Patrice Turcotte

Toxicity of silver nanoparticles to rainbow trout: a toxicogenomic approach.

Silver (Ag) nanoparticles are used as antimicrobial adjuvant in various products such as clothes and medical devices where the release of nano-Ag could contaminate the environment and harm wildlife. The purpose of this study was to examine the sublethal effects of nano-Ag and dissolved Ag on Oncorhynchus mykiss rainbow trout. Hepatic Ag contents and changes in gene expression were monitored to provide insights on bioavailability and mode of action of both forms of silver. Fish were exposed to increasing concentrations (0.06, 0.6 and 6 μg L(-1)) of nano-Ag (20 nm) and silver nitrate (AgNO(3)) for 96 h at 15°C. A gene expression analysis was performed in the liver using a DNA microarray of 207 stress-related genes followed by a quantitative polymerase chain reaction on a selection of genes for validation. The biochemical markers consisted of the determination of labile zinc, metallothioneins, DNA strand breaks, lipid peroxidation (LPO) and vitellogenin-like proteins. The analysis of total Ag in the aquarium water revealed that nano-Ag was mostly aggregated, with 1% of the total Ag being dissolved. Nevertheless, hepatic Ag content was significantly increased in exposed fish. Indeed, dissolved Ag was significantly more bioavailable than nano-Ag only at the highest concentration with 38 ± 10 and 11 ± 3 ng Ag mg(-1) proteins for dissolved and nano-Ag respectively. Exposure to both forms of Ag led to significant changes in gene expression for 13% of tested gene targets. About 12% of genes responded specifically to nano-Ag, while 10% of total gene targets responded specifically to dissolved Ag. The levels of vitellogenin-like proteins and DNA strand breaks were significantly reduced by both forms of Ag, but DNA break levels were lower with nano-Ag and could not be explained by the presence of ionic Ag. Labile zinc and the oxidized fraction of metallothioneins were increased by both forms of Ag, but LPO was significantly induced by nano-Ag only. A discriminant function analysis revealed that the responses obtained by biochemical markers and a selection of ten target genes were able to discriminate completely (100%) the effects of both forms of Ag. Exposure to nano-Ag involved genes in inflammation and dissolved Ag involved oxidative stress and protein stability. Hence, the toxicity of Ag will differ depending on the presence of Ag nanoparticles and aggregates.

Effects of cadmium telluride quantum dots on cadmium bioaccumulation and metallothionein production to the freshwater mussel, Elliptio complanata.

Nanotechnology has gained increasing commercial attention over recent years and its use has raised concerns about its potential release in the environment. The purpose of this study was to determine the size distribution of CdTe in freshwater, bioavailability and potential toxic effects of cadmium telluride quantum dots (CdTe QD) to the freshwater mussel Elliptio complanata. Mussels were exposed to increasing concentrations (0 to 8 mg Cd L(-1)) of CdTe and 0.5 mg/L CdSO4 for 24 h at 15 degrees C to examine the initial uptake and toxic effects of Cd from CdTe QDs and dissolved CdSO4. After the exposure period, Cd bioaccumulation in the gills, digestive gland and gonad tissues and metallothionein (MT) levels were determined. The results revealed that about 80% of Cd was retained by a 450 nm pore filter (aggregates) and that 14% of the Cd was in the dissolved phase (i.e., eluted through a 1 kDa ultrafiltration membrane) which suggested that uncoated CdTe QDs were not stable in freshwater. In mussels, Cd was accumulated principally by the gills and digestive gland and the bioaccumulation factors of Cd from CdTe were similar to that of dissolved Cd. Indeed, tissue-levels of Cd were below the proportion of dissolved Cd from CdTe which suggests that Cd rather comes from the dissociation of Cd from the ingested QDs than from the internalization of the QDs in mussel tissues. The levels of MT were induced in both the digestive gland and gonad but were readily decreased in the gills by both CdTe and CdSO4. The observed decrease in the metallic form of MT might result from the oxidative stress by CdTe and dissolved Cd. In conclusion, uncoated CdTe QD in freshwater leads to aggregates and a dissolved component of Cd where the latter explained the contribution of the observed accumulation pattern in mussel tissues and effects on MT levels in mussels.

Sublethal effects of silver nanoparticles and dissolved silver in freshwater mussels.

The increasing application of silver nanoparticles (nAg) in various consumer products has raised concerns regarding toxicological impacts in the environment. It is unclear at present whether the toxicity of nAg is mainly the result of the release of ionic Ag+ in mussels. The freshwater mussel Elliptio complanata was exposed to increasing concentrations of 20-nm nAg, 80-nm nAg, and dissolved Ag+ for 48 h at 15°C. The following biomarkers were used to determine the mode of action of nAg-induced adverse effects: metallothioneins (MT) (ionic Ag+ release), lipid peroxidation (LPO) (ionic Ag+ and nanosurface interactions), heat-shock proteins (HSP) (size-related effects), protein-ubiquitin levels (size-related effects), and DNA strand breaks (ionic Ag+ and size effects). Results revealed that the response pattern of 80 nm nAg was more closely related to ionic Ag+ than 20 nm nAg, suggesting a more important release of dissolved Ag from 80 nm nAg. Data showed that all forms of Ag were able to increase the levels of MT and LPO, which suggests the presence of ionic Ag+ leads to oxidative stress. However, nanoparticles were also able to induce changes in protein-ubiquitin and to a lesser extent actinomyosin-ATPase, MT, and DNA strand breaks in the digestive gland in a manner different from Ag+, which permitted discrimination of the forms of Ag. Moreover, LPO was closely associated with DNA strand breaks in the digestive gland and was not entirely explained by induction of MT, suggesting another type of toxic interaction. It was concluded that the presence of nAg not only increases the toxic loadings of released Ag ions but also generates other and perhaps cumulative effects of nanoparticle-induced toxicity related to size and surface properties.

Bioavailability and Immunotoxicity of Silver Nanoparticles to The Freshwater Mussel Elliptio complanata

The purpose of this study was to examine the effects of Ag nanoparticles (nAg) of two different sizes (20 and 80 nm) and Ag+ on the immune system of the freshwater mussel Elliptio complanata. Mussels were exposed to increasing concentrations of nAg and dissolved Ag (AgNO3) for 48 h at 15°C and concentration of 0, 0.8, 4, or 20 μg/L. Immunocompetence was determined by hemocyte viability, phagocytosis, and cell cytotoxicity. Ag tissue loadings and levels of metallothioneins (MT), lipid peroxidation (LPO), and labile zinc (Zn) were also determined. Results revealed first that 20- and 80-nm nAg readily formed aggregates in freshwater. Ag was detected in soft tissues with each form of Ag with bioconcentration factors of 20, 9, and 7 for Ag+, 20-nm nAg, and 80-nm nAg, respectively. Significant induction in phagocytosis and decreased cell cytotoxicity were observed. All forms of Ag were able to induce LPO in gills and digestive glands at concentrations below those from the initial fraction of dissolved Ag. The effects of nAg on MT levels in mussels were not discernible from those of dissolved Ag, but the 80-nm was 25-fold more potent than 20-nm nAg in inducing MT. Multivariate analysis revealed that the global responses of the 20- and 80-nm nAg were generally similar to those of dissolved Ag. Data also demonstrated that nAg are bioavailable for mussels where the immune system is a target during early exposure to nanoparticles.

In vitro immunotoxicology of quantum dots and comparison with dissolved cadmium and tellurium

The increasing use of products derived from nanotechnology has raised concerns about their potential toxicity, especially at the immunocompetence level in organisms. This study compared the immunotoxicity of cadmium sulfate/cadmium telluride (CdS/Cd‐Te) mixture quantum dots (QDs) and their dissolved components, cadmium chloride (CdCl2)/sodium telluride (NaTeO3) salts, and a CdCl2/NaTeO3 mixture on four animal models commonly used in risk assessment studies: one bivalve (Mytilus edulis), one fish (Oncorhynchus mykiss), and two mammals (mice and humans). Our results of viability and phagocytosis biomarkers revealed that QDs were more toxic than dissolved metals for blue mussels. For other species, dissolved metals (Cd, Te, and Cd‐Te mixture) were more toxic than the nanoparticles (NPs). The most sensitive species toward QDs, according to innate immune cells, was humans (inhibitory concentration [IC50] = 217 μg/mL). However, for adaptative immunity, lymphoblastic transformation in mice was decreased for small QD concentrations (EC50 = 4 μg/mL), and was more sensitive than other model species tested. Discriminant function analysis revealed that blue mussel hemocytes were able to discriminate the toxicity of QDs, Cd, Te, and Cd‐Te mixture (Partial Wilks λ = 0.021 and p < 0.0001). For rainbow trout and human cells, the immunotoxic effects of QDs were similar to those obtained with the dissolved fraction of Cd and Te mixture. For mice, the toxicity of QDs markedly differed from those observed with Cd, Te, and dissolved Cd‐Te mixture. The results also suggest that aquatic species responded more differently than vertebrates to these compounds. The results lead to the recommendation that mussels and mice were most able to discriminate the effects of Cd‐based NPs from the effects of dissolved Cd and Te at the immunocompetence level.

Size distribution effects of cadmium tellurium quantum dots (CdS/CdTe) immunotoxicity on aquatic organisms

The increasing use of products derived from nanotechnology has raised concern about their potential toxicity to aquatic life. This study sought to examine the comparative immunotoxicity of capped cadmium sulphide/cadmium telluride (CdS/CdTe) quantum dots (QDs) and possible impact of particle/aggregate size on two bivalves (Mytilus edulis and Elliptio complanata) and a fish (Oncorhynchus mykiss). The QDs were dispersed in sterile water and fractionated using a series of micro/ultrafiltration membranes of decreasing pore size: 450 nm, 100 nm, 50 nm, 25 nm, 100 kDa (6.8 nm), 30 kDa (4.6 nm), 10 kDa (3.2 nm) and 1 kDa (1.5 nm). The total concentrations of cadmium and tellurium were determined for the filtered material and for that retained on the filters (retentate). The immunotoxicity was determined by measuring cell viability and phagocytosis. Results revealed that nanoparticles retained on the ultrafilters had a higher Cd/Te ratio compared to the permeate fraction (ratio of 5 and 2 respectively) which could indicate that the CdS core was not associated with the permeable fraction of Cd. Our results demonstrate that the toxicity of CdS/CdTe QDs was concentration and size dependent. Large CdS/CdTe QD aggregates (25 nm < size < 100 nm) reduced phagocytosis more than did smaller nanoparticles (<25 nm). Moreover, our results revealed that the different species responded differently to these fractions. Mytilus edulis hemocytes were less sensitive to CdS/CdTe QDs than the Oncorhynchus mykiss macrophage and Elliptio complanata hemocytes.

Ecotoxicological impacts of effluents generated by oil sands bitumen extraction and oil sands lixiviation on Pseudokirchneriella subcapitata.

The exploitation of Athabasca oil sands deposits in northern Alberta has known an intense development in recent years. This development has raised concern about the ecotoxicological risk of such industrial activities adjacent to the Athabasca River. Indeed, bitumen extraction generated large amounts of oil sands process-affected water (OSPW) which are discharged in tailing ponds in the Athabasca River watershed. This study sought to evaluate and compare the toxicity of OSPW and oil sands lixiviate water (OSLW) with a baseline (oil sands exposed to water; OSW) on a microalgae, Pseudokirchneriella subcapitata, at different concentrations (1.9, 5.5, 12.25, 25 and 37.5%, v/v). Chemical analyses of water-soluble contaminants showed that OSPW and OSLW were enriched in different elements such as vanadium (enrichment factor, EF=66 and 12, respectively), aluminum (EF=64 and 15, respectively), iron (EF=52.5 and 17.1, respectively) and chromium (39 and 10, respectively). The toxicity of OSPW on cells with optimal intracellular esterase activity and chlorophyll autofluorescence (viable cells) (72h-IC 50%<1.9%) was 20 times higher than the one of OSW (72h-IC 50%>37.5%, v/v). OSLW was 4.4 times less toxic (IC 50%=8.5%, v/v) than OSPW and 4.5 times more toxic than OSW. The inhibition of viable cell growth was significantly and highly correlated (<-0.7) with the increase of arsenic, beryllium, chromium, copper, lead, molybdenum and vanadium concentrations. The specific photosynthetic responses studied with JIP-test (rapid and polyphasic chlorophyll a fluorescence emission) showed a stimulation of the different functional parameters (efficiency of PSII to absorb energy from photons, size of effective PSII antenna and vitality of photosynthetic apparatus for energy conversion) in cultures exposed to OSPW and OSLW. To our knowledge, our study highlights the first evidence of physiological effects of OSPW and OSLW on microalgae.

Fate of silver nanoparticles in wastewater and immunotoxic effects on rainbow trout.

Silver nanoparticles (AgNPs) are currently used in technology, medicine and consumer products, even though the fate and the ecotoxicological risks on aquatic organisms of these new materials are not well known. The purpose of this study was to investigate the fate, bioavailability of AgNPs and their effects on fish in presence of municipal effluents. Juvenile rainbow trout were exposed for 96h to 40μg/L of AgNPs or 4μg/L of dissolved silver (AgNO3) in diluted (10%) municipal wastewater. Silver (Ag) concentrations were measured both on water samples and fish tissues (liver and gills). Toxicity was investigated by following immunological parameters in the pronephros (viability, phagocytosis) and biomarkers in liver and gills (cyclooxygenase activity, lipid peroxidation, glutathione-S-transferase, metallothioneins, DNA strand breaks and labile zinc). Results indicated that AgNPs appeared as small non-charged aggregates in wastewaters (11.7±1.4nm). In gills, the exposure to AgNPs induced morphological modifications without visible nanoparticle bioaccumulation. Dissolved Ag(+) was bioavailable in diluted effluent and induced oxidative stress (lipid peroxidation), labile zinc and a marginal decrease in superoxide dismutase in fish gills. Ag(+) also increased significantly metallothionein levels and inhibited the DNA repair activity in the liver. Finally, the two silver forms were found in liver and induced immunosuppression and inflammation (increase in cyclooxygenase activity). This study demonstrated that both forms of Ag produced harmful effects and AgNPs in wastewater were bioavailable to fish despite of their formation of aggregates.

Impacts of municipal wastewater oxidative treatments: Changes in metal physical speciation and bioavailability

The environmental repercussions of the discharge of disinfected effluents are still poorly understood. This study assessed the impact of ozonation and UV oxidative treatment processes on metal forms - particulate, colloidal and permeable fractions - and bioavailability in disinfected wastewaters. In addition to wastewater analyses, mussels were placed in continuous flow-through aquaria and exposed for 4wk to wastewater, then metals in their tissues were analysed in parallel with exposure biomarkers. Metal size distribution was affected by oxidative processes; results showed that ozonation treatment generally increases the permeable fraction of some metals, particularly Cd and Cu, in treated waters, whereas UV treatment fosters the formation of permeable Zn. Ozone treatment of wastewater generally increased the bioavailability of specific metals. Metal bioaccumulation was in most cases significantly higher in mussels exposed to ozone-treated effluent compared to the UV treatment: 58%, 32%, 42% and 47% higher, respectively, for Ag, Cd, Cr and Cu. Physical metal speciation in these wastewaters comparatively measured the permeable fraction of metals to relate them to the bioaccumulation results for the exposed mussels. The levels of lipid peroxidation were significantly increased in gills but not in the digestive gland. The levels of metallothionein in the digestive gland were also significantly reduced suggest decreased input of particulate metals. Results of bioaccumulation in mussels suggested that metal bioavailability can be modified by the different oxidative processes. Despite this disadvantage, ozonation still represents a great choice of treatment considering the overall environmental benefits.

An investigation of the immunotoxicity of oil sands processed water and leachates in trout leukocytes

Increased oil sands (OS) mining activity has raised concerns about impacts on aquatic organisms. This study sought to examine the effects of single representative compounds from OS (benzo(a)pyrene, naphthalene), a mixture of naphthenic acids (NAs), OS-processed water (OSPW) and OS leachate (OSL) extracts on rainbow trout leukocytes. Primary cultures of trout leukocytes were exposed to increasing concentrations of benzo(a)pyrene, naphthalene, NAs, OSPW and OSL for 48h at 18°C. Immunocompetence was followed by measuring changes in lymphocyte and macrophage viability and phagocytosis. Changes in the expression of 10 transcripts were also followed: interleukin 1, 2 and 6 (Il-1, Il-2 and Il-6), calreticulin (CRT), caspase 9 (Cas9), aryl hydrocarbon receptor (AhR), cyclooxygenase-2 (COX2), glutathione S-transferase (GST), catalase (CAT) and p53 tumor suppressor. The results revealed that exposure to OSPW extracts decreased the capacity of macrophages to engulf three beads or more, while the other compounds generally increased phagocytosis activity. Lymphocyte apoptosis was increased by all compounds and mixtures except naphthalene. Both OSPW and OSL induced apoptosis in macrophages. At the gene expression level, Cas9, CRT, Il-1 (inhibition) and Il-2 were specifically influenced by OSPW, while CAT, p53, COX2 and Il-1 (induction) transcripts were specifically expressed by OSL. Leukocyte exposure to OSPW produced characteristic changes in immunocompetence and genes involved in proinflammatory, apoptosis and protein damage (CRT) pathways which could not be explained by OSL, benzo(a)pyrene, naphthalene and NA mixture.