A Comprehensive Analysis of Residual Antibiotics, Organochlorine Pesticides and Heavy Metals in Beef and Chicken

Abstract

Bangladesh is renowned for its agriculture-based economy. The farming of poultry and livestock is extremely popular in the country. Consumers in Bangladesh have a strong preference for beef and chicken meat, and these products are widely available. Toxic substances from various sources can permeate food animals through their feed and the environment, eventually making their way through the entire food chain via bioaccumulation and biomagnification. Additionally, these chemicals can enter the human body through the consumption of these foods, leading to significant public health concerns. This doctoral research focuses on identifying and measuring chemical pollutants such as leftover antibiotics, organochlorine pesticides, and heavy metals in beef and broiler chicken meat as well as liver samples, while assessing the health risks associated with each contaminant. Additionally, it aims to explore potential methods for reducing these contaminants. In this research, the presence of antibiotic residues (including tetracycline, oxytetracycline, chlortetracycline, amoxicillin, and patulin) in samples of beef meat, liver, and chicken meat, liver was examined utilizing reversed-phase High Performance Liquid Chromatography with a photodiode array detector (HPLC-PDA). Organochlorine pesticides and heavy metals were assessed using Gas Chromatography with an electron capture detector (GC-ECD) and Inductively Coupled Plasma Mass Spectrometry (ICP-MS), respectively. The beef and chicken meat and liver samples were extracted employing a modified version of the Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) method designed for antibiotics and organochlorine pesticides. A total of 180 biological samples, including beef meat, beef liver, and broiler chicken meat and liver, were collected for this study from local markets of Dhaka North and South City, Bangladesh. A total of one hundred and twenty samples (with 30 each of beef meat, beef liver, chicken meat, and chicken liver) were examined for each antibiotic. The correlation coefficients (r²) demonstrated a linear relationship, measuring 0.9978, 0.9980, and 0.9988 for oxytetracycline, tetracycline, and chlortetracycline, respectively, across six concentration levels. Matrix-matched calibration was performed for each matrix, resulting in linear correlation coefficients (r²) of 0.9980, 0.9990, and 0.9981 for beef meat; 0.9985, 0.9984, and 0.9973 for beef liver; 0.9988, 0.9982, and 0.9993 for chicken meat; and 0.9980, 0.9985, and 0.9991 for chicken liver concerning oxytetracycline, tetracycline, and chlortetracycline, respectively. The intra-day and inter-day recovery tests for each of the tetracyclines (TCs) were conducted, with relative standard deviation (RSD%) remaining below 10%. The Limit of Detection (LOD) for oxytetracycline, tetracycline, and chlortetracycline were recorded at 1.11, 1.15, and 1.19 μg/kg, respectively, with the associated Limit of Quantification (LOQ) being 3.17, 3.84, and 3.96 μg/kg. Residual oxytetracycline was V found and measured in eight beef liver samples, with levels varying from 86.76 to 368.97 μg/kg, all below the Maximum Residue Limit (MRL) set by Codex. However, one beef liver sample surpassed the MRL established by the European Union (EU), registering at 368.97 μg/kg. Oxytetracycline was detected in three chicken meat samples (220.94, 153.45, and 101.32 μg/kg), tetracycline was present in two samples (715.00 and 698.88 μg/kg), and chlortetracycline was found in one sample (677.35 μg/kg). Four of the positive samples exceeded the Codex recommended MRL, while six positive chicken meat samples were above the MRL limit set by the EU. The linear correlation coefficient (r²) for standard amoxicillin across six different concentrations was determined to be 0.9982. For amoxicillin in various samples, the respective linear correlation coefficients (r²) were 0.9979 for beef meat, 0.9980 for beef liver, 0.9995 for chicken meat, and 0.9981 for chicken liver. The intra-day and inter-day recovery experiments were conducted for amoxicillin, yielding a relative standard deviation (RSD%) within 10%. The limit of detection (LOD) and limit of quantification (LOQ) for the validated method were found to be 0.55 and 1.84 μg/kg for standard amoxicillin, respectively. The Codex Alimentarius Commission and the EU have established a maximum residue level (MRL) of 50 μg/kg for amoxicillin in beef and chicken meat and liver. Residual levels of amoxicillin were identified in seven beef meat samples, with concentrations ranging from 4.89 to 9.36 μg/kg, and in fifteen beef liver samples, which varied from 10.39 to 89.47 μg/kg. Among the beef liver samples, two exceeded the MRL, with values of 53.46 μg/kg (BL10) and 89.47 μg/kg (BL14). The linear correlation coefficient (r²) was determined to be linear at 0.9991 for standard patulin across six different concentrations. For patulin, the correlation coefficients (r²) were found to be linear at 0.9984, 0.9983, 0.9980, and 0.9990 in beef meat, beef liver, chicken meat, and chicken liver, respectively. An intra-day and inter-day recovery study was conducted for patulin, and the RSD% remained within 10%. The limit of detection (LOD) for the proposed method was established at 0.18 and 0.60 μg/kg for standard patulin. Residual levels of patulin antibiotic (as well as mycotoxin) were identified in twenty-five beef samples, with concentrations ranging from 47.72 to 193.91 μg/kg, and in eighteen chicken meat samples, with levels from 16.94 to 310.53 μg/kg. Patulin was detected in six beef liver samples, with concentrations between 43.31 and 166.91 μg/kg, and in eleven chicken liver samples, with levels ranging from 14.75 to 52.88 μg/kg. The health risk for each antibiotic was assessed for adults, and the hazard index (HI) was found to be less than 1. A total of one hundred and twenty samples (30 each of beef meat, beef liver, chicken meat, and chicken liver) were subjected to analysis for organochlorine pesticides (OCPs). To establish a standard calibration curve, six varying concentrations of a standard solution (comprising 20 OCPs) were injected into the gas chromatograph-electron capture detector VI (GC-ECD). The correlation coefficients (r²) showed a linear relationship, with values of 0.9999, 0.9994, 0.9990, 0.9988, 0.9980, 0.9997, 0.9991, 0.9994, 0.9993, 0.9996, 0.9983, 0.9978, 0.9995, 0.9985, 0.9990, 0.9997, 0.9981, 0.9991, 0.9994, and 0.9998 for alpha- BHC, gamma-BHC, beta-BHC, delta-BHC, heptachlor, aldrin, heptachlor epoxide, transchlordane, cis-chlordane, endosulfan I, 4, 4’-DDE, dieldrin, endrin, 4, 4’-DDD, endosulfan II, endrin aldehyde, 4, 4’-DDT, endosulfan sulfate, methoxychlor, and endrin ketone, respectively. Recovery experiments for intra-day and inter-day were conducted for 20 OCPs, and the relative standard deviation percentage (RSD%) remained within the acceptable limit of 20%. Out of thirty beef meat samples tested, alpha-BHC was detected in 11 samples (ranging from 1.01 to 62.49 μg/kg), gamma-BHC in 9 samples (with levels from 1.22 to 103.01 μg/kg), beta-BHC in 9 samples (from 1.13 to 8.94 μg/kg), and delta- BHC in 28 samples (with concentrations between 84.45 and 329.08 μg/kg). Heptachlor was found in 13 samples (ranging from 0.97 to 29.64 μg/kg), while aldrin was present in 20 samples (from 0.96 to 61.71 μg/kg). Heptachlor epoxide appeared in 28 samples (with levels from 57.87 to 304.25 μg/kg), trans-chlordane was found in 14 samples (from 0.86 to 7.90 μg/kg), and cis-chlordane in 18 samples (ranging from 0.61 to 6.22 μg/kg). Endosulfan I was detected in 26 samples (with concentrations between 1.23 and 22.86 μg/kg), and 4, 4´-DDE was found in 20 samples (ranging from 0.17 to 21.41 μg/kg). Dieldrin was present in 22 samples (from 0.47 to 36.50 μg/kg), endrin in 17 samples (ranging from 0.63 to 16.98 μg/kg), and 4, 4´-DDD was detected in 23 samples (with levels from 0.48 to 38.94 μg/kg). Endosulfan II was found in 27 samples (ranging from 1.61 to 187.29 μg/kg), and endrin aldehyde was present in 19 samples (from 0.98 to 24.70 μg/kg). Additionally, 4, 4´-DDT was detected in 15 samples (ranging from 0.24 to 76.81 μg/kg), endosulfan sulfate in 12 samples (with levels from 0.83 to 11.10 μg/kg), methoxychlor was found in 18 samples (ranging from 0.77 to 14.04 μg/kg), and endrin ketone was present in 4 samples (from 0.83 to 10.55 μg/kg), all measured in μg/kg. Among the thirty beef liver samples analyzed, alpha-BHC was detected in 28 samples (ranging from 17.40 to 340.42 μg/kg), gamma-BHC in 16 samples (from 1.75 to 15.79 μg/kg), beta- BHC in 16 samples (from 2.87 to 42.82 μg/kg), delta-BHC in 24 samples (from 2.24 to 26.41 μg/kg), heptachlor in 22 samples (ranging from 4.67 to 16.67 μg/kg), aldrin in 21 samples (from 0.66 to 20.93 μg/kg), heptachlor epoxide in 28 samples (ranging from 65.92 to 197.61 μg/kg), trans-chlordane in 16 samples (from 0.43 to 29.68 μg/kg), cis-chlordane in 10 samples (from 1.19 to 6.22 μg/kg), endosulfan I in 24 samples (ranging from 1.15 to 5.02 μg/kg), 4, 4´-DDE in 11 samples (from 0.10 to 3.38 μg/kg), dieldrin in 21 samples (ranging from 1.03 to 6.43 μg/kg), endrin in 20 samples (from 1.03 to 6.43 μg/kg), 4, 4´- DDD in 7 samples (ranging from 0.59 to 1.84 μg/kg), endosulfan II in 20 samples (from 0.22 to 25.55 μg/kg), endrin aldehyde in 26 samples (from 0.21 to 31.55 μg/kg), 4, 4´-DDT VII in 9 samples (ranging from 3.08 to 17.75 μg/kg), endosulfan sulfate in 2 samples (from 0.04 to 0.05 μg/kg), and methoxychlor in 8 samples (ranging from 0.26 to 11.67 μg/kg). In the thirty chicken meat samples, alpha-BHC was detected in 2 samples (at 1.00 and 2.87 μg/kg), gamma-BHC in 1 sample (at 4.94 μg/kg), delta-BHC in 27 samples (ranging from 5.91 to 201.65 μg/kg), heptachlor in 4 samples (ranging from 6.81 to 8.93 μg/kg), aldrin in 10 samples (from 1.60 to 33.93 μg/kg), heptachlor epoxide in 25 samples (ranging from 137.76 to 270.60 μg/kg), trans-chlordane in 13 samples (from 3.64 to 27.96 μg/kg), cischlordane in 7 samples (ranging from 0.44 to 2.24 μg/kg), endosulfan I in 25 samples (from 1.72 to 5.91 μg/kg), 4, 4´-DDE in 2 samples (at 0.41 and 1.69 μg/kg), dieldrin in 12 samples (ranging from 0.72 to 13.53 μg/kg), endrin in 10 samples (from 0.44 to 12.73 μg/kg), 4, 4´-DDD in 17 samples (ranging from 1.48 to 5.75 μg/kg), endosulfan II in 9 samples (from 0.97 to 56.96 μg/kg), endrin aldehyde in 16 samples (ranging from 0.93 to 5.03 μg/kg), 4, 4´-DDT in 7 samples (from 1.39 to 71.84 μg/kg), endosulfan sulfate in 6 samples (ranging from 0.21 to 3.14 μg/kg), and methoxychlor in 13 samples (from 1.59 to 5.95 μg/kg). In a study of thirty chicken liver samples, alpha-BHC was detected in 23 samples, with concentrations ranging from 2.12 to 159.13 μg/kg. Gamma-BHC was present in 3 samples, showing levels between 2.17 and 5.85 μg/kg, while beta-BHC was found in 10 samples, with values between 13.87 and 69.96 μg/kg. Delta-BHC was identified in 25 samples, displaying concentrations from 1.67 to 224.65 μg/kg. Heptachlor was detected in 3 samples, at levels ranging from 3.01 to 7.78 μg/kg. Aldrin appeared in 10 samples, with concentrations varying from 0.91 to 259.93 μg/kg. Heptachlor epoxide was found in 28 samples, with a range of 58.71 to 196.47 μg/kg. Trans-chlordane was present in 3 samples, with levels from 5.26 to 336.49 μg/kg, and cis-chlordane was identified in 4 samples, showing concentrations between 0.12 and 280.64 μg/kg. Endosulfan I was found in 26 samples, with values ranging from 1.23 to 22.86 μg/kg. The compound 4, 4'-DDE was detected in 9 samples (0.54 to 12.79 μg/kg), while dieldrin was present in another 9 samples, ranging from 0.84 to 9.09 μg/kg. Endrin was found in 9 samples, with concentrations between 1.24 and 40.29 μg/kg, and 4, 4'-DDD was detected in 10 samples, showing levels from 0.35 to 28.90 μg/kg. Endrin aldehyde appeared in 18 samples, with concentrations ranging from 1.12 to 307.65 μg/kg. 4, 4'-DDT was identified in 12 samples, at levels between 1.37 and 29.80 μg/kg, while endosulfan sulfate was found in 5 samples (0.97 to 204.26 μg/kg) and methoxychlor in another 5 samples, ranging from 0.72 to 5.68 μg/kg. Endrin ketone was present in 4 samples, with concentrations from 1.95 to 4.47 μg/kg. The health risks associated with these pesticides were assessed, revealing that the hazard indices for delta-BHC, heptachlor, aldrin, heptachlor epoxide, endrin, endrin aldehyde, and endrin ketone exceeded 1 for both adults and children. VIII A total of 120 biological samples were examined, comprising 30 each of beef meat, beef liver, chicken meat, and chicken liver for ten heavy metals. The samples underwent digestion with concentrated HNO3 and H2O2. In the ICP-MS, four varying concentrations of a standard solution (a mix of the ten metals) were injected to establish calibration curves. A linear correlation coefficient (r²) was determined for chromium (Cr), nickel (Ni), lead (Pb), cadmium (Cd), arsenic (As), manganese (Mn), cobalt (Co), copper (Cu), zinc (Zn), and selenium (Se), with values of 0.9988, 0.9989, 0.9990, 0.9991, 0.9991, 0.9994, 0.9991, 0.9994, 0.9998, and 0.9993, respectively. The Limits of Detection (LOD) and Limits of Quantification (LOQ) were calculated for each of the ten metals. Copper (Cu), manganese (Mn), cobalt (Co), zinc (Zn), and selenium (Se) were detected and quantified across all beef and chicken meat and liver samples. The concentration of Cu ranged from 2.82 x 10⁻⁵ to 1.24 x 10⁻⁴, 0.09 to 0.23, 0.93 to 4.68, and 3.94 to 12.52; Mn levels ranged from 1.77 x 10⁻³ to 6.69 x 10⁻², 7.62 to 57.28, 0.005 to 0.05, and 0.04 to 0.10; Co concentrations were between 2.89 x 10⁻³ to 2.95 x 10⁻², 5.33 to 11.82, 0.03 to 6.28, and 2.95 to 9.18; Zn levels varied from 0.11 to 0.55, 81.03 to 203.27, 23.70 to 41.20, and 41.26 to 152.73, while Se was found in a range of 5.02 x 10⁻⁵ to 4.40 x 10⁻⁴, 0.38 to 1.54, 0.48 to 0.81, and 1.12 to 2.54 in all beef meat, beef liver, chicken meat, and chicken liver samples, respectively. Cr concentrations were found to be between 2.63 x 10⁻⁵ to 3.20 x 10⁻⁴, 0.06 to 0.20, 0.58 to 1.68, and 0.32 to 1.42 in 30 beef meat, 9 beef liver, 30 chicken meat, and 30 chicken liver samples. Ni levels varied from 2.43 x 10⁻⁵ to 9.86 x 10⁻⁵, 0.04 to 0.92, 0.10 to 1.45, and 0.06 to 42.63 in 30 beef meat, 29 beef liver, 29 chicken meat, and 30 chicken liver respectively. Pb was found at concentrations ranging from 1.12 x 10⁻⁶ to 7.46 x 10⁻⁴, 0.05 to 24.36, 0.05 to 1.71, and 0.03 to 5.28 in 30 beef meat, 29 beef liver, 26 chicken meat, and 22 chicken liver. Cd levels were detected between 4.82 x 10⁻⁶ to 5.61 x 10⁻⁴, 0.04 to 1.17, 0.14 to 0.18, and 0.04 to 0.24 in 30 beef meat, 12 beef liver, 2 chicken meat, and 3 chicken liver respectively. As concentration ranged from 1.34 x 10⁻⁶ to 4.41 x 10⁻⁴, 0.01 to 0.69, 0.005 to 0.52, and 0.005 to 10.79 mg/kg in 30 beef meat, 28 beef liver, 19 chicken meat, and 24 chicken liver samples respectively. The health risk assessment (EDI, THQ, HI, TCR) for heavy metals was conducted for both adults and children. Chemical pollutants can build up in the fatty tissues of beef and poultry, potentially changing the fatty acid profile and causing genetic mutations. Therefore, the fatty acid composition and overall fat content were examined using gas chromatography with a flame ionization detector (GCFID). The average fat content (%) calculated was 1.57, 6.19, 0.78, and 2.95% for beef meat, beef liver, broiler chicken meat, and liver, respectively. This research underscores comprehensive evidence of the occurrence of antibiotic, pesticide, and heavy metal residues in raw beef and broiler chicken meat and liver in Bangladesh. Although most samples were within internationally accepted limits, several exceeded Codex and EU MRLs, posing potential public health concerns.