Research Report

Bioaccumulation of Persistent Environmental Pollutants (PEPs) in the Mud Catfishes (Clarias gariepinus and Heterobranchus longifilis) from the Ogu Creek, Rivers State, Nigeria  

Dike Henry Ogbuagu1 , Marcus Osezojie Idewele2 , Kingsley Prince Nzekwue2
1 Department of Biology, Federal University of Technology, Owerri, Nigeria
2 Department of Environmental Technology, Federal University of Technology, Owerri, Nigeria
Author    Correspondence author
International Journal of Marine Science, 2019, Vol. 9, No. 7   doi: 10.5376/ijms.2019.09.0007
Received: 05 Jul., 2019    Accepted: 13 Oct., 2019    Published: 02 Dec., 2019
© 2019 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Ogbuagu D.H., Idewele M.O., and Nzekwue K.P., 2019, Bioaccumulation of Persistent Environmental Pollutants (PEPs) in the mud catfishes (Clarias gariepinus and Heterobranchus longifilis) from the Ogu Creek, Rivers State, Nigeria, International Journal of Marine Science, 9(7): 54-65 (doi: 10.5376/ijms.2019.09.0007)


The long-term discharge of poorly treated petroleum refinery effluent into the Ekerekana Creek, proximal to the Ogu Creek in Ogu, Rivers State, Nigeria, as well as contributions from artisanal refining have created worries about safety to health of consumers of the catfish delicacies, Clarias gariepinus and Heterobranchus longifilis from this coastal water. This research investigated the presence and levels of trace elements (Zn, Cr, Cd, Pb, Mg, Ca, K, Na, Fe and Mn) and hydrocarbons (Total Petroleum Hydrocarbon-TPHs, Benzene, Toluene, Ethylbenzene and Xylenes-BTEX, and Polycyclic Aromatic Hydrocarbons-PAHs) and Phenol in tissues of the fishes. In C. gariepinus, mean maximum accumulations in mg/kg were as follows: Mg 15.01±2.38 (muscle), Zn 10.25±2.62 (muscle), Fe 9.51±0.53 (gills), K 7.01±0.88 (muscle), Ca 0.42±0.05 (muscle), Pb 0.06±0.01 (muscle), Mn 1.58±0.45 (liver), TPH 6.65±0.42 (muscle), PAHs 0.24±0.05 (muscle), BTEX 0.07±0.01 (muscle) and Phenol 0.007±0.001 (muscle). In H. longifilis, accumulations in mg/kg were as follows: Mg 17.21±1.84 (muscle), Zn 11.27±2.49 (muscle), Fe 11.39±1.64 (gills), K 7.66±1.24 mg/g (muscle), Cd 0.47±0.043 mg/g (muscle), Pb 0.056±0.006 mg/g (muscle) and Mn 1.16±0.268 (liver), TPH 8.69±0.60 (muscle), PAHs 0.49±0.085 (muscle), BTEX 0.09±0.005 (muscle) and Phenol 0.0016±0.0003 (muscle). The accumulations of Cd, Pb, Mg, Ca, K, Na, Fe and Mn in C. gariepinus (Sig. values=0.001, 0.002, 0.011, 0.003, 0.004, 0.008, 0.000, 0.025 respectively) and those of Zn, Cd, Pb, Mg, Ca, K, Na, Fe and Mn (Sig. values=0.024, 0.000, 0.002, 0.002, 0.002, 0.008, 0.024, 0.005, 0.012 respectively) in H. longifilis differed significantly between tissues from impacted and reference locations at p<0.05. TQ/HQ values of, especially Cd and Zn were high (6.80-11.00 and 1.38-3.24 respectively) and exceeded regulatory limits in both fish species.


Persistent environmental pollutants; Bioaccumulation; Ogu Creek; Catfish; Hydrocarbons; Trace elements


Beek (1999) and Neff (2002) explained that bioaccumulation is an increase in the concentration of a chemical in a biological organism over a period of time compared to the chemical's concentration in the environment. Compounds therefore accumulate in living things any time they are taken up and stored faster than they are metabolized or excreted, and occur when certain toxic chemicals and pollutants such as trace elements, pesticides or polychlorinated biphenyls (PCBs) are absorbed by terrestrial and aquatic animals. The major focus of bioaccumulation however is the accumulation of certain non-essential chemicals that are persistent in the environment. These compounds are highly soluble and can be stored in fats, and when the fatty tissues are used up for energy, the compounds are released and could cause acute poisoning which leads to eco-toxicological problems. The longer the biological half-life of the toxic substance the greater the risk of chronic poisoning, even if the environmental levels of the toxin are not very high (Bryan et al., 1979). 


Some of these persistent pollutants such as trace elements and hydrocarbons are introduced contributed in the environment basically by various anthropogenic activities, including oil exploration, bunkering activities, mining activities in the deep sea (which processes involve the extraction of minerals and metal ores like zinc, cobalt, silver, aluminum and gold), as well as other industrial and domestic activities in catchment areas. Agricultural activities also contribute to the pollution of the aquatic environment through runoffs into water bodies. Once in the environment, these toxic substances can endanger public health by being incorporated in the food chain (Di Leo et al; 2010; Ogbuagu et al; 2011). Two factors contribute to the deleterious effects of most toxic substances that constitute environmental pollution. Firstly, they cannot be destroyed through biological degradation as in the case of most organic pollutants (Egborge, 1994), thus, they are Persistent Environmental Pollutants (PEPs), and secondly, they are easily assimilated and can be bioaccumulated in the protoplasm of aquatic organisms. Aquatic organisms are thus, exposed to a myriad of chemicals in their environment. Though some of these chemicals occur in trace concentrations in the environment, yet they may be selectively accumulated by organisms to much larger concentrations that can cause toxicity. The concomitant effect of bioaccumulation can be the buildup (biomagnification) of large concentrations of persistent pollutants in aquatic organisms and ultimately human at the top of the trophic chain (Ogbuagu and Iwuchukwu, 2014). 


It is rather of great concern that over 80% of the industries in Nigeria discharge their solid wastes, liquid and gaseous effluents containing toxic concentration of heavy metals and other pollutants into the environment without any prior treatment, while just only 18% undertake rudimentary recycling prior to disposal (Ayejuyo et al., 2001). 


The Ogu creek located in Ogu-Bolo Local Government Area of Rivers State is a major habitat for aquatic organisms including fish, crabs, periwinkles, crayfish, and prawn that are consumed by inhabitants around and beyond the area. These aquatic foods are mostly consumed as local diet due to their high protein, low saturated fat and omega fatty acid contents that are known to contribute to good health (Kennedy et al., 2009). The Port Harcourt Refining Company Ltd (PHRC), sited on the Okrika Mainland discharges her poorly treated petroleum effluent into the Ekerekana Creek, and the effluent spreads onto the proximal Ogu and other creeks. An increasing population of inhabitants who are attracted by the presence of this hydrocarbon industry, especially of the artisanal refiners has included more domestic and petroleum effluents in the effluent stream discharges. Consequently, more aquatic foods are enmeshed in possible toxic pollutants which could threaten their fecundity and survival in the ecosystem. 


However, whether these trace elements in the aquatic environment are made bioavailable to the tissues and organs of the commonly consumed fish delicacies or not is still unconfirmed, as there exist paucity of researches on bioaccumulation status of the recalcitrant species of pollutants in the creek. This work therefore attempted to close the gap in knowledge, by investigating the presence of some toxic PEPs in tissues of the common mud catfishes, Clarias gariepinus and Heterobranchus longifilis sourced from the creek.


1 Results

1.1 Bioaccumulation of trace elements

At the impacted location and in C. gariepinus, Zn, Mg, and Fe concentrations ranged in the tissues from 6.88(liver)-8.45(gills) (10.25±2.62), 10.42(gills)-18.45(muscle) (15.01±2.38) and 8.5(liver)-10.41(gills) (9.51±0.53) mg/kg, and in H. longifilis they rangedfrom 8.08(liver)-16.20(muscles) (11.28±2.49), 13.55(gills)-19.40(muscle) (17.22±1.84) and 9.14(liver)-14.60(gills) (11.39±1.64) mg/kg respectively. However, at the reference location they range from 2.75(gills)-3.85(muscle) (3.25±0.32), 3.62(muscle)-4.88(liver) (4.08±0.40) and 2.08(muscle)-2.42(gills) (2.21±0.10) mg/kg in C. gariepinus and in H. longifilis, they ranged from 1.95(gills)-2.66(muscle) (2.37±0.21), 2.86(gills)-4.90(liver) (3.55±0.67) and 1.89(muscle)-2.22(liver) (2.03±0.97) mg/kg respectively (Figure 1). At the impacted location, Ca, K and Na ranged in the tissues from 9.41(gills)-11.08(muscle) (10.21±0.48), 5.88(liver)-8.75(muscle) (7.01±0.88) and 2.45(liver)-4.35(gills) (3.36±0.54) mg/kg in C. gariepinus and from 10.5(gills)-12.50(muscle) (11.65±0.80), 5.50(liver)-9.80(muscle) (7.67±1.244) and 2.20(liver)-5.25(gills) (3.68±0.88) mg/kg in H. longifilis respectively. At the reference location, they ranged from 5.84(gills)-6.92(liver) (6.32±0.31), 1.35(liver)-1.95(muscles) (1.57±0.18) and 0.22(liver)-0.84(gills) (0.47±0.18) mg/kg in C. gariepinus and from 4.60(gills)–5.90(liver) (5.43±0.417), 1.50(muscle)-1.75(gills) (1.49±0.153) and 0.21(liver)-0.75(gills) (0.50±0.157) mg/kg respectively in H. longifilis (Figure 2).



Figure 1 Accumulations of Zn, Mg and Fe in gills, muscle and liver of the African catfishes in the Ogu Creek



Figure 2 Accumulations of Ca, K and Na in gills, muscle and liver of the African catfishes in the Ogu Creek


At the impacted location, the concentrations of Cr ranged from 0.28mg/kg in the liver to 8.42mg/kg in the muscle (3.05±2.68 mg/kg) ofC. gariepinus, Mn ranged from 0.92mg/kg in the gills to 2.45mg/kg in the liver (1.58±0.45 mg/kg) (Figure 3), Cd ranged from 0.34mg/kg in the gills to 0.52mg/kg in the muscle (0.42±0.05)mg/kg, while Pb ranged from 0.05mg/kg in the gills to 0.08mg/kg in the muscles (0.06±0.01) mg/kg (Figure 4). At the reference location, they ranged from 0.04mg/kg in the muscle to 0.08mg/kg in the gills (0.06±0.01 mg/kg), 0.005mg/kg in the gills to 0.01mg/kg in the liver (0.01±0.00 mg/kg) (Figure 3), 0.002mg/kg in the liver to 0.006mg/kg in the gills (0.004±0.001mg/kg), and 0.002mg/kg in the liver to 0.004mg/kg in the muscle (0.003±0.001mg/kg) respectively (Figure 4).



Figure 3 Accumulations of Mn, Cr and Total Petroleum Hydrocarbons (TPH) in gills, muscle and liver of the African catfishes in the Ogu Creek



Figure 4 Accumulations of Cd, Pb and the mononuclear aromatic hydrocarbons (BTEX) in gills, muscle and liver of the African catfishes in the Ogu Creek


For H. longifilis at the impacted site, Cr ranged from 0.30 mg/kg in the liver of the fish to 8.80 mg/kg in the muscle (3.20±2.80 mg/kg), while at the reference location it ranged from 0.06 mg/kg in the muscle to 0.09 mg/kg in the liver (0.08±0.01 mg/kg) (Figure 3). Mn ranged from 1.70 mg/kg in the liver to 0.95 mg/kg in the muscle (1.17±0.27 mg/kg), while at the reference location it ranged from 0.003 mg/kg in the liver to 0.006 mg/kg in the gills (0.004±0.002 mg/kg) (Figure 3). However, Cd and Pb ranged from 0.40(gills)-0.55(muscle) (0.476±0.0433) and 0.05(liver)-0.07(muscles) (0.566±0.0066) mg/kg respectively at the impacted location (Figure 4). At the reference location, they range from 0.002(liver)-0.007(gills) (0.005±0.003) and 0.008(muscle)-0.01(gills) (0.0093±0.0006) mg/kg respectively (Figure 4).


The One-Way ANOVA test revealed that the accumulations of Cd, Pb, Mg, Ca, K, Na, Fe and Mn in C. gariepinus (Sig. values=0.001, 0.002, 0.011, 0.003, 0.004, 0.008, 0.000, 0.025 respectively)  and those of Zn, Cd, Pb, Mg, Ca, K, Na, Fe and Mn (Sig. values=0.024, 0.000, 0.002, 0.002, 0.002, 0.008, 0.024, 0.005, 0.012 respectively) in H. longifilis differed significantly in tissues sampled in the impacted and reference locations at p<0.05.


1.2 Bioaccumulation of the hydrocarbons and phenol

At the impacted location and in C. gariepinus, TPH ranged in the tissues from 5.84mg/kg in the gills to 7.25mg/kg in the muscle (6.65±0.42 mg/kg), while at the reference location it ranged from 0.000mg/kg in the liver to 0.004mg/kg in the gills (0.002±0.001 mg/kg) (Figure 3). PAHs and Phenol ranged from 0.18(Liver)-0.35(muscle) (0.24±0.05) and 0.005(liver)-0.008(gills) (0.007±0.001) mg/kg respectively at the impacted location (Figure 5), and at the reference location they range from 0.000(liver)-0.004(muscles) (0.002±0.001) and 0.000(liver)-0.002(gills) (0.007±0.001) mg/kg respectively (Figure 5). The concentration of the mononuclear aromatic hydrocarbons (consisting of Benzene, Toluene, Ethylbenzene and Xylenes- BTEX) was 0.05 (liver)-0.08(gills) (0.07±0.01) mg/kg at the impacted location, and 0.000(muscle/liver)-0.003(gills) (0.001±0.000) mg/kg at the reference location (Figure 5).



Figure 5 Accumulations of the polynuclear (PAHs) and mononuclear aromatic hydrocarbons (BTEX) in gills, muscle and liver of the African catfishes in the Ogu Creek


In H. longifilis, the accumulation of TPH ranged from 7.80mg/kg in the liver to 9.85mg/kg in the gills (8.69±0.60 mg/kg), while at the reference location it ranged from 0.003mg/kg in the liver to 0.01mg/kg in the gills (0.006±0.002 mg/kg) (Figure 3). PAHs and Phenol concentrations ranged from 0.33(Liver)-0.61(muscle) (0.49±0.085) and 0.001(muscle)-0.002(gills) (0.0016±0.0003) mg/kg respectively. At the reference location, they range from 0.001(liver)-0.002(muscles) (0.0016±0.0003) and 0.000(Liver)-0.001(muscles) (0.0003±0.0003) mg/kg respectively (Figure 5). That of the BTEX ranged from 0.08(liver)-0.10(muscle) (0.09±0.005) mg/kg at the impacted location and 0.001(liver)-0.004(gills) (0.002±0.001) mg/kg at the reference location (Figure 5).


The ANOVA test of homogeneity in mean variance revealed that the accumulation of all of the hydrocarbons measured in C. gariepinus(Sig values=0.000, 0.009, 0.005, 0.006 respectively) and in H. longifilis (Sig values=0.000, 0.004, 0.000, 0.04 respectively) differed significantly between the impacted and reference locations at p<0.05. With a marked Pearson correlationship (Sig. r=0.000), accumulations of the PEPs differed significantly between the tissues of C. gariepinus and H. longifilis (Sig. t=0.021) at the 95% confidence interval. 


1.3 Toxicity/Hazard Quotient (TQ/HQ)

Nested in Table 1 are the Maximum Permissible Limits (MPLs) of some PEPs measured in this study. Mean accumulations of the petroleum hydrocarbons (TPH and PAHs), Cd and Zn exceeded the MPLs provided by regulatory agencies in both fish species. Additionally, mean accumulations of Cr in muscle tissues of both fish species also exceeded the MPLs. Highest HQ values of 10.40 (in C. gariepinus) and 11.00 (in H.longifilis) were recorded for Cd in the muscle while least HQs of 0.25 was recorded for Pb in the gills of C. gariepinus and gills and liver tissues of H. longifilis. Generally, HQ values varied in the tissues as follows: Muscle; 0.40(Pb)-10.40(Cd), Gills; 0.25(Pb)-6.80(Cd), Liver; 0.06(Pb)-6.88(Zn) for C. gariepinus, and Muscle; 0.35(Pb)-11.00(Cd), Gills; 0.25(Pb)-8.00(Cd), Liver; 0.25(Pb)-9.60(Cd) for H. longifilis. Outrageous HQ values [5840.00(gills)-7250.00(muscle) in C. gariepinus and 7800.00(liver)-9850.00(gills) in H. longifilis] were recorded for total petroleum hydrocarbons. 



Table 1 Toxicity/Hazard Quotients (TQ/HQ) of some trace elements, hydrocarbons and phenol accumulated in tissues of C. gariepinus and H. longifilis sampled from the Ogu Creek (TQ values were calculated using MPL values nested below)

Note: a=WHO/UNEP/FAO (UNEP, 1986), b=WHO (Clinton et al., 2009), c=EU (2014), d=UNEP/ILO/WHO (IPCS, 1994)


2 Discussion

As evident in this work, water bodies are the ultimate recipient of wastes generated by industries and artisans. The presence of trace metals and hydrocarbons in the tissues of fish sampled therefore indicates allocthonous input of pollutants from industrial and artisanal sources proximal to the creek. Both local (Olowu et al., 2010; Oladele and Jenyo-Oni, 2015) and foreign researchers (Ikem et al., 2003; Alam et al., 2012) have observed that contaminants are introduced into aquatic ecosystems through many routes and sources such as industrial, domestic, municipal run-offs and leachates. Alam et al. (2012) and Akan et al. (2012) had also reported that effluents generated by various processes in factories and discharged untreated into nearby water bodies may affect water quality and may result in dramatic changes in the chemical quality of the water. Since fishes live in close contact with, and are dependent on aquatic environment, effect of the pollutants in water may be acute or chronic on them. Trace metals and hydrocarbons have been noted to cause persistent health implication such as cancer, respiratory disease, body fatigue, headache, and other health conditions when they get incorporated in the food chain and consumed by higher organisms (Copat et al., 2012).


Juxtaposed with a recent work by Anaero-Nweke et al. (2018) in the proximal Ekerekana Creek (among other coastline creeks in the Upper Bonny Estuary), the current work reveals very significant accumulations of Cr, Zn, Pb and Cd in the catfishes. Anaero-Nweke et al. had recorded only 0.007 mg/L of Zn, and less than 0.001mg/L each of Cd, Cr and Pb in water columns of the creek. Even though the strict definition of bioaccumulation is an increase in concentration of a pollutant in the body of an organism over its ambient levels, and so the observed petroleum hydrocarbon levels in the fishes sampled in this study may not be tagged to have accumulated in tissues of the organisms, both the upper concentrations recently recorded by Moslen and Aigberua (2018) in water columns of the nearby Azuabie Creek of same estuary (45.20±13.88mg/L) and mean values recorded in the current study were well above the regulatory limits. 


The significantly higher PEPs concentrations recorded in tissues of fish sampled from the impacted than reference locations clearly indicate that effluents from the nearby PHRC which are discharged directly into the nearby creek, as well as those from artisanal refining activities along the coastlines obviously contain a variety of pollutants ranging from inorganic metal pollutants to hydrocarbons. Although many of these metals have been reported to be essential for the growth of organisms at trace levels, they become essentially toxic when their concentrations exceed certain levels (Wangboje and Ikhuabe, 2015). Of great concern here are the accumulation levels of Cd and Zn, whose Toxicity/Hazard Quotients were above the recommended unity (1.00) by the WHO/UNEP/FAO (UNEP, 1986). Together with that of Cr in the muscle tissues of both fish species, reference to regulations of these metal species therefore indicate toxicity risks to, especially man as a tertiary consumer of the aquatic foods. 


Wangboje and Ikhuabe (2015) also observed that Zn had one of the greatest tendencies to bioaccumulate in fish tissues sampled from the River Niger at Agenebode, Nigeria. Giri and Singh (2013) recorded HQ values above unity for Cr in shrimps from the Subarmarekha River, India. However, Wangboje and Ikhuabe (2015) also recorded HQ values less than unity for Pb, while Bandowe et al. (2014) recorded similar less than unity values for several other metals, including Cd, Cu, Fe, Mn, and Zn. The presence and levels of the PEPs detected in tissues sampled from reference location, farther away from the point of discharge of industrial effluents reflects transport by tides causing mixture of the pollutants further upstream and from the surrounding lands. The coastal aquatic ecosystem studied is tidal in nature and so, pollutants introduced at the mouth of the creeks are carried further upstream- ocean-wards. Additionally, during rainfalls, runoffs which are usually rich in certain trace metals and other pollutants contributes to pollutant loading in the coastal water. The very high accumulations of the hydrocarbons, as well as their outrageous HQ values further affirm that the Niger Delta aquatic ecosystems are largely associated with petroleum hydrocarbon pollution, an observation also made by Clinton et al. (2009); even as the proximal PHRC introduces larger volumes of improperly treated hydrocarbon-laden effluents into the creek. Observations have also been made that mixture of organic materials, trace metals (Olowu et al., 2010) and hydrocarbons (Jackson et al., 1994) result to high toxicities which affect the ecosystem drastically. These high values indicate high toxicity predispositions to consumers of these seafoods. Incidents of endocrine disruption and carcinogenicity from heavy metals and hydrocarbon pollutants have severally been indicated (ATSDR, 1994;US EPA, 1998; Brooks et al., 2004; Giri and Singh, 2014; Martin, 2018).


Accumulations in the liver could be associated with its functions in detoxification. Charles et al. (2007) had reported that in organisms, metabolic conversion of compounds not essential for normal biological functions takes place mainly in the liver. On the other hand, the gills are the first point of contact with water during respiration. Samad et al (2015) had observed that fish and other organisms that respire through the gills can absorb metals through their respiratory surfaces. It is well documented that muscles of fish accumulate trace metals from their aquatic enmeshments (Chouba et al., 2007; Tiimub and Afua, 2013; Wangboje and Ikhuabe, 2015), and similar observations were made here. Accumulations were obviously higher in the larger H. longifilis than in C. gariepinus, a reflection of the relationship between body mass and surface area for accumulation. 



This study revealed bioaccumulation of some persistent environmental pollutant species, including the trace elements- Zn, Cr and Cd, and high tissue concentrations of the hydrocarbons in the mud catfishes, Clarias gariepinus and Heterobranchus longifilis. Accumulations varied according to proximity to pollution source, as well as fish species sampled. Accumulation levels of some of the pollutants in many of the tissues were above regulatory limits in edible fishes.



It could be concluded that the accumulation of Zn, Cr and Cd, together with very elevated levels of petroleum hydrocarbons predisposes consumers of the catfishes- Clarias gariepinus and Heterobranchus longifilis from the Ogu Creek in Rivers State, Nigeria to toxicity hazards. 



For the protection of aquatic lives and public health, effluents discharges from the nearby Port Harcourt Refinery Company should be properly treated before discharge into the nearby creek.


3 Materials and Methods

3.1 Study area

The Ogu Bolo Local Government Area (OLGA) is a predominantly low-lying pluvial location in the eastern part of the Niger Delta on the ocean ward extension of the Benue Trough. The typical Niger Delta environment features many mangrove swamps, and rainfall is generally seasonal, variable, heavy, and occurs between March and November; with a short dry season covering the rest of the year (December-February) (Osuji and Opiah, 2007).  Rainfall amounts of up to 2400-2600 mm are common, average temperatures are typically between 25 and 28°C, and relative humidity rarely dips below 80% and fluctuates between 90% and 100% for most of the year. Oil exploration and production operation have been ongoing for over 30 years in the area, even as the major activities of inhabitants includes fishing, farming, petty trading, artisanal labour, and in few cases civil service.


The Ogu creek (Figure 6) in Ogu Bolo; one of the creeks close to the Ekerekana creek in Okrika Mainland is also a popular area for riverine activities with enormous abundance of marine organisms, making it rich in fishing and so, a major area that support economic activities. It is also a source of transportation, tourism, and industrial activities to neighboring communities. The Ogu creek is impacted by petroleum hydrocarbon pollution spread mainly from the nearby Ekerekana Creek which receives direct effluent discharges from the Port Harcourt Refining Company (PHRC), and from artisanal refining by local operators. 



Figure 6 Ogu Creek is one of the creeks along the Upper reaches of the Bonny Estuary, between the Okrika Mainland and Ogu Bolo community in Rivers State, Nigeria


3.2 Sample collection

Adult fish samples of C. gariepinus (average length 80±2.3cm, weight 650±5.0g, n=20) and H. longifilis (average length=150±5.0cm, weight 1,500±1.0kg, n=10) were collected with nets and hooks from PHRC effluent contaminated/impacted (Imp) (nearest to the mouth of the creek) and a reference (C) (sited about 1km away from the mouth of the creek) locations around the creek (Figure 7). Samples of approximately uniform sizes were collected for each species in order to minimize possible error due to size differences. Samples were labeled, wrapped with aluminium foil and transported to the laboratory on the same day for confirmatory identification in the Department of Fisheries and Aquaculture Technology, Federal University of Technology, Owerri, Nigeria.



Figure 7 Fish samples were collected from locations designated as IMP1-IMP3 (Impacted) and Ref. (1b) (Reference, also designated as C in Figs. 1-5)


3.3 Laboratory analysis

3.3.1 Heavy metals

The method of Wangboje and Ikhuabe (2015) was employed. The fish samples were dissected and their muscle, gills and liver tissues removed. Tissues were oven-dried at 70ºC for 48hours, milled separately with a porcelain mortar and pestle, and kept in foils. Two grams, each of the tissue samples was weighed into 250mL conical flask, into which 5 mL of HClO4 and 15mL HNO3 were added. The mixture was heated until a clear solution was formed. Five mL of 20% HCl was added, the mixture filtered into a 100mL volumetric flask through a No. 42 Whatman filter paper, and the filtrate made up to mark with distilled water. The digest was stored in a 100mL plastic reagent bottle for subsequent atomic absorption spectrophotometric (Unicam® 696 Series with air acetylene flame) analysis. Standard solutions of each sample of the metals were prepared according to the manufacturer procedure.


3.3.2 Hydrocarbons

The method of Abdallah (2017) was employed. The tissue samples were homogenized with NaSO4 for 2-3 minutes for adequate dryness. The mixture was transferred to a pre-cleaned extraction thimble inside which the dehydrated tissue was extracted with 200 mL of n-hexane-dichloromethane in the ratio of 1:1 for 8 hours in a Soxhlet apparatus cycling 5-6 times/hr. Anhydrous NaSO4 was also extracted the same way as the sample and used as blank. The extracted solvents were concentrated with a rotary evaporator down to 2 mL at a maximum temperature of 35ºC, and then further concentrated with a pure nitrogen gas stream down to 2 mL. Cleanup and fractionation were conducted by passing the extract through a silica/alumina column. The first milliliter volume of the extract was passed through slurry packing of 20 mL (10g) silica, 10 mL (10g) of aluminium, and then 1g of anhydrous NaSO4. Elution was made with 40 mL of hexane (for the aliphatic fractions, F1), followed by 40 mL hexane/dichloromethane in the ratio 90:10, and then by 20 mL hexane/dichloromethane in the ratio 50:50 (for unsaturated and aromatic hydrocarbon fractions, F2). Eluted samples were then concentrated under a gentle stream of purified nitrogen to about 0.2 mL, before injection into a Gas Chromatograph interfaced with Flame Ionization Detector (GC-FID). 


3.3.3 Phenol

Preserved sample was acidified to a pH 4.0 with phosphoric acid. The sample was cooled at 5 to 10ºC and collected with hexane. A portion of the sample was diluted with 5 to 10cm of water, cooled, neutralized carefully with 15cm of concentrated ammonia, and made up to the mark with distilled water. On neutralization the colour deepened to a clear yellow, indicating the presence of phenol. Sample was then analyzed 24 hours with the HACH DR 2000 spectrophotometer at 260nm wavelength.


3.3.4 Statistical analysis

Data were analyzed with the SPSS© V.22.0 software. Descriptive statistics was used to compute means and standard errors of the data set, while the student’s t-test was used to compare mean accumulations of the trace elements, hydrocarbons, and phenol in the tissues of the fish samples from the impacted and Reference locations, as well as in tissues of the fish species at p<0.05. The test of homogeneity in mean variance of accumulations in the tissues was conducted with the One-Way ANOVA at the 95% confidence interval. Variation plots were used to represent accumulation of the PEPs in the tissues.

Toxicity/Hazard Quotient (TQ/HQ) was computed as follows: 

TQ/HQ = Measured concentration of chemical element of ecological matrix

                        Health based criteria                           (Newstead et al., 2002).


Authors’ contributions

Author DHO designed the study, wrote the protocol, performed the statistical analysis, and wrote the draft manuscript of the studyt. Authors DHO, MOI and KPN carried out the field sampling. Authors MOI and KPN managed the literature searches. All authors read and approved the final manuscript.



We acknowledge the assistance of Transcontinental Petrotech (Nigeria) Limited in the laboratory analysis of samples used in this study. We are also grateful to Dr. E. T. Adebayo of the Department of Fisheries and Aquaculture Technology, Federal University of Technology, Owerri for the identification of the fish species.



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International Journal of Marine Science
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