For the analysis of PFAAs in soil, the soil was dried in an oven at 40 C until no further weight loss was recorded. After homogenization, 1 g of dried soil was weighed in a 15 mL PP tube and spiked with internal standards. The soil was then extracted with 10 mL MeOH by vortex mixing for 1 min and sonication for 10 min. Phase separation was achieved by centrifugation . The supernatant was transferred to a new 15 mL PP tube and concentrated in the Rapidvap. The extraction was repeated twice with 5 mL MeOH. The extracts were combined and concentrated in the Rapidvap to a final volume of 1 mL. For pore water analysis 20 g of the soil was put in a 50 mL centrifugation filter tube with a 0.2 mm nylon filter. After 20 min of centrifugation at 2000 RPM, 0.5 mL of pore water was transferred to a vial. The internal standards and MeOH were added to achieve a final volume of 1 mL. All final extracts were passed through an Acrodisc LC 13 GHP Pall nylon filter into 2 mL PP vials and stored at 4 C until analysis.An HPLC system coupled with a tandem mass spectrometer was used to analyze the samples for PFAAs. A pre-column prior to the injection valve was used to remove potential background contamination from the LC system. Separation of the analytes was achieved using an ACE 3 C18-300 column maintained at 30 C with a mobile phase gradient consisting of two eluents A and B , both containing 2 mM ammonium acetate. The gradient used for separation and the mass transitions as well as other mass spectrometer settings can be found in the Supporting Information. The mass spectrometer was equipped with an electrospray ionization interface operating in the negative ionization mode, berry pots and it was run in a scheduled MRM-mode. The purified extracts were diluted 1:1 with water prior to analysis to match the injection conditions of the HPLC. A volume of 20 ml was injected. Raw data were processed with the Analyst 1.5 software .Each sample was extracted three times and each extract was injected in duplicate.
The relative standard deviation of the concentrations derived from these six injections was <10% for all analytes in all samples. Concentrations were quantified using a twelve-point calibration with fitted correlation lines that had r2 values of >0.99 for all analytes; no weighting was applied. Further information on quality assurance and quality control is provided in our previous studies . Recoveries were determined by comparison with a matrix free solution spiked with internal standard immediately prior to injection. Average recoveries of the internal standards in the samples were between 22% and 112% . Since mass labeled internal standards were used for quantifying the analytes, no correction for recovery was necessary. See Table S4 in the Supporting Information for detailed information on recoveries. Limits of quantification were calculated on the basis of the lowest validated calibration standard. They were derived from the amount injected back calculated to an extract volume of 1 mL and divided by the average extracted sample quantities. Method blanks were prepared repeatedly with the same extraction procedure as the samples, but showed no quantifiable contamination. Solvent blanks were injected every ten injections to check for contamination of the LC system and for memory effects, but no contamination or memory effects were observed during the study. All PFAA concentrations from the non-spiked lysimeters were subtracted from the concentrations in the spiked lysimeters. Any resulting concentrations below the LoQ were neglected. Since PFOS is the only compound for which branched isomers were included in the standards used for the calibration curve, branched isomers could only be quantified for PFOS.
All reported PFOS concentrations are sum concentrations of non-branched and branched isomers.The soil concentrations at the time of planting were generally within the intended concentration range . The soil concentrations at the harvest date show that the shortest chain PFAAs, the C4eC6 PFCAs and PFBS, were depleted. Less than 3% of the initial mass was left in the soil . Depletion occurred in both the upper and lower soil layers . In contrast, some 80e90% of the longer chain PFCAs dosed were still present in the soil at the harvest date. We analyzed the behavior of the PFAAs in the lysimeter soil in another paper in which we include data from 12 other lysimeters prepared in the same manner but planted with different crops . That work showed that the depletion of the shorter chained PFAAs was due to leaching, and that the leaching was greater than anticipated due to interactions between the PFAAs. This accelerated leaching increased with the initial PFAA contamination level of the soil. Lower precipitation towards the end of the growth period contributed to reduced leaching and more stable conditions; two weeks before harvest the lysimeters had already received 91% of the water input for the whole growth period . Hence, although the lettuce was exposed to changing PFAA concentrations in soil, the evidence indicates that the concentrations were more stable towards the end of the growth period when the plants were largest and transpiring most.To evaluate the plant uptake of the PFAAs, the PFAA concentrations in the plant tissues were compared with the PFAA concentrations in the sampled exposure media, soil and pore water, using uptake factors. Concentrations in soil were only available for the start of the experiment and at the time of harvest, and concentrations in pore water were only available at harvest. We chose to use the concentrations in exposure media measured at harvest because a much larger portion of the plant growth and transpiration occurred during the latter part of the growth period and because soil concentrations were judged to be more stable .
It is nevertheless possible that the uptake factors for the shortest chain PFAAs are somewhat overestimated due to the depletion of these chemicals in the soil over the course of the experiment.Downy mildew is one of the most severe and devastating diseases for grapevines . Although there are various plant protection products that can solve this issue, viticulture has traditionally used copper-based products as a fungicide, which is the most efficient way controlling it, and has become an indispensable product for a large number of grape growers worldwide . The reliance on this product is even higher for organic farming since there are no synthetical alternatives accepted to be used and copper-based pesticides are indispensable for organic vine cultivation . Therefore, the continued use of these kinds of products in viticulture has triggered the accumulation of this metal in several vineyard area soils around Europe and worldwide which may even affect the productivity of food crops . Although Cu is fixed by organic and clayey matter content in the soil, Sonoda et al. observed copper mobility due to organic matter degradation , suggesting possible groundwater contamination. The high potential ecotoxicity of copper in freshwater and its accumulation in European soils have raised concerns in the European Union, leading to a tightening of the legislation on the use of copper-based products, limiting the use to 28 kg/ha of copper for seven years . Even though other PPP such as synthetic organic fungicides could be considered to replace copper in conventional vineyard production, their use is also not exempt from environmental risks such as soil accumulation and surface or groundwater pollution depending on their composition and the site characteristics. For example, hydroponic grow system a recent publication study observed that synthetic organic fungicides used for grapevine cultivation in northern Italy were present in 80% of monitored water wells, exceeding the Environmental Quality Standards limits for groundwater in 30% of them. The toxicity of dimethomorph has been observed in several living organisms, such as fish and invertebrates, other soil organisms , soil and water microflora and aquatic plants . Concerning zoxamide, low risk was observed for non-target arthropods, soil microorganisms, and non-target terrestrial plants .
The increase of organic vine cultivation during recent years has increased the application of other kinds of organic fungicides, those named natural or biopesticides, produced from a natural source and with minimum adverse effects on the physiological processes of plants , as is the case of laminarin, which is considered to have low environmental toxicity by EFSA . However, although some ecological products are considered good candidates for reducing copper dependency in vineyards, there is no treatment as effective as copper for controlling grapevine downy mildew . The replacement of copper-based products can be done with the use of plant resistance-inducers or biological control agents,which have no environmental hazards. Henceforth, the use of PPPs such as copper-based and organic fungicides in agriculture can involve a human threat due to the intake of crops cultivated under those conditions and a negative impact on other non-target receptors such as water, other plants, and animals. In addition, the sustainable use of PPPs in agriculture must include an application rate according to the characteristics of the vegetation and adjusted spraying, in order to reduce product losses due to drift.Despite this, it is worth knowing that active substances of pesticides are washed away by rainfall, causing the deposition of pesticides on the soil , which means continuing to seek solutions for preventing groundwater contamination from agricultural activity. Bare soil, commonly used in low rainfall areas, allows the rainfall water to drag with it the PPPs losses remaining on the topsoil without any obstacles and can even be simulated and modelled . Moreover, erosion and nutrient loss in bare soil would be more pronounced due to torrential rainfall events that are expected to be more recurrent due to climate change. On the contrary, cover crops in vineyards have been proven to be effective in protecting the soil from erosion and nutrient loss, improving soil fertility, structure, soil microbial functional diversity, and balancing the productive, and vegetative parts of the vine.Although the phytoextraction effect of covers in viticulture is not sufficient to eliminate the entire volume of copper annually applied as a phytosanitary product against mildew , cover crops are suggested to reduce the amounts of pesticide leached and, consequently, the risk of groundwater contamination .
Despite this, there has been no research on the effect of covers on the soil leaching of the commonly used organic fungicides in vineyards . The main objective of this work is to demonstrate the capability of using cover crops in vineyards for the mitigation and prevention of soil and groundwater pollution caused by the application of fungicides. Also, to study if the biodiversity of this cover crops has an effect on it. Copper, 3 conventional/synthetic organic fungicides , and 1 ecological fungicide were selected. The specific objectives of this work were as follows: i) assessment of the soil type on fungicides leaching, ii) quantification of pollution reduction due to the use of cover crops, and iii) analysis of the vegetation effect on fungicides degradation. Five different PPPs were selected based on previous trials carried out at OPTIMA project according to fungicide efficacy against downy mildew in vineyards and lower environmental impact . Table 1 shows the commercial name, register number, the active ingredient, the molecule structure, the octanol-water partition coefficient and the environmental risk for each of the fungicides studied. Codimur 50 , which is a copper-based product used as preventive treatments, is used as a control/reference treatment due to its preferential use in viticulture against mildew. Forum and Zorvec Vinabel are novel synthetic organic fungicides, with a systemic action, with dual preventive and curative effect whereas Bion MX is a synthetic inducer and activator of plant self-defense mechanisms. Finally, Vacciplant is an ecological organic fungicide product. All experimental studies were performed by using commercial fungicides supplied by the manufacturer’s brands or purchased from Agrogava.During the spray application process carried out for crop protection, there is an inevitable loss of fungicides to the ground. According to Gil , the amount of ground losses due to the spraying application process in a vineyard was calculated as 4.62 μg cm− 2 for an application of 1 kg Cu⋅ha− 1 under the worst conditions .