Janeck J. Scott-Fordsmand

Biomarkers in Earthworms

Earthworms are believed to be so-called key species within ecosystems and are often exposed to a wide range of anthropogenic compounds released to the terrestrial environment. As a consequence, they may suffer from the toxicity of these compounds. For these and other reasons, earthworms have been used extensively in ecotoxicological studies. In recent years the use of other biological responses (biomarkers) to estimate either exposure or resultant effects of chemicals has received increased attention. Biomarkers address the question of bioavailability by only responding to the bioactive fraction. They may incorporate effects following exposure to a mixture of chemicals. Biomarkers may also reduce extrapolation of results from the laboratory to the field, as they may be applicable under both conditions. The present review has drawn together current knowledge on potential biomarkers in earthworms and appraised them in relation to basic requirements needed for supplying information relevant to devising satisfactory risk assessment. A wide range of potential biomarkers have been measured in earthworms, including DNA alteration, induction of metal-binding proteins (MTs and MBP), depression of ChE activity and other enzymatic responses, energy reserve responses, responses in neural impulse conductivity, lysosomal membrane stability, immunological responses, changes in sperm numbers, histopathological changes, and behavioral changes. Both organic and inorganic compounds have been included; however, for each biomarker the main emphasis historically has been placed on only a few chemicals. Dose-response relationships were in some cases observed. Little information is available on the linkage of the biomarker response to effects at population or community levels. The influence of other factors, biotic and abiotic, on the biomarker responses and their temporal duration have been only sporadically reported.

Effects of C60 fullerene nanoparticles on soil bacteria and protozoans

Nanotechnology should produce numerous new materials in the coming years. Because of the novel design of nanomaterials with new physicochemical characteristics, their potential adverse impact on the environment and human health must be addressed. In the present study, agglomerates of pristine C60 fullerenes (50 nm to microm-size) were applied to soil at 0, 5, 25, and 50 mg/kg dry soil to assess their effect on the soil microbiota by measuring total respiration; biomass, number, and diversity of bacteria; and total number and diversity of protozoans during 14 d. Respiration and microbial biomass were unaffected by the fullerenes at any time, whereas the number of fast-growing bacteria was decreased by three- to fourfold just after incorporation of the nanomaterial. Protozoans seemed not to be very sensitive to C60, because their number decreased only slightly in the beginning of the experiment. With polymerase chain reaction and denaturing gradient gel electrophoresis analysis of eubacteria and kinetoplastids from the soil, however, a difference between the fullerene treatments and nonamended controls was demonstrated. The fullerenes did not induce more than 20 to 30% of relative dissimilarity (with both bacteria and protozoans) between treatments, but this effect was persistent throughout the experiment. It therefore is recommended that fullerene nanomaterial not be spread deliberately in the environment and that their ecotoxicology be further clarified.

Effects of pesticides on soil invertebrates in laboratory studies: A review and analysis using species sensitivity distributions

Species sensitivity distributions (SSD) and 5% hazardous concentrations (HC5) are distribution-based approaches for assessing environmental risks of pollutants. These methods have potential for application in pesticide risk assessments, but their applicability for assessing pesticide risks to soil invertebrate communities has not been evaluated. Using data obtained in a systematic review, the present study investigates the relevance of SSD and HC5 for predicting pesticide risks to soil invertebrates. Altogether, 1950 laboratory toxicity data were obtained, representing 250 pesticides and 67 invertebrate taxa. The majority (96%) of pesticides have toxicity data for fewer than five species. Based on a minimum of five species, the best available endpoint data (acute mortality median lethal concentration) enabled SSD and HC5 to be calculated for 11 pesticides (atrazine, carbendazim, chlorpyrifos, copper compounds, diazinon, dimethoate, gamma-hexachlorocyclohexane, lambda-cyhalothrin, parathion, pentachlorophenol, and propoxur). Arthropods and oligochaetes exhibit pronounced differences in their sensitivity to most of these pesticides. The standard test earthworm species, Eisenia fetida sensu lato, is the species that is least sensitive to insecticides based on acute mortality, whereas the standard Collembola test species, Folsomia candida, is among the most sensitive species for a broad range of toxic modes of action (biocide, fungicide, herbicide, and insecticide). These findings suggest that soil arthropods should be tested routinely in regulatory risk assessments. In addition, the data indicate that the uncertainty factor for earthworm acute mortality tests (i.e., 10) does not fully cover the range of earthworm species sensitivities and that acute mortality tests would not provide the most sensitive risk estimate for earthworms in the majority (95%) of cases.

The toxicity testing of double-walled nanotubes-contaminated food to Eisenia veneta earthworms.

Nanotechnology is playing an increasing role in the society, but very little is known about the environmental toxicity of nanotechnological products. Few previous studies have been published with regard to the effect of nanoparticles on soil organisms. The present paper deals with the toxicity of carbon double-walled nanotubes (DWNT) and C60 fullerenes to the soil-dwelling earthworm Eisenia veneta, with the aim of evaluating the lethal and sub-lethal toxicity. Reproduction of E. veneta was affected by DWNT administered through food at concentrations above 37 mg DWNT/kg food. The most sensitive toxicological parameter was reproduction (cocoon production), with no effect on hatchability, survival or mortality at up to 495 mg DWNT/kg and 1000 mg C60/kg.

ITS-NANO - Prioritising nanosafety research to develop a stakeholder driven intelligent testing strategy

BackgroundTo assess the risk of all nanomaterials (NMs) on a case-by-case basis is challenging in terms of financial, ethical and time resources. Instead a more intelligent approach to knowledge gain and risk assessment is required.MethodsA framework of future research priorities was developed from the accorded opinion of experts covering all major stake holder groups (government, industry, academia, funders and NGOs). It recognises and stresses the major topics of physicochemical characterisation, exposure identification, hazard identification and modelling approaches as key components of the current and future risk assessment of NMs.ResultsThe framework for future research has been developed from the opinions of over 80 stakeholders, that describes the research priorities for effective development of an intelligent testing strategy (ITS) to allow risk evaluation of NMs. In this context, an ITS is a process that allows the risks of NMs to be assessed accurately, effectively and efficiently, thereby reducing the need to test NMs on a case-by-case basis.For each of the major topics of physicochemical characterisation, exposure identification, hazard identification and modelling, key-priority research areas are described via a series of stepping stones, or hexagon diagrams structured into a time perspective. Importantly, this framework is flexible, allowing individual stakeholders to identify where their own activities and expertise are positioned within the prioritisation pathway and furthermore to identify how they can effectively contribute and structure their work accordingly. In other words, the prioritisation hexagon diagrams provide a tool that individual stakeholders can adapt to meet their own particular needs and to deliver an ITS for NMs risk assessment. Such an approach would, over time, reduce the need for testing by increasing the reliability and sophistication of in silico approaches.The manuscript includes an appraisal of how this framework relates to the current risk assessment approaches and how future risk assessment could adapt to accommodate these new approaches. A full report is available in electronic format (pdf) at http://www.nano.hw.ac.uk/research-projects/itsnano.html.ConclusionITS-NANO has delivered a detailed, stakeholder driven and flexible research prioritisation (or strategy) tool, which identifies specific research needs, suggests connections between areas, and frames this in a time-perspective.

Concern-driven integrated approaches to nanomaterial testing and assessment - report of the NanoSafety Cluster Working Group 10

Abstract Bringing together topic-related European Union (EU)-funded projects, the so-called “NanoSafety Cluster” aims at identifying key areas for further research on risk assessment procedures for nanomaterials (NM). The outcome of NanoSafety Cluster Working Group 10, this commentary presents a vision for concern-driven integrated approaches for the (eco-)toxicological testing and assessment (IATA) of NM. Such approaches should start out by determining concerns, i.e., specific information needs for a given NM based on realistic exposure scenarios. Recognised concerns can be addressed in a set of tiers using standardised protocols for NM preparation and testing. Tier 1 includes determining physico-chemical properties, non-testing (e.g., structure–activity relationships) and evaluating existing data. In tier 2, a limited set of in vitro and in vivo tests are performed that can either indicate that the risk of the specific concern is sufficiently known or indicate the need for further testing, including details for such testing. Ecotoxicological testing begins with representative test organisms followed by complex test systems. After each tier, it is evaluated whether the information gained permits assessing the safety of the NM so that further testing can be waived. By effectively exploiting all available information, IATA allow accelerating the risk assessment process and reducing testing costs and animal use (in line with the 3Rs principle implemented in EU Directive 2010/63/EU). Combining material properties, exposure, biokinetics and hazard data, information gained with IATA can be used to recognise groups of NM based upon similar modes of action. Grouping of substances in return should form integral part of the IATA themselves.

Earthworms and humans in vitro: characterizing evolutionarily conserved stress and immune responses to silver nanoparticles

Little is known about the potential threats of silver nanoparticles (AgNPs) to ecosystem health, with no detailed report existing on the stress and immune responses of soil invertebrates. Here we use earthworm primary cells, cross-referencing to human cell cultures with a particular emphasis on the conserved biological processes, and provide the first in vitro analysis of molecular and cellular toxicity mechanisms in the earthworm Eisenia fetida exposed to AgNPs (83 ± 22 nm). While we observed a clear difference in cytotoxicity of dissolved silver salt on earthworm coelomocytes and human cells (THP-1 cells, differentiated THP-1 cells and peripheral blood mononuclear cells), the coelomocytes and differentiated (macrophage-like) THP-1 cells showed a similar response to AgNPs. Intracellular accumulation of AgNPs in the coelomocytes, predominantly in a phagocytic population, was evident by several methods including transmission electron microscopy. Molecular signatures of oxidative stress and selected biomarker genes probed in a time-resolved manner suggest early regulation of oxidative stress genes and subsequent alteration of immune signaling processes following the onset of AgNP exposure in the coelomocytes and THP-1 cells. Our findings provide mechanistic clues on cellular innate immunity toward AgNPs that is likely to be evolutionarily conserved across the animal kingdom.

Toxicity of Nickel to the Earthworm and the Applicability of the Neutral Red Retention Assay

The toxic effects of nickel on survival, growth, and reproduction of Eisenia veneta were investigated following 4 weeks of exposure to a nickel-chloride spiked loamy sand soil. The ability of a simple earthworm biomarker, the lysosomal membrane stability of coelomocytes, to reflect nickel exposure was also studied. Nickel caused a significant toxic effect on E.veneta at soil concentrations above 85 mg Ni/kg. Reproduction (cocoon production) was the most sensitive parameter being reduced at soil concentrations above 85 mg Ni/kg (EC10 = 85 mg Ni/kg). Survival of adults was only reduced at concentrations above 245 mg Ni/kg, while adult and cocoon wet weight were not affected by soil nickel concentrations up to 700 mg Ni/kg. The lysosomal membrane stability, measured as neutral-red retention time, was reduced at soil nickel concentrations similar to those that reduced reproduction, and demonstrated a dose-response relationship. The neutral-red retention time showed large individual variation for the earthworms within each exposure concentration. It was concluded that the lysosomal membrane stability, measured as neutral red retention time, has a potential role in risk assessment, but care should be taken conducting this test.

Grouping and Read-Across Approaches for Risk Assessment of Nanomaterials

Physicochemical properties of chemicals affect their exposure, toxicokinetics/fate and hazard, and for nanomaterials, the variation of these properties results in a wide variety of materials with potentially different risks. To limit the amount of testing for risk assessment, the information gathering process for nanomaterials needs to be efficient. At the same time, sufficient information to assess the safety of human health and the environment should be available for each nanomaterial. Grouping and read-across approaches can be utilised to meet these goals. This article presents different possible applications of grouping and read-across for nanomaterials within the broader perspective of the MARINA Risk Assessment Strategy (RAS), as developed in the EU FP7 project MARINA. Firstly, nanomaterials can be grouped based on limited variation in physicochemical properties to subsequently design an efficient testing strategy that covers the entire group. Secondly, knowledge about exposure, toxicokinetics/fate or hazard, for example via properties such as dissolution rate, aspect ratio, chemical (non-)activity, can be used to organise similar materials in generic groups to frame issues that need further attention, or potentially to read-across. Thirdly, when data related to specific endpoints is required, read-across can be considered, using data from a source material for the target nanomaterial. Read-across could be based on a scientifically sound justification that exposure, distribution to the target (fate/toxicokinetics) and hazard of the target material are similar to, or less than, the source material. These grouping and read-across approaches pave the way for better use of available information on nanomaterials and are flexible enough to allow future adaptations related to scientific developments.

Effects of pesticides on soil invertebrates in model ecosystem and field studies: a review and comparison with laboratory toxicity data

A systematic review was carried out to investigate the extent to which higher-tier (terrestrial model ecosystem [TME] and field) data regarding pesticide effects can be compared with laboratory toxicity data for soil invertebrates. Data in the public domain yielded 970 toxicity endpoint data sets, representing 71 pesticides and 42 soil invertebrate species or groups. For most pesticides, the most frequent effect class was for no observed effects, although relatively high numbers of pronounced and persistent effects occurred when Lumbricidae and Enchytraeidae were exposed to fungicides and when Lumbricidae, Collembola, and Arachnida were exposed to insecticides. No effects of fungicides on Arachnida, Formicidae, or Nematoda or of herbicides on Lumbricidae, Formicidae, or Nematoda were observed in any studies. For most pesticides, higher-tier no-observed-effect concentration or lowest-observed-effect concentration values cannot be determined because of a lack of information at low pesticide concentrations. Ten pesticides had sufficient laboratory data to enable the observed higher-tier effects to be compared with 5% hazardous concentrations (HC5) estimated from acute toxicity laboratory data (atrazine, carbendazim, chlorpyrifos, diazinon, dimethoate, gamma-hexachlorocy-clohexane, lambda-cyhalothrin, parathion, pentachlorophenol, and propoxur). In eight cases, higher-tier effects concentrations were within or below the 90% confidence interval of the HC5. Good agreement exists between the results of TME and field tests for carbendazim, but insufficient information is available for a comparison between TME and field studies for other pesticides. Availability and characteristics (e.g., taxonomic composition and heterogeneity) of the higher-tier effects data are discussed in terms of possible developments in risk assessment procedures.