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Repeating History: PHARMACEUTICALS in the ENVIRONMENT
Research and resource investments made to understand and assess endocrine-active chemicals can help scientists to define the ecological risks of pharmaceuticals. GER A LD T. A NKLEY U.S. EPA BRYA N W. BROOKS BAY LOR UNIV ERSIT Y DUA NE B. HUGGETT UNIV ERSIT Y OF NORTH TEX AS JOHN P. SUMPTER BRUNEL UNIV ERSIT Y (U.K.) © 2007 American Chemical Society
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s state-of-the-art analytical techniques become more sensitive and more widely deployed, an increasing number of human and veterinary drugs are being detected in environmental samples (1–3). However, comparatively little is known about the possible ecological risks of most of these chemicals. This lack of knowledge has resulted in a substantial amount of ongoing effort to develop data and approaches that might prove useful for assessing the impact of pharmaceuticals in the environment. The current situation with pharmaceuticals is analogous to one in the early 1990s, when several very visible reports about endocrine-active substances resulted in an enormous amount of public attention, scientific research, and regulatory activity. Many effects of endocrine-disrupting chemicals in humans and wildlife have been hypothesized (4). From an ecological perspective, one of the best-documented examples involves feminization of male fish exposed to municipal effluents (4). Pioneering work in this area was done in the U.K. (5), and in recent years feminized male fish have been found at effluent-impacted sites all over the world (4). December 15, 2007 / Environmental Science & Technology n 8211
JASON BERNINGER
The nature of the feminization observed in male fish indicates that the phenomenon is due to chemicals that activate the vertebrate estrogen receptor (4). Although many chemicals can interact with the estrogen receptor, the weight of evidence to date indicates that the substances most likely responsible for a substantial amount of the estrogenic activity of municipal effluents are natural and synthetic steroids, including ethinylestradiol (EE2), a synthetic estrogen used as a pharmaceutical in humans (6– 9). In retrospect, it is not surprising that EE2 would affect extant fish populations; it is widely used in developed countries, is relatively stable in the environment, is continuously introduced to aquatic systems from point sources, targets a specific (and
in a categorical exclusion from testing. In the case of veterinary drugs, only aquaculture-related medicines are subject to aquatic testing if the water PEC is >1 μg/L (10, 11). For other veterinary drugs, aquatic testing is required when soil PECs are >100 μg/kg (12, 13). The use of dissimilar approaches to initiate ecological effects testing of human versus veterinary drugs could result in different conclusions about the environmental safety of chemicals, depending on their use classification. One obvious problem with this approach is that some products used to treat humans and animals are the same—the nonsteroidal anti-inflammatory drug diclofenac, certain steroidal compounds, and many antibiotics are good examples. Although exposures may differ, their potential effects on nontarget organisms would be the same, so effects-testing approaches should be similar. Even when human and veterinary drugs are different chemicals, they can possess properties that are similar enough to warrant a common approach to ecological effects assessment. For example, EE2 and 17-trenbolone, a synthetic steroidal androgen used in livestock, are quite analogous in that they both impact the reproductive endocrine system of fish at very low yet environmentally relevant (nanograms-per-liter) concentrations in the water (8, 14, 15). However, current testing approaches would consider their ecological risks differently.
Production volume as a guide for pharmaceutical testing
Treated municipal wastewater is discharged into Pecan Creek, an effluent-dominated stream in Denton, Texas.
evolutionarily conserved) biological pathway, and is highly potent. Although other classes of pharmaceuticals affect different biological pathways than EE2 does, many have these same general properties. Therefore, we feel that the substantial research and resource investments made to understand and assess endocrine-active chemicals (such as EE2) can help to focus current efforts to define the ecological risks of other classes of pharmaceuticals. In this article, we identify several issues associated with assessing the risks of pharmaceuticals and discuss how experiences with endocrine-disrupting chemicals can help scientists address these issues.
Human vs veterinary drugs Approaches currently used to assess potential ecological effects of human and veterinary drugs are in some respects dissimilar (2, 3). In the U.S., for example, the expected environmental concentration (more commonly termed the predicted environmental concentration; PEC) is used differently to trigger ecological effects testing for human drugs versus those for livestock. A PEC for human pharmaceuticals of >0.1 μg/L necessitates aquatic ecotoxicity testing, whereas a lower concentration results 8212 n Environmental Science & Technology / December 15, 2007
Intuitively, it seems reasonable to base concern about possible toxicity (of any chemical) on production volume. The more of a chemical produced, the greater the concentration likely to be present in the environment, and therefore (the argument goes), the greater the possibility that adverse effects on organisms will occur. On the basis of this reasoning, most environmental legislation aimed at protecting wildlife from industrial chemicals sets production volumes below which either no testing is required, or the testing is tiered, becoming progressively more rigorous as production volume increases. A similar argument is used to exclude pharmaceuticals from testing when they are likely to be present in the aquatic environment at very low concentrations. Because the actual (measured) concentrations of a drug may not be known, and definitely will not be if the substance is new (i.e., yet to be used), this exclusion usually is based on the PEC (derived, in part, from production volume or prescribed dose per capita). Various hydrological models have been developed to allow calculation of PECs, and the resultant values are usually the maximum concentrations likely to occur. Many environmental regulations, such as the recently introduced European guidance on assessing the risks of human drugs (16), exclude from testing pharmaceuticals whose PEC is below an action limit of 0.01 μg/L (which is equivalent to a dose of 2 mg/day in the target population; 16). Although trigger values differ, analogous approaches are used in the U.S. (e.g., a 1 μg/L threshold that equates to a production volume
Persistence and fate of pharmaceuticals Characterization of fate and persistence is a critical component of the ecological risk assessment process for contaminants. Most currently marketed drugs have shorter half-lives in the environment than traditional pollutants (e.g., DDT, PCBs) that
have received intensive study in the past. However, the characterization of exposures of aquatic organisms to pharmaceuticals need not deviate conceptually from that for other groups of contaminants: the magnitude, frequency, and duration of the interaction of organisms with bioavailable toxicants in environmental matrices must be evaluated. However, some drugs, particularly human pharmaceuticals, differ from many other types of contaminants in that they are introduced relatively continually to aquatic systems via wastewater effluents, albeit in small quantities. When pharmaceuticals are constantly introduced to aquatic ecosystems, organisms may experience exposures similar to those of traditional pollutants, even for therapeutics with a limited persistence (Figure 1; 1). Consequently, pharmaceuticals may be FIGURE 1
Effluent influences Potential influence of effluent-dominated conditions on pharmaceutical exposures of aquatic organisms.
Effective exposure duration
In-stream pharmaceutical concentration
of 44,000 kg/yr; 10, 11). However, these trigger values (for production volume, PEC, or projected dose) may not be supportable for certain drugs. Hence, the European Medicines Agency (EMEA) (16) states that a PEC surface water value of 0.01 µg/L applies only if “no other environmental concerns are apparent.” In other words, if a drug (or one of its degradation products) is already known, or can be reasonably expected, to affect aquatic wildlife at concentrations 300,000 (2). Again, this is because of relatively low acute toxicity and extreme chronic potency in a biological pathway (i.e., the hypothalamic–pituitary–gonadal [HPG] axis) that is highly conserved across vertebrates. Limited data for other MOA classes of pharmaceuticals suggest that this phenomenon is not restricted to HPG-active steroids. For example, Huggett et al. (23) reported an ACR in fish of ~50,000 for propranolol, a -blocker. These examples illustrate that for pharmaceuticals with a targeted MOA in conserved biological pathways, acute toxicity test data can be inadequate for predicting ecological risks. Most test data currently available for pharmaceuticals also are limited relative to species (and life stage) representation. Basic tests often include only aquatic species—typically, a unicellular plant, one asexual invertebrate (cladocerans), and embryonic or larval fish (10, 11, 16). This limited focus would result in unanticipated ecological effects. For example, failure to consider effects of EE2 on fish at key developmental (sexual differentiation) and later reproductive life stages has contributed to underestimation of potential ecological risks of the steroid. Another well-documented example of an unanticipated ecological effect involves large-scale die-offs of vultures in Southeast Asia that have been linked to the birds’ consumption of dead cattle treated with diclofenac (24). Although renal problems are known to be an occasional side effect of diclofenac in hu8214 n Environmental Science & Technology / December 15, 2007
mans, no test data were available to indicate that birds might be exceptionally sensitive to kidney failure caused by the anti-inflammatory agent.
Testing pharmaceuticals for ecological effects To summarize, with the exception of a handful of drugs (largely endocrine-active substances), currently available ecotoxicity data could be inadequate for technically rigorous risk assessments. A brute-force fix to this problem would be to require an extensive suite of chronic, sublethal tests with multiple species for all new pharmaceuticals as well as a retrospective assessment of those currently detected in the environment. However, this increased testing would be set against a socioeconomic backdrop of limited resources and a desire to decrease use of animals in testing. Fortunately, the potential exists to develop strategic, resource-efficient approaches for collection of toxicity data for drugs of most concern. A major uncertainty for many types of new chemicals is lack of knowledge as to whether their properties predispose them to cause unacceptable environmental risks. However, for most human and many veterinary drugs, insights as to possible effects in the environment can be gleaned from existing data collected to determine drug efficacy and human-health safety (2, 3, 25, 26). Pharmaceuticals are developed purposely to affect specific biological pathways (and, hopefully, not others) in target species. Even when drugs cause undesirable consequences in target species, these side effects often are well documented. Given this type of information, it should be possible to focus testing in two ways: identification of drugs with the most potential to elicit adverse effects, and determination of which species and endpoints should be used for testing (Figure 2). One consideration that should weigh heavily into prioritizing pharmaceuticals for testing involves pathway specificity with respect to intended therapeutic action or established side effects (Figure 2). A critical component of this involves the degree to which the biological pathway of interest is conserved across species. For example, endocrine-active chemicals that affect discrete nodes in the highly conserved (across vertebrates) HPG axis would be obvious candidates for close scrutiny. Chemicals within this group include not only steroidal receptor agonists, such as EE2 and 17trenbolone, but pharmaceuticals that affect steroid hormone synthesis or act as nuclear hormone receptor antagonists (2). Classes of pharmaceuticals known to target other conserved pathways also should receive strong consideration for expanded ecological effects testing, especially if these pathways are directly involved in development and reproduction, endpoints associated with population-level responses. Some of these classes might include anticancer drugs that affect DNA, progesterone receptor agonists (used for both human and veterinary applications), drugs (such as statins) that alter lipid synthesis, pharmaceuticals that affect the highly conserved hypothalamic–pituitary–thyroid axis, and compounds such as con-
azoles (used as antimycotics in both humans and livestock) that inhibit a variety of cytochrome P450mediated reactions that are key to many physiological processes (2). A priori knowledge of target pathways for pharmaceuticals also can be used to identify tests, species, and endpoints most appropriate for assessing ecological risks (Figure 2). For example, in the case of HPG-active chemicals, assays with vertebrates (such as fish) that focus on critical windows during early development and active reproduction would be expected to produce the most sensitive indications of potential ecological risks. Conversely, relative toxicity of endocrine-active chemicals to plants and invertebrates would be expected to be less relevant to assessing risks, because little evidence exists in these phyla for a physiological role for vertebrate sex steroids. Segner et al. (27) tested several estrogenic chemicals, including EE2, in a variety of partial and full life-cycle assays with a model fish (zebrafish) and several aquatic invertebrate species. The fish was by far most sensitive to the effects of the estrogenic chemicals and was the only species that responded to EE2 at environmentally relevant concentrations. So, in terms of optimal resource use, it would be reasonable to focus ecotoxicity testing of HPG-active substances on sublethal endpoints in vertebrates as opposed to invertebrates. Parallel reasoning could be developed for other MOA groupings, including those to which invertebrates are likely to be more sensitive than vertebrates (e.g., organophosphate pesticides registered for veterinary use; 3). Knowledge of MOAs also can help scientists identify pharmaceuticals of lesser concern for chronic, sublethal effects in nontarget species. Examples here include chemicals likely to exert toxicity via narcosis, such as anesthetics (e.g., benzocaine, tri caine) and iodinated contrast media (2). For these types of compounds, short-term lethality assays should provide a reasonable data set for predicting ecological risks. Although a priori knowledge of biological pathways likely to be affected by different classes of pharmaceuticals can be used to strategically guide testing, inevitably in some instances a compound will cause toxicity in unexpected species via novel mechanisms. For example, blue-green algae are (perhaps unexpectedly) quite sensitive to some antibiotics, compared with animals (19). Because of this type of unanticipated response, recommendations have been made that all pharmaceuticals anticipated to enter the environment be subjected to some level of baseline testing (2, 3).
Biomarkers in pharmaceutical risk assessments Historically, biomarkers have seldom been used for decision making in regulatory ecotoxicology, but they could play a role in assessing risks of some human and veterinary pharmaceuticals. In particular, molecular or biochemical responses specific for certain pharmaceutical MOAs in nontarget species may serve as powerful tools for ecological risk assessments (2, 3). An excellent example of a MOA-spe-
cific biomarker in fish and other oviparous animals is vitellogenin (egg yolk protein), which is normally present in reproductively mature females but which can be induced in males exposed to estrogens such as EE2. The specificity and sensitivity of vitellogenin induction in males have made the response valuable for detecting exposure (28). Much recent biomarker discovery research has been fueled by new genomic tools that enable scientists to simultaneously measure literally thousands of genes, proteins, and metabolites in organisms exposed to different types of chemical stressors (29). However, aspects of the FIGURE 2
Selecting test species and endpoints Example decision tree for selection of test species and endpoints for assessing ecological risk of pharmaceuticals. Pharmaceutical effects characterization
Aquatic plants and algae
Vertebrates
Invertebrates
Target and pathway present?* Yes Physiological function understood?* Yes Conduct chronic studies – Select species with specific target and pathway present – Select related endpoints of physiological and ecological relevance
Risk characterization *May require baseline in vitro or in vivo studies to ascertain.
use of biomarkers in assessments of pharmaceuticals (or other chemicals) remain uncertain, particularly with regard to what molecular or biochemical changes might mean to the types of endpoints on which environmental regulations typically are based (i.e., survival, growth, reproduction). To address this uncertainty in a risk-based framework, characterization of the various sublethal responses to pharmaceuticals as biomarkers of either exposure or effect is useful. A biomarker of exposure provides information as to whether an organism has been exposed to a specific contaminant (or, more typically, an MOA class of contaminants). Biomarkers of effect differ from exposure biomarkers in that they are sublethal measures that can be indicative of toxicity, particularly when they can be linked directly to a physiologically and ecologically relevant December 15, 2007 / Environmental Science & Technology n 8215
endpoint (Figure 3). A recent example of an effective biomarker of effect for HPG-active chemicals was reported by Miller et al. (30), who linked toxicant-induced depressions in vitellogenin in female fish to reduced egg production and, subsequently, population declines. FIGURE 3
Biomarkers Logic framework for integrating biomarkers of effect into ecological risk assessments. Do target pathways exist in phyla of concern? Does exposure lead to a measurable biochemical response? Does biochemical change result in physiological response? Is physiological response an ecologically relevant effect?
Include biochemical and physiological response as measures of effect in problem formulation.
Future perspectives Over the past decade, tens, if not hundreds, of millions of dollars have been spent to conduct research and support development of regulatory strategies for assessing endocrine-disrupting chemicals. From these efforts, substantial progress has been made in optimizing testing of chemicals with varying degrees of persistence that target specific biological pathways, often with very high potency. We feel that the experience gained from work focused on a few classes of HPG-active chemicals (e.g., estrogen and androgen agonists) can be applied effectively to dealing with the broader issue of the ecological risks of many different MOA classes of veterinary and human drugs (box at right). A critical lesson that we have learned from HPGactive chemicals is how to develop focused assays and endpoints on the basis of MOAs of concern. In the case of pharmaceuticals, the use of a priori knowledge about target biological pathways can both identify chemicals that should receive priority for testing and determine which species, life stages, and endpoints are appropriate for testing. This type of information also can augment PEC data when decisions are made about the extent of ecotoxicity testing needed to assess potential risks. Use of a priori knowledge in these ways should ultimately lead to technically rational testing designs, which would be more environmentally protective as well as cost-effective. The current prospective ecological risk assessment process (i.e., product registration) for drugs involves a highly prescriptive set of environmental fate and toxicity studies that must be conducted before a new product is submitted. Although in all instances a base set of ecotoxicity tests is desirable, flexibility within the risk assessment process is criti8216 n Environmental Science & Technology / December 15, 2007
cal for an acceptable level of environmental protection to be achieved. Challenges exist, however, in moving away from a highly prescriptive approach for regulatory testing (31). For example, identifying appropriate suites of tests on the basis of anticipated or known MOA (and subsequently interpreting test results) would, at least initially, require more direct involvement of research scientists in the regulatory process than currently occurs. However, the potential benefits (i.e., better data, optimal resource use) of a shift from highly prescriptive testing to such an approach could be substantial. A couple of important final observations are worth noting about current efforts to better determine the ecological risks of pharmaceuticals in the environment. First, a significant impetus for research and regulatory activities involving endocrine-disrupting chemicals over the past few years emanated from public perceptions of risk, often fueled by highly charged or visible manifestations of possible effects in humans and wildlife. However, there has been little effort to date to determine the risk of endocrine-active chemicals relative to other chemical and nonchemical stressors. For example, in the case of ecological effects, populations of fish may be impacted far more by factors such as changes in habitat quality or competition from nonnative invasive species than, for example, by exposure to estrogenic chemicals. This highlights the need not only to determine risks of pharmaceuticals entering the environment but also to accurately assess their relative risks.
Summary recommendations for assessing the ecological risk of pharmaceuticals • • •
• •
•
Use similar effects assessment approaches for veterinary and human drugs. Reassess the basis of using production volume or PEC values as testing triggers. Evaluate persistence as a function of chemical stability and input relative to hydrodynamic regime. Allow MOA data to guide species and endpoint selection for testing. Support the development and use of MOA-specific biomarkers for both diagnostic and prospective applications. Build flexibility into the regulatory process to allow a priori knowledge to guide testing.
A second point relative to the ecological effects of pharmaceuticals involves mitigation options. Specifically, even if a drug (or class of drugs) is discovered (or predicted) to have substantial ecological impacts, the consequences of this need to be dealt with from a broader societal perspective. A comprehensive approach is needed to balance the environmental risks and human-health benefits of pharmaceuticals such that wastewater treatment alternatives, environmental degradation, relative ecological risks, and other considerations are brought together in a quantitative manner. To revisit EE2, we could consider how
mitigation strategies, such as a ban on EE2 or a shift from the therapeutic use of EE2 to that of conjugated or “natural” estrogens (e.g., 17-estradiol), may impact women of childbearing potential, while at the same time taking into account the desire to protect local fish populations. Similarly, increasing the capacity of treatment plants to reduce concentrations of EE2 to below those likely to impact fish may not be economically viable, especially when viewed against the larger backdrop of factors, such as increased energy use (and carbon emissions), associated with enhanced treatment. Gerald T. Ankley is a research toxicologist with the U.S. EPA. Bryan W. Brooks is an associate professor of environmental science and biomedical studies at Baylor University. Duane B. Huggett is an assistant professor in the department of biology at the University of North Texas. John P. Sumpter is a professor at the Institute for the Environment at Brunel University (U.K.). Address correspondence about this article to Ankley at ankley.
[email protected].
Acknowledgments Helpful comments were supplied by David Mount, Jim Lazorchak, and Carl Richards. We thank the many colleagues who have influenced our thinking on this topic through forums such as Pellston Workshops and other advisory groups.
Disclaimer This paper has been reviewed in accordance with U.S. EPA guidelines and approved for publication. However, views expressed herein are those of the authors and do not necessarily reflect EPA policy or opinion.
References (1) Daughton, C. G.; Ternes, T. A. Pharmaceuticals and Personal Care Products in the Environment: Agents of Subtle Change? Environ. Health Perspect. 1999, 107, 907–938. (2) Society of Environmental Toxicology and Chemistry. Human Pharmaceuticals: Assessing Impacts on Aquatic Ecosystems; Williams, R. T., Ed.; SETAC Press: Pensacola, FL, 2005. (3) Society of Environmental Toxicology and Chemistry. Veterinary Medicines in the Environment; Crane, M., Barrett, K., Boxall, A., Eds.; SETAC Press: Pensacola, FL, in press. (4) World Health Organization. Wildlife. In Global Assessment on the State-of-the-Science of Endocrine Disruptors; Damstra, T., et al., Eds.; WHO/PCS/EDC/02.2; 2002; Chapter 4. (5) Purdom, C. E.; et al. Estrogenic Effects of Effluents from Sewage Treatment Works. Chem. Ecol. 1994, 8, 275–285. (6) Gross-Sorokin, M. Y.; Roast, S. D.; Brighty, G. C. Assessment of Feminization of Male Fish in English Rivers by the Environment Agency of England and Wales. Environ. Health Perspect. 2006, 114 (Suppl. 1), 147–151. (7) Desbrow, C.; et al. Identification of Estrogenic Chemicals in STW Effluent. 1. Chemical Fractionation and In Vitro Biological Screening. Environ. Sci. Technol. 1998, 34, 1548–1558. (8) Brian, J. V.; et al. Accurate Prediction of the Response of Freshwater Fish to a Mixture of Estrogenic Chemicals. Environ. Health Perspect. 2005, 113, 721–728. (9) Sumpter, J. P.; et al. Modeling Effects of Mixtures of Endocrine Disrupting Chemicals at the River Catchment Scale. Environ. Sci. Technol. 2006, 40, 5478–5489. (10) U.S. Department of Health and Human Services, Food and Drug Administration. Guidance for Industry: Environmental Assessment of Human Drug and Biologics Applications; July 1998; www.fda.gov/cder/guidance/1730fnl. pdf.
(11) U.S. Department of Health and Human Services, Food and Drug Administration. Guidance for Industry: Environmental Impact Assessments for Veterinary Medicinal Products—Phase I; March 2001; www.fda.gov/cvm/ Guidance/guide89.pdf. (12) Veterinary International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products (VICH). Environmental Impact Assessment for Veterinary Medicinal Products—Phase I; June 2000; www.apvma.gov.au/guidelines/downloads/VICH_ %20GL6.pdf. (13) Veterinary International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products (VICH). Environmental Impact Assessment for Veterinary Medicinal Products—Phase II; June 2004; www.apvma.gov.au/guidelines/downloads/ VICH_GL38.pdf. (14) Länge, R.; et al. Effects of the Synthetic Estrogen 17-Ethinylestradiol over the Life-Cycle of the Fathead Minnow (Pimephales promelas). Environ. Toxicol. Chem. 2001, 20, 1216–1227. (15) Durhan, E. J.; et al. Identification of Metabolites of Trenbolone Acetate in Androgenic Runoff from a Beef Feedlot. Environ. Health Perspect. 2006, 114 (Suppl. 1), 65–68. (16) EMEA. Guideline on the Environmental Risk Assessment for Medicinal Products for Human Use; CHMP/SWP/4447/ 00; 2006; www.emea.eu.int/pdfs/human/swp/444700en. pdf. (17) Brooks, B. W.; Riley, T. W.; Taylor, R. D. Water Quality of Effluent-Dominated Stream Ecosystems: Ecotoxicological, Hydrological, and Management Considerations. Hydrobiologia 2006, 556, 365–379. (18) Fent, K.; Weston, A. A.; Caminada, D. Ecotoxicology of Human Pharmaceuticals. Aquat. Toxicol. 2006, 76, 122–159. (19) Webb, S. A Data-Based Perspective on the Environmental Risk Assessment of Human Pharmaceuticals I—Collation of Available Ecotoxicity Data. In Pharmaceuticals in the Environment—Sources, Fate, Effects and Risks, 1st ed.; Kümmerer, K., Ed.; Springer: Berlin, 2001; pp 17–201. (20) Jones, O. A. H.; Voulvoulis, N.; Lester, J. N. Potential Ecological and Human Health Risks Associated with the Presence of Pharmaceutically Active Compounds in the Aquatic Environment. Crit. Rev. Toxicol. 2004, 34, 335–350. (21) Crane, M.; Watts, C.; Boucard, T. Chronic Aquatic Environmental Risks from Exposure to Human Pharmaceuticals. Sci. Total Environ. 2006, 367, 23–41. (22) Cunningham, V. L.; et al. Effects of Human Pharmaceuticals on Aquatic Life: Next Steps. How Do Human Pharmaceuticals Get into the Environment, and What Are Their Effects? Environ. Sci. Technol. 2006, 40, 3457–3462. (23) Huggett, D. B.; et al. Toxicity of Select Beta Adrenergic Receptor Blocking Pharmaceuticals (-Blockers) on Aquatic Organisms. Arch. Environ. Contam. Toxicol. 2002, 43, 229–235. (24) Green, R. E.; et al. Diclofenac Poisoning as a Cause of Vulture Population Declines across the Indian Subcontinent. J. Appl. Ecol. 2004, 41, 791–800. (25) Huggett, D. B.; et al. Theoretical Model for Prioritizing Potential Impacts of Human Pharmaceuticals to Fish. J. Human Eco. Risk Ass. 2003, 9, 1789–1799. (26) Länge, R.; Dietrich, D. Environmental Risk Assessment of Pharmaceutical Drug Substances—Conceptual Considerations. Toxicol. Lett. 2002, 131, 97–104. (27) Segner, H.; et al. Identification of Endocrine-Disrupting Effects in Aquatic Vertebrates and Invertebrates. Ecotoxicol. Environ. Saf. 2003, 54, 302–314. (28) Wheeler, J. R.; et al. Vitellogenin: A Review of Analytical Methods To Detect (Anti) Estrogenic Activity in Fish. Toxicol. Mech. Methods 2005, 15, 293–306. (29) Ankley, G. T.; et al. Toxicogenomics in Regulatory Ecotoxicology. Environ. Sci. Technol. 2006, 40, 4055–4065. (30) Miller, D. H.; et al. Linkage of Biochemical Responses to Population-Level Effects: A Case Study with Vitellogenin in the Fathead Minnow. Environ. Toxicol. Chem. 2007, 26, 521–527. (31) Bradbury, S. P.; Feijtel, T. C. J.; Van Leeuwen, C. J. Meeting the Scientific Needs of Ecological Risk Assessment in a Regulatory Context. Environ. Sci. Technol. 2004, 38, 463A–470A. December 15, 2007 / Environmental Science & Technology n 8217
