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Intake of methyl mercury by the population of Mumbai, India
the Science of the Total Environment ELSEVIER
The Science of the Total Environment 205 (1997) 267-270
Short communication
Intake of methyl mercury by the population of Mumbai, India G.G. Pandit, S.K. Jha, R.M. Tripathi, T.M. Krishnamoorthy” EnvironmentalAssessment
Division,
Bhabha
Atomic
Research
Centre,
Bombay,
400 085, India
Received 18 March 1997; accepted 7 July 1997
Abstract Reversedphasehigh performanceliquid chromatography(HPLC) with ultra violet detection (UV) was optimised for separationand quantification of methyl mercury in coastalsedimentand fish samples.The extraction efficiency of methyl mercury from sedimentand biologicalsampleswasfound to be 56% with a detection limit of 0.5 ng for a 200 ~1 samplevolume. The concentrationsof methyl mercury and the relative fractions with respect to total mercury were distinctly lower, 5.9-65.5 rig/g (3-8%) in sedimentcomparedto biological samples,20.4-344.5 rig/g dry wt. (33-97%). The daily intake of methyl mercury by the Mumbai population through marine food is about 0.5 pg forming 62% of the total mercuxy intake from this route. 0 1997Elsevier ScienceB.V. Keywords:
Reversed phase high performance liquid chromatography; Methyl mercury; Coastal sediment; Fish
samples
1. Introduction
Mercury exists in the environment, both in inorganic and organic forms. Environmental studies have shown that the elemental and ionic mercury can be converted into highly toxic organ0 mercury compounds (Westoo, 1968). The contamination of methyl mercury in fish samples is a potential health hazard to human health. The concentrations of mercury in marine fishes are
* Corresponding author.
reported to range between 4 and 1000 rig/g total mercury, of which methyl mercury could form a significant fraction (Bloom, 1992) as high as 95%. Even though there are published data on the concentration of total mercury in environmental samples, there is a lack of data on the concentration distribution of methyl mercury in Indian marine ecosystems. Hempel et al. (1992) used a new HPLC method with UV detection of organomercurial compounds as their thio-ethanol complexes. This method has been adapted for the determination of methyl mercury in the Thane Creek ecosystem
0048-9697/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PZI SOO48-9697(97)00201-5
268
G.G. Pandit
et al. /The
Science of the Total Environment
to assess the daily intake of methylmercury and total mercury through the marine food pathway. 2. Experimental 2.1. Apparatus and chromatographic conditions
Measurements were carried out using a HPLC system Shimadzu (LC-10 AD) with UV-visible detection. A column (250 x 4.6 mm i.d. with a 10 X 4.6 mm i.d. guard column) packed with totally porous spherical silica particles with RP-18 material (particle size 5 pm) was used. The chromatographic data were processed with a C-R7A chromatopac data processor. The flow rate was 0.5 ml/min with UV detection at 230 nm. The methanol-water mixture (50:50) with 0.1 mM 2mercaptoethanol buffered to pH 5.0 with acetic acid and ammonium acetate (0.02 M) was used as the mobile phase. Typically, 200~~1 sample volumes in 0.1 mM sodium thiosulphate medium were injected onto the column. 2.2. Reagents
Analytical grade hydrochloric acid (6 M) was used for the leaching of sediment and fish samples. Sodium thiosulphate (0.1 mM) buffered with ammonium acetate (0.02 M) was used in the back extraction step. A stock solution of methyl mercury chloride (Merck) was prepared in distilled water. Standard solutions were prepared by diluting stock solution with sodium thiosulphate. 2.3. Sample collection and preparation
The sediment samples were collected from three locations, Airoli, Ghansoli and Koperkhairane of Trans Thane Creek Area (TICA). Samples were frozen as soon as they were brought to the laboratory and all the samples, except those fractions required for wet and dry weight analysis were freeze dried. A known weight (2.0 g) of sediment sample was treated with 5.0 ml of 6 M HCl and shaken for 12 h using a mechanical shaker. The methyl mercury chloride was extracted with toluene and back extracted with 2 ml of 0.1 mM sodium thiosul-
205 (1997) 267-270
phate solution. The concentrations of methyl mercury in the sediment samples were evaluated using the calibration curve generated with known standards as well as by the internal standard addition technique. About 1 kg of surface marine sediment was collected far away from the study area and was used for the spike studies. For the leach tests, 2 g of the sediment sample was spiked with 25 ~1 of methyl mercury at a concentration of 6.8 mg/dm3. A 5-ml volume of the leaching agent to be tested was added to the spiked sediment sample and the mixture was shaken for 12 h using a mechanical shaker. After sedimentation and filtration, the supernatant was treated according to the procedure described above. Another 2-g aliquot of the same sample was used to estimate the inherent methyl mercury for the blank (which showed a lower concentration of methyl mercury) subtraction. Fresh marine organisms (shrimp fish and crabs) were purchased from local fishermen who harvest marine organisms from the Creek. The samples were dissected for edible portions (muscle), freeze dried and then homogenized in a mixer grinder having plastic blades. The homogenized tissues were washed three times with acetone and once with toluene (Hight and Corcoran, 1987) as prescribed by the Association of Official Analytical Chemists (AOAC). A known weight (2.0 g> of the washed tissue was treated with 5.0 ml of 6 M HCl and was shaken for 12 h using a mechanical shaker. The methyl mercury chloride was extracted with toluene and back extracted with 2 ml of 0.1 mM sodium thiosulphate solution. The Table 1 Concentrations of methyl mercury in sediment samples collected at Trans-Thane Creek area (April-May, 1996) Sample
Location
Concentration of methyl mercury (rig/g dry wt.)
1 2 3 4 5 6
Airoli Airoli Ghansoli Ghansoli Koparkhairane Koparkhairane
46.8 f 49.6 + 65.5 + 36.7 f 5.9 + 15.5 k
6.7 5.8 5.8 3.5 1.5 2.3
G.G. Pandit et al. /The
Science
of the
Total Environment
Table 2 Concentration of methyl mercury in various marine organisms collected at TIC area (April-May, Sample number
9 10 11 12
269
205 (1997) 267-270
1996)
Marine organism
Concentration of methyl mercury (q/g dry wt.)
Boi, Mugil dussumieti Black fish, Tilapia mossambique Red prawns, Metapaenpios afJinis Cat fish, Macrones vitattus Prawns, Penaeus Salpa Dhoma, Sciaena dicanthus Muddskipper, Boleophthalmas boddaerti Crabs, S$la serrataShrimp, Acetis indicus Shrimp, Pineus indicus Tuna fish (IAEA-350)
20.4 + 2.2 80.2 + 6.7 42.3 f 4.0 123.0 + 11.2 310.1 + 26.2 94.2 f 11.2 60.5 + 7.9 50.9 + 9.5 344.4 + 25.4 118.5 f 7.3 90.0 + 9.3 3.4a (3.2-3.5)
aConcentration in pg/g. Certified value of IAEA-350 = 3.65 pg/g (3.32-4.01).
analytical efficiency of the procedure was found to be 56%. The analytical procedure was verified using the homogenized tuna fish containing methyl mercury, a certified reference material obtained from the International Atomic Energy Agency (IAEA). The results obtained through the complete methodology were in good agreement with the certified value. 2.4. Detection limits and reproducibility
The detection limit together with the retention time, its reproducibility, the measured peak areas (%) and the relative standard deviation (R.S.D.) when 1 mg/dm3 of methyl mercury was injected were: 1. Retention 2. R.S.D. of 3. Detection lute). 4. R.S.D. of
3. Results
time (t,) = 9.67 min. t, = 2.28%. limit = 2.5 pg/dm3 (0.5 ng absopeak area = 2.64%.
and Discussion
Methyl mercury is attached to clay minerals, sulfydryl groups and humic substances of sedi-
ment matrices and amino acids and protein components are able to attach methyl mercury in marine organisms. By acidifying the sediment and tissue samples with halogen0 and organic acids, the exchange sites are protonated and the methyl mercury fraction is detached from these sites. The methyl mercury chloride is formed during the leaching step and is soluble in toluene. The efficient leaching of methyl mercury in strong acids like 6 N HCl is mainly due to the formation of non-polar alkyl mercury halides which are readily soluble in toluene. This method was used in the present work for leaching methyl mercury components from sediment and biological matrices. Acetone washing of tissue samples was found to be necessary to remove lipid components which interfere in HPLC analysis. Sodium thiosulphate has been found suitable for back extraction owing to its high complexing affinity for methyl mercury Table 3 Daily intake of organic and total mercury from marine food Sample number
Mercury component
Concentration Wg
1 2
Total mercury Methyl mercury
62.5-548.8 (189.0) 20.4-344.4 (121.0)
Daily intake of Hg ( wg) 0.8 0.5
270
G.G. Par&it
et al. / The Science of the Total Environment
and in addition, provides a rapid quantitative clean up of toluene extracts. The method described above has been used to estimate the methyl mercury concentration levels in sediment and marine organisms collected from Thane Creek (Tables 1 and 2). The concentration of methyl mercury in sediment was found to be distinctly lower (6-60 rig/g) compared to is total mercury concentration which ranged from 75 to 2140 rig/g. The high values of total mercury in these sediment samples may be attributed to the presence of clay material (48-65%) having a mean grain size of 2-9 pm and the presence of montmorillonite with the minor amount of Kaolinite and ellite types of minerals (Ratha and Sahu, 1994). The concentration of total mercury in marine organisms collected from Thane Creek varied from 62.5 to 548 rig/g with a mean of 189 rig/g (on a dry weight basis). The daily mean intake of total mercury through marine food for the Mumbai population is be about 0.8 pg based on the marine food consumption rate of 14 g/day and a dry to wet weight ratio of 3.7 (Table 3). Krishnamurti (1987) reported a daily intake of total mercury through air, water and food including the marine component as 13.8 pg in which the marine component formed only 0.5 pg. The reported intakes of mercury cover a range of 3-25 pg/day with a median around 15 pg/day (Fergusson, 1990). The FAO/WHO permissible tolerable level has been set at 43 pg/day (0.3 mg/week) of which no more than 66% is methylmercury. The methyl mercury concentration in the marine food ranged between 20.4 and 344 rig/g with a mean of 121 rig/g (on a dry weight basis). The highest concentrations are found in the local delicacy such as crabs and prawns. Crab muscle with hepatopancreas which are a delicacy for fisher folk. The daily intake of methyl mercury component forms nearly 62% of the total mercury (i.e. 0.5 pg/day).
205 (1997) 267-270
4. Conclusions The leaching with 6 N HCI followed with extraction in toluene and back extraction in sodium thiosulphate; measurement with HPLC and UV detection was found to be an effective method for the determination of methyl mercury in complex matrices like sediment and marine organisms and the method can be used without any clean up step. The extraction efficiency of methyl mercury from sediment and biological samples was found to be 56% with the detection limit of 0.5 ng for a 200 ~1 sample volume. The concentrations of methyl mercury and the relative fractions with respect to total mercury were distinctly lower, 5.9-65.5 rig/g (3-8%) in sediment compared to 20.4-344.5 rig/g dry wt. (33-97%) in biological samples. The daily intake of methyl mercury through marine food is about 0.5 pg and forms 62% of the intake of total mercury from this route. References Bloom NS. On the chemical form of mercury in edible fish and marine invertebrate tissue. Can J Aquat Sci 1992;4:1010. Fergusson JE. The heavy elements chemistry: environmental impact and health effects. New York: Pergamon Press, 1990:533-567. Hempel M, Hintelmann H, Wilken RD. Determination of organic mercury species in soil by high performance chromatography with ultraviolet detection. Analyst 1992; 117:669. Hight SC, Corcoran MT. Rapid determination of methyl mercury in fish and shellfish. J Assoc Anal Chem 1987;70:22-28. Krishnamurti CR. Lead, mercury, cadmium and arsenic in environment. In: Hutchinson TC, editor. 1987:315-333. Ratha DS, Sahu BK. Statistical assessment of geochemical variable and size distribution characteristics of sediments from two estuaries in Bombay, India. Int J Environ Stud 1994;46:115-142. Westoo G. Determination of methylmercury salts in various kinds of biological samples. Acta Chem Stand 1968;22:2277.
The Science of the Total Environment 205 (1997) 267-270
Short communication
Intake of methyl mercury by the population of Mumbai, India G.G. Pandit, S.K. Jha, R.M. Tripathi, T.M. Krishnamoorthy” EnvironmentalAssessment
Division,
Bhabha
Atomic
Research
Centre,
Bombay,
400 085, India
Received 18 March 1997; accepted 7 July 1997
Abstract Reversedphasehigh performanceliquid chromatography(HPLC) with ultra violet detection (UV) was optimised for separationand quantification of methyl mercury in coastalsedimentand fish samples.The extraction efficiency of methyl mercury from sedimentand biologicalsampleswasfound to be 56% with a detection limit of 0.5 ng for a 200 ~1 samplevolume. The concentrationsof methyl mercury and the relative fractions with respect to total mercury were distinctly lower, 5.9-65.5 rig/g (3-8%) in sedimentcomparedto biological samples,20.4-344.5 rig/g dry wt. (33-97%). The daily intake of methyl mercury by the Mumbai population through marine food is about 0.5 pg forming 62% of the total mercuxy intake from this route. 0 1997Elsevier ScienceB.V. Keywords:
Reversed phase high performance liquid chromatography; Methyl mercury; Coastal sediment; Fish
samples
1. Introduction
Mercury exists in the environment, both in inorganic and organic forms. Environmental studies have shown that the elemental and ionic mercury can be converted into highly toxic organ0 mercury compounds (Westoo, 1968). The contamination of methyl mercury in fish samples is a potential health hazard to human health. The concentrations of mercury in marine fishes are
* Corresponding author.
reported to range between 4 and 1000 rig/g total mercury, of which methyl mercury could form a significant fraction (Bloom, 1992) as high as 95%. Even though there are published data on the concentration of total mercury in environmental samples, there is a lack of data on the concentration distribution of methyl mercury in Indian marine ecosystems. Hempel et al. (1992) used a new HPLC method with UV detection of organomercurial compounds as their thio-ethanol complexes. This method has been adapted for the determination of methyl mercury in the Thane Creek ecosystem
0048-9697/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PZI SOO48-9697(97)00201-5
268
G.G. Pandit
et al. /The
Science of the Total Environment
to assess the daily intake of methylmercury and total mercury through the marine food pathway. 2. Experimental 2.1. Apparatus and chromatographic conditions
Measurements were carried out using a HPLC system Shimadzu (LC-10 AD) with UV-visible detection. A column (250 x 4.6 mm i.d. with a 10 X 4.6 mm i.d. guard column) packed with totally porous spherical silica particles with RP-18 material (particle size 5 pm) was used. The chromatographic data were processed with a C-R7A chromatopac data processor. The flow rate was 0.5 ml/min with UV detection at 230 nm. The methanol-water mixture (50:50) with 0.1 mM 2mercaptoethanol buffered to pH 5.0 with acetic acid and ammonium acetate (0.02 M) was used as the mobile phase. Typically, 200~~1 sample volumes in 0.1 mM sodium thiosulphate medium were injected onto the column. 2.2. Reagents
Analytical grade hydrochloric acid (6 M) was used for the leaching of sediment and fish samples. Sodium thiosulphate (0.1 mM) buffered with ammonium acetate (0.02 M) was used in the back extraction step. A stock solution of methyl mercury chloride (Merck) was prepared in distilled water. Standard solutions were prepared by diluting stock solution with sodium thiosulphate. 2.3. Sample collection and preparation
The sediment samples were collected from three locations, Airoli, Ghansoli and Koperkhairane of Trans Thane Creek Area (TICA). Samples were frozen as soon as they were brought to the laboratory and all the samples, except those fractions required for wet and dry weight analysis were freeze dried. A known weight (2.0 g) of sediment sample was treated with 5.0 ml of 6 M HCl and shaken for 12 h using a mechanical shaker. The methyl mercury chloride was extracted with toluene and back extracted with 2 ml of 0.1 mM sodium thiosul-
205 (1997) 267-270
phate solution. The concentrations of methyl mercury in the sediment samples were evaluated using the calibration curve generated with known standards as well as by the internal standard addition technique. About 1 kg of surface marine sediment was collected far away from the study area and was used for the spike studies. For the leach tests, 2 g of the sediment sample was spiked with 25 ~1 of methyl mercury at a concentration of 6.8 mg/dm3. A 5-ml volume of the leaching agent to be tested was added to the spiked sediment sample and the mixture was shaken for 12 h using a mechanical shaker. After sedimentation and filtration, the supernatant was treated according to the procedure described above. Another 2-g aliquot of the same sample was used to estimate the inherent methyl mercury for the blank (which showed a lower concentration of methyl mercury) subtraction. Fresh marine organisms (shrimp fish and crabs) were purchased from local fishermen who harvest marine organisms from the Creek. The samples were dissected for edible portions (muscle), freeze dried and then homogenized in a mixer grinder having plastic blades. The homogenized tissues were washed three times with acetone and once with toluene (Hight and Corcoran, 1987) as prescribed by the Association of Official Analytical Chemists (AOAC). A known weight (2.0 g> of the washed tissue was treated with 5.0 ml of 6 M HCl and was shaken for 12 h using a mechanical shaker. The methyl mercury chloride was extracted with toluene and back extracted with 2 ml of 0.1 mM sodium thiosulphate solution. The Table 1 Concentrations of methyl mercury in sediment samples collected at Trans-Thane Creek area (April-May, 1996) Sample
Location
Concentration of methyl mercury (rig/g dry wt.)
1 2 3 4 5 6
Airoli Airoli Ghansoli Ghansoli Koparkhairane Koparkhairane
46.8 f 49.6 + 65.5 + 36.7 f 5.9 + 15.5 k
6.7 5.8 5.8 3.5 1.5 2.3
G.G. Pandit et al. /The
Science
of the
Total Environment
Table 2 Concentration of methyl mercury in various marine organisms collected at TIC area (April-May, Sample number
9 10 11 12
269
205 (1997) 267-270
1996)
Marine organism
Concentration of methyl mercury (q/g dry wt.)
Boi, Mugil dussumieti Black fish, Tilapia mossambique Red prawns, Metapaenpios afJinis Cat fish, Macrones vitattus Prawns, Penaeus Salpa Dhoma, Sciaena dicanthus Muddskipper, Boleophthalmas boddaerti Crabs, S$la serrataShrimp, Acetis indicus Shrimp, Pineus indicus Tuna fish (IAEA-350)
20.4 + 2.2 80.2 + 6.7 42.3 f 4.0 123.0 + 11.2 310.1 + 26.2 94.2 f 11.2 60.5 + 7.9 50.9 + 9.5 344.4 + 25.4 118.5 f 7.3 90.0 + 9.3 3.4a (3.2-3.5)
aConcentration in pg/g. Certified value of IAEA-350 = 3.65 pg/g (3.32-4.01).
analytical efficiency of the procedure was found to be 56%. The analytical procedure was verified using the homogenized tuna fish containing methyl mercury, a certified reference material obtained from the International Atomic Energy Agency (IAEA). The results obtained through the complete methodology were in good agreement with the certified value. 2.4. Detection limits and reproducibility
The detection limit together with the retention time, its reproducibility, the measured peak areas (%) and the relative standard deviation (R.S.D.) when 1 mg/dm3 of methyl mercury was injected were: 1. Retention 2. R.S.D. of 3. Detection lute). 4. R.S.D. of
3. Results
time (t,) = 9.67 min. t, = 2.28%. limit = 2.5 pg/dm3 (0.5 ng absopeak area = 2.64%.
and Discussion
Methyl mercury is attached to clay minerals, sulfydryl groups and humic substances of sedi-
ment matrices and amino acids and protein components are able to attach methyl mercury in marine organisms. By acidifying the sediment and tissue samples with halogen0 and organic acids, the exchange sites are protonated and the methyl mercury fraction is detached from these sites. The methyl mercury chloride is formed during the leaching step and is soluble in toluene. The efficient leaching of methyl mercury in strong acids like 6 N HCl is mainly due to the formation of non-polar alkyl mercury halides which are readily soluble in toluene. This method was used in the present work for leaching methyl mercury components from sediment and biological matrices. Acetone washing of tissue samples was found to be necessary to remove lipid components which interfere in HPLC analysis. Sodium thiosulphate has been found suitable for back extraction owing to its high complexing affinity for methyl mercury Table 3 Daily intake of organic and total mercury from marine food Sample number
Mercury component
Concentration Wg
1 2
Total mercury Methyl mercury
62.5-548.8 (189.0) 20.4-344.4 (121.0)
Daily intake of Hg ( wg) 0.8 0.5
270
G.G. Par&it
et al. / The Science of the Total Environment
and in addition, provides a rapid quantitative clean up of toluene extracts. The method described above has been used to estimate the methyl mercury concentration levels in sediment and marine organisms collected from Thane Creek (Tables 1 and 2). The concentration of methyl mercury in sediment was found to be distinctly lower (6-60 rig/g) compared to is total mercury concentration which ranged from 75 to 2140 rig/g. The high values of total mercury in these sediment samples may be attributed to the presence of clay material (48-65%) having a mean grain size of 2-9 pm and the presence of montmorillonite with the minor amount of Kaolinite and ellite types of minerals (Ratha and Sahu, 1994). The concentration of total mercury in marine organisms collected from Thane Creek varied from 62.5 to 548 rig/g with a mean of 189 rig/g (on a dry weight basis). The daily mean intake of total mercury through marine food for the Mumbai population is be about 0.8 pg based on the marine food consumption rate of 14 g/day and a dry to wet weight ratio of 3.7 (Table 3). Krishnamurti (1987) reported a daily intake of total mercury through air, water and food including the marine component as 13.8 pg in which the marine component formed only 0.5 pg. The reported intakes of mercury cover a range of 3-25 pg/day with a median around 15 pg/day (Fergusson, 1990). The FAO/WHO permissible tolerable level has been set at 43 pg/day (0.3 mg/week) of which no more than 66% is methylmercury. The methyl mercury concentration in the marine food ranged between 20.4 and 344 rig/g with a mean of 121 rig/g (on a dry weight basis). The highest concentrations are found in the local delicacy such as crabs and prawns. Crab muscle with hepatopancreas which are a delicacy for fisher folk. The daily intake of methyl mercury component forms nearly 62% of the total mercury (i.e. 0.5 pg/day).
205 (1997) 267-270
4. Conclusions The leaching with 6 N HCI followed with extraction in toluene and back extraction in sodium thiosulphate; measurement with HPLC and UV detection was found to be an effective method for the determination of methyl mercury in complex matrices like sediment and marine organisms and the method can be used without any clean up step. The extraction efficiency of methyl mercury from sediment and biological samples was found to be 56% with the detection limit of 0.5 ng for a 200 ~1 sample volume. The concentrations of methyl mercury and the relative fractions with respect to total mercury were distinctly lower, 5.9-65.5 rig/g (3-8%) in sediment compared to 20.4-344.5 rig/g dry wt. (33-97%) in biological samples. The daily intake of methyl mercury through marine food is about 0.5 pg and forms 62% of the intake of total mercury from this route. References Bloom NS. On the chemical form of mercury in edible fish and marine invertebrate tissue. Can J Aquat Sci 1992;4:1010. Fergusson JE. The heavy elements chemistry: environmental impact and health effects. New York: Pergamon Press, 1990:533-567. Hempel M, Hintelmann H, Wilken RD. Determination of organic mercury species in soil by high performance chromatography with ultraviolet detection. Analyst 1992; 117:669. Hight SC, Corcoran MT. Rapid determination of methyl mercury in fish and shellfish. J Assoc Anal Chem 1987;70:22-28. Krishnamurti CR. Lead, mercury, cadmium and arsenic in environment. In: Hutchinson TC, editor. 1987:315-333. Ratha DS, Sahu BK. Statistical assessment of geochemical variable and size distribution characteristics of sediments from two estuaries in Bombay, India. Int J Environ Stud 1994;46:115-142. Westoo G. Determination of methylmercury salts in various kinds of biological samples. Acta Chem Stand 1968;22:2277.