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Susceptibility of bacterial populations to organotin compounds and microbial degradation of organotin compounds in environmental water
Environmental Pollution, Vol. 98, No. 2, pp. 157±162, 1997 # 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain PII: S0269-7491(97)00133-4 0269-7491/97 $17.00+0.00
SUSCEPTIBILITY OF BACTERIAL POPULATIONS TO ORGANOTIN COMPOUNDS AND MICROBIAL DEGRADATION OF ORGANOTIN COMPOUNDS IN ENVIRONMENTAL WATER Hiroya Harino,a* Minoru Fukushima,a Yuko Kurokawab and Shin'ichiro Kawaib a
Osaka City Institute of Public Health and Environmental Sciences, Tojo-cho 8-34, Tennoji-ku, Osaka, 543, Japan b Department of Human Sciences, Kobe College, Okadayama 4-1, Nishinomiya, 662, Japan (Received 16 April 1997; accepted 6 September 1997)
TBT is widespread in aquatic environment and concentrations are especially high in marinas and shipyards (Fent and Hunn, 1991; Hugget et al., 1992). TBT concentrations in coastal water ranged from 2.3 to 18 g literÿ1 in Bahrain (Hasan and Juma, 1992). Additionally, MBT and DBT were found in water samples where TBT was detected, due to dealkylation of TBT. Therefore, studies on degradation process of butyl- and phenyltin compounds by bacteria are important in elucidating the fate of organotin compounds in aquatic environment. There are several published papers relating to microbial degradation of organotin compounds in water. For example, calculated half-lives of TBT in water collected from a yacht harbour and a clean-water site were reported to be 7 and 19 days, respectively (Seligman et al., 1986). Dowson et al. (1993) investigated the degradation of TBT in contaminated freshwater and estuarine sediments. As a result, TBT half lives in surface sediments were ranged from 360 to 775 days. On the contrary, little is known about the degradation of TPT in coastal waters (Kannan and Lee, 1996). In this paper, susceptibility of bacterial populations to butyltin and phenyltin compounds has been described. Additionally, in order to clarify the degradation mechanism of organotin compounds in estuarine water, biodegradation process of TBT, DBT and TPT were investigated by measuring concentration of these organotin compounds which were added to water throughout the study period (river die-away method).
Abstract Susceptibility of bacterial populations to organotin compounds was studied in river water. Organotin compounds including tributyltin (TBT), dibutyltin (DBT), monobutyltin (MBT), triphenyltin (TPT), diphenyltin (DPT) and monophenyltin (MPT) dissolved in dimethyl sulfoxide (DMSO) were added to river water. Number of colony forming units (CFU) of bacteria was measured using 1.5% agar plate. CFU count was not decreased at concentration less than 1.0 mg literÿ1 of organotin compounds. CFU was decreased markedly at 10 mg litreÿ1 of TBT, DBT or MPT, however, the populations increased after 2 days. Degradation of TBT, DBT and TPT by bacteria in estuarine water was studied using river dieaway method. TBT was degraded to DBT moderately, and DBT was degraded to MBT rapidly. When the initial concentration of TBT or DBT was adjusted to 9.3 and 8 g literÿ1, half-lives of TBT and DBT were 15 and 10 days, respectively. TPT was degraded scarcely during 60 days of culture. Growth of bacteria in estuarine waters containing organotin compounds was found throughout the study period. # 1998 Elsevier Science Ltd. All rights reserved INTRODUCTION Organotin compounds have been widely used as biocides, stabilizers in vinyl chloride, wood preservatives and so on. In particular, TBT is an eective biocide in antifouling paints. In 1976, deformity in oysters (Crassostrea gigas) was shown to be caused by the exposure of TBT released from antifouling paints (Laughlin and Linden, 1985). Since then, eects of organotin compounds on plankton, mussel and ®sh have been well studied (Roberts, 1987) and it was found that TBT can be toxic to these marine organisms at concentration of 1 g literÿ1 or less (U'ren, 1983). However, little known about the susceptibility of bacteria which are widely distributed in aquatic environment.
MATERIALS AND METHODS Site description Figure 1 shows the sampling area. Yodo River originates from the Lake Biwa and ¯ows into the Port of Osaka. Stations 1 and 2 are situated in Lake Biwa and Stations 3 and 4 are in the Yodo River basin. Station 5 is a marina and has berths for 121 sailing boats. Water samples collected at Stations 1±4 were used to test inhibitory eects of bacterial growth by organotin compounds. Water from Station 5 was used for the degradation study using river die-away method.
*To whom correspondence should be addressed. Fax:+81-6772-0676. 157
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H. Harino et al. lone. After extraction, moisture in the solvent layer was removed with anhydrous sodium sulfate. The organotin compounds in these extracts were propylated with 1 ml of n-propylmagnesium bromide and were cleaned with ¯orisil Sep-Pak (Waters Association Co. Ltd). Propylated organotin compounds were determined by gas chromatography equipped with ¯ame photometric detector. Concentrations of organotin compounds were expressed as organotin cation. When 0.4 g of each organotin compound was added to 50 ml of estuarine water, the recoveries of MBT, DBT, TBT, MPT, DPT and TBT in water samples were 94, 89, 98, 97, 100 and 80%, respectively. The detection limit of each organotin compound for a signal-to-noise ratio of three was 0.2 g literÿ1. RESULTS AND DISCUSSION
Fig. 1. Map of sampling locations.
Water sampling in Stations 1±4 and Station 5 was carried out in May 1991 and December 1991, respectively. Susceptibility of bacterial populations to organotin compounds TBT, DBT or TPT was dissolved in DMSO and 1000 or 10000 mg literÿ1 of stock organotin solution was prepared. By adding 0.5 ml of these solution to 500 ml of water sample, concentration of each organotin compound in water was adjusted to 1 or 10 mg literÿ1. Samples were incubated with shaking at 30 C in darkness. After 0, 1, 5, 12, 19 and 28 days, bacterial numbers were measured using agar plates purchased from Nissui Co. containing meat extract 5 g, peptone 10 g, sodium chloride 5 g and agar 15 g in one liter of distilled water. River die-away method TBT, DBT, or TPT in DMSO was added to each estuarine water sample (50 ml) at the initial concentration of 10 or 100 g literÿ1. Autoclaved estuarine water samples were used as control. Each sample was prepared individually because organotin compounds might be adsorbed on the glass. Water samples were analyzed after shaking at 30 C in darkness for 4, 9, 12, 15, 22, 30, 40, 50 and 60 days. Bacterial cell number was also measured after 0, 1, 5, 12, 19 and 28 days. Analytical methods The analytical method used for organotin compounds was modi®ed version of a procedure reported previously (Harino et al, 1992). Fifty millilitres of water was extracted with 1 N HCl and benzene containing tropo-
Susceptibility of bacterial populations to organotin compounds Initial log CFU in water samples of Stations 1±4 ranged from 4.1 to 5.2. Though the number of bacteria was dramatically declined at the initial concentration of 10 mg literÿ1, log CFU increased moderately after 5 days of incubation (Fig. 2). Only TBT-tolerant bacteria seemed to have increased. At the initial concentration of 0.01±1 mg literÿ1, decrease of CFU count was not found and after a day, the number of bacteria increased ten times more than the initial population. The eects of DBT and MBT were also studied at Stations 1 and 4 (Fig. 3). At an initial concentration of 10 mg literÿ1 DBT, log CFU of bacteria was decreased from 4.1 to 1.6 at Station 1. However at Station 4, the decrease of bacterial population was not observed at all. This discrepancy might be due to dierences of bacterial populations at each location. After 5 days, the number of bacteria increased moderately. At the concentration of MBT ranging from 0.01 to 10 mg literÿ1, the decrease of CFU count was not found. It can be concluded that indigenous bacteria can keep a population at 105±106 CFU mlÿ1 level in the presence of TBT less than l mg literÿ1. The eect of phenyltin compounds on bacterial growth was also investigated in Stations 1 and 4 (Fig. 4). Even the concentration of 10 mg literÿ1 of TPT and DPT, decrease of CFU was not found. While, at 10 mg literÿ1 of MPT, the number of bacterial population was decreased after a day. In particular, at Station 1 a remarkable decrease of log CFU was observed. It is clear that the sensitivity of bacteria to MPT is greatest among phenyltin compounds. Uchida (1993) reported that an inhibitory eects on colony forming ability of environmental bacteria occurred at 5±100 g literÿ1 of TBT and TPT. In our experiment, it was clear that bacteria which could be multiplied at concentration of TBT more than 10 mg literÿ1 were existed in river water. Accordingly, the sensitivity of bacteria to organotin compounds is varied by the population of bacteria collected from each area.
Susceptibility of bacterial populations to organotin compounds and microbial degradation
159
Fig. 2. Time course of number of bacteria cultured in the river water containing TBT. The each value is the average of CFU on four plates. *, 10 mg literÿ1; &, 1 mg literÿ1; ~, none.
Fig. 3. Time course of number of bacteria cultured in the river water containing DBT. The each value is the average of CFU on four plates. *, 10 mg literÿ1; &, 1 mg literÿ1; ~, none.
Degradation of organotin compounds by the bacteria in estuarine water Concentration of TBT in water at Station 5 was 0.063 g literÿ1. Organotin compounds except TBT
were not detected. It was reported that this station showed the highest level of TBT in Osaka harbour area (Harino et al., 1990). The time-course of butyltin concentrations during 60 days was shown in Fig. 5(a). In
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Fig. 4. Time course of number of bacteria cultured in the river water containing TPT. The each value is the average of CFU on four plates. *, 10 mg literÿ1; &, 1 mg literÿ1; ~, none.
this experiment, the initial TBT concentration was adjusted to the levels which CFU was not decreased throughout the study period. TBT decreased rapidly during the initial 20 days and was not detected after 40 days of incubation. DBT increased slightly during the initial 15 days, and then DBT also decreased slowly and remained undetected after 30 days. On the other hand, MBT increased slowly and a remarkable variation in concentration was not observed between 12 and 60 days. Maguire and Tkacz (1985) reported that in accordance with the decrease of TBT DBT and MBT increased, which is in agreement with our results. The compositions of butyltin compounds over 60 days are shown in Fig. 5(b). The ratio of TBT to total butyltin compounds decreased during 60 days of incubation. On the contrary, the ratio of MBT to total butyltin compounds increased. The ratio of DBT increased between 0 and 15 days and then decreased. This suggests that TBT is successively degraded to DBT, and DBT is debutylated
to MBT. TBT concentration of the autoclaved control decreased gradually, and it might be due to the chemical degradation or volatilization. When the concentration of total butyltins was calculated as inorganic tin, the concentration decreased from 4.3 to 1.0 g literÿ1 over 60 days. It is considered that MBT is further degraded to inorganic tin or it can be presumed that the other intermediates of butyltin compounds are produced. The degradation of DBT in water samples was studied (Fig. 6(a)). The experiment was carried out in the same way as TBT. DBT decreased rapidly during initial 11 days and disappeared after 22 days. The concentration of MBT increased for the ®rst 11 days, and then decreased moderately. The compositions of butyltin compounds over 60 days are shown in Fig. 6(b). The ratio of DBT to total butyltin compounds decreased from 100% to 19% between 0 and 15 days of incubation and was 0% after 22 days, and MBT accounted 100% of total butyltin compounds. The concentration of total
Susceptibility of bacterial populations to organotin compounds and microbial degradation
Fig. 5. Degradation of TBT by bacteria in estuarine water. (a) Concentration of butyltin compounds during 60 days; (b) changes of the composition (%) of butyltin compounds during 60 days.
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Fig. 7. Degradation of TPT by bacteria in estuarine water. Concentration of phenyltin compounds during 60 days; (b) changes of the composition (%) of phenyltin compounds during 60 days.
Table 1. Estimated half-lives of organotin compounds Organotin compound TBT DBT TPT
Fig. 6. Degradation of DBT by bacteria in estuarine water. (a) Concentration of butyltin compounds during 60 days; (b) changes of the composition (%) of butyltin compounds during 60 days.
butyltin compounds decreased from 4.4 to 0.2 g literÿ1. The degradation of TPT was also investigated by river die-away method. The initial concentration of TPT was 8 g literÿ1. The concentration of TPT scarcely changed after 60 days (Fig. 7(a)). The compositions of
Initial concentration (g literÿ1)
Half-life (days)
9.3 104 8.0 8.0
15 >60 10 >60
phenyltin compounds are shown Fig. 7(b). The ratio of TPT decreased only 15% between 0 and 60 days. The ratio of DPT and MPT changed only slightly during 60 days. It shows that the dephenylation of TPT does not occur or it is extremely slow. The half-lives of organotin compounds in water samples were estimated from time course of organotin compounds which were corrected with the values in autoclaved control (Table 1). At the concentration of 9.3 g literÿ1, the observed half-life of TBT was 15 days. When TBT was adjusted to 104 g literÿ1, the half-life of TBT could not be observed. The higher the TBT concentration, the slower the degradation rate. When the initial DBT concentration was 8 g literÿ1, the observed half-life was 10 days. It was obvious that debutylation rate of DBT occurred more rapid than that of TBT. In river water and seawater of the most polluted areas in Osaka Bay, DBT degraded more rapidly in comparison with TBT (Hattori et al., 1988), which is in agreement with our results. The half-life of TPT could not be determined, because this compound
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was scarcely degraded by the aquatic bacteria during 60 days of culture. CONCLUSIONS At concentrations of DBT, TBT and MPT of 10 mg literÿ1, decrease of the CFU count was observed after a day of incubation. However, the bacterial populations increased again after 5 days. Bacterial degradation of TBT, DBT and TPT was investigated in waters by river die-away method. TBT was degraded to DBT and MBT by a stepwise debutylation process. When the initial concentration of TBT, DBT and TPT was 9.3, 8 and 8 g literÿ1, respectively, the observed half-lives of TBT and DBT were 15 and 10 days, respectively. TPT was scarcely degraded during 60 days of incubation. It is concluded that the bacterial species that degrade organotin compounds exist in the aquatic environment and butyltin compounds are debutylated easily, however, TPT was not degraded. Further studies are needed to clarify biodegradation of phenyltin compounds by bacteria. REFERENCES Dowson, P. H., Bubb, J. M., Williams, T. P. and Lester, J. N. (1993) degradation of tributyltin in freshwater and estuarine marina sediments. Water Science Technology 28, 133± 137. Fent, K. and Hunn, J. (1991) Phenyltin in water, sediment, and biota of freshwater marinas. Environmental Science and Technology 25, 956±963.
Harino, H., Fukushima, M. Mori, Y. and Nakadoi, T. (1990) Distribution of organotins in the harbour area of Osaka City, Japan. IAWPRC International Symposium on Hazard Assessment and Control of Environment Contaminants in Waters, pp. 158±164. Harino, H., Fukushima, M. and Tanaka, M. (1992) Simultaneous determination of butyltin and phenyltin compounds in the aquatic environment by gas chromatography. Analytica Chimica Acta 264, 91±96. Hasan, M. A. and Juma, H. A. (1992) Assessment of tributyltin in the marine environment of Bahrain. Marine Pollution Bulletin 24, 408±410. Hattori, Y., Kobayashi, A., Nonaka, K., Sugimae, A. and Nakamoto, M. (1988) Degradation of tributyl tin and dibutyl tin compounds in environmental waters. Water Science Technology 20, 71±76. Huggett, R. J., Unger, M. A., Seligmann, P. F. and Valkirs, A. O. (1992) The marine biocide tributyltin. Environmental Science and Technology 26, 232±237. Kannan, K. and Lee, R. F. (1996) Triphenyltin and its degradation products in foliage and soil from sprayed pecan orchards and in ®sh from adjacent ponds. Environmental Toxicology and Chemistry 15, 1492±1499. Laughlin, R. B., Jr and Linden, O. (1985) Fate and eects of organotin compounds. AMBIO 14, 88±94. Maguire, R. J. and Tkacz, R. J. (1985) Degradation of the trin-butyltin species in water and sediment from Toronto harbor. Journal of Agricultural and Food Chemistry 33, 947±953. Roberts, M. H. Jr (1987) Acute toxicity of tributyltin chloride to embryos and larvae of two bivalve mollusks Crassostrea Virginia and Mercenaria mercenaria. Bulletin of Environmental Contamination and Toxicology 39, 1012±1019. Seligman, P. F., Valkirs, A. O. and Lee, R. F. (1986) Degradation of tributyltin in San Diego Bay, California waters. Environmental Science and Technology 20, 1229±1235. Uchida, M. (1993) Inhibitory activity of organotin compounds against colony formation of estuarine bacteria. Nippon Suisan Gakkaishi 59, 2037±2042. U'ren, S. C. (1983) Acute toxicity of bis(tri-butyltin) oxide to a marine copepod. Marine Pollution Bulletin 14, 303±306.
SUSCEPTIBILITY OF BACTERIAL POPULATIONS TO ORGANOTIN COMPOUNDS AND MICROBIAL DEGRADATION OF ORGANOTIN COMPOUNDS IN ENVIRONMENTAL WATER Hiroya Harino,a* Minoru Fukushima,a Yuko Kurokawab and Shin'ichiro Kawaib a
Osaka City Institute of Public Health and Environmental Sciences, Tojo-cho 8-34, Tennoji-ku, Osaka, 543, Japan b Department of Human Sciences, Kobe College, Okadayama 4-1, Nishinomiya, 662, Japan (Received 16 April 1997; accepted 6 September 1997)
TBT is widespread in aquatic environment and concentrations are especially high in marinas and shipyards (Fent and Hunn, 1991; Hugget et al., 1992). TBT concentrations in coastal water ranged from 2.3 to 18 g literÿ1 in Bahrain (Hasan and Juma, 1992). Additionally, MBT and DBT were found in water samples where TBT was detected, due to dealkylation of TBT. Therefore, studies on degradation process of butyl- and phenyltin compounds by bacteria are important in elucidating the fate of organotin compounds in aquatic environment. There are several published papers relating to microbial degradation of organotin compounds in water. For example, calculated half-lives of TBT in water collected from a yacht harbour and a clean-water site were reported to be 7 and 19 days, respectively (Seligman et al., 1986). Dowson et al. (1993) investigated the degradation of TBT in contaminated freshwater and estuarine sediments. As a result, TBT half lives in surface sediments were ranged from 360 to 775 days. On the contrary, little is known about the degradation of TPT in coastal waters (Kannan and Lee, 1996). In this paper, susceptibility of bacterial populations to butyltin and phenyltin compounds has been described. Additionally, in order to clarify the degradation mechanism of organotin compounds in estuarine water, biodegradation process of TBT, DBT and TPT were investigated by measuring concentration of these organotin compounds which were added to water throughout the study period (river die-away method).
Abstract Susceptibility of bacterial populations to organotin compounds was studied in river water. Organotin compounds including tributyltin (TBT), dibutyltin (DBT), monobutyltin (MBT), triphenyltin (TPT), diphenyltin (DPT) and monophenyltin (MPT) dissolved in dimethyl sulfoxide (DMSO) were added to river water. Number of colony forming units (CFU) of bacteria was measured using 1.5% agar plate. CFU count was not decreased at concentration less than 1.0 mg literÿ1 of organotin compounds. CFU was decreased markedly at 10 mg litreÿ1 of TBT, DBT or MPT, however, the populations increased after 2 days. Degradation of TBT, DBT and TPT by bacteria in estuarine water was studied using river dieaway method. TBT was degraded to DBT moderately, and DBT was degraded to MBT rapidly. When the initial concentration of TBT or DBT was adjusted to 9.3 and 8 g literÿ1, half-lives of TBT and DBT were 15 and 10 days, respectively. TPT was degraded scarcely during 60 days of culture. Growth of bacteria in estuarine waters containing organotin compounds was found throughout the study period. # 1998 Elsevier Science Ltd. All rights reserved INTRODUCTION Organotin compounds have been widely used as biocides, stabilizers in vinyl chloride, wood preservatives and so on. In particular, TBT is an eective biocide in antifouling paints. In 1976, deformity in oysters (Crassostrea gigas) was shown to be caused by the exposure of TBT released from antifouling paints (Laughlin and Linden, 1985). Since then, eects of organotin compounds on plankton, mussel and ®sh have been well studied (Roberts, 1987) and it was found that TBT can be toxic to these marine organisms at concentration of 1 g literÿ1 or less (U'ren, 1983). However, little known about the susceptibility of bacteria which are widely distributed in aquatic environment.
MATERIALS AND METHODS Site description Figure 1 shows the sampling area. Yodo River originates from the Lake Biwa and ¯ows into the Port of Osaka. Stations 1 and 2 are situated in Lake Biwa and Stations 3 and 4 are in the Yodo River basin. Station 5 is a marina and has berths for 121 sailing boats. Water samples collected at Stations 1±4 were used to test inhibitory eects of bacterial growth by organotin compounds. Water from Station 5 was used for the degradation study using river die-away method.
*To whom correspondence should be addressed. Fax:+81-6772-0676. 157
158
H. Harino et al. lone. After extraction, moisture in the solvent layer was removed with anhydrous sodium sulfate. The organotin compounds in these extracts were propylated with 1 ml of n-propylmagnesium bromide and were cleaned with ¯orisil Sep-Pak (Waters Association Co. Ltd). Propylated organotin compounds were determined by gas chromatography equipped with ¯ame photometric detector. Concentrations of organotin compounds were expressed as organotin cation. When 0.4 g of each organotin compound was added to 50 ml of estuarine water, the recoveries of MBT, DBT, TBT, MPT, DPT and TBT in water samples were 94, 89, 98, 97, 100 and 80%, respectively. The detection limit of each organotin compound for a signal-to-noise ratio of three was 0.2 g literÿ1. RESULTS AND DISCUSSION
Fig. 1. Map of sampling locations.
Water sampling in Stations 1±4 and Station 5 was carried out in May 1991 and December 1991, respectively. Susceptibility of bacterial populations to organotin compounds TBT, DBT or TPT was dissolved in DMSO and 1000 or 10000 mg literÿ1 of stock organotin solution was prepared. By adding 0.5 ml of these solution to 500 ml of water sample, concentration of each organotin compound in water was adjusted to 1 or 10 mg literÿ1. Samples were incubated with shaking at 30 C in darkness. After 0, 1, 5, 12, 19 and 28 days, bacterial numbers were measured using agar plates purchased from Nissui Co. containing meat extract 5 g, peptone 10 g, sodium chloride 5 g and agar 15 g in one liter of distilled water. River die-away method TBT, DBT, or TPT in DMSO was added to each estuarine water sample (50 ml) at the initial concentration of 10 or 100 g literÿ1. Autoclaved estuarine water samples were used as control. Each sample was prepared individually because organotin compounds might be adsorbed on the glass. Water samples were analyzed after shaking at 30 C in darkness for 4, 9, 12, 15, 22, 30, 40, 50 and 60 days. Bacterial cell number was also measured after 0, 1, 5, 12, 19 and 28 days. Analytical methods The analytical method used for organotin compounds was modi®ed version of a procedure reported previously (Harino et al, 1992). Fifty millilitres of water was extracted with 1 N HCl and benzene containing tropo-
Susceptibility of bacterial populations to organotin compounds Initial log CFU in water samples of Stations 1±4 ranged from 4.1 to 5.2. Though the number of bacteria was dramatically declined at the initial concentration of 10 mg literÿ1, log CFU increased moderately after 5 days of incubation (Fig. 2). Only TBT-tolerant bacteria seemed to have increased. At the initial concentration of 0.01±1 mg literÿ1, decrease of CFU count was not found and after a day, the number of bacteria increased ten times more than the initial population. The eects of DBT and MBT were also studied at Stations 1 and 4 (Fig. 3). At an initial concentration of 10 mg literÿ1 DBT, log CFU of bacteria was decreased from 4.1 to 1.6 at Station 1. However at Station 4, the decrease of bacterial population was not observed at all. This discrepancy might be due to dierences of bacterial populations at each location. After 5 days, the number of bacteria increased moderately. At the concentration of MBT ranging from 0.01 to 10 mg literÿ1, the decrease of CFU count was not found. It can be concluded that indigenous bacteria can keep a population at 105±106 CFU mlÿ1 level in the presence of TBT less than l mg literÿ1. The eect of phenyltin compounds on bacterial growth was also investigated in Stations 1 and 4 (Fig. 4). Even the concentration of 10 mg literÿ1 of TPT and DPT, decrease of CFU was not found. While, at 10 mg literÿ1 of MPT, the number of bacterial population was decreased after a day. In particular, at Station 1 a remarkable decrease of log CFU was observed. It is clear that the sensitivity of bacteria to MPT is greatest among phenyltin compounds. Uchida (1993) reported that an inhibitory eects on colony forming ability of environmental bacteria occurred at 5±100 g literÿ1 of TBT and TPT. In our experiment, it was clear that bacteria which could be multiplied at concentration of TBT more than 10 mg literÿ1 were existed in river water. Accordingly, the sensitivity of bacteria to organotin compounds is varied by the population of bacteria collected from each area.
Susceptibility of bacterial populations to organotin compounds and microbial degradation
159
Fig. 2. Time course of number of bacteria cultured in the river water containing TBT. The each value is the average of CFU on four plates. *, 10 mg literÿ1; &, 1 mg literÿ1; ~, none.
Fig. 3. Time course of number of bacteria cultured in the river water containing DBT. The each value is the average of CFU on four plates. *, 10 mg literÿ1; &, 1 mg literÿ1; ~, none.
Degradation of organotin compounds by the bacteria in estuarine water Concentration of TBT in water at Station 5 was 0.063 g literÿ1. Organotin compounds except TBT
were not detected. It was reported that this station showed the highest level of TBT in Osaka harbour area (Harino et al., 1990). The time-course of butyltin concentrations during 60 days was shown in Fig. 5(a). In
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Fig. 4. Time course of number of bacteria cultured in the river water containing TPT. The each value is the average of CFU on four plates. *, 10 mg literÿ1; &, 1 mg literÿ1; ~, none.
this experiment, the initial TBT concentration was adjusted to the levels which CFU was not decreased throughout the study period. TBT decreased rapidly during the initial 20 days and was not detected after 40 days of incubation. DBT increased slightly during the initial 15 days, and then DBT also decreased slowly and remained undetected after 30 days. On the other hand, MBT increased slowly and a remarkable variation in concentration was not observed between 12 and 60 days. Maguire and Tkacz (1985) reported that in accordance with the decrease of TBT DBT and MBT increased, which is in agreement with our results. The compositions of butyltin compounds over 60 days are shown in Fig. 5(b). The ratio of TBT to total butyltin compounds decreased during 60 days of incubation. On the contrary, the ratio of MBT to total butyltin compounds increased. The ratio of DBT increased between 0 and 15 days and then decreased. This suggests that TBT is successively degraded to DBT, and DBT is debutylated
to MBT. TBT concentration of the autoclaved control decreased gradually, and it might be due to the chemical degradation or volatilization. When the concentration of total butyltins was calculated as inorganic tin, the concentration decreased from 4.3 to 1.0 g literÿ1 over 60 days. It is considered that MBT is further degraded to inorganic tin or it can be presumed that the other intermediates of butyltin compounds are produced. The degradation of DBT in water samples was studied (Fig. 6(a)). The experiment was carried out in the same way as TBT. DBT decreased rapidly during initial 11 days and disappeared after 22 days. The concentration of MBT increased for the ®rst 11 days, and then decreased moderately. The compositions of butyltin compounds over 60 days are shown in Fig. 6(b). The ratio of DBT to total butyltin compounds decreased from 100% to 19% between 0 and 15 days of incubation and was 0% after 22 days, and MBT accounted 100% of total butyltin compounds. The concentration of total
Susceptibility of bacterial populations to organotin compounds and microbial degradation
Fig. 5. Degradation of TBT by bacteria in estuarine water. (a) Concentration of butyltin compounds during 60 days; (b) changes of the composition (%) of butyltin compounds during 60 days.
161
Fig. 7. Degradation of TPT by bacteria in estuarine water. Concentration of phenyltin compounds during 60 days; (b) changes of the composition (%) of phenyltin compounds during 60 days.
Table 1. Estimated half-lives of organotin compounds Organotin compound TBT DBT TPT
Fig. 6. Degradation of DBT by bacteria in estuarine water. (a) Concentration of butyltin compounds during 60 days; (b) changes of the composition (%) of butyltin compounds during 60 days.
butyltin compounds decreased from 4.4 to 0.2 g literÿ1. The degradation of TPT was also investigated by river die-away method. The initial concentration of TPT was 8 g literÿ1. The concentration of TPT scarcely changed after 60 days (Fig. 7(a)). The compositions of
Initial concentration (g literÿ1)
Half-life (days)
9.3 104 8.0 8.0
15 >60 10 >60
phenyltin compounds are shown Fig. 7(b). The ratio of TPT decreased only 15% between 0 and 60 days. The ratio of DPT and MPT changed only slightly during 60 days. It shows that the dephenylation of TPT does not occur or it is extremely slow. The half-lives of organotin compounds in water samples were estimated from time course of organotin compounds which were corrected with the values in autoclaved control (Table 1). At the concentration of 9.3 g literÿ1, the observed half-life of TBT was 15 days. When TBT was adjusted to 104 g literÿ1, the half-life of TBT could not be observed. The higher the TBT concentration, the slower the degradation rate. When the initial DBT concentration was 8 g literÿ1, the observed half-life was 10 days. It was obvious that debutylation rate of DBT occurred more rapid than that of TBT. In river water and seawater of the most polluted areas in Osaka Bay, DBT degraded more rapidly in comparison with TBT (Hattori et al., 1988), which is in agreement with our results. The half-life of TPT could not be determined, because this compound
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was scarcely degraded by the aquatic bacteria during 60 days of culture. CONCLUSIONS At concentrations of DBT, TBT and MPT of 10 mg literÿ1, decrease of the CFU count was observed after a day of incubation. However, the bacterial populations increased again after 5 days. Bacterial degradation of TBT, DBT and TPT was investigated in waters by river die-away method. TBT was degraded to DBT and MBT by a stepwise debutylation process. When the initial concentration of TBT, DBT and TPT was 9.3, 8 and 8 g literÿ1, respectively, the observed half-lives of TBT and DBT were 15 and 10 days, respectively. TPT was scarcely degraded during 60 days of incubation. It is concluded that the bacterial species that degrade organotin compounds exist in the aquatic environment and butyltin compounds are debutylated easily, however, TPT was not degraded. Further studies are needed to clarify biodegradation of phenyltin compounds by bacteria. REFERENCES Dowson, P. H., Bubb, J. M., Williams, T. P. and Lester, J. N. (1993) degradation of tributyltin in freshwater and estuarine marina sediments. Water Science Technology 28, 133± 137. Fent, K. and Hunn, J. (1991) Phenyltin in water, sediment, and biota of freshwater marinas. Environmental Science and Technology 25, 956±963.
Harino, H., Fukushima, M. Mori, Y. and Nakadoi, T. (1990) Distribution of organotins in the harbour area of Osaka City, Japan. IAWPRC International Symposium on Hazard Assessment and Control of Environment Contaminants in Waters, pp. 158±164. Harino, H., Fukushima, M. and Tanaka, M. (1992) Simultaneous determination of butyltin and phenyltin compounds in the aquatic environment by gas chromatography. Analytica Chimica Acta 264, 91±96. Hasan, M. A. and Juma, H. A. (1992) Assessment of tributyltin in the marine environment of Bahrain. Marine Pollution Bulletin 24, 408±410. Hattori, Y., Kobayashi, A., Nonaka, K., Sugimae, A. and Nakamoto, M. (1988) Degradation of tributyl tin and dibutyl tin compounds in environmental waters. Water Science Technology 20, 71±76. Huggett, R. J., Unger, M. A., Seligmann, P. F. and Valkirs, A. O. (1992) The marine biocide tributyltin. Environmental Science and Technology 26, 232±237. Kannan, K. and Lee, R. F. (1996) Triphenyltin and its degradation products in foliage and soil from sprayed pecan orchards and in ®sh from adjacent ponds. Environmental Toxicology and Chemistry 15, 1492±1499. Laughlin, R. B., Jr and Linden, O. (1985) Fate and eects of organotin compounds. AMBIO 14, 88±94. Maguire, R. J. and Tkacz, R. J. (1985) Degradation of the trin-butyltin species in water and sediment from Toronto harbor. Journal of Agricultural and Food Chemistry 33, 947±953. Roberts, M. H. Jr (1987) Acute toxicity of tributyltin chloride to embryos and larvae of two bivalve mollusks Crassostrea Virginia and Mercenaria mercenaria. Bulletin of Environmental Contamination and Toxicology 39, 1012±1019. Seligman, P. F., Valkirs, A. O. and Lee, R. F. (1986) Degradation of tributyltin in San Diego Bay, California waters. Environmental Science and Technology 20, 1229±1235. Uchida, M. (1993) Inhibitory activity of organotin compounds against colony formation of estuarine bacteria. Nippon Suisan Gakkaishi 59, 2037±2042. U'ren, S. C. (1983) Acute toxicity of bis(tri-butyltin) oxide to a marine copepod. Marine Pollution Bulletin 14, 303±306.