Amélie Châtel

A marine mesocosm study on the environmental fate of silver nanoparticles and toxicity effects on two endobenthic species: The ragworm Hediste diversicolor and the bivalve mollusc Scrobicularia plana

Silver nanoparticles are widely used in a range of products and processes for their antibacterial properties, electrical and thermal conductivity. The fate and effects of Ag nanoparticles were examined in two endobenthic species (Scrobicularia plana, Hediste diversicolor), under environmentally realistic conditions in outdoor mesocosms exposed to Ag at 10 μg L(-1) in nanoparticulate (Ag NPs) or soluble salt (AgNO3) forms for 21 days. Labile Ag was determined in water and sediment by using diffusive gradient in thin films. Ag levels were equivalent in contaminated Ag NPs mesocosms to those contaminated with the soluble form. Bioaccumulation of Ag was observed for both species exposed to either Ag in the nanoparticulate or ionic forms. Concerning biomarker responses, both soluble and nanoparticulate Ag forms, induced defenses against oxidative stress, detoxification, apoptosis, genotoxicity and immunomodulation. Nevertheless, DNA damages measured by the comet assay in the digestive gland of S. plana, and Phenoloxidase and lysozyme activities in S. plana and H. diversicolor, respectively, were higher in the presence of Ag NPs compared to soluble Ag suggesting a specific nano effect.

Fate and effects of metal-based nanoparticles in two marine invertebrates, the bivalve mollusc Scrobicularia plana and the annelid polychaete Hediste diversicolor

The objective of this paper is to synthesize results from seven published research papers employing different experimental approaches to evaluate the fate of metal-based nanoparticles (Ag NPs, Au NPs, CuO NPs, CdS NPs, ZnO NPs) in the marine environment and their effects on two marine endobenthic species, the bivalve Scrobicularia plana and the ragworm Hediste diversicolor. The experiments were carried out under laboratory (microcosms) conditions or under environmentally realistic conditions in outdoor mesocosms. Based on results from these seven papers, we addressed the following research questions: (1) How did the environment into which nanoparticles were released affect their physicochemical properties?, (2) How did the route of exposure (seawater, food, sediment) influence bioaccumulation and effects?, (3) Which biomarkers were the most responsive? and (4) Which tools were the most efficient to evaluate the fate and effects of NPs in the marine environment? The obtained results showed that metal‐based NPs in general were highly agglomerated/aggregated in seawater. DGT tools could be used to estimate the bioavailability of metals released from NPs under soluble form in the aquatic environment. Both metal forms (nanoparticulate, soluble) were generally bioaccumulated in both species. Among biochemical tools, GST and CAT were the most sensitive revealing the enhancement of anti-oxidant defenses in both species exposed to sub-lethal concentrations of metal-based NPs. Apoptosis and genotoxicity were frequently observed.

The integrated biomarker response: a suitable tool to evaluate toxicity of metal-based nanoparticles

Abstract Nanotechnology is a much promising field of science and technology with applications in a wide range of areas such as electronics, biomedical applications, energy and cosmetics. Metal-based engineered nanoparticles (ENPs) are common in many technological applications; some of the most common nanoparticles available commercially are silver, gold, copper oxide (CuO), zinc oxide (ZnO) and cadmium sulphide (CdS). The toxicity of metal-based NPs may be either due to their specific physical characteristics as NPs or to the specific toxicity of metals released from NPs under environmental conditions. In this study we evaluated the toxicity effects of a range of ENPs (Ag, Au, CuO, CdS, ZnO) along with a control containing equivalent quantities of dissolved metal on two endobenthic species: the ragworm Hediste diversicolor and the bivalve Scrobicularia plana. A suite of complementary biomarkers was used to reveal toxicity effects. A common challenge in multibiomarkers studies is to go beyond an independent interpretation of each one, and to assess a global response of individuals. The Integrated Biomarker Response (IBR) was calculated for both species exposed to the different metal-based ENPs studied or to their dissolved metal counterpart to provide efficient and easy tools for environmental managers. We evidence that metal-based NPs lead to an overall difference in biological responses from that of their dissolved counterparts. The IBR could thus be considered as an efficient tool to transfer research results to stakeholders with possible implementation for regulatory purposes.

Genotoxicity and activation of cellular defenses in transplanted zebra mussels Dreissena polymorpha along the Seine river.

The aim of the present study was to confirm the relevance of studying DNA adduct formation in a field study. In that context, freshwater mussels Dreissena polymorpha, collected in a reference station, were transplanted in different sites with a pollution gradient. After one and two months, mussels were collected and DNA adduct formation was analyzed using the (32)P post labelling technique on both gills and digestive glands. In addition, the expression of genes involved in the detoxification system (catalase (CAT), superoxide dismutase (SOD), glutathione S-transferase (GST), HSP70, aryl hydrocarbon receptor (AHR), P glycoprotein (PgP), metallothionein (MT)) was assessed by RT-PCR. DNA adducts were observed at amount comparable to data from literature. Increase of DNA adducts after two months of transplantation could be correlated with strong modulation of gene expression implicated in detoxification processes. Indeed, PgP and HSP70 gene expressions were similarly induced in gills and digestive glands while SOD and CAT expressions were down regulated in both tissues. AHR, GST and MT genes were differently regulated depending upon the tissue studied and the level of contamination in the different sites. We demonstrated that mussels transplanted in the different stations with pollution gradient were able to biotransform PAHs, assessed by DNA adduct formation and the high decrease of detoxification genes. Specific DNA adducts pattern obtained after one and two month mussel transplantations demonstrated the relevance of DNA adduct as biomarker of environmental pollution.

The influence of salinity on the fate and behavior of silver standardized nanomaterial and toxicity effects in the estuarine bivalve Scrobicularia plana

Because of their antibacterial properties, silver (Ag) engineered nanomaterials are included in many products. The present study used a standardized Ag nanomaterial (NM-300K, 20 nm) supplied with a stabilizing agent. The aim was to investigate the behavior of Ag nanomaterial in an estuarine-like medium at 2 salinities (15 psu and 30 psu). Uptake as well as sublethal effects of Ag nanomaterial (10 μg Ag/L), its stabilizing agent, and AgNO3 (10 μg Ag/L) were assessed in the clam Scrobicularia plana, after 7 d of exposure. The release of soluble Ag from Ag nanomaterial in the experimental media was quantified by using diffusive gradient in thin films and ultrafiltration. A multibiomarker approach was employed to reveal responses of clams at subindividual and individual levels. The bioaccumulation of Ag was significantly greater at 15 psu versus 30 psu, which could be explained by differences in Ag speciation. In conclusion, the present study showed different impacts of Ag nanomaterial that were not always explained by the release of Ag ions in clams at both salinities; such impacts were particularly characterized by induction of oxidative stress, cell damage, and impairment of energetic levels. Burrowing of clams was affected by the stabilizing agent depending on the salinity tested, with stronger effects at 15 psu. Finally, the present study highlighted salinity-dependent changes in the physiology of estuarine bivalves. Environ Toxicol Chem 2016;35:2550-2561.

A novel methodology for the determination of biomarker baseline levels in the marine polychaete Hediste diversicolor

Identifying environmental damage due to anthropogenic activities is a focal point for scientists and policy makers like those involved in the European Water Framework Directive (WFD). Many of these approaches focus on ecological endpoints for assessing environmental perturbations, which lead to policies emphasizing mitigation rather than prevention. Biomarkers provide early-warning indicators of stress but it is necessary to distinguish their natural variations from those induced by chemical stress. The global aim of this study was to establish a baseline assessment criterion (BAC) using historical data in a reference site to define toxicity thresholds. We have developed a multiple polynomial regression model (MPR) accounting the influence of salinity, temperature and size of individual on energetic reserves (glycogen and lipids) in the marine polychaete Hediste diversicolor. The model identified a complex, orthogonal relationship between confounding factors and glycogen and a linear relationship between lipids and size of individuals.

DNA adduct formation and induction of detoxification mechanisms in Dreissena polymorpha exposed to nitro-PAHs

Derived polycyclic aromatic hydrocarbons (PAHs) such as nitro-PAHs are present in the environment and are known to be much more toxic than PAHs compounds. However, very few studies have analysed their effects on the aquatic environment and none have investigated the freshwater environment. In the present study, we determined whether 1-nitropyrene (1-NP), a model of nitro-PAHs, can induce DNA adducts in gills and digestive glands of the freshwater mussel Dreissena polymorpha. Two concentrations of 1-NP (50 and 500 μM) were tested. In addition, in order to understand the metabolic pathways involved in 1-NP genotoxicity, mRNA expression of genes implicated in biotransformation mechanisms was assessed by quantitative reverse transcription-PCR. Results showed the presence of DNA adducts in both gills and digestive glands, with highest levels obtained after 5 days of exposure to 500 μM. Metallothionein mRNA levels were enhanced in digestive glands exposed to 50 μM. Surprisingly, at the higher concentration (500 μM), aryl hydrocarbon receptor and HSP70 genes were only up-regulated in digestive glands while PgP mRNA levels were increased in both tissues. Results suggested a cytotoxic and genotoxic effect of 1-NP. Mussels seemed to be able to partially detoxify this compound, in view of the low amount of DNA adducts observed after 5 days exposure to 50 μM. For the first time, 1-NP biotransformation and detoxification systems have been characterised in D. polymorpha.

Metallothionein mRNA induction is correlated with the decrease of DNA strand breaks in cadmium exposed zebra mussels

We have previously shown that cadmium (Cd) and benzo[a]pyrene (BaP) induced early DNA damages in zebra mussels, and that the level of DNA strand breaks (SB) returned to a basal level after 3 days of exposure to Cd. The aim of the present study was to go further in the mechanisms of Cd and BaP detoxification. For that purpose, expression of genes encoding for metallothionein (MT), aryl hydrocarbon receptor (AHR), P-gp, catalase, glutathione S-transferase and heat shock protein 70 (HSP70) proteins have been measured using RT-qPCR. Data reported here show that Cd is a strong inducer of MT and HSP70 genes, and that BaP is a strong inducer of P-gp and AHR genes. Exposure to Cd and BaP resulted in moderate changes in antioxidant enzymes mRNA. Since the increase of MT mRNA occurred when the DNA SB level returned to its basal level, we can suggest that MT is implicated in cadmium detoxification.

Spermatozoa: A relevant biological target for genotoxicity assessment of contaminants in the estuarine bivalve Scrobicularia plana

Evaluation of DNA quality of gametes is a relevant method to predict potential consequences of pollutants in the next generations, as it allows to define adverse outcome pathways implicated in pollutant-mediated toxicity for risk assessment. In the present study, a comet assay was developed for the spermatozoa of Scrobicularia plana exposed to 10 and 100μg/L of benzo[a]pyrene (B[a]P) for 24h and 5days. The induction of apoptosis and repair mechanisms was assessed by determining caspase-3 activity and polymerase cell nuclear antigen (PCNA) mRNA expression level. Results showed that B[a]P induced high levels of DNA breaks that were associated with apoptosis for all the conditions tested, indicating that the spermatozoa were sensitive to B[a]P. PCNA gene expression was induced in animals exposed to the highest concentrations of B[a]P, suggesting that defence mechanisms were enhanced in these animals. This preliminary study demonstrated the utility of spermatozoa as a relevant biological target for genotoxicity assessment of contaminants and will enable to predict the effect of contaminants on future generations.

The role of high-throughput screening in ecotoxicology and engineered nanomaterials

The field of environmental toxicology developed as a result of growing concerns about anthropogenic influences on the environment and how to ameliorate ecological impact. Many governmental bodies are beginning to emphasize prevention rather than mitigation when addressing novel products, leading to more of a focus on identifying potential toxicity prior to release. With the exponential advances in their development and sale, novel metamaterials and biotechnology are set to dramatically outpace the capabilities of current testing strategies. To address the need for a fast, cost-effective means of testing chemicals, high-throughput screening (HTS) is currently being used in toxicology and being adapted to ecotoxicology in projects such as ToxCast and Tox21. Despite the growth of research using HTS platforms, its role in ecotoxicology is still uncertain, particularly in how it should be applied in regulation. The aim of the present review is to discuss common test strategies used in designing HTS platforms, the current potential applications for ecotoxicological research, its role in regulatory policies, and its ability to address growing concerns such as engineered nanomaterials. Environ Toxicol Chem 2017;36:1704-1714.