Accumulation and effects of organotin compounds in oysters and mussels: Correlation with serum biochemical and cytological factors and tissue burdens

Oysters and mussels exposed to a concentration of 0·7 ppb (μg/liter) tributyltin from painted panels in flowing seawater accumulated tin in the digestive glands to comparable levels. The mussels experienced approximately 50% mortality during the 60-day test period, but the oysters suffered virtually no deaths. There was no evidence from either bivalve of elevated numbers of hemocytes during the test period and no evidence for cellular disruption as detected by increased levels of serum lysosomal hydrolases. Serum protein of exposed mussels relative to controls increased with time of exposure to the toxicant, while oyster serum protein, normally 10 x higher than in mussels, did not. No evidence was found for elevated stress proteins (heat shock proteins) or metallothioneins in the serum hemocytes of either bivalve. Responses by these animals to fatal or near fatal doses of TBT were thus very different from responses to copper that we have reported elsewhere.^1,2     

The effect of tributyltin upon steroid titres in the female dogwhelk, Nucella lapillus, and the development of imposex

Exposure of female Nucella lapillus to tributyltin (TBT) in seawater at a concentration of 40 ng Sn/litre led to accumulation of TBT in the tissues, and an increase in penis length compared to control animals. Investigation of the steroid concentrations by radioimmunoassay revealed a significant increase in testosterone after 28 days exposure to TBT. No significant effect was noted upon the amounts of progesterone or estradiol-17β. When testosterone was injected into female N. lapillus in the absence of TBT, penis length was found to increase significantly. These observation suggest that there may be an association between the change in testosterone titre in response to exposure to TBT and the development of imposex in the dogwhelk.     

Uptake and transformation of C-labelled tributyltin chloride by the dog-whelk, nucella lapillus: Importance of absorption from the diet

The importance of the diet as a source of tributyltin (TBT) in Nucella lapillus was studied using [¹⁴C]tributyltin chloride. In N. lapillus provided with prelabelled mussels, Mytilus edulis, in labelled water (mean 20·5 ng/litre TBT) the rate of accumulation of total ¹⁴C was 2–3 times that in unfed animals. Owing to its degradation in the tissues of both fed and unfed animals, concentrations of [¹⁴C]TBT tended to reach a plateau after only 28 days. However, total concentrations of ¹⁴C were still increasing after 49 days. Under experimental conditions (15°C, ample food, no disturbance) the diet accounted for about 50% of the body burden of TBT in N. lapillus after 49 days exposure: concentration factors (dry tissue/water) for [¹⁴C]TBT in both male and female N. lapillus were similar at about 60 000 in fed and 30 000 in unfed animals. It is concluded that the diet may contribute less than half of the body burden of TBT found in natural populations subjected to life-long exposure.     

Reductions in TBT concentrations in UK estuaries following legislation in 1986 and 1987

In 1986 the Government of the United Kingdom (UK) introduced legislation to control the sale of tribytltin (TBT)-based paints. From 1986 to 1989 monitoring was undertaken, whereby samples of water, sediment, oysters (Crassostrea gigas) and mussels (Mytilus edulis) were collected from six estuaries and water samples from five marinas/harbours, and analysed for TBT. In 1989 the concentrations of TBT in water, oysters and mussels were generally only one-third to one-quarter of those observed in 1986. The most marked decrease in concentrations of TBT in oysters and mussels occurred in 1987–1988, following the 1987 ban on the use of TBT-based paints on small boats and mariculture equipment. As the concentrations of TBT in oyster tissues had decreased there has been improvement in oyster growth in terms of both meat production and growth of thin (i.e. normally shaped) shells. Oysters with a normal shell shape can now be grown in five of the six estuaries which were monitored. In contrast to the reduction in concentrations of TBT noted in water and bivalves, changes in concentrations of TBT in sediments showed no clear trend from 1986 to 1989.     

Oxygen radical formation in anoxic transgression and anoxia-reoxygenation: Foe or phantom? Experiments with a hypoxia tolerant bivalve

Intertidal blue mussels, Mytilus edulis, experience hypoxia reoxygenation during tidal emersion and resubmersion cycles, and this is often suggested to represent a major stress for the animals, especially for their respiratory tissues, the gills. We exposed mussels to experimental short and prolonged anoxia and subsequent reoxygenation and analyzed the respiratory response in excised gill tissue and the effects of treatment on reactive oxygen species (mainly ROS: superoxide anion, O₂^·− and hydrogen peroxide, H₂O₂), formation using live imaging techniques and confocal microscopy. Our aim was to understand if this “natural stress” would indeed produce oxidative damage and whether antioxidant defenses are induced under anoxia, to prevent oxidative damage during reoxygenation. Exposure to declining pO₂ in the respiration chamber caused an increase of gill metabolic rate between 21 and 10 kPa, a pO₂ range in which whole animal respiration is reported to be oxyregulating. Exposure of the animals to severe anoxia caused an onset of anaerobiosis (succinate accumulation) and shifted high and low critical p_c values (p_c1: onset of oxyregulation in gills, p_c2: switch from oxyregulation to oxyconformity) to higher pO₂. Concentrations of both ROS decreased strongly during anoxic exposure of the mussels and increased upon reoxygenation. This ROS burst induced lipid peroxidation in the mantle, but neither were protein carbonyl levels increased (oxidative damage in the protein fraction), nor did the tissue glutathione concentration change in the gills. Further, analysis of apoptosis markers indicated no induction of cell death in the gills. To our knowledge, this is the first paper that directly measures ROS formation during anoxia reoxygenation in mussels. We conclude that hypoxia tolerant intertidal mussels do not suffer major oxidative stress in gill and mantle tissues under these experimental conditions.     

Tributyltin compounds in mussels,oysters and sediments of Chinhae Bay,Korea

To study the historical and current status of the environmental levels of organotin compounds in Chinhae Bay in southeast Korea, mussels (Mytilus edulis galloprovincialis), oysters (Crassostrea gigas) and sediment cores were analysed for tributyltin (TBT) compounds. TBT concentrations were higher in oysters (mean: 0.25 μg TBT-Sn/g dry wt) than in mussels (mean: 0.20 μg TBT-Sn/g dry wt). The major sources of TBT are in the inner bay, and the highest TBT concentrations of 0.50 μg TBT-Sn/g (mussels) and 0.74 μg TBT-Sn/g (oysters) were observed at the site adjacent to the port. Concentrations of TBT decreased sharply with distance from the inner bay toward the outer bay and there was little TBT in the outer bay. A significant correlation was observed between this distance and the tissue TBT content of oysters and mussels. The concentration and percentages of TBT in sediment cores decreased with depth as a result of debutylation. Half-lives of TBT and dibutyltin (DBT) in sediments were 6.9 and 11.6 y, respectively. The TBT concentrations in mussels were higher than those in sediments by a factor of 4–7.     

Assessing site-specific effects of TBT contamination with mussel growth rates

During nine field transplant tests in San Diego Bay (1987–1990), juvenile mussels were exposed to mean concentrations of tributyltin (TBT) in ambient seawater ranging from 2 to 530 ng liter⁻¹ for 12 weeks under natural conditions. A total of 79 cages with 18 mussels each were monitored at 18 different sites. Growth and seawater TBT concentrations were measured weekly or on alternate weeks (biweekly). Mean growth rates ranged from 17 to 505 mg week⁻¹ (0·2 to 2·5 mm week⁻¹). Accumulation of TBT in mussel tissues was measured at the end of each 12-week test exposure and ranged from 0·1 to 3·2 μg g⁻¹ TBT wet weight. The frequency of the measurements and the integration of chemical and biological measurements improved the accuracy of the assessment over more traditional approaches. Growth was significantly related to seawater and tissue TBT. The statistical relationships with growth effects were used to estimate chemical effect zones for TBT in San Diego Bay. Site-specific differences were distinguished by additional statistical analyses and consideration of environmental significance.     

Fate of tributyltin in sewage sludge treatment

We have shown that municipal wastewater and sewage sludge are contaminated by organotins. Here, we investigate the behaviour and fate of tributyltin (TBT) in laboratory-scale sewage sludge treatment at various conditions including both aerobic and anaerobic, and mesophilic and thermophilic. Organotins were determined by capillary GC-FPD. Fresh raw sewage sludge was stored in a tank that was connected to five chemostats maintained at different conditions. Raw sludge contained TBT, dibutyltin (DBT) and monobutyltin (MBT) residues in the range of 0·28 to 0·83 mg/kg (dry weight). The volumetric TBT concentration was 10·5 μg/litre in the influent, and ranged from 7·4 to 8·3 μg/litre in the effluent of the different chemostats. In the anaerobic mesophilic treatment, concentrations of TBT did not decrease with increasing sludge residence time, DBT increased and MBT showed no clear trend. The DBT increase is suggested to be due to leaching from the PVC walls of the chemostats, and not to degradation of TBT alone. The TBT reduction did not show significant differences between the mesophilic and thermophilic anaerobic fermentors. These findings indicate that the degradation of TBT during sludge treatment is only low under all conditions tested.     

Editorial

A flow-through system for exposure of seagrass to pollutants is described. Sea water with dissolved atrazine or PCP (pentachlorophenol) was pumped through a sealed 2-litre volume glass exposure vessel that contained either whole plants or leaves of Thalassia testudinum. Effects of the compounds on oxygen evolution and uptake by leaves were measured after exposure for 40 and 88 h. Rate of oxygen evolution was depressed strongly after 40 h by 1 ppm of atrazine or PCP. The rate of oxygen uptake was slightly depressed by atrazine but strongly depressed by PCP. Photosynthesis/respiration ratios were depressed to less than one by 1 ppm of atrazine or PCP and by 0·5 ppm atrazine. EC₅₀ values based upon depression of oxygen evolution after 40 hours exposure were: atrazine, 0·32 ppm PCP, 0·74 ppm. The data suggest that leaves may recover from, or adapt to, the presence of either toxicant.     

In vivo metabolism of aromatic amines by the bay mussel, Mytilus edulis

The hazard posed to aquatic organisms by exposure to aromatic amines will depend on their ability to activate or detoxify these potentially genotoxic compounds. We studied the metabolism of four aromatic amines (p-toluidine, aniline, 2-acetylaminofluorene (2-AAF) and 2-aminofluorene (2-AF)) in Bay mussels (Mytilus edulis) to gain an understanding of their metabolic capabilities in vivo. Mussels rapidly accumulated these compounds from the water column and depurated the majority of their steady-state body burdens as metabolites and unmetabolized parent compounds. The biotransformation pathways of [¹⁴C]-labeled parent compounds were determined by analyses of depurated metabolites and tissue residues. Biotransformation of p-toluidine and aniline resulted in the formation of their corresponding N-acetylated detoxification products. Mussels converted small amounts of 2-AAF into a deacetylated product (2-AF) and a mutagenically active metabolite (N-hydroxy-2-AF). 2-AF was extensively detoxified via N-acetylation, which limited the amount of primary amine available for activation.     

The role of oxyradicals in intracellular proteolysis and toxicity in mussels

Studies were performed on the common mussel, M. edulis L., to determine whether copper (Cu) exposure can affect the extent to which digestive cell proteins are oxidised and whether such oxidative damage is mediated by free radicals. Three age groups of mussels were exposed for 6 ^-days to environmentally realistic concentrations of Cu and then digestive gland homogenates were examined for evidence of protein carbonyl formation. Significant increases in carbonyls relative to untreated control mussels were seen for the youngest (2–4 year-old) and oldest (≥ 10 year-old) mussels only after exposure for 6 days, followed by recovery from exposure for a further 6 days. Untreated mussels also showed an age-related difference in protein oxidation, with a significantly lower concentration in the youngest animals (2–4 year olds). Copper did not affect the levels of modified tryptophan or tyrosine residues or the extent of total lipid peroxidation in digestive gland homogenate. Significant depletion of total vitamin E (a-tocopherol) was seen only in young and medium-aged mussels following exposure for 6 days. The levels of protein carbonyl groups were increased in digestive cell cytosol and lighter lysosomes but not in heavier lysosomes or digestive gland microsomes following 5 days exposure to Cu. Dihydrohodamine-123 was converted to fluorescent rhodamine-123 following sequestration into digestive cell lysosomes. The results suggest a link between the lysosomal sequestration of copper, a concomitant increase in the production of oxyradicals and the potential for intracellular oxidative damage, as well as an increased capacity for oxidative damage in older animals.     

高温胁迫下厚壳贻贝(Mytilus coruscus)消化腺代谢组学研究

在全球变暖的背景下, 海水温度不断升高、海洋暖化逐渐加剧, 高温严重影响着海洋生物的各种生命过程, 但对于海洋双壳贝类如何应对热应激的研究仍然不足。为此, 开展了以18 °C (CT)为对照组在26 °C (ST)和33 °C (HT)下对厚壳贻贝消化腺组织进行了急性热胁迫下的代谢组学分析,以便于研究其代谢反应。采用LC-MS/MS技术, 并结合生物信息分析手段对差异代谢物进行筛选, 并分析确定相关的代谢通路的变化, 共有2 532种代谢物在厚壳贻贝消化腺中被鉴定。KEGG富集分析用于探索差异代谢物的潜在代谢途径, 共有29条代谢通路被显著富集, 与对照组相比, ST组显著富集于牛磺酸和次牛磺酸代谢、神经活性配体-受体相互作用、鞘脂类代谢和视黄醇代谢等代谢通路; HT组显著富集于酪氨酸代谢、亚油酸代谢、丙氨酸新陈代谢、酪氨酸代谢、色氨酸代谢、苯丙氨酸代谢等代谢通路。研究结果显示, 厚壳贻贝消化腺主要通过调节色氨酸代谢、酪氨酸代谢、鞘脂代谢、苯丙氨酸代谢、氧化磷酸化, 脂肪酸、赖氨酸降解等信号通路应对热应激, 从而帮助维持身体内部环境的稳定状态。上述研究为多视角探究厚壳贻贝耐热机制与应对环境中温度变化的适应性进化提供理论基础。     

Effects of the suspension feeding mussel Mytilus trossulus on a brackish water macroalgal and associated invertebrate community

The importance of suspension-feeding mussels is particularly apparent in benthic communities; however, the role of this feeding strategy on the development of macroalgal and associated invertebrate communities is in general poorly known. The effect of suspension-feeding mussels Mytilus trossulus on benthic communities was studied in an in situ factorial field experiment in the Northern Baltic Sea over one ice-free season. The experiment was performed under different regimes of wave exposure (low and moderate) and on different sedimentary habitats (soft bottom with high organic content, soft bottom with low organic content, and hard bottom). In general the presence of mussels was associated with increased biomass of filamentous algae, herbivores and deposit feeders and decreased biomass of charophytes. The effect of M. trossulus interacted with the effect of exposure and substrate. Stronger responses were observed in moderately exposed than in sheltered areas. The presence of M. trossulus affected charophytes and deposit feeders on sand with low content of organic matter and filamentous algae on pebbles but not on other substrate types. The magnitude of the effects varied between months. The results suggest that (i) even in dynamic coastal systems the biodeposits and excretions of mussels are at least partly assimilated locally and are not flushed away to the open sea, (ii) the accumulation of faecal material induced elevated growth of deposit feeders, (iii) mussels enhanced the growth of ephemeral macroalgae and reduced the growth of perennial macroalgae, and (iv) together with increasing benthic primary production, mussels indirectly increase the production of herbivores.     

Enzymatic activities in spermatozoa and butyltin concentrations in Baltic turbot (Scophthalmus maximus)

Spermatozoal enzymes of fish (NAD+- and NADP-dependent dehydrogenases and creatine kinase (CK)) were previously determined to be sensitive to tributyltin (TBT) in laboratory experiments and were thus indicated for use as biomarkers for TBT exposure. However, the potential ability of spermatozoal enzymes as biomarkers of TBT exposure has never been recapitulated in a field study. For this purpose, the kinetic activities of spermatozoal enzymes of the natural turbot Scophthalmus maximus population from the Gulf of Gdańsk (GDA) and the Pomeranian Bay (POM) in the southern Baltic Sea were measured. Gas chromatography/high-resolution mass spectrometry was used to determine the concentrations of TBT and its breakdown products, dibutyltin (DBT) and monobutyltin (MBT), in the muscle, liver and testes of the male turbot. Males from GDA had significantly higher enzymatic activities and butyltin (BT) content in tissues than those from POM. A general linear model (GLM) showed that lactate dehydrogenase (LDH), malate dehydrogenase (MDH), glucose-6-phosphate dehydrogenase (G6PDH) and CK activities increased significantly with BT concentration in the testes and liver. We indicate the potential effects of TBT pollution on the spermatozoal enzymes of Baltic turbot.     

Effects of 17β-estradiol exposure in the mussel Mytilus galloprovincialis

Mussels Mytilus galloprovincialis were exposed to different concentrations of estradiol (20, 200, and 2000 ng/l) in a semi-static regime (1-day dosing intervals) for up to 7 days in an attempt to see how mussels dealt with exogenous estrogenic compounds. Sex hormone levels were determined in whole tissue. Free-estradiol was only significantly elevated at the highest exposure dose (up to 10-fold). Most of the estradiol was in the tissues as fatty acid esters (>78%), which sharply increased in a dose-dependent manner (from 4 ng/g in controls to 258 ng/g at the high exposure group). In contrast, neither free nor esterified testosterone levels showed significant differences between control and exposure groups. The results suggest the existence of mechanisms that allow mussels to maintain their hormonal status, and the important role that fatty acid esterification may play within those mechanisms. Synthesis and conjugation rates of estradiol were further investigated by measuring the activity of P450 aromatase, and palmitoyl-CoA:estradiol acyltransferase, in digestive gland microsomal fractions. Overall, the study contributes to the better knowledge of molluscan endocrinology, and defines new mechanisms of regulation of free steroid-levels in mussels.     

Exploring the use of caged Nucella lapillus to monitor changes to TBT hotspot areas: a trial in the River Tyne estuary (UK)

Caged dogwhelks (Nucella lapillus) and mussels (Mytilus edulis) were placed in situ at sites along the River Tyne (UK) in order to investigate the potential for monitoring any change in tributyltin (TBT) mediated effects. After 6 months the development of imposex was investigated in the dogwhelks and concentrations of TBT were determined in the whole tissues of both species. Sites within the estuary showed a high degree of imposex, whilst those at sites North and South of the estuary mouth exhibited significantly lower induction. Concentrations of TBT measured in the dogwhelk and mussel tissues showed the same pattern. The success of the caging technique, occurrence of imposex at other UK sites and the need for further monitoring at TBT impacted locations in the light of current international controls on the use of TBT-based antifouling paints on sea-going vessels are discussed.     

The impact of tributyltin from large vessels on dogwhelk (Nucella lapillus) populations around Scottish oil ports

Surveys of imposex in dogwhelk (Nucella lapillus) populations around oil terminals in Sullom Voe (Shetland) and Scapa Flow (Orkney) have confirmed that the effects of tributyltin (TBT) released from large vessels in coastal waters can be recognised. The impact of large vessels can be separated from the effects of other inputs of TBT in the areas studied. The intensity of the effects was greater in Shetland than in Orkney, reflecting the higher exposure to TBT resulting from the greater volume of traffic using the Shetland terminal, and the more restricted water exchange in that area. A large proportion of the female dogwhelks in Sullom Voe were unable to reproduce. Females in Scapa Flow were still able to reproduce, except for a small proportion close to two fish farm sites where TBT net-treatments were used prior to 1987.     

Sex-different effects of tributyltin on brain aromatase,estrogen receptor and retinoid X receptor gene expression in rockfish (Sebastiscus marmoratus)

Since the brain plays important roles in reproduction, the brain aromatase (Cyp19b), estrogen receptor (ER), retinoid X receptor (RXR) α and peroxisome proliferator-activated receptor γ were examined in rockfish after TBT exposure (1, 10, and 100 ng L⁻¹). The results showed that the Cyp19b expression was elevated in the male rockfish, while no effect was produced in the females. Inconsistently, serum testosterone and 17β-estradiol showed no change in the males, while an increase of testosterone and a decrease of 17β-estradiol were observed in the females. TBT affected the ER expression in the males depending on the concentrations, however, no change was observed in the females. In addition, TBT elevated the RXRα expression in the males but produced an opposite effect in the females. In conclusion, TBT might have had sex-different effects on the brain Cyp19b, ER and RXR expression in rockfish, indicating a complex endocrine disrupting effect of TBT.     

Effects of the toxic dinoflagellate Alexandrium tamarense on the greenshell mussel Perna canaliculus

Abstract

Oxygen consumption, grazing rate, and byssus production of the greenshell mussel Perna canaliculus were measured following exposure to bloom concentrations (10⁶ cells l^?1 ) of a toxic clone of the dinoflagellate AIexandrium tamarense (GT429), using anon‐toxic clone of the same species (PLY173) as control. Following feeding for 1 h on GT429, mussels (dry weight 54–127 mg) showed significantly increased oxygen uptake at 15°C (0.08 μl O₂ (mg dry tissue weight)^?1 ) compared with control mussels. However, following 24 h recovery, oxygen uptake was similar to initial values in all experimental conditions. The grazing rate of P. canaliculus fed upon toxic clone GT429 was 4.58 ± 0.76 cells mg^?1 h^?1 compared with 1.99 ± 0.47 cells mg^?1 h^?1 for PLY173. On exposure to A. tamarense, mussels maintained normal opening behaviour and there was no change in the byssus production over 24 h. It was concluded that P. canaliculus showed no dramatic physiological effects following short‐term feeding on the toxic strain of A. tamarense. During 2 weeks’ exposure to twice daily bloom concentrations of GT429, there was no mussel mortality. Toxicity in the tissues was 1295 μg STX equivalent per 100 g tissue, levels at which these mussels would be unsuitable for human or animal consumption.     

A model describing the interactive roles of predation,competition and tidal elevation in structuring mussel populations

The black mussel Choromytilus meridionalis can survive when exposed in air up to 50 per cent of the time, but it is normally restricted to about 30 per cent exposure by extremes of temperature and desiccation. Growth rates vary greatly with location and tidal elevation, but as they grow, the mussels compete intensively for space, consequent mortality rates being proportional to growth rate. Because growth declines with tidal elevation and as terminal size is approached, losses attributable to competition tend to be greatest in lowshore and sublittoral regions and early in life. There are many predators of mussels, each differing in its feeding rate and technique. The quantities and size distributions of C. meridionalis taken by communities of natural predators at selected sites are estimated. Total predator consumption is compared with rates of loss as individual mussels increase in size and compete for space. Fast-growing populations with large terminal sizes can support dense predator communities with little deleterious effect. Slower-growing ones with a smaller adult size have lower rates of competition-induced mortality and remain vulnerable to predators for longer. They are thus more likely to be predator-controlled. Predators such as gastropods (Natica and Nucella species), which preferentially select small mussels and have low consumption rates, have far less impact than those, such as Octopus spp. and birds, with higher energy demands and capable of taking larger mussels subject to less mortality from other causes.