Since their discovery and development, antibiotics have been frequently used as veterinary and human medicines to treat various infectious diseases (Samanidou et al., 2007). They can either have activity against a broad spectrum of microorganisms or can be active against a small extent of specific organisms (Samanidou et al., 2006; Samanidou and Evaggelopoulou, 2015). As veterinary drugs, antibiotics play a vital role in the maximal production of industrial farm animals and in reducing the morbidity and mortality rates among livestock (Page and Gautier, 2012). Ciprofloxacin (CIP), tetracycline (TC), and oxytetracycline (OTC) are the most frequently applied veterinary drugs in Bangladesh. The main causes of antibiotic residues include the unauthorized use and unrestricted administration of veterinary medications to healthy animals, as well as failing to consider the withdrawal period, not seeking professional advice, using medications beyond the recommended dosage, administering medications frequently, and increasing dosing (Baran et al., 2011; Croubels and Daeseleire, 2012; Bitas et al., 2018). These residues have the potential to be hazardous to human health and can trigger allergic reactions. In addition, the prolonged accumulation of CIP, TC, and OTC residues can lead to the growth of microorganisms that cause antibiotic resistance (Garcia et al., 2004; Fan et al., 2020). To ensure the reduction of the quantity of veterinary drugs from the edible parts of farm animals, a minimum withdrawal time between the administration of antibiotics and the slaughter has been established (Croubels and Daeseleire, 2012). The European Union (EU) has established maximum residual limits (MRLs) for residues of veterinary pharmaceuticals, including CIP, TC, and OTC, in animal tissues and derivative foods entering the human food chain in Council Regulation EEC 37/2010 and its later modification [Commission Regulation (EU) No. 37/2010 of 22 December 2009, L.15/1-72]. Monitoring is therefore required to ensure that antibacterial agents are not present in quantities that could endanger the health of the general public. Recently, multi-residue techniques have been created for a variety of residue classes observed in animal tissues, including fluoroquinolones (Schneider et al., 2005; Verdon et al., 2005) and TCs (Anderson et al., 2003; Cinquina et al., 2003a). HPLC has a reputation for being a reliable and highly selective separative tool (Almalki et al., 2021) for detecting antibiotic residues. Although a lot of initiatives have been taken from government regulatory bodies, the misuse of antibiotics is still occurring. The presence of antibiotic residues in poultry food has become a matter of concern as its resistance is considered as a health risk. Therefore, annual monitoring is mandatory. The aims of the present study were to determine CIP, TC, and OTC residues from poultry samples collected from Tangail Sadar Upazila using HPLC and to assess their risks to consumer health.

The present study used SPE in sample preparation, and this required conditioning in order to moisten the sorbent and permit the liquid to penetrate the pores before the sample is introduced (Lu et al., 2019). In our approach, the wavelength used for CIP was consistent with Yorke and Froc (2000). For TC and OTC, a silica-based C₁₈ analytical column, UV detection at 355 nm, flow rate of 1.0 ml/min, SPE C₁₈ cartridges, and a 0.22-μm syringe filter were used following De Ruyck et al. (1999), Miranda et al. (2009); Shalaby et al. (2011), and Zhao et al. (2010). Christodoulou et al., (2007) found 53.5%–83.7% CIP recovery in egg yolk using different extraction solvents. A higher recovery of CIP was obtained in chicken muscle and liver by changing the matrix, extraction solvent, and their ratio (Posyniak et al., 2001). The recovery of OTC reported by Olatoye and Kayode (2012) was 80.5%–87.8%, which was somewhat lower, while Guo et al. (2012) reported 80.9%–119.5% recovery for both TC and OTC, which was higher than our findings. Strong acids and precipitating agents are recommended for deproteinization to improve the extraction of antibiotics from complex matrices as TCs readily bind to proteins (Pérez-Rodríguez et al., 2018). Our recovery values for the edible parts of chicken were comparable to those obtained by Blasco et al. (2009); Yu et al. (2011), and Masawat and Slater (2007). The LOD and LOQ values for TC and OTC were comparable to those obtained by Cornejo et al. (2017), Ozdemir Olgun and Demirata (2020), and Shalaby et al. (2011), but were higher than that in the study by Guo et al. (2012). According to Schneider et al. (2005), the LOD and LOQ of CIP were 1 and 5 µg/kg, respectively, which were smaller than those in our study. The findings for the precision values had similarities to others. Shalaby (1996) revealed a higher recovery in chicken muscle (98.4%–100.3%) and egg yolk (99.3%–101.6%) and lower RSD values (1.9%–3.1%) in chicken muscle and egg yolk (0.4%–2.5%) for intra-day precision. Schneider et al. (2007) reported below 4% RSD and 75%–90% recovery from muscle tissue in his findings. Another study stated that the recovery of intra-day precision for CIP in milk sample was 85.4%–89.1% and that the RSD was below 6% (Christodoulou et al., 2007). Yorke and Froc (2000) disclosed a 67% recovery and an 8.4% RSD as inter-day precision in chicken muscle, whereas Cinquina et al. (2003b) obtained 4.1% RSD. Yu et al. (2011) reported within- and between-day precision values for chicken samples of 6.2%–8.2%, 5.9%–8.1%, and 8.1%–9.8%, and 8.7%–8.8% for both TC and OTC, respectively. Blasco et al. (2009) reported higher RSD values for intra-day (i.e., 14.3% and 11.2%) and inter-day precision (16.5% and 15.6%) for TC and OTC in chicken samples. Moudgil et al. (2019b) carried out a study on accuracy and precision for TC and OTC. He reported that the recovery of accuracy was in the range 90.2%–108.1% for TC and was 99.2%–105.6% for OTC. The RSDs of the intra- and inter-assay precision for TC were 4.7% and 6.7–9.2%, while those for OTC were 6.2% and 9.6–16.2%, respectively.

Numerous investigations have been conducted to identify the presence of antibiotic residues in animal products in Asia, such as in Vietnam and China (Baghani et al., 2019), and the prevalence of antibiotic residues in chicken meat has been reported (Vandenberge et al., 2012; Sarker et al., 2018; Gompo et al., 2020; Maharjan et al., 2020; Rahman et al., 2021). In this study, an alarming number of samples were found to be positive for CIP (quinolone group), with 14.3% liver, 17.9% skin, and 3.6% muscle being above the MRLs in the raw stage, hence a potential health risk for consumption. The presence of CIP residues in the samples indicated that farmers in poultry production used this antibiotic as a therapeutic agent, which might have been excessively used or misused (Widiastuti et al., 2022). In developing countries, antibiotics are available as livestock medication without a prescription, and imported illegal products sometimes lack information about their use in the local language (Yamaguchi et al., 2015). In another study, Pereira et al. (2018) also mentioned that the detection of high frequencies of fluoroquinolone (including CIP) residues indicates the random use of these antibiotics due to the lack of proper monitoring by an authorized body. In the study of Panzenhagen et al. (2016), enrofloxacin (fluoroquinolone group) was found positive by HPLC in the muscles (23%), livers (17%), and kidneys (17%) from chickens. The mean concentrations of CIP found by Hasanen et al. (2016) in chicken thigh, breast, liver, and kidney were 92.11, 131.7, 211.1, and 316 µg/kg, while those in turkey thigh, breast, liver, and kidney were 83.2, 119.2, 205.7, and 291.9 µg/kg, respectively, which were higher than our findings. The results showed that approximately 10.71% muscle, 12.66% liver, and 15.38% skin samples exceeded the MRL (EU) among the positive samples of CIP, which were markedly high. In Malaysia, Marni et al. (2011) found that 10 out of 37 samples were contained ciprofloxacin (3.42–238.11 ng/g) and enrofloxacin in 33 samples (3.51–1,734.61 ng/g). Moreover, 45.7% of samples in a study in Turkey (Kaynak Onurdă Er. et al., 2013) and 59.2% of chicken meat samples in another study in Iran (Mashak et al., 2017) were found to be quinolone-positive. In Pakistan, Aslam et al. (2016) found that 58.3% (21) of meat samples had enrofloxacin residues above the MRL (100 ng/g). The findings of chicken samples containing CIP residues above the MRL of 100 ng/g in developing countries revealed the indiscriminate use of antibiotics, which is different from the picture in developed countries such as Spain, the UK, Portugal (Pereira et al., 2018), and Korea (Lee et al., 2018), where no samples exceeded the MRL of antibiotic residues. To reiterate, the differences in the results between the present study and other studies may be due to the multiple methods of administration of antibiotics in chicken, such as with feed and drinking water, intramuscularly, or intravenously (Abdullah et al., 2012; Ramatla et al., 2017). Furthermore, the detection rate of antibiotics may depend on the length of their use in chickens, slaughter, and sampling (Yamaguchi et al., 2015). Little change was found in the concentration of CIP in the positive samples after freezing, whereas a significant difference was observed in the study of O Aboul El-Nile (2006), who stated that temperatures below −18°C can have a positive effect on the reduction of CIP residues. In this study, in the boiling stage, the mean concentration of CIP was reduced from 74.49 to 50.72 µg/kg in the muscle, from 184.6 to 140.4 µg/kg in the liver, and from 79.75 to 49.15 µg/kg in the skin. In contrast, Hasanen et al. (2016) found that the CIP concentrations in both chicken and turkey did not change after boiling, grilling, and roasting, whereas these were notably reduced after microwave treatment and freezing for 1 month at −20°C. In support of this, Botsoglou and Fletouris (2001) also found that CIP has a high heat stability and is not easily destroyed at cooking temperature. In this study, the TC-positive samples in the raw stage were from 7.1% to 71.4%, with 7.1% of these found to be above the MRL; the OTC-positive samples were 3.6%–21.4%, but no sample was above the MRL. Bahmani et al. (2020) reported that TC residues were present in 54.3–389.1 µg/kg of the chicken samples, which was comparable to our data (40–353.1 µg/kg). According to Miranda et al. (2009), the TC and OTC residues in poultry meat ranged from 197 to 2,564 µg/kg and from 651 to 1,003 µg/kg, respectively, which were higher than our findings. In a study in Nepal, TCs and other antibiotics exceeded the MRLs in 6 out of 66 samples (Raut et al., 2017). This could be explained by marketing representatives promoting the use of these medications with easy affordability to poultry farmers, who then administered them without the assistance of trained veterinary officers and with lack of compliance to the specified withdrawal periods (Odundo et al., 2023). TCs can induce the overproduction of Pseudomonas and Clostridium, which change the normal intestinal flora (Hassan et al., 2020) and cause diarrhea, nausea, and even death. TCs consumed during pregnancy could remain in the structure of newly formed teeth (Muhammad et al., 2019). In Southern Italy, low-frequency OTC levels were reported in the muscle (3%) and liver (7%), showing a similar trend to that of the present study (Cammilleri et al., 2019). In poultry farming, OTC is used for the prevention of mouth infections and pneumonia, which can vary with the change of season depending on humidity (Aalipour et al., 2013). In addition, weather and seasonal variations are important factors that change the rate of a drug’s metabolism in chicken (Cerveny et al., 2021). Al-Ghamdi et al. (2000) found that the TC and OTC levels in beef and liver were lowered after boiling, and according to the observation of Gratacós-Cubarsí et al. (2007), boiling and microwave cooking lower the initial quantities of TC and OTC residues by 56% to 82%, respectively. Similarly, Abou-Raya et al. (2013) observed that the residual concentrations of TC and OTC were reduced by 25.8%–28.0% and 45.35%–67.05%, respectively, after boiling. All these have similarities to our findings.

The percent EDI-to-ADI ratio in 60-kg adults is used to measure the human health risk, whereas ADI is a standard criterion for assessing the safety of chemical contaminants in edible animal tissues. The present data for health risk assessment showed that the HI values for CIP, TC, and OTC from all chicken samples in the three stages were below 1, indicating low-risk exposure for human health. Oyedeji et al. (2019) analyzed the antibiotic residues from imported turkey-chicken products in Nigeria and found that the concentrations of 19 antibiotic residues were within the permissible limits. The HI for CIP in all the samples analyzed was less than 1 (HI < 1), indicating no risk for an adult population in Indonesia (Widiastuti et al., 2022). Pena et al. (2010) in Portugal and Pereira et al. (2018) revealed that the EDIs for enrofloxacin in chickens were 0.029 and 0.46 ng/kg body weight per day, respectively. The estimated daily exposure dose was found below 1 µg/kg bw per day with the presence of TCs, quinolones (QNLs), and sulfonamide antibiotics (SAs) in poultry meat in Shanghai (Wang et al., 2017). From 2012 to 2017, Kyriakides et al. (2020) studied antibiotic exposure from the consumption of pork meat in children and adolescents and found a low HI (below 0.056). These trends were also observed in our results. However, Vragovi´c et al. (2011) obtained significantly higher EDIs for streptomycin than for TCs in meat samples. Antibiotic exposure, even in low doses, develops antibiotic-resistant microorganisms, which could have long-term consequences on human health (Karbiwnyk et al., 2007; Gomes and Kuyucu, 2017; Kyuchukova, 2020). Dietary exposure to antibiotic residues could cause therapeutic failure, particularly in children (Jahantigh and Dizaji, 2015). Individuals who inadvertently consume antibiotic residues through edible chicken parts could suffer from allergies, from mutagenic, teratogenic, and anaphylactic shocks, and digestive disorders (Mund et al., 2016; Jammoul and El Darra, 2019). Arsène et al. (2022) also reported adverse health effects such as reproductive disorders, mutagenicity, alteration of the gut flora, and carcinogenicity, which can be caused by exposure to antibiotic-containing food. Cross-resistance could be produced in a new group of microorganisms from the antibiotic-resistant group due to structural alterations in the enzyme from the mutated gene (Sköld, 2001; Baran et al., 2011; Dimitrios et al., 2018).

This study demonstrated two methods for the determination and validation of CIP, TC, and OTC. These methods were efficient in determining the CIP, TC, and OTC from different parts of poultry samples, which were confirmed by recovery studies. A great number of samples were found to be positive with antibiotics. The overall concentration of each antibiotic detected in broiler chicken was higher in raw samples than those in frozen and boiled samples. A few samples contained antibiotics in concentrations higher than their MRLs even after heat treatment, indicating a hazardous situation for human health. The results from the health risk assessment showed that low exposure to antibiotic residues may be a possible human health concern. In countries such as Bangladesh, where most of the poor and middle-class people are somehow dependent on broiler chicken as a cheaper source of protein and which has become a part of their consumption, it is important for authorities to pay attention to the use of veterinary drugs and to take necessary steps to avoid antibiotic resistance.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

The animal studies were approved by Ethical Review Committee (ERC), Mawlana Bhashani Science and Technology University, Tangail, Bangladesh. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent was obtained from the owners for the participation of their animals in this study.

SH: Data curation, Formal analysis, Investigation, Methodology, Software, Writing – original draft, Writing – review & editing. MJ: Data curation, Formal analysis, Investigation, Methodology, Software, Writing – original draft, Writing – review & editing. MK: Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing. MR: Data curation, Formal analysis, Investigation, Methodology, Resources, Software, Visualization, Writing – original draft, Writing – review & editing. ME: Data curation, Software, Writing – review & editing. MA: Data curation, Resources, Software, Writing – review & editing. MZ: Project administration, Validation, Visualization, Writing – original draft, Writing – review & editing. MS: Data curation, Resources, Software, Writing – review & editing. LB: Conceptualization, Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing.