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.

Environmental microplastic accumulation exacerbates liver ischemia-reperfusion injury in rat: Protective effects of melatonin.

Ischemia-reperfusion (IR) injury is an inevitable complication of liver transplantation and partial hepatectomy. Although the hazards of environmental microplastics (EMPs) have been well explored, data underlying their impact on IR-induced hepatotoxicity and how to alleviate these damages remain largely undefined. In this study, the involvement of melatonin (MT) in modulating EMPs toxicity in the liver undergoing ischemia-reperfusion injury was investigated. Male Wistar rats were exposed to MPs for 7 days and then subjected to 1 h of partial warm ischemia (70 %) followed by 24 h of reperfusion. We analyzed some parameters as the oxidative stress, the stability of cytoskeleton as well as inflammation, and autophagy. Our data suggested that EMPs elicited liver injury in ischemic animals. Data revealed several histological alterations caused by EMP and IRI, including cellular disorientation, cell necrosis, and microvacuolar steatosis, as well as inflammatory cell infiltration. EMPs increased blood transaminase (AST and ALT) and oxidative stress levels in the ischemic liver. In addition, RT-qPCR, immunofluorescence, and western blot analyses highlighted an increased expression of α-tubulin, IL-18, NFkB, and LC3. However, the ability of MT to reduce MPs and IRI toxicity was consistent with a significant decrease in the evaluated markers. The combined data not only document that melatonin is an effective agent to protect against hepatic IRI but also reduces cellular dysfunction caused by EMPs.

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.

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.

Impact of environmental microplastics alone and mixed with benzo[a]pyrene on cellular and molecular responses of Mytilus galloprovincialis.

The hazard of microplastic (MP) pollution in marine environments is a current concern. However, the effects of environmental microplastics combined with other pollutants are still poorly investigated. Herein, impact of ecologically relevant concentrations of environmental MP alone (50 µg/L) or combined with B[a]P (1 µg/L) was assessed in mussel Mytilus galloprovincialis after a short-term exposure (1 and 3 days) to environmental MP collected from a north-Mediterranean beach. Raman Microspectroscopy (RMS) revealed bioaccumulation in mussel hemolymph of MP, characterized by polyethylene (PE), polyethylene terephthalate (PET), polypropylene (PP), polyethylene vinyl acetate (PEVA) and high-density polyethylene (HDPE), with abundance of MP sized 1.22-0.45 µm. An increase of B[a]P was detected in mussels after 3-day exposure, particularly when mixed with MP. Both contaminants induced cytotoxic and genotoxic effects on hemocytes as determined by lysosomal membrane stability (LMS), micronuclei frequency (FMN), and DNA fragmentation rate by terminal dUTP nick-end labeling (TUNEL). About apoptosis/DNA repair processes, P53 and DNA-ligase increased at 1-day exposure in all conditions, whereas after 3 days increase of bax, Cas-3 and P53 and decrease of Bcl-2 and DNA-ligase were revealed, suggesting a shift towards a cell apoptotic event in exposed mussels. Overall, this study provides new insights on the risk of MP for the marine ecosystem, their ability to accumulate xenobiotics and transfer them to marine biota, with potential adverse repercussion on their health status.

Metabolomic disorders unveil hepatotoxicity of environmental microplastics in wild fish Serranus scriba (Linnaeus 1758).

Coastal areas are worldwide subject to large inputs of anthropogenic wastes that are discharged directly into inshore waters, where they will be weathered into small microplastics (MPs) of up to a size <20 µm. This study provides information about the presence of small environmental MPs (≤3 µm) in the liver of adult benthopelagic fish Serranus scriba (Linnaeus 1758), caught from three coastal regions in Tunisia distinguished by different patterns of human activity. Polymer composition in fish liver was identified using Raman microspectroscopy. Results revealed differences in the abundance, size distribution and presence of plastic additives over the investigated sites. Polyethylene-vinyl acetate (PEVA: 34% particles/g of tissue), high density polyethylene (HDPE: 24.4%) and the two smaller size classes, i.e. 3-1.2 µm and 1.2-0.45 µm, were the most abundant MPs types and size distribution found, respectively, in Bizerte channel (BC) site (Bizerte city, Tunisia). Moreover, at hepatic level data showed a significant site-dependent cytotoxicity expressed by changes in malondialdehyde (MDA) content, presence of reactive oxygen species (ROS) expressed by altered level of catalase (CAT) and glutathione-S-transferase (GST) activities and in the content of metallothioneins (MTs), as well as genotoxicity by changes in the amount of micronucleus (MN), and neurotoxicity by altered activity of acetylcholinesterase (AChE). A innovative metabolomics analysis was also performed to further investigate the distinct patterns of key metabolite changes in the liver of Serranus scriba. A total of 36 metabolites were significantly affected, mainly involved in energy, amino acid and osmolyte metabolism. These findings emphasised for the first time a close relationship between the source, abundance and size ranges of environmental MPs ≤ 3 µm and their hepatotoxicity in wild organisms.

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.

Autophagic event and metabolomic disorders unveil cellular toxicity of environmental microplastics on marine polychaete Hediste diversicolor.

Although the hazards of microplastics (MPs) have been quite well explored, the aberrant metabolism and the involvement of the autophagy pathway as an adverse response to environmental MPs in benthic organisms are still unclear. The present work aims to assess the impact of different environmental MPs collected from the south coast of the Mediterranean Sea, composed by polyethylene (PE), polyethylene vinyl acetate (PEVA), low-density polyethylene (LDPE), high-density polyethylene (HDPE), polypropylene (PP) and polyamide (PA) on the metabolome and proteome of the marine polychaete Hediste diversicolor. As a result, all the microplastic types were detected with Raman microspectroscopy in polychaetes tissues, causing cytoskeleton damage and induced autophagy pathway manifested by immunohistochemical labeling of specific targeted proteins, through Tubulin (Tub), Microtubule-associated protein light chain 3 (LC3), and p62 (also named Sequestosome 1). Metabolomics was conducted to further investigate the metabolic alterations induced by the environmental MPs-mixture in polychaetes. A total of 28 metabolites were differentially expressed between control and MPs-treated polychaetes, which showed elevated levels of amino acids, glucose, ATP/ADP, osmolytes, glutathione, choline and phosphocholine, and reduced concentration of aspartate. These novel findings extend our understanding given the toxicity of environmental microplastics and unravel their underlying mechanisms.

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.

Uptake, tissue distribution and toxicological effects of environmental microplastics in early juvenile fish Dicentrarchus labrax.

As the smallest environmental microplastics (EMPs), even at nanoscale, are increasingly present in the environment, their availability and physical and chemical effects on marine organisms are poorly documented. In the present study, we primarily investigated the uptake and accumulation of a mixture of environmental microplastics (EMPs) obtained during an artificial degradation process in early-juvenile sea bass (Dicentrarchus labrax). Moreover, we evaluated their hazardous effects using biochemical markers of cytotoxicity. Polymer distribution and composition in gill, gut, and liver were analyzed using polarized light microscopy (PLM) and Raman microspectroscopy (RMS). Our findings revealed the size-dependent ingestion and accumulation of smaller MPs (0.45-3 µm) in fish tissues even after a short-term exposure (3 and 5 days). In addition to MPs, our results showed the presence of plastic additives including plasticizers, flame retardants, curing agents, heat stabilizers, and fiber-reinforced plastic materials in fish tissues, which contributed mostly to the larger-sized range (≥ 1.2 µm). Our data showed that significant oxidative alterations were highly correlated with MPs size range. Our results emphasized that the toxicity of smaller EMPs (≤ 3 µm) was closely related to different factors, including the target tissue, exposure duration, size range of MPs, and their chemical properties.

Uptake, accumulation and associated cellular alterations of environmental samples of microplastics in the seaworm Hediste diversicolor.

The ubiquitous distribution of microplastics (MPs) in the marine environment raises global concern to understand their impact. Environmental MPs have been shown to exhibit different physicochemical properties during their life cycles. However, the body of knowledge regarding their accumulation and biological effects is still significantly limited compared to manufactured MPs. To evaluate the hazardous effects of a mixture of environmental MPs collected along the Tunisian beaches, their accumulation and cellular effects were investigated in Hediste diversicolor. MP sample was composed of polyethylene (PE), polyethylene vinyl acetate (PEVA), low-density polyethylene (LDPE), high-density polyethylene (HDPE), polypropylene (PP) and polyamide (PA) analyzed using Raman microspectroscopy (RM). The concentrations of MPs in seaworm tissues increased over time, following the order 1.2-0.45 µm > 3-1.2 µm > 100-3 µm. The ingestion of MPs by H. diversicolor reduced their survival and growth, affected the neuro-transmission and antioxidant pathways. Our data emphasised that the toxic effects of environmental MPs were closely related to the exposure dose and period. The results also demonstrated that the size distribution of MPs in seaworms was mainly correlated with biochemical markers. This study highlights the ecological risk in the ingestion and accumulation of environmental MPs by biota that threatens their functional parameters.

First report on the presence of small microplastics (≤ 3 µm) in tissue of the commercial fish Serranus scriba (Linnaeus. 1758) from Tunisian coasts and associated cellular alterations.

There is limited research on the ingestion of microplastic particles (MPs) by fish from the southern part of the Mediterranean Sea. This study provides the occurrence of small MPs (≤3 µm) in the gastrointestinal tract and muscle of adult benthopelagic fish Serranus scriba (L.1758), caught along Tunisian coasts. MPs were extracted from selected tissues using a potassium hydroxide digestion method (KOH 10%) and then quantified, and their chemical structure was characterized through Raman microspectroscopy. The results highlighted that MPs were present in all samples. The average abundance of MPs per gram of fish tissue identified through successive filters of 3 µm, 1.2 µm, and 0.45 µm differed significantly among the sites. The properties of the MPs extracted indicated that polyethylene-vinyl-acetate (PEVA: 33.45%), high density polyethylene (HD-PE: 17.33%), and fragments were the most abundant plastic types and shape found, respectively. Among those, most MPs were found at a size class of 3-1.2 µm (∼60%), especially in the muscle, suggesting a high transfer of MPs into the human diet. Our field work also aimed to explore the effects observed in the gastrointestinal tract with a battery of biomarkers assessing oxidative stress and neurotoxicity. The preliminary results of this study showed the existence of a link between small MPs, sites, and their associated urban activities and induced oxidative stress. However, more detailed studies are required to evaluate the transfer of MPs into tissues and the potential impacts of this transfer on human health.

Abundance and distribution of small microplastics (≤ 3 µm) in sediments and seaworms from the Southern Mediterranean coasts and characterisation of their potential harmful effects.

Microplastics (MPs) are an uncontrolled contaminant affecting marine ecosystems. Studying their undesirable effects has been an attractive field for scientists in recent years. This study is the first to investigate the uptake and distribution of small microplastics (≤3 µm) from several sites in the Southern Mediterranean coasts. This work primarilyaims to provide a qualitative and quantitative analysis of microplastics in sediments as well as in the seaworms (Hediste diversicolor) from eight sites from the Tunisian coasts using Fourier transform infrared spectroscopy and Raman microspectroscopy. The second aim is to evaluate the potential toxic effects of environmental microplastics using a set of biomarkers such as Catalase, Glutathione-S-Transferase, Malondialdehyde and Acetylcholinesterase. Our findings showed that microplastics (1 mm-1.2 µm) were present in all sediments with its abundance ranging from 129 to 606 items kg-1. Microplastic accumulation in seaworms (3 µm-0.22 µm) was 0.5-3.7 items g-1. The predominant polymer was polyethylene. Results also revealed a significant variation among sites in the parameters associated with oxidative stress. Thus, size abundance of microplastics in seaworms was mainly correlated with oxidative stress biomarkers. Our data should be carefully considered in view of the microplastic presence with several types and sizes in Tunisian coastal sites, their potential toxic effects, and their transfer into food web.

Ecotoxicity of trace elements to chicken GALLUS gallus domesticus exposed to a gradient of polymetallic-polluted sites.

Mining activity may cause heavy metal accumulation, which threatens human and animal health by their long-term persistence in the environment. This study aims to assess the impact of polymetallic pollution on chicken (Gallus domesticus) from old lead mining sites in northeast of Tunisia: Jebel Ressas (JR). Samples of soil and chickens were collected from five sites being ranked along a gradient of heavy metal contamination. Heavy metal loads were evaluated in soil samples and in chicken liver and kidney. Biochemical evaluation of oxidative stress parameters termed as Catalase (CAT), Glutathione-S-Transferase (GST), and Malondialdehydes (MDA) accumulation was monitored. Metallothionein protein level was assessed as a specific response to heavy metals. DNA alteration was achieved using MNi frequency in the investigated tissues. Finally, the evaluation of gene expression levels of CAT, GST, mt1, mt4, P53, bcl2, caspase3 and DNA-ligase was performed. Our data showed the highest loads of Cd, Cu, Zn and Pb in tissues of animals from site 3, being more pronounced in kidney. Biochemical data suggested a significant increase in antioxidant enzymes activities in all sites respect to control except in site 3 were CAT and GST were inhibited. DNA alteration was observed in all tissues being very pronounced in animals from site 3. Overall, transcriptomic data showed that genes involved in apoptosis were up-regulated in animals exposed to the most contaminated soils. Our data suggest that chicken and selected biomarkers offer a suitable model for biomonitoring assessment of heavy metals transfer through the food web in mining sites. Finally, the obtained results of heavy metals accumulation and related alterations should be carefully considered in view of the controversial relationship between distribution and toxicology of contaminants in exposed organisms.

Subacute cutaneous lupus erythematosus induced by nivolumab: two case reports and a literature review.

Nivolumab is widely used to treat several late-stage malignancies such as melanoma and non-small-cell lung cancer by inhibiting the interaction between the programmed cell death protein-1 and its ligand. By stimulating an antitumor immune response, it also leads to immune adverse events. Here. we report two cases of subacute cutaneous lupus erythematosus (SCLE) induced by nivolumab. Case 1: a 72-year-old woman with a stage IV melanoma. Two months after nivolumab discontinuation because of autoimmune hepatitis, the patient was in complete remission and pruritic nummular erythematous plaques appeared on the back and arms. Case 2: a 43-year-old man put under nivolumab for a metastatic non-small-cell lung cancer. After two cycles, an annular erythematous eruption appeared on the hands, arms, and chest. The hypothesis of SCLE was confirmed by biopsies showing lymphoid perivascular inflammatory infiltrates, with scarce C3 deposits along the basal layer of the epidermis in patient 2. Both patients tested positive for antinuclear antibodies and anti-SSA antibodies. Lesions were regressive under topical corticosteroids and hydroxychloroquine for the first patient and oral prednisone for the second patient. No systemic involvement was observed. The occurrence of SCLE 2 months after nivolumab discontinuation is evidence that the drug effect is prolonged because of the maintenance of programmed cell death protein-1 reception saturation for months. A causal relationship between SCLE and nivolumab is suggested by (i) the occurrence of SCLE after at least two cycles, (ii) the regression of lesions following treatment with corticosteroids and hydroxychloroquine, and (iii) the fact that it appeared after remission in our first patient.