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The productivity and sustainability of agricultural fields depend on the maintenance of relevant ecosystem functions as well as on the existence of a sufficiently complex species diversity. In this context, earthworms play a prominent role for soil ecosystem by influencing organic and inorganic matter breakdown, and carabid beetles are one of the most important arthropod predators on soil surface. So far, the environmental risk assessment of potential impacts of plant protection products on earthworms (EFSA 2017) and ground beetles (ESCORT 2000) is based on single compounds and does not take into account that organisms within and above the soil are exposed to a mixture of active ingredients from different plant protection products within one season and partly over successive seasons. In addition, the potential for secondary poisoning, as assessed in EFSA (2009), may increase if contaminated earthworms and carabid beetles are combined in the diet of a mixed-diet feeding bird or mammal. In this study, earthworms and carabid beetles were sampled at two seasons from eight fields in Croatia and analysed for up to 300 active ingredients. Based on publicly available draft assessment reports from EC and EFSA, degradation parameters (DT50, DT90) were used to calculate degradation curves and the current soil concentration at the date of earthworm sampling as well as the current residue concentration on soil surface at the date of carabid sampling. Subsequently, bioaccumulation factors were calculated by dividing the analysed pesticide residues in earthworms and carabid beetles by the calculated concentrations in soil and on soil, respectively. The aim of this survey was to check the applicability and reliability of this method and to examine (i) whether the re-calculated soil concentrations of individual active ingredients and mixtures of active ingredients with the same mode of action do pose a potential risk to earthworms and carabid beetles ; (ii) whether a potential of secondary poisoning has to take into account when a bird or mammal feed both on contaminated earthworms and carabid beetles.

Of all soil organisms earthworms are the most abundant representing approximately 60% of the total animal biomass in soil. They strongly influence degradation of organic matter, soil structure and microbial community. Their role in aeration, water infiltration and mixture of soil horizons has great ecological importance. Therefore they are often used as indicator species for soil pollution, ecological risks assessment and environmental impacts of cropping systems. The active period for earthworms is considered to be spring and autumn. Low temperatures cause hibernation, while too high temperatures lead to diapause or reduction of activity. The earthworm species richness can be linked to vegetation of the area. Intensive agriculture with high input of pesticides and fertilizers poses a risk for beneficial organisms causing a loss of biodiversity. In cultivated fields earthworms are exposed to frequent and different pesticide applications. Insecticides and fungicides appear to be the most toxic chemicals affecting survival and reproduction, although the herbicides are well represented in toxic chemicals as well. Previous studies focus mainly on the effect of three insecticides substances (cypermethrin, chlorpyrifos, dimethoate), two fungicides (carbendazim, mancozeb) and one herbicide (glyphosate) or metal compounds residues in earthworms. Majority of studies are conducted under laboratory-controlled conditions based on short- term experiments and standardized laboratory procedures that are not able to predict the real field effects of pesticides. Therefore the aim of this research was to determine the pesticide residues in earthworms collected in open field conditions and correlate the finding with soil characteristic, agronomic practices, crop rotation, pesticides applied and continuous duration of exposure to different pesticides. Samples were collected from two different counties in Croatia. Location Lukač represents the area where conservation tillage with less frequent pesticide use is applied. Soils in Lukač contain a great amount of fine sand and coarse silt. Location Tovarnik represents the area of intensive agriculture with very high application of pesticides, while soils contain a great amount of humus. Samples were collected from 4 different arable fields per location in autumn 2015, spring 2016 and autumn 2016 according to the ISO/TC 190/SC 4/WG 2 N0222 methodology. Earthworm samples were analyzed for residues on more than 300 different pesticides using LC-MS/MS and SPE-QuEChERS method (EN 15662:2008). Preliminary results showed 18 different pesticide residues present in earthworm samples including 7 fungicides, 6 herbicides and 5 insecticides. More pesticides with higher concentrations were identified in Tovarnik. Since the effect of mixtures of pesticides are insufficiently studied final results will determine the pesticide concentration in earthworms accumulated during the two vegetation seasons from different arable systems in Croatia.