Contrasting effects of fungicide and herbicide active ingredients and their formulations on bumblebee learning and behaviour.

Fungicides and herbicides are two of the most heavily applied pesticide classes in the world, but receive little research attention with regards to their potential impacts on bees. As they are not designed to target insects, the mechanisms behind potential impacts of these pesticides are unclear. It is therefore important to understand their influence at a range of levels, including sublethal impacts on behaviours such as learning. We used the proboscis extension reflex (PER) paradigm to assess how the herbicide glyphosate and the fungicide prothioconazole affect bumblebee olfactory learning. We also assessed responsiveness, and compared the impacts of these active ingredients and their respective commercial formulations (Roundup Biactive and Proline). We found that learning was not impaired by either formulation but, of the bees that displayed evidence of learning, exposure to prothioconazole active ingredient increased learning level in some situations, while exposure to glyphosate active ingredient resulted in bumblebees being less likely to respond to antennal stimulation with sucrose. Our data suggest that fungicides and herbicides may not negatively impact olfactory learning ability when bumblebees are exposed orally to field-realistic doses in a lab setting, but that glyphosate has the potential to cause changes in responsiveness in bees. As we found impacts of active ingredients and not commercial formulations, this suggests that co-formulants may modify impacts of active ingredients in the products tested on olfactory learning without being toxic themselves. More research is needed to understand the mechanisms behind potential impacts of fungicides and herbicides on bees, and to evaluate the implications of behavioural changes caused by glyphosate and prothioconazole for bumblebee fitness.

'Inert' ingredients are understudied, potentially dangerous to bees and deserve more research attention.

Agrochemical formulations are composed of two broad groups of chemicals: active ingredients, which confer pest control action, and 'inert' ingredients, which facilitate the action of the active ingredient. Most research into the effects of agrochemicals focusses on the effects of active ingredients. This reflects the assumption that 'inert' ingredients are non-toxic. A review of relevant research shows that for bees, this assumption is without empirical foundation. After conducting a systematic literature search, we found just 19 studies that tested the effects of 'inert' ingredients on bee health. In these studies, 'inert' ingredients were found to cause mortality in bees through multiple exposure routes, act synergistically with other stressors and cause colony level effects. This lack of research is compounded by a lack of diversity in study organism used. We argue that 'inert' ingredients have distinct, and poorly understood, ecological persistency profiles and toxicities, making research into their individual effects necessary. We highlight the lack of mitigation in place to protect bees from 'inert' ingredients and argue that research efforts should be redistributed to address the knowledge gap identified here. If so-called 'inert' ingredients are, in fact, detrimental to bee health, their potential role in widespread bee declines needs urgent assessment.

Environmental exposure assessment of co-formulants in plant protection products under REACH.

It is a regulatory requirement to assess co-formulants in plant protection products (PPP) under the European Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) legislation. The standard environmental exposure assessment framework for chemicals under REACH is a multicompartmental mass-balanced model and, at the local scale, is designed for use with urban (wide dispersive) or industrial (point source) emissions. However, the environmental release of co-formulants used in PPP is to agricultural soil and indirectly to waterbodies adjacent to a field and, for sprayed products, to the air. The Local Environment Tool (LET) has been developed to assess these specific emission pathways for co-formulants in a local-scale REACH exposure assessment, based on standard approaches and models used for PPP. As such, it closes a gap between the standard REACH exposure model's scope and REACH requirements to assess co-formulants in PPP. When combined with the output of the standard REACH exposure model, the LET includes an estimate of the contribution from other nonagricultural background sources of the same substance. The LET is an improvement over the use of higher tier PPP models for screening purposes because it provides a simple standardized exposure scenario. A set of predefined and conservatively selected inputs allows a REACH registrant to conduct an assessment without requiring detailed knowledge of PPP risk assessment methods or typical conditions of use. The benefit to the co-formulant downstream user (formulators) is a standardized and consistent approach to co-formulant assessment, with meaningful and readily interpretable conditions of use. The LET can serve as an example to other sectors of how to address possible gaps in the environmental exposure assessment by combining a customized local-scale exposure model with the standard REACH models. A detailed conceptual explanation of the LET model is provided here together with a discussion on its use in a regulatory context. Integr Environ Assess Manag 2023;19:1544-1554. © 2023 BASF SE, Bayer AG et al. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Oxidative Stress and Cytotoxicity Induced by Co-Formulants of Glyphosate-Based Herbicides in Human Mononuclear White Blood Cells.

The use of genetically modified, glyphosate-resistant crops has led to the widespread application of glyphosate-based herbicides (GBHs), making them one of the most widely used herbicide formulations on the market. To enhance the efficacy of the active ingredient, GBHs used in practice often contain other ingredients marked as inert "adjuvants" or "co-formulants", the toxic properties of which are poorly understood. The objective of this study was to compare the cytotoxic effects of pure glyphosate, three GBHs (Roundup Mega, Fozat 480 and Glyfos) and two co-formulants commonly used in GBHs as assessed via CCK-8 assay, and the extent of their potential oxidative damage as assessed via superoxide dismutase (SOD) assay, in order to reveal the role of adjuvants in the toxicity of the formulations. Our results showed that glyphosate alone did not significantly affect cell viability. In contrast, GBHs and adjuvants induced a pronounced cytotoxic effect from a concentration of 100 µM. SOD activity of cells treated with GBHs or adjuvants was significantly lower compared to cells treated with glyphosate alone. This suggests that the adjuvants in GBHs are responsible for the cytotoxic effects of the formulations through the induction of oxidative stress.

Cytotoxic effects of Roundup Classic and its components on NE-4C and MC3T3-E1 cell lines determined by biochemical and flow cytometric assays.

Cytotoxic effects of the market leading broad-spectrum, synthetic herbicide product Roundup Classic, its active ingredient glyphosate (in a form of its isopropylamine (IPA) salt) and its formulating surfactant polyethoxylated tallowamine (POE-15) were determined on two murine cell lines, a neuroectodermal stem cell-like (NE-4C) and a high alkaline phosphatase activity osteoblastic cell line (MC3T3-E1). Cytotoxicity, genotoxicity, effects on cell viability and cell cycles were examined in five flow cytometry tests, the two former of which were compared by the enzymatic-assay and the alkaline single cell gel electrophoresis (Comet) assay. All of the tests indicated the NE-4C cells being more sensitive, than the MC3T3-E1 cell line to the treatments with the target compounds. Higher sensitivity differences were detected in the viability test by flow cytometry (7-9-fold), than by the MTT assay (1.5-3-fold); in the genotoxicity test by the Comet assay (3.5-403-fold), than by the DNA-damage test (9.3-158-fold); and in the apoptosis test by the Annexin V dead cell kit (1.1-12.7-fold), than by the Caspase 3/7 kit (1-6.5-fold). Cell cycle assays indicated high count of cells (~70%) in the G0/G1 phase for MC3T3-E1 cells, than in NE-4C cell (~40%) after 24 h. The order of the inhibitory potency of the target substances has unequivocally been POE-15 > Roundup Classic > > glyphosate IPA salt.

Magnitude and decline of pesticide co-formulant residues in vegetables and fruits: results from field trials compared to estimated values.

BACKGROUND: The application of plant protection products (PPPs) leads to the formation of residues in treated crops. Even though PPPs contain considerable amounts of co-formulants, regulation and monitoring of residues normally focus on the active substances (a.s.) only. For our study we selected four commonly used co-formulants (three anionic surfactants and one organic solvent) and investigated the formation and decline of residues in vegetables and apples under field conditions. The aims were to characterize the behavior of co-formulant residues on crops and to provide a basis for future investigations on consumer exposure. RESULTS: The development of robust and sensitive analytical methods allowed the quantification of residues in the low µg/kg-level. After treatment with PPPs, co-formulants were detected up to approximately 10 mg kg-1 in vegetables. In general, these residues declined fast with half-lives of a few days. Wash-off and volatilization were identified as important removal processes for anionic surfactants and the organic solvent, respectively. However, in specific crops (parsley and celery), organic solvent residues were still considerable (≈2 mg kg-1 ) 2 weeks after treatment. We further demonstrate that it is feasible to estimate co-formulant residues using publicly available data on pesticide a.s. CONCLUSION: To date no information on co-formulant residues in food is available. The findings from our field trials, as well as the presented approach for the prediction of residues, provide key elements for future consideration of consumer exposure to PPP co-formulants. © 2020 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Assessment of occupational exposure to pesticide mixtures with endocrine-disrupting activity.

Occupational exposure to pesticide mixtures comprising active substance(s) and/or co-formulant(s) with known/possible endocrine-disrupting activity was assessed using long-term activity records for 50 professional operators representing arable and orchard cropping systems in Greece, Lithuania, and the UK. Exposure was estimated using the harmonised Agricultural Operator Exposure Model, and risk was quantified as a point of departure index (PODI) using the lowest no observed (adverse) effect level. Use of substances with known/possible endocrine activity was common, with 43 of the 50 operators applying at least one such active substance on more than 50% of spray days; at maximum, one UK operator sprayed five such active substances and 10 such co-formulants in a single day. At 95th percentile, total exposure was largest in the UK orchard system (0.041 × 10-2 mg kg bw-1 day-1) whereas risk was largest in the Greek cropping systems (PODI 0.053 × 10-1). All five cropping systems had instances indicating potential for risk when expressed at a daily resolution (maximum PODI 1.2-10.7). Toxicological data are sparse for co-formulants, so combined risk from complex mixtures of active substances and co-formulants may be larger in reality.

Improving Efficacy of Beauveria bassiana against Stored Grain Beetles with a Synergistic Co-Formulant.

The potential of a dry powder co-formulant, kaolin, to improve the control of storage beetles by the entomopathogenic fungus Beauveria bassiana, isolate IMI389521, was investigated. The response of Oryzaephilus surinamensis adults to the fungus when applied to wheat at 1 × 10(10) conidia per kg with and without kaolin at 1.74 g per kg wheat was assessed. Addition of kaolin increased control from 46% to 88% at day 7 and from 81% to 99% at day 14 post-treatment. Following this the dose response of O. surinamensis and Tribolium confusum to both kaolin and the fungus was investigated. Synergistic effects were evident against O. surinamensis at ≥0.96 g of kaolin per kg of wheat when combined with the fungus at all concentrations tested. For T. confusum, adult mortality did not exceed 55%, however, the larvae were extremely susceptible with almost complete suppression of adult emergence at the lowest fungal rate tested even without the addition of kaolin. Finally, the dose response of Sitophilus granarius to the fungus at 15 and 25 °C, with and without kaolin at 1 g per kg of wheat, was examined. Improvements in efficacy were achieved by including kaolin at every fungal rate tested and by increasing the temperature. Kaolin by itself was not effective, only when combined with the fungus was an effect observed, indicating that kaolin was having a synergistic effect on the fungus.

Co-Formulants in Glyphosate-Based Herbicides Disrupt Aromatase Activity in Human Cells below Toxic Levels.

Pesticide formulations contain declared active ingredients and co-formulants presented as inert and confidential compounds. We tested the endocrine disruption of co-formulants in six glyphosate-based herbicides (GBH), the most used pesticides worldwide. All co-formulants and formulations were comparably cytotoxic well below the agricultural dilution of 1% (18-2000 times for co-formulants, 8-141 times for formulations), and not the declared active ingredient glyphosate (G) alone. The endocrine-disrupting effects of all these compounds were measured on aromatase activity, a key enzyme in the balance of sex hormones, below the toxicity threshold. Aromatase activity was decreased both by the co-formulants alone (polyethoxylated tallow amine-POEA and alkyl polyglucoside-APG) and by the formulations, from concentrations 800 times lower than the agricultural dilutions; while G exerted an effect only at 1/3 of the agricultural dilution. It was demonstrated for the first time that endocrine disruption by GBH could not only be due to the declared active ingredient but also to co-formulants. These results could explain numerous in vivo results with GBHs not seen with G alone; moreover, they challenge the relevance of the acceptable daily intake (ADI) value for GBHs exposures, currently calculated from toxicity tests of the declared active ingredient alone.