NOTES
Determination of Submicrogram Quantities of Monomethyl Mercury in Aquatic Samples James J. Bisogni, Jr.,* and Alonzo Wm. Lawrence
Department of Environmental Engineering, Cornell University, Ithaca. N.Y. 14850
A method of measuring submicrogram quantities of monomethyl mercury is described. The procedure combines aspects of several published methods of mercury analysis. Methyl mercury is separated from inorganic mercury by benzene extraction. The extracted methyl mercury is then analyzed by a flameless atomic absorption procedure. Thin-layer chromatography is employed as a verification step. The procedure is applicable to a wide variety of aquatic samples containing monomethyl mercury. Interfering agents include elevated concentrations of chlorides, organic solvents, inorganic mercury, dimethyl mercury, and other forms of organic mercury. Methods to attenuate these interferences are discussed. Mean recovery efficiencies of greater than 90% were obtained with coefficients of variation less than 4.1% for monomethyl mercury spiked into microbial biomass. The appropriate analytical apparatus can be set up a t a relatively low cost. The recent discovery of high concentrations of mercury, particularly methyl mercury, in some aquatic life [Irukayama et al. ( I ) ] has initiated much investigation into methods of analyzing for organomercurials. Several different techniques have been utilized by various investigators [Gage ( 2 ) , Westoo ( 3 ) , Tatton and Wagstaffe ( 4 ) ] .These techniques are all concerned with: separating total mercury from its organic or inorganic matrix, separating the organomercurial from the inorganic mercury, and quantitatively measuring the segregated mercury forms. The method described here incorporates modifications of several schemes to analyze for monomethyl mercury in aqueous solution, biomass, or inorganic and organic sediment. The analsis is a modified combination of Westoo’s ( 3 ) benzene-cysteine extraction procedure and Hatch and Ott’s ( 5 ) cold vapor atomic absorption technique. The procedure involves essentially three steps: separation of the organic mercury from inorganic mercury, identification of the separated organomercury with thin-layer chromatography, and measuring the amount of separated organomercury. Step 1 includes a separation of the monomethyl mercury from its binding matrix (if present), as well as separation from inorganic forms of mercury. The chemistry of this extraction step is presented by West66 ( 3 ) .The West66 extraction procedure is accomplished by converting all monomethyl mercury forms to methyl mercuric chloride. The conversion is carried out by acidifying a homogenized sample with HC1. The methyl mercuric chloride is then extracted into benzene. This extraction separates the organomercurials from most of the inorganic mercury and from the original methyl mercury matrix. Following the extraction into benzene, methyl mercuric chloride is partitioned back into an aqueous phase. This is accomplished by converting the methyl-mercuric chloride to methyl mercuric cysteine [ C H ~ H ~ S C H Z C H ( N H Z ) C O O H The ]. methyl mercuric cysteine is partitioned favorably into the 850
Environmental Science & Technology
aqueous phase because of its carboxyl group’s affinity for water. Step 2 involves taking a portion of the benzene extract and identifying the organomercurials that are present. Organic mercury compounds, such as phenyl, methoxyethyl, and ethyl mercury, tend to be extracted in Step 1. It is the intent of this analysis to measure only monomethyl mercury, so it is necessary to verify that monomethyl mercury is the predominant extracted mercury form. This verification can be accomplished by employing the thinlayer chromatography technique developed by Westoo ( 3 ) . Step 3 of the analysis is concerned with measuring the amount of methyl mercury yielded from the extraction process of Step 1. This procedure is essentially the flameless atomic absorption (FAA) technique proposed by Hatch and Ott ( 5 ) and used by Kopp et al. (6). The analysis consists of a wet oxidation of all forms of mercury to the mercuric form, followed by a reduction of mercuric ion to metallic mercury. The metallic mercury is vaporized and pumped through an absorption cell of a photometer. Mercury vapor absorbs light a t a characteristic wavelength of 2537 nm. Absorbance is proportional to mercury vapor concentration according to Beer’s Law. Procedure The seven-step procedure for monomethyl mercury analysis is given here: Homogenize 50 ml of sample. This sample should contain less than 4 wg of methyl mercury as mercury. Solid samples should be suspended in 50 ml of deionized water. Place the homogenate in a 500-ml separatory funnel and acidify with 15 ml of concentrated hydrochloric acid. Mix the acidified sample thoroughly. Add 70 ml of A. R. grade thiophene-free benzene and shake vigorously for 5 min. Let the phases separate and drain off a portion of the organic solvent; centrifuge this extract to obtain a clear benzene layer. To a 60-ml separatory funnel, transfer an appropriate volume of the clear benzene solution. The volume used will depend on the initial concentration of methyl mercury in the sample and calibration range of the FAA instrument. Add 5.0 ml of 1.0% cysteine acetate solution to the benzene extract. The cysteine acetate solution is prepared by dissolving 1.0 gram of cysteine hydrochloride monohydrate, 0.744 gram of sodium acetate trihydrate and 12.5 grams of anhydrous sodium sulfate in 100 ml of deionized water. Shake the benzene-cysteine acetate mixture vigorously for 2-3 min. Allow the phases to separate. If a clear aqueous layer (bottom layer) is not obtained, this layer must be centrifuged. Care must be taken not to allow any benzene to be transferred with the aqueous phase, since benzene is an interfering agent in the FAA analysis. Submit an appropriate aliquot of clarified (clear) aqueous solution to the FAA total mercury analysis as described below.
The flameless atomic absorption method for total mercury analysis is given in five steps a s follows: To a 300-ml BOD bottle transfer the sample containing an amount of mercury which falls into the calibration range of the atomic absorption spectrophotometer. Add enough distilled water to bring the total volume to 100 ml. Mix thoroughly and add 5 ml of concentrated sulfuric acid and 5 ml of concentrated nitric acid. Add 1 ml of 5% (w/v) KMn04 solution to the bottle, shake, and add additional portions of potassium permanganate solution until the purple color persists for 15 min. Add 2 ml of 5% (w/v) KzSzOs and heat the bottle and contents for 2 hr a t 95°C. Remove bottle from heat source and bubble mercuryfree nitrogen or oxygen through the sample for 10-15 min. This is done to strip the sample of any benzene contamination. After the bottle and sample have cooled to room temperature, add 2 ml of 12% (w/v) hydroxylamine hydrochloride solution to reduce excess permanganate. Add 5 ml of 10% (w/v) stannous chloride solution and immediately attach the bottle to the aeration apparatus of the atomic absorption spectrophotometer. Record the peak absorbance. Remove the bubbler from the BOD bottle and vent the system into an exhaust hood. The quantity of mercury in the sample is determined from a calibration curve and adjustment of this value by appropriate dilution factors. A. R. grade chemical reagents are employed for all analyses. A careful check on blank values is necessary to ensure low background mercury levels. Glassware should be cleaned with hot soapy water, followed by a nitric acid wash, and a final rinse with deionized water.
Table I. Accuracy, Precision, and Recovery Efficiency of Direct FAATotal Mercury Analysis
Sample
True concn, @g/I,
Mean measured value, &g/l.
Std dev, n =4
Coeff of, variation, %
Mean %recovery
0.67
10.04
5.60
99.3
4.16
10.10
2.62
99.2
Inorganic mercurya 0.68 (HgCIJ Organic mercurya 4.20 (phenylmercuric acetate) Methylmercuric 2.00 chloride e
1.99
k0.06
3.37
99.9
EPA reference sample.
Table II. Accuracy, Precision, and Recovery Efficiency of Direct FAA Total Mercury Analysis for Inorganic and Organic Mercury Spiked in Microbial Biomass Sample
Mercuric chloride spiked in anaerobic microbial biomass Mercuric chloride spiked in aerobic microbial biomass Methylmercuric chloride spiked in anaerobic microbial biomass Methylmercuric chloride spiked i n aerobic microbial biomass
True concn, pg/l.
1.00
Mean measured concn, M e a n ’% pg/l. recovery
0.92
92.0
Results A series of mercury analyses was performed on several types of aquatic samples to determine the accuracy, precision, and recovery efficiency of the proposed method of analysis. Table I presents the results of a series of experiments used to evaluate the accuracy and precision of direct FAA total mercury analysis used in this research. Table I1 shows the recovery efficiency, precision, and accuracy of direct FAA total mercury analysis when the mercury was spiked into anaerobic or aerobic microbial biomass. The anaerobic and aerobic microbial biomass was obtained from laboratory microbial reactors used in aerobic and anaerobic mercury methylation studies ( 7 ) . Table I11 shows the overall accuracy, precision, and recovery efficiency for the benzene-cysteine extraction followed by the FAA total mercury analysis.
Discussion The combined extraction-atomic absorption technique for monomethyl mercury analysis is straightforward. The usual precautions must be observed when handling all forms of mercury. In addition. care must be taken to avoid prolonged contact and inhalation of benzene. Organic solvents, particularly benzene, absorb radiation a t a wavelength of 2537 nm and thus interfere with the FAA analysis. Chloride ions can be oxidized to chlorine in the oxidation step of the total mercury analysis. Chlorine also absorbs 2537 nm light. Both benzene and chloride interference can be eliminated by aerating the sample after the oxidation step in the FAA procedure. Inorganic mercury can interfere with the organic mercu-
Table 111. Recovery Efficiencies of Benzene-Cysteine Acetate Procedure for Organic Mercury Analysis T r u e concn.
@e/l.
Sample
Phenylmercuric 0.132 acetate i n deionized HiO CH?HgCI in de0.118 ionized H 2 0 4.00 CH3HgCI in 800 mg/ I . anaerobic rnicrobial solids suspension CHIHgCl in 800 rng/ I . aerobic microbial solids sus-
4.00
Mean measured concn,
C o e f f of variation
pg/l.
Mean % recovery
0.131
99.1
1.20
0.115
97.7
2.96
3.65
91.3
4.05
3.71
92.8
3.51
(n = 4),
%
Dension
C o e f f of variation (n = 4 ) ,
%
3.1
Table IV. Inorganic Mercury Interference in BenzeneCysteine Acetate Analysis for Organic Mercury l n o r ~ a n i cm e r c u r y concentration
1.00
0.94
94.0
2.8
3.00
2.77
92.3
6.8
3.00
2.83
94.3
5.6
1 m g / l . as Hg
5 mg/l. a s H g
0.002pg Hg/ rnl C 6 H s Not
0.001 pg Hg/ ml C s H 6
0.035pg Hg/ ml C 6 H c
Aerobic microbial Not solids suspendetectable detectable sion, 1000 mg/l. Anaerobic micro- Not Not bial solids susdetectable detectable pension, 1000 mg/l., total S = 10 mg/l.
0.006 pg Hg/ ml C 6 H s
Sample
Deionized water
100 m g / l . as H g
0.001 pg Hg/ ml C 6 H s
Volume 8 , Number 9, September 1974
851
ry analysis because it can be carried over during the benzene extraction. The degree of interference from inorganic mercury depends on the nature of the sample and the concentration of inorganic mercury in the sample. Table IV shows the effect of sample type and inorganic mercury concentration on inorganic mercury carryover into benzene. The magnitude of the error caused by inorganic mercury will depend on the amount of organic mercury that is being extracted. Error calculations should be performed for each analysis. If very small quantities of organic mercury are to be extracted in the presence of high background levels of inorganic mercury, addition of 10 grams of NaCl to the homogenized sample, before extraction, will minimize inorganic mercury interference. The formation of complexes, such as NaHgCl3 or NazHgC11, probably prevents the inorganic mercury from being partitioned into the benzene phase (8). A solution containing 10 mg/l. mercuric mercury was treated with S a c 1 and submitted to the benzene extraction procedure. Inorganic mercury carryover was not detected in the extract. It is apparent from Tables I and I11 that extraneous organomercurials (in this case phenylmercury) can be extracted and detected by the above analysis. This type of interference is difficult to overcome. Samples with high concentrations of extraneous organomercurials (not methyl mercury) should not be analyzed with the described procedure. In such situations the only practical recourse is to use the more expensive gas-liquid chromatography system. Fortunately, in many natural aquatic systems, not directly contaminated by specific organomercurials, monomethyl mercury appears to be the dominant organomercurial. For such situations the procedure described is attractive. Finally, dimethyl mercury can interfere with monomethyl mercury analyses if excess mercuric ions are present in the sample. Under acidic conditions and excess mercuric mercury, dimethyl mercury is converted to monomethyl mercury according to Equation l. CHjHgCH,
+
H C1
HgC1,
--+
BCH3HgCl
(1)
Dimethyl mercury interference should not present significant problems in most samples because of the relatively low water solubility and high volatility of (CH3)zHg. It
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Environmental Science & Technology
should be noted that dimethyl mercury analyses can be performed by adding excess mercuric ions to the sample, converting to monomethyl mercury (Equation l),then analyzing the monomethyl mercury according to the procedure described above. The recovery efficiencies reported here are higher than those reported by West% (3). Westoo explains that the relatively low recovery (
