Environmental impact and human health risk assessment of polycyclic aromatic hydrocarbons (pahs) in raw milk from free-ranging cattles in northwest nigeria

Graphical abstract

Polycyclic aromatic hydrocarbons (PAHs) were determined in raw milk from Northwest Nigerian cattle (cow, goat and sheep) as indicators in assessing the current pollution status of the region. In Northwest Nigeria, most animals are free rangers with the likelihood that PAHs have been taken up by these animals on the process of drinking PAHs contaminated surface waters, eating road-side grasses, and soils. A total of 42 composite milk samples were collected by milking 3–5 animals. PAHs were extracted after saponification of the milk fats through sonication using dichloromethane/hexane mixture and the five ascertained most abundant PAHs are: Phe > BaP > Chr > Fla. > Pyr using gas chromatography-flame ionization detector (GC-FID). The diagnostic ratios showed that combustion is the major pathway of PAHs emission in the study area which judging from the economic level of the area, could be attributed to vehicular exhaust emission, use of firewood, industrial fumes, waste incineration and bush burning at the beginning of the farming season. The values of PAHs showed no significant variations (p > 0.05), either between the milk types (cow, goat and sheep) or between the seven states’ milk samples, indicating a similar source of the PAHs. The European Food Safety Authority set 2 μg/kg w/w BaP as a marker for the occurrence of PAHs contamination and assessment of likely effect of carcinogenic PAH in foodstuffs, the value of BaP obtained was however much higher even though raw milk is about 80% water. Most PAHs have carcinogenic effects on humans and induce various cancers. Therefore there should be a reduction in the bush burning at the beginning of the farming season, use of firewood and other pronounced sources of PAHs in the region.

Introduction

Polycyclic aromatic hydrocarbons (PAHs) are organic compounds originating from natural and anthropogenic sources [2, 34, 51, 52]. PAHs emissions from anthropogenic sources significantly exceed natural sources [23]. Natural sources of emission are mainly from petroleum and coal exploration [23], while the anthropogenic emissions are mainly from the burning of organic material and fossil fuel. Because of PAHs widespread abundance, high toxicity, carcinogenic and mutagenic health effects [2, 28, 42] resulting from their ability to bind to deoxyribonucleic acid (DNA) [1], they are of great environmental concern [23, 25, 52]. Many studies involving different organs and PAH doses have shown a positive correlation between levels of polycyclic aromatic hydrocarbon-deoxyribonucleic acid (PAH-DNA) adduct formation [18, 43]. The ability of PAHs to induce short-term health effects in humans is not clear, but long-term exposure to PAHs may include decreased immune function, cataracts, kidney and liver damage, breathing problems, asthma-like symptoms, lung function abnormalities; and repeated contact with the skin may induce redness and skin inflammation [32]. Naphthalene, a specific PAH, can cause the breakdown of red blood cells if inhaled or ingested in large amounts. Studies have also shown routes of PAH exposure such as food, water, pharmaceutical products, tobacco smoke, etc. [10]. Food materials in the farm can be contaminated by PAHs through atmospheric deposition on soil, water deposition and transfer [2]. Other anthropogenic paths of PAHs include the burning of bushes and the spreading of contaminated sewage sludge on agricultural lands [11], surface layer (4–8 cm depth) close to an emission site such as a motorway. Concentrations in the range of 2–5 mg/kg can be found near the highways whereas, in unpolluted areas, the levels in the soil are in the range of 5–100 μg/kg [16]. Significant amount of PAHs is also emitted during the burning of automobile tires or creosote-treated wood [16]. Tobacco smoke also contains a certain amount of PAHs [16].

The release of PAHs into the Nigerian environment across the country arises mainly from heavy dependence on road transportation and petroleum as the major source of energy in the country [5]. Other sources of emission include oil spills, accidental discharges, and industrial activities. Certain types of cancer have been associated with unusually high exposure to PAHs in some industries such as coke, coal tar, and pitch as well as creosote, aluminium plants, mineral oil, soot and carbon black, iron and steel foundries, rubber manufacturing companies [47]. PAHs enter surface waters through discharges from industrial plants and wastewater treatment plants and they can be released to soils at hazardous waste sites and by escape from storage containers [6]. PAHs are present in the air as vapours or are stuck to surfaces of small solid particles [6]. PAHs can travel long distances in the air before they return to earth with rainfall [39]. Certain PAHs in the soil can contaminate underground water [36].

Milk and dairy products are important components of human diets and are widely consumed by children and adults especially growing children and elderly people [9, 15, 44]. In other words, milk could be contaminated by many hazardous compounds such as PAHs [6]. Contaminants such as PAHs have a lipophilic nature, accumulate in the food chain, mostly in foods with higher fat content [6]. Again, foods from animal sources are understood to be the major PAH sources, such as milk and milk-derived products [6]. Milk has been known to be contaminated [20, 33, 35]. Intake of contaminated feed and grass around industrialized areas or contaminated water and soil leads to a higher concentration of PAHs in milk from cattle [33, 35].

In Northwest Nigeria, most animals are free rangers with the likelihood that PAHs have been taken up by these animals on the process of drinking PAHs contaminated surface waters, eating road-side grasses, and soils in the region. Though PAHs have been assessed in selected commercial brands of milk [29] as well as in raw milk in other countries [24] but not raw milk from this region of which the current PAHs pollution status of the region is not known. Therefore, we sort to assess the PAHs contamination levels using cattle milks from the region.

Materials and method

Study area

The study area (Fig. 1) constitutes the seven states of the Northwest Nigeria which lies between latitudes 7°30’N and 20°N and longitudes 2°20′E and 10°E and with a landmass of about 216,694 km². The Fulani communities where the samples were collected are in Kazaure (KAZ), Roni (RRN), Gwiwa (GWW) in Jigawa, Zaria (ZRA), Makarfi (IGB), Soba (SBA) in Kaduna, Danbatta (DBT), Makoda (MKD), Dawakin tofa (DTF) in Kano, Birnin Kebbi (BKB), Jega (JEG), Maiyama (MYM) in Kebbi, Mai’adua (MDW), Zango (ZNG), Daura (DRA) in Katsina, Shagari (SGR), Yabo (YAB), Tambuwal (TWL) in Sokoto and Bungudu (BUG), Gusau (GUS), Maru (MAR) in Zamfara States. According to the 2006 national census [40], the area is the most populated among the six geopolitical zones in Nigeria with a total population of about 38,576,051 people. The economy is predominantly agrarian due to vast arable land, economically viable rivers and fine tropical climate. The area is the largest producer of grains and cattle in the country.

Fig. 1.

Map of the study area showing sampling stations

Sample collection and preservation

Samples of milk were obtained based on availability from Fulani herders who are the major cattle breeders in Nigeria, responsible for about 90% of fresh milk production in the country. These Fulani herders are predominant in Kazaure, Roni, Gwiwa, Zaria, Makarfi, Soba Danbatta, Makoda, Dawakin tofa, Birnin Kebbi, Jega, Maiyama, Mai’adua, Zango, Daura, Shagari, Yabo, Tambuwal, Bungudu, Gusau and Maru in North-western part of the Nigeria. Glass and plastic wares were soaked in dilute nitric acid overnight, washed with tap water, rinsed with de-ionized water, and finally with acetone. The glass wares were kept in an incubator to avoid contamination or dust [46]. All the chemicals were of analytical grade. Composite milk samples were obtained with the help of the Fulani breeders (Table 1), in 1 L plastic bottles previously soaked in dilute nitric acid, washed with tap water, and rinsed with acetone. A composite sample of each milk type was obtained by milking 3–5 lactating animals (cow-25, Goat-15 or Sheep-2). On the whole, 42 composite samples were collected, taken to the laboratory in ice box and analyzed within 4 days while refrigerated. Before sampling, 1 mL of nitric acid was added to the plastic bottles; the udder of each selected animal was washed with de-ionized water and stationed on the mouth of the sampling bottle. The samples were transported to the laboratory in an ice box and kept frozen until analyzed.

Table 1.

Fulani settlements/Communities where raw milk samples were obtained

State	Community	Milk Type and number of milk samples
		Cow	Goat	Sheep
Jigawa	Kazaure (KAZ)	1	1	–
	Roni (RRN)	2	–	–
	Gwiwa (GWW)	1	1	–
Kano	Danbatta (DBT)	1	1	–
	Makoda (MKD)	1	1	–
	Dawakin tofa (DTF)	1	1	–
Kaduna	Zaria (ZRA)	1	1	–
	Makarfi (MKF)	1	1	–
	Soba (SBA)	1	1	–
Katsina	Mai’adua (MDW)	1	1	–
	Zango (ZNG)	1	1	–
	Daura (DRA)	1	1	–
Kebbi	Birnin Kebbi (BKB)	1	1	–
	Jega (JEG)	2	–	–
	Maiyama (MYM)	1	1	–
Sokoto	Shagari (SGR)	2	–	–
	Yabo (YAB)		1	–
	Tambuwal (TWL)	2	–	–
Zamfara	Bungudu (BUG)	1	1	–
	Gusau (GUS)	1	–	1
	Maru (MAR)	1	–	1
Total		25	15	2

Sampling limitations

During the course of this research, several issues were encountered and this led to some limitations in the research. One among the limitations is the number of samples obtained for the study. The numbers of samples were limited resulting from the fewer number of lactating cattles at the time of sampling. Also, the inabilities of the cattle breeders to allow us express the udder of the cattles, even when we agreed for them to help in collecting the samples by themselves.

PAHs extraction

ASTM D3328–06 [7] and ASTM D3415–96 [8] modified methods were followed for the extraction of PAHs from the milk samples. Five gram (5 g) of homogenized milk sample was weighed into a 250 mL borosilicate beaker and 10 ml of 0.4 M NaOH was added. The beaker was covered with aluminium foil [31] and allowed to stand for 30 min in a water bath at 60 °C. 50 mL hexane:dichloromethane (3:1) mixture was added and the beaker was placed in a Sonicator (Shimadzu Model SALD-BS2, USA) to extract the PAHs for 2 h. The organic layer was separated using a separatory funnel and dried by filtering through a funnel packed with anhydrous sodium sulphate, into a 250 mL beaker. The filtrate was concentrated to 20 mL in a rotary evaporator (Searchtec RE52, England).

Clean up

For clean up, a 10 cm column of neutral alumina washed previously and cleaned with redistilled hexane was used. The extract was poured onto the alumina and eluted with the aid of redistilled hexane to remove the aliphatic profiles. The aromatic fractions were eluted with a 3:1 mixture of hexane and dichloromethane to recover the PAH fractions. The mixture was concentrated to 1 mL by a stream of nitrogen gas and gas chromatographic analysis coupled to flame ionization detector was performed on it [30].

Chromatographic analysis

The sixteen target PAHs were determined with a Hewlett Packed gas chromatograph, equipped with a flame ionization detector (GC-FID). The column used was HP-1932530, a non-polar, fused-silica capillary column (30 m length × 25 μm inner diameter × 0.25 μm film thickness). The injector temperature was programmed as follows: initial temperature 60 °C hejld for 5 min, raised to 250 °C at 15 °C/min for 14 min, followed by 3 min hold time; and finally raised to 320 °C at 10 °C/min with a hold time of 3 min. The initial oven temperature was 60 °C for 5 min, raised to 250 °C, first at a rate of 15 °C/min for 14 min, maintained for 3 min; then increase at 10 °C for 5 min, allowed for 4 min and finally elevated to 350 °C at 7 °C/min, held for 20 min. Nitrogen gas (ultra-pure, 99.99% purity) was used as the carrier gas at the flow rate of 1 cm³/min at a pressure of 30 psi. The retention time was used to characterize each PAH.

Recovery analysis

All PAHs and surrogate standards as well as other chemicals were of analytical grades and purchased from Sigma (Seelze, Germany). The silica gel used in column clean-up was purchased from Merck (Darmstadt, Germany). 5000 ppm stock solutions of surrogate standards (perylene D-12, phenanthrene –D, chrysene D-10 and acenaphthene D-12) were prepared and kept in a refrigerator. Mixed standard solutions comprising of equal concentrations of each surrogate were prepared in the range of 100 to 1000 mg/L. 1 mL of the 3 surrogates mixed standards were respectively used to spike a five gram portion of the milk sample for the recovery studies. The 3 spiked milk samples were then extracted using hexane/dichloromethane (3:1) mixture in a sonicator (Shimadzu Model SALD-BS2, USA). The extract was filtered into a 250 mL borosilicate beaker and dried by passing it through the funnel containing anhydrous sodium sulphate and concentrated in a rotary evaporator. It was further concentrated with a stream of nitrogen gas. The concentrated extract was cleaned using silica gel chromatography. Elution was achieved with redistilled hexane and concentrated to 1 mL under a gentle stream of nitrogen. The concentration of extracted PAHs was determined by GC-FID (Hewllet Parked Model 6890 (USA). Following good recoveries in the average of 97.46%, milk samples for analysis were extracted by sonication and determined using the same procedure. PAHs standards (100–1000 mg/L were used to recalibrate the GC-FID prior to the determination. A sample of 1 μL was taken by the auto sampler and injected into a capillary column, 30 m by 0.25 μm i.d. A splitless injection mode was used. Temperature programming was as follows: initial column temperature was 60 °C held for 5 min, followed by an increase of 15 °C/min for 14 min, and maintained for 3 min; second rate was an increase of 15 10 °C/min for 5 min and maintained for 4 min and finally increased to350 ^oC at 7 °C/min and held for 20 min at the maximum set temperature. Ultra-high purity nitrogen gas was used as the carrier gas at a flow rate of 1 mL/min.

Limit of detection (LOD) and limit of quantization (LOQ)

The limit of detection (LOD) is the lowest concentration leading to a signal-to-noise ratio of 3, whereas the limit of quantification (LOQ) is the concentration leading to a signal-to-noise ratio of 10 [4]. LOD was determined by continuous dilution and analysis of standard solution until the least concentration was obtained at the signal to noise ratio of 3. Likewise, LOQ was determined by continuous dilution and analysis of standard solution until the least concentration was obtained at a signal ratio of 10.

Statistical analysis

Analysis of variance (ANOVA), Duncan multiple range t-test and Pearson correlation were performed on the data at 0.05 significant level to understand the relationship between the determined PAHs and their possible source using SPSS version 20.00.

Toxic equivalency factor

Toxicity equivalency concentrations (TEQs) were calculated as the product of toxic equivalent concentration (TEF) values and concentrations of PAHs (Ci) as follows:

$$\mathit{\text{TEQs}}=\left(\mathit{Ci}\times \mathit{TEF}\right)$$ 1

PAH diagnostic ratios analysis

The sources of the PAH_S detected in this study were determined using various PAH diagnostic ratios; namely, Ant/(phe + Ant), Fla./(pyr + Fla), I[c,d]P/(I[cd]P + B[g,h,i]P), and B[a] A/B[a] A + Chr., Pry/B[a] P, B[a]B[a]P + Chr (full names in Table 3).

Table 3.

limits of detection and quantification

PAHs	LOD (mg/L)	LOQ (mg/L)
Naphthalene (Naph)	0.00000004	0.00000012
Acenaphthylene (Acenaphthy)	0.00000003	0.00000011
Acenaphthene (Acenaph)	0.00000003	0.00000023
Fluorene (Flu)	0.00000005	0.00000011
Phenanthrene (Phen)	0.00000006	0.00000014
Anthracene (Anth)	0.00000008	0.00000017
Fluoranthene (Fluoranth)	0.00000007	0.00000014
Pyrene (Pyr)	0.00000004	0.00000012
Benzo[a]anthracene (Baa)	0.00000003	0.00000024
Chrysene (Chry)	0.00000006	0.00000024
Benzo[b]fluoranthene (Bbf)	0.00000009	0.00000025
Dibenzo[a,h]anthracene (Dibenz)	0.00000006	0.00000017
Benzo[k]fluoranthene (Bkf)	0.00000008	0.00000016
Benzo[a]pyrene(Bap)	0.00000006	0.00000012
Benzo[g,h,i]perylene (Ben)	0.00000009	0.00000022
Indeno[1,2,3-cd]pyrene(Ind)	0.00000004	0.00000021

Results and discussion

Results

The mean recovery results for Acenaphthalene D-12, Phenanthrene D, Perylene D-12 and Chrysene D-12 are 98.03, 98.1, 96.2 and 97.5% respectively (Table 2). Samples were prepared based on these recoveries. The limit of detection and quantification were very low up to 0.0000003 mg/L and 0.000000011 mg/L respectively (Table 3).

Table 2.

Recovery results for 0.1, 0.5 and 1 ppm surrogates

Surrogate PAHs	0.1 ppm	0.5 ppm	1 ppm
Acenaphthalene D-12	97.7%	98.5%	97.9%
Phenanthrene D	97.5%	97.3%	99.5%
Chrysene D-12	98.5%	98.5%	98.7%
Perylene D-12	97.8%	94.1%	96.9%

The total PAHs in each cattle per state (Table 4) revealed that in Jigawa state, cow milk had the highest concentrations of all the PAHs except fluoranthene, chrysene, Benzo[b]fluoranthene and Benzo[k]fluoranthene which were found to be higher in goat milk, but in Kaduna state, goat milk recorded higher concentrations of the PAHs except acenaphthene, pyrene and Benzo[g.h,i]perylene which were higher in cow milk. All PAHs in cow milk were higher than their goat milk counterparts in Sokoto and Zamfara state, unlike Kano were Benzo[a]pyrene and Benzo[g.h,i]perylene were higher in cow milk while other PAHs were higher in goat milk. Goat milk in Katsina state had more of the PAHs except fluorene, Benzo[b]fluoranthene and Benzo[g.h,i]perylene which were more in cow milk. The total PAHs concentration followed the trend: Sokoto state (cow > goat) > Jigawa state (cow > goat) > Kebbi (sheep > goat > cow) > Kano (goat > cow) > Zamfara (sheep > goat) > Katsina (goat > cow) > Kaduna (goat > cow). Generally speaking, the PAHs concentrations in cow milk were higher than that of goat milk which was higher than sheep milk.

Table 4.

Total Concentrations of PAH in each Animal per state (mg/L)

PAH	Jigawa		Kaduna		Kano		Katsina		Kebbi			Sokoto		Zamfara
	Cow	Goat	Cow	Goat	Cow	Goat	Cow	Goat	Cow	Goat	Sheep	Cow	Goat	Cow	Goat	Sheep
Naphthalene	1.44	0.83	0.86	1.1	0.97	1.57	0.99	1.27	1.79	0.76	1.33	2.4	0.14	0.84	0.29	1.24
Acenaphthylene	6.9	3.87	4.5	5.62	6	7.26	3.64	5.65	31.69	4.19	6.99	6.52	1.23	7.89	3.96	10.77
Acenaphthene	11.99	7.47	11.74	9.454	3.33	18.97	6.26	9.45	8.34	8.25	18.24	30.64	6.62	9.93	4.10	13.25
Fluorene	3.42	0.84	0.82	0.96	2.03	7.08	1.4	1.11	0.32	0.49	5.63	6.69	0.61	1.81	0.30	3.25
Phenanthrene	126.7	83.9	81.5	93.5	107	119.9	77.3	120.5	30.66	42.8	131.4	155.3	36.9	92.6	39.4	101.2
Anthracene	27	4.02	7.68	9.92	6.26	7.44	9.72	27.06	5.06	3.72	4.48	10.02	4.92	14	2.43	6.01
Fluoranthene	54.95	62.5	57.53	75.9	75.2	78.5	33.9	58.35	29.6	31.1	82.5	47.48	35.5	42.52	15.4	61
Pyrene	97.7	51.1	19.78	0.13	60.23	65	38	73.41	19.03	12.87	72.6	138.3	13.9	44.3	6.13	63
Benzo[a]anthracene	111.5	30.9	17.87	20.74	58.75	67.82	25.58	56.04	15.28	10.01	67.8	99.51	11.7	35.92	4.71	47.4
Chrysene	69.58	81	63.1	91.1	60.2	70.8	51.28	83.5	26.7	41.6	91.2	130.7	35.0	69.6	18.1	85.8
Benzo[b]fluoranthene	37.62	72.2	42.7	45.6	18.62	35.15	51.33	33.71	15.21	32	82.5	63.61	36.5	36.13	14.5	71.1
Benzo[k]fluoranthene	63.28	66.5	12.79	20.52	57.93	58.72	16.45	53.71	13.49	8.22	67.4	93.1	15.9	34.69	3.69	54.3
Benzo[a]pyrene	113.6	61.5	83.8	98.1	72.1	71.7	68.7	99.5	57.7	67.5	76.3	152.8	31.8	89.5	35.1	78.1
Indeno[1,2,3-cd]pyrene	146.7	8.18	7.75	11.7	10.56	11.29	8.19	13.74	4.54	7.27	19	21.79	4.08	48.83	3.09	13.97
Dibenzo[a,h]anthracene	11.45	9.34	6.66	7.35	5.53	7.71	6.07	8.03	7.11	3.85	7.57	15.43	3.09	26.68	1.26	6.11
Benzo[g.h,i]perylene	13.22	7.32	3.46	0.04	17.16	12.02	7.66	7.41	3.67	11	11.24	17	2.30	14.63	1.12	8.18
Total	897.05	551.47	422.54	491.734	561.87	640.93	406.47	652.44	270.19	285.63	746.18	991.29	240.19	569.87	153.58	624.68

Table 5 presents the mean PAHs determined in the milk samples. All the sixteen USEPA priority PAHs were detected in all the samples analyzed. With exception of Kaduna state samples were Fluorene was observed to be the least concentrated PAH, Naphthalene was the least concentrated PAH in all the samples. The most abundant PAH in all the samples was Phenanthrene except in Kaduna state as well were Benzo[a]pyrene was the most abundant. The total concentration of PAHs was most in Jigawa (241.42 ± 106.32 μg/kg) while Kaduna had the least total PAHs (153.88 ± 56.45 μg/kg). There were no significant variations (p > 0.05).

Table 5.

Mean Concentrations and standard deviation of PAHs (μg/kg) in fresh milk

PAH	Jigawa	Kaduna	Kano	Katsina	Kebbi	Sokoto	Zamfara
Naphthalene	0.38 ± 0.08	1.83 ± 3.72	0.42 ± 0.19	0.38 ± 0.06	0.65 ± 0.46	0.42 ± 0.23	0.40 ± 0.20
Acenaphthylene	1.80 ± 1.09	1.69 ± 0.29	2.21 ± 1.07	1.55 ± 1.04	7.15 ± 11.22	1.29 ± 0.60	3.77 ± 1.88
Acenaphthene	3.24 ± 0.66	3.53 ± 1.94	3.72 ± 3.21	2.62 ± 2.04	5.81 ± 2.71	6.21 ± 1.70	4.55 ± 2.01
Fluorene	0.71 ± 0.38	0.30 ± 0.18	1.52 ± 1.06	0.42 ± 0.38	1.07 ± 1.35	1.22 ± 0.67	0.89 ± 1.00
Phenanthrene	35.10 ± 7.11	29.17 ± 11.29	37.8 ± 15.5	32.97 ± 15.25	34.14 ± 25.24	32.0 ± 8.00	38.87 ± 14.29
Anthracene	5.17 ± 3.88	2.93 ± 1.59	2.28 ± 1.10	6.13 ± 7.92	2.21 ± 0.89	2.49 ± 1.31	3.74 ± 2.69
Fluoranthene	19.58 ± 11.00	22.24 ± 9.61	25.6 ± 14.1	15.35 ± 14.69	23.87 ± 13.47	13.78 ± 11.86	19.82 ± 9.54
Pyrene	24.80 ± 6.33	3.30 ± 4.37	20.9 ± 15.2	18.57 ± 13.42	17.42 ± 14.92	25.37 ± 10.89	18.91 ± 12.30
Benzo[a]anthracene	23.73 ± 9.79	6.44 ± 3.06	21.1 ± 16.0	13.60 ± 12.61	15.52 ± 14.52	18.5 ± 14.1	14.67 ± 11.72
Chrysene	25.10 ± 12.73	25.70 ± 10.89	21.8 ± 8.71	22.46 ± 8.00	26.58 ± 15.16	27.6 ± 4.55	28.92 ± 13.27
Benzo[b]fluoranthene	18.30 ± 14.46	14.72 ± 0.74	8.96 ± 7.20	14.17 ± 10.46	21.62 ± 15.78	16.7 ± 11.7	20.29 ± 13.19
Benzo[k]fluoranthene	21.63 ± 12.28	5.55 ± 3.82	19.4 ± 13.20	11.69 ± 13.14	14.85 ± 14.83	18.2 ± 5.2	15.45 ± 11.84
Benzo[a]pyrene	29.18 ± 5.95	30.32 ± 3.10	23.97 ± 3.78	28.03 ± 9.86	33.58 ± 4.56	30.8 ± 1.6	33.78 ± 7.04
Indeno[1,2,3-cd]pyrene	25.81 ± 18.22	3.24 ± 0.87	3.63 ± 1.41	3.66 ± 1.19	5.14 ± 3.47	4.31 ± 0.50	10.98 ± 15.23
Dibenzo[a,h]anthracene	3.47 ± 1.53	2.34 ± 0.34	2.21 ± 1.10	2.35 ± 0.60	3.09 ± 1.69	3.09 ± 0.46	5.68 ± 8.12
Benzo[g.h,i]perylene	3.42 ± 0.83	0.58 ± 0.64	4.86 ± 3.00	2.51 ± 1.54	4.32 ± 2.99	3.22 ± 0.83	7.80 ± 9.12
Total	241.42 ± 106.3	153.88 ± 56.5	200.38 ± 105.8	176.46 ± 112.2	217.02 ± 143.3	205.2 ± 74.2	228.52 ± 133.4

Table 6 showed the descriptive statistics for total PAHs determined in the 42 milk samples obtained from the different animals (cow, goat, and sheep). It was observed that there are no significant variations (p > 0.05) in the concentrations of the PAHs. The mean distribution of PAHs in the three milk types are presented in figs. 2-4. In cow milk samples (Fig. 2), the order of abundance or rate of contamination were: phenanthrene > benzo[a]pyrene > chrysene > pyrene > benzoo[a]anthracene > fluoranthene > benzo[k]fluoranthene > benzo[b]fluoranthene > indeno[1,2,3,c-d]perylene. The order of abundance of the contaminants in goat milk followed the trend: phenanthrene > benzo[a]pyrene > chrysene > fluoranthene > benzo[b]fluoranthene > pyrene > benzo[k]fluoranthene > benzo[a]anthrancene > benzo[g,h,i]perylene (Fig. 3), while in sheep milk samples, the trend followed: phenanthrene > chrysene > benzo[a]pyrene > benzo[b]fluoranthene > fluoranthene > pyrene > benzo[a]anthracene > benzo[k]fluoranthene > benzo[g,h,i]perylene (Fig. 4). Generally, it was observed that phenanthrene was the most abundant PAH while the least was indeno[1,2,3c-d]pyrene. The observed abundance trend in the seven sampling states is phenanthrene > benzo[a]pyrene > chrysene > fluoranthene > pyrene > benzo[a]anthracene > benzo[b]fluoranthene > benzo[k]fluoranthene > indeno[1,2,3c-d]pyrene (Fig. 5).

Table 6.

Descriptive Statistics for total PAHs in the Seven States of Study

	N	Mean	Sum	Std. Deviation	Std. Error	95% Confidence Interval for Mean		Minimum	Maximum
						Lower Bound	Upper Bound
JIGAWA	6	238.5333	1431.20	36.76627	15.00977	199.9495	277.1172	183.80	291.60
KADUNA	6	153.84	923.10	35.07327	14.31860	117.0420	190.6563	109.70	206.66
KANO	6	200.4517	1202.71	90.55045	36.96706	105.4248	295.4785	120.50	326.40
KATSINA	6	176.4625	1058.78	90.66222	37.01270	81.3183	271.6067	106.50	351.50
KEBBI	6	217.	1302.00	122.16355	49.87306	88.7972	345.2028	109.13	380.31
SOKOTO	6	205.2013	1231.21	35.24244	14.38767	168.2167	242.1860	148.25	243.42
ZAMFARA	6	228.4983	1370.99	90.82653	37.07978	133.1817	323.8149	130.62	373.69
Total	42

Fig. 2.

Mean distribution of PAHs (μg/kg) in cow milk samples

Fig. 4.

Mean distribution of PAHs (μg/kg) in sheep milk samples

Fig. 3.

Mean distribution of PAHs (μg/kg) in goat milk samples

Fig. 5.

Mean distribution of PAHs (μg/kg) in the seven states

The results obtained showed that the populace residing in this area were at potential risk of exposure to the carcinogenic effect of various types of PAHs, which is in line with the observation of earlier studies [20, 33, 35] which demystified a probable risk for exposure to increased levels of PAHs for road users and urban dwellers of developing cities.

PAH emission profile of a given source depends on the processes producing the PAHs [37]. In high-temperature processes, such as the combustion of fuels in engines, higher molecular weight PAH compounds are emitted, but during low-temperature processes like wood burning, low molecular weight PAHs are usually formed [38]. At high temperatures, organic compounds are cracked to reactive radicals, which react to form stable PAHs during pyrosynthesis. These PAHs are less alkylated and their molecules contain more aromatic rings than petrogenic PAHs [26]. The diagnostic ratio indicates that the PAH in the study area is fuel combustion-related (Table 7). The Fla./(Fla + Pyr) and Ind/(Ind + Ben) ratios are more conservative than Ant/(Ant + Phe) and Baa/(Baa þ Chr), which are heavily sensitive to photodegradation. The Ant/(Ant + Phe) ratio is susceptible to environmental changes and its values for the identification of particular processes lie within a petty range, which makes it difficult to use [48]. This attributes the earlier mentioned heavy dependence on petroleum as the major source of energy in the country.

Table 7.

Diagnostic Ratios of PAHs in the Present Study

Diagnostic Ratio	Diagnostic ratio guidelines			Calculated ratios from this study
	Petrogenic	Fuel combustion	Coal, grass, wood-burning
				Jig	Kad	Kan	Kat	Keb	Sok	Zam
Ant/(Ant+Phe)	<0.1	<0.1 (0.16)	–	0.128	0.100	0.057	0.0157	0.061	0.072	0.088
Flu/(Flu+Pyr)	<0.4	0.4–0.5	>0.5 (0.52)	0.441	0.871	0.551	0.453	0.578	0.352	0.511
Baa/(Baa + Chry	<0.2	>0.35	0.2–0.35 (0.39)	0.430	0.581	0.428	0.587	0.543	0.572	0.585
Ind/(Ind] + Ben	<0.2	0.2–0.5	>0.5 (0.68)	0.485	0.200	0.491	0.377	0.368	0.401	0.337

Source: Domingueze, et al., [12]; Olajire and Brack, [41]

All the calculated toxicity equivalency factor values and toxicity equivalent concentration are less than 100 μg/kg (Table 8), which is stipulated by ATSDR for non-pollution and low toxicity [3], except for benzo[a]pyrene. The European Food Safety Authority (EFSA) in 2006, adopted BaP as a marker for the occurrence of PAHs contamination in food and assessment of the likely effect of carcinogenic PAHs and had set the maximum allowable value of 2 μg/kg w/w BaP for many foodstuffs including fats and oils [17]. This indicates that the PAHs in this area are of concern having exceeded the maximum allowable limit by ATSDR and EFSA.

Table 8.

TEFs of Total PAH Concentration

PAH	*TEF	Total	TEQ
Naphthalene	0.001	4.48	0.00448
Acenaphthylene	0.001	19.46	0.01946
Acenaphthene	0.001	29.68	0.02968
Fluorene	0.001	6.13	0.00613
Phenanthrene	0.001	240.10	0.24010
Anthracene	0.001	24.95	0.02495
Fluoranthene	0.001	140.26	0.14026
Pyrene	0.001	129.24	0.12924
Benzo[a]anthracene	0.100	113.60	11.36000
Chrysene	0.010	178.21	1.78210
Benzo[b]fluoranthene	0.100	114.75	11.47500
Benzo[k]fluoranthene	0.100	106.78	10.67800
Benzo[a]pyrene	1.000	209.63	209.63000
Indeno[1,2,3-cd]pyrene	0.100	56.77	5.67700
Dibenzo[a,h]anthracene	1.000	22.23	22.23000
Benzo[g.h,i]perylene	0.010	26.71	0.26710

*Vaneet [49]

Discussion

Northwest of Nigeria constitutes the major cattle breeding centre in Nigeria. The cattle breeders which are mainly Fulanis practice their trade by taking the animals from place to place for pasture and water. In this traditional method, the cattle are exposed to a high risk of contamination by environmental contaminants. As a result, such free-ranging animals could become veritable indicators of the level of contamination in the general environment. The PAHs concentrations indicate a range of 0.30–38.87 μg/kg which are much higher than values reported in raw milk in Turkey (from not detectable to 0.14 μg/kg w/w) according to Kazmaz, [31]. In Egypt, Gehad et al. [19] reported 0.083–182 μg/kg, while in Spain, Isabel et al. [27] reported a PAHs range of 0.89–18.18 μg/kg in various foodstuffs.

The PAHs determined in the analyzed milk samples did not show significant variations (p > 0.05) either between milk types or between the seven states. The spatial similarity is an indication that these compounds are widespread and evenly distributed in the study area. In the same vein, the lack of significant differences in the three milk types (cow, goat, and sheep) indicates that the animals were subjected to the same level of exposure in the same environment. However, there were slight variations in the abundance of PAHs in the three milk types. The most three abundant PAHs in cow and goat milk followed the order: Phe > BaP > CHR; but in sheep milk, CHR was more abundant than BaP. Moreover, PYR and BaA were among the five most abundant in cow milk while they did not occur so much in goat and sheep milk, instead, their positions were taken by Fla. and Bbf in the order of Fla. > BbF in goat and BbF > Fla. in sheep milk. Overall five most abundant PAHs were in the order: Phe > BaP > CHR > Fla. > PYR. The diagnostic ratios (Table 7) showed clearly that the major sources of PAHs compounds in the study area could be attributed to vehicular exhaust emission, domestic use of firewood, industrial fumes, waste incineration, and bush burning and petroleum. Kano and the Kaduna States is much more industrialized than the other states which have mainly agrarian economy, it is expected that industrial and vehicular emission as well as burning of urban wastes would likely be the major contributors to atmospheric PAHs; while in the other states, use of firewood and bush burning likely contributed most to the levels of PAHs. PAHs in the air eventually come down as dry and wet depositions.

In 2006, BaP has been adopted by the European Food Safety Authority (EFSA) as a marker for the occurrence of PAHs contamination in food and assessment of the likely effect of carcinogenic PAHs. The maximum allowable value of 2 μg/kg w/w was set for BaP in many foodstuffs including fats and oils [13]. However, no standard was set for milk, except that in 2008, a lower level of 1 μg/Kg w/w was set for infant foods including infant milk and follow-up milk [14]. The mean concentration of BaP in this study was in the range of 23.97–33.58 μg/kg w/w. EFSA, [13] further established the following eight PAHs for assessment of oral carcinogenicity in food due to PAHs contamination: BaA + CHR + BbF + BaP + IndP + BagA + Bghi, and suggested that these eight PAHs (PAHs8) or the subgroup of four (PAHs4): BaA + CHR + BbF + BaP or the subgroup of two PAHs (PAHs 2): CHR + BaP are better indicators of the occurrence of PAHs contamination in foods.

Nonetheless, PAHs are usually found as a mixture containing two or more of these compounds, e.g., soot [21]. However, PAHs affect organisms through various toxic actions [45]. The mechanism of toxicity is considered to be interference with the normal function of cellular membranes as well as with enzyme systems associated with the membrane [22]. The effects on human health depend mainly on the length and route of exposure, the amount or concentration of PAHs one is exposed to, and of course the innate toxicity of the PAHs [50]. The ability of PAHs to induce short-term health effects in humans is not clear, but long-term exposure to PAHs may include decreased immune function, cataracts, kidney and liver damage, breathing problems, asthma-like symptoms, lung function abnormalities; and repeated contact with the skin may induce redness and skin inflammation [32]. Naphthalene, a specific PAH, can cause the breakdown of red blood cells if inhaled or ingested in large amounts. With exposure to PAHs, the harmful effects that may occur largely depend on the way in which theindividual is exposed. However, it is not known which components of the mixture are responsible for the effects; and other compounds commonly found with PAHs may be the cause of these symptoms.

By applying the EFSA criteria, it was obvious that the values of PAHs determined in the raw milk samples far exceeded the value of 2 μg/kg, set generally as the allowable limit for PAHs in foods.

Levels of PAHs in the milk samples are high in comparison with the standard value (2 μg/kg) generally set for food by the European Food Safety Authority. The levels obtained for PAHs are good indicators of high contamination levels of these pollutants in the study area which would also be projected for the country as a whole which is atmospheric (ubiquitous). Sources of PAHs in the study area are mainly vehicular fumes, industrial discharges, bush burning, cooking smoke, incineration of waste, and petroleum.

Conclusion

Burning of materials is a common practice in the studied area and this has played out in the result of the milk analysis. Levels of PAHs in the milk samples are higher than the set value of 2 μg/kg generally for food by EFSA. The PAHs concentration levels obtained are good indicators of high contamination levels of these pollutants in the study area. Sources of PAHs in the study area are mainly vehicular fumes, industrial discharges, bush burning, cooking smoke, incineration of waste. The diagnostic ratios attribute the sources of PAHs to heavy dependence on petroleum being the major source of energy in the country. Milks from these sampled cattles in Northwest Nigeria are heavily contaminated with PAHs. The high content of PAHs in the raw milk sample is attributed to the levels in the environment. The animals being free rangers are exposed to these pollutants through eating of PAHs contaminated grasses and soil and so serve as good bio-indicators.

Declarations

Conflict of interest

There are no conflicts of interest.