Holistic evaluation of long-term earthworm field studies with a fungicide.

Plant protection products to be placed on the market in the European Union need to meet rigorous safety criteria including the testing of lumbricid earthworms, the functionally most important soil organism group in Central European agricultural ecosystems. To address uncertainties and investigate the potential long-term in-crop effects of the fungicide Cantus® containing 50% boscalid as an active substance, a series of standardized earthworm field studies with an overall duration of 5 years per study program was carried out in four German agricultural fields under realistic crop rotation conditions. A two-step approach was chosen to analyze the potential overall long-term effects on earthworms in agricultural fields: (i) an assessment of the earthworm abundance development in the course of the four study programs in relation to the determined actual content of boscalid in soil and (ii) an effect size meta-analysis of earthworm abundance 1 year after treatment for each consecutive year and study program. Measured boscalid concentrations in the soil after multiple applications were well above the maximum boscalid residues observed in agricultural soils across Central Europe. There were isolated statistically significant reductions of earthworm abundance for some species and groups at some time points during the studies, but no consistent relationship to the Cantus® treatments was observed. These results were supported by the meta-analysis, indicating no adverse effects on earthworm populations. Therefore, fluctuations of abundance reflect the natural variation of the populations rather than a concentration-related response. Based on this comprehensive analysis, we conclude that there is no application rate-related effect of the 5-year use of Cantus® on the development of the earthworm communities. The four study programs, paired with a comprehensive evaluation, directly address the concerns about the potential long-term effects of boscalid on earthworms in the field and suggest that multiyear applications do not adversely affect earthworm populations. Integr Environ Assess Manag 2022;18:1399-1413. © 2021 ECT Oekotoxikologie GmbH and BASF SE. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Application of the Closure Principle Computational Approach Test to Assess Ecotoxicological Field Studies: Comparative Analysis Using Earthworm Field Test Abundance Data.

Field studies to determine the effects of chemicals on earthworm communities are generally conducted according to International Organization for Standardization standard 11268-3 (and later comments). However, statistical test procedures suggested in the guideline are frequently criticized, mainly for 2 reasons: 1) Earthworm abundances are count data and often do not fulfill requirements for multiple t tests (normal distribution and homogeneity of variance), and 2) the resulting toxicity metrics of multiple testing procedures (no/lowest-observed-effect concentrations [NOEC/LOEC]) fail to adequately detect the actual level of effects. Recently, a new method to overcome these shortcomings was presented by the introduction of the closure principle computational approach test (CPCAT). We applied this statistical method to assess chemical effects on abundance in a large dataset of 26 earthworm field studies (with up to 3 test chemical application rates) and an additional extended study with 6 application rates. A comparative analysis was provided considering results of well-established multiple testing approaches (Dunnett's test) with particular consideration of the degree of overdispersion found in these data. It was shown that the CPCAT detects substantially more effects in earthworm field tests as statistically significant than standard t test approaches. This lowered the LOEC/NOEC for many chemical treatments to control comparisons. As a consequence, the statistically detected NOECs/LOECs were often set at lower percentage deviations between control and chemical treatment. This is the first time the performance of the CPCAT has been assessed within a comprehensive analysis of earthworm field study data. Environ Toxicol Chem 2021;40:1750-1760. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

A new ecotoxicological test method for genetically modified plants and other stressors in soil with the black fungus gnat Bradysia impatiens (Diptera): current status of test development and dietary effects of azadirachtin on larval development and emergence rate.

BACKGROUND: Few suitable and standardized test methods are currently available to test the effects of genetically modified plants (GMP) on non-target organisms. To fill this gap and improve ecotoxicological testing for GMP, we developed a new soil ecotoxicological test method using sciarid larvae as test organisms. RESULTS: Bradysia impatiens was identified as a candidate species. Species of the genus Bradysia occur in high numbers in European agroecosystems and B. impatiens can be reared in the laboratory in continuous culture. A functional basic test design was successfully developed. Newly hatched larvae were used as the initial life stage to cover most of the life cycle of the species during the test. Azadirachtin was identified as a suitable reference substance. In several tests, the effects of this substance on development time and emergence rate varied for different temperatures and test substrates. The toxicity was higher at 25 °C compared to 20 °C and in tropical artificial soil compared to coconut fiber substrate. CONCLUSIONS AND OUTLOOK: Results suggest that the developed test system is suitable to enter a full standardization process, e.g., via the Organisation for Economic Co-operation and Development. Such a standardization would not only assist the risk assessment of GMP, but could include other stressors such as systemic pesticides or veterinary pharmaceuticals reaching the soil, e.g., via spreading manure. The use of sciarid flies as test organisms supports recommendations of EFSA, which stressed the ecological role of flies and encouraged including Diptera into test batteries.

Possibilities of using the German Federal States' permanent soil monitoring program for the monitoring of potential effects of genetically modified organisms (GMO).

BACKGROUND: In the Directive 2001/18/EC on the deliberate release of genetically modified organisms (GMO) into the environment, a monitoring of potential risks is prescribed after their deliberate release or placing on the market. Experience and data of already existing monitoring networks should be included. The present paper summarizes the major findings of a project funded by the Federal Agency for Nature Conservation (Nutzungsmöglichkeiten der Boden-Dauerbeobachtung der Länder für das Monitoring der Umweltwirkungen gentechnisch veränderter Pflanzen. BfN Skripten, Bonn-Bad Godesberg 369, 2014). The full report in german language can be accessed on http://www.bfn.de and is available as Additional file 1. The aim of the project was to check if it is possible to use the German permanent soil monitoring program (PSM) for the monitoring of GMO. Soil organism communities are highly diverse and relevant with respect to the sustainability of soil functions. They are exposed to GMO material directly by feeding or indirectly through food chain interactions. Other impacts are possible due to their close association to soil particles. RESULTS: The PSM program can be considered as representative with regard to different soil types and ecoregions in Germany, but not for all habitat types relevant for soil organisms. Nevertheless, it is suitable as a basic grid for monitoring the potential effects of GMO on soil invertebrates. CONCLUSIONS: PSM sites should be used to derive reference values, i.e. range of abundance and presence of different relevant species of soil organisms. Based on these references, it is possible to derive threshold values to define the limit of acceptable change or impact. Therefore, a minimum set of sites and minimum set of standardized methods are needed, i.e. characterization of each site, sampling of selected soil organism groups, adequate adaptation of methods for the purpose of monitoring of potential effects of GMO. Finally, and probably most demanding, it is needed to develop a harmonized evaluation concept.

Checklist of earthworms (Oligochaeta: Lumbricidae) from Germany.

A checklist of the German earthworm fauna (Oligochaeta: Lumbricidae) is presented, including published data, data from reports, diploma- and PhD- theses as well as unpublished data from museum collections, research institutions and private persons. Overall, 16,000 datasets were analyzed to produce the first German checklist of Lumbricidae. The checklist comprises 46 earthworm species from 15 genera and provides ecological information, zoogeographical distribution type and information on the species distribution in Germany. Only one species, Lumbricus badensis Michaelsen, 1907, is endemic to Germany, whereas 41% are peregrine. As there are 14 species occurring exclusively in the southern or eastern part of Germany, the species numbers in German regions increase from north to south.

General recommendations for soil ecotoxicological tests suitable for the environmental risk assessment of genetically modified plants.

Before a genetically modified plant (GMP) can be placed on the market in the European Union (EU), an environmental risk assessment has to be conducted according to EU-Directive 2001/18/EC or Regulation (EC) No. 1829/2003 of the European Parliament and of the Council. However, no harmonized concept for ecotoxicological testing is available today that considers the characteristics of GMPs as a whole. In fact, to date, mainly ecotoxicological tests originally developed and standardized for pesticides are used for this purpose. Frequently in these tests, not the whole GMP is tested but only specific transgene products (mainly toxins). In this contribution, ecotoxicological methods developed for the testing of pesticides are evaluated for whether they are suitable for risk assessment of GMPs as well. In total, 105 test methods covering a wide range of terrestrial invertebrates, microbes, and plants (laboratory, semifield, and field levels) were assessed. Only 7 of them had already been used with GMPs, and in about 20 studies the existing tests methods were modified, mostly in a way such that nonstandard species were used. In the laboratory, few earthworm and nontarget arthropod (NTA) species as well as collembolans and isopods were tested, and, in the field, only the litter-bag test was used. Clearly, more species than these few standard organisms currently in use have to be selected for testing purposes. A more detailed analysis of GMP tests with soil invertebrates published in the literature revealed that some of the relevant GMP exposure routes, such as via bulk soil, soil porewater, and litter from GMPs, are well covered. However, studies addressing either consumption of GMPs themselves or secondary exposure after GMPs have been taken up by invertebrates that feed on living or dead GMPs are underrepresented.

Derivation of soil values for the path 'soil-soil organisms' for metals and selected organic compounds using species sensitivity distributions.

BACKGROUND, AIMS AND SCOPE: According to the German Federal Soil Protection Act, the natural function of soil as a habitat for human beings, animals, plants and soil organisms is, among other things, to be protected by deriving soil values for important chemicals regarding their amounts in the environment, their persistence and/or their toxicity. This contribution presents the results of the mathematical derivation of such values for nine metals and ten organic substances from soil ecotoxicological effect values available in the literature for microbial processes, plants and soil invertebrates. MATERIAL AND METHODS: Ecotoxicological data were mostly extracted from published papers and reports and had to originate from valid studies that were performed according to internationally standardised guidelines (e.g. ISO) or were otherwise well documented, plausible and performed according to accepted laboratory practice. As test results, both structural (i.e., effects on mortality, growth or reproduction) and functional (i.e., effects on microbial activity or organic matter breakdown) parameters were included. The derivation of soil values was performed using the distribution based extrapolation model (DIBAEX) and EC(50)s (Effective Concentration) as input data. RESULTS: For 19 compounds, soil values could be calculated. In 18 of these 19 cases clear laboratory ecotoxicological effects (i.e., EC50 values) below the calculated soil value have been found in the literature. DISCUSSION: In those few cases where a comparison with field studies is possible, effects have been observed in the same order of magnitude as the calculated soil values. A comparison with other similar approaches confirmed the plausibility of the calculated values. CONCLUSIONS: The DIBAEX-method is a feasible and widely accepted method for deriving soil values from ecotoxicological input data. Data availability was already satisfactory for some substances, but other substances, especially organics, were only poorly covered. The soil values presented here were based on EC50 input data. However, depending on the protection level aimed at by using soil values in legislation, it might be appropriate to use other input data such as NOECs in the derivation process. RECOMMENDATIONS AND PERSPECTIVES: It is recommended to generate an appropriate number of data for further relevant substances by means of a test battery or multi-species approaches such as terrestrial model ecosystems. These tests should also consider the influence of the bioavailability of substances. A final recommendation for legally binding soil values demands a plausibility check of the mathematically derived values. This should include a comparison with natural background concentrations, soil values for other pathways and soil values used in legislation of other countries. Finally, expert judgement always has to be considered.

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.

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.

Improvement of the applicability of ecotoxicological tests with earthworms, springtails, and plants for the assessment of metals in natural soils.

The environmental risk assessment of metals in the soil compartment is based mainly on tests performed in Organization for Economic Cooperation and Development (OECD) artificial soil, but ecologically, the use of natural soils would be more relevant. In this contribution, the reproduction and growth of three standard species (an earthworm, a collembolan, and a dicotyledonous plant, respectively) was evaluated in nine natural soils (covering a wide range of pH values, organic matter content, texture, and so on) and in OECD artificial soil. Afterward, the effects of the model chemical zinc nitrate were assessed in all soils that were identified as being suitable for these species. The test results indicate that the toxicity of zinc nitrate can be higher by a factor of approximately four compared to artificial soil for invertebrates (earthworms and collembolans), whereas plants are only slightly more sensitive in some natural soils than in artificial soil. When comparing the different endpoints, it could be confirmed that the median effective concentration (EC50) is the most robust compared to the highly uncertain 10% effective concentration. Decreasing toxicity of zinc nitrate to collembolans was significantly correlated with an increase in soil pH but not with cation exchange capacity (CEC) or organic carbon (OC) content. No significant correlation was found between the toxicity of zinc nitrate to earthworms or plants and soil pH, CEC, or OC content. Possible consequences of the results are discussed, such as the testing of natural soils in addition to the OECD artificial soil or the inclusion of an additional safety factor to use the EC50 in current risk assessment schemes focusing on no-observed-effect concentrations.