Research on the development of analytical method for methyl mercury in biological and environmental samples in than sa gold mine, Thai Nguyen

ovince 1.7.2. Gold mining activities in Than Sa commune, Vo Nhai district, Thai Nguyen province CHAPTER 2. EXPERIMENTAL 2.1. Instrument, chemicals 2.1.1. Instrument, apparatus  Instrument - Automatic trace mercury analyzer Model VAST-HG 01(Upgraded, designed and built by Institute of Chemistry). - Gas chromatography with electron capture detector (GC-ECD, 4 Shimadzu- GC 2010). - High performance liquid chromatography - Inductively coupled plasma - Mass spectrometry (HPLC-ICP-MS) by Perkin-Elmer Model Nexion 2000. - Analytical balance with accuracy of 10 -5 g (Satorious). - Hot plate: Stuart SB300 - Vortex mixer: Fisher brand Whirli Mixer - Bottle-top dispenser: Socorex Calibrex 520/530 - Centrifuge: Heraeus Multifuge 3SR, Thermo Fisher Scientific. - Oven: Memmert UN55 (Germany).  Apparatus - Centrifuge tube: 50 mL, 15 mL - Volumetric flasks: 50mL, 100mL, 500mL, 1000mL. - Sample digestion flask (quartz): 50 mL - Pipettes, glass beakers Because Hg in the samples has trace levels, the instruments and apparatuses must be cleaned carefully to avoid maximum contamination by soaking in KMnO4 0,5% + H2SO4 1N solution, and then washing and rinsing by distilled water. 2.1.2. Chemicals Due to strict requirements of analysis, distilled water and reagents must be of high purity. During the research, the following chemicals and reagents had been used: 1. Acid HNO3 65% Merck, PA 2. Acid H2SO4 98% Merck, PA 3. Acid HClO4 72% Merck, PA 4. Acid HCl 36% Merck, PA 5. Acid HBr 48% Sigma-Aldrich, PA 6. SnCl2.2H2O Merck, PA 7. KMnO4 Merck, PA 8. Hydroxylamine (NH2OH.HCl) Merck 9. Na4EDTA (C10H12N2O8Na4.4H2O) Merck 10. Dithizone (C6H5N:NCSNHNHC6H5) Merck 11. Stock solution Hg 2+ 1000 mg/L Merck 12. Methyl mercury salt (CH3HgCl) Merck 13. Toluene Merck 14. Hexane Merck 15. Acetic acid (CH3COOH) Merck 16. NaOH Merck 5 17. CuCl2.2H2O Merck 18. L-cysteine hydrochloride Merck 2.1.3. Chemical preparation and standard solutions 1. NaOH 10M: Dissolve 400g NaOH (analytical grade) in 1 L of distilled water. 2. NaOH 0.1M: Transfer 1 mL of NaOH 10M solution into a 100 mL volumetric flask and add distilled water to the mark. 3. HCl 1M: Transfer 82 mL of HCl 36% into a 1000 mL volumetric flask which already has 500 mL distilled water, add distilled water to make a final volume of 1000 mL. 4. H2SO4 20N: Gradually add 600 mL of H2SO4 98% into a volumetric flask of 1000 mL which already has 350 mL distilled water. Cool down to room temperature and then add distilled water to the mark. 5. HBr 5M: Transfer 271.5 mL concentrated HBr 48% into a 1000mL volumetric flask and add distilled water to make a final volume of 1000 mL. 6. CuCl2 2M: Dissolve 342 g of CuCl2.2H2O in 1 L of distilled water. 7. NH2OH.HCl 10%: Dissolve 10g of NH2OH.HCl in 100 mL of distilled water. 8. EDTA 10%: Dissolve 10 g of Na4EDTA (C10H12N2O8Na4.4H2O) in 100 mL of distilled water. 9. Dithizone 0.01% in toluene: Dissolve 0.01 g diphenylthiocarbazone (C6H5N:NCSNHNHC6H5) in 100 mL of toluene. 10. Purify Dithizone solution: Transfer 100 mL Dithizone 0.01% into 200 mL separatory funnel, add 50 mL of NaOH 0.1N and shake briefly for 5 minutes, discard toluene organic phase. The aqueous phase will be acidified with HCl 1N so that the solution will become green and re-extracted with 100 mL of toluene, discard the aqueous phase and store Dithizone-toluene solution in a brown color glass container. Prepare a fresh solution for each analysis. 11. L-cysteine 0.1%: Dissolve 10 mg of L-cysteine hydrochloride C3H7O2S.HCl.H2O in 10 mL of NaOH 0.1N. Prepare a fresh solution for each analysis. 12. Methyl mercury stock solution: Weight out and dissolve 12.5 mg of CH3HgCl in a 100 mL toluene, 1 mL of this solution contains 100 µg of Hg. 13. Methyl mercury standard solution: Dilute stock solution 100-fold with toluene to obtain a methyl mercury standard solution, 1 mL of this solution contains 1.0 µg of Hg. 14. Methyl mercury-cysteine solution: Transfer 0.5 mL of the 6 methyl mercury standard solution and 5 mL of the L-cysteine 0.1% solution into a 10-ml conical centrifuge tube fitted with a glass stopper. Shake for 3 minutes with a shaker to extract methyl mercury into the aqueous phase. Centrifuge at 1200 rpm for 3 minutes and draw off and discard the organic phase (upper phase). The obtained solution contains 0.1 µg Hg/mL, seal the tube and store in a cool dark place. Prepare a fresh solution monthly. 15. SnCl2 10%: Dissolve 10 g of SnCl2.2H2O in 9 mL of HCl and dilute to 100 mL with distilled water. 16. KMnO4 0.5%: Dissolve 0.5 g KMnO4 in 100 mL distilled water. 2.2. Validation of the analytical method for the determination of total Hg by CV-AAS 2.2.1. Calibration curve construction for total Hg determination 2.2.2. Analytical method for the determination of total Hg in soil and sediment 2.2.3. Analytical method for total mercury in water 2.2.4. Analytical method for total mercury in fish, hair and blood 2.3. Validation of the analytical method for the determination of Me- Hg in sediment by GC-ECD 2.3.1. Calibration curve construction for Me-Hg determination by GC- ECD 2.3.2. Analytical method for Me-Hg in sediment by GC-ECD 2.4. Research, development of analytical method for the determination of Me-Hg in biological samples by CV-AAS 2.4.1. Calibration curve construction for Me-Hg determination by CV- AAS 2.4.2. Analytical method for Me-Hg in biological samples by CV-AAS 2.5. Subjects and research methods 2.5.1. Research subjects - Research samples:  Environmental samples (sediment, water) and fishery collected in rivers and streams in the gold mining area in Than Sa commune, Vo Nhai district, Thai Nguyen province.  Human biological samples including hair, blood directly working in the exploitation and processing gold in Than Sa commune, Vo Nhai district, Thai Nguyen province. - Analytical method for mercury speciation in biological and 7 environmental samples:  Validation of the analytical method for the determination of total Hg by CV-AAS.  Validation of the analytical method for the determination of Me- Hg by GC-ECD.  Research, development of analytical method for the determination of Me-Hg in biological samples by CV-AAS. 2.5.2. Research method 2.5.2.1. Literature review method 2.5.2.2. Measurement, quantitation a. Atomic absorption spectroscopy - cold vapor CV-AAS b. Gas chromatography with electron capture detector (GC-ECD) c. High performance liquid chromatography - inductively coupled plasma mass spectrometry (HPLC-ICP-MS) 2.5.2.3. Data analysis The experimental results are processed by the software: Microsoft Excel 2010. 2.6. Sample collection and preparation 2.6.1. Sampling location Biological and environmental samples were collected from 2 villages Tan Kim and Thuong Kim (Bai Mo, Ha Kim, Thuong Kim) in the North of Than Sa commune, Thai Nguyen province. 2.6.2. Sample collection and storage 2.6.2.1. Environmental samples collection 2.6.2.2. Biological samples collection 2.7. Determination of mercury in environmental and biological samples Based on the researched and developed analytical procedures, the total mercury and methyl mercury content in environmental and biological samples taken in Than Sa commune, Vo Nhai district, Thai Nguyen province were determined. 8 CHAPTER 3. RESULTS AND DISCUSSION 3.1. Results of the validation of the analytical method for the determination of total Hg by CV-AAS 3.1.1. Calibration curve for the determination of total Hg Figure 3.1. Results of repeated measurements of standard concentration when constructing the calibration curve for the determination of total Hg (dependence of measuring the signal on concentration) Figure 3.2. Calibration curve for the determination of total Hg by CV- AAS (dependence of measuring the signal on concentration) Calibration curve in Figure 3.2 has the equation y = 1818.2 x + 40.698 with slope a = 1818.2 and correlation coefficient R 2 = 0.9994. With a sample volume of 5 mL, the linearity is within the range of 0.1 to 1.0 µg/L, so it is suitable for analyzing trace concentration of Hg in environmental and biological samples. 3.1.2. Limit of Detection (LOD) and Limit of Quantitation (LOQ) Determination of Limit of detection (LOD) and Limit of quantitation(LOQ) of the analytical method for the determination of total mercury were performed using the following samples: sediment sample, blood sample which has total mercury of 8.15 ng/g and 1.70 ng/g, respectively; according to the procedure described in section 2.2.4. The sample analysis results were repeated 10 times with , SD, LOD and LOQ summarized in Table 3.2 and Table 3.3. According to the results shown in the above tables, LOD and LOQ of sediment and biological (blood) samples are 1.04 and 3.47 ng/g; 0.22 and 0.75 ng/g, respectively. 9 The calculated coefficient R of both samples satisfied the requirements of AOAC (4 < R < 10) proving that the sample test concentration is suitable and calculated LOD, LOQ are reliable. 3.1.3. Accuracy of the method 3.1.3.1. Evaluation of accuracy based on certified reference materials (CRM) The replicate measurements of total mercury in certified reference materials MESS-3, DOLT-3, and DORM-2 were shown in Table 3.4, 3.5 and 3.6. The results show that the deviation value of the samples MESS-3, DOLT-3 and DORM-2 are 4.34, 3.92, and 5.68, respectively which are all less than 15%. In addition, the calculated relative errors are also less than the maximum accepted value according to AOAC. Thus, the analytical method for the determination of total mercury has high accuracy and good repeatability which can be applied to determine the total mercury in environmental and biological samples. 3.1.3.2. Evaluation of accuracy based on the spiked environmental samples For water samples, since there is no certified reference material for water sample, the accuracy must be evaluated based on the recovery. The result of total Hg in BM-III 01 water sample which was spiked with 3 different concentrations of 5 ng/L, 10 ng/L, and 20 ng/L, is shown in Table 3.7. The result in Table 3.7 shows that the recovery of spiked water samples was in the range from 86.60 - 103.00 %, which satisfies the requirements of AOAC regarding the acceptable recovery in the working concentration of 40 - 120%. The relative standard deviation of the samples is within 0.30 - 1.56%, which is less than the maximum accepted value according to AOAC (30% at a concentration of 1 ppb). The above evaluation results show that the analytical method for the determination of total mercury in water samples has good repeatability and high accuracy. 10 3.2. Results of the validation of the analytical method for the determination of Me-Hg in sediment samples by CV-AAS 3.2.1. Calibration curve for the determination of Me-Hg by GC-ECD Figure 3.3. Calibration curve for the determination of Me-Hg by GC-ECD The calibration curve in Figure 3.3 is the first order linear line with a slope of 3.4555 and correlation coefficient of 0.9992. For a sample volume of 5L, the linearity ranges from 1.0 to 10.0 g/L. 3.2.2. Limit of detection (LOD) and Limit of quantitation (LOQ) Limit of detection (LOD) and Limit of quantitation (LOQ) of the analytical method for the determination of methyl mercury was performed on sediment sample that has the methyl mercury of 0.767 ng/g and carried out according to the procedure described in section 3.2.1. The results of 10 repeated measurements and the values of , SD, LOD and LOQ are summarized in Table 3.9. According to the results shown in Table 3.9, the LOD and LOQ of the analytical method for the determination of methyl mercury by gas chromatography (GC-ECD) on sediment samples were 0.228 and 0.761 ng/g, respectively. The calculated R coefficient (4.582) satisfies the requirements of AOAC (4 <R <10) proving that the test solution concentration is suitable and LOD, LOQ calculated are reliable. 3.2.3. Accuracy of GC-ECD method The IAEA-405 certified reference material (Trace Elements and Methylmercury in Estuarine Sediment) was used to evaluate the accuracy of the analytical method for the determination of methyl 11 mercury in sediment samples by GC-ECD. The analytical results are shown in Table 3.10. From the results in Table 3.10, the difference of methyl mercury in the IAEA-405 certified reference material analyzed is 6.25% smaller than the maximum allowed value according to USFDA (15%). The calculated relative standard deviation value is also smaller than the maximum acceptable value according to AOAC. Thus, the analytical method for the determination of methyl mercury by GC-ECD has high accuracy and good repeatability, which can be applied to analyze methyl mercury in sediment samples. 3.3. Results of developing the analytical method for the determination of Me-Hg in biological samples by CV-AAS 3.3.1. Analytical method for Me-Hg in biological samples by CV-AAS 3.3.1.1. Sample pretreatment a. Effect of KOH concentration Figure 3.6. Effect of KOH concentration on the recovery of Me-Hg The results in Figure 3.6 shows that: when the KOH concentration is low, the biodegradability of biological tissues is not complete, thus the recovery of Me-Hg is low but when the KOH concentration increases, the recovery increases and reaches the maximum value when the concentration KOH is 2M. When the KOH concentration continues to increase to 5M, the recovery does not increase. Therefore, in subsequent studies, the concentration of KOH 2M was chosen to dissolve the sample. 12 b. Effect of sample dissolving time Figure 3.7. Effect of heating time on the recovery of Me-Hg The results in Figure 3.7 show that when the heating time increases, the recovery of Me-Hg increases and reaches the maximum value at 60 minutes. Therefore, in the following research experiments the heating time of 60 minutes was chosen. 3.3.1.2. Methyl mercury extraction Figure 3.9. Effect of complexing agents and solvent ratio on the recovery of Me-Hg According to the above results, when the ratio of extraction solvent (toluene) to the sample volume is equal to 1, the extraction efficiency reaches the maximum value for both 1M HCl and 1M HBr, however in 1M HCl maximum recovery is 80.6% even when the extraction solvent/sample volume ratio is 2. If using HBr 1M the extraction efficiency is over 97% and when using the extraction solvent/sample volume of 0.5, the recovery has reached 94.9%. 13 Higher methyl mercury extraction efficiency when using halide chelating agent Br - compared with Cl - is explained as follows: The stability constant of complex CH3HgBr is 10 6.62 which is higher than that of complex CH3HgCl of 10 5.51 , therefore when CuCl2 is added, the ion Cu 2+ will compete to form complex with Cysteine to release methyl mercury, but complex CH3HgBr is more stable than CH3HgCl and has better solubility in toluene solvent so the reaction occurs completely and the methyl mercury extraction efficiency is higher. With this extraction procedure, methyl mercury is separated from inorganic mercury and there are only inorganic mercury ions in the aqueous phase. The separation of methyl mercury was proved by HPLC-ICP-MS using 8 column with mobile phase of 0.6% 2-mercaptoethanol and 3% methanol in Perkin-Elmer Nexion 200 instrument, the mass to charge ratio (m/z) of mercury isotope was 202. Figure 3.10. Chromatogram of mercury speciation in aqueous phase after pretreatment Figure 3.11. Chromatogram of Me-Hg after extraction The chromatograms in Figure 3.10 and 3.11 show that after the pretreatment of the biological sample there were two peaks: Me-Hg with retention time of 0.45 min and Hg 2+ with retention time of 1.40 min. However, after complex formation and extraction into toluene phase, on chromatogram (Figure 3.11), there was only one peak of Me-Hg appeared with the retention time of 0.45 min. Therefore, it is possible to 14 completely separate methyl mercury from other forms of mercury this extraction technique. In order to determine the methyl mercury in the toluene phase by atomic absorption spectroscopy, methyl mercury needs to be extracted into the aqueous phase and then digested with HClO4-HNO3 and H2SO4 and then measured by CV-AAS. . Table 3.14 summarizes the results of experiments to study the effect of factors on the sample pretreatment to determine methyl mercury in biological samples by CV-AAS following steps (1) to (3). Table 3.14. Summary of the effect of factors on the sample pretreatment when determining Me-Hg in biological samples by CV-AAS No Factors Parameters 1 Effect of KOH concentration (M) on the recovery of Me-Hg 2.0 2 Effect of heating time T (min) on the recovery of Me-Hg 60 3 Effect of solvent volume ratio toluene/aqueous phase on the recovery of Me-Hg 1.0 4 Effect of complexing agent on the recovery of Me- Hg HBr (1M) 3.3.2. Construction of calibration curve for the determination of Me- Hg by CV-AAS 3.3.2.1. Calibration curve for the determination of Me-Hg Figure 3.13. The results of replicates of concentration when constructing the calibration curve Figure 3.14. Calibration curve for the determination of Me-Hg by CV-AAS The calibration curve in Figure 3.14 has a linear equation: y = 1454.6 x + 34.771 with a slope of a = 1454.6 and correlation coefficient R 2 = 0.9998. For a sample volume of 5 mL the linearity was obtained over the concentration range of 0.05 - 1.00 µg/L which is suitable for the determination of trace Hg in the environmental and biological samples. 15 3.3.2.2. Limit of detection (LOD) and Limit of quantitation (LOQ) Table 3.16. LOD and LOQ No Replicate Weight (g) mHg (ng) Me-Hg in biological sample (ng Hg/g) 1 1 1.0003 3.5279 1.6880 2 2 1.0035 3.3336 1.5950 3 3 1.0012 3.4506 1.6510 4 4 1.0015 3.4590 1.6550 5 5 1.0045 3.3273 1.5920 6 6 1.0053 3.4694 1.6600 7 7 1.0011 3.1308 1.4980 8 8 1.0035 3.3336 1.5950 9 9 1.0005 3.2729 1.5660 10 10 1.0002 3.3127 1.5850 Mean ( ) 1.6085 Standard deviation (SD) 0.0559 LOD 0.17 LOQ 0.56 R 9.58 The results in Table 3.16 show that: Limit of detection of the developed analytical method LOD=0.17ngHg/g, Limit of quantitation LOQ=0.56 ng Hg/g, when 1 g of certified reference material of fish was used for the analysis. The obtained HR was: 4 < HR = 9.58 < 10 which satisfies the requirement of AOAC, thus the obtained LOD, LOQ are accepted. 3.3.2.3. Accuracy of the method - Accuracy evaluation based on certified reference material (CRM) Table 3.17. The results of Me-Hg found in the certified reference material DOLT-3 Replicate Weight (g) Found (ng/g) Mean (ng/g) Certified value (ng/g) Deviation ∆ (%) RSD (%) 1 1.0003 1.6880 1,611 1,59 ± 0.12 Min: 0.83 Max: 7.54 4.64 2 1.0043 1.6703 3 1.0012 1.6512 4 1.0025 1.4890 5 1.0004 1.5974 6 1.0015 1.5685 According to the results in Table 3.14, the values of ∆ = 0.83 - 7.54%; RSD = 4.64 which satisfy the requirement for evaluating the accuracy of the method. 16 3.4. Results of the determination of total Hg and Me-Hg in environmental and biological samples 3.4.1. Analytical results of environmental samples 3.4.1.1. Sediment samples Figure 3.15. Total mercury and methyl mercury in sediment samples The methyl mercury in sediment samples in the research area has an average value of 3.41 ppb. The minimum and maximum concentration of Me-Hg was 0.31 ppb and 33.71 ppb, respectively. In particular, the highest mercury methyl was observed in the Ha Kim area which was in the downstream of the mining area. To assess the transformation of methyl mercury, the total mercury, the methyl mercury and the Me- Hg/T-Hg ratio were assessed in three areas. The results are shown in Figures 3.16, 3.17, 3.18 and 3.19. Figure 3.16. The average of T-Hg in sediment samples collected in different sampling locations Figure 3.17. The average of Me- Hg in sediment samples collected in different sampling locations 17 The chart in Figure 3.16 shows that the average value of total mercury in sediment samples in Bai Mo area (7.93 ppm) was the largest, followed by Thuong Kim (4.91 ppm) and Ha Kim (3.31). This was due to the fact that mining and processing activities are all carried out in Thuong Kim and Bai Mo areas, leading to a larger amount of mercury used and discharged to this area than in Ha Kim. However, the results obtained from Figure 3.17 show that the highest methyl mercury was in Ha Kim area (8.26 ppb) which was followed the Bai Mo area (1.90 ppb) and Thuong Kim (1.89 ppb). Therefore, it can be noticed that there was a transformation from inorganic mercury species to methyl mercury in sediments in the lower region of Ha Kim. The Me-Hg in Ha Kim area was 4 times higher than that in Thuong Kim and Bai Mo areas, while the total mercury in Ha Kim area was 2.4 times lower than that in Bai Mo area and 1.5 times higher than that in Thuong Kim area. Figure 3.18. Percentage ratio of methyl mercury to total mercury in sediment samples collected at different sampling locations Figure 3.19. Ratio of the average of T-Hg to the average of Me-Hg in sediment samples collected at different sampling locations The results obtained from Figure 3.18 show that the average ratio of methyl mercury/total mercury in sediment samples in Ha Kim (0.25%) was 6 to 12 times greater than that in Thuong Kim (0.04%) and Bai Mo (0.02%). Similarly, Figure 3.19 shows that the highest ratio of total mercury/methyl mercury in sediment samples was in Thuong Kim, followed by Bai Mo and Ha Kim. 3.4.1.2. Water samples Water samples were collected in 3 areas including Bai Mo, Thuong Kim, and Ha Kim. The sample was filtered with a 0.45 µm membrane filter and acidified to pH < 2. The total mercury was determined by CV- AAS. The analytical results of total mercury in water samples are presented in Figure 3.20. 18 Figure 3.20. Total mercury (T-Hg) in water samples The chart in Figure 3.20 shows that the total mercury in water samples was very small, the mean was 0.086 µg/L, the maximum was obtained in Ha Kim with concentration of 0.668 µg/L which is lower than the allowable value of 1.0 µg/L according to the National technical regulation on surface water quality (QCVN 08:2008/BTNMT). Because the total mercury in surface water samples is very small, in this study it is not possible to assess the level of methyl mercury transformation in water in the study area. 3.4.2. Analytical results of biological samples 3.4.2.1. Fishery samples Figure 3.21. Total mercury and methyl mercury in fishery samples The results presented in Figure 3.21 show that: 16/24 (66.67%) samples had the total mercury higher than the allowable limit (0.5 ppm) of that and 15/24 (62.50%) samples had the methyl mercury greater than the allowable limit (0.5 ppm) of that specified in specified in the National technical regulation on the limits of heavy metals contamination in food (QCVN 8-2:2011/BYT). 19 The average of total mercury, methyl mercury and the correlation between Me-Hg and T-Hg in fishery samples were assessed in three l0cations. The results are shown in Figure 3.22, 3.23 and 3.24. Figure 3.22. The average of total mercury in fishery samples collected in different sampling locations. Figure 3.23. The average of methyl mercury in fishery samples collected in different sampling locations. The charts in Figures 3.22 and 3.23 show that the average value of the total mercury in the fishery samples in the Ha Kim (1.13 ppm) is the largest, followed by Thuong Kim (0.79 ppm) and Bai Mo (0.59) is the lowest. Similarly, the methyl mercury in fishery samples has the same trend as the total mercury, the methyl mercury gradually increases from Bai Mo (0.52 ppm) to Thuong Kim (0.69 ppm) and Ha Kim (1.04 ppm). Figure 3.24. The percentage ratio of methyl mercury to total mercury in fishery samples collected in different sampling locations. The correlation between the total mercury and methyl mercury in fishery samples in Thuong Kim, Ha Kim, and Bai Mo is shown in Figure 3.25. 20 Figure 3.25. Correlation between total mercury and methyl mercury in fishery s