Polycyclic aromatic hydrocarbons (PAHs) and microplastics (MPs) may coexist in soil. Consequently, the toxicity of PAHs to “soil engineers” earthworms (vermitoxicity), may be influenced by various ...MPs. However, studies on this issue are scarce. In particular, the differential effect of MPs of different polymers on the vermitoxicity of PAHs remains unclear. Therefore, a series of microcosm experiments were conducted to evaluate the potential vermitoxicity of fluoranthene (Fla) in the presence of polyethylene (PE), polyvinyl chloride (PVC), and polystyrene (PS) MPs at an environmentally relevant concentration (125 mg kg−1). The results indicated that Fla exerted vermitoxicity and MPs enhanced the adverse effects. Specifically, after the introduction of MPs, the 14-d LC50s of Fla on earthworms decreased from 130.9 to 98.0–123.6 mg kg−1; in addition, the growth inhibition rates improved from 5.37%–15.34% to 15.63%–33.38%, and the avoidance rate increased by 10%–100% at the same exposure doses in most cases. In most cases, the neutral red retention time was shortened by 14.3%–47.9%, indicating that the integrity of the coelomocytes' lysosomal membrane had worsened. The affected antioxidant enzyme activity and improved malondialdehyde content indicated enhanced oxidative damage in the treatments containing MPs. However, the aggravation of the vermitoxicity varied by MPs type and toxicological endpoints. Overall, Fla + PS MPs exerted the greatest effect on the mortality of earthworms. On the contrary, PVC and PE MPs exhibited higher subacute effects on the vermitoxicity of Fla. Our study also demonstrated that MPs at environmentally relevant doses may directly induce vermitoxicity. In particular, damaged coelomocytes’ lysosomal membrane stability by MPs was second reported to the best of our knowledge. Our results revealed the differences in the effects of various MPs on the vermitoxicity of PAHs, which provides new data in assessing the ecological effects of PAHs and MPs in soils.
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•The impacts of three MPs on the vermitoxicity of Fla were systematically studied.•PS, PE, and PVC MPs enhanced the negative effects of Fla on earthworms.•Aggravation of the vermitoxicity of Fla varied by MPs type and toxicological endpoints.•Both Fla and MPs reduced the stability of worms' coelomocytes lysosomal membrane.
Despite its known positive impacts when added to soil, the negative effects of biochar on earthworms are not fully understood. Here, we investigated the toxicity of nine biochars of three feedstock ...origins, animal (cow dung), plant (corncob) and microorganism (sewage sludge), produced at three pyrolysis temperatures (350 °C, 550 °C and 750 °C) on earthworms. Vermitoxicity was first assessed using acute toxicity test, neutral red retention time (NRRT) assay and oxidative stress response assay. Furthermore, we evaluated whether the thermal treatment of biochars could reduce their vermitoxicity using an acute toxicity assay. We found that, according to LC50 and earthworm weight loss, cow dung biochar was more toxic than corncob or sewage sludge biochar; thus, production feedstock is apparently important to biochar vermitoxicity. Furthermore, NRRTs indicated cow dung biochar disrupted lysosomal membrane stability in earthworm coelomocytes, providing further evidence for the toxicity of this biochar to earthworms. Disturbed antioxidant enzyme activities and elevated malondialdehyde content showed that earthworm suffer oxidative stress, also implying a potential vermitoxicity. However, thermal treatment of cow dung biochar substantially improved its LC50 and decreased earthworm weight loss, implying that the PAHs in this biochar might be damage factors and that heating could reduce the potential toxicity of biochar. Besides, NRRT assay was first used to evaluate the effects of biochar on earthworms and clear dose-effect relationships indicated that NRRT assay might be a useful tool for assessing the potential negative effects of biochar. Overall, given the different effects of various biochars, including toxicity, reported here, our findings will help improve understanding of biochar vermitoxicity mechanisms, serve to improve biochar ecological risk assessments and provide a reference for the proper application of biochar amendments.
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•Feedstock but not pyrolysis temperature shapes the vermitoxicity of biochar.•Biochars could disrupt the lysosomal membrane stability in earthworm coelomocytes.•Thermal treatment of biochars could alleviate their vermitoxicity.•NRRT assay can assess biochars' sub-acute vermitoxicity at field application rates.
ABSTRACTDestabilisation of blood cell lysosomes in Mediterranean green crabCarcinus aestuarii was investigated using Neutral Red Retention Assay (NRRA). Crabs collected in Narta Lagoon, Vlora ...(Albania) during May 2014 were exposed in the laboratory to sub-lethal, environmentally realistic concentrations of copper. Neutral Red Retention Time (NRRT) and glucose concentration in haemolymph of animals were measured. The mean NRRT showed a significant reduction for the animals of the treatment group compared to the control one (from 118.6 ± 28.4 to 36.4 ± 10.48 min, p<0.05), indicating damage of lysosomal membrane. Haemolymph glucose concentration was significantly higher in the treatment group (from 37.8 ± 2.7 to 137.8.4 ± 16.2 mg/dL, p<0.05) than in control group, demonstrating the presence of stress on the animals. These results showed thatC. aestuarii could be used as a successful and reliable bioindicator for evaluating the exposure to contaminants in laboratory conditions. NRRA provides a successful tool for rapid assessment of heavy metal pollution effects on marine biota.
The problem of effective assessment of risk posed by complex mixtures of toxic chemicals in the environment is a major challenge for government regulators and industry. The biological effect of the ...individual contaminants, where these are known, can be measured; but the problem lies in relating toxicity to the multiple constituents of contaminant cocktails. The objective of this study was to test the hypothesis that diverse contaminant mixtures may cause a greater toxicity than the sum of their individual parts, due to synergistic interactions between contaminants with different intracellular targets. Lysosomal membrane stability in hemocytes from marine mussels was used for in vitro toxicity tests; and was coupled with analysis using the isobole method and a linear additive statistical model. The findings from both methods have shown significant emergent synergistic interactions between environmentally relevant chemicals (i.e., polycyclic aromatic hydrocarbons, pesticides, biocides and a surfactant) when exposed to isolated hemocytes as a mixture of 3 & 7 constituents. The results support the complexity-based hypothesis that emergent toxicity occurs with increasing contaminant diversity, and raises questions about the validity of estimating toxicity of contaminant mixtures based on the additive toxicity of single components. Further experimentation is required to investigate the potential for interactive effects in mixtures with more constituents (e.g., 50–100) at more environmentally realistic concentrations in order to test other regions of the model, namely, very low concentrations and high diversity. Estimated toxicant diversity coupled with tests for lysosomal damage may provide a potential tool for determining the toxicity of estuarine sediments, dredge spoil or contaminated soil.
Evidence for synergistic toxicity as a consequence of increasing diversity of harmful chemicals. Test matrix (circular symbols) for chemical molar concentrations versus chemical diversity (log10 scales) with the % lysosomal toxicity (shaded boxes) shown for the corresponding treatment. The conjectured curve for the subcriticality/supracriticality boundary employs the 50% isoeffective concentrations (open grey diamond shapes) for the 3 mixtures. The 50% additivity concentrations are shown as a dashed line. Display omitted
•Emergent synergistic interactions between toxic chemicals.•Toxicity increases with increasing chemical diversity.•Toxicant diversity as a potential tool for assessing mixture toxicity.
Synergistic interactions have been observed in mixtures of toxic chemicals and relatively non-toxic chemicals increase the toxicity of the mixture. Toxicity increases with chemical diversity.
Effects on earthworms in the contaminated floodplain area the Biesbosch, the Netherlands, were determined at different levels of organization using a combination of field and laboratory tests. The ...species
Lumbricus rubellus, collected from different polluted sites in the Biesbosch, showed reduced values for the biomarker neutral red retention time (NRRT), mainly explained by high metal concentrations in the soil and the resulting high internal copper concentrations in the earthworms. Organic pollutant levels in earthworms were low and did not explain reduced NRRTs. Earthworm abundance and biomass were not correlated with pollutant levels in the soil. Litterbag decomposition and bait-lamina feeding activity, measures of the functional role of earthworms, were not affected by metal pollution and did not show any correlation with metal concentrations in soil or earthworms nor with NRRT. Effects at the biochemical level therefore did not result in a reduced functioning of earthworm communities.
Metal pollution in floodplain soils does affect earthworm biomarker response but not their activity in decomposition processes.
Carbon capture and storage is a technology that has been widely determined to be one of the best choices for the short-term reduction of atmospheric CO2 emissions. The aim of this study was to ...analyze the effects of CO2 enrichment in the ocean on the mussel species Mytilus galloprovincialis using three different endpoints: mortality, embryo-larval development, and neutral red retention time assays (NRRT). Acute effects were found to be associated with a pH values of 6.0 while citotoxity effects and embryo-larval development were associated with a pH value of 7.0. The NRRT assay and embryo-larval development can be recommended as good endpoints for assessing the environmental risk associated with acidification by CO2 enrichment because they provide sensitive responses on the effects of changes in seawater pH on mussels in a short period of time. Moreover, this study may support policymakers in finding appropriate solutions for the conservation of marine ecosystems.
•The paper uses three endpoints to analyze the impacts of CO2 enrichment on mussels.•The results showed significant effects on the mussels associated with low pH values.•The increase in pCO2 was strongly correlated with the increase of certain metals.•LMS and embryo-larval assays provide sensitive results of ocean acidification.
► Compared toxicity of two glyphosate formulates on Eisenia andrei was determined. ► Based on LC50, Roundup FG (72% ea) was 4.5-fold more toxic than MON 8750 (85.4% ea). ► Roundup FG showed sublethal ...effects on weight change, DNA and lysosomal damage. ► MON 8750 affected NRRT and growth at high doses, close to its LC50. ► Differences found would be due to some components of the formulations.
Glyphosate-based products are the leading post-emergent agricultural herbicides in the world, particularly in association with glyphosate tolerant crops. However, studies on the effects of glyphosate-based formulations on terrestrial receptors are scarce. This study was conducted to evaluate the comparative toxicity of two glyphosate-based products: Roundup FG (monoammonium salt, 72% acid equivalent, glyphosate-A) and Mon 8750 (monoammonium salt, 85.4% acid equivalent, glyphosate-B), towards the earthworm Eisenia andrei. Median lethal concentration (LC50) showed that glyphosate-A was 4.5-fold more toxic than glyphosate-B. Sublethal concentrations caused a concentration-dependent weight loss, consistent with the reported effect of glyphosate as uncoupler of oxidative phosphorylation. Glyphosate-A showed deleterious effects on DNA and lysosomal damage at concentrations close to the applied environmental concentrations (14.4μg ae cm−2). With glyphosate-B toxic effects were observed at higher doses, close to its LC50, suggesting that the higher toxicity of formulate A could be attributed to the effects of some of the so-called “inert ingredients”, either due to a direct intrinsic toxicity, or to an enhancement in the bioavailability and/or bioaccumulation of the active ingredient. Our results highlight the importance of ecotoxicological assessment not only of the active ingredients, but also of the different formulations usually employed in agricultural practices.
Glyphosate-based herbicides are among the most produced and widely-used herbicides. Studies have shown that commercial formulations and adjuvants may be more toxic to non-target organisms than the ...active ingredients alone, but the mechanisms of action of these chemicals remain unclear. The aim of this study was to investigate the in vitro effects of glyphosate, a commercial formulation and adjuvant alone using primary culture of hemocytes from the European abalone Haliotis tuberculata, a commonly farmed shellfish. Glyphosate was found to have negligible effects on viability, phagocytic activities and lysosome stability even with very high doses (i.e. 100 mg L−1). By contrast, greater effects on viability were observed for the commercial formulation and adjuvant alone, with EC50 values of 41.42 mg L−1 and 1.85 mg L−1, respectively. These results demonstrate that the toxic sublethal effects (i.e. phagocytic activity and destabilization of lysosomal membranes) of formulated glyphosate came from adjuvants and suggest they may be related to cell and organelle membrane destabilization.
•Glyphosate by-itself is practically non-toxic on Haliotis tuberculata hemocytes.•Toxicity of glyphosate based herbicides comes from adjuvants.•Sublethal effects occur at non-realistic environmental concentrations.•The mechanisms of toxicity seem to be linked to biological membrane destabilization.
Intrinsic genotoxic and cytotoxic potential of titanium dioxide (TiO₂) engineered nanoparticles (ENPs) were evaluated in a metabolically competent, established fish cell line derived from rainbow ...trout (Oncorhyncus mykiss) gonadal tissue (i.e. RTG-2 cells). Prior to evaluation of the toxic potential, mean size of the ENPs was determined using transmission electron microscopy (TEM). As a prerequisite, an extensive characterisation of the ENPs was carried out following sonication which enabled the synthesis of an efficient dosing strategy for the cells in which exposure in phosphate buffered saline (PBS) gave an optimal agglomeration effects compared to distilled water (H₂O) and minimal essential media (MEM). Interaction of the ENPs with cells under scanning electron microscope (SEM) was also studied. The genotoxic and cytotoxic potential of the ENPs were determined either alone or in combination with ultraviolet radiation (i.e. UVA). Whilst genotoxic potential was determined by evaluating DNA strand breaks using single cell gel electrophoresis (SCGE) or the comet assay and induction of cytogenetic damage using cytokinesis-blocked micronucleus (MN) assay, cytotoxicity was determined by measuring the retention of supra vital stain, neutral red, by the lysosomes using the neutral red retention (NRR) assay. In addition, while performing the comet assay, lesion specific bacterial endonuclease, formamidopyrimidine DNA glycosylase (Fpg), which recognises oxidised purine bases, was used to determine oxidative DNA damage. The results suggested that the highest concentration of the ENPs (i.e. 50 μg ml⁻¹) did not produce elevations in DNA damage over 4 h (comet assay), 24 h (modified comet assay) or 48 h (MN assay) exposures in the absence of UVA irradiation, although there was a significant reduction in lysosomal integrity over 24 h exposure (NRR assay). The induction of MN did not show any enhanced levels as a function of ENP concentration. A significantly increased level of strand breaks was observed in combination with UVA (3 kJ m⁻²). In general, the NRR assay suggested elevated levels of cytotoxicity when the UVA exposure was carried out with MEM compared to PBS, although both showed an increase when in combination with the highest concentration of ENPs (i.e. 50 μg ml⁻¹). Overall, the study emphasises the need for adoption of an holistic approach while evaluating the potential toxic effects of ENPs in which appropriate measures should be taken to avoid agglomeration or aggregation to facilitate efficient cellular uptake to evaluate potential biological responses.
Environmental conditions and xenobiotic exposure can be sources of stress to living organisms. Biological markers are measurable indicator of changes which may happen at any biological level and ...which can be considered an early warning signal of some biological or environmental state or condition. A structured field study was undertaken to investigate the relationship between three biomarker assays and the spatial and temporal variation of each biomarker in samples of Mytilus edulis. The three biomarkers were the neutral red retention assay, micronucleus assay and comet assay, which indicate damage at different cellular/molecular levels. Three sites in Poole Harbour, an area on the South coast of the UK were sampled on six separate occasions at least three weeks apart. The results for the comet assay showed a significant difference between sites and between sampling dates whereas the results for the other two assays did not show a significant difference for either factor. There was no significant correlation between the results of any pair of the three biomarkers. The results of the micronucleus assay showed a significant correlation with water temperature. This temperature effect, as well as induced repair, may contribute to explain the lack of a strict correspondence between pollution gradients and biomarkers responses.
•The CA results, but not those of NRR or MN, showed a significant variation with respect to sampling site and date.•No significant correlation between NRR or CA and any of the measured environmental variables (temperature, salinity, pH).•The MN Index showed a significant correlation with water temperature but no significant correlation with salinity or pH.•There was no correlation reported between the results of the three biomarkers (NRR, MN and CA).
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