Higher Tier Studies Research Articles

Assessing Effects of Genetically Modified Plant Material on the Aquatic Environment Using higher-tier Studies.

Genetically modified organisms are used extensively in agriculture. To assess potential side effects of genetically modified (GM) plant material on aquatic ecosystems, only a very small number of higher-tier studies have been performed. At the same time, these studies are particularly important for comprehensive risk assessment covering complex ecological relationships. Here we evaluate the methods of experimental higher-tier effect studies with GM plant material (or Bt toxin) in comparison to those well-established for pesticides. A major difference is that nominal test concentrations and thus dose-response relationships cannot easily be produced with GM plant material. Another important difference, particularly to non-systemic pesticides, is that aquatic organisms are exposed to GM plant material primarily through their feed. These and further differences in test requirements, compared with pesticides, call for a standardisation for GM-specific higher-tier study designs to assess their potentially complex effects in the aquatic ecosystems comprehensively.

Comprehensive identification of gene expression fingerprints and biomarkers of sexual endocrine disruption in zebrafish embryo

Endocrine disruptors (EDs), capable of modulating the sex hormone system of an organism, can exert long-lasting negative effects on reproduction in both humans and the environment. For these reasons, the properties of EDs prevent a substance from being approved for marketing. However, regulatory testing to evaluate endocrine disruption is time-consuming, costly, and animal-intensive. Here, we combined sublethal zebrafish embryo assays with transcriptomics and proteomics for well-characterized endocrine disrupting reference compounds to identify predictive biomarkers for sexual endocrine disruption in this model. Using RNA and protein gene expression fingerprints from two different sublethal exposure concentrations, we identified specific signatures and impaired biological processes induced by ethinylestradiol, tamoxifen, methyltestosterone and flutamide 96 h post fertilization (hpf). Our study promotes vtg1 as well as cyp19a1b, fam20cl, lhb, lpin1, nr1d1, fbp1b, and agxtb as promising biomarker candidates for identifying and differentiating estrogen and androgen receptor agonism and antagonism. Evaluation of these biomarkers for pre-regulatory zebrafish embryo-based bioassays will help identify endocrine disrupting hazards of compounds at the molecular level. Such approaches additionally provide weight-of-evidence for the identification of putative EDs and may contribute significantly to a reduction in animal testing in higher tier studies.

Simulating the Effects of Pesticides on Honey Bee (Apis mellifera L.) Colonies with BeePop+

The US Environmental Protection Agency (USEPA) employs a tiered process for assessing risks of pesticides to bees. The model discussed in this paper focuses on honey bees (Apis mellifera L.). If risks to honey bees are identified at the first tier based on exposure and toxicity data for individual adult and larval honey bees, then effects are evaluated in higher-tier studies using honey bee colonies. Colony-level studies require large amounts of resources (to conduct and review) and can yield data complicated by the inherent variability of colonies, which are influenced by factors that cannot readily be controlled, including weather, pests, diseases, available forage, and bee management practices. To better interpret these data, the USEPA and the US Department of Agriculture (USDA) developed a simulation model, BeePop+, that assesses potential honey bee colony-level effects of pesticides. Here, we describe this model using the population model guidance, use, interpretation, and development for ecological risk assessment (Pop-GUIDE) framework, which is a conceptual framework for the development and evaluation of population models. Within the context of Pop-GUIDE, BeePop+ is considered a “realistic-precise” model and reflects the inherent variability of colony response to pesticide exposure by simulating many outcomes. This model meets the desired features needed for use in pesticide risk assessments as its required data inputs are typically available, it is applicable to different US locations, and the outputs are both relevant to USEPA’s protection goals for honey bees and are consistent with the outcomes of empirical studies. This model has also been evaluated using available empirical colony-level data; however, additional evaluation with other studies may still be done in the future prior to completing implementation.

The Extended Amphibian Metamorphosis Assay: A Thyroid-Specific and Less Animal-Intensive Alternative to the Larval Amphibian Growth and Development Assay.

The amphibian metamorphosis assay (AMA; US Environmental Protection Agency [USEPA] test guideline 890.1100 and Organisation for Economic Co‐Operation and Development test guideline 231) has been used for more than a decade to assess the potential thyroid‐mediated endocrine activity of chemicals. In 2013, in the context of the Endocrine Disruptor Screening Program of the USEPA, a Scientific Advisory Panel reviewed the results from 18 studies and recommended changes to the AMA test guideline, including a modification to a fixed‐stage design rather than a fixed‐time (i.e., 21‐d) design. We describe an extended test design for the AMA (or EAMA) that includes thyroid histopathology and time to metamorphosis (Nieuwkoop–Faber [NF] stage 62), to address both the issues with the fixed‐time design and the specific question of thyroid‐mediated adversity in a shorter assay than the larval amphibian growth and development assay (LAGDA; Organisation for Economic Co‐Operation and Development test guideline 241), using fewer animals and resources. A demonstration study was conducted with the EAMA (up to NF stage 58) using sodium perchlorate. Data analyses and interpretation of the fixed‐stage design of the EAMA are more straightforward than the fixed‐time design because the fixed‐stage design avoids confounded morphometric measurements and thyroid histopathology caused by varying developmental stages at test termination. It also results in greater statistical power to detect metamorphic delays than the fixed‐time design. By preferentially extending the AMA to NF stage 62, suitable data can be produced to evaluate thyroid‐mediated adversity and preclude the need to perform a LAGDA for thyroid mode of action analysis. The LAGDA remains of further interest should investigations of longer term effects related to sexual development modulated though the hypothalamus–pituitary–gonadal axis be necessary. However, reproduction assessment or life cycle testing is currently not addressed in the LAGDA study design. This is better addressed by higher tier studies in fish, which should then include specific thyroid‐related endpoints. Environ Toxicol Chem 2021;40:2135–2144. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Application of grouping and read-across for the evaluation of parabens of different chain lengths with a particular focus on endocrine properties

This article presents the outcomes of higher-tier repeated-dose toxicity studies and developmental and reproductive toxicity (DART) studies using Wistar rats requested for methyl paraben and propyl paraben under the European Union chemicals legislation. All studies revealed no-observed adverse effects (NOAELs) at 1000 mg/kg body weight/day. These findings (absence of effects) were then used to interpolate the hazard profile for ethyl paraben, further considering available data for butyl paraben. The underlying read-across hypothesis (all shorter-chained linear n-alkyl parabens are a ‘category’ based on very high structural similarity and are transformed to a common compound) was confirmed by similarity calculations and comparative in vivo toxicokinetics screening studies for methyl paraben, ethyl paraben, propyl paraben and butyl paraben. All four parabens were rapidly taken up systemically following oral gavage administration to rats, metabolised to p-hydroxybenzoic acid, and rapidly eliminated (parabens within one hour; p-hydroxybenzoic acid within 4–8 h). Accordingly, for ethyl paraben, the NOAELs for repeated-dose toxicity and DART were interpolated to be 1000 mg/kg body weight/day. Finally, all evidence was evaluated to address concerns expressed in the literature that parabens might be endocrine disruptors. This evaluation showed that the higher-tier studies do not provide any indication for any endocrine disrupting property. This is the first time that a comprehensive dataset from higher-tier in vivo studies following internationally agreed test protocols has become available for shorter-chained linear n-alkyl parabens. Consistently, the dataset shows that these parabens are devoid of repeated-dose toxicity and do not possess any DART or endocrine disrupting properties.

Inclusion of seasonal variation in river system microbial communities and phototroph activity increases environmental relevance of laboratory chemical persistence tests

Regulatory tests assess crop protection product environmental fate and toxicity before approval for commercial use. Although globally applied laboratory tests can assess biodegradation, they lack environmental complexity. Microbial communities are subject to temporal and spatial variation, but there is little consideration of these microbial dynamics in the laboratory. Here, we investigated seasonal variation in the microbial composition of water and sediment from a UK river across a two-year time course and determined its effect on the outcome of water-sediment (OECD 308) and water-only (OECD 309) biodegradation tests, using the fungicide isopyrazam. These OECD tests are performed under dark conditions, so test systems incubated under non-UV light:dark cycles were also included to determine the impact on both inoculum characteristics and biodegradation. Isopyrazam degradation was faster when incubated under non-UV light at all collection times in water-sediment microcosms, suggesting that phototrophic communities can metabolise isopyrazam throughout the year. Degradation rate varied seasonally between inoculum collection times only in microcosms incubated in the light, but isopyrazam mineralisation to 14CO2 varied seasonally under both light and dark conditions, suggesting that heterotrophic communities may also play a role in degradation. Bacterial and phototroph communities varied across time, but there was no clear link between water or sediment microbial composition and variation in degradation rate. During the test period, inoculum microbial community composition changed, particularly in non-UV light incubated microcosms. Overall, we show that regulatory test outcome is not influenced by temporal variation in microbial community structure; however, biodegradation rates from higher tier studies with improved environmental realism, e.g. through addition of non-UV light, may be more variable. These data suggest that standardised OECD tests can provide a conservative estimate of pesticide persistence end points and that additional tests including non-UV light could help bridge the gap between standard tests and field studies.

Evaluation of the Rhizosphere Contribution to the Environmental Fate of the Herbicide Prometryn.

Plant protection products (PPPs) undergo rigorous regulatory assessment to ensure that they do not pose unacceptable risks to the environment. Elucidation of their fate and behavior in soil is an integral part of this environmental risk assessment. The active substance degradation in soil of PPPs is first assessed in laboratory studies (typically following Organisation for Economic Co-operation and Development [OECD] test guideline 307). Conditions in guideline laboratory studies are far removed from those occurring under agricultural use, and the contribution of crop roots has currently not been assessed. We integrated viable plant root systems, representative of 3 different crop types, into the OECD test guideline 307 design to assess their impact on the dissipation of the herbicide prometryn. Significantly faster decline of parent residue and higher formation of nonextractable residues were observed in all 3 planted systems. This led to a reduction in the time required for 50% of the compound to dissipate (DT50) of approximately one-half in the presence of rye grass and hot pepper and of approximately one-third in the presence of red clover. These findings imply that plants and their associated root networks can have a significant influence on PPP dissipation. Based on these data, greater environmental realism could be added to the standardized laboratory study design by the inclusion of plant root systems into higher tier studies, which, in turn, could serve to improve the environmental risk assessment process. Environ Toxicol Chem 2020;39:450-457. © 2019 SETAC.

Overcoming Challenges of Incorporating Higher Tier Data in Ecological Risk Assessments and Risk Management of Pesticides in the United States: Findings and Recommendations from the 2017 Workshop on Regulation and Innovation in Agriculture.

ABSTRACTPesticide regulation requires regulatory authorities to assess the potential ecological risk of pesticides submitted for registration, and most risk assessment schemes use a tiered testing and assessment approach. Standardized ecotoxicity tests, environmental fate studies, and exposure models are used at lower tiers and follow well‐defined methods for assessing risk. If a lower tier assessment indicates that the pesticide may pose an ecological risk, higher tier studies using more environmentally realistic conditions or assumptions can be performed to refine the risk assessment and inform risk management options. However, there is limited guidance in the United States on options to refine an assessment and how the data will be incorporated into the risk assessment and risk management processes. To overcome challenges to incorporation of higher tier data into ecological risk assessments and risk management of pesticides, a workshop was held in Raleigh, North Carolina. Attendees included representatives from the United States Environmental Protection Agency, United States Department of Agriculture, National Oceanic and Atmospheric Administration, universities, commodity groups, consultants, nonprofit organizations, and the crop protection industry. Key recommendations emphasized the need for 1) more effective, timely, open communication among registrants, risk assessors, and risk managers earlier in the registration process to identify specific protection goals, address areas of potential concern where higher tier studies or assessments may be required, and if a higher tier study is necessary that there is agreement on study design; 2) minimizing the complexity of study designs while retaining high value to the risk assessment and risk management process; 3) greater transparency regarding critical factors utilized in risk management decisions with clearly defined protection goals that are operational; and 4) retrospective analyses of success–failure learnings on the acceptability of higher tier studies to help inform registrants on how to improve the application of such studies to risk assessments and the risk management process. Integr Environ Assess Manag 2019;15:714–725. © 2019 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals, Inc. on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Ecotoxicological characterization of the antiepileptic drug carbamazepine using eight aquatic species: baseline study for future higher tier tests

Ecotoxicological effects of the antiepileptic drug carbamazepine (CBZ) were investigated in one primary producer (Desmodesmus subspicatus) and seven invertebrate species (Daphnia magna, Daphnia pulex, Ceriodaphnia dubia, Gammarus fossarum, Potamopyrgus antipodarum, Lumbriculus variegatus and Chironomus riparius) using OECD and US EPA guidelines for chronic toxicity testing. The present data set was used to conduct a hazard assessment for CBZ including confirmatory data. While most of our results were in accordance with previous studies, published effect data for C. dubia and D. pulex could not be confirmed, even though they have previously been considered to be the most sensitive invertebrate species to CBZ exposure. The non-biting midge, C. riparius, was the most sensitive test organism in the present study. From an EC10 of 406 µg/L and a no observed effect concentration (NOEC) of 400 µg/L, a predicted no effect concentration (PNEC) of 8 µg/L was calculated. With regard to realistic predicted and measured environmental concentrations, the environmental risk can be considered as low for CBZ when the assessment is based on laboratory-based effect data. To conduct a refined and more realistic assessment, this study provides foundational data for two future, higher tier studies: one multiple-stressor experiment and one mesocosm study.

Bumble bee semi-field studies: choice and management of colonies to reduce variability in assessment endpoints

The publication of the proposed EFSA risk assessment for pollinators resulted in an increasing demand for experiments with non-Apis pollinators (EFSA 2013). However, no official guideline for the standardized semifield trials exists so far. To overcome this lack of guidance, the development of semi-field study designs are under way. The methodology is concurrently be developed by an ICPPR working group (non-Apis working group). A major challenge in higher tier studies is the variability of the different endpoints. Hive development and particularly the production of young queens are very variable (Cabrera et al. 2016). With the current knowledge it seemed crucial to select appropriate colonies for the tests to reduce variability. The aim was to evaluate different strategies for the selection of bumble bee colonies and to improve the data quality with regard to the most important endpoints in bumble bee semi-field studies.

Exposure by nesting material? – Investigation of potentially suitable methods for higher tier studies with solitary bees

The registration processes and risk assessment of plant protection products (PPPs) on bees resulted in an increasing need for experiments with non-apis pollinators to assess potential side effects of PPPs on this relatively new group of test organisms. Recently, numerous studies have been performed but there is still a wide range of ongoing challenges. One of the challenges is the risk from insecticide exposure to solitary bees (especially at larval stages) by contaminated nesting material (e.g. mud partitions – mason bees). In 2017, an experiment was performed with the horn-faced mason bee Osmia cornuta (Hymenoptera, Megachilidae) under modified field conditions. The aim of the experiment was to develop a suitable test method for higher tier risk assessments with solitary wild bees exposed to treated nesting material. The potential effect of an insect growth regulator (IGR) to bees and their brood was examined. The reproduction capacity and brood termination rate were observed in the study as endpoints. Furthermore, hatching success and flight activity were recorded as additional information at several occasions.The present results provide no evidence that the exposure has an effect on the development during the larval stages of Osmia cornuta, neither in pollen mass nor in the nesting material.

Bumble bee queen production in semi-field studies: assessment of endpoints and challenges

Bumble bees (Bombus terrestris L; Hymenoptera, Apidae) provide important pollination services and are commercially used, e.g. in greenhouse cultures. Consequently, the impacts of pesticides on bumble bees were already tested in the past. In the light of the newest EFSA guidance document on the risk assessment of plant protection products for pollinators standardized higher tier studies for pollinators are needed (EFSA 2013). For that reason a ringtest protocol for a bumble bee semi-field study design was developed in the ICPPR Non-Apis working group starting in 2015 to date. The central endpoint in a higher tier bumble bee study is the colony reproduction success (production of young queens, Cabrera et al. 2016). The endpoint is chosen because at the end of the annual life cycle of a bumble bee colony all workers die and only young queens overwinter. Queens that survive establish a new colony in the following year. However, assessing queen reproduction is challenging. Many variables can influence the number of produced queens, such as the right timing for the termination of the study or the condition of the colony at study start. Furthermore, young queen weights are measured. Weight is used as indicator of diapause survival. Literature values of average weight needed for survival before overwintering state 0.8 g for a young queen for successful overwintering (Beekman et al. 1998). Based on data from ring tests of 2016 and 2017 we tried to answer several open questions concerning queen reproduction, i.e. how can the experimental set-up influence queen weights and how high is the natural variation in queen numbers and queen weight/size?

Quantitative weight of evidence assessment of higher-tier studies on the toxicity and risks of neonicotinoid insecticides in honeybees 1: Methods

ABSTRACTA quantitative weight of evidence (QWoE) methodology was developed and used to assess many higher-tier studies on the effects of three neonicotinoid insecticides: clothianidin (CTD), imidacloprid (IMI), and thiamethoxam (TMX) on honeybees. A general problem formulation, a conceptual model for exposures of honeybees, and an analysis plan were developed. A QWoE methodology was used to characterize the quality of the available studies from the literature and unpublished reports of studies conducted by or for the registrants. These higher-tier studies focused on the exposures of honeybees to neonicotinoids via several matrices as measured in the field as well as the effects in experimentally controlled field studies. Reports provided by Bayer Crop Protection and Syngenta Crop Protection and papers from the open literature were assessed in detail, using predefined criteria for quality and relevance to develop scores (on a relative scale of 0–4) to separate the higher-quality from lower-quality studies and those relevant from less-relevant results. The scores from the QWoEs were summarized graphically to illustrate the overall quality of the studies and their relevance. Through mean and standard errors, this method provided graphical and numerical indications of the quality and relevance of the responses observed in the studies and the uncertainty associated with these two metrics. All analyses were conducted transparently and the derivations of the scores were fully documented. The results of these analyses are presented in three companion papers and the QWoE analyses for each insecticide are presented in detailed supplemental information (SI) in these papers.

Quantitative weight of evidence assessment of higher tier studies on the toxicity and risks of neonicotinoids in honeybees. 4. Thiamethoxam

ABSTRACTA quantitative weight of evidence (QWoE) methodology was used to assess several higher-tier studies on the effects of thiamethoxam (TMX) on honeybees. Assessment endpoints were population size and viability of commercially managed honeybee colonies and quantity of hive products. A higher-tier field toxicology study indicated a no-observed-adverse effect concentration (NOAEC) of 29.5 µg TMX/kg syrup, equivalent to an oral no-observed-adverse-effect-dose (NOAED) of 8.6 ng/bee/day for all responses measured. For exposures via deposition of dust, a conservative no-observed-adverse-effect-rate at the level of the colony was 0.1 g TMX/ha. There was minimal risk to honeybees from exposure to TMX via nectar and pollen from its use as a seed-treatment. For exposures via dust and dust/seed applications, there were no concentrations above the risk values for TMX in nectar and pollen. Although some risks were identified for potential exposures via guttation fluid, this route of exposure is incomplete; no apparent adverse effects were observed in field studies. For exposures via dust/seed and dust/foliar applications, few adverse effects were observed. Considering all lines of evidence, the quality of the studies included in this analysis was variable. However, the results of the studies were consistent and point to the same conclusion. The overall weight of evidence based on many studies indicates that TMX has no adverse effects on viability or survival of the colony. Thus, the overall conclusion is that the treatment of seeds with thiamethoxam, as currently used in good agricultural practices, does not present a significant risk to honeybees at the level of the colony.

Quantitative weight of evidence assessment of higher-tier studies on the toxicity and risks of neonicotinoids in honeybees. 2. Imidacloprid

ABSTRACTA quantitative weight of evidence (QWoE) methodology was used to assess higher-tier studies on the effects of imidacloprid (IMI) on honeybees. Assessment endpoints were population size and viability of commercially managed bees and quantity of hive products. A colony-level no-observed-adverse effect concentration (NOAEC) of 25 µg IMI/kg syrup, equivalent to an oral no-observed-adverse-effect-dose of 7.3 ng/bee/d for all responses, was measured. The overall weight of evidence indicates that there is minimal risk to honeybees from exposure to IMI from its use as a seed treatment. Exposures via dusts from currently used seed coatings present a de minimis risk to honeybees when the route of exposure is via uptake in plants that are a source of pollen or nectar for honeybees. There were few higher-tier observational (ecoepidemiological) studies conducted with IMI. Considering all lines of evidence, the quality of the studies included in this analysis was variable, but the results of the studies were consistent and point to the same conclusion – that IMI had no adverse effects on viability of the honeybee colony. Thus, the overall conclusion is that IMI, as currently used as a seed treatment and with good agricultural practices, does not present a significant risk to honeybees at the level of the colony.

The honey bee brood test under semi-field conditions for the assessment of positive reference chemicals in Korea

Honey bee brood tests under semi-field conditions are higher-tier studies for investigating the potential impact of pesticides on brood development in honey bee colonies. Semi-field studies on the effects of pesticides on honey bees have been mainly carried out in the EU and USA, with only a relatively small number conducted in Korea and other Asian countries. Here, we report the first semi-field test in Korea, which was carried out from April to May 2016. The experiment included three treatment groups (control and two toxic reference chemicals), each with three replicate tunnels. The honey bee colonies were placed in tunnels covering 70 m2 and containing Brassica napus. Flight activity, mortality, the condition of the colonies, and brood development were assessed during the 28 days of testing period. The toxic reference treatments were 400 g dimethoate a.i./ha and 600 g diflubenzuron a.i./ha. Brood termination rates for marked eggs were 31.3% in the control group, 83.5 and 68.0% for dimethoate and diflubenzuron, respectively. These results confirm the sensitivity of the test method and indicate that these two chemicals could be used as appropriate toxic reference compounds in future semi-field tests in Korea.

Effects of Organic Pesticides on Enchytraeids (Oligochaeta) in Agroecosystems: Laboratory and Higher-Tier Tests

Enchytraeidae (Oligochaeta, Annelida) are often considered to be typical forestliving organisms, but they are regularly found in agroecosystems of the temperate regions of the world. Although less known than their larger relatives, the earthworms, these saprophagous organisms play similar roles in agricultural soils (but at a smaller scale), e.g., influencing soil structure and organic matter dynamics via microbial communities, and having a central place in soil food webs. Their diversity is rarely studied or often underestimated due to difficulties in distinguishing the species. New genetic techniques reveal that even in anthropogenically highly influenced soils, more than ten species per site can be found. Because of their close contact with the soil pore water, a high ingestion rate and a thin cuticle, they often react very sensitively to a broad range of pesticides. Firstly we provide a short overview of the diversity and abundance of enchytraeid communities in agroecosystems. Afterwards, we explore the available data on enchytraeid sensitivity towards pesticides at different levels of biological organization, focusing on pesticides used in (mainly) European agroecosystems. Starting with non-standardized studies on the effects of pesticides on the sub-individual level, we compile the results of standard laboratory tests performed following OECD and ISO guidelines as well as those of higher-tier studies (i.e., semi-field and field tests). The number of comparable test data is still limited, because tests with enchytraeids are not a regulatory requirement in the European Union. While focusing on the effects of pesticides, attention is also given to their interactions with environmental stressors (e.g., climate change). In conclusion, we recommend to increase the use of enchytraeids in pesticide risk assessment because of their diversity and functional importance as well as their increasingly simplified use in (mostly standardized) tests at all levels of biological organization.

Initial recommendations for higher-tier risk assessment protocols for bumble bees, Bombus spp. (Hymenoptera: Apidae).

Global declines of bumble bees and other pollinator populations are of concern because of their critical role for crop production and maintenance of wild plant biodiversity. Although the consensus among scientists is that the interaction of many factors, including habitat loss, forage scarcity, diseases, parasites, and pesticides, potentially plays a role in causing these declines, pesticides have received considerable attention and scrutiny. In response, regulatory agencies have introduced more stringent pollinator testing requirements for registration and reregistration of pesticides, to ensure that the risks to pollinators are minimized. In this context, guidelines for testing bumble bees (Bombus spp.) in regulatory studies are not yet available, and a pressing need exists to develop suitable protocols for routine higher-tier studies with these non-Apis sp., social bees. To meet this need, Bayer CropScience LP, Syngenta Crop Protection LLC US, and Valent USA. Corporation organized a workshop bringing together a group of global experts on bumble bee behavior, ecology, and ecotoxicology to discuss and develop draft protocols for both semi-field (Tier II) and field (Tier III) studies. The workshop was held May 8-9, 2014, at the Bayer Bee Care Center, North Carolina, USA. The participants represented academic, consulting, and industry scientists from Europe, Canada, the United States, and Brazil. The workshop identified a clear protection goal and generated proposals for basic experimental designs, relevant measurements, and endpoints for both semifield (tunnel) and field tests. These initial recommendations are intended to form the basis of discussions to help advance the development of appropriate protocol guidelines.

Responses of earthworms to repeated exposure to three biocides applied singly and as a mixture in an agricultural field

The study aimed at investigating effects of three differently acting biocides; the insecticide esfenvalerate, the fungicide picoxystrobin and the bactericide triclosan, applied individually and as a mixture, on an earthworm community in the field. A concentration–response design was chosen and results were analyzed using univariate and multivariate approaches. Effects on juvenile proportions were less pronounced and more variable than effects on abundance, but effects in general were species- and chemical-specific, and temporal variations distinct. Esfenvalerate and picoxystrobin appeared to elicit stronger effects than triclosan at laboratory-based ECx values, which is in accordance with our previous laboratory study on Eisenia fetida. The mixture affected abundance and juvenile proportions, but the latter only at high mixture concentrations. Esfenvalerate and picoxystrobin appeared to be the main drivers for the mixture's toxicity. Species-specific toxicity patterns question the reliability of mixture toxicity predictions derived on E. fetida for field earthworms. Biocide concentrations equaling EC50s (reproduction) for E. fetida provoked effects on the field earthworms mainly exceeding 50%, indicating effect intensification from the laboratory to field as well as the influence of indirect effects produced by species interactions. The differing results of the present field study and the previous laboratory study imply that lower- and higher-tier studies may not be mutually exclusive, but to be used in complementary.

Potential use of an arthropod database to support the non-target risk assessment and monitoring of transgenic plants

Worldwide, plants obtained through genetic modification are subject to a risk analysis and regulatory approval before they can enter the market. An area of concern addressed in environmental risk assessments is the potential of genetically modified (GM) plants to adversely affect non-target arthropods and the valued ecosystem services they provide. Environmental risk assessments are conducted case-by-case for each GM plant taking into account the plant species, its trait(s), the receiving environments into which the GM plant is to be released and its intended uses, and the combination of these characteristics. To facilitate the non-target risk assessment of GM plants, information on arthropods found in relevant agro-ecosystems in Europe has been compiled in a publicly available database of bio-ecological information during a project commissioned by the European Food Safety Authority (EFSA). Using different hypothetical GM maize case studies, we demonstrate how the information contained in the database can assist in identifying valued species that may be at risk and in selecting suitable species for laboratory testing, higher-tier studies, as well as post-market environmental monitoring.