Exposure to silver and titanium dioxide nanoparticles at supra-environmental concentrations decreased sperm motility and affected spermatozoa subpopulations in gilthead seabream, Sparus aurata.

Marine pollution by nanoparticles (NPs) can be reprotoxic for fish and disturb successful reproduction of wild populations. In gilthead seabream (Sparus aurata), a mild effect on sperm motility was observed after exposure to high concentrations of silver NPs. Considering the great heterogeneity traits within a sperm sample, it is possible that NPs affect spermatozoa accordingly, modulating subpopulation profile. Thus, this work aimed to analyse NP effects in sperm motility in general and considering spermatozoa population structure, using a subpopulation approach. Seabream sperm samples from mature males were exposed for 1 h to increasing concentrations of titanium dioxide (1, 10, 100, 1000 and 10,000 µg L-1) and silver (0.25, 25 and 250 µg L-1) NPs, including Ag NP and Ag+, dissolved in a non-activating medium (0.9 % NaCl). Concentrations chosen include realistic (10-100 and 0.25 µg L-1, respectively, for TiO2 and Ag) and supra-environmental values. The mean particle diameter was determined as 19.34 ± 6.72 and 21.50 ± 8.27 nm in the stock suspension, respectively, for titanium dioxide and silver. After the ex vivo exposure, sperm motility parameters were determined using computer-assisted sperm analysis, and sperm subpopulations were later identified using a two-step cluster analysis. Results revealed a significant reduction in total motility after exposure to the 2 highest concentrations of titanium dioxide NPs, while curvilinear and straight-line velocities were not altered. Exposure to silver NPs (Ag NP and Ag+) lowered significantly total and progressive motilities at all concentrations, while curvilinear and straight-line velocities were significantly lower only at the highest concentration. Sperm subpopulations were also affected by the exposure to both titanium dioxide and silver NPs. In both cases, the highest levels of NPs triggered a decrease in the percentage of fast sperm subpopulations (38.2% in TiO2 1000 µg L-1, 34.8.% in Ag NP 250 µg L-1, and 45.0% in Ag+ 250 µg L-1 vs 53.4% in the control), while an increase on slow sperm subpopulations. A reprotoxic effect was proven for both NPs, but only at supra-environmental concentrations.

Mild Effects of Sunscreen Agents on a Marine Flatfish: Oxidative Stress, Energetic Profiles, Neurotoxicity and Behaviour in Response to Titanium Dioxide Nanoparticles and Oxybenzone.

UV filters are potentially harmful to marine organisms. Given their worldwide dissemination and the scarcity of studies on marine fish, we evaluated the toxicity of an organic (oxybenzone) and an inorganic (titanium dioxide nanoparticles) UV filter, individually and in a binary mixture, in the turbot (Scophthalmus maximus). Fish were intraperitoneally injected and a multi-level assessment was carried out 3 and 7 days later. Oxybenzone and titanium dioxide nanoparticles induced mild effects on turbot, both isolated and in mixture. Neither oxidative stress (intestine, liver and kidney) nor neurotoxicity (brain) was found. However, liver metabolic function was altered after 7 days, suggesting the impairment of the aerobic metabolism. An increased motility rate in oxybenzone treatment was the only behavioural alteration (day 7). The intestine and liver were preferentially targeted, while kidney and brain were unaffected. Both infra- and supra-additive interactions were perceived, with a toxicodynamic nature, resulting either in favourable or unfavourable toxicological outcomes, which were markedly dependent on the organ, parameter and post-injection time. The combined exposure to the UV filters did not show a consistent increment in toxicity in comparison with the isolated exposures, which is an ecologically relevant finding providing key information towards the formulation of environmentally safe sunscreen products.

Mercury Stable Isotopes Discriminate Different Populations of European Seabass and Trace Potential Hg Sources around Europe.

Our study reports the first data on mercury (Hg) isotope composition in marine European fish, for seven distinct populations of the European seabass, Dicentrarchus labrax. The use of δ202Hg and Δ199Hg values in SIBER enabled us to estimate Hg isotopic niches, successfully discriminating several populations. Recursive-partitioning analyses demonstrated the relevance of Hg stable isotopes as discriminating tools. Hg isotopic values also provided insight on Hg contamination sources for biota in coastal environment. The overall narrow range of δ202Hg around Europe was suggested to be related to a global atmospheric contamination while δ202Hg at some sites was linked either to background contamination, or with local contamination sources. Δ199Hg was related to Hg levels of fish but we also suggest a relation with ecological conditions. Throughout this study, results from the Black Sea population stood out, displaying a Hg cycling similar to fresh water lakes. Our findings bring out the possibility to use Hg isotopes in order to discriminate distinct populations, to explore the Hg cycle on a large scale (Europe) and to distinguish sites contaminated by global versus local Hg source. The interest of using Hg sable isotopes to investigate the whole European Hg cycle is clearly highlighted.

Shipping can be achieved but cargo arrives with damage - titanium dioxide nanoparticles affect the DNA of Pacific oyster sperm.

Titanium dioxide nanoparticles (TiO2 NP) were reported to be reprotoxic in humans and fish. However, the effects of these NP on the reproduction of marine bivalves, namely oysters, remain unknown. Thus, a short-term (1 h) direct exposure of sperm of the Pacific oyster (Crassostrea gigas) to two TiO2 NP concentrations (1 and 10 mg.L-1) was performed, and sperm motility, antioxidant responses, and DNA integrity were evaluated. Although no changes occurred in sperm motility and the activities of the antioxidants, the genetic damage indicator increased at both concentrations, demonstrating that TiO2 NP affects the DNA integrity of oyster sperm. Although DNA transfer can happen, it does not fulfill its biological mission since the transferred DNA is not intact and may compromise the reproduction and recruitment of the oysters. This vulnerability of C. gigas sperm towards TiO2 NP highlights the importance of studying the effects of NPs exposure to broadcast spawners.

Silver nanoparticles and silver ions indistinguishably decrease sperm motility in Pacific oysters (Magallana gigas) after short-term direct exposure.

The present study aimed to evaluate the reprotoxicity of environmental (0.25 µg.L-1) and supra-environmental (25 µg.L-1 and 250 µg.L-1) levels of silver nanoparticles (Ag NP) on the Pacific oyster (Magallana gigas), by determining sperm quality. For that, we evaluated sperm motility, mitochondrial function and oxidative stress. To determine whether the Ag toxicity was related to the NP or its dissociation into Ag ions (Ag+), we tested the same concentrations of Ag+. We observed no dose-dependent responses for Ag NP and Ag+, and both impaired sperm motility indistinctly without affecting mitochondrial function or inducing membrane damage. We hypothesize that the toxicity of Ag NP is mainly due to adhesion to the sperm membrane. Blockade of membrane ion channels may also be a mechanism by which Ag NP and Ag+ induce toxicity. The presence of Ag in the marine ecosystem is of environmental concern as it may affect reproduction in oysters.

Mercury-induced chromosomal damage in wild fish (Dicentrarchus labrax L.) reflecting aquatic contamination in contrasting seasons.

Ria de Aveiro (mainly Laranjo basin, Portugal) has been subjected to mercury contamination from a chlor-alkali plant, currently presenting a well-described mercury gradient. This study aimed to assess mercury genotoxicity in this area by measuring the frequency of erythrocytic nuclear abnormalities (ENA) in the European sea bass (Dicentrarchus labrax), addressing the relation with total mercury concentration in the blood and the modulatory role of seasonal variables. Fish were collected, in warm and cold periods, at three locations differing in their distances to the main mercury source: reference (R), moderately (M), and highly (H) contaminated sites. Genotoxicity was detected in both degrees of contamination (M and H) and in both periods of the year (warm and cold), which is in line with the greater levels of mercury measured in fish blood. No significant seasonal variations were observed for mercury bioaccumulation or ENA frequency. The apparent low imperviousness of ENA frequency to seasonal factors reinforced its consistency as a genotoxicity biomarker, thus enabling a clearer identification of cause-and-effect relationships. Overall, the results reflected a serious environmental risk to native ichthyofauna at Laranjo basin due to mercury contamination, showing a potential of mercury to induce genetic damage in fish blood cells through clastogenic and/or aneugenic actions.

Mercury organotropism in feral European sea bass (Dicentrarchus labrax).

The knowledge of mercury (Hg) burdens in a wide set of tissues and organs of exposed fish is crucial to understand the internal distribution dynamics and thus predict Hg bioavailability and implications for ecosystem and human health. Total Hg was measured in six tissues of Dicentrarchus labrax captured along an estuarine contamination gradient, revealing the following pattern: liver > kidney > muscle > brain ≈ gills > blood. All of the tissues displayed intersite differences, although brain and muscle seemed to better reflect the extent of contamination. Hg speciation showed that liver presented higher concentrations than muscle for both organic and inorganic forms. Furthermore, liver seemed to exert a protective action in relation to Hg accumulation in the other tissues and organs. This protection seems to be particularly marked in relation to the brain, whereas liver is assisted in that action by kidney and muscle.

Mercury distribution in key tissues of fish (Liza aurata) inhabiting a contaminated estuary-implications for human and ecosystem health risk assessment.

This study brings a new viewpoint based on multiple-tissue analyses to form the basis for a predictive mode of mercury accumulation dynamics in fish body under field conditions. Total mercury (T-Hg) was determined in key tissues of Liza aurata captured along an estuarine contamination gradient, displaying the following hierarchy: kidney > liver > muscle > brain > gills > blood. Brain was the tissue that better reflected the mercury contamination extent, closely followed by liver and muscle. Organic mercury (O-Hg) measured in muscle and liver represented more than 85% and less than 30% of the T-Hg, respectively. The lowest O-Hg percentage was found in the most contaminated area, for both muscle and liver. Mercury distribution and accumulation patterns showed dependence on the specific tissue. The high mercury levels found in organs involved in vital physiological processes point out the risk to autochthonous fish fauna. Human risk associated to the ingestion of fish living in the surveyed areas cannot be excluded.

Rare earth elements in mud volcano sediments from the Gulf of Cadiz, South Iberian Peninsula.

Eight gravity cores (GC) were retrieved from the deep mud volcanoes Sagres, Bonjardim, Soloviev and Porto in the Gulf of Cadiz. Cores with 137 to 317 cm long were sliced in intervals of 15 to 20 cm thickness, and 46 samples were analyzed for grain size distribution, loss on ignition, Al, Fe, Ca, Mg, Mn, Sr, Ba and the rare earth elements (REE) La, Ce, Pr, Nd, Sm, Eu, Gd, Dy, Ho, Er, Tm, Yb and Lu. REE profile normalized to Post-Archean Australian Average Shale (PAAS) was dominated by a mid-REE bulge with a pronounced Eu enhancement. Sediment reducing conditions resulting from the anaerobic oxidation of methane probably contributed to the positive-Eu anomaly (1.18-2.19, PAAS normalization). Most likely, reactions near the sulfate-methane transition zone such as the precipitation of barium sulfate found at layers around 50-cm depth explain the enhancement of Eu/Eu* ratios. The Ce anomalies (0.93-1.09) were almost absent.

Effect of historical contamination in the fish community structure of a recovering temperate coastal lagoon.

This study aimed to assess the impact of trace element concentrations in fish assemblages of a recovering coastal lagoon. Fish, water and sediment were sampled in winter and summer in the Ria de Aveiro (Portugal). Multivariate analyses were used to examine the relationship between fish assemblages and environmental variables (physical-chemical parameters, contaminants and sediment grain size). In winter, fish density and biomass were mainly affected by the water turbidity, while Li concentration in the water column was found to be significant for fish biomass. During summer, a significant relationship was found between fish density and temperature, Hg, Li and Zn concentration in the sediment. These contaminants were mainly associated with the historically contaminated area, were Liza spp. and Dicentrarchus labrax appeared as dominant species. Environmental variables were not significant for fish biomass. The historical contamination in the Ria de Aveiro still seems to exert some influence on fish community structure.

The significance of cephalopod beaks in marine ecology studies: Can we use beaks for DNA analyses and mercury contamination assessment?

Cephalopod beaks found in the diet of predators have been a major source of scientific information. In this study, we evaluated the usefulness of DNA and contaminants analysis (total mercury - T-Hg) in cephalopod beaks in order to assess their applicability as tools in marine ecology studies. We concluded that, when applying DNA techniques to cephalopod beaks from Antarctic squid species, when using flesh attached to those beaks, it was possible to obtain DNA and to successfully identify cephalopod species; DNA was not found on the beaks themselves. This study also showed that it is possible to obtain information on T-Hg concentrations in beaks: the T-Hg concentrations found in the beaks were 6 to 46 times lower than in the flesh of the same cephalopod species. More research on the relationships of mercury concentrations in cephalopod beaks (and other tissues), intra- and inter-specifically, are needed in the future.

Lipid peroxidation vs. antioxidant modulation in the bivalve Scrobicularia plana in response to environmental mercury--organ specificities and age effect.

This study aimed at the assessment of mercury burden and its association to damage vs. antioxidant protection in the bivalve Scrobicularia plana environmentally exposed to mercury. Inter-age and organ-specific approaches were applied by using different annual size classes (2+, 3+, 4+ and 5+ year old) and assessing specific organs (gills, digestive gland), respectively. Bivalves were collected from moderately and highly contaminated sites at Laranjo basin - Ria de Aveiro (Portugal), where a mercury gradient was identified, and compared with those from a reference site. Besides total and organic mercury accumulation, endpoints combining lipid peroxidation (LPO), as a damage sign, and antioxidant protection (catalase, CAT; glutathione peroxidase, GPX; glutathione S-transferase, GST, activities) were determined. The whole-body accumulation as total mercury changed according to the environmental gradient for all age classes, while in terms of organic mercury, only 5+-year-old animals showed increased accumulation with increased environmental mercury level. Mercury induced peroxidative damage, showing that antioxidative mechanisms were insufficient. The adaptive capacity to pro-oxidant challenge, expressed as antioxidant induction and lesser vulnerability to enzyme inhibition, increased with age. The specific analyses of gills and digestive gland revealed that both organs were able to mirror external levels of exposure in the accumulation of total and organic mercury. Nevertheless, gills displayed higher potential to accumulate organic forms. The organ specificity was evident for antioxidant response with a clear pattern of overall increase in gills and decrease in digestive gland. In addition, only gills displayed statistical correlations between oxidative stress responses and mercury accumulation. Besides the contribution to understand mercury toxicodynamics, specific organs approach is strongly recommended in order to avoid misinterpretations. The use of whole-body analyses can be particularly compromising when oxidative stress responses (rather than mercury accumulation) are addressed.