Methodology

       For all objectives

       Activity 1: Selection of sampling stations

The Pattani Bay, Thailand

       The sediment sampling stationswill be selected about 10stations (Figure 1) inside the PattaniBay 8 stations and outside the PattaniBay 2 stations.

       The shellfish (blood cockle) will be collected about 4 stations (around stations 1-4)

       The Setiu Wetland, Malaysia

       The sediment sampling stations will be selected from about 6 stations (Figure 2),with 5 stations inside the Setiu Wetland (lagoon area) and 1 station outside of the lagoon area.

       The shellfish (blood cockle) will be collected from 4 stations (around stations1,2,3 and 4)

Activity 2:Selection of sampling period

       The sediment samplings will be carried out 2 times:

       1. Post northeast monsoon period (around February to March 2014)

       2. Pre northeast monsoon period (around September to October 2014)

       Activity 3: Sediment and shellfish sampling

       The surface sediments will be collected using a grab sampler. About 1 kg. of sediment in each station will be packed tightly in the plastic bag then put on ice in cooler boxes and transported to the laboratory. The samples are stored frozen until used for analysis.

       The shellfish (blood cockle) will be sampling by hand about 10-20 individuals per station.

Activity 4: Trace metals analysis(Laboratory work at INOS/UMT, Malaysia)

1.1 Determination of Trace Metals (As, Cd, Cu, Pb, Zn) in sediment

The sediment sample will be digested and analyses for trace metals following the published methodologies with some modifications (Noriki, et al., 1980; Loring and Lantala, 1992) The digestion method are the combination of HNO₃ and HCL (1:3)  (aquaregia) and HF following standard procedure (Loring and Lantala, 1992). The trace metals will be determined by ICP-MS.

              1.2 Determination of Trace Metals (As, Cd, Cu, Pb, Zn)in Blood Cockle

Approximately 1-2 g of tissue will be digested with 10 ml mixture of HNO₃ and H₂O₂ (1:1 ratio) following standard procedure (Robisch and Clark 1993). The trace metals will be determined by ICP-MS.

1.3  Quality control

The sediment certified reference material (MESS-2) and the biological certified reference material (DORM-2) are used to check the accuracy and the analytical values are within the range of certified values, which demonstrate the validity of the methods applied.

       Activity 5: Characterization of these sediments (Laboratory work at PSU, Thailand)

1.1        Determination of Readily Oxidizable Organic Matter

The readily oxidizable organic matter contents of the sediment samples is determined by the Walkey-Black method (1947) as described in  Loring andRantala (1992) and Schumacher (2002)

                  i) Place 0.5 g of dried and sieved sediment sample in a 500 mL Erlenmeyer flask

ii) Add exactly 10 mL of 1 N K₂Cr₂O₇ solution by burette and 20 mL of concentrated H₂SO₄ with Ag₂SO₄ and mix by gently rotating the flask for about 1 min

                  iii)Allow the mixture to stand for 30 min, add 200 mL distilled water,

                  iv) After 30 min, add 200 mL distilled water; 10 mL of 85% H₃PO₄ and 0.2 g

Remove the tube from the heat source and cool it to room temperature

v) Add 15 drops (0.5 mL) of the diphenylamine indicator to the sample flask

vi) Back titrate the solution with the 0.5 N ferrous ammonium sulfate solution to a one drop end point (brilliant green)

1.2        Determination of sediment particle size

The particle size analysis is determined by using the hydrometer method (Gee and Bauder, 1986).

i)    Place 40 g of air dried and sieved (2 mm ferrous sieve) sediment sample in a 600  mL beaker

ii)       Add 100 to 150 mL of distilled water

iii)     Add H₂O₂  30% continuously until bubbles in sample disappeared

iv)     Heat sample at 90 °C on a hot plate for 1 hour before incubating in an oven at 105 ºC for 24 hours

v)       After cooling in a desiccator, weigh sample again

vi)     Add 100 mL of Calgon 50% and 50 mL of distilled water.

vii)   Sieved on  63 mm sieve

viii)  Transfer the portion that passed through the sieve to a 1 liter cylinder

ix) Make the solution up to 1 L with distilled water

x)  Disperse the sample with plunger after 50 second measure density and temperature of the sample by hydrometer and thermometer

xi)      Leave the sample for 2 hours without disturbance then measure density and temperature again