Assessing the Potential Ecotoxicological Risk of Different Organic Amendments Used in Agriculture: Approach Using Acute Toxicity Tests on Plants and Earthworms.

In Europe, spreading organic wastes to fertilize soils is an alternative commonly used instead of chemical fertilizers. Through their contributions of nutrients and organic matter, these wastes promote plant growth and thus agricultural production. However, these organic amendments can also contain mineral and organic pollutants requiring chemical and ecotoxicological analyses to guarantee their harmlessness on soil and its organisms during spreading. The purpose of this study was to assess the potential toxicity of three organic amendments from different sources (sewage sludge, dairy cow manure, dairy cow slurry) by performing chemical analyses and acute toxicity tests on three types of organism: earthworms, plants, soil microbial communities. Chemical analysis revealed a higher content of certain pharmaceuticals, polycyclic aromatic hydrocarbons and metals in sewage sludge in comparison with the two other types of organic wastes. The ecotoxicological assessment showed a dose-dependent effect on soil organisms for the three organic amendments with higher toxic effects during the exposure tests with a soil amended with dairy cow slurry. However, at realistic spreading doses (10 and 20 g kg-1 dry weight of organic amendments) on a representative exposed soil, organic amendment did not show any toxicity in the three organisms studied and had positive effects such as increased earthworm biomass, increased plant root growth and earthworm behavior showing attraction for organic amendment. On the contrary, exposure assays carried out on a limited substrate like sandy soil showed increased toxicity of organic amendments on plant germination and root growth. Overall, the ecotoxicological analysis revealed greater toxicity for soil organisms during the amendment of cow slurry, contrary to the chemical analysis which showed the potential high risk of spreading sewage sludge due to the presence of a higher quantity of pollutants. The analysis of the chemical composition and use of acute toxicity tests is the first essential step for assessing the ecotoxicological risk of spreading organic amendments on soil organisms. In addition to standard tests, the study suggests using a representative soil in acute toxicity tests to avoid overestimating the toxic effects of these organic amendments.

Inter-laboratory validation of an ISO test method for measuring enzyme activities in soil samples using colorimetric substrates.

The evaluation of soil quality requires the use of robust methods to assess biologically based indicators. Among them, enzyme activities are used for several decades, but there is a clear need to update their measurement methods for routine use, in combining feasibility, accuracy, and reliability. To this end, the platform Biochem-Env optimized a miniaturized method to measure enzyme activities in soils using colorimetric substrates in micro-well plates. The standardization of the method was carried out within the framework of ISO/TC 190/SC 4/WG 4 "Soil quality - Biological methods" workgroup, recommending an inter-laboratory evaluation for the publication of a full ISO standard. That evaluation, managed by the platform, was based on the measurement, in six soils of contrasted physicochemical properties, of the ten soil enzyme activities described in the standard. Eight laboratories were involved in the validation study. Only 2.7% of outliers were identified from the analyses of the whole dataset. The repeatability and reproducibility of the method were determined by computing, respectively, the intra-laboratory (CVr,) and inter-laboratory (CVR) coefficients of variation for each soil and enzyme. The mean CVr ranged from 4.5% (unbuffered phosphatase) to 9.9% (α-glucosidase), illustrating a reduced variability of enzyme activities within laboratories. The mean CVR ranged from 13.8% (alkaline phosphatase) to 30.9% (unbuffered phosphatase). Despite this large CVR noticed for unbuffered phosphatase, the method was repeatable, reproducible, and sensitive. It also proved to be applicable for measuring enzyme activities in different types of soils. These results have been found successful by ISO/TC 190/SC4 and resulted in the publication of ISO 20130:2018 standard.

Soil enzymatic activity data over eight years at the EFELE site, a long-term field experiment on effects of organic waste products and tillage practices.

Land application of organic waste products (OWPs), catch crops and reduced soil tillage are accepted as sustainable management practices in agriculture. They can optimize resources by supplying nutrients to plants and helping to maintain soil fertility. They also can influence soil functions in agricultural production systems. Soil microorganisms can feed on fresh organic matter by producing extracellular enzymes. Enzyme production responds to resource availability and soil C:N:P ratios, which could limit biogeochemical cycling. Allocating resources to produce nutrient-acquiring enzymes requires a large amount of energy to achieve optimal growth. In this context, studying the use of OWPs is important, as alternatives to long-term use of mineral fertilizers, to understand the dynamics of response and how the OWPs influence production of extracellular enzymes in the soil. Effects of OWPs on soil enzymatic activities have been studied widely, but long-term effects remain poorly understood, and no information is available about differences in dynamics among systems for each biogeochemical cycle. The data described here were collected during two trials from an initial state, and they allow assessment of long-term effects of OWP application, mineral nitrogen fertilization, tillage and vegetation cover on soil enzymatic activities. Data are presented for the activities of five soil enzymes measured from 2012 to 2019: ß-glucosidase, phosphatase, urease, arylamidase and arylsulfatase. Five additional enzymes were included in 2019 to supplement the analysis of biogeochemical cycles: alkaline phosphatase, phosphodiesterase, α-glucosidase, ß-galactosidase and n-acetyl-glucosaminidase. These activities were measured in two trials at the EFELE study site: PROs (five OWPs applied to a corn-wheat rotation) and TS/MO (four treatments that examine interactions between OWP and type of tillage). These data can be used as a reference for future studies of soil enzymes in France and other regions (e.g. for developing reduced-tillage systems and organic or inorganic amendments, and to assess dynamics of the systems).

Earthworms Mitigate Pesticide Effects on Soil Microbial Activities.

Earthworms act synergistically with microorganisms in soils. They are ecosystem engineers involved in soil organic matter degradation and nutrient cycling, leading to the modulation of resource availability for all soil organisms. Using a soil microcosm approach, we aimed to assess the influence of the earthworm Aporrectodea caliginosa on the response of soil microbial activities against two fungicides, i.e., Cuprafor Micro® (copper oxychloride, a metal) and Swing® Gold (epoxiconazole and dimoxystrobin, synthetic organic compounds). The potential nitrification activity (PNA) and soil enzyme activities (glucosidase, phosphatase, arylamidase, and urease) involved in biogeochemical cycling were measured at the end of the incubation period, together with earthworm biomass. Two common indices of the soil biochemistry were used to aggregate the response of the soil microbial functioning: the geometric mean (Gmean) and the Soil Quality Index (SQI). At the end of the experiment, the earthworm biomass was not impacted by the fungicide treatments. Overall, in the earthworm-free soil microcosms, the two fungicides significantly increased several soil enzyme and nitrification activities, leading to a higher GMean index as compared to the non-treated control soils. The microbial activity responses depended on the type of activity (nitrification was the most sensitive one), on the fungicide (Swing® Gold or Cuprafor Micro®), and on the doses. The SQI indices revealed higher effects of both fungicides on the soil microbial activity in the absence of earthworms. The presence of earthworms enhanced all soil microbial activities in both the control and fungicide-contaminated soils. Moreover, the magnitude of the fungicide impact, integrated through the SQI index, was mitigated by the presence of earthworms, conferring a higher stability of microbial functional diversity. Our results highlight the importance of biotic interactions in the response of indicators of soil functioning (i.e., microbial activity) to pesticides.

Biochem-Env: a platform of biochemistry for research in environmental and agricultural sciences.

Biochemical indicators are potent tools to assess ecosystem functioning under anthropic and global pressures. Nevertheless, additional work is needed to improve the methods used for the measurement of these indicators, and for a more relevant interpretation of the obtained results. To face these challenges, the platform Biochem-Env aims at providing innovative and standardized measurement protocols, as well as database and information system favoring result interpretation and opening. Its skills and tools are also offered for expertise, consulting, training, and standardization. In addition, the platform is a service of a French Research Infrastructure for Analysis and Experimentation on Ecosystems, for research in environmental and agricultural sciences.

Oxidoreductases provide a more generic response to metallic stressors (Cu and Cd) than hydrolases in soil fungi: new ecotoxicological insights.

The present study investigates the effect of metals on the secretion of enzymes from 12 fungal strains maintained in liquid cultures. Hydrolases (acid phosphatase, ß-glucosidase, ß-galactosidase, and N-acetyl-ß-glucosaminidase) and ligninolytic oxidoreductases (laccase, Mn, and lignin peroxidases) activities, as well as biomass production, were measured in culture fluids from fungi exposed to Cu or Cd. Our results showed that all fungi secreted most of the selected hydrolases and that about 50% of them produced a partial oxidative system in the absence of metals. Then, exposure of fungi to metals led to the decrease in biomass production. At the enzymatic level, Cu and Cd modified the secretion profiles of soil fungi. The response of hydrolases to metals was contrasted and complex and depended on metal, enzyme, and fungal strain considered. By contrast, the metals always stimulated the activity of ligninolytic oxidoreductases in fungal strains. In some of them, oxidoreductases were specifically produced following metal exposure. Fungal oxidoreductases provide a more generic response than hydrolases, constituting thus a physiological basis for their use as biomarkers of metal exposure in soils.

A coordinated set of ecosystem research platforms open to international research in ecotoxicology, AnaEE-France.

The infrastructure for Analysis and Experimentation on Ecosystems (AnaEE-France) is an integrated network of the major French experimental, analytical, and modeling platforms dedicated to the biological study of continental ecosystems (aquatic and terrestrial). This infrastructure aims at understanding and predicting ecosystem dynamics under global change. AnaEE-France comprises complementary nodes offering access to the best experimental facilities and associated biological resources and data: Ecotrons, seminatural experimental platforms to manipulate terrestrial and aquatic ecosystems, in natura sites equipped for large-scale and long-term experiments. AnaEE-France also provides shared instruments and analytical platforms dedicated to environmental (micro) biology. Finally, AnaEE-France provides users with data bases and modeling tools designed to represent ecosystem dynamics and to go further in coupling ecological, agronomical, and evolutionary approaches. In particular, AnaEE-France offers adequate services to tackle the new challenges of research in ecotoxicology, positioning its various types of platforms in an ecologically advanced ecotoxicology approach. AnaEE-France is a leading international infrastructure, and it is pioneering the construction of AnaEE (Europe) infrastructure in the field of ecosystem research. AnaEE-France infrastructure is already open to the international community of scientists in the field of continental ecotoxicology.

Is there a relationship between earthworm energy reserves and metal availability after exposure to field-contaminated soils?

Generic biomarkers are needed to assess environmental risks in metal polluted soils. We assessed the strength of the relationship between earthworm energy reserves and metal availability under conditions of cocktail of metals at low doses and large range of soil parameters. Aporrectodea caliginosa was exposed in laboratory to a panel of soils differing in Cd, Pb and Zn total and available (CaCl2 and EDTA-extractable) concentrations, and in soil texture, pH, CEC and organic-C. Glycogen, protein and lipid contents were recorded in exposed worms. Glycogen contents were not linked to the explaining variables considered. Variable selection identified CaCl2 extractable metals concentrations and soil texture as the main factors affecting protein and lipid contents. The results showed opposite effects of Pb and Zn, high inter-individual variability of biomarkers and weak relationships with easily extractable metals. Our results support the lack of genericity of energy reserves in earthworms exposed to field-contaminated soils.

ISTA 14--impact of antibiotics from pig slurry on soil microbial communities, including the basidiomycete Trametes versicolor.

Livestock slurry containing antibiotics is a source of contamination of agricultural soils, with possible effects on soil function and micro-organisms. Extracellular oxido-reductases and hydrolases from the fungus T. versicolor and fungal growth were monitored in liquid cultures in the presence of tetracycline, lincomycine, sulfadiazine and ciprofloxacin for 10 days, in order to assess the suitability of these enzymes as biomarkers. Among the conditions of treatment, statistical analysis demonstrated an increase in manganese-dependent peroxidase after exposure to sulfadiazine at 1 mg/L when compared with the control. Acid phosphatase activity was decreased by lincomycine at 1 or 10 mg/L. Conversely, ß-glucosidase activity increased in the presence of this antibiotic at 10 mg/L. In Terrestrial Model Ecosystems spiked with contaminated pig slurry, lincomycine at the concentration of 8 or 80 µg/kg dry soil, and ciprofloxacin at 250 ng/kg dry soil decreased the activity of soil dehydrogenase, when compared with a green slurry treatment, over 28-day incubations. Laccase activity was similarly decreased in the presence of the highest concentration of antibiotics. We determined bacterial and fungal biomasses using Q-PCR. Bacterial biomass was increased in the presence of lincomycine at 80 µg/kg whatever the time of exposure, and to a lesser extent in the presence of ciprofloxacin at 250 ng/kg, but only at day 28. In contrast, both antibiotics, whatever their concentrations, did not modify fungal biomass in soil. In conclusion, we were unable to demonstrate important effects of antibiotics at concentrations found in the agricultural environment.

Secretion profiles of fungi as potential tools for metal ecotoxicity assessment: a study of enzymatic system in Trametes versicolor.

The relationship between the expression of extracellular enzymatic system and a metal stress is scarce in fungi, hence limiting the possible use of secretion profiles as tools for metal ecotoxicity assessment. In the present study, we investigated the effect of Zn, Cu, Pb and Cd, tested alone or in equimolar cocktail, on the secretion profiles at enzymatic and protein levels in Trametesversicolor. For that purpose, extracellular hydrolases (acid phosphatase, ß-glucosidase, ß-galactosidase and N-acetyl-ß-glucosaminidase) and ligninolytic oxidases (laccase, Mn-peroxidase) were monitored in liquid cultures. Fungal secretome was analyzed by electrophoresis and laccase secretion was characterized by western-blot and mass spectrometry analyses. Our results showed that all hydrolase activities were inhibited by the metals tested alone or in cocktail, whereas oxidase activities were specifically stimulated by Cu, Cd and metal cocktail. At protein level, metal exposure modified the electrophoretic profiles of fungal secretome and affected the diversity of secreted proteins. Two laccase isoenzymes, LacA and LacB, identified by mass spectrometry were differentially glycosylated according to the metal exposure. The amount of secreted LacA and LacB was strongly correlated with the stimulation of laccase activity by Cu, Cd and metal cocktail. These modifications of extracellular enzymatic system suggest that fungal oxidases could be used as biomarkers of metal exposure.

The ecto-enzyme CD38 is a nicotinic acid adenine dinucleotide phosphate (NAADP) synthase that couples receptor activation to Ca2+ mobilization from lysosomes in pancreatic acinar cells.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca(2+)-mobilizing intracellular messenger and is linked to a variety of stimuli and cell surface receptors. However, the enzyme responsible for endogenous NAADP synthesis in vivo is unknown, and it has been proposed that another enzyme differing from ADP-ribosyl cyclase family members may exist. The ecto-enzyme CD38, involved in many functions as diverse as cell proliferation and social behavior, represents an important alternative. In pancreatic acinar cells, the hormone cholecystokinin (CCK) stimulates NAADP production evoking Ca(2+) signals by discharging acidic Ca(2+) stores and leading to digestive enzyme secretion. From cells derived from CD38(-/-) mice, we provide the first physiological evidence that CD38 is required for endogenous NAADP generation in response to CCK stimulation. Furthermore, CD38 expression in CD38-deficient pancreatic AR42J cells remodels Ca(2+)-signaling pathways in these cells by restoring Ca(2+) mobilization from lysosomes during CCK-induced Ca(2+) signaling. In agreement with an intracellular site for messenger synthesis, we found that CD38 is expressed in endosomes. These CD38-containing vesicles, likely of endosomal origin, appear to be proximal to lysosomes but not co-localized with them. We propose that CD38 is an NAADP synthase required for coupling receptor activation to NAADP-mediated Ca(2+) release from lysosomal stores in pancreatic acinar cells.

Exploring the regulation of the expression of ChAT and VAChT genes in NG108-15 cells: implication of PKA and PI3K signaling pathways.

Involvement of different protein kinases regulated by cAMP and implication of muscarinic receptors in the regulation of choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) mRNA levels and ChAT activity has been studied in NG108-15 cells. Dibutyryl cAMP enhanced both ChAT and VAChT mRNA levels and stimulated ChAT activity. Muscarinic stimulation or inhibition did not change ChAT activity or the receptor subtype mRNA pattern. MEK1/2 did not affect the regulation of ChAT and VAChT mRNA levels. However, PKA plays a major role in regulating ChAT and VAChT mRNA levels, because H89 decreased both. Strikingly, inhibition of PI3K by LY294002 had two opposite effects: ChAT mRNA level was decreased and VAChT mRNA level was increased. Such a result consolidates the observation that ChAT and VAChT genes, despite their unusual organization in a single "cholinergic locus," can be differentially or synergistically regulated, depending on the activated signaling pathways.