Polycyclic Aromatic Hydrocarbons in Ologe Lagoon and Effects of Benzo[b]fluoranthene in African Catfish

effluents from neighboring industries. These effluents may increase the levels of anthropogenic contaminants in the lagoon, thereby creating stressors for aquatic organisms. Objectives. To assess the occurrence of polycyclic aromatic hydrocarbons (PAHs) in Ologe Lagoon, along with the histopathological, biochemical and genotoxic effects of the most prevalent PAH compound. Methods. An initial field study was performed to determine the concentrations of PAHs in Ologe Lagoon, followed by a chronic toxicity test to assess the effects of the most prevalent PAH compound in a fish model ( Clarias gariepinus ). Results. High molecular weight PAHs were more predominant than low molecular weight PAHs in the lagoon, with B[b]F being the most predominant. The formation of micronuclei and binuclei was induced by a 10-fold increase over the present environmental concentration of B[b]F in Ologe Lagoon. Histopathological studies showed that epithelial necrosis, fused lamellae, shortened lamellae, and desquamation were the major histological anomalies induced by ERCs of B[b]F. Results from the biochemical assay indicated that ERCs of B[b] F increased aspartate aminotransferase and alanine transaminase levels in fish. Glutathione-S-transferase, superoxide dismutase and catalase were inhibited in the exposed fish, whereas malondialdehyde was significantly increased. Conclusions. Concentrations of fluoranthene, pyrene, benzo[a]anthracene, and benzo[a] pyrene in the surface water of Ologe Lagoon were above the Canadian Council of Ministers of the Environment’s (CCME) safe limits, suggesting that the water may not be safe for domestic uses, and the present concentration of B[b]F in Ologe Lagoon may be chronically toxic to aquatic organisms, in terms of oxidative stress and hepatotoxicity. Competing Interests. The authors declare no competing financial interests.


Introduction
Polycyclic aromatic hydrocarbons (PAHs) are a group of organic pollutants that contain ≥2 fused aromatic rings composed of hydrogen and carbon. 1 They are solids that have high melting and boiling points, and low vapor pressure and aqueous solubility. 2 There are pyrogenic, petrogenic and biological sources of PAHs. 3 Pyrogenic sources of PAHs are formed by the incomplete combustion of organic substances such as coal, wood, and petroleum, whereas petrogenic sources of PAHs are petroleum products such as kerosene, diesel fuel, gasoline, lubricating oil, and asphalt. 4 Biologically, PAHs can be formed by the degradation of vegetative matter. 5 Forest fires, volcanoes, algal synthesis, and petroleum seeps are natural sources of PAHs. 6 Anthropogenic sources of PAHs include automotive emissions, petroleum product spills, incineration, cigarette smoke, and sewage sludge. 3 An important route of PAH transport is through the atmosphere. 7 Some PAHs released into the atmosphere settle on surface waters by wet and dry deposition and are thereafter integrated with sediments though water currents. 7,8 Surface waters and sediments also receive PAHs from effluents and road runoffs. 9,10 Due to the persistence of PAHs in aquatic environments, they can bioaccumulate in aquatic organisms. 11 The occurrence of PAHs in aquatic foods is an important route of human exposure, particularly as some PAHs are known carcinogens. 12 The International Agency for Research on Cancer (IARC) has classified PAHs into Groups 1 (known carcinogens), 2A (possibly carcinogenic to humans) and 2B (probably carcinogenic to humans). 13 The highly investigated benzo [a]pyrene is in Group 1 whereas naphthalene, chrysene, benz [a] anthracene, benzo[k]fluoranthene and benzo [b]fluoranthene are Group 2B.
Research economically important aquatic ecosystems in Lagos State, as it is located near Agbara Industrial Estate, a major manufacturing hub, which discharges effluents into the lagoon via the central sewage treatment plant. There is limited information on the occurrence of organic compounds in the lagoon. 14 Moreover, there is no published literature on the occurrence of PAHs in the lagoon. Therefore, the present study aimed to quantify the occurrence of PAHs in the lagoon and determine the ecotoxicological effects of the predominant PAH compound in the lagoon.

Methods
The field study was carried out in May 2017 at Ologe Lagoon, a water body notable for the receipt of effluents from Agbara Industrial Estate. The lagoon is located between latitudes 6 o 26' N and 6 o 30' N, and longitudes 3 o 03' E and 3 o 07' E. Surface water samples were collected from three sampling stations in reference to the reception of effluents from Agbara Industrial Estate ( Figure 1) using 2.5 L amber glass bottles at the water surface. The samples were stored in a cooler with ice packs before transporting to the laboratory. Sediments were collected with a Van Veen grab sampler. The sediments were quickly transferred into aluminum foil. The samples were each labelled with masking tape and permanent markers and stored in a cooler containing ice. The tools and equipment were washed with methylene chloride and distilled water, and dried before use.
Fishes were collected from Ologe Lagoon with the aid of fishermen using cast nets. The fishes were wrapped in aluminum foil, labeled and placed inside a closed-glass vessel containing ice packs before they were taken for identification and laboratory analysis. All samples were sent to the laboratory for analysis within 8 hours of collection. Identified fish species included Liza falcipinnis, Cynoglossus senegalensis, Galeodes decadactylus, Chrysichthys nigrodigitatus, Tilapia mariae, Hemichromis fasciatus, Sarotherodon melanotheron, and Hyperopisus bebe. Sarotherodon melanotheron was chosen for PAHs analysis based on its abundance at the time of sampling, tolerance to pollution, and economic importance. 15 Five samples of fish tissues were pooled together to form a composite for the analysis.

Extraction and analysis of samples
Polycyclic aromatic hydrocarbons were extracted from water samples by adding 300 ml of dichloromethane, according to the method of Zeng and Vista. 16 Water extracts were Research concentrated to 10 ml using a rotary evaporator. Sediment and fish sample extractions were carried out following the method of Sojinu et al. 17 Identification and quantification of PAHs were determined in the extracts by Agilent gas chromatography-7890A with an HP-5MS fused silica column (30 m x 320 μm x 0.25 μm film thickness). Helium was used as the carrier gas. One (1) µl extract was injected with an autosampler in the splitless/split mode with a split time of 1 min after injection, and the injector temperature was set at 270 o C. The flow rate was 1.2 ml/min. The column temperature was initiated at 60 o C, then increased to 210 o C at 12 o C/ min, and finally increased to 320 o C at 10 o C/min (held for 5 minutes).

Laboratory bioassay
Juvenile African catfish, Clarias gariepinus (weight 34-41 g, total length 16-21 cm), belonging to the same parent stock were acquired from a fish farm. They were transported in aerated bags to the laboratory and kept in 50 L plastic tanks for acclimatization. The acclimatization of the fish was done for 15 days before the exposure period (photoperiod: 12 hours dark, 12 hours light).

Chronic toxicity test
Based on the prevalence of benzo[b] fluoranthene (B[b]F) in the lagoon, the surface water concentration (0.03 mg/L) and 10-fold concentration (0.3 mg/L) of B[b]F were selected for the chronic toxicity test to simulate chronic exposures that are likely to occur in the lagoon and mimic a worst-case scenario that may occur in the future, respectively. Two controls were included, an absolute control and vehicle control (0.2% acetone). A total of 42 acclimatized juvenile fish (six replicates of 7 fish per replicate) were transferred to each test concentration, as well as the controls in glass aquaria (40 cm × 30 cm × 30 cm). The semistatic (renewal) test was employed to renew the test media every 24 hours for a period of 28 days. Physicalchemical parameters of the test media were monitored after each renewal using digital instruments (Jenway). After 28 days of exposure, fish samples were collected for the biomarker studies.

Histopathological analysis
Fish gills were cut open and stored in Bouin's fluid in universal bottles before analysis. The histopathological analysis was conducted following the method employed by Amaeze et al. 18

Determination of metabolic enzymatic activities in the liver
Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were determined following the principle described by Reitman and Frankel. 19

Micronucleus assay
Blood samples were collected from fish using a 1 ml syringe. After the extraction, a drop of the blood sample was placed on a glass slide. The drop of sample blood on the glass slide was smeared by spreading with another slide at 45 o C until the smear became feathered in appearance. The slide was prepared based on the technique of Jiraungkoorskul et al. 20 Once the smear was dried, it was fixed using 70% ethanol for 30 minutes to ensure that the cell structure was not damaged. After the ethanol dried, a follow-up staining was carried out with May-Grünwald stain for 30 minutes which was then rinsed in water and allowed to dry. After the water dried, a counterstaining using 5% Giemsa stain was performed for Table 1

-Polycyclic Aromatic Hydrocarbon Levels in Sediments from Ologe Lagoon
Research 10 minutes and then gently rinsed in water and left to air dry. After staining, each slide was ready for microscopy using a drop of oil immersion on each sample glass to be viewed. The slide was placed under the microscope and its lens was adjusted to x100 and viewed using a hand-held counter. 21

Determination of oxidative stress markers in the gills of Clarias gariepinus
The levels of glutathione-Stransferase (GST), catalase (CAT), superoxide dismutase (SOD) and malondialdehyde (MDA) were measured in accordance with the methods of Habig and Jakoby, Sinha, Sun and Zigma, and Buege and Aust, respectively. 22-25

Statistics
One-way analysis of variance (ANOVA) was used to test for significant difference between means, where differences in means were considered significant when P < 0.05 and significant means were separated using the least significant difference test. All data were analyzed using Statistics Package for Social Sciences (SPSS) version 20 (IBM).

Results
Eleven PAH compounds were detected in the sediments from Ologe Lagoon.  Table 3). The level of ∑PAHs in the fish was 0.23580 mg/kg. The biotasediment accumulation factor was <1 whereas the bioaccumulation factor was >1. Generally, benzo(b) fluoranthene was the most Obanya et al

Table 2 -Polycyclic Aromatic Hydrocarbon Levels in Surface Water from Ologe Lagoon
Research predominant PAH compound, followed by benzo[a]pyrene.

Micronucleus induction
The results of the micronucleus assay are presented in Table 4. Exposure to 0.3 mg/L of B[b]F significantly (p = 0) induced micronuclei and binuclei in the blood erythrocytes of the fish. On the other hand, 0.03 mg/L of B[b]F did not significantly alter the number of micronucleated and binucleated cells in the blood of the fish (p = 0.198 and 0.061, respectively).

Superoxide dismutase
The results showed that the activity of the enzyme SOD was inhibited (p = 0) in the gills of C. gariepinus exposed to environmentally relevant concentrations (ERCs) (0.03 and 0.3 mg/L) of B[b]F (p = 0.002 and p = 0, respectively; Figure 2a). Gill SOD levels ranged from 3.38 ± 0.58 to 7.69 ± 0.69 U/mg protein in the control and treated groups.

Catalase
Exposure to ERCs (0.03 and 0.3 mg/L) of B[b]F caused a significant (p = 0) modification in the gill tissues of C. gariepinus, in terms of CAT activity ( Figure 2b). Gill CAT levels ranged from 14.00 ± 1.29 to 20.12 ± 2.10 U/ mg protein in the control and treated groups.

Glutathione-S-transferase
The gill tissues were affected in terms of GST activity by exposure to ERCs of B[b]F (p = 0; Figure 2c). Gill GST levels ranged from 10.94 ± 1.76 to 22.84 to 1.70 U/mg protein in the control and treated groups.

Induction of lipid peroxidation
The results of the lipid peroxidation Obanya et al

Table 4 -Micronucleus Induction in the Blood Erythrocytes of Clarias gariepinus
Research assay showed that the level of lipid peroxidation product, MDA, in the gills of C. gariepinus exposed to ERCs of B[b]F increased significantly (p = 0) compared to the vehicle control ( Figure  2d). Gill MDA levels ranged from 0.04 ± 0.02 to 0.21 ± 0.03 U/mg protein in the control and treated groups.

Metabolic enzymatic activities in the liver
The effects of ERCs of B[b]F on metabolic enzyme activities in the liver of C. gariepinus are presented in Figure  3. Hepatic AST and ALT activities were increased significantly (p = 0) in fish          Malondialdehyde is produced when the cells are damaged by these free radicals. 42 Therefore, MDA is used as a lipoperoxidation marker. Following the production of free radicals, due to exposure to chemicals or toxicants, anti-oxidative stress enzymes (SOD and CAT) work to tranform the free radicals into water and oxygen. 43 However, the high production of free radicals may overwhelm the activities of the enzymes, resulting in cellular damage. Polycyclic aromatic hydrocarboncontaining mixtures have been reported to induce histological alterations in the gills of fishes. Fish exposed to petroleum water-soluble fractions and refined products exhibited alterations in the gills, such as the lifting of respiratory epithelium, tissue necrosis, hyperplasia, hypertrophy, hemorrhage, and telangiectasia. 48 In Campos Bay, Brazil, crude oil induced histological effects in the gills of freshwater fish (Astyanax sp.). 49 Gills of Oncorhynchus mykiss chronically exposed to petroleum showed epithelial lifting and damage of secondary lamellae, associated with cellular hypertrophy. 50 Hyperplasia and hypertrophy of the lamellar epithelium were also observed in three species of marine flatfishes inhabiting an area near an oil refinery. 51 Brand et al. exposed pink salmon fry, Oncorhynchus gorbuscha, to sublethal concentrations of Alaska North Slope crude oil for over 10 days and found morphologic and stress-induced lesions in the gills, such as epithelial lifting, fusion, mucous cell hyperplasia, and vascular constrictions. 52 In the present study, tissue damage was observed in the gills of the fish exposed to ERCs of B[b]F in the form of epithelial necrosis, fused lamellae, shortened lamellae, and desquamation. This could be associated with the effect of oxidative stress caused by this compound.

Conclusions
In Ologe Lagoon, HMW PAHs were more predominant than LMW PAHs, suggesting that the source of the contamination was of pyrogenic origin.