The laboratory reproduction test with the predatory mite Hypoaspis aculeifer is currently a mandatory test in the new EU data requirements for prospective environmental risk assessment of Plant Protection Products (PPPs). However, the low sensitivity often shown by this mite towards PPPs, when compared to other invertebrates (namely Folsomia candida and Eisenia fetida), makes the test with this species not very useful in the lower tier test battery. However, the current test protocol only considers exposure to contaminants via contaminated soil, disregarding exposure via contaminated food and does not take into account the fact that H. aculeifer is a predatory species. Therefore, through this protocol, the toxicity of contaminants to soil mites might be underestimated and, thus, an adaptation of the test performance, by including exposure via contaminated food, may be necessary. With this aim, two reproduction tests with H. aculeifer were performed using copper chloride as model substance, artificial soil as test substrate and cheese mites as food. The OECD guideline was followed but, while in one test cheese mites from normal laboratory breeding cultures (clean prey mites) were provided, in the other test, cheese mites previously exposed to copper (Cu pre-exposed prey mites) were provided. Predatory mites were affected at lower concentrations in tests using Cu pre-exposed prey compared to test with clean-prey (NOEC = 1225 and 1508 mg kg⁻¹ and EC₁₀ = 1204 and 1903 mg kg⁻¹ using Cu pre-exposed and clean prey, respectively). However, this higher sensitivity was not detected by EC₅₀ values (EC₅₀ = 2634 and 2814 mg kg⁻¹ using Cu pre-exposed and clean prey, respectively). Further tests are needed in order to (i) investigate the relevance of oral exposure to different PPPs, (ii) optimize the contamination of prey mites according to the chemical properties of each substance and (iii) substantiate a proposal to adapt the standard protocol.

The dissipation of famoxadone as well as the behaviour of its metabolites in environmental samples such as water and soil is a major concern. In this study, the dissipation of the target compound in both matrices was carried out applying an analytical method based on ultra-high performance liquid chromatography coupled to Orbitrap mass spectrometry (UHPLC-Orbitrap-MS). The dissipation of famoxadone was monitored over a period of 100 days after the plant protection product, Equation Pro®, was administered to the target matrices. This study was performed at two doses, normal and double in the case of soils and fivefold instead of double dose in water. The concentration of famoxadone steadily decreased during the monitoring period in both matrices. Half-life (DT₅₀) values were lower than 30 days in most cases except for loam soils, for which it was 35 days. Therefore, persistence of this pesticide in both matrices was low. Famoxadone metabolites such as IN-KF015 ((5RS)-5-methyl-5-(4-phenoxyphenyl)-1,3- oxazolidine-2,4-dione) and IN-JS940 ((2RS)-2-hydroxy-2-(4- phenoxyphenyl)propanoic acid) were detected in both matrices and their concentration increased while the concentration of the parent compound decreased. Metabolite IN-JS940 was the compound detected at highest concentration for both matrices. In water the maximum concentration was 20% of the initial famoxadone content and in soils it was 50% of initial famoxadone content. In addition, another metabolite, IN-MN467 ((5RS)-5-methyl-3-[(2-nitrophenyl)amino]- 5-(4-phenoxyphenyl)-1,3-oxazolidine-2,4-dione), was detected in soils, following the same behaviour as the other metabolites. These results provided ample information about the behaviour of metabolites and the necessity of knowing their toxicity in both matrices in order to detect possible risks for living beings.

Despite the efforts of the European Commission to implement measures that offset the detrimental effects of agricultural intensification, farmland bird populations continue to decline. Pesticide use has been pointed out as a major cause of decline, with growing concern about those agro-chemicals that act as endocrine disruptors. We report here on the effects of flutriafol, a ubiquitous systemic fungicide used for cereal seed treatment, on the physiology and reproduction of a declining gamebird. Captive red-legged partridges (Alectoris rufa; n = 11–13 pairs per treatment) were fed wheat treated with 0%, 20% or 100% of the flutriafol application rate during 25 days in late winter. We studied treatment effects on the reproductive performance, carotenoid-based coloration and cellular immune responsiveness of adult partridges, and their relationship with changes in oxidative stress biomarkers and plasma biochemistry. We also studied the effect of parental exposure on egg antioxidant content and on the survival, growth and cellular immune response of offspring. Exposed partridges experienced physiological effects (reduced levels of cholesterol and triglycerides), phenotypical effects (a reduction in the carotenoid-based pigmentation of their eye rings), and most importantly, severe adverse effects on reproduction: a reduced clutch size and fertile egg ratio, and an overall offspring production reduced by more than 50%. No effects on body condition or cellular immune response of either exposed adult or their surviving offspring were observed. These results, together with previous data on field exposure in wild partridges, demonstrate that seed treatment with flutriafol represents a risk for granivorous birds; they also highlight a need to improve the current regulation system used for foreseeing and preventing negative impacts of Plant Protection Products on wildlife.

Twenty-four experiments where EDU was used to protect plants from ozone (O3) in Italy are reviewed. Doses of 150 and 450 ppm EDU at 2-3 week intervals were successfully applied to alleviate O3-caused visible injury and growth reductions in crop and forest species respectively. EDU was mainly applied as soil drench to crops and by stem injection or infusion into trees. Visible injury was delayed and reduced but not completely. In investigations on mode of action, EDU was quickly (<2 h) uptaken and translocated to the leaf apoplast where it persisted long (>8 days), as it cannot move via phloem. EDU did not enter cells, suggesting it does not directly affect cell metabolism. EDU delayed senescence, did not affect photosynthesis and foliar nitrogen content, and stimulated antioxidant responses to O3 exposure. Preliminary results suggest developing an effective soil application method for forest trees is warranted. EDU was successful as a tool in determining ozone effects on vegetation in Italy, but while progress was made, its mode of action remains unresolved.

The area of agricultural wastes valorisation to fertilizers is attracting growing attention because of the increasing fertilizer prices of fertilizers and the higher costs of waste utilization. Despite the scientific and political interest in the concept of circular economy, few studies have considered the practical approach towards the implementation of elaborated technologies. This article outlines innovative strategies for the valorisation of different biobased wastes into fertilizers. The present work makes a significant contribution to the field of new ideas for waste biomass management to recover significant fertilizer nutrients. These results emphasize the importance of the biomass use as a base of renewable resources, which has recently gained special importance, especially in relation to the outbreak of pandemia and war. Broken supply chains and limited access to deposits of raw materials used in fertilizer production (natural gas, potassium salts) meant that now, as never before, it has become more important and feasible to implement the idea of a circular economy and a green deal. We have obtained satisfactory results that demonstrate that appropriate management of biological waste (originating from agriculture, food processing, aquaculture, forest, pharmaceutical industry, and other branches of industry, sewage sludge) will not only reduce environmental nuisance (reducing waste heaps), but will also allow recovery of valuable materials, such as nitrogen (especially valuable amino acids), phosphorus, potassium, microelements, and biologically active substances with properties that stimulate plant growth. The results reported here provide information on production of biobased plant protection products (bioagrochemicals) from agri-food waste. This work reports an overview of biopesticides and biofertilisers production technologies and summarizes their properties and the mechanisms of action.

Pollinating bees are stressed by highly variable environmental conditions, malnutrition, parasites and pathogens, but may also by getting in contact with microorganisms or entomopathogenic nematodes that are used to control plant pests and diseases. While foraging for water, food, or nest material social as well as solitary bees have direct contact or even consume the plant protection product with its active substance (e.g., viruses, bacteria, fungi, etc.). Here, we summarize the results of cage, microcolony, observation hive assays, semi-field and field studies using full-size queen-right colonies. By now, some species and subspecies of the Western and Eastern honey bee (Apis mellifera, A. cerana), few species of bumble bees, very few stingless bee species and only a single species of leafcutter bees have been studied as non-target host organisms. Survival and reproduction are the major criteria that have been evaluated. Especially sublethal effects on the bees' physiology, immune response and metabolisms will be targets of future investigations. By studying infectivity and pathogenic mechanisms, individual strains of the microorganism and impact on different bee species are future challenges, especially under field conditions. Overall, it became evident that honey bees, bumble bees and few stingless bee species may not be suitable surrogate species to make general conclusions for biological mechanisms of bee-microorganism interactions of other social bee species. Solitary bees have been studied on leafcutter bees (Megachile rotundata) only, which shows that this huge group of bees (∼20,000 species worldwide) is right at the beginning to get an insight into the interaction of wild pollinators and microbial plant protection organisms.

Fungicide application for controlling fungal diseases can increase copper (Cu) accumulation in soil. More urgently, Cu released from fungicides can associate with soil clay and favour the mutual aggregation of Cu and soil clay, thereby potentially intensifying the accumulation of Cu. We investigated the effects of Cu salt and six common Cu-based fungicides on colloidal dynamics of a clay fraction from citrus cultivated soil. Batch experiments were carried out to provide the loading capacity of the clay fraction for Cu. The colloidal dynamic experiments were performed over a pH range from 3 to 8 following a test tube method, while surface charge, the key electrochemical factor of the solid-liquid interface, was quantified by a particle charge detector. It was found that all the studied fungicides, via releasing Cu²⁺, acted to effectively favour clay aggregation. The dissolved organic matter obtained from the dissolution of polymers in fungicides can theoretically stimulate clay dispersion. However, their effects were obscured due to the overwhelming effect of Cu²⁺. Therefore, Cu²⁺ appears as the most active agent in the fungicides that intensifies clay aggregation. These findings imply that the intensive application of fungicides for plant protection purposes can inadvertently reduce clay mobility, favour the co-aggregation of clay and fungicides, and hence potentially exacerbate the contamination of the citrus soil.

The neonicotinoid insecticide imidacloprid is used in Bangladesh for a variety of crop protection purposes. Imidacloprid may contaminate aquatic ecosystems via spray drift, surface runoff and ground water leaching. The present study aimed at assessing the fate and effects of imidacloprid on structural (phytoplankton, zooplankton, macroinvertebrates and periphyton) and functional (organic matter decomposition) endpoints of freshwater, sub-tropical ecosystems in Bangladesh. Imidacloprid was applied weekly to 16 freshwater microcosms (PVC tanks containing 400 L de-chlorinated tap water) at nominal concentrations of 0, 30, 300, 3000 ng/L over a period of 4 weeks. Results indicated that imidacloprid concentrations from the microcosm water column declined rapidly. Univariate and multivariate analysis showed significant effects of imidacloprid on the zooplankton and macroinvertebrate community, some individual phytoplankton taxa, and water quality variables (i.e. DO, alkalinity, ammonia and nitrate), with Cloeon sp., Diaptomus sp. and Keratella sp. being the most affected species, i.e. showing lower abundance values in all treatments compared to the control. The observed high sensitivity of Cloeon sp. and Diaptomus sp. was confirmed by the results of single species tests. No significant effects were observed on the species composition of the phytoplankton, periphyton biomass and organic matter decomposition for any of the sampling days. Our study indicates that (sub-)tropical aquatic ecosystems can be much more sensitive to imidacloprid compared to temperate ones.

Among the many factors responsible for the decline of bee populations are plant protection products such as neonicotinoids. In general, bees are exposed to not only one but mixtures of such chemicals. At environmental realistic concentrations neonicotinoids may display negative effects on the immune system, foraging activity, learning and memory formation of bees. Neonicotinoids induce alterations of gene transcripts such as nicotinic acetylcholine receptor (nAChR) subunits, vitellogenin, genes of the immune system and genes linked to memory formation. While previous studies focused on individual compounds, the effect of neonicotinoid mixtures in bees is poorly known. Here we investigated the effects of neonicotinoids acetamiprid, clothianidin, imidacloprid and thiamethoxam as single compounds, and binary mixtures thereof in honeybees. We determined transcriptional changes of nAChR subunits and vitellogenin in the brain of experimentally exposed honeybees after exposure up to 72 h. Exposure concentrations were selected on the basis of lowest effect concentrations of the single compounds. Transcriptional induction of nAChRs and vitellogenin was strongest for thiamethoxam, and weakest for acetamiprid. To a large extent, binary mixtures did not show additive transcriptional inductions but they were less than additive. Our data suggest that the joint transcriptional activity of neonicotinoids cannot be explained by concentration addition. The in vivo effects are not only governed by agonistic interaction with nAChRs alone, but are more complex as a result of interactions with other pathways as well. Further studies are needed to investigate the physiological joint effects of mixtures of neonicotinoids and other plant protection products on bees to better understand their joint effects.

We investigated how the adaptation to metalliferous environments can influence the plant response to biotic stress. In a metallicolous and a non-metallicolous population of Silene paradoxa the induction of oxidative stress and the production of callose and volatiles were evaluated in the presence of copper and of the PAMP fungal protein cerato-platanin, separately and in combination. Our results showed incompatibility between the ordinary ROS-mediated response to fungal attack and the acquired mechanisms of preventing oxidative stress in the tolerant population. A similar situation was also demonstrated by the sensitive population growing in the presence of copper but, in this case, with a lack of certain responses, such as callose production. In addition, in terms of the joint behaviour of emitted volatiles, multivariate statistics showed that not only did the populations respond differently to the presence of copper or biotic stress, but also that the biotic and abiotic stresses interacted in different ways in the two populations.Our results demonstrated that the same incompatibility of hyperaccumulators in ROS-mediated biotic stress signals also seemed to be exhibited by the excluder metallophyte, but without the advantage of being able to rely on the elemental defence for plant protection from natural enemies.