GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY TRINH THI THUY. Major: Analytical Chemistry Code: SUMARY OF DOCTORAL THESIS IN CHEMISTRY

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1 MINISTRY OF EDUCATION AND TRAINING VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY ***. TRINH THI THUY STUDY ON DETERMINATION OF MERCURY SPECIES IN SEDIMENT USING SELECTIVE EXTRACTION TECHNIQUE Major: Analytical Chemistry Code: SUMARY OF DOCTORAL THESIS IN CHEMISTRY Hanoi

2 The thesis has been completed at: Institute of Chemistry - Graduate university science and technology Vietnam Academy of Science and Technology Science supervisor: 1.Assoc.Prof.Dr Vu Duc Loi 2.Assoc.Prof.Dr Le Thi Trinh Reviewer 1: Reviewer 2: Reviewer 3: The thesis was defended at National level Council of Thesis Assessment held at Graduate University of Science and Technology Vietnam Academy of Science and Technology at, on, 201 Thesis can be further referred at: -The Library of Graduate University of Science and Technology -National Library of Vietnam

3 INTRODUCTION 1. Background Mercury and its compounds are chemicals which are great bio accumulative potential causing serious effects on human health and the environment. Mercury is used in many industries such as chemicals, fertilizers, plastics, electrical engineering, electronics, cement, paint, silver and gold in mineral ores, fluorescent lamps, barometer, thermometer, blood pressure monitor, cosmetics... According to the United Nations Environment Program (UNEP), Asia's rapid economic growth has accelerated the growth of industries that use mercury in production, making it the largest source of mercury, accounting for nearly 50% of the world's waste. According to the Ministry of Industry and Trade's 2016 national mercury inventory report, Vietnam has four major production sectors related to the use and emission of mercury: the manufacture and use of lighting equipment: fuel use coal in industrial activities; Use of mercury and compounds in the health sector and smallscale manual gold mining. Total amount of mercury imported into Vietnam in 2014 is about kg. However, there is no investigation to clarify the path and purpose of the use of mercury and mercury compounds sold in the domestic market. In October 2013, Vietnam signed the Minamata Convention on Mercury, which shows the concern and attention of state management agencies on mercury pollution, including monitoring, pollution control, minimize mercury using and emissions. The toxicity of mercury depends on its chemical form. In general, inorganic mercury is more toxic than organic mercury, mercury element and sulfide mercury are less toxic. The only form of mercury is methyl mercury, which can accumulate in fatty tissues, as well as in fish and other animals. Therefore, the determination of the content of different chemical forms of mercury in environmental samples, biological samples is very important, especially the sediment samples which accumulate many pollutants from the waste sources and are the habitats for many aquatic plants. Currently, there are a number of scientific studies on the method of determining mercury forms in different samples in the world, but there are not many comprehensive studies on sample processing to extract existing forms of mercury in sediment samples. The International Organizations and countries haven t also issued standards and guidelines for the determining mercury species in sediment samples except for the US Environmental Protection Agency (EPA) standard. In Vietnam, there is no standard procedure for the analysis of total mercury content and mercury species in sediment samples as there are very few studies evaluating the presence of mercury and its forms in the environment. Therefore, the study on Study on the determination of mercury species in sediment using selective extraction technique was conducted. 2. Objectives of this dissertation This study will be achieved by aiming to: - Build the analytical procedure of determination of mercury species in sediment using selective extraction technique - Assess of the reliability of the method of determination of mercury speciation in sediment - Apply the result of study to determine forms of mercury in sediment at a certain area. 3. The composition of the thesis - Investigate, select optimal conditions and validate of the analytical method of total mercury content in sediment; - Investigate and develop the procedure of determination of methyl mercury content in sediment by gas chromatography using Gas Chromatography - Electron Capture Detector (GC-ECD) with a capillary column instead of packed columns used previously; - Investigate and develop the procedure of determination of concentration of methyl mercury in sediment using selective extraction techniques and atomic absorption spectrometry. - Develop the procedure of selective extraction to determination of mercury species in sediment samples. 1

4 - Apply the analytical procedure to determine the content of total mercury and mercury speciation in surface sediment samples (ponds, lakes) in Minh Khai trade village, Van Lam, Hung Yen; sediment core at Han River estuary, Da Nang city and assess their pollution level in the environment. 2

5 CHAPTER 1: LITERATURE REVIEW 1.1 Mercury and mercury compounds: Introduce mercury and mercury compounds in terms of physical and toxicological properties, the metabolic pathways of mercury in the environment. 1.2 Source of Mercury, Mercury Compounds Emissions: Summarize sources, current status of mercury emissions into the environment in the world and in Vietnam. 1.3 Classification of the existence of mercury: Describes the classification of mercury forms in the environment, classify the forms of mercury in soil and sediment. 1.4 Methods of Determining Mercury Content: Overview the methods for quantifying mercury from postprocessing of Hg 2+ and methylmercury. 1.5 Research in and outside the country related to thesis: Summarize studies on methods of determining the total content of mercury in sediment, studies on sequential extraction methods, selection of mercury forms in sediment, some guidelines on the quantification of mercury and mercury forms in environmental samples. 1.6 Overview of sampling sites: Provide information on sampling sites for the study: The study was conducted on two matrix samples: surface sediment samples collected in the Minh Khai plastic recycling village, Nhu Quynh district, Hung Yen province and marine sediment collected from Han River estuary in Da Nang city. Base on references related to the method of determination of mercury species in sediment, show that: - The determination of total mercury content in sediments is not sufficiently scientific for evaluation the mobility, bioaccumulation potential, the impacts of mercury and its compounds the environment and ecosystem. In addition to the analysis of total mercury content, the concentration of mercury species in the sample should be determined in order to obtain a complete assessment. - For the procedures of determination of the total content of mercury, there have been many studies on methods of sample handling and quantitative techniques of total mercury content in sediment samples and other matrix samples. Several analytical method guidelines for determination of total mercury content in sediment were issued by US EPA, Japan. However, when applying these guidelines, laboratories have more or less changed or developed the methods. Therefore, the methods should be validated to ensure the reliability and accuracy of the results of the analysis under the actual conditions of existing laboratories. - Researches on sample preparation and quantification of methyl mercury in sediment is not very much, especially in Vietnam. According to international studies, methyl mercury in extract solution could be quantified by using GC instrument coupled with high sensitivity detector such as ECD, AAS, AFS, MS; there are small number of studies using CV-AAS or DMA to quantify MeHg. Therefore, it is very necessary to select, survey and evaluate the reliability of the analytical method in real experimental conditions. - There have been many studies on procedures of determination of some mercury species in soil and sediment samples, but there have been little agreement among authors on the classification and sequential extraction of species, also very few guideline standards for systematically validation of method. Hence, it is necessary to research on classification of mercury species, sequential extracting procedures, optimistically condition and evaluation of reliability as well as understanding of structural phase change of the sample after each extraction step to evaluate selectivity of extraction. Based on the above-mentioned research issues, we chose the research thesis: Study on the determination of mercury species in sediment using selective extraction technique 3

6 2.1 Research subjects CHAPTER 2: MATERIALS AND METHODS - Process analysis Mercury Species in sediment: total mercury, methyl mercury, organic mercury total, watersoluble and mercuric oxides, mercuric sulfide. - Sediment samples: + Quality control samples: method blank, matrix spike, duplicate and laboratory control sample + Environmental Samples: The sediment core samples from Han River estuary, Da Nang city; surface sediment samples were taken at a pond, lake and river in Minh Khai Plastic Recycling Village, Van Lam District, Hung Yen Province. 2.2 Research methods: The research methods used in the thesis: Literature review of publications Quantification method and equipment + Using the Cold Vapor Atomic Absorption Spectrometry (CV - AAS) method to measure and determine mercury Species after the sample digestion specializing Hg Gas Chromatography Electron Capture Detector: determine methyl mercury Data processing methods The experimental results were processed using Microsoft Excel 2010, Origin 8.5, SPSS - 20 software Optimize and validate analytical procedures: Design experiments for calculation of LOD, LOQ, accuracy, accuracy and uncertainty measurement. 2.3 Chemicals, tools: Presenting chemicals and devices for research in the full report. 2.4 Experiment Sample preparation for the study: present sample collection techniques, preparation of samples for research: Blank method samples, laboratory control sample (add the standard chemical to the cleaned sediment) Evaluation of the reliability of result of the total mercury content in sample: Method validation and determination Investigation and evaluation of the procedure for determination of methyl mercury content in sediment: To investigate the process of determination of methylmercury content in sediment by CV-AAS and GC- ECD method, then validate the use of two analytical processes Investigation and evaluation of selective sequential extraction of some forms of mercury in sediment: Investigation of selective sequential extraction of some forms of mercury in sediment, evaluation of the reliability of survey process. In this study we selected the following classification: Form F1: organic mercury Form F2: Soluble in water, HgO Form F3: mercuric sulfide Form F4: Residual form (fractional residue is a fraction of Hg bound to elements that can not be extracted by the previous reactants) Apply the procedures to determine the concentration of mercury speciation in the sediment: Determine the total mercury, methyl mercury and other forms in surface sediment and sediment cores taken at the Han estuary, Da Nang city; surface sediment samples were taken at a pond, lake and river in Minh Khai Plastic Recycling Village, Van Lam District, Hung Yen Province. 4

7 CHAPTER 3: RESULTS AND DISCUSSION 3.1 Result of confirming the use value of the total mercury content analysis The calibration curve was established using the standard solution of methyl mercury - Cysteine and carry out digestion of sample under the same conditions as for the environmental sample (calibration curve is built on blank sample matrix). First, measure the signal strength by repeatedly measuring each standard point at five times. The results show that the measurement signal is stable (the relative standard deviations (RSD) of signal of standard sample were under 15%). The linear range was from 0.05 to 1.4μg Hg/L. Validation result of the process of determining the total mercury content was shown in the following table: Table 3.6: Summarize the result of method validation of the T - Hg analysis process No Parameter Result Requirement of AOAC 1 Limit of detection and Limit of quantitation LOD = 1,04 ng/g LOQ = 3,45 ng/g 4< R = 5,52 < 10 2 Repeatability of the method RSD = 0,933-4,53 % RSD < 15% 3 4 A ccuracy of the method (Recovery productivity) Estimation of expanded measurement uncertainty U (%) R = 89,76 103,80%. 80 R 110% 13,72% 3.2 Investigation and evaluation of the determination of level of methyl mercury Determination of concentration of methyl mercury using CV-AAS a) Investigation of some extracted conditions The overall recoveries obtained from the experiments investigating the factors that affect the extraction efficiency of methyl mercury were shown in Figure 3.1. Figure 3.2: Summarize the results of investigating factors in the process of sample determination of methyl mercury by CV-AAS method 5

8 Based on the results of the study, we propose an experimental procedure to determine methyl mercury in sediment by the CV-AAS as follows: Weigh approximately 2.0 gram of sediment sample into 50mL glass centrifuge tube. Add 10.0mL of 6M HCl to the centrifuge tube, shake the mixture in a horizontal shaker, for 5 minutes. Centrifuge for 10 minutes at 2400 rpm, separate the water phase into a new centrifuge tube. Add 20.0 ml Toluene to the centrifuge tube, shake the mixture in a horizontal shaker, for 15 minutes. The mixture is then centrifuged for 20 minutes at 2400 rpm. Separate organic phase, repeat this step 2 times. Transfer the whole extract to a centrifuge tube, add 1 ml of L-Cyanine, shake for 20 minutes, centrifuge for 3 minutes at 2000 rpm, extract for L-Cystine. The concentration of methyl mercury in the final extract solution was determined by CV-AAS. b) Evaluate the reliability of the analysis procedure Determine the Limit of detection and Limit of quantitation of method: Limit of detection (LOD) and Limit of quantitation (LOQ) of established analytical procedure were calculated from result of the analysis of the sample in a given matrix containing small amount of MeHg. Ten replicates of MK8 sample were performed. The LOD andd LOQ of the built-up process were 0.34ng Hg/g and 1.12 ng Hg/g if using 2 grams of dry sediment samples for analysis, respectively. Evaluate the accuracy of the analytical process: Accuracy were evaluated at three levels as repeatability and precision using sediment samples and matrix sample spiked. Relative standard deviations (RSD) obtained were lower than 8.29%, it was lower than the acceptable RSD for the 10 ppb sample analyzes by AOAC (RSD Criteria <21%). Thus, the analytical procedure has been constructed to ensure the required repeatability. The determination of the trueness was considered through determination of recoveries using matrix spike. The recoveries from spiked samples at three concentration levels were ranged from 88.51% to %. This result is consistent with AOAC requirements (at the ng/g levels, the required recoveries are between 60 and 115%). Measurement uncertainty of method: expanded measurement uncertainty of method U = 24,34 (%) Determination of concentration of methyl mercury using GC/ECD a) Optimization of conditions of GC/ECD to determine methyl mercury Select the column When we used 03 column types DB - 608, DB - 5; DB - 17 to separated the analyst, realized that using the DB column give the pick signal more stable than the other two columns. Therefore, we choose the DB column (30m x 0.25mm x 0.25μm) for the next steps of the methyl mercury analysis on GC/ECD. Determination of operation conditions of equipment Survey methyl mercury determining conditions on GC/ECD equipment, column DB is carried out in accordance with parameters: temperature of detector, temperature of injector, program of furnace temperature in different conditions. From the experimental results, 3 rd condition was selected for quantitation of Methyl mercury by GC/ECD. The optimization control are: the injector temperature is C, the detector temperature is C, the gradient of temperature at column oven: initial temperature at 50 0 C (keep 1 minute) and increased to C with rate of temperature of 20 0 C/min (kept at the last temperature of 15 minutes). Under this condition, the retention time of methyl mercury is in the range of minutes (Figure 3.3) 6

9 MeHg MeHg MeHg MeHg MeHg MeHg 950, , , , , , , , , , , , , , , , , ,000 50, , ,000 µv MeHg200ppb2.DATA 7.15 RT [min] 7.2 2,200,000 2,100,000 µv MeHg500ppb1.DATA MeHg200ppb2.DATA 2,000,000 MeHg100ppb2.DATA 1,900,000 MeHg1000ppb Pha loang2.data MeHg50ppb2.DATA 1,800,000 1,700,000 1,600,000 1,500,000 1,400,000 1,300,000 1,200,000 1,100,000 1,000, , , , , , , , , , , ,000 RT [min] a) Diagram of standard at 200ppb b) Diagram of standard at different concentration Figure 3.3: Chromatographic of methyl mercury standard samples Next, the limit of detection and the limit of quantitative of the instrument with optimization condition. Five replicates of standard samples at 0.5ppb and signal-to-noise ratio (S/N) was calculated. Results of standard deviation and mean values of S/N, IDL and IQL values are shown in Table 3.2. Table 3.17: Results of IDL and IQL determination First Second Third 4 th time 5 th time Mean Concentration (ppb) 0,5 0,5 0,5 0,5 0,5 Pick area (µv) 117,7 93,4 88,1 76,3 102,1 Noise 69,27 48,90 45,70 51,20 71,30 S/N 1,699 1,910 1,928 1,490 1,432 1,69 IDL 0,883 0,785 0,778 1,007 1,048 0,90 IQL 2,943 2,618 2,594 3,355 3,492 3,00 According to the results, the Instrument detection limit (IDL) for MeHg is 0.90 ppb and the Instrument Quantitation limit (IQL) is 3.00 ppb. Those values allows the quantification of trace levels of methyl mercury in the sample after cleansing and enrichment. b) Optimization of conditions of sample preparation Synthesis results from the GC/ECD methylmercury determination process are shown in Figure 3.4 below. Thus, the research has selected the parameters for the process of determining methyl mercury by GC/ECD method as follows: Carefully weigh about 2 grams of sediment sample into 50mL glass centrifuge tube. Add 5.0 ml of KOH/CH 3 OH (25%), sonication for 45 minutes. Add 5mL of 4M H 2 SO 4 saturated with CuSO 4 solution, 5mL of KBr 4M solution and 3 ml of Toluene solution, shake for 3 minutes, then centrifuge at 2200 rpm for 10 minutes, muscle. Add 3 ml of solvent Toluene to the rest, repeating the extraction process twice. Collect the whole organic extracts to the new centrifuge tube. Add 1 ml of L-Cysteine solution 2%, shake for... minutes, extract the liquid phase (L-Cysteine extract) above. Repeat this process 03 times. Add 0.5 ml of 6M HCl solution to the L-Cysteine extracts, extracting methyl mercury by 0.5 ml of Toluene. The extraction is repeated twice. Collect whole extract, dry with anhydrous Na 2 SO 4. Extracts analysis on GC/ECD equipment, using column DB-08, with measurement conditions: - The temperature of the injector is C - The detector temperature is C - The column temperature program starts at 50 0 C (keep 1 minute) and increases to C with the temperature rise of 20 0 C/min (keep at the last temperature of 15 minutes). - Sample volume of 1μl - Carrier gases: N 2 (2 ml/min); Make up gases: N 2 (30 ml/min) 7

10 Figure 3.4: Summarize the results of investigating factors in the process of sample determination of methyl mercury by GC/ECD method c) Evaluate the analysis process Evaluation of signal stability, stability of calibration curve The stability of the signal, the stability of the calibration curve is carried out on the standard solution of methyl mercury with a concentration of Hg from 1 ppb 1000 ppb. Repeat the 5 μl of 1 μl of standard solutions at a concentration value on the GC / ECD with the quantitative conditions described above. Calculate the mean of the measured signals, SD, RSD. The measured signal RSD values of the benchmark must be less than 15%, according to the results show that the measured signal of the device under the selected condition is stable. Build the graph showing linear relationship between peak area and MeHg concentration on Origin 8.5 software. Figure 3.5: Graph of linear range of MeHg determination using GC/ECD method Results showed that, with a concentration of 1 to 200 ppb, the correlation coefficient R was R2> and the coefficient Sb (%) was less than 5%. However, to confirm that in this range the dependence between concentration and peak area is linear and Mandel's statistic must be compared with Fisher F (99%, 1, n-3). Standard Mandel results for a concentration range of 1 to 200 ppb. Determination of detection limits and quantitative limits of the method Experimentally, the LOD and LOQ values were calculated as ng Hg / g and ng Hg / g (sample size was 2 g), value 4 <R = Xtb / MDL = 4.2 <10 meets AOAC requirements. 8

11 The LOD value, LOQ of the identified process was similar to that of AM Caricchia, G. Minervini, P. Soldati using the GC / ECD method for the determination of MeHg, 2.0 g of dry sample for analysis, the LOQ value is 0.5 ng Hg / g. Evaluate the accuracy of the process Evaluate the accuracy of the procedure using the standard environmental and environmental sampling methods, the quantities evaluated as repeatability and recovery. The relative standard deviation of the analytical process was 13.53%. According to AOAC regulations, the acceptable RSD range for analysis samples is less than 21% lower than the acceptable RSD range for 10 ppb sample analyzes as required by AOAC. Repeatability criterion (<21%). Thus, the analytical process has been constructed to ensure the required repeatability. The recovery of the analytical procedure was performed on a standard additive sample at three concentrations ranging from %. This result is consistent with AOAC requirements (at the ng / g level, the required recovery is %). Estimation of uncertainty of method: Extinction width of method U = 25.26%. The reliability of the methyl mercury content analysis in sediment by the two methods is summarized in Table 3.24: Table 3.24: Summarize the results of the MeHg analysis using two CV-AAS and GC / ECD method No 1 Parameter Limit of detection and Limit of quantitation Phương pháp CV - AAS LOD = 0,347 ng/g LOQ = 1,156 ng/g R = 4,95 Result Phương pháp GC/ECD LOD = 0,215 ng/g LOQ = 0,716 ng/g R = 4,2 AOAC requirements 4< R< 10 2 Repeatability of the method RSD = 1,90-8,29 RSD = 1,82 13,53 RSD < 15% 4 5 A ccuracy of the method (Recovery productivity) Estimation of expanded measurement uncertainty U (%) R = 88,51% - 114,00% R = 72,01% -111,21%. 60 R 115% 22,69% 25,26% d) Compare two MeHg analysis methods To assess whether there are significant differences in the analysis results of the two methods, the method of comparing pairs according to the Student standard. The results show that there is no significant difference in the results. One of these methods can be used to analyze the content of methyl mercury in sediments Results from the examination and evaluation of the extraction process for selective mercury forms in sediment Examining the determination process of F1 species The blank sediment samples were spiked by addition of methylmercury at the concentration of 40 µg Hg/kg. Each experiment conducted repeatedly 3 times. The examining factors and result are shown respectively in Table 3.26 and Figure 3.6 Table Examining factors in F1 species determination process Examining Factors Quantities Varied conditions 9

12 (1) Volume of Chloroform extraction solvent (2) Agitation time for Chloroform solvent (3) Volume of Na 2 S 2 O 3 0,01M solution (4) Agitation time for Na 2 S 2 O 3 (Unit) Exp1 Exp2 Exp3 Exp4 Exp5 Volume (ml) Averaged recovery (%) 91,66 92,68 9,87 95,56 97,34 Time (mins) Averaged recovery (%) 50,82 65,58 75,56 92,27 99,01 Volume (ml) Averaged recovery (%) 92,17 102,03 98,55 97,97 Time (mins) Averaged recovery (%) 60,29 76,52 93,33 92,75 95,07 Examining factor (1): Recovery rates from all experimental volume were in the range of %, meeting the requirements of AOAC. However, with the volume of 5 ml and 10 ml, we observed a cloudy extraction fluid which can affect the subsequent organic mercury extraction from Chloroform into Na 2 S 2 O 3 solution. Therefore, the volume of 15 ml was selected. Examining factor (2): The results indicated that while agitation time within 2 minutes and 7 minutes had the recovery ranged from 50,6 75,3 %, the number for 10 min agitating extraction process was noticeably higher (91,9 98,7%). As a result, we chose 10 minutes as the agitation time for extracting organic mercury species from sediments into Chloroform solvent. Examining factor (3): When using different values of Na 2 S 2 O 3 0,01M volume, the recovery percentages ranged from 90,3 98,4 %. In practise, however, selecting 1 ml for extraction would be difficult during the phase separation of Na 2 S 2 O 3. Thus, it is more reasonable to use 2 ml or 3 ml of Na 2 S 2 O 3 0,01M. Examining factor (4): For the organic mercury extraction from Chloroform solvent using Na 2 S 2 O 3 solution, with the agitation time of 1 minute to 2 minutes, the maximum recovery rate of 76,7% did not satisfied the demand of AOAC. In contrast, with the time of 3 minutes to 5 minutes, the rate ranges significantly from 90,8 98,8 %. Therefore, the appropriate agitation time was 3 minutes. 10

13 Figure 3.6: The recovery data obtained in experiments for optimization of procedure of F1 selective extraction Examining the determination process of F2 species Specific factors and varying conditions are demonstrated in Table All experiments were carried out on the blank sediment samples which were added HgO spike. Each examining experiment conducted repeatedly 3 times. Table Examining factors in F2 species determination process Varied conditions Examining Factors Quantities (Unit) Exp1 Exp2 Exp3 Exp4 Exp5 (1) Concentration of H 2 SO 4 (2) Volume of H 2 SO 4 solution with optimum concentration (3) Agitation time Conc (mol/l) 0,02 0,05 0,1 0,2 0,5 Averaged recovery (%) 47,52 81,14 102,61 97,76 98,27 Volume (ml) Averaged recovery (%) 93,68 95,96 99,90 Time (mins) Averaged recovery (%) 71,07 98,18 99,66 103,17 Figure 3.7: The recovery data obtained in experiments for optimization of procedure of F2 selective extraction As illustrated in figure 3.7, at 0,05M the recovery rate was lower than 80%. However, increasing concentration between 0,1M to 0,5M gave a favourable rate, which ranged between 96,43 106,20 %. Thus, in order to ensure the required recovery, the concentration at 0,1M was chosen. Three initial experiments of H 2 SO 4 0,1M volume recorded moderately high recovery levels (> 80%). Starting from 15 ml, the recovery rate was considerably higher than 90%. Hence, to optimize the extraction process, out of several values, 15 ml of H 2 SO 4 0,1M was used for the research. 11

14 Recovery (%) from all examining agitation time with selected H 2 SO 4 volume and concentration were ranged from 65,37 to 97,76 %. The result for extraction which was shacked in 5 minutes (96,16 110,21%) was higher than that within 2 minutes (lower than 80%). For better efficiency and time saving, the optimal agitation time was clearly 5 minutes Examination of the determination process of F3 species concentration a) Examining the solubility of HgS The HgS solution was completely saturated when added CuCl and HCl + HNO 3 with the ratio of HCl:HNO 3 :H 2 O was 1:1:1; 1:1:2 (solubility was greater than 99,3%). Therefore, a mixture of HCl and HNO 3 with the ratio 1:1:1 and the addition of CuCl are considered to be the most suitable for mercuric sulfide extraction. b) Examining the conditions for mercury sulfide extraction in sediment Specific factors and varying conditions are summerized in Table All experiments were carried out on blank sediment samples which were added HgS spike. Each experiment conducted repeatedly 3 times. The examining factors and result are shown respectively in Table 3.28 and Figure 3.8. Table Examining factors in F3 species determination process Varied conditions Examining Factors Quantities (Unit) Exp1 Exp2 Exp3 Exp4 Exp5 (1) Volume of extraction solution (2) The amount of CuCl added (3) Agitation time Volume (ml) Averaged recovery (%) 61,87 93,27 97,01 99,04 99,67 Mass (gram) 0,1 0,1 0,3 0,4 0,5 Averaged recovery (%) 72,81 93,63 98,56 92,94 88,03 Time (mins) Averaged recovery (%) 63,96 97,08 94,21 96,86 12

15 Figure 3.8: The recovery data obtained in experiments for optimization of procedure of F3 selective extraction According to the experimental results, HgS concentration in sediments was determinated by using the HCl + HNO 3 extraction solution with the ratio of 1:1:2, 15 ml extraction solution for every 2,5 g sediment, 5 minutes of agitation and an 0,2 g additional amount of CuCl. Each extraction process repeated 3 times Evaluating the confidence level of the extraction of F1, F2 and F3 species a) Examining the variation of phase pattern through each extraction stage XDR spectrum of residue before F2 extraction (after F1 extraction) and afer F2 extraction (before F3 extraction) are demonstrated in Figure 3.10 and 3.11 Figure 3.10: The XDR spectrum of spike sample before F2 extraction 13

16 Figure 3.11: The XDR spectrum of spike sample after F2 extraction From Figure 3.10, the sediment samples contained several phases, namely HgCl2 (reached the main peaks of 2Ө at 20,36; 25,55 and 33,15); HgO (hit the main peaks of 2Ө at 30,11 and 32,45); HgS (hit main peaks of 2Ө at 26,55; 31,25; 43,76) and SiO 2 (hit main peaks of 2Ө at 20,36; 26,65; 36,55; 50,14 and other low - intensity peaks). After applying the examined extraction procedure for F1 species (methyl mercury chloride) with chloroform solvent, methyl mercury chloride was not displayed on the XDR spectrum. Thus, in the first step, the phase structure of sediment samples remained unchanged. F2 species extracted from H 2 SO 4 0,05M solution is easily soluble in acidic environment. And the XDR spectrum of residue after F2 extraction (Figure 3.11) indicated that phases in sediment samples still had HgS and SiO 2 (the main peaks at 2Ө are similar to those before F2 extraction conducted). It appeared that HgO and HgCl 2 were dissolved during F2 leaching period. As for HgS form (F3), this a durable form that is difficult to extract from sediment samples. Figure 3.12 shown the change in phase structure after F3 extraction. 14

17 Figure 3.12 The XDR spectrum of spike sample after F3 extraction As can be seen, the appeared peaks mainly belonged to SiO 2 phase. Compared to Figure 3.11, peaks of HgS have disappeared completely, which means HgS has dissolved entirely by a mix solution of HCl and HNO 3 with appropriate addition of CuCl. b) Evaluating the repeatability and trueness The reliability of the procedure is assessed through the repeatability and trueness Analysing the total mercury content and different species of mercury in environmental samples We applied the examined procedure to analyse total mercury content and level of other species in 2 types of sediment samples: - Pond and lake sediment samples: sediments were collected from ponds and lake of plastic recycling village Minh Khai, Nhu Quynh district, Hung Yen province. - Marine sediment samples: sediment cores were taken at the estuary of Han River and in the coastal area of Da Nang City. a. Total mercury content in the pond and lake sediments of Minh Khai trade village The results showed that the mercury concentration of sampling site was moderately high, ranging from 367,17 ng Hg/g to 1773,3 ng Hg/g. There is only one (MK8) out of eight samples was in compliance with the QCVN 43: 2012/ BTNMT - National Technical Regulation on Sediment Quality. This is alarming in terms of pollution levels which can affect community s health directly. The mercury level at Minh Khai village can be explained by the origin of raw materials for recycling activities. These materials are not only gathered from domestic market but also developing countries (Japan, Korea, Germany, ). Among them, electronic waste occupied a remarkable large proportion and generated heavy metals such as lead, mercury, chrome, in circuit board, batteries or electronic bulbs. b. Total mercury content in sediments of Han River Estuary, Da Nang City 15

18 The results are shown in Table Table 3.32 Total mercury content (ng/g dry weight) in sediment cores Depth (cm) Core SH1 Core SH2 Core SH3 Core SH4 Core SH5 From ,55 ± 0,7 128,62 ± 0,44 136,44 ± 2,73 170,08 ± 0,45 170,47 ± 1,90 From ,07 ± 0,69 198,52 ± 0,45 141,84 ± 2,74 171,85 ± 0,45 182,15 ± 1,96 From ,22 ± 0,73 151,64 ± 0,46 112,56 ± 2,73 176,67 ± 0,45 192,82 ± 2,02 From ,73 ± 0,73 178,70 ± 0,45 135,72 ± 2,72 174,24 ± 0,45 244,77 ± 2,01 From ,69 ± 0,77 247,60 ± 0,45 141,25 ± 2,73 179,37 ± 0,46 296,71 ± 2,01 From ,74 ± 0,74 130,60 ± 0,45 199,09 ± 2,72 199,33 ± 0,46 259,41 ± 1,01 From ,76 ± 0,69 88,73 ± 0,45 159,22 ± 2,72 250,23 ± 0,45 222,11± 2,04 From ,43 ± 0,81 111,49 ± 0,45 178,26 ± 2,79 207,71 ± 0,45 209,13 ± 1,90 From ,50 ± 0,67 115,36 ± 0,44 149,21 ± 2,75 182,62 ± 0,45 196,15 ± 1,84 From ,31 ± 0,76 96,01 ± 0,44 187,04 ± 2,78 214,30 ± 0,52 173,35 ± 176 From ,73 ± 0,83 227,39 ± 0,45 138,64 ± 1,83 125,42 ± 1,57 150,55 ± 1,68 From ,62 ± 0,76 131,68 ± 0,45 114,48 ± 1,84 158,17 ± 0,45 175,23 ± 0,93 From ,51 ± 0,70 137,88 ± 0,44 100,78 ± 1,83 118,23 ± 0,45 199,92 ± 1,86 From ,55 ± 0,72 124,22 ± 0,44 102,37 ± 1,83 130,58 ± 0,45 163,63 ± 0,93 From ,06 ± 0,75 173,27 ± 0,44 98,85 ± 1,85 143,68 ± 0,45 127,35 ± 01,86 From ,93 ± 0,66 128,91 ± 0,45 93,02 ± 1,80 174,39 ± 0,51 78,73 ± 1,88 From ,79 ± 0,68 130,12 ± 0,45 64,83 ± 1,80 163,53 ± 1,54 98,28 ± 1,88 From ,10 ±0,65 84,19 ± 0,44 89,24 ± 1,85 130,73 ± 0,45 117,26 ± 1,87 From ,81 ± 0,46 79,13 ± 1, From ,55 ± 0,7 128,62 ± 0,44 136,44 ± 2,73 170,08 ± 0,45 170,47 ± 1,90 As depicted in Table 3.32, mercury concentration tended to stabilize at the depth of cm above the surface, then decrease sharply at the cm depth, followed by a slight increase at the depth of 50 cm. After fluctuating slightly, a significant rise was recorded at depth of cm and the content declined gradually at surface sediment (depth less than 20 cm). In more intensive study, if sediment ages and the accumulative trend of mercury at depth could be assessed concurrently, it would be possible to evaluate the mercury pollution history of the area Analysing different species of mercury a. Forms of mercury in the pond and lake sediments of Minh Khai trade village Sampling sites Results of mercury species in pond and lake of plastic recycling village were shown in Table Table 3.34 Forms of mercury in pond and lake sediments of Minh Khai Village F1 F2 F3 F4 Total of 4 ng/g ng/g ng/g ng/g species 16 Total Devia tion MeHg (ng/g) MK1 41,77 ± 6,45 31,49 ± 2,47 438,97 ± 40,12 132,38 ± 18,32 644,60 ± 53,50 578,92 ± 79,43 11,35 3,55 ± 0,90 MK2 81,71 ± 12,62 93,63 ± 7,36 970,85 ± 88,74 556,67 ± 77, ,87 ± 141, ,30 ± 243,30 3,97 7,63 ± 1,93

19 Sampling sites F1 F2 F3 F4 Total of 4 ng/g ng/g ng/g ng/g species Total Devia tion MeHg (ng/g) MK3 45,44 ± 7,02 34,62 ± 2,72 778,95 ± 71,20 228,87 ± 31, ,87 ± 90, ,40 ± 168,67 11,51 8,99 ± 2,27 MK4 41,14 ± 6,36 47,53 ± 3,74 439,78 ± 40,20 181,88 ± 25,17 710,33 ± 58,96 669,18 ± 91,81 6,15 7,47 ± 1,89 MK5 79,54 ± 12,29 64,84 ± 5,10 449,56 ± 41,09 271,60 ± 37,59 865,53 ± 71,84 939,71 ± 128,93 7,89 3,81 ± 0,96 MK6 79,52 ± 12,29 35,65 ± 2,80 740,36 ± 67,67 57,50 ± 7,96 913,02 ± 75,78 846,32 ± 116,11 7,88 1,89 ± 0,48 MK7 74,45 ± 11,50 41,52 ± 3,26 451,99 ± 41,31 148,90 ± 20,61 716,86 ± 59,50 695,01 ± 95,35 3,14 6,60 ± 1,67 MK8 32,09 ± 4,96 25,76 ± 2,02 256,90 ± 23,48 97,50 ± 13,49 412,25 ± 34,22 367,17 ± 50,38 12,28 1,34 ± 0,34 MK9 33,45 ± 5,17 36,97 ± 2,91 678,72 ± 62,03 132,02 ± 18,27 881,15 ± 73,14 745,89 ± 102,34 18,13 2,41 ± 0,61 MK10 56,70 ± 8,76 45,90 ± 3,61 585,96 ± 53,56 212,38 ± 29,39 900,93 ± 74,78 865,32 ± 118,72 4,12 1,81 ± 0,46 According to 3.34 table, the mercury species in solution of F3 fraction was accounted with highest percentage (ranged from 51,94 % to 81,09 %), while those in F2 solution varied between 3,80% and 10,39% and in F1 and F4 solution, those only took up from 3,18 to 7,49% and from 6,79 to 31,39%, in respectively. The results here is considered to be consisted with several studies in the world. In research of Leonard Boszke et al., they had recorded the content of mercury species in the Vistula sediment of Netherlands: mercury sulphide contributed 55 82%, the highest percentage, followed by the organic mercury and water soluble mercury with 0,6 1,3% and 5,1 13% respectively. Prevalently, the percentage of methylmercury compared to total mercury in pond sediments made up less than 5%. Methylmercury content of our research ranged from 1,34 9,99 ng Hg/g (approximately 0,21 1,12%), which is also consistent with other research in the world. b. Analysing mercury species by depths in sediment cores of the Han River estuary The content of mercury forms in depth of the sediment columns collected at the mouth of the Han River and in coastal Danang is shown in Tables The percentaged distribution of different mercury species was indicated in figure and the distribution trend by depths was shown in the graphs

20 Depth (cm) Table 3.35 : Results of the analysis of the content of the types in sediment columns F1 F2 F3 F4 The sum of ng/g ng/g ng/g ng/g 4 forms Core SH1 Total Devia tion MeHg (ng/g) 0-5 2,67 ± 0,41 7,53 ± 0,59 46,67 ± 4,27 16,60 ± 2,30 73,47 ± 6,10 65,55 ± 8,99 12,08 0,29 ± 0, ,56 ± 0,55 8,83 ± 0,69 94,27 ± 8,62 24,56 ± 3,40 131,22 ± 10,89 141,22 ± 19,38 7,08 0,54 ± 0, ,68 ± 0,57 7,65 ± 0,60 85,24 ± 7,79 18,41 ± 2,55 114,98 ± 9,54 121,74 ± 16,70 5,55 0,26 ± 0, ,28 ± 0,51 8,28 ± 0,65 113,32 ± 10,36 13,54 ± 1,87 138,42 ± 11,49 124,50 ± 17,08 11, ,41 ± 0,53 7,09 ± 0,56 133,06 ± 12,16 18,40 ± 2,55 161,97 ± 13,44 153,62 ± 21,08 5, ,38 ± 0,52 7,05 ± 0,55 118,40 ± 10,82 17,25 ± 2,39 146,08 ± 12,12 156,06 ± 21,41 6, ,82 ± 0,44 7,26 ± 0,57 125,87 ± 11,50 12,19 ± 1,69 148,15 ± 12,30 134,10 ± 18,40 10,47 - Core SH ,94 ± 0,61 10,82 ± 0,85 100,91 ± 9,22 30,37 ± 4,02 146,04 ± 12,12 128,62 ± 17,65 13,54 0,87 ± 0, ,17 ± 0,80 14,40 ± 1,13 116,36 ± 10,63 30,83 ± 4,27 166,76 ± 13,84 151,64 ± 20,81 9,97 0,52 ± 0, ,58 ± 0,55 10,49 ± 0,82 110,11 ± 10,06 23,07 ± 3,19 147,26 ± 12,22 130,60 ± 17,92 12, ,19 ± 0,49 8,60 ± 0,68 98,56 ± 9,01 15,66 ± 2,17 126,01 ± 10,46 115,36 ± 15,83 9, ,72 ± 0,27 4,33 ± 0,34 94,40 ± 8,63 21,04 ± 2,91 121,49 ± 10,08 131,68 ± 18,07 7, ,54 ± 0,39 2,70 ± 0,21 135,23 ± 12,36 18,48 ± 2,56 158,95 ± 13,19 173,27 ± 23,77 8, ,14 ± 0,18 3,88 ± 0,30 76,27 ± 6,97 12,27 ± 1,70 93,55 ± 7,76 84,19 ± 11,55 11, ,92 ± 0,30 3,20 ± 0,25 120,59 ± 11,02 14,45 ± 2,00 140,16 ± 11,63 130,50 ± 17,90 7,40 - Core SH ,11 ± 0,63 10,06 ± 0,79 106,69 ± 9,75 25,89 ± 3,58 146,75 ± 12,18 136,44 ± 18,72 7,56 0,57 ± 0, ,33 ± 0,67 8,71 ± 0,68 90,88 ± 8,31 23,46 ± 3,25 127,38 ± 10,57 112,56 ± 15,44 13,16 0,32 ± 0, ,16 ± 0,80 11,23 ± 0,88 148,14 ± 13,54 33,78 ± 4,68 198,30 ± 16,46 199,09 ± 27,32 0,40 0,64 ± 0, ,92 ± 0,76 9,72 ± 0,76 130,17 ± 11,90 19,08 ± 2,64 163,89 ± 13,60 149,21 ± 20,47 9, ,09 ± 0,32 5,22 ± 0,41 87,77 ± 8,02 14,82 ± 2,05 109,90 ± 9,12 114,48 ± 15,71 4, ,46 ± 0,38 4,17 ± 0,33 90,88 ± 8,31 14,21 ± 1,97 111,71 ± 9,27 98,85 ± 13,56 13, ,54 ± 0,24 3,85 ± 0,30 80,14 ± 7,32 10,95 ± 1,51 96,47 ± 8,01 89,24 ± 12,24 8, ,47 ± 0,23 2,89 ± 0,23 68,53 ± 6,26 8,60 ± 1,19 81,49 ± 6,76 72,15 ± 9,90 12,94 - Core SH ,45 ± 1,00 11,76 ± 0,92 129,48 ± 11,83 31,20 ± 4,32 178,89 ± 14,85 170,08 ± 23,33 5,18 0,60 ± 0, ,80 ± 0,74 12,53 ± 0,98 138,27 ± 12,64 21,88 ± 3,03 177,48 ± 14,73 176,67 ± 24,24 0,46 0,74 ± 0, ,94 ± 0,92 10,45 ± 0,82 165,85 ± 15,16 25,75 ± 3,56 207,99 ± 17,26 199,33 ± 27,35 4,34 0,54 ± 0, ,58 ± 0,55 10,36 ± 0,81 163,23 ± 14,92 14,46 ± 2,00 191,63 ± 15,91 182,62 ± 25,06 4,93 0,42 ± 0, ,70 ± 0,42 6,88 ± 0,54 140,90 ± 12,88 20,34 ± 2,82 170,83 ± 14,18 158,17 ± ,01 0,30 ± 0,08 18

21 Depth (cm) F1 F2 F3 F4 The sum of ng/g ng/g ng/g ng/g 4 forms Total Devia tion ,70 ± 0,26 7,49 ± 0,59 123,58 ± 11,30 4,26 ± 0,59 137,03 ± 11,37 143,68 ± 19,71 4, ,02 ± 0,31 6,12 ± 0,48 120,79 ± 11,04 12,32 ± 1,71 141,26 ± 11,72 130,73 ± 17,94 8,05 - Core SH5 MeHg (ng/g) ,28 ± 1,90 12,17 ± 0,96 110,96 ± 10,14 49,09 ± 6,79 184,50 ± 15,31 170,47 ± 23,39 8,23 0,70 ± 0, ,19 ± 3,58 16,51 ± 1,30 145,20 ± 13,27 30,02 ± 4,16 214,92 ± 17,84 192,82 ± 26,45 11,46 0,56 ± 0, ,07 ± 3,41 16,21 ± 1,27 175,19 ± 16,01 62,95 ± 8,71 276,43 ± 22,94 259,41 ± 35,59 8,49 0,52 ± 0, ,73 ± 1,35 11,28 ± 0,89 151,48 ± 13,85 18,95 ± 2,62 190,44 ± 15,81 196,15 ± 26,91 2,91 0,35 ± 0, ,51 ± 0,48 10,08 ± 0,79 145,69 ± 13,32 13,61 ± 1,88 180,68 ± 15,00 175,23 ± 24,04 3,11 0,26 ± 0, ,06 ± 1,09 7,39 ± 0,58 117,62 ± 10,75 13,09 ± 1,81 145,16 ± 12,05 127,35 ± 17,47 13, ,27 ± 0,66 7,10 ± 0,56 111,81 ± 10,22 7,76 ± 1,07 130,94 ± 10,87 117,26 ± 16,09 11,67-19

22 Figure 3.15: Distribution of mercury species in sediment cores (%) 20

23 Figure 3.16: Distribution trends of T - Hg, Org. Hg, MeHg in sediment by depth As in Table 3.35 and Figure , it is clear that sulphide species made up the largest proportions of the mercury content (SH1: 63,53-86,89%; SH2: 69,01-86,04%; SH3: 71,34-84,1%; SH4: 72,38-90,18%; SH5: 67,56-85,39%). By contrast, organic mercury species occupied less than 5% for every sample. Similarly, HgO and water - soluble species were also low in percentage (less than 10%). The percentage of residues varied 21

24 from 7,92 to 22,59%. According to Hayao Sakamoto et al. the percentage distribution of organic mercury, HgO and mercury sulphide in sediments of Kagoshima Bay ranged from 0,26-11,12%; 1,0-42%; 38,4-96,1%, respectively. The graphs which presented the content of each mercury species by depth indicated a downward trend in the percentage of organic mercury and methylmercury from the surface. Meanwhile, the deeper sediment samples get, the higher mercury sulphide content are. In greater depth, methylmercury tended to below the regulation limit and others species had unclear trend. According to studies, researchers also noticed a downward trend by depth in methylmercury and organic mercury content. 22

25 CONCLUSIONS After studying on the thesis Study on the determination of mercury species in sediment using selective extraction technique, we reached the following conclusions: Investigation and evaluation of the reliability and application of analytical method for determination of total mercury content determination in sediment were studied. LOD and LOQ of the method was 1,04 ng/g and 3,45 ng/g respectively (sample size was 0,5 g). High repeatability and accuracy were assessed by using standard sample MESS3 and spiked sample. The result of total uncertainty measurement (6,68%) and expanded certainty measurement (13,72%) indicated that the process is highly reliable and in compliance with the requirement for the analysis of trace mercury in sediment. A number of mercury species in sediment samples have been studied and selected, including: organic mercury (F1); soluble mercury (F2); mercury sulphide (F3). The selectivity and solubility of the reagent for these mercury compounds was demonstrated by the change of phase structure before and after extraction through X-Ray Diffraction (XRD). The reliability was assessed through repeatability and trueness which were consistent with the AOAC guidelines. The procedure of methylmercury determination in sediment by 02 methods (CV AAS method and GC/ECD method) was studied, developed and assessed. Overall, both methods showed great repeatability, high accuracy and negligible difference in result. Thus, CV AAS and GC / ECD method can be used to analyse the content of methylmercury in sediment equally. Analytical procedures were applied to determine different species of mercury in sediment, which were sampled at coastal estuaries and ponds, lakes in Da Nang, Hung Yen Province. 23

26 NEW FINDINGS OF THE THESIS 1. The procedure of sequential selective extraction of different mercury species including organic mercury, soluble mercury, mercury oxide and mercury sulfide in sediment have been studied. The selectivity and solubility of the reagent for these mercury compounds were confirmed by the change of phase structure in X-Ray Diffraction (XRD) patterns before and after extraction. 2. Cold vapor Atomic Absorption Spectroscopy (CV-AAS) combined with the selective extraction have been proved to be an effective alternative method for chromatography methods to determine mercury species in sediments. 24

27 LIST OF WORKS HAS BEEN PUBLISHED 1. Trinh Thi Thuy, Vu Duc Loi, Le Thi Trinh, Dang Quoc Trung (2016). Research on Methods for Determination Organic Mercury in Sediments by Cold Vapor - Atomic Absorption Spectrometry (CV- AAS), VNU Journal of Science, Natural Science and Technology, Vol. 32, No. 2, page Trinh Thi Thuy, Vu Duc Loi, Le Thi Trinh, Nguyen Thi Van, Pham Thi Hong (2016). The distribution of mercury in Sediment cores of Han River Estuary, Da Nang City, VNU Journal of Science, Natural Science and Technology, Vol. 32, No. 2, page Trinh Thi Thuy, Vu Duc Loi, Le Thi Trinh (2017). Study on the method to determine the mercury sulfide content in sediment, Journal of Chemistry - Viet Nam Academy of Science and Technology, Vol. 55, No. 2e, page Trinh Thi Thuy, Le Thi Trinh, Vu Thi Mai, Pham Thi Hong (2016). Evaluation of the mercury accumulation in sediment of Han River Estuary, Da Nang City, Journal of Natural Resources and Environment, 6/2016, page Trinh Thi Thuy, Le Thi Trinh, Duong Tuan Hung, Vu Duc Loi. Research on Methyl Mercury Analysis Procedure in Sediments by Gas Chromatography GC/ECD), Jounal of Analytical Sciences, Vol. 22, No. 3/2017, page