ABSTRACT Tecnazene, 2,3,5,6-tetrachloronitrobenzene, a sprouting inhibitor and fungicide used on ... more ABSTRACT Tecnazene, 2,3,5,6-tetrachloronitrobenzene, a sprouting inhibitor and fungicide used on stored potatoes, has been found to enter the freshwater environment at specific sites in the United Kingdom due to the practice of bulk washing potatoes for supermarket presentation. Because of the paucity of toxicity data for aquatic organisms, the acute toxicity of tecnazene was determined for 7 freshwater species (3 fish, 2 crustaceans, 1 mollusc and 1 insect). A 28-day fish growth test and bioconcentration study were also conducted. Tecnazene is highly toxic to the aquatic species tested, with 96 h LC50 values ranging from 270 to 2340 μg 1−1; adverse effects on fish growth rates were found above 7.5 μg 1−1. Mean bioconcentration factors of between 2390 and 5170 were found after 28 days' exposure although the accumulated tecnazene depurated rapidly. A limited field investigation in the potato growing region of the Lincolnshire Fens, UK, revealed no traces of tecnazene in fish samples but concentrations of tecnazene and one of its breakdown products, 2,3,5,6-tetrachloroaniline, were detected in water at 18 and 5 μg 1−1 respectively, immediately downstream of one potato washing plant. Sporadic washing activity probably affected the frequency of occurrence of tecnazene.
The joint toxicity to rainbow trout fingerlings (Salmo gairdneri Richardson) of eleven tank-mixes... more The joint toxicity to rainbow trout fingerlings (Salmo gairdneri Richardson) of eleven tank-mixes composed of pairs of six fungicides and herbicides (prochloraz, fenpropimorph, diclofop-methyl, tridemorph, benzoylprop-ethyl and propiconazole) was measured using 96 h LC~o tests. The toxicity of the mixtures ranged from being half of that expected on the basis of additive toxicity of the components, to less than 1.4 times the expected value. In view of the degree of experimental error inherent in the LCs0 determinations, these data provide no evidence for the existence of synergistic (i.e. more-than-additive) toxicity of the respective tank-mixes.
Integrated Environmental Assessment and Management, 2015
The ecotoxicity testing of chemicals for prospective environmental safety assessment is an area w... more The ecotoxicity testing of chemicals for prospective environmental safety assessment is an area where a high number of vertebrates are used across a variety of industry sectors. Refining, reducing and replacing the use of animals such as fish, birds and amphibians for this purpose addresses the ethical concerns and the increasing legislative requirements to consider alternative test methods. Members of the UK-based National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) Ecotoxicology Working Group, consisting of representatives from academia, government organisations and industry, have worked together over the past six years to provide evidence bases to support and advance the application of the 3Rs in regulatory ecotoxicity testing. The group recently held a workshop to identify the areas of testing, demands and drivers that will impact on the future of animal use in regulatory ecotoxicology. As a result of these discussions we have developed a pragmatic approach to prioritise and realistically address key opportunity areas, to enable progress towards the vision of a reduced reliance on the use of animals in this area of testing. This paper summarises the findings of this exercise and proposes a pragmatic strategy towards our key long-term goals - the incorporation of reliable alternatives to whole organism testing into regulations and guidance, and a culture shift towards the reduced reliance on vertebrate toxicity testing in routine environmental safety assessment. This article is protected by copyright. All rights reserved.
Integrated environmental assessment and management, Jan 24, 2015
The trend in discharges of petroleum-related substances from refineries in Europe shows a consist... more The trend in discharges of petroleum-related substances from refineries in Europe shows a consistent picture of declining emissions, since first measured in 1969. This decline coincides with enhanced internal capture or recycling procedures and increasing use of physical and biological treatments. At the same time, and partly in response to legislative drivers, there has been an increase in the use of chronic (long-term) toxicity tests, and alternative methods for assessing the quality of effluent discharges. The Whole Effluent Assessment (WEA) approach has also driven the increased conduct of studies addressing the fate of effluent constituents. Such studies have included the use of biodegradation and Solid Phase Micro-Extraction-Biomimetic Extraction (SPME-BE) methods to address Potentially Bioaccumulative Substances (PBS). In this way it is then possible to address the persistence and toxicity of these PBS constituents of an effluent. The data collected in various case studies hi...
Large-scale toxicogenomic screening approaches offer great promise for generating a bias-free sys... more Large-scale toxicogenomic screening approaches offer great promise for generating a bias-free system-wide view of toxicological effects and modes-of-action of chemicals and ecotoxicants. However, early applications of microarray technology have identified relatively small groups of responding genes with which to define new targets for analysis by conventional means. We have trialled a more intensive approach to the design and interpretation of array experiments incorporating a balanced interwoven ANOVA design with higher levels of biological replication, a more thorough analysis of errors and false discovery rates, and an analysis of response patterns using gene network models. Zebrafish embryos were exposed from 1.5 h post-fertilization for 72 h to ecotoxicants representing different classes-2,4-dichlorophenol, 3,4-dichloroaniline, pentachlorophenol, and cadmium chloride-at low concentrations producing a developmental disturbance to 10% of embryos and half of this dose. Extracted whole embryo RNA was then analyzed on microarrays. Analysis revealed responses of 3000-5000 genes, which is 10-1000 times greater than previously reported, with significance at lower levels of fold change. Some gene responses were common to multiple toxicants, and others were restricted to just one or two toxicants. The gene expression profiles for the different toxicants were distinctive, and analysis using network-based models provided a high level of detail of affected processes, some of which were novel. This approach provides a more highly refined view of toxic effects, from which meaningful patterns of response can be discerned and related to functional deficits and from which more reliable indicators of toxicological effect can be predicted.
Integrated Environmental Assessment and Management, 2007
When addressing the use of fish for the environmental safety of chemicals and effluents, there ar... more When addressing the use of fish for the environmental safety of chemicals and effluents, there are many opportunities for applying the principles of the 3Rs: Reduce, Refine, and Replace. The current environmental regulatory testing strategy for bioconcentration and secondary poisoning has been reviewed, and alternative approaches that provide useful information are described. Several approaches can be used to reduce the number of fish used in the Organization for Economic Cooperation and Development (OECD) Test Guideline 305, including alternative in vivo test methods such as the dietary accumulation test and the static exposure approach. The best replacement approach would seem to use read-across, chemical grouping, and quantitative structure-activity relationships with an assessment of the key processes in bioconcentration: Adsorption, distribution, metabolism, and excretion. Biomimetic extraction has particular usefulness in addressing bioavailable chemicals and is in some circumstances capable of predicting uptake. Use of alternative organisms such as invertebrates should also be considered. A single cut-off value for molecular weight and size beyond which no absorption will take place cannot be identified. Recommendations for their use in bioaccumulative (B) categorization schemes are provided. Assessment of biotransformation with in vitro assays and in silico approaches holds significant promise. Further research is needed to identify their variability and confidence limits and the ways to use this as a basis to estimate bioconcentration factors. A tiered bioconcentration testing strategy has been developed taking account of the alternatives discussed.
Toxicity reduction evaluations (TREs) in the River Esk and Lower Tees Estuary were based on the a... more Toxicity reduction evaluations (TREs) in the River Esk and Lower Tees Estuary were based on the approach described by USEPA, but adapted to tackle the specific problems of the two sites. A combination of toxicity tracking and toxicity identification evaluation (TIE) was used at both locations to enhance the understanding of source and type of toxicants present. The assessment of toxicity at Langholm focussed on pesticides present in the sewerage network. The TIE programme indicated that the most likely toxic agents within the effluent were the organophosphate pesticides diazinon and to lesser extent propetamphos, although these did not account for all of the observed toxicity. The exact source of these toxicants was not clear although toxicity tracking identified two potential candidates. The TRE undertaken on the discharge to the lower Tees utilised high-throughput methods with standard test organisms to generate toxicity information throughout a complex sewerage network. The toxicity tracking information was used in conjunction with TIEs to identify a number of key sources of toxicity. Substantial toxicity was associated with a currently untreated industrial effluent. Chemical analysis and TIE highlighted cyanide as the likely toxicant in this effluent and its possible significance in the final discharge.
Animal alternatives research has historically focused on human safety assessments and has only re... more 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.
Tests with vertebrates are an integral part of environmental hazard identification and risk asses... more 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.
Integrated Environmental Assessment and Management, 2010
Legislation on the use of laboratory animals Numbers of fish used in the European Union Tests of ... more Legislation on the use of laboratory animals Numbers of fish used in the European Union Tests of fish required for regulatory purposes Political mandates for the reduction/refinement/replacement of animals for hazard and risk assessment
ABSTRACT Tecnazene, 2,3,5,6-tetrachloronitrobenzene, a sprouting inhibitor and fungicide used on ... more ABSTRACT Tecnazene, 2,3,5,6-tetrachloronitrobenzene, a sprouting inhibitor and fungicide used on stored potatoes, has been found to enter the freshwater environment at specific sites in the United Kingdom due to the practice of bulk washing potatoes for supermarket presentation. Because of the paucity of toxicity data for aquatic organisms, the acute toxicity of tecnazene was determined for 7 freshwater species (3 fish, 2 crustaceans, 1 mollusc and 1 insect). A 28-day fish growth test and bioconcentration study were also conducted. Tecnazene is highly toxic to the aquatic species tested, with 96 h LC50 values ranging from 270 to 2340 μg 1−1; adverse effects on fish growth rates were found above 7.5 μg 1−1. Mean bioconcentration factors of between 2390 and 5170 were found after 28 days' exposure although the accumulated tecnazene depurated rapidly. A limited field investigation in the potato growing region of the Lincolnshire Fens, UK, revealed no traces of tecnazene in fish samples but concentrations of tecnazene and one of its breakdown products, 2,3,5,6-tetrachloroaniline, were detected in water at 18 and 5 μg 1−1 respectively, immediately downstream of one potato washing plant. Sporadic washing activity probably affected the frequency of occurrence of tecnazene.
The joint toxicity to rainbow trout fingerlings (Salmo gairdneri Richardson) of eleven tank-mixes... more The joint toxicity to rainbow trout fingerlings (Salmo gairdneri Richardson) of eleven tank-mixes composed of pairs of six fungicides and herbicides (prochloraz, fenpropimorph, diclofop-methyl, tridemorph, benzoylprop-ethyl and propiconazole) was measured using 96 h LC~o tests. The toxicity of the mixtures ranged from being half of that expected on the basis of additive toxicity of the components, to less than 1.4 times the expected value. In view of the degree of experimental error inherent in the LCs0 determinations, these data provide no evidence for the existence of synergistic (i.e. more-than-additive) toxicity of the respective tank-mixes.
Integrated Environmental Assessment and Management, 2015
The ecotoxicity testing of chemicals for prospective environmental safety assessment is an area w... more The ecotoxicity testing of chemicals for prospective environmental safety assessment is an area where a high number of vertebrates are used across a variety of industry sectors. Refining, reducing and replacing the use of animals such as fish, birds and amphibians for this purpose addresses the ethical concerns and the increasing legislative requirements to consider alternative test methods. Members of the UK-based National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) Ecotoxicology Working Group, consisting of representatives from academia, government organisations and industry, have worked together over the past six years to provide evidence bases to support and advance the application of the 3Rs in regulatory ecotoxicity testing. The group recently held a workshop to identify the areas of testing, demands and drivers that will impact on the future of animal use in regulatory ecotoxicology. As a result of these discussions we have developed a pragmatic approach to prioritise and realistically address key opportunity areas, to enable progress towards the vision of a reduced reliance on the use of animals in this area of testing. This paper summarises the findings of this exercise and proposes a pragmatic strategy towards our key long-term goals - the incorporation of reliable alternatives to whole organism testing into regulations and guidance, and a culture shift towards the reduced reliance on vertebrate toxicity testing in routine environmental safety assessment. This article is protected by copyright. All rights reserved.
Integrated environmental assessment and management, Jan 24, 2015
The trend in discharges of petroleum-related substances from refineries in Europe shows a consist... more The trend in discharges of petroleum-related substances from refineries in Europe shows a consistent picture of declining emissions, since first measured in 1969. This decline coincides with enhanced internal capture or recycling procedures and increasing use of physical and biological treatments. At the same time, and partly in response to legislative drivers, there has been an increase in the use of chronic (long-term) toxicity tests, and alternative methods for assessing the quality of effluent discharges. The Whole Effluent Assessment (WEA) approach has also driven the increased conduct of studies addressing the fate of effluent constituents. Such studies have included the use of biodegradation and Solid Phase Micro-Extraction-Biomimetic Extraction (SPME-BE) methods to address Potentially Bioaccumulative Substances (PBS). In this way it is then possible to address the persistence and toxicity of these PBS constituents of an effluent. The data collected in various case studies hi...
Large-scale toxicogenomic screening approaches offer great promise for generating a bias-free sys... more Large-scale toxicogenomic screening approaches offer great promise for generating a bias-free system-wide view of toxicological effects and modes-of-action of chemicals and ecotoxicants. However, early applications of microarray technology have identified relatively small groups of responding genes with which to define new targets for analysis by conventional means. We have trialled a more intensive approach to the design and interpretation of array experiments incorporating a balanced interwoven ANOVA design with higher levels of biological replication, a more thorough analysis of errors and false discovery rates, and an analysis of response patterns using gene network models. Zebrafish embryos were exposed from 1.5 h post-fertilization for 72 h to ecotoxicants representing different classes-2,4-dichlorophenol, 3,4-dichloroaniline, pentachlorophenol, and cadmium chloride-at low concentrations producing a developmental disturbance to 10% of embryos and half of this dose. Extracted whole embryo RNA was then analyzed on microarrays. Analysis revealed responses of 3000-5000 genes, which is 10-1000 times greater than previously reported, with significance at lower levels of fold change. Some gene responses were common to multiple toxicants, and others were restricted to just one or two toxicants. The gene expression profiles for the different toxicants were distinctive, and analysis using network-based models provided a high level of detail of affected processes, some of which were novel. This approach provides a more highly refined view of toxic effects, from which meaningful patterns of response can be discerned and related to functional deficits and from which more reliable indicators of toxicological effect can be predicted.
Integrated Environmental Assessment and Management, 2007
When addressing the use of fish for the environmental safety of chemicals and effluents, there ar... more When addressing the use of fish for the environmental safety of chemicals and effluents, there are many opportunities for applying the principles of the 3Rs: Reduce, Refine, and Replace. The current environmental regulatory testing strategy for bioconcentration and secondary poisoning has been reviewed, and alternative approaches that provide useful information are described. Several approaches can be used to reduce the number of fish used in the Organization for Economic Cooperation and Development (OECD) Test Guideline 305, including alternative in vivo test methods such as the dietary accumulation test and the static exposure approach. The best replacement approach would seem to use read-across, chemical grouping, and quantitative structure-activity relationships with an assessment of the key processes in bioconcentration: Adsorption, distribution, metabolism, and excretion. Biomimetic extraction has particular usefulness in addressing bioavailable chemicals and is in some circumstances capable of predicting uptake. Use of alternative organisms such as invertebrates should also be considered. A single cut-off value for molecular weight and size beyond which no absorption will take place cannot be identified. Recommendations for their use in bioaccumulative (B) categorization schemes are provided. Assessment of biotransformation with in vitro assays and in silico approaches holds significant promise. Further research is needed to identify their variability and confidence limits and the ways to use this as a basis to estimate bioconcentration factors. A tiered bioconcentration testing strategy has been developed taking account of the alternatives discussed.
Toxicity reduction evaluations (TREs) in the River Esk and Lower Tees Estuary were based on the a... more Toxicity reduction evaluations (TREs) in the River Esk and Lower Tees Estuary were based on the approach described by USEPA, but adapted to tackle the specific problems of the two sites. A combination of toxicity tracking and toxicity identification evaluation (TIE) was used at both locations to enhance the understanding of source and type of toxicants present. The assessment of toxicity at Langholm focussed on pesticides present in the sewerage network. The TIE programme indicated that the most likely toxic agents within the effluent were the organophosphate pesticides diazinon and to lesser extent propetamphos, although these did not account for all of the observed toxicity. The exact source of these toxicants was not clear although toxicity tracking identified two potential candidates. The TRE undertaken on the discharge to the lower Tees utilised high-throughput methods with standard test organisms to generate toxicity information throughout a complex sewerage network. The toxicity tracking information was used in conjunction with TIEs to identify a number of key sources of toxicity. Substantial toxicity was associated with a currently untreated industrial effluent. Chemical analysis and TIE highlighted cyanide as the likely toxicant in this effluent and its possible significance in the final discharge.
Animal alternatives research has historically focused on human safety assessments and has only re... more 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.
Tests with vertebrates are an integral part of environmental hazard identification and risk asses... more 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.
Integrated Environmental Assessment and Management, 2010
Legislation on the use of laboratory animals Numbers of fish used in the European Union Tests of ... more Legislation on the use of laboratory animals Numbers of fish used in the European Union Tests of fish required for regulatory purposes Political mandates for the reduction/refinement/replacement of animals for hazard and risk assessment
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