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Immunomodulating effects of environmentally realistic copper concentrations in Mytilus edulis adapted to naturally low salinities
Abstract
The monitoring of organisms' health conditions by the assessment of their immunocompetence may serve as an important criterion for the achievement of the Good Environmental Status (GES) as defined in the Marine Strategy Framework Directive (EU). In this context, the complex role of natural environmental stressors, e.g. salinity, and interfering or superimposing effects of anthropogenic chemicals, should be carefully considered, especially in scenarios of low to moderate contamination. Organisms from the Baltic Sea have adapted to the ambient salinity regime, however energetically costly osmoregulating processes may have an impact on the capability to respond to additional stress such as contamination. The assessment of multiple stressors, encompassing natural and anthropogenic factors, influencing an organisms' health was the main aim of the present study. Immune responses of Mytilus edulis, collected and kept at natural salinities of 12‰ (LS) and 20‰ (MS), respectively, were compared after short-term exposure (1, 7 and 13 days) to low copper concentrations (5, 9 and 16μg/L Cu). A significant interaction of salinity and copper exposure was observed in copper accumulation. LS mussels accumulated markedly more copper than MS mussels. No combined effects were detected in cellular responses. Bacterial clearance was mostly achieved by phagocytosis, as revealed by a strong positive correlation between bacterial counts and phagocytic activity, which was particularly pronounced in LS mussels. MS mussels, on the other hand, seemingly accomplished bacterial clearance by employing additional humoral factors (16μg/L Cu). The greatest separating factor in the PCA biplot between LS and MS mussels was the proportion of granulocytes and hyalinocytes while functional parameters (phagocytic activity and bacterial clearance) were hardly affected by salinity, but rather by copper exposure. In conclusion, immune responses of the blue mussel may be suitable and sensitive biomarkers for the assessment of ecosystem health in brackish waters (10-20‰S).
Supplementary resource (1)
Data
January 2014
... defence. So far in Mytilus sp., available data indicates that metal exposure can result in increases or decreases in hemocyte immune parameters, depending on the exposure conditions (Parry and Pipe, 2004;Höher et al., 2013). In this light, the role of copper/zinc homeostasis in mussel immune response deserves further investigation. ...
... Environmental factors are known to significantly affect immune responses. Thus, immune parameters of mussels and oysters (total hemocyte counts, the proportion of hemocyte populations, immune gene expression) vary with temperature and exposure to pollutants, including heavy metals and nanoparticles (Parry and Pipe, 2004;Höher et al., 2013;Wendling and Wegner, 2015;Canesi and Corsi, 2016;Auguste et al., 2020), which may have important consequences on bivalve susceptibility to pathogens. In addition, recent studies have revealed that previous encounter with pathogens is a life-history trait that defines the resistance/tolerance of bivalves to infections (i.e. ...
In the marine environment, bivalve mollusks constitute habitats for bacteria of the Vibrionaceae family. Vibrios belong to the microbiota of healthy oysters and mussels, which have the ability to concentrate bacteria in their tissues and body fluids, including the hemolymph. Remarkably, these important aquaculture species respond differently to infectious diseases. While oysters are the subject of recurrent mass mortalities at different life stages, mussels appear rather resistant to infections. Thus, Vibrio species are associated to the main diseases affecting the worldwide oyster production. Here we review the current knowledge on Vibrio‐bivalve interaction in oysters (Crassostrea sp.) and mussels (Mytilus sp.). We discuss the transient versus stable associations of vibrios with these bivalves as well as technical issues limiting the precise monitoring of vibrios in health and disease. Based on the current knowledge of oyster/mussel immunity and their interactions with Vibrio species pathogenic for oyster, we discuss how differences in immune effectors could contribute to the higher resistance of mussels to infections. Finally, we review the multiple strategies evolved by pathogenic vibrios to circumvent the potent immune defenses of bivalves and how key virulence mechanisms could have been positively or negatively selected in the marine environment through interactions with predators. This article is protected by copyright. All rights reserved.
... Caspase-3 is a frequently activated death protease in apoptotic cells of both vertebrates and invertebrates , Motta et al., 2013. Using the FCM method, increases of caspase-3 activation have been reported in P. canaliculus haemocytes exposed to copper or LPS , and Mytilus edulis haemocytes exposed to copper (Höher et al., 2013). As an example, used the Muse ® Caspase-3/7 kit to quantitative measurements of apoptotic status in P. canaliculus haemocytes in vitro exposed to copper. ...
... Hence, activation of caspase-3 is considered to be an indicator of apoptosis . Caspase-3 activation has been reported in haemocytes of Crassostrea virginica oysters and Mytilus edulis mussels exposed to cadmium and copper (Höher et al., 2013), respectively. The caspase-3 activation has also been observed in mantle tissues of M. galloprovincialis mussels exposed to various heavy metals . ...
Molluscs form an important group in aquaculture as well as in coastal wild systems. However, high mortalities in molluscan species, specifically marine bivalves, have been encountered in the wild during summer times (summer mortality) as well as in aquaculture settings, which present a major economical challenge in many parts of the world. The complex interactions between host, environment and pathogens during these mortality events require new diagnostic tools and integrated approaches. Metabolomics is one of the newest and fastest growing omics. The sensitivity and specificity of metabolomics approaches make this a powerful tool for immunological studies, where it can provide insights into disease processes as well as the identification of metabolite biomarkers for early warning systems. This thesis was designed to provide, for the first time, a comprehensive understanding of the metabolic responses of mussel haemocytes and other tissues (e.g., gills, hepatopancreas, mantle) to external stimuli (Vibrio sp., lipopolysaccharides [LPS], Cu2+) using gas chromatography-mass spectrometry (GC-MS)-based metabolomics approach. Along with the core metabolomics tool, novel flow cytometry (FCM) protocols were developed in order to assess immunological parameters of the host upon stimulation. The combined method allows characterization of the mussel immune responses at both cellular and molecular levels and expands the number of biomarkers used to understand the animal’s response.
Initially, tissue-specific metabolic responses of gill, haemolymph and hepatopancreas were observed in mussels challenged with Vibrio sp. Then, haemolymph was chosen as the target tissue/organ for the rest of the experiments in the thesis (Chapter 4). FCM revealed sex-based differences in immune responses of mussels to Vibrio sp. challenge. In this case, female mussels had lower haemocyte mortality, production of reactive oxygen species (ROS) and apoptotic cells after pathogen exposure compared to male mussels (Chapter 5). This suggests that female mussels have more efficient defence system than male mussels. However, metabolite profiles of haemolymph showed no significant difference between males and females. Subsequently, metabolic profiles of mussel haemolymph were intensively investigated in response to Vibrio sp. challenge, LPS and copper exposure (Chapter 6, 7 & 8). The alterations of metabolite profiles along with changes in immune characteristics due to stimulation provided insights into a number of pathways involved in immune responses of the host to Vibrio sp. infection and copper exposure.
The study also identified a number of candidate biomarkers involved in mussel immune processes. Among these metabolites, the presence of itaconic acid (ITA) and its accumulation were observed in different tissues of mussels following Vibrio sp. challenges, suggesting the important role of this metabolite as an antimicrobial compound in the innate immune system of bivalves (Chapter 4, 5 & 6). In fact, the challenge experiment (Chapter 9) revealed the complete inhibition of ITA on Vibiro sp. growth at 6 mM, and Vibrio growth was partially inhibited at 3 mM ITA. This confirmed, for the first time, the antibacterial activity of ITA against marine Vibiro sp. and suggests that ITA could be used as an antimicrobial compound for antibiotic resistant bacteria in aquaculture. Subsequently, the ITA concentrations in different tissues of mussels challenged with Vibrio sp. were quantitatively measured (Chapter 10). Interestingly, the results revealed that mussels are able to produce an effective amount of ITA to support the internal defence system, suggesting that ITA could be a valuable biomarker for health assessment of bivalves. In addition, ITA may also involve in anti-inflammation activities and other unknown functions in the bivalve innate immune system, which need further studies to reveal.
In conclusion, this thesis has successfully demonstrated the use of novel metabolomics approaches for aquaculture and marine science, which contribute new information regarding the molluscan immune system. It is envisaged that metabolomics will continue to grow as a tool of choice in studies of marine molluscs, as well as the broader field of marine science.
Permanent link: http://hdl.handle.net/10292/13154
... The formation of micronuclei and other nuclear abnormalities served as markers for geno-and cytotoxicity (Baršienė et al., 2004(Baršienė et al., , 2006. Immunocompetence was evaluated by the performance of circulating haemocytes, particularly phagocytic activity as the predominant cellular defence mechanism of bivalve invertebrates against pathogens (Pipe and Coles, 1995;Höher et al., 2013). Finally, chemical analysis of tissue and water samples enabled an integrated assessment. ...
... Haemocytes are circulating within the mussel tissues and are involved in immune processes such as wound healing and pathogen elimination. Phagocytic activity as a measure of successful pathogen uptake by haemocytes has been shown to be reduced after exposure to high concentrations of contaminants (Höher et al., 2013). However, stimulation may be triggered at lower concentrations or acute exposure, as observed in the current study (N5eN10; except N6). ...
Baltic blue mussels (Mytilus trossulus) were implemented to assess potential toxicity, health impairments and bioaccumulation of dumped chemical warfare agents on marine benthic organisms. Mussels were collected from a pristine cultivation side and exposed under laboratory conditions to different mixtures of chemical warfare agents (CWAs) related phenyl arsenic compounds, Clark I and Adamsite as well as chloroacetophenone. Using a multi-biomarker approach, mussels were assessed thereafter for effects at different organisational levels ranging from geno-to cytotoxic effects, differences in enzyme kinetics and immunological responses. In an integrated approach, chemical analysis of water and tissue of the test organisms was performed in parallel. The results show clearly that exposed mussels bioaccumulate the oxidized forms of chemical warfare agents Clark I, Adamsite (DAox and DMox) and, to a certain extent, also chloroacetophenone into their tissues. Adverse effects in the test organisms at subcellular and functional level, including cytotoxic, immunotoxic and oxidative stress effects were visible. These acute effects occurred even at the lowest test concentration.
... Caspase-3 is a frequently activated death protease in apoptotic cells of both vertebrates [114] and invertebrates [94,110,115,116]. Using the FCM method, increases of caspase-3 activation have been reported in P. canaliculus haemocytes exposed to copper [40] or LPS [41], and Mytilus edulis haemocytes exposed to copper [117]. As an example, Nguyen et al. [40] used the Muse™ Caspase-3/7 kit to quantitative measurements of apoptotic status in P. canaliculus haemocytes in vitro exposed to copper. ...
Flow cytometry (FCM) is routinely used in fundamental and applied research, clinical practice, and clinical trials. In the last three decades, this technique has also become a routine tool used in immunological studies of molluscs to analyse physical and chemical characteristics of haemocytes. Here, we briefly review the current implementation of FCM in the field of molluscan immunology. These applications cover a diverse range of practices from straightforward total cell counts and cell viability to characterize cell subpopulations, and further extend to analyses of DNA content, phagocytosis, oxidative stress and apoptosis. The challenges and prospects of FCM applications in immunological studies of molluscs are also discussed.
https://www.sciencedirect.com/science/article/abs/pii/S105046481930885X?via%3Dihub
... Although the literature has few studies regarding L. fortunei exposure to HgCl 2 , studies have been conducted with other mollusks that were exposed to this metal. Höher et al. (2013) reported tissue injury associated with an increase in superoxide dismutase activity in Mytilus edulis exposed to 50 μg/L of HgCl 2 for up to 11 days. HgCl 2 (~15 μg/kg wet weight) provoked a decrease in phagocytic capacity and increased the oxidative burst activity in Scobicularia plana (Ahmad et al., 2011). ...
This study evaluated the Limnoperna fortunei (golden mussel) as a bioindicator of cytotoxicity and genotoxicity in aquatic environments contaminated by heavy metals. Five groups of 50 subjects each were exposed to different concentration of mercuric chloride (HgCl2) (0.001 mg/L, group I; 0.005 mg/L, group II; 0.01 mg/L, group II; 0.02 mg/L, group IV; and 0.1 mg/L, group V). The control group for both chronic and acute treatment did not receive HgCl2. For chronic exposure, the respective groups were placed in aquaria with water contaminated with the above concentrations of HgCl2. For acute exposure, the different concentrations of HgCl2 were injected into the posterior adductor muscle of the individuals belonging to the aforementioned groups. The biological matrix used in the tests was the whole body muscle. Tests (cell viability assay, alkaline comet test; enumeration of micronuclei and necrotic cells, quantification of Hg content in tissues and water, and histopathological analysis of tissues), were carried out on the 7th, 15th, and 30th treatment days or 2 h after injection. Our results demonstrated that L. fortunei showed cell damage in both chronic and acute exposure groups. Significant DNA damage was observed at both the 15th (0.1 mg/L) and 30th (0.01–0.1 mg/L) days of chronic exposure. However, in acute treatment all concentrations induced DNA breaks. The presence of necrosis increased at all concentrations tested for both acute and chronic exposure. Tissue mercury retention on the 15th day was higher than on the 30th day of exposure, while in the same period, there was a decrease in the mercury content of aquarium water. Taking the data together, it is concluded that L. fortunei as a possible bioindicator of the quality of aquatic environments.
... [61][62][63] Hence, activation of caspase-3 is considered to be an indicator of apoptosis. 60 Caspase-3 activation has been reported in haemocytes of Crassostrea virginica oysters and Mytilus edulis mussels exposed to cadmium 62 and copper, 64 respectively. The caspase-3 activation has also been observed in mantle tissues of M. galloprovincialis mussels exposed to various heavy metals. ...
Copper is widely used in industry and agriculture, and thus is a common chemical contaminant in aquatic environments. As filter feeders, bivalves are prone to accumulate certain heavy metals, such as copper. However, the toxicity mechanisms of copper in marine bivalves is not fully understood. In this study, we provide the first report on the combined approach of GC–MS-based metabolomics and novel flow cytometry techniques to characterize the toxic effects and elucidate the toxicity mechanism of copper on New Zealand GreenshellTMmussel (Perna canaliculus) haemocytes. Mussel haemolymph samples were exposed to increasing concentrations of Cu2+ (0.0, 25.0, 62.5, 125.0 and 187.5 μM) for 24 hours. Then, flow cytometric assays (mortality, ROS production and apoptosis) and metabolomics analyses were conducted for all samples. Flow cytometric results showed significant increases in a dose dependent manner in cell mortality, production of reactive oxygen species (ROS) and apoptosis (via alteration of caspase 3/7 activation and mitochondrial membrane potential) of haemocytes exposed to increasing Cu2+concentrations. For metabolomics, we observed alterations of 25 metabolites within metabolites profile of Cu2+-exposed haemolymph (125 μM) compared to those of control samples. Changes in levels of these metabolites may be considered important signatures of oxidative stress (e.g., glutathione, cysteine) and apoptosis processes (e.g., alanine, glutamic acid) that may be involve in the transsulfuration pathway, glutathione metabolism and taurine metabolism. In this model, Cu2+exposure led to excessive ROS production that caused the decreases of GSH, methionine, cysteine then taurine. The decrease of taurine, in turn, would have led to a decrease in glutamic acid and accumulation of alanine. This study provides insights into the cellular and molecular mechanisms of oxidative stress and apoptosis in marine bivalves and highlights the applicability and reliability of metabolomics and flow cytometry techniques for immunotoxicological studies in marine organisms.
... [61][62][63] Hence, activation of caspase-3 is considered to be an indicator of apoptosis. 60 Caspase-3 activation has been reported in haemocytes of Crassostrea virginica oysters and Mytilus edulis mussels exposed to cadmium 62 and copper, 64 respectively. The caspase-3 activation has also been observed in mantle tissues of M. galloprovincialis mussels exposed to various heavy metals. ...
Copper is a common contaminant in aquatic environments, which may cause immune dysfunction in marine organisms. However, the toxicity mechanisms of copper in marine bivalves is not fully understood. In this study, we applied an integrated approach that combines flow cytometry and Gas Chromatography–Mass Spectrometry (GC-MS)-based metabolomics to characterize cellular and molecular mechanisms of copper immunotoxicity in New Zealand Greenshell™ mussel (Perna canaliculus) haemolymph. Flow cytometric results showed significant increases in haemocyte mortality, production of reactive oxygen species and apoptosis (via alteration of caspase 3/7 and mitochondrial membrane potential) of haemocytes exposed to increasing total concentrations of Cu2+ (1.60, 3.20 and 4.80 μg/mL) compared to a low Cu2+ concentration (0.64 μg/mL) and control (0 μg/mL). These results reveal that Cu2+ induces oxidative stress and apoptosis in mussel haemocytes. In addition to flow cytometric data, our metabolomics results showed alterations of 25 metabolites within the metabolite profile of Cu2+-exposed haemolymph (3.20 μg/mL) compared to those of control samples. Changes in levels of these metabolites may be considered important signatures of oxidative stress (e.g., glutathione) and apoptosis processes (e.g., alanine, glutamic acid). This study provides insights into the cellular and molecular mechanisms of oxidative stress and apoptosis in marine bivalves and highlights the applicability and reliability of metabolomic techniques for immunotoxicological studies in marine organisms.
... For instances, elevated pCO 2 or POPs (Benzo[a]pyrene, 5 mg/L) can reduce THC and hamper phagocytic activity of the haemocytes of T. granosa [32,41]. Similar hampered immune responses were also observed when bivalves such as M. edulis and C. gigas were exposed to heavy metal contaminations such as cadmium, chromium, and mercury [44,45]. It has been suggested that more energy of marine organisms would be allocated to critical life processes under environmental stress, leading to a reduction in the energy budget for immune responses [46]. ...
The increasing production and extensive application of nanoparticles (NPs) inevitably leads to increased release of NPs into the marine environment and therefore poses a potential threat to marine organisms, especially the sessile benthic bivalves. However, the impacts of NPs on the immunity of commercial and ecological important bivalve species, Tegillarca granosa, still remain unknown to date. In addition, the molecular mechanism of the immunotoxicity of NPs still remains unclear in marine invertebrates. Therefore, the immunotoxicity of nTiO2 exposure to T. granosa at environmental realistic concentrations was investigated in the present study. Results obtained showed that the total number, phagocytic activity, and red granulocytes ratio of the haemocytes were significantly reduced after 30 days nTiO2 exposures at the concentrations of 10 and 100 μg/L. Furthermore, the expressions of genes encoding Pattern Recognition Receptors (PPRs) and downstream immune-related molecules were significantly down-regulated by nTiO2 exposures, indicating a reduced sensitivity to pathogen challenges. In conclusion, evident immunotoxicity of nTiO2 to T. granosa at environmental realistic concentrations was detected by the present study. In addition, the gene expression analysis suggests that the PRRs (both TLRs and RIG1 investigated) may be the molecules for NPs recognition in marine invertebrates.
... In this sense, no significant differences in Cu accumulation were observed in M. galloprovincialis exposed to Cu after 4 or 21 days, although Cu concentrations were found to double in the cases of Mytilus edulis and Mytilus trossulus (Brooks et al. 2015). Increasing Cu accumulation has also been reported by many authors (Boudjema et al. 2014;Fokina et al. 2013;Jorge et al. 2013) but in many cases environmental conditions and exposure times differ from those employed in the present study (Amiard-Triquet et al. 1991;Höher et al. 2013). On the other hand, statistically significant higher Cu concentrations were found in mussels exposed to Cd + Cu than in the control group. ...
Heavy metals often accumulate in complex mixtures in the environment and are currently a source of concern in many marine ecosystems. Pb, Cd, and Cu are regarded as priority hazardous metals due to their great persistence, bioaccumulation, and toxicity. The aim of the present study was to investigate the tissue accumulation and distribution of these heavy metals in Mediterranean mussels (Mytilus galloprovincialis) exposed to binary and ternary mixtures of metals as opposed to only single exposures. Heavy metal concentrations in the digestive gland, gills, and the other soft tissues were determined by inductively coupled plasma optical emission spectrometry (ICP-OES), and the distribution of each metal was analyzed according to compartments. The concentrations of Pb, Cd, and Cu increased significantly in the group exposed to the ternary mixture; however, there was no common response pattern to exposure in single and binary mixtures. Above all, the metals concentrated in the digestive gland, although the percentages of each element varied between compartments and varied between tissues according to the treatment.
Metabolomics (the broad-scale analysis of metabolites in biological samples) is one of the fastest growing “omics”. A dramatic increase of metobolomics applications in the field of life science, especially in aquaculture research has been observed in recent years (Alfaro and Young 2016; Young and Alfaro 2016). As metabolites are end-products of cellular regulatory processes, metabolomics could provide a novel phenotyping tool for understanding endogenous metabolic changes of an organism in the course of a biological process promoted by disease or environmental perturbations. In our recent studies, we have shown the successful application of integrated metabolomics approaches with novel flow cytometric techniques to provide insights into the molecular mechanisms of bivalve immune responses against pathogens and/or environmental contaminants. In this presentation, we showcase three examples where this integrative approach is used to characterize the metabolic and immunological responses of marine bivalves to pathogenic Vibrio sp. infection, lipopolysaccharides and copper exposure. Generation of metabolic signatures specific to different external stressors may provide valuable information to identify new intervention points and develop remediation strategies for infectious diseases and environment management in aquaculture. These studies also highlight the significant potential of metabolomics applications in studies of bivalve immunity that could be expanded to other aspects of marine science in the coming years.
[Reference:
Alfaro AC, Young T (2016) Showcasing metabolomic applications in aquaculture: a review Reviews in Aquaculture:n/a-n/a doi:10.1111/raq.12152
Young T, Alfaro AC (2016) Metabolomic strategies for aquaculture research: a primer Reviews in Aquaculture:n/a-n/a doi:10.1111/raq.12146]
Nanoparticles toxicity (1-100nm) to living organisms so far reported to affect different organismal levels, from whole organism, cellular to molecular levels. An integrative approach was followed in this thesis to investigate the role of nanoparticle size, exposure time and uptake routes in determining the immunotoxicity of silver nanoparticles to Mytilus galloprovincialis.
In so doing, in vivo exposures for 3, 6 and 12 hr to a single sublethal dose (100 µg/L) of two size of silver nanoparticles (AgNP: < 50 nm and < 100 nm) prior to and after inhibition of clathrin- and caveolae-mediated endocytosis uptake routes, were designed. Cytotoxicity assay and the hemocytic parameters used as markers to assess the sub-cellular responses. Histopathological analyses of inflammation in the gills and digestive gland were done at tissues level. Redox-based proteomic investigations conducted to assess the proteome status after exposure to AgNPs.
The results showed a significant cytotoxic effect on immune cells of mussels exposed to both sizes of AgNP for different timeframes (p < 0.01); the greater effect was with the smaller size. However, hemocytes seemed to be more sensitive to the larger size of AgNP after clathrin-mediated endocytosis was blocked (p < 0.01); this was not so with inhibition of caveolae-mediated endocytosis. The percentages of different hemocytes sub-populations showed to be varied after exposure to AgNP; these variations seemed to be decreased as exposure length progressed and noted to be delayed after blockade of either clathrin- or caveolae-mediated endocytosis (p < 0.01). Histological analyses showed that AgNP impacted on inflammation morphology and intensity dependently to exposure time, nanoparticle size (Highest response recorded with Ag-NPs <100nm alone) and uptake pathways (with Ag-NPs <50nm after blockade of uptake routes).
Overall, it was concluded that AgNP toxicity was size- as well as exposure time-dependent. It was proven that endocytotic routes were deeply involved in determining redox-based changes and immune response face to NP toxicity. Interestingly, NP uptake pathways found to be more impactful than the size. Lastly, a protective role of clathrin-mediated endocytosis route against NP toxicity was reported.
Nanomaterials have increasingly emerged as potential pollutants to aquatic organisms. Nanomaterials are known to be taken up by hemocytes of marine invertebrates including Mytilus galloprovincialis. Indeed, assessments of hemocyte-related parameters are a valuable tool in the determination of potentials for nanoparticle (NP) toxicity. The present study assessed the effects from two size-types of silver nanoparticles (AgNP: < 50 nm and < 100 nm) on the frequency of hemocytes sub-populations as immunomodulation biomarkers exposed in a mollusk host. Studies were performed using exposures prior to and after inhibition of potential NP uptake pathways (i.e., clathrin- and caveolae-mediated endocytosis) and over different durations of exposure (3, 6, and 12 hr). Differential hemocyte counts (DHC) revealed significant variations in frequency of different immune cells in mussels exposed for 3 hr to either AgNP size. However, as exposure duration progressed cell levels were subsequently differentially altered depending on particle size (i.e., no significant effects after 3 hr with larger AgNP). AgNP effects were also delayed/varied after blockade of either clathrin- or caveolae-mediated endocytosis. The results also noted significant negative correlations between changes in levels hyalinocytes and acidophils or in levels basophils and acidophils as a result of AgNP exposure. From these results, we concluded AgNP effects on mussels were size- as well as duration-of-exposure-dependent. This study highlighted how not only was NP size important, but that differing internalization mechanisms could be key factors impacting on the potential for NP in the environment to induce immunomodulation in a model/test sentinel host like M. galloprovincialis.
Coastal areas are complex environments frequently contaminated by numerous pollutants that represent a potential threat to marine organisms, especially bivalves. These pollutants may have major ecological consequences. Although effects of different environmental contaminants on the immune system in marine bivalves have been already reported, a few of reviews summarizes these effects. The main purpose of this chapter relies on summarizing recent body of data on immunotoxicity in bivalves subjected to contaminants. Immune effects of heavy metals, pesticides, HAP, PCB and pharmaceuticals are presented and discussed and a particular section is devoted to nanoparticle effects. A large body of literature is now available on this topic. Finally, the urgent need of a better understanding of complex interactions between contaminants, marine bivalves and infectious diseases is noticed.
Copyright © 2015. Published by Elsevier Ltd.
Oysters (Crassostrea virginica) and soft shell clams (Mya arenaria) were exposed to two different total copper concentrations (10 μg liter−1 and 30 μg liter−1) and two different cupric ion activities (10−10m and 10−9m) under high salinity and low salinity conditions. Cupric ion activities were adjusted by adding nitrilotriacetic acid (NTA) to the experimental media.Copper accumulation by both species was inversely related to salinity and positively associated with total copper and cupric ion activity. This salinity effect was proportionately greater for lower copper concentrations and activities and it was concluded that salinity would, therefore, be an important influence on metal accumulation in the natural environment. The salinity effect was still apparent when the cupric ion activity was held constant indicating that the effect is, at least in part, independent of the copper speciation in the medium.
American oysters, Crassostrea virginica, from a high-salinity (HS) and a low-salinity (LS) location in the Chesapeake Bay were acclimated to six salinities (6-36 ppt) in the laboratory for 3-4 weeks. After acclimation, hemolymph was drawn from oysters and granular hemocytes were tested in vitro. Measurements of time to hemocyte spreading (TTS) and rate of hemocyte locomotion (ROL) were made in six media ranging in salinity from 6-36 ppt. TTS measurements were fastest at the acclimation salinities and slowed with acute rises in salinity. The time to spreading may be a measure of the osmotic adjustment process. Locomotion was dependent on ameboid shape. ROL decreased with acute rises in salinity for both populations, and increased with acute reductions in salinity for all test conditions except HS hemocytes acclimated at 30 and 36 ppt salinity. ROL tested at the acclimation salinities showed no differences between HS oysters (complete acclimation to lower salinities) but LS oyster hemocytes at 30 and 36 ppt were still slower even after 27 days of acclimation. There were a greater number of agranular hemocytes for HS oysters at all salinities. These findings are discussed in relation to osmotic adjustment, ameboid locomotion, acclimation, and disease susceptibility.
Eight species of bivalve molluscs were exposed both to gradual and abrupt salinity fluctuations and the changes in free amino acids and ninhydrin-positive substances in their adductor muscles measured. In all the species there was an initial rise in the concentration of ninhydrin positive substances when exposed to decreasing salinities. After acclimation for one week (14 cycles) to a 30 % sea-water minimum sinusoidal salinity regime there was no difference in the concentration of ninhydrin positive substances at high and low salinities in the adductor muscles of Mytilus edulis L. Together, the changes in taurine and non-essential amino acids alanine, aspartic acid, glutamic acid, and glycine largely accounted for the changes in the free amino-acid pool. It was found that ‘shell-closing’ mechanisms may result in changes in the free amino-acid pool brought about by reductive amination of Krebs cycle and other keto-acids under anaerobic conditions. It is suggested that ninhydrin-positive substances and free amino acids are used as osmotic effectors in marine bivalves exposed to constantly lowered salinities, but are not used for the same purpose in animals exposed to cyclic salinity changes.
Numerous studies have demonstrated that environmental parameters affect bivalve immunomarkers. In the present study, we tested the hypothesis that clams (Venerupis philippinarum) collected in sites with different environmental conditions respond differently to experimental contaminant exposure. Clams were collected at two sites within the Lagoon of Venice that are influenced differently by both anthropogenic impact and natural conditions: Marghera, which is characterised by relatively high contamination levels and restricted clam fishing, and Chioggia, which is inside a licensed clam culture area that is characterised by lower contamination levels. Total haemocyte count, haemocyte diameter and volume, lysozyme activity in both haemocyte lysate and cell-free haemolymph, superoxide dismutase and catalase activities in gills and digestive glands were measured at time 0 (clam sampling time), after 7 days of acclimation in the laboratory and after 1, 3 and 7 days of copper exposure. Interestingly, statistical analyses (three-way ANOVA and Canonical Correlation Analysis) revealed persistent differences in the biological responses of clams from the two sampling sites before and after copper exposure. Conversely, the influence of copper on cellular and biochemical parameters was negligible. Overall, the results obtained indicated that animals with a different ecological history respond differently to experimental contaminant exposure. In addition, this study suggested that immunomarkers and other biomarkers might be used to determine the origin of fishing products.
Earlier studies demonstrate that blue mussels (Mytilus edulis) from the Baltic population are more sensitive, in terms of physiological response and survival when exposed to toxic substances,
compared to mussels from a more marine environment. The question whether this can be explained by environmental factors or
genetic differences in the ability to synthesise a common stress-inducible protein (HSP 70) was addressed in two experiments.
In the first experiment mussels from the North and Baltic Seas were acclimatised to an intermediate salinity of 15‰ S in the
laboratory. The physiological performance was studied when the heavy metal cadmium was added and accumulated by the animals
during a one week exposure. Tissue concentration was measured and related to physiological response. The level of expression
of HSP 70 was analysed by densitometry on Western blots. North Sea mussels rapidly induced high levels and multiple forms
of HSP 70, reached a comparatively low tissue concentration of cadmium, and showed only a minor physiological response and
low mortality rates. Baltic Sea mussels had low levels of HSP 70, induced at slow rate, reached higher tissue concentrations
of cadmium, and showed a more pronounced physiological response and higher mortality rates. High levels of stress proteins
and a rapid induction corresponded well with physiological fitness, and the mussels from the North Sea thus seem to have a
more efficient detoxification system, probably including stress-inducible proteins. In the second experiment, juvenile mussels
from the Baltic population were acclimatised for a month to North Sea conditions in a field transplant. The mussels were then
exposed to low-dose copper in the laboratory and the accumulation, physiological response and synthesis of stress inducible
proteins were assessed. The results indicate that the physiological differences described between the populations are to a
large extent explained by environmental factors. However, some differences can still be observed between the populations,
e.g., a lower rate of induction of a major stress protein in Baltic than in North Sea mussels. It can thus be suggested that
a reduced ability to stress protein induction, in their natural low saline habitat, might be a contributing factor to the
higher pollution sensitivity earlier demonstrated for Baltic blue mussels.
We review various aspects of the structure and functioning of Danish estuaries from data collected by the National Monitoring
Program and from information in published sources. We present data on the physical, chemical, and biological characteristics
of estuaries in Denmark, we evaluate the functioning of these systems as filters and transformers of nutrients and we evaluate
the outlook for Danish estuaries in the future. Danish estuarine systems are for the most part shallow (<3 m deep), have short
residence times, and tend to be heavily loaded with nutrients primarily from agricultural sources. Total average loads from
land per unit watershed area are 112 kg P km−2 yr−1 and 2,400 kg N km−2 yr−1 during the period 1989–1995. The total phosphorus (TP) load in estuaries has been significantly reduced over the last decade,
following implementation of the 1987 Action Plan for the Aquatic Environment (Vandmiljøplan in Danish) that prescribed that
nitrogen loads to the total aquatic environment should be reduced by 50% and phosphorus loads by 80%. Reductions in the total
nitrogen (TN) load have been more modest. Nutrient loading is one of the primary determinants of estuarine nutrient concentration
with 70% of the annual variation in TN concentration and 55% of the annual variation in TP concentration explained by variation
in the load. Many Danish estuaries have rich communities of macrophytes and benthic filter feeders, such asMytlis edulis andCiona intestinalis, that can control water column chlorophyll concentrations by their filter feeding activities. Many of the estuaries experience
hypoxia and anoxia, especially during warm and calm summer months. Further reductions in nutrient loading are expected following
implementation of the Action Plan for the Aquatic Environment II, with predicted improvements in oxygen concentrations and
in the functioning of these shallow, dynamic estuarine systems.
The Baltic Sea is a semi-enclosed sea with a steady salinity gradient (3‰–30‰). Organisms have adapted to such low salinities, but are suspected to be more susceptible to stress. Within the frame of the integrated environmental monitoring BONUS + project “BEAST” the applicability of immune responses of the blue mussel was investigated in Danish coastal waters. The sampling sites were characterised by a salinity range (11–19‰) and different mixtures of contaminants (metals, PAHs and POPs), according to chemical analysis of mussel tissues. Variation partitioning (redundancy analysis) was applied to decompose salinity and contamination effects. The results indicated that cellular immune responses (total and differential haemocyte count, phagocytic activity and apoptosis) were mainly influenced by contaminants, whereas humoral factors (haemolytic activity) were mainly impacted by salinity. Hence, cellular immune functions may be suitable as biomarkers in monitoring programmes for the Baltic Sea and other geographic regions with salinity variances of the studied range.
Programmed cell death leading to apoptosis is essential for normal development and homeostasis in plants and throughout the animal kingdom. Although there are differences in apoptotic mechanisms between lower animals and vertebrates, crucial biochemical components of the programmed cell death pathways remained remarkably conserved throughout evolution. Despite decades of studies on the neurobiology and development of mollusks, comparatively little is known about the mechanisms of apoptosis in this phylum. In this review, an attempt is made to summarize data obtained on mollusks so far, and to discuss the molecular mechanisms, the functional and ecological significance of apoptosis and the advantages of snail preparations as tools for programmed cell death research. A definitive comparison of the data obtained on mollusks with those obtained on the more widely studied vertebrates, will contribute to the better understanding of the apoptotic process in general and of its evolutionary development.
It is a well-known fact that the Baltic Sea blue mussels are very abundant but do not grow as large and fast as Mytilus edulis from the North Sea. This is often attributed to physiological differences due to the decline in salinities, although the mechanisms behind this are not yet fully understood.In order to elucidate this, the metabolism of mussels from the northern Baltic proper (Askö) and the North Sea (Tjärnö) was compared in the laboratory at ambient salinities of about 6.5 and 28.8 ‰, respectively, together with the magnitude of response to acute changes in the salinity.At 13°C and ambient salinities, respiration was higher for North Sea mussels in a non-reproductive stage, while after spawning respiration was higher for Baltic mussels.NH4-N excretion was always about two times higher for Baltic than for North Sea mussels at ambient salinities, and very sensitive to changes in salinity due to changes in the metabolism of free amino acids. While lowered salinities increased excretion, these decreased with raised salinities.The resulting O:N ratios (calculated from respiration and excretion) at ambient salinities were consistently lower for Baltic mussels, indicating that these have a less favourable energy metabolism, which may contribute to their smaller size.
The response of gill cell volume in Mytilus californianus and Mytilus trossolus (=edulis) to acute changes in salinity was assessed using three independent indicators: optical measurement of lateral cell height, measurement of intracellular water content using radiolabeled tracers and measurement of the contents of the major osmolytes of the gills. Optical measurements indicated significant variation in the response of individual lateral cells of M. californianus to acute low-salinity shock. Lateral cell height increased by approximately 20% shortly after abrupt exposure to 60% artificial sea water (ASW). Following this initial swelling, we estimate that a substantial regulatory volume decrease (RVD) was present in 25% of the trials. More commonly, however, an RVD was either absent or minimal: cell height remained elevated for at least 1 h, then returned to the control height when gills were re-exposed to 100% ASW. Changes in the combined water space of all cells in the gill, measured as the difference between total water space and extracellular space ([14C]polyethylene glycol space), indicated that cell volume regulation in the gill as an organ was also absent or minimal. Cell water space was 2.16 ml g-1 dry mass in isolated gills of M. californianus acclimated to 100% sea water in the laboratory and increased to 2.83 ml g-1 dry mass after a 6 min exposure to 60% ASW. Cell water space was still 2.81 ml g-1 dry mass after 1 h in 60% ASW and returned to 2.06 ml g-1 dry mass upon re-exposure to 100% ASW. Consistent with these observations, the gill contents of the principal cytoplasmic osmolytes (taurine, betaine and K+) were unchanged (approximately 450, 250 and 230 mu mol g-1 dry mass, respectively) following exposure of gills from 100% ASW-acclimated mussels to 60% ASW. A decrease in cell water space to 2.66 ml g-1 dry mass after 4 weeks of acclimation to 60% ASW corresponded with a 37% decrease in betaine content; taurine and K+ contents were unchanged. The changes in water space and solute content of gills from freshly collected M. californianus and M. trossolus were also consistent with the absence of volume regulation; cell water space remained elevated for at least 1 h after low-salinity exposure, and solute contents were unchanged after this period. We calculated the potential energetic cost of cell volume regulation for mussels exposed to 12 h of sinusoidal fluctuations between 100% and 50% sea water; solute uptake for full volume regulation in all tissues would cost a minimum of approximately 30% of the standard metabolic rate during the period of salinity increase. The routine absence of substantial cell volume regulation in Mytilus gill may reflect the potentially high energetic cost of volume regulation in the face of the large and frequent salinity fluctuations that are regularly encountered by estuarine bivalves.
Marginal populations are often isolated and under extreme selection pressures resulting in anomalous genetics. Consequently, ecosystems that are geographically and ecologically marginal might have a large share of genetically atypical populations, in need of particular concern in management of these ecosystems. To test this prediction, we analysed genetic data from 29 species inhabiting the low saline Baltic Sea, a geographically and ecologically marginal ecosystem. On average Baltic populations had lost genetic diversity compared to Atlantic populations: a pattern unrelated to dispersal capacity, generation time of species and taxonomic group of organism, but strongly related to type of genetic marker (mitochondrial DNA loci had lost c. 50% diversity, and nuclear loci 10%). Analyses of genetic isolation by geographic distance revealed clinal patterns of differentiation between Baltic and Atlantic regions. For a majority of species, clines were sigmoid with a sharp slope around the Baltic Sea entrance, indicating impeded gene flows between Baltic and Atlantic populations. Some species showed signs of allele frequencies being perturbed at the edge of their distribution inside the Baltic Sea. Despite the short geological history of the Baltic Sea (8000 years), populations inhabiting the Baltic have evolved substantially different from Atlantic populations, probably as a consequence of isolation and bottlenecks, as well as selection on adaptive traits. In addition, the Baltic Sea also acts a refuge for unique evolutionary lineages. This marginal ecosystem is thus vulnerable but also exceedingly valuable, housing unique genes, genotypes and populations that constitute an important genetic resource for management and conservation.
Aquatic invertebrates take up and accumulate trace metals whether essential or non-essential, all of which have the potential to cause toxic effects. Subsequent tissue and body concentrations of accumulated trace metals show enormous variability across metals and invertebrate taxa. Accumulated metal concentrations are interpreted in terms of different trace metal accumulation patterns, dividing accumulated metals into two components - metabolically available metal and stored detoxified metal. Examples of different accumulation patterns are described from crustaceans but have a general applicability to all aquatic invertebrates. Toxicity does not depend on total accumulated metal concentration but is related to a threshold concentration of internal metabolically available metal. Toxicity ensues when the rate of metal uptake from all sources exceeds the combined rates of detoxification and excretion (if present) of the metal concerned. The biodynamic model of trace metal bioaccumulation allows the prediction and explanation of widely differing accumulated trace metal concentrations in organisms, combining geochemical analyses of environmental metal concentrations with the measurement of key physiological parameters for a species from the site under consideration. The combination of the biodynamic model as a unified explanation of metal bioaccumulation with an understanding of the relationship between accumulation and toxicity sets the stage for a realistic understanding of the significance of trace metal concentrations in aquatic invertebrates.
The present text provides a review of waterborne as well as dietary copper (Cu) toxicity and Cu homeostasis in fish, and leads to suggestions for further research in this area. Copper, although essential for life, is a potent toxicant and as such, delicate homeostatic controls have evolved at the organismal and cellular level. During exposure to elevated levels of Cu in the water or the diet, homeostatic systems may become overwhelmed such that cellular Cu levels increase to a point where protein function becomes impaired. A range of potential cellular targets for Cu manifest in altered physiology and toxicity, at the organ and organismal level during Cu exposure; these targets and organismal responses are discussed. Copper toxicity is not simply a matter of ambient concentrations as water chemistry greatly influences not only the bioavailability of this metal but also the physiology and thus susceptibility of fish. This complexity has recently been realized and is being considered in current environmental regulations for Cu. Prolonged exposure to Cu elicits an acclimation response which includes a compensatory response of the functions impaired by Cu and adjustments in the homeostatic control of the metal, enabling fish to survive despite continued exposure and to tolerate subsequent exposures to higher concentrations. Although not quantified, these acclimation responses must occur at a cost to the organism, possibly explaining observations of reduced growth, reproductive output, and swimming performance. The literature review forming the basis for this chapter was completed by December 2010.
The rates of oxygen consumption, filtration and ammonia excretion by Mytilus californianus have been related to body size and to ration. The rate of oxygen consumption (VO2) by individuals while immersed, measured on the shore, resembled rates recorded for mussels starved in the laboratory. VO2 by M. californianus was relatively independent of change in temperature, with a Q 10 (13–22° C) of 1.20. In contrast, the frequency of heart beat was more completely temperature dependent [Q 10 (13–22° C)=2.10]. Filtration rate showed intermediate dependence on temperature change [Q 10 (13–22° C)=1.49] up to 22° C, but declined at 26° C. Both VO2 and filtration rate declined during starvation. The utilisation efficiency for oxygen was low (approx. 4%) between 13 and 22° C, but increased to 10% at 26° C. Three components of the “routine” rate of oxygen consumption are recognised and estimated; viz. a basal rate (0.136 ml O2 h-1 for a mussel of 1 g dry flesh weight), a “physiological cost” of feeding (which represented about 6% of the calories in the ingested ration), and a “mechanical cost” of feeding which was three times higher than the physiological cost. The ratio oxygen consumed to ammonia-nitrogen excreted was low, and it declined during starvation. These data are compared with previously published measurements on Mytilus edulis, and the two species of mussel are shown to be similar in some of their physiological characteristics, though possibly differing in their capacities to compensate for change in temperature. For M. californianus, the scope for growth was highest at 17–22° C and declined at 26° C; it is suggested that exposure to temperatures in excess of 22° C, as for example during low tides in the summer, might result in a cumulative stress on these populations of mussels by imposing a metabolic deficit which must be recovered at each subsequent high tide. The high “mechanical cost” of feeding imposes a more general constraint on the scope for activity of the species.
Mytilus californianus regulated its rate of oxygen consumption (VO2) during decline in oxygen tension, but did not acclimate VO2 when held at 58 mm Hg PO2. In spite of a capacity to consume oxygen while exposed to air (the average VO2 in air being equivalent to 0.74xthe standard rate of oxygen consumption in water), these mussels acquired an oxygen debt during aerial exposure which was discharged on subsequent reimmersion. During exposure to air the oxygen tension of the fluid in the mantle cavity was rapdly reduced to approximately 40 mm Hg, in animals both in the laboratory and on the shore. Heart rate was also reduced during air exposure, though not to the point of cardiac arrest. The concentration of ammonia in the fluid of the mantle cavity increased during aerial exposure, but the rate of excretion of ammonia was much lower than during immersion. Observations of mussels on the shore at low tide indicated that dehydration of the tissues was not a serious threat, possibly due to the large volume of fluid retained in the mantle cavity. During aerial exposure some end-products of anaerobic metabolism (alanine and malate) accumulated in the posterior adductor muscle. Malate accumulation was rapid during the first hour of exposure; alanine accumulated more gradually. It is concluded that during aerial exposure M. californianus resorts to anaerobiosis in spite of a capacity to extract some oxygen from the atmosphere. This results in a metabolic deficit during each period of low tide which, coupled with the reduced time available for feeding, imposes a physiological stress on mussels distributed on the shore.
The survival of humans as a species is intimately linked to the well-being of ecosystems and the resources they provide. The well-being of ecosystems depends, in turn, on minimizing the damaging impacts of human activities. Irrespective of the kinds of habitats we choose to protect or restore, we need to understand how ecosystems, and the organisms that inhabit them, respond to chemicals and radiation exposure. Recent technological advances in molecular biology and analytical science have allowed the development of rapid, robust, and sensitive diagnostic tests (biomarkers) that can be used to monitor exposure to, and the effects of, pollution. For the first time, we are able to make health assessments of individual organisms in much the same way that we evaluate human health. Here, we discuss how to best use this capability to ensure that pollution does not threaten the sustainability of our ecosystems and cause adverse effects in human and wildlife populations.
In vitroassays were performed to assess the phagocytic ability of mussel haemolymph cell types. Among the haemocyte types, the granulocytes showed an important phagocytic activity whereas the hyalinocytes did not, thus indicating that mussel haemocytes are functionally heterogeneous. Phagocytic activity of acidophilic granulocytes was higher than basophilic ones. Ultrastructural study showed thatVibrioP1 cells were internalised in phagosomes and then digested. As a result of the digestive process, concentric lamellae and other degraded materials were produced inside the phagosomes, and non degraded materials gave rise to residual bodies which could be discharged from the cells. Similar events were observed in the phagocytic process of zymosan particles. The study of influence of temperature on phagocytosis activity revealed that the percentage of phagocytic cells was lower at 10°C than at 20°C and 30°C. Phagocytosis activity was higher in the assays performed at 15ppt than those at 25ppt and 36ppt. No significant differences on percentage of phagocytic cells were found between young and adult mussels. Generation of superoxide anion by haemocytes using zymosan as a phagocytosis stimulant was demonstrated through the nitroblue tetrazolium reduction test.
Regulation of cell volume utilizing intracellular free amino acids has been studied in isolated ventricles from marine bivalves of the genus Modiolus. As in the intact animal, ventricles taken from Modiolus acclimated to various salinities show only a slight change in tissue hydration. This control over cell volume is accomplished by isosmotic intracellular regulation of taurine, alanine, glycine and proline concentrations. When stressed with decreased external salinities the isolated spontaneously beating ventricle becomes quiescent for a period, and then resumes activity. During the period of quiescence ninhydrin-positive substances (NPS) are released. The duration of quiscence and the amount of NPS released increase with increasing dilution of the external medium. The salinity-induced NPS efflux is composed of taurine, alanine, glycine and proline. In molluscs, the amino acids utilized for volume regulation are released from the cells unchanged and are not degraded into keto-acids and ammonia as they are in the crustaceans.
To resolve the sources of amino nitrogen which is accumulated as intracellular solute during hyperosmotic volume regulation, components of protein metabolism were monitored during compensation for a change from 15 to 30% salinity in the blue mussel, Mytilus edulis L. Net solute gain stemmed primarily from a marked reduction in total output from the metabolic pool of free amino acids, most of this ‘saving’ resulting from slower whole-body protein synthesis, and the remainder from lower nitrogenous excretion. Indeed, total inputs to the metabolic pool of free amino acids actually decreased over the period of net solute gain at 30%. Associated contributions from dietary assimilation, de novo synthesis and the direct uptake of dissolved amino acids were each negligible, indicating that breakdown products from endogenous body protein were the only significant source of amino nitrogen accumulated during hyperosmotic regulation. This accumulation represented more than 3% of soft-tissue protein nitrogen within the whole animal. We therefore impress that the excretion, during hyposaline adjustment, of all nitrogen previously accumulated as solute in response to equivalent hypersaline change, represents a major component cost of cell volume regulation, and which helps to explain stress and even mortality consequent upon what may be small but frequent fluctuations of salinity.
Although the nitrogenous excretory products of a number of marine bivalve molluscs have been identified (Hammen, Miller & Geer, 1966; Hammen, 1968; Emerson, 1969; Allen & Garrett, 1971; Bayne, 1973 a) and average values for rates of nitrogen excretion recorded for some species (Lum & Hammen, 1964; Hammen et al. 1966; Bayne, 1973a), there is little information available on the ways in which environmental factors may affect either the balance between the various nitrogenous end-products, or their rates of excretion. The most thorough investigations have dealt with the effects of reduced salinity, which causes an increase in the rates of excretion of ammonia by Macoma inconspicua (Emerson, 1969), Mya arenaria (Allen & Garrett, 1971) and Mytilus edulis (Bayne, 1975). Dilution of the medium is also known to accelerate the loss of amino acids from the body, as part of the mechanism of volume regulation (Pierce & Greenberg, 1972, 1973). There is some evidence of altered rates of excretion during starvation (Hammen, 1968; Emerson, 1969; Bayne, 1973 a, b) and differences in excretion rate due to differences in animal size have been documented for Donax vittatus by Ansell & Sivadas (1973) and for Mytilus californianus by Bayne, Bayne, Carefoot & Thompson (1976 a, b).
An acute toxicity threshold of 0·09–0·10 ppm added copper was determined for specimens of Mytilus edulis from the Menai Strait which were exposed to copper (as CuSO 4 ) in a flowing sea-water system.
Results are presented which show that the closure response of the mussel to added copper is a three-part process. First, a sharp adduction of the shell valves is seen at a mean total copper concentration of only 0·021 ppm, then as the copper concentration rises, ‘testing’ behaviour is observed and finally the shell valves close to isolate the animal from its environment. The complete valve closure mechanism only operates at added copper concentrations of 0·2 ppm or more. The initial behavioural reaction of valve adduction at low concentration occurred at a significantly higher mean total copper concentration (0·16 ppm) in mussels which had been previously acclimated to 0·02 ppm total copper in sea water for 10 days.
The accumulation and physico-chemical forms of metals were determined by atomic absorption spectrometry in the whole soft tissues of oysters, by histological and microanalytical techniques in tissue sections, by gel permeation chromatography of soft tissue homogenates. Oysters were reared according to four types of experimental conditions: exposed to silver (20 g Ag/l) or unexposed, in sea water (33) or brackish water (8). Copper, zinc and silver accumulation in oysters are inversely related to salinity. Amoebocytes, which play a key role in accumulating copper and zinc in natural sea water, are able to sequester an important part of added silver as Ag2S. In brackish water an increase of the number of amoebocytes may be considered as responsible for the enhancement of Cu and Zn concentrations in the whole soft tissues. In such conditions, additional silver is concentrated in these cells rather than in the basement membranes which are target structures for Ag2S accumulation in sea water. If the global fluctuations of metal concentrations in the soft tissues are ascribable mainly to changes at the histological level, the fate of metals in the soluble fraction must not be neglected since the speciation of metals influences their toxicity. The freshening of sea water induced a change in the distribution of cytosolic silver and zinc but in no case were the molecular masses of compounds associated with each metal perfectly identical. From these results, it is concluded that the accumulation of silver by oysters is not mediated by the same mechanisms as those for copper and zinc.
Patterns of nine intracellular free amino acids (FAA), which are utilized as organic osmolytes for salinity-induced cell volume regulation in marine osmoconformers, were compared in five Macoma balthica populations and seven Mytilus spp. populations along their European distribution. Three types of FAA patterns were classified within both taxa: a northern Baltic type, a southern Baltic type and an Atlantic/Mediterranean type which mainly differ regarding the share of alanine and taurine. Differences are discussed in relation to habitat salinity and population genetics. Along a salinity gradient, the total size of the intracellular FAA pool did not differ between sympatric M. balthica and Mytilus spp., and was significantly correlated with habitat osmolality in a range from 70 to 600mmolkg−1H2O (oligohaline to mesohaline) in both bivalves. In M. balthica, this correlation was mainly based on significant correlations of alanine (15–100mmolkg−1DW), glycine (30–100mmolkg−1DW) and taurine (0–70mmolkg−1DW) with habitat osmolality. In Mytilus spp., only glycine (25–100mmolkg−1DW) and taurine (4–180mmolkg−1DW) were significantly correlated with habitat osmolality. The concentration of alanine was three times lower in Mytilus spp. than in M. balthica and did not correlate with habitat osmolality. Within a habitat osmolality range from 600 to 1,100mmolkg−1H2O (mesohaline to marine) the concentration of FAA remained constant in both taxa. It is suggested that under marine conditions additional organic osmolytes must become more important for cell volume regulation in Macoma and Mytilus.
After the widespread ban of TBT, due to its severe impact on coastal biocoenoses, mainly related to its immunosuppressive effects on both invertebrates and vertebrates, alternative biocides such as Cu(I) salts and the triazine Irgarol 1051, the latter previously used in agriculture as a herbicide, have been massively introduced in combined formulations for antifouling paints against a wide spectrum of fouling organisms. Using short-term (60 min) haemocyte cultures of the colonial ascidian Botryllus schlosseri exposed to various sublethal concentrations of copper(I) chloride (LC(50)=281 μM, i.e., 17.8 mg Cu L(-1)) and Irgarol 1051 (LC(50)>500 μM, i.e., >127 mg L(-1)), we evaluated their immunotoxic effects through a series of cytochemical assays previously used for organotin compounds. Both compounds can induce dose-dependent immunosuppression, acting on different cellular targets and altering many activities of immunocytes but, unlike TBT, did not have significant effects on cell morphology. Generally, Cu(I) appeared to be more toxic than Irgarol 1051: it significantly (p<0.05) inhibited yeast phagocytosis at 0.1 μM (∼10 μg L(-1)), and affected calcium homeostasis and mitochondrial cytochrome-c oxidase activity at 0.01 μM (∼1 μg L(-1)). Both substances were able to change membrane permeability, induce apoptosis from concentrations of 0.1 μM (∼10 μg L(-1)) and 200 μM (∼50 mg L(-1)) for Cu(I) and Irgarol 1051, respectively, and alter the activity of hydrolases. Both Cu(I) and Irgarol 1051 inhibited the activity of phenoloxidase, but did not show any interactive effect when co-present in the exposure medium, suggesting different mechanisms of action.
The imminent ban of environmentally harmful tributyltin (TBT)-based paint products has been the cause of a major change in the antifouling paint industry. In the past decade, several tin-free products have reached the commercial market, and claimed their effectiveness as regards the prevention of marine biofouling on ships in an environmentally friendly manner. The main objective of this review is to describe these products in as much detail as possible based on the knowledge available in the open literature. This knowledge has been supplemented by means of performance data provided, upon request, by some of the paint-producing companies. An exhaustive review of the historical development of antifouling systems and a detailed characterisation of sea water are also included. The need for studies on the behaviour of chemically active paints under different sea water conditions is emphasised. In addition, the most common booster biocides used to replace TBT-containing compounds are listed and described. It must be stressed that there is still a lack of knowledge of their potential environmental side effects.
Results from a monitoring programme conducted in the Bay of Arcachon between 1979 and 1991 reveal an increase of copper content in oysters harvested in a zone located in the vicinity of marinas and mooring areas. This clearly perceptible increase recorded since 1982, may be attributable to the growing use of copper paints subsequent to the antifouling paint regulations adopted in 1982.
The increased observation of pollution induced disease conditions in marine organisms has led to a growing interest on the effects of environmental contaminants on the immune system. Most studies on modulation of the immune system in bivalves by pollutants have concentrated on the effects of heavy metals and polycyclic aromatic hydrocarbons (PAHs). The current literature on contaminant effects on specific components of the bivalve immune system is reviewed together with the effects on susceptibility to
infection. Data are presented showing the effects on immune parameters of exposure to Vibrio tubiashi following pre-exposure to copper or cadmium. Mussels exposed to cadmium for 7 days followed by 7 days exposure to V. tubiashi demonstrated significantly higher numbers of circulating haemocytes compared with non-Vibrio-exposed groups. Similar experiments conducted with copper exposure for both 7 days and 7 weeks followed by V. tubiashi for 7 days demonstrated a significant decrease in the percentage of circulating eosinophils compared with basophilic cells for both short and long term exposures. The intracellular release of superoxide (NBT reduction) by haemocytes was stimulated in Vibrio-challenged mussels with no copper pre-exposure but was significantly reduced in mussels pre-exposed to 0"2 ppm
of copper for 7 weeks. The mortalities for the copper experiments showed increased levels with increasing copper concentration and were consistently higher in the V. tubiashi challenged mussels which had also been exposed to copper.
An effort is ongoing to develop a biotic ligand model (BLM) that predicts copper (Cu) toxicity in estuarine and marine environments. At present, the BLM accounts for the effects of water chemistry on Cu speciation, but it does not consider the influence of water chemistry on the physiology of the organisms. We discuss how chemistry affects Cu toxicity not only by controlling its speciation, but also by affecting the osmoregulatory physiology of the organism, which varies according to salinity. In an attempt to understand the mechanisms of Cu toxicity and predict its impacts, we explore the hypothesis that the common factor linking the main toxic effects of Cu is the enzyme carbonic anhydrase (CA), because it is a Cu target with multiple functions and salinity-dependent expression and activity. According to this hypothesis, the site of action of Cu in marine fish may be not only the gill, but also the intestine, because in this tissue CA plays an important role in ion transport and water adsorption. Therefore, the BLM of Cu toxicity to marine fish should also consider the intestine as a biotic ligand. Finally, we underline the need to incorporate the osmotic gradient into the BLM calculations to account for the influence of physiology on Cu toxicity.
We report a quantitative structure-activity relationship study of a new class of pyrazole-pyridine copper complexes that establishes a clear correlation between the ability to promote copper accumulation and cytotoxicity. Intracellular metal accumulation is maximized when ligand lipophilicity allows the complex to rapidly cross the membrane. Copper and ligand follow different uptake kinetics and reach different intracellular equilibrium concentrations. These results support a model in which the ligand acts as an ionophore for the metal ion, cycling between intra- and extracellular compartments as dissociated or complexed entities. When treating cancer cells with structurally unrelated disulfiram and pyrazole-pyridine copper complexes, as well as with inorganic copper, the same morphological and molecular changes were reproduced, indicating that copper overload is responsible for the cytotoxic effects. Copper-based treatments drive sensitive cancer cells toward paraptotic cell death, a process hallmarked by endoplasmic reticulum stress and massive vacuolization in the absence of apoptotic features. A lack of caspase activation, as observed in copper-treated dying cells, is a consequence of metal-mediated inhibition of caspase-3. Thus, copper acts simultaneously as an endoplasmic reticulum (ER) stress inducer and a caspase-3 inhibitor, forcing the cell into caspase-independent paraptotic death. The establishment of a mechanism of action common to different copper binding agents provides a rationale for the exploitation of copper toxicity as an anticancer tool.
Copper is an important ionoregulatory toxicant in freshwater, but its effects in marine and brackish water systems are less well characterised. The effect of salinity on short-term copper accumulation and sublethal toxicity in two estuarine animals was investigated. The osmoregulating crab Hemigrapsus crenulatus accumulated copper in a concentration-dependent, but salinity-independent manner. Branchial copper accumulation correlated positively with branchial sodium accumulation. Sublethal effects of copper were most prevalent in 125% seawater, with a significant increase in haemolymph chloride noted after 96h at exposure levels of 510 microg Cu(II) L(-1). The osmoconforming gastropod, Scutus breviculus, was highly sensitive to copper exposure, a characteristic recognised previously in related species. Toxicity, as determined by a behavioural index, was present at all salinities and was positively correlated with branchial copper accumulation. At 100% seawater, increased branchial sodium accumulation, decreased haemolymph chloride and decreased haemolymph osmolarity were observed after 48h exposure to 221 microg Cu(II) L(-1), suggesting a mechanism of toxicity related to ionoregulation. However, these effects were likely secondary to a general effect on gill barrier function, and possibly mediated by mucus secretion. Significant impacts of copper on haemocyanin were also noted in both animals, highlighting a potentially novel mechanism of copper toxicity to animals utilising this respiratory pigment. Overall these findings indicate that physiology, as opposed to water chemistry, exerts the greatest influence over copper toxicity. An understanding of the physiological limits of marine and estuarine organisms may be critical for calibration of predictive models of metal toxicity in waters of high and fluctuating salinities.
The toxic effects of Aroclor 1254 (0.05, 0.5, 5 and 50 microg l(-1)) on scallop (Chlamys farreri) immune system in vivo were studied. The results showed that Aroclor 1254 had significant toxic effect on the parameters tested in this paper (P<0.05). The total number of haemocytes, the proportion of granulocytes, phagocytosis in all groups as well as the lysosomal membrane stability (LMS) in 5, 50 microg l(-1) and bacteriolytic activity 0.5, 5, 50 microg l(-1) treatments decreased significantly, while the proportion of hyalinocytes and the production of O2(-) in all treatments remarkably increased during the sampling time and tended to be stable gradually after 6-15 d. The bacteriolytic activity in 0.05 microg l(-1) treatments, LMS in 0.05, 0.5 microg l(-1) groups and the DNA damage (comet ratios and arbitrary values) in all treatments increased at the beginning of exposure and reached their peaks on day 1, day 1, day 6 and day 3, following that they all decreased gradually and became stable after 9-15 d. When the indices reached stability, except for DNA damage was higher than controls, the others were all significantly lower than those of controls (P<0.05). Thus, Aroclor 1254 has evident toxic effects on scallop immune system, which supports the view that a relationship exists between pollution and immunomodulation in aquatic organisms. Also it supports the speculation that the PCBs pollution is one of the important reasons of the mass mortality of the C. farreri.
The accumulation and physico-chemical forms of metals were determined by atomic absorption spectrometry in the whole soft tissues of oysters, by histological and microanalytical techniques in tissue sections, by gel permeation chromatography of soft tissue homogenates. Oysters were reared according to four types of experimental conditions: exposed to silver (20 micrograms Ag/l) or unexposed, in sea water (33%) or brackish water (8%). Copper, zinc and silver accumulation in oysters are inversely related to salinity. Amoebocytes, which play a key role in accumulating copper and zinc in natural sea water, are able to sequester an important part of added silver as Ag2S. In brackish water an increase of the number of amoebocytes may be considered as responsible for the enhancement of Cu and Zn concentrations in the whole soft tissues. In such conditions, additional silver is concentrated in these cells rather than in the basement membranes which are target structures for Ag2S accumulation in sea water. If the global fluctuations of metal concentrations in the soft tissue are ascribable mainly to changes at the histological level, the fate of metals in the soluble fraction must not be neglected since the speciation of metals influences their toxicity. The freshening of sea water induced a change in the distribution of cytosolic silver and zinc but in no case were the molecular masses of compounds associated with each metal perfectly identical. From these results, it is concluded that the accumulation of silver by oysters is not mediated by the same mechanisms as those for copper and zinc.
A variety of organisms and cell types spanning the five taxonomic kingdoms are exposed, either naturally or through experimental means, to osmotic stresses. A common physiological response to these challenges is maintenance of cell volume through changes in the concentration of intracellular inorganic and organic solutes, collectively termed osmolytes. Research on the mechanisms by which the concentration of these solutes is regulated has proceeded along several experimental lines. Extensive studies on osmotically activated ion transport pathways have been carried out in vertebrate cells and tissues. Much of our knowledge on organic osmolytes has come from investigations on invertebrates, bacteria, and protists. The relative simplicity of bacterial genetics has provided a powerful and elegant tool to explore the modifications of gene expression during volume regulation. An implication of this diverse experimental approach is that phylogenetically divergent organisms employ uniquely adapted mechanisms of cell volume regulation. Given the probability that changes in extracellular osmolality were physiological stresses faced by the earliest organisms, it is more likely that cell volume regulation proceeds by highly conserved physiological processes. We review volume regulation from a comparative perspective, drawing examples from all five taxonomic kingdoms. Specifically, we discuss the role of inorganic and organic solutes in volume maintenance and the mechanisms by which the concentrations of these osmolytes are regulated. In addition, the processes that may transduce volume perturbations into regulatory responses, such as stretch activation of ion channels, intracellular signaling, and genomic regulation, are discussed. Throughout this review we emphasize areas we feel are important for future research.
A number of environmental contaminants can suppress immune responses and enhance susceptibility to infectious and/or neoplastic disease. Most of the evidence for immunotoxicity of such contaminants has been obtained from laboratory animal studies and risk assessors must make decisions about risk to the human population based on these studies. Uncertainties associated with this process include determining what level of immune suppression is adverse, extrapolating across species from rodent to human, and across levels of biologic organization from effects on immune function at the cellular level to effects on incidence of disease at the population level, accounting for intra-species variability, and assessing the relationship between effects following acute, subchronic, and chronic exposure. This paper reviews immunotoxicity data that may be applied to the development of risk assessment methods and models designed to reduce some of these uncertainties.
Concern is growing regarding the impact of chemicals suspected of altering the function of the immune system in humans and wildlife. There are numerous examples of links between pollution and increased susceptibility to disease in wildlife species, including immunosuppression in harbour seals feeding on fish from contaminated sites, altered immune function in riverine fish and decreased host resistance in birds exposed to pollutants. Laboratory tests have identified potential immunological hazards posed by a range of anthropogenic chemicals in mammals and higher vertebrates. However, few reports have considered the ecological relevance of pollution-induced immunosuppression in invertebrate phyla, which constitute around 95% of all animal species and occupy key structural and functional roles in ecosystems. In this paper effects of chemicals on immune function in invertebrates are briefly reviewed and biomarkers of immunotoxicity are identified. Examples of new approaches for the measurement of immunological inflammatory reactions and stress in molluscan haemocytes are detailed. The relevance of defining the immune system as a target organ of toxicity in invertebrates is discussed and an integrated approach for the use of immunological biomarkers in environment management is proposed, combining measures of immune function and organismal viability at the biochemical, cellular and population level.
Criteria for scoring micronuclei and nucleoplasmic bridges in binucleated cells in the cytokinesis-block micronucleus assay for isolated human lymphocyte cultures are described in detail. Morphological characteristics of mononucleated cells, binucleated cells, and multinucleated cells as well as necrotic and apoptotic cells and nuclear buds are also described. These criteria are illustrated by a series of schematic diagrams as well as a comprehensive set of colour photographs that are of practical assistance during the scoring of slides. These scoring criteria, diagrams and photographs have been used in a HUman MicronNucleus (HUMN) project inter-laboratory slide-scoring exercise to evaluate the extent of variability that can be attributable to individual scorers and individual laboratories when measuring the frequency of micronuclei and nucleoplasmic bridges in binucleated cells as well as the nuclear division index. The results of the latter study are described in an accompanying paper. It is expected that these scoring criteria will assist in the development of a procedure for calibrating scorers and laboratories so that results from different laboratories for the cytokinesis-block micronucleus assay may be more comparable in the future.
The interactive effects of temperature and copper on immune function and consequently disease susceptibility of the marine mussel, Mytilus edulis were investigated. Two studies were carried out, the first involved sequential exposure to copper at 0.02 and 0.05 ppm followed by the bacterium Vibrio tubiashii. In the second study, mussels were simultaneously exposed to copper and V. tubiashii. Both studies were carried out at 10 and 15 degrees C, to ascertain whether temperature had an additional effect on immunocompetence. A multi-assay approach was used to obtain an overall view of immune function in the mussels. Assays carried out included total and differential haemocyte counts, production of intracellular superoxide and phagocytosis by haemocytes. Data are presented showing significant effects on immune parameters of sequential and simultaneous exposure to copper and V. tubiashii at 10 and 15 degrees C. Each of the factors considered were shown to have a significant effect on at least one of the immune parameters measured. There were also significant effects due to the interaction of these factors. The response of total and differential blood cell counts to copper were shown to alter, if mussels were exposed in a sequential manner as opposed to simultaneous exposure. The results confirmed that the immune system of M. edulis is susceptible to copper at relatively low concentrations. Furthermore, the effects of copper alter with environmental variables, including temperature and the presence of a potential pathogen. The complexity of the interactions demonstrate that extrapolation of data obtained from single stressor studies into field situations could give a misleading picture.
All cells face constant challenges to their volume either through changes in intracellular solute content or extracellular osmolality. Cells respond to volume perturbations by activating membrane transport and/or metabolic processes that result in net solute loss or gain and return of cell volume to its normal resting state. This paper provides a brief overview of fundamental concepts of osmotic water flow across cell membranes, mechanisms of cell volume perturbation, the role of inorganic ions and organic osmolytes in cell volume regulation and the signaling mechanisms that regulate the activity of cell volume-sensitive transport and metabolic pathways.
Organic osmolytes are small solutes used by cells of numerous water-stressed organisms and tissues to maintain cell volume. Similar compounds are accumulated by some organisms in anhydrobiotic, thermal and possibly pressure stresses. These solutes are amino acids and derivatives, polyols and sugars, methylamines, methylsulfonium compounds and urea. Except for urea, they are often called ;compatible solutes', a term indicating lack of perturbing effects on cellular macromolecules and implying interchangeability. However, these features may not always exist, for three reasons. First, some of these solutes may have unique protective metabolic roles, such as acting as antioxidants (e.g. polyols, taurine, hypotaurine), providing redox balance (e.g. glycerol) and detoxifying sulfide (hypotaurine in animals at hydrothermal vents and seeps). Second, some of these solutes stabilize macromolecules and counteract perturbants in non-interchangeable ways. Methylamines [e.g. trimethylamine N-oxide (TMAO)] can enhance protein folding and ligand binding and counteract perturbations by urea (e.g. in elasmobranchs and mammalian kidney), inorganic ions, and hydrostatic pressure in deep-sea animals. Trehalose and proline in overwintering insects stabilize membranes at subzero temperatures. Trehalose in insects and yeast, and anionic polyols in microorganisms around hydrothermal vents, can protect proteins from denaturation by high temperatures. Third, stabilizing solutes appear to be used in nature only to counteract perturbants of macromolecules, perhaps because stabilization is detrimental in the absence of perturbation. Some of these solutes have applications in biotechnology, agriculture and medicine, including in vitro rescue of the misfolded protein of cystic fibrosis. However, caution is warranted if high levels cause overstabilization of proteins.
Two waterborne Cu exposures were performed to investigate if Cu is an ionoregulatory toxicant at all salinities in the killifish, Fundulus heteroclitus. A 30-day flow through exposure in 0 (FW), 5, 11, 22, and 28 ppt (SW) and three [Cu]'s (nominal 0, 30, and 150 microg Cu L(-1)) revealed no apparent Cu induced mortality at the intermediate salinities and high mortality in FW and SW. Fish were sampled at 4, 12, and 30 days after the start of the exposure and both Na+/K+ adenosine triphosphatase (Na+/K+ ATPase) and carbonic anhydrase (CA) activity in the gill and intestine as well as whole body [Na+], and [Cl-] were measured. At the high [Cu] a reduction of whole body [Na+] after 4 days of exposure in FW was the only physiological parameter influenced. A second static 24h Cu exposure was performed in FW, 5, 13, and 29 ppt (SW) and two [Cu]'s (nominal 0 and 110 microg Cu L(-1)). In addition to the parameters listed above, ammonia flux was measured at all salinities and Na+ flux was measured in FW fish. Cu affected ionoregulation in FW where decreased Na+ uptake associated with inhibition of Na+/K+ ATPase led to decreased whole body [Na+] after 24h. The only affected parameter in SW was net ammonia excretion suggesting that Cu is not an ionoregulatory toxicant in SW at the concentrations employed. We propose that physiology rather than chemistry explain much of the variation in Cu toxicity seen across salinities.
The present study investigates the effects on immune-related parameters of various stress factors (air exposure, mechanical stress, high temperature and extreme salinity conditions) faced by the bivalve mollusc Mytilus galloprovincialis during marketing procedures. We observed that some stress typologies increase phagocytosis and the number of circulating immunocytes, while others can modify immunocyte response towards a further perturbation, i.e. the marine algal toxin yessotoxin. Our results suggest that non-lethal stress can be counteracted for sometime by increasing the level of some defence parameters. Moreover, our data indicate that fishing and transport procedures could interfere with mussel immunosurveillance.
In this study, three major hemocyte types were identified in the Sydney rock oyster. They were characterized primarily by light and electron microscopy based on the presence or absence of granules and nucleus to cytoplasm ratios. Hemoblast-like cells were the smallest cell type 4.0+/-0.4microm and comprised 15+/-3% of the hemocyte population. They had large nuclei and scanty basic cytoplasm. This cell type also had some endoplasmic reticuli and mitochondria. The second major type were hyalinocytes. Hyalinocytes represented 46+/-6% of all hemocytes. They were large cells (7.1+/-1.0microm) that had low nucleus:cytoplasm ratios and agranular basic or acidic cytoplasm. Hyalinocytes had the ability to phagocytose yeast cells and formed the core of hemocyte aggregates associated with agglutination. Four discrete sub-populations of hyalinocytes were identified. The third major cell type were the granulocytes, comprising 38+/-1% of the hemocyte population. These cells were large (9.3+/-0.3microm) and were characterized by cytoplasm containing many acidic or basic granules. Granulocytes were more phagocytic than hyalinocytes and they formed the inner layer of hemocytes during the encapsulation of fungal hyphae. Five discrete sub-populations of granulocytes were identified based on the types of granules in their cytoplasm. Flow cytometry showed that the hemocytes of rock oysters could be divided into between two and four major cell types based on their light scattering properties. The most common of the cell types identified by flow cytometry corresponded to hyalinocytes and granulocytes. Cytochemical assays showed that most enzymes associated with immunological activity were localized in granulocytes. Their granules contained acid phosphatase, peroxidase, phenoloxidase, superoxide and melanin. Hyalinocytes were positive only for acid phosphatase. All of these observations suggest that Sydney rock oysters have a broad variety of functionally specialized hemocytes, many of which are involved in host defense.
The use of copper in antifouling paints has increased in the UK in the last 20 years as TBT and several other organic biocides have been phased out. To assess the probable impact of copper on estuarine systems a survey was undertaken to measure the different fractions of copper present in the water column at current usage. The different fractions measured were; labile copper, (LCu) considered as both the free copper ions and inorganically bound copper, the total dissolved copper (TDCu) present, and the difference between them taken as the organically bound likely non-toxic copper fraction. The survey considered sites with different levels of boat use, namely marinas, harbours and estuaries, differing physical parameters of suspended and dissolved organic matter, different seasons of the year and different depths in the water column all of which control speciation behaviour. Suspended particulate matter (SPM) values were measured at all sites and increased from West to East coast locations (5.7-34.4 mg/l). Dissolved organic matter (DOM) values ranged from 0.58 to 2.2mg/l C. The total dissolved copper concentrations ranged from 0.30 to 6.68 microg/l, with labile fraction ranging from 0.02 to 2.69 microg/l, and most labile copper concentrations below 1 microg/l. None of the yearly mean copper measurements exceeded the 76/464/EEC EQS of 5 microg/l. Of the 306 measurements, only one dissolved copper value in one season was above 5 microg/l. This ratio of labile to total copper was between 10 and 30%. The results from this survey suggest that if toxicity of copper is due to the labile fraction then using the total dissolved copper concentrations as an indicator of impact overestimate the risk by a factor of four times.
The present paper presents original data and a review of the copper (Cu) toxicity literature for estuarine and marine environments. For the first time, acute Cu toxicity across the full salinity range was determined. Killifish, Fundulus heteroclitus, eggs were hatched in freshwater (FW), 2.5, 5, 10, 15, 22 and 35 ppt (seawater, SW) and juveniles were allowed to acclimate for 7 days prior to acute toxicity testing. Sensitivity was highest in FW (96 h LC50: 18 microg/l), followed by SW (96 h LC50: 294 microg/l) with fish at intermediate salinities being the most tolerant (96 h LC50 > 963 microg/l at 10 ppt). This approximately 50-fold, non-linear variation in sensitivity could not be accounted for by Cu speciation or competition among cations but can be explained by physiology. The relative Na(+) gradient from the blood plasma to the water is greatest in FW followed by SW and is smallest at 10 ppt. Regression of Cu toxicity versus the equilibrium potential for Na(+), which reflects the relative Na(+) gradient, revealed that 93% of the variation can be attributed to Na(+) gradients and thus osmoregulatory physiology. Examination of the existing literature on acute Cu toxicity in SW (defined as >25 ppt) confirmed that early life stages generally are most sensitive but this pattern may be attributable to size rather than developmental stage. Regardless of developmental stage and phylogeny, size clearly matters for Cu sensitivity. The existing literature on the influence of salinity on acute Cu toxicity as well as studies of mechanisms of Cu toxicity in fish and invertebrates are reviewed.