Stress responses in captive Crocodylus moreletii associated with metal exposure.

Environmental pollution by metals has repercussions on wildlife health. It is known that some metals can have an influence on the neuroendocrine stress response, and at the same time, metals have pro-oxidant effects that can overwhelm the antioxidant system and cause oxidative stress. This study evaluates the association of metals with neuroendocrine stress activity and biomarkers of oxidative stress in 42 captive female Morelet's crocodiles (Crocodylus moreletii). We measured five metals of ecotoxicological importance (Hg, Cd, Pb, Cu and Zn), and three biomarkers of the oxidative stress response in the liver: glutathione (GSH) and glutathione disulfide (GSSG) as markers for antioxidant system and thiobarbituric acid reactive substances (TBARS) for oxidative damage. We also measured one biomarker of the neuroendocrine response to stress: corticosterone (B) in blood plasma. The mean ± SD concentrations of metals in the liver expressed in µg/g (dw) were: Cd: 0.004 ± 0.003, Hg: 0.014 ± 0.019, Cu: 0.017 ± 0.013, Zn: 0.043 ± 0.035, Pb: 0.16 ± 0.256. The mean ± SD of GSH was 0.42 ± 0.35 nmol/mg protein, the mean ± SD of GSSG was 0.24 ± 0.20 nmol/mg protein, the mean ± SD concentrations of TBARS were 0.36 ± 0.21 nmol/mg protein, and the mean ± SD of B was 393.57 ± 405.14 pg/mL. Hg presented a significant negative relationship with corticosterone. Cd had a negative relationship with both GSH and GSSG; meanwhile, Zn showed a negative relationship with TBARS levels, could be a protective element against hepatic oxidative damage. Finally, B had negative relationship with oxidative damage. The connection found between Hg and the neuroendocrine stress response, as well as the correlations of Cd and Zn with oxidative damage and antioxidant activity should be studied further, given their toxicological importance and implications for the conservation of C. moreletii and other crocodilians.

Mercury distribution and enrichment in coastal sediments from different geographical areas in the North Atlantic Ocean.

This study evaluated mercury (Hg) in three coastal areas in the North Atlantic Ocean (Azores archipelago, Northwest Portugal and Southeast Mexico) and calculated their enrichment factor and geoaccumulation index. Hg presented greater affinity with fine sediment fraction. In all sampling areas the sites with proximity to possible Hg hotspots presented increased [Hg] in fine sediments. However, this fraction only represents less than 1% of the whole sediment, resulting in a significantly lower [Hg] in the total sediment fraction. After comparing [Hg] in the fine fraction and the values established by the Sediment Quality Guidelines, two sites in the Azores exceeded these values. Nevertheless, when considering the total sediment fraction, adverse biological effects are not expected to occur. Finally, both enrichment factor and geoaccumulation index indicated that the Hg present in 86% comes from natural sources and only sampling sites close to potential hotspots showed some degree of enrichment and contamination.

Water temperature modulates mercury accumulation and oxidative stress status of common goby (Pomatoschistus microps).

Mercury (Hg) is a widespread pollutant across estuarine and coastal areas, raising concern on its potential impact on aquatic organisms. Hg may origin from natural and anthropogenic sources, being persistent and potentially toxic to biota, ultimately representing a serious risk to human health. Hg accumulation and toxicity may also induce reactive oxygen species (ROS) production in marine organisms, responsible for cell and tissue damage. Additionally, the temperature is undoubtedly an important environmental factor to consider regarding accumulation, due to its marked influence on the physiology and ecology of aquatic organisms. This study aimed to investigate the effect of different temperature scenarios (15, 20 and 25 °C) on the Hg accumulation in Pomatoschistus microps (Krøyer, 1838) liver and muscle, as well as on oxidative stress responses and energy metabolism, after short-term exposure to a naturally contaminated sediment with an environmentally relevant [Hg] (1.2 µg g-1). The results showed that Hg accumulation tends to increase along the temperature gradient with higher values of Hg accumulated in liver than in muscle tissue. The action of antioxidant enzymes and stress proteins seems to be effective in combating oxidative stress in the liver. Despite the action of antioxidant defences in the muscle, oxidative damage was observed at the protein level concomitantly with a decrease in aerobic energy production after exposure to Hg at higher temperatures. These findings are ecologically relevant and highlight the importance of further investigation of combined effects of Hg and other stressors, especially in a scenario of a changing climate where events leading to rapid alterations on water parameters are more frequent.

Mercury bioaccumulation in the long-fin squid Loligo forbesi near the Mid-Atlantic Ridge: Implications to human exposure.

Due to growing commercial interest as a fishing resource and its intermediate position in the marine trophic chains as both prey and predator, cephalopods can play an important role in mercury (Hg) transfer along the marine food webs, since they can bioaccumulate this metal in its tissues. Our study aims to analyze Hg accumulation in Loligo forbesi caught in the Azores Archipelago (Portugal) quantifying Hg in different tissues (mantle and stomach), as well in the squid stomach content, in order to evaluate the efficiency of Hg transfer from prey to predator. Hg data from the tissues was used to estimate the weekly tolerable Hg intake due to squid consumption. Overall data indicate that Hg measured in the stomach tissue (0.1 ± 0.01 µg g-1) was significantly higher than Hg levels found in the mantle (0.04 ± 0.001 µg g-1) and stomach contents (0.01 ± 0.001 µg g-1). BMF (bioaccumulation factor) was >1 for all the samples, indicating a biomagnification process from prey to predator. Hg concentration in the mantle tissue was correlated with mantle size; although females present higher Hg levels than males, the difference was found to be not related to gender but rather to the fact that females had larger bodies. Finally, considering the Hg concentration found in the mantle and the permitted Hg levels, it is advisable to consume up to 1050-1890g of squid per week, according to the regulatory agencies. Thus, our results indicate that, since these doses are respected, consumption of squids from the Azorean waters do not pose a risk to humans.

Distribution of metals in tissues of captive and wild Morelet's crocodiles and the potential of metallothioneins in blood fractions as a biomarker of metal exposure.

The distribution of Hg, Cd, Cu, and Zn in keratinized tissues, blood fractions, and excretory organs, and MTs in blood fractions and excretory organs was determined in captive, semicaptive, and wild Morelet's crocodiles and they were compared to select the most useful non-destructive tissues for the monitoring of metal exposure and to assess the potential of MTs as a biomarker. Our results indicate blood plasma, claws, and caudal scutes altogether are suitable tissues for xenobiotic metals exposure, with concentrations in blood plasma being an indicator of recent exposure, whereas concentrations in claws and caudal scutes are indicators of chronic exposure. Results in keratinized tissues suggest they are an important detoxification strategy in crocodiles, and claws presented the highest concentrations of metals in both captive (Hg = 0.44 ± 0.23 µg g-1, Cd = 11.10 ± 5.89 µg g-1, Cu = 45.98 ± 23.18 µg g-1, Zn = 124.75 ± 75.84 µg g-1) and wild populations (Hg = 1.31 ± 0.32 µg g-1, Cd = 26.47 ± 21.15 µg g-1, Cu = 191.75 ± 165.91 µg g-1, Zn = 265.81 ± 90.62 µg g-1). Thus, they are an appropriate tool for assessing metal exposure in populations where scutes clipping as a marking technique is not allowed, and their collection is less complicated than with other tissues. MTs are a suitable biomarker in blood plasma, whereas in erythrocytes detoxification processes might depend on hemoglobin, rather than MTs. Future studies should consider the implementation of these tools for the monitoring of wild populations.

Ecotoxicological tools for the tropics: Sublethal assays with fish to evaluate edge-of-field pesticide runoff toxicity.

This study proposes short-term sublethal assays for the tropics using the fish Poecilia reticulata. Assays were evaluated under realistic exposure scenarios by simulating a runoff over an agricultural area sprayed with deltamethrin (Decis). In situ assays were performed inside microcosms set up to simulate runoff water entrance in lentic systems. Laboratory assays were conducted with water samples from the microcosms. In both assays the biochemical parameters were similarly responsive to Decis, with a significant inhibition of acetylcholinesterase (30-46%) and induction of lactate dehydrogenase (33-67%) and glutathione S-transferases (48-176%) activities, observed as of the lowest Decis dose. Postexposure feeding was more sensitive in the laboratory assay than in situ. Among the additional potential stressors, only acetylcholinesterase was significantly influenced by the runoff per se. The proposed methodologies were well adapted for assays with P. reticulata, as organisms were easily deployed and retrieved and enzymatic activities and postexposure feeding were sensitive endpoints.

Ras gene in marine mussels: a molecular level response to petrochemical exposure.

Mussels are susceptible to numerous toxicants and are often employed as bioindicators. This study investigated the status of the ras proto-oncogene in Mytilus galloprovincialis following petrochemical exposure. A M. galloprovincialis homologue of the vertebrate ras gene was isolated, showing conserved sequence in regions of functional importance and a high incidence of polymorphic variation. Mutational damage was investigated in mussels chronically exposed to the water-accommodated fraction of #4 fuel-oil (WAF), and in mussels collected along the NW coast of Portugal in sites with different levels of petrochemical contamination. A ras gene point mutation was identified in the codon 35 of one individual exposed to 12.5% WAF. No mutations were detected in mussels from the WAF control or environmental samples. This represents the first report of a ras gene mutation, experimentally-induced by petrochemical exposure, in an invertebrate species.

The pink shrimp Farfantepenaeus duorarum, its symbionts and helminths as bioindicators of chemical pollution in Campeche Sound, Mexico.

The pink shrimp Farfantepenaeus duorarum may acquire pollutants, helminths and symbionts from their environment. Statistical associations were studied between the symbionts and helminths of F. duorarum and pollutants in sediments, water and shrimps in Campeche Sound, Mexico. The study area spatially overlapped between offshore oil platforms and natural shrimp mating grounds. Spatial autocorrelation of data was controlled with spatial analysis using distance indices (SADIE) which identifies parasite or pollutant patches (high levels) and gaps (low levels), expressing them as clustering indices compared at each point to produce a measure of spatial association. Symbionts included the peritrich ciliates Epistylis sp. and Zoothamnium penaei and all symbionts were pooled. Helminths included Hysterothylacium sp., Opecoeloides fimbriatus, Prochristianella penaei and an unidentified cestode. Thirty-five pollutants were identified, including polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), pesticides and heavy metals. The PAHs (2-3 ring) in water, unresolved complex mixture (UCM), Ni and V in sediments, and Zn, Cr and heptachlor in shrimps were significantly clustered. The remaining pollutants were randomly distributed in the study area. Juvenile shrimps acquired pesticides, PAHs (2-3 rings) and Zn, while adults acquired PAHs (4-5 rings), Cu and V. Results suggest natural PAH spillovers, and continental runoff of dichlorodiphenyltrichloroethane (DDT), PCBs and PAHs (2-3 ring). There were no significant associations between pollutants and helminths. However, there were significant negative associations of pesticides, UCM and PCBs with symbiont numbers after controlling shrimp size and spatial autocorrelation. Shrimps and their symbionts appear to be promising bioindicators of organic chemical pollution in Campeche Sound.

In vivo evaluation of three biomarkers in the mosquitofish (Gambusia yucatana) exposed to pesticides.

In this study, the acute toxicity and the in vivo effects of commercial chlorpyrifos, carbofuran and glyphosate formulations on cholinesterase (ChE), glutathione S-transferase (GST) and lactate dehydrogenase (LDH) activities of the mosquitofish (Gambusia yucatana) were investigated. In a first phase of the study, head and muscle ChE were characterized with different substrates (acetylthiocholine iodide, s-butyrylthiocholine iodide and propionylthiocholine iodide) and the selective inhibitors eserine hemisulfate, 1,5-bis(4-allyldimethylammoniumphenyl)-pentan-3-one dibromide (BW284C51), and N,N'-diisopropylphosphorodiamic acid (iso-OMPA). The results obtained suggest that the enzyme present in both head and muscle of G. yucatana is mainly acetylcholinesterase (AChE). Acute toxicity was evaluated by exposing fish to several concentrations of single pesticides and of a mixture of chlorpyrifos/glyphosate. LC50 values were determined after 96 h of exposure, except in the case of carbofuran for which LC50 was calculated after 24 h since almost all the fish died within this period. LC50 values were 0.085 mg/l for chlorpyrifos, 17.79 mg/l for glyphosate, 0.636 mg/l for carbofuran and 0.011 mg/l for the chlorpyrifos/glyphosate mixture. A Toxic Unit approach was used to compare the toxicity of chlorpyrifos and glyphosate when occurring in a mixture with their toxicities as single compounds. Synergistic effects of chlorpyrifos and glyphosate when present in a mixture were found. At the end of each bioassay (24 h for carbofuran, 96 for the other substances/mixture), effects on biomarkers were analyzed. Muscle LDH activity was not altered by any of the three pesticides tested. Gill GST activity was significantly inhibited (40%) by carbofuran after 24 h of exposure to concentrations equal or higher than 0.06 mg/l. ChE muscle and head activity were significantly inhibited (50% and 30%, respectively) by carbofuran at concentrations equal or higher than 0.25 mg/l. Chlorpyrifos induced a significant inhibition of both muscle and head ChE (80% and 50%, respectively) after 96 h of exposure to concentrations equal or higher than 0.05 mg/l. Carbofuran did not induce significant alterations of fish ChE. The ChE EC50 determined for chlorpyrifos/glyphosate mixture (0.070 mg/l) was higher than the correspondent value calculated for chlorpyrifos alone (0.011 mg/l) suggesting an antagonistic effect of glyphosate on ChE inhibition by chlorpyrifos. ChE activity of G. yucatana seems to be a good biomarker to diagnose the exposure of wild populations of this species exposed to anticholinesterase pesticides.