Material and Methods

2.1 Materials

2.1.1 Apparatus and Equipment

The following apparatus were used in this study which includes

Beakers

Test-tube

Soxhlet extractor

Oven

Grinder

Polythene

Charcoal

Firewood

Drums

GC-MS spectrophotometer

Pyrex

Pyrex

Pyrex

Samsumg

Locally Produced

Locally Produced

Locally Available

Locally Available

Locally Produced

GC-MS-QP2010 plus, Shimadzu Japan

2.1.2. Chemicals

All the chemicals used in this study were of analytical grade and in their pure forms

n-Hexane

Dicholoromethane

Sodium Sulphate

Magnesium Silicate

Methanol

Sigma-aldrich

BDH, England

Aldrich chemie Germany

Aldrich chemie germany

99.5+% BDH, England

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Acetone

99.5+% BDH, England

PAHs solution catalog number

z-013-17, LOT 213061049 AccustandardInc,USA

200µg/ml Analyte

Acenaphthene

Acenaphthylene

Anthracene

1,2-Benzanthracene

Benzo (a) pyrene

Benzo (b) floranthene

Benzo (g,h,i)perylene

Benzo(k)fluoranthene

Chrsene

Dibenz (a,h) anthracene

Fluoranthene,

Fluorine

Indeno (1,2,3-cd)pyrene

Naphthalene

Phananthrene and

Pyrene

2.1.3 Fish Samples

The fishes used in this study were obtained from the Otuocha River in Anambra state within the periods of October 2013, November 2013 and January 2014.

2.1.4 Study Site

Eastern Nigeria stretches from the Atlantic Ocean covering wide expanse of the forest region up the lower boundary of the savannah forest belt. Otuocha in Anambra State is located between

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longitude 6.85000 and latitude 6.33330. The region land mass falls within several communities such as Ogurugu, Onitsha and Nsugbe all in Anambra State.

Fig.4: Map showing Otuocha River in Anambra State

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2.2 Methods

2.2.1. Collection of fish Samples and Drying

The fish samples used in this study were collected during the dry season months of October to January.

The fish samples, which was a collection of Tilapia spp., Arius heude loti and others are predominant during the months of October to January when the dry season is in session, were collected from Otuocha river, where relatively no explorative activity has taken place recently. The choice of Otuocha River was to avoid all chances of pollution originating from explorative activity (petroleum). There was no differentiation of the fish samples into different species since the work centred on determing the level of PAHs deposited on the fish samples from the different drying regimes. Nonetheless, the river water was collected for analysis to determine the amount of PAHs in the river and possibly from other sources which could affect the results obtained.

The fishes were divided into groups and the wet weight of each group was noted. These groups are:

Group A: homonized fresh fish

Group B: Sundried fishes

Group C: Oven dried fishes

Group D: Charcoal smoked fishes

Group E: Firewood smoked fishes

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Group F: Fishes smoked with charcoal augmented polythene material (20g)

Group G: Fishes smoked with firewood augmented of polythene material (20g)

The smoking of the fishes were for 3 days at 2 hours each day at high temperature of above

2500c.

The smoking process involved producing smoke from smouldering wood (hardwood) or charcoal placed directly below the hanging fishes laid out on mesh trays. A piece of cardboard is placed over the fishes as done locally to cover the fishes during the process. The piece of cardboard traps the smoke to enable it act directly on the fish samples.

The group dried under the sun was done in Uwelu Ibeku Opi in Nsulla local Government Area for 3 days.

The smoked and dried fishes were homogenized immediately using a very clean and dry grinder

and stored in a refrigerator at 40C prior to extraction and analysis. The extraction and analysis followed immediately to avoid ageing.

2.2.2. Sample Preparation for the Analysis of Dried Fishes:

Soxhlet Extraction:

The sample of fishes were homogenized, minced into smaller fillets and blended using a grinder. Twenty grams (20g) of the homogenized fish sample was thoroughly mixed with 60g of anhydrous sodium sulphate in an agate mortar (Wang et al., 1999) to absorb moisture. The homogenate was placed into an extraction cellulose thimble covered with a Whatman filter paper (125mm diameter) and inserted into a soxhlet extraction chamber of the soxhlet extraction unit. Extractions were then carried out with 200ml of n-hexane using EPA 3540C method (US EPA,

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1994) for 8 hours. The crude extract obtained was carefully evaporated using Ribby RE 200B

rotary vacuum evaporator at 400C, just to dryness. The residue was redisolved in 5ml of n-hexane and transferred onto a 10ml florisil column for clean up.

2.2.3. Preparation of Florisil for Clean-up:

This clean-up step to remove more polar substances was performed using activated florisil

(Magnessium silicate) and anhydrous Na2SO4. The florisil was heated in an oven at 1300c

overnight and transferred to a 250ml size beaker and placed in a desicator.

Anhydrous Na2SO4 (1.0g) was added to 2.0g of activated florisil (60-100mm mesh) on a 10ml

column which was plugged with glass wool. The packed column was filled with 5ml n-haxane for conditioning.

The stopcock on the set-up was opened to allow the n-hexane run out until n-hexane just reached the top of the sodium sulphate into a receiving vessel whilst taping gently the top of the column till the florisil settled well in the column. The extract was then transferred onto the column with a disposable Pasteur pipette from an evaporating flask. The crude extract was eluted on the column with the wide opening of the stopcock. Each evaporating flask was immediately rinsed twice with 1ml n-hexane and added to the column by the use of the Pasteur pipette. The eluate was collected into an evaporating flask and rotary evaporated to dryness. The dry eluate was then dissolved in 1ml n-hexane for Gas chromatographic analysis.

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2.2.4. Instrumental Analysis

Gases used are Helium and Hydrogen gases. Hydrogen and Helium with a purity of 99.999% were used as carrier gas at a constant flow of 30 and 300ml/min respectively. The determination of PAHs was performed on the samples and standards using a Buck 901 GC-FID equipped with a split /split-less injection port. 1.0g of extracted samples were dissolved in10ml n-hexane. Some quantity were taken into 2ml chromatographic vial and made up to 2ml with toluene, injected and separated on a Restek chrompack capillary column CP5860 with 95% methyl and 5%

phenylpolysiloxane phase, (oven max. temperature 3500C).

WCOT fused silica, 30m X 0.25mm id and 0.25µm film thickness with CP-sil 8 CB low bleeds/MS coating. Carrier gas was helium 26cm sec. Temperature profile during the

chromatographic analysis was 500C for 3minutes, 80C/min to 3200C hold for 15 minutes and

detector at 3200C. Fixed setting: Generally the operator must adjust gas flows to the column, the inlets, the detectors, and the split ratio. In addition, the injection and detector temperature must be set. The detectors are generally held at the high end of the oven temperature range to minimize the risk of analyte precipitation (Annual Book of ASTM standards, 2005). All of these parameters should have been set to the correct values. A double check was done on all the instrument: Agilent 6890 Gas chromatograph equipped with an on-column, automatic injector, flame ionization detector, HP 88 capillary column (100m X 0.25 µm film thickness) CA, USA.

Detector Temp: 2500c

Injector Temp: 220c

Integrator Chart Speed: 2cm/min

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Temperature Condition.

Table 4: Temperature condition of GC-MS

Initial Temp

Hold

Ramp

Final Temp

700c

5min

10min

2200c

2200c

2min

5min

2800c

When the instrument is ready, the “NOT READY” light turns off, and the run begins. Then 1µL sample was injected into column A using proper injector technique (US. EPA, 2003).