Stephanie K. Bopp

Development of a framework based on an ecosystem services approach for deriving specific protection goals for environmental risk assessment of pesticides

General protection goals for the environmental risk assessment (ERA) of plant protection products are stated in European legislation but specific protection goals (SPGs) are often not precisely defined. These are however crucial for designing appropriate risk assessment schemes. The process followed by the Panel on Plant Protection Products and their Residues (PPR) of the European Food Safety Authority (EFSA) as well as examples of resulting SPGs obtained so far for environmental risk assessment (ERA) of pesticides is presented. The ecosystem services approach was used as an overarching concept for the development of SPGs, which will likely facilitate communication with stakeholders in general and risk managers in particular. It is proposed to develop SPG options for 7 key drivers for ecosystem services (microbes, algae, non target plants (aquatic and terrestrial), aquatic invertebrates, terrestrial non target arthropods including honeybees, terrestrial non-arthropod invertebrates, and vertebrates), covering the ecosystem services that could potentially be affected by the use of pesticides. These SPGs need to be defined in 6 dimensions: biological entity, attribute, magnitude, temporal and geographical scale of the effect, and the degree of certainty that the specified level of effect will not be exceeded. In general, to ensure ecosystem services, taxa representative for the key drivers identified need to be protected at the population level. However, for some vertebrates and species that have a protection status in legislation, protection may be at the individual level. To protect the provisioning and supporting services provided by microbes it may be sufficient to protect them at the functional group level. To protect biodiversity impacts need to be assessed at least at the scale of the watershed/landscape.

Comparison of four different colorimetric and fluorometric cytotoxicity assays in a zebrafish liver cell line

BackgroundA broad spectrum of cytotoxicity assays is currently used in the fields of (eco)toxicology and pharmacology. To choose an appropriate assay, different parameters like test compounds, detection mechanism, specificity, and sensitivity have to be considered. Furthermore, tissue or cell line can influence test performance. For zebrafish (Danio rerio), as emerging model organism, cell lines are now increasingly used, but few studies examined cytotoxicity in these cell systems. Therefore, we compared four cytotoxicity assays in the zebrafish liver cell line, ZFL, to test four differently acting model compounds. The tests comprised two colorimetric assays (MTT assay using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide, and the LDH assay detecting lactate dehydrogenase activity) and two fluorometric assays (alamarBlue® using resazurin, and CFDA-AM based on 5-carboxyfluorescein diacetate acetoxymethyl ester). Model compounds were the pharmaceutical Tamoxifen, its metabolite 4-Hydroxy-Tamoxifen, the fungicide Flusilazole and the polycyclic aromatic hydrocarbon Benzo[a]pyrene.ResultsAll four assays performed well in the ZFL cells and led to reproducible dose-response curves for all test compounds. Effective concentrations causing 10% or 50% loss of cell viability (EC10 and EC50 values) varied by a maximum factor of 7.0 for the EC10 values and a maximum factor of 1.8 for the EC50 values. The EC values were not statistically different between the four assays, which is due to the assessed unspecific effects of the compounds. However, most often, the MTT assay and LDH assay showed the highest and lowest EC values, respectively. Nevertheless, the LDH assay showed the highest intra- and inter-assay variabilities and the lowest signal-to-noise ratios. In contrast to MTT, the other three assays have the advantage of being non-destructive, easy to handle, and less time consuming. Furthermore, AB and CFDA-AM can be combined on the same set of cells without damaging the cells, allowing later on their use for the investigation of other endpoints.ConclusionWe recommend the alamarBlue and CFDA-AM assays for cytotoxicity assessment in ZFL cells, which can be applied either singly or combined.

Transcriptomics Responses in Marine Diatom Thalassiosira pseudonana Exposed to the Polycyclic Aromatic Hydrocarbon Benzo[a]pyrene

Diatoms are unicellular, photosynthetic, eukaryotic algae with a ubiquitous distribution in water environments and they play an important role in the carbon cycle. Molecular or morphological changes in these species under ecological stress conditions are expected to serve as early indicators of toxicity and can point to a global impact on the entire ecosystem. Thalassiosira pseudonana, a marine diatom and the first with a fully sequenced genome has been selected as an aquatic model organism for ecotoxicological studies using molecular tools. A customized DNA microarray containing probes for the available gene sequences has been developed and tested to analyze the effects of a common pollutant, benzo(a)pyrene (BaP), at a sub-lethal concentration. This approach in diatoms has helped to elucidate pathway/metabolic processes involved in the mode of action of this pollutant, including lipid metabolism, silicon metabolism and stress response. A dose-response of BaP on diatoms has been made and the effect of this compound on the expression of selected genes was assessed by quantitative real time-PCR. Up-regulation of the long-chain acyl-CoA synthetase and the anti-apoptotic transmembrane Bax inhibitor, as well as down-regulation of silicon transporter 1 and a heat shock factor was confirmed at lower concentrations of BaP, but not the heat-shock protein 20. The study has allowed the identification of molecular biomarkers to BaP to be later on integrated into environmental monitoring for water quality assessment.

Development of a solvent‐free, solid‐phase in vitro bioassay using vertebrate cells

Miniaturized bioassays offer many advantages in exploring the toxic potential of chemicals, including small sample volumes and compatibility with high-throughput screening. One problem common to miniaturized systems, however, is the loss of test chemicals because of sorption. The idea of the current study was to use the sorption phenomenon in a positive way. It was found that contaminants sorbed to the growth surface in wells of tissue-culture plates or to the surface of selected sorbent bead materials are available to vertebrate cells growing in direct contact with the contaminant-coated surface. The use of beads provided more flexibility with regard to surface area, materials, and assay format. Biosilon, a bead cell-culture carrier made of polystyrene, was found to be most suitable. It supported cell adherence and allowed the detection of reproducible dose-response curves of an increase in cytochrome CYP1A enzyme activity by sorbed polycyclic aromatic hydrocarbons in the rainbow trout (Oncorhynchus mykiss) liver cell line, RTL-W1. The resulting bead assay provides a miniaturized, solvent-free exposure system. Potential future applications include the coupling to environmental sampling, in which the bead material is used as solid receiving phase before serving as a surface for vertebrate cells to attach and respond.

From the exposome to mechanistic understanding of chemical-induced adverse effects

The exposome encompasses an individuals exposure to exogenous chemicals, as well as endogenous chemicals that are produced or altered in response to external stressors. While the exposome concept has been established for human health, its principles can be extended to include broader ecological issues. The assessment of exposure is tightly interlinked with hazard assessment. Here, we explore if mechanistic understanding of the causal links between exposure and adverse effects on human health and the environment can be improved by integrating the exposome approach with the adverse outcome pathway (AOP) concept that structures and organizes the sequence of biological events from an initial molecular interaction of a chemical with a biological target to an adverse outcome. Complementing exposome research with the AOP concept may facilitate a mechanistic understanding of stress-induced adverse effects, examine the relative contributions from various components of the exposome, determine the primary risk drivers in complex mixtures, and promote an integrative assessment of chemical risks for both human and environmental health.

Gene biomarkers in diatom Thalassiosira pseudonana exposed to polycyclic aromatic hydrocarbons from contaminated marine surface sediments

Marine diatoms have a key role in the global carbon fixation and therefore in the ecosystem. We used Thalassiosira pseudonana as a model organism to assess the effects of exposure to environmental pollutants at the gene expression level. Diatoms were exposed to polycyclic aromatic hydrocarbons mixture (PAH) from surface sediments collected at a highly PAH contaminated area of the Mediterranean Sea (Genoa, Italy), due to intense industrial and harbor activities. The gene expression data for exposure to the sediment-derived PAH mixture was compared with gene expression data for in vitro exposure to specific polycyclic aromatic hydrocarbons. The data shows that genes involved in stress response, silica uptake, and metabolism were regulated both upon exposure to the sediment-derived PAH mixture and to the single component. Complementary monitoring of silica in the diatom cultures provide further evidence of a reduced cellular uptake of silica as an end-point for benzo[a]pyrene exposure that could be linked with the reduced gene and protein expression of the silicon transporter protein. However some genes showed differences in regulation indicating that mixtures of structurally related chemical compounds can elicit a slightly different gene expression response compared to that of a single component. The paper provides indications on the specific pathways affected by PAH exposure and shows that selected genes (silicon transporter, and silaffin 3) involved in silica uptake and metabolism could be suitable molecular biomarkers of exposure to PAHs.

Chapter 12 Use of ceramic dosimeters in water monitoring

Publisher Summary This chapter provides details on the functioning of ceramic dosimeter sampling devices with particular focus on the role of the ceramic membrane and requirements regarding the solid receiving phase. A number of practical considerations are discussed to highlight strengths and limitations of ceramic dosimeters in the field. The ceramic dosimeter consists of a ceramic tube with a diameter of 1 cm and a wall thickness of 1.5mm. The pore size of the inner wall coating of the tube is 5 nm. The length of the tube can be varied, but in most applications so far, a length of 5 cm was used. The tube is filled with water-saturated sorbent and closed at either end with caps made of polytetrafluoroethylene. The ceramic membrane serves both as a diffusion barrier and as a container to hold the sorbent material. Key characteristics of this membrane with regard to quantitative time-integrated passive sampling are its porosity and inertness, its inner pore size, and its thickness. Upon diffusion of solutes through the ceramic membrane, they need to be trapped onto a receiving phase. The role of this is to maintain a steep concentration gradient between the exterior and the interior of the sampler in order to ensure continuous diffusion along the gradient into the sampling device. To allow for chemical analysis after sampling, they are required to yield high recovery rates of the target chemicals by means of solvent extraction or, potentially, thermo-desorption. Three different bead materials have been identified to fulfill these criteria. These are Amberlite IRA-743; Biosilon; and chlorinated hydrocarbons.

IMPROVING SUBSTANCE INFORMATION IN USETOX®, PART 1: DISCUSSION ON DATA AND APPROACHES FOR ESTIMATING FRESHWATER ECOTOXICITY EFFECT FACTORS

The scientific consensus model USEtox® is recommended by the European Commission as the reference model to characterize life cycle chemical emissions in terms of their potential human toxicity and freshwater aquatic ecotoxicity impacts in the context of the International Reference Life Cycle Data System Handbook and the Environmental Footprint pilot phase looking at products (PEF) and organizations (OEF). Consequently, this model has been systematically used within the PEF/OEF pilot phase by 25 European Union industry sectors, which manufacture a wide variety of consumer products. This testing phase has raised some questions regarding the derivation of and the data used for the chemical-specific freshwater ecotoxicity effect factor in USEtox. For calculating the potential freshwater aquatic ecotoxicity impacts, USEtox bases the effect factor on the chronic hazard concentration (HC50) value for a chemical calculated as the arithmetic mean of all logarithmized geometric means of species-specific chronic median lethal (or effect) concentrations (L[E]C50). We investigated the dependency of the USEtox effect factor on the selection of ecotoxicological data source and toxicological endpoints, and we found that both influence the ecotoxicity ranking of chemicals and may hence influence the conclusions of a PEF/OEF study. We furthermore compared the average measure (HC50) with other types of ecotoxicity effect indicators, such as the lowest species EC50 or no-observable-effect concentration, frequently used in regulatory risk assessment, and demonstrated how they may also influence the ecotoxicity ranking of chemicals. We acknowledge that these indicators represent different aspects of a chemicals ecotoxicity potential and discuss their pros and cons for a comparative chemical assessment as performed in life cycle assessment and in particular within the PEF/OEF context. Environ Toxicol Chem 2017;36:3450-3462.

Strategies to improve the regulatory assessment of developmental neurotoxicity (DNT) using in vitro methods

Currently, the identification of chemicals that have the potential to induce developmental neurotoxicity (DNT) is based on animal testing. Since at the regulatory level, systematic testing of DNT is not a standard requirement within the EU or USA chemical legislation safety assessment, DNT testing is only performed in higher tiered testing triggered based on chemical structure activity relationships or evidence of neurotoxicity in systemic acute or repeated dose toxicity studies. However, these triggers are rarely used and, in addition, do not always serve as reliable indicators of DNT, as they are generally based on observations in adult rodents. Therefore, there is a pressing need for developing alternative methodologies that can reliably support identification of DNT triggers, and more rapidly and cost-effectively support the identification and characterization of chemicals with DNT potential. We propose to incorporate mechanistic knowledge and data derived from in vitro studies to support various regulatory applications including: (a) the identification of potential DNT triggers, (b) initial chemical screening and prioritization, (c) hazard identification and characterization, (d) chemical biological grouping, and (e) assessment of exposure to chemical mixtures. Ideally, currently available cellular neuronal/glial models derived from human induced pluripotent stem cells (hiPSCs) should be used as they allow evaluation of chemical impacts on key neurodevelopmental processes, by reproducing different windows of exposure during human brain development. A battery of DNT in vitro test methods derived from hiPSCs could generate valuable mechanistic data, speeding up the evaluation of thousands of compounds present in industrial, agricultural and consumer products that lack safety data on DNT potential.

Chapter 18 Use of passive sampling devices in toxicity assessment of groundwater

Publisher Summary This chapter illustrates the concepts and examples available to date on the use of passive sampling devices in assessing the toxicity of groundwater. It also discusses how additional groundwater-compatible passive sampling devices could be used in the future to derive information on the toxic potency of groundwater. There are currently two approaches to linking passive sampling of groundwater with toxicological analysis. The first is to build a passive sampler in such a way that it is directly compatible with toxicological tests. This has been accomplished in the taximeter that is based on adherence-dependent vertebrate cells to detect a toxicological response. The second approach is to employ a passive sampler commonly used for monitoring groundwater contaminants based on chemical analysis alone, prepare an extract of sampled contaminants, and apply this extract in a toxicity test. The toximeter is a recently developed passive sampling device. It is the first passive sampler to allow direct bioassay analysis of accumulated chemicals. It is also possible, by means of simple solvent extraction, to carry out a chemical analysis and link the information obtained on concentrations of chemicals present to the results of toxicity tests. The underlying principle of the toximeter is that sorbents used for sampling can be applied directly in toxicity tests. Although, technically, the design of the toximeter pertains to surface or pore-water sampling, its first development and application were focused on groundwater.