Metabolomics-Based Investigation on the Metabolic Changes in Crassostrea gigas Experimentally Exposed to Galvanic Anodes.

Cathodic protection is widely used to protect metal structures from corrosion in marine environments using sacrificial galvanic anodes. These anodes, either in Zinc, or preferentially nowadays in Al-Zn-In alloys, are expected to corrode instead of the metal structures. This leads to the release of dissolved species, Zn2+, Al3+, and In3+, and solid phases such as Al(OH)3. Few studies have been conducted on their effects on marine organisms, and they concluded that further investigations are needed. We therefore evaluated the effects of Zn and Al-Zn-In anodes on oysters stabulated in tanks, under controlled conditions defined through a comparison with those prevailing in a given commercial seaport used as reference. We analyzed the entire metabolome of gills with a non-targeted metabolomic approach HRMS. A modelling study of the chemical species, corresponding to the degradation products of the anodes, likely to be present near the exposed oysters, was also included. We identified 16 and two metabolites modulated by Zn- and Al-Zn-In-anodes, respectively, that were involved in energy metabolism, osmoregulation, oxidative stress, lipid, nucleotide nucleoside and amino acid metabolisms, defense and signaling pathways. The combination of chemical modelling and metabolomic approach, used here for the first time, enlightened the influence of Zn present in the Al-Zn-In anodes.

Multi-Year Monitoring of the Toxicological Risk of Heavy Metals Related to Fish Consumption by the Population of the Kendari Region (Southeast Sulawesi, Indonesia).

This study measured the concentrations of Hg, As, Ni, Cd, and Pb in six fish species commonly consumed in Kendari. Samples were bought within local markets from 2012 to 2017 at the end of the dry season. Results showed that mercury concentrations fluctuated between years and within species, except in the Caranx sexfasciatus, which showed no significant differences (Kruskall-Wallis, p-value > 0.05, df = 5) and an average concentration of 0.371 ± 0.162 µg g-1 DW. Arsenic was found in high concentrations across species and years and varied widely in C. sexfasciatus, the lowest value being 0.32 ± 0.01 µg g-1 DW in 2012 and the highest was 5.63 ± 1.89 µg g-1 DW in 2017. The highest nickel concentrations were found in 2016 across four of the six species. The fish samples displayed very low cadmium and lead concentrations throughout the study. In addition, the potential human health risk due to fish consumption was assessed. This showed that mercury is the only one of the five metals present in concentrations high enough to individually pose a potential hazard, the only metal likely to be accumulated beyond a safe concentration in Kendari. Chanos chanos never posed a toxicological risk based on the results of this research.

Biomonitoring of Mimachlamys varia transplanted to areas impacted by human activities (La Rochelle Marina, France).

The development of human activities on French Atlantic coastlines (La Rochelle) lead to chronic pollution of the environment by organic (pesticides, hydrocarbons, agrochemicals) and inorganic (heavy metals) contaminants. These past years, several regulations have been implemented to preserve coastal environments. The purpose of this study was to perform biomonitoring of bivalve species using an outdoor caging technique. The goal of our work was to assess the impact of harbour's trace elements on the state of health of the marine bivalve Mimachlamys varia. First, various molecular defence biomarkers were measured: SOD (oxidative stress), GST (detoxification process), MDA (lipid peroxidation), and Laccase (immune reaction). Thus, in April 2016, scallops were collected at three caging sites, which differ by their levels of pollution, after transplantation into port areas (fairing, rainwater) and a control site (marsh). Bivalve samples were taken at three sampling dates (D0, D07, D21). Biomarker assays were performed in the digestive glands due to their bioaccumulation properties. The second aim was to explore the impacts of inorganic pollutants placed in environmental harbour's sites. After 21 days, the biomarker response of transplanted bivalves revealed a SOD decrease, Laccase and GST stimulations, higher concentrations in Cu, Fe, As, Co, Mn, Zn, Sn and no significant variation of MDA concentration. Our ecological relevance of biomarker approaches opens interesting perspectives to identify M. varia such as a pertinent marine sentinel species. The several selected biomarkers determined could confirm their ability to appraise the water quality of hydro-systems located in French coastlines, such as port areas.

Polycyclic aromatic hydrocarbons (PAHs) in surface sediment and oysters (Crassostrea rhizophorae) from mangrove of Guadeloupe: levels, bioavailability, and effects.

Surface sediment and oysters (Crassostrea rhizophorae) from the coastlines of Guadeloupe were analysed for polycyclic aromatic hydrocarbons (PAHs) using GC/MS. Biomarkers of oxidative stress were used to assess the response of these oysters to hydrocarbons exposure. The total concentration of PAHs in the sediment ranged from 49 to 1065 ng/g dw, while concentrations in oyster ranged from 66 to 961 ng/g dw. Molecular indices based on isomeric PAHs ratios characterize the pollution sources and show that most of the contaminations in sediment originate from pyrolytic inputs. Bioaccumulation factors (BAFs) have been related to isomeric ratio calculated for oysters in order to refine PAHs sources. The variations of BAFs observed in the different compounds resulted from different uptake pathways in the mangrove oysters according to the type of inputs. Response of biomarkers showed inhibition of catalase and an increase of lipid peroxidation at the station where PAHs concentrations were the highest. Taken together, data obtained point to the relevance of considering environmental conditions as factors influencing biomarker responses in environmental monitoring programs. These data also indicate the need for regular environmental follow-up studies in Guadeloupe.

Impact of chronic cadmium exposure at environmental dose on escape behaviour in sea bass (Dicentrarchus labrax L.; Teleostei, Moronidae).

The effect of chronic exposure to a low concentration (0.5 microg l(-1)) of cadmium ions was investigated on escape behaviour of sea bass, Dicentrarchus labrax, using video analysis. Observations were also performed on the microanatomy of lateral system neuromasts. When fish were exposed for 4h per day over 8 days to the cadmium ions, most of both types of neuromasts observed remained intact. However, some of them presented damaged sensory maculae. Whereas before cadmium exposure, fish responded positively to nearly all the lateral system stimulations, after exposure they decreased by about 10% their positive responses to stimulations. From the 15th day after the beginning of cadmium exposure, neuromasts presented progressively less damage, cadmium accumulation in gills and scales decreased significantly and fish escape behaviour had recovered. This study presents a new concept in ecotoxicology: using behavioural change to reveal the effects of pollution levels, scarcely detectable by currently used techniques (physiological responses).

Impact of acute cadmium exposure on the trunk lateral line neuromasts and consequences on the "C-start" response behaviour of the sea bass (Dicentrarchus labrax L.; Teleostei, Moronidae).

Behavioural responses of sea bass Dicentrarchus labrax were investigated after exposure to cadmium ions in laboratory-controlled conditions. The aim of this study was to discover whether environmental exposure to cadmium ions inactivates fish lateral line system neuromasts, and to determine the behavioural consequences of such a sensory blockage. For this, fish escape behaviour in response to an artificial water jet was recorded using a 25-frames s(-1) analog video camera before and after cadmium exposure. Experimental set up was tested with fish whose lateral line system was artificially inactivated by antibiotics (gentamicin and streptomycin). Histological analyses with scanning electron microscopy showed antibiotic treatment destroyed lateral line system neuromasts. In addition, these fish did not respond to stimulations provoked by the water jet after antibiotic treatment. Fish escape behaviour was then recorded before and after cadmium exposure at two different concentrations. When fish were exposed to the first concentration of cadmium tested (0.5 microg l(-1), which represents the maximal cadmium concentration encountered in contaminated estuaries), no alteration in neuromast tissue was observed. In addition, before cadmium exposure, fish responded positively in 98.41 +/- 4.95% of lateral line system stimulations (escape behaviour in response to the water jet). After cadmium exposure, no behavioural modification could be detected: the fish responded positively in 95.16 +/- 9.79% of stimulations (chi(2) = 2.464, p = 0.116). In contrast, the high cadmium concentration used (5 microg l(-1), which represents 10 times the concentration occurring in highly polluted estuarine areas) involved severe neuromast tissue damage. Just after such cadmium exposure, fish showed only 41.67 +/- 35.36% of positive responses to their lateral line system stimulations, while they responded positively in 95.93 +/- 9.10% of stimulations under control conditions (chi(2) = 24.562, p < 0.0001). Their lateral line system neuromasts seemed to regenerate about 1 month after cadmium exposure. Associated with this regeneration, from the 21st day after cadmium exposure, their escape behaviour had recovered and was not significantly different from that recorded under control conditions (86.74 +/- 20.82%, chi(2) = 2.876, p = 0.090). This study shows that although 5 microg l(-1) cadmium is able to damage lateral line system neuromasts and causes fish behavioural alterations, fish exposed to 0.5 microg l(-1) cadmium displayed neither tissue neuromast nor behavioural modification.

Abnormal features of Macoma balthica (Bivalvia) in the Baltic Sea: alerting symptoms of environmental adversity?

Recent studies of the Baltic clam Macoma balthica (L.) from the southern Baltic (the Gulf of Gdansk) have revealed striking morphological, histological and cytogenetic features. Strong deformation of the shell, including elongation of the posterior end and the appearance of an easily visible flexure in this part, has been recorded. The population contribution of the deformed blunt shelled ("irregular") clams ranged from 0% to 65% and tended to increase with depth. The morphologically "irregular" clams had higher accumulated tissue concentrations of trace metals (As, Ag, Cd, Pb, Cu and Zn), indicating a different metal handling ability. Adverse conditions in deeper water regions of the Gulf (e.g. hypoxia, hydrogen sulphide, elevated bioavailability of contaminants) have been suggested as inducers of the phenotypical changes (morphological deformation) in part of the population and, in parallel, of the specific physiological adaptations that result in higher metal accumulation in the "irregular" clams. Cytogenetic and histological analyses showed the presence of tumours in gill cells and digestive system of the affected clams, the prevalence of disseminated neoplasia ranging from 0% to 94% depending on the site. The disease was manifested by a modified karyotype (i.e. an abnormal number and morphology of chromosomes), a higher activity of nucleolar organizer regions (AgNORs), and tissue lesions (enlarged cells, actively proliferative with pleomorphic nuclei). Bottom sediments showed acute toxicity and have been proposed as a source of an initialising carcinogenic factor. However, none of the ecotoxicological studies provided was successful in the clear demonstration of a single (or multifactorial) agent that can account for the disseminated neoplasia.

Application of trichloroacetic acid (TCA) to extraction of soft body for the determination of tissue Cd, Cu, Pb and Zn in the prosobranch Hydrobia ulvae (Pennant).

The application of trichloroacetic acid (TCA) as a shell extractant for preparation of soft body parts with reference to tissue metal concentrations (Cd, Cu, Pb, Zn) in shellfish has been evaluated on the example of the mud snail Hydrobia ulvae, a small marine prosobranch densely present in rocky and soft-bottom habitats of the eastern Atlantic. A solution of 0.1 M TCA was tested on individuals treated according to two different protocols: (1) thawed after freezing ("non-dried") and (2) thawed and air-dried to a constant weight ("dried"). Two points were investigated in detail to improve the method: individual soft tissue dry weight and tissue metal concentration following a standard digestion method. In both instances, the results were compared with those from manually dissected snails. Conditions for total shell decalcification of 60 individuals (3-4 mm long) were 5.5 h in 20 ml of 0.1 M TCA.No differences in individual soft tissue weight were observed between the treatments, indicating good efficiency of the TCA extraction with respect to weight of soft body parts. In contrast, tissue metal concentrations varied among treatments. The TCA extraction of the dried animals had a good recovery for Cd, most likely due to the lower solubility of Cd vital cellular components (proteins and mineral concretions) from the dried tissue. Satisfactory recoveries of the tissue concentrations of Cu and Pb were obtained for the non-dried individuals. This might be related to the specific distribution of metals in the organism (namely in the digestive glands and gonads) and their different chemical reactivity with TCA after the tissue was dried. Limited susceptibility of Zn-bearing protein bindings to complexing with TCA also accounts for significantly lower concentrations of Zn in the mud snail's soft tissue that was extracted. The 0.1 M TCA solution is therefore recommended for extraction of the shells of Hydrobia ulvae for tissue determination of Cd, Cu and Pb; however the treatment protocol does affect metal recovery and thus a consistent procedure should be followed. The extracted metals from the soft tissues and shells of the mud snails (on the basis of both metal concentrations and contents) were ranked in order of increasing contribution of soft body parts to the total (shell+tissue): Pb

The relationship between metal concentrations and phenotypes in the Baltic clam Macoma balthica (L.) from the Gulf of Gdansk, southern Baltic.

Soft tissue accumulated concentrations of nine metals (V, As, Se, Ag, Cd, Pb, Mn, Cu and Zn) were studied in two main phenotypes (1) according to external shell colour (white and pink), and (2) according to shell shape (shell with a rounded posterior end--"regular" and with an elongate posterior end and a notable flexure--"irregular") of the Baltic clam Macoma balthica from southern Baltic Sea off Poland. No differences in metal concentrations were observed between colour-based phenotypes. By contrast, "irregular" clams exhibited generally higher concentrations of all elements in their tissues than "regular" bivalves. This finding provides the first reference on a potential linkage of shell deformation with tissue metal concentrations within one entire population of clams living in the same habitat. Different ability of metal handling in the shape-based phenotypes is presumably related to different physiological capacity of the bivalves induced by the selection effect of specific environmental conditions. It is suggested that unfavourable conditions in deep waters of the Gulf of Gdansk (e.g. hypoxia/anoxia, hydrogen sulphide, elevated bioavailability of metals) induces, in a certain part of the population, morphological deformation of shell (thereby leading to irregular shape) and in parallel physiological adaptations which result in greater sensitivity to trace metals of "irregular" clams. This hypothesis however, requires further investigation with special focus on genetic divergences between phenotypes because till now we cannot exclude the co-occurrence of two types (semi-species) of clams in the Gulf: an Atlantic type and a Baltic type. Genetic analysis with a use of DALP technique revealed strong intrapopulational polymorphism but no fingerprints or intraspecific polymorphism characterising any of the phenotypes considered (both colour- and shape-based). Since eight polymorphic loci were clearly identified further studies of population genetic structure hold optimistic promise.