Polycyclic Aromatic Hydrocarbons in Sediment and Tissues of the Crab Callinectes pallidus from the Azuabie Creek of the Upper Bonny Estuary in the Niger Delta

The accumulation of Polycyclic Aromatic Hydrocarbons (PAH) in sediment and the flesh/carapace of the edible swimming crab Callinectes pallidus from Azuabie creek in the upper Bonny Estuary, Niger Delta and Nigeria were studied. Sediment and crab samples were collected from three sampling stations along the Azuabie creek in July (rainy season) and November (dry season) 2007. These stations were selected to reflect point of abattoir waste input (which includes ash from tyre used for roasting meat) into the creek as well as locations upstream and downstream of the point. Seasonal and spatial variations were observed in the concentrations of PAH. Significant differences in PAH (p<0.01) were observed between rainy season and dry season samples. The major classes of PAH found in sediment and C. pallidus were Naphthalene, Benzo (a) pyrene, Benzo (a) anthracene and Phenanthrene which are known to be carcinogenic. The PAHs were higher during the rainy season probably due to run-off of tyre ash from the nearby abattoir. The observations made in this study suggest that C. pallidus from the Azuabie creek might not be safe for human consumption because of the levels of PAHs found in the flesh of samples mostly in the rainy season. The abattoir appears to impact the creek because significantly higher levels of PAHs were recorded at the proximate sampling station.


INTRODUCTION
Polycyclic Aromatic Hydrocarbons (PAHs) are compounds that consist of fused aromatic rings and do not contain heteroatoms or carry substituent's (Fetzer, 2000).PAHs occur in oil, coal and tar deposits and are produced as a by-product of fuel burning.They are of concern as a pollutant because some of them are carcinogenic, mutagenic and teratogenic (causing malformation of an embryo).They are also found in foods like cereals, oils and fats with smaller amounts in vegetables and cooked meats (Larsson, 1983).According to Luch (2005) the PAHs known for their carcinogenic, mutagenic and teratogenic properties are benz (a) anthracene and chrysene, benzo (b) fluoranthene, benzo (j) fluoranthene, benzo (k) fluoranthene, benzo (a) pyrene, benzo (ghi) perylene, coronene, dibenz (a, h) anthracene (C 2 OH 12 ), indenol (1, 2, 3-Cd) pyrene (C 22 H 12 ) and ovalene.Their toxicity is structurally dependent with isomers varying from being non-toxic to extremely toxic.
Burning tyres can have serious environmental impact as they produce vast amount of harmful emissions that will pollute the atmosphere and water courses through run-offs.Burning tyres can have a serious environmental impact and tyre fires are quite common in Nigeria.The use of tyres for roasting meat in abattoirs is also a common practice.This burning results in palls of black smoke that is visible from quite a distance.They produce vast quantities of harmful emissions that will pollute the atmosphere and water courses by input of potentially harmful compounds, including PAHs through run-offs.
Uptake of hydrocarbons, particularly PAHs compound by aquatic biota is very rapid and may accumulate high tissue concentrations (Eisler, 1987;Varanasi et al., 1989).The PAHs are relatively stable constituent of petroleum and many of these compounds are potentially toxic, carcinogenic and mutagenic (Ghauch et al., 2000;Aderemi et al., 2003).Various species of crabs have been used to monitor PAH exposure around the world: Cancer magister in British Columbia, Canada (Eickhoff et al., 2003), Callinectes sapidus from Chesapeake Bay, (Mothershead et al., 1991;Hale, 1988), Cancer iroratus from New York Bight (Humason and Gadbois, 1982;Pancirov and Brown, 1977); Polybius henslowi on the north coast of France and Spain (Baumard et al., 1998); Scylla serrate from Brisbane River estuary in Australia (Kayal and Connell, 1995); Callinectes sapidus from Ondo Coastal area in Nigeria (Oladele et al., 2008).In this study, we report the concentrations of PAHs in sediment and tissues of the edible crab Callinectes pallidus Azuabie creek which receives run-off of effluents from tyre burning activities.Seasonal and spatial patterns of PAH levels in relation to the sources of contamination are discussed.

MATERIALS AND METHODS
The Azuabie creek is located between 48' E and latitude 7º 1' 30" 52" in the upper Bonny Estuary, the Niger delta showing location of the creek Southern Nigeria (Daka et al., 2007).The hydrodynamics of the estuarine creek and the presence of the point source of effluent run-off from the coastal abattoir were key considerations for the location of study stations along the creek.Three stations were established (Fig. 1) as follows: • Sediment samples were collected from each station using an Eckman grab.The swimming crab (Callinectes pallidus) was collected using a baited crab trap.The trap was set for a while at midstream and trapped crabs were pulled up and placed in labelled containers.Sediment and crab samples were preserved in ice in the field and then taken to the laboratory where sediment was air-dried while the crabs were stored at -19ºC until analysis.Sediment samples were air-dried, disaggregated and sieved.The crabs were dissected to separate the flesh (edible portion) from the carapace before the tissues were dried to constant weight in an oven and ground.Ten gram of dry sediment or ground tissue samples were mixed with 60 mL of xylene in a Sohxlet apparatus.Four mL of cyclohexane was further added to the mixture for extraction.The extracts were treated with Silica gel and Centrifuged before determination of PAH with Unicam PROGC gas chromatography.PAH was measured in ng/µL and converted to µg/g.Statistical analyses: Two-way Analysis of Variance (ANOVA) was used to test for significant differences between environmental matrices (sediment, tissues of Callinectes-flesh/carapace).One-way ANOVA (and Tukey tests for mean separation where ANOVA gave significant differences) were performed to determine spatial differences for each environmental matrix as two-way ANOVA showed no significant interaction between season and location for sediment/tissue.Data were log (x+1) transformed before input into the ANOAVA model.Statistical analyses were performed using MINITAB software.

RESULTS AND DISCUSSION
The concentrations of PAH were higher in sediment than crab tissues, with values in the flesh being higher than the levels in carapace (Fig. 2).The overall mean concentrations were: sediment (0.973±0.631 µg/g wet season, 0.020±0.014µg/g dry season), flesh (0.627±0.311 µg/g wet season, 0.019±0.008µg/g dry season), carapace (0.155±0.082 µg/g wet season, 0.007±0.003µg/g dry season).There was significant difference in the PAH values between the seasons (p≤0.01),but no significant difference was observable between sediment and the crab tissues, neither was there any significant interaction between environmental matrix and season (Table 1).
ANOVA showed significant differences between stations for both carapace (p<0.01,Table 3) and flesh (p<0.001,Table 4).Tukey test multiple comparisons for mean PAH in carapace showed significance where Stn A = Stn B>Stn C, while the pattern for flesh was Stn A>Stn B>Stn C. The PAH classes found in the samples include Phenanthrene, Benzo (a) anthracene, Benzo (a) pyrene, Naphthalene, Fluoranthrene, Fluorene, Anthracene (Fig. 3 to 5).All classes were found in sediment and crab tissues but anthracene was detected only the in sediment during the dry season.In sediment Polycyclic aromatic hydrocarbon concentrations were significantly higher during the rainy season in all compartments (sediment; flesh and carapace of Callinectes sapidus).The higher wet season values show that run-off from Fig. 3: Spatial and seasonal differences in the classes of PAH in sediment the ash of tyres used for smoking animals slaughtered at the abattoir was the main source of PAH.More so, PAHs in this study were highest at station A which receives the run-off from tyre ash.The consistency in the reduction for all matrices suggests a flux within the estuary tending towards attenuation during the dry season.Sediments were expected to act as sink that could have led to accumulation over time; however, frequent dredging in the area may have precluded the build-up in sediment.PAHs are formed by incomplete combustion of carbon containing fuels likes diesel, fat, tobacco and tyres.The predominant PAHs found in this study were Benzo (a) pyrene, Benzo (a) anthracene, naphthalene and Phenanthrene.They are known for their carcinogenic, mutagenic and teratogenic properties.Variations in concentration of PAHs can be attributed to variations in the content of soot particles of tyre burning which enter the Azuabie creek during periods of elevated surface run-offs (rainy season).The heterogeneity in the BSAF of PAHs has been attributed to variations in the content of soot particles which enter the aquatic system during periods of elevated surface run-offs and which tend to accumulate preferentially with other fine materials in the high inter tidal zone (Capuzzo, 1987).However, all samples in this study were however collected subtidal.PAHs have been reported to bioaccumulate differentially in benthic invertebrates (Maruya et al., 1997), varying over almost three orders of magnitude.Maruya et al. (1997) also reported that Biota-Sediment Accumulation Factor (BSAF) of PAHs varied with season and along an inter tidal gradient in a costal marsh in San Francisco Bay.
PAHs can enter the human body by many different ways.People near hazardous sites can breathe air containing PAHs.Drinking water and swallowing food is another route.Though absorption is slow; they can enter the body if the skin comes in contact with soil that contains a high level of PAH (ATSDR, 2008).The rate of entry of PAH into the body also depends on the presence of other compounds that one is exposed to at the same time.PAHs enter all the tissues of the body that contain fat and can be changed into various substances which could be harmful.Mice fed high levels of benzo (a) pyrene during pregnancy had difficulty reproducing and so did their offspring In conclusion, the highest values of PAH were found in sediments and the tissues of Calllinectes pallidus from stn A which receives run-off of tyre burning effluent.Significantly higher values in the rainy season also confirm the run-off as the major source of PAH in the estuarine creek.The major classes of PAHs found in the sediments and crab were naphthalene, benzo (a) pyrene, benzo (a) anthracene and phenanthrene, which are potential carcinogens, thus crabs and indeed other fish products from the estuary may constitute a potential health hazard for consumers.
Station A: Abattoir-Opposite abattoir representing point source of surface run-off from the tyre burning activity from the roasting of animal skins • Station B: Downstream-Ca. 1 km downstream of the abattoir • Station C: Upstream-Ca. 1 km upstream of the abattoir Sampling and analyses: Samples were collected from each station during the wet season-July 2007; and dry season-November 2007.

Fig. 4 :
Fig. 4: Spatial and seasonal differences in the classes of PAH in carapace Callinectes pallidus

Table 1 :
Two-way analysis of variance to test environmental matrix and season

Table 2 :
Summary of one-way analysis of variance and pair-wise comparisons with Tukey tests for PAH in sediment A: summary of ANOVA

Table 3 :
Summary of one-way analysis of variance and pair-wise comparisons with Tukey tests for PAH in carapace of Callinectes pallidus A: summary of ANOVA

Table 4 :
Summary of one-way analysis of variance and pair-wise comparisons with Tukey tests for PAH in flesh of Callinectes pallidus A: summary of ANOVA