Critical Review
Removal of Endocrine-Disrupting Chemicals in Activated Sludge Treatment Works A N D R E W C . J O H N S O N * ,† A N D J O H N P . S U M P T E R ‡ Centre for Ecology and Hydrology, Wallingford, Oxfordshire OX10 8BB, U.K., and Department of Biology and Biochemistry, Brunel University, Uxbridge, Middlesex UB8 3PH, U.K.
The release of endocrine-disrupting chemicals into the aquatic environment has raised the awareness of the central role played by sewage treatment in lowland water quality. This review focuses on the activated sludge process, which is commonly used to treat sewage in large towns and cities and which successfully removes the bulk of the organic compounds that enter the works. However, not all compounds are completely broken down or converted to biomass. For example, the estrogenic alkylphenols and steroid estrogens found in effluent are the breakdown products of incomplete breakdown of their respective parent compounds. Batch microcosm studies have indicated that estrone, ethinylestradiol, and alkylphenols will not be completely eliminated in activated sludge over typical treatment times. Field data suggest that the activated sludge treatment process can consistently remove over 85% of estradiol, estriol, and ethinylestradiol. The removal performance for estrone appears to be less and is more variable. Because of its relatively high hydrophobicity, the accumulation of alkylphenol in sludge has been observed. Although it has not been examined, accumulation of ethinylestradiol in sludge is a possibility due to its recalcitrance and hydrophobicity. A comparison between the concentrations of some of the major endocrineactive chemicals in effluents and their biological potencies has been made, to direct attention to the chemicals of most concern. While water purification techniques such as UV or activated charcoal could significantly remove these microorganic contaminants, the high costs involved suggest that research into the potential for treatment optimization should receive more attention.
Introduction The presence of endocrine-disrupting chemicals in the freshwater environment is regarded as a matter of concern by many scientists and water quality regulators (1-4). Estrogenic responses in fish exposed to sewage effluent were first reported by Purdom et al. (5), who found that caged fish exposed to effluent had very high plasma vitellogenin concentrations. Vitellogenin is an estrogenic responsive protein usually found only in female fish. Its presence in males suggested exposure to estrogenic chemicals. Subse* Corresponding author telephone: +44 (0) 1491 838800; fax: +44 (0) 1491 692424; e-mail:
[email protected]. † Centre for Ecology and Hydrology. ‡ Brunel University. 10.1021/es010171j CCC: $20.00 Published on Web 11/15/2001
2001 American Chemical Society
quent studies reported the presence of intersex roach, with oocytes in the testes, downstream of many domestic sewage effluents in the U.K. (6). The occurrence of oocytes in the testes can be induced in the laboratory by exposure to estrogenic chemicals, therefore suggesting that these wild roach had been exposed to estrogenic chemicals in the effluent. Thus, increasing attention is now being focused on the role played by sewage treatment works in preventing the release of these biologically active chemicals. Here, our knowledge of the present state of affairs based on published work is examined. While we are still at an early stage in our understanding of the fate and behavior of key endocrine disrupters and their removal efficiency, an attempt is made to predict the current situation. This paper is aimed to provoke discussion without being able to provide definitive answers. Principles of Sewage Treatment. The efficiency of modern sewage treatment is such that the overall organic loading of receiving waters is low, indeed significant improvements in water quality have been achieved over the past 50-100 yr, particularly with the introduction of the activated sludge process in 1913 (7). This, together with other improvements, has permitted the return of a range of previously absent flora and fauna to many freshwater systems (8, 9). Thus, environmental scientists have fauna to study and environmental regulators have something to protect again. Essentially, sewage treatment systems, such as activated sludge and biological (trickling) filters, rapidly convert aqueous organic compounds into biomass that is then separated from the aqueous phase by settlement. An influent biological oxygen demand of 300 mg/L may be converted to less than 10 mg/L in only a few hours (7, 10). Such rapid treatment of a semicontrolled tidal wave of waste must be acknowledged as a remarkable achievement. A sewage treatment works (STW) serving a large city will be treating as much as 30 000 ton of wastewater/h (11)! The biological treatment time in European activated sludge sewage treatment is often in the range of 4-14 h (hydraulic retention time, HRT), while biological filters, which normally serve smaller towns and villages, have an HRT of only 0.5 h. Because it is the system of choice for major urban areas, this paper will concentrate on the issue of activated sludge treatment system and microorganic contaminants, with special emphasis on endocrine-disrupting compounds. The effluent of STW does meet standards set to protect the general aquatic environment; however, these are not the same standards as met by drinking water purification plants. Scientists who work in the field of water quality in lowland rivers are becoming increasingly aware of a bewildering range of microorganic contaminants present in the water of urban and industrial catchments. A nonexhaustive list includes VOL. 35, NO. 24, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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chlorobenzene and hexane compounds (12), polyaromatic hydrocarbons (13), brominated flame retardants (14), synthetic musk fragrances (15), and dialkyl tetralinsulfonates, which are the byproducts of the linear alkylsulfonate surfactants (16). They represent the myriad waste products produced from our daily lives and industrial processes that are incompletely degraded/removed by the sewage treatment process. The study of endocrine disruption has forced scientists working in this area to consider the central role played by sewage treatment in the subject. For the purposes of this review, discussion will concentrate on two groups of chemicals suspected of causing estrogenic effects in fish; the xenobiotic estrogen mimics of the alkylphenol group and the steroid estrogens themselves. In both cases the parent compounds, the alkylphenol polyethoxylates and the estrogen conjugates, are not particularly estrogenic. However, partial breakdown in the sewer and treatment works yields some estrogenic intermediates (2, 17, 18). While increasing amounts of data on the fate of these compounds in sewage treatment are becoming available, one must always be aware of the difficulties of intercomparability between the data sets. The treatment conditions at the STW studied are often not completely described by the scientists involved. HRT, sludge retention time, temperature, denitrification, nitrification, and phosphate elimination will all have an important bearing on the works efficiency. Also sampling strategy and analysis may vary between different studies. Thus, apparently similar activated sludge treatment plants may give very different results. Similarly, where laboratory experiments are conducted using semicontinuous activated sludge (SCAS) techniques, often high spiking concentrations are used, which may select for an adapted microbial population that would not develop under normal conditions.
Fate of Alkylphenol Polyethoxylates For decades, alkylphenol polyethoxylates (APE) have been economically important as nonionic surfactants used in a variety of industrial and household applications. However, breakdown products such as the 4-tertiary isomer of nonyl and octylphenol have been shown to be estrogenic to fish (19). It was estimated that the nonylphenol polyethoxylates (NPEO) could represent between 4 and 10% of the total DOC entering a STW, although in many cases, this is less likely to be the case today due to use restrictions in many countries (20). Early research concentrated on the biodegradation of the parent polyethoxylates, which was clearly demonstrated to be successfully eliminated in an activated sludge environment (21). However, it was then realized that a wide variety of breakdown products were formed (20, 22). Apart from nonylphenol (NP) itself, these included shorter chain ethoxylates, particularly nonylphenol mono- and diethoxylates, and also nonylphenoxy carboxylated varieties, principally nonylphenol mono- and diethoxy carboxylates. Reducing the length of the ethoxylate chain has the effect of increasing the hydrophobicity of the molecule. Giger et al. (23) found that the concentration of NP and these intermediates decline following passage through the aeration tank and final settlement tank, while the more hydrophilic carboxylate products increased and hence escaped into the receiving water (20, 24). More recently, double carboxylated products have been discovered in sewage effluent (25) in which both the alkyl and ethoxy side chains become carboxylated to make carboxyalkylphenoxy ethoxy carboxylates (CAPECs), thus making a particularly hydrophilic product representing as much as 63% of the NPE breakdown products (22, 26). The finding of the double carboxylates only 5 yr ago demonstrates that we can be mislead by an incomplete understanding of the environmental fate of an apparently well-researched group of chemicals. Perhaps there are still 4698
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more metabolites to be identified? Despite the hydrophilic nature of the CAPECs, batch microcosm experiments indicated that these compounds were more persistent in the activated sludge environment than other NPE metabolites (22). With a log Kow (octanol-water partition coefficient) of 4.5, NP is a hydrophobic molecule (27), and at temperatures of 10-15 °C, which are quite typical for much of the year in Europe, it preferentially distributes into the sludge fraction (28, 29). Using the data from Tanghe et al. (29) in which 3-4 g of sludge/L was maintained in a SCAS system at 10-15 °C, it can be calculated that between 60 and >90% of the spiked NP went into the sludge. Collected primary and secondary sludge are normally treated by anaerobic digestion. However, NP appears to persist under these conditions (20, 30, 31), and similar studies of anaerobic biodegradation with OP appear to show persistence in the activated sludge environment (32), which is certainly the case with river sediments (33). Following further treatment, much sludge is now utilized on agricultural land. There are some encouraging data indicating that NP would not persist in sludge-amended soil, a removal of NP by more than 90% in soil under normal field conditions within 1-3 months being observed by Marcomini et al. (34) and Kirchmann et al. (35) using methanol or hexane/ ethanol, respectively, for their soil extractions. In a recent Danish study, NP was mineralized in homogenized sludgeamended soil in 38 days at 20 °C,; however, mineralization was greatly retarded when 2-cm sludge aggregates were mixed with the soil (36). Poor mineralization was associated with local anaerobic conditions within the sludge aggregations. Not all the NP becomes bound to sludge, and low microgram per liter concentrations can be found in the effluent; Ko¨rner et al. (37) examining modern STW in southern Germany estimated OP and NP running through the plant at between 0.1 and 3.6 µg L-1. In Switzerland, the much studied STW on the river Glatt near Zurich has had NP values of 1-14 (38), 2.7 (28), and 1-4 µg L-1 (20). In Italy, Di Corcia et al. (39) found NP values of 0.7-2.6 µg L-1. Much higher concentrations have been reported where particular industries, such as textiles, exist (1, 40). A proportion of these APE breakdown products can partition into the river sediment (13, 24) or marine sediments (41) and persist for longer than might be predicted purely on the basis of aerobic degradation rates. Theoretically, once in the receiving water, intermediates may be converted to the estrogenic alkylphenols in the river, thus increasing the overall estrogenicity with increasing distances downstream of the effluent pipe. However, in a microcosm experiment with river water spiked with NPEO, all the metabolites discussed above were formed with the exception of NP after a 33-d incubation (42). This example of the fate of APE is a classic example of the mixed benefit of the activated sludge process with regard to some organic contaminants. On a basic level, it has successfully eliminated the parent compound, but incomplete biodegradation and sorption release breakdown products into the effluent potentially more harmful to the environment than the original compound.
Fate of Steroid Estrogens While the breakdown products of APE compounds are routinely found in sewage effluent, a toxicity identification and evaluation procedure using an in vitro estrogen yeast assay identified the steroid estrogen component of the effluent as contributing the greatest proportion of the overall estrogenic activity (2). Similarly, an in vitro test using human breast cancer MCF-7 cells, which proliferate when exposed to estrogens, suggested that the xenobiotic estrogens, such as NP, in the sewage effluent constituted only 1-4% of the overall estrogenic potency (37). It should be noted of course that in vitro assays may not perfectly reflect the true in vivo
response of fish to the same compound. Nevertheless, overall, the steroid estrogens appear to be the most potent endocrine disrupters of sewage effluent, at least in vitro. As with APE, steroid estrogens mainly enter the sewage system in a form that is largely not estrogenic. The natural and synthetic steroid estrogens are excreted primarily as a variety of inactive glucuronide or sulfonide conjugates by the human population (43, 44). However, the deconjugated steroid estrogens are detected in the influent at concentrations close to the total expected based on excretion values (11). Despite specifically examining raw influent for the glucuronide conjugates, none were detected by Belfroid et al. (3). Indeed it might be predicted, given that the β-glucuronidase enzyme is common in bacteria found in sewage (45), that the glucuronide moiety would be rapidly cleaved and metabolized in such a biologically active matrix as raw sewage. Indeed Panter et al. (46) found that the estradiol-3-glucuronide conjugate of estradiol (E2) was very readily converted to the active hormone E2 both in a SCAS system and in the dechlorinated tap water used to dose the system. This strongly suggests that no glucuronide conjugates would survive the sewer system. However, a study by Nasu et al. (47) showed E2 concentrations still increasing from raw influent to primary effluent within Japanese STW (before falling during biological treatment), which suggested that perhaps some further deconjugation was still taking place. Estrone (E1) is believed to be more commonly excreted as a sulfonide conjugate (43), which might be expected to be more persistent since the arylsulfatase enzyme is likely to be less common (48). Thus, the origin of E1 in sewage influent is unclear; some or all may be the byproduct of biodegradation of E2 in the sewer, or alternatively it is largely due to deconjugation of the E1 sulfonide in the sewer. A simple prediction method suggested that the amounts of E2 and E1 reported in the literature arriving in sewage influent were close to that which would be excreted by an “ideal” population following complete deconjugation (11). Again it would appear that the sewage system may transform inactive compounds into ones that inadvertently are potentially more harmful than the original. But until there is further information, there remains the possibility that perhaps most estrone-3-sulfate is passing through the treatment system and into the receiving waters. Fate of Steroid Estrogens Determined by Laboratory Studies. The principal mechanisms for removal are likely to be sorption and biodegradation. In the absence of literature on sorption potential to sludge, only rough predictions can be made. E2 is considered to be weakly hydrophobic (log Kow 3.1; 49); a modeled value for E1 has been given as log Kow 3.4 (50). Estriol (E3) would be predicted to be more hydrophilic (log Kow 2.7; 50), having an additional alcohol (16R) group. So, binding to sludge would be unlikely to dominate their fate. However, the synthetic steroid 17R-ethinylestradiol (EE2) is somewhat more hydrophobic (log Kow ∼3.9; 49; log Kow 4.1; 50) than the other steroid estrogens, perhaps 10 times greater than E2. Therefore, removal from the aqueous phase by sorption to sludge is likely to play a more important role with this compound. It is possible to obtain information indirectly on the sorption potential of EE2 from data produced from batch degradation experiments. For example, when Layton et al. (51) used fresh sludge at a concentration of 2-5 g L-1, it was noted that at the first sampling point (1 h), when no mineralization had taken place, that only 20% of labeled EE2 remained in the aqueous phase, thus 80% would appear to have bound to the sludge. The more rapid degradation of the natural steroids in these systems make sorption deductions much more difficult. In the case of removal by biodegradation, Ternes et al. (45), using an activated sludge (diluted 1:5 in water) batch test system, witnessed little or no EE2 transformation over 20 h. Vader et al. (52) used activated sludge in a laboratory
reactor with ammonium and hydrazine as the energy sources and found good EE2 removal (approximately 28 h half-life) with sludges that nitrified. The sludges that failed to nitrify significantly also failed to degrade EE2. They predicted that STW that favor nitrifying bacteria with long sludge retention times and warm summer temperatures would successfully eliminate EE2. It is difficult to evaluate the importance of these observations given the absence of organic substrates in the experiments. However, there is some encouragement that sorbed EE2 would be eliminated by biodegradation on sludge within the aeration tank given normal sludge retention times. While not a direct measure of transformation, Layton et al. (51) noted only 20% EE2 mineralization as compared to over 75% for E2 over 24 h in experiments with undiluted mixed sewage liquor. Ternes et al. (45) similarly observed the rapid transformation of E2, with over 90% of E2 (initial concentration 1 µg L-1) converted to E1 within 30 min. E1 appeared to persist in the system over the duration of the 4-h test, but Ternes et al. (45) observed a 50% loss after 24 h. These laboratory data suggest that some EE2 and E1 should be expected to persist and be found in the effluent but that much less E2, if any, would be expected to emerge from the sewage works. However, the following should be noted: (i) The batch test system is likely to overestimate the true degradation rates. These experiments were carried out at 20 °C, whereas field conditions will be more frequently in the 10-15 °C range. Aeration would have been likely to be consistently high in a shaking flask, which may not always be the case within an activated sludge aeration tank. (ii) Perhaps not all the conjugates are transformed prior to entering the STW; instead a proportion may be deconjugated toward the end of the biological treatment, yielding the free steroids into the effluent. Ternes et al. (45) still detected 3% of the glucuronide conjugates after 28 h in their batch experiments. (iii) Perhaps some of the previously bound E2, possibly protected from degradation, dissociates from the large floc particles as “third phase” microparticles, which fail to settle in the final clarification stage and emerge in the final effluent. The analysis/filtration process may extract these microparticles inadvertently. Assessment of Treatment Steroid Estrogen Removal Based on Field Data. A review of steroid estrogen removal efficiency using data generated by other researchers using composite sampling protocols suggested that 88% of influent E2 and 74% of influent E1 was removed by activated sludge plants (11). A recent study of 27 STW in Japan gave a mean value of only 70% E2 removal using enzyme-linked immunosorbent assay (ELISA) (47), which is less than that predicted from most studies. Perhaps the ELISA method picked up more E2 in the effluent then can be detected by conventional methods? It has been argued that ELISA can be a robust method of analysis for these complex matrixes and compares well with gas chromatography/tandem mass spectrometry (53). Strictly one cannot give values for E1 elimination/ removal rates since it is also clearly generated in situ following E2 breakdown. A recent assessment of six STW around the city of Rome suggested on average 87% removal of E2, 61% removal of E1, 85% removal of EE2, and 95% removal of E3 (48). These data of Baronti et al. (48) are worthy of close examination and comparison with the available laboratory data. From five separate determinations, spike recoveries of 84-86% for EE2 and limits of quantification of 0.9 ng L-1 STW in the influent and 0.3 ng L-1 in the effluent were achieved (48). Routine detection of EE2 in effluents at concentrations below 1 ng L-1 have not previously been reported (2, 3, 54) presumably because of the great difficulties VOL. 35, NO. 24, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Mean Steroid Estrogen Removal Values with Standard Deviations Taken from Baronti et al. (48) STW Cobis Fregene Ostia Roma Sud Roma Est Roma Nord
E2% removal
EE2% removal
E1% removal
89 ((10, n ) 5) 87 ((11, n ) 5) 84 ((3, n ) 5) 76 ((13, n ) 5) 92 ((2, n ) 5) 92 ((3, n ) 5)
87 ((15, n ) 5) 84 ((19, n ) 5) 84 ((18, n ) 5) 83 ((15, n ) 5) 85 ((10, n ) 5) 87 ((9, n ) 5)
86 ((6, n ) 5) 94 ((1, n ) 5) 22 ((22, n ) 5) 19 ((36, n ) 5) 84 ((8, n ) 5) 65 ((33, n ) 5)
mean removal 87 ((9, n ) 30) 85 ((14, n ) 30) 61 ((38, n ) 30)
in handling low concentrations in such a complex matrix. A mean influent EE2 concentration of 3 ng L-1 and 0.4 ng L-1 effluent concentration was recorded by Baronti et al. (48). Thus, it must be acknowledged that the effluent values were at the limit of detection and quantification. The rate of EE2 removal across STWs was strikingly consistent, including Roma Sud where E2 and E1 removal performance was poor (Table 1). On the basis of biodegradation alone, less EE2 removal by the STW would have been predicted since minimal transformation was observed in the microcosm tests of Ternes et al. (45). However, as discussed earlier, the experiments of Layton et al. (51) with 14C-labeled EE2 indicated that as much as 80% of the EE2 could bind to the sludge component and thus be removed from the aqueous phase in this way. Therefore, a possible explanation of the consistency in the EE2 removal rate is that it is largely independent of fluctuations in the biological performance of individual STW, the greater hydrophobicity of EE2 making it more susceptible to the passive process of sorption than the other steroid estrogens (50). It must be admitted that, while other authors have used different techniques, no such consistent pattern with EE2 removal has previously been observed. Some Dutch STW showed removal of EE2 to below detection (11), some Italian STW removed 30-90% (11), a Rio de Janeiro STW removed 80% (54), whereas a Swedish (55) and a German STW (54) failed to remove EE2. In addition to EE2, a feature of the six STWs studied by Baronti et al. (48) was the overall consistent removal performance shown with E3 and E2 both between different STWs and also for individual STW sampled in different months. E2 and E3, with lower Kow values, would be more dependent on biodegradation for removal. Laboratory data indicated that E2 biodegradation would be fast (45), and we can observe that even where E1 removal was poor (Ostia and Roma Sud), good E2 removal occurred. Good E2 removal would appear to be fairly commonplace, a value of over 90% has previously been observed in Italian and Dutch STW (9) and Rio de Janeiro STW (54) but less so with a German STW with 64% removal (54), although it must be recorded that this plant in Frankfurt was operating when the air temperature was only -2 °C. With a mean removal rate of 95%, E3 would appear to be the most biodegradable of the studied steroid estrogens (48). Similar high removal values of 8095% were reported for E3 in Canadian STW (56). An extraordinary contrast between the removal of E1 and that of other estrogens (Table 1) was observed by Baronti et al. (48). Not only was performance between STWs very variable, but also an individual STW varied a great deal. The authors did not indicate that E1 analysis in their samples was any more difficult than that of the other steroids, suggesting that this variability was a real observation. Likewise in the Dutch STW (11), the mean E2 removal for three plants was 92% (n ) 5, SD 10) but for E1 was less; 82% and with a wider variation (n ) 6, SD 16). A German plant studied (54) removed 64% of E2 but only 10% of E1. In five Canadian STW while E2 was routinely removed to below detection limits, E1 removal appeared far more erratic with values of 34%, 68%, 75%, 77%, and 85-87% being observed (56). 4700
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As with E2, E1 would be predicted to sorb much less than EE2, thus making biodegradation the most important removal process. However, unlike E2, the lab batch studies have shown E1 to be much more resistant to biodegradation (45). On occasions, more E1 has emerged in the effluent than was found in the influent (48). However, this apparent anomaly may be explained if we assume that E2 not found in the effluent has been converted to E1. It has been observed previously that E1 is the first byproduct of E2 biodegradation (45, 49). For example, for Ostia STW in October, of 18 ng L-1 E2 in the influent, 3 ng L-1 was found in the effluent (48). So if we assume that the remaining 15 ng L-1 was converted initially to E1, this would change the E1 pool from 67 to 82 ng L-1, which is identical to the 82 ng L-1 actually found in the effluent. This would suggest that on these occasions no E1 biodegradation took place. Almost all the other cases where more E1 was found in the effluent than in the influent by Baronti et al. (48) can be made to balance in this way. It is an interesting exercise to compare the observed field removal rates for E1 of Baronti et al. (48) with a half-life (10 h) taken from the data of Ternes et al. (45) using activated sludge diluted 5-fold. For example, taking Cobis STW, the removal of influent E1 was 86%. Assuming first-order degradation of E1, we may write
Ct ) Coe-Kt where Ct is the concentration of E1 at time t, Co is the influent concentration, and K is the first-order degradation rate. A 10-h half-life equates to 0.0693 h-1 and would predict ∼62% degradation of E1 during treatment, which was what was observed at Roma Nord STW (Table 1). The removal performance for E1 (Table 1) at the Cobis STW exceeds that predicted, whether or not we include E2 conversion to E1. In fact, to obtain 88% degradation over 14 h would require an E1 half-life of 4.6 h. It is difficult to say at this stage whether specific microflora are required to achieve the high E1 removal rates or whether it is related to the conditions, nutrients, or toxic effects occurring at the different plants. Perhaps the removal success of E1 could be considered as a sensitive indicator of plant biodegradative performance. However, although these recent data have given some interesting pointers, more data are needed before robust conclusions can be reached.
Evaluating Plant Performance against Relative Potencies of Endocrine Disrupters in Effluent From the point of view of tackling endocrine disruption, which compounds should we be most concerned about? A preliminary assessment may begin by assessing typical effluent concentrations against what is known about biological potencies either in vivo or in vitro (Table 2). Recent data by Thorpe et al. (57) using juvenile rainbow trout suggest that both the steroid estrogens and alkylphenolic estrogens can affect fish together in an additive way, thus we can try to predict the total impact of each endocrine disrupter based on “typical” effluent concentrations (Table 2). This might lead us to the following assessments: E1. Although it may have only half the potency of E2, it is frequently found at concentrations in the effluent greater than double that of E2. It is also very consistently found in effluents. STWs appear less effective at removing this compound as compared to the other steroid estrogens. As EE2 concentrations are often very low, or perhaps in some cases undetectable, an increase in the ability of STW to remove this compound may have a significant impact on the overall estrogenicity of the effluent. On the basis of the in vitro estrogenic potency and concentration, this would be the most important endocrine disrupter. However, in terms
TABLE 2. Relating Endocrine Disrupter Concentrations in Sewage Effluent to Potential Impacts on Wildlife
compd
in vitro estradiol equiv
typical effluent concn (ng L-1)a
E1 E2 E3 EE2 4t-NP or 4t-OP NPEOs, NPECs, & CNPECs
0.5 (59) 1 (59) 0.005 (59), 0.04 (18) 1-2 (71, 72) 0.0001 (19) 0.00001 (17, 18)
5 1.5 20 0.5 2 000 20 000
totalb a
typical predicted E2 equiv (in vitro)b 2.5 1.5 0.1 0.5-1 0.2 0.2
in vivo VTG response in trout estradiol equiv 0.5 (66) 1 0.001 (18) 25 (58) 0.001 (66), 0.0006 (57) no significant NPEO mixture effect at 100 µg/L (18)
5-5.5
Typical steroid concentrations from refs 11 and 48; alkylphenols and metabolites from refs 22 and 24. work in an additive way.
of the environment, a greater weight should be given to in vivo potency and here, assuming we really are detecting it in the effluent, EE2 would come out as far more important. E2. Although it has the greatest potency of the endocrine disrupters, bar EE2, given typical effluent concentrations, its overall impact on the estrogenicity would appear to be less than E1. Many STW using current practices appear to have good removal performance with this compound; for example, in some Dutch, Canadian, and Brazilian STWs, its complete elimination has been reported (11, 54, 56). E3. While high effluent concentrations have been reported (48), its relatively low potency as compared to other steroidal estrogens gives rise to less concern. EE2. On the basis of in vitro potency and its low concentration, EE2 would not be selected as the key player. However, it is undoubtedly an extremely potent estrogen based on recent in vivo studies (58). Potentially EE2 could be the most important endocrine disrupter in sewage effluent, yet given difficulties in measuring this compound so close to the present detection limits of most analytical techniques, it is also the compound most difficult to evaluate. Thus, the reduction/elimination of EE2 could have the biggest single impact on the estrogenicity of the effluent. OP/NP. The in vitro tests suggest that in most cases they would not be important players, but where they are present in effluents at concentrations exceeding 1 µg L-1, the in vivo studies indicate that their impact could be at the same level as that of E1/E2 or higher. In rivers contaminated at concentrations above 1 µg L-1, these compounds could be the most important endocrine disrupters present. However, there is a central problem in assessing the significance of endocrine disruption due to these compounds that should be born in mind. Routledge and Sumpter (59) clearly indicated that only the 4-(para-substituted)-tertiary isomers had significant estrogen-mimicking potential (1000-10 000 less than E2), while the other isomers were a great deal less potent. It would appear that commercially available NPE products begin with the production of p-nonylphenol. This product has been assessed as containing a mixture of o-nonylphenol, p-nonylphenol, and decylphenol (60). The p-nonylphenol (also described as 4 or para) has been reported by Wheeler et al. (60) as containing 22 isomers, 17 of which are tertiary, thus only 75-80% of NP found in effluents would be predicted to be in the estrogenic category. It is not entirely clear if the steroid estrogens and xenobiotic estrogens would work together in fish in an additive way, although they may well do so (57). As these chemicals originate from man-made products, we do not soley have to look for treatment improvements to reduce the risk, as their use could be restricted as already the case in some countries.
typical predicted E2 equiv (in vivo)b
judgment
2.5 1.5 0.02 12 2-20? ?
concern concern little concern greatest concern concern less concern
18.1-36 b
Assuming the combined compounds
APE Metabolites. The most potent metabolites of the longchain ethoxylates are the alkylphenols OP and NP, as described above. In general, the shorter the ethoxylate/ carboxylate side chain, the more estrogenic the metabolite (59, 17). The short-chain ethoxylates (NP1EO, NP2EO, etc.) are less active but not radically so. There is little information and little agreement about the carboxylates. Jobling and Sumpter (19) and Jobling et al. (61) tested them both in vitro and in vivo and found them to be reasonably active. However, more recent in vivo data (18) reported little or no estrogenic activity with these metabolites using Japanese Medaka. Although the CAPECS have not so far been tested, it would appear that the most potent of the metabolites is NP1EC, which is 10 times less potent than 4-t-OP, at least in vitro (17). Given their reported high concentrations in effluent (26), CAPECs could be significant if they have an endocrinedisrupting potential. Reasonably pure carboxylates and CAPECs would need to be tested for endocrine activity to produce the required data. Therefore, if we made an assessment soley on in vitro potency, E1 and E2 would emerge as the key players (Table 2). However, if we based our assessment on the more relevant in vivo potencies, then EE2 and OP/NP could account for as much as 90% of the estrogenicity of a typical effluent. As these chemicals enter the aquatic environment, other issues would inevitably affect our assessment over which chemicals pose the greatest endocrine-disrupting risk. While wildlife are likely to possess serum proteins that can sequester steroid estrogens, this is less likely to be the case for the alkylphenol family (62), and bioconcentration (perhaps up to 300; 63) may play a magnifying role with the hydrophobic octyl and nonylphenols. The alkylphenol group would be relatively persistent in river water (DT50 for OP 7-50 d), and they also have a strong potential to bind to bed sediments (33, 50, 64). Thus, the impact of these xenoestrogens in a river system may be greater than that predicted purely from effluent in vitro tests. The natural steroid estrogens would be predicted to be more rapidly degraded in river systems than the alkylphenols, with DT50 values of 1-4 d (49), but EE2 would maintain its position as one of the endocrine disrupters of greatest concern because of its persistence with a DT50 in the order of 20-40 d (49). The impact of the estrogens will also depend on what target organisms are being considered. For example, if considering a mid-river (pelagic) fish, like the roach (Rutiliz rutiliz), we presume that the concentration of the estrogens in the water column will be critical. However, if considering a bottom-living (demersal) animal (such as barbell, chub, or in the marine environment, flatfish) or organisms that live in the sediments (e.g., molluscs) then estrogen distribution onto and persistence within the sediments may be the most VOL. 35, NO. 24, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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important factor. Hydrophobic estrogens such as OP and NP will bind to sediments (24, 64) and, under anaerobic conditions, persist (33). Uptake of estrogenic chemicals via food may also be a significant route of exposure. However, chemicals entering organisms via the oral route may be digested and/or metabolized before reaching target tissues (such as the gonads), which may well reduce the significance of this route of exposure.
Significance of Endocrine Disruption in Aquatic Environment There is good evidence that in a number of aquatic environments indigenous fish can be found that exhibit symptoms of endocrine disruption (6, 65). However, this is still a long way from demonstrating a significant threat exists to indigenous fish populations from this cause. Even so, many would consider the potential threat serious enough to warrant some action. The field data suggest that the European activated sludge treatment plant, with a hydraulic residence time not greater than 14 h, in most cases cannot completely eliminate all the steroid estrogens or alkylphenols from the effluent. However, this needs to be confirmed with more high quality studies. We do not yet have all the information we need to identify the most important endocrine-disrupting chemicals in the aquatic environment on the basis of plant removal performance versus potency. However, any current assessment would highlight E1 on the basis of its concentration, relative persistence in treatment, and potency. EE2 would also be highlighted for its even greater persistence in treatment and potency, although its concentration may often be too low. The alkylphenol NP must also remain a concern due to its concentration, persistence, and potency. The concern of environmental regulators is that even the small quantities of endocrine-disrupting compounds that do escape may be sufficient to cause disruption in fish, particularly where dilution is poor (6, 66). This is an important issue in the U.K. where some rivers are close to 100% effluent in summer (67) and may also be an issue in other countries with high population densities and insufficient large watercourses in which to dilute the effluents. Both changes in seasonal flow rates and possible fish vulnerability windows must also be considered. While winter temperatures may reduce biodegradation rates, the large increase in dilution due to winter rains may ensure that endocrine-disrupter concentrations would remain below the no effect level. While not all fish follow similar patterns, it might be predicted that fish may be vulnerable to lasting effects of endocrine disruption following exposure to the chemicals at uniquely sensitive periods of their life cycle. Aside from endocrine disrupters, the discovery of other potentially biologically active compounds, such as antibiotics (68) and other pharmaceuticals (69), in effluent will surely increase the focus on sewage treatment in the years to come. Removing many of these compounds from the effluent is technically possible today (70). However, the techniques of UV treatment, ozonation, and granulated activated charcoal, which may well be effective, would be costly to implement. While the general public is willing to pay for “pure” drinking water, it would be much less likely to welcome a significant increase in water bills to ensure that fish do not suffer endocrine disruption or become tainted with perfume! It is ironic that the sewage treatment system that permitted the return of fish to previously grossly polluted river systems may now be seen in some quarters as the culprit in releasing substances harmful to fish.
Identification of Research Needs On the basis of the current appreciation presented in this paper, a number of research priorities can be suggested: 4702
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(i) Improvements in chemical analysis to reduce detection levels, and hence improve accuracy, for the steroid estrogens, particularly ethinylestradiol. Perhaps further exploration of ELISA methods may be profitable here. (ii) More high-quality national studies of influent and effluent concentrations of endocrine disrupters in different wastewater treatments are needed. Evaluating those STW that already possess tertiary treatment would be of particular interest. Studies should be linked to concurrent comprehensive monitoring of overall STW performance. A series of studies on individual STWs should be included to take into account variations in treatment performance with time of day and season. (iii) Studies focused on the fate of estrone-3-sulfate in the sewer system and activated sludge environment either by monitoring in the field or by laboratory microcosm studies. (iv) More fate and behavior studies of steroid estrogens, including sorption potentials, in simulated sewage systems and in rivers downstream of STW. (v) Studies to determine the fate of EE2 bound to sludge under aerobic and anaerobic conditions and the fate of EE2 in soil amended with sludge. (vi) Better biological data on what chemical, or mixture of chemicals, is actually causing the effects observed in wild fish. This will ensure that the analytical and fate studies (outlined above) can be conducted on the right chemical(s). (vii) The hypothesis that the degree of endocrine disruption is primarily connected to the quantity of human-excreted steroid estrogens in a river reach should receive more field testing. This could be accomplished by a coordination of predictive STW modeling/hydrology with fish biology studies targeted at best and worst case river reaches.
Need for Cost-Effective Improvement in Sewage Treatment A desire for greater removal of microorganic contaminants from sewage effluent is something that the endocrine disruption issue may stimulate. The future must lie in a reassessment of the ubiquitous activated sludge process and an exploration of the existing potential to enhance its biodegradative and sorptive capacity. The field data indicate that on occasions 99% removal of steroid estrogens has occurred within existing activated sludge plants, so this objective (increased efficiency) is by no means a lost cause. Ideally the HRT could be doubled to allow much more complete biodegradation to occur; however, this would require much larger sewage works. The challenge is for water treatment scientists to design a “super-activated sludge works” that does not increase the plant’s current size or running costs yet removes the microorganic contaminants more efficiently.
Acknowledgments The authors are grateful to the EU COMPREHEND project for support and wish to thank Richard Williams for assistance with the manuscript.
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Received for review June 21, 2001. Revised manuscript received September 19, 2001. Accepted September 21, 2001. ES010171J
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