Linkages between cellular biomarker responses and reproductive success in oysters – Crassostrea virginica

The purpose of these studies was to evaluate if there were relationships between lysosomal destabilization or glutathione concentrations and gamete viability of oysters, Crassostrea virginica. Oysters were collected from field sites during the peak spawning period (May–June) during 2001 and 2002. Lysosomal destabilization rates and glutathione concentrations of hepatopancreas tissues (e.g. digestive gland) were determined. Eggs and sperm from the same adults were also used to conduct embryo development assays with reference seawater collected from a clean site, site water, and also a range of Cd concentrations (the Cd exposures were used to determine if there were differences in susceptibility to pollutants). Baseline embryo development success (e.g. percent normal development when the assays were conducted with reference seawater or site water) was related to lysosomal destabilization, but not glutathione status. However, the susceptibility of embryos to metal toxicity was related to glutathione status, i.e., sensitivity to Cd exposures increased with decreasing glutathione levels. These studies support the hypotheses that there are linkages between embryo development success and susceptibility to pollutant stress and cellular biomarker responses. These kinds of effects on reproductive success could lead to subtle but significant long-term effects on recruitment and viability of oyster populations.     

Seasonal variation in lysosomal destabilization in oysters,Crassostrea virginica

Lysosomal destabilization assays have been used as valuable biomarkers of pollutant exposures in a variety of bivalve and fish species. The responses of oysters, Crassostrea virginica, deployed at and native to various reference and degraded sites were evaluated for lysosomal destabilization during both summer and winter seasons. In both native and deployed oysters, lysosomal destabilization rates tended to be higher during the winter at both reference and polluted sites. There are at least two hypothetical explanations. Greater lysosomal destabilization rates may be related to physiological changes associated with mobilization of nutrient reserves during the winter and gametogenesis. However, lysosomal destabilization in deployed oysters was correlated with tissue metal concentrations. These data also support a second hypothesis that seasonal differences in physico-chemical factors (such as reduced levels of acid volatile sulfides) may increase the bioavailability of metals during the winter so that adverse effects are more pronounced.     

The effects of glutathione depletion on reproductive success in oysters, Crassostrea virginica.

Glutathione (GSH) is a ubiquitous tripeptide that functions as a very important modulator of cellular homeostasis, including detoxification of metals and oxyradicals. Therefore, depletion of GSH may predispose organisms to pollutant stress. Reproductively active oysters (Crassostrea virginica) were exposed to buthionine sulfoximine in the laboratory to deplete gonadal GSH. The effects of metal exposures (Cd and Cu) on fertilization and developmental assays were evaluated using gametes from control and GSH-depleted adults. Fertilization success was not affected by GSH status, i.e. the fertilization rates of gametes derived from GSH-depleted adults were the same or slightly higher. However, GSH depletion did increase the susceptibility of developing embryos to metal toxicity, i.e. adverse effects on embryonic development were observed at lower metal concentrations with gametes derived from GSH-depleted adults. These effects may be related to diminished removal of free radicals or increased availability of metals. Whereas sperm penetration of embryonic membranes and fertilization success may be facilitated by free radicals, the persistence of free radicals during subsequent developmental periods may adversely affect differentiation and normal development. GSH probably also plays an important role in scavenging toxic metals and reducing metal interactions with essential developmental processes. These results suggest that parental depletion of GSH may increase the susceptibility of embryos to metal toxicity.     

An ecotoxicological protocol with caged mussels, Mytilus galloprovincialis, for monitoring the impact of an offshore platform in the Adriatic Sea

An ecotoxicological protocol with caged mussels, Mytilus galloprovincialis, was developed to evaluate the potential impact of an offshore gas platform in the central Adriatic Sea. Reference organisms were collected on a seasonal basis from an unpolluted site and transplanted for four weeks in both the sampling area and to the investigated platform. Chemical analyses of trace metals in mussel tissues were integrated with a multi-biomarker approach for the early detection of biological responses at several cellular targets. Induction of metallothioneins, peroxisomal proliferation and activity of acetylcholinesterase were measured as markers for specific classes of chemicals. Special attention was given to oxyradical metabolism and appearance of oxidative-mediated toxicity to reveal a more general onset of cellular disturbance. In addition to individual antioxidants (superoxide dismutase, catalase, glutathione S-transferases, glutathione reductase, Se-dependent and Se-independent glutathione peroxidases, and levels of total glutathione), the total oxyradical scavenging capacity (TOSC) allowed a quantification of the overall capability to neutralize specific forms of intracellular reactive oxygen species (ROS; i.e. peroxyl and hydroxyl radicals). Cellular damages were evaluated as lysosomal destabilization (membrane stability, accumulation of lipofuscin and neutral lipids), lipid peroxidation products (malondialdehyde) and DNA integrity (strand breaks and micronuclei); the air survival test was finally applied to evaluate the overall physiological condition of mussels. Concentration of trace metals (As, Ba, Cd, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Zn) revealed only limited variations in transplanted mussels during various experimental periods and such changes appeared partly related to natural fluctuations. Among biological responses, variations of antioxidants and lysosomal stability were confirmed as sensitive early warning signals for biological disturbance of both natural and anthropogenic origin. The presented protocol with caged mussels allowed marked biological effects caused by the investigated platform to be excluded, and represented a useful approach that is easy to extend for monitoring the impact of offshore activities in the Adriatic sea.     

Biomonitoring of trace metals using transplanted mussels, <Emphasis Type="Italic">Mytilus galloprovincialis</Emphasis>, in coastal areas around Ulsan and Onsan Bays,Korea

Mediterranean (blue) mussels (Mytilus galloprovincialis) collected from a reference site were transplanted to 15 stations in coastal areas around Ulsan and Onsan Bays, an extensively metal polluted area in Korean coastal waters, to assess metal contamination in the coastal oceans of Korea. During the biomonitoring periods (June 30 to July 20, 2003; 21 days), transplanted mussels, seawater, and particulate materials were collected for analysis of 15 metals (Ag, As, Cd, Co, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Sb, Se, Sn, and Zn). Transplanted mussels showed metal enrichment compared to initial concentrations and spatial gradients consistent with dissolved and/or particulate metal concentrations in seawaters. Based on Q mode factor analysis, stations were clustered into three groups. The first group, located on Onsan Bay, showed high Ag, Cd, Cu, Hg, Pb, Sb and Zn enrichment, presumably arising from non-ferrous metal refineries and chemical industries in this area. The second group was located near the mouth of the Oehwang River and was enriched in Co from petrochemical industries. The third group comprised a site intermediate between Group 1 and Group 2, an isolated station with independent metal sources located in Jangsaengpo harbor, where a number of ship repairing and building companies operate, and a less contaminated station near a small fishing village. Metal accumulation rates (%·day^-1) in mussels were estimated to be between 8% (Cr) and 281% (Pb), based on accumulated metal concentrations over 21 days. The active biomonitoring technique using M. galloprovincialis demonstrated here is a useful monitoring method because it reflects the present status of seawaters; furthermore, physiological factors can be standardized, and bioavailable and time-integrated metal concentrations can be obtained. Furthermore, this method can be applied even in coastal seawaters so heavily contaminated that living organisms would not normally survive.     

Effect of exposure time on the bioaccumulation of Cd, Mg, Mn and Zn in Cystoseira abies-marina samples subject to shallow water hydrothermal activity in São Miguel (Azores)

Shallow water hydrothermal vents can be compared to polluted places due to high concentrations of heavy metals, and are thus good models for bioaccumulation studies. The present study intended to estimate the time of exposure required for the accumulation of certain elements to stabilize in specimens of Cystoseira abies-marina , to be used as a reference in future work. Cystoseira abies-marina intertidal specimens were transplanted from Mosteiros (a non-hydrothermal and pristine site) to Ferraria (with hydrothermal activity) and left there. Transplanted samples were collected after 1, 2, 4 and 8 weeks and the concentrations of Cd, Mg, Mn and Zn were measured through flame atomic absorption spectrophotometry. Although further studies with increased periods of exposure are needed because the concentration of these elements never stabilized in the collected samples, there is strong evidence that increased time of exposure led to increased concentrations of Cd, Mg, Mn, but not Zn. These results are consistent with the assumption that C. abies-marina is bioaccumulating some of the heavy metals and can thus be a good indicator for polluted waters.     

Sea urchin sperm bioassay for sewage and chlorinated seawater and its relation to fish bioassays

Bioassays were conducted with sea urchin and sand dollar sperm to determine the toxicity of chlorinated and unchlorinated sewage effluent and chlorinated and brominated seawater. The sperm cells were exposed to seawater dilutions of each toxicant for 5–15 min. The fertilisation of eggs served as the indicators of sperm viability. The effective concentrations which reduced fertilisation success by 50% (EC50) averaged 2·2 and 4·8% chlorinated and unchlorinated sewage in seawater, respectively. The sperm cells were extremely sensitive to chlorinated seawater at concentrations from 0·002 to 0·020 mg/litre total residual oxidant (TRO). Brominated seawater proved toxic to sperm in one test at 0·015 mg/litre TRO. Results of the sperm bioassays are compared with previous acute and chronic bioassays with fish.     

Lower Cd/P ratio in settling particles than in surface seawater in the Okinawa Trough

The characteristics of the ratios between cadmium (Cd) and phosphorus (P) in settling particles collected from the Okinawa Trough in the East China Sea were examined using a sediment trap, moored at a depth of 811 m for one year. The Cd/P ratios varied within a narrow range throughout the year, in spite of the large seasonal change in the total dry mass, Cd, and P fluxes. The average Cd/P ratio of settling particles was 0.062 (nmol/μmol), which was obviously lower than that of surface seawater around the study site (0.16). This lower ratio in the Okinawa Trough particles collected using the 811 m-moored trap certainly reflected the mixture of biologically produced organic matter around the study site and other components that were mainly transported as lateral flux from the shelf edge and slope area of the East China Sea.     

Oxidative stress in two populations of killifish (Fundulus heteroclitus) with differing contaminant exposure histories

A population of killifish (Fundulus heteroclitus) inhabiting a creosote-polluted inlet of the Elizabeth River demonstrates tolerance to the acute toxic effects exerted by contaminated sediments on reference site killifish. Previous data have suggested that upregulated antioxidant defenses contribute to short-term tolerance in killifish exposed to Elizabeth River sediments. This study investigated population differences in antioxidant defenses from wild caught Elizabeth River and reference population killifish in different seasons, and after being held in the laboratory. Parameters measured in the killifish were total glutathione concentrations (GSH(T)), activities of glutathione reductase (GR), glutathione peroxidase (GPx), and lipid peroxidation (LPO), all in adult hepatic tissues. The Elizabeth River population exhibited greater GSH(T), higher GPx activities, and increased LPO as compared to the reference population. Sex specific population differences were also observed in GSH(T) and GPx. Both populations displayed decreased GSH(T) and increased GR from early to late summer, as well as after being held in the laboratory. This study indicates that there are many factors that may contribute to differences in levels of antioxidant defenses in addition to exposure to contaminants, including reproductive status and environmental conditions.     

Behavior of Cu, Ni and Cd during nutrient depletion in a spring bloom in Funka Bay

The concentrations of Cu, Ni and Cd were determined in Funka Bay during a spring phytoplankton bloom, consisting of diatoms. Just after the bloom, both dissolved Cd and nutrients were removed in the euphotic zone. However, the removal ratio of Cd to phosphate was very different from that in seawater. The removal of Cd took place at a Cd/phosphate ratio of 0.07×10⁻³, which was lower than in seawater before the bloom (0.25×10⁻³), leading to an increase in this ratio in seawater exceeding 0.7×10⁻³ at the end of the bloom. Elevated concentrations of Cd and phosphate were observed in the deeper layer after the bloom due to the decomposition of detrital materials produced in the bloom. The ratio of Cd/phosphate in the regeneration step was 0.24×10⁻³ which was different from the removal ratio of 0.07×10⁻³. These observations suggest that the high Cd/phosphate ratio in the regeneration would reflect a relatively high regeneration rate of Cd than that of phosphate. No significant decrease in Cu and Ni concentrations was observed during the development of the bloom, suggesting that biological removal of these metals was not so significant during the spring bloom. The concentrations of Cd, Cu and silicate in surface waters increased after the bloom with decreasing salinity due to the influence of a spring thaw.     

大亚湾珊瑚礁痕量金属的研究

作者采用阳极溶出伏安法测定大亚湾珊瑚礁痕量金属铜、铅、镉的含量 ,并讨论了相关的影响因素。测定结果发现 :A组、B组珊瑚礁礁体中铜、铅、镉的含量从里到外有递增的趋势 ,这与近年来大亚湾水体中铜、铅、镉含量的变化是一致的。而 A组珊瑚礁变化明显 ;B组珊瑚礁礁体中铜、铅、镉含量呈现较复杂的变化。分析认为 ,这是因珊瑚礁的形成受多种因素影响所致 :(1)海水组成对珊瑚礁化学成分的影响 ;(2 )海洋生物种类、数量及其矿物组成是制约元素分布的主要因素 ;(3)陆源碎屑组分对大亚湾珊瑚礁的化学组成有一定的影响。各因素在不同情况下所起的作用不同。     

Significance of metallothioneins and lysosomes in cadmium toxicity and homeostasis in the digestive gland cells of mussels exposed to the metal in presence or absence of phenanthrene

Metallothioneins and lysosomes are known to be involved in cellular detoxication and sequestration of certain metals^1–3 and both have been identified in this role in elimination of copper from marine mussels (Mytilus edulis/galloprovincialis).³ Cadmium (Cd), however, has been shown to persist in the cells of the digestive gland for long periods with only minimal elimination. An experiment was designed to test the effects of Cd on the fragility of lysosomal membranes in the digestive cells as a measure of cellular injury,^4,5 metallothionein content of the digestive gland and cadmium concentration in this organ. Phenanthrene was used also to destabilise lysosomal membranes⁶ in order to test if increased lysosomal fragility interfered with cadmium metabolism and detoxication. The results demonstrated that Cd induced metallothionein synthesis and that elimination of Cd was minimal after 28 days in clear seawater. Lysosomal fragility was initially increased but this effect was soon reversed, even with continued exposure to Cd. The lysosomal destabiliser, phenanthrene, did not appear to affect accumulation of Cd or levels of metallothionein.     

Oxidative stress in two populations of killifish (Fundulus heteroclitus) with differing contaminant exposure histories

A population of killifish (Fundulus heteroclitus) inhabiting a creosote-polluted inlet of the Elizabeth River demonstrates tolerance to the acute toxic effects exerted by contaminated sediments on reference site killifish. Previous data have suggested that upregulated antioxidant defenses contribute to short-term tolerance in killifish exposed to Elizabeth River sediments. This study investigated population differences in antioxidant defenses from wild caught Elizabeth River and reference population killifish in different seasons, and after being held in the laboratory. Parameters measured in the killifish were total glutathione concentrations (GSH_T), activities of glutathione reductase (GR), glutathione peroxidase (GPx), and lipid peroxidation (LPO), all in adult hepatic tissues. The Elizabeth River population exhibited greater GSH_T, higher GPx activities, and increased LPO as compared to the reference population. Sex specific population differences were also observed in GSH_T and GPx. Both populations displayed decreased GSH_T and increased GR from early to late summer, as well as after being held in the laboratory. This study indicates that there are many factors that may contribute to differences in levels of antioxidant defenses in addition to exposure to contaminants, including reproductive status and environmental conditions.     

粤西湛江港海底沉积物重金属的分布特征与来源

2004年9月23日至10月5日对湛江港海洋沉积物中金属元素的含量及分布进行了调查。共采集样品20个,采用王水-氢氟酸消解,电感耦合等离子体发射光谱法测定样品中的金属元素As、Cd、Co、Cr、Cu、Ni、Pb、Zn、Al、Ca、Fe、Mg和Mn。结果发现,湛江港大多数区域的沉积物未受污染,但霞山区港务局码头附近海域受到Cd、Cu和Pb的轻度污染,特呈岛养殖区也受到Cu的轻度污染。主成分分析结果表明,Al、Cd、Co、Cr、Cu、Fe、Ni、Pb和Zn主要通过陆源输入进入近岸水域的沉积相中,Ca、As、Mg和Mn主要通过海洋环境中水相和沉积相的相互作用进入沉积相中。结合其它调查结果认为,湛江港的Cd污染在水相、沉积相和生物相中均已存在,应引起有关部门的高度重视。     

Stability of lysosomal and cell membranes in haemocytes of the common mussel (Mytilus edulis): effect of low temperatures.

Expanding industrial activities in the Arctic require an urgent assessment of the toxicity of chemicals at low temperatures. Organisms acclimatized to low temperature exhibit specific adaptations. For example, the amount of unsaturated lipids is increased to maintain the fluidity of the cell membranes. It has been hypothesized that such temperature-induced alterations in membrane lipid composition may affect the stability of lysosomal and cell membranes in the common mussel, Mytilus edulis, an organism exposed to seasonal temperature extremes. As mussels may be exposed to petroleum compounds along industrialized coastlines, we tested the combined effects of exposure to low temperature and the petroleum compound, phenanthrene, on haemocyte membrane stability. Test animals, acclimated to either 0 or 10 degrees C, were exposed to phenanthrene (0 = control or 500 micrograms l-1) and haemocytes were examined using the neutral red retention assay (lysosomal stability) and a fluorescence assay (cell membrane stability). At 0 degree C, lysosomal and cell membranes from uncontaminated mussels were destabilized compared with 10 degrees C (P = 0.0005). No significant effects (P > 0.05) of phenanthrene were detected at either temperature. Possible mechanisms underlying membrane destabilization include a weaker physical resistance of the membrane due to a higher amount of unsaturated lipids, a potentially higher level of reactive oxygen radicals at low temperature and the higher susceptibility of unsaturated lipids to oxidative stress. More work is required to better understand the consequences of this membrane destabilization at low temperature on the susceptibility of the organism to pollutants.     

Zinc concentration effect at the organismal,cellular and subcellular levels in the eastern oyster

The purpose of this research was to demonstrate a concentration effect of zinc exposure at organismal, cellular and sub-cellular levels in the eastern oyster and to find associated protein expression signatures (PES) for each concentration of zinc. Oysters were exposed to six concentrations of zinc for 48 h in a controlled environment. At the organismal level, fecal material was observed as a measure of physiological health during metal exposures. At the cellular level, lysosomal destabilization was measured using hemolymph. This cellular response was significant only at the highest concentration, when the fecal index was lowest. Protein responses were monitored in the oyster following exposure to zinc. Gill tissue was excised and homogenized, and then analyzed using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and digital image analysis. Protein expression signatures were found to be specific to each concentration. The protein responses were linked to the other biological parameters measured, each of which followed a concentration gradient of zinc.     

The distribution and preferential biological uptake of cadmium at 6°W in the Southern Ocean

The spatial and temporal distribution of cadmium (Cd) and phosphate in the Southern Ocean are related to biology and hydrography. During a period of 18 days between transects 5/6 and 11, a phytoplankton spring bloom developed in the Polar Frontal region. Upper water Cd concentrations were not depleted and ranged from 0.2 to 0.8 nM at about 10 m depth. These relatively high Cd concentrations are attributed to upwelling of Upper Circumpolar Deep Water (0.5–1.2 nM in the core) in combination with low biological productivity (0.2 to 0.3 mg m⁻³ chlorophyll-a, 0.3 g C m⁻² d⁻¹). Total particulate Cd concentrations at 40 m depth were between 0.02 and 0.14 nM with the maximum in concentration in the Polar Frontal region. Most of the particulate Cd at this depth (85–94%) was detected in the first phase of a sequential chemical leaching treatment which includes adsorbed Cd as well as Cd incorporated in algae. The Polar Frontal region was characterized by minima in Cd concentration and Cd/phosphate ratio of seawater at both transects; values were the lowest at transect 11 after development of the spring bloom which was dominated by diatoms. This decreasing Cd/phosphate ratio in seawater during spring bloom development was attributed to preferential Cd gross uptake which more than compensated the process of preferential Cd recycling. Within the Upper Circumpolar Deep Water, Cd showed a maximum in concentration similar to that of the major nutrients. Both the Cd concentration and the Cd/phosphate ratio of the deeper water increased in southern direction, from 0.4 to 0.7 nM and from 0.2 to 0.3 nM/μM, respectively. Antarctic Intermediate Water has a Cd concentration of 0.21 nM with a Cd/phosphate ratio of 0.10 nM/μM. In Antarctic Bottom Water, Cd concentrations ranged from 0.60 to 0.82 nM.     

Bioaccumulation and depuration of Zn and Cd in mangrove oysters (Crassostrea rhizophorae, Guilding, 1828) transplanted to and from a contaminated tropical coastal lagoon

In order to study Zn and Cd accumulation and depuration, a set of oysters, Crassostrea rhizophorae, were transplanted to a metal contaminated coastal lagoon and another one was harvested there and transplanted to a non-polluted site. C. rhizophorae oysters and Perna perna mussels native from both sites were collected in order to monitor variability of metal concentration in resident populations. After three months exposure, oysters transplanted to the polluted site accumulated fourfold Zn (307-1319 microgg(-1)) without reaching the concentration level of resident oysters (9770 microgg(-1)). Cadmium concentrations had a slight but significant decrease during the same period (1.25-0.54 microgg(-1)). Oysters transplanted to the non-polluted site, showed threefold Zn depuration (6727-2404 microgg(-1)), while Cd had no significant variation (0.90-1.45 microgg(-1)). Results showed that transplanted oysters do not reach heavy metal concentrations in indigenous populations suggesting transplanted organisms would be better used to evaluate bioavailability instead of environmental concentrations.     

Metal concentrations in the shell of Bathymodiolus azoricus from contrasting hydrothermal vent fields on the Mid-Atlantic Ridge

Specimens of Bathymodiolus azoricus were sampled along the Mid-Atlantic Ridge at the Menez Gwen, Lucky Strike and Rainbow hydrothermal fields. Individual shells (n = 51), through the weight range 0.62 to 15.70 g, were analyzed for their magnesium, strontium, iron, manganese, copper, zinc and cadmium concentrations. Amongst the marine molluscs the shell of B. azoricus is confirmed as being particularly impoverished in strontium (mean 943 microg g(-1)). Trace metal concentrations in the shells decreased in the order Fe> Mn> Zn> Cu> Cd. Despite originating from trace metal rich environments mean concentrations were low (37.9, 13.2, 10.7, 1.1 and 0.7 microg g(-1), respectively). Irrespective of geographical origin magnesium, strontium and copper concentrations were primarily dictated by shell weight. In contrast cadmium concentrations were elevated in shells from the Rainbow field and ambient seawater chemistry imparted site specific chemical fingerprints to the shells with respect to the iron to manganese ratio.     

海水酸化和磺胺甲恶唑复合暴露对大马蹄螺抗氧化酶活性和脂质过氧化的影响

为探讨海水酸化和磺胺甲恶唑环境残留对珊瑚礁区生物的影响,本文以南海珊瑚礁区关键礁栖生物大马蹄螺（Trochus niloticus） 为研究对象,探究了两种环境胁迫因素对大马蹄螺的氧化胁迫作用。分别采用两个 pH 水平 （pH 8.1、 pH 7.6） 和三个磺胺甲恶唑浓度水平 （0 ng/L、100 ng/L 和 1 000 ng/L） 单独和复合暴露 28 天,测定其对大马蹄螺鳃组织的超氧化物歧化酶 （SOD）、过氧化氢酶 （CAT） 和谷胱甘肽巯基转移酶 （GST） 活性以及谷胱甘肽 （GSH） 含量和脂质过氧化水平 （LPO） 的影响。结果发现：海水酸化和磺胺甲恶唑单独暴露能够导致大马蹄螺鳃组织出现不同程度的氧化应激现象,表现为抗氧化酶 （CAT、SOD 和 GST） 活性和谷胱甘肽含量的升高。在复合暴露条件下,大马蹄螺鳃组织的抗氧化酶（CAT、SOD 和 GST） 活性和谷胱甘肽含量均受到不同程度的抑制,同时伴随着脂质过氧化水平的显著升高。这表明短期海水酸化和磺胺甲恶唑复合暴露可导致大马蹄螺鳃组织出现氧化损伤现象,进而可能对个体存活和种群繁衍造成负面影响。