alkylphenols. The concentrations of APs in anaerobically digested sludge are often extremely high.

The average removal efficiencies (water phases) of nonylphenolic compounds of 53 % and 59 % have been found in examination of STPs with analytical programs including nonylphenolethoxylates and their metabolites inclusive mono- and dicarboxylates.

There are to our knowledges no studies of the fate of alkylphenolethoxylates (APnEOs) within STPs which include all the know metabolites of APnEOs.

Bisphenol A is expected to be easily degraded under aerobic conditions in STPs but not under anaerobic or anoxic conditions. Furthermore, bisphenol A may be removed from the water phase by sorption to sludge particles. Removal efficiencies from the water phase of 47-96 % have been observed in Canadian STPs and of ~ 0-96 % in Danish low technology plants.

Influence of the type of sewage treatment plant on the removal efficiency of the estrogens/estrogenic compounds

Generally, the removal efficiencies of estrogens and alkylphenolic compounds seem to increase and consequently the effluent concentrations to decrease by increasing upgrading of STPs. E.g. effluent concentrations of < 0.1-0.32 ng estradiol/l have been observed in a California STP after reverse osmosis.

The use of low technology plants in the so-called "open land" may result in relatively high local concentrations of estrogenic compounds in the aquatic recipient.

Different operation conditions in STPs as e.g. HRT, SRT, temperature and loading rate will have an important bearing on the plant efficiency and thereby the removal of estrogens and xenoestrogens. However, there is only little information about the operation conditions in the studies of STPs.

More knowledge concerning the fate of estrogenic compounds within STPs is needed. High quality studies should be performed which are linked to concurrent comprehensive monitoring of overall STP performance. STPs with different treatment processes should be studied and the monitoring of full-scale plants should be accompanied by studies in pilot-plants and on laboratory scale.

Influence of advanced treatment processes on removal efficiencies

Several advanced treatment processes have been investigated with the aim of reducing e.g. endocrine disrupters. Very high removal of > 90 % have been obtained with powdered activated carbon while removals from 40 to 70 % have been seen with magnetic ion exchange. Investigations of ozone treatment of an effluent from a municipal STP followed by UV treatment have shown promising results regarding the removal of endocrine disrupters.

Possible non-sewage related sources of estrogens to the aquatic environment

Other sources to estrogens in the environment besides sewage effluent might be the outbringing of manure from life stock and sludge from sewage treatment plants. Too little is still known to assess their possible contribution via drain water to the total estrogenic activity in surface waters.

 

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