Ecotoxicological effects of atmospheric particulate produced by braking systems on aquatic and edaphic organisms.

Vehicles generate particulate matter (PM) in significant amounts as their brake systems wear. These particles can influence air quality and their transport/deposition may affect the edaphic and aquatic ecosystems. As part of the LOWBRASYS H2020 project, new more eco-friendly brake disc and pad formulations were developed. PMs generated from traditional (FM1-BD1) and innovative (FM4-BD2, FMB-BD7) brake systems in bench tests were studied. The PMs' physical/chemical characteristics were preliminarily investigated. To study the possible environmental impact of the nano-micro particulate, we used a battery of ecotoxicological tests. We employed the microalga Pseudokirchneriella subcapitata, the crustacean Daphnia magna and the bacteria Vibrio fischeri as aquatic bioindicators, while for the edaphic ecosystem we used the seeds of Lepidium sativum and Sorghum saccharatum, the nematode Caenorhabditis elegans, the earthworm Eisenia andrei and the ameba Dictyostelium discoideum. The results showed a higher sensitivity of the freshwater organisms exposed to the soluble PM fraction, with respect to the edaphic ones. FM4-BD2 brake formulation was slightly more toxic for algae (200 mg/L) than FM1-BD1 (500 mg/L). The new system FMB-BD7 particulate was not harmful for crustacean survival, and resulted weakly toxic for algal reproduction only at 500 mg/L. The particulate material per se was found to affect the algal reproduction. No toxic effects were found on nematodes, earthworms and seeds up to 1000 mg/L. However, in D. discoideum the reproduction rate was significantly reduced starting from 100 mg/L; and the lysosomal membrane stability showed a relevant alteration also at minimal concentration (0.1 mg/L). The results demonstrated a minimal risk for biodiversity of the particulates from the different brake systems and highlighted a more eco-friendly performance the new brake-pad FMB-BD7. However, the occurrence of sublethal effects should be considered as a possible contribution of the particle toxicity to the biological effects of the environmental pollution. Keywords: Brake discs and pads, Particulate matter, Bioassays, Sublethal effects, Environmental ris

An integrated approach to determine interactive genotoxic and global gene expression effects of multiwalled carbon nanotubes (MWCNTs) and benzo[a]pyrene (BaP) on marine mussels: evidence of reverse ‘Trojan Horse’ effects

International audienc

Mixtures of Chemical Pollutants at European Legislation Safety Concentrations: How Safe Are They?

The risk posed by complex chemical mixtures in the environment to wildlife and humans is increasingly debated, but has been rarely tested under environmentally relevant scenarios. To address this issue, two mixtures of 14 or 19 substances of concern (pesticides, pharmaceuticals, heavy metals, polyaromatic hydrocarbons, a surfactant, and a plasticizer), each present at its safety limit concentration imposed by the European legislation, were prepared and tested for their toxic effects. The effects of the mixtures were assessed in 35 bioassays, based on 11 organisms representing different trophic levels. A consortium of 16 laboratories was involved in performing the bioassays. The mixtures elicited quantifiable toxic effects on some of the test systems employed, including i) changes in marine microbial composition, ii) microalgae toxicity, iii) immobilization in the crustacean Daphnia magna, iv) fish embryo toxicity, v) impaired frog embryo development, and vi) increased expression on oxidative stress-linked reporter genes. Estrogenic activity close to regulatory safety limit concentrations was uncovered by receptor-binding assays. The results highlight the need of precautionary actions on the assessment of chemical mixtures even in cases where individual toxicants are present at seemingly harmless concentration

Mixtures of chemical pollutants at European legislation safety concentrations: how safe are they?

The risk posed by complex chemical mixtures in the environment to wildlife and humans is increasingly debated, but has been rarely tested under environmentally relevant scenarios. To address this issue, two mixtures of 14 or 19 substances of concern (pesticides, pharmaceuticals, heavy metals, polyaromatic hydrocarbons, a surfactant, and a plasticizer), each present at its safety limit concentration imposed by the European legislation, were prepared and tested for their toxic effects. The effects of the mixtures were assessed in 35 bioassays, based on 11 organisms representing different trophic levels. A consortium of 16 laboratories was involved in performing the bioassays. The mixtures elicited quantifiable toxic effects on some of the test systems employed, including i) changes in marine microbial composition, ii) microalgae toxicity, iii) immobilization in the crustacean Daphnia magna, iv) fish embryo toxicity, v) impaired frog embryo development, and vi) increased expression on oxidative stress-linked reporter genes. Estrogenic activity close to regulatory safety limit concentrations was uncovered by receptor-binding assays. The results highlight the need of precautionary actions on the assessment of chemical mixtures even in cases where individual toxicants are present at seemingly harmless concentrations

Combined effects of n-TiO2 and 2,3,7,8-TCDD in Mytilus galloprovincialis digestive gland: a transcriptomic and immunohistochemical study

Despite the growing concern over the potential biological impact of nanoparticles (NPs) in the aquatic environment, little is known about their interactions with other pollutants. In the marine mussel Mytilus galloprovincialis, exposure to nanosized titanium dioxide (n-TiO2), one of the most widespread type of NPs in use, in combination with and 2,3,7,8-tetrachlorodibenzo-p-dioxins (TCDD), chosen as model organic xenobiotic, was shown to induce significant changes in different biomarkers in hemocytes, gills and digestive gland, with distinct effects depending on cell/tissue and type of response measured. In this work, the interactive effects of n-TiO2 and TCDD at the tissue level were further investigated in mussel digestive gland using an integrated approach transcriptomics/immunohistochemistry. Mussels were exposed to n-TiO2 (100\u3bcgL-1) and TCDD (0.25\u3bcgL-1), alone and in combination, for 96h. Transcriptomic analysis identified 48-, 49- and 62 Differentially Expressed Genes (DEGs) in response to n-TiO2, TCDD and n-TiO2/TCDD, respectively. Gene Ontology (GO) term analysis revealed distinct biological processes affected in different experimental conditions. n-TiO2 mainly up-regulated cytoskeletal genes, while TCDD up-regulated endocrine and signal transduction related processes. Co-exposure induced transcriptional changes common to individual treatments, and identified a newly generated process, response to chemical stimulus. Transcription of selected genes was verified by qPCR. Moreover, expression of tubulin, as an example of target protein of interest identified by gene transcription data, was evaluated in tissue sections by immunolabelling. Tissue TCDD accumulation was evaluated by immunofluorescence with an anti-dioxins antibody.The results demonstrate both distinct and interactive effects of n-TiO2 and TCDD in mussel digestive gland at the molecular and tissue level, identify the main molecular targets involved, and underline how exposure to the n-TiO2/TCDD mixture does not result in increased TCDD accumulation and overall stressful conditions in the tissue. These represent the first data on transcriptional responses of marine invertebrates to exposure not only to n-TiO2 as a model of NP, but also to a legacy contaminant like TCDD

Application of Biotests for the Determination of Soil Ecotoxicity after Exposure to Biodegradable Plastics

Biodegradable plastics are mostly applied in packaging materials (e.g. shopping bags), waste collection bags, catering products, and agricultural applications. In this last case, degradation takes place directly in soil where biodegradable plastic products are intentionally left after use (e.g. mulch films for weeds control). Due to the growing volumes of biodegradable polymers and plastics, interest in their environmental safety is increasing and more research is carried out. Some attempt has been made to apply biotests, used in other sectors of environmental sciences, in the assessment of biodegradable plastics safety. In this work, the quality of soils after biodegradation of the bioplastics Mater-Bi has been assessed with a large array of biotests based on model organisms representative of the different trophic levels in the food chains of the edaphic and aquatic ecosystems. Mater-Bi was degraded under controlled conditions for 6 months at a 1% concentration. The selected organisms included bacteria and protozoa (V. fischeri and D. discoideum, respectively), the green alga P. subcapitata, plants (the monocotyledon S. saccharatum and the dicotyledon L. sativum), and invertebrates animals (D. magna, a freshwater crustacean, and the Oligochaeta earthworm E. andrei), using both acute and chronic endpoints. The results of the applied ecotoxicological tests showed that the Mater-Bi materials tested at very high doses did not affect the soil quality. Soil exposed to Mater-Bi has no noxious effects on edaphic organisms; in particular, mono and dicotyledon plants results, indicate that Mater-Bi plastic products are innocuous for agricultural uses. The use of more sensitive chronic endpoints allows to exclude possible effects at population level. This is the first time that such a comprehensive approach is applied to the assessment of possible ecotoxicity effects induced by biodegradable plastics in soil and represents a possible starting point for improved standardized testing schemes

Exposure to anti-mosquito insecticides utilized in rice fields affects survival of two non-target species, Ischnura elegans and Daphnia magna

Insecticides are commonly utilized to control mosquito larvae in rice fields. They can, however, have negative effects on both vertebrates and non-target invertebrate species. In this study, we examined the effects of pulse exposition to different concentrations of cypermethrin (0.15, 0.015, 0.0015 mg/L) and diflubenzuron (0.15, 0.015, 0.0015 mg/L) on egg hatching rate, larval growth, and larval survival in a damselfly, Ischnura elegans, and on survival of a crustacean, Daphnia magna. Insecticide exposure had significant negative effects on hatching rate in damselfly eggs. Exposed damselfly larvae also grew less and showed a higher mortality than control larvae. In Daphnia, the acute toxicity test (ISO 6341 in Water quality\u2014determination of the inhibition of the mobility of Daphnia magna Straus (Cladocera, Crustacea)\u2014acute toxicity test, Int Organ Stand Geneve, Geneva, 2012) showed an increased inhibition of mobility in the presence of insecticides. We observed a proportional response in relation to insecticide concentration, such that the highest exposure levels showed the largest reduction of vital performances. Our highest tested values correspond to those currently employed in agriculture. This study suggests that exposure to two common insecticides strongly affects non-target invertebrates even at very low concentration levels (cypermethrin 0.0015 mg/L and diflubenzuron 0.0015 mg/L)

Effects of dioxin exposure in Eisenia andrei: integration of biomarker data by an Expert System to rank the development of pollutant-induced stress syndrome in earthworms.

A battery of biomarkers has recently been developed in the earthworm Eisenia andrei. In this study, different biomarkers (i.e. Ca²⁺-ATPase activity, lysosomal membrane stability-LMS, lysosomal lipofuscin and neutral lipid content) were utilized to evaluate the alterations in the physiological status of animals, induced by exposure for 3d to different sublethal concentrations of TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) (1.5 × 10⁻³, 1.5 × 10⁻², 1.5×10⁻¹ ng mL⁻¹) utilizing the paper contact toxicity test. Lysosome/cytoplasm volume ratio and DNA damage were also evaluated as a biomarker at the tissue level and as a biomarker of genotoxicity, respectively. Moreover, the NR retention time assay conditions were optimized for the determination of in vivo LMS in earthworm coelomocytes. The results demonstrate that LMS and Ca²⁺-ATPase activity were early warning biomarkers able to detect the effects of minimal amounts of TCDD and that biomarkers evaluated at the tissue level are important for following the evolution of the stress syndrome in earthworms. To evaluate the health status of the animals, an Earthworm Expert System (EES) for biomarker data integration and interpretation was developed. The EES proved to be a suitable tool able to rank, objectively, the different levels of the stress syndrome in E. andrei induced by the different concentrations of TCDD

Molecular and cellular effects induced in Mytilus galloprovincialis treated with oxytetracycline at different temperatures

The present study evaluatedthe interactive effects of temperature (16\ub0C and 24\ub0C) and a 4- day treatment with the antibiotic oxytetracycline (OTC) at 1 and 100\u3bcg/L on cellular and mo- lecular parameters in the mussel Mytilus galloprovincialis. Lysosomal membrane stability (LMS), a sensitive biomarker of impaired health status in this organism, was assessed in the digestive glands. In addition, oxidative stress markers and the expression of mRNAs encod- ing proteins involved in antioxidant defense (catalase (cat) and glutathione-S-transferase (gst)) and the heat shock response (hsp90, hsp70, and hsp27) were evaluated in the gills, the target tissue of soluble chemicals. Finally, cAMP levels, which represent an important cell signaling pathway related to oxidative stress and the response to temperature chal- lenges, were also determined in the gills. Exposure to heat stress as well as to OTC rendered a decrease in LMS and an increase in malonedialdehyde accumulation (MDA). CAT activity was not significantly modified, whereas GST activity decreased at 24\ub0C. Cat and gst expres- sion levels were reduced in animals kept at 24\ub0C compared to 16\ub0C in the presence or ab- sence of OTC. At 16\ub0C, treatment with OTC caused a significant increase in cat and gst transcript levels. Hsp27 mRNA was significantly up-regulated at all conditions compared to controls at 16\ub0C. cAMP levels were increased at 24\ub0C independent of the presence of OTC. PCA analysis showed that 37.21% and 25.89% of the total variance was explained by tem- perature and OTC treatment, respectively. Interestingly, a clear interaction was observed in animals exposed to both stressors increasing LMS and MDA accumulation and reducing hsp27 gene expression regulation. These interactions may suggest a risk for the organisms due to temperature increases in contaminated seawaters

Combined effects of n-TiO2 and 2,3,7,8-TCDD in Mytilus galloprovincialis digestive gland: a transcriptomic and immunohistochemical study

Despite the growing concern over the potential biological impact of nanoparticles (NPs) in the aquatic environment, little is known about their interactions with other pollutants. In the marine mussel Mytilus galloprovincialis exposure to nanosized titanium dioxide (n- TiO2), one of the most widespread type of NPs in use, in combination with and 2,3,7,8-tetrachlorodibenzo-p-dioxins (TCDD), chosen as model organic xenobiotic, was shown to induced significant changes in different biomarkers in hemocytes, gills and digestive gland, with distinct effects depending on cell/tissue and type of response measured. In this work, the interactive effects of n-TiO2 and TCDD at the tissue level were further investigated in mussel digestive gland using an integrated approach transcriptomics/ immunohistochemistry. Mussels were exposed to n-TiO2 (100 μg L-1) and TCDD (0.25 μg L-1), alone and in combination, for 96 h. Transcriptomic analysis identified 48-, 49- and 62 Differentially Expressed Genes (DEGs) in response to n-TiO2, TCDD and n-TiO2/TCDD, respectively. Gene Ontology (GO) term analysis revealed distinct biological processes affected in different experimental conditions. n- TiO2 mainly up-regulated cytoskeletal genes, while TCDD up-regulated endocrine and signal transduction related processes. Co-exposure induced transcriptional changes common to individual treatments, and identified a newly generated process, response to chemical stimulus. Transcription of selected genes were verified by qPCR. Moreover, expression of tubulin, as an example of target protein of interest identified by gene transcription data, was evaluated in tissue sections by immunolabelling. Tissue TCDD accumulation was evaluated by immunofluorescence with an anti-dioxins antibody. The results demonstrate both distinct and interactive effects of n-TiO2 and TCDD in mussel digestive gland at the molecular and tissue level, identify the main molecular targets involved, and underline how exposure to the n-TiO2/TCDD mixture does not result in increased TCDD accumulation and overall stressful conditions in the tissue. These represent the first data on transcriptional responses of marine invertebrates to exposure not only to n-TiO2 as a model of NP, but also to a legacy contaminant like TCDD