ANALYST, SEPTEMBER 1991, VOL. 116 957 Application of a Microwave Oven for Drying and Nitric Acid Extraction of Mercury and Selenium From Fish Tissue Suei Y. LamLeung, Vincent K. W. Cheng and Yuet W. Lam Department of Chemistry, Hong Kong Baptist College, 224 Waterloo Road, Kowloon, Hong Kong The drying and nitric acid digestion of fish tissue using a microwave oven for the determination of Hg and Se were studied. For the drying process, the method was compared with the freeze-drying method. The water content found in the muscle of tilapia, Oreochromis mossanicus, by using a freeze dryer at -40 "C and a pressure of ~ 1 0 pmHg for 24 h, and a microwave oven (convection mode) at 70 "C for 3 h was 78.16 and 78.09% m/m, respectively. The Hg and Se levels found in fish tissues dried by the two methods were consistent with a 95% confidence level.The efficiency of dissolution of Hg and Se from dried muscle of tilapia, using concentrated HN03 in the closed vessel microwave digestion method was found to be much greater than that of traditional open vessel digestion. The microwave heating digestion method was tested satisfactorily using two certified reference materials. The recovery of added standards of Hg and Se was found to be 84.8 and 96.6%, respectively. Keywords: Microwave heating and drying; fish tissue; nitric acid ashing; selenium determination; mercury determination The process of drying and dissolution of biological materials is a lengthy task. The application of a microwave oven for wet ashing in a closed vessel containing geological and biological materials has been found to be an efficient method for the preparation of sample solutions for metal determination.1-3 A study of the application of a commercial microwave oven in drying and wet ashing of fish tissue for the determination of Hg and Se is reported here. Only HN03 was used in this ashing process, as it is a strong oxidizing agent and also the nitrates of most metals are soluble in water. Low microwave power was used for safety reasons. A sample of muscles of small individuals of tilapia of length <19 cm was used for the investigation of the microwave and freeze-drying processes. The muscles of large individuals of tilapia of length 26-31 cm were used for the study of the wet ashing processes in a closed vessel by microwave heating and in an open vessel by thermal heating.Recovery of added standards using the microwave method of digestion in a closed vessel was also studied. The microwave digestion method was tested by its application to two certified reference materials. Experimental Apparatus A commercial microwave oven (Sharp, Model R-9H10) with a frequency of 2450 MHz (power output of 750 W for microwave mode and 1500 W for convection heating) and a turntable was used for digestion and drying without modifica- tion. Sample digestion was carried out in capped 60 ml polytetrafluoroethylene (PTFE) vessels (Savillex, Model 561R2). A block digestor (Techne, Model DG-1) with a temperature controller (Techne, Model TC-D1 A) was used for open vessel digestion of samples. For the wet ashing process, a wide-mouthed earthenware vessel with a lid was used as it protects the microwave oven from the corrosive vapour released or from any explosion of the digestion vessel.All containers were treated successively with detergent (washing), tap water (rinsings), 70% m/m HN03 for glassware (rinsing) or 0.5% m/v HN03 for plastics (soaked for at least 24 h). An atomic absorption spectrometer (Varian, Model SpectrAA-10) equipped with a hydride vapour generation accessory (Perkin-Elmer, Model MHS- 10) was used for the determination of Hg and Se. A freeze dryer (Labconco 75034) was used for drying the fresh muscle of tilapia collected from a local river. Reagents All chemicals used were of analytical-reagent grade. A stock solution of 1000 pg ml-1 of Hg was prepared by dissolving 1.080 g of HgO in the minimum volume of HC1 (1 + l ) , and diluting to 1 1 with 1.5% m/v HCI.A stock solution of 1000 pg ml-1 of Se was prepared by dissolving 1.000 g of Se in approximately 5 ml of 70% m/m HN03, and diluting to 1 1 with Working standard solutions were prepared immediately before use by serial dilution of the stock solutions. The working ranges were as follows: Hg, 2-16 ppb for sample analysis and 50-200 ppb for recovery investigation; and Se, 2-12 ppb for sample analysis and 50-120 ppb for recovery investigation. The reducing solution was prepared fresh daily by dissolving 6 g of NaBH4 and 2 g of NaOH in 200 ml of distilled water. 5% m/v H2S04. Samples The axial muscle of tilapia, Oreochromis mossanicus (Peters), collected from a local river, and National Research Council Canada (NRCC) certified reference materials (DORM-1 , Dogfish Muscle and DOLT-1, Dogfish Liver) were studied.Analytical Procedure During wet digestion in a closed vessel by microwave heating the following precautions must be considered: the mass of sample used for digestion should not be greater than 0.2 g; the total volume of reactants should not exceed one tenth of the volume of the container;4 the heating times at high power should not be longer than 5 min; the digestion vessel must be cooled before opening; and a digestion vessel with a pressure releasing cap should be used. Drying of sample A collection of axial muscles of small individuals (<19 cm in length) of tilapia caught from a local river was homogenized in a stainless-steel blender. About half of the sample, 50 g, was spread thinly on several clean watch-glasses and placed in a microwave oven.The samples were dried (convection mode) at 70 "C for 2-3 h until constant mass was obtained. The958 ANALYST, SEPTEMBER 1991, VOL. 116 samples were removed from the oven and turned over every 20 min to ensure thorough drying. The remaining portion of the sample was dried in a freeze dryer at -40 "C and <lo pmHg for 24 h. The axial muscles of large individuals (2631 cm) of tilapia were treated in the same manner using a microwave oven. Dissolution of sample The microwave digestion of tilapia tissues or certified refer- ence material was carried out using four replicates of about 0.2 g of the dried muscle weighed into a dried, cleaned PTFE digestion vessel. A 4 ml aliquot of 70% m/m HN03 was added to each digestion vessel.Four tightly capped digestion vessels and one small beaker filled with 30 ml of water were placed in a wide-mouthed earthenware vessel. The lidded vessel was placed in the microwave oven. The samples were treated using three heating stages: low power (10%) for 8 min, medium-low power (30%) for 8 min and medium power (50%) for 4 min.5 The earthenware vessel was removed from the microwave oven and then uncovered and cooled to room temperature. The cap of the digestion vessel was opened slightly to release any pressure before being removed. The digest was quantitat- ively transferred into a 100 ml beaker and evaporated to <1 ml by heating to decompose any unused HN03.The solution was filtered through Whatman No. 42 filter-paper and quantita- tively transferred into a 25 ml calibrated flask. It was diluted to volume with 0.1% m/v HN03. The solution was then stored in a polyethylene bottle. For open vessel thermal digestion, four replicates of a sample (about 2 g) of dried muscle of tilapia were weighed into four 50 ml boiling tubes. The sample was pre-digested at 20 "C for 24 h with 10 ml of 70% m/m HN03. The sample was then heated at 100 "C for.3 h. Three additional portions of 5 ml of 70% m/m HN03 were introduced into the boiling tube successively at intervals of 30 min. When a clear solution was obtained, the temperature was raised to 130 "C to decompose all the unused HN03. After cooling, the digest was filtered through Whatman No.42 filter-paper then quantitatively transferred into a 25 ml calibrated flask and diluted to volume with 0.1% m/v HN03. The sample was stored in a poly- ethylene bottle for elemental determination. Recovery of added standard A known mass of the analyte, 100,200,300 and 400 ng of Hg and 100,140,200 and 240 ng of Se, together with 4 ml of 70% m/m HN03 were introduced into the Teflon digestion vessel. The digestion process in the microwave oven was the same as for the fish sample. Pa. The absorbance at 253.7 nm was measured for the determination of Hg and at 196.0 nm for Se.6 An acid blank solution was used in the determination of the analytes. Results and Discussion The data given in Table 1 indicate that the quality of the microwave-dried tissue was satisfactory.The colour of the microwave-dried tissue was similar to that of sun-dried tissue. The water content found in the species studied was 78.09% m/m (by microwave drying) and 78.16% (by freeze-drying). There was no significant difference (P > 0.05) in the values of Hg and Se found in specimens dried by the two methods. These results were consistent with results reported by K0h.7 The recovery of the added Hg and Se standards digested by microwave heating in a closed vessel was also found to be satisfactory (Table 2). The recovery of added standard was Table 1 Water content and concentration of Hg and Se in muscle of small species of tilapia dried by a freeze dryer and microwave oven Analyte Microwave oven Freeze dryer Water content (% d m ) 78.09 78.16 Hg/pg g- dry mass 3.777 f 0.198* 3.846 f 0.127* Setpg g- dry mass 2.224 k 0.021* 2.176 k 0.022* nations.* Mean values with standard deviations for four replicate determi- Table 2 Recovery of added standard after microwave heating under pressure in the presence of 70% m/m HN03 Element Addedhg 100 200 300 400 Se 100 140 200 240 Hg Found*/ng Recovery (YO) Mean (YO) 84.5 f. 0.0 84.5 t- 0.0 177.9 f. 0.9 89.0 f 0.5 252.8 k 1.4 84.3 k 0.5 325.0 f 2.4 81.3 f 0.6 84.8 f. 3.2 98.1 f 0.0 98.1 f 0.0 139.5 k 4.4 99.6 f 3.1 197.4 f 3.4 98.7 f 1.7 215.6 _+ 2.7 89.8 k 1.1 96.6 _+ 4.5 * Mean values with standard deviations for triplicate deterrnina- tions. Table 3 Comparison of the dissolution of dried muscle of large species of tilapia by using closed vessel microwave digestion and open vessel digestion in the presence of 70% m/m HN03, shown by recovery of added analytes Recovery/pg g-1 Mercury and selenium determination In each trial, 10 ml of the sample solution or working standard solution were used.A 1 ml volume of 30% HCI and 2 ml of reducing agent (a mixture of 3% m/v NaBH4 and 1% m/v NaOH) were added to the vapour generator. The solution was continuously purged with a stream of N2 at a pressure of 250 Microwave digestion* Open vessel 3.91 t- 0.29 1.24 k 0.02 1.71 f 0.08 1.25 f. 0.00 Element (closed vessel) digestion Hg Se * Mean values with standard deviations for four replicate determi- nations. Table4 Comparison of the found and certified values of Hg and Se in the NRCC certified reference materials DORM-1 and DOLT-1 (reference 9) Hg Found*/ Certified Sample g- value/pg g-1 DORM-1 Trial 1 0.759 k 0.030 Trial 2 0.754 t 0.019 Mean 0.757 f 0.025 0.798 f 0.074 DOLT-1 Trial 1 0.185 * 0.026 Trial 2 0.194 f.0.012 Mean 0.190 f 0.019 0.225 f 0.037 Se ~~~~~ Found?/ Certified Pg g- value/pg g- 1 1.44 f 0.03 1.43 k 0.01 1.44 f 0.02 6.64 t 0.16 6.60 f 0.15 6.62 f 0.16 1.62 k 0.12 7.34 f 0.42 * Mean value with standard deviation for four replicate determinations using cold vapour atomic absorption spectrometry. t Mean value with standard deviation for four replicate determinations using hydride generation atomic absorption spectrometry.ANALYST, SEPTEMBER 1991, VOL. 116 959 found to be 84.8 & 3.2 and 96.6 & 4.5% for Hg (100-400 ng) and Se (100-240 ng), respectively.The dissolution of the more volatile Hg and Se from fish tissue was superior to the traditional open vessel digestion (Table 3). The recovery of Hg and Se from samples of the large tilapia by open vessel digestion was only 35 and 68%, respectively, of the recovery by microwave heating under pressure. The results show that the closed digestion vessel can contain the samples tightly and the sample tissue is digested more completely at an elevated temperature and under pressure by microwave heating8 The microwave method was tested by studying two NRCC certified reference materials. A comparison of the values of Hg and Se found in DORM-1 (Dogfish Muscle) and DOLT-1 (Dogfish Liver) with the certified values is given in Table 4.9 The mean concentration of Hg found was within one standard deviation of the certified value in both the Dogfish Muscle and Dogfish Liver. For Se, the mean concentration was within two standard deviations of the certified value.The recovery of Hg from Dogfish Muscle and Dogfish Liver was 94.9 and 84.4%, respectively. For Se, the recovery was 88.9 and 90.2%, respectively. In conclusion, a method of microwave heating has been applied for the effective determination of the more volatile elements (Hg and Se) in biological samples. Microwave heating can be used for drying and wet ashing of fish tissue more efficiently than thermal heating in an open vessel in terms of a shorter time and smaller amounts of chemicals consumed. References Fischer, L. B . , Anal. Chem., 1986, 58, 261. Bettinelli, M., Baroni, U., and Pastorelli, N . , Anal. Chim. Acta, 1989, 225, 159. Aysola, P., Anderson, P., and Langford, C. H . , Anal. Chem., 1987, 59, 1582. Gedye, R., Smith, F., and Westaway, K., Educ. Chem., 1988, 25, 55. Vermeir, G., Vandecasteele, C., and Dams, R., Anal. Chirn. Acta, 1989, 220, 257. Tsalex, D. L., Atomic Absorption Spectrometry in Occupational and Environmental Health Practice, CRC Press, Boca Raton, FL, 1986, vol. 2, pp. 127-145 and 167-178. Koh, T. S . , Anal. Chem., 1980.52, 1978. Kingston, H. M., and Jassie, L. B . , Anal. Chem., 1986, 58, 2534. Marine Analytical Chemistry Standards Program, Dogfish Muscle and Liver Reference Materials for Trace Metals, National Research Council Canada, Ottawa, 1986. Paper 1101249E Received March 15th, 1991 Accepted May 20th, 1991