Environ Earth Sci (2015) 74:5077–5086 DOI 10.1007/s12665-015-4528-y
ORIGINAL ARTICLE
Antibiotic contamination in animal manure, soil, and sewage sludge in Shenyang, northeast China Jing An1 • Hongwei Chen3 • Shuhe Wei1 • Jian Gu2
Received: 9 August 2014 / Accepted: 10 May 2015 / Published online: 28 May 2015 Ó Springer-Verlag Berlin Heidelberg 2015
Abstract The occurrence and pollution characteristics of common antibiotics in manure, soil, and sewage sludge in Shenyang, the biggest city in northeastern China, were investigated. Commonly used antibiotics tetracyclines (TCs), including tetracycline, chlortetracycline, oxytetracycline, and deoxytetracycline; and sulfonamides (SAs), including sulfadiazine, sulfamerazine, sulfadimidine, and sulfamethoxazole, were measured by high-performance liquid chromatography–tandem mass spectrometry via solid-phase extraction. Multiple antibiotics could be simultaneously measured in a single manure sample. The highest concentration of antibiotics in manure was 143.97 mg kg-1 (chlortetracycline). There were no significant differences in concentrations of antibiotics between large-scale farms and individual household farms. The concentrations of TCs were significantly higher than those of SAs in manure and soils. However, the concentrations and detection frequencies of antibiotics in soils were significantly lower than in manure. The concentrations of antibiotics varied from 2.56 lg kg-1 of sulfadimidine to 1590.16 lg kg-1 of chlortetracycline, and
& Shuhe Wei
[email protected] Jing An
[email protected] 1
Key Laboratory of Pollution Ecology and Environmental Engineering, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China
2
Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110164, China
3
University of Chinese Academy of Sciences, Beijing 100039, China
the detection frequencies were 27.14–64.29 % for TCs and 5.71–28.57 % for SAs in soils. The concentrations of antibiotics in the vegetable field fertilized with manure were higher than those in the corn field not fertilized with manure. Various kinds of antibiotics were detected in sewage sludge, although concentrations were not high. Concentrations of antibiotics were higher in winter than in summer, The concentrations of antibiotics observed in this study were similar to those observed in other regions of China, but, in general, higher than those in other countries. Keywords Veterinary antibiotics Tetracyclines Sulfonamides Manure Soil Sewage sludge
Introduction Pharmaceuticals and personal care products (PPCPs) have recently been attracting attention, and in 1999 were designated as the most important emerging environmental contaminants by the EPA (Daughton and Ternes 1999). Among PPCPs, antibiotics are a primary contaminant because of their widespread consumption, numerous sources, continuous inputs, and potential ecotoxicity (Kemper 2008). Antibiotics are physiologically active substances widely used in livestock farms to promote animal growth and to prevent or treat animal diseases (Lin et al. 2012). However, the excessive emphasis on the economic benefits and the unquestioning reliance on antibiotics have resulted in a continuous increase in their consumption and application in unreasonable dosages and frequencies (Sarmah et al. 2006). Many veterinary antibiotics are poorly adsorbed in animal guts, resulting in as much as 30–90 % of the parent compound being excreted unchanged via feces or urine (Alcock et al. 1999; Bound and Voulvoulis 2004;
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Aust et al. 2008). The antibiotics then enter the environment in a variety of ways. Many veterinary antibiotics have been detected in surface water, soil, sediment, and ground water (Aga et al. 2005; Hu et al. 2010; Luo et al. 2011; Leung et al. 2012; Yan et al. 2014a). Antibiotics are believed to be intrinsically bioactive and bioavailable in the environment, resulting in toxic effects on nontarget organisms and on human health, the extent and nature of which are unclear (Dong et al. 2012). China is the largest producer and consumer of antibiotics in the world. It was reported that about 28,000 tons of penicillin (60 % of the global total) and 10,000 tons of terramycin (65 % of the global total) were produced in China in 2003, and the average usage of veterinary antibiotics has reached approximately 6000 tons annually (Richardson et al. 2005; Zhao et al. 2010). However, few studies have evaluated the residues and distribution of antibiotics released into surrounding environments, and the research has mainly focused on antibiotic contamination of water and sediments (Chen and Zhou 2014; Yan et al. 2014b). China is a country with a vast geographical area; climate, soil, and farming methods vary widely between the north and the south, resulting in variations in pollution characteristics of antibiotics in different areas. Shenyang, with an area of 12,980 km2, is the biggest city in northeastern China, and is the most important agricultural production base in China. In 2011, the total value of the agricultural output in Shenyang was 54.55 billion US dollars. Stockbreeding (68.8 % of the total agricultural) and farming (23.2 % of the total agricultural) were the key industries in the rural economy. With stable and rapid development of the agricultural industry, the dosage of agrochemicals, including veterinary antibiotics, will continue to increase. However, there are few published reports on residual antibiotics and pollution characteristics in this area. Tetracyclines (TCs) and sulfonamides (SAs) are groups of broad-spectrum antibiotics, widely used as antibacterial agents in veterinary medicine worldwide, especially in China (Motoyama et al. 2011; Jiang et al. 2013; Awad et al. 2014). They are active against a range of organisms, such as mycoplasma and chlamydia and a number of Gram-positive and Gram-negative bacteria. In recent years, the occurrence and fate of TCs and SAs in environments has received increasing attention. Aust et al. (2008) reported that up to 9990 lg kg-1 sulfamethazine and 401 lg kg-1 chlortetracycline on a dry matter basis were present in feedlot manure in Lethbridge, Alberta, Canada. Haller et al. (2002) found that up to 20 mg kg-1 sulfonamide were determined in six grab samples from manure pits on farms in Switzerland where medicinal feed had been applied. Kim and Carlson (2007) found that the
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concentrations of TCs and SAs in sediments collected from the Cachela Poudre River in northern Colorado, United States, ranged from 4.5 to 32.8 lg kg-1 and from 1.2 to 3.4 lg kg-1, respectively. Li et al. (2011) found that the total concentrations of TCs in 35 % of soil samples exceeded 100 lg kg-1, and the highest value was 243 lg kg-1. The main objectives of this study were (1) to determine the concentration and distribution of TCs and SAs in manure, soil, and sewage sludge in Shenyang, and (2) to investigate the factors that influence the occurrence of antibiotic residues in different samples from farms of different scales, in different crop types, and in different sampling seasons.
Materials and methods Chemicals Tetracyclines [oxytetracycline (OTC), tetracycline (TC), chlortetracycline (CTC), doxycycline (DOX)], and SAs [sulfadiazine (SDZ), sulfamerazine (SMR), sulfadimidine (SM2), sulfamethoxazole (SMZ)] were obtained from Sigma-Aldrich (St. Louis, MO, USA). The purity of all standard reagents was at least 98 %. Acetonitrile and methanol (HPLC grade) were purchased from Fisher Scientific (Fairlawn, NJ, USA). Ultrapure water was prepared with a Milli-Q water purification system (Millipore, Billerica, MA, USA). Oasis HLB (200 mg, 6 cc) cartridges for hydrophilic–lipophilic balances were purchased from Waters Corporation (Milford, MA, USA). All other reagents were of analytical reagent grade. Sampling The sampling sites in the city of Shenyang are shown in Fig. 1. 51 manure samples and 70 soil samples were collected from May to June 2013, while 18 sludge samples were collected from three large sewage treatment plants in January and August 2013. Fifteen pig feces samples were collected from seven pig feeding farms (more than 500 pigs in total), 16 dung samples from eight chicken feeding farms (more than 10,000 chicken in total), and 20 dung samples from individual household farms (30–50 pigs or 500–1000 chickens in total). Soil samples were collected at a depth of 0–15 cm with a small shovel. Five sampling sites were distributed as the ‘‘S’’ type, then were fully mixed into one sample. Each sewage sludge sample was placed into a brown glass bottle and immediately chilled in a carry-home icebox. All samples were transferred to the laboratory and stored at -18 °C until extraction.
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Fig. 1 Map of sampling locations in Shenyang city, northeast China
Sample preparation and analysis For antibiotic analysis, the solid-phase extraction method was used to enrich and clean up the samples. Triplicates were used for extracting each sample (n = 3). Lyophilized manure (0.5 g) was extracted with 30 ml of 0.1 M EDTA– McIlvaine buffer solution (pH 4.0). Each lyophilized soil and sludge sample (1.0 g) was extracted with 30 ml of acetonitrile and 0.1 M EDTA-McIlvaine (V:V = 1:1). The mixtures were agitated in a vortex agitator for 30 s, followed by ultrasonic (KQ250B, Kunshan Ultrasonic Bath Plant, China) for 15 min. Extracts were centrifuged at 4000 rpm for 15 min at 4 °C. The mixture concentrated to half using a rotary evaporator (EYEL4 Rotary Vacuum Evaporator N-N series, Tokyo, Japan), then passed through a Water Oasis HLB cartridge (previously conditioned with 10 ml of methanol and 15 ml of ultrapure water) at a speed of 3 ml min-1. The analytes were eluted from the cartridge with 10 ml of methanol, then concentrated to dryness under a flow of nitrogen. Finally, the residue was dissolved in 1 ml of methanol for LC–MS–MS analysis (Hu et al. 2010). To separate antibiotic residues, Alliance 2695 HPLC (Waters, Manchester, UK) and a Waters MicromassÒ Quattro MicroTMdetector with electrospray ionization were used. Samples were separated on an Agilent ZORBAX C18
(3.0 lm, 2.1 9 150 mm) maintained at 30 °C. Mobile phase A was 0.5 % formic acid and mobile phase B was acetonitrile. The gradient elution was set as follows: 0 min, 90 % A; 14 min, 85 % A; 24 min, 80 % A; 35 min, 90 % A. The injection volume was 10 ll and the flow rate was 0.25 ml min-1. For MS detection, the ionization was performed in positive mode. The electrospray source settings were optimized with the gas temperature adjusted to 350 °C, source temperature at 100 °C, and capillary voltage at 4.0 kv. The response for each of the antibiotics detected with the methods was evaluated for linearity, and the limits of detection and quantification for the instrument (LOD and LOQ) were determined, using calibration curves containing series of concentration levels. LOD and LOQ were determined using the standard deviation of the response (r) and the slope of the calibration curves (S) (ICH Steering Committee 1996). Precision was defined as the relative standard deviation (RSD) of a triplicate analysis of samples spiked at two different concentrations (10 lg kg-1 and 1 mg kg-1). Recovery studies for samples were carried out with a residue-free dung sample and soil sample at 1 mg kg-1 level for SAs and TCs. The recovery rate was calculated as an average of eight experiments at the spiked concentration listed above for each antibiotic. Validations of the analytical method are listed in Table 1.
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Table 1 Recoveries (%), RSD, detection and quantification limits (LOD and LOQ, lg kg-1) for the determining method in manure, soil, and sludge Manure Recovery (RSD)
Soil LOD
Sludge
LOQ
Recovery (RSD)
LOD
LOQ
Recovery (RSD)
LOD
LOQ 3.1
OTC
71.4 (1.7)
5
22
93.1 (1.2)
0.6
2.7
90.5 (1.1)
0.7
TC
75.2 (2.0)
6
25
92.7 (1.8)
0.6
2.2
87.3 (1.6)
0.7
2.7
CTC DOX
68.5 (2.2) 63.7 (2.1)
12 7
50 32
75.1 (2.3) 69.2 (2.0)
1.0 0.8
3.7 3.4
69.3 (2.4) 70.8 (2.2)
0.8 1.0
3.5 3.6
SDZ
74.9 (2.7)
8
37
92.2 (1.9)
0.5
2.3
80.4 (1.7)
0.8
3.3
SMR
72.6 (2.0)
15
42
85.1 (2.4)
1.1
4.7
79.6 (2.8)
0.7
3.0
SM2
77.4 (1.8)
13
47
87.6 (2.2)
0.9
3.8
83.7 (2.2)
0.5
2.1
SMZ
90.2 (2.1)
5
19
83.1 (1.8)
0.4
1.9
80.3 (2.0)
0.6
2.8
Statistical analysis Residual concentrations of selected antibiotics were statistically determined using SPSS software. The data were subjected to the analysis of variance (ANOVA) with different factors of antibiotics. The standard deviations were also calculated.
Results and discussion Descriptive statistics for the residual levels of veterinary antibiotics in manure, soil, and sludge samples are summarized in Table 2. Generally, the frequency and concentration of antibiotics were higher in manure and sludge than in soil. The detection frequency and concentration of TCs in manure, soil, and sludge were higher than that of SAs. Antibiotics in manures The highest concentration of antibiotics in manure was 143.97 mg kg-1 of CTC, and the lowest concentration was 0.02 mg kg-1 of SMZ. The concentrations of TCs and SAs were in the range of the concentrations observed in different research areas, according to the previous literatures (Table 3). That is, general concentration of various antibiotics were little different in various regions. Unlike the antibiotics in water and soil, the antibiotics in manure were slightly influenced by environmental factors (Hu et al., 2010). The usage mode and doses are the main factors that influence antibiotic residual concentrations. Results of this study, along with those of other published reports, showed that the detection rates and the highest concentrations of antibiotics in manure samples were significantly higher in China than in other countries, especially for TCs. This phenomenon is mainly caused by the extensive production and use of antibiotics in China. China is the country that most severely overuses antibiotics, both for clinical and
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agricultural purposes. Approximately 2.1 9 105 tons of antibiotics are produced in China annually, and about 46 % is used for livestock. In China, antibiotics can be easily purchased in pharmacies or hospitals, and there are no restrictions on its use in animal feed, while antibiotics were prohibited in animal feed in the European Union in 2006 and 16 antibiotics including TC and CTC were also prohibited by the US Food and Drug Administration in 2014. In terms of the two types of antibiotics, the concentrations of TCs were significantly higher than the concentrations of SAs in the manure samples, which is in contrast to the situation in other countries. This is consistent with the results in the previous literature (Pan et al. 2011). This may be connected with the different selection of antibiotics in livestock farming in different countries and regions. In China, TCs are most commonly used as the feed additives to promote the growth of animals. Besides, animals are always injected with TCs for disease prevention in different growing period. It is reported that the consumption of TCs accounted for about 45 % of the total antibiotics veterinary in breeding enterprises (Pei et al. 2015). Besides the using habits and weak regulation of antibiotics, lower prices of TCs are also an important reason that why they are used in breeding industry frequently. High concentrations of antibiotics in manure can enter environmental through many pathways, especially into the soil due to the manure always being used as fertilizer (Hamscher et al. 2002; Wang and Yates 2008). Animal operations may vary widely in their administration of veterinary antibiotics because of different breeding performances and differences in farm scale. Figure 2 shows the variations of levels of antibiotics present in manures from different farms. Manures from large-scale breeding feedlots did not have higher concentrations of the veterinary drugs in manure. Statistical analyses suggest that there are no significant differences in the concentrations of antibiotics between large-scale farms and individual household farms. Indeed, the smaller units showed the
Environ Earth Sci (2015) 74:5077–5086 Table 2 Frequency, concentration and standard deviation of antibiotics in manure (mg kg-1), soil (lg kg-1) and sludge (lg kg-1)
Compound
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Frequency (%)
Min
Median
Max
Standard deviation
Manure OTC
94.12
0.57
18.54
47.25
10.31
TC
80.39
0.06
4.69
56.95
11.90
CTC
96.08
1.24
45.12
143.97
35.15
DOX
62.75
0.03
1.74
6.5
1.84
SDZ
54.90
0.12
0.8
4.98
1.29
SMR
27.45
0.07
1.17
4.59
1.55
SM2
35.29
0.05
0.33
1.95
0.56
SMZ
41.17
0.02
1.14
18.00
3.81
OTC
48.57
17.62
608.82
1398.47
367.73
TC
38.57
29.51
240.69
976.17
246.91
CTC
64.29
8.29
717.57
1590.16
426.14
DOX
27.14
11.05
120.21
870.45
233.29
SDZ SMR
28.57 17.14
1.93 11.07
71.51 65.83
760.09 311.26
181.03 94.64
Soil
SM2
5.71
2.56
3.49
11.45
4.18
SMZ
18.57
6.36
19.35
671.52
186.20
Sludge OTC
100
174.21
1553.50
7369.67
2260.64
TC
100
297.12
1007.76
2174.46
662.48
CTC
100
197.39
850.45
3843.79
1114.42
DOX
58.33
127.45
754.47
2104.27
801.03
SDZ
75
1.27
21.06
56.32
17.27
SMR
50
0.87
14.37
37.21
12.46
SM2
41.67
0.73
16.45
27.14
9.94
SMZ
58.33
11.45
26.47
665.00
241.93
highest levels of some antibiotics, especially OTC. This trend was very similar to that observed for the residual characteristics of antibiotics in manure in eastern China (Chen et al. 2012). Drug administration strategies and operational experience may explain the variation in occurrence of antibiotics in manures among the farms (Chen et al. 2012). According to the investigation, large-scale farms choose the fine varieties of animals and use manufactured feedstuff with strict anti-epidemic measures and normative culturing times. Therefore, the types, dose, and frequency of admission of veterinary antibiotics are relatively consistent, although the number of animals and the consumption of antibiotics are large. Individual household farm always choose native species and use traditional home mixes with a long feeding time. Although the breeding scale is small, the doses and frequency of veterinary antibiotics used in individual household farms cannot be ignored, because disease resistance of the animals is generally poor and there is less supervision. And, the antibiotics were always re-used in wrong ways due to lack of technical guidance. For example, when the animals
get the flu, common antibiotics such as CTC and OTC were administered to the animal immediately. However, these antibiotics did not suit the flu most of the time. So the farmers had to increase the dosage or change to other types of antibiotics. CTC was the dominant residue no matter in intensive operations or in individual household farms, implying that CTC was the most widely used antibiotic in this area. And, the concentration of OTC in the individual household farm was significantly higher than that in the large-scale farm. The lower price of OTC is one of the reasons that farmers are more willing to use it in household breeding operations. For example, the price of CTC is 1.27 US dollars per kg, while the price of OTC is 0.50 US dollars per kg in China. Antibiotics in soil The concentration range of antibiotics in soils in the study area was 0.29–1590.16 lg kg-1, and the maximum concentration was observed for CTC. The concentrations of antibiotics in soil samples were two to three orders of
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Table 3 The concentrations of antibiotics in manure reported in other countries and regions (mg kg-1) Sample sites
Manure type
Antibiotics, concentration (mg kg-1)
References
Australia
Pig and chicken manure
TC: 0.36–23, OTC: 0.21–29, CTC: 0.1–46, SDZ: 90, SM2: 20
Martinez-Carballo et al. (2007)
Germany
Pig manure
OTC: 1.6–136, CTC: 1.1–26
Winckler et al. (2003)
TC: 4.0, CTC: 0.1
Hamscher et al. (2002)
Alberta, Canada
Feedlot manure
CTC: 0.40;
Aust et al. (2008)
North Marmara region, Turkey
Poultry manure
Denmark
Swine manure
OTC: 0.04–1.6, TC: 0.01–1.9, CTC: 0.95–24.4, DOX: 0.29–3.5
Tianjin, China
Swine and chicken manure
TC: 10.2–41.5, OTC: 9.7–173.2, CTC: 0.6–24.3, DOX: 8.6–59.8
SM2: 9.99 OTC: 0.06–0.45, CTC: 0.25–0.4, Sulfachloropyridazine(SCP): 35.53, SMZ: 3.76
Karci and Balciog˘lu (2009) Jacobsen et al. (2006)
SDZ: 0.49–3.2 Hu et al. (2008)
SDZ: 4.5–18.7, SM2: 3.3–24.8, SMZ: 2.3–5.2, SCP: 0.6–1.8 Eight provinces of China
Shandong, China
Pig, chicken and cow dung Swine manure
OTC: 0.15–59.59, CTC: 0.16–27.59, DOX: 0.23–13.5,
Zhao et al. (2010)
SMZ: 0.12–2.80, SM1: 0.10–0.66; SM2: 0.06–6.04, SCP: 0.09–3.51 CTC: 764.4
Pan et al. (2011)
SMR: 28.7 Zhejiang, China
Swine manure
TC: 98.2, OTC: 354.0, CTC: 139, DOX: 37.2
Chen et al. (2012)
SDZ: 7.1
Fig. 2 Variations in antibiotics levels in different farms
magnitude lower than in manure samples and the detection frequency of antibiotics in soil was generally lower than in manure. This phenomenon may be caused by many factors, such as dilution in the soil, degradation, leaching, and uptake by plants (Hawker et al. 2013; Srinivasan and Sarmah 2014). Moreover, the concentration of antibiotics varied greatly in soil between regions, in this study and previous studies, which is in sharp contrast to the results from manure samples. In Malaysia, DOX concentrations in soil from different sample locations were 62.6–728.4 lg kg-1, while no SAs were found in soils (Ho et al. 2012). In Italy, OTC concentrations were
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127–216 lg kg-1 in arable soil (Brambilla et al. 2007). In Denmark, concentrations of CTC in soil ranged from 0.6 to 15.5 lg kg-1, while OTC and SDZ were not detected in soil samples (Jacobsen et al. 2004). In China, the concentrations of antibiotic residues in soils were much higher than in other countries. For example, in Tianjin, northern China, concentrations of up to 2683 lg kg-1 OTC and 1079 lg kg-1 CTC were detected in organic vegetable farmland soil (Hu et al. 2010). In the Pearl River Delta, southern China, the total concentrations of TCs and SAs were up to 242.6 and 321.4 lg kg-1, respectively (Li et al. 2011). Except for the different types and dosage of antibiotics used in various regions, the environmental characteristics and soil texture may affect the fate of antibiotics in the soil (Zhang et al. 2014). For example, soil pH is critical to the residual of antibiotics in soil. Avisar et al. (2010) found that the distribution coefficient (Kd) for SAs was pH-dependent and decreased with increasing pH, which means that the soil with low pH had stronger absorption ability of SAs. Kulshrestha et al. (2004) observed that the adsorption of OTC in two selected montmorillonite clays decreased with increasing pH in the order pH 1.5 [ 5.0 [ 8.7 [ 11. Moreover, the residual concentrations of TCs were significantly higher than that of SAs in soil, manure, and sludge (Table 2). Difference in consumption is one reason for the observed discrepancy between TCs and SAs in soil,
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manure, and sludge. In China, about 20,000 tons of SAs were produced in 2006, while the production of OTCs alone were high up to 10,000 tons. Another reason is that physicochemical properties of antibiotics have impacts on their fate in the soil. TCs and SAs have different mobility characteristics in soil. It was found that the Kd values of TCs were 417–1026 mg g-1, indicating that they can strongly bind with soil particles, while the Kd values of SAs were 0.9–18.1 mg g-1, suggesting that SAs easily move downward from the surface soil (Lunestad and Goksoyr 1990; Boxall et al. 2002). So, higher concentrations of TCs in soils may be the result of their poor mobility in soil. It was also found that the frequency and concentration of antibiotics in vegetable fields were significantly higher than in crop fields in the research area. The detection frequency and the concentration of antibiotics in vegetable soil ranged from 9.30 to 83.02 % and 0.29–1590.16 lg kg-1, respectively (Fig. 3). On the contrary, only CTC was detected in four soil samples from crop fields, with a detection frequency of 14.81 %, and the highest concentration of CTC was 24.17 lg kg-1. A major reason for this finding may be that organic manure is commonly used as fertilizer in vegetable fields. In the present study, the most frequent detection and the highest concentration of the antibiotics were observed in the vegetable soil sampled near the poultry farm, which only uses manure as the fertilizer. And these soil samples were found to contain at least two members of the eight antibiotics investigated. Previous studies also indicated the manure applied to vegetable fields is often considered to be the most important sources of antibiotics to soil (Hamscher et al. 2002; Brambilla et al. 2007; Karci and Balciog˘lu 2009). And the research of Wand and Yates (2008) confirmed that antibiotic residuals can persist in soil for long periods of time once they are released from manure. In the research area, corn and soybean were the most important crops in the crop fields with
Fig. 3 The frequency and concentration of antibiotics in vegetable field
5083
wide cultivating areas. And chemical fertilizer is always used instead of manure in the crop fields. So the antibiotics detected in the soils sampled were very few. Antibiotics in sewage sludge Eight target antibiotics were detected in all sludge samples from three main sewage treatment plants. The results demonstrate that the detection frequency of antibiotics in sludge was similar to that in manure, but the concentrations of antibiotics in sludge were significantly lower than in manure (Table 2). The concentrations of TCs and SAs in sludge ranged from 127.45 to 7369.67 and 0.73–26.47 lg kg-1, respectively. OTC, TC, and CTC were the predominant antibiotics in all sludge samples. In contrast, SAs made up a minor proportion of the total antibiotics present. The higher sludge–water partitioning coefficients of TCs compared with SAs may one of the most important reasons for this phenomenon (Kim et al. 2005; Okuda et al. 2009). There were no significant differences in antibiotic concentrations between the three sewage treatment plants, mainly because they all receive municipal wastewater, a result different from a previous work in which industrial wastewater contained small quantities of antibiotics (Li et al. 2013a; Cheng et al. 2014). Generally, the concentrations of antibiotics in sludge samples observed in this study were in the range of the published concentrations in China according to the previous reports on Chinese sludge samples (Gao et al. 2012; Chen et al. 2013; Li et al. 2013b). However, the concentrations of TCs in sludge in China were much higher than in Europe and the United States (McClellan and Halden 2010; Cheng et al. 2014). A possible explanation is that the use of TCs is controlled very strictly both for humans and animals because of drug resistance and side effects in the developed countries, but TCs are still used as prescription drugs and for poultry production in China. Of course, China’s weak regulatory environment of antibiotics is also an important factor that cannot be neglected. For SAs, the difference in the concentrations in sludge between China and other countries was not significant (Xu et al. 2007; Spongberg and Witter 2008). The concentrations of SMZ and SDZ were higher than SMR and SM2, which corresponds with findings from other studies in China (Cheng et al. 2014). Seasonal changes play an important role in the distribution of organic pollutants, including antibiotics, from sewage treatment plants and hospitals; these have been documented in various published reports (Hu et al. 2010; Pena et al. 2010; Awad et al. 2014). Results from this study were similar to these findings. In Shenyang, the temperature changes greatly between winter and summer. The daily coldest temperatures in Shenyang are below -30 °C in winter (December to February), while the hottest
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temperatures in summer are up to 35 °C (July to September). As shown in Fig. 4, the concentrations of RTCs and RSAs in sludge ranged from 729.72 to 11,722.88 lg kg-1 and from 2.14 to 117.40 lg kg-1, respectively. The mean concentration of TCs and SAs in sludge sampled in January was 8415.18 and 68.98 lg kg-1, respectively, while the values in August were 2561.87 and 20.12 lg kg-1, respectively. The high concentration of antibiotics in winter might be due to their widespread use in human health and livestock breeding. It has been proven that humans and livestock fall ill more easily and therefore need more antibiotics for the treatment of diseases in winter (Hu et al. 2010). Moreover, the accelerated biodegradation of antibiotics at high temperatures and in response to strong bacterial activity in summer may be another important reason to explain the phenomenon (Vaclavik et al. 2004). The amount of raw sewage is also an ignored factor affecting the concentration of antibiotics in sludge. On average, the annual precipitation of Shenyang is about 721 mm, 65 % of which is in summer. With higher rainfall in summer compared with winter, dilution of raw sewage by heavy rain has been observed to cause a reduction in the concentration of antibiotics in sewage (Vieno et al. 2007). Additionally, water consumption by urban residents usually declines in winter (Gao et al. 2012).
Conclusions In this study, we investigated the detection frequency and concentrations of antibiotics in manure, soil, and sewage sludge in Shenyang, northeastern China. Multiple antibiotic compounds could be simultaneously detected in individual samples. The concentrations of TCs and SAs in manure in the study area were in the range reported from other regions in China, but were generally higher than those in other
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countries. The usage mode and doses, type of animal operations, and type of animal are the main factors influencing the residual concentrations of antibiotics in manure. The detection frequency of antibiotics in soil was generally lower than in manure; this may be the result of many factors, such as dilution with soil, degradation, leaching, and uptake by plants. The concentrations of antibiotics varied greatly in soil between different regions, which was in sharp contrast to results obtained for manure samples. The detection frequency and concentrations of antibiotics in soils in which organic manure was used as fertilizer were much higher. Although the concentrations of antibiotics in sludge were not high, the detection frequency was high; this indicates that antibiotic use is widespread. Lower temperatures in winter, resulting in lower levels of microbial activity, along with higher consumption of antibiotics and lower quantities of sewage, resulted in higher detection frequencies and higher concentrations of antibiotics. The concentration of TCs was significantly higher than those of SAs, not only because of differences in consumption, but also because of differences in their mobility characteristics in soil. Residual concentrations of antibiotics in manure, soil, and sludge were significant, and these compounds may act as an important source of antimicrobial contamination in terrestrial environments, which threaten ecological safety and human health. This study serves as a first step toward the assessment of antibiotics in Shenyang. Future studies are needed to predict the actual fate of antibiotics in terrestrial environments as well as their environmental effects. Acknowledgments This research was jointly supported by the National Natural Science Foundation of China (No. 21277150, 31370523, and 41001340), a research fund for the public welfare granted by the Ministry of Environmental Protection of PRC (No. 201209030), and a research fund for science and technology Project of Shenyang (No. F13-146-3-00).
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Fig. 4 The concentration of antibiotics in sludge in different season
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