Comparative effects of trace metal elements released from dissolution of aluminum-based galvanic anodes, aluminum chloride, zinc chloride, and their mixture on the development of the Pacific oyster D-larvae, Crassostrea gigas.

As the most abundant metal in the earth's crust, aluminum (Al) is used in many sectors, and nowadays, there is an increase in anthropogenic releases to aquatic ecosystems. This is particularly true in the context of corrosion protection systems involving galvanic anodes, which are mostly made of Al. Corroded instead of the steel structures they protect, galvanic anodes are described as sacrificial anodes. In contact with seawater, they undergo oxidation and release various metals in the form of ions or oxy-hydroxides into the marine environment, mainly Al and zinc (Zn). Several studies agree that Al increases the incidence of abnormal development in bivalve larvae from 150 µg L-1 which is close to the highest Al concentrations recorded in coastal waters. Therefore, we studied the impact of the cocktail of metals released by aluminum-based galvanic anodes on the development of Crassostrea gigas larvae, which we compared to the effects of aluminum chloride hexahydrate and zinc chloride alone and their mixture. The anode solution was realized thanks to an experimental device simulating the dissolution of a galvanic anode in the marine environment in order to reproduce the cocktail of metal species. We calculated an EC50 of 193.55 µg L-1 and 100.05 µg L-1 for Al and Zn chloride alone, respectively, and we highlighted an EC50 of 190.22 µg L-1 for the galvanic anode based on Al concentration. The mixture of the two metals in their chloride form resulted in the observation of additive and synergistic effects, which underlines the importance of considering the cocktail effect in ecotoxicological studies.

Impact assessment of metals realeased by aluminium-based galvanic anode on the physiology of the abalone Haliotis tuberculata in controlled conditions.

To protect metal structures immersed in the sea from corrosion, the galvanic anode cathodic protection system (GACP) is often applied. However, this association leads to continuous oxidation of the galvanic anode and therefore to a release of a metal cocktail in the forms of ions or oxy-hydroxides. Therefore, the main objective of our study was to investigate the toxicity of elements released from the dissolution of an aluminium-based galvanic anode (∼95% Al, ∼5% Zn, <0.1% for In, Cu, Cd, Mn, Fe) on a grazing gastropod, the abalone Haliotis tuberculata. The present study was carried out in complement to other research currently in submission. Gastropods were exposed for 16 weeks (12 weeks of exposure and 4 weeks of decontamination phase) to 6 conditions including a control, 4 concentrations based on total aluminium level (86, 425, 1096 and 3549 µg L-1) and a trophic control, corresponding to abalones placed in non-contaminated natural seawater but fed with contaminated algae. The effects of metals on growth, glycogen levels, brix index of hemolymph, MDA levels in digestive gland and gills, hemocyte phagocytic activity, ROS production, lysosomal system and the progress of gametogenesis were investigated throughout the entire exposure allowing the realization of kinetics. The results revealed that the aluminium-based anode does not seem to have an effect on the health status of the individuals for environmentally realistic concentrations. However, in extreme conditions strong effects were reported on the growth, immune system and reproduction of abalone.

Aluminium-based galvanic anode impacts the photosynthesis of microphytobenthos and supports the bioaccumulation of metals released.

Very few studies have looked at the potential biological effects of degradation products of galvanic anodes particularly on primary producers which are central to food webs in marine ecosystems. The galvanic anode cathodic protection system (GACP) is widely used to protect submerged metallic structures from corrosion. Aluminium (Al) and zinc (Zn) are the main constituents of galvanic anodes and are therefore released in the marine environment by oxidation process to form ions or oxy-hydroxides. The main objective of our study was to evaluate the effects of the metals released from an aluminium-based galvanic anode on microphytobenthos performance in term of biofilm growing through the analysis of photosynthetic parameters, the determination of chlorophyll and extracellular polymeric substances (EPS). The bioaccumulation of Al and Zn were measured in the microphytobenthic compartment collected at the surface of polyvinyl chloride (PVC) plates exposed during 13 days to seawaters enriched in different concentrations of metals released from dissolution of one anode. Determination of bioconcentration factors confirmed that the microphytobenthos has incorporated Al. A significative effect was observed on the Chl a concentration for the higher tested concentration ([Al] = 210.1 ± 60.2 µg L - 1; [Zn] = 20.2 ± 1.4 µg L - 1). The seawater exposed to the anode affected the MPB productivity (ETRIImax) with consequences on acclimatation light (Ek), absorption cross section of PSII (σPII), Fv/Fm and NPQ. Regarding the EPS production, the anode degradation presented an impact on high and low molecular weight of both carbohydrates and protein fractions of microphytobenthos suggesting that EPS play an essential role in sequestering metal contaminants to maintain the integrity of the biological membranes and the functionality of the cellular organelles. The accumulation of Al released by GACP in microphytobenthos cells could lead to physiologic problems in photosynthetic organisms.

Effects on Growth of Juvenile Abalones Haliotis tuberculata Under Chronic Exposition to Metals Released from the Dissolution of an Aluminium-Based Galvanic Anode.

In the marine environment, the galvanic anode cathodic protection system (GACP) undergoes oxidation and releases metals in the forms of ions or oxy-hydroxides into the environment. The objective of the present study was to investigate the toxicity of a cocktail of metals released from the dissolution of an aluminium-based galvanic anode (~ 95% Al, ~ 5% Zn) on the abalone Haliotis tuberculata. Juveniles were exposed for 16 weeks (i.e. 12 weeks of exposure and 4 weeks of decontamination phase) and their growth, intake rate, conversion rate and metallic concentrations were monitored. A total of 6 conditions were tested: a control, 4 concentrations based on Al and a trophic control. Results showed that the mortality reached 57% for individuals exposed to 1125 µg L-1 of Al, and the abalone growth significantly decreased for an Al concentration greater than 495 µg L-1. At the highest exposure concentration, intake rate measurements revealed that the appetite of abalones was affected, supported by the large increase in the conversion rate which was indicative of a poor feed efficiency. The monitoring of metallic concentrations showed that H. tuberculata strongly bioconcentrated Al relative to zinc. The diet did not appear to be the primary pathway for metal entry. Concentrations that significantly impacted abalone growth and survival during the experiment were higher than those found in natural environment, but the bioconcentration of Al into the tissues of a primary consumer such as abalone may be a potential pathway for Al to enter food webs.

Effects of chronic exposure of metals released from the dissolution of an aluminium galvanic anode on the Pacific oyster Crassostrea gigas.

Among the anthropogenic sources releasing metallic species into the marine environment, the galvanic anode cathodic protection system (GACP) is widely used to protect submerged metallic structures from corrosion. Galvanic anodes are an alloy of metals of which the main component is aluminum or zinc. Very few studies were performed to study their potential biological effects. We investigated the chronic toxicity of an aluminum-based galvanic anode on the Pacific oyster, Crassostrea gigas. Oysters were exposed for 84 days to three concentrations of aluminum (50, 100 and 300 µg L-1) obtained with an electrochemical experimental device simulating the dissolution of a galvanic anode. At different exposure times, we studied a battery of biomarkers of the immune system, reproductive parameters and the metabolic state of the oysters. Results demonstrated a sensitivity of oysters at the highest concentration and some biological effects were observed especially for the malondialdehyde content in the digestive gland after 84 days of exposure. In addition to these biomarkers, the bioaccumulation of the different metals composing the anode was measured in oysters' tissues. Bivalves bioaccumulated more zinc than aluminum, even if aluminium was present in greater concentrations during exposures. Moreover, exposure time did not influence the bioaccumulation of aluminum in contrast to zinc.

Toxicological effects of CdSe nanocrystals on the marine diatom Phaeodactylum tricornutum: The first mass spectrometry-based proteomic approach.

In the marine environment, benthic diatoms from estuarine and coastal sediments are among the first targets of nanoparticle pollution whose potential toxicity on marine organisms is still largely unknown. It is therefore relevant to improve our knowledge of interactions between these new pollutants and microalgae, the key players in the control of marine resources. In this study, the response of P. tricornutum to CdSe nanocrystals (CdSe NPs) of 5 nm (NP5) and 12 nm (NP12) in diameter was evaluated through microscopic, physiological, biochemical and proteomic approaches. NP5 and NP12 affected cell growth but oxygen production was only slightly decreased by NP5 after 1-d incubation time. In our experimental conditions, a high CdSe NP dissolution was observed during the first day of culture, leading to Cd bioaccumulation and oxidative stress, particularly with NP12. However, after a 7-day incubation time, proteomic analysis highlighted that P. tricornutum responded to CdSe NP toxicity by regulating numerous proteins involved in protection against oxidative stress, cellular redox homeostasis, Ca2+ regulation and signalling, S-nitrosylation and S-glutathionylation processes and cell damage repair. These proteome changes allowed algae cells to regulate their intracellular ROS level in contaminated cultures. P. tricornutum was also capable to control its intracellular Cd concentration at a sufficiently low level to preserve its growth. To our knowledge, this is the first work allowing the identification of proteins differentially expressed by P. tricornutum subjected to NPs and thus the understanding of some molecular pathways involved in its cellular response to nanoparticles. SIGNIFICANCE: The microalgae play a key role in the control of marine resources. Moreover, they produce 50% of the atmospheric oxygen. CdSe NPs are extensively used in the industry of renewable energies and it is regrettably expected that these pollutants will sometime soon appear in the marine environment through surface runoff, urban effluents and rivers. Since estuarine and coastal sediments concentrate pollutants, benthic microalgae which live in superficial sediments will be among the first targets of nanoparticle pollution. Thus, it is relevant to improve our knowledge of interactions between diatoms and nanoparticles. Proteomics is a powerful tool for understanding the molecular mechanisms triggered by nanoparticle exposure, and our study is the first one to use this tool to identify proteins differentially expressed by P. tricornutum subjected to CdSe nanocrystals. This work is fundamental to improve our knowledge about the defence mechanisms developed by algae cells to counteract damage caused by CdSe NPs.

Ability of the marine bacterium Pseudomonas fluorescens BA3SM1 to counteract the toxicity of CdSe nanoparticles.

UNLABELLED: In the marine environment, bacteria from estuarine and coastal sediments are among the first targets of nanoparticle pollution; it is therefore relevant to improve the knowledge of interactions between bacteria and nanoparticles. In this work, the response of the marine bacterium Pseudomonas fluorescens BA3SM1 to CdSe nanocrystals (CdSe NPs) of 3nm (NP3) and 8nm (NP8) in diameter was evaluated through microscopic, physiological, biochemical and proteomic approaches. Transmission electron microscopy images showed that NP3 were able to penetrate the bacteria, while NP8 were highly concentrated around the cells, embedded in large exopolysaccharides. In our experimental conditions, both CdSe NP sizes induced a decrease in respiration during the stationary growth phase, while only NP8 caused growth retardation and a decrease in pyoverdine production. Proteomic analyses highlighted that the strain responded to CdSe NP toxicity by inducing various defence mechanisms such as cell aggregation, extracellular CdSe NP sequestration, effective protection against oxidative stress, modifications of envelope organization and properties, and cadmium export. In addition, BA3SM1 presented a biosorption capacity of 1.6×10(16)NP3/g dry weight and 1.7×10(15)NP8/g dry weight. This strain therefore appears as a promising agent for NP bioremediation processes. Proteomic data are available via ProteomeXchange with identifier PXD004012. BIOLOGICAL SIGNIFICANCE: To the best of our knowledge, this is the first report focussing on the effects of CdSe colloidal nanocrystals (CdSe NPs) on a marine strain of Pseudomonas fluorescens. CdSe NPs are extensively used in the industry of renewable energies and it is regrettably expected that these pollutants will sometime soon appear in the marine environment through surface runoff, urban effluents and rivers. Bacteria living in estuarine and coastal sediments will be among the first targets of these new pollutants. The pseudomonads are frequently found in these ecosystems. They are involved in several biogeochemical cycles and are known for their high resistance to pollutants. Consequently, this study focussing on the effects of CdSe NPs on the marine strain P. fluorescens BA3SM1 is highly relevant for several reasons. First, it aims at improving knowledge about the interactions between bacteria and NPs. This is fundamental to effectively use NPs against pathogenic bacteria. Secondly, in spite of CdSe NP interactions with the bacterial cells, the strain BA3SM1 can develop various strategies to counteract CdSe NP toxicity and ensure its growth. It exhibits interesting properties to sequester CdSe NPs and it retains its ability to form biofilm. The strain therefore appears as a promising agent for NP bioremediation thanks to biofiltration processes. Finally, this study shows that CdSe NPs of 8nm in diameter cause a decrease in the secretion of siderophore pyoverdine, a secondary metabolite playing a key role in microbial ecology since it drives bacterial survival and competitiveness in ecosystems. Bacteria producing effective siderophores survive better in a Fe-deficient environment where they antagonize the growth of other microbes thought iron deprivation. Furthermore, siderophores are also employed as virulence factors in human pathogenic strains such as P. aeruginosa. Consequently, this study highlights that NPs can impact the secondary metabolism of bacteria with environmental and medical implications. In addition, in this work, Data-Dependant Acquisition (DDA) provided state of the art Mass Spectrometry data by Spectral Counting and MS1 Label-Free. The combination of these two well-known proteomic techniques including manual validations strengthened the identification and quantification of regulated proteins. Moreover, numerous correlations between proteomic analyses and other observations (physiological, biochemical, microscopic) consolidated our interpretations.

Physiological perturbations in juvenile cuttlefish Sepia officinalis induced by subchronic exposure to dissolved zinc.

Although cephalopod early life stage development often occurs in coastal areas where contamination is real and continuous, the physiological perturbations induced by contaminants have been rarely investigated. This study focused on the Zn as it is one of the trace metals the most concentrated in coastal waters, worldwide. As Zn-tolerance limits were unknown in juvenile Sepia officinalis, the aim of this study was to estimate the threshold inducing mortality during the 2-first weeks post-hatching, and to determine its sensitivity using digestive and immune enzymatic assays, as well as growth and behavior follow-up during the first 5weeks post-hatching. Our study highlighted a Zn-mortality threshold lying between 185 and 230µgl(-1), and growth reductions occurring after 5-week at 108µgl(-1) and above, associated with enzymatic perturbations. These results underline a relatively important sensitivity of juvenile cuttlefish to Zn, pointed out by a wide diversity of biomarkers.

Trace metal concentrations in post-hatching cuttlefish Sepia officinalis and consequences of dissolved zinc exposure.

In this study, we investigated the changes of 13 trace metal and metalloid concentrations (i.e. Ag, As, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, Se, V, Zn) and their subcellular fractionation in juvenile cuttlefish Sepia officinalis reared in controlled conditions between hatching and 2 months post-hatching. In parallel, metallothionein concentrations were determined. Our results highlighted contrasting changes of studied metals. Indeed, As and Fe concentrations measured in hatchlings suggested a maternal transfer of these elements in cuttlefish. The non-essential elements Ag and Cd presented the highest accumulation during our study, correlated with the digestive gland maturation. During the 6 first weeks of study, soluble fractions of most of essential trace metals (i.e. Co, Cr, Cu, Fe, Se, Zn) slowly increased consistently with the progressive needs of cuttlefish metabolism during this period. In order to determine for the first time in a cephalopod how metal concentrations and their subcellular distributions are impacted when the animals are trace metal-exposed, we studied previously described parameters in juveniles exposed to dissolved Zn at environmental (i.e. 50 µg l(-1)) and sublethal (i.e. 200 µg l(-1)) levels. Moreover, oxidative stress (i.e. glutathione S-transferase (GST), superoxide dismutase (SOD) and catalase activities, and lipid peroxidation (LPO)) was assessed in digestive gland and gills after 1 and 2 months exposures. Our results highlighted no or low ability of this stage of life to regulate dissolved Zn accumulation during the studied period, consistently with high sensitivity of this organism. Notably, Zn exposures caused a concentration-dependent Mn depletion in juvenile cuttlefish, and an increase of soluble fraction of Ag, Cd, Cu without accumulation modifications, suggesting substitution of these elements (i.e. Mn, Ag, Cd, Cu) by Zn. In parallel, metallothionein concentrations decreased in individuals most exposed to Zn. Finally, no perturbations in oxidative stress management were detected in gills, whereas modifications of GST, SOD and catalase activity levels were recorded in digestive gland, resulting in an increase of LPO content after a 6-week exposure to low Zn concentration. Altogether, these perturbations are consistent with previously described high sensitivity of juvenile cuttlefish towards Zn. Our results underlined the need to study deeply contamination impact on this animal at this stage of life.

The effect of cold stress on the proteome of the marine bacterium Pseudomonas fluorescens BA3SM1 and its ability to cope with metal excess.

This study examined the effect of cold stress on the proteome and metal tolerance of Pseudomonas fluorescens BA3SM1, a marine strain isolated from tidal flat sediments. When cold stress (+10 °C for 36 h) was applied before moderate metal stress (0.4 mM Cd, 0.6 mM Cd, 1.5 mM Zn, and 1.5 mM Cu), growth disturbances induced by metal, in comparison with respective controls, were reduced for Cd and Zn while they were pronounced for Cu. This marine strain was able to respond to cold stress through a number of changes in protein regulation. Analysis of the predicted differentially expressed protein functions demonstrated that some mechanisms developed under cold stress were similar to those developed in response to Cd, Zn, and Cu. Therefore, pre-cold stress could help this strain to better counteract toxicity of moderate concentrations of some metals. P. fluorescens BA3SM1 was able to remove up to 404.3 mg Cd/g dry weight, 172.5 mg Zn/g dry weight, and 11.3 mg Cu/g dry weight and its metal biosorption ability seemed to be related to the bacterial growth phase. Thus, P. fluorescens BA3SM1 appears as a promising agent for bioremediation processes, even at low temperatures.

Relaxation of the non-photochemical chlorophyll fluorescence quenching in diatoms: kinetics, components and mechanisms.

Diatoms are especially important microorganisms because they constitute the larger group of microalgae. To survive the constant variations of the light environment, diatoms have developed mechanisms aiming at the dissipation of excess energy, such as the xanthophyll cycle and the non-photochemical chlorophyll (Chl) fluorescence quenching. This contribution is dedicated to the relaxation of the latter process when the adverse conditions cease. An original nonlinear regression analysis of the relaxation of non-photochemical Chl fluorescence quenching, qN, in diatoms is presented. It was used to obtain experimental evidence for the existence of three time-resolved components in the diatom Phaeodactylum tricornutum: qNf, qNi and qNs. qNf (s time-scale) and qNs (h time-scale) are exponential in shape. By contrast, qNi (min time-scale) is of sigmoidal nature and is dominant among the three components. The application of metabolic inhibitors (dithiothreitol, ammonium chloride, cadmium and diphenyleneiodonium chloride) allowed the identification of the mechanisms on which each component mostly relies. qNi is linked to the relaxation of the ΔpH gradient and the reversal of the xanthophyll cycle. qNs quantifies the stage of photoinhibition caused by the high light exposure, qNf seems to reflect fast conformational changes within thylakoid membranes in the vicinity of the photosystem II complexes.

The porifera Hymeniacidon perlevis (Montagu, 1818) as a bioindicator for water quality monitoring.

Because sponges are promising bioindicators, we present here a multispecies comparison of the bioconcentration capacity for copper, zinc and the hydrocarbon fluoranthene. The spatial distribution of sponge populations was studied in 17 areas in intertidal zones on the Lower Normandy coast (France) to determine the most common species with the highest bioaccumulation capacity. Results are compared with published data on blue mussels Mytilus edulis from the Réseau d'Observation de la Contamination Chimique biomonitoring network. A total of 720 sponge samples were collected to assess species richness. Samples were analysed for metal concentrations by flame-mode atomic absorption spectrometry. Analyses of polycyclic aromatic hydrocarbon were sub-contracted. Species richness varies according to the water mass concerned. The most common species in the study area showing the highest bioconcentration in its soft tissues is Hymeniacidon perlevis, which contains about 20 times the zinc, 44 times the copper and 16 times the fluoranthene levels found in mussels. The variability of contaminant concentrations in H. perlevis is also systematically higher than those in mussels. The results obtained for this sponge closely reflect the heterogeneous distribution of contaminants. This study demonstrates that H. perlevis has a much higher capacity to accumulate in situ contaminants than the blue mussel M. edulis. H. perlevis meets all the requirements of a good bioindicator suitable for use in an integrated monitoring programme. In the near future, controlled cultivation of H. perlevis will allow us to produce sufficient quantities of this species to carry out ecotoxicological tests and in situ biomonitoring by caging.

Effect of chronic exposure to zinc in young spats of the Pacific oyster (Crassostrea gigas).

The marine coastal environment is exposed to a mixture of environmental pollutants of anthropogenic origin, resulting in chronic low concentrations of contaminants. As a consequence, most coastal marine species are exposed to low doses of such pollutants during their entire life. Many marine species live for years in their natural environment, whereas they do not under laboratory exposure conditions. Using early stages of development in laboratory work allows animals to be chronically exposed from an early age over a reasonable experiment period. In the present study, the authors investigated the effect of chronic exposure to zinc in spats of the Pacific oyster (Crassostrea gigas), from metamorphosis up to 10 weeks. The authors investigated integrated biological endpoints that would account for the apparent general health of the animals as well as molecular markers showing more subtle effects that could potentially go unnoticed at a biologically integrated level. The authors measured in parallel both growth and the transcriptional level of target stress genes. Growth was monitored by image analysis of large samples to avoid high variability and ensure statistical robustness. A dose-response relationship was derived from growth data, yielding a median effective concentration (EC50) of 7.55 µM. Stress genes selected on the basis of available RNA sequences in C. gigas included genes involved in chaperone proteins, oxidative stress, detoxification, and cell cycle regulation. Out of nine stress target genes, only metallothionein displayed overexpression in response to high levels of zinc.

Effect of zinc sacrificial anode degradation on the defence system of the Pacific oyster, Crassostrea gigas: chronic and acute exposures.

Two types of exposures were performed to assess the effects of zinc released from sacrificial anode degradation: a chronic exposure, in which oysters were exposed to 0.53±0.04 mg Zn L(-1) for 10 weeks, and an acute exposure, where oysters were exposed to 10.2±1.2 mg Zn L(-1) for 1 week. At the end of the acute exposure experiment, 81.8% mortality was recorded. In contrast, no mortality was detected after 10 weeks exposure. Moreover, all of the immune system biomarkers studied, except the number of circulating haemocytes, were stimulated by a moderate level of zinc and inhibited by a high level. Our exposure conditions did not induce SOD or MXR mRNA expression in gills and digestive gland. However, an increase of MT mRNA is observed in these tissues. The results indicate that oysters are sensitive to acute zinc toxicity but are only moderately affected by a mild zinc concentration.

Impact of a sacrificial anode as assessed by zinc accumulation in different organs of the oyster Crassostrea gigas: results from long- and short-term laboratory tests.

Sacrificial anodes made of zinc are currently used in marine environments to mitigate marine corrosion as part of CP systems of immerged metallic structures. The aim of this work was to study zinc bioconcentration in the oyster Crassostrea gigas by performing two in vivo tests during different time periods and at different zinc concentrations. The first test was conducted during a period of 10 weeks at a concentration of 0.53 ± 0.04 mg Zn L(-1) to simulate long-term exposure, and a second test was conducted during a 168-hour period at a concentration of 10.2 ± 1.2 mg Zn L(-1) to reproduce short-term exposure. In these experiments, the zinc source was an electrochemical device that included a sacrificial anode to mimic the in situ conditions. During the first 14 days of the long-term experiment, digestive glands of C oysters exhibited bioaccumulation of zinc that varied according to the oysters' reproductive cycle. Both a bioconcentration factor (BCF) of ≤ 13,397 and a zinc accumulation percentage of +297% of zinc occurred in this organ after 10 weeks. The results obtained from the short-term test showed a lower BCF of 405 but a faster bioaccumulation of zinc (starting from the first day) in the same organ. No mortality was observed in long-term assay, but 81.8% of the oysters died at the end of the short-term assay. These results demonstrate the great capacity of C. gigas to accumulate zinc released from the anode, especially when low concentrations are released, as in the case of anode dissolution used as CP. This study confirmed the necessity to monitor this zinc-contamination source in marine environments in relation to the usual oyster consumption by humans (especially in France). No implication for human health of this zinc-contamination source was demonstrated until now, and this was not the purpose of this study; however, zinc remains one of the most abundant nutritionally essential elements in the human body that may affect the human immune system at high-level uptake.

Assessment of sacrificial anode impact by aluminum accumulation in mussel Mytilus edulis: a large-scale laboratory test.

Since the early 1960s, the application of aluminum alloy sacrificial anodes to mitigate marine corrosion has been well known. The aim of this work was to study aluminum bioconcentration in Mytilus edulis by an in vitro test performed in two tanks: the first containing non-contaminated water (NCW) and the second containing aluminum-contaminated water (CW) (530 µg L(-1)) released by sacrificial anode. The mussels were collected and examined over a period of 8 weeks. A comparison between the aluminum concentrations in the digestive glands of mussels from the CW and NCW tanks shows that the highest value (1700 mg/kg d.w.) was found in the CW mussels collected after 13 days. In NCW, the mean aluminum concentration in digestive glands during the test was 281 mg/kg d.w. The rapid concentration decrease in digestive glands is probably due to the inhibition of filtering activity due to valve closure at the high concentration as well as the induction of the detoxification response.

Comparative toxicities of aluminum and zinc from sacrificial anodes or from sulfate salt in sea urchin embryos and sperm.

The toxicity of aluminum or zinc from either sacrificial anodes (SA) or their sulfate salts (SS) was evaluated in sea urchin (Paracentrotus lividus) embryos or sperm exposed to Al(III) or Zn(II) (SA or SS, 0.1-10 microM), scoring developmental defects (DDs), fertilization rate (FR), and mitotic abnormalities. A significant DD increase was observed in SS, but not SA Al(III)- and Zn(II)-exposed embryos vs. controls. Both Al(III) and Zn(II), up to 10 microM, from SA and SS, inhibited mitotic activity and induced mitotic aberrations in exposed embryos. SA-Al(III)-exposed sperm displayed a significant FR increase, unlike Al(III) sulfate overlapping with controls. Both SA-Zn(II) and Zn(II) sulfate sperm exposure resulted in a significant FR increase. The offspring of SA-Al(III)-exposed sperm displayed a significant DD decrease, unlike Al(III) sulfate exposure. Zinc sulfate sperm exposure resulted in a significant increase in offspring DDs, whereas SA-Zn(II) sperm exposure decreased DDs. Together, exposures to SA-dissolved Al(III) or Zn(II) resulted in lesser, if any toxicity, up to hormesis, compared to SS. Studies of metal speciation should elucidate the present results.

Effect of in vitro exposure to zinc on immunological parameters of haemocytes from the marine gastropod Haliotis tuberculata.

Environmental pollutants such as heavy metals exert immunotoxic effects on aquatic organisms. The immune defence of molluscs is comprised of cell-mediated and humoral mechanisms, in which haemocytes play a key role. In this study, a model based on primary cultured haemocytes from the gastropod mollusc Haliotis tuberculata was established to investigate the effects of zinc in vitro. Cells were exposed for 24 h to ZnCl(2) concentrations of 0, 10, 100 or 1000 microM. The effects of zinc on haemocyte parameters were investigated using morphological, spectrophotometric and flow cytometry analysis. Immunotoxicity was reflected by a significant decrease in the number of viable haemocytes (LC(50)(24 h) = 314 microM). Moreover, the cell area was dramatically reduced, and the percentage of rounded cells increased with increasing zinc concentrations. Exposure to 1000 muM zinc induced a significant reduction in acid phosphatase activity, phagocytic activity and reactive oxygen species production in haemocytes. However, several haemocyte parameters increased significantly after 24 h of zinc exposure. In response to a 1000 microM exposure, the phenoloxidase level was 26-fold higher than that of the control, and non-specific esterase activity was increased by 69% above that of the control. These results suggest a relationship between zinc exposure and alterations in the functional responses of haemocytes from H. tuberculata.