International audience | Medicago sativa was cultivated at a former harbor facility near Bordeaux (France) to phytomanage a soil contaminated by trace elements (TE) and polycyclic aromatic hydrocarbons (PAH). In parallel, a biotest with Phaseolus vulgaris was carried out on potted soils from 18 sub-sites to assess their phytotoxicity. Total soil TE and PAH concentrations, TE concentrations in the soil pore water, the foliar ionome of M. sativa (at the end of the first growth season) and of Populus nigra growing in situ, the root and shoot biomass and the foliar ionome of P. vulgaris were determined. Despite high total soil TE, soluble TE concentrations were generally low, mainly due to alkaline soil pH (7.8–8.6). Shoot dry weight (DW) yield and foliar ionome of P. vulgaris did not reflect the soil contamination, but its root DW yield decreased at highest soil TE and/or PAH concentrations. Foliar ionomes of M. sativa and P. nigra growing in situ were generally similar to the ones at uncontaminated sites. M. sativa contributed to bioavailable TE stripping by shoot removal (in g ha−1 harvest−1): As 0.9, Cd 0.3, Cr 0.4, Cu 16.1, Ni 2.6, Pb 4, and Zn 134. After 1 year, 72 plant species were identified in the plant community across three subsets: (I) plant community developed on bare soil sowed with M. sativa; (II) plant community developed in unharvested plots dominated by grasses; and (III) plant community developed on unsowed bare soil. The shoot DW yield (in mg ha−1 harvest−1) varied from 1.1 (subset I) to 6.9 (subset II). For subset III, the specific richness was the lowest in plots with the highest phytotoxicity for P. vulgaris.

Medicago sativa was cultivated at a former harbor facility near Bordeaux (France) to phytomanage a soil contaminated by trace elements (TE) and polycyclic aromatic hydrocarbons (PAH). In parallel, a biotest with Phaseolus vulgaris was carried out on potted soils from 18 sub-sites to assess their phytotoxicity. Total soil TE and PAH concentrations, TE concentrations in the soil pore water, the foliar ionome of M. sativa (at the end of the first growth season) and of Populus nigra growing in situ, the root and shoot biomass and the foliar ionome of P. vulgaris were determined. Despite high total soil TE, soluble TE concentrations were generally low, mainly due to alkaline soil pH (7.8–8.6). Shoot dry weight (DW) yield and foliar ionome of P. vulgaris did not reflect the soil contamination, but its root DW yield decreased at highest soil TE and/or PAH concentrations. Foliar ionomes of M. sativa and P. nigra growing in situ were generally similar to the ones at uncontaminated sites. M. sativa contributed to bioavailable TE stripping by shoot removal (in g ha⁻¹ harvest⁻¹): As 0.9, Cd 0.3, Cr 0.4, Cu 16.1, Ni 2.6, Pb 4, and Zn 134. After 1 year, 72 plant species were identified in the plant community across three subsets: (I) plant community developed on bare soil sowed with M. sativa; (II) plant community developed in unharvested plots dominated by grasses; and (III) plant community developed on unsowed bare soil. The shoot DW yield (in mg ha⁻¹ harvest⁻¹) varied from 1.1 (subset I) to 6.9 (subset II). For subset III, the specific richness was the lowest in plots with the highest phytotoxicity for P. vulgaris.

Most of the detrimental effects of using conventional insecticides to control crop pests are now well identified and are nowadays major arguments for replacing such compounds by the use of biological control agents. In this respect, the bacterium Bacillus thuringiensis var. kurstaki and Trichogramma (Hymenoptera: Trichogrammatidae) parasitic wasp species are both effective against lepidopterous pests and can actually be used concomitantly. In this work, we studied the potential side effects of B. thuringiensis var. kurstaki on Trichogramma chilonis females. We first evidenced an acute toxicity of B. thuringiensis on T. chilonis. Then, after ingestion of B. thuringiensis at sublethal doses, we focused on life history traits of T. chilonis such as longevity, reproductive success and the time spent on host eggs patches. The reproductive success of T. chilonis was not modified by B. thuringiensis while a significant effect was observed on longevity and the time spent on host eggs patches. The physiological and ecological meanings of the results obtained are discussed.

Most of the detrimental effects of using conventional insecticides to control crop pests are now well identified and are nowadays major arguments for replacing such compounds by the use of biological control agents. In this respect, the bacterium Bacillus thuringiensis var. kurstaki and Trichogramma (Hymenoptera: Trichogrammatidae) parasitic wasp species are both effective against lepidopterous pests and can actually be used concomitantly. In this work, we studied the potential side effects of B. thuringiensis var. kurstaki on Trichogramma chilonis females. We first evidenced an acute toxicity of B. thuringiensis on T. chilonis. Then, after ingestion of B. thuringiensis at sublethal doses, we focused on life history traits of T. chilonis such as longevity, reproductive success and the time spent on host eggs patches. The reproductive success of T. chilonis was not modified by B. thuringiensis while a significant effect was observed on longevity and the time spent on host eggs patches. The physiological and ecological meanings of the results obtained are discussed.

International audience | Environmental controls of 2-methyl-4-chlorophenoxyacetic acid (MCPA) degradation are poorly understood. We investigated whether microbial MCPA degraders are stimulated by (maize) litter and whether this process depends on concentrations of MCPA and litter. In a microcosm experiment, different amounts of litter (0, 10 and 20 g kg(-1)) were added to soils exposed to three levels of the herbicide (0, 5 and 30 mg kg(-1)). The treated soils were incubated at 20 A degrees C for 6 weeks, and samples were taken after 1, 3 and 6 weeks of incubation. In soils with 5 mg kg(-1) MCPA, about 50 % of the MCPA was dissipated within 1 week of the incubation. Almost complete dissipation of the herbicide had occurred by the end of the incubation with no differences between the three litter amendments. At the higher concentration (30 mg kg(-1)), MCPA endured longer in the soil, with only 31 % of the initial amount being removed at the end of the experiment in the absence of litter. Litter addition greatly increased the dissipation rate with 70 and 80 % of the herbicide being dissipated in the 10 and 20 g kg(-1) litter treatments, respectively. Signs of toxic effects of MCPA on soil bacteria were observed from related phospholipid fatty acid (PLFA) analyses, while fungi showed higher tolerance to the increased MCPA levels. The abundance of bacterial tfdA genes in soil increased with the co-occurrence of litter and high MCPA concentration, indicating the importance of substrate availability in fostering MCPA-degrading bacteria and thereby improving the potential for removal of MCPA in the environment.

Environmental controls of 2-methyl-4-chlorophenoxyacetic acid (MCPA) degradation are poorly understood. We investigated whether microbial MCPA degraders are stimulated by (maize) litter and whether this process depends on concentrations of MCPA and litter. In a microcosm experiment, different amounts of litter (0, 10 and 20 g kg⁻¹) were added to soils exposed to three levels of the herbicide (0, 5 and 30 mg kg⁻¹). The treated soils were incubated at 20 °C for 6 weeks, and samples were taken after 1, 3 and 6 weeks of incubation. In soils with 5 mg kg⁻¹ MCPA, about 50 % of the MCPA was dissipated within 1 week of the incubation. Almost complete dissipation of the herbicide had occurred by the end of the incubation with no differences between the three litter amendments. At the higher concentration (30 mg kg⁻¹), MCPA endured longer in the soil, with only 31 % of the initial amount being removed at the end of the experiment in the absence of litter. Litter addition greatly increased the dissipation rate with 70 and 80 % of the herbicide being dissipated in the 10 and 20 g kg⁻¹ litter treatments, respectively. Signs of toxic effects of MCPA on soil bacteria were observed from related phospholipid fatty acid (PLFA) analyses, while fungi showed higher tolerance to the increased MCPA levels. The abundance of bacterial tfdA genes in soil increased with the co-occurrence of litter and high MCPA concentration, indicating the importance of substrate availability in fostering MCPA-degrading bacteria and thereby improving the potential for removal of MCPA in the environment.

[Departement_IRSTEA]Eaux [TR1_IRSTEA]ARCEAU | International audience | Subsurface lateral flow in both texture-contrast soils and catchments with shallow bedrock is suspected to be a non-point source of contamination of watercourses by pesticides used in agriculture. As a case study, the north of the Beaujolais region (eastern France) provides a favorable environment for such contamination due to its agro-pedoclimatic conditions. Environments seen in the Beaujolais region include intense viticulture, permeable and shallow soils, steep hillslopes, and storms that occur during the periods of pesticide application. Watercourse contamination by pesticides has been widely observed in this region, and offsite pesticide transport by subsurface lateral flow is suspected to be involved in diffuse and chronic presence of pesticides in surface water. In order to confirm and quantify the potential role of such processes in pesticide transfer, an automated trench system has been designed. The trench was set up on a steep farmed hillslope in a texture-contrast soil. It was equipped with a tipping bucket flow meter and an automatic sampler to monitor pesticide concentrations in lateral flow at fine resolution, by means of a flow-dependent sampling strategy. Four pesticides currently used in vine growing were studied to provide a range of mobility properties: one insecticide (chlorpyrifos-methyl) and three fungicides (spiroxamine, tebuconazole, and dimethomorph). With this system, it was possible to study pesticide concentration dynamics in the subsurface lateral flow, generated by substantial rainfall events following pesticide applications. The experimental design ascertained to be a suitable method in which to monitor subsurface lateral flow and related transfer of pesticides.

Subsurface lateral flow in both texture-contrast soils and catchments with shallow bedrock is suspected to be a non-point source of contamination of watercourses by pesticides used in agriculture. As a case study, the north of the Beaujolais region (eastern France) provides a favorable environment for such contamination due to its agro-pedo-climatic conditions. Environments seen in the Beaujolais region include intense viticulture, permeable and shallow soils, steep hillslopes, and storms that occur during the periods of pesticide application. Watercourse contamination by pesticides has been widely observed in this region, and offsite pesticide transport by subsurface lateral flow is suspected to be involved in diffuse and chronic presence of pesticides in surface water. In order to confirm and quantify the potential role of such processes in pesticide transfer, an automated trench system has been designed. The trench was set up on a steep farmed hillslope in a texture-contrast soil. It was equipped with a tipping bucket flow meter and an automatic sampler to monitor pesticide concentrations in lateral flow at fine resolution, by means of a flow-dependent sampling strategy. Four pesticides currently used in vine growing were studied to provide a range of mobility properties: one insecticide (chlorpyrifos-methyl) and three fungicides (spiroxamine, tebuconazole, and dimethomorph). With this system, it was possible to study pesticide concentration dynamics in the subsurface lateral flow, generated by substantial rainfall events following pesticide applications. The experimental design ascertained to be a suitable method in which to monitor subsurface lateral flow and related transfer of pesticides.

International audience | Impacts of subsurface biogeochemical processes over time have always been a concern for the long-term performance of zero valent iron (Fe0) based Permeable Reactive Barriers (PRB’s). To evaluate the biogeochemical impacts, laboratory experiments were performed using flow-through glass columns for 210 days at controlled temperature (20 °C). Two different particle size of Fe0 were used in the columns, and to simulate indigenous microbial activity, extra carbon source was provided in two columns (biotic columns) and remaining two columns were kept abiotic. Heavy metals (Zn, As) were removed efficiently in all the columns, and no exhaustion of treatment capability or clogging were observed during our experimental duration. Newly formed Fe- mineral phases and precipitates were characterized using XRD, SEM-EDX and micro-XRF techniques in solid phase at the end of the experiment. In addition, 16S rRNA gene extraction was used for microbial community identification in biotic columns. During the incubation, microbial population shifted in favour of Desulforsporosinus species (sulfate-reducing bacteria) from initial dominance of Acidithiobacillus ferrooxidans in sediments. Dominant mineral phases detected in biotic columns were mackinawite (FeS) and sulfate green-rust while in abiotic columns magnetite/maghemite phases were more prevalent.

Impacts of subsurface biogeochemical processes over time have always been a concern for the long-term performance of zero valent iron (Fe⁰)-based permeable reactive barriers (PRBs). To evaluate the biogeochemical impacts, laboratory experiments were performed using flow-through glass columns for 210 days at controlled temperature (20 °C). Two different particle sizes of Fe⁰ were used in the columns, and to simulate indigenous microbial activity, extra carbon source was provided in the two columns (biotic columns) and the remaining two columns were kept abiotic using gamma radiations. Heavy metals (Zn, As) were removed efficiently in all the columns, and no exhaustion of treatment capability or clogging was observed during our experimental duration. Newly formed Fe mineral phases and precipitates were characterized using X-ray diffraction (XRD), scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDX), and micro-XRF techniques in solid phase at the end of the experiment. In addition, 16S rRNA gene extraction was used for microbial community identification in biotic columns. During the incubation, microbial population shifted in favor of Desulfosporosinus species (sulfate-reducing bacteria) from initial dominance of Acidithiobacillus ferrooxidans in sediments. Dominant mineral phases detected in biotic columns were mackinawite (FeS) and sulfate green rust, while in abiotic columns, magnetite/maghemite phases were more prevalent.

Avec nos remerciements à Jodie Thénard, Virginie Grondin, Jean-Pierre Pétraud, Amélie Trouvé, Christelle Marrauld et Françoise Poiroux | Earthworms play a key role in agroecosystem soil processes. This study aims to assess the effects of different doses of a commercial formulation of epoxiconazole (Opus®), a persistent and widely used fungicide, on the earthworm Aporrectodea icterica. A laboratory study was conducted in a natural soil in order to measure effects of Opus® on earthworm mortality, uptake, weight gain, enzymatic activities (catalase and glutathione-S-transferase), and energy resources (lipids and glycogens). The estimated LC50 was 45.5 mg kg−1, or 268 times the recommended dose. Weight gains were 28, 19, and 13 % of the initial weight after 28 days of exposure in the control and D1 and D10 (1 and 10 times the recommended dose) treatments, respectively. No difference was observed for catalase activity between the three treatments, at 7, 14, or 28 days. The glutathion-S-transferase (GST) activity was two times as high in D1 as in D0 at 14 days. At 28 days, glycogen concentration was lower in D10 than in the D1 treatment. This study highlighted moderate sublethal effects of the commercial formulation Opus® for earthworms. Considering that these effects were observed on a species found in cultivated fields, even at recommended rates, much more attention should be paid to this pesticide.

Avec nos remerciements à Jodie Thénard, Virginie Grondin, Jean-Pierre Pétraud, Amélie Trouvé, Christelle Marrauld et Françoise Poiroux | Earthworms play a key role in agroecosystem soil processes. This study aims to assess the effects of different doses of a commercial formulation of epoxiconazole (Opus®), a persistent and widely used fungicide, on the earthworm Aporrectodea icterica. A laboratory study was conducted in a natural soil in order to measure effects of Opus® on earthworm mortality, uptake, weight gain, enzymatic activities (catalase and glutathione-S-transferase), and energy resources (lipids and glycogens). The estimated LC50 was 45.5 mg kg−1, or 268 times the recommended dose. Weight gains were 28, 19, and 13 % of the initial weight after 28 days of exposure in the control and D1 and D10 (1 and 10 times the recommended dose) treatments, respectively. No difference was observed for catalase activity between the three treatments, at 7, 14, or 28 days. The glutathion-S-transferase (GST) activity was two times as high in D1 as in D0 at 14 days. At 28 days, glycogen concentration was lower in D10 than in the D1 treatment. This study highlighted moderate sublethal effects of the commercial formulation Opus® for earthworms. Considering that these effects were observed on a species found in cultivated fields, even at recommended rates, much more attention should be paid to this pesticide.

Evaluation of epoxiconazole bioavailability in soil to the earthworm Aporrectodea icterica

In soil, the determination of total concentration using an exhaustive extraction method has little relevance to evaluate the exposure of an organism to a chemical, because of sorption processes. This study aims to propose a mild extraction method to evaluate the bioavailability of the fungicide epoxiconazole to the earthworm Aporrectodea icterica. Experiments were conducted in soils presenting various textures and organic carbon contents, spiked with formulated epoxiconazole 7 to 56 days prior to their extraction. In parallel, the epoxiconazole concentration was determined in exposed earthworms and the fungicide’s effects were evaluated by measuring weight gain, enzymatic activities and total protein contents. Among the various mild chemical solvents tested to evaluate the environmental availability of the fungicide, the 50 mM hydroxypropyl-β-cyclodextrin solution allowed to extract around 30 % of epoxiconazole. This percentage corresponded to the ratio determined in exposed A. icterica under similar soil conditions. Furthermore, this mild method was demonstrated to be sensitive to soil sorption capacities and to ageing. The mild extraction method was then applied to explore the relationship between total and (bio)available concentrations in soil and in A. icterica, over 7- or 28-day exposure time. This demonstrated the proportionality between epoxiconazole concentration in earthworm and available in soil (up to 96 %, with regression coefficient R ² = 0.98). Sublethal effects on earthworm remained not significant.

International audience

Phytoextraction of Cd is a growing biotechnology although we currently know few Cd hyperaccumulators, i.e., plant species able to accumulate at least 0.1 mg Cd g⁻¹ dry weight in aerial organs. Owing their deep root system and high biomass, trees are more and more preferred to herbaceous species for phytoextraction. Assuming that conifers could be relevant models under cold climates, we investigated cadmium tolerance of the hybrid larch Larix × eurolepis Henry (Larix decidua × Larix kaempferi) and the efficiency of this species to store this metal. In vitro grown larches were chosen in order to reduce time of exposure and to more rapidly evaluate their potential efficiency to accumulate Cd. One-month-old plantlets were exposed for 2 and 4 weeks to 250 and 500 μM Cd. Results showed that they tolerated a 4-week exposure to 250 μM Cd, whereas the content of photosynthetic pigment strongly dropped in plantlets growing in the presence of 500 μM Cd. In the presence of 250 μM Cd, shoot growth slightly decreased but photosynthetic pigment and total soluble carbohydrate contents were not modified and no lipid peroxidation was detected. In addition, these plantlets accumulated proline, particularly in shoots (two to three times more than control). In roots, Cd concentration in the intracellular fraction was always higher than in the cell wall fraction contrary to shoots where Cd concentration in the cell wall fraction increased with time and Cd concentration in the medium. In shoots, Cd concentration was lower than in roots with a ratio of 0.2 after 4 weeks of exposure but stayed around 0.2 mg g⁻¹ dry weight, thus a value higher than the threshold requested for Cd hyperaccumulators. Hybrid larch would thus be a relevant candidate for field test of Cd phytoextraction.

Phytoextraction of Cd is a growing biotechnology although we currently know few Cd-hyperaccumulators, i.e. plant species able to accumulate at least 0.1 mg Cd g-1 dry weight in aerial organs. Owing their deep root system and high biomass, trees are more and more preferred to herbaceous species for phytoextraction. Assuming that conifers could be relevant models under cold climates, we investigated cadmium tolerance of the hybrid larch Larix × eurolepis Henry (L. decidua × L. kaempferi) and the efficiency of this species to store this metal. In vitro grown larches were chosen in order to reduce time of exposure and to more rapidly evaluate their potential efficiency to accumulate Cd. One-month-old plantlets were exposed for 2 and 4 weeks to 250 and 500 µM Cd. Results showed that they tolerated a four-week exposure to 250 µM Cd whereas the content of photosynthetic pigment strongly dropped in plantlets growing in presence of 500 µM Cd. In presence of 250 µM Cd, shoot growth slightly decreased but photosynthetic pigment and total soluble carbohydrate contents were not modified and no lipid peroxidation was detected. In addition, these plantlets accumulated proline, particularly in shoots (2 - 3 times more than control). In roots, Cd concentration in the intracellular fraction was always higher than in the cell wall fraction contrary to shoots where Cd concentration in the cell wall fraction increased with time and Cd concentration in the medium. In shoots, Cd concentration was lower than in roots with a ratio of 0.2 after 4 weeks of exposure but stayed around 0.2 mg g-1 dry weight, thus a value higher than the threshold requested for Cd-hyperaccumulators. Hybrid larch would thus be a relevant candidate for field test of Cd-phytoextraction.

International audience | Phytoextraction of Cd is a growing biotechnology although we currently know few Cd-hyperaccumulators, i.e. plant species able to accumulate at least 0.1 mg Cd g-1 dry weight in aerial organs. Owing their deep root system and high biomass, trees are more and more preferred to herbaceous species for phytoextraction. Assuming that conifers could be relevant models under cold climates, we investigated cadmium tolerance of the hybrid larch Larix × eurolepis Henry (L. decidua × L. kaempferi) and the efficiency of this species to store this metal. In vitro grown larches were chosen in order to reduce time of exposure and to more rapidly evaluate their potential efficiency to accumulate Cd. One-month-old plantlets were exposed for 2 and 4 weeks to 250 and 500 µM Cd. Results showed that they tolerated a four-week exposure to 250 µM Cd whereas the content of photosynthetic pigment strongly dropped in plantlets growing in presence of 500 µM Cd. In presence of 250 µM Cd, shoot growth slightly decreased but photosynthetic pigment and total soluble carbohydrate contents were not modified and no lipid peroxidation was detected. In addition, these plantlets accumulated proline, particularly in shoots (2 - 3 times more than control). In roots, Cd concentration in the intracellular fraction was always higher than in the cell wall fraction contrary to shoots where Cd concentration in the cell wall fraction increased with time and Cd concentration in the medium. In shoots, Cd concentration was lower than in roots with a ratio of 0.2 after 4 weeks of exposure but stayed around 0.2 mg g-1 dry weight, thus a value higher than the threshold requested for Cd-hyperaccumulators. Hybrid larch would thus be a relevant candidate for field test of Cd-phytoextraction.