Wong Wai Khet

SKA6014
ASSIGNMENT 1

PROCEDURE TO DETERMINE
CONCENTRATION OF MERCURY IN FISH

In the process of determine the concentration of mercury in fish, all the apparatus and reagent used
must be prepared according to standard procedure.
Apparatus and reagent

Glassware and plastic containers

Elga, Ultra Pure Water System, Maxima.

Important:
Glassware and plastic containers were soaked in 2 % nitric acid and left overnight before they were
rinsed thoroughly with ultrapure water(obtain from Elga). All reagents used were of analytical grade.

During the sampling process, the sample should handle carefully and follow the correct procedure to
minimize the damage to the sample.
Sampling and sample preparation
Minimum number of sample: 383 (For analysis purposes).
Measurement of the fish samples:
Total length - measured to the nearest millimeter.
Total weight - measured to the nearest gram.
Fish samples are packed in polyethylene bags, labeled, and put into an icebox before they are
transported to the laboratory.
Samples are kept frozen at 21 C in the laboratory.
Procedure for sample preparation:
1. Fish samples are thawed at room temperature.
2. The edible portion of fish is filleted, cut into small pieces, and homogenized.
3. Dry the homogenized muscles in the laboratory oven at 65 C to constant dry weight and ground
using mortar.
Important:
The food items and feeding habits of the fish sample should referenced from the Global Information
System on Fishes. http://www.fishbase.us

Digestion procedures

Samples for mercury analysis

(including blanks) are digested in
a microwave digestion system
(Multiwave 3000Anton Paar)

Seal the vessels and placed

into the rotor for
microwave digestion.

Use the power profile for

the digestion of samples
with the Multiwave

Weigh accurately 0.5g dried

fish samples and put into the
digestion vessels.

Add a total of 5.0-ml

concentrated nitric acid
and 2.0 ml of hydrogen
peroxide to each vessel.

Phase 1
Power of the digestion
system : 600W
5-min ramping
5-min holding time.

Phase 3
Power of the digestion
system : 0W
holding time of 15 min.

Phase 2
Power of the digestion
system : 1,400W 5-min
ramping
10-min holding time.

Filter samples from the

clear solutions through a
0.45-m acid resistant
membrane.

Transfer the solution into

a 25-ml volumetric flask
and diluted with ultrapure
water.

Note: Prepare the analytical reagent blanks in the same manner but without the dried fish samples.

Mercury analysis
Technique:
Use cold vapor atomic absorption spectrometry (AAS) technique with PerkinElmer Flow Injection
Mercury System (FIMS) instrument equipped with FIMS-400 and a programmable sample dispenser
following the method of Mohd Fairulnizal et al. (1998).
Procedures
Obtain stock standard solution of
mercury, 1,000 g/ml from
PerkinElmer.

Dilute stock standard solution to

prepare a sub-stock solution of
10 mg/l

Calibration:
Correlation coefficient, r = 0.9995.

Dilute sub-stock standard

solution to prepare the working
standard solutions of 0, 2, 5, and
10 g/l

Injection of samples, collect result.

(The results are expressed in dry
weight basis. )

Convert mercury concentrations in fish samples to a wet basis values using the formula:
Dry weight concentration = wet weight concentration(100/100 moisture percentage).

Calculate the amount of moisture content based on

the works of Tee et al.(1997)and Nurnadia et al.(2011).

Compare the results with the national and international guidelines

for the purpose of public health perspective.
Grouped the result into five categories:
0.05 to 0.15 g/g as very low,
0.150.25 g/g as low,
0.250.35 g/g as medium,
0.350.45 g/g as high
above 0.45 g/g as very high.

Important
Calibration method:
A linear range calibration method. Control correlation coefficient at r = 0.9995.
Detection limit:
Based on the mercury concentration corresponding to three times the standard deviation of ten reagent
blanks.
Validated analysis:
For every ten injection of samples, two different concentrations of mercury standard solutions of utilized
is injected as quality control.
The acceptable range is set between 85 and 110 %.
Analytical control is accompanied by analysis of reagent blanks and standard reference samples.
The result is grouped following Chvojka et al. (1990) as cited by Al-Majed and Preston (2000).
The recommended guideline levels by the joint FAO/WHO Expert Committee on Food Additives
(FAO/WHO 2006) was set at 0.5 mg/kg methylmercury in fish.
The maximum permitted proportion of methylmercury set in Malaysia, under the Fourteenth
Schedule of Regulation 38, Malaysian Food Regulation 1985 (Food Act 1983, (Act 281) and
Regulations 2006) is same as the guideline levels by the joint FAO/WHO Expert Committee on Food
Additives
Statistical analysis
1. Clean and check data for discrepancies before analysis.
2. Calculate the medians, interquartile range, and percentile range using SPSS (version 11.5 for
Windows, 2002, SPSS Inc.).
3. Assess the statistical significance of difference using:
a) Mann - Whitneys (MW) - Test for two groups.
b) Kruskall - Walliss (KW) - Test for three groups or more.
4. Use Spearman correlation analysis to study the correlation coefficients.
5. Design the level for significance, p.
6. Obtain the final result and make conclusion.
Important Note:
There are several method can be used to determine the concentration of mercury in fish. The technique
stated above, cold vapor atomic absorption spectrometry (AAS) is one of the most frequently used
method in determine the concentration of mercury in biological sample.
For determine the mercury in fish for a large area such as Peninsular Malaysia, fishes should be
collected from several places within the area.
The Sub-stock standard solution must prepared fresh daily
The mercury concentrations in fish samples must be convert to a wet basis values in order to compare
the results with the national and international guidelines