Copper toxicity on Eisenia fetida in a vineyard soil: a combined study with standard tests, genotoxicity assessment and gut metagenomic analysis.

Copper (Cu) toxicity is a pressing concern for several soils, especially in organic viticulture. The objective of this work was to assess Cu toxicity on the non-target organism Eisenia fetida, employing both traditional and novel tools for early identification of Cu-induced damages. In addition to traditional tests like avoidance and reproductive toxicity experiments, other tests such as the single cell gel electrophoresis (SCGE) and gut microbiome analysis were evaluated to identify early and more sensitive pollution biomarkers. Four sub-lethal Cu concentrations were studied, and the results showed strong dose-dependent responses by the earthworm avoidance test and the exceeding of habitat threshold limit at the higher Cu doses. An inverse proportionality was observed between reproductive output and soil Cu concentration. Bioaccumulation was not detected in earthworms; soil concentrations of potentially bioavailable Cu were not affected by E. fetida presence or by time. On the contrary, the SCGE test revealed dose-dependent genotoxicity for the 'tail length' parameter already at the second day of Cu exposition. Gut microbiome analysis a modulation of microbial composition, with the most aboundant families being Pectobateriaceae, Comamonadaceae and Microscillaceae. Bacillaceae increased over time and showed adaptability to copper up to 165 mg/kg, while at the highest dose even the sensitive Acetobacteriaceae family was affected. The research provided new insights into the ecotoxicity of Cu sub-lethal doses highlighting both alterations at earthworms' cellular level and changes in their gut microbiota.

Copper Monitoring in Vineyard Soils of Central Italy Subjected to Three Antifungal Treatments, and Effects of Sub-Lethal Copper Doses on the Earthworm Eisenia fetida.

The extensive employment of copper-based fungicides has increased copper concentration in vineyard soils. The present study's objectives were to monitor copper concentration in two vineyard soils during two cropping seasons and study the ecotoxicological effects on the earthworm Eisenia fetida. Total, soluble, and bioavailable copper fractions were measured at the end of two cropping seasons and different depths in two vineyards of central Italy, characterised by three anticryptogamic control methods: copper compounds, chitosan, and combined treatments of them. A laboratory experiment to assess the effects on Eisenia fetida was conducted with soil samples collected in the vineyards with a mean copper concentration of 60 mg/kg and two higher concentrations of 90 and 150 mg/kg. Results showed low levels of total copper concentration in the first 20 cm of soils, regardless of antifungal treatment, highlighting prudent management of the vineyards under study, but the soluble fractions showed a significant increase in all samples during the two cropping seasons. At the dose of 150 mg/kg, earthworms suffer during the first two days, showing weight loss and DNA damage, but they are able to recover until day 28, showing no permanent harm at this copper concentration in soil.

Losses and dissipation of penconazole in vineyard soil as affected by mid-row management system.

A field experiment was performed with the aim to assess the amount of penconazole losses during field application and the spatial variability of penconazole concentration in a vineyard soil, under two different management techniques (tilled and grass covered). The field dissipation of penconazole under the two techniques was also followed for 114 days, highlighting the effects of spatial variability of the initial concentration and of the grass covering in terms of the different soil metabolic activity. Data found show that a high percentage of penconazole, from 42.5 % to 67.43 %, can reach the soil during the treatments, despite the fact that penconazole is applied to the foliage. The high values of the coefficient of variation for data within rows show considerable variability in all applications, ranging from 30 to 65 in the first application, from 35 to 79 in the second and from 36 to 75 in the third. Since the applications of penconazole occurred under almost same climatic conditions in terms of wind speed and direction, the high variability of concentration of penconazole found within mid-rows was attributed to the uneven slope of the vineyard causing an irregular speed of the sprayer equipment. However, least significant differences of the mean values did not end in a significant difference of penconazole concentration among mid-rows for all applications, indicating that the variability between rows does not contribute to the overall variability. The calculated half-life values for penconazole in tilled soil were 62.4 days for tilled and 33.0 for grassed soil, highlighting the tendency of penconazole to faster dissipate in grassed than in tilled soil. Grassed soil was characterised by a higher metabolic activity in terms of microbial biomass carbon content, basal respiration and total hydrolytic activity, than tilled soil. A significant correlation between penconazole dissipation and total hydrolytic activity, was found. The faster dissipation of penconazole in grassed soil was attributed to the higher amount of the total hydrolytic activity which, in grassed soil, was 1.5 fold-higher than in tilled soil.

Zeolite and bentonite as nickel sequestrants in carbonation lime coming from the sugar industry.

A laboratory trial was performed to test the sequestration capacity of two minerals (bentonite and zeolite) at three initial concentrations (2.5, 5 and 10%) in order to counter water-soluble nickel (Ni) exceeding the Italian legal limit (10 µg L-1) in carbonation lime disposed of in a field and sampled for an 85-day lab study. The results show a noticeable reduction in water-soluble and bioavailable Ni in lime after the addition of sequestrants, especially at the dose of 5% bentonite or zeolite, thereby indicating a "ceiling effect" of the sequestrant, i.e., an increasing dose could reduce the adsorption capacity and be less effective. The alkaline pH and the presence of organic matter could be the main factors affecting the good performance of sequestrant addition, causing an increase in the negative charge of the organic and mineral colloids and the formation of unavailable Ni precipitates. The 85-day experiment seems to be sufficient to reach an adsorption equilibrium for water-soluble nickel, while for the bioavailable form a longer period appears to be necessary.

Phytoremediation Potential of Crop Plants in Countering Nickel Contamination in Carbonation Lime Coming from the Sugar Industry.

The phytoremediation potential of four crop species cultivated on carbonation lime coming from the sugar industry with water-soluble nickel (Ni) exceeding the Italian legal limit of 10 µg L-1 was assessed. Two autumn-winter species (spinach and canola) were tested with and without the addition of bentonite in a greenhouse experiment in order to overcome prolonged unfavourable weather conditions. Two spring-summer species (sunflower and sorghum) were grown in outdoor boxes. Plant species were selected among crops of interest for phytoremediation and their rotation throughout the year enable to maintain a permanent vegetation cover. Nickel concentration in different plant tissues and the concentrations of soluble and bioavailable Ni in lime were measured. In the greenhouse study, soluble Ni decreased below the legal limit in all the tests, and the combined effect of bentonite and plants reduced Ni in lime mainly in the bioavailable fraction. Spinach and sunflower emerged to be more suitable for phytoextraction than canola and sorghum, because of the higher concentration of the metal in the epigeal portions. The results from the outdoor experiment highlighted that sorghum has a good phytostabilisation potential since its ability to accumulate Ni mainly at the root level and to attract a significant amount of bioavailable Ni in the rhizosphere. This study arose from a real scenario of environmental contamination and investigated the potential of different approaches on the bioremediation of a specific industrial waste product.

Sub-Lethal Effects of Pesticides on the DNA of Soil Organisms as Early Ecotoxicological Biomarkers.

This review describes the researches performed in the last years to assess the impact of pesticide sub-lethal doses on soil microorganisms and non-target organisms in agricultural soil ecosystems. The overview was developed through the careful description and a critical analysis of three methodologies based on culture-independent approaches involving DNA extraction and sequencing (denaturing gradient gel electrophoresis, DGGE; next-generation sequencing, NGS) to characterize the microbial population and DNA damage assessment (comet assay) to determine the effect on soil invertebrates. The examination of the related published articles showed a continuous improvement of the possibility to detect the detrimental effect of the pesticides on soil microorganisms and non-target organisms at sub-lethal doses, i.e., doses which have no lethal effect on the organisms. Considering the overall critical discussion on microbial soil monitoring in the function of pesticide treatments, we can confirm the usefulness of PCR-DGGE as a screening technique to assess the genetic diversity of microbial communities. Nowadays, DGGE remains a preliminary technique to highlight rapidly the main differences in microbial community composition, which is able to give further information if coupled with culture-dependent microbiological approaches, while thorough assessments must be gained by high-throughput techniques such as NGS. The comet assay represents an elective technique for assessing genotoxicity in environmental biomonitoring, being mature after decades of implementation and widely used worldwide for its direct, simple, and affordable implementation. Nonetheless, in order to promote the consistency and reliability of results, regulatory bodies should provide guidelines on the optimal use of this tool, strongly indicating the most reliable indicators of DNA damage. This review may help the European Regulation Authority in deriving new ecotoxicological endpoints to be included in the Registration Procedure of new pesticides.

Retention capacity of an organic bio-mixture against different mixtures of fungicides used in vineyards.

A laboratory experiment was carried out to test the efficiency of a bio-mixture made up of pruning residues at two (PR2) and five (PR5) years of composting and wheat straw (STW) in the biological cleaning of water contaminated by different mixtures of fungicides usually employed in vineyards. The experiment was conducted and reproduced at a scale of 1:100 of operating field conditions. Commercial formulates of penconazole (PC), (RS)-1-[2-(2,4-dichlorophenyl)pentyl]-1H-1,2,4-triazole), dimetomorph (DM), (EZ)-4-[3-(4-chlorophenyl)-3-(3,4-dimethoxyphenyl)acryloyl]morpholine), azoxystrobin (AZ), (methyl (E)-2-{2-[6-(2-cyanophenoxy)pyrimidin-4-yloxy]phenyl}-3-methoxyacrylate), iprovalicarb (IP), (isopropyl 2-methyl-1-[(RS)-1-p-tolylethyl]carbamoyl-(S)-propylcarbamate), metalaxyl (MX), (methyl N-(methoxyacetyl)-N-(2,6-xylyl)-DL-alaninate), fludioxonil (FL), (4-(2,2-difluoro-1,3-benzodioxol-4-yl)-1H-pyrrole-3-carbonitrile) and cyprodinil (CY), (4-cyclopropyl-6-methyl-N-phenylpyrimidin-2-amine) were mixed in water and discharged into the bio-mixture following the time schedule of the treatments carried out in the grapevine in real field conditions. At each treatment, contaminated water with fungicides was circulated repeatedly through the bio-mixture to enhance the sorption of fungicides. In fact, it retained them between 98-100% with the exception of MX of which it was able to retain only 90.5%. The adsorption/desorption experiment showed that repeated circulation of water, instead of enhancing MX retention, can easily remove about 30% of MX already adsorbed by the bio-mixture. This finding suggests that water contaminated by very mobile pesticides should be discharged at the end of field treatments without re-circulating the water in order to avoid the release of pesticides weakly adsorbed on the bio-mixture.

The effect of initial concentration, co-application and repeated applications on pesticide degradation in a biobed mixture.

A 180 d laboratory experiment was conducted to investigate the degradation rates of chlorpyrifos (10 and 50 mg kg(-1)) and metalaxyl (100 mg kg(-1)) separately and co-applied in a biomix constituted by topsoil, vine-branches and urban-waste-garden compost. The effect of repeated application of metalaxyl was also investigated. Microbial biomass-C (MBC) content and metabolic quotient (qCO(2)) were measured to evaluate changes in microbial biomass size and activity induced by the presence of the two pesticides. Degradation rate decreased with increasing concentration of chlorpyrifos in all treatments. Metalaxyl half-life was significantly reduced in co-application with chlorpyrifos indicating a synergic interaction between the two pesticides in favour of enhanced degradation rate for metalaxyl but not for chlorpyrifos. Furthermore, repeated application resulted in a sharp reduction of metalaxyl half-life from 37 d after first application to 4 d after third application. MBC content was negatively influenced by the addition of pesticides but it started to recover immediately, in both separate and co-applied treatments, reaching the control value when pesticide residues were about 50% of the initial concentration. The qCO(2) reached a steady-state after about 20 d in separately applied and 40 d in co-applied treatments, indicating a tendency to arrive at a new metabolic equilibrium. In conclusion, the biomix tested has been shown to degrade pesticides relatively fast and to have a microbial community that is varied enough to allow selection of those microorganisms able to degrade metalaxyl and chlorpyrifos.

Biomassbed: a biological system to reduce pesticide point contamination at farm level.

A potential method for cleaning water from point-source pollution by organic compounds is using biological reactors. In this study, four reactors were tested for their ability to retain and degrade pesticides. The pesticides tested were the insecticide chlorpyrifos, the fungicide metalaxyl and the herbicide imazamox. The reactors were filled with differing mixtures of vine-branch, citrus peel, urban waste and public green compost. The reactor volume was 188 l. Forced circulation of the contaminated solution was programmed to decontaminate the solution. Both retention and degradation of the compounds by the reactors was studied. Chlorpyrifos was the best retained, due to its physico-chemical characteristics, while only one substrate effectively retained metalaxyl and imazamox (citrus peel+urban waste compost). Degradation of the pesticides in the reactors was faster than published values for degradation in soil. The half-life of all pesticides in the reactors was less than 14 days, compared to literature values of 60-70 days in soil. The combined retention and fast degradation make the biofilter a feasible technique to reduce spill-related and point environmental contamination by pesticides. The technique is most effective against persistent pesticides, while for mobile pesticides, the efficiency can be improved with several passages of the contaminated solution through biofilters.

Effects of mercury on microbial biomass and enzyme activities in soil.

Mercury (Hg) is a persistent soil pollutant that affects soil microbial activity. We monitored the changes in soil microbial biomass and activity of enzymes, including alkaline phosphatase, arylsulfatase, fluorescein diacetate (FDA) hydrolytic activity, and o-diphenol oxidase (o-DPO) in three soils contaminated with different concentrations of Hg. Increasing levels of Hg, from 0.5 to 10 micromol/g of dried soil, generally depressed microbial activity; however, the effects of Hg on soil microbial activity depended on soil type and composition, particularly organic matter content. o-DPO was less affected by Hg than the other three enzymes tested. Our results indicate that the analysis of microbial biomass content and soil-enzyme activities may be used to predict the soil quality contaminated with Hg.

Fungicides degradation in an organic biomixture: impact on microbial diversity.

Biological systems are being developed all over EU countries to protect water-bodies from pesticide contamination at farm level. A laboratory experiment was carried out to test the efficiency of a mixture of compost and straw in bio-degrading different mixtures of fungicides usually applied in vineyards. At the same time the effects of fungicide applications on microbial community of biomixture were also evaluated. Results showed that the biomixture had a good capability of degrading pesticides. Indeed, at the end of the experiment (112 days), the concentration of most of the pesticides was close to complete degradation. Denaturing gradient gel electrophoresis (DGGE) analysis showed an evident modification of microbial diversity after the addition of fungicides. However, at the end of degradation process, no significant changes in the composition of microbial community were seen. In this specific substrate used in the biomixture, yeast flora and ascomycete filamentous fungi seemed to be involved in the degradation activity.

Moist olive husks addition to a silty clay soil: influence on microbial and biochemical parameters.

To investigate the effects of moist olive husks (MOH-residues) on soil respiration, microbial biomass, and enzymatic (o-diphenoloxidase, beta-glucosidase, dehydrogenase and alkaline phosphatase) activities, a silty clay soil was incubated with 0 (control), 8 x 10(3) (D), 16 x 10(3) (2D) and 80 x 10(3) (10D) kg ha-1 of MOH-residues on a dry weight basis. Soil respiration and microbial biomass data indicated that the addition of MOH-residues strongly increased microbial activity proportionally to the amounts added. Data of qCO2 suggested that the respiration to biomass ratio of the microbial population was strongly modified by MOH-residues additions during the first 90 days of incubation. The qCO2 data suggested a low efficiency in energy yields from C oxidation during the first 2 months of soil incubation. qFDA seemed to be relatively unaffected for treatments D and 2D as compared to the control, but was significantly lowered by the application of 10D, showing the lowest hydrolytic activity of microbial biomass in this treatment up to 360 days of incubation. o-Diphenoloxidase activity was delayed, and this delay was extended with the addition of larger quantities of MOH-residues. Alkaline phosphatase, beta-glucosidase and dehydrogenase activities were in line with the findings on microbial biomass changes and activities. The biological and biochemical data suggest that the addition of a large quantity of MOH-residues (80 x 10(3) kg ha-1) strongly modifies the soil characteristics affecting the r- and K-strategist populations, and that these changes last for at least the 360 days of incubation. The data also suggest that application rates exceeding 16 x 10(3) kg ha-1 are not recommended until the agro-chemical and -physical functions of the soil are further studied.