Concentration Assessment And Source Evaluation of Polycyclic Aromatic Hydrocarbons (PAHs) In Soil And Crops of The Agro-Industrial Tobacco Production Area of Igboho, Nigeria

The contribution of tobacco smoking and cigarette butts to global environmental pollution has been given signicant attention. However, little is known about tobacco-related agricultural activities on environmental pollution by polycyclic aromatic hydrocarbons (PAHs). In this study, the spatial distributions, composition, source, and toxicity potential of polycyclic aromatic hydrocarbons (PAHs) in soil and food crops within the vicinity of the agro-industrial tobacco production area of Igboho, Nigeria was investigated. Soil and food crop (Zea mays, Dioscorea alata, and Manihot esculenta) samples collected from the tobacco curing site and the surrounded farmlands were analyzed for the PAHs concentrations. The identication and quantication of priority PAHs in the samples were carried out using a gas chromatograph equipped with a ame-ionization detector. The total concentration of the priority PAHs in the soil ranged between 136.70 ng.g -1 to 889.30 ng.g -1 . The total concentration of carcinogenic PAHs ranged from 6.07 ng.g -1 to 321.04 ng.g -1 , and the total concentration of toxic PAHs ranged from 6.27 ng.g -1 to 254.37 ng.g -1 . The PAHs level was highest in crops from farmlands closest to the tobacco curing site. The distribution of PAHs ring size is in the order of 6-rings (cid:0) 4-rings (cid:0) 5-rings (cid:0) 3-rings (cid:0) 2, and the diagnostic indices showed that the sources of PAHs in the samples were mainly pyrogenic and associated with tobacco curing activities in the area. the values wt. 13.6 wt. The maximum TOC (13.6 wt. %) recorded at the tobacco curing site, while the farmland FL recorded the lowest value of 8.3 wt. %. These results are in line with 1.0 wt. % detention limit of Soil Guidelines Values (SGVs) in CLEA Model by and the Environment Agency (E.A.) which sets a framework for the appropriate assessment of risks human health from contaminated land, This is also justied with a 0.5 wt% (US limit. The soil from the tobacco curing site showed a relatively higher TOC percentage (13.6 wt. %) than the soil samples. The farmland FL 1 closest to the curing site recorded the second-highest value with (10.5 wt. %) while all the food crop samples (yam, cassava, maize) obtained from FL 1 recorded the highest values with 10.4 wt. %, 10.1 wt. %, 8.9 wt. % respectively. The farmland FL 3, the distant farmland from the curing site, recorded the smallest value with 8.3. wt. % while all the food crop samples (yam, cassava, maize) obtained from FL 3 recorded the lowest values with 3.1 wt. %, 5.4 wt. % and 1.9 wt. %, respectively.


Introduction
The investigation of toxic and carcinogenic compounds present in the environmental media and food items is a critical step towards reducing the incidence and mortality rate due to cancer. Tobacco use is a well-known threat PAHs in soil and crops within the vicinity of an agro-industrial tobacco plantation in Igboho, Nigeria, and assesses the probable source of the PAHs contamination toxicity potential. Priority PAHs in the samples including Naphthalene (Nap); Acenapthene, (Ace-Nap), Acenaphtalene, (Ace-Nap), Fluorene, (Flu), Phenanthrene, (Phen), Anthracene, (Anth), Flouranthene (Flo), Pyrene (pyr), benzo(a)pyrene, (B(a)P), Benzo(a)anthracene (B(a)A), Benzo(b) uoranthene (B(b)F), Benzo(k) uoranthene (B(k)F), Indeno(123-cd)pyrene (IP), Dibenzo(a,h)anthracene (Dib(ah)A and Benzo(ghi)pyrene (B(ghi)P) were identi ed and quanti ed. The study provides a base-line report on the level of the identi ed PAHs in the study area.

Description of the study area and sample collection
Igboho is one of the largest towns sprawling over a vast grassland area in the northern part of Oyo State, Nigeria. It is located within 8.83784 latitudes and 3.75628 longitudes, situated at an elevation of 405 meters above sea level. The town is bounded by Igbeti town in the south, Saki town in the west, and Kisi town is in the north. Igboho is the headquarters of the Orelope Local Government Area of Oyo State, Nigeria, and has an area of 907 km 2 and has a population of 104,441 according to the 2006 Nigerian population census result (National Population Census). Igboho community adopted commercial ue-cured tobacco production in 1950, and industrial tobacco agriculture becomes the main activity in the area. The soil and food crop samples were collected from the farmland and the tobacco curing site. The study area was divided into three sampling stations based on the distance of the farmland from the tobacco curing sites and was identi ed as TBS for the tobacco curing site and FL 1, FL 2, and FL 3 for farmland at a distance of 20 meters, 50 meters, and 1.0 km away from the tobacco curing site respectively. The soil samples (0-10 cm depth) were collected using a stainless-steel hand auger. Each sample was the composite of soil samples collected within 10 cm x 10 cm at each sampling station. The soil samples were packaged in aluminum foil and kept in plain paper bags for transportation to the laboratory. Similarly, food crops were harvested from ten points within each sampling station. Soil samples were dried at room temperature, crushed and sieved through 2 mm mesh size, and stored in an amber glass container at -4 o C before laboratory analysis. The soil samples were allowed to dry at room temperature after removal from the freezer and freeze-dried. The crop samples (maize, yam, and cassava) described and labeled as FL 1 M, FL 2 M, FL 3 M, FL 1 C, FL 2 C, FL 3 C, FL 1 Y, FL 2 Y, and FL 3 Y were collected from the farmlands at the distance of 20 m, 50 m, and 1.0 km away from the tobacco curing site respectively. The samples were wrapped with aluminum foils and kept at a temperature below − 4 o C. The crop samples were unwrapped, peeled, sliced, and dried at room temperature. Each sample was blended with a grinder and sieved with a 2 mm mesh size and wrapped with aluminum foil, and stored at temperature before extraction.

Determination of Total Organic Carbon
The total organic carbon was determined by the Walkley-Black method (Kakad and Thakare 2017; Matus et al. 2009). About 5g of the dried soil and food crop samples were grounded to pass through a 0.5 mm sieve. 0.5 g of each sample was measured in triplicate and transferred into different 250 mL Erlenmeyer ask. 10 mL of 1 N K 2 Cr 2 O 7 solution was added into each ask and swirled gently to disperse the samples. 20 mL of concentrated H 2 SO 4 was rapidly added. The ask was gently swirled immediately until the sample and reagents were mixed and then swirled more vigorously for 60 seconds. The ask was rotated again and allowed to stand on a sheet of asbestos for about 30 minutes. After standing for 30 minutes, 100 mL of distilled water was added to the mixture to provide a suspension for viewing the endpoint, three drops of ferroin indicator were then added, and the resulting solution titrated with 0.5 N ferrous sulphate solution. As the endpoint was approached, the solution took on a greenish cast and then changed to dark green, at which point ferrous sulphate was added drop by drop until the color changes rapidly from blue to maroon red. This procedure was then repeated for the remaining samples. The blank titration was carried out. The result of the TOC was then calculated as Equ. (1) A is the normality of K 2 Cr 2 O 7 x ml of solution, B is the normality of Fe 2 SO 4 x volume of solution, C is the mass of air-dried soil (g), and F is the correction factor (1.33). (1)

Determination of PAH in soil and food crops
Sample extraction and cleanup were carried out according to a previously reported method with modi cation (Adedosu et al., 2015). In brief, 10 g of dried soil sample was mixed with 10 g of anhydrous sodium sulfate and were extracted with 100 mL of 1:1 (v/v) acetone-dichloromethane mixture for 2 hours. The extract for each sample was collected into a pre-treated conical ask. The extraction was repeated with a new proportion of 100 mL of 1:1 (v/v) methanol-dichloromethane mixture for 2 hours. The supernatant for each sample was added to the previous ones obtained. The process was also repeated for all the food crop samples. A rotary evaporator concentrated the extracts to 2.0 mL and evaporated to dryness at room temperature after that. A prepared glass column was packed with 3.0 g of activated alumina adsorbent and 12 g of activated silica gel. The extracts were fractionated with 25 mL of n-hexane to obtain aliphatic fraction, 25 mL of n-hexane and dichloromethane (2:3) to obtain aromatic fraction (PAHs fraction), and 25 mL of methanol to obtain polar fraction. The PAHs fraction was concentrated by rotary evaporation to about 1 mL and carefully transferred into a sample vial and kept in the refrigerator until required for analysis. The concentration of PAHs in the sample extracts was determined according to USEPA Method 8100 using a Gas Chromatograph coupled with a Flame Ionization Detector (Agilent 5890 GC-FID) little modi cations. The separation was carried out on an HP5 (30 m x 0.25 mm id) fused silica capillary column. 1.0 µL of the extract was injected using split/splitless (25:1) injection mode. Helium was used as the carrier gas at a ow rate of 1 mL.min − 1, and nitrogen was used as the makeup gas. The oven temperature was programmed from 60 ºC (held for 2 min) to 180 ºC at a rate of 3 ºC min − 1 , from 180 ºC to 250 ºC at a rate of 5 ºC min − 1 and from 250 ºC to 330 ºC at a rate of 15 ºC min − 1 and then held for 8 mins. The USEPA 16 priority PAHs standard mix was analyzed using the same instrumental conditions to obtain the calibration curve. The identi cation of PAHs was by comparison of sample retention times with that of the USEPA standard mix. The quanti cation of PAHs in the samples was achieved using the external standard calibration curve method. The peaks of all the reference standards are well resolved.

Quality control and assurance
The laboratory and analytical procedures were monitored with strict quality assurance and control measures.
Laboratory quality control procedures include analysis of method blanks (Solvents), spiked blanks, and sample duplicates. The spiked sample analysis and recovery studies of PAHs were achieved by adding known concentrations of PAHs standards to previously analyzed soil samples of known PAHs concentrations. The average recoveries of the PAHs were between 92 % -120 % for the 19 PAHs.    PAHs, uoranthene, Chrysene, benzo(a)anthracene, and pyrene was 32.46%, and the abundance of ve-ring PAHs benzo(k) uoranthene, benzo(k) uoranthene, benzo(a)pyrene was 29%. The three rings sized PAHs; acenaphthene, uorene, phenanthrene, anthracene, acenaphthylene, have a relative abundance of 3.53%, while two rings PAH has the least concentration in the soil samples of the area. To be more speci c, as summarized in Table 2

Level and compositions of PAHs in food crops
The total concentration of PAHs in the food crop samples from the agricultural farmland was also evaluated.   As summarized in Table 3.0, PAHs' concentration in the Manihot esculenta decreased from the closest farmland FL 1 C to the distant farmland FL 3 C.  matter (NOM) that have a strong a nity for contaminants. As the contaminants are released in the soil matrix, they bind to the surface and become sequestrated into the soil matrix. The hydrophobicity of these compounds constitutes the main factor determining their persistence in the environment, and they tend to be strongly absorbed by soil particles with low bioavailability and possibly accumulate in the food chain. The scatter plot in Fig. 3 (b) was used to assess the relationship between the percentage total organic carbon and the total concentration of PAHs in the soil sample were shown by the linear regression curve in Fig. 3 (b). The R 2 value was 0.937, showing that there is a positive correlation between the TOC and PAHs. This con rmed that the soil sample's organic carbon content increases the PAHs' adsorption at the study site.  Table 4. The values of LMW/HMW PAHs for the TBS (0.46), FL 1 (0.30), FL 2 (0.12), and FL 3 (0.19) were less than 1.0, suggesting a pyrolytic source of PAHs contamination. The Phe/Ant ratio allowed the separation of the pyrolytic (combustion origin) and petrogenic (unburned petroleum products) PAH sources. A Phe/Ant ratio lower than 15.0 is assumed to be of a pyrolytic origin from the combustion of plants, wood, grass, and others, whereas a value higher than 15.0 is assumed to be of combustion of petroleum hydrocarbons such as coal, crude oil, and others. The values of Phe/Ant at TBS, FL 1, FL 2, and FL 3 were lower than 15.0 and con rmed that the PAHs were from the combustion of plants, wood, and leaves, possibly from the tobacco-curing activities in the area. Similarly, an Flt/Pyr ratio PAHs greater than 0.4 indicate the in uence of pyrolytic PAHs, and the ratio of PAHs lower than 0.4 indicates petrogenic sources. The BaA/Chr ratio PAHs that more signi cant than 0.25 are assumed to be pyrolytic sources, and the BaA/Chr ratio PAHs lower than 0.25 are assumed to be petrogenic sources. The TBS, FL 1, FL 3 but FL 2 was 0.93, which was greater than 0.25. The BeP/BaP ratio PAHs greater than 2.0 is assumed to be of pyrolytic origin. Similarly, other PAHs ratio in Table 4 con rmed that the source of PAHs contamination in the study area is from the combustion of biomass, woods, and other organic matter due to the tobacco curing process.   (Table 5). The B(a)P concentrations were lower than the European Union's maximum contaminant level (MCL) of 25 ng.g -1 . The B(a)P equivalent (B(a)Peqv) was calculated using the toxicity equivalent (T.E.) for each PAH, as presented in Table 5. The calculated total B(a)Peqv at the TBS and surrounded farmlands FL 1, FL 2, and FL 3 ranged from 13.84 ng.g -1 to 268.85 ng.g -1 . The highest B(a)Peqv was found at the TBS with 268.85 ng.g -1, and the distant farmland FL 3 recorded the least value of 13.84 ng.g -1 . It is observed that the tobacco curing site TBS (268.85 ng.g -1 ) has been polluted while the farmland FL 1 (112.21 ng.g -1 ), FL 2 (92.29 ng.g -1 ), and FL 3 (13.84 ng.g -1 ) were slightly polluted.   In Table 7, the concentration of carcinogenic compounds in the Dioscorea alata ranged from14.02-142.12 ng.g -1 .
As previously observed for other crops, the farmlands closer to the curing site FL 1 recorded the highest concentration, and the distant farmland FL 3 Y recorded the lowest concentrations of PAHs in the Dioscorea alata.
The concentrations of PAHs in the soils determined the amount adsorbed by plants and stored in the roots. This process is called phytoaccumulation. The calculated total B(a)Peqv obtained from samples at the sampling farmlands was within the range 10.14 -51.01 ng.g -1 . The European Union above the legally permissible limit (1.00 ng.g-1) processed this range for processed cereal-based foods (Dennis et al.; 1984). Table 8 shows that the concentration of carcinogenic PAHs in the Zea mays samples ranges from 0.00 ng.g -1 to 95.17 ng.g -1 . The highest concentration was recorded at the farmland closer to the curing site, and the concentration at other farmlands was below the detection limit. It was observed that maize could undergo phytovolatilization by mineralizing carcinogenic PAHs into harmless products such as carbon (iv) oxide, methane, and water, making the maize from FL 2 and FL 3 free from contamination (Brady, 1990).

Conclusion
The study has determined the distributions, sources, and toxicity potential of polycyclic aromatic hydrocarbon (PAHs) in the soils and food crops from the farmlands within the tobacco local processing vicinity industry. The result of total organic carbon in the soil and food samples established the accumulation of polycyclic aromatic hydrocarbon in the vicinity of the tobacco curing site and the surrounded farmlands because it was above 0.5% US EPA detection limit. The total number of PAHs identi ed in the study area was nineteen (19) priority polycyclic aromatic hydrocarbon compounds in which fourteen (14) were generally recorded in all samples. The total concentration of PAHs recorded in all the soil samples were above 100.0 ng.g -1 , USEPA warning limit. The concentration and spatial distribution of PAHs in all the food crop samples collected from farmland FL 1 were above 100.00 ng.g -1 limits of the USEPA. The concentration PAHs found in food crop samples from farmlands FL 2 and FL 3 was relatively close to the USEPA detection limit. However, there is a point that the vicinity of the curing site was more concentrated than the distant farmland.
Molecular matrix was used to distinguish between petrogenic and pyrogenic sources by comparing the ratio of the concentration species of PAHs obtained according to the statistical principles. The calculated values obtained depicted that almost all the PAHs were from pyrogenic sources. The concentration of higher molecular weight (HMW) was of a higher percentage than lower molecular weight (LMW), and this is an indication that the signi cant source of PAHs in the study area was pyrolytic. Based on the available evidence, both the International Agency for Research on Cancer (IARC, 1987) and US EPA (1994), the PAHs' carcinogenic potency in the study area was moderately high and posed a threat to human existence. The correlations coe cient matrix of concentration of individual PAHs and the total concentration of polycyclic aromatic hydrocarbon revealed that all samples from the farmlands originated from the same source.

Declarations
Authors declare no con ict of interest Figure 1 Map of Oorelope local government of Oyo state showing the study area's location and the sampling points. Note: The designations employed and the presentation of the material on this map do not imply the expression of any opinion whatsoever on the part of Research Square concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. This map has been provided by the authors.

Figure 2
Percentage distribution of PAHs in soils samples