Scott E. Belanger

Pharmaceuticals and Personal Care Products in the Environment: What Are the Big Questions?

Background: Over the past 10–15 years, a substantial amount of work has been done by the scientific, regulatory, and business communities to elucidate the effects and risks of pharmaceuticals and personal care products (PPCPs) in the environment. Objective: This review was undertaken to identify key outstanding issues regarding the effects of PPCPs on human and ecological health in order to ensure that future resources will be focused on the most important areas. Data sources: To better understand and manage the risks of PPCPs in the environment, we used the “key question” approach to identify the principle issues that need to be addressed. Initially, questions were solicited from academic, government, and business communities around the world. A list of 101 questions was then discussed at an international expert workshop, and a top-20 list was developed. Following the workshop, workshop attendees ranked the 20 questions by importance. Data synthesis: The top 20 priority questions fell into seven categories: a) prioritization of substances for assessment, b) pathways of exposure, c) bioavailability and uptake, d) effects characterization, e) risk and relative risk, f ) antibiotic resistance, and g) risk management. Conclusions: A large body of information is now available on PPCPs in the environment. This exercise prioritized the most critical questions to aid in development of future research programs on the topic.

The Fish Embryo Toxicity Test as an Animal Alternative Method in Hazard and Risk Assessment and Scientific Research

Animal alternatives research has historically focused on human safety assessments and has only recently been extended to environmental testing. This is particularly for those assays that involve the use of fish. A number of alternatives are being pursued by the scientific community including the fish embryo toxicity (FET) test, a proposed replacement alternative to the acute fish test. Discussion of the FET methodology and its application in environmental assessments on a global level was needed. With this emerging issue in mind, the ILSI Health and Environmental Sciences Institute (HESI) and the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) held an International Workshop on the Application of the Fish Embryo Test as an Animal Alternative Method in Hazard and Risk Assessment and Scientific Research in March, 2008. The workshop included approximately 40 scientists and regulators representing government, industry, academia, and non-governmental organizations from North America, Europe, and Asia. The goal was to review the state of the science regarding the investigation of fish embryonic tests, pain and distress in fish, emerging approaches utilizing fish embryos, and the use of fish embryo toxicity test data in various types of environmental assessments (e.g., hazard, risk, effluent, and classification and labeling of chemicals). Some specific key outcomes included agreement that risk assessors need fish data for decision-making, that extending the FET to include eluethereombryos was desirable, that relevant endpoints are being used, and that additional endpoints could facilitate additional uses beyond acute toxicity testing. The FET was, however, not yet considered validated sensu OECD. An important action step will be to provide guidance on how all fish tests can be used to assess chemical hazard and to harmonize the diverse terminology used in test guidelines adopted over the past decades. Use of the FET in context of effluent assessments was considered and it is not known if fish embryos are sufficiently sensitive for consideration as a surrogate to the sub-chronic 7-day larval fish growth and survival test used in the United States, for example. Addressing these needs by via workshops, research, and additional data reviews were identified for future action by scientists and regulators.

A European perspective on alternatives to animal testing for environmental hazard identification and risk assessment

Tests with vertebrates are an integral part of environmental hazard identification and risk assessment of chemicals, plant protection products, pharmaceuticals, biocides, feed additives and effluents. These tests raise ethical and economic concerns and are considered as inappropriate for assessing all of the substances and effluents that require regulatory testing. Hence, there is a strong demand for replacement, reduction and refinement strategies and methods. However, until now alternative approaches have only rarely been used in regulatory settings. This review provides an overview on current regulations of chemicals and the requirements for animal tests in environmental hazard and risk assessment. It aims to highlight the potential areas for alternative approaches in environmental hazard identification and risk assessment. Perspectives and limitations of alternative approaches to animal tests using vertebrates in environmental toxicology, i.e. mainly fish and amphibians, are discussed. Free access to existing (proprietary) animal test data, availability of validated alternative methods and a practical implementation of conceptual approaches such as the Adverse Outcome Pathways and Integrated Testing Strategies were identified as major requirements towards the successful development and implementation of alternative approaches. Although this article focusses on European regulations, its considerations and conclusions are of global relevance.

Use of fish embryo toxicity tests for the prediction of acute fish toxicity to chemicals

The fish embryo test (FET) is a potential animal alternative for the acute fish toxicity (AFT) test. A comprehensive validation program assessed 20 different chemicals to understand intra- and interlaboratory variability for the FET. The FET had sufficient reproducibility across a range of potencies and modes of action. In the present study, the suitability of the FET as an alternative model is reviewed by relating FET and AFT. In total, 985 FET studies and 1531 AFT studies were summarized. The authors performed FET-AFT regressions to understand potential relationships based on physical-chemical properties, species choices, duration of exposure, chemical classes, chemical functional uses, and modes of action. The FET-AFT relationships are very robust (slopes near 1.0, intercepts near 0) across 9 orders of magnitude in potency. A recommendation for the predictive regression relationship is based on 96-h FET and AFT data: log FET median lethal concentration (LC50) = (0.989 × log fish LC50) - 0.195; n = 72 chemicals, r = 0.95, p < 0.001, LC50 in mg/L. A similar, not statistically different regression was developed for the entire data set (n = 144 chemicals, unreliable studies deleted). The FET-AFT regressions were robust for major chemical classes with suitably large data sets. Furthermore, regressions were similar to those for large groups of functional chemical categories such as pesticides, surfactants, and industrial organics. Pharmaceutical regressions (n = 8 studies only) were directionally correct. The FET-AFT relationships were not quantitatively different from acute fish-acute fish toxicity relationships with the following species: fathead minnow, rainbow trout, bluegill sunfish, Japanese medaka, and zebrafish. The FET is scientifically supportable as a rational animal alternative model for ecotoxicological testing of acute toxicity of chemicals to fish.

Saltatory ontogeny of fishes and sensitive early life stages for ecotoxicology tests.

Fish display a wide range of developmental ontogenies. These distinctions have taxonomic, evolutionary, and ecological importance in addition to practical implications on the use of fish in aquatic toxicity tests. With respect to animal welfare, vertebrates are afforded protected or non-protected status in the European Union based upon whether they feed endogenously off the yolk or exogenously by procurement and ingestion of food. The concept of saltatory ontogeny suggests development is not gradual but proceeds in leaps separated by a series of stable developmental states. In this context, endogenous/exogenous feeding also distinguishes the developmental phases of embryo (egg), eleutheroembryo (feeding off the yolk sac) and larvae (exogenous feeding) in fish. The recent proposal for the Fish Embryo Test (FET) as an animal alternative to the standard fish acute toxicity test (OECD 203 and equivalent tests) puts a clear focus on the need to identify the non-protected and protected life intervals in test species as well as their sensitivities which coincides with the developmental phases identified in saltatory ontogeny. In this paper we described a method to quantify embryo, eleutheroembryo, and larva phases in Danio rerio, the zebrafish. Danio eleutheroembryos preyed upon 5 different protozoan species (Euglena, Euplotes, Paramecium aurelia, Paramecium bursaria and Paramecium multimicronucleatum) between 24 and 48hr following hatching (85-95% of fish, n=20 per species, 25 degrees C). Based upon these data it is recommended that testing of developing zebrafish embryos should be terminated between 24 and 48hr after hatching in order to be compliant with existing animal welfare legislation within Europe.

Adverse outcome pathways during early fish development: a conceptual framework for identification of chemical screening and prioritization strategies.

The fish early life-stage (FELS) test guideline (OECD 210 or OCSPP 850.1400) is the most frequently used bioassay for predicting chronic fish toxicity and supporting aquatic ecological risk assessments around the world. For each chemical, the FELS test requires a minimum of 360 fish and 1 to 3 months from test initiation to termination. Although valuable for predicting fish full life-cycle toxicity, FELS tests are labor and resource intensive and, due to an emphasis on apical endpoints, provide little to no information about chemical mode of action. Therefore, the development and implementation of alternative testing strategies for screening and prioritizing chemicals has the potential to reduce the cost and number of animals required for estimating FELS toxicity and, at the same time, provides insights into mechanisms of toxicity. Using three reference chemicals with well-established yet distinct adverse outcome pathways (AOPs) in early life stages of fish, we proposed FELS-specific AOPs as conceptual frameworks for identifying useful chemical screening and prioritization strategies. The reference chemicals selected as case studies were a cardiotoxic aryl hydrocarbon receptor agonist (2,3,7,8-tetrachlorodibenzo-p-dioxin), neurotoxic acetylcholinesterase inhibitor (chlorpyrifos), and narcotic surfactant (linear alkylbenzene sulfonate). Using qualitative descriptions for each chemical during early fish development, we developed generalized AOPs and, based on these examples, proposed a three-tiered testing strategy for screening and prioritizing chemicals for FELS testing. Linked with biologically based concentration-response models, a tiered testing strategy may help reduce the reliance on long-term and costly FELS tests required for assessing the hazard of thousands of chemicals currently in commerce.

Environmental properties and aquatic hazard assessment of anionic surfactants: Physico-chemical, environmental fate and ecotoxicity properties

This paper summarizes the environmental hazard assessment of physicochemical properties, environmental fate and behavior and the ecotoxicity of a category of 61 anionic surfactants (ANS), comprised of alkyl sulfates (AS), primary alkane sulfonates (PAS) and alpha-olefin sulfonates (AOS) under the High Production Volume Chemicals Program of the Organisation for Economic Co-operation and Development (OECD). The most important common structural feature of the category members examined here is the presence of a predominantly linear aliphatic hydrocarbon chain with a polar sulfate or sulfonate group, neutralized with a counter-ion. The hydrophobic hydrocarbon chain (with a length between C(8) and C(18)) and the polar sulfate or sulfonate groups confer surfactant properties and enable the commercial use of these substances as anionic surfactants. The close structural similarities lead to physico-chemical properties and environmental fate characteristics which follow a regular pattern and justify the applied read-across within a category approach. Common physical and/or biological properties result in structurally similar breakdown products and are, together with the surfactant properties, responsible for similar environmental behavior. The structural similarities result in the same mode of ecotoxic action. Within each of the three sub-categories of ANS the most important parameter influencing ecotoxicity is the varying length of the alkyl chain. Although the counter-ion may also influence the physico-chemical properties, there is no indication that it significantly affects chemical reactivity, environmental fate and behavior or ecotoxicity of these chemicals. Deduced from physico-chemical and surfactancy properties, the main target compartment for the substances of the ANS category is the hydrosphere. They are quantitatively removed in waste water treatment plants, mainly by biodegradation. Quantitative removal in biological treatment plants is reflected by low AS concentrations measured in effluents of waste water treatment plants (mostly below 10 μg/L). In addition, bioaccumulation of ANS does not exceed regulatory triggers based upon experimental data. A considerable number of reliable aquatic toxicity data for the whole ANS category are available, including chronic and subchronic data for species of all trophic levels. Based upon the highest quality data in hand, there appears to be no singularly most sensitive trophic level in tests on the toxicity of alkyl sulfates, with a large degree of overlap among algae, invertebrates and fish. Algae proved to be more variable in sensitivity to alkyl sulfate exposure compared to fish and daphnia. The key study for the aquatic hazard assessment is a chronic test on Ceriodaphnia dubia, which covers a range of the alkyl chain length from C(12) to C(18). A parabolic response was observed, with the C(14) chain length being the most toxic (7d-NOEC=0.045 mg/L). Responses of aquatic communities to C(12) AS and C(14-15) AS have been studied in high quality stream mesocosm studies containing a broad range of species and ecological interactions. These studies are regarded as a better approximation to reality when extrapolating to the environment. The 56-d chronic NOEC for C(12) AS and C(14-15) AS were 0.224 and 0.106 mg/L, respectively, based on integrated assessments of periphyton (algal, bacterial and protozoan) and invertebrate communities. Taking into account the rapid biodegradation of the ANS compounds as well as the low concentrations measured in different environmental compartments, this category of surfactants is of low concern for the environment.

OECD validation study to assess intra- and inter-laboratory reproducibility of the zebrafish embryo toxicity test for acute aquatic toxicity testing

The OECD validation study of the zebrafish embryo acute toxicity test (ZFET) for acute aquatic toxicity testing evaluated the ZFET reproducibility by testing 20 chemicals at 5 different concentrations in 3 independent runs in at least 3 laboratories. Stock solutions and test concentrations were analytically confirmed for 11 chemicals. Newly fertilised zebrafish eggs (20/concentration and control) were exposed for 96h to chemicals. Four apical endpoints were recorded daily as indicators of acute lethality: coagulation of the embryo, lack of somite formation, non-detachment of the tail bud from the yolk sac and lack of heartbeat. Results (LC50 values for 48/96h exposure) show that the ZFET is a robust method with a good intra- and inter-laboratory reproducibility (CV<30%) for most chemicals and laboratories. The reproducibility was lower (CV>30%) for some very toxic or volatile chemicals, and chemicals tested close to their limit of solubility. The ZFET is now available as OECD Test Guideline 236. Considering the high predictive capacity of the ZFET demonstrated by Belanger et al. (2013) in their retrospective analysis of acute fish toxicity and fish embryo acute toxicity data, the ZFET is ready to be considered for acute fish toxicity for regulatory purposes.

Investigating Alternatives To The Fish Early-Life Stage Test: A Strategy For Discovering And Annotating Adverse Outcome Pathways For Early Fish Development

The fish early-life stage (FELS) test (Organisation for Economic Co-operation and Development [OECD] test guideline 210) is the primary test used internationally to estimate chronic fish toxicity in support of ecological risk assessments and chemical management programs. As part of an ongoing effort to develop efficient and cost-effective alternatives to the FELS test, there is a need to identify and describe potential adverse outcome pathways (AOPs) relevant to FELS toxicity. To support this endeavor, the authors outline and illustrate an overall strategy for the discovery and annotation of FELS AOPs. Key events represented by major developmental landmarks were organized into a preliminary conceptual model of fish development. Using swim bladder inflation as an example, a weight-of-evidence–based approach was used to support linkage of key molecular initiating events to adverse phenotypic outcomes and reduced young-of-year survival. Based on an iterative approach, the feasibility of using key events as the foundation for expanding a network of plausible linkages and AOP knowledge was explored and, in the process, important knowledge gaps were identified. Given the scope and scale of the task, prioritization of AOP development was recommended and key research objectives were defined relative to factors such as current animal-use restrictions in the European Union and increased demands for fish toxicity data in chemical management programs globally. The example and strategy described are intended to guide collective efforts to define FELS-related AOPs and develop resource-efficient predictive assays that address the toxicological domain of the OECD 210 test. Environ Toxicol Chem 2014;33:158–169.

Environmental Safety of the Use of Major Surfactant Classes in North America

This paper brings together over 250 published and unpublished studies on the environmental properties, fate, and toxicity of the four major, high-volume surfactant classes and relevant feedstocks. The surfactants and feedstocks covered include alcohol sulfate or alcohol sulfate (AS), alcohol ethoxysulfate (AES), linear alkylbenzene sulfonate (LAS), alcohol ethoxylate (AE), and long-chain alcohol (LCOH). These chemicals are used in a wide range of personal care and cleaning products. To date, this is the most comprehensive report on these substances chemical structures, use, and volume information, physical/chemical properties, environmental fate properties such as biodegradation and sorption, monitoring studies through sewers, wastewater treatment plants and eventual release to the environment, aquatic and sediment toxicity, and bioaccumulation information. These data are used to illustrate the process for conducting both prospective and retrospective risk assessments for large-volume chemicals and categories of chemicals with wide dispersive use. Prospective risk assessments of AS, AES, AE, LAS, and LCOH demonstrate that these substances, although used in very high volume and widely released to the aquatic environment, have no adverse impact on the aquatic or sediment environments at current levels of use. The retrospective risk assessments of these same substances have clearly demonstrated that the conclusions of the prospective risk assessments are valid and confirm that these substances do not pose a risk to the aquatic or sediment environments. This paper also highlights the many years of research that the surfactant and cleaning products industry has supported, as part of their environmental sustainability commitment, to improve environmental tools, approaches, and develop innovative methods appropriate to address environmental properties of personal care and cleaning product chemicals, many of which have become approved international standard methods.