Sumary of doctoral thesis in chemistry: Study on the determination of mercury species in sediment using selective extraction technique

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.

TRINH THI THUY

STUDY ON DETERMINATION OF MERCURY SPECIES IN SEDIMENT

USING SELECTIVE EXTRACTION TECHNIQUE

Major: Analytical Chemistry

Code: 9.44.01.18

SUMARY OF DOCTORAL THESIS IN CHEMISTRY

Hanoi - 2018

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

Thesis can be further referred at:

-The Library of Graduate University of Science and Technology

-National Library of Vietnam

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 small-scale manual gold mining Total amount of mercury imported into Vietnam in 2014 is about 14000 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

- 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

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

post-processing of Hg2+ 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”

CHAPTER 2: MATERIALS AND METHODS 2.1 Research subjects

- Process analysis Mercury Species in sediment: total mercury, methyl mercury, organic mercury total, soluble and mercuric oxides, mercuric sulfide

water 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:

2.2.1 Literature review of publications

2.2.2 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 Hg2+

+ Gas Chromatography – Electron Capture Detector: determine methyl mercury

2.2.3 Data processing methods

The experimental results were processed using Microsoft Excel 2010, Origin 8.5, SPSS - 20 software

2.2.4 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

2.4.1 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)

2.4.2 Evaluation of the reliability of result of the total mercury content in sample: Method validation and

determination

2.4.3 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 ECD method, then validate the use of two analytical processes

GC-2.4.4 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)

2.4.5 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

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

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 A ccuracy of the method (Recovery

productivity) R = 89,76 ÷ 103,80% 80 ≤ R ≤ 110%

4 Estimation of expanded measurement

uncertainty U (%) 13,72%

3.2 Investigation and evaluation of the determination of level of methyl mercury

3.2.1 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

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 114.00% 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 (%) 3.2.2 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 - 608 column give the pick signal more stable than the other two columns Therefore, we choose the DB

- 608 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 - 608 is carried out

in accordance with parameters: temperature of detector, temperature of injector, program of furnace temperature

7.2 7.15 7.1 7.05 7 6.95 6.9 6.85 6.8 6.75 6.7 6.65 6.6 6.55 6.5

6.82 6.8 6.78 6.76 6.74 6.72 6.7 6.68 6.66 6.64 6.62 6.6 6.58 6.56 6.54 6.52 6.5 6.48 6.46 6.44 6.42 6.4

2,000,000 1,800,000 1,600,000 1,400,000 1,200,000 1,000,000 900,000 700,000 500,000 300,000 100,000 0 -100,000

MeHg1000ppb Pha loang2.DATA

MeHg50ppb2.DATA

µV

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

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/CH3OH (25%), sonication for 45 minutes Add 5mL of 4M H2SO4 saturated with CuSO4 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 Na2SO4

Extracts analysis on GC/ECD equipment, using column DB-08, with measurement conditions:

- The temperature of the injector is 2200C

- The detector temperature is 2800C

- The column temperature program starts at 500C (keep 1 minute) and increases to 2400C with the temperature rise of 200C/min (keep at the last temperature of 15 minutes)

- Sample volume of 1μl

- Carrier gases: N2 (2 ml/min); Make up gases: N2 (30 ml/min)

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> 0.995 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 0.215 ng Hg / g and 0.716 ng Hg / g (sample size was 2 g), value 4 <R = Xtb / MDL = 4.2 <10 meets AOAC requirements

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 72.01 ÷ 111.21% This result is consistent with AOAC requirements (at the ng / g level, the required recovery is 60 -115%)

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

Result

AOAC requirements Phương pháp CV - AAS Phương pháp GC/ECD

1 Limit of detection and

Limit of quantitation

LOD = 0,347 ng/g LOQ = 1,156 ng/g

R = 4,95

LOD = 0,215 ng/g LOQ = 0,716 ng/g

R = 4,2

4< R< 10

2 Repeatability of the method RSD = 1,90 - 8,29 RSD = 1,82 – 13,53 RSD < 15%

4 A ccuracy of the method

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

3.3 Results from the examination and evaluation of the extraction process for selective mercury forms in sediment

3.3.1 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 3.26 Examining factors in F1 species determination process

(Unit) Exp1 Exp2 Exp3 Exp4 Exp5

(1) Volume of Chloroform

extraction solvent

Volume (mL) 5 10 15 20 25 Averaged recovery (%) 91,66 92,68 9,87 95,56 97,34 (2) Agitation time for Chloroform

solvent

Averaged recovery (%) 50,82 65,58 75,56 92,27 99,01 (3) Volume of Na2S2O3 0,01M

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 Na2S2O3 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 Na2S2O3 Thus, it is more reasonable to use 2 ml or 3 ml of Na2S2O3 0,01M

Examining factor (4): For the organic mercury extraction from Chloroform solvent using Na2S2O3

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

Figure 3.6: The recovery data obtained in experiments for optimization of procedure of F1 selective

extraction

3.3.2 Examining the determination process of F2 species

Specific factors and varying conditions are demonstrated in Table 3.27 All experiments were carried out

on the blank sediment samples which were added HgO spike Each examining experiment conducted repeatedly

3 times

Table 3.27 Examining factors in F2 species determination process

(1) Concentration of H2SO4

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 (2) Volume of H2SO4

solution with optimum

concentration

Volume (mL) 10 15 20 Averaged recovery (%)

Three initial experiments of H2SO4 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 H2SO4 0,1M was used for the research