摘要:

Evaluation of the state-of-the-art of butyl- and phenyltin compound determinations in freshwater sediment prior to certification of a reference material F. Ariese,a W. Cofino,a J. L. Go�mez-Ariza,b G. Kramerc and Ph. Quevauvillerd aVrije Universiteit, Instituut voor Milieuvraagstukken, de Boelelaan 1115, NL -1081 HV Amsterdam, The Netherlands bUniversidad de Huelva, Departamento de Quý�mica, Campus Universitario de la Rabida, E-21819 Palos de la Frontera, Spain cInstitute for Reference Materials and Measurements, Retieseweg, B-2440 Geel, Belgium dEuropean Commission, Standards, Measurements and Testing Programme, 200 rue de la Loi, B-1049 Brussels, Belgium Received 12th October 1998, Accepted 29th January 1999 In order to control the quality of butyl- and phenyltin compound determinations in sediment, the Standards, Measurements and Testing Programme (formerly BCR) of the European Commission has started a project, the final aim of which is to certify a freshwater sediment for its contents of a range of organotin compounds (mono-, di- and tributyltin, and mono-, di- and triphenyltin). The first part of this project involved an interlaboratory study which aimed to test the feasibility of preparation of candidate freshwater sediment reference material and to detect and remove most of the pitfalls observed in organotin determinations.This paper presents the main results of this interlaboratory study carried out prior to the certification campaign. The agreement obtained among laboratories for the six compounds determined was considered to reflect the state-of-the-art and was encouraging enough to decide upon the organisation of a certification campaign, which will be concluded in June 1999. Introduction Preparation of the reference material The sediment material used in the interlaboratory study was Butyl- and phenyltin compounds, particularly trisubstituted species, are known to be very toxic to aquatic biota at very collected from a side arm of the North Sea Canal (northwest of Amsterdam, The Netherlands).First, a small batch was low concentrations.1,2 Tributyltin (TBT) is released in the aquatic (marine and freshwater) environment from the leaching collected to test the stability of organotin compounds after diVerent drying procedures, and part of this material was used of TBT-based antifouling paints used on boats and ships, whereas triphenyltin (TPhT) is used both as an antifouling for the interlaboratory study.One year later, a larger batch was collected at the same location for the preparation of the agent and a herbicide formulation; both compounds are present in the water phase and can be released from polluted candidate reference material.The wet material was dispatched to the Institute for Reference Materials and Measurements sediments and harbour dredges. The monitoring of tin compounds is required by the European Community (EC) legis- (IRMM) in Geel (Belgium) for further treatment. The wet material was dried at 60 °C in Teflon-protected stainless steel lation, e.g. under the Council Decisions 75/437/EEC (Marine Pollution from Land-based Sources), 77/585/EEC trays, crushed using a stainless steel jaw crusher and subsequently sieved (<1 mm).As the material was extremely wet, (Mediterranean Sea) and 77/586/EEC (Rhine River), and the Council Directive 80/68/EEC (Groundwater). Compliance to it was necessary to repeat the drying and crushing procedure several times. The moisture content of the sieved material this legislation and the need for a worldwide comparability of data require that the analyses are accurate. However, the (determined by Karl–Fischer titration) was less than 3.5%.Fine grinding of the sediment was carried out with the fraction methods currently used for the determination of organotin compounds involve various analytical steps, such as extraction, of less than 1 mmusing a jetmilling/ultrafine classification system based on the following principles.At the bottom of the grinding derivatisation, separation and final detection which multiply the risks of analytical errors.3 In order to improve and ensure chamber, three nozzles were mounted through which jets of air (6 bar) were blown; the three-dimensional nozzle arrangement a good quality control of tin speciation analysis, a series of interlaboratory studies (including certifications) has been enabled the feed material to be ground completely without residue.The air accelerated thematerial particles, which impacted organised in the past few years.4 The project described in this paper is a continuation of this programme; it aims to evaluate on each other and generated a size reduction without any contamination risks. Fine particles were collected through the classi- the state-of-the-art of butyl- and phenyltin compound determinations in freshwater sediment prior to a certification cam- fying wheel on top of the chamber.Coarse particles flowed back along the walls into the grinding chamber. The grinding process paign. Several drying procedures were tested in order to select the method that would lead to a suitably stable reference was performed with a classifier speed of 4000 rpm.Based on previous experience,4 heat sterilisation tests were material. In addition, a complete set of organotin calibrants was prepared, purified in large batches and made available to carried out at 100 and 130 °C under air to verify the final organotin content, i.e.to determine whether phenyltin all participating laboratories. J. Environ. Monit., 1999, 1, 191–196 191compounds were still present in measurable amounts in the final The excess of Grignard reagent was removed with 0.5 mol L-1 sulfuric acid, and the ether extract was reduced to 1 mL and material. Subsamples of the jet-milled sediment were heat sterilised in Teflon protected stainless steel trays (30 mm powder passed through a Florisil column.The organotin compounds were eluted with pentane, concentrated to dryness under a layers) for 2 h and dispatched to the Institute for Environmental Studies (Amsterdam, The Netherlands) for analytical control. nitrogen stream and dissolved in a suitable solution containing the internal standard.The quantification was by GC-FPD. The organotin contents of the sediment treated at 60, 100 and 130 °C are presented in Table 1. The heat sterilisation at 100 °C The variations in amounts of organotin in the sediment under the diVerent storage conditions are depicted in Fig. 1. All the was considered to be the most adequate treatment for the whole batch, i.e.a suYciently high temperature to sterilise the sediment organotin species were found to be stable in samples stored at -20 °C over the entire duration of the experiment (540 d); (which would not be likely at 60 °C), but not modifying drastically the organotin pattern by degradation (which ismuch higher this result confirms previous studies which demonstrated the stability of organotins in frozen sediments.6–8 Other storage at 130 °C for some compounds, e.g.TPhT). Homogenisation was carried out in a multipurpose cone temperatures (+4 and+25 °C) are also possible for maintaining the integrity of the sediment material during the same period of mixer with semi-automatic filling equipment. After an additional treatment of 2 h at 100 °C, the powder was directly time; however, a temperature of +40 °C drastically aVects the stability of the sediment, and significant losses of TBT were introduced into the cone mixer by a central filling nozzle on top of the mixer.All drives and gearing were placed outside noted after 365 days of storage (see Fig. 1). It was decided to store the material at -30 °C to ensure maximum long-term the mixing chamber so that contamination by oil was excluded.All parts of the mixer in contact with the powder were made stability. The material is suYciently stable to allow for shipment at ambient temperature without the risk of degradation. of polished stainless steel. A feed screw, 100 mm from the bottom of the mixer, enabled the filling of the vials without stopping mixing with given masses of the material.Directly Preparation of calibrants after filling of atle, the feeder turned to the opposite direction to push the powder back into the cone mixer. The purpose of this subproject was to prepare highly purified butyltin and phenyltin compounds (in the form of chlorides), Samples were bottled in amber glass bottles with a polyethylene insert and a screw cap, each containing about 40 g of material.as well as their ethylated and pentylated derivatives, for use as recovery standards and calibration standards. The purity Finally, a shrink sleeve was sealed on top of the bottle around the screw cap. The material was stored at -30 °C. of commercially available organotin compounds is often not adequate, while the alkylated derivatives usually need to be prepared in-house.It was felt that, by providing ultrapure Homogeneity and stability checks standards synthesised and purified in large quantities by an expert laboratory, some errors due to impure calibrants could The homogeneity of the sediment reference material was verified by determining the analytical variability (10 determi- be avoided. The calibrants prepared were mono-, di- and tributyltin and mono-, di- and triphenyltin; these six com- nations of the same extract), the within-bottle variability (10 independent determinations from the same bottle) and the pounds were made available as chloride salts, as pentyl derivatives and as ethyl derivatives. Commercial organotin salts were between-bottle variability (45 independent determinations, corresponding to 3% of the total number of bottles).The tests used as starting materials; purification was carried out by a number of recrystallisation and/or distillation steps. The alkyl- did not reveal any significant inhomogeneities, i.e. the withinand between-bottle variabilities were in the same range as the ated derivatives were prepared via a Grignard reaction using the purified organotin chlorides as starting materials.The analytical variability. One of the most critical aspects of the preparation of a resulting products were again purified by repeated recrystallisations and/or distillations. The purity was checked by elemen- reference material is related to the risks of instability, and a stability study was therefore carried out at various tempera- tal analysis, by 1H and 13C NMR and, in the case of the alkylated derivatives, also by GC-MS.For all 18 compounds, tures. Four aliquots of the heat sterilised material were stored in the dark at -20 °C, +4 °C, +25 °C and +40 °C, and the purity was better than 98%. The organotin chlorides were distributed as pure com- analyses were performed in triplicate after 30, 60, 90, 120, 180, 360 and 540 days of storage.The sediment was pre-treated pounds; the ethylated and pentylated standards were dissolved in hexane and sealed in ampoules. The nominal concentrations with 50 mL of a solution of 151 water–hydrogen bromide, and the organotins were extracted with 50 mL of 0.04% (w/v) were calculated by weighing. It could be shown, using propagation error theory, that the uncertainty in the calibrant tropolone solution in dichloromethane for 2 h.The organic phase was dried with anhydrous sodium sulfate and reduced concentrations was dominated by the uncertainty in the material purity (1%). Other possible sources of error, such as to 1 mL. The solid phase was washed with 5 mL of hexane, then added to the dichloromethane extract and reduced again weighing errors, were found to be insignificant.Participants were instructed to transfer the hexane solutions from the to 1 mL to substitute the solvent for final derivatisation.5 The organotins were derivatised with 4 mL of 1 mol L-1 pentylmag- ampoules to screw-cap vials and to store them in the refrigerator after opening. Participants were also informed that the nesium bromide solution in ether for 1 h at room temperature.Table 1 Organotin results in subsamples of the freshwater sediment resulting from pasteurisation experiments. Organotin contents are expressed in mg kg-1 (as Sn) dry mass Air dried Sn species 60 °C 60° C+2 h at 100 °C 60° C+2 h at 130 °C MBT 270 300 330 320 290 310 DBT 170 250 250 250 220 260 TBT 460 430 340 340 100 110 MPhT 120 93 87 86 59 74 DPhT 38 34 28 30 28 37 TPhT 41 100 21 28 8 21 192 J.Environ. Monit., 1999, 1, 191–196Fig. 1 Stability of organotin species in air-dried sediments followed by pasteurisation under diVerent storage conditions (in the dark at various temperatures). The symbols and bars correspond to the mean and standard deviations of organotin compounds obtained from triplicate analyses.pure salts had been stored under nitrogen and should be summarises the diVerent methods used by these laboratories for the diVerent compounds. Calibration was either by cali- handled under nitrogen, especially phenyl SnCl3 which is very sensitive to moisture in the air. bration graph or standard additions. Samples of one of the test batches produced during the feasibility study were dispatched on dry ice to the participating Analytical techniques used in the interlaboratory laboratories.Pure organotin chloride calibrants were also study provided, as well as ethylated or pentylated derivatives for calibration and/or recovery measurements. Each laboratory Sixteen laboratories from eight European states participated in the interlaboratory study (see Acknowledgements).Table 2 which participated in the exercise was requested to make a J. Environ. Monit., 1999, 1, 191–196 193Table 2 Summary of methods used in the interlaboratory study (extraction, derivatisation, separation and detection) Lab. 1: acetic buVer, methanol extraction, sonication, NaBEt4 derivatisation, GC-QFAAS Lab. 2: acetic acid addition, toluene extraction, microwave leaching, NaBEt4 derivatisation, GC-MIP Lab. 3: HCl addition, hexane/ethyl acetate/tropolone extraction, sonication, NaBEt4 derivatisation, GC-MIP Lab. 4: HCl/NaCl addition, ethyl acetate/tropolone extraction, fluorogenic derivatisation, HPLC-FLUO Lab. 5: addition of HBr/H2O/MeOH, dichloromethane/tropolone extraction, NaBEt4 derivatisation, GC-FPD Lab. 6: addition of HBr/H2O/MeOH, tropolone extraction, Grignard pentylation, GC-FPD Lab. 7a: acetic acid extraction, shaking overnight, NaBH4 derivatisation, GC-QFAAS Lab. 7b: acetic acid extraction, shaking overnight, NaBEt4 derivatisation, GC-FPD Lab. 8: addition of acetic acid/MeOH, hexane extraction, NaBEt4 derivatisation, GC-AED Lab. 9a: HCl addition, MeOH/tropolone extraction, sonication, Grignard pentylation, GC-MS Lab. 9b: HCl addition, MeOH/tropolone extraction, sonication, Grignard pentylation, GC-FPD Lab. 10: acetic acid extraction using microwave, NaBEt4 derivatisation, GC-MIP Lab. 11a: acetic acid/toluene extraction, sonication, Grignard pentylation, GC-MS Lab. 11b: addition of HCl, diethyl ether/tropolone extraction, sonication, Grignard pentylation, GC-MS Lab. 12: addition of acetic acid, MeOH/hexane extraction, sonication, NaBEt4 derivatisation, GC-QFAAS Lab. 13: addition of MeOH, hexane extraction, NaBH4 derivatisation, GC-FPD Lab. 14: acetic acid/toluene extraction, sonication, Grignard pentylation, GC-FPD Lab. 15: acetic acid/tropolone extraction, dichloromethane back-extraction, sonication, post-column NaBEt4 derivatisation, HPLC-ICPAES Lab. 16: HBr/H2O/dichloromethane extraction, tropolone back-extraction, Grignard pentylation, GC-FPD AED, atomic emission detection; HPLC, high performance liquid chromatography; GC, gas chromatography; FLUO, fluorimetry; FPD, flame photometric detection; ICPAES, inductively coupled plasma emission spectrometry; QFAAS, quartz furnace atomic absorption spectrometry; MIP, microwave inductively coupled plasma atomic emission spectrometry; MS, mass spectrometry.minimum of five replicate determinations on at least two some improvements are deemed necessary, the participants considered that certification would be feasible. diVerent days. The results submitted in the interlaboratory study were discussed amongst all participants in a technical The results of dibutyltin (DBT) were subject to systematic errors due to, for example, poor separation (Lab. 15), poor meeting; they were presented in the form of bar graphs showing the laboratory codes and the methods used, the individual extraction (Lab. 1), diVerence in behaviour of spiked and non-spiked compounds during standard addition leading to means and standard deviations and the mean of laboratory means with its standard deviation; Fig. 2 gives an example of low results (Lab. 16), and possible underestimation of the hydride generation response leading to overestimated contents such a bar graph. Table 3 summarises the results obtained in the interlaboratory study. (Lab. 7). The same RSD range as observed for MBT was obtained (Table 3), with similar conclusions on possible certification.Intercomparison results The situation for TBT mirrored that observed for DBT and the same rationale was applied to the results from Labs. 1 and In the case of monobutyltin (MBT), two groups of results were apparent. Lab. 10 had used microwave-assisted extrac- 16. The spread of the results (between-laboratory RSDs of 38% for all data and 27% for selected sets) was felt to be tion, which apparently resulted in a higher extraction eYciency, thus explaining the high results. Lab. 12 also produced a somewhat disappointing in comparison with previous interlaboratory studies (e.g. certification of CRM 462). Participants higher result, although a diVerent extraction procedure had been used, and no explanation could be found. Lab. 9 sus- were recommended to carefully look at their methods prior to certification and to thoroughly follow quality control pro- pected its high results to be due to a pre-extraction during the spiking process; the three sets of data were withdrawn on the cedures to make sure that a better agreement will be obtained at the certification stage.basis of the doubts expressed. The RSD between laboratories ranged from 57% (raw data) to 26% (selected values); while For the three phenyltin compounds, Lab. 9 experienced Fig. 2 Example of bar graph used for the evaluation of the results of the interlaboratory study (MPhT in mg kg-1). The laboratory codes are indicated along with the methods used (abbreviations defined in Table 2). The results plotted correspond to five replicate determinations. M is the mean of laboratory means. 194 J.Environ. Monit., 1999, 1, 191–196Table 3 Summary of the results of the interlaboratory study. The results are given in mg kg-1 of the respective cations Number Number of Mean±s RSD (%) RSD (%) Compound of sets sets accepted (accepted sets) raw data accepted data MBT 16 11 154±40 57.0 25.9 DBT 18 14 540±138 51.8 25.6 TBT 19 17 1283±348 38.0 25.2 MPhT 11 9 56.0±10.3 38.0 18.4 DPhT 9 8 51.2±11.7 82.7 22.8 TPhT 12 10 80.6±19.1 43.1 23.7 instrumental problems. Other problems were noted for Labs. 3 ment of spiked samples along with candidate reference materials was found to be an excellent means to evaluate extraction and 7 which withdrew their results. With the exception of these obvious outliers, the agreement was considered to be recoveries on the same basis. reasonable (ranging from 18 to 24% for the three compounds) and promising with respect to future certification.Conclusions One of the most critical aspects to be considered in the Technical discussion preparation of a possible CRM is the long-term organotin stability. Previous experience with sediment (CRM 462)10 and A systematic comparison of the extraction methodologies demonstrated that a high range of recoveries were obtained, mussel tissue (CRM 477)11 demonstrated the diYculty in stabilising reference materials with respect to their organotin depending on the types of reagent and procedure.9 The six organotin compounds considered in this study do not behave contents.In particular, long-term storage at +4 °C was not suYcient to preserve the butyltin compounds in CRM 462, in the same way for diVerent extraction methods and it is clear that there is no ‘universal’ method which can be optimised for which required re-certification;12 in the case of CRM 477, the stability of the butyltin contents was achieved at -20 °C, but all compounds; in other words, no firm recommendation on the use of one single method was given and participants were phenyltins were not found to be stable under any of the conditions tested.11 Participants recommended that some free to use their own method provided that it was properly validated.Clean-up may result in the degradation of com- samples of the candidate CRM be deep frozen (at a temperature of -70 °C or lower) to enable future long-term stability pounds depending on the conditions employed, and this risk should be faced.checks of the CRM (which will be stored at -30 °C). It was stressed that, while heat sterilisation will eliminate bacterial Some comments on the extraction methods were made by the participants: (i) it was noted that HCl concentrations action, the change of temperature when handling the material (from refrigerator to ambient temperature) may be quite above 1 mol L-1 lead to the degradation of trisubstituted tin species; (ii) extraction with glacial acetic acid, followed by critical for its long-term stability, and this should be stressed in the recommendation of the use of the future reference back-extraction into toluene of deep frozen samples, spiking with isotopically labelled tin and then analysis by HPLC- material.The stability of the MBT and MPhT chloride calibrants is another point of concern, and practical ways should ICPMS, indicated some degradation consistent with a molecular rearrangement; (iii) microwave-assisted extraction of sedi- be developed to prevent degradation. With respect to the results of the interlaboratory study, the ments could lead to the rapid degradation of TPhT unless precautions were taken to avoid the high temperature ramp participants agreed that this range of RSDs corresponded to the state-of-the-art and recommended the organisation of the eVect. Derivatisation is another critical step in tin speciation, and certification campaign. A candidate CRM of freshwater sediment (CRM 646) was prepared in June–October 1997; it was a systematic study was carried out to test the influence of reaction temperature, reagent concentration, reaction time and shipped to the participating laboratories in January 1998.The certification project was conducted during the first half of shaking mixture.6 The reagent concentration was found to be a critical factor governing side reactions, whereas the length 1998 and the results were discussed in September 1998.The results of the certification will be discussed by the ‘BCR of the alkyl substituents played a role in the derivatisation yield, particularly for the less substituted tin compounds, due Certification Committee’ who will report on the suitability of the material as a CRM in June 1999; provided that this possibly to volatilisation. Pre-concentration to near dryness gave rise to variations between substituted forms, and should material is accepted, the new CRM could be on the market by the end of 1999.be avoided when short chain alkyl derivatives are used (cf. pre-concentration to 0.3 mL). Ethylation via Grignard reaction gave slightly better results than the use of sodium Acknowledgements tetraethylborate.General observations on the methods were as follows: This project was carried out under EC contract nr. MAT1-CT94–0071. The collection, preparation, homogeneity GC-AED provided an excellent separation, good sensitivity and flat baselines; GC-MS oVered a good separation and peak and stability studies of the material used in the interlaboratory study were carried out by the Institute for Environmental shapes; GC-FPD showed a poor baseline performance that could not be corrected; GC-AAS gave a poor sensitivity but Studies, Free University of Amsterdam (The Netherlands).The following laboratories participated in the interlaboratory was otherwise satisfactory; LC provided variable peak shapes and potential overlap problems leading to unresolved compo- study: CID-CSIC, Departamento de Quý�mica Ambiental, Barcelona (Spain); ENEA, Divisione di Chimica Ambientale, nents/interferences; polarography oVered a limited sensitivitand selectivity. Rome (Italy); GKSS-Forschungszentrum, Geesthacht (Germany); Kernforschungsanlage, Ju� lich (Germany); RIKZ, With respect to extraction recoveries, it was decided that a protocol would be drawn up for possible guidelines.However, Haren (The Netherlands); MAFF, Burnham-on-Crouch (UK); Technical University of Athens (Greece); Tin Research the participants recommended that laboratories be free to adopt their own (documented) spiking procedures; the ship- Institute, Uxbridge (UK); Universidad de Huelva, J. Environ. Monit., 1999, 1, 191–196 195Speciation in Bioinorganic Chemistry, ed.S. Caroli, John Wiley, Departamento de Quý�mica Analý�tica (Spain); Universitaire New York, 1996, p. 195. Instelling Antwerpen, Analytische Scheikunde, Wilrijk 4 Ph. Quevauviller, M. Astruc, L. Ebdon, H. Muntau, W. Cofino, (Belgium); Universidad de Barcelona, Departamento de R. Morabito and B. Griepink, Mikrochim. Acta, 1996, 123, 163. Quý�mica Analý�tica (Spain); Universita` di Genoa (Italy); 5 J.L.Go� mez-Ariza, E.Morales, I. Gira�ldez and R. Beltra�n, Int. J. Universite� de Bordeaux, Laboratoire de Photophysique et Environ. Anal. Chem., 1997, 66, 1. 6 P.H. Dowson, J. M. Bubb and J. N. Lester, Estuar. Coast. Shelf Photochimie Mole�culaire, Talence (France); Universite� de Pau Sci., 1996, 42, 551. et des Pays de l’Adour, Laboratoire de Chimie Analytique 7 Ph. Quevauviller and O. F. X. Donard, Fresenius’ J. Anal. Chem., (France); Universidad de Santiago de Compostela, 1991, 339, 6. Departamento de Quý�mica Analý�tica (Spain); Vrije 8 J.L.Go� mez-Ariza, E. Morales, R. Beltra�n, I. Gira�ldez and M. Universiteit, Instituut Milieuvraagstukken, Amsterdam (The Ruiz-Bený�tez, Quý�m. Anal., 1994, 13, S76. 9 M. B. De la Calle, R. Scerbo, S. Chiavarini, Ph. Quevauviller and Netherlands). R. Morabito, Appl. Organomet. Chem., 1997, 11, 693. 10 Ph. Quevauviller, M. Astruc, L. Ebdon, V. Desauziers, P. M. Sarradin, A. Astruc, G. N. Kramer and B. Griepink, Appl. Organomet. Chem., 1994, 8, 629. References 11 R. Morabito, P. Soldati, M. B. de la Calle and Ph. Quevauviller, Appl. Organomet. Chem., 1994, 12, 621. 1 R. J.Maguire, Appl. Organomet. Chem., 1987, 1, 475. 12 A. Lamberty, Ph. Quevauviller and R. Morabito, EUR Report, 2 Ph. Quevauviller and O. F. X. Donard, in Element Speciation in EN 18406, EC, Brussels, 1998. Bioinorganic Chemistry, ed. S. Caroli, John Wiley, New York, 1996, p. 331. 3 Ph. Quevauviller, E. A. Maier and B. Griepink, in Element Paper 8/07886F 196 J. Env

ISSN:0960-7919    

DOI:10.1039/a807886f

出版商:RSC    

年代:1999

数据来源: RSC

摘要:

Stability and storage problems in organotin speciation in environmental samples J. L. Go�mez-Ariza,*a I. Gira�ldez,a E. Morales,a F. Ariese,b W. Cofinob and Ph. Quevauvillerc aDepartamento de Quý�mica y Ciencia de los Materiales, Escuela Polite�cnica Superior, Universidad de Huelva, La Rabida, Huelva, Spain. E-mail: ariza@uhu.es bVrije Universiteit, Instituut voor Milieuvraagstukken, de Boelelaan 1115, NL-1081 HV Amsterdam, The Netherlands cEuropean Commission, Standards, Measurements and Testing Programme, 200 rue de la Loi, B-1049 Brussels, Belgium Received 16th October 1998, Accepted 23rd February 1999 The stability of both tributyltin (TBT) and triphenyltin (TPT) in water, sediment, oysters and cockles was studied over a period of 18 months using several storage conditions. Butyltins were stable in unacidified sea-water stored in polycarbonate bottles in the dark at 4 °C for 7 months, but half of the TBT concentration was lost after 540 d.A comparable preservation time was achieved for butyltins stored on C18 cartridges at room temperature. However, phenyltins extracted from sea-water were stable for only 60 d stored on cartridges and even more pronounced losses (about 90% after 540 d) occurred when they were stored in either polycarbonate or Pyrex glass bottles.Losses of organotins were observed in sediments after air drying and pasteurization treatments using a freeze-dried sediment as a comparator, whereas both butyltin and phenyltin species remained stable in sediments stored at -20 °C for the 18 months tested, irrespective of the treatment used for stabilization.Air drying followed by pasteurization was shown to be superior to other treatments for the stabilization of organotin compounds in sediments stored at higher temperatures, but 30% of TBT was lost after 540 d at 25 °C. Finally, butyltins were stable in both frozen cockles and oysters in the dark over a 7 month period and in freeze-dried samples stored at 4 °C for 5 months, but TBT losses of about 70% were observed after 540 d.Only a few studies have been specifically devoted to Introduction organotin stability in environmental samples during storage.7 The main inputs of tributyltin (TBT) and triphenyltin (TPT) No losses of diVerent organotin compounds in acidified disinto the marine environment are by release from marine tilled water were observed for at least 20 d in 1 l brown glass antifouling paints containing these species, which are then bottles stored at 25 °C.8 TBT and monobutyltin (MBT) were dissolved in sea-water and partially adsorbed on suspended found to be stable in natural filtered sea-water for 4 months solids or bioaccumulated by living organisms.Several eVects when stored at 4 °C in the dark, but the stability of dibutyltin of these compounds in organisms exposed to extremely low (DBT) was more doubtful.9 Otherwise, freezing followed by levels of both TBT and TPT are well documented: shell oven drying at 50 °C has been shown to be suitable to preserve thickening in oysters1 and mussels,2 imposex in gastropods,1 the stability of butyltins in sediments for 4 months9 and both poor growth in bivalves,1 etc.Therefore, monitoring of tin freezing and storage at 4 °C preserved butyltins for 1 year.10 compounds is required by EC legislation, and comparability However, degradation of TPT was observed after 3 months.10 of data produced by diVerent laboratories is necessary. Finally, good stability of butyltins was achieved for mussel The diVerent toxicities of organotin compounds4 necessitate samples stored for 44 months at -20 °C in the dark.11,12 the use of sophisticated approaches for analytical speciation.5 The aim of this study was to assess the stability of TBT, These procedures generally involve several analytical steps.DBT, MBT, TPT, diphenyltin (DPT) and monophenyltin Therefore, the validation of these techniques and the improve- (MPT) in natural samples (sea-water, sediments, oysters and ment of quality control in tin determination require the use cockles) during storage under diVerent conditions such as pH, certified reference materials (CRMs).These materials are temperature and type of container, in order to select the best products of very high added value.Therefore, rigorous control preservation conditions for both environmental studies and of their stability over long periods of time is necessary. Oven- for the preparation of CRMs. or freeze-drying and gamma irradiation pre-treatments have been used to reduce chemical and microbiological changes in Experimental solid materials and for achieving long-term stability.6 The accuracy of organotin determinations in natural samples Reagents, standards and apparatus not only depends on the measurement step, but also sampling, The reagents used in the experiments were of analytical-reagent storage and sample preparation aVect the reliability of the grade and obtained from Merck (Darmstadt, Germany) and results.Several processes such as contamination, volatilization, Aldrich (Milwaukee, WI, USA).C18 cartridges (Sep-Pak adsorption and degradation due to microbial activity or UV Classic cartridges, 600 mg of sorbent) were obtained from irradiation may alter the initial composition of the sample. Waters (Milford, MA, USA). Pesticide grade solvents were Many of the above-mentioned eVects are highly dependent on temperature, which has to be monitored during sample storage.purchased from Romil (Loughborough, UK). Organotin com- J. Environ. Monit., 1999, 1, 197–202 197pounds as chlorides were obtained from Aldrich (purity higher Organotin concentrations were deduced from calibration curves derived from derivatized calibrant solutions using peak than 95%, evaluated by FAAS) and were used without further purification, but analysis did not reveal detectable impurities.areas. The calibration curves were linear for Sn amounts less than 1.4 ng. All results produced in these experiments are Water used in the experiments was doubly distilled and deionized and gave blank readings in all the analyses. The expressed as Sn, ng l-1 for water and ng g-1 dry mass for sediments and animal tissues. glassware used for experiments was previously soaked in saturated Na2Cr2O7 in H2SO4 for 24 h, rinsed carefully with The analytical quality was monitored by preparing a calibration graph each week and by injecting a pentylated doubly distilled water and then with methanol and dried.organotin calibrant solution with all the tin species every day Stock standard calibrant solutions of each organotin species to test the instrument signal.Quality control diagrams were were prepared gravimetrically in diethyl ether at about constructed and rejection thresholds for analytical samples 100 mg l-1 (as Sn). They were stored refrigerated in the dark were set at three standard deviations of the mean, generated and diluted in hexane to give working calibrant solutions.An for 10 sample solutions analyzed at the beginning of the internal standard, dimethyldipentyltin (DPeT), was prepared experiments. The same calibrant solution stored at -20 °C from dimethyltin chloride (Aldrich) by Grignard derivawas used for all calibrations. This solution was verified with tization.13 a fresh calibrant solution prepared every 6 months and was A gas chromatograph (Star 360, Varian, San Fernando, found to be stable.Two Certified Reference Materials, a CA, USA) fitted with an SPI injector, a glass capillary column sediment (CRM 462) and a mussel sample (CRM 477), both (SPB-1; Supelco, Bellefonte, PA, USA) (15 m×0.53 mm id, obtained from BCR (Brussels, Belgium) were used for vali- film thickness 1.5 mm) and a pulsed flame photometric detector dation of the procedures.Recovery tests were performed for operating with a 610 cut-oV interference filter was used. The each kind of sample every month and ranged from 85±8% injector temperature was programmed as follows: initial tem- (for MPT in water) to 99±6% (for TBT in biota). All perature 40 °C held for 0.1 min, 300 °Cmin-1 ramp to 250 °C experiments for each organotin preservation study were per- and a final isotherm for 10 min.The injelume was formed by the same operator. The detection limits (evaluated 1 ml. The oven temperature started at 75 °C held for 0.85 min, as 3s of the blank) in water, biota and sediment analysis followed by a heating ramp of 10 °Cmin-1 to 250 °C and a (including the extraction step) are given in Table 1. Samples final isotherm for 5 min.Helium served as the carrier gas at a were analyzed at least three times with relative standard flow rate of 9.5 cm3 min-1. The detector was operated at deviations (RSD) in the range 4–10% for all the organotin 300 °C. compounds evaluated in water and biota samples and for TBT and DBT in sediment. However, higher RSD values, ranging from 2 to 37%, were obtained for both MBT and phenyltins Organotin determination calibration and quality control in sediments, particulary in the pasteurized sample. These poor Water samples were extracted using both liquid–liquid and results may be caused by the presence of an interfering solid phase extractions following methods described in the chromatographic peak close to MBT and by the low levels of literature.14,15 Briefly, a 1000 ml portion of sample acidified phenyltins in the sediment samples, being close to the detecwith 10 ml of HBr was extracted by shaking vigorously in the tion limits.dark with 300 ml of a 0.07% m/v solution of tropolone in pentane for 10 min. The organic extract was dried with anhy- Sample collection and preservation drous Na2SO4 and reduced in volume to 0.5 ml by rotary Two water samples collected in Cadiz harbor (southwest evaporation.The extract was derivatized by pentylation. Solid Spain) on diVerent days were used to study the stability of phase extraction was carried out as follows: for cartridge organotin compounds in polycarbonate and Pyrex glass bottles conditioning, 10 ml of methanol followed by 10 ml of distilled and on C18 cartridges, respectively.Previous analyses carried water were passed through a C18 cartridge with the aid of a out using liquid–liquid extractions revealed that phenyltin vacuum pump at a flow rate of 5 ml min-1. The water sample compound concentrations were below the detection limits in (1000 ml ) was passed through the cartridge at a flow rate of these samples.Therefore, the samples had to be spiked with 8 mlmin-1. Then the cartridge was dried under a stream of known amounts of phenyltin chlorides in order to assess the nitrogen for 5 min. Organotins were eluted using 2 ml of preservation of these species. One sample was spiked with 380, 1% v/v HBr and 0.1% tropolone solution in methanol. Prior 800 and 960 ng l-1 (as Sn) of MPT, DPT and TPT, respect- to the derivatization step, the solvent was removed under a ively, and two subsamples were stored in 2.5 l bottles.However, stream of nitrogen and the residue was solubilized in 1 ml these concentrations are high compared with those usually of hexane. found in environmental samples. These subsamples were stabil- Sediment and biota samples were digested using 50 ml of ized using diVerent treatments: (i) storage in polycarbonate water–hydrogen bromide mixture (1+1) and extracted with bottles at 4 °C both without acid addition and filtering and 50 ml of 0.04% m/v tropolone solution in dichloromethane for (ii) acidification with 0.5% HBr and storage in Pyrex glass 2 h.The organic phase was dried with anhydrous sodium bottles at 4 °C in the dark.The other sample was spiked to sulfate and reduced to 1 ml. The solid phase was washed with final concentrations of 380, 700 and 770 ng l-1 (as Sn) of 5 ml of hexane and was then added to the dichloromethane MPT, DPT and TPT, respectively, and was passed through extract and reduced again to 1 ml to substitute the solvent for the cartridges, which were then stored at 25 °C in the dark.final derivatization.16,17 The organotins were derivatized with 4 ml of 1 mol l-1 pentylmagnesium bromide solution in diethyl ether for 1 h at room temperature. The excess of Grignard reagent was Table 1 Detection limits for organotin species in water, biota and removed with 4 ml of 0.5 mol l-1 sulfuric acid and the ether sediment samples, with values given as ng Sn l-1 for water and ng extract was reduced to 1 ml and passed through a Florisil Sn g-1, dry mass basis, for biota and sediment column (4 g).The organotin compounds were eluted with Sample TBT DBT MBT MPT DPT TPT pentane, concentrated to dryness under a stream of nitrogen and dissolved in a suitable solution containing the internal Water 4.6 5.3 5.3 12 12 11 standard (ranging between 0.05 and 1.0 ml ).They were quant- Biota/sediment 0.30 0.32 0.33 1.4 1.6 1.3 ified by GC-FPD. 198 J. Environ. Monit., 1999, 1, 197–202stored in 250 ml brown glass bottles with plastic screw-caps. The initial concentrations of organotins were determined by five replicate analyses. No phenyltin compounds were found in the samples. Phenyltins were not added to the samples because of the physical and/or chemical binding of these analytes to the matrix, hence their analytical behavior may diVer considerably from bioaccumulated organotins.The subsequent analyses at 15, 30, 60, 90, 150, 210, 270, 365 and 540 d were made in triplicate. The samples were manually homogenized prior to each analysis. All samples were stored in the dark since butyltin and phenyltin photodegradation has been demonstrated.18 Statistical treatment The data were analyzed statistically for diVerences using factorial analysis of variance (ANOVA).Prior to analysis, all the data were tested for homogeneity of variance using the Barlett and Levene tests. Student’s t-test was applied to test diVerent hypotheses. An a value of 0.05 was adopted as the critical level for all statistical testing giving a 95% confidence level (CSS: STATISTICA).Results and discussion Organotin stability in water samples The results for diVerent concentrations of both butyl- and phenyltin species as a function of the storage time are shown in Fig. 1. No significant changes in concentration of TBT were Fig. 1 Stability of organotin species in sea-water stored in the dark observed during the first 210 d for samples stored in both under the following conditions: (a) and (b) unacidified samples in polycarbonate bottles and adsorbed on C18 cartridges polycarbonate bottles at 4 °C; (c) and (d) acidified samples in Pyrex (ANOVA, p=0.33 and 0.24, respectively).However, a slight glass bottles at 4 °C; (e) and (f ) on C18 cartridges at room temperature.decrease in TBT concentration was observed after 90 d in Symbols and error bars represent means and standard deviation of samples stored in Pyrex glass bottles (t-test, p=0.002). organotin concentrations in triplicate analyses. DiVerent results were obtained for TPT, whose preservation was more diYcult to achieve than that of TBT, showing a decrease in concentration from the first month of storage both A sediment sample was collected in a side arm of the North Sea Canal (northwest of Amsterdam, The Netherlands), homo- in polycarbonate and in Pyrex glass bottles (t-test, p<0.008).The decrease in both TBT and TPT was not followed by an genized and divided into three subsamples. Each subsample was subject to diVerent stabilization procedures at the Institute increase in the concentration of dialkyltin or monoalkyltin species, which indicated that losses of TBT and TPT may not for Reference Materials and Measurements (IRMM): (i) freeze-drying, (ii) air drying at 40 °C and (iii) air drying at necessarily be due to degradation, and adsorption on the bottle surface is also possible.Better preservation of this 40 °C followed by pasteurization at 120 °C.Some of these processes may alter the composition of the sample. However, species was obtained in cartridges and no changes in concentration were observed during the first 60 d (ANOVA, p= since this sample will be used as a candidate certified reference material, the main purpose was to obtain the maximum 0.57). Therefore, the use of cartridges for organotin determination has undoubted advantages: (i) it requires a smaller stability of the sample during a long-term period. Each subsample was divided into four aliquots and stored at four diVerent volume of solvent than traditional liquid–liquid extraction; (ii) it involves simple manipulations which are not time temperatures in the dark: (i) 40±0.1, (ii) 25±1, (iii) 4±0.5 and (iv) -20 °C.Analyses of the organotins at 30, 60, 90, consuming and allow for in situ treatment of samples; and (iii) the cartridges can be used for storage of organotins for 2 120, 180, 360 and 540 d were performed in triplicate on diVerent days. The purpose was to test the stability of organot- months at room temperature. The stability of butyltins in both acidified, filtered natural ins with regard to eVects of storage temperature and drying procedure in a sediment sample.samples and non-acidified synthetic aqueous solutions stored at 4 °C in Pyrex glass bottles for 4–5 months has been The study of the stability of butyltins in biota during storage was performed on Crassostrea gigas oyster samples and reported.9,19,20 The disagreement between these longer preservation times and those obtained in this work could be explained Cerastoderma edulis cockle samples.They were collected in three estuarine areas of southwest Spain: Piedras River (oys- by the use of unfiltered samples with 30–35 mg l-1 of suspended matter, which has been proved to decrease the stability ters), Carreras River (oysters) and San Pedro River (two samples of cockles, collected downstream and upstream).of butyltin compounds stored in Pyrex glass bottles.9 Moreover, we spiked the samples with high phenyltin concen- Oysters and cockles were depurated in clean 0.45 mm filtered sea-water for 24 h and then the animal tissues were removed trations and the losses obtained in the present study may be lower than those obtained in samples with more realistic from their shells and drained.The edible part of each sample was homogenized using a mechanical mixer and divided into environmental levels because the biological activity may be slowed, aVecting the stability of both butyl- and phenyltins, two parts. One half was stored at -20 °C and the other was lyophilized (48 h at -60 °C), ground in a Teflon-coated and the adsorption on the container walls is also higher at lower concentrations.grinding mill and stored at 4 °C. The material was finally J. Environ. Monit., 1999, 1, 197–202 199Fig. 3 Stability of organotin species in air-dried sediments stored in Fig. 2 Stability of organotin species in freeze-dried sediments stored the dark at the following temperatures: (a) and (b) 40 °C; (c) and (d) in the dark at the the following temperatures: (a) and (b) 40 °C; (c) 25 °C; (e) and (f ) 4 °C; (g) and (h) -20 °C.Symbols and error bars and (d) 25 °C; (e) and (f ) 4 °C; (g) and (h) -20 °C. Symbols and represent means and standard deviation of organotin concentrations error bars represent means and standard deviation of organotin in triplicate analyses. concentrations in triplicate analyses.Organotin stability in sediment samples natural sediments,10 these treatments may be used to achieve long-term stabilized reference materials and as a consequence The integrity of the organotin levels in the sediment sample during the stabilization treatment was evaluated by triplicate they were considered for further experiments. Figs. 2–4 summarize the trends of the contents of the analyses of each sample.The results are given in Table 2. Freeze-drying treatment was selected as a comparator to check organotin species as a function of the storage time for the diVerent storage conditions tested. All organotin species were the eVects of both air-drying and pasteurization processes on organotin levels as it does not produce changes in either shown to be stable in sediment samples stored at -20 °C for the 540 d checked and the stability was not aVected by the use butyltin or phenyltin species in sediments.9,10 Air drying treatment caused a decrease of 27.5% in the TBT content, of diVerent drying treatments (ANOVA, p>0.05).This con- firms previous studies demonstrating the stability of these with a simultaneous increase in MBT content.Pasteurized samples displayed a larger decrease of the TBT content (54.8%) compounds in frozen sediments.9,10,20 Moreover, losses or interconversion of species were prevented in both freeze-dried and a simultaneous increase in both DBT and MBT (26.2 and 110%, respectively). These results indicated that both air and pasteurized sediments stored at 4 °C in the dark for the 540 d tested (ANOVA, p>0.07 for both samples), showing drying and pasteurization degraded TBT to DBT and MBT. Similar results were obtained for phenyltin species in pasteur- the longest stability reached with both treatment methods in comparison with air-dried samples. ized samples, in which a decrease of TPT and a corresponding increase of DPT contents were observed (68.3 and 25.9%, Temperature aVected the stability of the organotin compounds.A significant decrease in the TBT content was respectively). Although a CRM should be representative of real samples, one of the most important requirements for the observed after 3 months in both air-dried and freeze-dried sediments stored at 25 °C (t-test, p<0.01) and after 1 month production of this kind of material is its long-term stability.Consequently, some compromise has to be found between the when stored at 40 °C (t-test, p<0.01). A less pronounced influence of the temperature on the organotin stability was matrix resembling natural samples and stability. Although neither air drying nor pasteurization is not advisable for observed in pasteurized sediments, in which marked TBT losses were only observed after 1 year of storage at 40 °C.It environmental studies as they change the composition of the Table 2 Influence of the stabilization treatment on the organotin concentrations in a candidate CRM sediment Concentration (ng g-1 as Sn, dry mass basis)±one standard deviation Treatment TBT DBT MBT MPT DPT TPT Freeze-drying 418±14 225±12 58.5±5.3 32.9±3.1 10.8±0.5 22.0±0.5 Air drying 303±12 241±11 105±35 44.3±7.3 17.3±0.7 27.2±2.2 Air drying+pasteurization 189±9.7 284±2.4 123±10 20.4±1.5 13.6±1.7 6.98±1.54 200 J.Environ. Monit., 1999, 1, 197–202Fig. 4 Stability of organotin species in air-dried sediments followed by pasteurization stored in the dark at the following temperatures: (a) Fig. 5 Stability of butyltin species in: (a) frozen C.gigas; (b) frozen and (b) 40 °C; (c) and (d) 25 °C; (e) and (f ) 4 °C; (g) and (h) -20 °C. C. edulis; (c) C. gigas freeze-dried at 4 °C; (d) C. edulis freeze-dried at Symbols and error bars represent means and standard deviation of 4 °C. All samples were stored in the dark. Symbols and error bars organotin concentrations in triplicate analyses. represent means and standard deviation of organotin concentrations by triplicate analyses.is advisable to prepare the CRM in such way that the samples in the dark, the three butyltin species being stable for only 5 remain stable at room temperature, avoiding special storage months (ANOVA, p>0.15). This behavior indicated that TBT conditions because of the large amount of CRM to be prodegraded by a stepwise debutylation mechanism to DBT, cessed.Moreover, they should not be aVected by short expo- MBT and inorganic tin. A higher stability has been obtained sure to extreme conditions during shipment of the material to for freeze-dried mussels stored under experimental conditions customers. Therefore, air drying followed by pasteurization similar to those used in this study, no changes in butyltin was considered to be the optimum treatment.contents being found after 44 months of storage.12 Possibly the diVerent nature of the biological organism tested could Organotin stability in biota samples account for these diVerences. Prior to studying the stability of the butyltin compounds in biota samples, the possibility of losses of these compounds Conclusions during the lyophilization process was evaluated.Analyses were performed on fresh and freeze-dried samples and the results Only a few preservation studies concerning aqueous, sediment and biota samples containing organotin compounds have been were compared. Five replicate analyses were performed for each sample and no significant diVerences were found (by reported in the literature.Storage of unfiltered and nonacidified sea-water in polycarbonate bottles at 4 °C in the dark using the regression line test, the correlation coeYcient was 0.997 and the calculated slope and intercept did not diVer is suitable to achieve good stability for butyltins and phenyltins for 7 and 3 months, respectively, which allows a considerable significantly from the values of 1 and 0, respectively), which indicated that lyophilization is an excellent procedure to dry period of time between sampling and the final determination.However, better preservation was achieved using C18 cartridges the samples without losses or interconversions of butyltin compounds. even at ambient temperature with the additional advantage of the small space necessary to store the samples, which is The results for butyltin contents as a function of the storage time are shown in Fig. 5. According to these results, butyltin important if a large number of samples have to be analyzed. Both butyl- and phenyltin species were stable in air-dried, compounds were stable in both cockles and oysters stored at -20 °C in the dark over a 7 month period (ANOVA, p>0.11).freeze-dried and pasteurized sediments stored at -20 °C for at least 540 d, which allows the use of the three treatments to However, a decrease in the TBT content of about 14% was detected after 270 d followed by a increase in DBT content (t- obtain suitable stability for a candidate CRM. However, although the organotin content changed during pasteurization, test, p<0.007).A longer storage period produced a decrease in both TBT and DBT levels and a corresponding increase of the long-term stability of this kind of sample was less aVected by relatively high temperatures (40 °C), which is important MBT and finally, after 540 d, a decrease in all the butyltin compounds was observed (t-test, p<0.001). Comparable when extreme conditions are present during CRM shipment.Finally, the maximum time of storage for oyster and cockle results were obtained for freeze-dried samples stored at 4 °C J. Environ. Monit., 1999, 1, 197–202 2017 M. Abalos, J. M. Bayona, R. Compan�o� , M. Granados, C. Leal samples at either-20 or 4 °C after freeze-drying was 6 months, and M. D. Prat, J. Chromatagr., 1997, 788, 1. which allows adequate preservation for both environmental 8 K.Bergmann, U. Ro�hr and B. Neidhart, Fresenius’ J. Anal. studies and interlaboratory quality control analysis. However, Chem., 1994, 349, 815. other types of biological organisms such as mussels are more 9 Ph. Quevauviller and Q. F. X. Donard, Fresenius’ J. Anal. Chem., useful for preparing CRMs owing to the long-term stability 1991, 339, 6. 10 J. L. Go� mez-Ariza, E. Morales, R. Beltra�n, I. Gira�ldez and of butyltin species. M. Ruiz-Bený�tez, Quý�m. Anal., 1994, 13, s76-s79. 11 A. M. Caricchia, S. Chiavarini, C. Cremisini, R. Morabito and R. Scerbo, Anal. Chim. Acta, 1994, 286, 329. Acknowledgement 12 Ph. Quevauviller, R. Morabito, L. Ebdon, W. Cofino, H. Muntau and M. J. Campbell, EUR Report, EN 17921, European The authors express their thanks to the Measurements and Commission, Brussels, Belgium, 1997. Testing Programme (BCR) Project MAT1-CT94–071 and to 13 J. L. Go�mez-Ariza, E. Morales and M. Ruiz-Benitez, Analyst, DGICYT (Direccio�n General de Investigacio�n Cientý�fica y 1992, 117, 641. Te�cnica), Grant No. Pb95–0731. 14 J. L. Go�mez-Ariza, E. Morales and M. Ruiz-Benitez, Appl. Organomet. Chem., 1992, 6, 279. 15 J. L. Go� mez-Ariza, R. Beltran, E. Morales, I. Gira�ldez and M. Ruiz-Benitez, Appl. Organomet. Chem., 1994, 8, 553. References 16 J. L. Go�mez-Ariza, E. Morales, I. Gira�ldez and R. Beltra�n, Inter. 1 C. Alzieu, Mar. Environ. Res., 1991, 32, 7. J. Environ. Anal. Chem., 1997, 66, 1. 2 M. D. Stephenson, D. R. Smith, J. Goetz, G. Ichikawa and M. 17 J. L. Go� mez-Ariza, E. Morales, I. Gira�ldez, R. Beltran and Martin, in Proceedings of the Organotin Symposium of the Oceans J. A. Pozas-Escobar, Fresenius’ J. Anal. Chem., 1997, 357, 1007. ’86 Conference, Washington, DC, Marine Technology Society, 18 J. A. Navý�o, C. Cerrillos, M. A. Pradera, E. Morales and 1986, p. 1246. J. L. Go�mez-Ariza, Langmuir, 1998, 14, 388. 19 C. A. Dooley and V. Homer, Naval Oceans Systems Technical 3 E. Oberdo� rster, D. Rittschof and P. McClellan-Green, Mar. Report No. 197, 1983, San Diego. Pollut. Bull., 1998, 36, 144. 20 W. R. Blair, G. H. Olson, F. E. Brinckman, R. C. Paule and D. A. 4 J. Widdows and D. S. Page, Mar. Environ. Res., 1993, 35, 233. Becker, Natural Bureau of Standards, Gaithersburg, MD, 1986. 5 F. Pannier, A. Astruc and M. Astruc, Appl. Organomet. Chem., 1994, 8, 595. 6 Ph. Quevauviller, Mikrochim. Acta, 1996, 123, 3. Paper 8/08043

ISSN:0960-7919    

DOI:10.1039/a808043g

出版商:RSC    

年代:1999

数据来源: RSC

摘要:

EVect of freezing on the length of the penis in Nucella lapillus (L.)† Annagh Minchin* and Ian M. Davies FRS Marine Laboratory, PO Box 101, Victoria Road, Aberdeen, UK AB11 9DB. E-mail: minchina@marlab.ac.uk; daviesim@marlab.ac.uk; Fax: +44 1224 295511; Tel:+44 1224 876544 Received 16th December 1998, Accepted 1st February 1999 When quantifying imposex in Nucella lapillus, two indices are used: the Vas Deferens Sequence Index (VDSI) and the Relative Penis Size Index (RPSI).Freezing and thawing increase the length of the penis in both male and female Nucella lapillus. In the population studied, this had no significant eVect on the RPSI, but was potentially an important source of additional variance in the estimation of the mean penis length. The sex and VDS of each snail were recorded.The penis Introduction length of each snail was measured immediately after dissection The dogwhelk, Nucella lapillus, has been widely used as a bio- to the nearest 0.01 mm by microscope eyepiece graticule. The indicator of the eVects of tri-butyl-tin (TBT) on marine variance of these measurements was estimated by repeated ecosystems.1–4 Exposure to TBT results in the superimposition observations of a series of snails and, for example, penis length of male characteristics on females, known as imposex.5 The ranged from 2.4 mm to 2.6 mm for a single snail with four quantification of imposex involves two indices: the Vas measurements out of six of 2.5 mm.The snails were then Deferens Sequence Index (VDSI) and the Relative Penis Size placed into numbered segmented trays and stored for 19 h at Index (RPSI).VDSI gives an indication of the reproductive -20 °C. The trays were wrapped in polythene bags while in competency (mean of the females in the population) of the the freezer to prevent the snails from drying out. The snails population, and the RPSI expresses the relative development were then allowed to thaw for 2.5 h and two measurements of of the penis in males and females: penis length were repeated, firstly soon after thawing and secondly a further 4 h later.RPSI= (Mean female penis length)3 (Mean male penis length)3 ×100% Results Imposex has been adopted by the Oslo and Paris The VDSI values for adults and juveniles at Skatey Shore Commission (OSPAR) as a component of their international were 3.95 and 3.88 respectively. The RPSI value in adults was Joint Assessment and Monitoring Programme (JAMP), rec- 9.4%.The mean shell height of toothed adults was 24.96 mm ommending that snails should be examined alive (without (s=1.45) and of juveniles 24.53 mm (s=1.79). narcotisation). The use of Nucella lapillus has been adopted The VDS of individual Nucella ranged from 3 to 4.The for Laboratory Performance Studies under the international VDS of each individual was identified before and after freezing. QUASIMEME quality assurance programme using the revised Freezing therefore had no eVect on VDSI determination. JAMP Guidelines.6 Freezing caused an increase in penis length in both males and It is sometimes impractical, for example during extended females (Fig. 1) and an increase in the variance of the penis periods of fieldwork, to maintain Nucella alive after collection. length measurements. There were only small changes in the Preservation of samples is then necessary. Chemical preserv- RPSI values (Table 1). atives, such as formalin, commonly change the texture of the Linear regressions were fitted to the relationships (Figs. 2 tissues of the snail making them diYcult to handle. Freezing and 3) between the initial penis length (PL0) and the penis is a convenient form of preservation which has less eVect on length immediately after thawing (PLF) and, for adults, after the texture of the tissues. However, it is not known what eVect a further 4 h (PLF4): freezing may have on imposex measurements. This paper examines the eVects of freezing on the length of the penis and, juveniles: PLF=1.133PL0+0.165 (R2=0.851, n=97) consequently, on the RPSI.adults: PLF=1.28PL0+0.171 (R2=0.842, n=196) PLF4=1.32PL0+0.154 (R2=0.835, n=196) Methods The two regression lines for adults were not significantly diVerent (Student t-test). Nucella lapillus were collected from below mid-shore level at Skatey Shore, Stonehaven, east Scotland on 22 January (97 juveniles) and 4 June (200 toothed adults) 1998.Snails were Discussion maintained at ambient temperature in an aquarium at the Freezing Nucella resulted in an increase in penis length in both laboratory for no more than 4 d prior to analysis. males and females, but had no eVect on VDSI and only a The shell height of each snail was measured to the nearest small eVect on RPSI (Table 1).The penis length in adult males 0.1 mm, using electronic callipers. The snails were then crushed and females increased by factors which were approximately with the aid of a vice and the soft tissues carefully removed. log normally distributed. The median value of the factor for males was 1.36 (interquartile range, 1.23–1.50) and for females was 1.36 (interquartile range, 1.10–1.57).No significant †© Crown copyright. J. Environ. Monit., 1999, 1, 203–205 203Table 1 Summary table of the eVects of freezing on Nucella lapillus Mean penis length/mm s VDSI RPSI (%) Adult Live male 2.72 0.50 Live female 1.24 0.37 3.95 9.4 Thawed male 3.73 0.69 Thawed female 1.67 0.42 3.95 8.9 Thawed for 4 h male 3.82 0.75 Thawed for 4 h female 1.69 0.41 3.95 8.7 Juvenile Live male 1.50 0.54 Live female 0.69 0.19 3.88 9.7 Thawed male 1.87 0.71 Thawed female 0.95 0.26 3.88 13.0 Fig. 1 Ratio of mean penis lengths after thawing (PLF) and 4 h later (PLF4) to initial mean penis length (PL0) in adult Nucella lapillus. Fig. 4 Predicted ratios of RPSI values calculated after thawing (PLF and PLF4) to the initial RPSI value (RPSI0) for adult and juvenile Nucella lapillus.Also shown is the predicted eVect of 6 h delay in measuring live dogwhelks (PL6) from Minchin and Davies.7. changes in penis length occurred during a further 4 h of storage after thawing. The marked changes in penis length with time observed in live dogwhelks by Minchin and Davies7 did not occur in frozen specimens (Fig. 4).Application of the regression equations above to a range of initial mean penis lengths can be used to estimate the potential eVect of freezing on RPSI measurements on populations showing a range of initial RPSI values. This eVect is relatively minor at initial RPSI values greater than 1%. The increases in penis length after freezing and thawing show considerable diVerences between individual dogwhelks.Fig. 2 Relationship between penis lengths after thawing (PLF) and In the population studied, the consequential increase in the initial penis length (PL0) for adult Nucella lapillus. standard deviation of the mean penis length in the adults was from 15% (females) to 40% (males). Snails of equal initial penis length showed wide ranges of penis lengths after thawing.For example, after thawing, the penis length in males of initial penis length 2.7 mm ranged from 2.97 to 5.13 mm. To investigate the importance of freezing as a source of additional variance of penis length, subgroups of males (or females) of equal initial mean penis length, but diVerent variance, were selected from the data set. The relationships between the standard deviation of the penis lengths of these subgroups before and after freezing (Fig. 5) show that freezing increases the variance of penis length in both males and females. The eVect is most marked in subgroups of low initial variance, and relatively small in subgroups of females where the initial standard deviation is >0.25 mm (RSD >~20%). While narcotisation leads to an underestimation of the RPSI,8 and a delay in the measurement of untreated snails Fig. 3 Relationship between penis lengths after thawing (PLF4) and initial penis length (PL0) for adult Nucella lapillus. leads to an overestimation,7 freezing introduces little bias to 204 J. Environ. Monit., 1999, 1, 203–205Acknowledgements We would like to thank Patricia Barrett for her help with the preparation of the samples.References 1 P. E. Gibbs, G. W. Bryan, P. L. Pascoe and G. R. Burt, J. Mar. Biol. Ass. UK, 1987, 67, 507. 2 G. W. Bryan, P. E. Gibbs, L. G. Hummerstone and G. R. Burt, J. Mar. Biol. Ass. UK, 1988, 66, 611. 3 G. W. Bryan and P. E. Gibbs, in Metal Ecotoxicology: Concepts and Applications, ed. M. C. Newman and A. W. McIntosh, Lewis, Ann Arbor, 1991, pp. 323–361. 4 E.Stroben, J. Oehlman, U. Schulte-Oehlmann and P. Fioroni, Malacol. Rev., 1996, Suppl. 6 (Molluscan Reproduction), 173. 5 B. S. Smith, Proc. Malacol. Soc. London, 1971, 39, 377. Fig. 5 The eVect of freezing on the standard deviation of the penis 6 I. M. Davies, A. Minchin and M. Harding, Marine Laboratory, length in adult Nucella lapillus. Aberdeen Report No 9/97, FRS Marine Laboratory, Aberdeen, 1997. 7 A.Minchin and I. M. Davies, Marine Laboratory, Aberdeen Report the RPSI measurement, but is potentially a source of consider- No 4/98, FRS Marine Laboratory, Aberdeen, 1998. able increased uncertainty in the estimation. 8 M. Huet, P. Fioroni, J. Oehlmann and E. Stroben, Hydrobiologia, 1995, 309, 29. Conclusion Paper 8/09806I Freezing and thawing increase the length of the penis in both male and female Nucella lapillus. In the population studied, this had no significant eVect on the RPSI, but was potentially an important source of additional variance in the estimation of the mean penis length, particularly in populations showing relatively narrow ranges of initial penis length. J. Environ. Monit., 1999, 1, 203–205 205

ISSN:0960-7919    

DOI:10.1039/a809806i

出版商:RSC    

年代:1999

数据来源: RSC