Adjuvants in fungicide formulations can be skin sensitizers and cause different types of cell stress responses.

New approaches based on -omics technologies can identify biomarkers and processes regulated in response to xenobiotics, and thus support toxicological risk assessments. This is vital to meet the challenges associated with "cocktail effects", i.e. combination effects of chemicals present simultaneously in a product, our environment, and/or our body. For plant protection products (PPPs), investigations largely focus on active ingredients such as herbicides and fungicides. In this study, we have analyzed agricultural chemicals, two surfactants (poly(oxy-1,2-ethanediyl), alpha-sulfo-omega-[2,4,6-tris(1-phenylethyl)phenoxy]-, ammonium salt, POL; N,N-dimethylcapramide, NND), and one preservative, 1,2-benzisothiazol-3(2 H)-one (BEN) used as adjuvants in PPPs, and further three fungicide PPPs, Proline EC 250, Shirlan, Folicur Xpert, containing the adjuvants, and other major individual constituents (fluazinam (FLU), prothioconazole (PRO), tebuconazole (TEB)) as well as defined mixtures ("mixes") thereof using several in vitro approaches. All investigated single agricultural chemicals were predicted as skin sensitizers using an in vitro transcriptomic assay based on a dendritic cell model. For selected chemicals and mixes, also skin sensitization potency was predicted. The preservative BEN induced significant changes in cytokine secretion and dendritic cell activation marker CD86 expression. The surfactant NND changed cytokine secretion only and the POL only affected CD86 expression. Proteomic analyses revealed unique response profiles for all adjuvants, an oxidative stress pattern response in BEN-treated cells, and differentially abundant proteins associated with cholesterol homeostasis in response to POL. In summary, we find responses to agricultural chemicals and products consistent with the dendritic cell model reacting to chemical exposure with oxidative stress, ER stress, effects on autophagy, and metabolic changes especially related to cholesterol homeostasis. After exposure to certain mixes, novel proteins or transcripts were differentially expressed and these were not detected for any single constituents, supporting the occurrence of cocktail effects. This indicates that all chemicals in a PPP can contribute to the toxicity profile of a PPP, including their skin sensitizing/immunotoxic properties.

Oral anti-diabetic drugs as endocrine disruptors in vitro - No evidence for additive effects in binary mixtures.

Diabetes is one of the World's most concerning health problems and millions of patients are using anti-diabetic drugs (ADDs) in order to control blood glucose. The in vitro H295R steroidogenesis assay was implemented to investigate endocrine effects of three ADDs, metformin (MET), glimepiride (GLIM), sitagliptin (SIT) and the cholesterol-lowering drug simvastatin (SIM) individually and in three binary mixtures. Steroid hormones were analyzed using LC-MS/MS. Mixture effects were assessed by applying the Concentration Addition (CA) model. All tested drugs and binary mixtures interrupted the H295R steroidogenesis with different potency. The effects of MET:GLIM on the steroidogenesis were overall similar to the steroidogenic profile of GLIM, however effects were less pronounced. The binary mixture of MET:SIT showed overall minor effects on steroid production and only at very high concentrations. The SIM:SIT mixture showed inhibition downstream from cholesterol, which was attributed to the effects of SIM. The CA model partly predicted the effect of MET:SIT on some steroids but significantly overestimated the effects of MET:GLIM and SIM:SIT. Thus, the applicability of the CA model was limited and cocktail effects appeared to be intermediate responses of individual drugs, rather than additive. The complexity of dynamic pathways such as steroidogenesis appears to significantly reduce the use of the CA model. In conclusion, more dynamic models are needed to predict mixture effects in complex systems.

Mixtures of an insecticide, a fungicide and a herbicide induce high toxicities and systemic physiological disturbances in winter Apis mellifera honey bees.

Multiple pesticides originating from plant protection treatments and the treatment of pests infecting honey bees are frequently detected in beehive matrices. Therefore, winter honey bees, which have a long life span, could be exposed to these pesticides for longer periods than summer honey bees. In this study, winter honey bees were exposed through food to the insecticide imidacloprid, the fungicide difenoconazole and the herbicide glyphosate, alone or in binary and ternary mixtures, at environmental concentrations (0 (controls), 0.1, 1 and 10 µg/L) for 20 days. The survival of the honey bees was significantly reduced after exposure to these 3 pesticides individually and in combination. Overall, the combinations had a higher impact than the pesticides alone with a maximum mortality of 52.9% after 20 days of exposure to the insecticide-fungicide binary mixture at 1 µg/L. The analyses of the surviving bees showed that these different pesticide combinations had a systemic global impact on the physiological state of the honey bees, as revealed by the modulation of head, midgut and abdomen glutathione-S-transferase, head acetylcholinesterase, abdomen glucose-6-phosphate dehydrogenase and midgut alkaline phosphatase, which are involved in the detoxification of xenobiotics, the nervous system, defenses against oxidative stress, metabolism and immunity, respectively. These results demonstrate the importance of studying the effects of chemical cocktails based on low realistic exposure levels and developing long-term tests to reveal possible lethal and adverse sublethal interactions in honey bees and other insect pollinators.

Effects of monoamine manipulations on the personality and gene expression of three-spined sticklebacks.

Among-individual behavioral differences (i.e. animal personality) are commonly observed across taxa, although the underlying, causal mechanisms of such differences are poorly understood. Animal personality has been correlated with physiological functions as well as fitness-related traits. Variation in many aspects of monoamine systems, such as metabolite levels and gene polymorphisms, has been linked to behavioral variation. Therefore, here we experimentally investigated the potential role of monoamines in explaining individual variation in personality, using two common pharmaceuticals that respectively alter the levels of serotonin and dopamine in the brain: fluoxetine and ropinirole. We exposed three-spined sticklebacks, a species that shows animal personality, to either chemical alone or to a combination of the two chemicals, for 18 days. During the experiment, fish were assayed at four time points for the following personality traits: exploration, boldness, aggression and sociability. To quantify brain gene expression on short- and longer-term scales, fish were sampled at two time points. Our results show that monoamine manipulations influence fish behavior. Specifically, fish exposed to either fluoxetine or ropinirole were significantly bolder, and fish exposed to the two chemicals together tended to be bolder than control fish. Our monoamine manipulations did not alter the gene expression of monoamine or stress-associated neurotransmitter genes, but control, untreated fish showed covariation between gene expression and behavior. Specifically, exploration and boldness were predicted by genes in the dopaminergic, serotonergic and stress pathways, and sociability was predicted by genes in the dopaminergic and stress pathways. These results add further support to the links between monoaminergic systems and personality, and show that exposure to monoamines can causally alter animal personality.

Can the pharmaceutically active compounds released in agroecosystems be considered as emerging plant stressors?

Pharmaceutically active compounds (PhACs) entering agroecosystems as a result of various human activities may be taken up by and accumulated within crop plants, with potential human health implications. Despite their extensive metabolism by a sophisticated enzyme-based detoxification system in plant cells, PhACs and their transformation products (TPs) may result in adverse effects on plants' physiology. PhACs-mediated phytotoxic effects, as well as plants' defense responses have been depicted on plants exposed to individual or low number of PhACs under controlled conditions. We highlight the need to consider the cocktails effects and synergistic interactions of PhACs present in mixtures in actual agroecosystems, towards phytotoxicity and agricultural sustainability in general. Considering PhACs as emerging plant stressors will better facilitate the understanding of their phytotoxic effects.