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Chapter 2
Ecotoxicology Data Requirements and Evaluation in California Alexander Kolosovich* and Richard Bireley California Department of Pesticide Regulation, California Environmental Protection Agency, Sacramento, California 95812, United States *E-mail:
[email protected].
The overall goal of the Ecotoxicology Station in the Department of Pesticide Regulation’s Pesticide Registration Branch is to evaluate the risks to nontarget organisms and ensure that significant risks are mitigated when the pesticide product is used in compliance with the directions for use. The two components that must be considered when evaluating risk are toxicity and exposure. Additionally, persistence and bioaccumulation are also important considerations when determining whether a pesticide product will have significant adverse effects on nontarget organisms.
The California Department of Pesticide Regulation (CDPR), much like the U.S. Environmental Protection Agency (US EPA) and other national and international regulatory agencies, is charged with protecting human health and the environment from risks associated with the use of pesticides. One component of this mission is evaluating the hazards and risks of pesticide products containing new active ingredients (AIs) to nontarget fish, wildlife, terrestrial and aquatic invertebrates (including honey bees), pets, and livestock. This is accomplished by a detailed analysis of scientific data submitted by the registrant of the pesticide as well as by researching any other available information. Registrant-submitted data are typically conducted by contract laboratories under Good Laboratory Practice Standards (1). At the CDPR, environmental fate and product chemistry data are first evaluated by the Chemistry Station (2). The results of this evaluation and the chemistry endpoints (e.g., degradation half-lives, partition coefficients) are then submitted to the Ecotoxicology Station. The Ecotoxicology Station evaluates © 2019 American Chemical Society Goh et al.; Pesticides in Surface Water: Monitoring, Modeling, Risk Assessment, and Management ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
toxicity data and, using the chemistry endpoints and product label information, determines risks to various taxa. The label information includes precautionary statements, crops and locations on which a pesticide may be used, and the application rate, timing, number, and methods of applications. Toxicity is assessed based on toxicity tests conducted on various organisms that serve as surrogates for different classes of animals (e.g., birds, fish, and invertebrates). The data requirements for the registration of a new pesticide product containing a new AI are extensive and differ based on the classification of the AI and the use pattern. In general, conventional chemicals require the most toxicity testing, with AIs that are classified as microbial or biochemical having fewer required tests. The use patterns outlined on the pesticide product label are also taken into consideration when determining which specific tests are required. Pesticide products with broader use patterns, such as terrestrial, aquatic, forestry, and residential, require more nontarget organism toxicity tests than do products limited to only indoor or greenhouse use. The full nontarget organism data requirements for conventional, microbial, and biochemical AIs are specified in Title 40 of the Code of Federal Regulations (40 CFR) § 158.630, § 158.2150, and § 158.2060, respectively (3–5). The first step in the evaluation of a new pesticide product is to review the data submitted in support of registration (Table 1). The data is in the form of study reports of toxicity tests, which must be thoroughly evaluated to ensure the studies are scientifically sound. Good Laboratory Practice Standards (40 CFR § 160 (1)) and the associated laboratory accreditation and inspection procedures help ensure the studies are of high quality and integrity. All toxicity tests have been standardized, so that the results are reproducible regardless of where they are being conducted. Study parameters such as the duration of exposure, observation points (i.e., when observations for mortality and sublethal effects must be conducted and recorded), light regimes (i.e., how many hours the test organisms are exposed to light and darkness), temperature, concentrations and types of solvents used for AIs with low solubility, and so forth are written into the US EPA’s Office of Chemical Safety and Pollution Prevention’s Ecological Effects Test Guidelines (OCSPP Series 850) for each toxicity test (6). Studies are generally determined to be acceptable if the endpoints (e.g., LC50 [the concentration that is lethal to 50% of the test population], EC50 [the concentration that causes a defined non-lethal effect in 50% of the test population], NOEC [the no observed effect concentration]) are scientifically sound. All study reports must contain details describing the conditions under which the studies were conducted as well as raw data to allow for independent statistical analysis and determinations of study endpoints. If the study endpoints cannot be independently evaluated by CDPR scientists, then the study is determined to be unacceptable and must be repeated or additional data or information must be submitted to upgrade the study to acceptable. Toxicity tests, especially acute toxicity tests, are often conducted with 10 to 20 test organisms per treatment group (6). The test organism sample sizes may be too small to properly assess if the data is normally distributed. This requires the use of nonparametric statistical tests, which have reduced power to detect differences compared with parametric tests (7). Thus, the ecotoxicology evaluator must consider biological relevance in addition to statistical significance. An effect 12 Goh et al.; Pesticides in Surface Water: Monitoring, Modeling, Risk Assessment, and Management ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
that is not statistically significant could be due to low sample size or a statistical test’s lack of power to detect differences, but it could still produce population-level effects in the real world. Conversely, an effect might be statistically significant but not biologically relevant. A dose–response can be one avenue to determine whether an effect is treatment-related in lieu of statistics. Statistical tests are powerful tools, but they are not the only way to determine whether effects are treatment-related. A variety of issues can result in a study not being acceptable. Poor control performance can make studies unacceptable. Excessive mortality in the control groups can make it difficult or impossible to determine whether mortality in the treatment groups is treatment-related. Excessive precipitate in aquatic toxicity tests conducted with low-solubility compounds can also complicate interpretation of the studies because of uncertainties with the concentrations to which the test organisms were exposed. In addition, validation of the dose or concentration must be conducted. Significant differences in the nominal versus mean measured concentration can result in studies that are difficult to interpret or not acceptable. Other factors that increase the ability of the assessor to interpret a study include odd or unusual symptoms or necropsy findings that may or may not be treatmentrelated. Overly complex studies can result in outcomes that appear contradictory or endpoints that are difficult to interpret. Inconsistencies in the write-up by the study authors can result in confusion or an unacceptable study. Finally, the typical goal of most toxicity testing is to see a dose–response such that effects are more severe at increasing exposures. In practice, there are occasions in which there is no dose–response. When this occurs, it is usually the result of there being no observed treatment effect to the test organism. However, on rare occasions, there are effects that appear to be attributable to exposure, including mortality, but no discernable dose–response. Once the full suite of toxicity tests are evaluated and determined to be acceptable, the challenge of relating toxicity values to environmental concentrations can be initiated. This involves understanding the environmental fate of the chemical and utilizing the proposed product label to determine where, how, how much, and how often the product is applied. In addition, the stability of the compound in various environmental matrices is critical to understanding and quantifying the potential for exposure. Chemicals may be highly labile and degrade rapidly in all environmentally relevant matrices. They may degrade under some conditions but be relatively stable under others, whereas a few compounds are relatively stable under most environmental conditions. In addition to the chemical itself, knowing which degradation breakdown products occur under which conditions is critical. Additional toxicity and environmental fate data may be required for major metabolites or degradates that are expected to be present in the environment in significant concentrations or are expected to be more persistent, toxic, or bioaccumulative than the parent chemical. There is a common misconception that one species or class of organisms is always the most sensitive to chemicals. In reality, it is not unusual for a fish species that resides in relatively murky and low-oxygen water, such as carp, to be more sensitive than trout, which are most often associated with clear mountain streams. Different chemicals affect various animals differently, and the current state of 13 Goh et al.; Pesticides in Surface Water: Monitoring, Modeling, Risk Assessment, and Management ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
knowledge of the specific biological receptors that interact with chemicals on a molecular basis is much more advanced for humans than it is for other animals. The CDPR uses a deterministic approach to assessing risks. This is done by dividing an expected exposure concentration (EEC) by a toxicity value such as an NOEC or an LC50 for chronic and acute tests, respectively. The resulting risk quotient has no units and is compared to a Level of Concern, which is normally 0.5 for acute toxicity tests and 1.0 for chronic tests. Unmitigated risks have the potential to occur when the toxicity of the new chemical is at or exceeds the concentration of the chemical in the environment following a labeled use. For example, if use of the product in compliance with the labeled directions results in an EEC value of 20 parts per billion (ppb) and the acute fish toxicity test results in an LC50 value of 10 ppb, then the risk quotient would be 2.0 and the risks are unmitigated. The US EPA has many models that are available to the public and can be used to calculate EEC values. The Pesticide in Water Calculator is used to calculate EEC values in water (8). Variables such as the application method (e.g., foliar, aerial, chemigation), application rate, reapplication intervals, the crop that the product is being applied to, various physicochemical parameters (e.g., degradation half-lives, partition coefficients), and a weather file for various locations and climatic scenarios are entered into the model to calculate the EEC value when the product is used under various labeled application scenarios. The Bee-REX model is used for estimating exposure to honey bees and the T-REX model is used to estimate exposure to mammals and birds (9, 10). Most pesticide products containing a new AI are conventional chemicals intended for agricultural (i.e., terrestrial) use. Products containing these AIs will often be applied to large agricultural fields, so there is potential for nontarget organism exposure and the list of required toxicity tests is extensive (Table 1). Foliar applications generally have the greatest potential to move offsite, whereas applications to soil, through drip irrigation, or as a seed treatment have less potential to drift. Many avenues are available for mitigation if an AI is determined to have unmitigated risks to nontarget organisms when the product is used in compliance with the label directions. Individual application rates as well as maximum annual application rates can be decreased if the product is still efficacious. Reapplication intervals can be extended to allow the chemical to degrade before additional applications are made, thus reducing the expected environmental concentrations for nontarget organisms (i.e., the concentrations to which nontarget organisms can potentially be exposed when the product is used as directed). Limits can be placed on the number of applications permitted. Buffer zones can be made a requirement for ground or aerial foliar applications. Aerial applications, which have higher potential to drift in comparison with foliar applications made using ground equipment, can be prohibited. All of these mitigation options can serve to decrease the expected environmental concentrations to which nontarget organisms are exposed. The chemical companies are also required to submit labels that are reviewed by CDPR’s Ecotoxicology Unit. The label is the law, and use of pesticide products in ways that violate the directions for use can carry heavy penalties. The US EPA has sole authority over pesticide label language. CDPR cannot require 14 Goh et al.; Pesticides in Surface Water: Monitoring, Modeling, Risk Assessment, and Management ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
specific language on labels, but it can refuse to register a product determined to have unmitigated risks. Companies can choose to voluntarily modify their product labels to reduce risks to acceptable levels for nontarget organisms. In cases in which CDPR determines an AI is toxic, persistent, or has high potential for bioaccumulation, it may be difficult to mitigate the risks. Most nontarget organism toxicity tests are conducted on the AI alone, but there are some conditions that trigger the requirement for companies to submit toxicity tests conducted with formulated pesticide products. These toxicity tests expose the test organisms to all the inert ingredients that will be mixed with the AI when the product is sold to the end user. For example, the freshwater fish toxicity tests (OCSPP 850.1075) and acute toxicity to freshwater invertebrates (OCSPP 850.1010) toxicity tests conducted with the AI alone must always be submitted to register new products containing new AIs (11, 12). However, if the directions for use allow for application directly to the aquatic environment, such as for some mosquito abatement products and aquatic herbicides, then the companies are also required to submit those same tests conducted with the formulated end product (3). The requirement for formulated end-product testing is also triggered if the maximum expected environmental concentrations exceed one half of the LC50 or EC50 of the most sensitive species tested. The determination of whether these conditions are triggered is at the discretion of CDPR. Overall, this assessment allows CDPR scientists to distinguish those actions that are likely to have significant adverse effects on populations of nontarget fish, wildlife, aquatic and terrestrial invertebrates (including honey bees), livestock, and pets.
Table 1. Terrestrial and Aquatic Nontarget Organism Data Requirements (40 CFR § 158.630 (3)) Test Type
OCSPP guideline number
Avian toxicity tests Avian oral toxicity a
850.2100
Avian dietary toxicity b
850.2200
Avian reproduction b
850.2300
Aquatic organism toxicity tests Freshwater fish toxicity test c
850.1075
Acute toxicity to freshwater invertebrates d
850.1010
Acute toxicity to estuarine and marine organisms e
850.1025; 850.1035; 850.1075
Aquatic invertebrate life cycle (freshwater) d
850.1300
Aquatic invertebrate life cycle (saltwater) f,g
850.1350
Fish early-life stage (freshwater) h
850.1400 Continued on next page.
15 Goh et al.; Pesticides in Surface Water: Monitoring, Modeling, Risk Assessment, and Management ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
Table 1. (Continued). Terrestrial and Aquatic Nontarget Organism Data Requirements (40 CFR § 158.630 (3)) Test Type
OCSPP guideline number
Fish early-life stage (saltwater) Fish bioconcentration factor
g,i
850.1400
j
850.1730
Freshwater invertebrate spiked whole sediment 10-day toxicity test k
850.1735
Insect pollinator tests Honey bee acute contact toxicity
850.3020
Honey bee acute oral toxicity
OECD 235
a Required tests include one passerine species (e.g., canaries or zebra finches) and one upland bird species (e.g., bobwhite quail) or one waterfowl species (e.g., mallard ducks). b Required tests include one upland bird species (e.g., bobwhite quail) and one waterfowl species (e.g., mallard ducks). c Required tests include one cold water fish species (e.g., rainbow trout) and one warm water fish species (e.g., bluegill sunfish, fathead minnow, carp). d Required test includes one freshwater aquatic invertebrate species (e.g., Daphnia magna). e Required tests include oyster shell deposition, mysid shrimp acute toxicity, and one estuarine/marine fish species (e.g., sheepshead minnow). f Required test includes one estuarine or marine invertebrate species (e.g., mysid shrimp). g Conditionally required when the product is intended for direct application to the estuarine or marine environment, is expected to enter estuarine or marine environments in significant concentrations because of expected use or mobility patterns, or if the acute LC50 or EC50
