The quantum of polycyclic aromatic hydrocarbon in Pyxicephalus edulis and sediments in

: Polycyclic aromatic hydrocarbons (PAHs) are organic compounds composed of two or more fused aromatic rings. PAHs can be found in a variety of foods and beverages, including drinking water, vegetables, fruits, cereals, oils, seafood, and meats. The current study investigated the pollution levels of the quantum of polycyclic aromatic hydrocarbon in Pyxicephalus edulis and river sediments. Twenty-five sediment samples and male and female specimens of Pyxicephalus edulis were collected from Oluwa River near Igbekebo, Ondo State Nigeria. The sediment samples were then dried in the open air for three days while the identified frog species were dried separately for 6 hours at 105°C before being powdered. The physicochemical parameters of sediment samples examined using standard physical and chemical analytical techniques and polycyclic aromatic hydrocarbons in frog and sediment samples were examined using gas chromatography-flame ionization analysis (GC-FID). The toxicity equivalent (TEQ) values of PAHs in sediment samples were determined by comparing the findings of this study to the Sediment Quality Guideline (SQG) with effects range from viz ., low (ERL), median (ERM) and threshold-effects level (TEL)/probable-effects level (PEL) to assess the toxicity of PAHs to aquatic organisms living in sediments. Male frogs had significantly higher concentrations of Acenaphthylene, Phenanthrene, Anthracene, Pyrene, and total PAHs than females, and lower concentrations of Naphthalene, Acenaphthylene, Fluorene, and Fluoranthene. BaAnt, Benzo(b) fluoranthene, Benzo(k)fluoranthrene, Benzo(a)pyrene, Indeno(1,2,3-cd) pyrene, Dibenz(a,h)anthracene, Benzo(g,h,i) perylene were reported only among males. The cause of this variation is unknown, but it could be due to differences in the genetic make-up of male and female frogs.


INTRODUCTION
Polycyclic aromatic hydrocarbons (PAHs) are organic chemicals composed of two or more fused benzene and/ or pentacyclic rings arranged in a linear, angular or cluster configuration (Alomirah et al., 2009;Iwegbue et al., 2014;2015a, 2015c, Ossai et al., 2015Iwegbue et al., 2016a). Man-made activities and natural processes contribute to PAH formation in the environment (Iwegbue et al., 2014). PAHs are present in a wide range of foods and beverages, including seafood, and meat vegetables, fruits, cereals, water and atmospheric particles (Kazerouni et al., 2001;Dost et al., 2012;Ediagbonya et al., 2012;2013b;Ediabonya et al., 2013a). Contamination from sediment, soil, atmosphere, water and manufacturing industry are the most likely sources of PAHs in food. PAHs are highly lipophilic and have a low water solubility (Llobet et al., 2006). Owing to the lipophilicity of these compounds, they can easily cross lipid membranes and creating fatty tissues of a wide range of aquatic organisms (Ramalhosa et al., 2009;Squadrone et al., 2014). PAHs dispersed widely in the environment as a result of their mechanism of synthesis (Ossai et al., 2015;Iwegbue et al., 2015b, d) and are also notorious for their long-lasting, genotoxic, mutagenic and carcinogenic properties (Kishikawa et al., 2003;Iwegbue et al., 2015b;Ossai et al., 2015;Iwegbue et al., 2016b). PAHs are dispersed across the gas and particle phases of the atmosphere and undergo long-distance transport and deposition (Cabuk et al., 2013). PAHs are almost all hydrophobic and biodegradable. As a result, they are thought to be easily absorbed by organisms and sediments (Das et al., 2008;Guo et al., 2010). PAHs are detrimental to animal, humans and other organisms near or far away from their origin (Naccari et al., 2011). These compounds are of concern not only because they are among the most common types of environmental pollutants but because they are carcinogenic and mutagenic (Fernandez et al., 2000;Xue & Warshawsky, 2005;Usenko et al., 2007). The amount and constituents of PAHs found in sediments, air, organisms and water reveal source characteristics as well as the physicochemical properties of individual PAHs with significant impacts on the partitioning of various phases (Deb et al., 2000;Colombo et al., 2006;Ozaki et al., 2006). Based on their frequency of occurrence and toxicological characteristics, the US Environmental Protection Agency (USEPA) has designated 16 PAHs as priority control pollutants, with seven of them known to cause human cancer (Qin et al. 2013;Zhao et al. 2014;Qin et al. 2014). Furthermore, ultraviolet light exposure can alter the carcinogenicity of PAHs, and 11 of the 16 USEPA priority PAHs have photo-mutagenic properties (Yang et al., 2004). Apart from cigarette smoking and exposure to PAH in workplaces, consuming polluted food, particularly aquatic lives and plant are also possible sources of PAH exposure in humans (Phillips, 1999;Falcó et al., 2005). PAHs cause growth retardation, endocrine disturbances, reproductive system failure, and DNA damage in marine creatures (Nkpaa et al., 2013). Frogs are members of the vertebrate class known as amphibians (Omonona & Ekpenko, 2011). Warketin et al. (2009 summarized alarming data on the number of Asian frogs taken for human consumption. Human interference in the environment has resulted in the decline of frogs (Cohen 2001;Hayes et al. 2006;Simon et al. 2012). Frogs are more vulnerable to environmental contaminants than other animals because their egg membranes and skins do not obstruct the passage of foreign substances (Snodgrass et al. 2003). Eggs and tadpoles have been used as bioindicators for pollution evaluation (Berzins and Bundy 2002;Haywood et al. 2004;Simon et al. 2012).
The goal of this study is to assess the concentration of PAHs in Pyxicephalus edulis (frogs) and sediments in the Igbekebo area of Oluwa River as well as determine bio availability in order to compare values in sediment to other studies and standards.

Study Area
Igbekebo is the Administrative Headquarter of Ese -Odo Local Government Area in Ondo State, Nigeria. It is home to the Ijaw (Izon) ethnic subgroups of the Western Apoi and the Arogbo tribes. It has a land area of 762 km 2 and a population of 154,978 people according to the 2006 census. Oluwa River stretches along Okitipupa, Ilaje and Ese-Odo Local Governments with Igbekebo being one of the communities along the river bank. The river is usually heavily polluted due to several anthropogenic activities such as oil spills which might lead to large amounts of PAHs in the river and sediments. The map of the sampling location (Ediagbonya et al. 2022a;2022b) is shown in Figure 1.

Collection of samples
Sediment and frog (male and female) samples were taken from the Igbekebo area of Oluwa River. Plastic bottles were used to collect samples from five (5) separate locations at a depth of 10 cm. Samples A and B were assigned to the frog samples. Ten (10) equal-sized samples of male and female frogs were taken, as were 25 samples of silt. Figure  1 shows the sampling locations and Table 1 contains the coordinates of the locations.

Location
Latitude Longitude

Preparation of sample
The frogs were dried for 6 hours at 105°C and then crushed into fine powder for analysis. The sediment samples were air-dried for three days before being prepared for analysis.

Sample analyses
GC-FID 5890 series paired with a Flame ionization detector was used for the GC/FID analysis. The GC had a Helium gas carrier with a split less inlet mode and a linear velocity of 30cm/sec. The GC separation was performed on a capillary column HP-5 (cross linked PH ME siloxane) 19091J-413 (30 m 0.32 mm 0.25 m, phase ratio 320). The following temperature programs were used for the analysis: initial temperature of 100°C, initial time of 1 minute with rate 1: 4°C/minute, final temperature of 310°C, and detector temperature of 300 o C. A flame Ionization Detector holding 35ml/minute of hydrogen and 350ml/minute of air (EPA, 1984;NIOSH 1994;Cai et al. 2009).

Statistical Analysis
The data was analyzed using the Statistical Package for Social Sciences (SPSS) version 26.0 for Windows. The data was described using means and standard deviation. The independent t-test was used to compare mean differences between frog types (water and land) and sexes, while spatial variation was compared using One Way Analysis of Variance (ANOVA) with Scheffe post hoc. The relationship between physicochemical parameters and PAHs was investigated using Pearson's correlation. The distribution of physicochemical characteristics was represented using box plots.

Quality Control
A method blank, a method blank spike, a Diesel/Lube standard, a sample duplicate, and a sample matrix spike were all prepared in order for exactness and precision of the PAHs determination process in this investigation. The quality control standard was done by using the method blank with only reagents (hexane: acetone 1:1) and treating it in the same manner as the sample. Using reagents (hexane: acetone 1:1) and spiked with 200L (150,000 ppm) of Diesel/Lube standard, a technique blank spike was created. It was processed in the same way as the sample. With each analysis, a sample duplicate (SD) and a sample matrix spike (SS) were also prepared. The SD is the same sample made exactly, while the SS is prepared exactly and then spiked with 200L of Diesel/Lube.

Toxicity equivalency factors
The toxic equivalent factor (TEF) and mutagenic factor (MF) were used to assess the carcinogenic and mutagenic potential of PAHs (MEF). The total of the products of the amounts of each individual PAH and their associated poisonous equivalent factors or mutagenic factors, yields the carcinogenic and mutagenic equivalents respectively.
The BaP eq was calculated using the toxic equivalent factor value for each PAH according to Nistbet and Lagoy (1992) while the mutagenic factor value for 7 PAH was according to Durant et al., (1999) and used for the calculation of the mutagenic potency (BaP MEQ ) of the individual PAH. The TEF values (Chaber & Gworek, 2020;Ediagbonya et al., 2022b).
The mutagenic and carcinogenic equivalents can be calculated by multiplying the levels of each PAH and their corresponding toxic equivalent factors or mutagenic factors, Where C i was the concentration of individual PAHs (ng/g d.w.) and TEF i was its corresponding toxic equivalency factor.

RESULTS AND DISCUSSION
The mean comparisons of PAHs of male and female frogs are given in Table 2. The results revealed that the PAH parameters ranged 0.41 ± 0.02 -47.85 ± 0.00 (ng/g -1 ) for the male frogs and 1.56 ± 0.00 -22. 15 ± 0.30 (ng/g -1 ) for the female frogs. Dibenz(ah)anthracene (47.85 ± 0.00 ng/g -1 ) and Anthracene (22. 15 ± 0.30 ng/g -1 ) were highest for male and female frogs respectively. Also, except for Fluoranthene, Acenaphthene and Fluorene, all the PAHs were higher in the male frogs than the females. The order of concentrations of the PAHs in frogs were; DahA > Ant > BghiP > Bap > Pry > InP > Acy > Nap > Fl > Phe > BaA > BkF > Ace > BbF > Chr > Fla. The detection of PAHs in frogs suggests their accumulation in the aquatic animal. Result of this study corroborates the assertion that aquatic animals are known bio-indicators and they accumulate heavy metals and persistent organic pollutants (Iwebue et al 2016b; Ediagbonya et al 2019;2020b;2022a). However, the concentration of the following Chr BaA, BbF, BkF, Bap, InP, DahA and BghiP were detected in the male frogs but all values were below the detection levels in female frogs.
The variation in PAH concentrations might be due to their genetic compositions, ecological or biological factors. PAHs are known carcinogens. PAHs and especially their metabolic products are therefore of great concern. Savinov et al. (2003) reported that BaA, BaP, BbF, BkF, DahA, and InP are some of the potentially carcinogenic PAHs (CPAHs). Total CPAH concentrations in Jialu River sediments ranged from 192.8 to 856.0 ng/g d.w., with an average of 489.1 ng/g, accounting for 20.07-51.58 percent of total PAH concentrations.
The concentration of the PAHs in sediment for Nap in location C 253.46 ± 0.30 has the highest mean concentration (253.46 ± 0.30) while location B had the lowest (250.85±0.00). Locations A, D, E were approximately the same 252.36±2.14, 252.25±1.97, 252.21±2.35 respectively. Acy in location C has the highest mean concentration (197.81±0.00), locations A, B, D, E, are spatially the same 192. 56±7.42, 192.56±7.37, 192.57±7.41, 192.54±7.40, for Ace Location A, C, D, E, were higher than location B 100.11±0.00. The concentration of Fl ranged 213.09±0.00 -213.68±0.83 for all the locations with location B having the least value (213.09±0.00). Table 3 shows that for Benzo(a)pyrene, Acenaphthylene, Fluoranthene Acenaphthene, recorded maximum values at location B, while Napthalene, Chrysene, Benzo(k), fluoranthrene recorded maximum values at location C, Fluorene, Benzo(g,h,i) perylene reported maximum values  Table also shows that there is no significant (p>0.05) spatial variation in the mean PAHs. Table 4 above shows that Naphthalene, Indeno (1,2,3-cd) pyrene, Dibenz (a,h) anthracene, Acenaphthene, Fluorene, Benzo(g,h,i)perylene, Pyrene, Benz(a)anthracene, Benzo(b)fluoranthene, Benzo(a)pyrene, Phenanthrene and Total PAH shows negative relationship with pH, while the other PAHs showed positive relationship with pH. Also, Naphthalene, Phenanthrene, Pyrene, Acenaphthene, Fluorene, Benz(a)anthracene, Benzo(b)fluoranthene, Benzo(a)pyrene, Indeno(1,2,3-cd) pyrene, Dibenz (a, h) anthracene, Benzo(g,h,i)perylene, and Total PAH show positive relationship with EC, while the other PAHs parameters show negative relationship with EC. However, none of the relationships is statistically significant (P >0.05). PAH concentrations in surface sediments collected from the Oluwa water body are compared to those reported in other studies (Table 5). The PAH levels found in the surface sediments are similar to those seen in India's River Gomti (Tripathi et al., 2009) and the Yellow River in China (Xu et al., 2007) but higher than those reported in Yalujiang River in China (Wu et al., 2003), Taiwan's Gao-Ping River (Doong and Lin, 2004), China's Daliao River (Guo et al., 2007), Serbia's Sava River (Crnkovic et al., 2008) and China's Luan River (Bai et al., 2008). Therefore, the levels were lower than those found in the United States' Passaic River (Huntley et al., 1995), the Czech Republic's Morava River (Huntley et al., 1995;Vondracek et al.2001) and the China's Zhujiang River (Mai et al., 2002). Table  6 summarizes the 16 PAH concentrations in surface sediments. The total concentrations of the 16 USEPA priority PAHs ranged from 100.1 to 6,008.89 ng/g dry weight, with a mean concentration of 1147.4 ng/g. Wang et al. (2002) used semipermeable membrane devices (SPMD) to analyze the normalized concentrations of 13 PAHs (without Acy, Inp, and BghiP) in sediment samples from Xinyang City and Huainan City in the Huaihe River. They found that the total concentrations of 13 PAHs were 7.7 and 9.7 ng/mg organic carbon (OC), respectively, with a mean concentration of 8.7 ng/m. This is significantly lower than the pollution levels observed in our study (424.3-4185.6 ng/OC), and the use of SPMD may have influenced the analyzed results. Huang et al. (2004) looked at PAHs in surface sediments in the Jiangsu section of the Huaihe River and found that total concentrations of 12 PAHs (without Nap, Acy, BaA, and BbF) were 690-6,630 ng/g d.w., with a mean concentration of 3,740 ng/g, which is higher than our observation.
The PAH concentrations in surface sediments from the Jialu River were similar to those found in the River Gomti in India (Tripathi et al. 2009) and the Yellow River in China (Xu et al., 2007), and higher than those found in the Yalujiang River in China (Wu et al., 2003), the Sava River in Serbia (Crnkovic' et al., 2008), the Luan River in China Bai (Doong & Lin 2004). The levels were, however, lower than those reported in the Zhujiang River in China 259 Ediagbonya et al.  (Mai et al., 2002), the Passaic River in the United States (Huntley et al., 1995), and the Morava River in the Czech Republic (Huntley et al., 1995;Vondracek et al., 2001). The harm of contaminants in sediments is determined by measuring the biological effects on organisms and animals, which frequently includes the application of sediment quality guidelines (SQGs). (Long et al., 1995;Wenning et al., 2002;Apitz et al., 2005) assessed the toxicity of PAHs to aquatic organisms living in the sediment of the Akaki River, Lake Awassa, and Lake Ziway in Ethiopia using SQGs based on effects range-low (ERL)/ effects range-median (ERM) and threshold-effects level (TEL)/probable-effects level (PEL). As a result, the potential toxicity of PAHs in the sediments of the Oluwa River to sediment dwelling organisms was assessed using the SQGs and the ERL and ERM target values. The comparisons of PAH levels with SQGs in the current study are shown in the Table 6 above. The ERL and ERM values for a chemical define three concentration ranges, including those that were rarely used (below the ERL). A biological effect is unlikely to occur at concentrations lower than the ERL.
In contrast, concentrations greater or equal to ERL and less than or equal to ERM, a biological effect occurs on occasion, at concentrations greater or equal to ERM, a negative biological effect occurs on a regular basis, and sediments were expected to be toxic (Nasher et al., 2013, Yuan et al., 2013Ediagbonya & Ayedun 2018a;Ediagbonya & Gbolahan 2018b;Ediagbonya & Balogun 2020). Table 6 shows the mean concentration of each PAH in sediments from the river sample. Some sampling sites had concentrations lower than the ERL while others had concentrations higher than the ERL. Except for Fln, Pyr, and BAnt, which recorded average values between the ERL and ERM, the levels of PAHs quantified in all sediment samples from the river were higher than the ERL. Except for Fln, Pyr, and BAnt, these values showed that the PAHs in the Oluwa River sediment samples have no adverse biological effects. These may occasionally impose negative toxic effects, but not acute effects. Therefore, all individual PAHs in sediment samples from the Oluwa River were below the ERM, indicating that biological activities are unlikely to occur in this study area and that the sediments are not toxic. Only Bkf, DahAnt, and BghiP are greater than ERM, suggesting that biological activities may occur.
A number of approaches have been developed to set numerical sediment quality guidelines (SQGs) based on the available ecotoxicology data on PAHs (Chapman 1989). Sediment assessors may find it difficult to select the most appropriate SQGs for specific applications.    PAHs in sediment samples have been used to assess risk all over the world. (Nasher et al., 2013;Zhang et al., 2018) However, there is no data on the quantitative risk assessment of PAHs. According to Yang et al., (2014), soils with BaPE values less than 0.1 mg/kg are considered uncontaminated, soils with values between 0.1 and 1.0 mg/ kg BaPE are considered slightly contaminated, and soils with 1 to 10 mg/kg BaPE are considered significantly contaminated. The value of the DahHBaP eq (6008µgg-1 ) in this study was very low compared to this range which indicated that toxicity level of the area was low and does not pose a significant risk to human.

CONCLUSION
The results conclude that the concentrations of PAHs in male and female frogs were significantly higher than the WHO limits, thus confirming that frogs from this study area to be harmful for consumption. PAHs that are produced by human activities such as burning of coal, oil, gas, wood, garbage, and tobacco industrial food processes could impose major impacts on the wildlife, and eventually on human health. In particular, oil spills may have contributed to the presence of polycyclic aromatic hydrocarbons in high concentrations in rivers.