Evaluation of Dioxin/Furan and Elements in Poultry from Zarqa Governorate, Jordan

This study is the first to determine the concentration for 17 congeners of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDD/Fs) and element contamination in poultry that is close to petroleum refinery at Al-Hashemiya Municipality, Zarqa Governorate, Jordan. Ten different samples (chicken) were collected to cover ten different locations of poultry farms in Al-Hashemiya Municipality. These locations are considered polluted areas as a result of exhaust gases produced from the refinery. The 17 PCDD/Fs congeners and elements of Pb, Cd, As, Zn, Cu, Se, Hg, Cr, and Ni were determined for three parts of each sample (liver, muscle, and gizzard). All samples were analyzed for PCDD/Fs after a Soxhlet extraction procedure and cleanup by column chromatography; then, all compounds were identified and determined using GC-MS techniques. The elements were analyzed after digestion and measured using an inductively coupled plasma optical emission spectrometer (ICP-OES) and validated with the Lab Mix24 RM NCS ZC73016 reference material. The highest total sum concentration of PCDD/Fs was found in liver samples to be 214.07 ng/kg (dry weight), while the highest sum of toxicity equivalent to PCDD/Fs of 22.54 ng TEQ/kg was found in gizzard samples. For element concentrations, the highest total sum of 16.89 mg/kg (dry weight) was found in liver samples. The concentration level of the elements of Se, Hg, Cr, and Ni for all parts of the chicken was within an acceptable range according to Jordanian standards and therefore the measured level of heavy and trace elements in the poultry samples (chicken) does not pose a danger to public health. The chickens found in poultry farms near the refinery are more likely to contain a higher concentration of PCDD/Fs congeners due to exhaust gas exposure.


Introduction
Various industrial activities contribute to widespread contamination of the environment by diferent classes of persistent organic pollutants (POPs), such as polychlorinated dibenzo-p-dioxins and furans (PCDD/Fs).Among these, the oil industry has shown to have a considerable environmental impact on diferent ecosystems [1].PCDD/Fs compounds can bioaccumulate and biomagnify in the food chain and the human body.Some of these compounds, such as PCBs, may persist in the environment for periods of years and may bioconcentrate by factors up to 70,000 times [2].PCDD/Fs belong to the most toxic environmental organic pollutants and can cause serious public health problems [3,4].Te measurements of industry-posed PCDD/Fs pollutants were considered in many studies, and the negative health efects have been presented [3,5].Te qualitative and quantitative evaluation of PCDD/Fs in urban air and soils has been studied, and results demonstrated the need of decreasing both the concentration and toxicity of PCDD/Fs [6].
In addition, the concentration levels of elements, namely, lead (Pb), cadmium (Cd), arsenic (As), zinc (Zn), copper (Cu), selenium (Se), mercury (Hg), chromium (Cr), and nickel (Ni), should always be monitored to assess the risk to human health and dietary intake.Tese elements can be taken up from soil to plants to animals, and fnally to humans.Poultry is a major food source in the Middle East, especially in Jordan, and considered as a signifcant source of human exposure to elements.Food rarely causes acute intoxications, but As, Pb, and Cd accumulate in the body with possible subclinical adverse efects [7].According to the World Health Organization (WHO), low levels of some elements such as Hg, Cd, Pb, and As can cause diseases in humans (WHO 1977(WHO , 1992(WHO , and 2001)).Trace elements, such as manganese (Mn), cobalt (Co), selenium (Se), molybdenum (Mo), and antimony (Sb), may also be present in high concentrations in food and in excessive dietary intake.Te dietary intake of metals in the diet of major food products was determined by the National Library of Medicine [8], and these elements can also have adverse efects on health [9,10].

Standards and Internal Standards. Standard solutions:
for the metal calibration curve, the multielement standard solutions (100 μg/mL) (PerkinElmer, Waltham, MA, USA) were used, while for validation, the certifed reference material NCS ZC73016 (Lab Mix24, Germany) was used.While AOAC 2015.01 method was used for element analysis.Although the 1613 EPA method stock solution of PCDD/Fs and the internal standard for PCDD/Fs (1,2,3,4-tetrachloro [ 13 C 12 ] dibenzo-p-dioxin), 200 ng/mL, were purchased from Willington Laboratory, Canada.

Instrumentation.
Inductively coupled plasma optical emission spectrometer (ICP-OES) (PerkinElmer) was used for the element determination and interfaced with the computer for calculations, integration, and calibration.A microwave digestion system (Bioevopeak, USA) was used for sample digestion.Te instrument gas chromatographmass spectrometer (GC/MS), type Shimadzu QP 2010, was used for the determination of OCPs, PCBs, and PCDD/Fs.Tis instrument was equipped with a split-splitless injector, auto sampler, and quadruple mass selective detector.GC/MS was interfaced with computer for calculations, integration, and calibration.
Analytical balance, hot plate with stirrer, drying oven, water bath, and sonicator were used.All glassware was washed with detergent and water, soaked with 2% (v/v) nitric acid overnight, and rinsed with water and dried.

Sampling and Study Area.
Ten samples were collected from ten poultry farms located in diferent places in the Al-Hashemiya Municipality, which is considered a polluted area due to the exhausted gases produced from the refnery.Elements and PCDD/Fs were determined for the liver, muscles, and gizzard of each chicken.Te locations and surroundings of the samples are shown in Figure 1.

Sample Preparation.
For elements: 0.25 g wet sample was introduced into a microwave digestion vessel, then 4 mL of concentrated HNO 3 and 1 mL of 30% hydrogen peroxide were added to each digestion vessel.Te samples were digested at a minimum temperature of 190 °C for a minimum time of 10 min gradually until the vapor and acid fuid inside the vessel became clear.Te vessel was cooled to room temperature and the content was poured into an acid-free 50 mL HDPE centrifuge tube and diluted with deionized water, acidifed with 1% (v/v) HNO 3, to a fnal volume of 20 mL.Pb, Cd, As, Zn, Cu, Se, Hg, Cr, and Ni were analyzed using ICP-OES using an internal standard solution of 40 μg/ mL rhodium (Rh).Te dynamic range for the calibration curve was obtained with concentrations between 0.05 and 20 µg/mL.Recoveries were tested on spiked samples at 100, 200, and 500 µg/kg.
For PCDD/Fs [2]: 1.0 g of each homogenized sample was weighed in a Soxhlet thimble, spiked with 200 μL IS (100 ppb), and then extracted in a Soxhlet apparatus with 200 mL toluene for 16 h.Te volume of the extracts was reduced to 3 mL and evaporated using the rotary evaporator at 40 °C and 240 mbar.Te residues were cleaned up on three columns as follows: Macro alumina column: A glass column (30 × 0.8 cm) was flled with 25 g of Alumina B, super I (basic), and 10 g of sodium sulfate.Te residues from the extraction step were added and washed with 80 mL n-hexane, 100 mL n-hexane/ dichloromethane (98 : 2%), and 180 mL n-hexane/ dichloromethane (50 : 50%) successively.Te last fraction was evaporated using a rotary evaporator (40 °C, 280 mbar) at a few milliliters.
Mixed silica gel column: A glass column (30 × 0.8 cm) was flled from the bottom upward with 10 g of anhydrous sodium sulfate, 2 g of neutral silica gel, 10 g of acidic silica gel, 2 g of neutral silica gel, 5 g of basic silica gel, and 2 g of neutral silica gel.Te residues from the last step were added, and the column was washed with 250 mL n-hexane.Te eluates were evaporated using a rotary evaporator at 40 °C and 280 mbar to 1-2 mL.
Mini alumina column: A small glass column (10 × 0.8 cm) was flled with 4 g of basic alumina and 2.5 g of sodium sulfate and then the residues from the last step were added.Te column was washed with 20 mL of n-hexane/dichloromethane (98 : 2) and then with 35 mL n-hexane/dichloromethane (1 :1).Te last fraction was evaporated using the rotary evaporator to 1-2 mL at 40 °C and 280 mbar.Te last few milliliters were evaporated using a gentle stream of nitrogen, and the residues were dissolved in 200 µL n-nonane.

Method Validation
3.1.Detection Limits and Limits of Quantitation.Te method limit of detection (MLOD) was obtained for each element taking 0.25 g of blank sample and spiked with multistandard solution and measured 10 times (n � 10).While for PCDD/ Fs, by taking 1.0 g of dry weight of blank chicken samples and mixed with 5.0 g of anhydrous sodium sulfate and spiked with the standard solution.From the average value, the method limit of detection (MLOD) and method limit of quantitation (MLOQ) were calculated when S/N ratio is equal to 3 and 10, respectively.Tables 1 and 2 show the MLOD and MLOQ for elements and PCDD/Fs, respectively.

Results and Discussion
4.1.Elements.Zn was found in high concentrations for all samples and the highest sum was 53.37 mg/kg in muscle samples.In liver samples, the highest sum of elements was found to be 16.89 mg/kg for sample S6, and the ranges of element concentration (in mg/kg) are as follows: Pb (0.05-0.24),Cd (0.03-0.04),As (0.04-0.06),Zn (1.42-5.85),Cu (1.46-4.00),Se (0.37-0.95), Hg (0.02-0.53),Cr (0.42-3.12), and Ni (0.94-4.42).Table 4 shows the average concentration and the sum of all elements for each liver sample, while the distribution of the elements is shown in Figure 2. In muscle samples, the highest sum of elements was found for sample S3 16.85 mg/kg, and the ranges of element concentration (in mg/kg) found in Pb (0.05-0.23),Cd (0.03-0.05),As (0.04-0.07),Zn (2.44-6.69),Cu (1.74-4.04),Se (0.40-0.94),Hg (0.10-0.54),Cr (0.39-2.31), and Ni (0.70-5.40).Te highest sum of all elements found for Zn in muscle samples 53.37 mg/kg.Table 5 shows the average concentration and the sum of the nine elements for each muscle sample, while the distribution of the elements is shown in Figure 3.
Te calculated MLOD and MLOQ for Pb, Cd, As, Zn, Cu, Se, Hg, Cr, and Ni are between 0.03 and 0.1 (mg/kg) and between 0.11 and 0.35 (mg/kg), respectively, as shown in Table 1, while the precision was found to be between 5.07%     Journal of Analytical Methods in Chemistry and 9.75%, which falls within the acceptable values for trace analysis (CV < 15%) [22].
All recoveries for all elements analyzed were found to fall within the reported acceptable range of 80-120% [22].In liver samples, recovery values were as follows: 89. 8 in all samples were compared to the Lab Mix24 NCS ZC73016 reference material and were found to be within <10%, as shown in Table 3, indicating the validity of the analysis method and the results obtained.
Table 7 shows a comparison between the level of elements found in chicken meat from Jordan and other countries [9,[23][24][25].
Table 8 shows the average concentration of all congeners for each liver sample, while the distribution is shown in Figure 5.
Te calculated MLOD and MLOQ for PCDD/F congeners are between 0.02 and 0.03 (ng/kg) and between 0.07 and 0.10 (ng/kg), respectively, as shown in Table 2   8 Journal of Analytical Methods in Chemistry precision was found to be between 5.07% and 9.75%, which falls within the acceptable values for the trace analysis (CV < 15%) [22].
Te TEQ (ng/kg) for PCDD/Fs congeners was found as follows: in liver samples, the highest sum was found for sample S6 20.75 ng TEQ/kg.For muscle samples, the highest sum was 12.58 ng TEQ/kg for sample S7.While, in gizzard, the highest sum of TEQ values for PCDD/Fs congeners was found to be 22.54 ng TEQ/kg for sample S3 and was considered the highest between all samples.Tables 11-13 show the average TEQ concentrations (ng TEQ/kg) for liver, muscle, and gizzard samples, respectively.Journal of Analytical Methods in Chemistry   Journal of Analytical Methods in Chemistry  Journal of Analytical Methods in Chemistry

Conclusions
Te average concentration levels of Se, Hg, Cr, and Ni elements in samples S3, S5, and S6 show a higher value in all parts of chicken, while the average concentrations for PCDD/Fs congers for samples S6, S2, and S4 found the highest sum concentrations due to the proximity of the farms to the refnery and lies to the northeast of wind direction.Te level of pollution obtained in chickens in poultry farm samples, for metals, according to Jordanian standards of Codex General Standard for Contaminants and Toxins in Food and Feed (Codex Stan 1993-1995) did not pose a danger to public health in the Al-Hashemiya Municipality in Jordan with a range of 0.01-1.0mg/kg for poultry meat.Te measured PCDD/Fs pollution level, expressed as the sum of all 17 congeners, against the WHO-TEQ approved limit (< 1.0 μg/kg) shows chickens close to the refnery, where more exhaust gas exposure, contain a the higher concentration of congeners.Te preliminary results of this study will be available to environmentalists ofcials in Jordan to reduce the emission of exhaust gases from the petroleum refnery.Te fndings of this work suggest that more studies should be conducted within the study area to mitigate the health efect on people who live around the refnery region.

Data Availability
Te data that support the fndings of this study are available within the article.

Figure 1 :
Figure 1: Sample locations and environment.

Table 1 :
Method limit of detection (MLOD) and method limit of quantitation (MLOQ) for elements a .
a Concentric nebulizer with axial view optical system.

Table 3 :
Determination of elements in chicken with comparison with reference material (Lab Mix24 NCS ZC73016).

Table 5 :
Te average concentration and the sum of elements in muscle samples (mg/kg).

Table 7 :
Comparison between the levels of elements in chicken meat found in Jordan and other countries.

Table 11 :
TEQ ng/kg for PCDD/Fs congeners in liver samples.
Te number given in bold is to show the highest sum between all.

Table 12 :
TEQ ng/kg for PCDD/F congeners in muscle samples.Te number given in bold is to show the highest sum between all.

Table 13 :
14Q ng/kg for PCDD/F congeners in gizzard samples.Te number given in bold is to show the highest sum between all.14Journal of Analytical Methods in Chemistry