Summary of doctoral thesis in Chemistry: Research on the development of analytical method for methyl mercury in biological and environmental samples in Than Sa gold mine, Thai Nguyen

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Research to develop an analytical method for methyl mercury in biological samples with high sensitivity, selectivity, and accuracy; research and assessment of the transformation and bioaccumulation of mercury in sediment and biological samples in Than Sa gold mining area, Vo Nhai district, Thai Nguyen province.

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Nội dung Text: Summary of doctoral thesis in Chemistry: Research on the development of analytical method for methyl mercury in biological and environmental samples in Than Sa gold mine, Thai Nguyen

MINISTRY OF EDUCATION VIETNAM ACADEMY OF AND TRAINING SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY PHAN THANH PHƢƠNG RESEARCH ON THE DEVELOPMENT OF ANALYTICAL METHOD FOR METHYL MERCURY IN BIOLOGICAL AND ENVIRONMENTAL SAMPLES IN THAN SA GOLD MINE, THAI NGUYEN Major: Analytical Chemistry Code: 9.44.01.18 SUMMARY OF DOCTORAL THESIS IN CHEMISTRY HANOI - 2019
The work was completed at: Graduate University of Science and Technology – Vietnam Academy of Science and Technology Scientific advisors: 1. Assoc. Dr. Vu Duc Loi 2. Assoc. Dr. Le Lan Anh Reviewer 1:……………………………………………… Reviewer 2:……………………………………………… Reviewer 3:………………………………………………. The thesis will be defended in front of Academy-level Doctoral Committee at: Graduate University of Science and Technology – Vietnam Academy of Science and Technology At … hour … date… month … year 2019 Thesis can be further referred at: - The Library of Graduate University of Science and Technology - National Library of Vietnam
LIST OF PUBLICATIONS 1. Vũ Đức Lợi, Dương Tuấn Hưng, Nguyễn Thị Vân, Phan Thanh Phƣơng, “Phân tích thủy ngân oxit (HgO) và thủy ngân sunfua (HgS) trong trầm tích thuộc lưu vực sông Nhuệ và sông Đáy”, Tạp chí phân tích Hóa, Lý và Sinh học, 2015, Tập 20 (4), 135-142 2. Mineshi Sakamoto, Nozomi Tatsuta, Kimiko Izumo, Phuong Thanh Phan, Loi Duc Vu, Megumi Yamamoto, Masaki Nakamura, Kunihiko Nakai, Katsuyuki Murata, "Health Impacts and Biomarkers of Prenatal Exposure to Methylmercury: Lessons from Minamata, Japan", Toxics, 2018, 6 (3), 45 3. Vu Duc Loi, Duong Tuan Hung, Phan Thanh Phuong, “Mercury pollution due to gold mining activities in Thai Nguyen province”, Food Control Conference 2018, 4-5th October 2018, Hanoi, Vietnam. 4. Vũ Đức Lợi, Dương Tuấn Hưng, Nguyễn Thị Vân, Phan Thanh Phƣơng. “Nghiên cứu phương pháp xác định hàm lượng metyl thủy ngân trong mẫu sinh học bằng phương pháp quang phổ hấp thụ nguyên tử kỹ thuật hóa hơi lạnh (CV - AAS)”, Tạp chí phân tích Hóa, Lý và Sinh học, 2019, Tập 24 (3), 111-117. 5. Phan Thanh Phƣơng, Vũ Đức Lợi, Dương Tuấn Hưng, Nguyễn Thị Vân. “Đánh giá mức độ ô nhiễm metyl thủy ngân trong trầm tích suối Nước Đục thuộc xã Thần Sa, huyện Võ Nhai, tỉnh Thái Nguyên”, Tạp chí phân tích Hóa, Lý và Sinh học, 2019, Tập 24 (3), 123-129.
1 INTRODUCTION Environmental pollution is a global problem that is concerned by all countries and many scientists. Among the pollutants present in the environment, heavy metals, especially mercury, play an important role in the processes of transformation and bioaccumulation, when penetrating into the body, heavy metals will cause a great negative impact on human health, so they are considered as one of the causes of cancer and other serious diseases. Mercury toxicity depends on its speciation; organic mercury is more toxic than inorganic mercury and the most toxic form of mercury is methyl mercury (CH3Hg+), which accumulates in fish and animal cells. Methyl mercury is soluble in fat, the fat fraction of membranes and in the cerebrospinal fluid. The most dangerous property of methyl mercury is its ability to move across the blood - brain membrane and penetrate into the fetal tissue through the placenta. There have been many cases of mercury poisoning occurring on a large scale in the world. During 1953 - 1960 in Minamata City, Japan about 2955 people had been poisoned with mercury. Among those infected, 45 died. Genetic defects have been observed in newborn children whose mothers ate seafood harvested from the bay. In 1972, in Iraq, 459 farmers died after ingesting pesticides contaminated with mercury. Mercury is used in many industries such as chemicals, fertilizers, plastics, electrical engineering, electronics, paint, gold splitting in placer ores, manufacture of fluorescent lamps, batteries, thermometers, sphygmomanometer, cosmetics… According to a report of the Vietnam Chemical Agency - Ministry of Industry and Trade, in 2016, Vietnam had 4 main industries related to mercury use and emissions, including the manufacture and use of lighting equipment, burning coal from the factory, using it in the field of health and artisanal and small-scale gold mining, our country annually emits about 49,131 kg of mercury into the environment. In the environment, mercury is transformed into its other speciation by the activities of nature and humans, mercury is released into the atmosphere by various sources, then dispersed and deposited into land, mercury is stored and metabolized in soil and water. Biological conversion of inorganic mercury compounds to methyl mercury compounds can occur in sediments, in water, and in organisms. The process of mercury methylation is the most important factor that contributes to the introduction of mercury into the food chain. Artisanal and small-scale gold mining uses metal mercury to create amalgams, but
2 methyl mercury has been discovered in sediments in the mining area. The transformation of mercury speciation in the gold mining area is very complicated, the research on methylation and bioaccumulation of mercury in fish and benthos in these areas is limited. On the other hand, there is no manual procedure for methyl mercury analysis in sediments and biological samples in Vietnam. Therefore, we have chosen the thesis research entitled “Research on the development of analytical method for methyl mercury in biological and environmental samples in Than Sa gold mine, Thai Nguyen”. The objectives of the thesis are: - Research to develop an analytical method for methyl mercury in biological samples with high sensitivity, selectivity, and accuracy. - Research and assessment of the transformation and bioaccumulation of mercury in sediment and biological samples in Than Sa gold mining area, Vo Nhai district, Thai Nguyen province. To achieve above objectives, the main research contents of the thesis include: - Investigate, select optimal conditions and validate the method of determination of total mercury in sediment and biological samples. - Research, select optimal conditions and validate the method of determination of methyl mercury in sediment by the gas chromatography with electron capture detector (GC-ECD). - Research, investigate and develop the analytical procedure for the determination of methyl mercury in biological samples by atomic absorption spectroscopy - improved cold vapor technique combined with liquid-liquid extraction techniques. - Apply the developed analytical procedure to determine the total mercury and methyl mercury in sediment, biological samples in Than Sa gold mining area, Vo Nhai district, Thai Nguyen province and assess the transformation and accumulation of mercury in the above samples. CHAPTER 1. OVERVIEW 1.1. Mercury in nature and the cause of environmental pollution 1.1.1. Mercury in nature 1.1.2. Transformation cycle of mercury in the environment 1.1.3. Application of mercury 1.1.4. Causes of mercury pollution in the environment 1.2. Property of mercury
3 1.2.1. Physical, chemical properties of mercury 1.2.2. Characteristic properties of mercury 1.2.3. Toxicity of mercury and its compounds 1.3. Standards for evaluating Hg pollution in the environment 1.3.1. National technical regulation on sediment quality 1.3.2. National technical regulation on the limits of heavy metals contamination in food 1.3.3. National technical regulation on water quality 1.4. Mercury analysis methods 1.4.1. Analytical methods for total mercury 1.4.1.1. Atomic absorption spectroscopy - cold vapor (CV-AAS) 1.4.1.2. Atomic fluorescence spectroscopy 1.4.1.3. Atomic emission spectroscopy 1.4.1.4. Mass spectrometry (ICP-MS) 1.4.2. Analytical methods for methyl mercury 1.4.2.1. Selective extraction 1.4.2.2. Gas chromatography 1.4.2.3. Liquid chromatography 1.5. Validation of analytical method 1.5.1. Limit of detection LOD, limit of quantitation LOQ 1.5.2. Method for the determination of LOD and LOQ 1.5.3. Accuracy of the analytical method 1.6. Domestic and foreign research on the analysis of Hg, Me-Hg 1.6.1. Research on the accumulation and transformation of mercury 1.6.2. Research on the analytical methods for determination of mercury 1.7. Overview of the research area 1.7.1. Natural and socio-economic conditions of Than Sa commune, Vo Nhai district, Thai Nguyen province 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 10-5g (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 Hg2+ 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 Figure 3.2. Calibration curve for the measurements of standard determination of total Hg by CV- AAS concentration when constructing the (dependence of measuring the signal calibration curve for the determination on concentration) of total Hg (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 R2 = 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
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 106.62 which is higher than that of complex CH3HgCl of 105.51, therefore when CuCl2 is added, the ion Cu2+ 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 Figure 3.11. Chromatogram of mercury speciation in aqueous Me-Hg after extraction phase after pretreatment 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 Hg2+ 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 Effect of KOH concentration (M) on the recovery 1 2.0 of Me-Hg Effect of heating time T (min) on the recovery of 2 60 Me-Hg Effect of solvent volume ratio toluene/aqueous 3 1.0 phase on the recovery of Me-Hg Effect of complexing agent on the recovery of Me- 4 HBr (1M) Hg 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 Figure 3.14. Calibration curve for replicates of concentration when the determination of Me-Hg by constructing the calibration curve 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 R2 = 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 Me-Hg in biological No Replicate Weight (g) mHg (ng) 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 Certified Weight Mean Deviation RSD Replicate Found (ng/g) value (g) (ng/g) ∆ (%) (%) (ng/g) 1 1.0003 1.6880 2 1.0043 1.6703 3 1.0012 1.6512 1,59 ± Min: 0.83 1,611 4.64 4 1.0025 1.4890 0.12 Max: 7.54 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 Figure 3.17. The average of Me- in sediment samples collected in Hg in sediment samples collected different sampling locations 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 Figure 3.19. Ratio of the average methyl mercury to total mercury in of T-Hg to the average of Me-Hg sediment samples collected at in sediment samples collected at different sampling locations 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.