Developmental toxicity of cypermethrin in embryo-larval stages of zebrafish

Cypermethrin, a type II pyrethroid insecticide, is widely used throughout the world in agriculture, forestry, horticulture and homes. Though the neurotoxicity of cypermethrin has been thoroughly studied in adult rodents, little is so far available regarding the developmental toxicity of cypermethrin to fish in early life stages. To explore the potential developmental toxicity of cypermethrin, 4-h post-fertilization (hpf) zebrafish embryos were exposed to various concentrations of cypermethrin (0, 25, 50, 100, 200 and 400 μg L⁻¹) until 96 h. Among a suite of morphological abnormalities, the unique phenotype curvature was observed at concentrations as low as 25 μg L⁻¹. Studies revealed that 400 μg L⁻¹ cypermethrin significantly increased malondialdehyde production. In addition, activity of antioxidative enzymes including superoxide dismutase and catalase were significantly induced in zebrafish larvae in a concentration-dependent manner. To further investigate the toxic effects of cypermethrin on fish, acridine orange (AO) staining was performed at 400 μg L⁻¹ cypermethrin and the result showed notable signs of apoptosis mainly in the nervous system. Cypermethrin also down-regulated ogg1 and increased p53 gene expression as well as the caspase-3 activity. Our results demonstrate that cypermethrin was able to induce oxidative stress and produce apoptosis through the involvement of caspases in zebrafish embryos. In this study, we investigated the developmental toxicity of cypermethrin using zebrafish embryos, which could be helpful in fully understanding the potential mechanisms of cypermethrin exposure during embryogenesis and also suggested that zebrafish could serve as an ideal model for studying developmental toxicity of environmental contaminants.     

Polyaniline nanofibers: acute toxicity and teratogenic effect on Rhinella arenarum embryos

The fate and effect of nanomaterials in the environment is of paramount importance towards the technological application of the materials. This work shows the ecotoxicological potential of polyaniline (PANI) nanofibers in the larvae Rhinella arenarum by means of AMPHITOX test. Acute toxicity of PANI nanofibers towards embryos of the common South American toad R. arenarum (Anura: bufonidae) was evaluated in the premetamorphosis (stage 25) larvae. The exposure of R. arenarum larvae to at dose of 150, 250 and 400 mg L⁻¹ resulted in 100% viability within 96 h exposure. The embryos at 2-4 blastomers stage (early life stage teratogenic test) revealed that embryos were not killed and no teratogenic effects were observed when embryos were incubated with PANI nanofibers (150 and 250 mg L⁻¹), while only a growth retardation of embryos was induced at levels of 250 mg PANI nanofibers L⁻¹. On the other hand, at 400 mg L⁻¹ concentration, a reduction in the body length of larvae and tail malformation was observed. This results suggest that a concentration-dependent toxicity is operative, typified by phenotypes that had abnormal body axes. The presence of PANI nanofibers in gut contents and its excretion by larval stages of R. arenarum was confirmed by UV-visible spectroscopy.     

Acute effects of Fe₂O₃, TiO₂, ZnO and CuO nanomaterials on Xenopus laevis

Metal oxide nanomaterials have exhibited toxicity to a variety of aquatic organisms, especially microbes and invertebrates. To date, few studies have evaluated the toxicity of metal oxide nanomaterials on aquatic vertebrates. Therefore, this study examined effects of ZnO, TiO₂, Fe₂O₃, and CuO nanomaterials (20-100 nm) on amphibians utilizing the Frog Embryo Teratogenesis Assay Xenopus (FETAX) protocol, a 96 h exposure with daily solution exchanges. Nanomaterials were dispersed in reconstituted moderately hard test medium. These exposures did not increase mortality in static renewal exposures containing up to 1000 mg L⁻¹ for TiO₂, Fe₂O₃, CuO, and ZnO, but did induce developmental abnormalities. Gastrointestinal, spinal, and other abnormalities were observed in CuO and ZnO nanomaterial exposures at concentrations as low as 3.16 mg L⁻¹ (ZnO). An EC₅₀ of 10.3 mg L⁻¹ ZnO was observed for total malformations. The minimum concentration to inhibit growth of tadpoles exposed to CuO or ZnO nanomaterials was 10 mg L⁻¹. The results indicate that select nanomaterials can negatively affect amphibians during development. Evaluation of nanomaterial exposure on vertebrate organisms are imperative to responsible production and introduction of nanomaterials in everyday products to ensure human and environmental safety.     

Comparative acute freshwater hazard assessment and preliminary PNEC development for eight fluorinated acids

Short-term 48, 72 and 96-h aquatic toxicity tests were conducted to evaluate the acute toxicity of eight fluorinated acids to the cladoceran, Daphnia magna, the green alga, Pseudokirchneriella subcapitata, and the rainbow trout, Oncorhynchus mykiss or the fathead minnow, Pimephales promelas. The eight fluorinated acids studied were tridecafluorohexyl ethanoic acid (6:2 FTCA), heptadecafluorooctyl ethanoic acid (8:2 FTCA), 2H-dodecafluoro-2-octenoic acid (6:2 FTUCA), 2H-hexadecafluoro-2-decenoic acid (8:2 FTUCA), 2H,2H,3H,3H-undecafluoro octanoic acid (5:3 acid), 2H,2H,3H,3H-pentadecafluoro decanoic acid (7:3 acid), n-perfluoropentanoic acid (PFPeA) and n-perfluorodecanoic acid (PFDA). The results of the acute toxicity tests conducted during this study suggest that the polyfluorinated acids, 8:2 FTCA, 8:2 FTUCA, 6:2 FTCA, 6:2 FTUCA, 7:3 acid and 5:3 acid, and the perfluorinated acids PFPeA and PFDA, are generally of low to medium concern based on evaluation of their acute freshwater toxicity (EC/LC50s typically between 1 and >100 mg L⁻¹) using the USEPA TSCA aquatic toxicity evaluation paradigm. For the polyfluorinated acids, aquatic toxicity generally decreased as the number of fluorinated carbons decreased and as the overall carbon chain length decreased from 12 to 8. Acute aquatic toxicity of the 5 and 10 carbon perfluorocarboxylic acids (EC/LC50s between 10.6 and >100 mg L⁻¹) was greater or similar to that of the 6-9 carbon perfluorocarboxylic acids (EC/LC50s > 96.5 mg L⁻¹). This study also provides the first report of the acute aquatic toxicity of the 5:3 acid (EC/LC50s of 22.5 to >103 mg L⁻¹) which demonstrated less aquatic toxicity than the 7:3 acid (EC/LC50s of 0.4-32 mg L⁻¹). The cladoceran, D. magna and the green alga, P. subcapitata had generally similar EC50 values for a given substance while fish were typically equally or less sensitive with the exception that PFPeA was most toxic to fish. Predicted no-effect concentrations (PNECs) were estimated using approaches consistent with REACH guidance and when compared with available environmental concentrations, these PNECs suggest that the fluorinated acids tested pose little risk for aquatic organisms.     

Interference with xenobiotic metabolic activity by the commonly used vehicle solvents dimethylsulfoxide and methanol in zebrafish (Danio rerio) larvae but not Daphnia magna

Organic solvents, such as dimethylsulfoxide (DMSO) and methanol are widely used as vehicles to solubilise lipophilic test compounds in toxicity testing. However, the effects of such solvents upon innate detoxification processes in aquatic organisms are poorly understood. This study assessed the effect of solvent exposure upon cytochrome P450 (CYP)-mediated xenobiotic metabolism in Daphnia magna and zebrafish larvae (4 d post fertilisation). Adult D. magna were demonstrated to have a low, but detectable, metabolism of ethoxyresorufin in vivo and this activity was not modulated by pre-exposure to DMSO or methanol (24 h, up to 0.1% and 0.05% v/v, respectively). In contrast, the metabolism of ethoxyresorufin in zebrafish larvae was significantly reduced by both solvents (0.1% and 0.05% v/v, respectively) after 24 h of exposure. In zebrafish, these observed decreases in activity towards ethoxyresorufin were accompanied by decreased expression of a variety of genes coding for drug metabolising enzymes (corresponding to CYP1, CYP2, CYP3 and UDP-glucuronyl transferase [UGT] family enzymes), measured by quantitative PCR. Reduction of gene expression and CYP1 enzyme activities by methanol (0.05% v/v) in zebrafish larvae was partially reversed by co-exposure with Aroclor 1254 (100 μg L⁻¹). Overall this study suggests that relatively low concentrations of organic solvents can impact upon the biotransformation of certain xenobiotics in zebrafish larvae, and that this warrants consideration when assessing compounds for metabolism and toxicity in this species.     

Cypermethrin has the potential to induce hepatic oxidative stress, DNA damage and apoptosis in adult zebrafish (Danio rerio)

Cypermethrin (CYP), a widely used Type II pyrethroid pesticide, is one of the most common contaminants in the freshwater aquatic system. We studied the effects of CYP exposure on the induction of hepatic oxidative stress, DNA damage and the alteration of gene expression related to apoptosis in adult zebrafish. Hepatic mRNA levels for the genes encoding antioxidant proteins, such as Cu/Zn-Sod, Mn-Sod, Cat, and Gpx, were significantly upregulated when zebrafish were exposed to various concentrations of CYP for 4 or 8 days. In addition, the main genes related to fatty acid β-oxidation and the mitochondrial genes related to respiration and ATP synthesis were also significantly upregulated after exposure to high concentrations (1 and 3 μg L⁻¹) of CYP for 4 or 8 days. Moreover, in a comet assay of zebrafish hepatocytes, tail DNA, tail length, tail moment and Olive tail moment increased in a concentration-dependent manner. The significant induction (p < 0.01) of all four parameters observed with CYP concentrations of 0.3 μg L⁻¹ or higher suggests that heavy DNA damage was induced even at low levels. Furthermore, several apoptosis- related genes, such as p53, Apaf1 and Cas3, were significantly upregulated after CYP exposure, and Bcl2/Bax expression ratio decreased, especially in groups treated with 1 and 3 μg L⁻¹ CYP for 8 days. Taken together, our results suggested that CYP has the potential to induce hepatic oxidative stress, DNA damage and apoptosis in zebrafish. This information will be helpful in fully understanding the mechanism of aquatic toxicology induced by CYP in fish.     

Derivation of aquatic predicted no-effect concentration (PNEC) for 2,4-dichlorophenol: comparing native species data with non-native species data

2,4-Dichlorophenol (2,4-DCP) is known as an important chemical intermediate and an environmental endocrine disruptor. There is no paper dealing with the predicted no-effect concentration (PNEC) of 2,4-DCP, mainly due to shortage of chronic and site-specific toxicity data. In the present study, toxicity data was obtained from the tests using six Chinese native aquatic species. The HC₅ (hazardous concentration for 5% of species) was derived based on the constructed species sensitivity distribution (SSD), which was compared with that derived from literature toxicity data of non-native species. For invertebrates, the survival no-observed effect concentrations (NOECs) were 0.05 and 1.00 mg L⁻¹ for Macrobrachium superbum and Corbicula fluminea, respectively. NOECs based on fishes’ growth were 0.10, 0.20 and 0.40 mg L⁻¹ for Mylopharyngodon piceus, Plagiognathops microlepis and Erythroculter ilishaeformis, respectively. For aquatic plant Soirodela polyrhiza, NOEC based on concentration of chlorophyll was 1.00 mg L⁻¹. A final PNEC calculated using the SSD approach with a 50% certainty based on different taxa ranged between 0.008 and 0.045 mg L⁻¹. There is no significant difference between HC₅ derived from native and that from non-native taxa.     

Toxicity assessment of a common laundry detergent using the freshwater flagellate Euglena gracilis

Synthetic detergents are among the commonly used chemicals in everyday life. Detergents, reaching aquatic environments through domestic and municipal wastewater, can cause many different effects in aquatic organisms. The present study was aimed at the toxicity evaluation of a commonly used laundry detergent, Ariel, using the freshwater flagellate Euglena gracilis as a biotest organism. Different parameters of the flagellate like motility, swimming velocity, cell shape, gravitactic orientation, photosynthesis and concentration of light harvesting pigments were used as end points for the toxicity assessment. No Observed Effect Concentration (NOEC) and EC₅₀ values were calculated for the end point parameters at four different incubation times, i.e. 0, 6, 24 and 72 h. After 72 h incubation, swimming velocity of the cells was found to be the most sensitive parameter giving NOEC and EC₅₀ values of 10.8 and 34 mg L⁻¹, respectively. After 72 h exposure to the detergent, chlorophyll a and total carotenoids were significantly decreased in cultures treated with Ariel at concentrations of 50 mg L⁻¹ and above while chlorophyll b significantly decreased at concentrations above 750 mg L⁻¹. The maximum inhibitory effect on the quantum yield of photosystem II was observed after 24 h exposure and thereafter a recovery trend was observed. Motility, gravitaxis and cell shape were strongly impaired immediately upon exposure to the detergent, but with increasing exposure time these parameters showed acclimatization to the stress and thus the NOEC values obtained after 72 h were higher than those immediately after exposure.     

Derivation of predicted no effect concentrations (PNEC) for 2,4,6-trichlorophenol based on Chinese resident species

2,4,6-Trichlorophenol (2,4,6-TCP) is a common chemical intermediate and a by-product of water chlorination and combustion processes, and is a priority pollutant of the aquatic environment in many countries. Although information on the toxicity of 2,4,6-TCP is available, there is a lack of information on the predicted no-effect concentration (PNEC) of 2,4,6-TCP, mainly due to the shortage of chronic and site-specific toxicity data. In the present study, acute and sub-chronic toxicity of 2,4,6-TCP on six different resident Chinese aquatic species were determined. PNEC values were calculated and compared by use of two approaches: assessment factor (AF) and species sensitivity distribution (SSD). Values for acute toxicity ranged from 1.1 mg L⁻¹ (Plagiognathops microlepis) to 42 mg L⁻¹ (Corbicula fluminea) and the sub-chronic no observed effect concentrations (NOECs) ranged from 0.05 mg L⁻¹ (Mylopharyngodon piceus) to 2.0 mg L⁻¹ (C. fluminea). PNECs obtained by the assessment factor approach with acute (AF = 1000, 0.001 mg L⁻¹) or chronic (AF = 10, 0.005 mg L⁻¹) toxicity data were one order of magnitude less than those from SSD methods (0.057 mg L⁻¹). PNEC values calculated using SSD methods with a 50% certainty for 2,4,6-TCP was less than those obtained by use of the USEPA recommend final chronic value (FCV) method (0.097 mg L⁻¹) and the one obtained by use of the USEPA recommend acute-to-chronic (ACR) methods (0.073 mg L⁻¹). PNECs derived using AF methods were more protective and conservative than that derived using SSD methods.     

Assessing the toxicity of sodium chloride to the glochidia of freshwater mussels: implications for salinization of surface waters

Chloride concentrations in surface waters have increased significantly, a rise attributed to road salt use. In Canada, this may be a concern for endangered freshwater mussels, many with ranges limited to southern Ontario, Canada’s most road-dense region. The acute toxicity of NaCl was determined for glochidia, the mussel’s larval stage. The 24 h EC50s of four (including two Canadian endangered) species ranged from 113-1430 mg Cl L⁻¹ (reconstituted water, 100 mg CaCO₃ L⁻¹). To determine how mussels would respond to a chloride pulse, natural river water (hardness 278-322 mg CaCO₃ L⁻¹) was augmented with salt. Lampsilis fasciola glochidia were significantly less sensitive to salt in natural water (EC50s 1265-1559 mg Cl L⁻¹) than in reconstituted water (EC50 285 mg L⁻¹). Chloride data from mussel habitats revealed chloride reaches levels acutely toxic to glochidia (1300 mg L⁻¹). The increased salinization of freshwater could negatively impact freshwater mussels, including numerous species at risk.     

Effect of chronic exposure to acetaminophen and lincomycin on Japanese medaka (Oryzias latipes) and freshwater cladocerans Daphnia magna and Moina macrocopa, and potential mechanisms of endocrine disruption

Chronic toxicity of acetaminophen and lincomycin were evaluated using freshwater organisms including two crustaceans (Daphnia magna and Moina macrocopa) and a fish (Oryzias latipes). H295R, a human adrenal cell was also used to understand the effects on steroidogenesis. In 21 d D. magna exposure, survival NOEC was found at 5.72 mg L⁻¹ and no reproduction related effects were noted at this level of exposure to acetaminophen, while 21 d survival or growth effects were not observed even at the highest exposure levels (153 mg L⁻¹) for lincomycin. In the chronic fish toxicity test, significant reduction in juvenile survival was observed at 30 d post-hatch (dph) at 95 mg L⁻¹ of acetaminophen, and 0.42 mg L⁻¹ of lincomycin. After the exposure to both pharmaceuticals, vitellogenin levels tended to increase in male fish at 90 dph. In the eggs which were prenatally exposed to 9.5 mg L⁻¹ of acetaminophen, reduced hatchability was observed. The results of H295R cell assay showed that both pharmaceuticals could alter steroidogenic pathway and increase estrogenicity. Endocrine disruption potentials and their ecological implication may deserve further studies. Our observations suggest however that ecological risks of both pharmaceuticals are negligible at the concentrations currently found in the environment.     

Converging hazard assessment of gold nanoparticles to aquatic organisms

The gold nanoparticles (Au-NPs) are being increasingly used because of their huge diversity of applications, and consequently, elevated levels in the environment are expected. However, due to their physico-chemical properties and functionalization a high variety of Au-NPs can be found, and complete toxicological information for each type of Au-NPs still lacks, and even, the toxicological information for the same species is sometimes contradictory. Therefore, hazard assessment should be done case by case. Hence, the objective of this study was to obtain ecotoxicological information of the same Au-NPs in aquatic organisms and to find a rationale for Au-NPs toxicity. For such a purpose, bare and hyaluronic acid capped Au-NPs (12.5 nm) along with Au-NPs bulk material were tested on freshwater algae, Daphnia and zebrafish. Results showed that while gold nanoparticles were found to be harmless to the tested organisms, the soluble gold showed to be toxic to algae and Daphnia, with an LC₅₀ between 1 and 2 mg L⁻¹. Comparing our results with those gathered in the literature, it appears that a common hazard assessment of Au-NPs on the studied organisms can be elucidated.     

Effects of four commonly used UV filters on the growth,cell viability and oxidative stress responses of the Tetrahymena thermophila

UV filters are increasingly used in sunscreens and other personal care products. Although their residues have been widely identified in aquatic environment, little is known about the influences of UV filters to protozoan. The growth inhibition effects, cell viability and oxidative stress responses of four commonly used UV filters, 2-ethylhexyl 4-methoxycinnamate (EHMC), benzophenone-3 (BP-3), 4-methyl-benzylidene camphor (4-MBC) and octocrylene (OC), to protozoan Tetrahymena thermophila were investigated in this study. The 24-h EC₅₀ values with 95% confidence intervals for BP-3 and 4-MBC were 7.544 (6.561–8.675) mg L⁻¹ and 5.125 (4.874–5.388) mg L⁻¹, respectively. EHMC and OC did not inhibit the growth of T. thermophila after 24 h exposure at the tested concentrations. The results of cell viability assays with propidium iodide (PI) staining were consistent with that of the growth inhibition tests. As for BP-3 and 4-MBC, the relatively higher concentrations, i.e. of 10.0 and 15.0 mg L⁻¹, could lead to the cell membranes impairment after 4 h exposure. With the increase of the exposure time to 6 h, their adverse effects on cell viability of T. thermophila were observed at the relatively lower concentration groups (1.0 mg L⁻¹ and 5.0 mg L⁻¹). In addition, it is noticeable that at environmentally relevant concentration (1.0 μg L⁻¹), BP-3 and 4-MBC could lead to the significant increase of catalase (CAT) activities of the T. thermophila cells. Especially for the BP-3, the oxidative injuries were further confirmed by the reduction of glutathione (GSH) content. It is imperative to further investigate the additive action of UV filters and seek other sensitive endpoint, especially at environmentally relevant concentration.     

Toxic effects of Hydrilla verticillata exposed to toluene, ethylbenzene and xylene and safety assessment for protecting aquatic macrophytes

Little information is available about the toxicity of toluene, ethylbenzene and xylene acting on macrophytes, and their toxicity data are rarely used in regulation and criteria decisions. The results extended the knowledge on toxic effects of toluene, ethylbenzene and xylene on aquatic plants. The responses of Hydrilla verticillata to these pollutants were investigated. Chlorophyll levels, lipid peroxidation, and antioxidant enzymes (superoxide dismutase and guaiacol peroxidase) showed diverse responses at different concentrations of toluene, ethylbenzene and xylene. The linear regression analyses were performed respectively, suggesting the concentrations of toluene, ethylbenzene and xylene expected to protect aquatic macrophytes were 7.30 mg L⁻¹, 1.15 mg L⁻¹ and 2.36 mg L⁻¹, respectively. This study emphasized that aquatic plants are also sensitive to organic pollutants as fishes and zooplanktons, indicating that macrophytes could be helpful in predicting the toxicity of these pollutants and should be considered in regulation and criteria decisions for aquatic environment protection.     

Food utilization and growth of cutworm Spodoptera litura Fabricius larvae exposed to nickel,and its effect on reproductive potential

Food utilization and growth of the 5th and 6th instar Spodoptera litura Fabricius larvae, and its effect on reproduction potential was evaluated by feeding larvae diets with different doses of Ni for 3 generations. Dose-dependent relationships between Ni levels and food consumption and growth were variable with different larval developmental period and Ni exposure duration. RCR, AD and RGR of the 6th instar larvae were much more affected by Ni exposure than those of 5th instar larvae, and the effects were strongest in the 3rd generation. It was found that RCR was significantly stimulated after 1 and 20 mg kg⁻¹ Ni exposure, while AD was significantly inhibited after 1, 5, 10 and 40 mg kg⁻¹ Ni exposure. However, lower levels of Ni (?5 mg kg⁻¹) significantly increased and higher levels of Ni (?10 mg kg⁻¹) significantly decreased RGR. In 3 successive generations, 10 mg kg⁻¹ Ni significantly increased the ECI and ECD of the 5th instar larvae, and 5 mg kg⁻¹ Ni significantly increased the ECD of the 6th instar larvae. However, ECD were all significantly inhibited with 20 mg kg⁻¹ Ni exposure. Results also revealed that durations of larvae were shortened at low levels of Ni, but extended at high levels of Ni. Fecundity was inhibited by the highest Ni doses in each generation, while improved by low Ni doses in the 3rd generation. Hatching rates in all treatments were significantly decreased in a Ni dose-dependent manner. Study indicated that effects of Ni on these parameters were predominant with the increasing Ni exposure period.     

The effect of oil sands process-affected water and naphthenic acids on the germination and development of Arabidopsis

Oil sands mining in the Athabasca region of northern Alberta results in the production of large volumes of oil sands process-affected water (OSPW). We have evaluated the effects of OSPW, the acid extractable organic (AEO) fraction of OSPW, and individual naphthenic acids (NAs) on the germination and development of the model plant, Arabidopsis thaliana (Arabidopsis). The surrogate NAs that were selected for this study were petroleum NAs that have been used in previous toxicology studies and may not represent OSPW NAs. A tricyclic diamondoid NA that was recently identified as a component of OSPW served as a model NA in this study. Germination of Arabidopsis seeds was not inhibited when grown on medium containing up to 75% OSPW or by 50 mg L⁻¹ AEO. However, simultaneous exposure to three simple, single-ringed surrogate NAs or a double-ringed surrogate NA had an inhibitory effect on germination at a concentration of 10 mg L⁻¹, whereas inhibition of germination by the diamondoid model NA was observed only at 50 mg L⁻¹. Seedling root growth was impaired by treatment with low concentrations of OSPW, and exposure to higher concentrations of OSPW resulted in increased growth inhibition of roots and primary leaves, and caused bleaching of cotyledons. Treatment with single- or double-ringed surrogate NAs at 10 mg L⁻¹ severely impaired seedling growth. AEO or diamondoid NA treatment was less toxic, but resulted in severely impaired growth at 50 mg L⁻¹. At low NA concentrations there was occasionally a stimulatory effect on root and shoot growth, possibly owing to the broad structural similarity of some NAs to known plant growth regulators such as auxins. This report provides a foundation for future studies aimed at using Arabidopsis as a biosensor for toxicity and to identify genes with possible roles in NA phytoremediation.     

Comparative study of the accumulation and detoxification of Cu (essential metal) and Hg (nonessential metal) in the digestive gland and gills of mussels Mytilus galloprovincialis, using analytical and histochemical techniques

The aim of the present study is the comparative examination of accumulation and detoxification of Cu and Hg in digestive gland and gills of mussels Mytilus galloprovincialis, using atomic absorption spectrophotometry and autometallography. Mussels were exposed to 0.08 mg L⁻¹ Cu, 0.08 mg L⁻¹ Hg, as well as to a mixture of 0.08 mg L⁻¹ Hg and 0.08 mg L⁻¹ Cu for 11 d. After the experimental exposure, animals were kept under laboratory conditions for a detoxification period of 7 d. An antagonistic effect of Cu against to Hg accumulation was noted in the digestive gland of mussels after the experimental exposure, as well as after the detoxification period, supporting the protective role of Cu against to Hg toxicity in this tissue. Digestive gland was suggested as a main organ for Hg accumulation and gills as a target position for Cu accumulation. Additionally, lower time was evaluated for Hg detoxification in the digestive gland and gills of mussels, in relation to those addressed for Cu detoxification in the same tissues. The evaluation of black silver deposits (BSD) extent performed in digestive gland and gills was suggested as a less sensitive approach, in relation to atomic absorption spectrophotometry (AAS), to indentify the concentration of heavy metals in tissues of mussels. The toxic effects of Hg, Cu and a mixture of them on lysosomal system of the digestive cells are also discussed.     

Copper oxide nanoparticles can induce toxicity to the freshwater shredder Allogamus ligonifer

Increased commercialisation of nanometal-based products augments the possibility of their deposition into aquatic ecosystems; this, in turn, may pose risks to aquatic biota and associated ecological functions. Freshwater invertebrate shredders mostly use microbially-colonized plant litter as food resource and play an important role in aquatic detritus food webs. We assessed lethal effects of nanoCuO on the shredder Allogamus ligonifer (Trichoptera, Limnephilidae) by determining the concentration that induced 50% of death (LC₅₀), and sublethal effects of nanoCuO on the feeding behaviour and growth of the shredder by exposing the animals to: (i) stream water supplemented with nanoCuO and microbially-colonized leaves, and (ii) stream water (without nanoCuO) and microbially-colonized leaves pre-exposed to nanoCuO. Results from acute lethal tests showed that the 96 h LC₅₀ of nanoCuO was very high (569 mg L⁻¹). In the absence of nanoparticles, leaf consumption rate was 0.27 mg leaf DM mg⁻¹ animal DM d⁻¹ and the shredder growth rate was 56 μg animal DM mg⁻¹ animal DM d⁻¹. A significant inhibition in leaf consumption rate (up to 47%) and invertebrate growth rate (up to 46%) was observed when shredders were exposed to the higher tested sublethal concentration of nanoCuO (75 mg L⁻¹) through either contaminated stream water or pre-contaminated food. The exposure to increased nanoCuO concentration via water or pre-contaminated food led to higher accumulation of copper in the larval body. Leached water-soluble ionic copper from the nanoCuO adsorbed or accumulated in the shredder (up to 10.2% of total Cu) seemed to influence the feeding behaviour and growth of the shredder.     

The freshwater planarian Polycelis felina as a sensitive species to assess the long-term toxicity of ammonia

Behavioural endpoints are a good link between physiological and ecological effects. However long-term behavioural endpoints are not uniformly studied over all different organism groups. For example behaviour has been scarcely studied in planarians. Unionized ammonia (NH₃) is one of the most widespread pollutants in developed countries, and is known to alter animal behaviour. In this study a long-term (30 d) bioassay was conducted to assess the effect of this pollutant on survival and behavioural activity (e.g. locomotion activity) of the freshwater planarian Polycelis felina. One control and three environmentally-realistic concentrations of unionized ammonia (treatments of 0.02, 0.05, and 0.09 mg N-NH₃ L⁻¹) were used in quintuplicate. The behaviour of planarians was measured after 0, 10, 20 and 30 d of ammonia exposure. Mortality was recorded every 2 d. Unionized ammonia increased mortality in the two highest NH₃ concentrations and the locomotory activity was depressed in all treatments after 20 d of exposure. Behavioural effect was observed at concentrations 20 times lower than the short-term LC50 for this species. Previous studies proposed safe concentrations of unionized ammonia of 0.01-0.10 mg N-NH₃ L⁻¹ to aquatic ecosystems, but our study has shown that these concentrations will affect planarians. Because planarians play a key role in streams (as predator/scavenger), safe concentrations should be below 0.02 mg N-NH₃ L⁻¹ to protect this species in the freshwater community. Our results can contribute to improve the knowledge about ammonia toxicity to freshwater ecosystems, we recommend that safe concentrations of unionized ammonia should be based on very sensitive species.