Abstract
This study was carried out to determine the occurrence of tetracyclines, sulfonamides, fluoroquinolones and florfenicol residues in rainbow trout muscle samples (n = 138) obtained from Iranian trout farms. Concentrations of the antibiotics were determined using competitive enzyme-linked immunosorbent assay method. At measurable levels, 63.1%, 16.7%, 19.6% and 40.6% of the samples contained the residues of tetracyclines, sulfonamides, fluoroquinolones and florfenicol, respectively. The detected range of concentrations for positive samples was 1.43–101.4 µg/kg for tetracyclines, 4.03–90.4 µg/kg for sulfonamides, 6.75–99.8 µg/kg for fluoroquinolones and 0.11–172.6 µg/kg for florfenicol. The residues of the antibiotics in trout samples did not exceed the maximum residue levels recommended by the Institute of Standards and Industrial Research of Iran. However, the high occurrence of tetracyclines and florfenicol in the samples is alarming and could be a potential hazard for public health. Further investigations should be performed to determine the residues of antibiotics in other farmed fish species in Iran.
1. Introduction
Antibiotics are natural or synthetic compounds used in food-producing animals in order to prevent and treat the infectious diseases or promote the animal growth. Sulfonamides, tetracyclines, quinolones and florfenicol are common antibiotics approved for use in animal husbandry (Cañada-Cañada, Muñoz de la Peña, & Espinosa-Mansilla, Citation2009; Chander, Gupta, Goyal, & Kumar, Citation2007).
Aquaculture plays an important role in global food production. In order to enhance productivity, intensive cultivation systems are developed, which may lead to greater susceptibility of aquatic animals to diseases caused by micro-organisms. In these conditions, the therapeutic agents such as antibiotics are used to treat affected animals. On the other hand, the improper or extensive application of antibiotics without considering the withdrawal time for treated animals can lead to inappropriately high residue levels in the edible tissues (Mirzargar, Soltani, & Rostami, Citation2000; Peyghan, Najafzadeh Varzi, & Jamzadeh, Citation2012). This can be a potential hazard for public health due to the emergence of drug-resistant pathogenic micro-organisms in the environment, as well as the occurrence of allergic reactions in the consumers (Bilandžić et al., Citation2011; Chander et al., Citation2007; Reig & Toldrá, Citation2008).
Several health authorities have legislated permissible levels of antibiotics in foodstuffs of animal origin to ensure food safety for consumers. According to the European Commission (Citation2010), the maximum residue levels (MRLs) of some antibiotics in fish muscle are as follows: florfenicol, 1000 µg/kg; sum of enrofloxacin and ciprofloxacin, 100 µg/kg; oxytetracycline, 100 µg/kg; tetracycline, 100 µg/kg; chlortetracycline, 100 µg/kg and sulfonamides, 100 µg/kg. The Institute of Standards and Industrial Research of Iran (ISIRI, Citation2011) has also established MRLs for tetracyclines (200 µg/kg), fluoroquinolones (100 µg/kg), sulfonamides (100 µg/kg) and florfenicol (1000 µg/kg) in fish muscle.
Conventional analytical methods used to determine the antibiotic residues in seafood products are microbiological inhibition tests (Jeya Shakila, Saravanakumar, Shanmugapriya, & Jeyasekaran, Citation2008; Mirzargar et al., Citation2000), high-performance liquid chromatography with UV or fluorescence detection (Ansari, Raissy, & Rahimi, Citation2014; Ueno, Sangrungruang, & Miyakawa, Citation1999), enzyme-linked immunosorbent assay (ELISA; Kilinc, Cakli, & Meyer, Citation2010; Zhao et al., Citation2008), liquid chromatography-tandem mass spectrometry (Cháfer-Pericás, Maquieira, Puchades, Miralles, & Moreno, Citation2011; Cháfer-Pericás et al., Citation2010; Li, Hu, Huo, & Xu, Citation2006; Tittlemier et al., Citation2007) and immunochromatographic assay (Le, He, Niu, Chen, & Xu, Citation2013). ELISA method is mostly used for routine screening due to its convenience, rapidity and cheapness. Moreover, the method does not require sample pre-concentration and clean-up steps (Cañada-Cañada et al., Citation2009; Li et al., Citation2006).
Rainbow trout (Oncorhynchus mykiss) is widely used as a farmed fish in many countries around the world. It is also the main freshwater fish species farmed in Iran, with a production of 99,580 metric tonne in 2012 (Annual Fishery Statistics, Citation2013). Referring to the existing scientific literature, no comprehensive study is performed on the occurrence of drug residues in fish and seafood products in Iran. Therefore, this study is aimed to investigate the residue levels of tetracyclines, sulfonamides, fluoroquinolones and florfenicol in rainbow trout obtained from Iranian trout farms.
2. Materials and methods
2.1. Samples
During the year 2012, 138 trout farms were selected randomly from a total number of 665 certified farms in central, northern, western and north-west parts of Iran. In each geographical area, at least 20% of the farms were selected for sampling using the random number table. The mentioned areas produce more than 95% of rainbow trout in Iran market. From each farm, three market-size (250–350 g) rainbow trouts were obtained and transported to the laboratory inside an insulated ice chest. Upon arrival, the fish were eviscerated, beheaded, deboned and filleted. The fillets of three individual fish from each trout farm were pooled and homogenised as a sample. The samples (n = 138) were stored at −20°C prior to analyses.
2.2. Methods and reagents
Concentrations of tetracyclines, sulfonamides and fluoroquinolones in fish samples were determined using competitive ELISA test kits provided by EuroProxima B.V. (Arnhem, the Netherlands). The quantitative analysis of florfenicol was performed by competitive enzyme immunoassay using MaxSignal® florfenicol ELISA Test Kit provided by Bioo Scientific Corporation (Austin, TX, USA). Most of the used reagents were supplied by the manufacturers. The other reagents such as ethyl acetate, methanol, n-hexane, isooctane, trichloromethane, hydrochloric acid, sodium chloride, trisodium citrate dihydrate, sodium phosphate dibasic and potassium phosphate monobasic were of analytical grade and obtained from Merck KGaA (Darmstadt, Germany).
2.3. Preparation of fish samples
2.3.1. Tetracyclines
One gram of the homogenised sample and 3 ml of Mcllvain buffer (pH = 7) were transferred into a tube, vortexed for 2 min and mixed for 10 min by shaking. After centrifugation (2000×g, 10 min), 50 µl of the supernatant was mixed with 200 µl of sample dilution buffer. This mixture was used in the test.
2.3.2. Sulfonamides
One gram of the homogenised sample and 5 ml of ethyl acetate were placed in a tube, vortexed for 45 s and mixed for 35 min by shaking. After centrifugation (2000×g, 5 min), 1 ml of supernatant was transferred into a tube and evaporated to dryness under a stream of nitrogen at 50°C. The residue was dissolved in 1 ml of phosphate buffer saline (PBS); then 1 ml of a solution containing isooctane:trichloromethane (2:3, v/v) was added and vortexed for 1 min. After centrifugation (2000×g, 5 min), 100 µl of supernatant was mixed with 300 µl of PBS. This mixture was used in the test.
2.3.3. Fluoroquinolones
One gram of the homogenised sample and 3 ml of 80% methanol in sample dilution buffer were transferred into a test tube and mixed for 15 min by shaking. After centrifugation (2000×g, 10 min), 2 ml of the supernatant was placed in a tube and evaporated to dryness under a stream of nitrogen at 50°C. The residue was dissolved in 1 ml of 8% methanol in sample dilution buffer. Subsequently, 1 ml of n-hexane was added and vortexed for 1 min. After centrifugation (2000×g, 15 min), the underneath layer was taken and used in the test.
2.3.4. Florfenicol
Three grams of the homogenised sample and 6 ml of ethyl acetate were placed in a tube and vortexed for 3 min. After centrifugation (4000×g, 5 min), 4 ml of the supernatant was transferred into a vial and evaporated to dryness under a stream of nitrogen at 60°C. The residue was dissolved in 2 ml of n-hexane. Subsequently, 1 ml of sample extraction buffer was added and vortexed for 2 min. After centrifugation (4000×g, 10 min), the upper hexane layer was discarded, and the lower aqueous layer was used in the test.
2.4. ELISA test procedure
For ELISA tests, 50 µl from each standard solution or prepared sample was added into separate microtitre wells. Immunoassays were carried out according to the instructions described by manufacturers. The absorbance for all assays was measured at λ = 450 nm using an ELISA plate reader (ELX800, Bio-Tek Instruments, USA). The absorbance values obtained from the standards and the samples were divided by the absorbance value of the zero standard, and multiplied by 100. The zero standard is thus made equal to 100%, and the other absorbance values are quoted in percentages. To construct a calibration curve for each antibiotic, the values calculated for the standards were plotted on the Y-axis versus the antibiotic concentrations (µg/l) on a logarithmic X-axis. In case the antibiotic concentration in a sample (real or spiked sample) was higher than the maximum concentration that can be read from the calibration curve, the sample was further diluted and tested again. The detection limits were 1.3 µg/kg for tetracyclines, 4 µg/kg for sulfonamides, 0.3 µg/kg for fluoroquinolones and 0.1 µg/kg for florfenicol.
2.5. Recovery evaluation
In order to validate our methods, trout samples (10 g each) were spiked with tetracycline (10, 20, 50, 100, 200 and 300 µg/kg), sulfamethazine (10, 20, 50, 100 and 150 µg/kg), enrofloxacin (10, 20, 50, 100 and 150 µg/kg) and florfenicol (10, 50, 100, 200, 500, 1000 and 1500 µg/kg) just before the test. The three spiked levels (0.5 × MRL, 1 × MRL and 1.5 × MRL) were according to the described approach in Commission Decision 2002/657/EC (European Commission, Citation2002). The other spiked levels were selected to evaluate the accuracy of the methods more efficiently. Spiking was carried out in five samples for each level. The preparation of the samples and ELISA tests were done as described above. Under these conditions, the recovery values were in the range of 90.4–95.7% for tetracycline, 90.1–97.3% for sulfamethazine, 90.6–97.1% for enrofloxacin and 90.0–96.6% for florfenicol. The relative standard deviations were less than 7.5% ().
Table 1. Validation of ELISA methods for determination of antibiotic residues in farmed rainbow trout.
2.6. Statistical analyses
The statistical analyses were performed using chi-square test of the SPSS software Version 9 for windows (SPSS Inc., Chicago, IL, USA) to compare the detection rates of each antibiotic among the different geographical regions. The differences were considered significant at P < 0.05.
3. Results and discussion
The occurrence and levels of tetracyclines, sulfonamides, fluoroquinolones and florfenicol are presented in . From the total of 138 samples obtained from Iranian trout farms, 87 (63.1%), 24 (17.4%), 27 (19.6%) and 56 (40.6%) samples contained detectable residues of tetracyclines, sulfonamides, fluoroquinolones and florfenicol, respectively. Also, the residues of the antibiotics in trout samples did not exceed the MRLs recommended by the ISIRI (Citation2011). The detected range of concentrations for positive samples was 1.43–101.4 µg/kg for tetracyclines, 4.03–90.4 µg/kg for sulfonamides, 6.75–99.8 µg/kg for fluoroquinolones and 0.11–172.6 µg/kg for florfenicol ().
Table 2. Occurrence and levels of antibiotic residues in farmed rainbow trout in Iran.
Tetracyclines were detected in 66.7%, 67.7%, 52% and 61.8% of the samples obtained from trout farms in central, northern, western and north-west parts, respectively. There was no statistically significant difference (P > 0.05) on the occurrence of tetracyclines among the geographical regions. Most of the positive samples contained tetracyclines at the range of 1.3–10 µg/kg (). In a recent survey conducted in Nigeria (Olatoye & Basiru, Citation2013), 30% of the catfish fillet samples contained oxytetracycline, ranging between 22.5 and 553.2 µg/kg, and 18.8% of the samples exceeded the limit of 200 µg/kg established by the Codex Alimentarius Commission (Citation2011). In Croatia, 87.6% of the analysed fish products contained tetracyclines, with a maximum value of 9.4 µg/kg (Vragović, Bažulić, Jakupović, & Zdolec, Citation2012). In contrast, shrimp samples obtained from the farms in southern states of India were free of tetracyclines (Swapna, Rajesh, & Lakshmanan, Citation2012).
In this study, the occurrence of sulfonamides in trout samples of the central (28.9%) and northern (20.6%) parts was significantly higher (P < 0.05) than those from north-west (11.8%) and western (0.0%) parts. Most of the positive samples contained sulfonamides at the range of 4–10 µg/kg (). In a recent study performed in India (Palaniyappan et al., Citation2013), sulfonamides were not detected in sea prawn samples, while the residues were detected in all of the farmed prawn samples (24 samples) at the concentrations ranging from 12 to 134 µg/kg. Moreover, in seven samples, concentrations of sulfonamides exceeded the MRL of 100 µg/kg set by the European Commission (Citation2010). In South Korea, sulfonamide residues were identified in three samples out of 309 marine products, and only one sample contained sulfamethoxazole above the MRL of 100 µg/kg (Won et al., Citation2011).
The occurrence of fluoroquinolones in trout samples of the central part (37.8%) was significantly higher (P < 0.05) than those from northern (17.7%), western (16%) and north-west (0.0%) parts. Most of the positive samples contained fluoroquinolones at the range of 10–100 µg/kg (). In Canada, enrofloxacin was detected in 3 out of 30 seafood composite samples, ranging between 0.30 and 0.73 µg/kg (Tittlemier et al., Citation2007). In another study (Fadaeifard, Citation2012), fluoroquinolones were detected in 23.3% of farmed rainbow trout samples obtained from Chaharmahal-va-Bakhtiari province of Iran, and none of the samples exceeded the limit of 100 µg/kg recommended by the ISIRI (Citation2011).
In the present study, florfenicol was detected in 40%, 50%, 44% and 29.4% of the trout samples obtained from the farms in central, northern, western and north-west parts, respectively. There was no statistically significant difference (P > 0.05) on the occurrence of florfenicol among the geographical regions (). Considering the short depletion time of florfenicol (Mirzargar et al., Citation2000), its presence in trout samples may be due to the drug usage even to final days of culture period. In a recent survey performed in Chaharmahal-va-Bakhtiari province of Iran (Ansari et al., Citation2014), 14 out of 40 samples (35%) of farmed rainbow trout contained florfenicol, but only five samples (12.5%) exceeded the limit of 1000 µg/kg recommended by the ISIRI (Citation2011) and European Commission (Citation2010).
Frequencies of co-occurrence of the analysed antibiotics in farmed rainbow trout are shown in . This study demonstrated that 53 out of 138 samples (38.4%) contained more than one antibiotic. The co-occurrence of tetracyclines-florfenicol (11.6%) in analysed trout samples was the highest, followed by tetracyclines-fluoroquinolones-florfenicol (7.25%), tetracyclines-sulfonamides (5.07%) and tetracyclines-fluoroquinolones (4.35%). It was found that only one sample from a trout farm in central part of Iran contained simultaneously four antibiotics (). Although the levels of antibiotics in the samples did not exceed the MRLs, their co-occurrence could be hazardous for public health.
Table 3. Frequency of antibiotics co-occurrences in farmed rainbow trout (n = 138) in Iran.
As notified by the Rapid Alert System for Food and Feed (RASFF, Citation2014) of the European Commission, the residues of veterinary medicines were found in fish and seafood products available in the EU markets, and that is a great concern for consumers health. In the period from 2002 to 2014, the RASFF recorded 1003 notifications of the drug residues in fish and seafood products; among them, 15 records were the residues of tetracyclines, sulfonamides and quinolones above the admissible levels (). Most of the reported cases (more than 70%) were the residues of tetracyclines in shrimp and in fish products imported from the Asian countries. The highest detected levels were as follows: (1) 2065 µg/kg of oxytetracycline in frozen shrimp, (2) 506 µg/kg of quinolones in frozen cobbler fillets and (3) 300 µg/kg of sulfadiazine in frozen tiger shrimp ().
Table 4. Occurrence of antibiotic residues higher than MRLs in seafood products reported by RASFF of the European Commission.
4. Conclusions
This study demonstrated a high occurrence of tetracyclines and florfenicol and low occurrence of fluoroquinolones and sulfonamides in rainbow trout samples obtained from Iranian trout farms. The residues of the antibiotics in trout samples did not exceed the MRLs recommended by the ISIRI (Citation2011). However, the high occurrence of some antibiotics in trout samples is alarming and could be a potential hazard for public health. Therefore, monitoring and inspection programmes on the antibiotic residues in various aquaculture products should be conducted in a regular manner. The fish farms with the antibiotics residue levels higher than regulation limits should be restricted, and their products should not enter the food chain. Because this survey is limited to farmed rainbow trout, further investigations should be performed to determine the residues of antibiotics in other farmed fish species in Iran.
Acknowledgement
This study was financially supported by the Faculty of Veterinary Medicine, Shahrekord University, Iran.
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