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Response to Comment on “Occurrence of Butyltin Compounds in Human Blood” SIR: The intent of our study (1), as has been stated in our paper, was to examine the possibility of occurrence of butyltin compounds in human blood. This has been justified by the presence of butyltins in a variety of consumer products including foodstuffs. It is documented by a large number of studies from various parts of the world that seafood such as fishes, oysters, and clams contain butyltins (see ref 1 for selected references). Studies have also estimated human daily intake of butyltins through seafood consumption and market basket surveys (2-4). Cooking is ineffective in eliminating butyltins from foods (2, 3, 5). Similarly, various researchers have reported the presence of butyltins, although at small concentrations (a few parts per billion; µg/L), in common food items/beverages such as fruit juices, beer, wine, and tap water (6-10). In addition to foodstuffs, various household items such as baking parchments, polyester fabrics, and plastics including polyvinylchloride (PVC) pipes contain butyltins (5, 11). A recent study reported the presence of mono-(MBT), di-(DBT), and tributyltins (TBT) in cookies, that were prepared using baking parchment papers purchased from local stores, at 260, 720, and 15 ng/g, respectively (5). These results unequivocally documented the existence of a variety of sources of butyltins to which humans can be exposed. Following exposure, studies have documented bioaccumulation of butyltins in various organisms including mammals (see refs 12 and 13 for selected references). In addition to our study on the occurrence of the butyltins in human blood collected from Michigan (1), a Japanese study has reported the concentrations of MBT and DBT of up to 22 and 78 ng/g, wet wt, respectively, in livers of Japanese subjects (5). Similarly, an earlier study has reported the occurrence of butyltins in livers of humans in Poland (12). Thus, the existence of sources and exposure of humans to butyltin compounds are indisputable, and our results of their occurrence in human blood (1) have been interpreted in the grounds of several logical evidences. The comments raised by Robinson and Kluck are centered on the methods applied. Following are our explanations to the comments of Robinson and Kluck: Robinson and Kluck claim that “the method used for the analysis of butyltins in blood is developed for mammal blubber, not blood”. They also state that “to our knowledge, butyltins have not been reliably analyzed in blood samples”. It should be noted that this method was developed not just to analyze butyltins in the blubber, rather it is developed for application in all biological tissues. Moreover, this method has been used earlier for the analysis of butyltins in the blood of marine mammals and fish (13, 14). The method that we used is a state-of-the-art analytical technique involving derivatization and high-resolution capillary gas chromatography-flame photometric detection (HRGC-FPD) with a tin-specific filter at 610 nm, which is selective for tincontaining compounds. Robinson and Kluck have indicated that the analysis of butyltins in water requires a sample volume of 2 L, which is concentrated to 1 mL. They are dubious about how 2 mL of blood could generate a detection limit of 1 ppb (µg/L). In our study, the blood volume used was 2-3 mL, and the final extract volume was 0.5-3 mL. Robinson and Kluck claim that the detection limit of our method is 1 µg/L, without specifically mentioning the values that we have reported for 10.1021/es002003h CCC: $19.00 Published on Web 03/24/2000
2000 American Chemical Society
different butyltin compounds in the paper. We have mentioned that our method detection limits are 7 µg/L for MBT, 2.5 µg/L for DBT, and 1 µg/L for TBT. The detection limit that can be obtained with 2 L of water, after 1000-fold concentration to a final volume of 1 mL, is 1 ng/L (ppt) or less (15). This detection limit is required for water because TBT causes “imposex” (development of male sex characteristics/organs in females) in gastropods at a concentration as little as 2 ppt (ng/L) (16). Therefore, the water quality criterion for TBT in seawater is 2 ng/L in several countries (see ref 17). Thus, 2 L volume is required for water samples to achieve a detection limit of 1 ng/L or less. As mentioned above, the detection limits that we obtained for TBT with 2-3 mL volume of blood is 1 µg/L. The “ppb” detection limit in our samples and “ppt” limit in water samples is clearly explained by the differences in the volume of samples used for the analyses. In other words, to be toxicologically relevant a method detection limit (MDL) of 1 ng/L is necessary for water. A MDL of 1 µg/L is adequate for samples of blood because the threshold for toxic effects seems to be greater than that concentration (18). Furthermore, since most of the samples contained concentrations in excess of this value, a lesser MDL was not necessary. Robinson and Kluck speculate that our results are artifacts and represent false positives in blood analysis, but provide no valid support for this speculation. We have analyzed several procedural blanks, which were passed through the whole procedure with every set of eight samples to check for interfering compounds (1). The blanks were concentrated to the same extent as the samples. MBT was present at trace levels, and that is the reason for its greater detection limit (7 ppb) as compared to TBT, which has a detection limit of 1 ppb. TBT was never found in blanks. In fact, we have clearly mentioned that MBT, DBT, and TBT were detected only in 53, 81, and 70% of the samples analyzed. If there were false positives due to interferences, butyltin compounds could have been present in all the samples (100%) analyzed. Moreover, the tin-specific filter (610 nm) used in the detector is selective for tin-containing compounds. In addition, as mentioned earlier, other studies also have reported the occurrence of butyltins in human tissues (5, 12). Robinson and Kluck suggested that we have not described the quantitation techniques in detail. The method used in this study was developed in Japan (13, 15), and several publications have detailed the analytical procedures. Using this method, our laboratories in the United States and several national and academic research laboratories in Japan have published over 100 research papers in more than 20 different peerreviewed journals. This method is also used in leading organotin monitoring research laboratories (14, 15, 19), and similar methods using derivatization and capillary gas chromatography-flame photometric detection techniques are used in several research laboratories in Europe (see ref 17). Since the details of quantification have been described in previous publications, they were not repeated in our paper due to the space limitations. Robinson and Kluck also discuss the risks of the occurrence of butyltins in human blood. We have never discussed or assessed the risks of butyltins in human blood. Risk assessment of butyltins in human blood was beyond the scope of our investigation, and thus, the comments of Robinson and Kluck relate issues not addressed in our paper. Moreover, some of their comments on in vitro assays, free and bound residues, suggest that they have not examined the experimental procedures closely. For example, the in vitro assays conducted by Whalen et al. (18) used whole blood VOL. 34, NO. 9, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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collected from human volunteers; therefore, they should reflect the “real” exposure and “bioavailable” concentrations. Robinson and Kluck also suggest that an outlier value of TBT concentration of 85 ppb in human blood should not be discussed because it is an outlier. This value should not be ignored because this suggests the occurrence of great butyltin exposures in certain individuals. In fact, we have reports of occupational exposure (unpublished reports) of butyltin compounds in humans, which indicate that some individuals are exposed to relatively great concentrations of butyltins. The implication is that the two high values are statistically outliers but do represent a real world scenario. We have confirmed the result to be accurate and feel that it indicates that certain individuals in the population may be more exposed. This should not be ignored but should be treated as a subject for more detailed studies in the future.
Literature Cited (1) Kannan, K.; Senthilkumar, K.; Giesy, J. P. Environ. Sci. Technol. 1999, 33, 1776-1779. (2) Short, J. W.; Thrower, F. P. Mar. Pollut. Bull. 1986, 17, 542545. (3) Tsuda, T.; Inoue, T.; Kojima, M.; Aoki, S. J. AOAC. Int. 1995, 78, 941-943. (4) Kannan, K.; Tanabe, S.; Iwata, H.; Tatsukawa, R. Environ. Pollut. 1995, 90, 279-290. (5) Takahashi, S.; Mukai, H.; Tanabe, S.; Sakayama, K.; Miyazaki, T.; Masuno, H. Environ. Pollut. 1999, 106, 213-218. (6) Forsyth, D. S.; Sun, W. F.; Daglish, K. Food Addit. Contam. 1994, 11, 343-350.
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(7) Forsyth, D. S.; Weber, D.; Barlow, L. Appl. Organomet. Chem. 1992, 6, 579-585. (8) Forsyth, D. S.; Weber, D.; Cle´roux, C. Food Addit. Contam. 1992, 9, 161-169. (9) Forsyth, D. S.; Weber, D.; Daglish, K. J. AOAC Int. 1992, 75, 964-973. (10) Sadiki, A.-I.; Williams, D. T. Chemosphere 1999, 38, 15411548. (11) Yamada, S.; Fujii, Y.; Mikami, E.; Kawamura, N.; Hayakawa, J.; Aoki, K.; Fukaya, M.; Terao, C. J. AOAC Int. 1993, 76, 436-441. (12) Kannan, K.; Falandysz, J. Mar. Pollut. Bull. 1997, 34, 203-207. (13) Iwata, H.; Tanabe, S.; Mizuno, T.; Tatsukawa, R. Environ. Sci. Technol. 1995, 29, 2959-2962. (14) Oshima, Y.; Nirmala, K.; Go, J.; Yokota, Y.; Koyama, J.; Imada, N.; Honjo, T.; Kobayashi, K. Environ. Toxicol. Chem. 1997, 16, 1515-1517. (15) Harino, H.; Fukushima, M.; Tanaka, M. Anal. Chim. Acta 1992, 264, 91-96. (16) Bryan, G. W.; Gibbs, P. E.; Hummerstone, L. G.; Burt, G. R J. Mar. Biol. Assoc. U.K. 1986, 66, 611-640. (17) Fent, K. Crit. Rev. Toxicol. 1996, 26, 1-117. (18) Whalen, M. M.; Loganathan, B. G.; Kannan, K. Environ. Res. 1999, 81, 108-116. (19) Horiguchi, T.; Shiraishi, H.; Shimizu, M.; Morita, M. J. Mar. Biol. Assoc. U.K. 1994, 74, 651-669.
K. Kannan*, K. Senthilkumar, and J. P. Giesy National Food Safety and Toxicology Center Michigan State University East Lansing, Michigan 48824 ES002003H
