Effects of tributyltin (TBT) and testosterone on the female genital system in the mesogastropodLittorina littorea (Prosobranchia)

Experiments were performed with the mesogastropodLittorina littorea on Helgoland, in Roscoff, and in the laboratory in order to evaluate the reaction of the female genital system to TBT, an environmental toxicant. The snails were either injected with 50 or 100 ng tributyltin (TBT) soluted in ethanol or exposed to artificial sea water treated with 5, 50, 100, and 200 ng TBT/l, and 33 ng testosterone/l. The duration of the experiments was either four or eight weeks. None of the analysed femaleL. littorea showed signs of imposex. Compared to results for the control groups, the size of the female glandular complex was significantly reduced if the pre-experimental toxication was already high, as is the case in snails collected around Helgoland. TBT-related gland complex reduction occurs also in femaleL. littorea from other sampling sites. In addition, injection of ethanol also causes a decrease in gland size. The experimental results demonstrate that the distal female genital system responds with significantly lower sensitivity to TBT than that of other prosobranchs. This behaviour is ascribed to the lack of an androgen receptor at the ovipositor. The results further strengthen the case of the extreme rarity of imposex described for femaleL. littorea in natural habitats. However, strong TBT-toxication may affectL. littorea populations significantly because of increasing masculinization of the females, which reduces reproduction ability.     

TBT effects on the female genital system of Littorina littorea: a possible indicator of tributyltin pollution

Specimens of the prosobranch Littorina littorea (L., 1758) collected along the East Frisian North Sea coast in summer 1993 exhibited alterations of the pallial oviduct termed as intersex in response to tributyltin (TBT) pollution. The range of TBT body burden was between 150.9 and 1289.5 μg as Sn kg⁻¹ (dry wt.). Five stages of intersex development (0–4) could be distinguished and are documented with scanning electron micrographs. In stages 2–4, which can be found in the direct vicinity of harbours and marinas, the morphological malformations of the oviduct inhibit successful copulation and capsule formation, resulting in sterilization. The intersex index (ISI, calculated as the average intersex stage of a population) and the average prostate length of females were used as parameters for the determination of intersex intensities in the populations. Both indices show significant and positive correlations to the TBT body burden of L. littorea and are promising parameters for TBT biomonitoring. A comparison of TBT bioconcentration factors with populations from England and France indicates that the threshold concentration for intersex development is in the range of 15 ng TBT as Sn/l. Morphometric analyses of the midgut gland revealed no significant differences between sampling stations. In the ovary a retardation and blockage of maturation (atresia) was observed in populations close to harbours. Lytic processes in ovary follicles were observed not only at TBT exposed sites but also at reference stations.     

三丁基锡(TBT)对罗氏沼虾的毒性效应

将罗氏沼虾(Macrobrachium rosenbergii)幼虾和成虾暴露在不同浓度的三丁基锡(tributyhin,TBT)溶液中,进行急性和慢性毒性实验,通过组织病理学观察研究TBT对罗氏沼虾的毒性效应。结果表明:TBT对罗氏沼虾幼虾和成虾的96h半致死浓度(96h LC50)分别为51和376μg.L-1,安全浓度(SC)分别为5.1和37.6μg.L-1;罗氏沼虾幼虾暴露在浓度分别为1.25、2.5和5μg.L-1的TBT溶液中30d后,其体重和体长均极显著低于对照组(P<0.05);罗氏沼虾成虾暴露在浓度分别为2、8和32μg.L-1的TBT溶液中30d后,生长速度虽未见明显差异,但鳃和肝胰脏组织结构均出现异常;TBT浓度高于8μg.L-1时,处理组鳃上皮细胞肿胀,支持细胞中的细胞核萎缩,鳃血窦中血细胞聚集,肝胰脏细胞肿大,出现空泡化,细胞中出现细小颗粒;TBT浓度达32μg.L-1时,罗氏沼虾肝胰脏细胞空泡化程度严重,细胞核消失,部分细胞解体并出现坏死区;表明一定剂量的TBT对罗氏沼虾的鳃和肝胰脏有明显的毒性效应。     

Cellular effects of tributyltin (TBT) on the penis epithelium cells of prosobranchs (Hinia reticulata andOcinebrina aciculata)

Cytopathological effects on organelles of penis epithelium cells were investigated in prosobranchs that had been exposed for two weeks to three months to high TBT-concentrations in artificial seawater. TBT exposure damaged cell organelles, such as mitochondria, Golgi dictyosomes, endoplasmatic reticulum, and injured the cell membranes. In addition, atypical intercellular spaces were observed between the cells of the epithelial layer. Further cell alterations included the increase of residual bodies within the cells as well as structural changes of the basal lamina. The ultrastructural changes were compared with cell alterations of specimens which had been collected in a polluted environment on the coast of Brittany (France).     

Hermit crabs as bioindicators of recent tributyltin (TBT) contamination

Evaluation of the assimilation pathway and depuration time of a given pollutant by aquatic species is important to understand the dynamics of this substance in the biota, and to search for potential ecological indicators. In the present study, the uptake pathway and depuration time and rate of the pollutant tributyltin (TBT) were investigated in the omnivorous hermit crab (Clibanarius vittatus). The assimilation and uptake pathway were investigated using hermit crabs collected in an area free of TBT. The crabs were held in the laboratory for 45 days, under one of four treatments: procedural control (PC) - water and food without TBT; T1 - water with and food without TBT; T2 - water without and food with TBT; and T3 - water and food with TBT. To determine the depuration time, the crabs were collected in a contaminated area, maintained in the laboratory with clean water, and removed every 15 days for 120 days. The concentrations of TBT and DBT (dibutyltin) were determined by chromatographic analysis. The TBT was taken up by the crabs mainly via food, and the presence of DBT in crab tissues was hypothesized to result from internal TBT degradation. TBT (as well as DBT) was depurated rapidly by C. vittatus. After approximately 30 days, the initial concentration of 111 ± 36 ng Sn g⁻¹ w. w. decreased to 3 ± 3 ng Sn g⁻¹ w. w., and after 75 days the TBT concentration was below the detection limit. The same pattern was recorded for DBT, which showed a higher depuration rate than TBT. The rapid TBT and DBT depuration is useful information, since C. vittatus and possibly other hermit crabs may be used as indicators of recent or recycled environmental contamination.     

Mate discrimination in Littorina littorea (L.) and L. saxatilis (Olivi) (Mollusca:Prosobranchia)

The ability of males of Littorina littorea and L. saxatilis to discriminate between mates of different sex, species and size was examined. In partner choice experiments males of L. littorea had the possibility to initiate a copulation with either a female or a male. The males did not show a preference for either sex. There was therefore no evidence that they could determine the sex of a conspecific prior to copulation. The duration of intrasexual copulation was considerably shorter than for intersexual copulation, both in the field and in laboratory experiments. For the two species, intersexual copulations were far more frequent than intrasexual ones. This can partly be explained by the difference in copulation time.Few interspecific copulating pairs were found on the shore. This may reflect a low interspecific encounter rate rather than a mechanism of species recognition. On all of these occasions, however, the active male was of L. saxatilis. It is argued that selection against precopulatory species and sex recognition is a more likely explanation than an hypothesis that states that the required mutations for precopulatory mate identification has not yet occurred. L. littorea males copulated longer with large than with small females. Copulation time was short with parasitized females, which are sterile or of low fecundity. The allocation of mating effort by males is discussed.     

The veliger larva of Hydrobia ulvae with observations on the veliger of Littorina littorea (Mollusca: Prosobranchia)

The veliger larva of Hydrobiu ulvue (Pennant) from the west coast of Wales is described and illustrated from veligers hatched in the laboratory and collected in plankton samples. The larvae are planktotrophic and it is estimated that the pelagic phase is of about four weeks duration. In terms of density they form an important constituent of the plankton of inshore waters. The veliger larva of Littorina littoreu (L.) is also described and illustrated, and it is suggested that some of the conflicting reports on the length of the pelagic phase of H. ulvae result from confusion in indentification of the larvae of these two species.     

Rediae of echinostomatid and heterophyid trematodes suppress phagocytosis of haemocytes in Littorina littorea (Gastropoda: Prosobranchia)

A modulation of the phagocytic activity of hemocytes from the common periwinkle Littorina littorea by secretory-excretory products (SEP) released by trematode rediae during axenic in vitro cultivation was studied. The SEP released by the parasites Himasthla elongata (Echinostomatidae) and Cryptocotyle lingua (Heterophyidae) were found to inhibit the phagocytosis of zymozan particles by periwinkle hemocytes. The specificity of SEP effects was assessed: SEP of Himasthla militaris and Cryptocotyle concavum, two trematodes belonging to the same genera but infecting another closely related prosobranch snail Hydrobia ulvae, were also shown to be able to suppress L. littorea hemocytes phagocytic activity. However, no decrease in phagocytosis rate was observed when SEP of H. elongata and C. lingua were applied to monolayers of hemocytes from the bivalve mollusc Mytilus edulis. SEP from H. elongata was fractionated; only those fractions containing proteins of molecular weight more than 50 kDa were shown to possess inhibitory activity. Different H. elongata SEP concentrations were tested in for their ability to suppress phagocytosis by L. littorea hemocytes. Even very low SEP concentrations were shown to retain their ability to decrease phagocytosis rate, the inhibitory effect being dose-dependent. Hemocytes derived from snails naturally infected with H. elongata were also found to have lower phagocytic ability as compared to healthy individuals.     

Phospholipids and protein adaptation of Pseudomonas sp. to the xenoestrogen tributyltin chloride (TBT)

A tributyltin (TBT)-resistant strain of Pseudomonas sp. isolated from an overworked car filter was tested for its adaptation to TBT. The isolate was checked for organotin degradation ability, as well as membrane lipid and cellular protein composition in the presence of TBT. The phospholipid profiles of bacteria, grown with and without increased amounts of TBT, were characterized using liquid chromatography/electrospray ionization/mass spectrometry. The strain reacted to the biocide by changing the composition of its phospholipids. TBT induced a twofold decline in the amounts of many molecular species of phosphatidylglycerol and an increase in the levels of phosphatidic acid (by 58 %) and phosphatidylethanolamine (by 70 %). An increase in the degree of saturation of phospholipid fatty acids of TBT exposed Pseudomonas sp. was observed. These changes in the phospholipid composition and concentration reflect the mechanisms which support optimal lipid ordering in the presence of toxic xenobiotic. In the presence of TBT the abundances of 16 proteins, including TonB-dependent receptors, porins and peroxidases were modified, which could indicate a contribution of some enzymes to TBT resistance.     

An easy,rapid and inexpensive method to monitor tributyltin (TBT) toxicity in the laboratory

Tributyltin (TBT) contamination remains a major problem worldwide. Many laboratories are committed to the development of remediation methodologies that could help reduce the negative impact of this compound in the environment. Furthermore, it is important to have at hand simple methodologies for evaluating TBT toxicity in the laboratory, besides the use of complex and costly analytical instrumentation. With that purpose, a method was adapted that is based on the inhibition of growth of an indicator strain, Micrococcus luteus ATCC 9341, under TBT. Different types of matrices, of TBT concentrations and sample treatments were tested. The results herein reported show that the bioassay method can be applied for both aqueous and soil samples and also for a high range of TBT concentrations (at least up to 500 μmol/L). Besides being cheap and easy to perform, it can be performed in any laboratory. Additionally, one possible application of the method to monitor TBT degradation is presented as an example.     

The toxic effects of bis (tributyltin) oxide on the rat thoracic aorta.

The toxic effects of bis (tributyltin) oxide (TBTO) on the ultrastructure and permeability of rat thoracic aorta were studied electron microscopically and the accumulation sites of tin were determined with an X-ray microanalyzer. Male Wistar rats received 0.05ml/kg of TBTO as an emulsion in 1 ml of distilled water through a stomach tube. After time intervals of 2, 4, 6, 8, 10, 12 h after intubation, thoracic aortae were isolated and prepared for electron microscopy. Marked swelling of mitochondria in the aortic endothelial cells appeared at 4 h after TBTO treatment. By x-ray microanalysis, tin L-alpha peaks (3.44 keV) were obtained from these swollen mitochondria. Subendothelial edema progressed between 6 and 8 h after TBTO treatment. By tracer experiment, it was seen that large amounts of peroxidase reaction products filled the expanded subendothelial space. At 12 h after TBTO treatment, degenerative changes of the endothelial cells were prominent. These results indicated that orally administered TBTO accumulated in the mitochondria of the endothelial cells of thoracic aorta. The direct toxic effects of TBTO on mitochondria might induce severe damage to the endothelial cells and cause disturbance of the permeability barrier function of the endothelial layer and subendothelial edema.     

Aeromonas molluscorum Av27 is a potential tributyltin (TBT) bioremediator: phenotypic and genotypic characterization indicates its safe application

Aeromonas molluscorum Av27 is an estuarine bacterium highly resistant to tributyltin (TBT). Also, the strain is able to degrade TBT into the less toxic compounds dibutyltin and monobutyltin. Therefore, this bacterium has potential to be employed in bioremediation processes. In this context, defining its biological safety is crucial. With that purpose a number of intrinsic characteristics, usually present/associated with virulent strains, were investigated. Few virulence factors were detected in strain Av27. For instance, a DNase gene is present, but it is not apparently expressed in vitro. Motility, adherence factor and phospholipase activity were also detected. Additionally, cytotoxicity to Vero cells was negative. Resistance to penicillin (10 μg ml^?1), amoxicillin/clavulanic acid (30 μg ml^?1) and cephalothin (30 μg ml^?1) and also to the vibriostatic agent O/129 was observed. Five plasmids (4, 7, 10, 100 kb and one greater than 100 kb) were identified. No Class I and II integrons were detected. Study of the optimal growth conditions showed that Av27 easily adapts to different environmental conditions. Overall, the results suggest that A. molluscorum Av27 can be considered safe to use to bioremediate TBT in contaminated environments.     

Rhythmic egg-release in Littorina littorea (Mollusca: Gastropoda)

Batches of female Littorina littorea from four estuarine sites were kept in aquaria throughout their breeding season (December to July), some permanently submerged in circulating sea-water and others in a regime of simulated semi-diurnal tides of constant small amplitude. Females without the tidal experience released few eggs with little relation to the lunar month. Those in the tidal regime released more than four times the total number of eggs and released far more in periods of new and full moon than in periods of half-moon. Over the whole season, release was 1–5–3–25 times faster (at different sites) during full and new moon but the difference was more accentuated at the height of the season in February to early March and in early May. While individual females differed considerably and there were minor variations between the four sites, it is concluded that a two-weekly rhythm relates egg release to the spring-neap tide cycle and that this rhythm requires a tidal cycle of immersion and emersion for its maintenance. The special significance of such a rhythm for a species with pelagic eggs and larvae in an estuary is discussed.     

Revision of the Arctic-Atlantic Rissoidae (Gastropoda, Prosobranchia)

The present paper contains a revision of the Arctic-Atlantic Rissoidae (Gastropoda, Prosobranchia). Twenty-two species are recognized. Two new species, Alvania obliqua and Alvania pseudoareolata are described, both previously wrongly identified. One new genus, Frigidoalcania , is established with Rissoa janmayeni as type-species.