Genotoxic and mutagenic effects of chlorothalonil on the estuarine fish Micropogonias furnieri (Desmarest, 1823).

Chlorothalonil is a fungicide widely used in agriculture as well as an active ingredient in antifouling paints. Although it causes toxic effects on non-target organisms and can accumulate in fish tissues, little is known about its sublethal effects. Thus, genotoxic and mutagenic effects of intraperitoneal injected chlorothalonil in Micropogonias furnieri, an estuarine fish of frequent human consumption and a promising test-organism for ecotoxicological assays, were assessed. Chlorothalonil showed to be genotoxic (DNA damage by comet assay) and mutagenic (micronuclei, nuclear buds, apoptotic fragments, and bilobed cells) even at the lowest dose tested (0.35 µg g-1) and in a dose-dependent manner (0.35 and 3.5 µg g-1) for micronuclei, apoptotic fragments, and bilobed cells. As genomic instability may lead to carcinogenesis, the present evidence can assist decision-makers in banning this compound since any benefit toward food production is outweighed by the hazard to aquatic ecosystems and human health.

Response of biomarkers to metals, hydrocarbons and organochlorine pesticides contamination in crabs (Callinectes ornatus and C. bocourti) from two tropical estuaries (São José and São Marcos bays) of the Maranhão State (northeastern Brazil).

Response of biomarkers to chemical contamination was evaluated in crabs of the Callinectes genus (Callinectes ornatus and C. bocourti) from two tropical estuaries (São José and São Marcos bays) of the Maranhão State (northeastern Brazil). Biomarkers evaluated included hepatopancreatic metallothionein-like proteins (MTLP) and lipid peroxidation (LPO), as well as muscle acetylcholinesterase (AChE). Tissue concentrations of metals (pereiopod muscle and hepatopancreas), hydrocarbons (hepatopancreas) and organochlorine pesticides (hepatopancreas) were also evaluated. Crab samples were collected in three sites of each estuary (São Marcos Bay and São José Bay). Sampling was performed in August/2012 (dry season), January/2013 (rainy season), August/2013 (dry season), and January/2014 (rainy season). Concentrations of chemical contaminants and responses of biomarkers showed significant spatial (São Marcos Bay and São José Bay) and/or seasonal (dry and rainy seasons) and annual (2012-2014) variability. However, a general higher Zn concentration was observed in hepatopancreas of crabs from São José Bay. In turn, a general higher Cd concentration paralleled by oxidative damage (LPO) was observed in hepatopancreas of crabs from São Marcos Bay. As expected, these findings support the idea that this bay is more intensively or chronically impacted by industrial activities while the São José Bay is likely more affected by domestic activities. Interestingly, LPO level in crab hepatopancreas showed to be the most reliable and adequate biomarker to distinguish the two bays.

Negative metal bioaccumulation impacts on systemic shark health and homeostatic balance.

Contamination by metals is among the most pervasive anthropogenic threats to the environment. Despite the ecological importance of marine apex predators, the potential negative impacts of metal bioaccumulation and biomagnification on the health of higher trophic level species remains unclear. To date, most toxicology studies in sharks have focused on measuring metal concentrations in muscle tissues associating human consumption and food safety, without further investigating potential impacts on shark health. To help address this knowledge gap, the present study evaluated metal concentrations in the gills, muscle, liver and rectal gland of coastal sharks opportunistically sampled from Brazilian waters and tested for potential relationships between metal bioaccumulation and general shark health and homeostatic balance metrics. Results revealed high metal concentrations in all four tissue types, with levels varying in relation to size, sex, and life-stage. Metal concentrations were also associated with serum biomarkers (urea, lactate, ALT, triglycerides, alkaline phosphatase, and phosphorus) and body condition, suggesting potential negative impacts on organismal health.

Biochemical and Histological Biomarkers in Crassostrea sp. (Bivalvia, Ostreidae) for Environmental Monitoring of a Neotropical Estuarine Area (São José Bay, Northeastern Brazil).

This study aimed to compare biochemical and histological biomarkers in oysters to identify impacted areas in a Brazilian port region. Oysters belonging to the Crassostrea genus were collected in two points in São José Bay (Brazil): (A1) Curupu Island (control area) and (A2) Braga Port (impacted area). Digestive glands from oysters were used to analyze the enzymatic activity of glutathione S-transferase and Catalase. The gills were used for standard histology analyses. Water samples were collected for metal analyses. Our results indicated that there was a change in the activity of oyster GST and CAT enzymes, especially in A2. Histological gill analysis indicated more frequent changes in A2. The analyzed metals presented higher values in A2. The results of this study suggest that enzymatic alterations, histological changes and higher metal values are indicative of initial stress caused by contaminants in São José Bay, especially in the port region.

Biomarkers (glutathione S-transferase and catalase) and microorganisms in soft tissues of Crassostrea rhizophorae to assess contamination of seafood in Brazil.

The goal of this study was to evaluate biomarkers (glutathione S-transferase and catalase) and microorganisms in soft tissues of Crassostrea rhizophorae to assess possible contamination of seafood in Brazil. The oysters were sampled from a reference area (Ports 1 and 2) and an impacted area (Ports 3 and 4) in Brazil (São Luís Island, Maranhão). Six attributes were examined in sampled oysters: glutathione S-transferase activity, catalase activity, concentrations of total coliforms and thermotolerant coliforms, and levels of Escherichia coli and Aeromonas hydrophila. Water samples were analysed for aluminium, cadmium, iron, manganese, lead, mercury, phenolics, and polychlorinated biphenyls. We found that Ports 3 and 4 are impacted by several contaminants (mercury, phenolics, and polychlorinated biphenyls), while Ports 1 and 2 are still relatively free of these contaminants. Changes in enzymes activity as well as the highest tissue bacterial concentrations were recorded in oysters from Ports 3 and 4 during the rainy season.

Life-time exposure to waterborne copper II: Patterns of tissue accumulation and gene expression of the metal-transport proteins ctr1 and atp7b in the killifish Poecilia vivipara.

The involvement of transporting proteins on copper (Cu) bioaccumulation was evaluated in the killifish Poecilia vivipara chronically exposed to environmentally relevant concentrations of waterborne Cu. Fish (<24 h-old) were maintained under control condition or exposed to different waterborne Cu concentrations (5, 9 and 20 µg/L) for 28 and 345 days in saltwater. Following exposure periods, Cu accumulation and the expression of genes encoding for the high affinity Cu-transporter (ctr1) and the P-type Cu-ATPase (atp7b) were evaluated. Whole-body metal accumulation and gene expression were evaluated in fish exposed to 28 days. Similarly, in fish exposed to 345 days, liver, gills and gut were also evaluated. No fish survival was observed after exposure to 20 µg/L for 345 days. Whole-body Cu accumulation was significantly higher in fish exposed to 20 µg/L Cu for 28 days and in fish exposed to 9 µg/L for 345 days in comparison to control animals. Similarly, tissue Cu accumulation was significantly higher in fish exposed to 9 µg/L for 345 days in comparison to control animal. However, no significant accumulation was observed in fish muscle. Following exposure for 28 days, whole-body ctr1 expression was slightly induced in fish exposed to 9 µg/L. In turn, no significant change in ctr1 expression was observed following exposure to Cu for 345 days. Differently, whole-body atp7b expression was markedly up-regulated in the whole-body of fish exposed Cu for 28 days and in tissues of fish exposed to Cu for 345 days. These findings indicate the expression of atp7b is more responsive to Cu accumulation in P. vivipara than ctr1 expression and, therefore, more suitable to be used as a biomarker of exposure to this metal. Also, we argue that the expression of atp7b is sustained at elevated levels for as much time as fish are maintained in Cu contaminated water.

Physiological effects of marine natural organic matter and metals in early life stages of the North Pacific native marine mussel Mytilus trossulus; a comparison with the invasive Mytilus galloprovincialis.

The role of seawater NOM in reducing metal toxicity for marine organisms is not well understood. We investigated the effects of five different marine NOMs (two autochthonous, one allochthonous, two of mixed origin, at 8 mg C/L), three metals (6 µg Cu/L; 20 µg Pb/L; 25 µg Zn/L), and combinations between them, to early life stages of Mytilus trossulus (a North Pacific native) in 48-h tests. Endpoints were whole body Ca2++Mg2+-ATPase activity, carbonic anhydrase (CA) activity and lipid peroxidation. Comparisons were made with previously reported tests (identical conditions) on the invasive M. galloprovincialis. Unexposed M. trossulus had lower Ca2++Mg2+-ATPase but similar baseline CA activity and lipid peroxidation to unexposed M. galloprovincialis. NOMs alone induced increased enzyme activities, and increased lipid peroxidation, but the latter did not occur with NOMs of mixed origin in M. trossulus. There was no clear difference in the sensitivity to various NOMs between species. In M. trossulus, all three metals by themselves caused increases in lipid peroxidation, as did many metal-NOM combinations. The origin of the NOMs influenced the nature of the responses to NOM-metal combinations in both species, but no clear relationship to NOM chemistry was apparent. Overall, M. trossulus was more sensitive to metals and NOM-metal combinations, with a greater number of significant responses (27 versus 22 treatment endpoints, out of a total of 72) and a greater proportion of negative effects (81% versus 50%) than in M. galloprovincialis. Therefore, marine NOMs by themselves, as well as metals by themselves and NOM-metal combinations, can induce both positive and negative physiological responses. Lipid peroxidation appears to be a particularly common negative response. In future studies, NOM quality and mussel species should be considered since native M. trossulus and invasive M. galloprovincialis exhibited markedly different responses after exposure to the same environmental conditions.

Physiological effects of five different marine natural organic matters (NOMs) and three different metals (Cu, Pb, Zn) on early life stages of the blue mussel (Mytilus galloprovincialis).

Metals are present in aquatic environments as a result of natural and anthropogenic inputs, and may induce toxicity to organisms. One of the main factors that influence this toxicity in fresh water is natural organic matter (NOM) but all NOMs are not the same in this regard. In sea water, possible protection by marine NOMs is not well understood. Thus, our study isolated marine NOMs by solid-phase extraction from five different sites and characterized them by excitation-emission fluorescence analysis-one inshore (terrigenous origin), two offshore (autochthonous origin), and two intermediate in composition (indicative of a mixed origin). The physiological effects of these five NOMS alone (at 8 mg/L), of three metals alone (copper, lead and zinc at 6 µg Cu/L, 20 µg Pb/L, and 25 µg Zn/L respectively), and of each metal in combination with each NOM, were evaluated in 48-h exposures of mussel larvae. Endpoints were whole body Ca2++Mg2+-ATPase activity, carbonic anhydrase activity and lipid peroxidation. By themselves, NOMs increased lipid peroxidation, Ca2++Mg2+-ATPase, and/or carbonic anhydrase activities (significant in seven of 15 NOM-endpoint combinations), whereas metals by themselves did not affect the first two endpoints, but Cu and Pb increased carbonic anhydrase activities. In combination, the effects of NOMs predominated, with the metal exerting no additional effect in 33 out of 45 combinations. While NOM effects varied amongst different isolates, there was no clear pattern with respect to optical or chemical properties. When NOMs were treated as a single source by data averaging, NOM had no effect on Ca2++Mg2+-ATPase activity but markedly stimulated carbonic anhydrase activity and lipid peroxidation, and there were no additional effects of any metal. Our results indicate that marine NOMs may have direct effects on this model marine organism, as well as protective effects against metal toxicity, and the quality of marine NOMs may be an important factor in these actions.

The Effects of Acute Copper and Ammonia Challenges on Ammonia and Urea Excretion by the Blue Crab Callinectes sapidus.

Copper (Cu) is a persistent environmental contaminant that elicits several physiological disturbances in aquatic organisms, including a disruption in ammonia regulation. We hypothesized that exposure to Cu in a model crustacean (blue crab, Callinectes sapidus) acclimated to brackish water (2 ppt) would lead to hyperammonemia by stimulating an increase in ammonia production and/or by inhibiting ammonia excretion. We further hypothesized that urea production would represent an ammonia detoxification strategy in response to Cu. In a pilot experiment, exposure to 0, 100, and 200 µg/L Cu for 6 h caused significant concentration-dependent increases in ammonia excretion (J amm). Based on these results, an acute 24-h 100 µg/L Cu exposure was conducted and this similarly caused an overall stimulation of J amm during the 24-h period, indicative of an increase in ammonia production. Terminal haemolymph total ammonia content (T amm) was unchanged, suggesting that while ammonia production was increased, there was no inhibition of the excretion mechanism. In support of our second hypothesis, urea excretion (J urea) increased in response to Cu exposure; haemolymph [urea] was unaffected. This suggested that urea production also was increased. To further test the hypothesis that J urea increased to prevent hyperammonemia during Cu exposure, crabs were exposed to high environmental ammonia (HEA; 2.5 mmol/L NH4HCO3) for 12 h in a separate experiment. This led to a fourfold increase in haemolymph T amm, whereas J urea increased only transiently and haemolymph [urea] was unchanged, indicating that urea production likely does not contribute to the attenuation of hyperammonemia in blue crabs. Overall, Cu exposure in blue crabs led to increased ammonia and urea production, which were both eliminated by excretion. These results may have important implications in aquaculture systems where crabs may be exposed to elevated Cu and/or ammonia.

Impaired regulation of divalent cations with acute copper exposure in the marine clam Mesodesma mactroides.

The mechanism of copper (Cu) toxicity in marine invertebrates remains unclear. Therefore, marine clams (Mesodesma mactroides) were exposed (96h) to a concentration of dissolved Cu (1.6µmolL(-1)) inducing 10% mortality in sea water (30ppt). After in vivo exposure, tissue Cu accumulation (hemolymph, gill and digestive gland); hemolymph ionic (Na(+), K(+), Mg(2+) and Ca(2+)) and osmotic concentrations; tissue (gill and digestive gland) ionic concentration, enzyme (Na(+),K(+)-ATPase and carbonic anhydrase) activity, and oxygen consumption; and whole-body oxygen consumption were analyzed. Succinate dehydrogenase activity was evaluated in mitochondria isolated from gills and digestive gland and exposed (1h) in vitro to different concentrations of dissolved Cu (0.8, 7.7 and 78.7µmolL(-1)). In vivo exposure induced Cu accumulation in hemolymph, gills and digestive gland; increased Mg(2+) and decreased Ca(2+) concentration in hemolymph; decreased Mg(2+) concentration, increased Na(+),K(+)-ATPase activity and reduced carbonic anhydrase activity in gills; decreased Mg(2+) concentration, increased Ca(2+) concentration and increased Na(+),K(+)-ATPase activity in digestive gland; and reduced gill, digestive gland and whole-body oxygen consumption. Succinate dehydrogenase activity was inhibited after in vitro exposure to 78.7µmolL(-1) Cu. These findings indicate that Cu is an ionoregulatory toxicant in the marine clam M. mactroides. However, toxicity is related to disturbances in regulation of divalent cations (Mg(2+) and Ca(2+)) without effect on regulation of major monovalent cations (Na(+) and K(+)), as opposed to that observed in osmoregulating invertebrates exposed to Cu. However, other mechanism(s) of toxicity cannot be ruled out. Future studies must be performed to evaluate the consequence of the Cu-induced respiratory disturbances observed in M. mactroides.

Effects of copper exposure on the energy metabolism in juveniles of the marine clam Mesodesma mactroides.

In freshwater osmoregulating mollusks, Cu can cause toxicity by inducing ionoregulatory disturbances. In mussels, it inhibits the activity of key enzymes involved in Na(+) uptake and consequently induces ionic and osmotic disturbances. In snails, Cu induces disruption of the Ca(2+) homeostasis leading to effects in shell deposition and snail growth. However, the mechanisms involved in Cu toxicity in osmoconforming sweater mollusks remain unclear. Recent findings from our laboratory have suggested that Cu toxicity in marine invertebrates can be associated with both ionic and respiratory disturbances. In the present study, metabolic changes induced by waterborne Cu exposure were evaluated in the osmoconforming clam Mesodesma mactroides, a bivalve species widely distributed along the South American sandy beaches. Juvenile clams were kept under control conditions (no Cu addition in the water) or acutely (96h) exposed to Cu (96-h LC10=150µgL(-1)) in artificial seawater (30ppt). ATP, protein, lipid, glycogen and glucose contents were analyzed in gills, digestive gland, pedal muscle and hemolymph. Dinucleotide (NAD(+) and NADH) content was also analyzed in gills, digestive gland and pedal muscle while pyruvate and lactate content was determined in pedal muscle and hemolymph. In all tissues analyzed, Cu exposure did not affect ATP content and NAD(+)/NADH ratio, except in the hemolymph, where a decrease in ATP content was observed. These findings indicate that clam cells, except those from hemolymph, were able to maintain a constant level of free energy. A significant increase in total protein content was observed in the digestive gland, which could be a compensatory mechanism to counteract the higher level of protein oxidation previously observed in M. mactroides exposed to Cu under the same experimental conditions. Finally, reduced glucose content in the pedal muscle paralleled by increased lactate content in the pedal muscle and hemolymph was observed in Cu-exposed clams. Overall, these findings indicate that Cu exposure is leading to an increased reliance upon the anaerobic energy production to maintain the overall cellular ATP production in the clam M. mactroides.

Infrared radiation influence on molt and regeneration of Neohelice granulata Dana, 1851 (Grapsidae, Sesarminae).

This paper analyzes the influence of infrared radiation (IR) on regeneration, after autotomy of limb buds of Neohelice granulata and consequently the time molt. Eyestalks were ablated to synchronize the start of molt. Afterward, animals were autotomized of five pereopods and divided into control and irradiated groups. The irradiated group was treated for 30 min daily until molt. Limb buds from five animals of days 4, 16 and 20 were collected and histological sections were made from them. These sections were photographed and chitin and epithelium content measured. Another group was made, and after 15 days limb buds were extracted to analyze mitochondrial enzymatic activity from complex I and II. The irradiated group showed a significant reduction in molt time (19.38+/-1.22 days) compared with the control group (32.69+/-1.57 days) and also a significant increase in mitochondrial complex I (388.9+/-27.94%) and II (175.63+/-7.66%) in the irradiated group when compared with the control group (100+/-17.90; 100+/-7.82, respectively). However, these effects were not accompanied by histological alterations in relation to chitin and epithelium. This way, it was possible to demonstrate that IR increases complex I and II activity, reduces the time molt and consequently increases the appendage regeneration rate.