To Tributyltin Research Articles

Imposex development in response to TBT pollution in Hinia incrassata (Ström, 1768) (Prosobranchia, Stenoglossa)

The stenoglossan prosobranch Hinia incrassata, collected along the coast of Brittany and Normandy between 1988 and 1997, exhibits imposex also known as pseudohermaphroditism (the occurrence of male parts in addition to the female genital system) in response to tributyltin (TBT) pollution. Excluding some normal females (stage 0), different imposex stages as defined by Fioroni et al. (1991a)(1a, 1b, 2a, 3a and 4) can be distinguished and have been documented for the first time using scanning electron micrographs. Additional malformations of the genital tract are described. Neither TBT-induced sterilization nor sex change occur in the analyzed samples. TBT accumulation in soft parts was analyzed and bioconcentration factors (BCF) were found to be between 4.68 · 10 4 and 1.22 · 10 5. The VDS (vas deferens sequence) index, RPL (relative penis length) index and the average female penis length (FPL) in populations exhibit a significant dependence on both TBT concentrations in ambient sea water and TBT body burden. An interspecific comparison between imposex intensities in sympatrically living populations of Hinia incrassata, Nucella lapillus and Hinia reticulata reveals that H. incrassata exhibits the lowest TBT sensitivity of the three species. But even in this relatively unsensitive species the threshold concentration for imposex development is below 1.5 ng TBT as Sn l −1. The VDS index is recommended as the most suited biological parameter for effect monitoring purposes because this index is characterized by the lowest deviation of data points from calculated concentration-effect-equations and shows no seasonal variations. Hinia incrassata is proposed for large large-scale TBT surveys in the Mediterranean being planned by OSPARCOM for the North Atlantic. Furthermore the species can be used for TBT temporal trend monitoring. Studies in France, for example, where TBT was partially banned in 1982, show little or no recovery from TBT contamination in H. incrassata and two other prosobranch species between 1989 and 1997.

Effects of tributyltin and hypoxia on the progression of Perkinsus marinus infections and host defence mechanisms in oyster, Crassostrea virginica (Gmelin)

AbstractOysters, Crassostrea virginica (Gmelin), naturally infected by the protozoan parasite Perkinsus marinus in the field were exposed for 6 weeks to tributyltin (TBT), hypoxia, or to both stressors simultaneously. The TBT‐exposed oysters continuously bioaccumulated TBT, reaching about 4 mg kg−−1 dry weight by 6 weeks; hypoxic oysters were exposed to water containing an average dissolved oxygen level of about 3 mg L−−1. Untreated control oysters suffered about 30% cumulative mortality by 6 weeks as a result of the progression of their P. marinus infections. The TBT treatment alone produced no additional mortality; however, cumulative mortality in hypoxic oysters was elevated. Mortality among oysters receiving both TBT and hypoxia significantly exceeded that caused by either stressor alone, suggesting a synergistic effect. In an attempt to identify immunotoxicological mechanisms underlying stress‐related augmentation of P. marinus infections, defence‐related immune functions were measured at 3 and 6 weeks in control and treated oysters. In general, the total number of haemocytes increased as the infections progressed, and the TBT and hypoxic treatments also caused significant additional increments in some samples. However, oxygen‐dependent (reactive oxygen species) and oxygen‐independent (lysozyme) antimicrobial host defence mechanisms appeared to be largely unaffected by TBT and/or hypoxia. This may be explained by the death of those oysters with marked immunological lesions prior to sampling or by the actual lack of treatment effects.

High Tolerance to Tributyltin in Embryos and Larvae of the Horseshoe Crab, Limulus polyphemus

The effects of acute and chronic exposure to tributyltin (TBT) were examined in bioassays using horseshoe crab (Limulus polyphemus) embryos and “trilobite” larvae. Larvae had>95% survival after 24-h exposure to nominal concentrations of 1–500 μg l−1 TBT. Survival was also high following 48-h and 72-h exposure to ≤100 μg l−1 TBT; >50% mortality was seen only after 48-h and 72-h exposure to 500 μg l−1 TBT. Estimated median lethal concentrations (LC50) were >1000 μg l−1, 742 μg l−1, and 594 μg l−1 for 24-h, 48-h, and 72-h exposure, respectively. Much higher toxicity LC50=42 μg l−1) was seen following chronic exposure of larvae to TBT. Acute exposure to TBT significantly increased the time required by larvae to molt into the first-tailed stage. LC50 for horseshoe crab embryos exposed to TBT were 44 μg l−1, 20 μg l−1, and 14 μg l−1 for 24, 48, and 72 h acute exposure, indicating that this earlier developmental stage was about 30–40 fold more susceptible to TBT than larvae. Horseshoe crabs are highly tolerant of TBT in comparison to early developmental stages of other marine arthropods. The ability of horseshoe crab embryos and larvae to survive in the presence of organotin pollution suggests the possibility of bioaccumulation and movement into the estuarine food chain via shorebirds, gulls, and fish.

Evidence of the Differential Sensitivity of Neogastropods to Tributyltin (TBT) Pollution, with Notes on a Species (Columbella Rustica) Lacking the Imposex Response

Five neogastropod species collected from a tributyltin (TBT)-polluted site near the port of Faro within the Ria Formosa on the Algarve coast of Portugal were examined in April 1996 for ‘imposex’. All females of four of these species - Hexaplex trunculus, Ocenebra erinacea, Ocinebrina aciculata (all Muricidae) and Nassarius reticulatus (Nassariidae) - exhibited the characteristic development of male sex organs (penis plus vas deferens) but no female of the fifth - Columbella rustica (Columbellidae) - showed any sign of being similarly masculinised. Only two neogastropods have been previously described as lacking the imposex response to TBT exposure. These two species, along with C. rustica, can be considered as ‘zero-response’ forms. Other species can be grouped according to the maximum level of masculinisation they exhibit: level I species develop just a penis and vas deferens; in level II forms oviduct structure and function are disrupted, and in level III species ovary transformation to testis is observed. Such a comparative scheme of ordering can be used as a guide to the differential sensitivity of species to TBT pollution.

A Mechanistic Approach to Tributyltin (TBT) Sorption by Marine Microflagellated AlgaPavlova lutheri

The sorption of tributyltin (TBT) by phytoplankton cells was studied by exposing the marine microflagellated alga Pavlova lutheri grown in batch culture to TBT chloride concentrations ranging from 0.37 to 74 nM (0.1 to 21 μg l−1) for a 24-hour period. The phytotoxicity of TBT was indicated by a decrease in cell density at all TBT concentrations used. TBT was tightly bound to the surface cell and was only washed out by a strong acidic solution (pH⩽3) while inner cell-absorbed TBT was only recovered by means of an organic solvent. The sorption of TBT by P. lutheri occurred via two mechanisms: a passive ionic surface adsorption followed by a facilitated intracellular absorption. The coupling/uncoupling of these two mechanisms was dependent upon the contamination level used. The cell surface adsorption of TBT was best described by the Freundlich adsorption model whereas the obstruction of the facilitated absorption mechanism was apparently related to the toxic activity of TBT towards cell components. © 1997 John Wiley & Sons, Ltd.

The metabolism of testosterone by the periwinkle ( Littorina littorea) in vitro and in vivo: Effects of tributyl tin

Exposure to tributyl tin (TBT) at concentrations as low as 0.5 ng/liter has been associated with disrupted sexual physiology (imposex) in 40–50 species of marine gastropod. Imposex is a state of pseudohemaphrodism in which females exhibit nonfunctional secondary male characteristics. It has been suggested that the mechanism underlying imposex involves disrupted metabolism of endogenous sex hormones and in particular inhibition of cytochrome P450-dependent aromatization of androgens to estrogens. In the current study, the effects of TBT on the ability of the Periwinkle ( Littorina littorea) to metabolize the androgenic sex steroid testosterone was examined in vitro and in vivo. Microsomes were prepared from ‘digestive gland’ (visceral complex including the gonads) and combined kidney and gill fractions. CO-ligated reduced difference spectra contained peaks at 459 nm and calculated P450 contents of 0.3 and 0.05 nmol/mg. Digestive gland microsomes were found to be capable of oxidative metabolism of testosterone in either the presence or absence of NADPH. In the absence of NADPH, testosterone was metabolized to androstenedione, 6β-, 6α- and 15β-hydroxytestosterone, 17β-estradiol and an unidentified metabolite possibly 3α-androstene-17β-diol. In the presence of NADPH the same products were produced at a higher rate but the major products formed were the products of the NADPH-dependent steroid 5α-reductase-3α(β)hydroxysteroid dehydrogenase pathway; dihydrotestosterone (DHT) and 3α(β)androstane-17β-diols (DHT diols). TBT, even at high concentrations up to 100 μM, had only modest effect on P450-dependent testosterone metabolism in vitro, producing increases in androstenedione formation and only 30–40% inhibition of aromatase. In vivo, [ 14C] testosterone was injected directly into the cephalopedal sinus of adult snails and animals maintained in sea water at 15 °C for 42 h in the presence of 0, 0.5 and 5 mM TBT. Testosterone was almost completely metabolized during this time, predominantly to water-soluble sulfur conjugates of testosterone, the 5α-reduced products and 6α-hydroxytestosterone. However, with increasing concentrations of TBT, more radioactivity was retained within the animal and was increasingly associated with organic extractable unmetabolized testosterone and its phase I products androstenedione, dihydroandrostenedione (DHA), DHT and DHT-diols. Thus it appears that, in vivo, TBT inhibits sulfur conjugation of testosterone and its phase I metabolites and their excretion resulting in a build-up of pharmacologically active androgens in the tissues. This data are consistent with the hypothesis that TBT-induced imposex in sensitive gastropods, such as stenoglossans, may arise from peturbations in sex steroid metabolism. However, the major biochemical targets of TBT appear to be steroid conjugation and excretory transport mechanisms rather than P450-dependent oxidative pathways such as aromatase.

Enhancement of Perkinsus marinus disease progression in TBT-exposed oysters ( Crassostrea virginica)

In vitro exposure of Crassostrea virginica hemocytes to tributyltin (TBT) can inhibit antimicrobial defense mechanisms dependent on reactive oxygen intermediates, as measured by chemiluminescence (CL). However, tissue TBT concentrations attained in this in vivo study (~ 7500 ppb, dry weight) did not inhibit hemocyte recruitment, phagocytosis, CL activity or serum lysozyme. This TBT level was nevertheless effective in enhancing progression and lethality of Perkinsus marinus infections. Immune stimulation was found to be associated with advanced P. marinus infections, as indicated by increased hemocyte recruitment into the circulation and increased CL activity. This played no apparent role in controlling the infection and was insensitive to TBT. Using this battery of standard assays of defense-related functions, the exacerbation of P. marinus infections by TBT could not be attributed to its putative immunosuppresive activity.

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.

An evaluation of the potential of gastropod imposex as a bioindicator of tributyltin pollution in Port Phillip Bay, Victoria

Imposex, the imposition of male sexual characteristics on female marine snails, was used as an indicator of past and present exposure to tributyltin (TBT) in the abundant muricid whelk Thais orbita (Gmelin). Wild populations of Thais orbita were surveyed for frequency and intensity of imposex throughout Port Phillip Bay and at two sites on the adjacent oceanic coast. Imposex indices correlated strongly with proximity to marinas or harbours. Average TBT body burdens for each population were mostly below 10 ng g −1 Sn (as TBT, wet wt) and failed to correlate with imposex indices. Even the highest body burdens were indicative of low contemporary ambient TBT levels. Thais orbita females from a population with negligible levels of imposex were transplanted to two sites where the highest imposex indices were found. Transplants failed to develop a significantly higher frequency or intensity of imposex than controls after 11 weeks. These results confirm that, as with many other gastropod species, imposex in T. orbita is irreversible, and may be found in populations where TBT contamination is no longer present. Nevertheless, controlled transplantation of T. orbita shows promise as a routine bioindicator of contemporary TBT contamination in southern Australia. Imposex in other gastropod species in Port Phillip Bay is described and discussed.

Sensitivity ofselenastrum capricornutum, Daphnia magnaand submttochondrial particles to tributyltin

Abstract The purpose of this study was to evaluate the sensitivity to tributyltin (TBT) of two aquatic organisms belonging to two different levels of the feeding chain: Selenastrum capricornutum alga and Daphnia magna crustacean. The EC50 value at 96 h for Selenastrum capricornutum and the ones at 24 h and 45 h for Daphnia magna with relative confidence intervals and NOEC values were calculated. A partial stimulus effect was observed both for alga and crustacean at very low TBT concentrations. The data obtained were compared both with toxicity TBT values, obtained from bibliography, concerning invertebrates, fishes and algae and with the results obtained by a new in vitro test utilizing submitochondrial particles (SMP). The response of SMP to TBT confirmed that the inhibition of the respiratory functions is one of the mechanisms by which the TBT causes toxicity to intact cells as well as to more complex organisms.

Effects of tributyltin and dibutyltin on the physiological energetics of the mussel, Mytilus edulis

Sublethal physiological responses (clearance rate, oxygen uptake, absorption efficiency and scope for growth) of mussels ( Mytilus edulis) were measured following exposure to tributyltin (TBT) and dibutyltin (DBT). Effects were related to the concentration of TBT and DBT in the mussel tissues. Rate of oxygen uptake increased two-fold with increasing TBT concentration from 0·5 to 10 μg TBT g −1 (dry weight). Clearance (= feeding) rate was significantly reduced above a threshold concentration of 3 to 4 μg TBT g −1. Absorption efficiency was unaffected at concentrations below 10 μg TBT g −1. The overall effect, in terms of scope for growth, indicated a severe inhibition of growth above 4 μg TBT g −1. DBT was shown to be an order of magnitude less toxic than TBT, when expressed in terms of the tissue concentration inducing toxic effects. The threshold concentrations causing a reduction in the rates of feeding and oxygen uptake were 23 and 36 μg DBT g −1, respectively. These sublethal effects are discussed in relation to mechanisms of toxicity, previous alkyltin studies and environmental concentrations.

Differential sensitivity of marine infaunal amphipods to tributyltin

Three species of infaunal gammaridean amphipods, Rhepoxynius abronius (Phoxocephalidae), Eohaustorius washingtonianus, and E. estuarius (Haustoriidae) were tested in a water-only system to assess their sensitivity to tributyltin (TBT) without the influence of factors that could affect bioavailability. When mortality (LC50) was the endpoint, the results indicated that R. abronius was ≈20 times more tolerant to tributyltin than either haustoriid species; however, when mortality plus reburial behavior (EC50) was assessed, the difference was only about 10 times. The bioconcentration factor (BCF) was also consistently lower in R. abronius (11.1 to 16.5 times) than in the haustoriids; however, when the LD50 was calculated, the concentration in the tissues associated with 50% mortality for each species was not significantly different. The large disparity in species' response is attributed to reduced uptake and a potentially greater ability to metabolize this compound by R. abronius. An analysis of TBT uptake confirmed that R. abronius was able to accumulate less TBT and hence maintain a low body burden for a given water concentration. The results of a separate uptake study were used to formulate a hypothesis for observed differences in reburial behavior. Because the rate of TBT uptake was lower in R. abronius, we propose that the slower rise in toxicant body burden allowed for a gradual response in this species which included a sublethal effect (non reburial), compared to a rapid rise in the body burden for E. estuarius which caused the response to quickly proceed from no effect to death.

The morphological expression of imposex in Hinia reticulata (Gastropoda: Buccinidae): a potential indicator of tributultin pollution

Specimens from the prosobranch Hinia reticulata collected along the coast of Brittany and Normandy from 1988 to 1991 exhibited imposex (occurrence of male parts in addition to the female genital duct) in response to tributyltin (TBT) pollution. Four stages of imposex development (1 to 4) with two different types in the Stages 1, 3 and 4 could be distinguished and have been documented with scanning electron micrographs for the first time. Furthermore, three additional alterations of the genital tract are shown. Neither TBT-induced sterilization nor sex change occurred. TBT accumulation in whole body and selected tissues is described and sex-related differences are shown. The VDS (vas deferens sequence) index, cubed and uncubed RPS (relative penis size) index and average female penis length of a population were analysed with regard to their quality as indices for TBT biomonitoring. Based on the way imposex develops in this species the VDS is recommended as the most valid index. Only in highly polluted areas should the uncubed RPS be used as a second index.

The reversibility of tributyltin-induced toxicity in vitro as a function of concentration and duration of exposure ( C × T)

The toxicity exhibited by murine erythroleukemic cells (MELC) exposed to tributyltin (TBT) is a function of both concentration (C) and duration of exposure (T). At or above a critical C x T product value (CPV) (e.g., 0.5-1.0 microM TBT, 6 hr), exposed MELC exhibit severe, irreversible toxicity: decreased membrane integrity (viability, measured by propidium iodide [PI] exclusion), grossly perturbed cell-cycle distributions, and fixation of the plasma membrane/cytoplasm complex. Below the CPV, exposed cells exhibit retention of carboxyfluorescein (CF) fluorescence (indicative of decreased plasma membrane permeability) and decreased cell proliferation, a result of retardation of progression into, through, and out of the S (DNA synthetic) phase of the cell cycle. However, following washout and recovery, mean CF fluorescence, cell proliferative capacity, and cell-cycle kinetics return to control levels. These results suggest that the toxic changes induced by TBT exposure may be reversible if exposure conditions do not exceed the CPV. To assess whether the CPV has been exceeded, a multiparameter flow cytometric analysis of membrane integrity and cell-cycle kinetics is useful.

Hinia reticulata and Nucella lapillus. Comparison of two gastropod tributyltin bioindicators

Hinia reticulata and Nucella lapillus (collected from 1989 to 1991 at Pointe de Pleneuf and Mean Melen, France, respectively) exhibit imposex in response to tributyltin (TBT) pollution leached from antifouling paints and proved to be good TBT bioindicators. H. reticulata was kept for 18 mo under TBT-free conditions in the laboratory, but no evidence for imposex remission was found. A second series of experiments showed that food chain uptake of TBT from a contaminated diet is an important mode of TBT exposure in H. reticulata. Comparative tank experiments with H. reticulata and N. lapillus demonstrated that the same type of TBT exposure resulted in comparable TBT body burdens, biological concentration factors, and imposex development in both species [measured as increase of VDS (vas deferens sequence index), uncubed RPS (relative penis size index) and average female penis length]. Differences in imposex development of natural and laboratory populations are discussed against the background of different types of TBT contamination of their food. A statistical study, based on an analysis of natural populations of both prosobranch species, makes a comparison of the specific TBT sensitivity of the two bioindicators possible. As a consequence it is proposed that TBT biomonitoring programs in Europe should use both prosobranchs as indicator species.

Tributyltin-resistant bacteria from estuarine and freshwater sediments

Resistance to tributyltin (TBT) was examined in populations from TBT-polluted sediments and nonpolluted sediments from an estuary and from fresh water as well as in pure cultures isolated from those sediments. The 50% effective concentrations (EC50s) for populations were higher at a TBT-polluted freshwater site than at a site without TBT, suggesting that TBT selected for a TBT-resistant population. In contrast, EC50s were significantly lower for populations from a TBT-contaminated estuarine site than for those from a site without TBT, suggesting that other factors in addition to TBT determine whether populations become resistant. EC50s for populations from TBT-contaminated freshwater sediments were nearly 30 times higher than those for populations from TBT-contaminated estuarine sediments. We defined a TBT-resistant bacterium as one which grows on trypticase soy agar containing 8.4 microM TBT, a concentration which prevented the growth of 90% of the culturable bacteria from these sediments. The toxicity of TBT in laboratory media was influenced markedly by the composition of the medium and whether it was liquid or solid. Ten TBT-resistant isolates from estuarine sediments and 19 from freshwater sediments were identified to the genus level. Two isolates, each a Bacillus sp., may be the first gram-positive bacteria isolated from fresh water in the presence of a high concentration of TBT. There was a high incidence of resistance to heavy metals: metal resistance indices were 0.76 for estuarine isolates and 0.68 for freshwater isolates.(ABSTRACT TRUNCATED AT 250 WORDS)

Continuing impact of TBT, previously used in mariculture, on dogwhelk (Nucella lapillus L.) populations in a Scottish Sea Loch

Abstract The degree of development of a penis and vas deferens in adult female dogwhelks as a result of exposure to tributyltin (TBT) compounds, and concentrations of total tin in homogenised soft parts of these individuals, have been investigated for three consecutive years (1986–1988) in a Scottish sea loch where TBT anti-foulants were used in mariculture until 1987. Results of these surveys, which included the examination of sub-adults and juveniles, and of adults transplanted from a site with low imposex levels to two sites within Loch Laxford, have indicated that, even though the use of TBT paints in mariculture was prohibited in the UK in 1987, low residual concentrations of TBT were still present in the loch during 1988–1989. A mathematical model describing the penis-length-shell-length relationships in individuals of all size ranges, from selected populations in 1987 and 1988, has shown that penis growth was stimulated in males, as well as females, in response to exposure to TBT; and that there was a smaller degree of imposex development in the sub-adult and juvenile dogwhelks in 1988, than in 1987. The decrease in imposex development suggests that although TBT was present in 1988, the concentrations were lower than in the previous year, suggesting that the 1987 legislation curtailing the use of TBT in mariculture was beginning to be reflected in the imposex condition of dogwhelk populations.

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

Abstract 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.

TBT-induced imposex in the dogwhelk, Nucella lapillus: Geographical uniformity of the response and effects

The imposex response of the dogwhelk, Nucella lapillus, to tributyltin (TBT) pollution has been investigated in adults taken from four UK populations geographically separated over 10° of latitude. Exposure to TBT water concentrations of c.1 and 17 ng Sn/litre over a period of 12 weeks resulted in similar levels of accumulation of TBT within the body tissues and similar increases in female penis length in all four populations. The features are imposex in N. lapillus populations in south-west Brittany are described; they follow the same pattern as found in earlier surveys of the southern England coast, including sterilisation of females, population decline and disappearance of the species close to TBT sources. The evidence of these two investigations indicates that the level of sensitivity and development of imposex as a response to TBT pollution is uniform in N. lapillus throughout its distributional range.

Reproductive abnormalities in female Ocenebra erinacea (Gastropoda) resulting from tributyltin-induced imposex

Imposex (the development of male sex organs on the female) has been studied in the European sting winkle, Ocenebra erinacea. In common with studies of other gonochoristic stenoglossan gastropods, the evidence gained from surveys of the south-west regions of England and Brittany (France) indicates that the phenomenon is a response to tributyltin (TBT) pollution. Close to sources of TBT, O. erinacea females exhibit structural abnormalities of the oviduct as a result of advanced imposex, including absence of a genital papilla and a normal vulva, a deformed bursa copulatrix and the presence of a longitudinal split. Copulation and capsule formation would appear to be inhibited in the worst-affected females. Such curtailment of breeding activity would result in population decline: direct evidence of the extinction of O. erinacea close to harbours, as is known for N. lapillus, is generally wanting for British shores, but the decline of the species in the Bay of Arcachon in France is attributable to TBT pollution.