Co-exposure to environmental microplastic and the pesticide 2,4-dichlorophenoxyacetic acid (2,4-D) induce distinctive alterations in the metabolome and microbial community structure in the gut of the earthworm Eisenia andrei.

Microplastics (MPs) are recognized as emergent pollutants and have become a significant environmental concern, especially when combined with other contaminants. In this study, earthworms, specifically Eisenia andrei, were exposed to MPs (at a concentration of 10 µg kg-1 of soil), herbicide 2,4-D (7 mg kg-1 of soil), and a combination of the two for 7 and 14 days. The chemical uptake in the earthworms was measured, and the bacterial and archaeal diversities in both the soil and earthworm gut were analyzed, along with the metabolomic profiles. Additionally, data integration of the two omics approaches was performed to correlate changes in gut microbial diversity and the different metabolites. Our results demonstrated that earthworms ingested MPs and increased 2,4-D accumulation. More importantly, high-throughput sequencing revealed a shift in microbial diversity depending on single or mixture exposition. Metabolomic data demonstrated an important modulation of the metabolites related to oxidative stress, inflammatory system, amino acids synthesis, energy, and nucleic acids metabolism, being more affected in case of co-exposure. Our investigation revealed the potential risks of MPs and 2,4-D herbicide combined exposure to earthworms and soil fertility, thus broadening our understanding of MPs' toxicity and impacts on terrestrial environments.

Heavy metal accumulation, biochemical and transcriptomic biomarkers in earthworms Eisenia andrei exposed to industrially contaminated soils from south-eastern Tunisia (Gabes Governorate).

Heavy metal pollution is causing harmful consequences on soil fertility, and earthworms are frequently employed as test organisms to evaluate the ecotoxicity of polluted soils. In this study, Eisenia andrei was exposed for 7 and 14 days to polymetallic contaminated soils collected from an industrial zone in the south-eastern Tunisia. Earthworm growth, heavy metal accumulation, genotoxicity, cytotoxicity, biochemical and transcriptional responses were determined. Results revealed a higher accumulation of heavy metals in earthworms after 14 than 7 days of soil exposure, a reduction in lysosomal membrane stability (LMS), besides an increase in micronuclei frequency (MN). Moreover, earthworm oxidative status was affected in terms of increases in malondialdehyde (MDA) and metallothionein (MTs) content, and enhancement of catalase (CAT) and glutathione-S-transferase (GST) activities. An inhibition of acetylcholinesterase (AChE) activity was also observed in treated earthworms, whereas transcriptional data demonstrated an up-regulation of cat, gst, mt, p21 and topoisomerase genes. Overall, these findings support the use of earthworms as suitable bioindicator species for pollution monitoring and assessment, advance our understanding of the interaction between heavy metals and earthworms, and provide valuable information about the harmful impact of biota exposure to naturally contaminated soils.

Combined toxicity of Cd and 2,4-dichlorophenoxyacetic acid on the earthworm Eisenia andrei under biochar amendment.

Due to anthropogenic activities, various pollutants can be found in agricultural soil, such as cadmium (Cd) and 2,4-dichlorophenoxyacetic acid (2,4-D). They are highly toxic and can have a negative impact on soil fertility. For remediation strategies, biochar has acquired considerable attention due to its benefits for agriculture. However, we should recognize the ecological risk posed by biochar use. In addition, little is known about its non-desirable effects on soil organisms such as earthworms, especially in the case of soil remediation. In this study, earthworms (Eisenia andrei) were exposed to soil contaminated with Cd (0.7 mg/kg), (2,4-D) (7 mg/kg), and a mixture of the two in the presence and absence of biochar (2%). A 7- and 14-day incubation experiment was carried out for this purpose. Cd and 2,4-D uptakes in earthworms' tissues, oxidative stress, cytotoxic response, DNA damage, histopathological changes, and gene expression level were assessed. Results suggested that biochar increased the bioavailability of Cd and 2,4-D and the frequency of micronuclei (MNi) and decreased the lysosomal membrane stability (LMS) in earthworms. Also, histopathological examination detected numerous alterations in animals exposed to the contaminants without any amelioration when biochar was added. The biochemical response of earthworms in terms of oxidative stress demonstrates that in the presence of biochar, animals tend to alleviate the toxicity of Cd and 2,4-D. This was also supported by transcriptomic analyses where expression gene levels related to oxidative stress were upregulated in earthworms exposed to Cd and 2,4-D + biochar. The present investigation brought new insights concerning the use of biochar in agriculture.

The Effect of Nickel Exposure on Oxidative Stress of Vicia faba Plants.

Heavy metal contamination is a serious threat for terrestrial ecosystems. Thus, they could be accumulated in living organisms leading consequently to harmful consequences. In this context, the present work aims to evaluate the effects of four increasing Nickel (Ni) nominal concentrations (T: 0 mg/kg, C1: 150 mg/kg, C2: 250 mg/kg, C3: 500 mg/kg) on agronomic and biochemical parameters in bean (Vicia faba) plants. The measured exposure concentrations were in the range of 96.69%-104.18% of the nominal concentrations. Bean's responses were evaluated at biometric levels, chlorophyll content and biochemical parameters namely catalase glutation-S-transferase activities and malondialdehyde content, in booth parts of plants. Our data revealed a marked negative effect of Ni exposure on bean plant development and chlorophyll content. Biochemical biomarkers reported that plants anti-oxidative defense system has been significantly affected specially in roots at the high Ni concentration. Briefly, resistance mechanisms of Vicia faba to Ni seem to imply an activation of the antioxidant system and a limitation of the reactive oxygen species.

Interactive effects of environmental microplastics and 2,4-dichlorophenoxyacetic acid (2,4-D) on the earthworm Eisenia andrei.

Given the wide use of plastic and pesticides in agriculture, microplastics (MP) and the herbicide 2,4 dichloro-phenoxy-acetic acid (2-4-D) can be present simultaneously in soil. Nevertheless, little is known about their combined toxicity. In this study, Eisenia andrei was exposed to environmental MP (100 µg kg-1 soil) and 2,4-D (7 mg kg-1 soil) for 7 and 14 days. Bioaccumulation, genotoxicity, oxidative stress and gene expression level were assessed. Results revealed that MP increased 2,4-D bioaccumulation in earthworms. Simultaneous exposure to both these pollutants caused a significant reduction in lysosomal membrane stability (LMS) and an increase in micronuclei (MNi) frequency. Biochemical analysis revealed oxidative alterations in earthworms exposed to all treatments; being very pronounced in earthworms exposed to the mixture in terms of increase in glutathione-S-Transferase (GST), catalase (CAT) and malondialdehydes accumulation (MDA). Furthermore, an up-regulation in cat and gst expression level was recorded in worms exposed to single or mixture treatment, except MP in case of gst. Our data highlight the toxicity of the combined exposure to MP and 2,4-D and afford new insights into the potential ecological risks posed by MP in terrestrial ecosystems.

Assessing the presence of microplastic particles in Tunisian agriculture soils and their potential toxicity effects using Eisenia andrei as bioindicator.

In the present study, we investigated microplastics (MPs) in agricultural soils with different agronomic practices (organic farming, soil under greenhouses, soil under mulching and soil irrigated with treated wastewater (TWW)). Plastic particles from each site were collected and characterized by FTIR and Raman microspectroscopy. Plastic particles were then ground and added (size rage under 100 µm) at a concentration of 100 µg kg-1 to soils from organic farming containing Eisenia andrei for 7 and 14 days. MPs accumulation in earthworms was quantified and characterized. Oxidative stress was assessed by evaluating the activities of catalase, glutathione-S-transferase, and acetylcholinesterase as well as malondialdehyde accumulation. Our results revealed higher quantity of MPs in soils ranging from 13.21 ± 0.89 to 852.24 ± 124.2 items kg-1 with the dominance of small sizes (0.22-1.22 µm). Polyethylene (PE) and polybutyrate adipate terephtalate (PBAT) were the dominant MPs. Moreover, our results revealed a significant ingestion of MPs in earthworms with values ranging from 1.13 to 35.6, characterized mostly by PE, PBAT and polypropylene (PP). Biochemical data revealed an important alteration in worms exposed to MPs from soils with mulching and irrigated with TWW. Our study provides new insights into the effects of microplastic in earthworms and thus the vulnerability of terrestrial ecosystem to this emergent contaminant.

Morphological effect of dichloromethane on alfalfa (Medicago sativa) cultivated in soil amended with fertilizer manures.

In this work, we investigated the morphological effect of dichloromethane (DCM) on alfalfa (Medicago sativa) plant. We studied in vitro the influence of its concentration on alfalfa germination. The plants were placed in pots for 15 weeks, and exposed to increasing concentrations of DCM (50 µg L-1 and 84 mg L-1). In addition, we examined the effect of two manures (cow and sheep), which were applied to a contaminated soil, on alfalfa plant growth. The effect of the presence of dichloromethane is obvious even in plant-soil manure system. In fact, in the event of contamination, the soil-cow manure mixture represents the best setting medium for the Alfalfa plant compared to other environments, regardless of the contamination level. Indeed, the presence of two types of manure does not allow the suppression of the inhibitory effect of dichloromethane on the mass of the dry matter of the aerial part which is 18.38% for the cow manure-amended soil and 13.96% for the sheep manure-amended soil.

Impact of Intensive Farming on Soil Heavy Metal Accumulation and Biomarkers Responses of Earthworms Eisenia andrei.

In this work, we propose to evaluate the effect of agriculture intensification under greenhouses on the biochemical and transcriptomic responses of the earthworms Eisenia andrei. This work was conducted on two sites in Téboulba and Sahline (Monastir governorate) and a control site in an experimental plot that is undergoing organic farming. For this purpose, the earthworms Eisenia andrei were exposed to the soils during 7 and 14 days. The physicochemical properties of the soils were analyzed. The biochemical biomarkers of metallothioneins (MTs) and malondialdehyde (MDA) accumulations were also assessed. Moreover, the gene expression level of the MTs was analyzed. The results of our study revealed a significant trace element accumulation accompanied by a high level of MDA and MT proteins. Moreover, a significant expression of the MT gene was observed in earthworms exposed to the soils from Sahline and Téboulba. Hence, this work reveals that intensive agriculture can affect the biological responses of earthworms and consequently, the soil's biofertility.

Physiological, biochemical and transcriptomic responses of Medicago sativa to nickel exposure.

Metal accumulation in soil could lead to severe damage to plants, animals, and humans. The present work aims to evaluate the effects of nickel (Ni) exposure on Medicago sativa at physiological, biochemical, and transcriptomic levels. Plants were exposed to five increasing concentrations of Ni (0, 50, 150, 250, and 500 mg/kg) for 60 days. Agronomic parameters (fresh and dry matter) and chlorophyll content (Chl) were determined in an alfalfa plant. Chemical analyses were conducted, involving the determination of Ni loads in plants (roots and shoots). Moreover, malondialdehyde accumulation (MDA), glutathione-S-transferase (GST), and peroxidase activities, termed as oxidative stress biomarkers, were measured. The gene expression levels of Prx1C, GST, and phytochelatins (PCs) were determined at different nickel concentrations. Our results showed that Ni concentration in plants increased significantly along with Ni concentration in the soil. Regarding oxidative stress biomarkers, Ni contamination caused an increase in peroxidase and GST activities, with a remarkable accumulation of MDA, especially for the highest Ni concentration (500 mg/kg of Ni). Our data showed also a significant upregulation of Prx1C and GST genes in shoots and roots. The PCs' gene expression was significantly enhanced in response to the different nickel concentrations, suggesting their important role in Ni detoxification in alfalfa plants. Our data provided evidence about the clear toxicity of Ni, an often-underestimated trace element.

Identification of two pesticide-tolerant bacteria isolated from Medicago sativa nodule useful for organic soil phytostabilization

Plant-microbe interactions such as rhizobacteria legumes are interesting in organic farming that has undergone significant expansion in the world. The organic agriculture is as an environment-friendly technique and a sustainable alternative to intensive agricultural system. Three types of soil were chosen, organic (ORG), conventional (CON), and fallow land (NA) to isolate soil bacteria-nodulating Medicago sativa, in order to develop microbial inoculants for use in agricultural sustainable system. Soil analysis revealed significant higher amounts of total nitrogen, organic carbon, total phosphorus, and matter detected in ORG. As for heavy metals, ORG showed high Cu content due to the authorized chemical use in organic farming. A sample of 130 bacteria was isolated from Medicago sativa nodule, genetically characterized by PCR/RFLP of ribosomal 16S RNAs, and a great dominance of Sinorhizobium meliloti (88.4%, 73.8%, and 55.5%) is obtained among NA-, CON-, and ORG-managed soils, respectively. The ORG showed the high bacterial diversity with 13.3% of non-identified strains. The resistance against five pesticides (Prosper, Cuivox, Fungastop, Nimbecidine, and Maneb) revealed a maximum of inhibitory concentration about 10 mg l−1 of Prosper, 12 mg l−1 of Cuivox, 6 ml l−1 of Fungastop, 7.5 ml l−1of Nimbecidine, and 25 ml l−1 of Maneb. The analysis of the symbiotic properties and plant growth-promoting potential revealed two efficient strains significantly increased alfalfa dry weight through producing siderophores, phosphorus, and indole acetic acid (13.6 mg ml−1 and 19.9 mg ml−1 respectively). Hence, we identify two tolerant and efficient strains, Achromobacter spanium and Serratia plymuthica, isolated from Medicago sativa nodule with valuable potential able to phytostabilize pesticide-contaminated soils

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Identification of two pesticide-tolerant bacteria isolated from Medicago sativa nodule useful for organic soil phytostabilization.

Plant-microbe interactions such as rhizobacteria legumes are interesting in organic farming that has undergone significant expansion in the world. The organic agriculture is as an environment-friendly technique and a sustainable alternative to intensive agricultural system. Three types of soil were chosen, organic (ORG), conventional (CON), and fallow land (NA) to isolate soil bacteria-nodulating Medicago sativa, in order to develop microbial inoculants for use in agricultural sustainable system. Soil analysis revealed significant higher amounts of total nitrogen, organic carbon, total phosphorus, and matter detected in ORG. As for heavy metals, ORG showed high Cu content due to the authorized chemical use in organic farming. A sample of 130 bacteria was isolated from Medicago sativa nodule, genetically characterized by PCR/RFLP of ribosomal 16S RNAs, and a great dominance of Sinorhizobium meliloti (88.4%, 73.8%, and 55.5%) is obtained among NA-, CON-, and ORG-managed soils, respectively. The ORG showed the high bacterial diversity with 13.3% of non-identified strains. The resistance against five pesticides (Prosper, Cuivox, Fungastop, Nimbecidine, and Maneb) revealed a maximum of inhibitory concentration about 10 mg l-1 of Prosper, 12 mg l-1 of Cuivox, 6 ml l-1 of Fungastop, 7.5 ml l-1of Nimbecidine, and 25 ml l-1 of Maneb. The analysis of the symbiotic properties and plant growth-promoting potential revealed two efficient strains significantly increased alfalfa dry weight through producing siderophores, phosphorus, and indole acetic acid (13.6 mg ml-1 and 19.9 mg ml-1 respectively). Hence, we identify two tolerant and efficient strains, Achromobacter spanium and Serratia plymuthica, isolated from Medicago sativa nodule with valuable potential able to phytostabilize pesticide-contaminated soils.

Metals and micronutrients in some edible crops and their cultivation soils in eastern-central region of Tunisia: A comparison between organic and conventional farming.

In the present study, we evaluated the concentrations of several micronutrients and heavy metals (Fe, Mg, Mn, K, Ca, Na, Zn, Cu, Ni and Cd) in soils and in edible parts of two vegetables (Solanum lycopersicum cv. Amal) and (Lactuca sativa L. cv Augusta) and a fruit (Fragaria x ananassa cv. Sabrina) from conventional and organic farming in the eastern-central region of Tunisia. A principal component analysis (PCA) was conducted to investigate correlations between metals in the edible part of the crops and metals in soil. The bio-concentration factors (BCF) were calculated to evaluate the ability of plants to absorb nutrients from the soil. Our data indicated a significant decrease in micronutrients in the edible part of crops from organic farming. However, toxic metals loads significantly increased in crops from conventional agriculture. Our data should be carefully considered in view of the potential health risk assessment for Tunisian consumers.

Impact of heavy metal contamination on oxidative stress of Eisenia andrei and bacterial community structure in Tunisian mine soil.

The aims of this work were firstly to study the effect of heavy metal-polluted soils from Tunisian mine on earthworm biochemical biomarkers and on bacterial communities and therefore to analyze the interaction between earth worms and bacterial communities in these contaminated soils. For this purpose, we had introduced earthworm Eisenia andrei in six soils: one from mine spoils and five from agricultural soils, establishing a gradient of contamination. The response of worms to the presence of heavy metal was analyzed at the biochemical and transcriptional levels. In a second time, the impact of worm on bacterial community structure was investigated using automated ribosomal intergenic spacer analysis (ARISA) fingerprinting. An impact of heavy metal-contaminated soils on the oxidative status of E. andrei was observed, but this effect was dependent of the level of heavy metal contamination. Moreover, our results demonstrate that the introduction of earthworms E. andrei has an impact on bacterial community; however, the major change was observed in the less contaminated site. Furthermore, a significant correlation between earthworm oxidative status biomarkers and bacterial community structure was observed, mainly in the mine spoils. Therefore, we contribute to a better understanding of the relationships between epigenic earthworms and bacterial communities in heavy metal-contaminated soils.

Biomarker responses of Eisenia andrei to a polymetallic gradient near a lead mining site in North Tunisia.

Eisenia andrei earthworms were exposed for 7 and 14 days to six samples of soil taken from around an abandoned lead (Pb) mine and characterized by different levels of metal contamination (S6-S1, this latter being the most contaminated soil). The organisms were analyzed for metal bioaccumulation and for biological parameters as biomarkers of stress (lysosomal membrane stability; lipofuscin lysosomal content; lysosomal/cytoplasmic volume ratio) and genotoxicity (Micronucleus frequency). Chemical analysis showed the loads of Pb, Cd, Zn, and Cu in the worms following exposure. Among the stress biomarkers, lysosomal membrane stability was significantly affected in the coelomocytes of the earthworms exposed already 7 days to different contaminated soils. Organisms exposed for 14 days to S1 showed in the cells of the chloragogenous tissue, a particularly relevant increase in lipofuscin, a biomarker of oxidative stress, and an increase in the lysosome/cytoplasm volume ratio, indicating stressful condition at the tissue level. Moreover, in the same conditions, a decrease in total body weight was observed. At the longer exposure time, the coelomocytes of worms exposed to S1, S2, and S3 (soils with higher metal concentrations) showed a significant increase in micronuclei (MNi) frequency. Expressions of the P21 and topoisomerase genes, which are involved in DNA repair, showed significant up-regulation in the cells of worms exposed to S1, S2, S3, S4 and to a less extend S6. This may indicate that the worms were only able to successfully reduce the level of DNA damage in S4 and S5 if considering MN frequency data. The biomarker data was integrated by the Earthworm Expert System, allowing an objective interpretation of the complex biological data and clearly defining the areas in which the presence of chemicals is toxic for the edaphic organisms.

Transcriptional expression levels and biochemical markers of oxidative stress in the earthworm Eisenia andrei after exposure to 2,4-dichlorophenoxyacetic acid (2,4-D).

This study investigated the stress response of earthworms (Eisenia andrei) to exposure to a commonly used herbicide, 2,4 dichloro-phenoxy-acetic acid (2,4-D). We evaluated both stress biomarkers and the transcriptional expression levels and activity of three enzymes involved in oxidative stress responses. Earthworms were exposed to three sublethal concentration of 2,4-D (3.5, 7, and 14 mg kg(-1)) for 7 and 14 days. Exposure to 7 and 14 mg kg(-1) 2,4-D significantly reduced both worm body weight and lysosomal membrane stability (LMS); the latter is a sensitive stress biomarker in coelomocytes. Exposure to 2,4-D caused a pronounced increase in the accumulation of malonedialdehyde (MDA), a marker of oxidative stress, and significantly increased the activity of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD),and glutathione-S-transferase (GST). Compared to expression in controls, the expression levels of the sod, cat, and gst genes increased in worms exposed to all three 2,4-D doses for 7 days. However, after 14 days of exposure, only the expression of the gst gene remained higher than controls. These data provide new insights into the cytotoxicity of 2,4-D in the earthworm E. andrei and should be carefully considered in view of the biological effects of herbicides in soils organisms.

Specific mechanisms of tolerance to copper and cadmium are compromised by a limited concentration of glutathione in alfalfa plants.

The induction of oxidative stress is a characteristic symptom of metal phytotoxicity and is counteracted by antioxidants such as glutathione (GSH) or homoglutathione (hGSH). The depletion of GSH│hGSH in fifteen-day-old alfalfa (Medicago sativa) plants pre-incubated with 1mM buthionine sulfoximine (BSO) affected antioxidant responses in a metal-specific manner under exposure to copper (Cu; 0, 6, 30 and 100µM) or cadmium (Cd; 0, 6 and 30µM) for 7 days. The phytotoxic symptoms observed with excess Cu were accompanied by an inhibition of root glutathione reductase (GR) activity, a response that was augmented in Cd-treated plants but reverted when combined with BSO. The synthesis of phytochelatins (PCs) was induced by Cd, whereas the biothiol concentration decreased in Cu-treated plants, which did not accumulate PCs. The depletion of GSH│hGSH by BSO also produced a strong induction of oxidative stress under excess Cu stress, primarily due to impaired GSH│hGSH-dependent redox homeostasis. In addition, the synthesis of PCs was required for Cd detoxification, apparently also determining the distribution of Cd in plants, as less metal was translocated to the shoots in BSO-incubated plants. Therefore, specific GSH│hGSH-associated mechanisms of tolerance were triggered by stress due to each metal.

Photosynthesis and growth responses of pea Pisum sativum L. under heavy metals stress.

The present work aimed to study the physiological effects of cadmium (Cd) and copper (Cu) in pea (Pisum sativum). Pea plants were exposed to increasing doses of cadmium chloride (CdCl2) and copper chloride (CuCl2) for 20 d. The examined parameters, namely root and shoot lengths, the concentration of photosynthetic pigments and the rate of photosynthesis were affected by the treatments especially with high metals concentrations. The analysis of heavy metals accumulation shows that leaves significantly accumulate cadmium for all the tested concentrations. However, copper was significantly accumulated only with the highest tested dose. This may explain the higher inhibitory effects of cadmium on photosynthesis and growth in pea plants. These results are valuable for understanding the biological consequences of heavy metals contamination particularly in soils devoted to organic agriculture.