Mining the effect of the neonicotinoids imidacloprid and clothianidin on the chemical homeostasis and energy equilibrium of primary mouse neural stem/progenitor cells using metabolomics.

The projection of plant protection products' (PPPs) toxicity to non-target organisms at early stages of their development is challenging and demanding. Recent developments in bioanalytics, however, have facilitated the study of fluctuations in the metabolism of biological systems in response to treatments with bioactives and the discovery of corresponding toxicity biomarkers. Neonicotinoids are improved insecticides that target nicotinic acetylocholine receptors (nAChR) in insects which are similar to mammals. Nonetheless, they have sparked controversy due to effects on non-target organisms. Within this context, mammalian cell cultures represent ideal systems for the development of robust models for the dissection of PPPs' toxicity. Thus, we have investigated the toxicity of imidacloprid, clothianidin, and their mixture on primary mouse (Mus musculus) neural stem/progenitor (NSPCs) and mouse neuroblastoma-derived Neuro-2a (N2a) cells, and the undergoing metabolic changes applying metabolomics. Results revealed that NSPCs, which in vitro resemble those that reside in the postnatal and adult central nervous system, are five to seven-fold more sensitive than N2a to the applied insecticides. The energy equilibrium of NSPCs was substantially altered, as it is indicated by fluctuations of metabolites involved in energy production (e.g. glucose, lactate), Krebs cycle intermediates, and fatty acids, which are important components of cell membranes. Such evidence plausibly suggests a switch of cells' energy-producing mechanism to the direct metabolism of glucose to lactate in response to insecticides. The developed pipeline could be further exploited in the discovery of unintended effects of PPPs at early steps of development and for regulatory purposes.

Overview on legislation and scientific approaches for risk assessment of combined exposure to multiple chemicals: the potential EuroMix contribution.

This article reviews the current legislative requirements for risk assessment of combined exposure to multiple chemicals via multiple exposure routes, focusing on human health and particularly on food-related chemicals. The aim is to identify regulatory needs and current approaches for this type of risk assessment as well as challenges of the implementation of appropriate and harmonized guidance at international level. It provides an overview of the current legal requirements in the European Union (EU), the United States and Canada. Substantial differences were identified in the legal requirements for risk assessment of combined exposure to multiple chemicals and its implementation between EU and non-EU countries and across several regulatory sectors. Frameworks currently proposed and in use for assessing risks from combined exposure to multiple chemicals via multiple routes and different durations of exposure are summarized. In order to avoid significant discrepancies between regulatory sectors or countries, the approach for assessing risks of combined exposure should be based on similar principles for all types of chemicals. OECD and EFSA identified the development of harmonized methodologies for combined exposure to multiple chemicals as a key priority area. The Horizon 2020 project "EuroMix" aims to contribute to the further development of internationally harmonized approaches for such risk assessments by the development of an integrated test strategy using in vitro and in silico tests verified for chemical mixtures based on more appropriate data on potential combined effects. These approaches and testing strategies should be integrated in a scientifically based weight of evidence approach to account for complexity and uncertainty, to improve risk assessment.

Dermal exposure of pesticide applicators as a measure of coverall performance under field conditions.

In this study, the field performance of two coverall designs used by pesticide applicators was determined. Two coverall types were selected based on data from previously conducted comfort testing under field conditions in southern Europe. Dermal exposure was measured during 22 applications conducted with 11 operators using similar hand-held spray guns in greenhouse pepper crops in the Ierapetra region of Crete, Greece. One of the coverall designs studied was made from a cotton/polyester material treated with a water-repellent Resist Spills(R) finish, which was compared in the field study to a coverall of similar design, but using a woven, untreated cotton material. An in-house analytical method was developed and validated for determining residues of the active substance (a.s.) malathion on the dosimeters. The derived levels of dermal exposure were used as a measure of the protection provided by the two types of coveralls. In addition, by comparing the total amount of the a.s. recovered from outer and inner dosimeters (potential dermal exposure = 238.8 mg kg(-1) a.s. for the cotton coverall and 160.44 mg kg(-1) a.s. for the Resist Spills coverall), a value could be determined for the degree of coverall penetration. The mean penetration (milligrams per kilogram a.s.) of the outer coveralls, calculated as a percentage of the total contamination, was 0.4% for the water-repellent coverall and 2.3% for the cotton coverall. The mean recovery from the laboratory and field-fortified samples was >91 and 74%, respectively and used as the main criterion for quality control of the analytical data. Under the field trial conditions evaluated, both the coverall designs gave better protection than the default values used in the most relevant predictive exposure model. Therefore, they could be considered as appropriate tools of personal protection when both comfort and field performance is taken into account under the specific application scenario.

Comparative toxicities of fungicide and herbicide formulations on freshwater and marine species.

The estimation of the toxic effects of plant protection products on non-target aquatic organisms is essential for risk assessment evaluation. In this study the acute toxicity of two fungicide and two herbicide formulations was determined in four marine species in comparison with the toxicity assessed for the freshwater crustacean Daphnia magna. From the study it is indicated that the marine crustacean species are effectively protected when acute toxicity data on Daphnia magna are used as surrogate for risk assessment while the comparative sensitivity of the unicellular green algae may vary considerably, depending on the mode of action of the specific formulation.

Perceptions of pesticides exposure risks by operators, workers, residents and bystanders in Greece, Italy and the UK.

The EU Directive on the sustainable use of pesticides (EU128/2009/EC) requires European Member States to develop training activities targeting occupational exposure to pesticides, and communication material aimed at residents and bystanders. Risk perceptions, knowledge and attitudes associated with passive and occupational exposure to pesticide potentially influence the extent to which different stakeholders adopt self-protective behaviour. A methodology for assessing the link between attitudes, adoption of self-protective behaviours and exposure was developed and tested. A survey was implemented in the Greece, Italy and the UK, and targeted stakeholders associated with pesticide exposure linked to orchards, greenhouse crops and arable crops respectively. The results indicated that the adoption of protective measures is low for residents and bystanders, with the exception of residents in Greece, when compared to operators and workers, who tend to follow recommended safety practices. A regression analysis was used to examine the factors affecting the probability of adopting protective measures as well the as the level of exposure in the case of operators and workers where data are available. The results indicate that the likelihood of engaging in self-protective behaviour is not significantly affected by perceptions of own health being affected by pesticides for residents and bystanders. However, operators who perceive that their heath has been negatively affected by the use of pesticides are found to be more likely to adopt self-protective behaviours. Gender and country differences, in perceptions, attitudes and self-protection are also observed. Recommendations for improved communication, in particular for vulnerable groups, are provided.

Determination of operator exposure levels to insecticide during bait applications in olive trees: study of coverall performance and duration of application.

In this study the operator exposure levels during bait applications of an insecticide in olive groves were determined using a whole body dosimetry method for dermal exposure. The study design allowed the roles of application task duration and coverall type to be evaluated as factors influencing operator exposure. Twenty applications were carried out with knapsack sprayers in the Tanagra region of Viotia, Greece, ten of which were for a 1h and ten for a 3h duration. An in-house GC-NPD analytical method was developed and validated for the determination of malathion, the active substance (a.s.) of the insecticide formulation used in field trials. The mean recovery of field-fortified samples was 84% (%RSD=3.0). Field trial results generally indicated lower operator exposure levels than indicated by the most relevant operator exposure predictive model. Residues of malathion on internal dosimeters were compared to those measured on the respective outer coveralls (potential dermal exposure) to evaluate the protective factor of each one of the two coverall types used. Both coverall types provided satisfactory levels of protection and can be considered as suitable protection for the conditions of the application scenario studied. Furthermore, the results indicated that there is not a strong correlation between exposure levels and duration of application.

Comparative toxicity of the phytotoxins (8R,16R)-(-)-pyrenophorin and (5S,8R,13S,16R)-(-)-pyrenophorol on aquatic organisms.

The acute toxicities of the fungal phytotoxins (8R,16R)-(-)-pyrenophorin and (5S,8R,13S,16R)-(-)-pyrenophorol on Vibrio fischeri, Oscillatoria perornata, Pseudokirchneriella subcapitata, Lemna minor and Artemia fransiscana were evaluated. (8R,16R)-(-)-pyrenophorin was more toxic than (5S,8R,13S,16R)-(-)-pyrenophorol to V. fischeri, O. perornata, L. minor and A. fransiscana. The highest acute toxicity of (8R,16R)-(-)-pyrenophorin was exhibited on V. fischeri (5 min median effective concentration of 3.57 M 10(-5)) whereas the corresponding value for (5S,8R,13S,16R)-(-)-pyrenophorol was 801 M 10(-5). P. subcapitata exhibited a lack of sensitivity (median inhibitory concentration of >10 M 10(-5)) to both phytotoxins.

Determination of penconazole on personal protection equipment after field applications.

A simple analytical method, validated in-house and intra-laboratory, has been developed for the determination of penconazole on personal protection equipment (PPE) used by operators during field applications of Topas 20EW. The analytical determination of penconazole was performed by GC-ECD. Sample preparation was based on a liquid extraction procedure. The possible effect of different personal protection equipment matrices on the analytical determination of penconazole was studied and evaluated with regression analysis. No evidence of significant (at 95% CI level) effect was observed at several fortification levels. The percentage recovery of penconazole from the different PPE was in the range 59-100% with adequate correlation with the level of fortification (r2>0.99). The RSD% was in the range of 1-15% and the LOD and LOQ for penconazole were 0.84 and 2.5 ng mL(-1), respectively.

Determination of potential dermal and inhalation operator exposure to malathion in greenhouses with the whole body dosimetry method.

One of the steps during the authorization process of plant protection products (PPP) in the European Union is to evaluate the safety of the operator. For this purpose, information on the probable levels of operator exposure during the proposed uses of the PPP is required. These levels can be estimated by using existing mathematical models or from field study data. However, the existing models have several shortcomings, including the lack of data for operator exposure levels during spray applications by hand lance, especially in greenhouses. The present study monitored the potential dermal and inhalation operator exposure from hand-held lance applications of malathion on greenhouse tomatoes at low and high spraying pressures. The methodology for monitoring potential exposure was based on the whole body dosimetry method. Inhalation exposure was monitored using personal air pumps and XAD-2 sampling tubes. For the monitoring of hand exposure, cotton gloves were used in two trials and rubber gloves in another three. The total volumes of spray solution contaminating the body of the operator were 25.37 and 35.83 ml/h, corresponding to 0.05 and 0.07% of the applied spray solution, respectively, in the case of low pressure knapsack applications and from 160.76 to 283.45 ml/h, corresponding to 0.09-0.19% of the spray solution applied, in the case of hand lance applications with tractor-generated high pressure. Counts on gloves depended on the absorbance/repellency of the glove material. The potential inhalation exposures were estimated at 0.07 and 0.09 ml/h in the case of low pressure knapsack applications, based on a ventilation rate of 25 l/min. Both potential dermal operator exposure (excluding hands) and potential inhalation exposure were increased by a factor of approximately 7 when the application pressure was increased from 3 to 18 bar in greenhouse trials with a tractor-assisted hand lance, the rest of the application conditions being very similar.