Environ Sci Pollut Res DOI 10.1007/s11356-015-5002-9

RESEARCH ARTICLE

Assessment of oxytetracycline and tetracycline antibiotics in manure samples in different cities of Khuzestan Province, Iran Nadali Alavi 1,2 & Ali Akbar Babaei 1,2 & Mohammad Shirmardi 2,3 & Abolfazl Naimabadi 2,4 & Gholamreza Goudarzi 1,2

Received: 17 April 2015 / Accepted: 30 June 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract Tetracyclines (TCs), a class of antibiotics with a broad spectrum, are the most frequently used antibiotics in animal production. The major concern is that the widespread use of the antibiotics may lead to the emergence of new strains of bacteria that are resistant to these antibiotics. The objective of this study was to determine the residual levels of oxytetracycline and tetracycline in 80 animal manure samples that were collected from the livestock and poultry feedlots in Khuzestan Province. The residual levels of the antibiotics in the samples were extracted by using solid-phase extraction (SPE) method and subsequently were measured by liquid chromatography. Recoveries from the spiked poultry manure samples ranged from 65 to 113 % for tetracycline and 86 to 132 % for oxytetracycline. Relative standard deviations of the recoveries were less than 5.7 % within the same day. Method detection limit (MDL) measured for oxytetracycline and tetracycline in the manure were 0.011 and 0.01 mg/kg,

respectively. Analysis of the collected 50 chickens and 30 cow manure samples showed that the highest concentration of tetracycline was related to Behbahan City (5.36 mg/kg) and the lowest concentration was detected for Ramhormoz (0.05 mg/kg). The highest and lowest concentrations of oxytetracycline were respectively observed for Behbahan (13.77 mg/kg) and Ramhormoz (0.047 mg/kg). Based on the results, in chicken manure, there was significant statistical difference between the residual TC concentrations among five cities (pvalue 0.05). Keywords Antibiotic . Poultry . Livestock . Liquid chromatography . Khuzestan Province

Introduction Responsible editor: Ester Heath * Abolfazl Naimabadi [email protected] 1

Environmental Technologies Research Center (ETRC), Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran

2

Department of Environmental Health Engineering, Health Faculty, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran

3

Student Research Committee, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran

4

School of Public Health, Department of Environmental Health Engineering, Jundishapur University of Medical Sciences, P.O. Box: 61357-15751, Ahvaz, Islamic Republic of Iran

Antibiotics were discovered in the first half of the twentieth century as a cure against bacterial infections; nowadays, antibiotics are used in animals for therapeutic and non-therapeutic purposes. Non-therapeutic applications of veterinary antibiotics include animal growth promotion and metaphylactic use (Addison 1984). Antibiotics were introduced in veterinary medicine shortly after their application in human medicine for the treatment of bacterial infections. They were firstly used as veterinary drugs to treat mastitis in dairy cows (Dahshan et al. 2015). According to a report by the Union of Concerned Scientists (UCS), 16 million kilograms of antibiotics are used annually in the USA, and approximately 70 % are used for non-therapeutic purposes (Li et al. 2015a, b; Sarmah et al. 2006). Tetracyclines were discovered in the 1940s and

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exhibited activity against a wide range of microorganisms including gram-positive and gram-negative bacteria, chlamydiae, mycoplasmas, rickettsiae, and protozoan parasites. They are inexpensive antibiotics, which used extensively in human and animal prophylaxis, infectious therapy, and at subtherapeutic levels as animal growth promoters (Chopra and Roberts 2001). In European Union, tetracycline used in animal feeding has increased to 11 million pounds in 2007 (Animal Health Institute 2014). Although this union banned the use of antibiotics as growth agent since 2006, its use has not changed much until recently (Millet and Maertens 2011). Based on the results of a study carried out by Aalipour et al. (2014) in Iran, the consumption of antibiotics in livestock and poultry is higher than developed countries. Tetracycline and oxytetracycline had the highest consumption of livestock. It should be noted that about 80 % of 1806.896 t of the antibiotics distributed in animal farms in Iran in 2010 was related to tetracycline. Tetracycline antibiotics are the most common antibacterial sold to both livestock and poultry farms. The major concern about the widespread use of the antibiotics is that they may lead to the emergence of new strains of bacteria, which are resistant to these antibiotics, and consequently results in untreatable humans and livestock diseases (Hirsh et al. 1979). This is mainly due to the fact that the antibiotics administered to animals by means of oral or intramuscular injection are not fully metabolized. Tetracycline and ciprofloxacin have been detected in poultry manure at the concentrations of 2.125 and 1.401 mg/kg, respectively (Tenhagen et al. 2006; Dahshan et al. 2015). According to a study carried out in Turkey, the most frequently detected antimicrobial compound in animal manure was oxytetracyclin (OTC), while no ciprofloxacin was detected in manure samples at measurable amounts (Karcı and Balcıoğlu 2009). As antibiotics are poorly absorbed in the gut of the animals, the majority is excreted unchanged in feces and urine (Sarmah et al. 2006), resulting in as much as 30–90 % of the parent compound being excreted (Alcock et al. 1999; Elmund et al. 1971; Feinman and Matheson 1978). For instance, quantitative bioassays of fresh feedlot manure revealed that approximately 75 % of dietary chlortetracycline (CTC) was excreted (Elmund et al. 1971). In addition, antibiotic metabolites are bioactive and can be transformed back to the parent compound after excretion (Kemper 2008). Thus, a significant percentage of the administered antibiotics may be excreted into the environment in active forms (Berger et al. 1986; Warman and Thomas 1981). It is therefore likely that when animal wastes are applied as supplement to fertilizer, they can find their way into the receiving environment and can be present either as metabolite or as the parent compound (Mitscher 1978). Fertilization of agricultural soils with animal manure can lead to a nonpoint source that contaminates the terrestrial environment with these substances. In studies which evaluated the occurrence of

various antimicrobials in different soils fertilized with animal manure, the maximum concentrations for TCs, sulfonamides, and fluoroquinolones were 0.3, 0.015, and 0.37 mg/kg, respectively (Hamscher et al. 2005; Christian et al. 2003; Martínez-Carballo et al. 2007). In Iran, to the best of our knowledge, no study has been carried out to evaluate the residual levels of antibiotics in manure; therefore, this study was aimed to measure the residual concentrations of TC and OTC, which are widely used in Iran as animal feed additives, in manure samples.

Materials and methods Chemicals All chemicals used in this study were of analytical reagent grade unless otherwise specified. The antibiotics, OTC and TC, were purchased from Sigma-Aldrich. At first, we prepared a stock OTC and TC solution, 10 mg/L, by dissolving 1 mg of the related antibiotics in 100 mL methanol and stored it for a maximum of 3 months at −20 °C in a freezer. We prepared McIlvaine buffer by weighing and dissolving 12.9 g anhydrous citric acid (Mallinckrodt Baker Inc., Phillipsburg, NJ) and 10.9 g dibasic sodium phosphate (Fisher Scientific, Fair Lawn, NJ) in 1 L water (Arikan et al. 2009; Hamscher et al. 2005; O’Connor and Aga 2007). In addition, McIlvaine–EDTA buffer was prepared by dissolving of 37.2 g EDTA disodium salt dehydrate (Sigma-Aldrich, St. Louis, MO) to 1 L of McIlvaine buffer with gentle heating. Prior to use, the McIlvaine–EDTA buffer was filtered through a 0.2-μm nylon filter and stored at 4 °C. HPLC grade methanol (Mallinckrodt Baker Inc.), acetonitrile (Honeywell Burdick and Jackson, Morristown, NJ), and oxalic acid dehydrate (Sigma-Aldrich) were used in this study as the mobile phase for HPLC analysis (O’Connor and Aga 2007; MartínezCarballo et al. 2007). Double-distilled water was used throughout this study to prepare working solutions as well as the standards with predetermined concentrations. Sample collection Five cities in the north and south of Khuzestan Province were selected as the sampling sites: Shadegan, Dezful, Ahvaz, Behbahan, and Ramhormoz. The map of Khuzestan Province and the sampling sites is shown in Fig. 1. A total of 40 farms in the cities were chosen to sample manure. We selected five chicken farms in each city; therefore, we sampled manure from 25 chicken farms (20,000–60,000 chickens). Because Behbahan and Ramhormoz are the main husbandry centers of Khuzestan Province, we selected 15 cow farms (more than 100 cows) in these two cities to sample cow manure. Sampling was carried out in November 2014 and

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Fig. 1 Map of the study area and sampling locations

January 2015 because the Khuzestan Province is located in an arid area and has a very hot climate, and poultry production is mostly accomplished during this period. Thus, overall 80 manure samples were collected from the five cities. Based on the time of production, we selected the chicken farms that had chicken with the same age. However, for the cow farms, there were a few month difference in the age of cows. The manure samples were collected together with the straw from the surface to 5-cm depth at three different locations in the barn. Then, for each barn, the samples were mixed completely, and the final manure sample was placed into a plastic container and immediately transferred to the laboratory for further processing. The straw was removed from the samples by screening; then, the samples were stored at −20 °C until extraction. Before extraction, a specific amount of each manure sample was dehydrated to determine dry matter of the manure sample. To do this, the initial temperature was held at 45 °C and then was increased gradually to 105 °C over 2 h until a constant mass was attained (Zhao et al. 2010). All samples were analyzed within a week after collection.

Sample preparation The procedure used to extract TC and OTC from the manure samples was based on the method described in the literature (Arikan et al. 2009; O’Connor and Aga 2007). Briefly, 1.0 g equivalent dry weight of the fresh manure sample was placed into a 15 mL polypropylene tube, and then, 5 mL of McIlvaine–EDTA buffer was added to the tube. The sample

was vortexed at a high speed for 60 s and then sonicated in a sonication bath (Bandelin, Germany) for 15 min. After centrifuging for 5 min at 5000 rpm, the supernatant was decanted in to a clean 15-mL tube. This procedure was repeated three times as described above to obtain 15 mL extract solution. Afterward, the extracted solution was filtered through a 0.45-μm Whatman filter paper and passed through the prewashed Waters SPE-Pak C-18 Oasis HLB cartridges. The cartridges were prewashed with 2 mL of methanol followed by 2 mL HPLC grade water. After the extracts were loaded, the cartridges were washed with 2 mL HPLC grade water, followed by sample elution using 4 mL of methanol and 2 mL 0.01 M oxalic acid. The eluents were dried under a nitrogen flow, and then, 1 mL methanol was added prior to analysis by HPLC (Arikan et al. 2009; Hamscher et al. 2005; O’Connor and Aga 2007). To ensure the accuracy, the samples were extracted and analyzed in duplicate.

HPLC analysis Instrumental determinations of OTC and TC were performed using an HPLC device (Knauer, Germany) with a Eurospher column (5 μm 4.6 mm×250 mm), which equipped with ultimate variable wavelength UV detection 2500 set at 360 nm wavelength for TC and OTC. The mobile phase composed of oxalic acid (0.01 M)/methanol/acetonitrile with the volume ratio of 70/20/10, and the flow rate and temperature were 1 mL/min and 25 °C, respectively. Quantification of residues in samples was obtained and calculated from the areas under

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the curves extrapolated automatically by the software. Analytical standards of TC and OTC were purchased from Sigma-Aldrich to set up these two antibiotics on the HPLC instrument as well as to identify the related peak. Subsequently, seven mixed standards of OTC and TC (initial concentration of 0.01, 0.1, 1.0, 2.5, 5, and 10 mg/L) were prepared in methanol and analyzed by the HPLC at operating conditions to form the calibration curves. The residual concentration of the antibiotics in the samples was calculated according to the calibration curves. Some samples were analyzed by MS detector to ensure that the peaks were related to TC and OTC (Dahshan et al. 2015; Blackwell et al. 2004; Li et al. 2015a, b). The correlation coefficients for OTC and TC were obtained to be 0.999 and 0.9999, respectively. Method detection limit (MDL) was determined by spiking seven replicates of matrix with stock solutions to yield samples containing 1 μg/g. MDL was then calculated (Eq. (1)) by using a statistical approach established by the USEPA (Brown and Mac Berthouex 2002). The replicates of each spiked blank matrix went through all extraction procedures and instrumental analysis to determine MDL. M DL ¼ 3:143  SD

ð1Þ

where SD is the standard deviation of the analysis replicates. Method quantification limit (MQL) was mathematically defined as equal to 10 times the standard deviation of the results for a series of replicates used to determine a justifiable MDL (Ripp 1996). Recovery studies for the animal feces were carried out on a sample without any residual level of the antibiotics by spiking a 5 mg/kg level of mixed TC and OTC to it. The recovery rate (%) was calculated by comparing the predefined concentration of each analyte spiked before SPE to its concentration spiked after SPE in the same sample matrix using Eq. (2). The recovery rates of the spiked manure samples ranged from 73 to 111 % for oxytetracycline and 64 to 113 % for tetracycline. The relative standard deviations of the recoveries were less than 5.7 % within the same day. Results for the MDL, MQL, and recovery rates of the selected veterinary antibiotics are summarized in Table 1.  Cmeasured μg kg‐1  Recoveryð%Þ ¼  100 ð2Þ Cspiked μg kg‐1

Table 1 Results of method detection limit (MDL), method quantification limit (MQL), recovery and relative standard deviation (RSD) for OTC and TC antibiotics

Statistical analysis The residual concentrations of the antibiotics were analyzed using SPSS software version 17 (SPSS, Inc. Chicago, IL). The Kolmogorov–Smirnov test was employed to evaluate the normality of the data. Kruskal–Wallis test was used for the nonparametric to observe the differences between the antibiotic residual concentrations among the cities as well as among animal species manure.

Results and discussion Occurrence of tetracycline in manure As shown in Table 2, tetracycline was commonly detected in the chicken manure samples analyzed, indicating that the compound had been fed to the animals. However, the concentration of the antibiotics in the manure varied greatly in different regions. Based on dry weight, the average TC concentration in the chicken manure samples for the selected cities of Khuzestan Province was in the range of 0.16–0.763 mg/kg. The highest concentration of TC detected was up to 1.116 mg/kg for the manure sampled from Shadegan, and the lowest concentration was only 0.05 mg/kg for the manure sampled from Ramhormoz. The difference in the residual levels of the antibiotic in the five studied cities may be attributed to different geographical locations of the cities as well as different prescribing habits (Zhao et al. 2010). In cow manure, the highest concentration of TC was 5.36 mg/kg for the manures sampled from Behbahan and the lowest concentration was 0.052 mg/kg for the manures sampled from Ramhormoz (see Table 3). The differences in residue levels of the antibiotic among the animal species could be attributed to variations in different dose levels in the feed, and different metabolic characteristics included age, weight, and the health status of animals (Wenk 1998). The effect of antibiotics is pronounced in young growing animals especially under unfavorable climatic and management conditions. Li et al. (2015a) reported in Beijing that TCs has been the most frequently detectable compounds with a mean concentration of 4.976 mg/kg. MartínezCarballo et al. (2007) reported that in Austria, the tetracycline was found in 22 of 30 pig manure samples in a concentration range from 0.36 to 23 mg/kg. The results of the present study for the concentrations of TC are consistent with the results of Martínez-Carballo et al. (2007) and Li et al. (2015a). Occurrence of oxytetracycline in manure

Substance

MDL mg/kg

MQL mg/kg

Recovery %

RSD %

TC OTC

0.01 0.011

0.031 0.039

74.87 88.74

5.67 1.88

In this study, it was found that oxytetracycline can be detected in the chicken manure samples (Table 2). Based on dry weight, the highest and lowest concentrations of OTC detected in the sampled chicken manure were observed for

Environ Sci Pollut Res Table 2

The concentrations of tetracycline and oxytetracycline in chicken manure samples

City

Substance

Sample size

Average (mg/kg)

Minimum (mg/kg)

Maximum (mg/kg)

Standard deviation (mg/kg)

Ahvaz

TC OTC TC OTC TC OTC TC OTC TC OTC

10 10 10 10 10 10 10 10 10 10

0.186 1.87 0.41 0.24 0.76 0.55 0.49 0.13 0.16 6.18

0.1 0.34 0.37 0.08 0.58 0.54 0.05 0.047 0.08 0.34

0.26 5.87 0.53 0.49 1.11 0.55 0.93 0.17 0.24 13.77

0.7 2.29 0.08 0.18 0.31 0.006 0.62 0.07 0.06 6.76

Dezful Shadegan Ramhormoz Behbahan

Behbahan and Ramhormoz cities, respectively. The corresponding values were 13.77 and 0.047 mg/kg, respectively. The maximum average antibiotic concentration in chicken manure was detected in Behbahan, with a value of 6.18 mg/kg. In cow manure, as the results presented in Table 3, the highest concentration of OTC with the value of 0.62 mg/kg was observed for the manures sampled from Ramhormoz, and the lowest concentration with the value of 0.07 mg/kg was detected for the manures sampled from Ramhormoz. The results showed that the average concentrations of the antibiotic in cow manure were lower than those in chicken manure except for Ramhormoz because no detectable concentration of oxytetracycline was observed in some cow farms because some livestock are not administered the antibiotic. The results of this study are consistent with the results obtained by the other researchers. For example, Zhang et al. (2008) reported that OTC could be measured in significant residue levels in pig, chicken, and cow manure samples collected from north Zhejiang Province. In that study, the geometric means of OTC detected in the manures of pigs, chickens, and cows were 8.37, 4.32, and 2.18 mg/kg, respectively. Hu et al. (2008) stated that the detected concentrations of OTC in all animal dung samples investigated ranged from 9.7 to 173.2 mg/kg. In a study, Zhao et al. (2010) measured the OTC geometric mean ranges of 1.55 and 1.24 mg/kg, for the chickens and cows manures, respectively. In an investigation accomplished by Hamscher et al. (2002), no OTC was detected in any liquid manure, while Karcı and Balcıoğlu (2009) detected OTC in animal manure samples in the range of 0.06–0.50 mg/kg. Table 3

Effect of the sampling site on levels of antibiotics Based on the statistic of 2011, Khuzestan Province had 320 dairy industries with about 40,000 cows. In addition, there were approximately 660 poultry production farms with 2, 659,637 chickens. In this study, to sample chicken manure, the sampling sites were selected on the basis of their different geographical locations in Khuzestan Province: north, east, central, and south. However, for cow manure sampling, two cities (Behbahan and Ramhormoz) were selected as the sampling sites. It should be noted that these five cities are the main livestock centers in Khuzestan Province. Dezful is a city, which is located in the north of the province; Behbahan and Ramhormoz cities are located in the east of the province. Ahvaz and Shadegan are located in the central and south of the province, respectively. Among the five sampling districts, Ahvaz is the most developed economic city in Khuzestan, and there are 50 large-scale poultry production farms in this city. The Kruskal–Wallis test was used to compare the difference in the residual levels of the two antibiotics, TC and OTC, in chicken manures sampled from different five cities. The residual levels of TC were statistically highly different between five cities (pvalue 0.05). This indicates that OTC has been widely used in all five cities. The statistical difference among TC residual concentrations between the five cities could also be attributed to the number of chickens per farm, the season of the sampling, and age of the chickens during the study. Thus, because of the

Tetracycline and oxytetracycline in cow manure samples

City

Substance

Sample size

Average (mg/kg)

Minimum (mg/kg)

Maximum (mg/kg)

Standard deviation (mg/kg)

Ramhormoz

TC OTC TC OTC

14 14 16 16

0.14 0.23 0.94 0.21

0.05 0.07 0.14 0.10

0.22 0.62 5.36 0.45

0.11 0.26 1.79 0.13

Behbahan

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fluctuations in the price of antibiotics, cheaper or a specific class of antibiotics may be used in each city. This test was also used to compare the difference between residual concentrations of TC and OTC in cow manure samples. The results showed that there was no significant relationship between the residual concentration of TC and OTC among two cities (pvalue >0.05). This could be attributed to the similar cow production practices as well as small number of cow farms.

animal manure, and some control measures are urgently required to prevent the associated risks from the excessive use of antibiotics to humans. Acknowledgments The authors are grateful to the Vice Chancellory for Research Development and Technology of Ahvaz Jundishapur University of Medical Sciences for funding and providing necessary facilities to perform this research with project no. ETRC-9212.

Effect of manure types on residual antibiotic levels

References

Results showed that the concentration of the antibiotics in the chickens manure was higher than that found for the cows, and the maximum OTC with 13.76 was related to Behbahan. A relative fast degradation of TC is supposed, and studies about the persistence of this analyte showed also a rapid decline in manure (Mitscher 1978). In addition, antibiotic digestive and absorptive capacity for any species and the type of the animal could affect the residual level in different manures (Hu et al. 2010). A statistical test was also performed to compare the residues of each antibiotic among different animal species. Results of the Kruskal–Wallis test showed that there was no significant relationship between two animal species (pvalue > 0.05). Zhao et al. (2010) observed vast differences in antibiotic concentrations among the various manure categories. The mean concentrations of total antibiotics in chicken, duck, pig, and cow manure, as well as commercial organic fertilizer were 23, 17, 16, 0.043, and 0.459 mg/kg, respectively. These residual characteristics were consistent with the fact that antibiotics were widely used in the livestock industry, especially for the poultry and swine industries and a large portion of these compounds had been excreted as feces.

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Conclusions This study analyzed the residual levels of oxytetracycline and tetracycline in chicken and cow manure samples collected from large-scale livestock and poultry feedlots. Using a combination of ultrasonic extraction and liquid chromatography, the average recoveries of OTC and TC from the spiked chicken samples were 88.74 and 74.87 %, respectively, with relative standard deviation less than 6 %. The MQL for TC and OTC in the samples were 0.031 and 0.039 mg/kg, respectively. The highest concentration of OTC was 13.76 mg/kg dry weight for chicken manure sampled from Behbahan, and the lowest concentration was only 0.047 mg/kg dry weight for manure sampled from Ramhormoz. Based on the results, in chicken manure, there was significant statistical difference between the residual TC concentrations among five cities (pvalue 0.05). These results suggest that regular monitoring of antibiotics in

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Assessment of oxytetracycline and tetracycline antibiotics in manure samples in different cities of Khuzestan Province, Iran.

Tetracyclines (TCs), a class of antibiotics with a broad spectrum, are the most frequently used antibiotics in animal production. The major concern is...
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