97 5 Assessing the Aquatic Hazards of Veterinary Medicines Bryan W. Brooks, Gerald T. Ankley, James F. Hobson, James M. Lazorchak, Roger D. Meyerhoff, and Keith R. Solomon 5.1 INTRODUCTION In recent years, there has been increasing awareness of the widespread distribu- tion of low concentrations of veterinary medicine products and other pharmaceu- ticals in the aquatic environment. Although aquatic hazard for a select group of veterinary medicines has received previous study (e.g., aquaculture products and sheep dips), until very recently less information has been available in the pub- lished literature for other therapeutic groups (Halling-Sørensen 1999; Jørgensen and Halling-Sørensen 2000; Ingerslev and Halling-Sørensen 2001; Koschorreck et al. 2002; Boxall et al. 2003, 2004b). The majority of available aquatic ecotox- icity information for veterinary medicines was generated from short-term (e.g., 24 to 96-hour) bioassays to meet requirements for product registrations (Boxall et al. 2004b). Limited information is available for partial life cycle or life cycle exposure scenarios and on hazards in lentic systems and lotic systems, particu- larly in arid to semiarid regions (Brooks et al. 2006, 2007). Although aquatic hazard information for veterinary medicines is largely lim- ited to acute toxicity data, the various classes of veterinary medicines are gener- ally known to have specic biological properties, which are selected during the drug development process. It is possible that such information may be leveraged to focus future research and the screening of the potential hazards these com- pounds present to specic groups of nontarget organisms. For example, Huggett et al. (2003) describe a theoretical model that may be used to estimate impacts of selected veterinary medicines to sh, based on pharmacological information from other vertebrates. This chapter considers the utility and applications of existing techniques (e.g., standardized toxicity tests), developing approaches (e.g., ecotoxicogenomics), and technologies or methods that may be used in the future with the existing knowl- edge of physiochemical (e.g., log K ow ) and pharmacological properties (e.g., mode of action) to characterize potential impacts of veterinary medicines in aquatic systems. © 2009 by the Society of Environmental Toxicology and Chemistry (SETAC) 98 Veterinary Medicines in the Environment The chapter includes a critical evaluation of the state of veterinary medicine aquatic hazard assessment, and a characterization of available information for veterinary medicine impacts in aquatic systems. Furthermore, we identify data gaps and regula- tory uncertainties or deciencies, and provide recommendations for research needs. 5.2 PROTECTION GOALS When assessing the risk of a compound to the environment and selecting aquatic testing strategies, it is essential to have clear protection goals. The protection goals developed during a recent SETAC Pellston Workshop on Science for Assessing the Impact of Human Pharmaceuticals on Aquatic Ecosystems (Williams 2005) would appear to be appropriate for veterinary medicines. The previous workshop concluded, The key aspects of aquatic ecosystems that should be protected are 1) Ecosystem functionality and stability — including ecosystem primary productivity (based on algae and plants) and the key phyla of primary consumers (especially invertebrates) that are essential to the sustainability of aquatic food webs; 2) Biodiversity — espe- cially the potential to affect populations of potentially endangered species, taking into account both local and regional contexts; and 3) Commercially and socially important species, including shellsh (crustaceans and mollusks), sh, and amphib- ian populations. Finally, it is important to recognize the importance of linkages between ecosystem components. If an ecosystem component (population or group of populations) is strongly linked to other components, effects on that component have greater potential to cause secondary effects elsewhere in that ecosystem. In the following sections we discuss potential approaches that can be used for environmental assessment of veterinary medicines to help achieve these goals. 5.3 APPROACHES TO ASSESS EFFECTS OF VETERINARY MEDICINES Aquatic toxicity studies may be used in a number of ways (Chapter 3): they may contribute to the development of a risk assessment for a new product (prospective assessment), they may be used for routine monitoring of aquatic ecosystems such as in ecopharmacovigilance programs (retrospective or compliance assessment), or they may be used to help identify the causes of an observed impact on an ecosystem using approaches such as toxicity identication evaluation (retrospec- tive assessment). In the following sections we describe existing and novel aquatic toxicity testing approaches that are appropriate for veterinary medicines and that could be used for any one of these purposes. 5.3.1 CURRENT METHODS OF ASSESSING AQUATIC EFFECTS FOR RISK ASSESSMENT To date most developments in the area of toxicity of veterinary medicines to aquatic organisms have focused on prospective risk assessment, and several © 2009 by the Society of Environmental Toxicology and Chemistry (SETAC) Assessing the Aquatic Hazards of Veterinary Medicines 99 guidelines are now available on how the aquatic hazard of a veterinary medicine to aquatic organisms should be assessed. The most inuential of these guidelines are those developed by VICH and are discussed in more detail in Chapter 3. The approach is a 2-phase process, and during phase 2 aquatic hazard testing is per- formed using a tiered approach. 5.3.1.1 Lower Tier Approaches During the VICH phase I process (described in Chapter 3), compounds do not require additional study if they have a Predicted Environmental Concentration (PEC) or Environmental Introduction Concentration (EIC) of <1 Ng L –1 in aquatic systems, or <100 Ng kg –1 in soil (International Cooperation on Harmonization of Technical Requirements for Registration of Veterinary Products [VICH] 2000). Exceptions to this are a few therapeutic groups. compounds used in aquaculture, and endo- and actoparasiticides. For example, veterinary medicines applied to companion animals are not considered important because the mass potentially entering the aquatic environment is considered too small to result in exposures of ecological signicance. When an assessment of a veterinary medicine does not stop at phase 1 of the VICH process, acute algal, daphnid, and juvenile sh toxicity studies are per- formed at tier A of the VICH phase II process to estimate EC50 and LC50 values (VICH 2004). Predicted no-effect concentrations (PNEC) are then estimated by applying an assessment factor of 100 to the algal data and 1000 to the daphnid and sh data. The PNECs are then compared to the predicted exposure concentra- tions (see Chapter 4 for derivation) to generate a hazard quotient (HQ). If the HQ is < 1, the assessment is terminated. If an HQ > 1 is identied, tier B toxicity tests are performed that can include algal, cladoceran, sediment invertebrate, and sh assays to consider standardized sublethal responses such as growth or reproduc- tion (VICH 2004). 5.3.1.2 Higher Tier Testing The tier B tests (Table 5.1) incorporate responses to chronic exposures that dif- fer in terms of the life cycle of the test organisms and the organisms for which they are surrogates. Only some of these tests allow observations of effects on all aspects of the life cycle, including reproduction, and of these, some only assess 1 type of reproduction (Table 5.1). Assessment factors of 10 are applied to no observed effect concentrations (NOECs) generated from these tier B tests, and the HQ calculation is repeated. If the HQ remains > 1, the specic hazard of the compound can be further assessed during a tier C process in countries such as the United States, or risk management regimes can be considered. These additional tests may be required to address specic questions and test hypotheses related to the likely effects of the veterinary medicines on nontarget aquatic organisms. Specic recommendations © 2009 by the Society of Environmental Toxicology and Chemistry (SETAC) 100 Veterinary Medicines in the Environment for this testing are not currently included in VICH and FDA regulatory guidance documents. However, approaches described elsewhere in this chapter (e.g., tests and bioassays based on specic responses, such as hormonal activity) may be appropriate. For example, when the mode of action of a medicine in the target animal is known to be via hormone modulation, effects of reproductive function should be tested in an appropriate surrogate species such as sh. Some test proto- cols are available for this type of assessment (Ankley et al. 2001), and others are under development. TABLE 5.1 Tier B tests proposed by the International Cooperation on Harmonization of Technical Requirements for Registration of Veterinary Products (VICH) Test organism Test guideline Comments Freshwater algae growth inhibition OECD 201 Includes several life cycles and would likely allow the observation of subtle effects on growth, development, and reproduction. However, the form of reproduction may not include sexual modes. Freshwater Daphnia magna reproduction OECD 211 Includes 1 life cycle and would likely allow the observation of subtle effects on growth, development, and reproduction. However, the form of reproduction does not include sexual modes. Freshwater sh, early life stage OECD 210 A developmental bioassay that includes early stages of development and components of sexual differentiation, but not reproduction. Freshwater sediment invertebrate species toxicity OECD 218 and OECD 219 Includes survival and growth, but not reproduction. Saltwater algae growth inhibition ISO 10253 Includes several life cycles and would likely allow the observation of subtle effects on growth, development, and reproduction. However, the form of reproduction may not include sexual modes. Saltwater crustacean chronic toxicity or reproduction NA Not specied but would include 1 life cycle and would likely allow the observation of subtle effects on growth, development, and reproduction. Sexual reproduction would likely be observed if the correct species is selected. Saltwater sh chronic toxicity NA Not specied but could include reproduction. Saltwater sediment invertebrate species toxicity NA Not specied but could include reproduction. Note: NA = Not specied at this time. Source: International Cooperation on Harmonization of Technical Requirements for Registration of Veterinary Products (VICH 2004). © 2009 by the Society of Environmental Toxicology and Chemistry (SETAC) Assessing the Aquatic Hazards of Veterinary Medicines 101 5.3.1.3 Limitations to Current Approaches Single-species bioassays have greatly supported the improvement of water quality in many parts of the world. However, only relying on the endpoints employed in these standardized aquatic toxicity tests for prospective or retrospective contami- nant decisions may not be sufcient (Cairns 1983), because these studies are not intended to predict structural or functional ecological responses to contaminants (Dickson et al. 1992; La Point and Waller 2000) and may not represent the most sensitive species responses (Cairns 1986). Furthermore, standardized test end- points do not provide information on biochemical, developmental, behavioral, or transgenerational responses to veterinary medicine exposures. Although assessment factors are applied in order to account for some of these issues, the assessment factors applied to toxicity results from tiers A and B have not been derived from empirical information for aquatic organisms exposed to veterinary medicines. This omission may have important implications for more sensitive species and ecologically relevant sublethal responses with high acute:chronic ratios (ACRs). For example, ACRs greater than 1000 have previ- ously been reported for a number of pharmacologically active compounds in the environment (Huggett et al. 2002; Ankley et al. 2005; Crane et al. 2006). In recent years, selection of appropriate measures of effect has been dis- cussed for human medicines and personal care products and veterinary medi- cines (Daughton and Ternes 1999; Brooks et al. 2003; Crane et al. 2006). Because veterinary medicines are generally present in the environment at trace (ng L –1 ) concentrations, traditional standardized ecotoxicity tests and endpoints may not be appropriate to characterize risk associated with aquatic exposures to certain compounds (Brooks et al. 2006). This problem is illustrated for 3 veterinary med- icines with different modes of action (Table 5.2). Diazinon is used in sheep dips as an organophosphorus insecticide to kill targeted terrestrial invertebrates species that are considered to be pests. Because Daphnia magna is an aquatic invertebrate species that is also sensitive to cholin- esterase inhibition caused by compounds such as diazinon, a standardized toxicity test with this species using mortality and reproduction as the primary endpoints TABLE 5.2 Example scenarios for veterinary medicines where aquatic hazards might or might not be found by current regulatory toxicity-testing approaches with standard endpoints Compound Bioassay organism Hazard present Hazard detected Diazinon Daphnia Yes Yes Trenbolone Juvenile sh Yes No Oxytetracycline Green algae Yes Possibly © 2009 by the Society of Environmental Toxicology and Chemistry (SETAC) 102 Veterinary Medicines in the Environment will likely produce a sensitive measure of the potential hazard from this com- pound in surface waters. Oxytetracycline is a molecule that was selected to inhibit the growth of certain bacteria that result in disease conditions. There are no stan- dard toxicity tests designed to assess the hazards of antibiotics to a community of microbes in surface waters. The combined results of studies with algae, espe- cially blue-green algae, and soil microbes can provide an estimate of the potential hazards to aquatic microbes. So these standard tests may, or may not, allow an appropriate estimation of the hazard from an antibiotic in surface waters. The toxicity of the androgen trenbolone would not be appropriately characterized by the endpoints from an early life stage study with sh, a standard study conducted in tier B testing. Trenbolone can masculinize female sh (Ankley et al. 2003), but gender and reproduction are not determined in standard early life stage studies with sh. To account for biological hazards associated with unique compounds like veterinary medicines, Ankley et al. (2005) recommended that test selection for a compound consider ecological attributes and appropriate species and endpoint relevance. Therefore, in the next section we review relevant approaches that may be used in conjunction with knowledge of the mode or mechanism of action of veterinary medicines to focus further investigations of their hazards to aquatic organisms and the development of postauthorization assessment methodologies. 5.3.2 NOVEL APPROACHES TO AQUATIC EFFECTS ASSESSMENT 5.3.2.1 Use of Chemical Characteristics, Target Organism Efficacy Data, Toxicokinetic Data, and Mammalian Toxicology Data Veterinary medicines are chemicals that are extensively evaluated for targeted efcacy, the safety of treated animals, and human safety. A signicant number of studies are conducted to understand the physical, chemical, and structural characteristics of the molecules. Studies are also done to document the nature of the effects on the therapeutic target; the adsorption, distribution, metabolism, and excretion (ADME) of the chemical in the treated animal; and also potential unwanted toxicities in the treated animal. In order to protect humans from expo- sure to trace residues of the molecule in food sources, mammalian toxicology studies are conducted to characterize any reproductive or developmental effects, chronic whole organism and organ system toxicities, and cellular abnormalities. This information is interpreted by understanding the daily dose in the tested organisms, the resulting plasma exposure to the parent material, and the presence of metabolites. The basic environmental tests that are needed for the registered use of veterinary medicines also provide an important environmental hazard pro- le for these molecules. The extensive testing of veterinary medicines for efcacy, safety of treated animals, and human safety may provide a signicant amount of data that could be used to help identify information gaps in the environmental testing prole and to target appropriate testing to close these gaps. In the following sections, we © 2009 by the Society of Environmental Toxicology and Chemistry (SETAC) Assessing the Aquatic Hazards of Veterinary Medicines 103 describe potential applications for effects and bioaccumulation and bioconcentra- tion assessment. 5.3.2.1.1 Effects Assessment Information on the target mode of action of a veterinary medicine could poten- tially be used to select the most appropriate aquatic effect testing strategy (e.g., selection of the most appropriate test species and endpoints for use in ecological risk assessment and postauthorization monitoring). Examples of how the approach can be used are summarized in Table 5.3 and discussed in more detail below. Treatments for microbial diseases such as antibiotics, antifungals, and anti- coccidiostats are typically used to control specic types of microbes that can lead to respiratory, intestinal, and systemic infections as well as foot rot. The veteri- nary medicines developed for these purposes target the disease microorganisms by directly causing microbial cell death or by impeding the life cycle of targeted microbes through a variety of modes of action. Unless the veterinary product acts to improve the immune response in the host, the treatment does not achieve efcacy through a direct effect on the dosed animal. Understanding the modes of action for direct effects on disease microbes can help focus attention on possible TABLE 5.3 Examples of how the results from mammalian tests can be used to target environmental effects testing Target animal and mammalian results Trigger for further evaluation Taxa of interest Endpoint of interest Growth, development, and reproduction Estrogen agonist activity Fish Development and reproduction PEC/Cmax at lowest result dose > 1 (especially when receptor mediated) Fish Development and reproduction, especially if receptor conserved in sh Inhibition of cellular processes (e.g., ion transport or enzyme kinetics) PEC/Cmax at lowest result > 1 Fish Survival and growth, especially of cellular processes conserved Thyroid effects Hormone mediated Frogs Morphological transformation Antibiotic efcacy PEC/Efcacy Cmax >1 Similar microbial taxa or algae Maximum inhibition concentrations, population growth Ecto- and endoparasiticides PEC/efcacy concentration > 1 Arthropods Survival, growth ADME kinetics slow with high K ow Long half-life and little metabolism Fish, sediment, invertebrates Possible signicant bioaccumulation © 2009 by the Society of Environmental Toxicology and Chemistry (SETAC) 104 Veterinary Medicines in the Environment hazards for related taxa in aquatic ecosystems with potential sensitivities to the same modes of action. Veterinary medicines are also developed to have direct effects on parasites. These products can be delivered orally, topically, or by injection, but they are tar- geted at achieving a high enough exposure to kill or interrupt the life cycle of the parasite. Again, the treatment does not usually achieve efcacy through a direct effect on the dosed animal. Understanding the modes of action for direct effects on these parasites can provide the context for judging the adequacy of ecological hazard testing with invertebrate species. Promotion of feed utilization efciency and/or growth can be targeted through several very different modes of action. Some antimicrobials aim to modify the gut ora for more efcient digestion of feedstuffs and, therefore, better energy efciency and growth for the treated host. Some antimicrobials target a reduction in the bacterial load in the host, resulting in less animal stress and better growth. A few veterinary medicines act through receptor-mediated modes of action to modify basal metabolism or augment hormonal action on growth. Known modes of action might be extrapolated to evaluate ecological hazards for similar aquatic taxa, or species with similar receptor-mediated physiological responses. Treatment of veterinary medical conditions and aids for handling animals by veterinarians may act through a variety of modes of action. Some may be receptor mediated. Some might occur through direct modication of cellular processes; for example, through inhibition of enzyme kinetics or ion transporter activity. Other medicines may rely directly on the physical-chemical properties of the treatments (e.g., antifoaming agents for bloat). Again, knowledge of the mode of action tar- geted for these types of veterinary medicines can be useful in evaluating the types of hazards and species at risk when the chemical moves into aquatic ecosystems. Mammalian toxicology studies can also reveal important clues to the potential effects of veterinary medicines in aquatic species. If developmental or reproduc- tive effects occur at low doses, it may be important to evaluate further the poten- tial for these effects to occur in sh. Chronic effects or unusual pathology noted in chronic mammalian studies could be used to identify important endpoints to evaluate in aquatic vertebrates. Tissue changes resulting from hormone-mediated effects could suggest sensitive species to test. For example, frogs might be tested for temporal changes in the transformation from tadpoles to air-breathing adults when a chemical is known to have thyroid receptor activity in mammals. In order to assess the level of sensitivity at which these modes of action or toxicological endpoints occur, it is also important to relate the ADME charac- teristics of the veterinary medicines to their effects. The pharmacokinetic and toxicokinetic proles of the molecules usually provide an understanding of the maximal plasma concentrations (C max ) and total exposure (area under the expo- sure curve) for the parent material and the primary metabolites to help explain the pharmacodynamics and toxicodynamics of the treatment in mammals. The plasma concentrations also help explain the activity of antimicrobial agents in vivo in relationship to their activity in vitro. These exposure–effect relationships © 2009 by the Society of Environmental Toxicology and Chemistry (SETAC) Assessing the Aquatic Hazards of Veterinary Medicines 105 can be used to evaluate directly the potential for related effects on environmen- tal species that are exposed to predicted environmental concentrations (PECs) calculated for the parent material (Huggett et al. 2003; and see Figure 5.1). If the predicted environmental concentrations of a veterinary medicine could cause concentrations in sh plasma levels that are near the C max in mammalian plasma, resulting in efcacy or toxicity, it could be important to evaluate the endpoint further in a toxicity test with an appropriate environmental species. 5.3.2.1.2 Use of Chemical Characteristics and ADME Data in the Assessment of Bioaccumulation Potential The ADME of a veterinary medicine in mammals can also, in conjunction with physical-chemical properties such as pK a and solubility, provide some basis for estimating uptake and depuration characteristics in sh. Distribution within an aquatic vertebrate and the types of metabolism can parallel those found in mam- mals, although the kinetics and excretion routes in sh can be quite different. Absorption across the gut in mammals could lead to rst-pass metabolism through the liver, whereas the route of exposure to somewhat soluble molecules is prob- ably dominated in sh by absorption across the respiratory surfaces. Molecules              FIGURE 5.1 Screening assessment approach to target aquatic effects testing with sh from water exposure. Note: EIC = environmental introduction concentration. © 2009 by the Society of Environmental Toxicology and Chemistry (SETAC) 106 Veterinary Medicines in the Environment that are found to be deeply distributed into the fatty tissue in mammals, or that are poorly metabolized and excreted, could also tend to accumulate in aquatic organ- isms and, perhaps, sediments. Extraordinary concentration of residues in particu- lar tissues, such as reproductive organs, might also lead to concern for maternal deposition of active material in eggs for an environmental species. Other mol- ecules that could appear to have the potential to bioaccumulate in sh, based on low solubility or high K ow , might actually be as easily metabolized and excreted by sh as they are demonstrated to be through ADME studies in mammals. An illustration of the use of ADME data to design testing strategies for the aquacul- ture parasiticide emamectin benzoate is provided below. Emamectin benzoate is synthetically derived from the natural product abam- ectin. Data have been developed for several applications in addition to aquacul- ture. Existing data include physicochemical properties, pharmacokinetics and metabolism data in sh and mammalian species, and bioaccumulation data for invertebrate species in the laboratory and eld studies (Hobson 2004). Physicochemical properties for emamectin benzoate are presented in Table 5.4. The vapor pressure, 4 × 10 –3 mPa, indicates that the material is unlikely to enter or TABLE 5.4 Physicochemical characteristics of emamectin benzoate 4”-epimethylamino- 4”-deoxyavermectin B1a benzoate (MAB1a) 4”-epimethylamino- 4”-deoxyavermectin B1b benzoate (MAB 1b) Composition (%) > 90 < 10 Empirical formula C 49 H 75 NO 13 C 7 H 6 O 2 C 48 H 73 NO 13 C 7 H 6 O 2 Molecular weight 1008.26 994.23 Technical material Solubility (mg L -1 ) pH 5.03 320 ± 30 pH 7.04 24 ± 2 pH 9.05 0.1 ± 0.1 Seawater 5.5 Dissociation constant (pK a ) Benzoic acid group 4.2 ± 0.1 Methylamino group 7.6 ± 0.1 Log K ow pH 5.07 3.0 ± 0.1 pH 7.00 5.0 ± 0.2 pH 9.04 5.9 ± 0.4 Vapor pressure (mPa) 4 × 10 –3 Melting point (°C) 141–146 Density g cm –3 1.20 ± 0.03 © 2009 by the Society of Environmental Toxicology and Chemistry (SETAC) [...]... to understand further those factors and processes affecting the bioavailability and trophic transfer of veterinary medicines in aquatic systems 5) Studies to examine influences of enantiomer-specific fate and effects of racemic veterinary medicines 6) Consideration of the potential impacts of mixtures of veterinary medicines and mixtures containing veterinary medicines and other contaminant classes (e.g.,... recommended for veterinary medicines involve tests on single organisms They therefore ignore the complex interactions (including indirect effects) that can occur in the real environment Multispecies responses and indirect effects that are mediated through species interactions can be © 2009 by the Society of Environmental Toxicology and Chemistry (SETAC) 120 Veterinary Medicines in the Environment TABLE 5. 6 Typical... exposure to veterinary medicines may be episodic in nature; 2) due to matrix effects, the bioavailability in the environment may be very different than in the laboratory; 3) the parent medicine may be metabolized in the treated animal or be degraded in the environment, and it may be the metabolites or degradates that pose the risk; 4) veterinary medicines are highly unlikely to exist in the environment. .. medicines 5. 5.6 SORPTION TO SEDIMENT There is increasing evidence from monitoring studies that some classes of veterinary medicines can concentrate in aquatic sediments (see Chapter 4) This clearly is an important observation in terms of ascertaining the overall fate and transport of veterinary medicines in the environment However, occurrence of veterinary medicines in sediments also has potential repercussions... veterinary medicines As further discussed in Section 5. 5.7, lotic and lentic mesocosms are useful for higher tiered assessment of contaminant mixture hazards in aquatic ecosystems 5. 5 .5 ENANTIOMER-SPECIFIC HAZARD In recent years, increased attention has been given to chiral molecules in the environment (Garrison 2006) A number of environmental contaminants including representatives from human and veterinary. .. including human and veterinary medicines (Brain et al 2004, 2005a, 2005b; Richards et al 2004; Wilson et al 2004; Van den Brink et al 20 05) A number of cosm studies have been performed on active ingredients used as veterinary medicines Indirect effects mediated through the food chain have been observed for pyrethroids, some of which are used in veterinary applications (Kaushik et al 19 85; Giddings et... identify the effects of major metabolites and use that information as a starting point for identifying effects from metabolites in aquatic vertebrates using structure-activity relationship analysis 5. 5.4 MIXTURES It is highly unlikely that veterinary medicines will exist in aquatic systems alone, so it is important that the potential interactions with other veterinary medicines and contaminants are... reproduction) in the laboratory However, veterinary medicines are biologically © 2009 by the Society of Environmental Toxicology and Chemistry (SETAC) 122 Veterinary Medicines in the Environment active substances; there is an increasing body of evidence that exposure to select medicine groups could result in effects not identified using standard methodologies, and it is possible that indirect effects...Assessing the Aquatic Hazards of Veterinary Medicines 107 persist as a vapor in the atmosphere Solubility is pH dependent, ranging from 0.1 to 320 mg L –1, and is 5. 5 mg L –1 in seawater It is reported to be soluble in chloroform, acetone, and methanol but insoluble in hexane The pKa values of 4.2 and 7.6 indicate that at the pH of seawater, the molecule will be in an ionized form, which may lead to the. .. be present alongside other medicines as well as other aquatic contaminants, such as human medicines, pesticides, and nutrients; 5) veterinary medicines may be distributed as racemic mixtures, which may result in enantiospecific fate, exposure, and toxicity; and 6) aquatic ecosystems comprise interlinked communities of organisms, and exposure to veterinary medicines may result in indirect effects on a . impacts of veterinary medicines in aquatic systems. © 2009 by the Society of Environmental Toxicology and Chemistry (SETAC) 98 Veterinary Medicines in the Environment The chapter includes a. generated ngerprints. © 2009 by the Society of Environmental Toxicology and Chemistry (SETAC) 110 Veterinary Medicines in the Environment In either case, testing in addition to baseline assays (e.g.,. intraspecies variation in cal- culation of the PNEC. © 2009 by the Society of Environmental Toxicology and Chemistry (SETAC) 112 Veterinary Medicines in the Environment scale used in presenting