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Feminisation of fish

1. Summary (English)

Feminisation of male fish in freshwater and marine environments

During the past ten years feminisation of male fish has been detected in several European countries and in USA and Japan (1-12). These cases are examples of disturbances of the male reproductive system which are believed to be consequences of endocrine disruption caused by female hormones, estrogens, or chemicals mimicking estrogens present in the aquatic environment. The majority of estrogens are believed to reach the aquatic environment by sewage effluent. In Europe feminisation of male fish exposed to sewage effluent has been seen in England (1), Sweden (3), Norway (4), Germany (5), The Netherlands (6), France (7;13), Spain (8;14) and Denmark (2). Further, the observations of feminisation in wild populations of fish have been made in a range of different species including the freshwater species roach (1), gudgeon (15), carp (8;14), bream (5;6), chub (7;13) and brown trout (2) and the saltwater species flounder(16-22), eelpout (18) and two species of sand gobies (23). Thus, these kinds of disruption of the male reproductive system have both been found in freshwater and marine environments though most cases have been reported from freshwater environments. Controlled exposure of fish to sewage effluent i.e. in cage experiments have further added proof to the estrogenic nature of the sewage effluent and estrogens or estrogenic chemicals as causative agents of the disruptions.

The signs of feminisation in male fish are the production of a female yolk protein which is known only to be produced as a response to an estrogen exposure, and the occurrence of intersex – an abnormal form of hermaphroditism. Males with the intersex condition have early stages of egg cells in the testis and in some cases they have also developed the female duct which leads eggs to the oviduct. Feminisation has been found in varying degrees among individual fish from mild to very severe disturbances of the male reproductive system.

England is the country in which feminisation of fish has been most widely detected and where the most severe degrees of feminisation have been seen, both compared to countries inside and outside Europe. The feminisation of fish which has been detected in Denmark does not, however, differ markedly from the extent found in other countries. In these a lower occurrence and severity of feminisation compared to England have generally been seen though feminisation has been detected at several of the examined sites. There are, however, also sites where the fish populations appear unaffected.

It should be pointed out that short-term exposure experiments, where fish are exposed to sewage effluent for a short period of time, do not always give a correct estimate of the risk for wild populations of fish which live their entire life in the stream or river. It has been demonstrated that increasing the exposure time for sewage effluent lowers the concentration of sewage effluent needed to cause feminisation of fish (24).

Estrogenicity of sewage effluent

Support to the theory of estrogenic compounds in sewage effluent as the cause of the feminising effects, which have been found among male fish, has also come from a number of studies using cell culture assays designed to detect estrogenic activity. These have demonstrated the estrogenicity of sewage effluent from a number of countries (England, Germany, The Netherlands, Belgium, China, Korea and USA) and quantified the estrogenic potential in relation to the potency of 17ß-estradiol (6;25-33). Further, chemical analysis of the composition of sewage effluent and determinations of concentrations of estrogens and estrogenic compounds in the effluent have in a large number of cases demonstrated the natural estrogens, 17ß-estradiol, estrone and the synthetic estrogen, ethinylestradiol used in contraceptives as likely candidates for the observed disturbances in fish species from the sewage effluent receiving rivers. In single cases alkylphenols were also suggested as possible causative agents (34).

Sewage effluent and surface water concentrations of estrogens.

Internationally, the three natural estrogens, 17ß-estradiol, estrone and estriol have been detected in sewage effluent at concentrations of < 0.1 – 88 ng/l, < 0.1 – 220 ng/l and < 0.1 – 42 ng/l, respectively. The even more potent synthetic estrogen, ethinylestradiol has been found at concentrations of < 0.053 – 62 ng/l. The typical range for the steroids are in the range 1-10 ng/l and 5-20 ng/l for 17ß-estradiol and estrone, respectively, while ethinylestradiol often is below detection limits (0.1 – 1 ng/l). When ethinylestradiol is detected it is mostly below 10 ng/l. The concentrations measured in Danish sewage effluent lie within the typical range of estrogen concentrations reported from other Europeans countries. In surface waters, concentrations of estrogens are lower compared to sewage effluent and have been detected at concentrations of 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. For both sewage and surface water estrone is the most and ethinylestradiol the least frequently detected estrogen.

An important thing which has to be remembered whenever concentrations of estrogens are mentioned in the present report is, however, that it seldom is specified whether the chemical analysis measures only free or both free and conjugated estrogens. There must probably also be expected to be larger uncertainties related to measurements of estrogen concentrations in influent than in effluent due primarily to interference from large contents of organic matter (matrix effect). Further, the detection limits of the analyses, especially for ethinylestradiol, are close to and sometimes even above the concentrations which have been demonstrated to cause reproductive disturbances in fish.

Fate of estrogens in the aquatic environment

Knowledge on the fate, behaviour and persistence of estrogens in the environment is still relatively limited. The synthetic ethinylestradiol seems, however, to be more persistent than the natural estrogens both in water and sediment. Under aerobic conditions mean half-lives in water of 17ß-estradiol and estrone has been calculated to 2.8 and 3.0 days, respectively (35). Ethinylestradiol has been demonstrated to have a half-life ten times that of estradiol under the same incubation conditions (1.2 versus 17 days for estradiol and ethinylestradiol, respectively) (35;36). Estrogens have in general a medium sorption potential to sediment (37) and low degradation of both estrone and particularly ethinylestradiol in sediment with low oxygenation might indicate a risk for sediment as a sink for these estrogens (35).

Occurrence and fate of selected estrogenic compounds in sewage effluent and surface water

Alkylphenols and bisphenol A are two of the more potent of the estrogenic chemicals which might be released with sewage effluent and which therefore receive some attention in the present report. The most important alkylphenols with regard to estrogenicity are nonylphenol and octylphenol. These are in general detected at concentrations below 10 µg/l in sewage effluent and below 1 µg/l in surface water although examples of concentrations above 300 µg/l in sewage effluent and above 600 µg/l in river water have been reported from some countries. Bisphenol A is less frequently encountered than alkylphenols and has seldom been detected at concentrations above 1 µg/l in either sewage effluent or surface water.

As for estrogens, alkylphenols and bisphenol A will partition between the water phase and the sediment, and indications exist that both have a great potential for accumulating in sediment with anoxic conditions (38;39).

The feminising potential of estrogens, alkylphenols and bisphenol A

In light of the observations of reproductive disturbances in fish populations exposed to sewage effluent, a number of controlled exposure experiments have been performed to assess the lowest concentration of estrogens and estrogenic chemicals needed to obtain these observed disturbances. This aids in assessing which compounds might be responsible for the observed effects such as production of yolk protein in males, development of intersex or other disturbances in the male testis.

17ß-estradiol has induced the production of yolk protein at a concentration of 5 ng/l (40;41) and has induced intersex at 10 ng/l (42). A range of other testicular effects have been seen at concentrations between 10 – 50 ng/l (43-45) and also at concentrations above 100 ng/l (44). Examples of other effects are inhibition of the normal development of male germ cells, which can be seen as a lower relative weight of the testis and/or a presence of a larger proportion of early stages of germ cells (44). The presence of degenerated germ cells has also been seen (44).

A similar or slightly lower estrogenic activity has been detected for estrone. Induction of yolk protein and intersex in male fish has been seen at concentrations of 30 (46;47) and 10 ng/l (42), respectively. Estriol is the least estrogenic of the three natural estradiols. In vitro it has been demonstrated to be 30 times less potent than estradiol (42) but in regard to induction of intersex in vivo it seems to be 100 times less potent (42). In general, knowledge on the estrogenic capacity of this estrogen is limited.

Ethinylestradiol is even more potent than the natural estrogens in regard to inducing disruptions in the male reproductive system. Induction of yolk protein and induction of intersex have been seen at 0.1 ng/l (42) and changed sex ratio at 0.6 ng/l (48). A range of other reproductive effects such as inhibition of normal sperm cell development has been seen at concentrations below 10 ng/l.

Both the alkylphenols, nonylphenol and octylphenol, and bisphenol A have a lower potency than both natural and synthetic estrogens. Effects are seen in the µg/l concentration range. Induction of yolk protein has been detected at 5 µg/l nonylphenol or octylphenol (49;50), though at long exposure time with nonylphenol, the lowest effect concentration was decreased to 1 µg/l (51;52). Intersex, changed sex ratio, degenerated testes and inhibited growth of the testes have been seen at nonylphenol concentrations between 30 and 100 µg/l (50;53;54). Concentrations down to 2 µg/l octylphenol have caused reproductive disorders in male fish (49). Bisphenol A has been demonstrated to exert effects at concentrations between 10 and 40 µg/l (42;55;56).

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

When comparing the actual sewage effluent and surface water concentrations of the estrogens, alkylphenols and bisphenol A with the lowest concentrations which in controlled laboratory studies can induce reproductive disturbances in male fish, it is seen that concentrations of estradiol, estrone and ethinylestradiol in some cases have been high enough to explain the feminisation of fish. The same holds in some cases for nonylphenol and octylphenol. Reports on environmental concentrations of estriol are too sparse to make a reliable estimation on the possible contribution of this estrogen to feminising effects, while bisphenol A in general has been detected in concentrations below the lowest effect concentration for inducing reproductive disorders in male fish.

In Denmark very few measurements have been performed on water concentrations of estrogens and it is not yet possible to point out the compounds responsible for the observed feminisations of male fish in Danish streams.

When assessing the possible implications for the reproductive health of male fish of the estrogenic compounds in the aquatic environment it is, however, important to remember that the estrogens present in the water will act in an additive manner (40;57). Therefore the concentration of a single compound which can exert an effect on males will be lower when present in a mixture of estrogens and xenoestrogens. Further, different fish species show different sensitivities towards endocrine disruption with estrogens, and the timing of the exposure is also important for the resulting effects. The early life stages are generally considered to be the most sensitive stage since the development of the sex takes place at this time (58). There might, however, also be certain periods in the reproductive cycle of sexually mature male fish in which they are more susceptible to endocrine disruption.
Intermittent exposure to high concentrations of estrogens have further been demonstrated to result in larger effects than obtained with a continuous exposure to a lower concentration, so release of short pulses of very high concentrations of estrogens and/or estrogenic compounds might have great importance (59).

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

The observations of feminised fish in many parts of the world have raised the question whether the fertility or reproductive capacity of the fish is reduced and populations in danger of decreasing. Based on the present knowledge this is not yet possible to answer. New results have, however, implicated reduced fertility among intersex roach in England (57;60). An asynchrony in the development of germ cells in males and females has been demonstrated due to a delay in the development of male germ cells. Only 50 % of the males were capable of spawning. Further, a reduced milt volume and a reduced density and motility of sperm cells were seen among the other males. This indicates reduced fertility among the severely intersexed males. The implications of a milder degree of feminisation are harder to estimate.

Controlled exposure studies with estrogens and estrogenic compounds have also demonstrated a reduced fertilisation success as well as altered sexual behaviour of exposed male fish (49;61-63).

The impact of production of the female yolk protein in male fish, which is a very widespread phenomenon, on the reproductive capacity is uncertain but could take place possibly via an indirect route. This can be via reduced energy sources to reproduction due to demonstrated effects on liver, kidney and other health conditions (64-67).

Most studies both in the field and in the laboratory have focused on the effects of estrogens and estrogenic compounds on the reproductive health of male fish since these are considered to be the most sensitive gender due to their low body concentrations of estrogens. There are, however, also studies which have demonstrated that the reproductive success of females can be reduced by estrogen exposure i.e. via reduced egg spawning. This seems to take place via disturbance of the normal maturation of egg cells (61;68;69).

Potential sources of estrogens to sewage

The three natural estrogens are female steroid sex hormones, which are produced naturally, in humans and other vertebrates. Both female and male vertebrates produce estrogens. The production and excretion varies throughout life and between the two sexes. Estradiol is both metabolised reversibly and irreversibly. In the reversible metabolism, estradiol is transformed to estrone, while estradiol is transformed to catechol estrogens or estriol in the irreversible metabolism. The main part of the produced metabolites are finally conjugated with sulphate and glucuronides and excreted in the urine and are as such an important source of natural estrogens in the municipal sewage system. A minor amount is excreted via faeces as unconjungated metabolites (70;71).

The production of estrogens in adult men varies in different age groups but the variations are small compared to the variation in mature women. The production and then the excretion of estrogens from the mature women vary during the menstrual cycle and the pregnancy until the menopause is reached. The daily excretion of estrogens in urine by menstruating women is on average: 4.8 µg estriol, 8.0 µg estrone and 3.5 µg estradiol whereas the average daily excretion by pregnant women is: 6,000 µg estriol, 600 µg estrone and 259 µg estradiol (72) (73). The production of estrogens decline at the menopause and the post menopause production of estrogens is very low (74). The average excretion of the three estrogens in urine by postmenopausal women is approx. 7 µg/day. This is at the same level as the average excretion by adult men (72). Hormone and estrogen replacement therapies are other sources of estriol, estrone and estradiol in raw sewage. Thus, approx. 65 % and 15% of orally administered estradiol or estrone are excreted in urine and faeces, respectively.

Hormone contraceptives (birth control pills) contain ethinylestradiol, which is found mainly as sulphate conjungates (80 %) in plasma shortly after administration. A large part is excreted in un-metabolised but conjungated form. A total excretion of 26 % has been assumed (72). The total human excretion of estrogens in Denmark (Table 1) was calculated using excretion patterns of estrogens and the demographic figures for Denmark 2001 (75).

Table 1
Estimated excretion of estrogens in Denmark 2001

Potential sources of alkylphenols to sewage

Commercially available alkylphenols are generally mixtures of alkylphenols with different degrees of branching but with the same number of C-atoms in the alkyl chain (76). Alkylphenols are mainly used in the production of alkylphenolethoxylates (APnEO), tris(nonylphenyl)phosphite and alkylphenol-formaldehyde condensation resins (77). However, unreacted alkylphenols can be used as plasticisers in plastics. Alkylphenolethoxylates are relatively easily degraded to alkylphenols and, therefore, important sources of alkylphenols (78). Nonylphenol is the most commercially prevalent of the alkylphenol family, representing approx. 85 % of the alkylphenol market. The remaining 15 % are assumed to be octylphenol.

Nonylphenolethoxylates and nonylphenol may be released from formulation and from use in Denmark in e.g. cleaning processes. Estimates for the release to waste water in Denmark have been calculated on the basis of the EU risk assessment of nonylphenol (76) and figures for former Danish releases, where available. The Danish release is estimated to between 37 and 996 t/year¹.

Potential sources of bisphenol A to sewage

The major consumption of bisphenol A is as a chemical building block in the production of polycarbonate plastic and epoxy resins accounting for approx. 71 % and 25 %, respectively, of the total use in the EU (79). The Danish release is, with some uncertainty, estimated to 735 kg/year.

General considerations regarding establishment of the fate of estrogens and estrogenic compounds in sewage treatment plants

Evaluation of the fate of estrogens, alkylphenols, the parent compounds of alkylphenols and bisphenol A in municipal sewage treatment plants (STPs) show that a considerable amount of the studies presented in the literature only includes analyses of effluents. Thus, only few studies include simultaneous monitoring of influents, sewage samples and intermediate streams in STPs, which are needed for a proper assessment of the fate of a compound within a STP. The most extensive investigations are performed of nonylphenolethoxylates (NPnEO). Furthermore, there are only very few studies of Danish STPs. Another drawback is that variable procedures of sampling have been used from one study to another, and in some cases within the same study. Also the applied analytical methods including their limits of detection and determination vary between different studies. All these things limit the possibilities of an exhaustive evaluation of the fate of the compounds in STPs and of an assessment of the impact of different treatment processes. Despite these facts it has been possible to extract some general aspects concerning the fate of the compounds of concern in STPs and the impact of the type of treatment plants on the removal efficiencies.

Fate of estrogens in sewage treatment plants

The estrogens are mainly excreted as conjugates. However, there are only very few studies which have included analysis of conjugated estrogens. Laboratory experiments have shown that the glucuronide conjugates of estradiol may be deconjugated relatively fast in suspensions of activated sludge (80) (81). It has been questioned whether these inactive conjugates are cleaved in the STP and perhaps already in the raw sewage and thereby released to the environment as active estrogens. An examination of both un-conjugated and conjugated estrogens (estradiol, estrone and ethinylestradiol) in raw sewage and sewage effluent from STPs in Germany showed that the conjugates contributed with up to approx. 50 % of the total steroid concentration in raw sewage containing 25.5 ng estrogen/l (82). The concentration of the total estrogens was reduced during the treatment process giving a total median estrogen concentration of 9.3 ng/l. However, the concentration of conjugates were still relatively high with a median value of 6.3 ng/l.

Investigations of the aerobic transformation of natural estrogens in activated sludge have shown that the possible initial degradation steps of a glucuronide conjugate of estradiol are: Estradiol-glucuronide ® estradiol ® estrone

The conjugates of estradiol seem to be de-conjugated relatively fast and the estradiol oxidised into estrone that is further eliminated. Biodegradation studies of estrogens with activated sludge form different sources showed a higher removal rate in sludge from a municipal STPs compared to the removal rate in sludge from an industrial STPs. This confirms the importance of an adapted microbial population in the biological removal of estrogens. Determination of mineralisation rates of (4-¹⁴C)-estradiol in aerated activated sludge from STPs in Måløv, Denmark showed a first order rate constant of 0.031 ± 0.003 l/d/g suspended solid (SS) at 15 °C when the concentration was less than 2.5 µg/l. No significant degradation of (4-¹⁴C)-estradiol was observed in an anoxic test system with the same type of sludge. Average sludge distribution coefficients K_d for ¹⁴C labelled compounds in the aerobic and anoxic test system with Måløv sludge were estimated to 0.25 l/g SS and 0.96 l/g SS, respectively (83). The mineralisation rate of ethinylestradiol is low compared to estradiol. Degradation of both compounds has been seen at temperatures down to 5 °C but the rate was significantly reduced at low temperature (84).

Investigations of samples taken from either the influent or the effluent from primary sedimentation tanks and final effluent from STPs in Canada, Germany, Italy, the Netherlands, Brazil and Japan show that the concentrations of estrogens are within the same ranges (85) (86) (87) (88) (89). The removal of estradiol and estriol are generally more extensive than the removal of estrone and ethinylestradiol. The removal efficiency of the four estrogens in the study of the Italian STPs, i.e. the percentage removal from the water phase, showed following average removal efficiencies: 61 ± 38 % of estrone, 87 ± 9 % of estradiol, 96 ± 6 % of estriol and 85 ± 14 % of ethinylestradiol (87). It is not possible to make any conclusion concerning the amount removed by biodegradation. It is obvious from the results obtained in batch tests that removal of the estrogens from the water phase in STPs may occur by degradations as well as sorption to sludge particles.

Fate of alkylphenols in sewage treatment plants

The alkylphenols (AP) in STPs are mainly a result of the biodegradation of alkylphenolethoxylates (APnEO). The degradation of APnEO is initiated by sequential cleaving of ethoxylated units. Under aerobic conditions the resulting products are alkylphenols, mono- and diethoxylates and the more hydrophilic carboxylates. Carboxylation may occur in both the alkyl and ethoxy side chains of the molecules. AP seems to be degradable under aerobic conditions. Thus, no detectable amounts of NP and OP were found after 35 days in a batch test inoculated with activated sludge from a STP in USA (90). The transformation of APnEO under anaerobic conditions (oxygen free conditions) as under anaerobic digestion of sludge results in production of mono- and diethoxylates and finally APs. APs are not further degraded under anaerobic conditions (91). This results, generally, in extremely high concentration of APs in anaerobically digested sludge, which may be reduced by introducing a post-aeration step (92). The mono- and diethoxylates as well as APs are hydrophobic compounds, which also are removed from the water streams within STPs by sorption to sludge particles.

Examinations of the fate of NPnEOs and their metabolites inclusive mono- and dicarcarboxylates have showed average elimination of nonylphenolic compounds (NP-c) from the water phase of 53 % and 59 % in traditional STPs mainly consisting of primary sedimentation, activated sludge processes, secondary sedimentation and anaerobic digestion (93) (94). The primary sedimentation did not significantly alter the distribution between the different metabolites (alkylphenol, alkylphenolethoxylates and carboxylated alkylphenols (APnEC)). However, APnEC was the most abundant group of the metabolites in the effluent after the activated sludge treatment. Investigations of eleven STPs in Switzerland showed that approx. 20 % of the amount of NP-c let to the STPs ended up in the digested sludge. Approx. 40 % was released with the effluent to the recipient (93).

There are no studies on the fate of APnEOs within STPs in the evaluated literature, which include all the known metabolites of APnEO and there might be even more not yet identified metabolites (95). Therefore, the potential pool of AP in the effluent from STPs may be considerably higher than known today.

Fate of bisphenol A in sewage treatment plants

Biodegradation studies of bisphenol A have shown that the compound should be easily degraded under aerobic condition in the activated sludge tank of STPs. However, degradation under anaerobic or anoxic conditions is much more unlikely to occur. It can also be expected that an amount of the bisphenol A received by STPs will be removed from the water phase by sorption. Examinations of samples from influent, effluents and sludge of Canadian STPs showed removal efficiencies from the water phase of 47-96 %. Concentration in samples of digested sludge were of 316-12,500 ng/g dry weight (96).

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

The fate of the compounds in STPs is not only correlated with the intrinsic properties of the compounds, i.e. physico-chemical properties and degradability but also the type of sewage treatment process and the operation conditions. However, treatment conditions of the STPs studied are often not completely described. Hydraulic retention time (HRT), sludge retention time (SRT), temperature, denitrification, nitrification, and phosphate elimination will all have an important bearing on the plants efficiency (95). Comparing data within single studies, in which the sampling techniques and analytical methods are identical, was thought to give the best basis for evaluation of the importance of different treatment processes. The present assessment of the effects of the type of STPs was therefore mainly based on studies with investigations of STPs with different processes.

Generally, the effluent concentrations of estrogens and alkylphenol compounds (AP-c) seem to decrease by upgrading the STPs to nutrient removal and by using other tertiary treatment process as e.g. tertiary lagoons, micro-filtration, reverse osmosis and ultra violet light (UV-light). E.g. effluent concentrations of < 0.1-0.32 ng estradiol/l have been observed in Californian STPs after reverse osmosis (97). Danish investigations of both low and high technology STPs in Århus showed that the lowest effluent concentration of both estrogens and alkylphenolic compounds (AP, AP1EO, AP2EO) generally were found in the high technology plants (98;99). The highest effluent concentration was found in a STPs with only mechanical treatment, which also showed the lowest observed removal efficiency of bisphenol A (20 – 38 %). However, effluent concentrations of nonylphenolic compounds were seen at the same level in one of the high technology plants, Søholt STP. Analysis of influent samples from this STP showed correspondingly high concentrations. Identification of the sources of NPnEOs in the catchment area showed that 52 % of the NPnEOs (n = 0-15) in the influent were discharged from a single industry. The concentrations of NPnEO (n = 0-2) in the effluent from the other high technology plants were in the range of approx. 0.2 to 1.2 µg/l. It can be concluded that the use of low technology plants, especially plants of very low technology in the so-called "open land" can be expected to result in relatively high concentrations of estrogens and xenoestrogens in the mixing zone of a recipient compared to plants of higher technology.

The highest reported effluent concentration of bisphenol A was found in the Danish Randers STP at 4,000 ng/l. The plant is upgraded to nutrient removal. It has not been possible to explain this high concentration on the basis of the available data (98).

Only few of the investigators have reported HRT and SRT of the studied STPs. However, long retention in the biological treatment step should increase the time of degradation. Furthermore, a long SRT of e.g. 25 days compared to a short retention time of e.g. 5 days may increase the possibility of establishing and maintaining a microbial flora capable of degrading the estrogens and xenoestrogens. The effect of HRT and SRT cannot be evaluated on the basis of the available data in the literature. However, an investigation of influent and effluent samples from three Dutch STPs based on the activated sludge system showed that the highest removal efficiencies of estrone and estradiol were obtained in the two plants with the highest HRTs (18 and 26 h) and SRTs (11 and 20 d) (88).

Apparently, there is a tendency to higher concentrations of especially estrogens in effluents from the U.K. compared to the other European countries. However, the data in the literature do not allow any final conclusions regarding potential differences between countries.

Influence of advanced sewage treatment processes on removal efficiencies

Conventional sewage treatment is not an effective barrier to trace contaminants like estrogens and xenoestrogens. There has, therefore, been an increasing focus on the use of more advanced treatment processes with the objective to remove the contaminants. Pilot scale studies have showed that both ozone/hydrogen peroxide and reverse osmosis effectively reduced estrogenicity of the secondary effluent from a full scale STP. No significant differences were observed using sand filtration or microfiltration on the same type of effluent (100). Laboratory experiments with estrone, estradiol, and ethinylestradiol standards showed removal in the range of 4-24 % after UV-treatment, and application of activated carbon (50 mg/l) has resulted in mean removal of estrogens from a pilot plant of 94.4 % (101). Two ongoing three years projects both initiated in 2000 are testing methods with the object of removing endocrine disrupters:

An Australian project: "Optimised Use of Membrane Hybrid Processes for Water Recycling" (ARC SPIRT Project) (102)

The EU-project POSEIDON working with the development of possible clarification techniques for increasing the removal of endocrine disrupters (103).

The key findings in the Australian project until now have been a negligible removal (< 10 %) of estrone with ferric chloride coagulation and very high removal (> 90 %) with powdered activated carbon. Magnetic ion exchange varied from 40 to 70 % removal depending on solution chemistry and dissociation of the hormone. Nanofiltration showed an initial retention of 70-95 % but, for most membranes, this retention dropped significantly after an initial filtration period. For some reverse osmosis membranes, retention was similar to nanofiltration, but others showed a very high and stable retention of the compounds. Microfiltration also showed initial almost complete retention followed by a drop as expected. The obtained results are currently being confirmed on larger scale systems (102).

The application of ozone and UV treatment of effluent from STPs is studied in the EU project POSEIDON. The use of an initial ozone treatment has been seen to improve the conditions for a following UV-treatment by decreasing absorption coefficients, which eventually improves the UV transmittance from 59 % to 84 %. The preliminary results of a pilot-scale process show a positive treatment effect regarding endocrine disrupters. The operating costs of the production of ozone were estimated to be between 0.90 and 1.60 EURO per kg ozone depending on the energy prices and the system capacity (103).

It can be concluded that more knowledge concerning the fate of both estrogens and xenoestrogens within STPs are needed. There should be high quality studies of influent, effluent, sludge and internal streams of STPs using standardized sampling procedures and validated analytical methods. The studies shall be linked to concurrent comprehensive monitoring of overall STP performance e.g. loading rate, removal efficiency of nutrient, HRT and SRT, as already suggested by Johnson et al. (2001). STPs with different treatment processes should be studied with the purpose of understanding the influence of different process designs and operation criteria. The monitoring of full-scale plants should be accompanied by studies in pilot- and laboratory-scale.

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

Besides sewage effluent other sources of estrogens to the aquatic environment might have to be considered. Two potential sources are the use of manure from life stock and sludge from sewage treatment plants as fertilisers on fields. Insufficient knowledge exist, however, on the extent of runoff of estrogens with drain water and it is not possible yet to assess whether these are considerable sources of estrogenic activity to the surface waters.

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