Levels of polycyclic aromatic hydrocarbon in the soil around typical automobile repair workshops in Nigeria

Background This study determined the levels of polycyclic aromatic hydrocarbons (PAHs) in the soil around typical automobile repair workshops in Nigeria. Risk assessment associated with human contact with the soil was carried out using hazard quotient (HQ) and incremental life cancer risk (ILCR) from human unconscious ingestion and dermal contact with the soil. Methods Soil samples were obtained at different automobile workshops in Ado-Ekiti, Western Nigeria. The PAHS in the samples were extracted using dichloromethane and extracts were cleaned up using silica-alumina gel open column chromatography. Analysis of PAHs in the soil was done using a gas chromatograph coupled to a mass selective detector operated on electron ionization mode. Results The results showed the mean PAHs concentration at the sampling locations ranged from 5.58 – 6.4 μg/g and the mean ∑ carcinogenic PAHs was 58.4 μg/g, equivalent to 59.39 % of total PAHs observed. The mean Toxicity equivalence (TEQs) ranged from 0.02 - 6.680 μg/g. Benzo (a) pyrene and dibenzo(a,h)anthracene have the highest toxicity equivalent. The total ILCRs from accidental ingestion and dermal contact in adults were 1 ×10^(-3) and 9.8 ×10^(-5), for adults and children respectively; both are higher than the permissible limit stipulated by the World Health Organization.The HQs obtained are several folds higher than 1. This implies high carcinogenic and non-carcinogenic risks for children and adults. Conclusions The study revealed the levels of PAHs and also revealed the risks associated with human contact with the soil around automobile repair workshops.


Introduction
Indiscriminate discharging of used engine oil into the surroundings, especially around automobile workshops, remains a major problem in most parts of Nigeria and this constantly affects the ecosystem (Odjegba and Sadiq, 2002;Emoyan et al., 2020).The soil around automobile workshops is usually contaminated with spent engine oil which contains heavy metals and polycyclic aromatic compounds (PAHs) and other pollutants.PAHs constitute two or more fused aromatic rings solely made from carbon and hydrogen (Arey and Atkinson, 2003).PAHs are emitted from the exhaust of automobiles as well as by-products from petroleum ranging from lubricating oil, gasoline, diesel, and several others put to constant use in automobile workshops, and these have high chances of spilling into and degrading the soil (Kidman and Boehlecke, 2011;Muze et al., 2020).
PAHs, which are the products of incomplete combustion, are transported into the crankcase and concentrated in lubricating oil in the engine of the car (Pruell and Quinn, 1988;Akintunde et al., 2015).Additionally, emissions from the exhausts of the cars being repaired at these workshops can be washed down through rain to the soil around the workshop.Attention has been drawn to the health risks associated with human contact with soil contaminated with PAHs (Wcisło, 1998;Adeniyi and Afolabi, 2002;Dong and Lee, 2009;Kwon and Choi, 2014).PAHs are found to have a negative effect on human health as they can lead to cancer, as well as having a mutagenic and teratogenic effect (Boström et al., 2002).
The presence of PAHs in the spent engine oils disposed in the environment during maintenance poses a serious threat to the environment (Ololade, 2014).The lack of required knowledge on proper handling of these hydrocarbon products during repairs and vehicle servicing in automobile workshops is a major problem, especially in Nigeria (Sharifi et al., 2007).Akintunde et al. (2015) studied the reproductive effects of used engine oil on male rats, and their results showed used engine oil has the potential to hamper male rats' germ cell development and also affect other testicular activities in producing viable spermatozoa.Their study also concluded that soil contaminated with spent engine oil poses a great reproductive risk to humans in areas where there is high exposure.The Agency of Toxic Substances and Diseases Registry rates PAHs as ninth on the list of compounds dangerous to human health.Organisms are also affected by the toxic effect of PAHs as they affect the functioning process of cellular membranes (Abdel-Shafy and Mansour, 2016).Clients and workers at automobile workshops are exposed to PAHs through contact with the soil and ambient around the workshop.
This study focuses on determining the levels of PAHs around automobile repair workshops, this is with a view to determining the risk associated with human contact with these pollutants.

Study area
The study area locations are shown in Table 1.In order to assess the implication of automobile repair workshops' activities on the soil levels of PAHs, soil samples were taken from five different local automobile repair workshops (locations A-E) in Ado-Ekiti, Southwestern Nigeria.Ado-Ekiti is the capital of Ekiti state in Southwestern Nigeria and is a city on 7°37 0 15.9996 00 N and 5°13 0 17.0004 00 E. The samples were collected between September -December 2022.

Soil sampling of PAHs
The top layer of soil samples (0-15 cm) was collected around the automobile workshops using stainless hand held auger.The sample was collected in the afternoon during the rush hour at workshop.The samples were wrapped in foil papers after collection and transported to the laboratory for analysis.At the laboratory, the impurities were then removed by hand picking, and then sieved through a 2 mm sieve shaker.The samples were then oven dried using a universal drier at 80 o C to constant mass, and they were then kept in aluminum foil bags and stored at -20 °C in the freezer.

REVISED Amendments from Version 2
In the latest review by the reviewer, it was pointed out that sampling period and year/regimen used was not included in the manuscript.This has been included in revised version of the manuscript.It was also pointed out that source identification was not included.Authors, however made it clear that, the objective of the work does not involve source identification and we are sure that littering of the spent engine oil is the source of PAHs from observed in the soil.It was also pointed out that the health implications were not stated in regard to adverse health effects from exposure to humans, which has now been added to the manuscript and the permissible limit of ILCR and HQ has now been included in the revised manuscript.

Sample pretreatment
The soil samples were initially spiked with recovery standards of PAHs to monitor the integrity of the treatment.The sample was then extracted with 30 ml dichloromethane (DCM) using a Soxhlet extractor at 30°C for 8 hrs to enable PAHs trapped on the PUF disk and soil to dissolve in DCM.A clean-up procedure was done to remove unwanted compounds in the matrix by using silica-alumina column chromatography.The column was prepared by adding about 10 to 15 mm plug of glass wool to a chromatograph column and stuffing it down using a glass rod, then alumina and silica were added (1:2).The column packing was partially deactivated using a methanol-DCM solution (1:3) for better recovery.The sample extract was decanted into the column and eluted with 40 mL 1:1, DCM: hexane (Adesina et al., 2018).The extracted samples were then concentrated using a rotary evaporator (Büchi ® ), to gently remove the solvent from the extract in order to bring down the volume of extract to 5ml.The resultant extracts were later analyzed for PAHs.

PAHs analysis
Quantification of PAHs present in the sample was carried out using gas chromatography (GC) (Agilent 7890) with a mass detector (Agilent 5975) operated in a selected ion monitoring mode and using electron impact ionization (EI).The chromatographic column dimensions are 30 m Â 0.25 mm, and the internal diameter of 0.25 μm film thickness.The temperature program for the analysis was set as 90°C (1.0 min), 30°C/min, 250°C, 4°C/min, and 330°C (5 min).Determination of the concentration of PAHs was done using the internal standard method.The internal standards are naphthalene-d 8 , acenaphthene-d 10 , phenanthrene-d 10 , chrysene-d 12 , and perylene-d 12 , used to quantify the amount of PAHs in the extract.
Quality assurance/quality control (QA/QC) Apart from normal samples, field and laboratory blanks were also taken and treated the same way as the samples to ensure high integrity of the data.Soil blank samples were taken at locations far from the workshops, that have not been contaminated by automobile repair activities.Determination of instrument detection limits (IDLs) and method detection limits (MDLs) followed Norlock et al. (2011) procedure.Standard solutions were prepared for PAHs and six surrogates which were analyzed four times in the SIM mode using GC-MS.The PAH concentrations in each calibration solution were recalculated from the regression equation obtained using all six calibration standards.The average of the four replicates with the lowest detectable concentration was taken as the IDL.MDLs were calculated by multiplying the standard deviation of the replicates by the one-side t statistic at the 99%.Before the extraction, 20 ng of phenanthrene d10 recovery standard (RS) was used to spike the sample and the recovery range of the PAH was between 80% and 90%.Concentrations of PAHs in the field blanks were below the detection limit for all targeted compounds and no blank correction was carried out.

Health implications Toxic equivalent
The potential toxicity of the PAHs is calculated by multiplying the individual concentration, C, with the toxicity equivalence factor (TEF) (eq.i) (Van den Berg et al., 2006).
ILCR from ingestion, inhalation, and dermal contact with PAHs contaminated soil are calculated using eq.ii, iii, iv, respectively.Also, the non-carcinogenic associated risk is assessed using the hazard quotient index (HQ) which is the ratio of the estimated to the reference dose using eq.v (USEPA, 1991(USEPA, , 2011)).
C is the PAH concentration in the soil solid residue (mg kg À1 ).IR ing is the soil ingestion rate (100 mg d À1 for adults and 200mg d À1 for children).IR inh is the inhalation rate (20 m 3 /day was assumed for adults while 9.6 m 3 /day was assumed for children).Table 2 shows the exposure parameters and factors used for the study.

Health implications of PAHs in the soil sample
The TEQ approach is used to determine the toxic potency of complex mixtures, the ILCR is used to determine the probability of developing cancer as the result of exposure to a specific carcinogen, and the hazard quotient is the ratio of the potential exposure to a substance and the level at which no adverse effects are expected.Table 3 shows the TEQ of the PAHs in all the soil samples analyzed.DhA has the highest toxicity with a mean value of 6.68 μg/g.BaP, which is usually used as an indicator of PAHs contamination, has a toxicity of 5.4 μg/g.Other compounds with high TEQ are BbF and BkF with mean values of 1.73 and 1.75 μg/g, respectively.
The ILCR is used to assess the probability of developing cancer by having contact with soil from the automobile workshop.Table 4 shows the ILCR and HQ values obtained from the study.The ILCR values from accidental ingestion of soil from these workshops are 3:9 Â 10 À4 and 4:4 Â 10 À4 for adults and children respectively, while ILCR values for dermal contact with the soil are 6:9 Â 10 À4 and 5:4 Â 10 À4 : The value is higher than the permissible limit of 10 À6 stipulated by the World Health Organization.This implies that, with exposure to this soil for a particular period, there is a probability of developing cancer.However, the ILCR values for accidental inhalation of the soil are 3:2 Â 10 À8 and 8 Â 10 À9 , for adults and children respectively.This indicates the chances of developing cancer from inhalation of this soil    are slim.The combination of ILCR from accidental ingestion, inhalation and dermal contact gives 1 Â 10 À3 and 9:8 Â 10 À5 , for adults and children respectively.These values are higher than the permissible limit of 1 Â 10 À6 .
Table 4 also shows the hazard quotient, which is several folds higher than 1, the permissible limit, which implies that there is great non-carcinogenic risk associated with exposure to this soil.

Conclusion
This work studied the levels of polycyclic aromatic hydrocarbons in the soil around typical automobile repair workshops in Nigeria.The study also assessed the risks associated with human contact with the soil.The results showed the mean PAHs concentration at the sampling locations ranged from 5.58 -6.4 μg=g.The total toxicity equivalents at various locations range from 8.57 to 16.6 μg=g.The value of summation of ILCR and HQs from ingestion, inhalation, and dermal contact with PAHs contaminated soil is higher than the permissible limit stipulated by the World Health Organization of 1 Â 10 -6 .This study revealed the soil around automobile workshops is highly contaminated with PAHs and there is high risk associated with human contact with soil around automobile workshops in Nigeria.It is therefore recommended contamination of soil with spent oil around automobile repair workshop should be avoided.

Loai Aljerf
Faculty of Pharmacy, Al-Sham Private University, Damascus, Damascus Governorate, Syria General Assessment: The manuscript, "Levels of polycyclic aromatic hydrocarbon in the soil around typical automobile repair workshops in Nigeria," presents a valuable investigation into the contamination of soil with polycyclic aromatic hydrocarbons (PAHs) around automobile repair workshops in Nigeria.The study's objectives, methodology, and results are well-structured, and the manuscript provides a comprehensive overview of the problem, highlighting the risks associated with human contact with PAH-contaminated soil.
Strengths: 1) Relevance: 1-The study addresses a critical environmental and health issue in Nigeria, where the indiscriminate disposal of used engine oil and other petroleum products is a significant concern.2-The study's focus on PAH levels in soil around automobile repair workshops in Nigeria is timely and significant, given the potential health risks associated with PAH exposure.3-The study addresses a critical environmental issue, investigating the levels of PAHs in soil around automobile repair workshops in Nigeria.The findings have important implications for human health and environmental protection.
2) Methodology: 1-The authors employed a robust methodology, including dichloromethane extraction, silica-alumina gel open column chromatography, and gas chromatography-mass spectrometry (GC-MS) analysis, to determine the levels of PAHs in soil samples.2-The authors have provided a detailed description of their sampling, extraction, and analytical procedures, which demonstrates a good understanding of the research design and protocols.
3) Comprehensive analysis: The authors have conducted a thorough analysis of 16 different USEPA priority PAHs in soil samples, providing a detailed understanding of the distribution and concentration of these pollutants.4) Inclusion of quality control measures: The authors have incorporated quality assurance and quality control measures, such as the use of field and laboratory blanks, recovery standards, and instrument detection limits, to ensure the integrity of their data.5) Risk Assessment: The study's risk assessment, which included the calculation of hazard quotients (HQs) and incremental life cancer risks (ILCRs), provides valuable insights into the potential health risks associated with human contact with PAH-contaminated soil.
Weaknesses: Literature Review: 1-Although the authors provide a good overview of the problem, the literature review could be more comprehensive, covering recent studies on PAH contamination in soil and its health risks.2-Lack of contextualization: The study would benefit from a more extensive introduction, providing context on the environmental and health impacts of PAHs, as well as the current state of knowledge on this topic in Nigeria. 1.
Limited study area: The study is limited to a single city in Southwestern Nigeria, which may not be representative of the entire country or other regions.Future studies should consider expanding the study area to increase generalizability.

2.
Methodology: The authors employed a robust methodology, including the use of the Toxic Equivalence (TEQ) approach, ILCR, and HQ to assess the toxicity and carcinogenic potential of PAHs in the soil samples.

3.
Sampling Strategy: 1-The manuscript does not provide sufficient information on the sampling strategy, including the number of samples, sampling locations, and sampling depth.This information is essential to understand the representativeness of the results.2-The sampling strategy, although described in detail, may benefit from a more systematic approach, such as a stratified random sampling design, to minimize bias and ensure a more representative sample.

4.
Analytical methods: Despite, the authors have used a well-established analytical method (GC-MS), it would be beneficial to provide more information on the method's performance, such as the limit of detection, limit of quantitation, and precision.

5.
Quality Control and Assurance: The authors should provide more details on the quality control and assurance measures taken during the analysis, such as the use of certified reference materials, blanks, and duplicates.

6.
Data Presentation: 1-The results could be presented more effectively, with clearer tables and figures that facilitate the comparison of results across different sampling locations.

7.
Health implications: The calculation of TEQ and ILCR is a strength of the study, but the authors could further explore the health implications of their findings, including a discussion of the potential risks to local residents and workers in the automobile repair industry.

8.
The study would benefit from a more comprehensive comparison with existing literature on PAH levels in soil and their health implications, which would help to contextualize the findings and identify knowledge gaps.

9.
Discussion and Conclusion: The discussion and conclusion sections could be more comprehensive, highlighting the implications of the findings and providing recommendations for policymakers, automobile workshop owners, and the general public. 10.
Aspects Not Assessed: 1) Soil Characteristics: The manuscript does not provide information on the soil characteristics, such as pH, texture, and organic matter content, which could influence the fate and transport of PAHs in soil.
2) PAH Source Apportionment: The study does not attempt to apportion the sources of PAHs in the soil, which could be an important aspect to understanding the contamination pathways and identifying effective mitigation strategies. Recommendations: 1) Compare with existing literature: Conduct a more comprehensive review of existing literature on PAH levels in soil and their health implications to contextualize the findings and identify knowledge gaps.
2) Provide more context: Expand the introduction to provide a more comprehensive overview of the environmental and health impacts of PAHs, as well as the current state of knowledge on this topic in Nigeria.
3) Expand the study area: Consider increasing the study area to include multiple cities or regions to enhance the generalizability of the findings.4) Refine the sampling strategy: Develop a more systematic sampling strategy to minimize bias and ensure a representative sample.5) Provide more analytical method details: Include more information on the performance of the analytical method, such as the limit of detection, limit of quantitation, and precision.6) Enhance data analysis and presentation: Use more visual aids to present the results and facilitate easier interpretation and comparison of the data.7) Explore health implications further: Discuss the potential health risks associated with PAH exposure and explore ways to mitigate these risks, including policy recommendations and public health interventions.8) Enhance data interpretation and discussion: Expand the discussion to explore the implications of the findings, potential sources of PAHs contamination, and potential strategies for mitigation and remediation.9) Consider additional analyses: Consider conducting additional analyses, such as principal component analysis (PCA) or cluster analysis, to explore the relationships between different PAHs compounds and their potential sources.10) Include a section on limitations: Acknowledge the limitations of the study, including potential sources of bias, and discuss how these limitations may have impacted the findings.11) Consider including a section on policy implications: Discuss how the findings of this study can inform policy decisions related to environmental regulation, public health, and sustainable development in Nigeria.12) Provide more information about the workshops: Describe the characteristics of the automobile repair workshops, including their size, location, and operations, to provide a better understanding of the context in which the study was conducted.13) Explore the feasibility of remediation strategies: Discuss potential remediation strategies that could be employed to mitigate the impact of PAHs contamination in soil around automobile repair workshops.

Conclusion:
In conclusion, the manuscript presents a valuable study on the contamination of soil with PAHs around automobile repair workshops in Nigeria.Although, the study has some strengths, it also has some weaknesses that need to be addressed.By revising the manuscript to address these weaknesses and incorporating additional information on soil characteristics and PAH source apportionment, the authors can strengthen the study's contributions to the field of environmental science and public health.
in indigenous crab species and human health risk implications.Emerging Contaminants.2022; 8: 4. The conclusion is not adequate, as there are no recommendation or summary of the study in tandem to adults to children influences and how these automobile workshops can work effectively and dispose waste oils.

Response:
This has been modified in the revised manuscript. Thanks.
Competing Interests: No competing interests were disclosed.
Author Response 02 Nov 2023

Olusola Adesina
The statement "Few or no studies are available on the level of PAHs around automobile repair workshops, especially in Sub-Saharan Africa countries such as Nigeria" is not actually true because there are a lot of recent data on the subject matter such as Ekanem et al.

Response:
This part has been deleted in the revised manuscript.
The methodology is not explicit.In the soil sampling section, the apparatus used, sampling method and time of sampling is not mentioned.In the sample pretreatment section, quantity of solvent used for extraction is not mentioned.The references in the methods section are old.The author should review literature for the latest references. 1.

Response:
Authors appreciate this comment apparatus used for sampling has now been included, quantity of solvent used has now been included in the revised manuscript.Authors want to state that standard method for extraction and analysis of PAHs were duly followed 1.The last sentence of your introduction "Few or no studies are available... in sub-Saharan African countries" is not correct because a clear check on google scholar states that over 100 papers have been published and data are available in Nigeria and sub-Saharan countries.Remodifying that statement is advised.
2. The materials and methods are okay within the scope of the work.
3. The results and discussion does not provide a relationship and impact of these PAHs components to cause health based impact.More discussion is needed in relation to previous works available.
4. The conclusion is not adequate, as there are no recommendation or summary of the study in tandem to adults to children influences and how these automobile workshops can work effectively and dispose waste oils. Thanks.

Is the work clearly and accurately presented and does it cite the current literature? Partly
Is the study design appropriate and is the work technically sound?Yes

Are sufficient details of methods and analysis provided to allow replication by others? Yes
If applicable, is the statistical analysis and its interpretation appropriate?Yes Are all the source data underlying the results available to ensure full reproducibility?Partly Are the conclusions drawn adequately supported by the results?No [e] pyrene (BeP), benzo [a] pyrene (BaP), benzo [b] fluoranthene (BbF), benzo [a] anthracene (BaA), chrysene (CHR), benzo [k] fluoranthene (BkF), indeno[1,2,3-cd] pyrene (InP), and dibenzo [a,h] anthracene (DAh).
Distribution of PAHs based on aromatic rings and molecular weightPAHs are also classified based on the number of aromatic rings in each compound.The following ring compounds were present in this analysis: 2-ring PAHs (naphthalene), 3-ring PAHs (acenaphthylene, acenaphthene, fluorene, anthracene, and phenanthrene), 4-ring PAHs (pyrene, fluoranthene, chrysene, and benzo [a]anthracene), 5-rings PAHs (benzo [b] fluoranthene, benzo [a] pyrene, benzo [e] pyrenes, dibenzo [a,h] anthracene, and benzo [k]fluoranthene) and 6-rings PAHs (indeno[1,2,3-cd]pyrene).Figure1shows the distribution of PAHs based on the number of rings.5-ring PAHs have the highest percentage at 39.37%, followed by 4-ring PAHs at 23.77%.3-ring PAHs have a percentage contribution of 23.23%, 6-ring PAHs have a percentage contribution of 11.67%, and 2-ring PAHs have the lowest contribution at 1.97%.PAHs classification can also be by molecular weight: low molecular weight (LMW), consisting of 2-and 3-ring PAHs, middle molecular weight (MMW) consisting of 4-ring PAHs, and high molecular weight (HMW), consisting of 5-and 6-ring PAHs.Based on the results in Figure2, LMW accounts for 25.2% of the total PAHs, MMW accounts for 23.77% and HMW accounts for the highest percentage of 51.03%.

Figure 2 .
Figure 2. Distribution of PAHs based on molecular weight.

Figure 1 .
Figure 1.Distribution of PAHs based on the number of aromatic rings.
Table3shows the concentrations of PAHs at different automobile workshops.The results showed BbF and Bkf have the highest concentrations.The concentration of BbF ranged from 4.0 -31.03 μg=g with a mean concentration of 17.30 μg=g.BkF's concentration ranged from 4.12-27.92μg=g with a mean concentration of 17.53.These two compounds contribute 36 % of the total PAHs found in the soil.A high concentration of this compound implies that the source of the PAHs contamination of the soil is largely from petroleum products such as spent engine oil.Another compound with a high concentration is Fln, with a range of 3.96 -18.13 μg=g and a mean concentration of 10.81 μg=g, which is 11 % of the total PAHs.Fln is one of the Middle Molecular Weight PAHs formed by the combustion of petroleum products such as emission from the exhaust of the vehicle.This could be the reason for the high concentration of this compound around the soil analyzed.BaP concentration is usually used as an indicator of PAH carcinogenic activity due to its stability.The result obtained showed a BaP mean concentration of 5.39 μg=g with a range of 0.26-11.5 μg=g, which shows carcinogenic activities.The mean concentration of other carcinogenic PAHs observed in the soil is benzo [a] anthracene (2.45 μg=gÞ, chrysene (3.24 μg=gÞ, benzo [b] fluoranthene (17.30μg=gÞ benzo [k] fluoranthene

Table 2 .
Exposure parameters and factors used for the study.

Table 3 .
Mean concentration and TEQs of different PAH compounds in the soil.