Adsorption and degradation of five selected antibiotics in agricultural soil

Adsorption and degradation of five selected antibiotics in agricultural soil

Science of the Total Environment 545–546 (2016) 48–56 Contents lists available at ScienceDirect Science of the Total Environment journal homepage: w...

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Science of the Total Environment 545–546 (2016) 48–56

Contents lists available at ScienceDirect

Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Adsorption and degradation of five selected antibiotics in agricultural soil Min Pan, L.M. Chu ⁎ School of Life Sciences, The Chinese University of Hong Kong, Shatin, NT, Hong Kong SAR, China

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• The persistence of antibiotics in soil was calculated • Sterilized and non-sterilized soils under aerobic and anaerobic conditions were examined • Higher concentration slowed down degradation and prolonged persistence in soil • Degradation was related to their properties, microbial activities and soil aeration • An equation was proposed to predict the fate of antibiotics in different soils

a r t i c l e

i n f o

Article history: Received 7 October 2015 Received in revised form 8 December 2015 Accepted 8 December 2015 Available online 30 December 2015 Editor: Kevin V. Thomas Keywords: Antibiotics adsorption affinity microbial degradation persistence soil physicochemical properties aerobic conditions sterilized soil

a b s t r a c t Large quantities of antibiotics are being added to agricultural fields worldwide through the application of wastewater, manures and biosolids, resulting in antibiotic contamination and elevated environmental risks in terrestrial environments. Most studies on the environmental fate of antibiotics focus on aquatic environments or wastewater treatment plants. Little is known about the behavior of antibiotics at environmentally relevant concentrations in agricultural soil. In this study we evaluated the adsorption and degradation of five different antibiotics (tetracycline, sulfamethazine, norfloxacin, erythromycin, and chloramphenicol) in sterilized and nonsterilized agricultural soils under aerobic and anaerobic conditions. Adsorption was highest for tetracycline (Kd, 1093 L/kg), while that for sulfamethazine was negligible (Kd, 1.365 L/kg). All five antibiotics were susceptible to microbial degradation under aerobic conditions, with half-lives ranging from 2.9 to 43.3 d in non-sterilized soil and 40.8 to 86.6 d in sterilized soil. Degradation occurred at a higher rate under aerobic conditions but was relatively persistent under anaerobic conditions. For all the antibiotics, a higher initial concentration was found to slow down degradation and prolong persistence in soil. The degradation behavior of the antibiotics varied in relation to their physicochemical properties as well as the microbial activities and aeration of the recipient soil. The poor adsorption and relative persistence of sulfamethazine under both aerobic and anaerobic conditions suggest that it may pose a higher risk to groundwater quality. An equation was proposed to predict the fate of antibiotics in soil under different field conditions, and assess their risks to the environment. © 2015 Elsevier B.V. All rights reserved.

1. Introduction For decades, antibiotics have been widely used worldwide to treat diseases and to protect the health of humans and animals (Sapkota ⁎ Corresponding author. E-mail address: [email protected] (L.M. Chu).

http://dx.doi.org/10.1016/j.scitotenv.2015.12.040 0048-9697/© 2015 Elsevier B.V. All rights reserved.

et al., 2008). The consumption of antibiotics has been increasing in both industrialized and developing countries. In the US, veterinary antibiotics used in animal feeds increased from 90 t in 1950 to 9300 t in 1999 (AHI, 2002), and to 130,000 t for factory farms in 2009 (USFDA, 2012). In China, the production of total antibiotics was nearly 1,470,000 t in 2009 (Yang et al., 2010). Tetracyclines, sulfonamides, fluoroquinolones, macrolides and others are the most commonly used

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antibiotic types for both human and animals, and their usage was 12,000, 7920, 27,300, 42,200 and 38,400 t in 2013, respectively (Zhang et al., 2015). These antibiotics may be excreted as parent compounds and/or metabolites, and may enter the environment through the spreading of manures and biosolids on agricultural lands, direct deposition by grazing livestock, or discharge of wastewater, resulting in routine detection in soil, surface water, ground water, sediment and treated municipal wastewater (Chen et al., 2014; Yan et al., 2013). The presence of antibiotics in the environment can adversely affect the soil system. Tetracycline, sulfamethazine, norfloxacin, erythromycin, and chloramphenicol were studied as representatives of the commonly used antibiotic types, as they are widely used in human and animal medicines, and are frequently detected with relatively high concentrations in the environment (Sarmah et al., 2006; Yan et al., 2013). They are ionizable and can occur as neutral, zwitterionic, or charged (negative or positive) species under environmental conditions (Kahle and Stamm, 2007; Zhang and Dong, 2008; Zhao et al., 2011). These species have different chemical properties and mechanisms of adsorption and degradation in soil (Site, 2001). An understanding of their adsorption and degradation will provide vital insights into their persistence in soil and potential mobility from soil to the water column (through leaching or runoff). However, most of the studies have been carried out in aqueous environments and mainly focus on the degradation of antibiotics (Dorival-Garcia et al., 2013; Hektoen et al., 1995). Studies that examined both adsorption and degradation of antibiotics in agricultural soil are too few with most using unrealistically high concentrations (in mg/kg levels) to overcome limitations in measurement (Yang et al., 2009a, 2012; Zhao et al., 2011). Also, no model has been developed for speculating the adsorption and degradation of different types of antibiotics in agricultural soil and the environmental risks they may pose. A comprehensive investigation of antibiotic adsorption and degradation is needed for a more complete understanding of the downward movement of antibiotics in soil. The objectives of this study were (1) to evaluate the adsorption and degradation of selected antibiotics in soil at environmentally relevant concentrations, (2) to study the relationship between their persistence in soil and their physicochemical properties, and (3) to derive an equation to predict their fate in soil under different field conditions. In the present study, the adsorption isotherm of selected antibiotics in the soil was determined. The mechanisms of degradation were investigated through comparative experiments using sterilized and non-sterilized soils under aerobic and anaerobic conditions with environmentally relevant concentrations in agricultural soil. Furthermore, based on the parameters obtained in the adsorption and degradation experiments, an equation would be derived to calculate the proportions of antibiotic adsorbed, degraded, and remained in the soil. The behaviors of other compounds in soil can also be evaluated to assess their environmental risks. This should be the first study to develop a model for the prediction of antibiotics in soil. Moreover, the estimated persistence in soil and environmental risks of the selected antibiotics were summarized. The results from the present study should help assess the environmental risks of antibiotics and other organic compounds in agricultural soil. 2. Materials and methods 2.1. Chemicals and soil Tetracycline, sulfamethazine, norfloxacin, erythromycin, and chloramphenicol, which belong to five different types of antibiotics, were examined in this study (Table 1). All the standards used and some of the internal standards (sulfamethazine-d4, norfloxacin-d5, and erythromycin-13C2) were obtained from Sigma-Aldrich (USA); chloramphenicol-d5 was purchased from Dr. Ehrenstorfer GmbH (Germany) and tetracycline-d6 from Toronto Research Chemicals

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(Canada). Oasis HLB extraction cartridges (6 mL, 500 mg) (Waters Corporation, USA) were used to extract and purify target compounds. All organic solvents used were of HPLC grade and purchased from Merck Corporation (Germany). Individual stock solutions and internal standards were prepared at 100 mg/L in methanol and stored in amber glass vials at −20 °C. The agricultural soil used in this study was collected from an organic farm (Produce Green, Hong Kong) at the 0–20 cm depth, which was sieved through a 2 mm sieve after air drying. It was a clay loam with a pH of 6.45, 0.80% organic carbon content (foc) and 50% maximum water holding capacity. The bulk density (ρb) was 1.39 g/cm3 and porosity (θ) 0.45. Soil texture, bulk density and porosity were determined according to the methods by American Society for Testing and Materials (ASTM, 2006, 2007; ATSM, 2009). Soil pH value was measured by a pH meter in distilled water (1:1). Organic carbon was analyzed using a TOC analyzer (Shimadzu, Japan). The maximum water holding capacity was determined according to the guideline of International Organization for Standardization (ISO, 2012). To determine the role of soil microorganisms in antibiotic degradation, a subsample was sterilized by autoclaving at 121 °C for 45 min over three consecutive days. 2.2. Adsorption experiment Batch adsorption experiments were performed according to the guideline of the Organization for Economic Co-operation and Development (OECD, 2000). A total of 25 mL CaCl2 solution (0.01 M) containing the five selected antibiotics and 5 g agricultural soil (dry weight) was added to 50 mL centrifuge tubes. The soil samples were spiked with 20 μL antibiotic solutions to yield initial nominal concentrations of 10, 20, 40, 60, 80, and 100 μg/L for each antibiotic in the aqueous phase. To inhibit microbial activity, sodium azide (25 μg) was added into each sample. The initial pH of the solution was adjusted by HCl and NaOH solutions to 6.45 ± 0.50. All the centrifuge tubes were shaken in an orbital shaker (Lab-line 3527–1 Environ shaker) at 180 rpm and 25 ± 1 °C for 24 h to obtain equilibrium. All procedures were conducted in the dark to avoid photodegradation. After equilibrium was achieved, the tubes were centrifuged at 8820 × g for 10 min. The supernatant was then filtered through a 0.2 μm syringe filter before HPLC-MS/MS analysis. All experiments were conducted in triplicates. Blank samples containing an equivalent amount of soil and a total aqueous solution volume of 25 mL (without antibiotics), and soil-less, antibiotic-only controls were subjected to the same experimental procedures. These were used as background controls to detect any interfering compound or contamination originating from the soil or the aqueous solution. The concentration of an antibiotic (Cw, μg/L) in the supernatant was determined after equilibrium was achieved and Cs (μg/kg) is the equilibrium concentration of antibiotic adsorbed in soil. The adsorption coefficient Kd (L/kg) was calculated using the following equation (Xu et al., 2009): Kd ¼ Cs =Cw

The organic carbon adsorption coefficient Koc value for each antibiotic was calculated by the equation: Koc = Kd/foc. Freundlich isotherm was used to test the linearity of adsorption coefficient over a range of concentrations (Xu et al., 2009): Cs ¼ K f  Cw n

where Kf is the Freundlich adsorption coefficient of a given substance and n is the Freundlich exponent which indicates whether the

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Table 1 The physicochemical parameters of the five antibiotics examined in this study.

Molecular formula Molecular weight Log Kow pKa

Tetracycline

Sulfamethazine

Norfloxacin

Erythromycin

Chloramphenicol

C22H24N2O8 444.43 −1.3 3.3;7.68; 9.69 a

C12H14N4O2S 278.33 0.76 2.07; 7.49a

C16H18FN3O3 319.33 −1.03 3.11; 6.1; 8.6 b

C37H67NO13 733.93 na 8.88; 12.91c

C11H12Cl2N2O5 323.13 0.92 5.5;9.61d

a Sarmah et al., 2006; b Zhang and Dong, 2008; c Yang et al., 2010; d Peng et al., 2008. na = not available. Log Kow: the logarithm of the octanol/water partition coefficient.

adsorption is linear. Both values are dimensionless Freundlich isotherm constants. The retardation factor, RF, was calculated using the measured Kd values, to estimate the travel time of antibiotics in soil. The variable of ρb is soil bulk density and θ soil porosity (Xu et al., 2009). R F ¼ 1 þ ρb  Kd =θ

2.3. Degradation experiment Degradation of selected antibiotics in sterilized and non-sterilized soils was examined under both aerobic and anaerobic conditions. In the aerobic incubation experiment, 5 g agricultural soil (dry weight) were weighed into 50 mL screw capped centrifuge tubes; the soil water content was maintained at 70% of maximum water-holding capacity by adding in a given amount of deionized water. The soil samples were spiked with 10 μL mixed antibiotic solutions to yield an initial nominal concentration of 100 μg/kg for each compound. The centrifuge tubes were weighed every other day for water loss and deionized water was added if necessary. The tubes were wrapped with aluminum foil to minimize any possible photodegradation. The treated samples were incubated in a dark and airy cabinet at room temperature (25 ± 1 °C). Three different initial antibiotic concentrations (50, 100, and 200 μg/kg) were used by adding into each tube 10 μL of mixed antibiotics solutions. The initial concentrations of antibiotics were prepared in accordance with their detections in soils and manure (Hu et al., 2010; Li et al., 2015). In the anaerobic incubation experiment, the anaerobic conditions were induced following the procedures for a previous study (Lin and Gan, 2011). Briefly, the sample-containing centrifuge tubes were opened and transferred into a gastight, inflatable plastic glove chamber filled with nitrogen gas (99.99%). The centrifuge tubes were flushed with nitrogen and equilibrated in the glove box for one day, and then sealed with screw caps. The soil samples were then removed from the glove chamber and immediately spiked with 10 μL methanol solution containing 0.5 μg of each antibiotic. After vortexing for 30 s, the treated soil samples were returned to the nitrogen-filled chamber for incubation at room temperature (25 ± 1 °C). For both aerobic and anaerobic degradation experiments, samples were collected on 0, 1, 7, 14, 21, 28, 35, 42, 49, 56, 63, and 90 d after treatment, and stored at −22 °C until analysis. All experiments were conducted in triplicates. Data from the degradation experiments were fitted to the exponential decay model (Ho et al., 2013): Ct = C0e-kt where Ct is the time-varying concentrations of antibiotics (μg/kg), C0 is the initial antibiotics concentration (μg/kg), k is the degradation rate constant (1/d), and t is degradation time (d). Half-lives (DT50, d) were calculated by the equation: DT50 = 0.693/k. SigmaPlot v.12.5 (Systat Software, San Jose, CA) was used for all the model fitting work in this study.

2.4. Extraction and analysis The antibiotics in supernatant and soil were determined according to the procedures described in our previous study (Pan et al., 2014). Briefly, one gram of freeze-dried soil was ground and spiked with 100 μL of each of the internal standards (1.0 mg/L). The soil samples were extracted three times with 30 mL acetonitrile and 0.2 M citric acid buffer (pH 4.4) (v:v = 1:1) through vortexing (60 s each time) and ultra-sonication (15 min each time). The mixture was centrifuged in air-cooled conditions at 1370 × g for 10 min and concentrated; the extract for each soil sample was evaporated to near dryness, and 0.2 g of Na2EDTA was added and diluted to 200 mL with Milli-Q water. The extracts were then passed through HLB cartridges for purification. The analytes were eluted from each cartridge with 10 mL methanol, and dried under a gentle nitrogen stream. The residue was finally redissolved in 1 mL methanol and filtered through a 0.2 μm syringe filter for analysis. The target antibiotic compounds were analyzed by HPLCMS/MS (Agilent Liquid Chromatography 1100 series HPLC system coupled to an Agilent 6410 triple quadrupole MS, and equipped with an electrospray ionization (ESI) source (Agilent, USA)) in a multiplereaction monitoring (MRM) mode. The rates of recovery of the target compounds ranged between 79% and 124%. The LOQ of the target compounds ranged from 0.70 to 4.63 ng/g. Ten concentrations (0.5, 1, 2, 5, 10, 20, 50, 100, 200, and 500 μg/L) of individual antibiotics were used to construct the calibration curves (r2 N 0.999).

3. Results and discussion 3.1. Soil adsorption Fig. 1 shows the adsorption isotherms of the five antibiotics in agricultural soil. Adsorption of these antibiotics conforms to the Freundlich equation over a range of equilibrium concentrations, with r2 ranging from 0.97 to 1.00 (Table 2). The Freundlich adsorption coefficients Kf, which reflect the affinity of each antibiotic to soil, are listed in Table 2. As all the n values derived from Freundlich equations were found to be close to 1, Kd values were calculated by fitting adsorption data to a linear equation. The adsorption coefficients of the five antibiotics suggest that sulfamethazine had the lowest Kd (1.365 L/kg) while tetracycline had the highest (1093 L/kg). Similar Kd values were obtained in previous studies with nearly soil organic matter (1140 L/kg for tetracycline and 2.0 L/kg for sulfamethazine) (Langhammer, 1989; Sithole and Guy, 1987). There were significant differences in Kd values between the antibiotics (p b 0.05). The log Koc value for each antibiotic ranged from 0.23 (sulfamethazine) to 3.14 (tetracycline). The five antibiotics exhibited adsorption affinities on soil in the descending order of tetracycline N norfloxacin N erythromycin N chloramphenicol N sulfamethazine. Sulfamethazine was the most mobile antibiotic in soil among the five, while tetracycline was the least mobile. Hamaker and Thompson (1972) postulated that sorption kinetics

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Fig. 1. Adsorption of the five antibiotics in agricultural soil (n = 3).

depend on the sorption process and the transport of the compound to the sorption sites, which consists of transport to outer sorption sites (macrotransport) and diffusion into micropores and capillaries. For most antibiotics, adsorption does not only depend on polarity and solubility in water, but also on the soil pH which affects the fraction of

ionized antibiotics in the soil (Figueroa-Diva et al., 2010; Srinivasan et al., 2013). Sulfamethazine was converted from its cationic form to neutral form in natural soil environments, with cation bridging playing a less significant role in its adsorption in soil, which resulted in lower adsorption (Figueroa-Diva et al., 2010). In this study, the adsorption of

Table 2 Adsorption isotherms parameters (mean ± standard error) for target antibiotics.

Tetracycline Sulfamethazine Norfloxacin Erythromycin Chloramphenicol

Kf, mg1-n Ln/kg

n

r2

Kd, L/kg

Log Koc

RF

1288 ± 102 1.96 ± 0.28 632 ± 28.6 198 ± 11.9 5.47 ± 0.84

1.01 ± 0.08 1.08 ± 0.29 1.01 ± 0.08 1.05 ± 0.12 1.15 ± 0.11

0.974 0.994 0.998 0.948 0.997

1093 ± 91.6 1.37 ± 0.16 591 ± 35.0 130 ± 9.33 2.50 ± 0.15

3.14 0.23 2.87 2.21 0.50

3377 5.22 1827 404 8.73

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sulfamethazine was found to be in contrast with that of tetracycline and norfloxacin which are known to interact with soils primarily through cation exchange, surface complexation, and cation bridging sorption mechanisms. Cation exchange reactions and surface complexation of zwitterions onto the clay surface have been proposed as two major mechanisms for tetracycline adsorption in neutral soil environment (Figueroa et al., 2004; Li et al., 2010; Wang et al., 2010). Norfloxacin has two proton-binding sites (carboxyl and piperazinyl groups) with pKa values of 6.1 and 8.6, respectively. Therefore, norfloxacin predominantly exists in the zwitterionic form and to a lesser extent cationic form in soil (Zhang and Dong, 2008), which results in lower adsorption than tetracycline. The adsorption affinity of erythromycin (Kd = 130.3) was found to be lower than that of norfloxacin. The neutral chloramphenicol can be absorbed by soil via hydrophobic interactions, while its low Kd suggests a low sorption affinity to soil particles and high mobility in soil. Besides these adsorption reactions of antibiotics in soil,

other factors involved include dissolved organic matter, humic acid, cation exchange capacity and metal cations (Site, 2001; Zhao et al., 2011). The retardation factor RF was calculated for each antibiotic to estimate its travel time in the soil. The RF values of tetracycline and norfloxacin were larger than 1000, while those of sulfamethazine and chloramphenicol were relatively low. This suggests that compared to sulfamethazine and chloramphenicol, tetracycline and norfloxacin were adsorbed more readily to soil, which substantially increased their residence. 3.2. Degradation in soil Fig. 2 shows the measured concentrations for each antibiotic at various time points following one of the four treatments. The degradation curves for each antibiotic were fitted to the exponential decay model; the degradation rate constant k (1/d) and half-lives (d) were calculated.

Fig. 2. Degradation of the five antibiotics in soil incubated under various conditions. The points and error bars represent the mean and standard deviation of replicates, respectively (n = 3).

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As shown in Table 3, most of the fits were excellent with r2 N 0.95, except for the case of erythromycin with non-sterilized soil and aerobic incubation (r2 = 0.85). This suggests that the degradation of the five antibiotics in agricultural soil under various conditions generally conformed to the exponential decay model. 3.2.1. Degradation of antibiotics in sterilized soil and non-sterilized soil under various conditions In sterilized soil, all the antibiotics exhibited moderate to high persistence in both aerobic and anaerobic conditions, with half-lives ranging from 40.8 to 86.6 d (Table 3). Under anaerobic condition, tetracycline was found to exhibit the highest persistence (DT50, 86.6 d), followed by sulfamethazine and erythromycin (same DT50, 57.8 d), suggesting that tetracycline, sulfamethazine, and erythromycin were preferably retained in the sterilized agricultural soil. The degradation of sulfamethazine in sterilized soil under anaerobic condition was relatively faster than that under anaerobic fermentation reported by Mohring et al. (2009), where 100% of sulfamethazine was detected even after 34 d. The half-lives of sulfamethazine in sterilized soil (49.5 d under aerobic condition and 57.8 d under anaerobic condition) were comparable to the respective results obtained by Yang et al. (2009a, 2009b), where half-lives of sulfonamides were 19–265 d in soils sterilized through different means (Yang et al., 2009a). In non-sterilized soil, all antibiotics except tetracycline and sulfamethazine were degraded by at least 50% after 11 d of incubation (Table 3). The half-life of tetracycline in non-sterilized soil was 31.5– 43.3 d, which was longer than the 23 d in manure-amended soil (Aga et al., 2005). The degradation of sulfamethazine in non-sterilized soil was slower than in manure compost reported by Ho et al. (2013) (DT50 = 1.4 d). For norfloxacin, erythromycin, and chloramphenicol, degradation appears to be very fast, and persistence in non-sterilized soil was low. The degradation of norfloxacin in non-sterilized soil was faster than in mineral salts medium (Amorim et al., 2014), but not as rapidly as in manure compost (Ho et al., 2013). Most antibiotics were degraded by more than 92% in non-sterilized soil after 28 d of incubation (Fig. 2). Tetracycline and sulfamethazine appeared to be more resistant to degradation across all the tested conditions, with DT50 of 31.5–86.6 and 24.8–57.8 d, respectively. Exponential decay equations were fitted to the degradation of tetracycline and sulfamethazine, with r2 of 0.94– 0.99 and 0.96–0.99, respectively (Table 3). After 90 d of incubation, more than 80% of the spiked tetracycline and sulfamethazine was degraded in non-sterilized soil, but only 44.4% for tetracycline and 66.1%

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for sulfamethazine in sterilized soil under anaerobic condition (Fig. 2). Tetracycline had the longest half-life than the other antibiotics, and also the strongest adsorption in the agricultural soil studied. As a result of kinetic adsorption, the bioaccessibility and bioavailability of tetracycline decrease with increasing contact time in the soil; secondary sorption reactions and diffusion into micro- and nano-pores that are too small for microorganisms and enzymes keep tetracycline away temporarily from biological degradation or uptake (Förster et al., 2009; Jechalke et al., 2014). The sequestration prolongs the residence of tetracycline in the soil by providing transient storage in a form that is not bioavailable. Other studies have found that the half-lives of sulfamethazine in non-sterilized and sterilized soil range from 5 to 30 d and 58.7–265 d, respectively (Accinelli et al., 2007; Kümmerer and Henninger, 2003; Lin and Gan, 2011; Mohring et al., 2009). Limited or no degradation of sulfamethazine has been detected in marine sediments, heated water, and sterilized systems (Accinelli et al., 2007; Hektoen et al., 1995), the last bit of which is consistent with the results in the present study. Under aerobic conditions, the fungi in non-sterilized soil play an important part in sulfamethazine degradation (Garcia-Galan et al., 2011), whereas under anaerobic conditions only little of the sulfamethazine in sterilized soil is degraded. This could be related to its chemical and metabolic stability towards hydrolysis (Teeter, 2003), as well as its negligible adsorption in soil. Norfloxacin, erythromycin, and chloramphenicol exhibited noticeable dissipation in non-sterilized soil, with half-lives 2.9–5.6, 6.4–11, and 6.7–8.6 d, respectively (Table 3). Most of the observed degradation occurred during the first 10 d and became less rapid thereafter (Fig. 2). For erythromycin and chloramphenicol, the degradation was so complete in non-sterilized soil under aerobic conditions by the end of the 90 d. Norfloxacin was the most rapidly degraded one in nonsterilized soil. Its half-lives at 24–98 d in non-sterilized soil and 153 d in sterilized soil under aerobic conditions by Yang et al. (2012) were much longer than those obtained in the present study. While the degradation of norfloxacin takes place at a similar rate in sludge under aerobic conditions (DT50 = 144.4 h) (Dorival-Garcia et al., 2013), an initial decrease of norfloxacin is probably due to microbial transformation (Adjei et al., 2006; Prieto et al., 2011). The degradation abilities of microorganisms can be attributed to their resistance to norfloxacin. For erythromycin the half-lives in non-sterilized soil were 6.4–11 d which was shorter than those in manure-treated soil (20 d) or those in manure storage (41 d) (Schlusener and Bester, 2006; Schlusener et al., 2006). The degradation of erythromycin is more efficient in sludge under nitrifying conditions, owing to its higher affinity for nitrifying bacteria (Suarez et al., 2010). Our results were similar to those of Suarez et al.

Table 3 Data of the exponential decay model for each antibiotic. Compound Tetracycline

Incubation conditions aerobic anaerobic

Sulfamethazine

aerobic anaerobic

Norfloxacin

aerobic anaerobic

Erythromycin

aerobic anaerobic

Chloramphenicol

aerobic anaerobic

k (1/d) sterilized non-sterilized sterilized non-sterilized sterilized non-sterilized sterilized non-sterilized sterilized non-sterilized sterilized non-sterilized sterilized non-sterilized sterilized non-sterilized sterilized non-sterilized sterilized non-sterilized

0.012 0.022 0.008 0.016 0.014 0.028 0.012 0.020 0.017 0.242 0.013 0.124 0.017 0.108 0.012 0.063 0.016 0.104 0.013 0.013

Equation -0.012t

Ct = 88.24 × e Ct = 82.36 × e-0.022t Ct = 89.20 × e-0.008t Ct = 83.57 × e-0.016t Ct = 94.29 × e-0.014t Ct = 94.57 × e-0.028t Ct = 99.95 × e-0.012t Ct = 95.98 × e-0.020t Ct = 95.94 × e-0.017t Ct = 107.0 × e-0.242t Ct = 93.09 × e-0.013t Ct = 111.9 × e-0.124t Ct = 74.95 × e-0.017t Ct = 44.59 × e-0.108t Ct = 69.45 × e-0.012t Ct = 72.55 × e-0.063t Ct = 100.2 × e-0.016t Ct = 79.25 × e-0.104t Ct = 103.9 × e-0.013t Ct = 98.78 × e-0.081t

r2

DT50 (days)

0.9818 0.9869 0.9407 0.9681 0.9841 0.9864 0.9545 0.9853 0.9903 0.9829 0.9977 0.9629 0.9933 0.8514 0.9784 0.9852 0.9934 0.9776 0.9903 0.9916

57.8 31.5 86.6 43.3 49.5 24.8 57.8 34.7 40.8 2.91 53.4 5.60 40.8 6.4 57.8 11.0 43.3 6.70 53.3 8.60

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(2010), which suggest erythromycin is highly biodegradable under aerobic conditions but more persistent under anaerobic conditions. Finally, for chloramphenicol, its degradation in non-sterilized soil was equally fast, with more than 99% degraded within 90 d. The half-lives in nonsterilized soil were significantly shorter than those in sterilized soil (p b 0.05). The value for the non-sterilized, aerobic condition (6.7 d) was comparable with those identified for aquaculture pond sediments (2.4–18.4 d) (Chien et al., 1999). The degradation rate of the five antibiotics under aerobic conditions followed the order of norfloxacin N erythromycin N chloramphenicol N sulfamethazine N tetracycline, which was slightly different from that under anaerobic conditions (norfloxacin N chloramphenicol N erythromycin N sulfamethazine N tetracycline). All five antibiotics had higher persistence in sterilized soil under anaerobic conditions,

with DT50 N 40 d. None of the antibiotics showed appreciable or fast degradation in sterilized soil, which indicates that microorganisms play an important role in the degradation. The degradation behavior of the antibiotics is believed to be a function of the chemical characteristics as well as the microbial activities and oxygen status in the soil. 3.2.2. Degradation of antibiotics present in various initial concentrations Each antibiotic was administered at three different initial concentrations in soil (50, 100, and 200 μg/kg) and subjected to the aerobic/nonsterilized condition. Their half-lives varied: 14.1–69.3 d for tetracycline, 16.90–53.31 d for sulfamethazine, 1.83–6.93 d for norfloxacin, 3.01– 16.9 d for erythromycin, and 3.19–19.3 d for chloramphenicol (Fig. 3). An increase in initial concentration would result in an increase in halflife. Schlusener and Bester (2006) found that the half-life of

Fig. 3. Degradation of the five antibiotics administered at various initial concentrations in soil. The points and error bars represent the mean and standard deviation of replicates, respectively (n = 3).

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erythromycin present at an initial concentration of 2000 μg/kg and under aerobic conditions is 20 d. Yang et al. (2012) found the halflives for norfloxacin in non-sterilized soil under aerobic conditions to be 24–98 d, a range much higher than 1.83–6.93 d obtained in the present study. The higher half-lives may be due to the extremely high initial concentrations (5–30 mg/kg) used. Again these results suggest that the higher the initial concentration of an antibiotic in soil, the lower the degradation rate constant and hence the longer the degradation time. Higher concentrations of antibiotics as a result of wastewater and/or manure application will decrease the rate of antibiotic degradation and prolong their persistence in the soil, as it will inhibit the activity of the degrading microorganisms (Xu et al., 2009; Yang et al., 2009b). The present study used concentrations that are environmentally relevant to determine the half-life of degradation as a function of the initial concentration in soil; use of unrealistically high concentrations tend to overestimate half-lives, which would deviate from genuine situations. The varied half-lives may also be attributed to the differences in the soils used in the experiments. Thus, the degradation behavior of the antibiotics is not only related to the initial concentration of antibiotics, but also to the physicochemical properties of the soil to which the antibiotics are applied. 3.3. Persistence in soil The inclusion of different types of antibiotics in this study allows an in-depth analysis of their adsorption and degradation in soil as well as an assessment of their persistence and environmental risks to the terrestrial and water environments. The persistence of a given antibiotic can be calculated using the following equation (the derivation of the equation is listed in the supplementary information): Ct ¼

Kd C ekt ð1 þ 2Kd Þ i

where Ct is the time-varying concentrations of the antibiotic and Ci is its initial spiked concentration in soil. The Kd values (L/kg) were obtained from the adsorption experiments, while the k values (1/d) were derived from the degradation experiments, and the maximum water holding capacity of the clay loam soil was 50%. Antibiotic photolysis and hydrolysis are not considered in the calculation. The equation can also be used to estimate the specific partition of adsorption and degradation of an antibiotic. When 100 μg/kg antibiotics are applied to soil (e.g. through wastewater irrigation or manure application), assuming the soil is aerobic and non-sterilized, there will be 49.9, 36.6, 50.0, 49.8, and 41.7 μg/kg of tetracycline, sulfamethazine, norfloxacin, erythromycin, and chloramphenicol adsorbed in the soil, respectively, after 10 days, and a respective amount of 9.87, 8.94, 45.5, 32.9, or 26.9 μg/kg degraded in the soil. These calculated values were comparable to the values derived from the adsorption experiments and the degradation experiments carried out under the aerobic/ non-sterilized condition. After being adsorbed or degraded in the soil, some of the antibiotic will be retained in its active form; these fractions vary between antibiotics: 40.1 μg/kg for tetracycline, 27.7 μg/kg for sulfamethazine, 4.44 μg/kg for norfloxacin, 16.9 μg/kg for erythromycin, and 14.7 μg/kg for chloramphenicol.

55

These active forms may pose further environmental risks to terrestrial organisms or groundwater. The persistence equation also applies to other compounds in soil. Based on the experimental data by Xu et al. (2009), the specific partition of adsorption and degradation of bisphenol A in the Arlington sandy loam can be calculated. Given the Kd and k values from their study, the compound is estimated to be initially present in the soil in 49.1 μg/kg, from which 45.2 μg/kg is estimated to be degraded after 8 days, leaving behind a residue of 3.90 μg/kg in the soil. These estimations are consistent with their experimental data. This equation would provide more information on the adsorption and degradation, as well as environmental risks of different compounds in soil. Table 4 is a summary of the antibiotics in terms of their estimated persistence in soil and the environmental risks they may pose. The phytotoxicity data were obtained from our previous study in which different plants were examined (data not shown). Among the five antibiotics, tetracycline was characterized by the highest level of adsorption and phytotoxicity, as well as the lowest rate of degradation in soil, suggesting that tetracycline was preferentially retained in soil and hence further toxic effects to terrestrial organisms. Sulfamethazine was more mobile and degradable in soil and therefore moved downward readily and caused risks to the water environments (e.g. groundwater and/or surface water). Norfloxacin and erythromycin were not as phytotoxic or well adsorbed in soil as tetracycline, but they still exhibited greater adsorption in soil and phytotoxicity than sulfamethazine and chloramphenicol. Nonetheless, they were degraded more rapidly than tetracycline and sulfamethazine.

4. Conclusions The present study suggests that the adsorption of antibiotics in soil depends on the physicochemical properties of both antibiotics and soil. The degradation of the five tested antibiotics at environmentally realistic concentrations under aerobic or anaerobic conditions appears to be influenced by soil microbial activities and oxygen status, as well as the initial concentrations of antibiotic compounds. The strongest adsorption and the slowest degradation behavior of tetracycline imply that the antibiotic was more persistent in the surface soil and resulted in higher environmental risks to organisms in the topsoil. The poor adsorption and slow degradation of sulfamethazine suggest that it moved downward readily during wastewater irrigation and/or manure application, leading to greater risks of groundwater contamination. All the antibiotics degraded faster under aerobic conditions than anaerobic conditions, indicating that a higher percentage of antibiotics would be degraded in a given time period in soil under aerobic conditions. Higher initial concentrations of antibiotics seem to prolong their persistence than environmentally relevant concentrations in soil. An equation was successfully developed to predict the concentrations of antibiotic in soil, and was validated by experimental results, which estimated the proportions of antibiotic adsorbed, degraded, and remained in soils. Based on our knowledge, this should be the first study to develop a model for the prediction of antibiotic persistence in soil, which is valuable for the investigation of the fate of antibiotics in the terrestrial environment.

Table 4 Persistence and environmental risk profile of the five studied antibiotics. Antibiotics

Tetracycline

Sulfamethazine

Norfloxacin

Erythromycin

Chloramphenicol

Adsorption DT50 (aerobic) DT50 (anaerobic) Phytotoxicity Environmental risk

***** * * ***** *****

* ** ** ** *

**** ***** ***** **** ***

*** **** *** *** **

** *** **** * *

The asterisks express the level of each attribute: a larger number of asterisks indicate a higher level.

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M. Pan, L.M. Chu / Science of the Total Environment 545–546 (2016) 48–56

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