TORT-I, DORM-1 and DOLT-1 - are determined by gas chromatography with electron ... and is one of the most toxic substances found in the environ- ment.
Fresenius Zeitschriftfiir Fresenius Z Anal Chem (1989) 333:641 -644
9 Springer-Verlag1989
Marine biological reference materials for methylmerc analytical methodologies used in certification
y:
S. S. Berman, K. W. M. Siu, P. S. Maxwell, D. Beauchemin, and V. P. Clancy Division of Chemistry, National Research Council of Canada, Montreal Road, Ottawa, Ontario K1A OR9, Canada Meeresbioligische Referenzmaterialien fiir Methylquecksilber: Analytische Methoden der Zertifizierung Summary. The methylmercury concentrations in three existing marine biological certified reference materials TORT-I, DORM-1 and DOLT-1 - are determined by gas chromatography with electron capture detection, cold vapour atomic absorption spectrometry and inductively coupled plasma mass spectrometry after selective isolation of methylmercury. Two such procedures were used. These and the three analytical techniques are evaluated and compared. The certified methylmercury concentrations are: TORT-I, 0.128• DORM-1, 0.731_+0.060; and DOLT-I, 0.080 _+ 0.011 gg Hg/g dry weight.
Introduction Methylmercury is a poison of the central nervous system and is one of the most toxic substances found in the environment. It is bioaccumulated and found to be present in relatively high concentrations in predators of the upper trophic levels. Despite being highly toxic itself, inorganic mercury is deemed comparatively less dangerous than methylmercury. Consequently, considerable effort has been spent on developing analytical methodologies that can differentiate the mercury species - the most common being some form of selective extraction followed by either gas chromatography with electron capture detection (GC-ECD) or cold vapour atomic absorption spectrometry (CVAAS). Although the toxicity of methylmercury and the difficulties of its determination are well recognized, there are currently no certified reference materials (CRMs) available for methylmercury. Aside from playing a major role in quality assurance in an analytical laboratory, CRMs also serve to validate the accuracy of newly developed analytical methodologies. One of the mandates of this laboratory is to produce and make available CRMs for the marine science community in accordance with the Marine Analytical Chemistry Standards Program [1]. Certification of methylmercury in three currently available marine biological tissues - the dogfish muscle (DORM-I) and liver (DOLT-I), and the lobster hepatopancreas (TORT-I) - is now complete to compli-
Offprint requests to."K. W. M. Siu
ment currently certified total mercury concentrations. The methylmercury concentrations are in the range of submicrogram Hg per gram material and span approximately an order of magnitude from high to low. Certification requires the use of at least two independent techniques. This report describes and critically compares some of the analytical methodologies used in this certification.
Experimental
Reagents. Methylmercuric chloride, bromide and iodide were purchased from commercial sources (ICN, methylmercuric chloride; Pfaltz & Bauer, all three salts) and were standardized against mercuric chloride by using inductively coupled plasma mass spectrometry (ICP-MS). Both methylmercuric chloride standards contained the expected percentage of Hg, whereas the bromide and the iodide standards contained non-stoichiometric amounts of mercury. As a result, only the chloride salts were used in subsequent work. The species purity of the methylmercury chloride standards was verified by using GC-ECD. Solvents were 'Distilled-in-Glass' grade (Caledon). Acids were purified by sub-boiling distillation in a quartz still from reagent grade stocks. Water was distilled and deionized (DDW). Other chemicals were reagent grade or better. Certified reference materials. The three marine biological reference materials - DORM-1, DOLT-1 and TORT-I are currently available from the National Research Council of Canada. Their collection, production and certification have been detailed elsewhere [2, 3]. Briefly, DORM-I and DOLT-1 were produced respectively from the flesh and the liver of spiny dogfish (Squalus acanthias); TORT-1 was derived from 'edible grade' lobster tomalley. To produce the reference biological tissues, the raw materials were homogenized, spray dried, acetone extracted three times, screened, tumbled, bottled and radiation sterilized.
Extraction of methylmercury. Two procedures for methylmercury isolation were employed: a simplified version of the official method of analysis for methylmercury recommended by the Association of Official Analytical Chemists (AOAC) [4] and a modified procedure of Uthe and coworkers [5, 6]. In the first procedure, the modifications were replacing benzene with toluene and omitting the concentration step which was deemed too time consuming [4]: 2 g of material was washed three times with acetone and once with toluene. Any required methylmercury spike (e.g. for standard
642 additions) was added at this stage followed by 10 ml of 5 tool/1 HC1 to liberate methymercury (plus any other organomercury species), which was then extracted into 3 x 20 ml aliquots of toluene. The combined toluene aliquots was diluted to 100 ml with toluene. This solution was amenable to gas chromatography. For the other techniques which accept only aqueous samples, methylmercury was extracted from the toluene solution with a cysteine acetate solution, 4/1 v/v. To prepare the cysteine acetate solution, 0.5 g of cysteine hydrochloride monohydrate, 0.34 g of sodium acetate and 6.25 g of anhydrous sodium sulphate were dissolved in 50 ml of DDW. In the second procedure, the principal modification was the substitution of sodium iodide with bromide resulting in formation of the relatively more stable methylmercuric bromide [5]. Two grams of material plus an appropriate methylmercury spike, 8 ml of a copper sulphate solution (1.25 g of copper sulphate pentahydrate in 50 ml DDW) and 4 ml of 2.5 mol/1 HBr were extracted with 3 x 8 ml aliquots of toluene. The methylmercury (and any other organomercury) was backextracted into 7 ml of a sodium thiosulphate solution (0.12 g sodium thiosulphate pentahydrate in 200 ml 50/50 DDW/ethanol). This solution was ready for subsequent analyses except gas chromatography, which required an additional extraction step involving addition of 4 ml of 2.5 tool/1 HBr and 8 ml of toluene. This last toluene extract was suitable for GC. Gas chromatography - electron capture detection. The gas chromatograph was a Varian model 3330 equipped with an electron capture detector. Two columns were used, a 1 5 m x 0 . 3 2 m m ID Carbowax 20M-typed fused silica capillary column (DB-wax, J & W) and a 1 m x 2 m m ID borosilicate column packed with 5% DEGS-PS on 1 0 0 120mesh Supelcoport (Supelco). Splitless injection was performed when using the capillary column, which entailed an initial column temperature of 110 ~ a linear temperature ramp of 20~ and a final temperature of 190~ for 2 rain. Methylmercury eluted at approximately 167 ~ when a carrier flow rate of 8 ml/min was used. When the DEGS packed column was used, injections were made on column at a temperature of 140~ At a carrier flow rate of 17 ml/ rain, methylmercury eluted between 4 and 5 min. Nitrogen (Ultra High Purity grade, Linde) further purified by sequential passage through molecular sieve 5A and a hea~ed oxygen scavenger (Supelco) was used as both the carder and the ECD purge gas. The ECD was operated in the constant current variable frequency pulsed mode and at a temperature of 220~ The usual purge flow rate was about 38 ml/min. The injector temperature was 200~ and 160~ for the DBwax and the DEGS column, respectively. To successfully elute methylmercury, the columns needed to be passiva~ed daily by injecting a few microlitres of a 1,000 ppm mercuric chloride solution in toluene prior to analysis [7]. One microliter of the toluene extract was injected for determination. Cold vapour atomic absorption spectrometry. Five millilitre of aqueous methylmercury extract was digested with 15 ml nitric, 2 ml of perchloric and 5 ml of sulphuric acid. After evaporation of nitric and perchloric acids, the whole sulphuric acid solution was then subjected to CVAAS. An all glass mercury cold vapour generator was used; the reducing agent was a solution of stannous chloride :and
Table 1. Minimum detectable amounts ~ Technique
GC-ECD CVAAS ICP-MS
Absolute (ng Hg)
0.001 20 1.8
Relative (rig Hg/g) AOAC
Uthe
50 40 225
4 17
" GC-ECD, 2 • peak height of blank; others, 2 x standard deviation of blank
b
4,A
l[lllL A I A ~ I,,
Fig. 1. Analysis of DOLT-I : a tissue extract; b tissue extract spiked with 0.1 ~g Hg/g tissue of methylmercury; c 16 pg Hg standard as methylmercury, the expected amount of Hg in the spike per injection. Large arrows indicate the methylmercury peaks. Small arrows show the points of injection. Column, DEGS; method of extraction, Uthe
hydroxylamine hydrochloride (2/1). Other experimental details have previously been reported [8]. Inductively coupled plasma mass spectrometry (ICP-MS). Analyses were carried out by means of the method of isotope dilution using the mercury isotope pair of 201 and 202 as respectively the spike and the reference isotope. Since the mercury 201-enriehed spike was inorganic mercury, it was added only after isolation of methylmercury. One hundred microliter of the spiked aqueous methylmercury extract was injected into the ICP mass spectrometer by means of flow injection in a carrier solution of DDW. ICP-MS operation details are available elsewhere [9].
Results and discussion
Extraction
Both extraction procedures worked well for methylmercury isolation and required approximately the same processing time. For samples where both procedures were used, agreement between analytical results was excellent. Two major differences between the two procedures were the dilution factor and concomitant substances' removal. In the simplified AOAC procedure, methylmercury was extracted from 2 g of tissue into 100 ml of toluene whereas in
643 Table 2. Analyses of CRMs for methylmercury (gg Hg/g dry weight) This study
TORT-I DORM-I DOLTq
GC-ECD
CVAAS
ICP-MS
0A4 • (17) 0.730• 0.035 (t0) 0.077 • 0.008 (6)
--
0.13 • (3) 0.721• 0.032 (6)
0.728• 0.061 (6) -
GC-ECD
Anion exchange CVAAS
Certified methylmercury concentration
Certified total mercury concentration
0.119+0.002 (6) 0.81 • 0.01 (6) 0.081 • 0.002 (6)
0.123+0.003 (6) 0.710 • 0.014 (6) 0.076• 0.003 (6)
0.128• 0.014 ~
0.33 •
0.731• 0.060"
0.798• 0.074 ~
0.080• 0.011"
0.225• 0.037 a
95% confidence intervals; others are standard deviations ( ) Number of replicate analyses
a
the modified Uthe procedure it was from 2 g into 8 ml. For this reason, the relative detection limit of methylmercury when the AOAC method was used was about twelve times higher than that when the Uthe procedure was employed. The minimum detectable amount (MDA) of the AOAC procedure may be improved by adding the solvent evaporation step recommended in the official method. This, however, will roughly double the sample processing time. Concomitant substances were removed in the simplified AOAC procedure by consecutive extractions with acetone and toluene prior to acid addition. This is deemed less effective than the modified Uthe procedure's extraction/backextraction strategy employed after acid addition, which liberated not only methylmercury but other substances as well; a lot of these substances were electron capturing. Results of a side project revealed that a combination of the isolation strategies of the two methods, i.e. washing with acetone and toluene before plus extraction/backextraction after acid addition is most effective against especially oily samples such as fresh lobster tomalley. Isolation/extraction of methylmercury is quantitative in all but one step in the two procedures. The exception is the cysteine backextraction step used in the simplied AOAC procedure, which is about 80% efficient. Consequently, only standard additions were performed when cysteine backextraction was involved. GC-ECD
Of the three analytical techniques used in tandem with the extraction procedures, GC-ECD is arguably the most sensitive. Its absolute detection limit was by far the best while its relative detection limit was comparable to the other two. The minimum detectable amounts of the three techniques are summarized in Table 1. Aside from sensitivity, GC-ECD is superior in that it is the only technique capable of differentiating methylmercury from other organomercury compounds, such as ethyl- and phenylmercury which occasionally occur in environmental samples [10]. CVAAS and ICP-MS measure the total extractable organomercury, and may only be used for methylmercury determination after presence of other organomercury compounds has been ruled out. The DEGS column is preferred over the DB-wax column, which is less effective in resolving methylmercury from co-injected substances. Of the three biological materials analyzed, baseline resolution of methyhnercury on DBwax was only achieved, despite efforts in running under high
resolution conditions, for DORM-1, which yielded chromatographically the cleanest extract. On the contrary, baseline resolution was accomplished for all three materials on DEGS. This is all the more surprising as DB-wax was a capillary column while DEGS was a packed column. As well, the packed DEGS column was compatible with isothermal operation; this allowed a shorter methylmercury retention time than that on the capillary DB-wax column which required temperature programming to facilitate splitless injection. Figure 1 shows consecutive injections of a DOLT-1 extract (modified Uthe procedure), an extract of DOLT-1 plus a 0.1 p~g Hg/g tissue spike as methylmercury, and a methylmercury standard containing 16 pg Hg as methylmercury. Analytical results from standard additions and simple calibration were indistinguishable. Methylmercury was the only organomercury species found in all three materials analyzed. CVAAS
Despite its relatively high absolute detection limit (Table 1), CVAAS is suitable for mercury determination by virtue of its ability to accomodate a very large sample volume (25 ml versus 5[ gl for GC-ECD) and, as a result, a very large concentration factor. The analytical procedures adopted for CVAAS relied on selective extraction of methylmercury and its quantitative conversion to inorganic mercury via total acid digestion. While methylmercury is the predominant organomercury species in the environment, it is not the only one [10]. The two extraction procedures employed are expected to also extract ethyl- and phenylmercury efficiently. As a consequence, CVAAS really measures methylmercury plus other toluene extractable organomercury species, and its results should not be attributed to methylmercury alone unless the presence of other organomercury species has been ruled out by species specific techniques, e.g. GC-ECD, as in the cases here. Although it was not employed in these experiments, a gold sorber/desorber for mercury may be added to improve detection limits. This is a common practice for the determination of mercury in natural waters [11]. ICP-MS
The absolute ICP-MS M D A for mercury extracts in this work is estimated to be about 10 times worse than that for aqueous solutions run under flow injection. This is resulting
644 from mercury signal suppression due to the presence of excessive concentrations of concomitant substances (e.g. about 4% sodium). For samples such as these, analysis is only possible by means of flow injection [9]. The isotope dilution work performed here was unconventional - an inorganic mercury spike was used for methylmercury. True isotope dilution analysis requires the enriched-isotope spike to be the same chemical species as the analyte; in this way, the analyte and the spike are affected equally by sample processing, and accurate quantitation is possible irrespective of non-quantitative recovery. This condition is easily met in inorganic analysis, but becomes non-trivial in organometallic determination as it necessitates custom synthesis of isotope enriched organometallic compounds. By adding the enriched-isotope spike after methylmercury isolation but prior to analysis, difficulties associated with matrix suppression were largely eliminated but problems of non-quantitative recovery in the cysteine backextraction step remained. Consequently, non-isotoped enriched methylmercury additions had to be made to the samples prior to isolation for the assessment of total sample recovery. This was done in every batch of analysis to obtain a sample recovery correction factor, which was about 80 + 4%. Like CVAAS, ICP-MS relies on selective sample processing to achieve speciation; as a result, it measures not only methylmercury but other toluene extractable organomercury species as well.
Analyses of TORT-I, DORM-1 and DOLT-1 The analytical results of the three methods described here are summarized in Table 2 along with those obtained by collaborating laboratories. One of them used a similar Uthe extraction procedure plus GC-ECD [6] and the other employed anion exchange to retain the tetrachloro complex of inorganic mercury and analyzed the eluted methylmercury (plus any other organomercury) by CVAAS [12]. Agreement amongst results obtained by different techniques and laboratories is excellent, proving that methylmercury is the only significant organomercury present. The certified values for methylmercury resulted from these analyses and
the certified total mercury concentrations are also tabulated for comparison. Methylmercury constitutes about 39%, 92% and 36% of total mercury in TORT-l, DORM-1 and DOLT-l, respectively. The remaining fraction is presumed to be inorganic mercury.
Acknowledgements. Collaboration from B. Averty and D. Cossa of Institut Frangais de recherche pour l'exploitation de lamer, Nantes, France and M. Stoeppter of the Institute of Applied Physical Chemistry, Jfilich, Federal Republic of Germany are gratefully acknowledged.
References
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