Science of the Total Environment 479–480 (2014) 284–291

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Dissipation of sulfamethoxazole in pasture soils as affected by soil andenvironmental factors

Prakash Srinivasan a,b, Ajit K. Sarmah a,⁎a Department of Civil & Environmental Engineering, Faculty of Engineering, The University of Auckland, Private Bag 92019, Auckland, New Zealandb Landcare Research, Private Bag 3127, Hamilton, New Zealand

H I G H L I G H T S

• Sulfamethoxazole dissipation was a combined effect of biotic and abiotic factors, with microbes being the major contributors.• SMO dissipation rate in soils was independent of initial spiked concentration.• Phospholipid fatty acid analysis was indicative of higher bacterial presence as compared to fungal community.• Sulfamethoxazole is unlikely to persist more than 5–6 months in pasture soils at either depth.

⁎ Corresponding author at: Department of Civil & EnviroEngineering, Private Bag 92019, Auckland 1142, New Zfax: +64 9 3737462.

E-mail address: a.sarmah@auckland.ac.nz (A.K. Sarma

http://dx.doi.org/10.1016/j.scitotenv.2014.02.0140048-9697/© 2014 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 9 January 2014Received in revised form 5 February 2014Accepted 5 February 2014Available online 22 February 2014

Keywords:SulfamethoxazoleDissipationKineticsDehydrogenasePLFA

The dissipation of sulfamethoxazole (SMO) antibiotic in three different soils was investigated through laboratoryincubation studies. The experiments were conducted under different incubation conditions such as initial chem-ical concentration, soil depth, temperature, andwith sterilisation. The results indicate that SMO dissipated rapid-ly in New Zealand pasture soils, and the 50% dissipation times (DT50) in Hamilton, Te Kowhai and Horotiu soilsunder non-sterile conditions were 9.24, 4.3 and 13.33 days respectively. During the incubation period for eachsampling event the soil dehydrogenase activity (DHA) and the variation inmicrobial communityweremonitoredthorough phospholipid fatty acid extraction analysis (PLFA). The DHA data correlated well with the dissipationrate constants of SMO antibiotic, an increase in the DHA activity resulted in faster antibiotic dissipation. ThePLFA analysis was indicative of higher bacterial presence as compared to fungal community, highlighting thetype of microbial community responsible for dissipation. The results indicate that with increasing soil depth,SMO dissipation in soil was slower (except for Horotiu) while with increase in temperature the antibiotic losswas faster, and was noticeable in all the soils. Both the degree of biological activity and the temperature of thesoil influenced overall SMO dissipation. SMO is not likely to persist more than 5–6 months in all three soils sug-gesting that natural biodegradationmay be sufficient for the removal of these contaminants from the soil. Its dis-sipation in sterile soils indicated abiotic factors such as strong sorption onto soil components to play a role in thedissipation of SMO.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

An estimated 9000 tonnes of antibiotics is annually used in thelivestock industry by the US, 5000 tonnes by the European Union, and6000 tonnes by China. After administration, a high proportion(30–90%) of the antibiotics is excreted by livestock animals in un-changed form and/or sometimes as metabolite/s (Sarmah et al., 2006).Occurrences of antibiotic residues are common in many parts of theworld and have been detected in environmental media such as soils,

nmental Engineering, Faculty ofealand. Tel.: +64 9 9239385;

h).

surface water, and ground water (Hamscher et al., 2003; Luo et al.,2011; Perret et al., 2006; Zuccato et al., 2005). Although the use of anti-biotics in livestock industry in New Zealand (NZ) is not as widespreadas in many other parts of the world, intra-mammary injectable antibi-otics dominate the dairy industry (Srinivasan et al., 2013). Accordingto the New Zealand Food Safety Authority, the sulfonamide group con-tributes ~17% of total antibiotic usage, and is a common class of antibi-otics widely used in livestock industries in NZ. Free pasture grazing bymillions of cattle is common in many parts of NZ, and dairy industryhas been also expanding at a rapid rate especially in South Island ofNZ. Because of direct excretal inputs by grazing animals and permittedactivity such as land-application of animal waste by farmers, there is aconcern that antibiotic residues may be entering the environment andcould potentially impact the aquatic and terrestrial ecosystems.

285P. Srinivasan, A.K. Sarmah / Science of the Total Environment 479–480 (2014) 284–291

In the last decade several studies have been conducted on thebiodegradation of sulfonamide antibiotics such as sulfadiazine (SDZ),sulfamethazine (SMZ), and sulfachloropyridazine (SCP) in soils underdiverse laboratory conditions (Accinelli et al., 2007; Fan et al., 2011;Halling-Sørensen et al., 2003; Kreuzig and Holtge, 2005; Thiele-Bruhnand Peters, 2007; Wang et al., 2006b; Yang et al., 2009). Most of thepublished studies reported in the literature are difficult to compare, asno two studies were similar in terms of the antibiotics investigated,and the experimental conditions and environmental matrices used.Limited studies on sulfonamide degradation in soils have shown that50% dissipation half-life (DT50) values for sulfonamides ranged fromas low as 1 day to 2 weeks under varied initial concentration and tem-perature (SI. Table 1). These studies are not appropriate to compareeach other because of the differences in their experimental approachesand objectives. Some of themajor findings from previous studies whichfocused on degradation of SMO or other compounds within the samegroup in soils suggest that sulfonamides may be more persistent thanwould be predicted from laboratory controlled studies (Bialk et al.,2005). Studies conducted by Accinelli et al. (2007) found that high ini-tial concentration (100 mg kg−1) did not affect the dissipation rate ofSMZ and SCP suggesting that at environmental concentrations (ppb orppt level) there would be little or no effects. Elsewhere, SMO and tri-methoprim showed higher dissipation than tylosin in soils, owing togreater sorption potential for the latter in soils (Liu et al., 2010). At aspiked concentration of 10 mg kg−1, SDZ half-lives in aerobic non-sterile soils ranged from 12 to 18 days while it was more persistent inanoxic non-sterile soils with half-lives ranging between 57 and237 days (Yang et al., 2009).

While there have been a number of studies on sulfonamide deg-radation in manure, sludge amended soils, studies on pasture soilshave hitherto been neglected. NZ pasture soils are high in organiccarbon content and minerals such as allophane, and these propertieshave been found to have pronounced effects on the sorption of thesecompounds which could indirectly influence their degradation be-haviour in soils (Srinivasan et al., 2012). Dissipation half-life is animportant input parameter required in antibiotic fate modelling ex-ercises and for risk assessment purposes. Sulfamethoxazole (SMO),which belongs to the sulfonamide group of antibiotics, was chosenfor this study, as limited information exists about its fate in soil(Holtge and Kreuzig, 2007). Given the varied soil and climatic condi-tions of NZ, extrapolating degradation data obtained from overseasstudies to NZ conditions may not reflect the true nature of SMO deg-radation behaviour. Although the terms ‘dissipation’ and ‘degrada-tion’ have been used interchangeably in the literature, it would beappropriate to use the term ‘dissipation’ instead of ‘degradation’ inthe work presented here as we did not attempt to identify metabo-lites formed during the experimental period.

The main objective of this study was to conduct laboratory incu-bation experiments to investigate the dissipation kinetics of SMOantibiotic in topsoils and subsoils collected from three pasturesoils (Te Kowhai, Hamilton and Horotiu). The use of subsoils inthis study was necessitated by the marked differences in the valuesfor pH, organic carbon, and microbial biomass of the top and sub-soils, which could affect the overall dissipation behaviour of SMO.The incubation conditions were maintained at 60% maximumwater holding capacity (MWHC) and with varying initial antibioticspiked concentrations, different depth profiles, temperature re-gimes (7.5 °C and 25 °C) and with sterilisation at 60% MWHC. Theprincipal focus was to derive the dissipation times (50%, 90%, and99%) of SMO under each condition, and compare them to valuesthat were reported in the literature. In order gain a better under-standing about the dynamics of the SMO degradation under variedtreatment conditions, we also performed phospholipid fatty (PLFA)analysis of samples and discuss our results in relation to the microbialcommunity composition and their effects on the fate of SMO in the se-lected soils.

2. Materials and methods

2.1. Chemicals

SMO (N98% purity), triphenyl tetrazolium chloride, Tris (hydroxy-methyl) aminomethane (TRIS) buffer and triphenyl formazan wereobtained from Sigma Aldrich, Australia. Acetonitrile (MallinckrodtChromAR, ≥99.8% purity), chloroform, acetone, methanol and di-chloromethane (Mallinckrodt UltimAR,≥99.9% purity) were obtain-ed from Thermo Fischer Scientific Ltd. NZ. High Performance LiquidChromatography (HPLC) grade deionised water was obtained froman onsite Arium® 61316 high performance reverse osmosis system(Sartorius Stedim Biotech GmbH, Germany).

2.2. Soils

Topsoil and subsoil of three soils (Te Kowhai silt loam, Hamilton clayloam, and Horotiu silt loam) representative of dairy farming areas ofWaikato region in the North Island of NZ were collected fresh fromtwo depths (0–10, 30–40), sieved (2 mm), and stored at 4 °C untiluse. The soil pH was measured using a PHM62 standard pH meter,and organic carbon (OC) content was determined using an IL550 TOC-TN analyser. The microbial biomass carbon (MBC) of the soils wasmeasured by the fumigation extraction method (Wu et al., 1990). Themoisture content (MC) of soils was determined gravimetrically at 105°C and the water content was adjusted to 60% of MWHC. The soil waspre-incubated at 25 °C and 7.5 °C for 2 days before spiking with the an-tibiotic. These two temperatures were selected based on the typicalsummer and winter temperatures observed in the regions where thesoils were collected from. The soils varied in their pH, OC, clay contentand MBC as shown in Table 1. A full description of the soils and themethods used to determine their physico-chemical properties can befound elsewhere (Blakemore et al., 1987).

2.3. Dissipation experiments

For all analyses, destructive soil samples (5 g) were performed to in-vestigate the effect of each factor (temperature, soil depth, sterile vsnon-sterile, concentration, and DHA) on SMO dissipation. Overall theexperimental protocol in the dissipation experiment involved a totalof 36 samples (12 each for temperature, depth and concentration effect)each with 2 replicates. Furthermore, another individual 18 samples (8for sterile control and 10 for DHAmeasurement) each with 2 replicateswere set up separately. Soil samples (5 g) were placed in 35 mL Kimaxcentrifuge tubes and appropriate amounts of SMO stock solution(1000 mg L−1) prepared in methanolic solution earlier were spikedonto the soil to obtain an initial concentration of 5 or 0.5 mg kg−1.The amount of methanol present in the antibiotic solution spiked ontosoil was unlikely to have any effect on soil microorganisms as weallowed the methanol to evaporate immediately after spiking inside afume cupboard. The contents were then thoroughlymixed by vortexingbefore incubating in the dark at 25 °C and 7.5 °C respectively. The mois-ture content in each vial was maintained gravimetrically to 60% of itsfield capacity (−33 kPa) by adding de-ionised water once every3 days during the experiment, and the tubeswere also aerated everydayto ensure a constant oxygen atmosphere. The entire experiment wasconducted in closed incubatorswith temperature control, andwrappingindividual tubeswith aluminum foil in order to avoid photodegradation.To establish the role of microorganisms in the degradation of the antibi-otic, the experiments were also conducted on sterile soils. Sterilisationwas achieved by means of autoclaving twice (121 °C, 103 kPa for30 min). Spiking procedure in sterile control treatment was similar towhat was used in non-sterile treatments, except that sterile de-ionised water was used to maintain the moisture content at 60% of itsfield capacity during the fortification of soil samples in sterile experi-ment. All equipment used during sterile treatment was swabbed with

Table 1Selected properties of soils used in the study.

Soils pH 1:2 (water) OC (%) Sand (%) Silt (%) Clay (%) MC (%) MWHC 60 (%) MBC (μg C g−1) DHA (μg g−1 h−1)

Horotiu TS 5.7 8.2 34.0 48.0 17.0 49.0 121.2 816 28.66Horotiu SS 6.6 1.7 34.0 48.0 17.0 60.9 134.8 584 3.18Te Kowhai TS 6.7 5.0 9.0 54.0 37.0 23.9 79.6 1126 13.78Te Kowhai SS 5.7 0.5 12.3 62.8 24.9 41.4 84.5 536 1.42Hamilton TS 5.8 4.0 13.7 51.0 30.4 23.7 77.6 1724 16.69Hamilton SS 5.1 0.8 13.4 40.3 46.2 23.8 75.5 620 1.28

TS = topsoil; SS = subsoil.

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methanol, and autoclaved. All handling and operations in relation to thesterile treatment were performed inside a laminar flow cabinet.

2.4. Extraction and HPLC analysis

Extraction of the antibiotic contained in the whole 5 g soils in eachtube was performed at selected time intervals using sonication. Briefly,duplicate samples (5 g) at each treatment were extracted with 10 mLdichloromethane (DCM), vortexed (1 min), followed by 15 min ofsonication, and shaken for 12 h in a rotary drum shaker. The tubeswere centrifuged at 1750 g for 5 min, and an aliquot of 1.5 mL of DCMextract was evaporated to dryness under a gentle stream of nitrogen,reconstituted in methanol (0.5 mL), and immediately analysed usingHPLC-UV/Fluorescence detection. The isocratic elution scheme usedfor the analysis of SMO antibiotic has been described in detail in an ear-lier study (Srinivasan et al., 2012). The use of DCM as a solvent to extractSMO from the soils resulted in acceptable recoveries ranging from 72 to88% in the three topsoils and subsoils, with SMO recovery being thelowest (72%) for the Te Kowhai topsoil (SI. Fig. 4). Similar recovery of76–85% (Blackwell et al., 2007) and 65% (Kay et al., 2004) was reportedfor sulfachloropyridazine in soils.

2.5. Measurement of soil bioactivity

Dehydrogenase is a cellular enzyme only active in living organismsand thus is an indicator for soil microbial activity (Friedel et al., 1994).Experimental protocol earlier developed by Ghaly and Mahamoud(2006) was used to determine DHA of soils at selected sampling timesduring the incubation study. Full description of DHA determination isgiven in Appendix A and the Supplementary data.

2.6. Phospholipid fatty acid extraction (PLFA)

For Hamilton top and subsoil (spikedwith 5mg kg−1) at selected in-cubation times, microbial community structure was characterised bydetermining the phospholipid fatty acid (PLFA) composition. Soil lipidswere extracted from soil samples (5 g) at various selected samplingtimes using the method of Bardgett et al. (1996), and detailed experi-mental protocol has been described in the Supplementary data. PLFAswere separated by Gas Chromatography on a SGE 25QC3 BP-5 25 m× 0.32 μm film thickness with flame ionisation detector (at 150 °C,400 mL min−1, hydrogen at 30 mL min−1 for 25 min). The separatedfatty acids were identified by and quantified from chromatographic re-tention time comparison to bacterial methyl esters (Supelco BacterialAcid Methyl Esters CP Mix 47080-U) as external standard. For eachsoil, the abundance of individual fatty acids was expressed as relativenmol g−1 of dry soil and standard nomenclature. The branched phos-pholipids i15:0, a15:0, i16:0, i17:0, and a17:0 were used as indicatorsfor gram-positive bacteria markers, while the PLFAs 18:1 ω7, cy17:0,18:1 ω9c, and cy19:0 were considered as gram-negative bacteriamarkers. The unsaturated PLFAs 18:1 ω6 and 18:2 ω6 were used as afungal-biomass indicator (Frostegård et al., 1993; Hammesfahr et al.,2011).

2.7. Data and statistical analysis

The dissipation of SMO in soils under varied treatments wasmodelled using simple first-order kinetics (SI. Fig. 3). Eq. (1) below rep-resents the most simple form of the concentration–time relationship,where t is time (days), k1 is the dissipation rate constant (day−1) andM0 andMt are the initial and final concentrations respectively, at time t

Mt ¼ M0 exp−k1tð Þ

: ð1Þ

Thefirst-order dissipation rate and the correspondingDT50 (time re-quired for 50% of the initial dose of sulfamethoxazole to be degraded)values were determined using Eq. (1). In order to compare the 90%and 99% dissipation times, we also calculated DT90 and DT99 to providean overall perspective on the fate of compounds in the environment.

A two-sample t-test was used to statistically examine two differentkinetic data sets and to distinguish whether there was any significantdifference in measured SMO concentration on sampling days betweendifferent soil types, initial concentrations used, soil depth, incubationtemperature, and sterile treatment. In addition, Pearson's correlationcoefficients (R) were also calculated to evaluate the influence of soil pa-rameters on the rate of antibiotic dissipation. Other statistical analysisperformed was a single one-way analysis of variance (ANOVA) to eval-uate the influence of the factors soil depth and temperature on the de-pendent DT50 values. All significant differences were accepted at levelp b 0.05.

3. Results and discussion

3.1. Dissipation in soils

No lag phase was observed in the SMO dissipation in soils underany of the treatments investigated, implying the absence of acclimationperiod for the microbial population involved in the dissipation process.However, initial rapid dissipation observed could also account for someabiotic loss of SMO. Dissipation of SMO at 60%MWHC (−10 kPa) undervarying initial concentrations (0.5 and 5mg kg−1), soil depth (0–10 cmand 30–40 cm), temperature (25 °C and 7.5 °C) and under sterile andnon-sterile conditions followed simple first order kinetics. In general,the SMO dissipation rate was found to be rapid irrespective of the soildepth at both initial concentrations, during the initial incubation time.The coefficients of determination (R2) for the first-order model fitsranged from 0.80 to 1.00 (Table 2) with the exception of Hamiltontopsoil under sterile treatment, where dissipation deviated from thefirst-order kinetic as evident with low R2 value (0.61). No statisticallysignificant difference (p b 0.05) was observed for the dissipation kineticdata sets for all the three soils between the experimental conditionswithin the incubation period. A simple Pearson's correlation matrix(SI. Table 2) was obtained using with the soil variables taken fromTable 1, and using k values from Table 2. The significance test was car-ried out at 0.05 levels for the Pearson's correlation coefficient (R). Thecorrelation matrix shows that pH, % clay, MBC and DHA to be positivelycorrelated to the rate constant (k). An increase in MBC and DHA valuesimplied greater biological activity, hence faster dissipation of SMO in thesoils. Both pH and % clay content reduce SMO bioavailability as they

Table 2Average first-order rate constants (day−1) and associated dissipation times (days) for SMO in three different soils under varying treatment conditions.

Soils Parameters 25 °C non-sterile5 mg kg−1

25 °C non-sterile0.5 mg kg−1

7.5 °C non-sterile0.5 mg kg−1

25 °C sterile0.5 mg kg−1

TS SS TS SS TS SS TS SS

Hamilton DT50 11.36 12.38 9.24 11.75 25.39 29.88 11.00 34.66DT90 37.75 41.12 30.70 39.03 84.34 99.25 36.55 115.13DT99 75.49 82.24 61.40 78.05 168.69 198.50 73.10 230.26k1 0.06 0.06 0.08 0.06 0.03 0.02 0.06 0.02M0 3.10 4.62 0.28 0.39 0.36 0.42 0.40 0.35R2 0.99 0.94 0.84 0.97 0.97 0.91 0.93 0.61

Te Kowhai DT50 NA* 4.31 14.15 20.69 20.95 13.00 22.43DT90 14.30 46.99 68.73 69.56 43.20 74.52DT99 28.60 93.98 137.47 139.13 86.40 149.03k1 0.16 0.05 0.04 0.03 0.05 0.03M0 0.31 0.40 0.31 0.40 0.32 0.40R2 0.97 0.96 0.94 1.00 0.93 0.90

Horotiu DT50 NA* 13.33 12.38 23.18 19.69 18.10 22.65DT90 44.28 41.12 77.01 65.41 60.12 75.25DT99 88.56 82.24 154.02 130.83 120.24 150.50k1 0.05 0.06 0.03 0.04 0.04 0.03M0 0.22 0.36 0.26 0.35 0.37 0.38R2 0.98 0.97 0.91 0.96 0.89 0.88

k1 is thefirst order rate constant;M0 is the initial observed concentration; DT50, DT90, and DT99 are the degradation endpoints for 50, 90 and 99% antibiotic dissipation; R2 is themeasure ofthe goodness of fit of the model. TS = topsoil; SS = subsoil. NA* = not applicable (For Te kowhai and Horotiu soil, data are not available, as the experiment on the effect of initialconcentration was performed only in Hamilton soil).

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enhance the process of sorption leading to permanently bound non-extractable residues in soils, and thus affecting the overall recoveriesand leading to underestimation of k values.

Hamilton TS

% r

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Hamilton SS

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Fig. 1.Dissipation of SMO as a function of time in Hamilton soil (TS = topsoil andSS = subsoil) at initial spiked concentrations of 0.5mg kg−1 (circle, red) and 5mg kg−1

(triangle, blue). Error bars show deviation of the duplicate samples. The plots also showtheDHA activity for 0.5mgkg−1 (circle, red) and 5mg kg−1 (triangle, blue) in the second-ary axis.

3.2. Effect of initial concentration

The concentration dependency of SMO dissipation was studied onlyin Hamilton soil (top and subsoils) under non-sterile and sterile treat-ments, at 25 °C using initial concentrations of 0.5 mg kg−1 and5 mg kg−1 and the results are summarised in Table 2. There was nomarked difference on DT50 values for Hamilton top and subsoils at5 mg kg−1 which were 11.4 and 12.4 days respectively. Ten-fold-reduction in the initial spiked concentration down to 0.5 mg kg−1 alsodid not have a marked effect on the DT50 values for SMO (9.2 and11.8 days respectively for top and subsoils). Though there was a15–20% increase in the rate constant (k1) of SMO at low concentration,it was inconclusive whether the persistence of SMOwas affected by ini-tial chemical concentration given conflicting results reported in theliterature which is discussed later.

Thiele-Bruhn and Beck (2005) showed that the extractability of an-tibiotic sulfapyridine depended on its initial concentration and that itwas most effective at smaller spiking levels. An examination of litera-ture data suggests that SMO dissipation kinetics are dependent on theinitial concentration and dissipation being slower at a higher concentra-tion. A plausible explanation is that, since the higher initial concentra-tion of antibiotic often involves spiking larger volumes of methanolicstock solution, the stock itself could indirectly inhibit microbial activityor even the high dosage of the antibiotic itself could be lethal to soil mi-crobes (Ma et al., 2001), thus lowering the microbial activity of the soilin both cases.

As shown in Fig. 1, the DHAactivity of the topsoil (5 μg g−1 h−1 TPF),and subsoils (at about 0.5 μg g−1 h−1 TPF) at both initial concentrations(low and high)was similar indicating that microbial activity was not af-fected by the increase in the chemical concentration. Similar findingswere also reported earlier involving other sulfonamides such assulfachloropyridazine and sulfamethazine (Accinelli et al., 2007). Theauthors concluded that the concentrations greater than 100 mg kg−1

would be necessary to affect the microbial process involved in sulfon-amide degradation. Thiele-Bruhn and Beck (2005) also reported thatthe antibiotic sulfapyridine and oxytetracycline had no effect on DHA,even at concentrations 1000 mg kg−1, which are several orders of

magnitude greater than the typical environmental values for these com-pounds. The authors attributed this to more specific effects of the com-pounds on single microbial species that were possibly compensated by

288 P. Srinivasan, A.K. Sarmah / Science of the Total Environment 479–480 (2014) 284–291

the remainder of the microbial community. In contrast to our findings,Wang et al. (2006a) observed that the degradation rate constant de-creased with increasing sulfadimethoxine concentration in manure,suggesting that the activity of degrading microorganisms was inhibitedat high concentrations. In a separate studyWang et al. (2006b) observeddecreasing bioactivity of the microorganisms with increases in theinitial sulfamethoxine concentrations in manure thereby leading tolessened antibiotic degradation. Yang et al. (2009) varied the initial con-centration for antibiotic sulfadiazine (1, 10 and 25mg kg−1) and foundhalf-life values of 2, 18 and 34days respectively. TheDT50 values obtain-ed in our study for SMOwere in agreement to values obtained for sulfa-diazine by Yang et al. (2009), however, the initial concentrations usedby the authorswere 2 to 5 fold greater than those in our study (Table 2).

3.3. Effect of soil type and depth

Figs. 1 and 2 show the general trend in the dissipation kinetics ofSMO in three soils at two depths, and the associated DHA at each sam-pling event. SMO dissipated at a faster rate in Te Kowhai topsoil withN95% of the applied amount being lost within the half of the incubationperiod. The dissipation rate for SMOwas initially slower in subsoils thanthe topsoils, especially in Te Kowhai soil; however, only 10% of the SMOremained on the last sampling day. Subsoil properties (clay content, OC,and MBC) correlated well with the lower dissipation rate for SMO in allsoils except Horotiu soil (Table 1). However, the ANOVA results showed

Horotiu

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Fig. 2.Dissipation of SMO as a function of time inHorotiu and Te Kowhai topsoils (triangle,blue) and subsoils (circle, red), at initial spiked concentration of 0.5 mg kg−1. Error barsshow deviation of the duplicate samples. The plots also show the DHA activity for topsoils(triangle, blue) and subsoils (circle, red) in the secondary axis.

that DT50 values were not influenced by soil depth (Fcrit = 7.7; p =0.23). It is important to note that the testing could have been biasedgiven the small sample size in this case (n = 3).

The DHA activity for subsoils was low at about 0.5 μg g−1 h−1TPF ascompared to the topsoils which had an order of magnitude greater(N5 μg g−1 h−1TPF) values across the three soils (Figs. 1 and 2). Howev-er, despite low DHA in subsoils, SMO continued to dissipate at a similarrate as observed in the topsoil, implying that factors other than bioticprocesses influenced SMO dissipation rate in subsoils. There was agood correlation between the dissipation rate of SMO and the bioactiv-ity of topsoils in our study. For instance, the Hamilton soil having thehighest MBC gave the lowest DT50 values as compared to other soils.The opposite was true for Horotiu soil, which had the lowest MBCamongst other soils that gave high DT50 values. This was in contrast tosome earlier studies (Monteiro and Boxall, 2009; Thiele-Bruhn andBeck, 2005), where no correlationwas observed between the rate of dis-sipation and the soil bioactivity.

Fig. 3 shows the fungal to bacterial PLFA ratios for Hamilton soil(5 mg kg−1). Analysis of the relative abundance of the major microbialgroups during the incubation time revealed higher proportion of bacte-rial biomass over fungal biomass (fungal to bacterial ratio b1) for eachsampling event. PLFA markers i-15:0, a-15:0, 15:0, i-16:0, i-17:0,cy-17:0, 17:0, and cy-19:0 showed bacterial community, whilePLFA marker 18:2 ω6 was indicative of fungal presence (Antizar-Ladislao et al., 2008). For the topsoil, the ratio of fungal to bacterialPLFAswas higher and ranged between 0.12 and 0.21 when comparedto subsoil which ranged between 0.05 and 0.15 respectively. Most PLFAmarkers for bacteria and fungi increased around the 12th and 7th daysof incubation period for TS and SS (Fig. 3). The dissipation kinetic dataalso correlated well with the PLFA data, while an increased presencein microbes resulted in faster rate of dissipation for SMO, which wasin agreement with studies conducted by Frostegård et al. (1993) indi-cating the relative importance of the bacterial and fungal energy chan-nels in antibiotic dissipation.

An interesting aspect of this workwas the slower dissipation of SMOin Horotiu topsoil compared to its subsoil which could be attributed to amultitude of factors. Horotiu soil is volcanically derived and contains al-lophane (an alumino-silicate clay mineral), and has the highest % OCamongst the other soils studied. A recent work by Srinivasan et al.(2014) found that SMO sorption by Horotiu surface soil was muchhigher than the Te Kowhai and Hamilton soils. Thus high sorption ofHorotiu soil could play a role in contaminant bioavailability, and influ-ence the overall dissipation behaviour of SMO. The soil microbial activ-ity in the Horotiu subsoil was 10-fold lower than the topsoil and ingeneral, reduced levels of OC and low MBC are common characteristics

Days

0 1 3 7 12 24

F:B

rat

io

0.00

0.05

0.10

0.15

0.20

0.25

Hamilton-TSHamilton-SS

0.333 3631

Fig. 3. Distribution of the fungal:bacterial community ratio for Hamilton topsoil and sub-soil as a function of the incubation time. Spiked concentration of SMO was 5 mg kg−1

while the incubated temperature was 7.5 °C.

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of subsoils (Sarmah et al., 2009). Thus, it is conceivable that relative in-crease in the rate of dissipation for Horotiu subsoil compared with theother two subsoils could be due to decreased sorption, and greater bio-availability. Another plausible explanation for the faster dissipation inthe subsoil despite a lower biological activity as shown by reducedDHA is that dissipation might be due to the existence of microbialspecies which are more specific in degrading the target compoundin the subsoil (Di et al., 1998). In the present study, DT50, DT90 andeven DT99 for all the three topsoils were obtained within the lengthof sampling time (40 days), except in subsoils, where DT90 and DT99values occurred outside the sampling period. In general, DT50 valuesfor SMO in subsoil increased when compared with the topsoils,which is consistent with the earlier assumption that reduced organiccarbon and lower microbial activity at increased depths hinder deg-radation process.

0 10 20 30 400

20

40

60

80

100

120

25oC

7.5oC

Sterile

Hamilton TS

0 10 20 30 40

% r

emai

nin

g (

init

ial c

on

cen

trat

ion

)

0

20

40

60

80

100

120

Days

0 10 20 30 400

20

40

60

80

100

120

Horotiu TS

Te Kowhai TS

Fig. 4.Dissipation kinetics of SMO inHamilton, Te Kowhai andHorotiu topsoils (TS) and subsoilscontrol datasets at 7.5 °C. Error bars show deviation of the duplicate samples.

3.4. Effect of incubation temperature

SMO dissipation in soils as affected by the incubation temperatureallowed a relative assessment of the effect of two temperatures inboth top and subsoils (Fig. 4). Summarised datasets in Table 2 showthat the dissipation rate constants (k1 day−1) for SMO were higher inall soils incubated at 25 °C than at 7.5 °C. For example, in TeKowhai top-soil at 7.5 °C, DT99 for SMO was nearly 5-fold greater than at 25 °C.Overall, lower temperature resulted in reduced dissipation rate irre-spective of the soil depth, and such a result is consistent either withmi-crobial or chemical degradation. The ANOVA results also confirmed thatDT50 values for SMO were most influenced by temperature (Fcrit = 7.7;p b 0.001). More than 80–90% of the applied antibiotic dissipated in thetopsoils and subsoils incubated at 25 °C by days 20 and 40 respectively.However, at 7.5 °C it required nearly the whole duration of the

0 10 20 30 40

Hamilton SS

0 10 20 30 40

Days

0 10 20 30 40

Horotiu SS

Te Kowhai SS

(SS) at 25 °C and 7.5 °C, at initial spiked concentration of 0.5mg kg−1 togetherwith sterile

290 P. Srinivasan, A.K. Sarmah / Science of the Total Environment 479–480 (2014) 284–291

experiment (40 days) to reach N70% dissipation in topsoils and subsoils.Wang et al. (2006a) reported an increase in the dissipation rate constantof the antibiotic sulfadimethoxine from 0.332 to 0.777 day−1in manurewhen the temperature was increased from 25 °C to 40 °C. Elsewhere, at6 °C, dissipation rates of four sulfonamides in activated sludge werefound to be three to four-fold slower than at 20 °C (Ingerslev andHalling-Sørensen, 2000).

In general, our study showed thatwhen incubation temperaturewasdecreased, DT50 values increased nearly two-fold for all soils irrespec-tive of the soil depth. The soil biological activity as measured by theDHA indicated little or no bioactivity for soils incubated at 7.5 °C as op-posed to the soils incubated at 25 °C, emphasising the role of micro-organisms in the degradation of SMO and the role of temperature inmoderating this. A likely explanation for this could be that during thedissipation experiment the contaminant bioavailability remains con-stant, and the overall dissipation follows first order kinetics. However,when the bioactivity is significantly altered by reducing the incubationtemperature, the rate constant, which is temperature-dependent, alsodecreases resulting in greater persistence of the compound in soils.

3.5. Effect of sterilisation

In order to investigate the relative role of microorganisms in theSMO dissipation, soils were sterilised, and Fig. 4 displays the dissipationpattern of SMO in non-sterile and sterile soils at both depths. An exam-ination of data in Table 2 reveals that overall, rates of dissipation wereslower and the associated DT50, DT90 and DT99 values were higher insterile soil compared with non-sterile soils, and this was evident in allthree soil types, except in Horotiu soil where dissipation rate for SMOwas similar to non-sterile treatment (Fig. 4).

Relatively smaller variation in dissipation parameters for SMO insterile andnon-sterile Horotiu soilswas indeed surprising. The apparentconsistency in the results between the two treatments in Horotiu soilled us to postulate that factors other than biotic processes may haveplayed a role in the dissipation of SMO.

Fig. 4 shows that 99% of the applied antibiotic disappeared by 73–120 days in sterile topsoils, and within 149–230 days in sterile subsoils.Several explanations can be offered to support these incongruent find-ings; the possibility of chemical hydrolysis, chemical reduction, photol-ysis, or sorption to glass bottles. Given sulfamethoxazole contains nitro-aromatic moiety like many other sulfa drugs, it is conceivable that thechemical species such as reduced sulphur, or iron compounds could po-tentially play a role on the abiotic dissipation of SMO (Mohatt et al.,2011; Zeng et al., 2012; Zhang and Weber, 2013). For example,Mohatt et al. (2011) demonstrated microbially-mediated abiotic trans-formation of SMO under ion-reducing soil conditions in a soil micro-cosm study. Remarkable dissipation of SMO under Fe(III)-reducingconditions with as much as N95% loss within 24 h was attributed tothe abiotic reactions between SMOand Fe(II) generated bymicrobial re-duction of Fe(III) soil minerals. Furthermore, a recent study by ZhangandWeber (2013) involving p-cyanonitrobenzene (pCNB) demonstrat-ed that surface-associated Fe(II) and reduced dissolved organic carbon(DOC) acted as the key reductants in natural sediments. Although a jus-tifiable explanation in our study could not be offered because of the ab-sence of data for soilmineral such as ferrous, sulphur or DOC, the studiesconducted in recent years shed lights on the fate of somenitro-aromaticcompounds in the environment, and these findings can serve as a basisfor understanding the abiotic loss mechanisms for other relatedcompounds.

Even though all precautions were taken to avoid photolysis, duringmanipulation of the samples (e.g. shaking, extraction, and analysis),some photolysis is bound to occur. Furthermore, dissipation of SMOcould have also occurred as a result of other means. For example,given the presence of high amounts of allophane (alumina-silicatemineral) in Horotiu soil, it is conceivable that surface-induced abiotictransformation of SMO could have occurred due to the catalytic effect

with various clay minerals. The pH of soils used in the present studyranged from 5.1 to 6.7, and pH measurements of soils before and afterautoclaving showed an increase by 0.2–0.4 log units, making sterilisedsoils slightly more alkaline. However, alkaline hydrolysis at this pHrange is highly unlikely, and SMO has no structural features that canbe hydrolysed (Loftin et al., 2008).

Very little microbial activity was observed for sterile soils comparedwith non-sterile soils as evident by the DHA measurements (SI. Fig. 5).Even though the autoclaving was monitored by using autoclave tapeand sterilisation indicators, the possibility of an artefact during the pro-cess of sterilising the soils cannot be ruled out. Autoclaving the soils canalter soil chemistry, and is known to change the physical, chemical, andmicrobiological properties of the soil due to the high treatment temper-ature and pressure involved (Fletcher and Kaufman, 1980; Wolf et al.,1989). For instance, autoclaving the soils has been found to increasethe concentrations of dissolved organic carbon dramatically, providinga good environment for those bacterial spores that had survived steriletreatment (Tuominen et al., 1994). Since autoclaving kills the bacteriaand not the spores (Nowak and Wronkowska, 1987), it is possible thatthe one autoclaving performed in this study may not have been suffi-cient to sterilise the soils. Other possible explanation for the abioticloss of SMO in the soils could be attributed to the irreversible bindingwith the soil components through cross-coupling mechanisms formingnon-extractable residues (Bialk et al., 2005), and binding onto the ke-tonic, carboxylic and phenolic carbon as well as aromatic C\H andmethoxy/N-alkyl C sites (Kahle and Stamm, 2007). Regardless of thefactors responsible for dissipation of SMO in the soils investigated, ourfindings suggest that SMO is unlikely to persist for a long duration onthese NZ dairy farm soils.

4. Conclusion

Concentration dependency of the SMO dissipation rate in the soilsinvestigated remained unclear with disparity between the results ob-tained in this study with those reported so far in the literature. Overalldissipation rate of SMO varied with soil type, soil depth, and incubationtemperature. The rate of dissipationwas faster in topsoil as compared tosubsoil in incubated samples collected from all the three sites, clearlydemonstrating the prominence of microbes and their role in SMO dissi-pation. Both the degree of biological activity and the soil temperatureinfluenced the overall SMO dissipation, and data revealed that this anti-biotic is unlikely to persistmore than 5–6 months in the three soils sug-gesting that natural attenuation may be sufficient for the removal ofthese contaminants from these pasture soils. There was a strong corre-lation between soil bioactivity (DHA) and soil MBC, consistent with thedissipation rate of SMO in all three soils. The PLFA analysis was indica-tive of higher bacterial presence as compared to fungal community,highlighting the type of microbial community responsible for dissipa-tion. The dissipation rate constants were higher in soils incubated athigher temperature (25 °C), which was supported by the measuredDHA showing little or no activity for soils incubated at 7.5 °C comparedwith 25 °C, emphasising the microorganism's role in the dissipationprocess that warmer temperatures can enhance SMO biodegradation.It was postulated that microbially mediated abiotic transformation ofSMO under sulphur or iron reducing soil conditions could be one ofthe mechanisms of abiotic loss for SMO in the soils, however, muchwork is warranted to validate this argument under controlled laborato-ry conditions.

Acknowledgements

This work was funded by the Foundation for Science, Research andTechnology (New Zealand), through contract CO9X0705 (LandcareResearch).

291P. Srinivasan, A.K. Sarmah / Science of the Total Environment 479–480 (2014) 284–291

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.scitotenv.2014.02.014.

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