COMPREHENDing. Endocrine Disrupters in. Aquatic Environments

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1 COMPREHENDing Endocrine Disrupters in Aquatic Environments Downloaded via on June 29, 2018 at 14:19:55 (UTC). See for options on how to legitimately share published articles. Additional projects arose from a three-year, multinational, multidisciplinary research program in the European Union. JAYNE BRIAN ALAN D. PICKERING NATIONAL ENVIRONMENT RESEARCH COUNCIL CENTRE FOR ECOLOGY AND HYDROLOGY, WINDERMERE (U.K.) JOHN P. SUMPTER BRUNEL UNIVERSITY (U.K.) (U.K.) Endocrine disruption became a topical issue during the past decade, largely because of concerns over risks to human health. The popular perception is that modern life exposes us to a cocktail of anthropogenic chemicals, some of which might interact directly with one or more components of the endocrine system, thereby causing some form of malfunction and ultimately health problems. The seriousness of this threat to humankind remains to be elucidated. However, the scientific community has somewhat misused the term endocrine disruption to cover a range of effects that are probably best described in terms of classical toxicology. Any toxic pollutant will eventually cause a secondary response in an animal s endocrine system; indeed, it would be astonishing if this were not true. Endocrine disruption should be reserved for those cases in which the primary effect of the chemical is on the endocrine system. This may include effects at the level of receptor-mediated hormone action, hormone synthesis, or clearance. Under these circumstances, extremely low concentrations of pollutants sometimes below the limits of detection for current analytical techniques may trigger very large biological effects in a susceptible animal American Chemical Society SEPTEMBER 1, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 331 A

2 FIGURE 1 In the aquatic environment, several well-documented examples describe endocrine disruption impacts on wildlife. Perhaps the clearest of these is the case of tributyltin (TBT), a component of certain antifouling paints in worldwide use in shipping and related industries, which has been shown to cause imposex in a range of marine gastropod mollusks, including the dogwhelk Nucella lapillus. Imposex is a condition in which male sex organs (penis and vas deferens) are superimposed in a female gastropod, effectively preventing the animal from reproducing. The precise nature of this form of endocrine disruption is still the subject of discussion but is believed to involve the suppression of the enzyme aromatase, which converts androgens to estrogens (1). The net effect is a shift in the hormonal balance of these animals to androgens. A marked decline in dogwelks at heavily contaminated sites is also the most convincing case of endocrine disruption causing damaging changes at the population level (2). Effluent assessment This experimental design served for the in vivo assessment of estrogenic activity of effluents from The Netherlands. River water 0% 25% 50% 100% 0% 25% 50% 100% Buffer tanks Return to river 200-L tanks carp 500-L tanks rainbow trout Effluent Meanwhile, evidence of feminized male cyprinid fish has been found in freshwaters near treated wastewater discharges (3). Both domestic and industrial effluents have been implicated in such feminizing effects, which include the presence of high levels of vitellogenin in the blood of male fish and the occurrence of oocytes in the testes of presumed male fish. Vitellogenin is a protein synthesized in the liver of fish under estrogenic stimulation and secreted into the bloodstream for incorporation, as the precursor of some of the yolk proteins, into developing oocytes. Vitellogenin production is a normal characteristic of sexually maturing female fish, but immature fish of either sex also have the genetic and molecular prerequisites necessary for its synthesis what males and immature fish of both sexes normally lack is estrogenic stimulation. Thus, significant levels of vitellogenin in the blood of male fish (or immature fish of either sex) form the basis of a useful bioassay to detect estrogenic chemicals in the water. It has not yet been established whether such signs of feminization in individual fish result in impacts at the population level, although this is a key question to investigate. To better understand these issues, the European Union (EU) commissioned a three-year research program entitled COMPREHEND (Community Programme of Research on Environmental Hormones and Endocrine Disrupters). The goal was essentially to establish whether the estrogenicity so evident in certain U.K. rivers was characteristic of the broader European aquatic environment. If estrogenic effluents could be located, the aim would be to identify the principal, active ingredients; understand their behavior and distribution in the environment; and look for evidence of impacts on aquatic wildlife. Clearly, such a challenge would require a range of skills and expertise, and it necessitated work in several European countries. Accordingly, a team of scientists was assembled from 13 laboratories across 7 countries with expertise in environmental chemistry, molecular biology, physiology and endocrinology, fisheries science, and population ecology. COMPREHEND began in January 1999, and this article highlights the final report, which was completed in December Effluent survey Twenty-four wastewater effluents from eight European countries (Belgium, Finland, France, Germany, The Netherlands, Norway, Sweden, and Switzerland) were selected for study. Most were domestic or municipal sewage effluents from a range of treatment systems, with the remaining wastewater discharges from industrial facilities. These industries included chemical manufacturing, pharmaceuticals, textiles, wood pulp, and paper. Effluent estrogenicity was assessed by an in situ bioassay. Groups of juvenile fish in cages or tanks were exposed to effluents, preferably as a series of dilutions, for a period of two to three weeks (Figure 1). Blood samples from control and exposed fish were assayed for vitellogenin, using either radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). For discharges to freshwater, rainbow trout Oncorhynchus mykiss (and, to a lesser extent, the carp Cyprinus carpio) were the preferred test species; however, the Atlantic cod Gadus morhua was used for marine outfalls. Figure 2 shows a typical estrogenic response, in which vitellogenin levels in juvenile male and female cod increased markedly following exposure to a 50% dilution of the effluent from a Norwegian domestic sewage treatment works. Curiously, not all individuals responded to the experimental exposure, a phenomenon that was also noted for the rainbow trout. Another source of variation was the choice of assay for measuring vitellogenin levels. Undoubtedly, the RIA approach was the most sensitive, but ELISAs do not require radioisotope handling facilities and were easier to perform. Heterologous ELISAs, which rely on significant antibody cross-reactivity, were not as sensitive as homologous assays that used specific antibodies raised against purified vitellogenin from the fish species 332 A ENVIRONMENTAL SCIENCE & TECHNOLOGY / SEPTEMBER 1, 2003

3 FIGURE 2 Individual differences in fish Blood vitellogenin levels of juvenile cod increased following 21 days of exposure to a 50% dilution of a Norwegian domestic sewage effluent. Note the logarithmic scale of the vitellogenin axis and the failure of three fish (in red) to respond. Blood vitellogenin (ng/ml) 1,000, ,000 10,000 1, Replicate 1 Replicate 2 Cod exposed to 50% effluent from a Norwegian sewage treatment plant under consideration. However, even when homologous ELISAs were used, it was clear that there is an urgent requirement to intercalibrate purified vitellogenin standards across various laboratories in order to compare results from different research groups. Bearing all these difficulties in mind, researchers could still categorize all effluents as strongly estrogenic, moderately estrogenic, or non-estrogenic. Moreover, it became apparent that strongly estrogenic domestic and industrial effluents are relatively commonplace in Europe, although there is considerable variation, even within a single country (Figure 3). Thus, we conclude that the earlier reports of strongly estrogenic effluents in the United Kingdom are simply part of a broader European picture. Indeed, recent research reports suggest that estrogenicity in the aquatic environment is probably a worldwide phenomenon. Whether it is considered a problem depends largely on the impact to aquatic wildlife and on the perspective of the viewer. What are the active ingredients? Having established that estrogenic effluents are currently being discharged into the aquatic environment throughout Europe, we need to identify the chemicals responsible for their biological activity. This sounds simple enough, but considering that wastewater may contain tens of thousands of individual chemicals and their degradation products, and that most analytical chemical techniques are based on pure solutions or, at most, simple mixtures, the true scale of the problem is revealed. The approach that COMPREHEND adopted combined the discriminatory abilities of analytical techniques such as gas chromatography, mass spectrometry, and high-performance liquid chromatography with the exquisite sensitivity of the biological response to estrogenic chemicals. We refer to this combination as bioassay-directed fractionation because the principle of this technique is to fractionate the complex effluent and test each fraction for estrogenicity using a bioassay. Estrogenic fractions are then pooled, further fractionated, and tested again for biological activity. This process can be repeated again, if necessary, each stage splitting the complex mixture into simpler fractions that can then be analyzed using modern, chemical analysis. The key to success is a robust, yet sensitive, bioassay that can be used to test a small volume of liquid. Of course, various checks for false negative results are necessary; however, in theory, it should be possible to identify and quantify the principal active ingredients in a complex mixture and create a synthetic mixture of these ingredients to confirm the level of overall biological activity. We examined several in vitro bioassays for their suitability in this work and eventually selected the yeast estrogen screen (YES) because it has the requisite combination of robustness and estrogenic sensitivity. This bioassay uses a genetically modified strain of yeast with an incorporated gene for the human estrogen -receptor, linked to a reporter construct that triggers the secretion of the enzyme -galactosidase into the yeast growth medium. The enzyme, in turn, converts a yellow substrate, chlorophenol red- -Dgalactopyranoside, into a red end product, the intensity of which is a quantitative estimation of the initial estrogenic activity. However, other assays could be used in this context. For example, bioassays based FIGURE 3 Effluent estrogenicity across Europe Relative estrogenic activity of different effluents examined during the COMPREHEND survey. Municipal Industrial Strongly estrogenic Moderately estrogenic No estrogenicity detected SEPTEMBER 1, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 333 A

4 The European Union and funding for environmental research The European Union (EU) consists of 15 member countries, and more will join in the next few years. EU countries and some associated countries like Switzerland contribute to funding European research projects. EU research is funded in three-year blocks referred to as frameworks. For example, Framework 6, which runs from 2003 to 2006, will provide more than 13.3 billion over the next three years for research covering a wide range of areas. Of this amount, about 2.1 billion will be allotted to environmental science studies that focus primarily on climate change. When funding research, the EU decides its priorities and then seeks proposals. The funding offices provide detailed guidance for applicants on the type of proposals the EU wishes to support. For example, early in 2002, the EU put out a call for proposals in the area of endocrine disruption, with an overall budget of 20 million ( index_en.html). In that case, the EU funded four proposals (see the Where do we go from here? section), which will collectively address the EU s priorities in endocrine disrupter research. The EU is taking a progressive approach in supporting large, integrated projects that address key issues in a particular subject area, such as endocrine disruption. These projects often involve contributions from scientists with different backgrounds, coming from many different European countries, and recently, collaboration with North American and Asian scientists has been possible. For example, EDEN (see p 426A) will involve clinicians, comparative and mammalian reproductive physiologists, toxicologists, epidemiologists, fisheries biologists, biostatisticians, and chemists. Because many of the key issues in endocrine disruption, such as quantifying exposure and understanding how organisms respond to mixtures of endocrine-active chemicals, are equally important for studying humans or wildlife, EDEN will bring experts from both camps together under one program. on human breast cancer cell lines, fish gonad cell lines, and primary fish liver cells (hepatocytes) also showed promising results. In some domestic sewage effluents, estrogenic steroids (almost certainly from human excretion) were present at sufficient concentrations to account for most of the estrogenic activity. Maximum measured concentrations were 14 nanograms per liter (ng/l) for the natural vertebrate estrogen estradiol (E2); 51 ng/l for its principal metabolite estrone (E1); 17 ng/l for another metabolite of estradiol, estriol (E3); and 2 ng/l for the synthetic steroid ethinylestradiol (EE2), which is the active ingredient in most human contraceptive pills. Although EE2 is present at extremely low concentrations, its biological potency and relative resistance to biodegradation mean it could still be one of the major factors in determining the estrogenicity of the final effluent from domestic sewage treatment works. Further work to reduce the analytical limits of detection for this steroid in complex effluents is urgently needed. Within the COMPREHEND laboratories, the limits of quantitation for EE2 were approximately 1 ng/l (concentration varied slightly with the nature of the wastewater treatment plant effluent). Yet, as we have shown, some species of fish are responsive to the steroid at such low concentrations. We were unable to detect estrogenic steroids in any of the industrial effluents examined during COM- PREHEND. However, several known industrial chemicals with weak estrogenic activity were measurable. These included nonylphenol (NP), a breakdown product from some industrial surfactants, at concentrations up to 3 micrograms per liter (µg/l); NP monoand diethoxylates at concentrations up to 7 µg/l; and bisphenol-a (BPA), a chemical used in the manufacture of plastics and epoxy resins, at concentrations up to 1 µg/l. We know from previous experimental studies on fish that such concentrations are near the lower limits of those known to have estrogenic effects on fish. However, if we are to assess the real risk of such industrial chemicals on aquatic biota, we urgently need independent verification of recent work reporting significant biological effects of octylphenol (OP) and BPA on freshwater mollusks at concentrations <1 µg/l (4). NP, OP, and BPA were also detectable in many of the domestic sewage effluents, probably reflecting wide usage of these compounds and the fact that most municipal effluent treatment facilities service both domestic and nonspecialized industrial wastes. The bioassay-directed fractionation approach also resulted in the identification of a new environmental estrogen, a form of hydroxyphenyl hexanoic acid found in the wastewater effluent from a chemical manufacturer. This can now be added to the growing list of industrial chemicals that have been shown to exhibit weak estrogenic activity. Thus it seems that the most pervasive estrogens in the aquatic environment are steroids derived from human excretion, but industrial chemicals may play a significant role in certain situations. COMPREHEND demonstrated that both the estrogenic activity and the concentration of estrogenic steroids in domestic effluents vary quite markedly among the different treatment facilities. This implies that some treatment facilities are more effective than others in minimizing (or even eliminating) estrogenic activity, a point that we will return to later in this article. Are aquatic biota affected? Within the COMPREHEND program, much of the focus was on freshwater fish. Observations of wild fish have been complemented by laboratory-based, multigeneration studies using zebrafish (Danio rerio) as the test species. The laboratory studies have demonstrated the remarkable sensitivity of fish to estrogenic steroids and, in particular, to EE2. Chronic exposure of juvenile zebrafish to very low, nominal concentrations of EE2 (1.5 ng/l) not only stimulates vitellogenesis but also sex-reverses a significant proportion of genetic males to phenotypic females. Predictably, ex- 334 A ENVIRONMENTAL SCIENCE & TECHNOLOGY / SEPTEMBER 1, 2003

5 perimental exposure to low concentrations of the androgenic steroid, methyltestosterone (MT), has a masculinizing effect (suppression of vitellogenesis and sex-reversal of females to males) but, paradoxically, high concentrations of MT have a feminizing effect, resulting in intersexuality in some fish. The explanation may lie in the conversion of androgens to estrogens by the enzyme aromatase, when fish are exposed to high androgen levels. The zebrafish studies have illustrated the potential for endocrine disruption in fish that are exposed to environmentally realistic levels of estrogenic substances; of course, they do not tell us whether such effects are actually occurring in the real world. To tackle this problem, we examined samples of wild fish taken from the areas known to have estrogenic effluents in The Netherlands and found clear evidence of elevated vitellogenin levels in male bream (Abramis brama) and carp, as well as cases of intersexuality in presumed male bream (Figure 4) and roach (Rutilus rutilus). Thus, individual fish showed signs of endocrine disruption, but how does this translate into effects at the population level? That is undoubtedly one of the most difficult questions to answer, partly because freshwater fish population structures are so strongly influenced by stochastic variables, such as water temperature, during the first few weeks of life. Fisheries statistics are expensive to collect and were beyond the budget of the COM- PREHEND program. As an alternative, we undertook a specific analysis of one of the largest existing fisheries data sets to see whether there were any indications of feminization at the level of the population. FIGURE 4 Intersex fish An example of an intersex condition is seen here in a (presumed) male bream captured in the vicinity of an estrogenic sewage effluent in The Netherlands. The left arrow indicates a primary oocyte within a lobule that consists mainly of cysts of male germ cells at various stages of spermatogenesis, such as the spermatocytes indicated by the right arrow. The Dutch database on freshwater bream populations consists of detailed information on some 25,000 fish from approximately 25 different populations. Assuming that the normal sex ratio should be equal, we examined these populations and found that 11 of them had significantly more females than males. In most cases, between 60 and 65% of the fish were female, but in one case, more than 70% of the fish were female. No case showed a significant preponderance of males. We are aware of the many factors that can influence sex ratio data in fish populations and of the risks around the assumption of sexual parity, but exposure to environmental estrogens could be one interpretation of these data. Clearly, this type of analysis can never show cause and effect, but it does at least raise the possibility of impacts of endocrine disruption at the population level. We conclude that the question of deleterious impacts of estrogenic effluents on fish populations is one of the most important that still needs to be answered. Management of estrogenic effluents The decision regarding whether such effluents need to be managed depends largely upon their impacts. It might be argued, for example, that unless or until damaging effects at the population level can be demonstrated, no environmental management is required. On the other hand, we know that high levels of vitellogenin in male fish and intersexuality are abnormal conditions, and some might consider this to be sufficient evidence on which to base mitigation strategies. The U.K. Environment Agency, for example, leans toward management. If, for the sake of argument, we adopt a precautionary approach to this problem, what management options are available? Basically, these consist of diluting effluent with the receiving water, controlling active substances at the source, or end-of-pipe technology. Effluent dilution depends, of course, upon an adequate volume or flow in the receiving water body. In many countries, this strategy would work during the wetter parts of the year but not during periods of drought. Moreover, in temperate countries the drier seasons often coincide with the main spawning period for many species of freshwater fish, thus exacerbating their estrogenic exposure during sensitive, early life stages. Controlling estrogenic chemicals at their source has been used successfully in some countries for alkylphenols and related compounds (and, to some extent, for TBT) but this, of course, cannot be applied to natural steroids of human origin. In this context, the issue of the contraceptive pill is more controversial. Theoretically, the pill could be controlled at a source, but the social implications of this would be totally unacceptable. However, whether the pharmaceutical industry could develop an effective but environmentally less persistent alternative to EE2 remains an open question. The third approach to effluent management treatment prior to discharge in the environment is worthy of further consideration. COMPREHEND has shown, using direct in vitro screening techniques, that most raw sewage influents are strongly estrogenic, whereas the estrogenicity of the final, treated effluent varies quite markedly. The implication of this finding is that some wastewater treatment systems are indeed capable of removing most, if not all, of the estrogenic activity. A closer examination of these results reveals that secondary biological treatment is the key process and that hydraulic residence time is a major factor in determining the removal efficiency. For ex- SEPTEMBER 1, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 335 A

6 ample, in activated sludge treatment plants with a residence time in excess of 15 hours, the removal of estrogenic activity was almost complete, but most plants we studied operate on much shorter residence times, typically 4 6 hours. To more than double the hydraulic residence time in sewage treatment works would not be a trivial task it would involve major expenditure and probably a complete redesign of the system. Thus, additional tertiary treatment such as membrane filtration, activated carbon, or ozonation may be more practical alternatives to consider. Having looked at some of the options for managing estrogenic effluents, we need to examine the issue of environmental targets or environmental quality standards. Working with mixtures of estrogenic compounds within the COMPREHEND program, we have found clear evidence that effects can be additive or suppressive, depending on the chemical mixture. More tantalizing still, we also obtained preliminary indications of supra-additivity in some in vitro experiments. This controversial finding requires further research before it can be substantiated. However, the overall implication is that measurement of chemical targets alone is not sufficient. Some estimation of total estrogenic activity in the effluent or receiving water is needed, and this will require bioassay testing. We have shown that in vitro techniques are feasible for such purposes but are not true surrogates for in vivo studies. For example, the potencies of estradiol and EE2 in the YES are similar, but EE2 is at least an order of magnitude more potent than estradiol in intact fish. Careful thought must be given to combining analytical chemistry and bioassay when setting environmental targets. Where do we go from here? COMPREHEND researchers provided answers to some of the questions they originally posed but, inevitably, raised additional concerns. The issue of the effects of mixtures of chemicals, particularly at low doses, requires much more research. The EU commissioned two new programs in this area (see The European Union and funding for environmental research ): Endocrine Disrupters: Exploring Novel Endpoints, Exposure, Low-Dose- and Mixture-Effects in Humans, Aquatic Wildlife and Laboratory Animals (EDEN; coordinated by Andreas Kortenkamp, School of Pharmacy, University of London, U.K.; November 2002 October 2006); and Analysing Combination Effects of Mixtures of Estrogenic Chemicals in Marine and Freshwater Organisms (ACE; coordinated by John Sumpter, Department of Biological Sciences, Brunel University, U.K.; December 2001 November 2004). COMPREHEND researchers also found evidence of androgenic activity both in sewage and in pulp mill effluents. The significance of such compounds for environmental and human health will be assessed in Comparative Research on Endocrine Disrupters Phylogenetic Approach and Common Principles Focussing on Androgenic/Antiandrogenic Compounds (COMPRENDO; led by Ulrike Schulte-Oehlmann at Johann Wolfgang Goethe University, Germany; January 2002 December 2005). In combination with the Dutch national program, National Investigation on Estrogenic Compounds (LOES), on potential endocrine disrupters in the environment, COMPREHEND reported extensive evidence of brominated flame retardants in the environment, with bioaccumulation in some aquatic organisms. The endocrine-disrupting activity of such compounds requires further investigation, and this will be undertaken in a new program called Risk Assessment of Brominated Flame Retardants as Suspected Endocrine Disrupters for Human and Wildlife Health (FIRE, coordinated by Joseph Vos at the National Institute of Public Health and the Environment in The Netherlands; December 2002 May 2006). Finally, demonstrating effects of endocrine disruption at the population level is important. Members of the COMPREHEND consortium believe that opportunities exist to apply modern molecular techniques, such as genetic sex probes for fish and the use of population genetics, to detect what might be quite subtle, but important, impacts of estrogenic effluents on fish populations. However, we are not aware of any major, new research initiatives in this area. The COMPREHEND consortium: Bengt-erik Bengtsson, Institute of Applied Environmental Research, Stockholm University (Sweden); Tim Bowmer, TNO, (The Netherlands); Anton Gerritsen, Institute for Inland Water Management and Waste Water Treatment (RIZA) (The Netherlands); Michel Gibert, Compagnie Generale des Eaux (France); Ketil Hylland, Norwegian Institute for Water Research (Norway); Andrew Johnson, Centre for Ecology and Hydrology (CEH), Wallingford (U.K.); Pim Leonards, Department of Environment, Quality, and Nutrition at The Netherlands Institute for Fisheries Research (RIVO-DLO) (The Netherlands); Tarja Nakari, Finnish Environmental Institute (Finland); Leif Norrgren, Swedish University of Agricultural Sciences (SLU) (Sweden); Alan Pickering, CEH, Windermere (U.K.); John Sumpter, Brunel University (U.K.); Mark Suter, EAWAG (Switzerland); and Anders Svenson, Swedish Environmental Research Initiative (IVL). Acknowledgment COMPREHEND was one of the last projects supported from the European Union s Programme Environment and Climate , Topic , Contract number ENV4-CT Alan Pickering recently retired as director and professor at CEH Windermere, one of the nine sites of the NERC Centre for Ecology and Hydrology. John Sumpter is a professor at Brunel University. Direct correspondence about this article to him at Department of Biological Sciences, Brunel University, Uxbridge, Middlesex, UB8 3PH, U.K. References (1) Bettin, C.; Oehlmann, J.; Stroben, E. Helgoländer Meeresunters 1996, 50, (2) Bryan, G. W.; Gibbs, P. E.; Hummerstone, G. L.; Burt, G. R. Journal of the Marine Biological Association of the United Kingdom, 1986, 66, (3) Jobling, S.; Nolan, M.; Tyler, C. R.; Brighty, G.; Sumpter, J. P. Environ. Sci. Technol. 1998, 32, (4) Oehlmann, J.; Schulte-Oehlmann, U.; Tillmann, M.; Markert, B. Ecotoxicology 2000, 9, A ENVIRONMENTAL SCIENCE & TECHNOLOGY / SEPTEMBER 1, 2003