Influence of calcium and EDTA on copper ion bioavailability in copper nanoparticle toxicity tests improves understanding of nano-specific effects.

Metal-based nanoparticles (NPs) can release metal ions that are toxic to aquatic organisms; however, whether the toxicity is from metal ions rather than unique "nano-scale" effects of the NPs is unresolved. The present study aimed to compare the toxicity of Cu2+ and Cu-NPs in larval zebrafish (Danio rerio) to clarify whether toxic effects are attributable to release of Cu ions and to determine the effect of the chelating agent ethylenediaminetetraacetic acid (EDTA) and calcium hardness (as CaCO3) on the Cu toxicity. First, the acute toxicity (96-h lethality) of Cu-NPs was determined in comparison to aqueous Cu in larvae exposed to CuSO4, and subsequently, sublethal tests with Cu-NPs and CuSO4 were conducted with additions of EDTA or calcium ions to evaluate alterations in expression of metallothionein-2 (MT2) gene transcripts (quantitative real-time polymerase chain reaction). Acute toxicity of Cu in larvae exposed to CuSO4 was greater (LC50 = 226 µg Cu/L) than for larvae exposed to Cu-NPs (LC50 = 648 µg Cu/L). The expression of MT2 increased with Cu concentration (p < 0.05), and the slope of the linear regression was significantly greater in fish exposed to CuSO4 (slope = 0.090) compared to Cu-NPs (slope = 0.011). Cu2+ was 2.9-fold more toxic than Cu-NPs. The presence of 5 mg/L EDTA and 220 mg/L CaCO3 significantly reduced the expression of MT2 (1.8-fold for EDTA, 2.3-fold for CaCO3) in larvae exposed to CuSO4. For larvae exposed to Cu-NPs, the presence of EDTA reduced the expression of MT2 (1.7-fold) relative to Cu-NP concentration. While Cu-NPs induced MT2 expression, the differences in concentration relationships of MT2 expression between Cu-NPs and CuSO4 indicated that factors other than release of Cu ions from Cu-NPs influenced acute toxicity of Cu-NPs. The conclusion drawn from this ecotoxicological risk assessment was that EDTA and calcium significantly decreased Cu toxicity in freshwater fish.

Transcriptome alterations and genotoxic influences in zebrafish larvae after exposure to dissolved aluminum and aluminum oxide nanoparticles.

Manufactured nanoparticles (NPs) can potentially cause negative effects on molecular (proteins and nucleic acids), subcellular, cellular, tissue, organ, and organism due to their unusual physicochemical characteristics. Ionizable NPs in water (e.g., Al2O3-NPs) may create toxic effects on aquatic animals. The present research determined the influences of Al2O3-NPs and appropriate concentrations of ionizing Al(III) using water-soluble AlCl3 in zebrafish larvae (72 h post-fertilization, Danio rerio) by analyzing transcriptional alterations of stress-associated genes (rad51, p53, mt2) with quantitative real-time PCR (qRT-PCR). In addition, genotoxic effects of Al(III) and Al2O3-NPs were evaluated. The lethal concentrations that cause death of 50% (LC50) of zebrafish larvae when exposed to 0-50 mg/l Al(III) and 0-500 mg/l Al2O3-NPs were 3.26 ± 0.38 and 130.19 ± 5.59 mg/l, respectively, for 96 h. A concentration-dependent increase was observed in the genotoxicity in cells of larvae exposed to Al(III) and Al2O3-NPs for 96 h. DNA damage was more severe in larvae exposed to Al(III) (41.0% tail) than that of Al2O3-NPs (21.8% tail). A complex induction of stress-associated genes was observed in fish and this induction was not directly related to the concentrations of Al(III) and Al2O3-NPs, although a significant induction was detected in mt2 gene of larvae exposed to Al(III) and Al2O3-NPs relative to control group. The induction levels of mt2 were 4.13 ± 0.1 and 2.13 ± 0.1-fold change (mean ± S.E.M.) in larvae at 15 mg/l of Al(III) and 100 mg/l of Al2O3-NP concentrations, respectively.

Bis(2-ethylhexyl) phthalate induces DNA strand breaks and gene expression alterations in larval zebrafish Danio rerio.

Contamination of the aquatic environment by plastic industrial products and their by-products is remarkable. Because of their physical, chemical, and biological degradation resistance, plasticizers can enter the food chain of living organisms, accumulate in the body and generate toxic effects. Here we determined the potential toxic effects of bis(2-ethylhexyl) phthalate (DEHP) plasticizer to larval (72 h post fertilization) zebrafish (Danio rerio) by analyzing changes in expression levels of stress-related genes (p53, rad51, and xrcc5) by the quantitative real-time polymerase chain reaction. Also, possible DNA damage by DEHP in larvae was determined. The concentration of DEHP (0-160 mg/l) that killed 50% of the larval zebrafish within 96 h was 54.02 mg/l. There was a concentration-related increase in DNA damage in cells from larvae exposed (96 h) to DEHP. DNA damage of 31.13% (mean ± standard error of the mean) was observed in larvae at the highest sublethal DEHP concentration (10 mg/l). Some significant differences in the induction of stress-related genes were also observed in larvae exposed to DEHP relative to control (p < 0.05). The conclusion drawn from this ecotoxicological risk assessment is that, under present use and exposure patterns, DEHP presents a small hazard to zebrafish larvae.

Comparison of Dissolved Nickel and Nickel Nanoparticles Toxicity in Larval Zebrafish in Terms of Gene Expression and DNA Damage.

With the use of nanoparticles (NPs) in many industrial activities and consumer products, it is important to evaluate the effects of their release into the environment. Metal NPs (e.g., Ni-NPs or Cu-NPs) can release metal ions that are toxic to aquatic organisms; however, whether the toxicity is from metal ions rather than unique "nano-scale" effects of the NPs is unresolved. This research investigated Ni-NP toxicity in zebrafish Danio rerio larvae to clarify whether toxic effects are attributable to release of Ni ions. First, the acute (96-h lethal) toxicity of Ni-NPs was determined in comparison to aqueous Ni in fish exposed to Ni(II) by water-soluble NiCl2. Subsequently, sublethal experiments with Ni-NPs and Ni(II) were conducted to assess changes in expression of stress-related genes (mt2, rad51, and p53) by quantitative PCR. Acute toxicity of Ni in fish exposed to Ni(II) was higher (96-h LC50 = 32.6 mg/L) than for fish exposed to Ni-NPs (96-h LC50 = 122.2 mg/L). Also, DNA strand breaks were higher in Ni(II)- than Ni-NPs-exposed larvae. Induction of stress-related genes in larvae was complex and was not directly related to Ni-NPs and Ni(II) concentration, although there was a significant induction in the mt2 and p53 gene of the larvae exposed to Ni-NPs and Ni(II) relative to controls. Results indicated that while Ni-NPs induced gene expression (presumably by the release of Ni ions), the differences in concentration relationships of gene expression between Ni-NPs and Ni(II) suggest that factors in addition to the release of Ni ions from Ni-NPs influence acute toxicity of Ni-NPs.

Stress-Induced Transcriptional Changes and DNA Damage Associated with Bis(2-ethylhexyl) Adipate Exposure in Zebrafish (Danio rerio) Larvae.

The present study evaluates potential toxic effects of bis(2-ethylhexyl) adipate (DEHA) plasticizer to larval (72 h post fertilization) zebrafish (Danio rerio) by analyzing changes in expression levels of stress-related genes (p53, rad51 and xrcc5) and assessing possible DNA damage of DEHA in larvae. The lethal concentration for 50% mortality (LC50) in larval zebrafish exposed for 96 h to 0-200 mg L-1 DEHA was 89.9 ± 8.03 mg L-1. A concentration-dependent increase in DNA strand breaks was detected in cells from larvae exposed for 96 h to DEHA. There were some significant differences in induction of stress-related genes in larvae exposed to DEHA relative to control.

Aqueous Hg(2+) associates with TiO2 nanoparticles according to particle size, changes particle agglomeration, and becomes less bioavailable to zebrafish.

Engineered nanoparticles (NPs) have unique physicochemistry and potential to interact with other substances in the aqueous phase. Here, gene [metallothionein 2 (mt2)] expression changes in larval zebrafish were used to evaluate the association between aqueous Hg(2+) and TiO2 (NPs and bulk particle size control) to investigate the relationship between changes in Hg(2+) behavior and TiO2 size. During 24h exposures, TiO2 agglomerates increased in size and in the presence of 25µg Hg(2+)/L, greater increases in size were observed. The concentration of Hg(2+) in suspension also decreased in the presence of TiO2-NPs. Mercury increased expression of mt2 in larval zebrafish, but this response was lessened when zebrafish were exposed to Hg(2+) in the presence of TiO2-NPs, and which suggests that TiO2-NPs alter the bioavailability of Hg(2+) to zebrafish larvae. This ameliorative effect of TiO2 was also likely due to surface binding of Hg(2+) because a greater decrease in mt2 expression was observed in the presence of 1mg/L TiO2-NPs than 1mg/L TiO2-bulk. In conclusion, the results show that Hg(2+) will associate with TiO2-NPs, TiO2-NPs that have associated Hg(2+) will settle out of the aqueous phase more rapidly, and agglomerates will deliver associated Hg(2+) to sediment surfaces.

Genotoxic effects and gene expression changes in larval zebrafish after exposure to ZnCl2 and ZnO nanoparticles.

Engineered nanoparticles (NPs) can potentially generate adverse effects at the tissue, organ, cellular, subcellular, DNA, and protein levels due to their unique physico-chemical properties. Dissoluble NPs (e.g. nZnO) can be toxic in aquatic organisms. We compared effects of nZnO and corresponding concentrations of released Zn(II) by water-soluble ZnCl(2) on larval zebrafish Danio rerio (72 h post fertilization) by analyzing changes in expression levels of stress-related genes (p53, rad51, mt2) by qRT-PCR. Additionally, genotoxicity of nZnO and Zn(II) was assessed. The lethal concentrations for 50% mortality (LC(50)) in larval zebrafish exposed for 96 h to 0 to 70 mg l(-1) nZnO and Zn(II) were 21.37 ± 1.81 mg l(-1) (95% CI) and 4.66 ± 0.11 mg l(-1), respectively. A concentration-dependent increase in DNA strand breaks was detected in cells from larvae exposed (96 h) to nZnO and Zn(II). DNA damage was higher in Zn(II)- than nZnO-exposed larvae. Induction of stress-related genes in larvae was complex and was not directly related to nZnO and Zn(II) concentrations, although there was significant induction in the mt2 gene of larvae exposed to Zn(II) and nZnO relative to controls. mt2 induction of 20.5 ± 1.9-fold and 2.5 ± 0.8-fold change (mean ± SEM) was observed in larvae at the highest Zn(II) and nZnO concentrations (3 and 6 mg l(-1)), respectively. The results suggest that toxicity associated with nZnO is primarily due to the release of Zn(II).

Use of an exposure chamber to maintain aqueous phase nanoparticle dispersions for improved toxicity testing in fish.

A novel chamber for maintaining aqueous phase dispersions of nanoparticles (NPs) to enable improved toxicity testing in larval zebrafish was developed. Aqueous concentrations were within 80% of initial NP concentrations, and the 96-h median lethal concentration (LC50) values were highly reproducible (coefficient of variation <0.16, n = 3 tests). Significantly lower toxicity for each NP tested (Ag, Cu, and TiO2 NPs) in static beakers suggested that traditional acute toxicity tests may underestimate aqueous phase toxicity of NPs.

Association of Hg2+ with aqueous (C60)n aggregates facilitates increased bioavailability of Hg2+ in Zebrafish (Danio rerio).

Manufactured nanoparticles (NPs) can associate with toxicants in the aqueous phase and these associations can influence the environmental fate, transport, and bioavailability of these toxicants in organisms. Dissolved metals (e.g., Hg(2+)) can be toxic in aquatic organisms, and, if metals associate with NPs in the aqueous phase, changes in bioavailability and toxicology may result. Here we demonstrated that Hg(2+) (25 µg/L) can associate with aqueous (C60)n (termed nC60) and increase aggregate size and settlement of nC60 aggregates out of the water column over 24 h. The concentration of C60 was directly related to concentration of Hg for nC60 aggregates that settled to the bottom of the container. Bioavailability of Hg(2+) in larval zebrafish Danio rerio, evaluated by assessment of metallothionein gene (mt2) expression, was reduced in the water column when nC60was present. However, zebrafish residing at the container bottom and exposed to nC60 aggregates with associated Hg(2+) had elevated expression of mt2 when compared to fish exposed to 25 µg/L Hg(2+) preparations without nC60, which indicated nC60 led to a localized increase in Hg(2+) bioavailability. Results indicate that aqueous nC60 can sorb Hg(2+), transport Hg(2+) to substrate surface, and increase concentrations of bioavailable Hg(2+) in organisms located where settled nC60 aggregates accumulate.

Assessment of acute toxicity and histopathology of the fungicide captan in rainbow trout.

Acute toxicity of the fungicide, captan, to juvenile rainbow trout was evaluated under static-renewal test condition. Actual concentrations of captan ranged from 0.05 to 1.00 mg/L. The concentrations of captan that killed 50% of the rainbow trout (3.11±0.8 g) within 24 (24 h; LC(50)), 48, 72 and 96 h were 0.57±0.09, 0.49±0.10, 0.44±0.11 and 0.38±0.13 mg/L (95% confidence limits), respectively. None of the unexposed control fish died and the first fish died 6 h after exposure to captan (≥0.65 mg/L). Hypertrophy, separation of epithelium from lamellae, lamellar fusion, and epithelial cell necrosis were observed on captan exposed fish. Gills also had scattered areas of focal lamellar hyperplasia. Fish exposed to fungicide had inflammation and necrosis in liver, trunk kidney and spleen. In order, the most affected organs were gill, trunk kidney and liver.

Influence of bioassay volume, water column height, and octanol-water partition coefficient on the toxicity of pesticides to rainbow trout.

Effects of water volume and water column height on toxicity of cypermethrin, carbaryl, dichlorvos, tetradifon, maneb, captan, carbosulfan endosulfan and HgCl2 to juvenile rainbow trout (Oncorhynchus mykiss, 3.2 ± 0.7 g) were evaluated in different glass aquaria under static conditions. When fish were exposed to the chemical compounds in 23 cm water column height (25 L), their mortality ranged between 0% and 58%. At the same water volume, but lower water column height (9 cm), mortality of fish increased significantly and was in a range from 60% to 95%. At the same water column height, toxic effects of chemicals were significantly higher in 25 L water volume than that of 8.5 L, water except maneb which has lowest (-0.45) octanol-water partition coefficient value. Mortality rates ratio of 9 and 23 cm water column height ranged between 1.12 and 90 while mortality rates ratio of 9 and 25 L water volume ranged between 1.20 and 4.0. Because actual exposure concentrations were not affected by either water volume or water column height, we propose that increased pesticides' toxicity was related to an increase in bioassay volume, since more pesticide molecules were able to interact with or accumulate the fish. However, there seem to be no relationship between the effects of water volume, water column height and Kow value of chemicals with regard to toxicity in juvenile rainbow trout.

Effects of some pesticides on the vital organs of juvenile rainbow trout (Oncorhynchus mykiss).

Gill, trunk kidney, spleen, and liver of rainbow trout (Oncorhynchus mykiss) were examined after exposure to different sublethal concentrations of carbosulfan (25, 50 and 200 µgL(-1)), propineb (3, 6 and 24 mgL(-1)), and benomyl (2, 5 and 20 mgL(-1)) for 14 days. Lesions were observed in gill, trunk kidney, spleen, and liver of rainbow trout exposed to either concentration of pesticides. The most important lesions were determined in the highest concentrations of pesticides. Lamellar fusion, lamellar hyperplasia, epithelial lifting, vacuolization of epithelial tissue, epithelial necrosis, hypertrophy and sloughing of epithelium were observed on fish exposed to carbosulfan, propineb and benomyl. Fish had cell necrosis, degeneration and oedemas in liver, trunk kidney and spleen. None of these lesions were seen in control fish.

Histopathological changes induced by maneb and carbaryl on some tissues of rainbow trout, Oncorhynchus mykiss.

Acute toxicity of the pesticides, maneb and carbaryl, to juvenile rainbow trout were evaluated under static-renewal test conditions. Actual concentrations of maneb ranged from 0.10mg/L to 2.00mg/L and carbaryl ranged from 0.20mg/L to 3.90mg/L. The concentrations of maneb that killed 50% of the rainbow trout (3.27+/-0.9g) within 24-h (24-h; LC(50)), 48-h, 72-h and 96-h were 1.19+/-0.12, 1.04+/-0.11, 0.92+/-0.12 and 0.81+/-0.14mg/L (95% confidence limits), respectively. LC(50) values of carbaryl for 24-h, 48-h, 72-h and 96-h were 2.52+/-0.71, 2.16+/-0.63, 1.71+/-0.46 and 1.39+/-0.15mg/L, respectively. None of the unexposed control fish died and the first fish died 6h after exposure to maneb (>or=1.30mg/L), and carbaryl (>or=2.60mg/L). Lamellar edema, separation of epithelium from lamellae, lamellar fusion, swelling of the epithelial cells and epithelial cell necrosis were observed on maneb and carbaryl exposed fish. Gills also had scattered areas of focal lamellar hyperplasia. Fish exposed to pesticides had inflammation and focal necrosis in liver, trunk kidney and spleen. Maneb and carbaryl had similar histopathological lesions. In order, the most affected organs were gill, trunk kidney and liver.