Removal of ionophoric antibiotics in free water surface constructed wetlands

Ecological Engineering 41 (2012) 13–21

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Removal of ionophoric antibiotics in free water surface constructed wetlands Syed Azfar Hussain, Shiv O. Prasher ∗ , Ramanbhai M. Patel Bioresource Engineering Dept., McGill University, Montreal 21111, Lakeshore Road, Ste. Anne de Bellevue, QC, Canada H9X 3V9

a r t i c l e

i n f o

Article history: Received 2 April 2011 Received in revised form 27 October 2011 Accepted 10 December 2011 Available online 3 February 2012 Keywords: Monensin Salinomycin Narasin Pharmaceuticals Free water surface wetlands Ionophores

a b s t r a c t Pharmaceuticals are organic compounds that are being widely considered as emerging contaminants. Among pharmaceuticals used exclusively for veterinary purposes, ionophore group of compounds form a prominent class. Based on the usage and detected environmental concentrations, ionophores are considered as high-risk compounds. This study was conducted to determine the removal efficiency of monensin, salinomycin and narasin in two free water surface constructed wetlands with different substrates: one with a sandy clay loam soil and another with a sandy soil. Three concentrations of each antibiotic in water were used. A significantly higher removal occurred with the sandy (vs sandy clay loam) soil. This enhanced removal was construed to be attained because water was able to infiltrate more in the sandy soil, providing greater solute-to-substrate interaction. The correlations obtained for removal with parameters like oxygen-reduction potential, temperature and pH indicated that sorption and degradation processes could be working together in both soils. Among the three compounds, monensin and narasin were found to be, respectively, the most and least mobile. Removal efficiencies were significantly affected by the antibiotic concentration in the influent. © 2011 Elsevier B.V. All rights reserved.

1. Introduction The ever-increasing demand for food and fiber has pushed agricultural industry toward using more and more organic and inorganic chemicals. These compounds are finding their way into fresh water resources, and potable waters. Concern on pollution by pharmaceuticals has grown after confirmation of their presence and ability to pseudo-persist in the environment (Heberer et al., 2002). These low-level environmental concentrations have the ability to directly affect exposed biota (Glassmeyer et al., 2008); they can also instigate resistance in some bacteria that can potentially be transferred to pathogens (Srinivasan et al., 2008). On account of simultaneous presence of a wide variety of these compounds, there is also a possibility of additive or synergistic effect on the exposed environment (Hansen et al., 2009a). A recent survey across US reported the detection of drugs in the drinking water supplies of 24 major metropolitan, potentially affecting more than 13% of the country’s population (Donn et al., 2008). On account of their antimicrobial characteristics, antibiotics can also detrimentally affect pollutant specific bacterial strains thus increasing the persistence of such contaminants in the environment (Kim et al., 2011). The drugs found in the environment can be traced back to two sources: human consumption and veterinary usage. It is estimated

∗ Corresponding author. Tel.: +1 514 398 7775; fax: +1 514 398 8387. E-mail address: [email protected] (S.O. Prasher). 0925-8574/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ecoleng.2011.12.006

that 75% of antibiotics administered to animals are not absorbed, and are excreted in waste (Chee-Sanford et al., 2009). Antimicrobials used in the livestock industry have been detected in surface waters in Canada (Lissemore et al., 2006; Smyth et al., 2008), the USA (Watkinson et al., 2009), Europe (Feitosa-Felizzola and Chiron, 2009) and Asia (Managaki et al., 2007; Minh et al., 2009). The predominant sources of these drugs are Concentrated Animal Farming Operations (CAFOs) and freshly manured agricultural soils (Pruden, 2009). Moreover, the possibility of direct leaching/runoff in high concentrations from manure stockpiles has also been reported (Dolliver and Gupta, 2008; Khan et al., 2008). Among pharmaceuticals used exclusively for veterinary purposes, ionophore group of compounds form a prominent class. Despite the fact that some studies have found these compounds to be somewhat susceptible to microbial degradation (Ramaswamy et al., 2010; Hussain et al., 2011b), based on the usage and detected environmental concentrations, ionophores are considered as high risk compounds (Hansen et al., 2009b). Recent studies have detected these compounds in environmental matrices (Onesios et al., 2009 Watanabe et al., 2008). These compounds comprise of complex, high molecular weight molecules, derived from various streptomyces species. Among this group, monensin, salinomycin and narasin are the most commonly administered compounds. All three have been detected in environmental waters (Kim and Carlson, 2006). Only recently Hussain et al. (2011a) reported the removal efficiency of horizontal subsurface wetlands for these three compounds. The molecular structures and

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Table 1 Physical and chemical characteristics of monensin, salinomycin and narasin ionophoric antibiotics. Compound

Monensin

Salinomycin

Narasin

CAS No. Mw (g mol−1 ) Formula

17090-79-8 671 C36 H62 O11

53003-10-4 751 C42 H70 O11

55134-13-9 765 C43 H72 O11

Streptomyces cinnamonensis 4.8 to 8.9b , <100k 4.2e , 6.65b Unstable in acidic condition, stable in alkaline condition 2.1 to 3.8h or >5.6b 2.8 to 4.2b , >6.3b , 5.4 to 8.5l 3.8h , 7.5m , 13 to 18 daysb

Streptomyces albus 57 to 905d , 302 to 7685c 4.4j , h Unstable in acidic condition, stable in alkaline condition 2.2 to 2.8d 5.15d , >6.2c 5i , 8 to 18 daysd

Streptomyces auriofaciens 102–681g 4.4e , f or 7.9g Unstable in acidic condition, stable in alkaline condition 6.06 to 6.88g , >5.63j 4.85 to >6.2g 8.8, 21 to 49 daysj

Structurea

Produced by Solubility in water (mg L−1 ) pKa Stability Log Koc Log Kow Soil DT50 a b c d e f g h i j k l m

Kim and Carlson (2006). Anonymous (2004a). Anonymous (2004b). EFSA (2004). Calculated using Marvinsketch (www.chemaxon.com/marvin/index.html). Carmosini and Lee (2008). EFSA (2007). Sassman and Lee (2007). Schlüsener et al. (2006). Elanco (2004). Dolliver and Gupta (2008). Thiele-Bruhn (2003). Carlson and Mabury (2006).

salient characteristics of these three antibiotics are presented in Table 1. In context of agricultural pollution, control techniques like vegetative buffer strips and/or constructed wetlands (CWs) have been found to be viable treatment technologies that blend-in well with the rural environment. On account of the highly unpredictable and variable loadings of agricultural effluents, wetlands, with greater ability to handle immoderations and extremes, seem to be the better treatment option. CWs can arguably perform with little loss of efficiency under variable volumes of water and varying contaminant levels that are typical of agricultural runoffs (Sim et al., 2008). Vymazal (2011) in a review of CWs in Czech reported that these wetlands were very effective in removing various contaminants at a steady rate throughout the year. These complex ecosystems of plants, microorganisms and substrate act together as a biogeochemical filter, efficiently removing low levels of contamination from a large volume of water (Kosolapov et al., 2004). Although the high usage and environmental persistence of monensin, salinomycin and narasin is known, control and removal techniques for these antibiotics have been sparsely explored (Ramaswamy et al., 2010). The present study was conducted to evaluate the pharmaceutical removal efficiency of two Free Water Surface (FWS) wetlands 2/3rd filled with the selected soil substrates (a sandy soil and a sandy clay loam soil). The specific objective was to quantify and compare the removal of salinomycin, monensin, and narasin in these systems. 2. Materials and methods 2.1. Chemicals Analytical standards of monensin, salinomycin and narasin were bought from Sigma–Aldrich, St. Louis, MO, USA, in the form

of sodium salts with 95–98% purity. Mobile phase chemicals were procured as follows: HPLC-grade methanol from EMD Chemicals, Gibbstown, NJ, USA; acetic acid (glacial) and ammonium hydroxide from Fisher Scientific, Fair Lawn, NJ, USA. Double-deionized water (Milli-Q, Millipore, Molsheim, France) was used in the study. Nigericin was used as internal standard and was obtained from Sigma–Aldrich, St. Louis, MO, USA. Stock solutions of 100 mg L−1 of the three ionophoric compounds: monensin, salinomycin and narasin were made in HPLC grade methanol (Fisher Scientific) on trimonthly basis whereas working standards were prepared in HPLC grade methanol on biweekly basis at concentrations of 0.01, 0.1, 1, 5, and 10 mg L−1 . Both the stock solution and working standards were stored under dark in amber colored glass bottles at 4 ◦ C. The buffer for the LC/MS mobile phase was prepared by mixing 0.05 M glacial acetic acid with 0.05 M ammonium hydroxide to attain a pH of 5.0. The solution was autoclaved and stored for a maximum period of 1 week. 2.2. Wetland system and field setup The wetlands were located at Macdonald Campus of McGill University, Ste. Anne de Bellevue, Quebec, Canada. These FWS systems were above-ground structures, made of high-density polyethylene (HDPE) half-pipes, each 6 m in length and 1.5 m in diameter. Of a total of six half-pipe tanks, three were filled with a sandy soil while another three were filled with a sandy clay loam soil to a depth of 0.6 m leaving 0.15 m for surface water flow. Each CW tank had a volume of approximately 4.17 m3 and a surface area of 9.29 m2 . Textural analysis and organic matter contents of the two soils are provided in Table 2. At the inlet-end of the FWS systems, a transverse deep zone was provided to stabilize incoming flow, induce lateral mixing and help in attaining a better distribution of water by offsetting the adverse impact of short-circuiting, thus maximizing hydraulic residence

S.A. Hussain et al. / Ecological Engineering 41 (2012) 13–21 Table 2 Textural analysis and organic matter percentage of soils used in wetlands. Soil type

Sandy clay loam

Sandy

Sand % Silt % Clay % OM %

62 18 20 9.2

99 0.2 0.8 4.7

time. A recent modeling study suggested that deep zones when properly sized and located may enhance wetland performance (Lightbody et al., 2009). The shallow zone occupied approximately 66% of the surface area and was vegetated with alternate bands of reed canary grass (Phalaris arundinaceae L.) and cattails (Typha latifolia L.) (Fig. 1). A tipping bucket was installed at the outlet of each of experimental unit to record actual flow. All tipping buckets were connected to dataloggers. The effluent from treatments was drained to a sump from where water was pumped out to be spread over a large uncultivated field.

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A gravity-driven delivery system was established to supply the artificially prepared agricultural wastewater at a daily mean flow rate of 1 L min−1 . A constant concentration of 10 mg L−1 nitrateN, 0.3 mg L−1 dissolved reactive phosphorous as orthophosphate and 25 mg L−1 of dissolved organic carbon added as cane sugar was maintained in the supplied water. The mean hydraulic residence time for the CWs, as ascertained through a bromide tracer study, was 2.2 days (Yates and Prasher, 2009). The prepared mixture was stocked in the two mixing tanks at ambient temperature, each having a capacity of 9000 L and delivered to CWs via the distribution tanks. Water was supplied to the CWs through an inflow manifold. Inflow distribution manifolds improve water distribution in the wetland and increase hydraulic retention times (Shilton and Prasad, 1996). Constant flow was maintained by using multiple control valves. These systems were established in 2005–2006 however, data collection from the study only started in 2007. The vegetation in these CWs was 1 year old. The study was carried out mainly from July to September, 2007 with each of the three antibiotic concentration levels being run for a period of 4 weeks.

Fig. 1. Dimensions, top and side sectional view of the constructed wetlands. Source: Adapted from Yates and Prasher (2009).

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2.3. Influent preparation

2.6. Liquid chromatography/mass spectrometry

High-concentration discharges can be expected from CAFOs. Dolliver and Gupta (2008) reported a maximum concentration of 3175 ␮g L−1 for monensin in runoff from manure stockpiles that are typically present near CAFOs. However, considering that normal operational concentrations would be lower than this elevated value, the current study used three levels of antibiotic concentrations: 100, 500 and 1000 ␮g L−1 . To prepare the solution of desired concentration, feed additive premixes of salinomycin, monensin and narasin, containing 60, 200 and 70 g kg−1 of active ingredient, respectively, were used. Pre-weighed amounts of these supplements were mixed with ACS grade methanol, procured from Fisher Scientific, Fair Lawn, NJ, USA, in 1:5 ratio and left for 4 h on a rotary shaker at 150 rpm. The mixture was then subjected to repeated washing with ACS grade methanol to extract the active ingredients. A recovery of 60–65% was achieved by this method.

A 1100-series Agilent LC with quadrupole mass spectrometer was used. An isocratic run was made with a ratio of 83:7:10 for mobile phase A (HPLC grade methanol), mobile phase B (buffer of glacial acetic acid 0.05 M and ammonium hydroxide 0.05 M), and mobile phase C (Milli-Q water), respectively. All aqueous solutions were filtered through a 0.22 ␮m nylon membrane filter (Fisher Scientific, Fair Lawn, NJ, USA). The flow rate was maintained at 1 mL min−1 . Injection volume was 10 ␮l for the analytes. Separation was achieved on a Discovery Supelco C-18 column (Bellefonte, PA, USA) at a temperature of 40 ◦ C. Concentration was calculated against a five point calibration. Electrospray Ionization (ESI) positive complete scan mode was used for MS detection. A 10-min post-time was allocated for re-equilibration of the column. All MS parameters were optimized before actual analysis.

2.4. Sampling Water samples were collected using a custom-built Avensys auto-sampler, equipped with a sample storage refrigerator, maintained at 4 ◦ C. The auto-sampler was provided with an auto-channel switcher to collect samples in sequence from different treatments at desired time intervals. The sampler was programmed to take batch samples after every 42 h. The samples were combined to make a composite sample on a weekly-basis. Composite samples were collected in 1-L amber colored glass bottles and a pre-specified amount of internal standard Nigericin was added (Hussain and Prasher, 2011). Grab samples were also collected at each sampling point and date, to verify the accuracy of the sampling procedure used. Dissolved oxygen (DO), pH, oxidation-reduction potential (ORP) and temperature data was also collected weekly in the deep zone and shallow zone of CWs. These data were collected using a Horiba D-52 portable pH meter with corresponding DO probe, ORP probe, and a pH/temperature probe. 2.5. Filtration and solid phase extraction After collection, samples were subjected to a two-step filtration process: first with 1.2 ␮m glass fiber filter, and then with 0.45 ␮m glass fiber filter (both from Fisher Scientific, Fair Lawn, NJ, USA). The step of adding a Na source to convert ionophores into a single sodium adduct species (Cha et al., 2005) was not followed as no notable effect of this step was detected in the actual analysis of the antibiotics. As a final cleaning and concentration step, solid phase extraction (SPE) was performed using 60 mg/3 mL Oasis HLB cartridges on a 20-slot waters vacuum manifold, Waters Ltd., Lachine, QC, Canada. Following protocol was adopted for conditioning the cartridges: 5 mL of HPLC grade methanol, 5 mL 50:50 methanol water, 5 mL water at pH 3 (using hydrochloric acid), 5 mL of double-deionized milli-Q water. A flow rate of 5 mL min−1 was maintained for the SPE step. After loading, the cartridges were air-dried. The ionophores were eluted with two 5 mL volumes of HPLC grade methanol at a flow rate of 0.5 mL min−1 and concentrated to 1 mL volume in amber auto-sampler vials for further analysis. A flow rate of 5 mL min−1 was maintained for SPE. After loading, the cartridge was air-dried and ionophores were eluted with 5 mL of HPLC-grade methanol at a flow rate of 0.5 mL min−1 , and concentrated to 1-mL volume over a stream of nitrogen gas, in amber-colored auto-sampler vials for further analysis.

2.7. Statistical analysis Results were analyzed applying General Linear Model (GLM) procedure of Least Squares Means adjustment for Multiple Comparisons (Tukey test) using SAS version 9.2. SAS Institute Inc., Cary, NC, USA. CORR procedure of SAS was used to develop Covariance Matrix between five variables: pharmaceutical removal, temperature, dissolved oxygen (DO), oxidation-reduction potential and pH. 3. Results and discussion Antibiotic removal was determined by calculating the difference between the inflow and outflow concentrations of the ionophoric antibiotics, while taking into account the flow variability observed in each treatment. The bromide tracer study only gave marginally higher retention times for the sandy soils (Table 3); this lack of clear difference could be because the greater penetration of water into the soil profile in case of sandy soils might have been compensated by the impediment to flow provided by the denser vegetation in sandy clay loam soil wetlands, resulting in a comparable retention time for the two wetlands. Despite this similarity in retention times, the two substrates removed antibiotics in a significantly different way (P < 0.01). 3.1. Effect of influent concentration levels Antibiotic removal rates for the three inflow concentrations are shown in Fig. 2a–c. For the same substrate, removal efficiencies varied significantly with concentration level (P < 0.01). Monensin was found to exhibit a significantly different behavior, compared to salinomycin and narasin, for the two substrates. The relative removal efficiency of each substrate observed the following order: monensin < salinomycin < narasin. In level 1 (100 ␮g L−1 ), monensin removal varied significantly in both substrates. Average removal Table 3 Bromide mass recovery and calculated retention time for the constructed wetlands. Wetlands

Mass recovery of bromide (%)

Retention time (days)

Sandy soil 1 Sandy soil 2 Sandy soil 3 Sandy clay loam soil 1 Sandy clay loam soil 2 Sandy clay loam soil 3

**

**

62.67 61.92 No data 84.25 72.45

2.22 2.38 No data 2.12 2.26

Source: Adapted from Yates and Prasher (2009). ** Erroneous data/excluded from calculations.

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Fig. 2. Percent removal achieved for concentration levels used for the three ionophoric pharmaceuticals evaluated: (a) level 1 (100 ␮g L−1 ), (b) level 2 (500 ␮g L−1 ), (c) level 3 (1000 ␮g L−1 ). Bars denote standard error.

was lowest in the sandy clay loam soil (21%, SD ± 3.68) and highest sandy soil (29%, SD ± 1.59). A similar removal trend was observed for salinomycin and narasin. Removal percentage for salinomycin was 28% (SD ± 3.47), and 39% (SD ± 2.13) for sandy clay loam soil and sandy soil, respectively, and those for narasin were respectively 30% (SD ± 5.06) and 47% (SD ± 2.60). In level 2 (500 ␮g L−1 ), higher removal rates were observed in both soil treatments as compared to those in level 1. For sandy clay loam soil, removal percentages for monensin,

salinomycin and narasin were 34.92% (SD ± 3.5), 41.18% (SD ± 3.0) and 41.77 (SD ± 2.9), respectively, they were 36.83% (SD ± 3.2), 41.66% (SD ± 4.31) and 42.31 (SD ± 6.27) for sandy soil. For level 2, the removal efficiency did not differ significantly between the two soil treatments (P > 0.05). Level 3 (1000 ␮g L−1 ) showed an overall reduction in removal percentages for all three systems. Corresponding antibiotic removal figures for sandy clay loam and sandy soils were 21.93% (SD ± 4.9), 22.03% (SD ± 1.81) and 22.15 (SD ± 6.88) and, 29.71%

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Table 4 Correlation matrix: antibiotic removal efficiency vs wetland environmental parameters.

Sandy clay loam soil Sandy soil a

Temp

DO

ORP

pH

0.46052a 0.42074a

0.0836 0.24794

0.42794a 0.46622a

0.49492a 0.0548

Levels of significance P < 0.0001.

(SD ± 4.13), 29.83% (SD ± 5.37) and 30.06% (SD ± 4.37), respectively, for monensin, salinomycin, and narasin (Fig. 2). While comparing compound-to-compound removal for the two CWs, monensin depicted a significantly lower removal as compared to the other two compounds (P < 0.001), whereas the difference between the removal efficiencies of salinomycin and narasin was significant only for level 1 of sandy soil (P < 0.01), with narasin removal being greatest. The general trend of antibiotic removal for all three levels in sandy soil was comparatively more evident; monensin was removed at the lowest rate and narasin at the highest. Fig. 2 provides the respective removal efficiencies on weekly basis for each antibiotic level. This study suggests that the significant difference in removal efficiencies for the three concentrations in both soils should not be solely attributed to the concentrations of pharmaceuticals used. The significant correlation of removal percentage with various environmental parameters (Table 4) shows the possible influence of microbial activity on antibiotic removal, as affected by temperature and oxidation-reduction potential (ORP).

3.2. Temperature effect The significant correlation of antibiotic removal with soil temperature indicates that microbial degradation was most likely involved in the attenuation of compounds achieved in the wetlands. Removal trends observed for the concentration levels can be partially explained on this premise. The high and low removal rates, recorded for levels 2 and 3, could have been largely temperature induced, rather than a concentration effect, as the comparable seasonal temperatures were positively related with the observed removal rates (Table 4). Average daily temperatures for the periods corresponding to levels 1, 2 and 3 were 21.51 ◦ C, 22.73 ◦ C and 17.08 ◦ C, respectively (Fig. 3).

3.3. Aerobic vs anaerobic environment A possible microbial role in the removal of these compounds has already been reported (Hussain et al., 2011b). The significant positive correlation between removal rates and ORP suggests that the active soil microbial population was shifted toward an aerobic environment. It has previously been shown that oxic or anoxic conditions, prevailing under a typical wetland setup, can influence the system’s mitigation efficiency. Zhang et al. (2011), while investigating the removal of some pharmaceutical compounds in constructed wetlands, pointed out that oxidized environment can improve the chances of biodegradation of the tested drugs. Lack of significant correlation with dissolved oxygen (DO) can be explained on the grounds that major microbe-mediated removals occur within the soil in biofilms, usually at the soil–root interface (White et al., 2006); as measurements of DO were taken on the surface, they may not be able to truly relate to processes occurring below the soil surface. The temporal trends of ORP, DO, temperature and pH are provided in Fig. 3.

Fig. 3. (a) Weekly measurements of dissolved oxygen (DO) in mg L−1 , temperature (Temp) ◦ C, pH and oxidation-reduction potential (ORP) in mV for sandy clay loam soil wetland. Bars denote standard error. (b) Weekly measurements of dissolved oxygen (DO) in mg L−1 , temperature (Temp) ◦ C, pH and oxidation-reduction potential (ORP) in mV for sandy soil wetland. Bars denote standard error.

3.4. Influence of pH pH is known to influence the behavior of ionophoric compounds (Kümmerer, 2009). An earlier laboratory study had shown that soil pH has an effect on sorption of these compounds (Hussain and Prasher, 2011). However, the range of pH change over the study period was small (6.8–8.0), making it difficult to verify and authenticate the observed correlation between pH and removal percentage (Fig. 3). Nevertheless, the higher correlation of removal with pH for sandy clay loam soil suggests a role for the inorganic soil fraction in the sorption process, since the polarity of these compounds can increase with pH, making them susceptible to adsorption through anion exchange phenomenon on clay complexes. In a study on pharmaceuticals, sorption to the inorganic portion of soil was found to be only an order of magnitude lower than to organic matter (Kahle and Stamm, 2007). Strong pH dependency for sorption of certain pharmaceuticals has been reported for sand and loam soils (Kurwadkar et al., 2007). In defining the behavior of ionophoric antimicrobials in environmental matrices, it will be appropriate to give consideration to these compounds’ specific physicochemical characteristics. While in soil, chemicals are known to be affected by pH, particularly when it exceeds the compounds’ pKa . They can then take up a

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charge and become ionized. In this state, they are more likely to behave like polar compounds and also depict tendency to sorb on anion exchange sites present in soil matrix or organic matter (Sassman and Lee, 2007). However, on account of their inherent preference for taking up cations like K+ , Na+ , Mg2+ , etc., there is a possibility that they are unable to remain polar and revert back to being neutral. The occurrence of this phenomenon can increase with a rise in pH, as the relative availability of cations in soil solution is proportional to pH, making it more likely for ionophores to turn into neutral species. These neutral species can then be possibly sorbed by the organic phase, resulting in a higher Koc value than what could be expected based on simple Kow -based calculations. Under field conditions, within a pH range of 6.8–8.0, both ionized and non-ionized forms of these compounds are expected to be present, thus their actual removal in response to sorption phenomena could be substantially complicated and difficult to explain accurately without proper quantification of the prevalent species. 3.5. Substrate effect The relatively higher removal in sandy soil, despite having lower organic matter content, could have been because of the higher relative hydraulic conductivity of sandy soils. The mean calculated retention time from bromide tracer study for both surface flow CWs was approximately 2.2 days (Yates and Prasher, 2009). Sandy soil CWs, however, gave slightly higher retention time than the CW filled with sandy clay loam soil (Table 3). The higher relative hydraulic conductivity of sand could have been instrumental in facilitating water movement within the soil profile. On the other hand, sandy clay loam soils generally have lower infiltration rates and are categorized under Class C in Hydrological Soil Grouping, thus the restricted movement within the soil profile of sandy clay loam soils could have provided the sandy soil treatment a comparative advantage. The ability to infiltrate to greater depths of the sandy soil profile is likely to provide greater opportunity of soil-tosolute interaction, possibly resulting in higher attenuation mainly through the twin removal mechanisms of sorption and microbial degradation. 3.6. Role of sorption and degradation Laboratory batch experiments were carried out to investigate both sorption and degradation processes. The high Koc values (4.26–5.03 L kg−1 ), determined earlier for these compounds in the laboratory (Hussain and Prasher, 2011), indicate that sorption can be the dominant pollutant removal pathway for these compounds. Degradation of organic compounds in the environment can occur both from the action of microbes and through physical dissipation processes such as photodegradation and hydrolysis. On account of the low retention times of the two substrates and, the relative stability of these compounds against photolysis and hydrolysis, the prime degradation pathway was expected to be biotic. Calculated photodegradation half-lives were 55.1, 40.1 and 37.2 days for monensin, salinomycin and narasin, respectively (unpublished data). Considering the small retention time of the CWs (2.2 days), it can be presumed that mitigation through photodegradation pathway would be minimal in the current constructed wetland setup. While evaluating the degradation of various pharmaceuticals compounds, it was reported that hydrolysis is not a prominent removal process for such chemicals in the environment (Loftin et al., 2008). In a typical wetland scenario aerobic biodegradation is usually confined to biofilms and soil–root interface. The relative anaerobic conditions prevalent in deeper horizons may not be conducive

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for microbial-mediated degradation of these drugs. Donoho (1984) reported that the degradation of ionophores occurs fairly rapidly under aerobic conditions, but is slow under anaerobic conditions. This reasoning further entails that, in sandy soil treatments, it can be expected that the dominant removal mechanism may be sorption, with biodegradation representing a strong but secondary process. It was expected that the sandy clay loam soil, with a higher organic matter content, will experience greater biodegradation. However, on observing removal trends across the three levels, it is clear that the relatively lower removal of drugs for the sandy clay loam (vs sandy) soil was more evident in the lower concentration range (level 1). It is conjectured that since level 1 was employed at the study’s inception, the microbial population may not be sufficiently acclimatized to these compounds at that early point in the study, thereby causing lesser biodegradation. This reasoning is supported by the lower differences observed between removal rates for the two soils for the later employed higher concentrations (levels 2 and 3). Microbial degradation seems to be compensating for the lower sorption trend anticipated for sandy clay loam soil in the case of levels 2 and 3. The low removal of monensin and high removal of narasin followed the sorption and degradation trends observed in laboratory studies. Mean log Koc values across both soils, were 4.27, 4.36 and 4.96 L kg−1 , respectively, for monensin, salinomycin and narasin (Hussain and Prasher, 2011). These calculated values are within the log Koc range reported in the literature for these compounds (Table 1). Salinomycin removal did not follow the expected pattern based on the log Koc values. Although it depicted a higher removal than monensin and lower than narasin, however, the large difference reported in the laboratory-determined log Koc values between salinomycin and narasin was not fully manifested in the removal observed for the two soils used in CWs. A lesser removal of salinomycin, compared to narasin, was found to be significant only for level 1 in sandy soils (P < 0.01), when sorption was possibly substantially dominating degradation as the primary removal mechanism. At levels 2 and 3, salinomycin removal was not significantly different from that of narasin for either of the CWs, implying a relatively larger microbial contribution in the overall dissipation of the compound than what was presumed based on the sorption and biodegradation results obtained from laboratory studies.

3.7. Quantitative removal by the substrates The quantitative removal achieved for each concentration level with respect to the hydraulic loadings of the antibiotics is given in Fig. 4. The removal is observed to follow the trend depicted in Fig. 2. The overall mean removal efficiency for the sandy clay loam and sandy soils was determined to be 26.77% and 31.76%; 28.86% and 34.21%; 29.73% and 36.01% for monensin, salinomycin and narasin, respectively. Previous treatment wetland studies on different pharmaceuticals using FWS (Matamoros et al., 2008), HSSF (Ávila et al., 2010) and VF systems (Hijosa-Valsero et al., 2010) have shown variable removal efficiencies, depending upon the compounds studied. Similar to the weekly analysis results, level 2 showed a highest percent removal of the compounds for the two soil treatments. On the basis of overall removal, sandy soil performed better than sandy clay loam soil in removing selected ionophoric antibiotics, affirming the use of this substrate in constructed wetlands for agricultural wastewaters. Possible reasons for this trend have been discussed in previous sections.

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Fig. 4. Mean total removal of the three antibiotics for each wetland/substrate vs their mean mass loading for the three concentration levels used.

4. Conclusions Among the two CW systems supporting texturally different soils removal efficiency of sandy soil was greater for the three ionophoric antibiotics. Enhanced removal observed in sandy soil could possibly be on account of its higher hydraulic conductivity. The phenomena of sorption and microbial degradation were proposed to be the dominant removal mechanisms for the attenuation of the selected compounds in the wetlands studied. Removal rates were significantly correlated with temperature and ORP, implying a microbial role in contaminant mitigation. On the other hand, the correlation with pH might have stemmed from the sorption processes. The average removal efficiency across levels for sandy clay loam and sandy soils was determined to be 26.77% and 31.76%; 28.86% and 34.21%; 29.73% and 36.01% for monensin, salinomycin and narasin, respectively. Monensin and narasin ionophores were found to be the most and least mobile compounds, respectively. References Anonymous, 2004a. Opinion of the scientific panel on additives and products or substances used in animal feed on the request of the commission on the reevaluation of coccidiostat Elancoban in accordance with article 9G of council directive 70/524/EEC. EFSA J. 42, 1–61. Anonymous, 2004b. Opinion of the scientific panel on additives and products or substances used in animal feed on a request from the commission on the safety and the efficacy of product BIO-COX 120G as feed additive in accordance with council directive 70/524/EEC. EFSA J. 75, 1–51. Ávila, C., Pedescoll, A., Matamoros, V., Bayona, J.M., García, J., 2010. Capacity of a horizontal subsurface flow constructed wetland system for the removal of emerging pollutants: an injection experiment. Chemosphere 81, 1137–1142.

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