Environ. Sci. Technol. 3909, 23 740-743 I
Tributyltin in Bay Mussels (Myfilus edulis) of the Pacific Coast of the United States Jeffrey W. Short" and Julle L. Sharp Auke Bay Laboratory, National Marine Fisheries Service, NOAA, P.O. Box 210155, Auke Bay, Alaska 99821
Tissue concentrations of tributyltin (TBT) were measured by graphite furnace atomic absorption spectrophotometry (GFAA) and by gas chromatography coupled with an atomic absorption spectrophotometer as the detector (GCAA) in bay mussels (Mytilus edulis) collected from San Diego Bay (CA), Monterey Bay (CA), Puget Sound (WA), Auke Bay (AK), and Kodiak harbor (AK) in 1986 and 1987; results from both methods were not significantly different ( P < 0.871). Concentrations of TBT in mussels ranged from less than 0.005 pg of TBT/g of wet tissue weight to 1.08 pg/g of wet tissue weight. Concentrations of TBT in mussels sampled from Puget Sound were significantly lower ( P < 0.033) in March 1987 than in June 1986, possibly due to the influence of boats freshly painted with marine antifoulants launched during the spring. Our results may be useful as a benchmark to gauge the efficacy of recently enacted legislation restricting the use of marine antifoulants containing TBT on the Pacific coast of the United States. Concern about adverse effects of tributyltin (TBT) compounds to marine life has resulted in widespread legislation restricting the use of TBT, including limitations enacted in 1987 by California, Oregon, Washington, and Alaska. Although marine antifoulant paints containing TBT are particularly effective, TBT has been shown to exert unintended adverse effects on marine fauna at concentrations near 0.020 pg of TBT/L of seawater. Thain et al. (1)reported that spat of the oyster Crussostrea gigas exposed in the laboratory to 0.020 pg of TBT/L for 49 days had thickened shells that were not commercially viable, and that C. gigas exposed in the field to similar concentrations of TBT appeared to corroborate their laboratory results. Gibbs et al. (2) reported that concentrations of TBT greater than 0.007-0.012 pg of TBT/L suppressed oogenesis and induced spermatogenesis in female dog whelks (Nucellu lapillus) in the laboratory. These effects are part of a syndrome termed imposex that incapacitates reproduction and has been observed in the field in N. lapillus ( 3 ) and in Nucella lima (4). The legislation enacted is intended to reduce the introduction of TBT to coastal marine waters, but will not eliminate it. In most cases, antifoulant paints containing TBT will still be allowed on large vessels and on aluminum-hulled vessels. It is, therefore, of interest to estimate the decline of the environmental TBT burden that should result from the enacted legislation. Changes in environmental TBT burden may be estimated by monitoring concentrations of TBT in water, sediment, or tissues of sentinel organisms such as bay mussels (Mytilus edulis). Methods that identify TBT in seawater at concentrations of 0.010 pg/L or less are described in Matthias et al. (5),but are relatively expensive. Sediment monitoring is especially well suited for evaluating long-term changes in pollutant burden, whereas sentinel organism monitoring is well suited for shorter term changes (1-5 years). Mussels are particularly convenient sentinel organisms because they have a cosmopolitan distribution, a propensity to concentrate many pollutants in their tissues, and provide a biologically relevant indication of exposure integrated over time. Mussels have been shown to 740
Environ. Sci. Technol., Vol. 23,
No. 6, 1989
concentrate TBT by a factor of --5ooo times in laboratory experiments (6) and so are well suited for monitoring changes in marine environmentalTBT burdens. Although TBT concentrations have been measured in mussels in California prior to enactment of legislation restricting use of TBT antifoulants (7,8), no data have been published on concentrations in mussels in the other three states. All of the mussels in this study were analyzed by two methods to verify concentrations of TBT in mussels: Method one (GCAA) employs gas chromatographic separation and atomic absorption detection of pentylated butyltin species in tropolone-complexedbenzene extracts of HC1-digested tissue and method two (GFAA) employs graphite furnace atomic absorption determination of tin in hexane extracts of HC1-digested tissue. We have used method two in an interlaboratory comparison study of TBT in mussel tissue and found very good agreement with values reported by the other participants in that study (7). We present information on TBT concentrations in mussels collected from California, Washington, and Alaska. These collections were taken from a wide geographic base including relatively clean sites and polluted sites in these states. Mussels were collected twice from washington with a 9-month interval between collections to ascertain seasonal effects there. Future analyses of TBT in mussels at these collection locations may help evaluate the efficacy of enacted legislation restricting the use of TBT antifoulants on the Pacific coast of the United States. Methods Mussel Collection. Mussels were collected from five locations along the Pacific coast of the United States: San Diego Bay, CA; Monterey Bay, CA; Puget Sound, WA; Auke Bay, AK; and Kodiak harbor, AK. The sampling sites at each location are given in Figure 1, where the site numbers are assigned according to the relative GCAAdetermined concentrations of TBT found in mussels at that location (1= highest, etc.), except sites 4-6 at Puget Sound where such ranking is ambiguous. The latitude, longitude, and collection dates for each site are listed in Table I, where the site numbers correspond with those in Figure 1. Sampling sites were chosen to include a range of expected TBT contamination levels in the mussels. All of the mussels from San Diego Bay and from site 2 at Monterey Bay were collected as part of an interlaboratory calibration exercise; the details of mussel collection and storage are given in Stephenson (7). Mussels from each site were combined and homogenized, and an aliquot was shipped to our Laboratory at Auke Bay. Mussels from the remaining sites were collected at low tide, blotted dry, wrapped with tissue paper, and kept refrigerated at 4 "C for up to 48 h prior to freezing. Mussel samples were kept frozen at -20 "C for up to 4 months prior to analysis. We attempted to collect the largest mussels available at these latter sites; shell lengths ranged from 16 to 85 mm. A t the time of analysis, the ratio of tissue wet weight to dry weight in mussels collected near Auke Bay was 11.6 f 2.1 (95% CI, n = 6). Tissue Preparation. The mussels from the interlaboratory calibration study had been homogenized prior to receipt and were subsampledjust before analysis by GFAA
Not subject to US. Copyright. Published 1989 by the American Chemical Society
Edmon
Canada
E D
F@UIO1. Sampling locations and sites wimh each location along the Pacific coast 01 the United States Site numbers are used in Tables I and I I Site numbers rank the G C A A d e t m i n e d wncentration 01 TBT b u n d wtthln each kcation except for sites 4 6 at puget Sound. where ranking
is ambiguous.
Table I. Location of Mussel Collection Sites and Dates of Collection (Site Numbers Refer to Those in Figure I) longitude San Diego Bay
collectn dates
site
latitude
1 2 3
32O42'3W' N 32O42'42" N 32"41'39" N
117°14'12" W 117O13'42" W 117O14'27" W
1 2
36O48'04" N 36'48'51'' N
Monterey Bay 121'47'08'' W 22 Oct 86 121O94'42" W 22 Oct 86
1
47O35'21" N 47O35'29" N 48O48'50" N 47O35'43" N 48O48'15" N 47'34'25'' N
122'22'43'' W 122'22'5W' W 122O22'54" W 122O22'06" W 122°50'00" W 122°25'M)" W
26 Jun 86,20Mar 81 26 Jun 86,20 Mar 87 25 Jun 86, 20 Mar 87 26 Jun 86, 20 Mar 87 24 Jun 86, 20 Mar 87 26 Jun 86, 20 Mar 87
58O23'05" N 58O23'08" N 58O22'52" N 58O22'17" N 58O19'52" N
Ante R av -------, 134O38'38" W 134O38'56" W 134'38'28'' W 134O41'52" W 134O44'05" W
24 Feb 87 24 Feb 87 23 Feb 87 9 Mar 87 16 Jun 87
Kodiak 152O24'34" W 152'24'19"W 152O24'25" W 152O24'31" W 152O24'36" W
3 Mar 81 3 Mar 87 3 Mar 87 3 Mar 87 3 Mar 87
Puget Sound 2 3
4 5 6 1
2 3 4 5 1 2 3
A.
5
57'47'18'' 57'47'17" 5l04I'2W' 57O47'11" 57O47'15"
N N N N N
22 Oct 86 22 Oct 86 22 Oct 86
or by GCAA. The mussels that had not been previously homogenized were dissected into acid-washed glass containers and mechanically homogenized just prior to
analysis. Five individual mussels were separately homogenized for analysis by GFAA. Five individual mussels were combined and homogenized for analysis by GCAA. T w o determinations of TBT in the homogenate prepared for GCAA analysis were made. GFAA Method. The GFAA method used was developed by M & T Chemical Co. (9)and modified by Short (10).A total of 1-5 g of an individual mussel homogenate was digested in 1&50 mL of concentrated hydrochloric acid for 2 h a t room temperature. The digest was extraded with hexane, and the hexane extract was washed with 3% NaOH to remove dibutyltin. The hexane extract was evaporated to dryness at 20 O C on a rotary evaporator. The residue was dissolved in a solution consisting of 0.1 mL of concentrated nitric acid and 4.9 mL of glacial acetic acid and analyzed by GFAA on a Perkin-Elmer Model 5000 spectrophotometer with a Zeeman background corrector and a tin electrodeless discharge lamp. Instrument settings followed those suggested by the manufacturer for tin. Aliquots (1-5 g) from each of six mussel homogenates were analyzed as a group. A known amount of TBT chloride was added to two additional 1-5-g aliquots selected a t random from the six mussel homogenates in the group. Two reagent blanks and two reagent blanks containing a known amount of TBT were also analyzed with each group. The increase of detector response due to TBT added to mussel homogenates or to reagent blanks was not significantly different (n = 23, P < 0.001), so the average increase of detector response due to TBT added to the two mussel homogenates was used to calculate the amount of TBT present in the six mussel homogenates as per the method of standard additions. The mean detector reEnvlron. Sci. Technol., Voi. 23,No. 6, 1989
741
sponse to reagent blanks was 0.003 f 0.002 absorbance unitss ( n = 46). The detection limit for this method was -0.005 kg of TBT/g of wet tissue, based on the mean value obtained for the reagent blank plus 5 times the standard deviation. GCAA Method. The GCAA method was developed by Maguire (11) and modified by Short (IO). Briefly, 1-5 g of a five-mussel homogenate was digested in concentrated hydrochloric acid for 2 h and extracted with 0.25% tropolone in benzene. Tetrabutyltin was added as an internal standard after the extracts were reduced in volume and pentylated via Grignard reaction. The pentylated extracts were purified with a 3% deactivated silica gel column, reduced in volume on a rotary evaporator at 20 "C to -0.1 mL, and analyzed with a gas chromatograph interfaced with an atomic absorption spectrophotometer. Four mussel homogenates were analyzed as a group. A 1-5-g aliquot was taken from one of the homogenates and spiked with TBT chloride for recovery determinations. A reagent blank was also included in the analysis. The concentration of TBT was determined from the area ratio of TBT to that of the internal standard. Recoveries of TBT chloride added to the mussel homogenates ranged from 89% to 101% in 15 determinations. Using the criterion that a peak was not considered real unless it was at least 3 times as large as the range of detector response at the retention time of pentyltributyltin in the reagent blank, the detection limit was determined to be 0.010 pg of TBT/g of wet tissue. No recognizable pentyltributyltin peaks were detected in any of the 15 reagent blanks analyzed for this study. Data Analysis. Student's t test was used to calculate 95% confidence intervals associated with the means of the GFAA results. Two-way analysis of variance (ANOVA) was used to determine differences among analytical methods, where classificationsincluded analytical methods and sampling sites. Results of all repetitions of analyses by both GFAA and by GCAA at all sites examined in this study (except sites 5 at Auke Bay and at Kodiak Harbor, where data were either missing or below the detection limits of both methods) were used for this two-way ANOVA (total df = 174). Two-way ANOVA was also used to determine differences among the two sampling dates at Puget Sound, where classifications included sampling data and sampling sites, and where the ANOVA was applied to the GFAA results. Data for these ANOVA included repetitions of all GFAA data from all sites and sampling dates of Puget Sound (total df = 59). Materials. Tetrabutyltin, tributyltin chloride, dibutyltin dichloride, and butyltin trichloride were purchased from Ventron (Danvers, MA). Tropolone was purchased from Aldrich (Milwaukee, WI). Silica gel, grade 923, was purchased from Davison Chemical (Baltimore, MD). Sodium hydroxide was purchased from Sigma (St. Louis, MO). Pentylmagnesium bromide was synthesized from metallic magnesium and bromopentane (Aldrich). Organic solvents were glass distilled. Hydrochloric acid was reagent grade but washed with tropolone in benzene in order to remove traces of inorganic tin. Nitric and glacial acetic acids were manufactured for pesticide analysis. Sodium hydroxide was reagent grade.
Results Tributyltin was detected in mussels at all of the sampling locations examined (Table 11). Concentrations of TBT ranged from near or below detection limits in all three states to 1.08 kg of TBT/g of wet tissue weight at Kodiak harbor, Alaska. The highest concentrations of 742
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Table 11. Concentrations of TBT in Mussels Collected from the Pacific Coast of the United States As Measured by GFAA and by GCAA (Site Numbers Refer to Figure 1)" TBT concn as measured by GFAA GCAA San Diego 0.38 f 0.03 0.28 f 0.05 0.25 f 0.05
0.44, 0.47 0.25, 0.26 0.19, 0.24
Monterey Bay 0.28 f 0.14 0.02 f 0.003
0.24, 0.36
