Estimating ecotoxicological risk and impact using indigenous aquatic microbial communities

Emphasis has increased on accuracy in predicting the effect that anthropogenic stress has on natural ecosystems. Although toxicity tests low in environmental realism, such as standardized single species procedures, have been useful in providing a certain degree of protection to human health and the environment, the accuracy of such tests for predicting the effects of anthropogenic activities on complex ecosystems is questionable. The use of indigenous communities of microorganisms to assess the hazard of toxicants in aquatic ecosystems has many advantages. Theoretical and practical aspects of microbial community tests are discussed, particularly in related to widely cited problems in the use of multispecies test systems for predicting hazard. Further standardization of testing protocols using microbial colonization dynamics is advocated on the basis of previous studies, which have shown these parameters to be useful in assessing risk and impact of hazardous substances in aquatic ecosystems.     

Utility and relevance of aquatic oligochaetes in Ecological Risk Assessment

Ecological risk assessment (EcoRA) provides both a process and a framework to evaluate the potential for adverse ecological effects occurring as a result of exposure to contaminants or other stressors. EcoRA begins with problem formulation/hazard identification, progresses to effects and exposure assessment, and culminates with risk characterization (an estimate of the incidence and severity of any adverse effects likely to occur). Key components of EcoRA include determining: stressors/contaminants of concern; sensitive, exposed biota; and, appropriate tests and organisms for evaluating effects. Aquatic oligochaetes are not generally used directly in EcoRA because of three major perceptions. First, EcoRA personnel are generally not familiar with or comfortable using this group of organisms. Second, there is believed to be a paucity of widely accepted toxicity tests with these organisms. Third, their taxonomy is considered difficult and uncertain. In fact, aquatic oligochaetes potentially have great utility and relevance to EcoRAs because of factors including: their importance in the aquatic food chain (e.g. prey to fauna including fish and waterfowl; as a vector for contaminant movement through the food chain from bacteria); many species are widely distributed and well studied; representatives include fresh, estuarine and marine species; as a group, they range from sensitive to insensitive over a wide range of environmental insults; they have a long history of use in pollution monitoring and assessment; and, relevant toxicity and biaccumulation tests exist. Toxicity testing under defined conditions is appropriate for problem formulation while more realistic testing for effects assessment (e.g. microcosms) is logistically easier with this group of organisms than with others due to their relatively small size. The importance of aquatic oligochaetes for EcoRA, in particular of sediments, is particularly compelling.     

A Novel Approach to Determining a Population-Level Threshold in Ecological Risk Assessment: A Case Study of Zinc

A novel approach to population-level assessment was applied in order to demonstrate its utility in estimating and managing the risk of zinc in a water environment. Much attention has been paid to population-level risk assessment, but there have been no attempts to determine a “safe” population-level concentration as an environmental criterion. Based on the published results of toxicity tests for various species, we first theoretically derived a threshold concentration at which a population size is unchanged due to the adverse effects of zinc exposure. To derive a zinc concentration that will protect populations in natural environments, we adopted the concept of species sensitivity distribution. Assuming the threshold concentrations of a set of species are log-normally distributed, we calculated the 95% protection level of zinc (PHC₅ :population-level hazardous concentration of 5% of species), which is 107 μg/L. Meanwhile, the 95% protection criterion (HC₅) based on conventional individual-level chronic toxicity, was calculated to be 14.6 μg/L. The environmentally “safe” concentration for a population-level endpoint is about 7 times greater than that for an individual-level endpoint. The proposed method provides guidance for a pragmatic approach to population-level ecological risk assessment and the management of chemicals.     

Ecological Risk Assessment and Regulation for Genetically-Modified Ornamental Plants

Classic plant breeding has increased the beauty and utility of ornamental plants, but biotechnology can offer completely new traits for plants used in homes and gardens. The creation of blue petal color in carnations and roses are examples where biotechnology has created novelty that conventional hybridization cannot match. However, all innovations have benefits and risks, and future commercialization of transgenic ornamental plants raises complex questions about potential negative impacts to managed landscapes and natural ecosystems. Predictive ecological risk assessment is a process that uses current knowledge to estimate future environmental harms or benefits arising from direct or indirect exposure to a genetically-modified (GM) plant, its genes, or gene products. This article considers GM ornamental plants in the context of current ecological risk assessment principles, research results, and current regulatory frameworks. The use of ecological risk assessment by government agencies to support decision-making is reviewed in the context of ornamental plants. Government risk assessments have usually emphasized the potential for pollen-mediated gene flow, weediness in managed areas, invasion of natural areas, and direct harm to nontarget organisms. Some of the major challenges for predictive risk assessment include characterizing gene flow over time and space, plant fitness in changing environments, and impacts to nontarget organisms, communities and ecosystems. The lack of baseline information about the ecology and biodiversity of urban areas, gardens, and natural ecosystems limits the ability to predict potential hazards, identify exposure pathways, and design hypothesis-driven research. The legacy of introduced ornamental plants as invasive species generates special concern about future invasions, especially for GM plants that exhibit increased stress tolerance or adaptability. While ecological risk assessments are a valuable tool and have helped harmonize regulation of GM plants, they do not define the acceptable level of risk or uncertainty. That responsibility belongs to regulators, stakeholders and citizens.     

Application of a direct toxicity assessment approach to assess the hazard of potential pesticide exposure at selected sites on the Crocodile and Magalies rivers,South Africa

The potentially hazardous effects of agricultural pesticide usage in the Crocodile (west) Marico catchment were evaluated using the Danio rerio and Daphnia pulex lethality, Selenastrum capricornutum growth inhibition and the Ames mutagenicity plate incorporation assays. Hazard assessment categories are proposed to standardise the output of the different toxicity assessments. Sites were categorised according to the toxicity hazard indicated and were related to the gradients of agricultural land-use intensity at each site. Intensive agricultural sites showed the highest effects to all tested biota. Receiving water at urban sites associated with increased nutrients and lowest pesticide usage showed few adverse effects, while the relatively unimpacted site indicated no hazard to any organism, and only a slight stimulation to algal growth. Weighted hazard scores indicated that the unimpacted sites were least hazardous, falling within a B category, the urban sites were moderately hazardous (C category), and the agricultural sites (D category) had the highest potential impacts on aquatic organisms. This study demonstrated the usefulness of using the hazard assessment approach and the role it could play in assessing site-specific potential toxicity hazards of river water impacted by agrochemicals. It can be used together with other assessment methods, such as biological indices, in a tiered approach.     

The role of laboratory and field leaching tests in hazard identification for solid materials

The use of various in vitro toxicity assays for testing environmental solid samples is dependent on the availability of reliable methods for the sampling and pretreatment of the material. This study focuses on the evaluation of leaching behaviour as a first step in the context of the toxicity testing of solid environmental matter. Spent shale, from oil shale retorting, was chosen as a suitable example of deposited solid waste material. For the generation of leachate in the laboratory setting, a standard two-stage batch-leaching test was applied to the samples of technogenic waste. In the field, a new type of lysimeter, which does not disturb the surface, was used for in situ leachate collection. The chemical composition of water extracts was found to be different under field conditions, as compared with the laboratory experiments. Thus, the hazard identification of a solid technogenic waste by in vitro toxicological tests applied to laboratory leachates would not be the best solution. The content of hazardous ingredients could be underestimated if only laboratory tests are used. For risk assessment concerned with solid waste materials, the generation of leachate by using field lysimeters is recommended.     

Hazard identification for 3,5-dichlorophenol in the aquatic environment

The risk assessment of substances in various environmental compartments is essential for their proper, safe and environmentally friendly production, handling, use and final deposition or destruction. Hazard identification is an important part of risk assessment. The aim of our research was to present a methodology for the hazard identification of substances dangerous to the aquatic environment according to the 93/21/EEC Directive relating to the classification, packaging and labelling of dangerous substances, from the adverse effect assessment of chemicals in European Union. A battery of toxicity tests and biodegradability studies with 3,5-dichlorophenol were performed. The substance was classified as toxic to aquatic organisms with possible long-term adverse effects. The presented methodology assures reliable data for the classification and labelling of substances according to their harmful effect on aquatic biota, suitable for the competent authorities at the national and EU level.     

Invited contribution: Acute toxicity testing,public responsibility and scientific challenges

Knowledge of the acute toxic effects of chemicals is important for the protection of exposed humans. Since sufficient information in humans is often lacking, experiments on laboratory animals must be performed. The LD₅₀ test, which requires large numbers of animals, has become the preferred procedure. It is now widely criticized on scientific and ethical grounds. This paper reviews the possibilities of using fewer animals to obtain relevant information on the acute hazards of chemical substances, but it also identifies the reasons why the traditional testing approaches cannot be changed immediately. An important problem is the practice of basing legal decisions on classification of chemicals in official lists of hazardous substances and for labeling purposes on LD₅₀ values. Proposals are presented on how pain and suffering of the animals included in acute toxicity tests can be reduced. The use of in vitro systems for the evaluation of the hazardous properties of chemicals is discussed.Abbreviations LD₅₀ lethal dose 50%, median lethal dose     

Comet assay: a reliable tool for the assessment of DNA damage in different models

New chemicals are being added each year to the existing burden of toxic substances in the environment. This has led to increased pollution of ecosystems as well as deterioration of the air, water, and soil quality. Excessive agricultural and industrial activities adversely affect biodiversity, threatening the survival of species in a particular habitat as well as posing disease risks to humans. Some of the chemicals, e.g., pesticides and heavy metals, may be genotoxic to the sentinel species and/or to non-target species, causing deleterious effects in somatic or germ cells. Test systems which help in hazard prediction and risk assessment are important to assess the genotoxic potential of chemicals before their release into the environment or commercial use as well as DNA damage in flora and fauna affected by contaminated/polluted habitats. The Comet assay has been widely accepted as a simple, sensitive, and rapid tool for assessing DNA damage and repair in individual eukaryotic as well as some prokaryotic cells, and has increasingly found application in diverse fields ranging from genetic toxicology to human epidemiology. This review is an attempt to comprehensively encase the use of Comet assay in different models from bacteria to man, employing diverse cell types to assess the DNA-damaging potential of chemicals and/or environmental conditions. Sentinel species are the first to be affected by adverse changes in their environment. Determination of DNA damage using the Comet assay in these indicator organisms would thus provide information about the genotoxic potential of their habitat at an early stage. This would allow for intervention strategies to be implemented for prevention or reduction of deleterious health effects in the sentinel species as well as in humans. IITR Communication No. 2656     

Transcriptome profiling in crustaceans as a tool for ecotoxicogenomics

Chemicals released into the environment have the potential to affect various species and it is important to evaluate such chemical effect on ecosystems, including aquatic organisms. Among aquatic organisms, Daphnia magna has been used extensively for acute toxicity or reproductive toxicity tests. Although these types of tests can provide information on hazardous concentrations of chemicals, they provide no information on their mode of action. Recent advances in toxicogenomics, the integration of genomics with toxicology, have the potential to afford a better understanding of the responses of aquatic organisms to pollutants. In a previous study, we developed an oligonucleotide-based DNA microarray with high reproducibility using a Daphnia expressed sequence tag (EST) database. In this study, we increased the number of genes on the array and used it for a careful ecotoxicogenomic assessment of Daphnia magna. The DNA microarray was used to evaluate gene expression profiles of neonate daphnids exposed to beta-naphthoflavone (bNF). Exposure to this chemical resulted in a characteristic gene expression pattern. As the number of the genes on an array was increased, the number of genes that were found to respond to the chemicals was also increased, which made the classification of the toxic chemicals easier and more accurate. This newly developed DNA microarray can be useful for a obtaining a better mechanistic understanding of chemical toxicity effects on a common freshwater organism.     

Risk assessment issues in breast cancer

Breast cancer is the most common malignancy affecting women, and its incidence has been increasing in many countries. The aetiology of breast cancer is poorly understood, so there is concern as to which factors in our environment or lifestyle are responsible for the increase. There is a need for reliable risk assessment, which involves the steps of hazard identification, hazard evaluation, exposure evaluation and risk estimation. Short-term laboratory tests and long-term tests in animals are useful for priority-setting, but quantitative human risk assessment should preferably involve observations of humans. Epidemiological studies vary in the degree of reliance that can be placed on their results. The main types of epidemiological investigation are illustrated by recent examples from the literature on breast cancer. Careful judgement is required in assessing whether any association between a factor and a disease is likely to be causal. The injectable contraceptive, depot medroxyprogesterone acetate (DMPA, ‘Depo-Provera’), has been controversial because it caused malignant mammary tumours in beagle dogs. Two recent case-control studies found no overall association between DMPA and the risk of breast cancer in women. There was some evidence of increased risk in certain sub-groups of women, which could be interpreted with more confidence if there were a better understanding of the biology of human breast cancer. Nevertheless, the results do not support the prediction from beagle experiments that DMPA might increase the overall risk of breast cancer.     

Soil ecotoxicology: state of the art and future directions

Developments in soil ecotoxicology started with observations on pesticide effects on soil invertebrates in the 1960s. To support the risk assessment of chemicals, in the 1980s and 1990s development of toxicity tests was the main issue, including single species tests and also more realistic test systems like model ecosystems and field tests focusing on structural and functional endpoints. In the mean time, awareness grew about issues like bioavailability and routes of exposure, while biochemical endpoints (biomarkers) were proposed as sensitive and potential early-warning tools. In recent years, interactions between different chemicals (mixture toxicity) and between chemical and other stressors attracted scientific interest. With the development of molecular biology, omics tools are gaining increasing interest, while the ecological relevance of exposure and effects is translating into concepts like (chemical) stress ecology, ecological vulnerability and trait-based approaches. This contribution addresses historical developments and focuses on current issues in soil ecotoxicology. It is concluded that soil ecotoxicological risk assessment would benefit from extending the available battery of toxicity tests by including e.g. isopods, by paying more attention to exposure, bioavailability and toxicokinetics, and by developing more insight into the ecology of soil organisms to support better understanding of exposure and long-term consequences of chemical exposure at the individual, population and community level. Ecotoxicogenomics tools may also be helpful in this, but will require considerable further research before they can be applied in the practice of soil ecotoxicological risk assessment.     

Assessing the Risk of Herbicides to Terrestrial Non-Target Plants Using Higher-Tier Studies

Risk assessment for non-target plants is based on single species phytotoxicity tests. This approach may not reflect relevant ecological processes in terrestrial ecosystems. The current risk assessment scheme is based on endpoints measured at the species level and the assessment of ecological effects relies on the extrapolation from one species to another or from a single species to a community. This extrapolation contains many uncertainties that may be reduced by adopting more realistic testing approaches. However, currently higher-tier plant studies are not obligatory in herbicide risk assessment. We reviewed the published literature and found that potential higher-tier approaches for terrestrial non-target plants are extremely limited. Sixteen studies were found that assessed the effects of herbicides on non-target plants by performing microcosms, mesocosms, or field studies. These studies showed that microcosms might provide useful data and help to reduce uncertainties associated with single-species tests. However, due to the limited number of available studies, much work is required to develop appropriate testing methods for regulatory processes. In addition, field experiments are necessary to establish baseline knowledge concerning the effects of herbicides on natural plant communities and to compare data generated in tiered testing approaches with data obtained from natural systems.     

Mysids in toxicity testing — a review

The use of mysid shrimp, particularly the genusMysidopsis, along with specific testing procedures, has become accepted in aquatic toxicology. Investigators have developed methodologies for both culture and testing of these organisms. Acute and chronic (life cycle) toxicity tests in addition to dredge spoil and effluent tests with mysids are now becoming common. Attempts have been made to use mysids as test organisms in behavioral, physiological, nutritional, and food-chain studies. In general,Mysidopsis spp. have been shown to be as sensitive or more sensitive to toxic substances than other marine species tested. The ease of handling and culture, relative sensitivity to toxicants, short life cycle, small size and direct larval development make these organisms desirable for research purposes. Continued research using mysid species will probably demonstrate even greater usefulness of these organisms in assessment of pollutant impacts on estuarine or marine communities.     

An integrated biomarker-based strategy for ecotoxicological evaluation of risk in environmental management

Environmental impacts by both natural events and man-made interventions are a fact of life; and developing the capacity to minimise these impacts and their harmful consequences for biological resources, ecosystems and human health is a daunting task for environmental legislators and regulators. A major challenge in impact and risk assessment, as part of integrated environmental management (IEM), is to link harmful effects of pollution (including toxic chemicals) in individual sentinel animals to their ecological consequences. This obstacle has resulted in a knowledge-gap for those seeking to develop effective policies for sustainable use of resources and environmental protection. Part of the solution to this problem may lie with the use of diagnostic clinical-type laboratory-based ecotoxicological tests or biomarkers, utilising sentinel animals as integrators of pollution, coupled with direct immunochemical tests for contaminants. These rapid and cost-effective ecotoxicological tools can provide information on the health status of individuals and populations based on relatively small samples of individuals. In the context of ecosystem status or health of the environment, biomarkers are also being used to link processes of molecular and cellular damage through to higher levels (i.e., prognostic capability), where they can result in pathology with reduced physiological performance and reproductive success. Complex issues are involved in evaluating environmental risk, such as the effects of the physico-chemical environment on the speciation and uptake of pollutant chemicals and inherent inter-individual and inter-species differences in vulnerability to toxicity; and the toxicity of complex mixtures. Effectively linking the impact of pollutants through the various hierarchical levels of biological organisation to ecosystem and human health requires a pragmatic integrated approach based on existing information that either links or correlates processes of pollutant uptake, detoxication and pathology with each other and higher level effects. It is further proposed here that this process will be facilitated by pursuing a holistic or whole systems approach with the development of computational simulation models of cells, organs and animals in tandem with empirical data (i.e., the middle-out approach). In conclusion, an effective integrated environmental management strategy to secure resource sustainability requires an integrated capability for risk assessment and prediction. Furthermore, if such a strategy is to influence and help in the formulation of environmental policy decisions, then it is crucial to demonstrate scientific robustness of predictions concerning the long-term consequences of pollution to politicians, industrialists and environmental managers; and also increase stakeholder awareness of environmental problems.     

Predictive Value of Species Sensitivity Distributions for Effects of Herbicides in Freshwater Ecosystems

In this article we present a review of the laboratory and field toxicity of herbicides to aquatic ecosystems. Single-species acute toxicity data and (micro)mesocosm data were collated for nine herbicides. These data were used to investigate the importance of test species selection in constructing species sensitivity distributions (SSDs), and in estimating hazardous concentrations (i.e., HC5) protective for freshwater aquatic ecosystems. A lognormal model was fitted to toxicity data (acute EC50s and chronic NOECs) and the resulting distribution used to estimate lower (95% confidence), median (50% confidence), and upper (5% confidence), HC5 values. The taxonomic composition of the species assemblage used to construct the SSD does have a significant influence on the assessment of hazard and only sensitive primary producers should be included for the risk assessment of herbicides. No systematic difference in sensitivity between standard and non-standard test species was observed. Hazardous concentrations estimated using laboratory-derived acute and chronic toxicity data for sensitive freshwater primary producers were compared to the response of herbicide-stressed freshwater ecosystems using a similar exposure regime. The lower limit of the acute HC5 and the median value of the chronic HC5 were protective of adverse effects in aquatic micro/mesocosms even under a long-term exposure regime. The median HC5 estimate based on acute data was protective of adverse ecological effects in freshwater ecosystems when a pulsed or short-term exposure regime was used in the microcosm and mesocosm experiments. There was also concordance between the predictions from the effect model PERPEST and the concentrations at which clear effects started to emerge in laboratory and field studies. However, compared to the SSD concept, the PERPEST model is able to provide more information on ecological risks when a common toxicological mode of action is evaluated as it considers both recovery and indirect effects.     

Equivalence tests to support environmental biosafety decisions:theory and examples

A major role of ecological risk assessment (ERA) has been to provide scientific guidance on whether a future human activity will cause ecological harm, including such activities as release of a genetically modified organism (GMO), exotic species, or chemical pollutant into the environment. This requires the determination of the likelihoods that the activity:would cause a harm, and would not cause a harm. In the first case, the focus is on demonstrating the presence of a harm and developing appropriate management to mitigate such harm. This is usually evaluated using standard hypothesis analysis. In the second case, the focus is on demonstrating the absence of a harm and supporting a decision of biosafety. While most ERA researchers have focused on finding presence of harm, and some have wrongly associated the lack of detection of harm with biosafety, a novel approach in ERA would be to focus on demonstrating directly the safety of the activity. Although, some researchers have suggested that retrospective power analysis can be used to infer absence of harm, it actually provides inaccurate information about biosafety. A decision of biosafety can only be supported in a statistically sound manner by equivalence tests, described here. Using a 20% ecological equivalence standard in GMO examples, we illustrated the use of equivalence tests for two-samples with normal or binomial data and multi-sample normal data, and provided a spreadsheet calculator for each. In six of the eight examples, the effects of Cry toxins on a non-target organism were equivalent to a control, supporting a decision of biosafety. These examples also showed that demonstration of equivalence does not require large sample sizes. Although more relevant ecological equivalence standards should be developed to enable equivalence tests to become the main method to support biosafety decision making, we advocate their use for evaluating biosafety for non-target organisms because of their direct and accurate inference regarding safety.     

Assessment of the genotoxic potential of Hoechst 33342, SYBR-14 and PI using the SOS ChromoTest™

Abstract

Fluorochromes in combination with flow cytometry can be used for laboratory assessment of semen quality in humans and domestic animals. Some studies have reported the potential toxicity of these fluorochromes toward the cells analyzed, but not toward the laboratory technician who operates the analytical instrument. We tested the genotoxic potential of three fluorochromes, SYBR-14, propidium iodide, and Hoechst 33342, using the colorimetric SOS ChromoTest^?. The test revealed no genotoxic potential for any of the three fluorochromes within the dilution ranges investigated. We conclude that occasional direct contact with these fluorescent probes does not necessarily pose a genotoxic hazard.     

Assessment of the genotoxic potential of Hoechst 33342, SYBR-14 and PI using the SOS ChromoTest(?)

Fluorochromes in combination with flow cytometry can be used for laboratory assessment of semen quality in humans and domestic animals. Some studies have reported the potential toxicity of these fluorochromes toward the cells analyzed, but not toward the laboratory technician who operates the analytical instrument. We tested the genotoxic potential of three fluorochromes, SYBR-14, propidium iodide, and Hoechst 33342, using the colorimetric SOS ChromoTest(?). The test revealed no genotoxic potential for any of the three fluorochromes within the dilution ranges investigated. We conclude that occasional direct contact with these fluorescent probes does not necessarily pose a genotoxic hazard.     

Assessing the risks of insect resistant transgenic plants on entomophagous arthropods Bt-maize expressing Cry1Ab as a case study

One of the primary concerns related to theadoption of insect resistant transgenic plantsin the environment is the detrimental effectthat these may pose on non-target organisms,including entomophagous arthropods (parasitoidsfand predators) which have an important functionin regulating pests. Despite the fact thatregulatory bodies require information regardingthe potential risk of releasing transgenicplants in the environment, to date, no specificprotocols have been designed for assessing therisks of insect resistant transgenic crops onentomophagous arthropods. Here a framework forrisk assessment is proposed to evaluate theeffects of insect resistant plants onentomophagous arthropods. Using maizeexpressing the Bacillus thuringiensisgene which codes for the Cry1Ab toxin, weillustrate the procedure necessary forassessing the risks. As a first step, it isrequired to determine which entomophagousarthropods play a major role in regulatingmaize pests, and which may be at risk. Becausethe risk which transgenic plants pose toentomophagous arthropods depends on both, theirexposure, and their sensitivity to theinsecticidal protein, it is essential todetermine, as a second step, if and at whatlevel organisms are exposed to the transgenecompound. Exposure will be associated with thefeeding behaviour of phytophagous andentomophagous arthropods together with thetissue and cell specific temporal and spatialexpression of the insecticidal protein. Forthose organisms which could potentially beexposed to the insecticidal protein,sensitivity tests, as a third step, should beperformed to assess toxicity. The testingprocedure and the type of tests which should beadopted to quantify the effects of insectresistant plants on natural enemies aresubsequently illustrated. Taking the greenlacewing Chrysoperla carnea as anexample, we propose a procedure on how toperform tests and give evidence that Bt-maizeposes no risk to this predator.