Ilaria Caliani

The global pollinator decline is linked to intensive farming and the high use of plant protection products (PPPs), necessitating risk assessment and mitigation. This study investigates the potential negative impacts of agricultural practices on pollinator health, specifically focusing on the effects of PPPs used in viticulture on the honey bee, Apis mellifera, despite grapevines’ lack of reliance on bee pollination. The beehives sampled were from two farms with vineyards under different management regimes: one transitioning from conventional to organic practices and an organic–biodynamic site with pollinator mitigation measures. Sampling was conducted during three phases, pre-, during, and post-PPP application, to evaluate biomarkers of neurotoxicity (AChE), detoxification enzymes (CaE, GST), metabolic stress (ALP), and immune markers (Lys, PO, proPO). Comparison between the organic–biodynamic farm and the transitioning one revealed a pattern suggesting significant neurotoxic effects in the transitioning farm characterised by a trend of decreased AChE activity during treatments and the subsequent induction of GST post exposure. Crucially, both PO and proPO were induced post treatment, but with a lower PO/proPO ratio compared to previous seasons, suggesting inefficient proPO activation and potentially weakened immune competence that could favour pathogen proliferation. Bee health appeared to deteriorate most at the transitioning farm post treatment, while the biodynamic site remained relatively stable; these differences are likely associated with legacy residues and drift, exacerbated by overwintering stress and summer heat. Given the specific environmental and management characteristics of these two farms, the results provide an indicative comparison of how different agronomic approaches may influence bee health. Moreover, these results support the multi-biomarker approach for detecting potential PPP impacts, suggesting that organic transitions and mitigation strategies could play a role in pollinator conservation.

This study investigated the sub-lethal effects of four commercial fungicides—two foliar (Amistar^®Xtra and Mirador^®) and two ear fungicides (Prosaro^® and Icarus^®)—applied alone and in combination to wheat crops on caged earthworms (Eisenia fetida). We measured biomarkers that included detoxification responses (glutathione S-transferase, GST), oxidative stress levels (lipid peroxidation, LPO, and catalase, CAT), DNA damage (comet assay), energy reserves (lactate dehydrogenase, LDH), and immune response (lysozyme activity, LYS). The absence of significant differences in catalase and lipid peroxidation levels suggested no oxidative stress due to fungicide exposure. However, the foliar fungicide Amistar^®Xtra induced the highest GST activity and DNA fragmentation, suggesting synergistic effects between its active ingredients and undisclosed co-formulants. Similar effects observed with the Amistar^®Xtra-Prosaro^® mixture confirmed the greater toxicity of Amistar^®Xtra. This study provides novel insights into the sub-lethal effects of single and combined commercial fungicides on a standard toxicity test organism, shedding light on the ecological implications of fungicide use in agroecosystems and reinforcing the need for pesticide reduction.

Agroecosystems provide habitats, food, and water for many pollinators and insects, but they are also heavily exposed to threats from the widespread use of pesticides and fertilizers. Managed honeybees and wild bees encounter pesticides in vineyards by collecting morning dew from vine leaves and accessing sugars from grapes, particularly during dry periods. This study assessed the toxicological effects of the commercial fungicide formulations Fantic FNCWG^® and Ramedit combi^®, both individually and in combination, on honeybees. Using a multi-biomarker approach, we evaluated neurotoxicity, metabolic disturbances, phase II detoxification processes, and immune system function. Our findings revealed that commercial fungicide mixtures with multiple active ingredients affect bees differently than single active compounds. Biomarker responses highlighted how these complex mixtures disrupt various enzymatic pathways; including immune function; altering critical enzyme kinetics involved in detoxification and potentially impairing essential bee functions. This study emphasizes the need for more comprehensive research into the sublethal effects of commercial pesticides, particularly those used in vineyards, which are understudied compared to pesticides used in orchards.

Brodifacoum is the most common rodenticide used for the eradication of invasive rodents from islands. It blocks the vitamin K cycle, resulting in hemorrhages in target mammals. Non-target species may be incidentally exposed to brodifacoum, including marine species. A case study conducted on the Italian Marine Protected Area of Tavolara Island was reported after a rodent eradication using the aerial broadcast of a brodifacoum pellet. Brodifacoum presence and effects on non-target marine organisms were investigated. Different fish species were sampled, and a set of analyses was conducted to determine vitamin K and vitamin K epoxide reductase concentrations, prothrombin time, and erythrocytic nuclear abnormalities (ENA) assay. In all the examined organisms, brodifacoum was not detected. The results obtained showed differences in vitamin K and vitamin K epoxide concentrations among the samples studied, with a positive correlation for three species between vitamin K, vitamin K epoxide, and fish weight. The prothrombin time assay showed a good blood clotting capacity in the fish. Higher abnormality values were recorded for four species. The results of this study suggest that it is possible to hypothesize that the sampled fish were not likely to have been exposed to brodifacoum and that consequently there are no negative issues concerning human consumption.

The principal aim of the present study was to develop and apply novel ex vivo tests as an alternative to cell cultures able to evaluate the possible effects of emerging and legacy contaminants in Caretta caretta. To this end, we performed ex vivo experiments on non-invasively collected whole-blood and skin-biopsy slices treated with chrysene, MEHP, or PBDE-47. Blood samples were tested by oxidative stress (TAS), immune system (respiratory burst, lysozyme, and complement system), and genotoxicity (ENA assay) biomarkers, and genotoxic and immune system effects were observed. Skin slices were analyzed by applying a 2D-PAGE/MS proteomic approach, and specific contaminant signatures were delineated on the skin proteomic profile. These reflect biochemical effects induced by each treatment and allowed to identify glutathione S-transferase P, peptidyl-prolyl cis-trans isomerase A, mimecan, and protein S100-A6 as potential biomarkers of the health-threatening impact the texted toxicants have on C. caretta. Obtained results confirm the suitability of the ex vivo system and indicate the potential risk the loggerhead sea turtle is undergoing in the natural environment. In conclusion, this work proved the relevance that the applied ex vivo models may have in testing the toxicity of other compounds and mixtures and in biomarker discovery.

Honey bees and the pollination services they provide are fundamental for agriculture and biodiversity. Agrochemical products and other classes of contaminants, such as trace elements and polycyclic aromatic hydrocarbons, contribute to the general decline of bees’ populations. For this reason, effects, and particularly sublethal effects of contaminants need to be investigated. We conducted a review of the existing literature regarding the type of effects evaluated in Apis mellifera, collecting information about regions, methodological approaches, the type of contaminants, and honey bees’ life stages. Europe and North America are the regions in which A. mellifera biological responses were mostly studied and the most investigated compounds are insecticides. A. mellifera was studied more in the laboratory than in field conditions. Through the observation of the different responses examined, we found that there were several knowledge gaps that should be addressed, particularly within enzymatic and molecular responses, such as those regarding the immune system and genotoxicity. The importance of developing an integrated approach that combines responses at different levels, from molecular to organism and population, needs to be highlighted in order to evaluate the impact of anthropogenic contamination on this pollinator species.