Tissue Distribution of Radiolabeled 110mAg Nanoparticles in Fish: Arctic Charr (Salvelinus alpinus).

This study presents the first whole-body tissue distributions of dissolved (AgI) and 20 nm silver nanoparticles (Ag0NPs20) in fish (Arctic charr, Salvelinus alpinus). The distributions are provided for fish exposed to three different treatments: (i) intravenous (IV), (ii) dietary, and (iii) waterborne. Quantitative whole-body autoradiography (QWBA) analyses obtained on high-resolution images reveal distinct silver distribution patterns according to the treatments. The IV exposures showed that AgNPs20 were mainly located in bile and kidney after 8 d, while AgI was distributed through the whole body and reached particular tissues such as bones, eyes, skin, liver, spleen, kidney, and intestine. The Ag0NPs20 distribution with the dietary exposures suggests that some dissolution occurred within fish organs. We propose that dissolved silver could later precipitate as chloride, sulfide, or selenide and be incorporated in bones during the growth. Consequently, it is yet difficult to state if Ag0NPs20 cross biological barriers. Finally, the waterborne exposures revealed that the gills can capture Ag0NPs20, but in small quantities. This suggests that the stability of Ag0NPs20 in water is critical for the uptake via the gills.

Diffuse sources of contamination in freshwater fish: Detecting effects through active biomonitoring and multi-biomarker approaches.

Aquatic organisms are usually exposed to a mixture of xenobiotics that may exert a large effect even in low concentrations, and when information is obtained exclusively from chemical analyses the prediction of the deleterious effects is potentially hindered. Therefore, the application of complementary monitoring methods is a priority. Here, in addition to chemical analyses, an active biomonitoring study using multiple biomarker responses in Nile tilapia Oreochromis niloticus was conducted to assess the effects of a contamination gradient along four reservoirs in Iguaçu River. Chemical analysis in the muscle showed high levels of metals in fish from the reservoir closest to an industrialized and environmentally degraded area, however fish exposed to all studied reservoirs showed hepatic alterations (necrosis and inflammatory processes). Also, significant variations of biochemical biomarkers were observed with no clear indication of contamination gradient, since an indicative of higher impact was found in an intermediary reservoir, including high concentrations of biliary polycyclic aromatic hydrocarbons (PAHs). However, nuclear morphological alterations (NMA) were less frequent at the same reservoir. Thus, the multi-biomarker approach allied to active biomonitoring is a practical and important tool to assess deleterious effects of contamination in freshwater, providing data for monitoring and conservation protocols.

Complex metabolic interactions between benzo(a)pyrene and tributyltin in presence of dichlorodiphenyltrichloroethane in South American catfish Rhamdia quelen.

In an attempt to explore complex metabolic interactions between toxicants present in polluted freshwater, hepatic metabolism of benzo(a)pyrene (BaP) and tributyltin (TBT) in fish was investigated when these compounds were administrated alone, mixed together and along with dichlorodiphenyltrichloroethane (DDT). Ten Rhamdia quelen per group were treated with a single intra-peritoneal (IP) dose (5-day experiment) or three successive doses (15-day experiment) either containing BaP (0.3; 3 or 30mgkg(-1)) or TBT (0.03; 0.3 or 3mgkg(-1)) or a combination of BaP+TBT, BaP+DDT, TBT+DDT and BaP+TBT+DDT under their respective lower doses, with DDT dose kept at 0.03mgkg(-1). Tetrahydroxy-benzo(a)pyrene (BaP-tetrol-I), and dibutyltin (DBT) and monobutyltin (MBT) were analyzed to assess BaP and TBT hepatic metabolism, respectively. A significant difference in BaP-tetrol-I concentration was observed in liver and bile between the lowest and the highest doses of BaP in both 5 and 15-day experiments. In the 15-day experiment, the presence of TBT with BaP reduced the amount of BaP-tetrol-I in bile compared to the BaP alone. The time of exposure and the number of doses affected BaP-tetrol-I concentration in the bile of fish exposed to BaP 0.3mgkg(-1) and BaP+DDT. TBT and its metabolites concentrations showed a dose-dependent increase in the liver in both experiments and in the bile in the 5-day experiment. TBT at its lowest dose was completely metabolized into DBT and MBT in the liver in the 15-day experiment. No TBT metabolites were detected in the bile of fish exposed to the mixtures in the 5-day experiment, except for a small MBT amount found in BaP+TBT+DDT. This study strengthens the hypothesis of a metabolic interaction between BaP and TBT in fish and suggests DDT as an important third player when present in the mixture.

Chronic genetic damages in Geophagus brasiliensis exposed to anthropic impact in estuarine lakes at Santa Catarina coast--southern of Brazil.

Biological monitoring through animals exposed to pollutants using biomarkers provides a promising tool for the identification of pollutants that may cause damage to human health and/or to sustainability of ecosystems. The effects of pollutants in fish tissues are important tools to understand the impact of human activities in natural ecosystems. The aim of this work was to study the water quality of two estuarine lakes in Santa Catarina, Brazil (Camacho Lake and Santa Marta Lake). Geophagus brasiliensis is a species widely distributed in Brazil and was used in this work. Comet assays in peripheral red blood and kidney cells, micronucleus tests in peripheral red blood cells, measurements of acetylcholinesterase activity in axial muscle and histopathological analysis of liver were used as biomarkers. Three sampling campaigns were undertaken in November 2004, June 2005 and November 2005. Thirty adult animals were sampled from each of three different sites (P1--Santa Marta Lake, P2 and P3--Camacho Lake). A negative control was sampled in a non-polluted site at Costa Ecological Park, Paraná. The positive control for genotoxicity was obtained by treating animals with copper sulphate. The results showed that both studied lakes are impacted by potential genotoxic substances. Severe lesions in liver of G. brasiliensis were also observed. The inhibition of acetylcholinesterase activity suggests the presence of pesticides or metals in the studied sites. This work shows that the water quality of Santa Marta and Camacho Lakes have been compromised and further control source of pollutants into these ecosystems is required.

Behavioral and neurochemical effects in mice after one-generation exposure to low doses of manganese: Focus on offspring development.

The risk of exposure to toxic metals is a known concern to human populations. The overexposure to Mn can lead to a pathological condition, with symptoms similar to Parkinson's disease. Although toxicity of Mn has been reported, studies in neonates are scarce but necessary, as Mn can cross biological barriers. The present study evaluated if chronic perinatal exposure to Mn at low doses lead to neurotoxic effects in mice, after direct and indirect exposure. Couples of mice were exposed to Mn (0.013, 0.13, and 1.3 mg kg-1.day-1) for 60 days prior to mating, as well as during gestation and lactation. The offspring was distributed into two groups: animals that were not exposed after weaning - parental exposure only (PE); and animals subject to additional 60-day exposure through gavages after weaning - parental and direct exposure (PDE). Neurological effects were evaluated by Mn quantification, behavior tests and biochemical markers in the brain. PDE animals had alterations in short/long-term memory and increased anxiety-like behavior. Exposure to Mn triggered a decrease of glutathione-s-transferase and increase of cholinesterase activity in different regions of the brain. These findings highlight the risk of exposure to low doses of Mn over a generation and at early stages of development.

Structures related to pheromone storage in alar androconia and the female abdominal scent gland of Heliconius erato phyllis, Heliconius ethilla narcaea, and Heliconius besckei (Lepidoptera: Nymphalidae: Heliconiinae).

We describe the morphology of alar androconia and the female abdominal scent gland of Heliconius erato phyllis, Heliconius ethilla narcaea, and Heliconius besckei. Androconial scales of Heliconius, which are arranged in overlapping wing bands, release pheromones during courtship, probably through vibratory movements of male wings over the female to induce her to mate. An antiaphrodisiac is produced by glands located in the valves of the male and is transferred during copulation to the yellow dorsal abdominal sac present in the virgin female, causing this sac to emit a scent that reduces the attractiveness of the female for courtship with other males. Stereomicroscopy, SEM, and TEM analyses were conducted to describe the morphology of the internal and external scales and the external abdominal scent sac. The findings revealed different sizes of external androconial scales and an internal group of porous structural vesicles that are probably related to the preservation of internal space, reception and storage of secretions, and elimination of volatiles when the male is actively involved in courtship. Translucent projections on the female abdominal scent sac create open reservoirs for the reception, storage, and emission of antiaphrodisiac volatiles along with stink clubs. Male valve denticles vary in form and probably attach securely to the female sac during mating, thus ensuring secretion transfer. These features are discussed in the context of a comparative analysis.

Multigenerational analysis of the functional status of male reproductive system in mice after exposure to realistic doses of manganese.

The present study aimed to analyze the effect of multigenerational exposure to Mn at realistic doses on the functional quality of the male reproductive system of mice. Females and males (generation F0) were treated for 60 days with MnCl2, via gavage, at the doses of 0, 0.013, 0.13, and 1.3 mg/kg/day. Treatment of F0 dams continued throughout gestation and lactation periods. At the time of weaning, the offspring (F1 generation) was divided into: animals that were not exposed after weaning - parental exposure (PE); and those exposed via parental generation and directly (PDE) for additional 60 days, at the same dose of F0 generation. F0 and F1 males were euthanized for assessment of sperm parameters and redox changes in the reproductive system. There was a decrease in the sperm concentration of the F0 generation. In addition, the sperm parameters of F1 generation were drastically affected. The activity of antioxidant enzymes was significantly reduced in PE animals. It was possible to verify that the biochemical damages were higher in the PE individuals, as demonstrated by the integrated biomarker response index. Our results show that Mn, even at low doses, is able to promote a reduction in sperm quality over a generation.

Biomarker responses in fish exposed to polycyclic aromatic hydrocarbons (PAHs): Systematic review and meta-analysis.

Biomarkers of antioxidant and biotransformation systems are commonly used to assess polycyclic aromatic hydrocarbons (PAHs) pollution in fish. Despite their extensive application of biomarkers, contradictory results are vastly reported in the literature, even for the same species in similar contamination scenarios. This study aims to verify response patterns of biomarkers in fish exposed to PAHs. Through systematic reviews and meta-analyses, we were able to evaluate: (i) overall magnitude of PAHs effects on biotransformation and oxidative stress biomarkers; (ii) patterns of response among experimental approaches (laboratory, field and active biomonitoring), environment (marine and freshwater) and fish habitat (pelagic, demersal, etc.); (iii) effects of exposure route, time and concentration of PAHs; and (iv) which biomarkers respond best to PAHs exposure. Overall, biomarker responses were significantly affected by PAHs exposure. The activities of ethoxyresorufin-O-deethylase (EROD), glutathione S-transferase (GST), superoxide dismutase (SOD), glutathione peroxidase (GPx) and levels of oxidized glutathione (GSSG) and lipid peroxide (LPO) significantly increased in fish exposed to PAHs, whereas catalase (CAT) and glutathione reductase (GR) activities and levels of reduced glutathione (GSH) were not affected. Amongst responsive biomarkers, EROD and GST activities significantly differed among approaches and between marine and freshwater environments, but were not affected by fish habitat. GSSG levels were higher in fish from laboratory bioassays compared to the field, but did not differ between environments nor habitats. Exposure route played a major role only for GST and GPx responses. Finally, increasing PAHs concentration and exposure time had a significant effect on all assessed biomarkers, except for CAT. We conclude that EROD and GST are robust biomarkers to assess PAHs effects in fish. Contrarily, CAT is an inadequate biomarker of PAHs exposure since no significant response was observed. Our study also highlighted some research gaps in PAHs contamination studies, such as a clear lack of active biomonitoring experiments.

Immunological responses, histopathological finding and disease resistance of blue mussel (Mytilus edulis) exposed to treated and untreated municipal wastewater.

This study provides new information on the response of the immune system of Mytilus edulis exposed to untreated and treated sewage, linking immune response to ecologically relevant endpoints, such as disease resistance. Our goal was to assess the potential effects of sewage on the immune system (phagocytic activity and production of cytotoxic metabolites, disease resistance) and gills (light microscope) of mussels through a bioassay and field study in an estuarine receiving environment (RE). A semi-static experiment was developed in a wastewater treatment plant in New Glasgow, NS Canada. Mussels were exposed for 21 days to 12.5%, 25%, 50% and 100% of untreated sewage influent and artificial seawater control. Sampling occurred after 7, 14 and 21 days of exposure. In the field study, eight sites were selected in East River and Pictou Harbour, NS, positioned upstream and downstream of sewage effluents outfalls. Caged mussels were exposed to the RE for 90 days (May-July 2005). Mussels were challenged to test their efficiency at eliminating the bacteria, Listonella anguillarium in the bioassay and field studies. The bioassay results showed that higher concentrations of untreated sewage could modulate the immune system of mussels through increased of phagocytic activity (PA), nitric oxide (NO) and hydrogen peroxide (H(2)O(2)) production during 14 days of exposure, and decreased activity and production at 21 days, with the exception of H(2)O(2) production which was high even at 21 days. Mussels exposed to untreated sewage RE also presented a high PA, NO and H(2)O(2) production and lower number of haemocytes compared to mussels from reference sites. In the bacterial challenge, mussels pre-exposed to 100% sewage died 24h after being infected with L. anguillarium, while mussels pre-exposed to 50% eliminated bacteria had a mortality rate of 30%. Mussels from the control, 12.5% and 25% groups eliminated bacteria and no mortality was observed. No significant difference was observed in bacterial clearance in mussels exposed to effluents in the RE. The lesions observed in gills in both studies were: infiltration of haemocytes in the tissue, epithelium proliferation, lamellar fusion and dilated haemolymphatic sinus. In summary, untreated municipal wastewater affected the immune system of blue mussels during 21 days of exposure and the effects were reflected in their capability to resist pathogens. And an immune modulation was observed in mussels exposed to untreated sewage in a RE, but this modulation was not reflected in the mussel's capability in eliminating pathogens.

Silver nanoparticles and dissolved silver activate contrasting immune responses and stress-induced heat shock protein expression in sea urchin.

Using immune cells of sea urchin Strongylocentrotus droebachiensis in early development as a model, the cellular protective mechanisms against ionic and poly(allylamine)-coated silver nanoparticle (AgNPs; 14 ± 6 nm) treatments at 100 µg L-1 were investigated. Oxidative stress, heat shock protein expression, and pigment production by spherulocytes were determined as well as AgNP translocation pathways and their multiple effects on circulating coelomocytes. Sea urchins showed an increasing resilience to Ag over time because ionic Ag is accumulated in a steady way, although nanoAg levels dropped between 48 h and 96 h. A clotting reaction emerged on tissues injured by dissolved Ag (present as chloro-complexes in seawater) between 12 h and 48 h. Silver contamination and nutritional state influenced the production of reactive oxygen species. After passing through coelomic sinuses and gut, AgNPs were found in coelomocytes. Inside blood vessels, apoptosis-like processes appeared in coelomocytes highly contaminated by poly(allylamine)-coated AgNPs. Increasing levels of Ag accumulated by urchins once exposed to AgNPs pointed to a Trojan-horse mechanism operating over 12-d exposure. However, under short-term treatments, physical interactions of poly(allylamine)-coated AgNPs with cell structures might be, at some point, predominant and responsible for the highest levels of stress-related proteins detected. The present study is the first report detailing nano-translocation in a marine organism and multiple mechanisms by which sea urchin cells can deal with toxic AgNPs. Environ Toxicol Chem 2017;36:1872-1886. © 2016 SETAC.

A multibiomarker evaluation of urban, industrial, and agricultural exposure of small characins in a large freshwater basin in southern Brazil.

Iguaçu River is the second most polluted river of Brazil. It receives agrochemicals and contaminants of urban and industrial sources along its course. A multibiomarker approach was employed here to evaluate the health of a small characin (Astyanax spp.) at two sites along the river, sampled during a dry (autumn) and a rainy (spring) season. Biomarkers were condition factor and somatic indices (gonads and liver); genetic damage (comet assay and micronucleus test); enzyme activities such as hepatic catalase (CAT) and glutathione S-transferase (GST), lipoperoxidation (LPO), branchial and renal carbonic anhydrase (CA), acetylcholinesterase (AChE) in the muscle and the brain, histopathology of the liver and gills, and concentrations of polycyclic aromatic hydrocarbons (PAHs) in bile. There were no consistent differences in biomarker responses between the two study sites. Some biomarkers revealed greater potential impact in the rainy season, when increased amounts of contaminants are washed into the river (combined CAT inhibition and LPO increase, CA upregulation). Other biomarkers, however, revealed potential greater impact in the dry season, when contaminants potentially concentrate (GST induction, AChE inhibition, and liver histopathological alterations). Although of a complex nature, field experiments such as this provide rich data for monitoring protocols and assessment of general risk of exposure to pollutants of river systems.

Analyses of paralytic shellfish toxins and biomarkers in a southern Brazilian reservoir.

The Alagados Reservoir (Brazil) is an important source for the supply of water, recreation and fishery. Since 2002, the occurrence of cyanobacterial blooms (paralytic shellfish toxins - PST producers) have been noted. This study was aimed at the monitoring of PST occurrence in the Reservoir's water and fish. Biomarkers such as ethoxyresorufin-O-deethylase (EROD), glutathione S-transferase (GST), catalase (CAT), and acetylcholinesterase (AchE) activities, lipoperoxidation (LPO), histopathology, and comet assay were analyzed in fish. Water and fish were sampled in spring, summer and autumn. The PST concentrations in water were 5.15, 43.84, and 50.78 ng equiv Saxitoxin/L in the spring, summer and autumn, respectively. The PST muscle concentration was below the limit for shellfish. Gonyautoxins (GTX) were found in water samples and fish muscle, and GTX 5 was the major analogous found in muscle. In the summer samples, the LPO, genetic damage, and the GST and AchE activities increased while in the autumn an increase in EROD activity and genetic damage were observed. In all samplings, histopathological alterations in the fish gills and liver were found. The results showed a seasonal variation in the fishes health, which could be related also to farming activities and to the contaminants bioavailability during the year.