Mortality Pattern of Poecilus cupreus Beetles after Repeated Topical Exposure to Insecticide─Stochastic Death or Individual Tolerance?

The mortality of organisms exposed to toxicants has been attributed to either stochastic processes or individual tolerance (IT), leading to the stochastic death (SD) and IT models. While the IT model follows the principles of natural selection, the relevance of the SD model has been debated. To clarify why the idea of stochastic mortality has found its way into ecotoxicology, we investigated the mortality of Poecilus cupreus (Linnaeus, 1758) beetles from pesticide-treated oilseed rape (OSR) fields and unsprayed meadows, subjected to repeated insecticide treatments. We analyzed the mortality with the Kaplan-Meier estimator and general unified threshold model for survival (GUTS), which integrates SD and IT assumptions. The beetles were exposed three times, ca. monthly, to the same dose of Proteus 110 OD insecticide containing thiacloprid and deltamethrin, commonly used in the OSR fields. Kaplan-Meier analysis showed that the mortality of beetles from meadows was much higher after the first treatment than after the next two, indicating the IT model. Beetles from the OSR displayed approximately constant mortality after the first and second treatments, consistent with the SD model. GUTS analysis did not conclusively identify the better model, with the IT being marginally better for beetles from meadows and the SD better for beetles from OSR fields.

Effects of agricultural landscape structure and canola coverage on biochemical and physiological traits of the ground beetle Poecilus cupreus.

The intensifications in the agricultural landscape and the application of pesticides can cause adverse effects on the fitness of organisms in that landscape. Here, we investigated whether habitats with different agricultural pressures influenced acetylcholinesterase (AChE) activity - a biomarker for exposure to pesticides, respiration rate, and resistance to starvation in the ground beetle Poecilus cupreus. Two differently structured landscapes were selected for the study, one dominated by small (S) and another by large (L) fields. Within each landscape three habitat types were selected: in the S landscape, these were habitats with medium (M), small (S) and no canola (meadow, 0) coverage (i.e., SM, SS, S0), and in the L landscape habitats with large (L), medium (M) and no canola (meadow, 0) coverage (i.e., LL, LM, L0), representing different levels of agricultural pressure. The activity of AChE was the highest in beetles from canola-free habitats (S0 and L0), being significantly higher than in beetles from the SM and SS habitats. The mean respiration rate corrected for body mass was also the highest in S0 and L0 beetles, with significant differences between populations from L0 vs. SS and from S0 vs. SS. Only beetles from S0, SS, L0, and LM were numerous enough to assess the resistance to starvation. Individuals from the LM habitat showed better survival compared to the canola-free habitat in the same landscape (L0), whereas in S landscape the SS beetles survived worse than those from S0, suggesting that characteristics of L landscape may lead to developing mechanisms of starvation resistance of P. cupreus in response to agricultural pressure.

Could a city-dwelling bat (Pipistrellus kuhlii) serve as a bioindicator species for trace metals pollution?

Bats are an endangered group of mammals that are very sensitive to environmental stresses. One of such stress factor is trace metals pollution which threatens populations of insectivorous bats due to their top position in the food webs and exceptionally long life span. In our research Pipistrellus kuhlii was tested as a promising indicator species (urban-dwelling, sedentary, with limited daily home-range) for trace metal exposure of bats. We measured concentrations of Pb, Cu, Zn and Cd in internal and external tissues of bats from the industrial city Mariupol and the village Karlovka, Ukraine, to answer the following questions: (1) Do metal concentrations in soil samples differ between urban and rural areas, and between populations of P. kuhlii from those areas? (2) Does metal contamination differ between individuals of different sexes and ages? (3) Whether fur and/or wing membrane, the two tissues that can be collected from live bats, can be used as proxies of metal contamination in internal tissues (liver, kidney, lung, forearm bones) of P. kuhlii? Metal concentrations in soil samples were significantly higher in the city. Bats from the city accumulated significantly more Cd, Pb and Zn in external tissues than those from the rural area. Females accumulated more Cd than males, and this-year-born did not differ significantly from adult individuals. We did not find, however, significant positive correlations between metal concentrations in external and internal tissues, indicating that external tissues cannot serve as an indicator of the metal contamination of internal tissues in P. kuhlii.

Homogeneity of agriculture landscape promotes insecticide resistance in the ground beetle Poecilus cupreus.

The intensification of agriculture leads to increased pesticide use and significant transformation from small fields towards large-scale monocultures. This may significantly affect populations of non-target arthropods (NTA). We aimed to assess whether the multigenerational exposure to plant protection products has resulted in the evolution of resistance to insecticides in the ground beetle Poecilus cupreus originating from different agricultural landscapes. Two contrasting landscapes were selected for the study, one dominated by small and another by large fields. Within each landscape the beetles were collected at nine sites representing range of canola coverage and a variety of habitat types. Part of the collected beetles, after acclimation to laboratory conditions, were tested for sensitivity to Proteus 110 OD-the most commonly used insecticide in the studied landscapes. The rest were bred in the laboratory for two consecutive generations, and part of the beetles from each generation were also tested for sensitivity to selected insecticide. We showed that the beetles inhabiting areas with medium and large share of canola located in the landscape dominated by large fields were less sensitive to the studied insecticide. The persistence of reduced sensitivity to Proteus 110 OD for two consecutive generations indicates that either the beetles have developed resistance to the insecticide or the chronic exposure to pesticides has led to the selection of more resistant individuals naturally present in the studied populations. No increased resistance was found in the beetles from more heterogeneous landscape dominated by small fields, in which spatio-temporal diversity of crops and abundance of small, linear off-crop landscape elements may provide shelter that allows NTAs to survive without developing any, presumably costly, resistance mechanisms.

Toxicokinetics of three insecticides in the female adult solitary bee Osmia bicornis.

The worldwide decline of pollinators is of growing concern and has been related to the use of insecticides. Solitary bees are potentially exposed to many insecticides through contaminated pollen and/or nectar. The kinetics of these compounds in solitary bees is, however, unknown, limiting the use of these important pollinators in pesticide regulations. Here, the toxicokinetics (TK) of chlorpyrifos (as Dursban 480 EC), cypermethrin (Sherpa 100 EC), and acetamiprid (Mospilan 20 SP) was studied for the first time in Osmia bicornis females at sublethal concentrations (near LC20s). The TK of the insecticides was analysed in bees continuously exposed to insecticide-contaminated food in the uptake phase followed by feeding with clean food in the decontamination phase. The TK models differed substantially between the insecticides. Acetamiprid followed the classic one-compartment model with gradual accumulation during the uptake phase followed by depuration during the decontamination phase. Cypermethrin accumulated rapidly in the first two days and then its concentration decreased slowly. Chlorpyrifos accumulated similarly rapidly but no substantial depuration was found until the end of the experiment. Our study demonstrates that some insecticides can harm solitary bees when exposed continuously even at trace concentrations in food because of their constant accumulation leading to time-reinforced toxicity.

Effects of agricultural landscape structure, insecticide residues, and pollen diversity on the life-history traits of the red mason bee Osmia bicornis.

Agricultural landscapes have changed substantially in recent decades, shifting from the dominance of small fields (S) with diverse cropping systems toward large-scale monoculture (L), where landscape heterogeneity disappears. In this study, artificial nests of the red mason bee, Osmia bicornis, were placed in S and L landscape types on the perimeter of oilseed rape fields representing different oilseed rape coverages (ORC, % land cover). The local landscape structure around each nest was characterised within a 100, 200, 500, and 1000 m radius using ORC and 14 landscape characteristics, which were then reduced by non-metric multidimensional scaling (nMDS) to two axes: nMDS1 characterised the dataset primarily according to land fragmentation and the main crop, whereas nMDS2 captured the prevalence of more natural areas in the landscape. Pollen diversity and insecticide risk levels in the pollen provisions collected by the bees were analysed, and their dependence on the landscape structure was tested. Thereafter, the effects of pollen diversity, insecticide risk, and landscape structure on the life-history traits of bees and their sensitivity to topically applied Dursban 480 EC were determined. Pollen taxa richness in a single nest ranged from 3 to 12, and 34 pesticides were detected in the pollen at concentrations of up to 320 ng/g for desmedipham. The O. bicornis foraging range was relatively large, indicating that the landscape structure within a radius of ~1000 m around the nest is important for this species. Pollen diversity in the studied areas was of minor importance for bee performance, but the ORC or landscape structure significantly affected the life-history traits of the bees. Contamination of pollen with insecticides affected the bees by decreasing the mass of newly emerged adults but their sensitivity to Dursban 480 EC was not related to environmental variables.

Physiological and biochemical response of the solitary bee Osmia bicornis exposed to three insecticide-based agrochemicals.

The physiological and biochemical stress induced by pesticides need to be addressed in economically and ecologically important non-Apis solitary bees, particularly at lower than field-applied concentrations. Thus, the aim of the present study was to analyse the physiological and biochemical changes in female adult Osmia bicornis bees upon continuous oral exposure to three insecticide-based agrochemicals - i.e. Dursban 480 EC (active ingredient - a.i. chlorpyrifos), Sherpa 100 EC (a.i. cypermethrin), and Mospilan 20 SP (a.i. acetamiprid), in a toxicokinetic manner (feeding with either insecticide-contaminated food or uncontaminated food (controls) for 8 d in the contamination phase followed by 8 d of decontamination (i.e. feeding with uncontaminated food)). All three tested agrochemicals altered the energetic budget of bees by the deprivation of energy derived from lipids and carbohydrates (but not proteins) and/or a decrease in respiration based metabolic rate (energy consumption) compared to the controls. The activities of acetylcholinesterase and glutathione-S-transferase enzymes were not altered by insecticides at tested concentrations. These results show that chronic exposure to at least some pesticides even at relatively low concentrations may cause severe physiological disruptions that could potentially be damaging for the solitary bees.

Wing membrane and Fur as indicators of metal exposure and contamination of internal tissues in bats.

All European bats are protected by the EU and Associated Members legal regulations. Being insectivorous and top predators, they can be particularly exposed to persistent organic and inorganic pollutants. It is surprising how little is known about the impact of environmental pollutants on bats from physiological to populational levels. In this study we focused on contamination with trace metals of first-year bats from Kharkiv city, NE Ukraine. Tissues from the carcasses of two species, Nyctalus noctula (n = 20) and Eptesicus serotinus (n = 20), were used for metal analysis. The samples of external (wing membrane, fur) and internal (liver, lung, kidney, bones) tissues were analysed for contents of Pb, Cu, Zn, and Cd to see whether fur or wing membrane can be used as proxies for metal contamination of the vital internal tissues. In E. serotinus, significant positive correlations in Pb concentrations were found between all external and internal tissues. For Cd only, correlation between the fur and lung was found, for Cu between the fur and liver, and for Zn between the fur and kidney. In contrast, for N. noctula, only one such correlation was found - between Zn concentrations in the fur and kidney. The tissues differed significantly in concentrations of all studied metals, with no difference between the species. The results showed that the fur and wing membrane can be used as good proxies for Pb concentrations in internal organs of E. serotinus, but not necessarily for other metals or for N. noctula. The results for Pb are, however, encouraging enough to conclude that the topic is worth further studies, covering more species, a wider age range and more diverse environments.

Supporting non-target arthropods in agroecosystems: Modelling effects of insecticides and landscape structure on carabids in agricultural landscapes.

Intensification of agricultural practices is one of the most important drivers of the dramatic decline of arthropod species. We do not know, however, the relative contribution to decline of different anthropogenic stressors that are part of this process. We used high-resolution dynamic landscape models and advanced spatially-explicit population modelling to estimate the relative importance of insecticide use and landscape structure for population dynamics of a widespread carabid beetle Bembidion lampros. The effects of in-crop mitigation measures through the application of insecticides with reduced lethality, and off-crop mitigation measures by increasing abundance of grassy field margins, were evaluated for the beetle along the gradient of landscape heterogeneity. Reducing the insecticide-driven lethality (from 90 to 10%) had larger positive impacts on beetle density and occupancy than increasing the abundance of field margins in a landscape. The effects of increasing field margins depended on their width and overall abundance in the landscape, but only field margins 4 m wide, applied to at least 40% of fields, resulted in an increase in beetle population density comparable to the scenario with the smallest reduction of insecticide-driven lethality we considered. Our findings suggest the importance of field margins rather as a supporting not stand-alone mitigation measure, as they generally improved effects of reduction of insecticide-driven lethality. Therefore, adding sufficiently broad off-field habitats should help to maintain viable beetle populations in agricultural landscapes even with moderate use of insecticides. In general, the less persistent the insecticides are in the environment, the larger positive impacts of applied mitigation measures on beetle populations were found. We also showed that the effectiveness of applied mitigation measures strongly depends on landscape and farmland heterogeneity. Thus, to achieve the same management or mitigation target in different landscapes might require different strategies.

The development of the solitary bee Osmia bicornis is affected by some insecticide agrochemicals at environmentally relevant concentrations.

Solitary bees provide essential pollination services for many arable crops, but are prone to global decline. Agricultural intensification, which is connected with pesticide usage, is among major threats to bees and, thus, to the food security and ecosystem stability. As it may not be possible to cease pesticide usage currently because of the growing demand for food, it is crucial to understand the pesticide toxicities to bees for better protection of pollinator populations. The majority of studies have focused on social bees, and those on solitary bees studied effects of adult exposure, whereas these bees are also likely to be exposed as larvae via the consumption of contaminated pollen. Here, the effects of three commonly used insecticide-based plant protection products on the development of the solitary bee, Osmia bicornis (red mason bee), were studied by exposing larvae to insecticide-contaminated multifloral pollen. The tested insecticides were: Dursban480EC, containing the organophosphate chlorpyrifos (CHP), Sherpa100EC, containing the pyrethroid cypermethrin (CYP), and Mospilan20SP with the neonicotinoid acetamiprid (ACT). When compared to the control larvae fed with uncontaminated-pollen, both CHP and CYP significantly reduced the O. bicornis larval survival and their body mass at all tested concentrations. In contrast, ACT did not affect either larval survival or body mass, but the length of larval stage to cocoon formation was significantly shortened compared to controls. None of studied insecticides affected the mass of cocooned individuals. However, at least 80% of individuals exposed to any of the tested insecticides died before reaching the adult stage, whereas 43% of the controls emerged successfully after overwintering. Although no clear monotonic dose-response relationships were found, our study showed that at least some insecticide formulations affect the development of O. bicornis even at concentrations actually found in pollen in the field, indicating an urgent need for revising current pesticide usage recommendations.

Guidance on harmonised methodologies for human health, animal health and ecological risk assessment of combined exposure to multiple chemicals.

This Guidance document describes harmonised risk assessment methodologies for combined exposure to multiple chemicals for all relevant areas within EFSA's remit, i.e. human health, animal health and ecological areas. First, a short review of the key terms, scientific basis for combined exposure risk assessment and approaches to assessing (eco)toxicology is given, including existing frameworks for these risk assessments. This background was evaluated, resulting in a harmonised framework for risk assessment of combined exposure to multiple chemicals. The framework is based on the risk assessment steps (problem formulation, exposure assessment, hazard identification and characterisation, and risk characterisation including uncertainty analysis), with tiered and stepwise approaches for both whole mixture approaches and component-based approaches. Specific considerations are given to component-based approaches including the grouping of chemicals into common assessment groups, the use of dose addition as a default assumption, approaches to integrate evidence of interactions and the refinement of assessment groups. Case studies are annexed in this guidance document to explore the feasibility and spectrum of applications of the proposed methods and approaches for human and animal health and ecological risk assessment. The Scientific Committee considers that this Guidance is fit for purpose for risk assessments of combined exposure to multiple chemicals and should be applied in all relevant areas of EFSA's work. Future work and research are recommended.

Concentrations of cadmium and lead, but not zinc, are higher in red fox tissues than in rodents-pollution gradient study in the Malopolska province (Poland).

The main purpose of our research was to assess the chronic exposure of red foxes to Cd, Pb and Zn. We have determined concentrations of these metals in the kidney, liver and muscle of 36 red foxes hunted between December 2002 and March 2003 in differently polluted areas in southern Poland. Tissue concentrations of Pb and Cd in the red foxes significantly co-varied with concentrations of these elements in the soil, and differed between the tissues. We compared concentrations of Pb, Cd, and Zn in red foxes with two rodent species, Apodemus flavicollis and Myodes glareolus, trapped simultaneously in the same area. Concentrations of Pb and Cd appeared significantly higher in the red foxes than in the rodents, but the slopes of the relationship between metal concentrations in tissues and in soil were similar in all species. This indicates that extrapolation from results of monitoring studies conducted on rodents to mammalian carnivores is possible but requires applying an extrapolation factor.

Scientific Opinion on the state of the science on pesticide risk assessment for amphibians and reptiles.

Following a request from EFSA, the Panel on Plant Protection Products and their Residues developed an opinion on the science to support the potential development of a risk assessment scheme of plant protection products for amphibians and reptiles. The coverage of the risk to amphibians and reptiles by current risk assessments for other vertebrate groups was investigated. Available test methods and exposure models were reviewed with regard to their applicability to amphibians and reptiles. Proposals were made for specific protection goals aiming to protect important ecosystem services and taking into consideration the regulatory framework and existing protection goals for other vertebrates. Uncertainties, knowledge gaps and research needs were highlighted.

Scientific opinion on pesticides in foods for infants and young children.

Following a request from the European Commission, the EFSA Panel on Plant Protection Products and their Residues (PPR Panel) prepared a scientific opinion to provide a comprehensive evaluation of pesticide residues in foods for infants and young children. In its approach to develop this scientific opinion, the EFSA PPR Panel took into account, among the others, (i) the relevant opinions of the Scientific Committee for Food setting a default maximum residue level (MRL) of 0.01 mg/kg for pesticide residues in foods for infants and young children; (ii) the recommendations provided by EFSA Scientific Committee in a guidance on risk assessment of substances present in food intended for infants below 16 weeks of age; (iii) the knowledge on organ/system development in infants and young children. For infants below 16 weeks of age, the EFSA PPR Panel concluded that pesticide residues at the default MRL of 0.01 mg/kg for food for infants and young children are not likely to result in an unacceptable exposure for active substances for which a health-based guidance value (HBGV) of 0.0026 mg/kg body weight (bw) per day or higher applies. Lower MRLs are recommended for active substances with HBGVs below this value. For infants above 16 weeks of age and young children, the established approach for setting HBGVs is considered appropriate. For infants below 16 weeks of age the approach may not be appropriate and the application of the EFSA guidance on risk assessment of substances present in food intended for infants below 16 weeks of age is recommended. The contribution of conventional food to the total exposure to pesticide residues is much higher than that from foods intended for infants and young children. Because of the increased intake of conventional food by young children, these have the highest exposure to pesticide residues, whereas infants 3-6 months of age generally have lower exposure. The impact of cumulative exposure to pesticide residues on infants and young children is not different from the general population and the EFSA cumulative risk assessment methodology is also applicable to these age groups. Residue definitions established under Regulation (EC) No 396/2005 are in general considered appropriate also for foods for infants and young children. However, based on a tier 1 analysis of the hydrolysis potential of pesticides simulating processing, the particular appropriateness of existing residue definitions for monitoring to cover processed food, both intended for infants and young children as well as conventional food, is questionable.

Scientific Opinion on the state of the art of Toxicokinetic/Toxicodynamic (TKTD) effect models for regulatory risk assessment of pesticides for aquatic organisms.

Following a request from EFSA, the Panel on Plant Protection Products and their Residues (PPR) developed an opinion on the state of the art of Toxicokinetic/Toxicodynamic (TKTD) models and their use in prospective environmental risk assessment (ERA) for pesticides and aquatic organisms. TKTD models are species- and compound-specific and can be used to predict (sub)lethal effects of pesticides under untested (time-variable) exposure conditions. Three different types of TKTD models are described, viz., (i) the 'General Unified Threshold models of Survival' (GUTS), (ii) those based on the Dynamic Energy Budget theory (DEBtox models), and (iii) models for primary producers. All these TKTD models follow the principle that the processes influencing internal exposure of an organism, (TK), are separated from the processes that lead to damage and effects/mortality (TD). GUTS models can be used to predict survival rate under untested exposure conditions. DEBtox models explore the effects on growth and reproduction of toxicants over time, even over the entire life cycle. TKTD model for primary producers and pesticides have been developed for algae, Lemna and Myriophyllum. For all TKTD model calibration, both toxicity data on standard test species and/or additional species can be used. For validation, substance and species-specific data sets from independent refined-exposure experiments are required. Based on the current state of the art (e.g. lack of documented and evaluated examples), the DEBtox modelling approach is currently limited to research applications. However, its great potential for future use in prospective ERA for pesticides is recognised. The GUTS model and the Lemna model are considered ready to be used in risk assessment.

Scientific Opinion about the Guidance of the Chemical Regulation Directorate (UK) on how aged sorption studies for pesticides should be conducted, analysed and used in regulatory assessments.

The EFSA Panel on Plant Protection Products and their Residues reviewed the guidance on how aged sorption studies for pesticides should be conducted, analysed and used in regulatory assessment. The inclusion of aged sorption is a higher tier in the groundwater leaching assessment. The Panel based its review on a test with three substances taken from a data set provided by the European Crop Protection Association. Particular points of attention were the quality of the data provided, the proposed fitting procedure of aged sorption experiments and the proposed method for combining results obtained from aged sorption studies and lower-tier studies on degradation and adsorption. Aged sorption was a relevant process in all cases studied. The test revealed that the guidance could generally be well applied and resulted in robust and plausible results. The Panel considers the guidance suitable for use in the groundwater leaching assessment after the recommendations in this Scientific Opinion have been implemented, with the exception of the use of field data to derive aged sorption parameters. The Panel noted that the draft guidance could only be used by experienced users because there is no software tool that fully supports the work flow in the guidance document. It is therefore recommended that a user-friendly software tool be developed. Aged sorption lowered the predicted concentration in groundwater. However, because aged sorption experiments may be conducted in different soils than lower-tier degradation and adsorption experiments, it cannot be guaranteed that the higher tier predicts lower concentrations than the lower tier, while lower tiers should be more conservative than higher tiers. To mitigate this problem, the Panel recommends using all available higher- and lower-tier data in the leaching assessment. The Panel further recommends that aged sorption parameters for metabolites be derived only from metabolite-dosed studies. The formation fraction can be derived from parent-dosed degradation studies, provided that the parent and metabolite are fitted with the best-fit model, which is the double first-order in parallel model in the case of aged sorption.

Scientific Opinion of the PPR Panel on the follow-up of the findings of the External Scientific Report 'Literature review of epidemiological studies linking exposure to pesticides and health effects'.

In 2013, EFSA published a comprehensive systematic review of epidemiological studies published from 2006 to 2012 investigating the association between pesticide exposure and many health outcomes. Despite the considerable amount of epidemiological information available, the quality of much of this evidence was rather low and many limitations likely affect the results so firm conclusions cannot be drawn. Studies that do not meet the 'recognised standards' mentioned in the Regulation (EU) No 1107/2009 are thus not suited for risk assessment. In this Scientific Opinion, the EFSA Panel on Plant Protection Products and their residues (PPR Panel) was requested to assess the methodological limitations of pesticide epidemiology studies and found that poor exposure characterisation primarily defined the major limitation. Frequent use of case-control studies as opposed to prospective studies was considered another limitation. Inadequate definition or deficiencies in health outcomes need to be avoided and reporting of findings could be improved in some cases. The PPR Panel proposed recommendations on how to improve the quality and reliability of pesticide epidemiology studies to overcome these limitations and to facilitate an appropriate use for risk assessment. The Panel recommended the conduct of systematic reviews and meta-analysis, where appropriate, of pesticide observational studies as useful methodology to understand the potential hazards of pesticides, exposure scenarios and methods for assessing exposure, exposure-response characterisation and risk characterisation. Finally, the PPR Panel proposed a methodological approach to integrate and weight multiple lines of evidence, including epidemiological data, for pesticide risk assessment. Biological plausibility can contribute to establishing causation.

Scientific Opinion addressing the state of the science on risk assessment of plant protection products for in-soil organisms.

Following a request from EFSA, the Panel on Plant Protection Products and their Residues developed an opinion on the science behind the risk assessment of plant protection products for in-soil organisms. The current risk assessment scheme is reviewed, taking into account new regulatory frameworks and scientific developments. Proposals are made for specific protection goals for in-soil organisms being key drivers for relevant ecosystem services in agricultural landscapes such as nutrient cycling, soil structure, pest control and biodiversity. Considering the time-scales and biological processes related to the dispersal of the majority of in-soil organisms compared to terrestrial non-target arthropods living above soil, the Panel proposes that in-soil environmental risk assessments are made at in- and off-field scale considering field boundary levels. A new testing strategy which takes into account the relevant exposure routes for in-soil organisms and the potential direct and indirect effects is proposed. In order to address species recovery and long-term impacts of PPPs, the use of population models is also proposed.