indefinitely if the sample is made 0.05 N in HC1. Arsenite is slowly oxidized to arsenate; in samples below 0.05 ppb As(II1) a loss of arsenite becomes detectable after about one week. Acidification of the sample increases the oxidation rate, and arsenite loss can be detected after one day. If the samples are stored in a freezer below -15 OC or under dry ice, an initial loss of arsenite corresponding to about 0.02 ppb is experienced. The sample then remains unchanged with prolonged storage.
ACKNOWLEDGMENT The author expresses his sincere thanks to E. D. Goldberg for his material and intellectual support. The Ansul Corporation donated highly purified standards for methylarsonic and dimethylarsinic acid.
LITERATURE CITED (1) (2) (3) (4)
(5) (6) (7) (8) (9)
S. Gohda, Bull. Chem. SOC. Jpn., 48, 1213 (1975). R. S. Braman and C. C. Foreback, Science, 182, 1247 (1973). Y. Talmi and D. T. Bostick, Anal. Chem., 47, 2145 (1975). Y. Talmi and D. T. Bostick, J . Chromatogr. Sci., 13, 231 (1975). Y. K. Chau, P. T. S. Wong, and P. D. Goulden, Anal. Chem., 47, 2279 (1975). J. Aggett and A. C. Aspell, Analyst (London), 101, 341 (1976). R. S. Braman, L. L. Justen, and C. C. Foreback, Anal. Chem.,44, 2195 (1972). A. E. Smith, Analyst(London), 100, 300 (1975). F. D. Pierce and H. R. Brown, Anal. Chem., 48, 693 (1976).
RECEIVED for review December 28,1976. Accepted February 22, 1977. This investigation was sponsored by the Office of Naval Research, Contract USN N O 0 0 14-75-C-0152.
Determination of Tetraalkyllead Compounds in Biological Materials G. R. Sirota" and J. F. Uthe Environment Canada, Fisheries and Marine Service, 1707 Lower Water Street, P . O . Box 429, Halifax, Nova Scotia, Canada
A fast, sensltive, atomic absorption spectroscopic method for determinlng common tetraaikyliead compounds In fish tissue is described. Tissue homogenates were extracted by shaking with a benrenelaqueous EDTA solution, a measured portion of the benzene was removed, and, after dlgestlon, the residue was defatted tf necessary. The resultant Pb2+was detemined by flameless atomic absorption spectroscopy using a heated graphite atomizer. Using a sample welght of 5 g, 10 ppb of lead as PbR4 can be determined wlth a relative standard deviation of 5 %. No other forms of lead that were tested, e.g., PbR3X, PbR2X2,were found to partition into the benzene layer under these condltlons.
The acute toxicity of lead alkyls is greater than that of inorganic lead compounds (1). Relatively little is known either of the effect of chronic exposure to small amounts of such compounds or the levels of organic lead compounds, such as the tetraalkylleads, in biological and food material. While there are methods for the determination of tetraalkyllead in air using gas-liquid chromatography (2) and atomic absorption spectroscopy (3), they have not been applied to small tissue samples. The present paper describes a fast, simple method for determining tetraalkyllead compounds in biological matrices using selective extraction, digestion, and atomic absorption spectroscopy.
EXPERIMENTAL Instruments. A Perkin-Elmer model 306 Atomic Absorption Spectrophotometer equiped with deuterium arc background correction was used. Samples were run in the normal flameless mode (argon purge) using the HGA 2100 controller and HG 74 graphite cell. The temperature program used was as follows: drying 100 "C/40 s; charring 500 OC/60 s; atomization 2000 "C/5 s. Samples were analyzed using the 283.3-nm resonance line. Reagents. Tetraethyllead (99%) and tetramethyllead (68% in toluene) were provided by the Ethyl Corporation (Ferndale,
Mich.) and were used as working standards at lead concentrations of 10 pg/mL and 1.0 pg/mL. Inorganic lead standards (in N HNOJ were prepared from a 1000 pg/mL stock solution of lead nitrate (Fisher Atomic Absorption Standard). Redistilled Fisher Scintillation grade benzene was used throughout. Disodium ethylenediaminetetraacetic acid (0.4% w/v, pH 6-7) was made up in glass distilled water. Di- and tri-substituted lead alkyls were obtained from Alfa Ventron Corp., Danvers, Mass. Procedure. Benzene (10 mL) and EDTA reagent (10 mL) were added to a homogenized tissue sample ( 5 g) in a 50-mL glass centrifuge tube fitted with a screw cap, and shaken on a Burrell wrist action shaker fitted with extension clamps to give 21/2-3-inch strokes for 10 min, after which the phases were separated by centrifugation at 2200 rpm for 30 min to give two layers. More discrete separation occurred if the sample was allowed to sit undisturbed overnight after centrifugation. An accuratelymeasured 3-mL portion of the benzene layer was transferred to a 50-mL calibrated Folin-Wu digestion tube and the contents were acidified with 3 mL of concentrated HN03 (Fisher ACS). The benzene layer was evaporated under a stream of nitrogen (high purity) at room temperature, and the residue was then digested for at least 2 h in a heated aluminum block at 80-90 O C or until evolution of large amounts of NO2 ceased. The sample was then made up to 10 mL with glass distilled water and shaken with approximately 2 mL of hexane. After removal of the hexane layer, the aqueous phase was analyzed by the method of standard additions, and lead concentration calculated following linear regression analysis of peak heights using a Hewlett-Packard HP-25 programmable calculator.
RESULTS AND DISCUSSION Previous experience in the flameless atomic absorption analysis of biological tissues has shown the influence of matrix on the signal response (4). The extent of background absorption on this work was evaluated by analysis of the samples a t the non-absorbing lead line of 280.3 nm (5). Background absorption of various samples a t this wavelength was found to vary approximately from 10 to 50% of the lead signal. Broad-band nonspecific background absorption was minimized by using deuterium arc background correction (6). Background correction and the method of standard additions were ANALYTICAL CHEMISTRY, VOL. 49, NO. 6, MAY 1977
823
Table I. Recovery of Tetraalkyllead Compounds from Cod Liver Homogenate
Compound Tetramethyllead
Amount added, wg Pb
Amount added, PPb
0.10
20 20 100 100
0.10 0.50 0.50
Tetraethyllead
0.10
20 20 100 100 100
0.10 0.50 0.50 0.60
Total Pb present prior to spike, wg
Total Pb found after spike,
Amount of spike found,
Pg
Pg
%
0.25 0.27 0.06 0.06 0.14 0.16 1.056 0.053 0.045
0.38 0.40 0.575 0.625
0.13 0.13 0.515 0.565
130 130 103 113
0.21 0.26 1.548 0.65 0.42
0.07 0.10 0.492 0.542 0.375
70 100 98 119 75
Recovery,
Table 11. Concentrations of Total Lead and Tetraalkyllead in Various Marine Tissues Concentration total Pb,ppm
Tissue Frozen cod (liver homogenate) Large, freshly killed cod (liver homogenate) Small, freshly killed cod, (2 separate lobes analyzed) Lobster digestive gland (homogenate) Frozen mackerel muscle (homogenate) Flounder meal a
0.39 0.52
?I
0.04 0.05
A. 0.21
0.04‘“
0.037 0.010 0.125
t ?I
f
of total lead
24
0.20
f
0.02
0.14
?:
0.02
0.054
*
5.34
f
1.02
4.79
0.32
f
% tetraalkyllead
0.003 0.001 0.005
0.028 0.044 0.162
t
B.
9.5
?I
0.004
13.3 20.9 81
0.005
38.6 89.7
For total lead determination, both lobes blended.
Table 111. Extraction of Di- and Tri-Substituted Alkyl Lead Compounds into Benzene Phase
Compound Diethyllead dichloride Trimethyllead acetate
Percent extraction into benzene phase After After 1st 2nd extrac- extraction tion 7.1
0.4
0 0
both used throughout the analysis. The recovery and selectivity of the method was evaluated by adding known amounts of different lead compounds to previously analyzed tissue samples. The results obtained are summarized in Table I and indicate a satisfactory recovery and selectivity for tetraalkyllead compounds: Various marine tissues were sampled for total lead and tetraalkyllead. Resulta are summarized in Table 11. Di- and tri-substituted alkyl leads were also evaluated in this system and the results are shown in Table 111. As can be seen from Table 111, there was no evidence of extraction of lead into the benzene phase after the second extraction, and it is postulated that the substituted alkyl compounds were contaminated with small amounts of tetraalkyllead, which were extracted into the benzene phase in the first extraction. Ionic lead, as both the chloride and nitrate, also remained in the aqueous phase. Earlier work indicated that traces of fat left after the digestion would give rise to erratic results, presumably by interfering with pipetting delivery. Therefore, the digested samples were shaken with hexane to remove any fat. Analysis of the hexane showed that no lead was extracted from the digest. 824
?r
Concentration PbR,, ppm
ANALYTICAL CHEMISTRY, VOL. 49, NO. 6, MAY 1977
To determine the effect of added lipid on possible extraction of ionic lead into the benzene phase, 25% by weight of an oil extracted from herring meal was added to spiked and unspiked samples of cod liver homogenate. Analysis showed that there was no co-extraction of lead into the benzene phase. As can be seen from Table I, samples were spiked at levels of only two times and five times the detection limit, and average recoveries at these levels were 108% and 102%, respectively. All levels of both totallead and tetraalkyllead in the marine samples analyzed were quite variable, and the percent of lead present as the tetraalkyl species, ranged from 9.5% in a cod liver to 89.7% in a flounder meal. The relatively high concentration of lead compounds in flounder meal is a result of the large water loss, approximately 75%, during production of this meal. The finding of a high tetraalkyllead to total lead ratio in the single available fish meal may either be a species specific phenomenon or a result of the manufacture and subsequent storage of this meal. This is being investigated further. CONCLUSIONS Organolead compounds are more toxic than ionic forms (1) and thus, the occurrence of tetraalkyllead compounds in fish tissue is highly significant. Since Chau (7) has shown that certain microorganisms present in lake sediments can convert ionic lead compounds to organic species, the possibility of methylation of ionic lead in vivo or in stored tissue cannot be discounted. It has also been shown (8) that an enzyme system which converts tetraethyllead to the more toxic triethyl species is found in liver. The finding of relatively high ratios of tetraalkyllead to total lead in certain fishery products may require re-evaluation of the current Canadian Food and Drug Act allowable level of 10 ppm. LITERATURE CITED (1) National Academy of Sciences, “Lead: Airborn Lead in Perspectlve”, National Academy of Sciences, Washington D.C., (1972).
(2) Y. K. Chau, P. T. S. Wong, and H. Saitoh, J . Chromatogr. Sci., 14, 162 (1976). (3) S. Hancock and A. Slater, Ana/yst(London), 100, 422 (1975). (4) . , Kaarr Julshamn and Olaf R. Braekkan. At. Absorot. News/.. 14(3). 49 ( 1975). (5) "Analytical Methods Using The HGA Graphite Furnace", Perkin-Elmer Corp., Norwalk, Conn., 1974. (6) H. L. Kahn, At Absorpt. News/., 7 , 2 (1968). 1
.
(7) Y. K. Chau, P. T. S. Wong, and P. L. Luxon, Nature(London), 253, 264 (1975). (8) J. E. Cremer, Occup. Health Rev., 17, 14 (1965).
,I
RECEIVED for review November 15,1976. Accepted January 28, 1977.
Determination of Traces of Arsenic in Siliceous Materials Cyrus Feldman Analytical Chemistry Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830
Finely ground rocks, slags, and fly-ash are completely converted to soluble form by HF-HN03 vapor phase attack at -70 OC. The treatment chamber is a comrnerclally available 1-L Teflon jar. Arsenic Is not volatlllzed by this procedure il In the As(V) form. Arsenlc contaminating the acld mlxture Is carrled over to the sample if the arsenic Is trivalent. Potassium permanganate, added to the acid mixture In advance, converts any As(II1) present to As(V), thus preventing contamination of the sample. Accurate results were obtained wlth NBS Coal Fly Ash (SRM 1633) and standard rocks.
Siliceous materials are usually dissolved for analysis with the aid of hydrofluoricacid, or are fused with alkali carbonates, borates, or fluoborates. These reagents are often contaminated with traces of foreign elements and, since the quantity of reagent used is much greater than the quantity of sample taken, the reagent blank may be unacceptably high for the determination of a given trace constituent. In 1957, N. N. Semenov (1) suggested the use of HF vapor, confined with the sample in a closed container, to circumvent the above difficulty in preparing quartz samples for the spectrographic determination of trace impurities. Morachevskii et al. (2) and Zilbershtein et al. ( 3 , 4 )subsequently used the vapors of a 1:l mixture of concentrated H F and HNOBin a similar way to eliminate the silicon from elemental silicon and/or high purity quartz. Piriutko (5)has shown that if this type of attack is used on elemental silicon, part of the nitrogen is reduced all the way to N(-111) and remains with the residue as (NH&3F6. Most of that part of the nitrogen which is reduced, however, is converted to NO and/or NOz. When it became necessary in this laboratory to determine low concentrations (10.05 ppm) of arsenic in coal slags, fly ash, and rocks, vapor phase HF-HN03 treatment was considered as a possible way to avoid contamination by reagents. The prospects seemed poor, however: the boiling point of AsFS is -53 "C, and that of AsF3,63 "C. Little encouragement was given by previous work in this field; according to Morachevskii et al. ( 2 ) ,76Astracer tests showed that if arsenic is added to HF or to 1:l HF + "03, the arsenic is volatilized completely when the liquid is evaporated to dryness a t 105-110 "C. Zilbershtein et al. ( 4 ) essentially confirmed this result, adding that all of the arsenic was also lost if added to elemental silicon which was then subjected to the vapor phase HF-HN03 treatment. Unfortunately, neither paper mentions the valence of the arsenic in the tracer or carrier used. It is well known, however, that traces of a given element may behave in one way in a dilute aqueous solution of carrier plus tracer, but in quite another way when the element exists
in a complex matrix. It therefore still appeared worthwhile to see whether conditions could be found under which the vapor phase HF-HN03 procedure could be used to make silicates soluble without either increasing or decreasing their arsenic content. Two types of experiment were necessary to test the applicability of the proposed procedure. First, it was necessary to demonstrate that a typical silicate sample of relatively high and accurately known As content does not lose arsenic when treated by this procedure. Such a sample was available in Standard Reference Material 1633 (Coal Fly Ash) (U.S. National Bureau of Standards, Washington, D.C., 20234). Several standard rock samples were also available from the U.S. Geological Survey and from the Zentrales Geologisches Institut, Berlin, DDR. The arsenic contents of these samples are known with greater accuracy in some cases than in others (6). Second, it would have to be shown that any arsenic which might be present originally in the H F and/or HNOB is not transferred to the sample during the vapor treatment. This could be determined by analyzing low-arsenic coal slags and U.S. Geological Survey standard rock samples (As 5 1 ppm in all cases) and comparing results obtained by the regular procedure with results obtained when substantial quantities of arsenic were added to the acid mixture before use. The reaction chamber was simple. Commercially available Teflon jars were employed to avoid use of the more expensive chambers machined from solid blocks of Teflon by Mitchell and Nash (7) and Wooley (8). Preliminary experiments indicated that -70 "C would be a suitable temperature for the present purposes. This temperature gave essentially complete conversion of the samples to fluorides and fluosilicates in a reasonable length of time, and was also considered low enough to minimize losses of arsenic and/or acids by volatilization.
EXPERIMENTAL Apparatus. A wide-mouth 32-02. cylindrical Teflon jar, with Teflon screw-on cap (Cole-ParmerCo., Chicago, Ill., Catalogue No. 6044-40) was modified slightly for use in the present procedure. The jar, with cap off, was laid on its side on a hotplate whose surface temperature was -350 O C . Pressure was exerted from above until the side wall of the jar softened to give an approximately flat area -1 in. X 61/2 in. Hotplate surface temperatures were measured with a bimetallic surface thermometer (Model 314C, Pacific Transducer Co., Los Angeles, Calif. 90064). All samples were held in 25-45 mL platinum dishes during exposure to the acid vapors. Heating was performed in an ordinary drying oven. See Figure 1. Reagents. All reagents (48% HF, 71% "Os, 72% HC104, and solid KMn04) were analytical reagent grade. The arsenic content of the HClO,, was found to be negligible compared with ANALYTICAL CHEMISTRY, VOL. 49,
NO. 8, MAY
1977
825
