The Science of the Total Environment 258 2000.

81 88

Analytical aspects of the gas chromatographic determination of tributyltin and metabolites in environmental samples
Alfred J. Vellaa, , Bernardette Mintoffa , Victor Axiakb
b a Department of Chemistry, Uni ersity of Malta, Msida MSD 06, Malta Marine Ecotoxicology Laboratory, Department of Biology, Uni ersity of Malta, Msida MSD 06, Malta

Accepted 14 April 2000

Abstract Tributyltin and its metabolites were determined in environmental samples by gas chromatography using a ame photometric detection method. The analytical method involved the propylation of the organotins and the use of a recovery standard to correct for inefcient recovery. A detection limit of 5 ng Sn ly1 for seawater, and 5 ng Sn gy1 for sediments, was obtained when the detector was equipped with an optical lter; however, this concentration limit, which is higher than the environmental quality standard for TBT, could be improved by a factor of 100 on removing the lter. The limitations imposed by the choice of recovery and internal standards were discussed and it was shown that the possible presence of SnII. had no effect on the total extractable inorganic tin, as determined by the analytical protocol. The identication of unexpected mixed methylbutyltins in TBT-contaminated sediments was described, and the implications of the existence of such compounds were discussed. 2000 Elsevier Science B.V. All rights reserved.
Keywords: Tributyltin TBT.; Gas chromatography; Methylbutyltins.

1. Introduction Organotin pollution is normally associated with TBT. The environmental quality standard EQS. for TBT, established in the UK, is currently 2 ng TBT ly1 , equivalent to 0.8 ng Sn ly 1 Cleary, 1991.. Most research work on organotins has conU

centrated on the presence and fate of TBT compounds in an aquatic environment Clark et al., 1988.. TBT is believed to degrade by a stepwise debutylation into dibutyltin DBT., monobutyltin MBT. and inorganic tin IV. Blunden et al., 1984; Maguire et al., 1983; Maguire et al. 1984.: Bu3 Snq Bu 2 Sn2q BuSn3q Sn4q The ionic forms are only formal representa-

Corresponding author.

0048-9697r00r$ - see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 8 - 9 6 9 7 0 0 . 0 0 5 0 8 - 8

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tions; when dispersed in seawater, the structures probably exist in equilibrium mixtures involving hydrated cations, chloride, carbonate and hydroxide Guard et al., 1982.. The decomposition scheme is probably an oversimplication, and other reactions and important organotin species may be involved in the geochemical cycling of tin. In this paper, we have discussed a number of issues concerning the analytical technique which we employed in determining the presence of organotin species in environmental samples from the Malta coastal zone. The conclusions, however, were not site specic and should serve to illustrate the general difculties encountered in generating data in this area of environmental chemistry.

ferred quantitatively to a vial where its volume was further reduced to approximately 0.5 ml under a stream of nitrogen. The vial was sealed and stored at y20C, pending derivatisation and gas chromatographic analysis. The sediments were wet sieved through a ne mesh, 4 g were spiked with 700 ng tripentyltin chloride in methanol and treated with 20 ml water and 5 ml hydrochloric acid, and left to stand overnight. The mixture was extracted with 25 ml dichloromethane containing 0.0125% tropolone Ashby and Craig, 1989.. The sediment was removed by vacuum ltration and the extract, after drying with sodium sulphate, was further treated as for the aqueous samples. 2.1. Deri atisation of extract and gas chromatography The extract was dissolved in 2 ml hexane and derivatised using propylmagnesium bromide 2 M in ether.. After quenching the reaction with HCl, the aqueous layer was re-extracted with another 1 ml of hexane, and the two organic layers were combined. The hexane extract was applied to a column containing 5 g silica gel 60, capped by 1 cm3 sodium sulphate and eluted with 15 ml hexane. The eluate was evaporated under nitrogen to approximately 2 ml, and transferred quantitatively to a vial containing 600 ng of tetrabutyltin, where the total volume was further reduced to 100 l. This product was analysed by gas chromatography using a Perkin Elmer 8000 gas chromatograph, equipped with a Grob injector and a ame photometric detector with a 610-nm lter. The carrier gas was helium and, for the detector, the ow rates of air and hydrogen were optimised for maximum sensitivity. The main analytical column was a 25-m fused silica narrow bore capillary column with a non-polar bonded phase BP1, SGE Australia.. For conrmation of the peak identities, co-chromatography with authentic standards was employed using the two different columns BP1 and BP10. and, where possible, the samples were also analysed by gas chromatography-mass spectrometry using an Ion Trap Detector Finnigan Matt.. A chromatographic peak was not accepted as real unless it had a height of not less than 10% of that of the internal standard. Appropriate reagent blanks were analysed in a

2. Materials and methods Samples were collected from stations along the coastline of Malta and Gozo, and consisted of bulk seawater, surface microlayer and sediments. Seawater samples 2 l. were collected at 1-m depth using a Van Dorn sampler, and were placed, unltered, in amber coloured glass bottles to which 5 ml of acetic acid were added. Microlayer samples 100 ml. were collected using either a glass or a Teon plate 0.2 m2 and 4 mm thick. introduced perpendicularly to the water surface Harvey and Burzell, 1972. and were treated in a similar manner to the seawater samples. Sediments were collected by an Ekman-Birge stainless steel bottom sampler: the top 2 3-cm layer of sediment was collected and stored in a polycarbonate bottle at y20C pending analysis. The analytical technique is summarised in the scheme shown in Fig. 1. Seawater samples 2 l. were spiked with a methanolic solution containing 700 ng tripentyltin chloride to serve as a recovery standard, and then extracted with 100 ml dichloromethane containing 0.0125% tropolone Waldock et al., 1986.. For the surface microlayer, the volumes of sample and extractant were 100 and 30 ml, respectively. The extract from the aqueous samples was dried over sodium sulphate and reduced to approximately 5 ml by rotary evaporation at room temperature, and then trans-

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Fig. 1. Analytical scheme for butyltins in environmental samples.

similar manner. Quantitation was performed by comparison with the peak height of tetrabutyltin as an internal standard. A mixture containing propylated butyltins, prepared daily from concentrated standard solutions, was used to test the performance of the gas chromatograph. 2.2. Quality assurance Quality assurance of the analytical technique was established by a comparison of the results from aqueous samples between our laboratory and two other independent laboratories.

3. Results and discussion Very often, an analytical technique is designed

to be practically blind to all chemical species other than that which is being sought. The advantage of using high-resolution capillary column gas chromatography in the analysis for TBT is that, in addition to being a sensitive method, the technique also permits the identication of other organotin forms that may be present. Moreover, once the identity of these additional organotins is established, their abundance may also be measured from the same gas chromatograms. Unfortunately, before the environmental sample can be converted into a form that can be presented to the gas chromatograph, several steps are required and this renders the protocol costly both in time and materials. Also, the larger the number of manipulations on the sample, the greater the experimental error and the risk of contaminating the sample.

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Table 1 Recovery efciencies of butyltins from water and sediment Butyltin Recovery from water % Run c a BuSn3q Bu 2 Sn2q Bu 3 Snq
a b

Recovery from sediment % Rcorb 59 73 92 %R unc 9 17 23 %R cor 27 58 76

48 62 77

Percentage recovery, uncorrected. Percentage recovery, corrected with a scaling factor based on recovery of tripentyltin chloride

In the following sections, we discuss specic problems associated with the analytical protocol and discuss their implications on the study of TBT in the marine environment in the light of our observations on eld samples from the Malta coastal zone. 3.1. The Grignard reagent Several Grignard reagents have been used as derivatising agents for TBT and its metabolites, including ethyl, propyl and pentyl magnesium bromide. The smaller the alkylating group, the more volatile the products of derivatisation, and the greater the losses during transfers and work up. On the other hand, the larger the carbon number of the alkyl group, the longer the residence time on the column, and this can signicantly increase the total instrument time per sample. We adopted the propyl reagent as it seemed to offer a suitable compromise, although it did introduce other problems that we had not anticipated ide infra.. We found that commercial ether solutions of the Grignard reagent were generally not pure enough for our purposes, hence the reagent was always prepared in-house in batches of 50 ml of a 2 M solution. We found that, even at y20C, the reagent cannot be stored for periods longer than a few weeks. 3.2. Use of a reco ery standard The peak corresponding to the derivatised recovery standard Pe3 PrSn. was used to correct the concentration values of analytes for losses during manipulation, extraction inefciency, incomplete conversion during derivatisation, evaporative

losses, etc. Page, 1989.. The recovery efciencies of tripentyltin from water and sediment were found to be 48 " 15. and 26 " 11.%, respectively. As regards the extraction efciency and loss by evaporation, the use of tripentyltin corrects best for Bu3 Snq, since this species has similar molecular dimensions and electrical charge to tripentyltin. The more polar dibutyltin DBT. and monobutyltin MBT. compounds partition less effectively in the organic solvent than Pe3 Snq, even though the presence of tropolone as the complexing agent should mitigate this problem somewhat. Moreover, MBT and DBT form more volatile products of derivatisation, and hence, losses during the workup will be greater. Indeed, these conclusions were veried experimentally using standards. The recovery efciencies from solutions of butyltin chloride in acidied CH3 COOH. distilled water 8000 ng Sn ly1 . were as reported in Table 1, and these represent mean values from triplicate runs. Recoveries from spiked sediments 5000 ng Sn gy1 . were even less efcient Table 1., ranging from corrected values of 27% for MBT to 76% for TBT. This suggests that while the use of a recovery standard corrects quite adequately for TBT in aqueous media, it underestimates the concentration of TBT in sediments by a signicant factor. We have used a recovery factor, as given in Table 1, in order to correct the concentrations of TBT as found in eld samples, but this approach assumes that different sediments have the same afnity for the species. In fact, this is probably not so: thus, in organic-rich sediments, butyltin species could be bound to ionic sites associated with mineral matter, but also to polymeric organic matter includ-

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ing humic materials, as may be found in matrices. This aspect needs to be kept in when environmental quality standards for pertaining to sedimentary materials are sidered. 3.3. Detection limits

such mind TBT con-

We have found that the ame photometric detector FPD. is a more sensitive device for organotin compounds than the ion trap mass spectrometric detector. The performance of the FPD depends critically on the ow rates of hydrogen and air used for the ame. A hydrogen-rich ame favours higher sensitivity. The optical lter, while favouring selectivity, signicantly reduces the sensitivity of the detector. We have opted to work with the lter in place and as a result, the detection limit for aqueous samples, i.e. that concentration which produces a signalrnoise ratio of approximately 3, was 5 ng Sn ly1 . This value is somewhat above that required to detect levels of TBT in environments conforming with the quality standard of 0.8 ng Sn as TBT. ly1 . For sediments, the detection limit was approximately 5 ng Sn gy1 . We could improve our detection limits by a factor of approximately 100 by removing the optical lter, but this was clearly at the cost of analyte discrimination. Other workers report detection limits which are similar to ours Tolosa and Readman, 1996.. It thus appears that, in order to detect TBT in natural waters at levels equal to or less than the EQS, the lterless operation of the FPD is essential. This implies that sulphur or phosphorus compounds, which are also detected by the ame photometer, can potentially interfere in the analysis and this clearly lowers the condence in the results. This problem needs to be considered when decisions relating to the setting of environmental quality standards are taken. 3.4. Choice of internal standard The use of tetrabutyltin as an internal standard Tolosa et al., 1992. presumes an absence of this organotin compound in environmental materials. Unfortunately, this presumption is being eroded

by reports, albeit infrequent, of the occurrence of this substance in such materials Matthias et al., 1986; Jantzen et al., 1993.. None of these reports pertained to Mediterranean sites, and in all samples analysed, the gas chromatographic peak height of tetrabutyltin was similar to that obtained from the mixture of standards that was used as a control. This suggests that none of the eld samples contained appreciable amounts of tetrabutyltin, although the choice of internal standard did create a blind spot for lower amounts of environmentally-derived Bu4 Sn. 3.5. The nature of sol ent extractable inorganic tin Current practice requires that solvent-extractable tin, which is not attached to organic groups, be reported as total recoverable inorganic tin TRISn., and is formally represented as Sn4q. Since inorganic tin is not particularly toxic, the interest in this species is not very impelling. However, if the tin burden of a sediment or water column is to be assessed, say with a view to establishing environmental fate of organotin compounds or calculating geochemical cycling uxes from estimated inputs of particular toxicants, then inorganic tin may well need to be considered. We investigated the recovery of inorganic tinIV. compounds by extracting distilled water spiked with an aqueous tinIV. solution 2785 ng Sn ly1 ., prepared by dissolving tin metal in hydrochloric acid and oxidising the product with potassium permanganate. Recovery from sediments was investigated by spiking dry beach sand, ground to pass through a 50- m sieve with aqueous tinIV.. In both cases, tripentyltin chloride was employed as the recovery standard. The mean corrected recovery rate of SnIV. from aqueous solution was 9 " 6.%, while that from sediments was 60 " 47 . %. These values compare with those of Maguire et al. 1982. who, using benzenertropolone as solvent, obtained recovery efciencies from water and sediment samples of 35 " 23.% and 55 " 26.%, respectively. As anticipated, tinIV. was extracted from the aqueous medium even less efciently than MBT, and this is consistent with the higher formal ionic charge of SnIV.. However, the greater extractability of

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SnIV. from sediment is unexpected, since one would have expected the adsorption onto the mineral matrix to reduce the availability of the metal ions to the organic solvent. We suggested that the formation of neutral wSnCl4 x, which is favoured in the presence of HCl-derived chloride ions, could be responsible for this phenomenon. Gas chromatographic determination of SnIV., coupled with the atomic absorption spectrophotometric determination of total tin for the same environmental sample, is an approach which would allow the speciation of inorganic tin into Sn2q and Sn4q, assuming that all and only the tinIV. present is convertible into the tetraalkyltin. Although SnII. is a mild reducing agent, liable to convert into SnIV. in oxic environments, it could conceivably be found in organicrich reducing environments. We tested whether, in the analytical protocol, SnII. could convert into tetrapropyltin, since it is known that tinII. salts react with borohydride to produce SnH4 Brinckman et al., 1981.. This was accomplished by extracting distilled water spiked with tinII. chloride 6 g Sn ly1 . and analysing the extract in the usual way. The percentage recovery of inorganic tin based on the formation of Pr4 Sn was found to be 1.1 " 0.7%. This nding is similar to that obtained by Maguire 1984. who attempted the butylation of tinII. with the same result. It appears, therefore, that any tinII. present in environmental material would not be incorporated to any signicant degree in the analysis for TRISn and, moreover, that the suggested method for speciating tin into SnII. and SnIV. could be possible. 3.6. Presence of en ironmental tetraalkyltins and its implications In several chromatograms from different types of environmental samples, but especially those from surface microlayer and TBT-contaminated sediments, we encountered peaks which did not correspond to any of the anticipated tin species, namely, TBT, DBT, MBT and Sn4q. We reanalysed those extracts where these peaks appeared using gas chromatography-ion trap detector mass spectrometry GC-ITDMS. and found

that a particularly abundant peak corresponded to a mixed butylmethyltin which, from its mass spectrum and relative gas chromatographic retention time, we interpreted as being trimethylbutyltin Me3 BuSn. We prepared the authentic compound and found it to have the same ion trap detector mass spectrum as the unknown peak, and to co-elute with it on co-injection using two different analytical columns. This organotin compound was rst reported by Maguire et al. 1986. in a few sediments from freshwater Canadian sediments. In Malta, we encountered the compound in approximately half of the sediments analysed 14 samples. and in two surface microlayer samples, but not in any of the 52 bulk seawater samples. In the sediments, the compound was present at concentrations which varied from 0.080 to 9.0 g Sn gy1 ; in these sediments, the TBT concentrations ranged from 0.020 to 1.5 g Sn gy1 . We considered the possibility that this compound and others similar to it, see below. could represent an analytical artifact arising from the use of microlitre quantities of methanol as a solvent for the internal recovery standard. We carried out several control experiments to test this hypothesis, but no peaks corresponding to the species were observed. Also, the fact that several eld samples were found to contain butyltins but had no peaks corresponding to trimethylbutyltin argues strongly against its formation as an artifact of the analytical procedure. By a similar procedure, we established that another unknown chromatographic peak belonged to dimethyldibutyltin, Me2 Bu2 Sn. In this case, however, the mass spectral evidence suggested that this peak had contributions from another organotin species, namely, methyltripropyltin, MePr3 Sn, which being an isomer of dimethyldibutyltin, co-elutes with it. Methyltripropyltin is the product of propylation of monomethyltin, a well-known species in the marine environment. On the other hand, dimethyldibutyltin has only been reported by Maguire 1984. in a few sediments from the Canadian Great Lakes. Again, this compound was abundant in sediments contaminated with TBT six samples: 0.080 0.30 g Sn gy1 . and in the microlayer

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seven samples: 0.40 21 g Sn ly1 .. None of these microlayer samples contained TBT. The presence of two mixed butylmethyltins suggested to us that a third, namely methyltributyltin, MeBu3 Sn, could also be present. However, since this molecule is isomeric with BuPr3 Sn, the propylated product from MBT, it is not possible to detect its presence since it co-elutes with this expected substance. Indeed, when we reanalysed the extracts using GC-ITDMS, we found that the peak corresponding to the retention time of MBT had a mass spectrum which, in most instances, revealed the presence of MeBu3 Sn. The problem of co-elution can be resolved by replacing the propyl group of the Grignard reagent with a larger alkyl group. In view of the possible occurrence of other, as yet unknown, tetraalkyltins in the environment, it may be preferable to investigate the possible use of a Grignard reagent having a distinctive alkyl structure e.g. internally branched as in 3,3-dimethylpentylmagnesium bromide. in order to allow for the easy recognition of the analytical label introduced during derivatisation. Butylmethyltins presumably result from the methylation of butyltin species in water or sediment. Their frequent occurrence in the Malta marine environment has interesting implications. Judging by the fact that very few reports exist concerning the presence of such compounds in the environment, one may be led to conclude that methylbutyltins are not particularly widespread. On the other hand, however, it may be that their presence has gone unnoticed and unreported due to the fact that attention may have been reserved solely for specic i.e. targeted. organotin forms. This may be especially the case when chemical analysis is performed in support of environmental quality assessment, where specic ecotoxicants e.g. TBT. are sought. It is not uncommon to see reports in the literature where FPD-generated gas chromatograms are presented that contain several more peaks than are identied and discussed by the authors. Mixed methylbutyltins could well be among such unidentied peaks. Methylbutyltins are expected to be less toxic than TBT Cooney and Wuertz, 1989., although it does not appear that their toxicology has received

much attention. Being neutral molecules, methylbutyltins are more volatile than the formally ionic TBT, DBT and MBT and this leads one to speculate on the possibility that the dredging of polluted sediments could cause these compounds to mobilise into the water column and eventually, into the atmosphere. Indeed, their presence in the surface microlayer suggests that the prospect of the ex-solution of these organotins into marine air is likely. The coastal atmosphere in areas proximate to TBT-polluted environments would appear to be an ideal candidate for an analysis for such compounds.

Acknowledgements This work was partly supported by the Commission of European Communities through its MedSPA programme under contract number MedSPA-91-1rUKr002rINTr06. References
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A.J. Vella et al. r The Science of the Total En ironment 258 (2000) 81 88 butylmethyltin species and ultratrace levels using simultaneous hybridizationrextraction with GC-FPD detection. Environ Sci Technol 1986;20:609 615. Page DS. An analytical method for butyltin species in shellsh. Mar Pollut Bull 1989;20:129 133. Tolosa I, Merlini L, De Bertrand N, Bayona JM, Albaiges J. Occurence and fate of tributyl- and triphenyltin compounds in western Mediterranean coastal enclosures. Environ Toxicol Chem 1992;11:145 155. Tolosa I, Readman JW. Simultaneous analysis of the antifouling agents tributyltin, triphenyltin and IRGAROL 1051 in marina water samples. Anal Chim Acta 1996;335:267 274. Waldock MJ, Thain JE, Waite ME. The distribution and potential toxic effects of tributyltin in UK estuaries during 1986. Appl Organomet Chem 1987;1:287 301.

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