International Journal of Marine Science, 2017, Vol.7, No.26, 260-271
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1.3 Sample analysis
The total mercury in the sediment was detected by Cold Vapour Atomic Fluorescence Spectrophotometer (CVAFS,
Brooks Rand, USA) following bromine monochloride (BrCl) oxidation (USEPA- method 1631, revision E, 2002).
The sediment samples were oven dried at 35
o
C and are ground to fine powder using an agate mortar. Then it is
sieved for <63 µm granulometric fraction with the help of ASTM standard sieves and digested with Suprapur
Nitric acid before BrCl oxidation. Utmost care has been taken during the sieving. Among the 33 sediment samples,
14 samples were undergone fractionation studies followed by Bloom’s five step sequential extraction scheme
(Bloom et al., 2003). The various fractions were (a) water soluble (F1), (b) ‘human stomach acid’ soluble (F2), (c)
organo-chelated (F3), (d) elemental Hg (F4), and (e) mercuric sulphide (F5) (Bloom et al., 2003; Boszke et al.,
2003). The sequential extraction scheme was already explained in our earlier paper (Ramasamy et al., 2012)
(Table 1). The mercury fractions F1 and F2 are weakly bound to sediments compared to other fractions. F3
fraction is the mercury bound to organic matter especially humic acids, fluvic acids mainly through sulphur
bonding. Elemental form of mercury (F4) is mainly coming from the anthropogenic activities like gold mining. F5
fraction is the residual mercury and is not easily available for chemical or biological transformations. Fraction-1
was obtained by shaking with ultrapure water. The residue after centrifugation was subjected to shaking with 0.1
M CH
3
COOH + 0.01M HCl for the second fraction. Centrifuged and the residue was extracted for third fraction
with 1 M KOH. The elemental mercury fraction (F4) was extracted with 12 M HCl. The final residue was treated
with Conc. HCl and HNO
3
for the last fraction. All the extractants were made up with 0.2 M bromine
monochloride.
Table 1 Bloom’s (2003) five step sequential extraction procedure (Ramasamy et al., 2012)
Fraction of Hg Chemical Extraction method
Step 1
Water –soluble 1g of sediment was mixed with 25 mL of ultra pure water in a centrifuging tube and shaken for 18±2
hour in an end-over-end shaker at 30 rpm. Separate the extract from the solid residue by
centrifugation at 3,000 rpm for 20 min. Centrifuged extracts were then filtered and 1 mL of 0.2 M
BrCl was added. As a rinse step, the extraction vials containing the sediment residue were refilled
with 20 mL of the same extractant, shaken vigorously to resuspend the sediment, re-centrifuged and
filtered. The rinse was then added to the extract from the same sample and the combined sample
diluted to 100 mL with ultrapure water.
Step 2
Human stomach
acid soluble
To Step 1-residue add 25 mL 0.1 M CH
3
COOH + 0.01M HCl (pH= 2). Shake for 18±2h. Centrifuge
extract as per Step 1. Again rinse with 20 mL of the same extractant. The detailed operation
conditions were the same as those of step 1.
Step 3
Organo-chelated The residue from the Step 2 was added with 25 mL of 1M KOH and centrifuged. Because the
solution (extract) has high acid neutralizing capacity, 10 mL of 0.2 M BrCl was added. The detailed
operation conditions were the same as those of step 1.
Step 4
Elemental
mercury
The Step 3 residue was extracted with 25 mL of 12 M HNO
3
solution and then rinsed with another
20 mL HNO
3
solution. The detailed operation conditions were the same as those of step 1. But no
filtration step was employed since the solution can destroy the filter paper.
Step 5
Mercuric sulfide 10 mL of conc. HCl was added to the sediment residue remaining in the vial. After swirling the
sample to dislodge the sediment, 3 mL of conc. HNO
3
was added. Then the vials were loosely
capped and kept in room temperature for 12 hours. Separate the extract from the solid residue by
centrifugation at 3,000 rpm for 20 min and the final volume was made up to 50 ml using ultrapure
water.
1.4 Quality assurance
High quality acids and ultrapure water was used for the analysis. Blank was determined for every run. Precision
was checked in between the runs with different concentration of mercury. The certified reference material
‘Estuarine sediment’ (ERMCC 580) was used for validating the total mercury determination. The percentage of
recovery was 97.07 ±0.21%. The method detection limit was 5.6 ng/L.
