Environmental microplastic interact with heavy metal in polluted soil from mine site in the North of Tunisia: Effects on heavy metal accumulation, growth, photosynthetic activities, and biochemical responses of alfalfa plants (Medicago saliva L.).

As emerging persistent pollutants, microplastic (MPs) pollution attracted increasing attention worldwide since it is posing several environmental concerns. MPs interact with heavy metals in soil and may provoke damages on soil properties and ultimately impaired plants and human health. The present study aims to evaluate alfalfa plants (Medicago sativa) response after exposure to heavy metal polluted soils from mine site in the North of Tunisia in presence of environmental microplastic. For that, soils were sampled from two sites of Jebel Ressass mine in addition to a control soil. Plants were exposed to the three soils in presence of two increasing rates of microplastics D1 (1 mg/kg of soil) and D2 (100 mg/kg of soil) for 60 days. After harvest, agronomic parameters, chlorophyll content as well as heavy metal accumulation in plants were analyzed. Furthermore, oxidative status was evaluated in terms of malondialdehyde accumulation (MDA), catalase (CAT) activities and glutathion-S-transferase (GST). Overall, our finding highlights that MPs disrupted agronomic parameters and the photosynthetic activities of alfalfa plants. Additionally, our results revealed that the presence of MPs in polluted soils cause an increase on heavy metal accumulation in alfalfa shoots. Biochemical analyses demonstrated that the combined exposure to MPs and heavy metal induced oxidative stress in alfalfa plants by increasing CAT activity and MDA accumulation. The present investigation highlights the ecological risks of microplastics in terrestrial environment.

Toxicity assessment of animal manure composts containing environmental microplastics by using earthworms Eisenia andrei.

Nowadays, animal manure composting constitutes a sustainable alternative for farmers to enhance the level of nutrients within soils and achieve a good productivity. However, pollutants may be present in manures. This study focuses on the detection of environmental microplastics (EMPs) into composts, as well as on the assessment of their potential toxicity on the earthworm Eisenia andrei. To these aims, animals were exposed to two types of compost, namely bovine (cow) and ovine (sheep) manure, besides to their mixture, for 7 and 14 days. The presence and characterization of EMPs was evaluated in all the tested composts, as well as in tissues of the exposed earthworms. The impact of the tested composts was assessed by a multi-biomarker approach including cytotoxic (lysosomal membrane stability, LMS), genotoxic (micronuclei frequency, MNi), biochemical (activity of catalase, CAT, and glutathione-S-transferase, GST; content of malondialdehyde, MDA), and neurotoxic (activity of acetylcholinesterase, AChE) responses in earthworms. Results indicated the presence of high levels of EMPs in all the tested composts, especially in the sheep manure (2273.14 ± 200.89 items/kg) in comparison to the cow manure (1628.82 ± 175.23 items/kg), with the size <1.22 µm as the most abundant EMPs. A time-dependent decrease in LMS and AChE was noted in exposed earthworms, as well as a concomitant increase in DNA damages (MNi) after 7 and 14 days of exposure. Also, a severe oxidative stress was recorded in animals treated with the different types of compost through an increase in CAT and GST activities, and LPO levels, especially after 14 days of exposure. Therefore, it is necessary to carefully consider these findings for agricultural good practices in terms of plastic mitigation in compost usage, in order to prevent any risk for environment health.

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.

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.

Assessing the effects of nickel on, e.g., Medicago sativa L. nodules using multidisciplinary approach.

Industrial wastes and fertilizers can introduce excessive levels of nickel (Ni) into the environment, potentially causing threats to plants, animals, as well as human beings. However, the number of studies on the effects of Ni toxicity on nodules is fairly limited. To address this issue, the effects of increasing Ni concentration on alfalfa nodules were assessed at chemical, biochemical, and transcriptomic levels. For this purpose, plants were grown in soils supplied with Ni (control, 0 mg/kg; C1, 50 mg/kg; C2, 150 mg/kg; C3, 250 mg/kg; and C4, 500 mg/kg) for 90 days. Ni loads in leaves, roots, and nodules were monitored after the exposure period. A set of biochemical biomarkers of oxidative stress was determined in nodules including antioxidants and metal homeostasis as well as lipid peroxidation. Gene expression levels of the main targets involved in oxidative stress and metal homeostasis were assessed. Our data indicated a high concentration of Ni in leaves, roots, and nodules where values reached 25.64 ± 3.04 mg/kg, 83.23 ± 5.16 mg/kg, and 125.71 ± 4.53 mg/kg in dry weight, respectively. Moreover, a significant increase in nodule biomass was observed in plants exposed to C4 in comparison to control treatment and percentage increased by 63%. Then, lipid peroxidation increased with a rate of 95% in nodules exposed to C4. Enzymatic activities were enhanced remarkably, suggesting the occurrence of oxidative stress, with increased superoxide dismutase (SOD), glutathione reductase (GR), and ascorbate peroxidase (APX). Our results showed also a significant upregulation of SOD, GR and APX genes in nodules. Nodule homoglutathione (HGSH) levels increased with the different Ni concentrations, with a remarkable decrease of glutathione S-transferase (GST) activity and glutathione (GSH) content for the highest Ni concentration with 43% and 52% reduction, respectively. The phytochelatin (PC) and metallothionein (MT) concentrations increased in nodules, which implied the triggering of a cellular protection mechanism for coping with Ni toxicity. The results suggested that Ni promotes a drastic oxidative stress in alfalfa nodules, yet the expression of MT and PC to reduce Ni toxicity could be used as Ni stress bioindicators. Our findings provide new insights into the central role of alfalfa nodules in limiting the harmful effects of soil pollution. Therefore, nodules co-expressing antioxidant enzymes may have high phytoremediation potential.

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.

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.

Earthworm Eisenia andrei modulates oxidative stress in bean plants Vicia faba irrigated with treated wastewater.

With respect to reducing the pressure on freshwater resources, treated wastewater (TWW) irrigation represents a sustainable alternative in agriculture. Due to their low quality and variable composition, TWW could entail harmful consequences for living organisms in terrestrial ecosystems. This study aims to evaluate how earthworm (Eisenia andrei) can modulate oxidative stress in bean plants (Vicia faba) that are irrigated over a course of 60 days with two doses of TWW (50 and 100%) in addition to a control condition (0%) irrigated with distilled water. This is achieved by measuring glutathione-S-transferase (GST) activity and malondialdehyde accumulation (MDA) in plants. Furthermore, catalase (CAT), GST, MDA, and acetylcholinesterase (AChE) activities of the earthworms are also assessed. Our results show that growth and physiological parameters are modified when applying TWW irrigation. Moreover, oxidative stress apprehended by GST activity and MDA accumulation is exacerbated in V. faba plants after exposure to increased TWW doses. Similarly, TWW irrigation enhances oxidative stress parameters in earthworms with a crucial decrease in AChE activity. In addition, the presence of earthworms increases growth and physiological parameters; it also results in a significant reduction in GST activity and MDA rate in V. faba plants. Our results provide new insights into the impact of TWW irrigation on soil organisms and the importance of earthworms in the reduction of oxidative stress in plants.

The aggregation pheromone phenylacetonitrile: Joint action with the entomopathogenic fungus Metarhizium anisopliae var. acridum and physiological and transcriptomic effects on Schistocerca gregaria nymphs.

The combined use of entomopathogenic fungi and sublethal rate of chemical insecticides or other biological control agents have been proposed as an environmentally and sustainable strategy in the control of locust pests. In this paper, the quarter and the half of the recommended dose of Metarhizium anisopliae var. acridum (» and ½ Ma) and the aggregation pheromone (Phenylacetonitrile: PAN) were applied simultaneously and sequentially to Schistocerca gregaria fifth-instar nymphs. In addition, the physiological effects of PAN on locusts were assessed at the behavior, immune response, and biochemical level by evaluating for glutathione-S-transferase (GST), acetylcholinesterase inhibition (AChE), and malondialdehyde accumulation (MDA). Results showed that simultaneous application of PAN and the entomopathogenic fungus exhibited additive interaction. Synergistic interaction was also demonstrated when nymphs were exposed to PAN first, then treated with M. anisopliae var. acridum. Behavioral bioassay revealed that fifth-instar nymphs avoided the PAN odour and tended to remain away from the stimulus cup. In the choice assay, the pheromone significantly repelled the locusts at 2, 4, and 6 h of exposure which selected the PAN-free arena chamber. Moreover, treated nymphs become hyperactive and disoriented as evidenced by the cumulative distance travelled and the trajectory of locusts during the experiment. Immunological studies showed that PAN significantly decreased the differential haemocyte counts (prohemocytes and plasmatocytes) with a dose-response relationship. Data of biochemical analyzes showed that the PAN exposure reduced the activity of acetylcholinesterase and induced significantly the glutathione S-transferases and MDA concentration in the desert locust fifth-instar nymphs. Moreover, transcriptomic responses to the PAN exposure were evaluated using gene expression levels of CYP540 and GST. The transcript levels showed an up-regulation in GST expression level particularly in nymphs exposed for 4 and 6 h. A significant increase in CYP450 transcript level was also observed after 2 h of exposure, which decreased significantly after 4 and 6 h.

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.

Biochemical and transcriptomic response of earthworms Eisenia andrei exposed to soils irrigated with treated wastewater.

In order to ensure better use of treated wastewater (TWW), we investigated the effect of three increasing doses of TWW, 10%, 50%, and 100%, on biochemical and transcriptomic statuses of earthworms Eisenia andrei exposed during 7 and 14 days. The effect of TWW on the oxidative status of E. andrei was observed, but this effect was widely dependent on the dilution degree of TWW. Results showed a significant decrease in the catalase (CAT) activity and an increase in the glutathione-S-transferase (GST) activity, and considerable acetylcholinesterase (AChE) inhibition was recorded after 14 days of exposure. Moreover, malondialdehyde (MDA) accumulation was found to be higher in exposed animals compared to control worms. The gene expression level revealed a significant upregulation of target genes (CAT and GST) during experimentation. These data provided new information about the reuse of TWW and its potential toxicity on soil organisms.

Effect of treated wastewater irrigation in East Central region of Tunisia (Monastir governorate) on the biochemical and transcriptomic response of earthworms Eisenia andrei.

Treated wastewater (TWW) reuse for irrigation has become an excellent way to palliate water scarcity in Mediterranean arid regions. However, the toxicological effects of these effluents on the soil's organisms, especially earthworms, have not been well studied as yet. In this paper, earthworms Eisenia andrei were exposed for 7 days and 14 days to five agricultural soils irrigated with TWW for different periods: 1 year, 8 years, and 20 years. In addition, they were also exposed to soil from one reference site sampled from the Ouardenin perimeter in the Monastir Governorate in Tunisia. The effect on earthworms was assessed at the biochemical level by evaluating for catalase (CAT), glutathione-S-transferase (GST), malondialdehyde accumulation (MDA) and acetylcholinesterase inhibition (AChE). On the other hand, genotoxicity and transcriptomic responses were evaluated using micronuclei test (MNT) and gene expression level of CAT and GST. Moreover, metals uptake by earthworms was analyzed. Results showed that CAT and GST activity in the earthworm increased significantly when they were exposed to soils irrigated with TWW for 1, 8 and 20 years. Furthermore, MDA concentration also increased significantly with the increase in exposure period. However, AChE activity decreased and MNi frequency increased in earthworms after 7 and 14 days of exposure to soils irrigated with TWW for more than a year. The gene expression level of CAT and GST showed a significant variability, thus data are discussed in relation to the studied biomarkers (CAT and GST). These data provide new insights into the effect of toxicity of TWW on the soil's macro fauna, which is strongly affected by the trace elements and other organic compounds accumulated in soils after 20 years of TWW irrigation.