Tri-n-butyltin)oxide Research Articles

Immunotoxicity of tri‐n‐butyltin oxide (TBTO) and tri‐n‐butyltin chloride (TBTC) in the rat

In a 1-month feeding trial, pure and commercial tri-n-butyltin oxide (TBTO) and tri-n-butyltin chloride (TBTC) were fed to rats at concentrations of 5 ppm and 25 ppm. At all times, the mean body weight gain and the food consumption was significantly less in rats treated with 25 ppm pure TBTO or pure TBTC as compared to control rats or rats receiving commercial TBTO. Histological examination of the thymus of rats treated for 7 days with TBTO showed atrophy with severe lymphocytic depletion in the cortex. After 28 days of exposure, most of the lesions reversed and the thymus became markedly smaller than in control rats, both in absolute terms and in relation to body weight. Seven days of exposure to TBTO increased liver weight but this change was reversed during a further 3-week exposure. Tin concentrations were the highest in livers and kidneys. Concentrations in the thymus were less than one-fifth of hepatic values. Changes in the rats treated with the commercial TBTO were very similar. Rats treated with TBTC showed lower tin levels and less immunotoxicity as compared to those treated with TBTO.

Immunotoxic1ty of bis(tri‐n‐butyltin) oxide in the rat

Previous studies in the rat have shown that bis(tri-n-butyltin) oxide (TBTO), used as a biocide, was immunotoxic at dose levels that did not affect other organs. In order to determine a no-effect level, weanling rats were treated for at least 28 consecutive days with TBTO at 0, 0.5, 2, 5, or 50 mg/kg of diet. Studies on clinical chemistry, hematology, pathology, and immune function, that is, plaque-forming cell (PFC) assay, delayed-type hypersensitivity (DTH) response, and the splenic clearance of Listeria monocytogenes, were performed at the end of treatment. No treatment-related effects were noted on clinical chemistry and hematology parameters and on PFC and DTH response, whereas thymic atrophy and impaired clearance of L. monocytogenes were noted only at a dietary concentration of 50 mg/kg. These results confirm the thymus as a target organ of TBTO immunotoxicity. Under the conditions of these experiments the dietary concentration of 5 mg/kg, equivalent to a dose of 0.5 mg/kg body weight, represents a no observed effect level (NOEL) for immunotoxicity in the Sprague-Dawley rat.

Contact sensitivity (allergic contact dermatitis) to bis(tri‐n‐butyltin) oxide in mice

Contact sensitivity (allergic contact dermatitis) to bis(tri-n-butyltin) oxide (TBTO) was demonstrated in the mouse. TBTO (in an acetone:olive olive oil vehicle) or acetone:olive oil alone (as a control) were applied to the shaved flank under an occlusive patch and animals were challenged on the dorsum of the ear 6 days later. Ear swelling was then measured using an engineer's micrometer, 2, 24, 48 or 72 h thereafter. Significant differences in ear swelling between control and TBTO-sensitized animals were found 24 h after challenge: thereafter the elicitation reaction declined rapidly whilst irritant swelling in control animals increased. Maximal elicitation of TBTO sensitivity could only be elicited by concentrations of TBTO that caused irritation. In a separate experiment, a single application of TBTO to the ears of naive animals provoked increase in auricular lymph node weight, cell yield and proliferation of lymph node cells during overnight culture. These data support the conclusion that TBTO is a contact sensitizer and illustrate the potential usefulness of the quantitative methods of contact sensitivity assessment which have been developed in the mouse.

Bis(tri-n-butyltin)oxide induces programmed cell death (apoptosis) in immature rat thymocytes

In order to characterise the mechanism of cytotoxicity of the immunotoxic organotin compound bis(tri-n-butyltin)oxide (TBTO) to lymphoid cells, isolated thymocytes from immature rats were exposed to TBTO (0.1-5 microM) for up to 6 h. At lower TBTO concentrations (0.1 and 1 microM) vital staining showed that only marginal loss of viability occurred, although morphological studies demonstrated increased numbers of cells with abnormal features indicative of programmed cell death (apoptosis). These changes included nuclear chromatin condensation (which was associated with increased DNA fragmentation), cytoplasmic contraction and formation of membrane bound apoptotic bodies. When visualised by agarose gel electrophoresis, genomic DNA appeared as a series of fragments with a repeat multiple of 180-200 base pairs. Comparable morphological changes and cleavage of DNA into oligonucleosomal fragments were evident in thymocytes incubated with 10 microM methyl prednisolone hemisuccinate (MPS); a glucocorticoid hormone known to induce programmed cell death in thymocytes. Marked cytotoxicity associated with degenerative changes indicative of necrosis was observed in thymocytes incubated with 5 microM TBTO. These findings indicate that, at levels which are not overtly cytotoxic, TBTO is capable of inducing programmed cell death in rat thymocytes. This suggests a possible mechanism for the T-cell immunodeficiency previously reported for TBTO in vivo.

Reversibility of thymic atrophy induced by 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) and bis(tri- n-butyltin)oxide (TBTO)

We studied the reversibility of thymic atrophy induced by intubation of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), 10 days after a single dose of 50 μg/kg, or bis(tri- n-butyltin)oxide (TBTO), 4 days after a single dose of 75 mg/kg. This was done by an experimental design in which the atrophic thymus was placed in an in vivo situation in which the toxic chemical was no longer present, e.g. by transplantation of atrophic thymic lobes in untreated normal rats with connection to the vasculature of the recipient. At 20 days after the transplantation, the atrophic thymus showed the morphology and architecture of a normal uninvoluted thymus: lymphocyte counts and phenotypic expression of markers on lymphocytes, epithelium, and macrophages in the transplanted lobe did not differ from those in untreated donor rats or those in the normal uninvoluted thymus. Considering the mechanism of action of the toxic chemicals, TBTO has been claimed to affect preferentially (passenger) lymphocytes in the thymus: the recovery after transplantation therefore is explained on the mere influx of newly-recruited precursor cells from the bone marrow. For TCDD a toxic action on the stationary epithelial component of the thymus has been claimed. We conclude that this epithelial damage is reversible within the 3-week period of the present experiment, with respect to both the morphology and immunologic phenotype of epithelium and other cell populations, as well as the recruitment of lymphocytes.

Structural characterization of sulphur compounds in petroleum fractions by 119Sn nuclear magnetic resonance spectrometry

A method has been developed for the identification of mercaptans in petroleum fractions by reaction with trialkyltin reagents. The derivatives formed have been characterized and quantified by 119Sn n.m.r. spectrometry. Sulphides in petroleum fractions were first reduced to thiols by calcium in liquid ammonia. The thiols were then derivatized with bis(tri-n-butyltin) oxide (TBTO) and characterized in situ by 119Sn n.m.r. spectroscopy. This study also shows that a unique chemical shift may be attributed to each class of sulphydryl-containing compound.

Immune alterations in rats following subacute exposure to tributyltin oxide

Adult male Fischer 344 rats were dosed by oral gavage with bis(tri- n-butyltin)oxide (TBTO) in peanut oil for 10 consecutive days, at dosages ranging from 1.25 to 15 mg/kg/day. Other groups of rats were dosed daily for 10 days by oral gavage with cyclophosphamide (CY) at dosages ranging from 0.75 to 6 mg/kg/day. These rats served as positive controls for the immune assays employed. The immune function parameters examined included the following: delayed-type hypersensitivity (DTH) and antibody responses to bovine serum albumin (BSA), primary antibody responses to sheep red blood cells (SRBC) and trinitrophenyl lipopolysaccaride (TNP-LPS) and enumeration of splenic lymphocyte populations. The DTH and antibody responses to BSA were not affected by TBTO exposure; however these responses were suppressed in rats dosed with CY at 6 g/kg/day. The plaque forming cell (PFC) response to the T cell-dependent antigen SRBC was enhanced in rats dosed with TBTO at from 5 to 15 mg/kg/day. On the other hand, the PFC response to the T cell-independent antigen TNP-LPS was unaffected by TBTO exposure. Rats dosed with CY had suppressed PFC responses to SRBC and TNP-LPS at dosages of 3 and 6 mg/kg/day, respectively. Enumeration of splenic lymphocyte populations from TBTO-exposed rats revealed a reduction in OX8- but not W3/25- or IgG-positive cells. These results, as well as results from an earlier study from this lab, suggest that T lymphocytes are a primary target for TBTO-induced immune alterations and that the enhancement of the PFC response to SRBC in TBTO-exposed rats may be mediated by alterations in the suppressor (OX8-positive) T lymphocyte population.

Immunotoxicity of Bis(tri- n-butyltin)oxide in the rat: Effects on thymus-dependent immunity and on nonspecific resistance following long-term exposure in young versus aged rats

To investigate whether immune function suppression observed in an earlier study after short-term bis(tri- n-butyltin)oxide (TBTO) exposure also occurred after long-term treatment, function studies for specific and nonspecific resistance were performed after exposure of weaned male rats to diets containing 0, 0.5, 5, or 50 mg TBTO/kg for 4–6 and 15–17 months. Treatment for 4.5 months had no effect on body weight but reduced thymus weight at 50 mg/kg. Regarding the thymus-dependent immunity, delayed-type hypersensitivity reactions to ovalbumin and tuberculin were not depressed, in contrast to the results of the short-term study. The resistance to the nematode Trichinella spiralis was dose-relatedly suppressed at the 5 and 50 mg/kg levels, in both experiments (5.5 and 16.5 months exposure), as shown by increased counts of muscle larvae and depressed serum IgE titers. Also the inflammatory reaction around cysts in parasitized musculature was reduced. No significant reduction was found in IgM and IgG titers to T. spiralis, ovalbumin, and sheep red blood cells as determined by enzyme-linked immunosorbent assay. TBTO exposure at 50 mg/kg for 4.5 months significantly reduced thymus weight, but the response of thymocytes to T-cell mitogens was unaltered. TBTO treatment for 4.5 or 16 months did not influence the response of spleen cells to T- and B-cell mitogens and neither influenced spleen weight. A dose-related shift was observed in T- and B-cell numbers in mesenteric lymph nodes as shown by flow cytometry using monoclonal antibodies: treatment for 6 and 18 months reduced the relative count of T-lymphocytes and consequently increased the percentage of B-lymphocytes. As a result, the T:B ratio was reduced in the 5 and 50 mg/kg groups. Concerning the nonspecific resistance, TBTO exposure for 5 and 17 months reduced macrophage function at 50 mg/kg as shown by impaired splenic clearance of Listeria monocytogenes bacteria. Natural cell-mediated cytotoxicity of spleen and peritoneal cells was investigated in a 51Cr-release assay with YAC-lymphoma target cells. TBTO treatment significantly suppressed natural killer (NK) activity in spleen cells. Significant suppression was noted in all treatment groups following 16 months TBTO exposure; in contrast to treatment for 4.5 months. No significant alterations were observed in the spontaneous cytotoxicity of nonadherent and adherent peritoneal cells following 4.5 months treatment. Treatment of aged (i.e., 1-year-old) male rats for 5 months with the 50 mg/kg diet reduced thymus weight but had no effect on body or spleen weight. At this dose level the resistance to T. spiralis was suppressed as shown by a retarded expulsion of adult worms from the small intestine and increased counts of muscle larvae. Also the splenic clearance of L. monocytogenes bacteria was reduced at the 50 mg/kg dietary level. It is concluded that long-term TBTO exposure of young and aged rats suppressed host resistance to a bacterial ( L. monocytogenes) and especially to a parasitic ( T. spiralis) infection, although the effects in aged rats were less pronounced. These data demonstrate the sensitivity of these challenge models since alterations were detected at dose levels at which no other effects were found.

Embryotoxic evaluation of bis (tri- n-butyltin)oxide (TBTO) in mice

Pregnant mice were treated on days 6–15 of gestation with 5, 20 and 40 mg/kg/d bis (tri- n-butyltin)oxide (TBTO), and sacrificed on gestational day 17. At the highest dose TBTO caused a significant reduction of maternal body weight gain and also proved to be highly embryotoxic. Necropsy showed a dose-related decrease in spleen weight while a dose-dependent increase in placental weight was observed.

The toxicity of bis(tri- n-butyltin)oxide (TBTO) and di- n-butyltindichloride (DBTC) in the small fish species Oryzias latipes (medaka) and Poecilia reticulata (guppy)

Toxicity studies were carried out on the Japanese medaka ( Oryzias latipes) with bis(tri- n-butyltin)oxide (TBTO) and di- n-butyltindichloride (DBTC), with emphasis on histopathological effects. Freshly fertilized eggs were exposed for 1 and 3 months to a concentration range of 0.1–32 μg TBTO/l in tank water. The range for DBTC was 320–3200 μg/l (1 month only). The histopathological effects included vacuolation of hepatocytes, tubulonephrosis and glomerulopathy, vacuolation of the retinal pigment epithelium, keratitis, hyperplasia and inflammation of oral and skin epithelium, hyperplasia and swelling of the gas gland epithelium and thyroid activation. No atrophy of the thymus was found. Except for the retinal and liver lesions, these changes occurred predominantly in the highest concentration with no essential differences in the lesions between 1 and 3 months, or between TBTO and DBTC exposure. Liver cell vacuolation was apparently caused by glycogen accumulation, as was confirmed in an additional experiment with guppies, a more expedient species. It is concluded that TBTO and DBTC have effects on the liver and gas gland, probably as a result of impaired glycogen breakdown. Effects on superficial epithelia are attributed to the irritative properties of these compounds. In addition, these compounds were toxic to kidney and retina. No-observed-effect concentrations using histopathology were 0.32 μg/l for TBTO, and lower than 320 μg/l for DBTC. Total tin and butyltins were determined by molecular absorption spectrometry, and gas chromatography with flame photometry, respectively, in guppies exposed to TBTO and DBTC. Considerable amounts of TBT were measured in DBTC-exposed fish, and chemical analysis indicated that DBTC contained TBT at a level that may account for the observed toxicity of DBTC.

Effects of ozone, hexachlorobenzene, and bis(tri- n-butyltin)oxide on natural killer activity in the rat lung

The respiratory tract is a major route of exposure to noxious agents as well as pathogens such as viruses. Natural killer (NK) activity is an important first line of defense to virally infected cells as well as certain neoplasms; therefore, testing the effects of exposure to toxic compounds on this activity is important in understanding the immunotoxic potential of the compound. Lymphoid cell suspensions, obtained after enzymatic dispersion of rat lungs and purification over nylon wool columns, showed in vitro natural killer activity toward YAC lymphoma cells. Validation of the test with well-known NK activity stimulators such as Bacillus Calmette-Guérin (BCG), interleukin-2 (IL-2), interferon (IFN), and inhibitors like anti-asialo-GM1 (ganglio- n-tetrasylceramide) antibody confirmed the reliability of the test as an assay for detecting NK activity in rat lungs. Using this assay, we studied the effects of exposure to ozone (O 3), hexachlorobenzene (HCB), and bis(tri- n-butyltin)oxide (TBTO) on NK activity in rat lung. Inhalation exposure to O 3 for 7 days at 0.4 and 0.8 mg/m 3 resulted in stimulation, and exposure at 1.6 mg O 3/m 3 resulted in suppression of NK activity. Oral exposure to HCB in concentrations of 150 and 450 mg/kg food for 6 weeks suppressed NK activity in rat lungs in a dose-related manner. This was also true for 6 weeks of oral exposure of rats to 20 and 80 mg TBTO/kg food, but to a lesser extent. In summary, we have developed and validated a method to measure the effects of (toxic) substances on NK activity in rat lung.

Toxicity of bis (tri-n-butyltin) oxide on learning and development in the rat.

The toxicity of bis (tri-n-butyltin) oxide (TBTO) was studied in THA rats. Doses of 0 (control), 10 or 15 mg/kg/day of TBTO were administered to adult rats for 12 consecutive days, and 0, 5 or 10 mg/kg/day was given to pregnant rats from day 6 to day 20 of gestation by gastric intubation. In the adult rat exposure experiment, the 15 mg/kg group showed slightly decreased body weight and spontaneous motor activity, and a low avoidance rate in the Sidman avoidance test in the early stage of administration. In the pregnant-rat exposure experiment, no effect of TBTO was seen in either the 5 mg/kg or 10 mg/kg groups in dams while all pups in the 10 mg/kg group died by postnatal day 3. Little effect of TBTO on body weight, pinna detachment, incisor eruption, eye opening, surface righting, cliff avoidance, pivoting, negative geotaxis and auditory startle was found in the 5 mg/kg pups. In the Sidman avoidance test, the avoidance rate of the 5 mg/kg pups was lower than that of control pups in the early stage of the session. In a water E-maze test, the swimming time to the goal and the number of errors were increased in the 5 mg/kg pups. In a reversal water E-maze test, increases of the swimming time to the goal and the number of errors made by the 5 mg/kg pups were marked. These results suggest that TBTO administration disrupts the learning acquisition of rats, and that the toxicity of TBTO is greater in prenatally exposed pups than in adult rats.

Chronic toxicity and carcinogenicity of bis(tri- n-butyltin)oxide (TBTO) in the rat

In a 106-wk toxicity and carcinogenicity study, groups of 60 male and 60 female weanling Wistar rats were fed 0, 0.5, 5 or 50 mg bis(tri- n-butyltin)oxide (TBTO)/kg diet. In males, feed consumption was increased in all treated groups and increased water consumption occurred at 5 and 50 mg/kg. During the second year, body weight decreased in the 50-mg/kg males, while the females in that group showed no weight gain. Excess mortality was confined to the 50-mg/kg group towards the end of the study. Haematological changes, comprising anaemia, lymphocytopenia and thrombocytosis were noted mainly at the high-dose level. Also, signs of decreased kidney function and increased plasma enzyme activities (alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase) were noted. No effects on serum hormone concentrations (thyrotropin, follicle stimulating hormone, luteinizing hormone or insulin) were observed, except for a decrease in the free thyroxin:thyroxin ratio in both sexes at the high-dose level. Higher serum IgM and IgA levels were present at 50 mg/kg, while, in females, IgG was decreased. At 50 mg/kg, the ovaries, adrenals, spleen (females), heart (males), pituitary, liver and kidneys were increased in weight, but the thyroid weight was decreased in females. The total tin concentrations in liver and kidneys showed a dose relationship and, in general, the concentrations were similar after 1 and 2 yr. Non-neoplastic histological alterations after 1 yr consisted of a decrease in the cell height of the thyroid follicles in all dose groups, with a reduced number of psammoma bodies at 50 mg/kg, a decrease in splenic iron content at 5 (females only) and 50 mg/kg, and a slight bile-duct activation. After 2 yr, only the thyroid changes were still present. In addition, at 2 yr, vacuolation and pigmentation of the proximal tubular epithelium and nephrosis were enhanced at 50 mg/kg. The incidence of benign tumours of the pituitary was significantly elevated and enhanced at 0.5 and 50 mg/kg. At 50 mg/kg increases in pheochromocytomas in the adrenal medulla and in parathyroid adenomas (males) were noted, while adrenal cortical tumours were decreased (males). There was a low, non-dose-related incidence of pancreatic carcinoma. Other tumour rates were in line with control data. It is concluded that lifetime feeding of 50 mg TBTO/kg diet induces toxicity in various organ systems. An increase in some common tumours was found at the high dose, probably due to hormonal or immunological changes. Effects on the incidence of some of these tumours were observed also in the low-dose groups, but not at the intermediate dose. Because of the high spontaneous incidence of the tumours in this strain of rats, the variable incidence in the treated groups, and the absence of a dose-effect relationship, the significance of the increased incidence of these tumours is questionable. On the basis of the marginal effects at 5 mg/kg, a no-adverse-effect level of 0.5 mg TBTO/kg can be established.

Immunotoxicity of tributyltin oxide in rats exposed as adults or pre-weanlings

A comparison was made between adult and pre-weanling rats of the immunotoxic effects of subacute dosing with bis(tri- n-butyltin) oxide (TBTO). Adult (9 weeks old) male Fischer rats were dosed by oral gavage with TBTO for 10 consecutive days at 1.25–10 mg/kg per dose or 3 times/week for a total of 10 doses at 5–20 mg/kg per dose. Adults rats similarly dosed by oral gavage with 6 mg/kg per dose cyclophosphamide (CY) served as positive controls. Pre-weanling rats (3–24 days old) were dosed 3 times/week for a total of 10 doses at 2.5, 5 or 10 mg/kg per dose. At various times after dosing rats were evaluated for alterations in body and lymphoid organ weights, mitogen and mixed lymphocyte reaction (MLR) lymphoproliferative (LP) responses, natural killer (NK) cell activity, cytotoxic T lymphocyte (CTL) responses and primary antibody plaque-forming cell (PFC) responses. In adult rats given 10 daily doses of TBTO, thymic involution was observed at a dosage of 2.5 mg/ kg and mitogen responses to Con A and PHA were suppressed at 5 mg/kg. The PFC response was enhanced in adult rats dosed daily at 2.5 mg/kg. A dosage of 5 mg/kg given intermittently (3 times/week) to adults or pre-weanlings resulted in thymic involution. Reductions in mitogen responses were observed in adults dosed intermittently at 10 and 20 mg/kg and in pre-weanlings at 5 and 10 mg/kg. The MLR response was suppressed in adult rats dosed intermittently at 20 mg/kg and in pre-weanling rats at 10 mg/kg. NK cell activity was suppressed only in pups dosed intermittently at 10 mg/ kg. CTL responses were not affected in either age group. Within 3 weeks following the last exposure of adult rats to TBTO all parameters returned to normal. On the other hand, LP responses to mitogens were suppressed in 10-week-old rats that were dosed with 10 mg/kg TBTO as pre-weanlings. However, this exposure regimen resulted in reductions in body weight that persisted for up to 13 weeks of age, which suggests that TBTO may be a developmental toxicant. These data indicate that while exposure of young rats to TBTO resulted in immune alterations at doses lower than those required to suppress responses in adults, the observed effects may also be influenced by the developmental toxicity of this compound.

Actions of orally administered organotin compounds on heme metabolism and cytochrome P-450 content and function in intestinal epithelium

The gastrointestinal tract is a major route by which humans are exposed to environmental chemicals. We have examined in these studies the effects of oral administration of organotin compounds in the small intestinal epithelium, an organ which exhibits highly active drug and other chemical metabolism. A series of n-butyltin compounds was administered by gavage to male Sprague-Dawley rats (225–275 g) in single doses up to 250 μmol/kg body weight. Bis(tri- n-butyltin)oxide (TBTO) produced dose- and time-dependent decreases in the content and functional activity of intestinal cytochrome P-450, together with an elevation (3-fold) in the activity of musomal heme oxygenase. The effects of di- n-butyltin dichloride on heme oxygenase and cytochrome P-450 were pronounced in the small intestine and extended to the liver and kidneys within 24 hr after oral exposure, whereas TBTO did not affect the liver until much later (6 days), when cytochrome P-450 content was reduced markedly (30%). Furthermore, the effects produced by tetra- n-butyltin on cytochrome P-450 at 24 hr were localized in the intestinal epithelium. These studies indicate important pharmacological effects of organotin compounds in the gut, and raise the possibility that concurrent oral ingestion of organotins with other environmental pollutants may alter the cytochrome P-450-dependent metabolism of xenobiotics and natural substrates of this monooxygenase system in the small intestine.

A technique for assessing the preventative efficacy against decay fungi of preservative treatments applied to wood

A method is described in which test blocks with envelope preservative treatments can be challenged by selected test fungi previously established on an untreated feeder block. The progress of the test fungus through the treated zone is monitored using novel baits or sensors inserted in holes drilled into blocks to within predetermined distances of the face being challenged. Preliminary results show the method to be discriminating. Three-minute immersion treatment with 5% pentachlorophenol retarded colonisation for a longer period than 1% tri n-butyltin oxide, particularly when penetration was in the tangential direction.

Kinetics and mechanism of oxidation of tri-n-butylstannyl alkoxides of benzhydrol and substituted benzhydrols by bromine in carbon tetrachloride

The tri-n-butylstannyl alkoxides (TBSA) of benzhydrols were prepared by treating the alcohols with tri-n-butyltin oxide (TBTO) and azeotropically removing the water. They were oxidised by bromine in carbon tetrachloride to the corresponding ketones. The oxidation was found to be a second order process. The effect of substituents on the rate was studied and the Hammett ϱ was found to be − 2.55 at 308 K. A mechanism is proposed.

Quantitative Analysis of Bis(tri-n-butyltin) Oxide in Cultured Fresh Fish by High Performance Liquid Chromatography

A simple method was developed for quantitative analysis of bis(tri-n-butyltin) oxide (TBTO) in cultured fresh fish by high-performance liquid chromatography (HPLC). The sample was decomposed with alkaline alcohol and TBTO was converted into tri-n-butyltin chloride (TBTC), which was then purified by a cation exchange method. TBTC was determined by HPLC with a cation exchange column and an ultraviolet absorption detector.The recoveries of TBTO added to the muscle of yellowtail and sea bream at the level of 2ppm were 87±4 and 87±3%, respectively, and that of TBTO added to the muscle of spanish mackerel at the level of 0.2ppm was 90±5%. The detection limit of TBTO was 0.1ppm.The concentrations of TBTO in cultured fresh fishes could be easily quantified by this method.

Evaluation of the genetic and embryotoxic effects of bis(tri- n-butyltin)oxide (TBTO), a broad-spectrum pesticide, in multiple in vivo and in vitro short-term tests

The genetic and embryotoxic effects of bis(tri- n-butyltin)oxide (TBTO) were evaluated in multiple in vivo and in vitro short-term tests preparatory to its potential wide use as a molluscicide in control of schistosomiasis. When tested in the rec assay in Bacillus subtilis, TBTO was not mutagenic and it did not induce reverse mutations in Klebsiella pneumoniae. Neither in the presence nor in the absence of rat liver activation system did TBTO produce point mutations in Salmonella typhimurium strains TA1530, TA1535, TA1538, TA97, TA98 or TA100. TBTO was mutagenic in strain TA100 in a fluctuation test, but only in the presence of rat liver S9 (Aroclor-induced). TBTO did not induce gene mutations in the yeast Schizosaccharomyces pombe, mitotic gene conversions in the yeast Saccharomyces cerevisiae, nor sister-chromatid exchange in Chinese hamster ovary cells in the presence or absence of rat or mouse liver S9. In the latter cells, structural chromosomal aberrations, endoreduplicated and polyploid cells were induced. TBTO did not induce gene mutations in V79 Chinese hamster cells (to 8-azaguanine-, ouabain- or 6-thioguanine-resistance) in the presence of a rat liver postmitochondrial fraction or in cell (hamster embryo cells and human and mouse epidermal keratinocyte)-mediated assays. In mouse lymphoma cells, TBTO did not induce 6-thioguanine- or BUdR-resistant mutations. As many tumour promoters inhibit metabolic cooperation between V79 Chinese hamster 6-thioguanine-resistant/-sensitive cells, TBTO was tested but showed no such activity. TBTO was examined for the induction of recessive lethal mutations in adult Berlin K male Drosophila melanogaster, either by feeding or by injection. Doses of 0.37 or 0.74 mM did not increase the number of X-linked recessive lethal mutations. An increased number of micronuclei was observed in the polychromatic erythrocytes of male BALB/c mice 48 h after a single oral dose of TBTO (60 mg/kg bw), while a lower dose (30 mg/kg bw) was ineffective. Neither of the two doses had induced micronuclei 30 h after treatment. The reproductive toxicity of TBTO was studied in NMRI mice. In a 10-day toxicity study, the LD 50 and LD 10 were 74 and 34 mg/kg bw, respectively. An increased frequency of cleft palates was seen in the fetuses of mice (compared with controls, 0.7%) treated orally during pregnancy with 11.7 mg/kg TBTO (7%), 23.4 mg/kg (24%) or 35 mg/kg (48%). The two highest doses of the drug also increased the frequencies of irregular ossification centres of sternebrae and of minor abnormalities, such as fusion of the bases of the os occipitalis. Doses of 6 mg of TBTO/kg bw do not produce teratogenic effects in mice. Teratogenic effects were only seen at TBTO doses that were toxic to the maternal organism. Electron microscopy 26 and 48 h after TBTO treatment showed no evidence that embryos were damaged. In contrast, the maternal liver was greatly affected. The tin contents of different organs of the maternal organism and of the whole fetus were determined after TBTO treatment. After a single oral treatment with 117 mg TBTO/kg bw, the total tin content in 11.5-day-old embryos 12 h after TBTO administration was 9.5 nmoles Sn/g wet weight — 70% greater than the normal value. The total concentration of tin in the maternal liver was increased to a similar extent (by approximately 60%) over the normal level between 6 and 24 h after treatment. No other organ studied showed similarly high increases in tin content. These results show that TBTO gives negative results in short-term tests using various genetic endpoints. However, at cytotoxic concentrations, it was mutagenic in one bacterial strain, clastogenic in CHO cells in vitro and produced micronuclei in mouse bone marrow cells in vivo. In view of these adverse biological effects, and its high capacity to interfere with morphogenetic differentiation processes in vitro careful evaluations must be made before TBTO is released in large quantities into the aquatic environment, and levels to which humans are exposed must be strictly controlled when it is used as a molluscicide.

Histopathological study of Poecilia reticulata (guppy) after long-term exposure to bis(tri- n-butyltin)oxide (TBTO) and di- n-butyltindichloride (DBTC)

The histopathologic effects in the small fish species Poecilia reticulata (guppy) were investigated after exposure to the biocide bis(tri- n-butyltin)oxide (TBTO), a contaminant of the aquatic environment. At the age of 3–4 wk animals were exposed to concentrations ranging from 0.01–32 μg TBTO/l over 1 and 3 months. In a comparative study young guppies were exposed to di- n-butyltindichloride (DBTC) for 1 month at a concentration range of 320–3200 μg/l. Both compounds induced thymus atrophy, hyperplasia of hemopoietic interstitial tissue of the kidney, and increase in number of free peritoneal cells in the body cavity, which were probably neutrophilic granulocyte analogs. In the liver vacuolation was increased, predominantly due to accumulation of fat and of glycogen; also muscle glycogen was increased. Effects of DBTC occurred at concentrations which were almost three orders of magnitude higher than of TBTO, and there were no indications that the TBTO-induced thymus atrophy is caused by the DBT-metabolite. In addition, at the higher concentrations of TBTO epidermal hyperplasia and keratitis occurred, as well as changes in the rods and cones layer of the retina. The ‘no-observed-effect’ concentration of TBTO was 0.01 μg/l, based on thymus atrophy, liver vacuolation or hyperplasia of the hemopoietic tissue, and this was 30-fold lower than when based upon classical toxicological criteria (growth). In the DBTC study, thymus atrophy was still present in the lowest concentration tested (320 μg/l) whereas the NOEC for mortality and behavior was 1800 μg/l. Atrrophy of the thymus, keratitis and skin irritation seem to be similar effects as found in mammals. It is concluded that long-term low dose TBTO-exposure of guppies causes thymus atrophy, increase in granulocytes, accumulation of glycogen and fat in the liver, and changes in cornea, retina and skin; except for the changes in the eye and skin, similar effects were induced by DBTC, although at manyfold higher concentrations.