COMPREHENDing Endocrine Disrupters in Aquatic Environments
JAYNE BRIAN
Additional projects arose from a three-year, multinational, multidisciplinary research program in the European Union.
ALAN D. PICKERING N AT I O N A L ENVIRONMENT RESEARCH COUNCIL CENTRE FOR ECOLOGY AND H Y D R O LO G Y, WINDERMERE (U.K.) J O H N P. S U M P T E R BRUNEL UNIVERSITY (U.K.) (U.K.)
© 2003 American Chemical Society
ndocrine 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.
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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). FIGURE 1
Effluent assessment This experimental design served for the in vivo assessment of estrogenic activity of effluents from The Netherlands.
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 2001.
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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 332 A ■ ENVIRONMENTAL SCIENCE & TECHNOLOGY / SEPTEMBER 1, 2003
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
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. 1,000,000
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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.
cause 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
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 beSEPTEMBER 1, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY ■ 333 A
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 (http://europa.eu.int/comm/research/endocrine/ index_en.html). In that case, the EU funded four proposals (see the “Where do we go from here?” section),
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 COMPREHEND. 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 334 A ■ ENVIRONMENTAL SCIENCE & TECHNOLOGY / SEPTEMBER 1, 2003
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.
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
