- Email: [email protected]
An analytical method for butyltin species in shellfish
"v\)lume 21)/Number 3/March 1989 which are k n o w n to o c c u r in the Tees d i s p o s a l a r e a in s u m m e r , the rate of d i l u t i o n w o u l d b e m u c h s l o w e r a n d the d i l u t i o n a t t a i n e d after 48 h w o u l d b e 3 . 7 x 106 times. This is still a p p r e c i a b l y g r e a t e r t h a n the d i l u t i o n a c h i e v a b l e in the s a m e time in the s a m e s m a l l e r d i s p o s a l area off the H u m b e r . 3. If the residual drift a l o n g the c o a s t is s t e a d y a n d small, it is p r e d i c t e d that the usual t i m e b e t w e e n s u c c e s sive d i s c h a r g e s o f 2 - 3 d a y s w o u l d b e insufficient for either the Tees o r H u m b e r areas to b e c o m p l e t e l y c l e a r e d of waste b e f o r e the next discharge. H o w e v e r , the k n o w n variability o f the r e s i d u a l flows a l o n g the east c o a s t o f E n g l a n d a n d the high d i l u t i o n s p r e d i c t e d by the m o d e l s suggest that c o n c e n t r a t i o n s w o u l d n o t b u i l d up to levels which w o u l d cause c h r o n i c a l l y toxic effects to the m a r i n e life. Admiralty. (1975,/76). Tidal stream atlas: North Sea-Northern portion NP 252 & Southern portion NP 251. Bowden, K. F. & Lewis, R. E. (1973). Dispersion in flow from a continuous source at sea. WaterRes. 7, 1705-1722. Byrne, C. D., Law, R. J., Hudson, P. M., Thain, J. E. & Fileman, T. W. (I989). Measurements of the dispersion of liquid industrial waste discharged into the wake of a dumping vessel off the River Tees. WaterRes. (In press.)
Davies, A. M. (1983). In Flushh,g times of the North Sea. ICES 1983/ CRR: 123. Delvigne, G, A. L. (1987). Experiments on the dilution capacity of wakes from dumping tankers in the North Sea. In Oceanic Processes in Marine Pollution, Vol. 2 (T. P. O'Connor, W. V. Burt & 1. W. DuedaU, eds), pp. 000-000. Robert E. Krieger Publishing Co., Malabar, Florida. Durance, J. A. & Jones, S. R. (1976). Spatial variability of residual drift offthe east coast of England in 1975. ICES CM 1976/C:3. Huthnance, J. (1987). NERC North Sea Project. NERC News, October, 1987. Lewis, R. E. (1985). Dilution of waste in the wake of a ship. Water Res. 18, 941-945. Lewis, R. E. (1984). Circulation and mixing in estuary outflows. Cont. Shelf Res. 3,201-214. Medler, K. J. (1977). Residual drift regimes in the west central North Sea during JONSDAP 76. ICES 1977/C:5. Okubo, A. (1971). Oceanic diffusion diagranls. Deep Sea Res. 18,789802. Pasquill. F. & Smith, E B. (1983). Atmospheric DiffiLsion. Third edition. Ellis Horwood Ltd., Chichester. Ramster, J. W. (1977). Residual drift reNmes off the north-east coast of England. ICES CM 1977/C:8. Talbot, J. W. & Talbot, G. A. (1974). Diffusion in shallow seas and in English coastal and estuarine waters. Rapp. P-v. Rdun. Cons. inc Explor. Mer. 167, 93-110. van Pagee, J. A., Gerritsen, H. & de Ruijter, W. R M. (1986). Transport and water quality modelling in the southern North Sea in relation to coastal pollution research and control. WaterSci. Tech. 18, 245-256. Webb, A. J. (1982). A random walk model of the dispersion of caesium-137 in the Irish Sea. M.Sc. thesis. University of Wales.
Marine Pollution tJtdletin, Volume 20, No. 3, p p 129-133, 1989 Printed in Great Britain.
0 0 2 5 - 3 2 ( , X / 8 9 S3.00+0 0c) ~) 1989 Pergamon Prcs~ plc
An Analytical Method for Butyltin Species in Shellfish D A V I D S. P A G E B o w d o i n College H y d r o c a r b o n Research Center, B r u n s w i c k , M a i n e 04011, U S A
A method for analysing tributyltin (TBT) and dibutyltin (DBT) in shellfish is presented that places modest requirements on equipment and supplies. It is based on capillary gas chromatographic analysis of hexyl derivatives of any extractable organotin species present. Two internal standards are used. Data are presented showing that the precision and accuracy of the method are generally better than _+20%.
T r i b u t y l t i n (TBT) c o n t a i n i n g m a r i n e a n t i f o u l a n t s h a v e seen w i d e s p r e a d use for o v e r 20 years. C o n c e r n a b o u t the a d v e r s e effects on m a r i n e life o f T B T l e a c h i n g f r o m t r e a t e d surfaces has resulted in strict c o n t r o l s of its use in F r a n c e , the U n i t e d K i n g d o m , the U n i t e d States, a n d o t h e r j u r i s i d i c t i o n s (see C h a m p & Pugh, 1987; W a l d o c k et al., 1987a; C l a r k et al., 1988). C u r r e n t
c o n c e r n a b o u t the a d v e r s e effects of T B T o n shellfish b e g a n with the i d e n t i f i c a t i o n of T B T - l e a c h a t e f r o m antifouling p a i n t s as b e i n g r e s p o n s i b l e for the c o l l a p s e of the o y s t e r fishery a r o u n d A r c a c h o n , F r a n c e in 1980 (see A l z i e u , 1986). It is n o w r e c o g n i z e d that w a t e r c o n c e n t r a t i o n s of T B T in the 1 0 - 5 0 ng 1-1 r a n g e can exert lethal a n d s u b l e t h a l effects on a wide variety of m a r i n e o r g a n i s m s p a r t i c u l a r l y for the case of sensitive j u v e n i l e life f o r m s ( W a l d o c k et al., 1987b; C a r d w e l l & S h e l d o n , 1986). M i c r o b i a l d e g r a d a t i o n is p r o b a b l y the d o m i n a n t p r o c e s s for the b r e a k d o w n of T B T in n e a r s h o r e waters ( S e l i g m a n et al., 1986; L e e et al., 1987) with d i b u t y l t i n ( D B T ) the m a j o r d e g r a d a t i o n p r o d u c t ( L e e et aL, 1987). W h i l e D B T a p p e a r s to b e at least a n o r d e r of m a g n i t u d e less toxic to m a r i n e a n i m a l s ( L a u g h l i n & L i n d e n , 1985; T h a i n et al., 1987), its w i d e s p r e a d use as a s t a b i l i s e r for P V C plastics ( B l u n d e n et al., 1985) a n d its r e l a t i o n s h i p to T B T as a d e g r a d a t i o n p r o d u c t m a k e s 129
Marine Pollution Bulletin
it important to analyse for both TBT and DBT in a marine monitoring program. In the marine environment, leachate from TBT may originate from a variety of sources such as treated hulls of commercial or military vessels and pleasure boats; dockyard painting and hull washing facilities. Table 1 summarizes some reported mussel tissue TBT concentrations for various locations. In assessing the impact of pollutants on indicator organisms such as mussels in bays and estuaries affected by marine activities, it is important to examine a range of potential stressors (Gilfillan & Page, 1986). Due to its toxic effect at low concentrations, TBT should be included in such assessments. In addition, analysis of TBT in filter-feeding bivalves such as mussels provides a time-integrated estimation of TBT in water consistent with the Mussel Watch Program approach for pollution monitoring in general (Farrington et al., 1983). A variety of methods for analysing TBT in tissues has been reported. They fall into two major categories: methods based on the analysis of organotin by atomic absorption spectrophotometric (AAS) quantitation of the Sn present; and methods based on the analysis of organotin derivatives by gas chromatographic (GC) techniques. In most cases, DBT was analysed either directly for the GC-based methods or indirectly for many of the AAS-based procedures. Atomic absorption-based methods include hydride-generation/AAS TABLE 1 Some reported concentrations of TBT in mussel tissues from field samples. The data are given as gg g-~ TBT on a dry tissue wt. basis. For those data reported on a wet tissue wt basis, the following conversion factor was used: 5×(wet wt ppm)=dry wt ppm. For those data reported on a weight of tin basis, the following conversion was used: 2.74 × (ppm as Sn) = ppm as TBT. Location
Type
gg g-~ TBT
Reference
San Diego, CA (USA) (3 sites)
2.90 2.90 1.60 0.25
Stephenson et aL, 1987
Monterey, CA (USA)
H/M H H R
Marina del Rey, CA
M
17.1
Stephenson etal., 1988
R
0.2
(USA) Humbolt, CA (USA) San Diego, CA (USA) (3 sites)
H/M N R Pearl Harbor, HI (USA) N H/M R Honolulu, HI (USA) H/M Norfolk, VA (USA) H/M R Little Creek, VA (USA) N H/M R -
2.69* 1.50" 0.93* 0.23* 0.30* 0.33* 7.35* 3.98" 1.18" 0.90* 1.41 * 0.69*
Ireland, Muh'oy Bay Galway Bay Banlry Bay
-
0.75 <0.25 <0.25
Sweden, W. coast E. coast
-
0.8-10.2 <0.2-3.9
Grovhoug el aL, 1986
Minchin et al., 1987
Linden, 1987
Data marked * are given as total solvent-extractable tin. Where replicate analyses are reported, the results are summarized as the average value. The type of sampling location is also given when identified in the source literature. The abbreviations are: H=harbour; M = marina; N = navy; R = reference; - = not indicated.
130
(Donard et al., 1986); direct solvent extraction/AAS (Short & Thrower, 1986); solvent extraction/NaOH wash to remove di- and monobutyltin/AAS {%Valdock & Miller, 1983; Bryan et al., 1986; Stephenson & Smith, 1988). Gas chromatographic methods generally involve solvent extraction of treated or untreated tissue homogenates followed by formation of a tetra-alkyl Sn derivative with a Grignard reagent and analysis by: GC/ AAS (Maguire et al., 1986); GC/flame photometric detection (Rice et al., 1987); GC/mass spectrometric detection (Humphrey & Hope, 1987); GC/flame ionization detection (Dooley & Vafa, 1986). In an intercalibration exercise between seven laboratories (Stephenson et al., 1987), reasonable agreement was found for the analyses of replicate mussel samples irrespective of the method used. The present work was undertaken to develop a method for analysing TBT and DBT in tissues that can be performed with a minimal investment in equipment and supplies by laboratories normally engaged in trace organic analysis of environmental samples.
Materials and Methods Analytical procedltre The method is based on the analytical approach used by Dooley & Vafa, (1986) and by Unger et al., (1986). In summary, it consists of a wet extraction of a spiked homogenate with methylene chloride. This is followed by treatment with hexyl magnesium bromide to form the hexyl derivatives of any extractable organotin species present. These tetra-alkyltins have low volatilities and separate well in the gas chromatograph. The resulting derivatized extract is cleaned up by liquid chromatography on silica gel followed by addition of a second internal standard, concentration, and quantitation using capillary GC/FID. Ordinarily, three sets of duplicate samples are analysed at a given time. If the results for a given duplicate are not sufficiently precise (s>_+25%), then a second duplicate from the remaining homogenate is analysed. A detailed description of the method and the preparation of standards and reagents is available from the author on request. Many of the small-scale manipulations used in this procedure are described by Mayo et al (1985). Frozen mussels were thawed, shucked, and thoroughly homogenized using a high-speed homogenizer. In the case of archived samples of frozen mussel tissue homogenate, the thawed samples were rehomogenized prior to analysis. Duplicate 0.5 g subsamples of the homogenate were taken for dry weight determination. Duplicate 7-10 g subsamples of the homogenate were taken for analysis. Each analytical replicate was weighed to __+i mg in a tared 25X 150 mm culture tube. To each replicate, 15 ml of 1:1 HC1 (aq) and 15 ml saturated NaCt (aq) was added, followed by 2000-3000 ng of the recovery standard, tripentyltin chloride, in methanol. Each replicate was divided between the original and a second 25× 150 mm culture tube, mixed thoroughly using a vortex mixer, and allowed to stand for 15 min. The homogenate in each of the two tubes was then extracted with 15 ml methylene
Volume 20/Number 3/March 1989
chloride with vortex mixing at 15 min. intervals over a period of 1 h. The organic layer was separated by centrifugation, concentrated, and dried through sodium sulphate. The total lipid extract was treated with 4 ml 2.0 M hexvl magnesium bromide (see below) and allowed to react at 30°C in foil-covered tubes for 30 min. The reaction mixture was quenched by adding crushed ice, followed by 10 ml 1:4 HCI (aq), with vortex mixing. The organic layer was separated by centrifugation and washed a second time with water as above. The derivatized extract was concentrated to less than 1 ml, brought up to a volume of 2 ml with n-hexane and applied to a lX30 cm glass chromatography column containing 5 gm of 5% deactivated 100-200 mesh silica with a 1 cm layer of anhydrous sodium sulphate on top. The nonpolar fraction containing the tetra-alkyltin derivatives was obtained by eluting with three column volumes (25 ml) of n-hexane. This eluate was concentrated to several ml. An aliquot of the butyltripentyltin GC standard in hexane (6000 ng) was then added and the extract concentrated to 0.2-0.5 ml. The final extract was analysed by capillary gas chromatography using a 30 mX0.32 mm ID fused silica SE-30 column and an SGE splitless injector on a Pye 104 gas chromatograph with a flame ionization detector. The carrier gas was helium at a flow rate of 3 ml rain -~ and an injector septum bleed rate of 0.5 ml min-L The temperature program was 150-250°C at 3°C rain -1 with a final bake-out at 280°C for 10 rain. The integrated signal was analysed by peak height with reference to a tetra-alkyltin calibration standard mixture run twice each day. The quantitation of each alkyltin component in the GC was based on the known amount of butyltripentyltin GC standard present. The final concentration of TBT and DBT in the sample was corrected for recovery of the tripentyltin chloride recovery standard. Figure 1 shows a representative set of gas chromatographic data for a mussel tissue extract, an oyster tissue extract, and an alkyltin standard mixture.
equal to the increment of TBTCI added, namely 1.00 ~g. In the case of the present study, the slope was 1.01 ~,g, well within the overall precision of the method.
Standards and Reagents The accuracy of the method depends on the two standards. The tripentyltin chloride recovery standard compensates for incomplete extraction and sample loss. The butyltripentyltin GC standard compensates for run-to-run fluctuations in the gas chromatographic equipment and enables one to obtain satisfactory results using older instruments. Chemicals for the synthesis of standards and Grignard reagents were obtained from Aldrich Chemical Co.
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,~
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I
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I
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Quality assurance~quality control All solvents were triple-distilled from reagent grade. Cotton wool and sodium sulphate were extracted with methylene chloride prior to use. Aqueous reagents were extracted with methylene chloride prior to use. Analytical precision was routinely determined by duplicate analyses. Higher levels of replication were also done periodically. A quadruplicate sample of partially-depurated March, 1988 Lynher River (Cornwall, UK) mussels was analysed. The results were: 0.71_+0.08 Ixg g-~ for TBT and 0.48+0.02 gg g-t for DBT. Accuracy of the method was tested by analyses of three sets of duplicate subsamples of a mussel homogenate spiked with 0, 1.00, and 2.00 gg of tributyltin chloride. The six samples were of equal weight to _+ 1% permitting direct comparison of the analytical data as a group. The results of the analyses and of a linear least squares fit of the data are given in Table 2. The slope of the least squares line should be
Fig. I Gas chromatograms of (a) a calibration of standard of tetraalkyl tins; (b) a derivatized mussel tissue extract; (c) a derivatized oyster tissue extract. All three chromatograms are at the same attenuation. The nanograms of each component in a given peak are noted. The abbreviations are: Bu~butyl; Hex=hexyl; Pen=pentyl. Tetrapentyl tin is present as an impurity in the tripentytttin recovery standard and has the same number of carbon atoms as dibutyldihexyl tin. It therefore serves as a useful means of judging the resolution of the gas chromatographic column on a daily basis.
131
Marine Pollution Bulletin TABLE 2 Results of replicate analyses of spiked mussel homogenate samples of very nearly equal weight to permit the direct comparison of the data. The mussels were collected from Whitsand Bay, Cornwall on 14 April 1988. The data were fitted to a linear least squares regression line. The results of the least squares regression are given below. Sample No.
gg TBTC added
total gg TBT found
total p.g DBT found
QAIA QA1B QA2A QA2B QA3B QA3B
0.00 0.00 1.00 1.00 2.00 2.00
0.36 0.38 1.27 1.57 2.39 2.38
0.17 0.13 0.16 0.11 0.17 0.17
Regression line: (g~,~ TBT)=I.01 (ggTBTCladded)+0.38
r2=0.988
Average gg DBT recovered: 0.15_+0.03 (n=6) Sample dry wt: 1.669 g_+0.013
The tripentyltin chloride recovery standard is chemically very similar to tributyltin chloride and can be expected to provide an accurate estimation of overall recovery. In the method above, the single extraction generally yields an overall recovery of 50-80% depending on sample size and loss during the procedure. Sample sizes in excess of 2 g dry wt give poorer overall recoveries. Tripentyl tin chloride was prepared from the reaction between 3 equivalents of tetrapentyltin and 1 equivalent of tin(IV) chloride at 200°C (see Unger et al., 1986). The precursor, tetrapentyl tin, was prepared from the reaction between a 10% molar excess of pentyl magnesium bromide and tin(IV) chloride. The identity and purity of the final tripentyltin chloride product were confirmed by gas chromatographic/mass spectrometric (GC/MS) analysis of the heyxl derivative. A direct synthesis of trialkyl tins from tin metal and the appropriate alkyl halide has also recently been reported (Holland, 1987). A working stock solution of tripentyltin chloride in methanol was prepared every 2-3 weeks from a concentrated stock solution in glacial acetic acid. Both were kept in a freezer between uses. The GC standard, butyltripentyltin (BPT), with one carbon more than the hexyl derivative of TBT and one carbon less than the dihexyl derivative of DBT, has a retention time approximately midway between the two (see Fig. 1) thus making it a useful GC standard for DBT and TBT. This was prepared from the reaction between 3+ molar equivalents of pentyl magnesium bromide and 1 equivalent of monobutyltin trichloride. The product was purified by passage through a silica column as above and the identity confirmed by G C / MS. Standard solutions in hexane were prepared and stored under refrigeration. Other tetra-alkyltin standards were prepared in pure form from the reaction of hexyl magnesium bromide with the appropriate butyl tin species and used as stock solutions in hexane. As above, the identities and purities of these standards were confirmed by GC/MS. Grignard reagents were prepared in the laboratory under nitrogen using the appropriate alkyl bromide, magnesium turnings, and anhydrous ether. Hexyl magnesium bromide (0.2 M) was prepared in 100 ml 132
quantities and stored under nitrogen in a bottle with a teflon-faced septum. Quantities were withdrawn with a syringe under positive nitrogen pressure.
Discussion The equipment and supplies required for this method are available in most laboratories engaged in environmental chemical analysis. Once standards are prepared and satisfactory gas chromatographic separations can be achieved, the unit cost per sample is quite low. For the analysis of four samples, reagents required include 120 ml of methylene chloride, 240 ml of n-hexane, 40 gm of silica, 20 gm of sodium sulphate, and 16 ml of hexyl magnesium bromide. Petroleum ether with a 6080°C boiling range may be substituted for the n-hexane. The use of capillary gas chromatography is essential to the method. The hexyl-organotin derivatives have retention times falling between the normal alkanes, n-C18 and n-C22. If interferences are a problem with the hexyl derivatives of specific samples, pentyl or heptyl derivatives can be readily prepared instead, with the use of appropriate standards. The temperature programme given is arbitrary and the analyst has considerable latitude concerning temperature programme parameters. In many cases, published gas chromatographic methods for TBT use a more specific flame photometric detector (see Rice et al., 1987) which could be used in the present method as well. However, a non-specific flame ionization detector was used because of its wider availability, wider (10 s) linear response range, and greater sensitivity compared with a flame photometric detector (Grob, 1985). The butyltripentyltin (BPT) GC standard, not used in other reported methods, enables satisfactory results to be obtained from older gas chromatographs given that satisfactory separations are possible. Because the sample peaks are quantitated in terms of known concentration/peak height relationships for the BPT standard, injection volume and sample volume are cancelled out, thus eliminating a possible source of error.
Conclusions The analysis of TBT and DBT in mussel tissues provides useful means for making an assessment of TBT inputs. The analytical method presented here, while labour-intensive and requiring some synthetic skill in setting up, provides good precision and accuracy with relatively unsophisticated instrumentation and modest amounts of supplies.
This work was performed while a visiting scientist at the Plymouth Marine Laboratory, West Hoe, Plymouth, England. The author would like to thank Dr. J. Readman for gas chromatographic/mass spectrometric analyses and Dr. R Donkin and Mr. J. Clearly for helpful discussions and support.
Alzieu, C. (1986). The detrimental effects on oyster culture in France-Evolution since antifouling paint regulation. In Proceedings of the Organotin Symposium of the Oceans 86 Conference, pp. 1130-1134. Marine Technology Society, Washington, D.C.
Volume 20/Number 3/March 1989 Blunden, S., Cusack, P. A. & Hill, R. (1985). The Industrial Uses of Organotin Compounds. Royal Society of Chemistry, London. Bryan, G. W., Gibbs, P. E., Hummerstone, L. G. & Burt, G. R. (1986). The decline of the gastropod Nucella lapiUus around south-west England: evidence for the effect of tributyltin from antifouling paints. J. Mar. Biol. Assoc. U.K. 66,611-640. Cardwell. R. D & Sheldon, A. W. (1986). A risk assessment concerning the fate and effects of tributyltins in the aquatic environment. In Proceedings of the Organotin Symposium of the Oceans 86 Confererzce, pp. 1117-1129. Marine Technology Society, Washington, D.C. Champ, M. A. & Pugh, W. L. (1987). Tributyltin antifouling paints: introduction and overview. In Proceedings of the Organotin 51vmposium of the Oceans 87 Conference, pp. 1296-1308. Marine Technology Society, Washington, D.C. Clark, E. A., Sterrit, R. M. & Lester, J. N. (1988). The fate of tributyltin in the aquatic environment. Environ. Sci. Technol. 22,600-604. Donard, O. F. X., Rapsomanikis, S. & Weber, J. H. (1986). Speciation of inorganic tin and alkyltin compounds by atomic absorption spectrometry using electrothermal quartz furnace after hydride generation. Anal. Chem. 58, 772-777. Dooley, C. A. & Vafa, G. (1986). Butyl tin compounds and their measurement in oyster tissues. In Proceedings of the Organotin &'mposium of the Oceans 86 Conference, pp. 1171-1176. Marine Technology Society, Washington, D.C. Farrington, J. W., Goldberg, E. D., Risebrough, R. W., Martin, R. W. & Bowen, V. T. (1983). U.S. Mussel Watch, 1976-1978: an overview of the trace metal, DDE, PCB, hydrocarbon, and artificial radionuclide data. Environ. ,';ci. Tec'hnol. 17,490-496. Gilfillan, E. S. & Page, D. S. (1986). Use of multivariate statistical techniques to associate biologically active stressors with observed effects in multiple stressor situations. In Proceedings of the Oceans 80 Conference, pp. 953-959. Marine Technology Society, Washington, D.C. Grob, R. L. (1985). Modern Practice of Gas Chromatography. WileyInterscience, New York. Grovhoug, J. G., Seligman, R F., Vafa, G. & Fransham, R. L. (1986). Baseline measurements of butyhin in U.S. harbors and estuaries. In Proceedings of the Organotin Symposium of the Oceans 86 ConJereace, pp. 1283-1288. Marine Technology Society, Washington, D.C. Holland, F. S. (1987). The direct synthesis of triorganotin compounds: proce~,s and reaction mechanism. Appl. Organomet. Chem. 1, 449458. Humphrey. B. & Hope, D. (1987). Analysis of water, sediments, and biota for organotin compounds, in Proceedings of the Organotin ,~vrnposiam of the Oceans 87 Conference, pp. 1348-1351. Marine Technology Society, Washington, D.C. Laughlin, R. B. & Linden, O. (1985). Fate and effects of organotin compounds. Ambio 14, 88-94. Lee, R. F., Valkirs, A. O. & Seligman, P. E (1987). Fate of Tributyltin in estuarine waters. In Proceedings of the Organotin Symposium of the
Oceans 87 Conference, pp. 1410-i415. Marine Technology Society, Washington, D.C. Linden, O. (1987). The scope of the organotin issue in Scandinavia. In Proceedings of the Organotin Synposium of the Oceans 87 Conference, pp. 1320-1323. Marine Technology Society, Washington, D.C. Maguire, R. J., Tkacz, R. J., Chau, Y. K., Bengert, G. A. & Wong, P. T. S. (1986). Occurrence of organotin compounds in water and sediments of Canada. Chemosphere 15,253-274. Mayo, D. W., Pike, R. & Butcher, S. S. (1985). Microscale Organic Laborato~. John Wiley and Sons, New York. Minchin, D., Duggan, C. B. & King, W. (1987). Possible effects of organotins on scallop recruitment. Mar. Pollut. Bull. 18,604-608. Rice, C. D., Espourteille, F. A. & Huggett, R. J. (1987). Analysis of Tributyltin in estuarine sediments and oyster tissue, Crassostrea virginica. Appl. Organomet. Chem. 1,541-544. Seligman, R F., Valkirs, A. O. & Lee, R. F. (1986). Degradation of tributyltin in marine and estuarine waters. In Proceedings of the Organotin Symposium of the Oceans 86 Conference, pp. i 189-1195. Marine Technology Society, Washington, D.C. Short, J. W. & Thrower, F. P. (1986). Accumulation of butyltins in muscle tissue of Chinook salmon reared in sea pens treated with trin-butyltin. Mar. Pollut. Bull. 17,542-545. Stephenson, M. D., Smith, D. R., Wall, L. W., Johnson, E., Michel, P., Short, J., Waldock, M., Huggett, R. J., Seligman, P. & Kola, S. (1987). An International intercomparison of butyhin determinations in mussel tissue and sediments. In Proceedings of the Organotin Symposium of the Oceans 87 ConJerence, pp. 1334-138. Marine Technology Society, Washington, D.C. Stephenson, M. D. & Smith, D. R. (1988). Determination of tributyltin in tissues and sediments by graphite furnace atomic spectrometry. Anal. Chem. 60,696-698. Thain, J. E., Waldock, M. J. & Waite, M. E. (1987). Toxicity trod degradation studies of tributyltin (TBT) and dibutyltin (DBT) in the aquatic environment. In Proceedings of the Organotin Symposium of the Oceans 87 Conference, pp. 1398-1404. Marine Technology Society, Washington, D.C. Unger, M. A., MacIntyre, W. G., Greaves, J. & Huggett, R. J. (1986). GC determination of butyltins in natural waters by flame photometric detection of hexyl derivatives with mass spectrometric confirmation. Chemosphere 15, 461-470. Waldock, M. J. & Miller, D. (1983).. The determination of total and tributyltin in seawater and oysters in areas of high pleasure craft activity. ICES paper CME:12 (mimeo), Int. Council Explor. Sea, Copenhagen. Waldock, M. J., Waite, M. E. & Thain, J. E. (1987a). Changes in concentrations of organotins in U.K. rivers and estuaries following legislation in 1986. In Proceedings of the Organotin Symposium of the Oceans 87 C))njerence, pp. 1352-1356. Marine Technology Society, Washington, D.C. Waldock, M. J., Thain, J. E. & Waite, M. W. (1987b). The distribution and potential toxic effects of TBT in UK estuaries during 1986. AppL Organomet. Chem. 1,287-3(/l.
1.~3
Davies, A. M. (1983). In Flushh,g times of the North Sea. ICES 1983/ CRR: 123. Delvigne, G, A. L. (1987). Experiments on the dilution capacity of wakes from dumping tankers in the North Sea. In Oceanic Processes in Marine Pollution, Vol. 2 (T. P. O'Connor, W. V. Burt & 1. W. DuedaU, eds), pp. 000-000. Robert E. Krieger Publishing Co., Malabar, Florida. Durance, J. A. & Jones, S. R. (1976). Spatial variability of residual drift offthe east coast of England in 1975. ICES CM 1976/C:3. Huthnance, J. (1987). NERC North Sea Project. NERC News, October, 1987. Lewis, R. E. (1985). Dilution of waste in the wake of a ship. Water Res. 18, 941-945. Lewis, R. E. (1984). Circulation and mixing in estuary outflows. Cont. Shelf Res. 3,201-214. Medler, K. J. (1977). Residual drift regimes in the west central North Sea during JONSDAP 76. ICES 1977/C:5. Okubo, A. (1971). Oceanic diffusion diagranls. Deep Sea Res. 18,789802. Pasquill. F. & Smith, E B. (1983). Atmospheric DiffiLsion. Third edition. Ellis Horwood Ltd., Chichester. Ramster, J. W. (1977). Residual drift reNmes off the north-east coast of England. ICES CM 1977/C:8. Talbot, J. W. & Talbot, G. A. (1974). Diffusion in shallow seas and in English coastal and estuarine waters. Rapp. P-v. Rdun. Cons. inc Explor. Mer. 167, 93-110. van Pagee, J. A., Gerritsen, H. & de Ruijter, W. R M. (1986). Transport and water quality modelling in the southern North Sea in relation to coastal pollution research and control. WaterSci. Tech. 18, 245-256. Webb, A. J. (1982). A random walk model of the dispersion of caesium-137 in the Irish Sea. M.Sc. thesis. University of Wales.
Marine Pollution tJtdletin, Volume 20, No. 3, p p 129-133, 1989 Printed in Great Britain.
0 0 2 5 - 3 2 ( , X / 8 9 S3.00+0 0c) ~) 1989 Pergamon Prcs~ plc
An Analytical Method for Butyltin Species in Shellfish D A V I D S. P A G E B o w d o i n College H y d r o c a r b o n Research Center, B r u n s w i c k , M a i n e 04011, U S A
A method for analysing tributyltin (TBT) and dibutyltin (DBT) in shellfish is presented that places modest requirements on equipment and supplies. It is based on capillary gas chromatographic analysis of hexyl derivatives of any extractable organotin species present. Two internal standards are used. Data are presented showing that the precision and accuracy of the method are generally better than _+20%.
T r i b u t y l t i n (TBT) c o n t a i n i n g m a r i n e a n t i f o u l a n t s h a v e seen w i d e s p r e a d use for o v e r 20 years. C o n c e r n a b o u t the a d v e r s e effects on m a r i n e life o f T B T l e a c h i n g f r o m t r e a t e d surfaces has resulted in strict c o n t r o l s of its use in F r a n c e , the U n i t e d K i n g d o m , the U n i t e d States, a n d o t h e r j u r i s i d i c t i o n s (see C h a m p & Pugh, 1987; W a l d o c k et al., 1987a; C l a r k et al., 1988). C u r r e n t
c o n c e r n a b o u t the a d v e r s e effects of T B T o n shellfish b e g a n with the i d e n t i f i c a t i o n of T B T - l e a c h a t e f r o m antifouling p a i n t s as b e i n g r e s p o n s i b l e for the c o l l a p s e of the o y s t e r fishery a r o u n d A r c a c h o n , F r a n c e in 1980 (see A l z i e u , 1986). It is n o w r e c o g n i z e d that w a t e r c o n c e n t r a t i o n s of T B T in the 1 0 - 5 0 ng 1-1 r a n g e can exert lethal a n d s u b l e t h a l effects on a wide variety of m a r i n e o r g a n i s m s p a r t i c u l a r l y for the case of sensitive j u v e n i l e life f o r m s ( W a l d o c k et al., 1987b; C a r d w e l l & S h e l d o n , 1986). M i c r o b i a l d e g r a d a t i o n is p r o b a b l y the d o m i n a n t p r o c e s s for the b r e a k d o w n of T B T in n e a r s h o r e waters ( S e l i g m a n et al., 1986; L e e et al., 1987) with d i b u t y l t i n ( D B T ) the m a j o r d e g r a d a t i o n p r o d u c t ( L e e et aL, 1987). W h i l e D B T a p p e a r s to b e at least a n o r d e r of m a g n i t u d e less toxic to m a r i n e a n i m a l s ( L a u g h l i n & L i n d e n , 1985; T h a i n et al., 1987), its w i d e s p r e a d use as a s t a b i l i s e r for P V C plastics ( B l u n d e n et al., 1985) a n d its r e l a t i o n s h i p to T B T as a d e g r a d a t i o n p r o d u c t m a k e s 129
Marine Pollution Bulletin
it important to analyse for both TBT and DBT in a marine monitoring program. In the marine environment, leachate from TBT may originate from a variety of sources such as treated hulls of commercial or military vessels and pleasure boats; dockyard painting and hull washing facilities. Table 1 summarizes some reported mussel tissue TBT concentrations for various locations. In assessing the impact of pollutants on indicator organisms such as mussels in bays and estuaries affected by marine activities, it is important to examine a range of potential stressors (Gilfillan & Page, 1986). Due to its toxic effect at low concentrations, TBT should be included in such assessments. In addition, analysis of TBT in filter-feeding bivalves such as mussels provides a time-integrated estimation of TBT in water consistent with the Mussel Watch Program approach for pollution monitoring in general (Farrington et al., 1983). A variety of methods for analysing TBT in tissues has been reported. They fall into two major categories: methods based on the analysis of organotin by atomic absorption spectrophotometric (AAS) quantitation of the Sn present; and methods based on the analysis of organotin derivatives by gas chromatographic (GC) techniques. In most cases, DBT was analysed either directly for the GC-based methods or indirectly for many of the AAS-based procedures. Atomic absorption-based methods include hydride-generation/AAS TABLE 1 Some reported concentrations of TBT in mussel tissues from field samples. The data are given as gg g-~ TBT on a dry tissue wt. basis. For those data reported on a wet tissue wt basis, the following conversion factor was used: 5×(wet wt ppm)=dry wt ppm. For those data reported on a weight of tin basis, the following conversion was used: 2.74 × (ppm as Sn) = ppm as TBT. Location
Type
gg g-~ TBT
Reference
San Diego, CA (USA) (3 sites)
2.90 2.90 1.60 0.25
Stephenson et aL, 1987
Monterey, CA (USA)
H/M H H R
Marina del Rey, CA
M
17.1
Stephenson etal., 1988
R
0.2
(USA) Humbolt, CA (USA) San Diego, CA (USA) (3 sites)
H/M N R Pearl Harbor, HI (USA) N H/M R Honolulu, HI (USA) H/M Norfolk, VA (USA) H/M R Little Creek, VA (USA) N H/M R -
2.69* 1.50" 0.93* 0.23* 0.30* 0.33* 7.35* 3.98" 1.18" 0.90* 1.41 * 0.69*
Ireland, Muh'oy Bay Galway Bay Banlry Bay
-
0.75 <0.25 <0.25
Sweden, W. coast E. coast
-
0.8-10.2 <0.2-3.9
Grovhoug el aL, 1986
Minchin et al., 1987
Linden, 1987
Data marked * are given as total solvent-extractable tin. Where replicate analyses are reported, the results are summarized as the average value. The type of sampling location is also given when identified in the source literature. The abbreviations are: H=harbour; M = marina; N = navy; R = reference; - = not indicated.
130
(Donard et al., 1986); direct solvent extraction/AAS (Short & Thrower, 1986); solvent extraction/NaOH wash to remove di- and monobutyltin/AAS {%Valdock & Miller, 1983; Bryan et al., 1986; Stephenson & Smith, 1988). Gas chromatographic methods generally involve solvent extraction of treated or untreated tissue homogenates followed by formation of a tetra-alkyl Sn derivative with a Grignard reagent and analysis by: GC/ AAS (Maguire et al., 1986); GC/flame photometric detection (Rice et al., 1987); GC/mass spectrometric detection (Humphrey & Hope, 1987); GC/flame ionization detection (Dooley & Vafa, 1986). In an intercalibration exercise between seven laboratories (Stephenson et al., 1987), reasonable agreement was found for the analyses of replicate mussel samples irrespective of the method used. The present work was undertaken to develop a method for analysing TBT and DBT in tissues that can be performed with a minimal investment in equipment and supplies by laboratories normally engaged in trace organic analysis of environmental samples.
Materials and Methods Analytical procedltre The method is based on the analytical approach used by Dooley & Vafa, (1986) and by Unger et al., (1986). In summary, it consists of a wet extraction of a spiked homogenate with methylene chloride. This is followed by treatment with hexyl magnesium bromide to form the hexyl derivatives of any extractable organotin species present. These tetra-alkyltins have low volatilities and separate well in the gas chromatograph. The resulting derivatized extract is cleaned up by liquid chromatography on silica gel followed by addition of a second internal standard, concentration, and quantitation using capillary GC/FID. Ordinarily, three sets of duplicate samples are analysed at a given time. If the results for a given duplicate are not sufficiently precise (s>_+25%), then a second duplicate from the remaining homogenate is analysed. A detailed description of the method and the preparation of standards and reagents is available from the author on request. Many of the small-scale manipulations used in this procedure are described by Mayo et al (1985). Frozen mussels were thawed, shucked, and thoroughly homogenized using a high-speed homogenizer. In the case of archived samples of frozen mussel tissue homogenate, the thawed samples were rehomogenized prior to analysis. Duplicate 0.5 g subsamples of the homogenate were taken for dry weight determination. Duplicate 7-10 g subsamples of the homogenate were taken for analysis. Each analytical replicate was weighed to __+i mg in a tared 25X 150 mm culture tube. To each replicate, 15 ml of 1:1 HC1 (aq) and 15 ml saturated NaCt (aq) was added, followed by 2000-3000 ng of the recovery standard, tripentyltin chloride, in methanol. Each replicate was divided between the original and a second 25× 150 mm culture tube, mixed thoroughly using a vortex mixer, and allowed to stand for 15 min. The homogenate in each of the two tubes was then extracted with 15 ml methylene
Volume 20/Number 3/March 1989
chloride with vortex mixing at 15 min. intervals over a period of 1 h. The organic layer was separated by centrifugation, concentrated, and dried through sodium sulphate. The total lipid extract was treated with 4 ml 2.0 M hexvl magnesium bromide (see below) and allowed to react at 30°C in foil-covered tubes for 30 min. The reaction mixture was quenched by adding crushed ice, followed by 10 ml 1:4 HCI (aq), with vortex mixing. The organic layer was separated by centrifugation and washed a second time with water as above. The derivatized extract was concentrated to less than 1 ml, brought up to a volume of 2 ml with n-hexane and applied to a lX30 cm glass chromatography column containing 5 gm of 5% deactivated 100-200 mesh silica with a 1 cm layer of anhydrous sodium sulphate on top. The nonpolar fraction containing the tetra-alkyltin derivatives was obtained by eluting with three column volumes (25 ml) of n-hexane. This eluate was concentrated to several ml. An aliquot of the butyltripentyltin GC standard in hexane (6000 ng) was then added and the extract concentrated to 0.2-0.5 ml. The final extract was analysed by capillary gas chromatography using a 30 mX0.32 mm ID fused silica SE-30 column and an SGE splitless injector on a Pye 104 gas chromatograph with a flame ionization detector. The carrier gas was helium at a flow rate of 3 ml rain -~ and an injector septum bleed rate of 0.5 ml min-L The temperature program was 150-250°C at 3°C rain -1 with a final bake-out at 280°C for 10 rain. The integrated signal was analysed by peak height with reference to a tetra-alkyltin calibration standard mixture run twice each day. The quantitation of each alkyltin component in the GC was based on the known amount of butyltripentyltin GC standard present. The final concentration of TBT and DBT in the sample was corrected for recovery of the tripentyltin chloride recovery standard. Figure 1 shows a representative set of gas chromatographic data for a mussel tissue extract, an oyster tissue extract, and an alkyltin standard mixture.
equal to the increment of TBTCI added, namely 1.00 ~g. In the case of the present study, the slope was 1.01 ~,g, well within the overall precision of the method.
Standards and Reagents The accuracy of the method depends on the two standards. The tripentyltin chloride recovery standard compensates for incomplete extraction and sample loss. The butyltripentyltin GC standard compensates for run-to-run fluctuations in the gas chromatographic equipment and enables one to obtain satisfactory results using older instruments. Chemicals for the synthesis of standards and Grignard reagents were obtained from Aldrich Chemical Co.
A
o
U'?
,~
I..0
I
,_:, III[llll
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I
I
I
E
I
f
I
I
I
Quality assurance~quality control All solvents were triple-distilled from reagent grade. Cotton wool and sodium sulphate were extracted with methylene chloride prior to use. Aqueous reagents were extracted with methylene chloride prior to use. Analytical precision was routinely determined by duplicate analyses. Higher levels of replication were also done periodically. A quadruplicate sample of partially-depurated March, 1988 Lynher River (Cornwall, UK) mussels was analysed. The results were: 0.71_+0.08 Ixg g-~ for TBT and 0.48+0.02 gg g-t for DBT. Accuracy of the method was tested by analyses of three sets of duplicate subsamples of a mussel homogenate spiked with 0, 1.00, and 2.00 gg of tributyltin chloride. The six samples were of equal weight to _+ 1% permitting direct comparison of the analytical data as a group. The results of the analyses and of a linear least squares fit of the data are given in Table 2. The slope of the least squares line should be
Fig. I Gas chromatograms of (a) a calibration of standard of tetraalkyl tins; (b) a derivatized mussel tissue extract; (c) a derivatized oyster tissue extract. All three chromatograms are at the same attenuation. The nanograms of each component in a given peak are noted. The abbreviations are: Bu~butyl; Hex=hexyl; Pen=pentyl. Tetrapentyl tin is present as an impurity in the tripentytttin recovery standard and has the same number of carbon atoms as dibutyldihexyl tin. It therefore serves as a useful means of judging the resolution of the gas chromatographic column on a daily basis.
131
Marine Pollution Bulletin TABLE 2 Results of replicate analyses of spiked mussel homogenate samples of very nearly equal weight to permit the direct comparison of the data. The mussels were collected from Whitsand Bay, Cornwall on 14 April 1988. The data were fitted to a linear least squares regression line. The results of the least squares regression are given below. Sample No.
gg TBTC added
total gg TBT found
total p.g DBT found
QAIA QA1B QA2A QA2B QA3B QA3B
0.00 0.00 1.00 1.00 2.00 2.00
0.36 0.38 1.27 1.57 2.39 2.38
0.17 0.13 0.16 0.11 0.17 0.17
Regression line: (g~,~ TBT)=I.01 (ggTBTCladded)+0.38
r2=0.988
Average gg DBT recovered: 0.15_+0.03 (n=6) Sample dry wt: 1.669 g_+0.013
The tripentyltin chloride recovery standard is chemically very similar to tributyltin chloride and can be expected to provide an accurate estimation of overall recovery. In the method above, the single extraction generally yields an overall recovery of 50-80% depending on sample size and loss during the procedure. Sample sizes in excess of 2 g dry wt give poorer overall recoveries. Tripentyl tin chloride was prepared from the reaction between 3 equivalents of tetrapentyltin and 1 equivalent of tin(IV) chloride at 200°C (see Unger et al., 1986). The precursor, tetrapentyl tin, was prepared from the reaction between a 10% molar excess of pentyl magnesium bromide and tin(IV) chloride. The identity and purity of the final tripentyltin chloride product were confirmed by gas chromatographic/mass spectrometric (GC/MS) analysis of the heyxl derivative. A direct synthesis of trialkyl tins from tin metal and the appropriate alkyl halide has also recently been reported (Holland, 1987). A working stock solution of tripentyltin chloride in methanol was prepared every 2-3 weeks from a concentrated stock solution in glacial acetic acid. Both were kept in a freezer between uses. The GC standard, butyltripentyltin (BPT), with one carbon more than the hexyl derivative of TBT and one carbon less than the dihexyl derivative of DBT, has a retention time approximately midway between the two (see Fig. 1) thus making it a useful GC standard for DBT and TBT. This was prepared from the reaction between 3+ molar equivalents of pentyl magnesium bromide and 1 equivalent of monobutyltin trichloride. The product was purified by passage through a silica column as above and the identity confirmed by G C / MS. Standard solutions in hexane were prepared and stored under refrigeration. Other tetra-alkyltin standards were prepared in pure form from the reaction of hexyl magnesium bromide with the appropriate butyl tin species and used as stock solutions in hexane. As above, the identities and purities of these standards were confirmed by GC/MS. Grignard reagents were prepared in the laboratory under nitrogen using the appropriate alkyl bromide, magnesium turnings, and anhydrous ether. Hexyl magnesium bromide (0.2 M) was prepared in 100 ml 132
quantities and stored under nitrogen in a bottle with a teflon-faced septum. Quantities were withdrawn with a syringe under positive nitrogen pressure.
Discussion The equipment and supplies required for this method are available in most laboratories engaged in environmental chemical analysis. Once standards are prepared and satisfactory gas chromatographic separations can be achieved, the unit cost per sample is quite low. For the analysis of four samples, reagents required include 120 ml of methylene chloride, 240 ml of n-hexane, 40 gm of silica, 20 gm of sodium sulphate, and 16 ml of hexyl magnesium bromide. Petroleum ether with a 6080°C boiling range may be substituted for the n-hexane. The use of capillary gas chromatography is essential to the method. The hexyl-organotin derivatives have retention times falling between the normal alkanes, n-C18 and n-C22. If interferences are a problem with the hexyl derivatives of specific samples, pentyl or heptyl derivatives can be readily prepared instead, with the use of appropriate standards. The temperature programme given is arbitrary and the analyst has considerable latitude concerning temperature programme parameters. In many cases, published gas chromatographic methods for TBT use a more specific flame photometric detector (see Rice et al., 1987) which could be used in the present method as well. However, a non-specific flame ionization detector was used because of its wider availability, wider (10 s) linear response range, and greater sensitivity compared with a flame photometric detector (Grob, 1985). The butyltripentyltin (BPT) GC standard, not used in other reported methods, enables satisfactory results to be obtained from older gas chromatographs given that satisfactory separations are possible. Because the sample peaks are quantitated in terms of known concentration/peak height relationships for the BPT standard, injection volume and sample volume are cancelled out, thus eliminating a possible source of error.
Conclusions The analysis of TBT and DBT in mussel tissues provides useful means for making an assessment of TBT inputs. The analytical method presented here, while labour-intensive and requiring some synthetic skill in setting up, provides good precision and accuracy with relatively unsophisticated instrumentation and modest amounts of supplies.
This work was performed while a visiting scientist at the Plymouth Marine Laboratory, West Hoe, Plymouth, England. The author would like to thank Dr. J. Readman for gas chromatographic/mass spectrometric analyses and Dr. R Donkin and Mr. J. Clearly for helpful discussions and support.
Alzieu, C. (1986). The detrimental effects on oyster culture in France-Evolution since antifouling paint regulation. In Proceedings of the Organotin Symposium of the Oceans 86 Conference, pp. 1130-1134. Marine Technology Society, Washington, D.C.
Volume 20/Number 3/March 1989 Blunden, S., Cusack, P. A. & Hill, R. (1985). The Industrial Uses of Organotin Compounds. Royal Society of Chemistry, London. Bryan, G. W., Gibbs, P. E., Hummerstone, L. G. & Burt, G. R. (1986). The decline of the gastropod Nucella lapiUus around south-west England: evidence for the effect of tributyltin from antifouling paints. J. Mar. Biol. Assoc. U.K. 66,611-640. Cardwell. R. D & Sheldon, A. W. (1986). A risk assessment concerning the fate and effects of tributyltins in the aquatic environment. In Proceedings of the Organotin Symposium of the Oceans 86 Confererzce, pp. 1117-1129. Marine Technology Society, Washington, D.C. Champ, M. A. & Pugh, W. L. (1987). Tributyltin antifouling paints: introduction and overview. In Proceedings of the Organotin 51vmposium of the Oceans 87 Conference, pp. 1296-1308. Marine Technology Society, Washington, D.C. Clark, E. A., Sterrit, R. M. & Lester, J. N. (1988). The fate of tributyltin in the aquatic environment. Environ. Sci. Technol. 22,600-604. Donard, O. F. X., Rapsomanikis, S. & Weber, J. H. (1986). Speciation of inorganic tin and alkyltin compounds by atomic absorption spectrometry using electrothermal quartz furnace after hydride generation. Anal. Chem. 58, 772-777. Dooley, C. A. & Vafa, G. (1986). Butyl tin compounds and their measurement in oyster tissues. In Proceedings of the Organotin &'mposium of the Oceans 86 Conference, pp. 1171-1176. Marine Technology Society, Washington, D.C. Farrington, J. W., Goldberg, E. D., Risebrough, R. W., Martin, R. W. & Bowen, V. T. (1983). U.S. Mussel Watch, 1976-1978: an overview of the trace metal, DDE, PCB, hydrocarbon, and artificial radionuclide data. Environ. ,';ci. Tec'hnol. 17,490-496. Gilfillan, E. S. & Page, D. S. (1986). Use of multivariate statistical techniques to associate biologically active stressors with observed effects in multiple stressor situations. In Proceedings of the Oceans 80 Conference, pp. 953-959. Marine Technology Society, Washington, D.C. Grob, R. L. (1985). Modern Practice of Gas Chromatography. WileyInterscience, New York. Grovhoug, J. G., Seligman, R F., Vafa, G. & Fransham, R. L. (1986). Baseline measurements of butyhin in U.S. harbors and estuaries. In Proceedings of the Organotin Symposium of the Oceans 86 ConJereace, pp. 1283-1288. Marine Technology Society, Washington, D.C. Holland, F. S. (1987). The direct synthesis of triorganotin compounds: proce~,s and reaction mechanism. Appl. Organomet. Chem. 1, 449458. Humphrey. B. & Hope, D. (1987). Analysis of water, sediments, and biota for organotin compounds, in Proceedings of the Organotin ,~vrnposiam of the Oceans 87 Conference, pp. 1348-1351. Marine Technology Society, Washington, D.C. Laughlin, R. B. & Linden, O. (1985). Fate and effects of organotin compounds. Ambio 14, 88-94. Lee, R. F., Valkirs, A. O. & Seligman, P. E (1987). Fate of Tributyltin in estuarine waters. In Proceedings of the Organotin Symposium of the
Oceans 87 Conference, pp. 1410-i415. Marine Technology Society, Washington, D.C. Linden, O. (1987). The scope of the organotin issue in Scandinavia. In Proceedings of the Organotin Synposium of the Oceans 87 Conference, pp. 1320-1323. Marine Technology Society, Washington, D.C. Maguire, R. J., Tkacz, R. J., Chau, Y. K., Bengert, G. A. & Wong, P. T. S. (1986). Occurrence of organotin compounds in water and sediments of Canada. Chemosphere 15,253-274. Mayo, D. W., Pike, R. & Butcher, S. S. (1985). Microscale Organic Laborato~. John Wiley and Sons, New York. Minchin, D., Duggan, C. B. & King, W. (1987). Possible effects of organotins on scallop recruitment. Mar. Pollut. Bull. 18,604-608. Rice, C. D., Espourteille, F. A. & Huggett, R. J. (1987). Analysis of Tributyltin in estuarine sediments and oyster tissue, Crassostrea virginica. Appl. Organomet. Chem. 1,541-544. Seligman, R F., Valkirs, A. O. & Lee, R. F. (1986). Degradation of tributyltin in marine and estuarine waters. In Proceedings of the Organotin Symposium of the Oceans 86 Conference, pp. i 189-1195. Marine Technology Society, Washington, D.C. Short, J. W. & Thrower, F. P. (1986). Accumulation of butyltins in muscle tissue of Chinook salmon reared in sea pens treated with trin-butyltin. Mar. Pollut. Bull. 17,542-545. Stephenson, M. D., Smith, D. R., Wall, L. W., Johnson, E., Michel, P., Short, J., Waldock, M., Huggett, R. J., Seligman, P. & Kola, S. (1987). An International intercomparison of butyhin determinations in mussel tissue and sediments. In Proceedings of the Organotin Symposium of the Oceans 87 ConJerence, pp. 1334-138. Marine Technology Society, Washington, D.C. Stephenson, M. D. & Smith, D. R. (1988). Determination of tributyltin in tissues and sediments by graphite furnace atomic spectrometry. Anal. Chem. 60,696-698. Thain, J. E., Waldock, M. J. & Waite, M. E. (1987). Toxicity trod degradation studies of tributyltin (TBT) and dibutyltin (DBT) in the aquatic environment. In Proceedings of the Organotin Symposium of the Oceans 87 Conference, pp. 1398-1404. Marine Technology Society, Washington, D.C. Unger, M. A., MacIntyre, W. G., Greaves, J. & Huggett, R. J. (1986). GC determination of butyltins in natural waters by flame photometric detection of hexyl derivatives with mass spectrometric confirmation. Chemosphere 15, 461-470. Waldock, M. J. & Miller, D. (1983).. The determination of total and tributyltin in seawater and oysters in areas of high pleasure craft activity. ICES paper CME:12 (mimeo), Int. Council Explor. Sea, Copenhagen. Waldock, M. J., Waite, M. E. & Thain, J. E. (1987a). Changes in concentrations of organotins in U.K. rivers and estuaries following legislation in 1986. In Proceedings of the Organotin Symposium of the Oceans 87 C))njerence, pp. 1352-1356. Marine Technology Society, Washington, D.C. Waldock, M. J., Thain, J. E. & Waite, M. W. (1987b). The distribution and potential toxic effects of TBT in UK estuaries during 1986. AppL Organomet. Chem. 1,287-3(/l.
1.~3