Phytoaccumulation of antimicrobials from biosolids: Impacts on environmental fate and relevance to human exposure

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Phytoaccumulation of antimicrobials from biosolids: Impacts on environmental fate and relevance to human exposure Niroj Aryal, Dawn M. Reinhold* Department of Biosystems and Agricultural Engineering, Michigan State University, 216 A.W. Farrall Agricultural Engineering Hall, East Lansing, MI 48824, USA

article info

abstract

Article history:

Triclocarban and triclosan, two antimicrobials widely used in consumer products, can

Received 5 January 2011

adversely affect ecosystems and potentially impact human health. The application of

Received in revised form

biosolids to agricultural fields introduces triclocarban and triclosan to soil and water

7 August 2011

resources. This research examined the phytoaccumulation of antimicrobials, effects of

Accepted 9 August 2011

plant growth on migration of antimicrobials to water resources, and relevance of phy-

Available online 23 August 2011

toaccumulation in human exposure to antimicrobials. Pumpkin, zucchini, and switch grass were grown in soil columns to which biosolids were applied. Leachate from soil columns

Keywords:

was assessed every other week for triclocarban and triclosan. At the end of the trial,

Triclosan

concentrations of triclocarban and triclosan were determined for soil, roots, stems, and

Triclocarban

leaves. Results indicated that plants can reduce leaching of antimicrobials to water

Phytoremediation

resources. Pumpkin and zucchini growth significantly reduced soil concentrations of tri-

Plant uptake

closan to less than 0.001 mg/kg, while zucchini significantly reduced soil concentrations of triclocarban to 0.04 mg/kg. Pumpkin, zucchini, and switch grass accumulated triclocarban and triclosan in mg per kg (dry) concentrations. Potential human exposure to triclocarban from consumption of pumpkin or zucchini was substantially less than exposure from product use, but was greater than exposure from drinking water consumption. Consequently, research indicated that pumpkin and zucchini may beneficially impact the fate of antimicrobials in agricultural fields, while presenting minimal acute risk to human health. ª 2011 Elsevier Ltd. All rights reserved.

1.

Introduction

Antimicrobials, specifically triclocarban (3,4,40 -trichlorocarbanilide) and triclosan (5-chloro-2-(2,4-dichlorophenoxy)phenol), are widely used in personal care products (Ying et al., 2007). Triclocarban and triclosan primarily enter the environment through domestic sewage discharge to wastewater treatment plants, where removal is predominantly due to sorption (78  11% for triclocarban and 80  22% for triclosan) to wastewater particulate matter (Chu and Metcalfe, 2007; Heidler and Halden, 2007; Heidler et al., 2006; Sapkota et al., 2007). Consequently, digested municipal sludge accumulates

51  15 mg triclocarban and 30  11 mg triclosan per g dry sludge (Heidler and Halden, 2007; Heidler et al., 2006). Greater than 97% of triclocarban and triclosan in sewage is discharged to water resources and biosolids, leading to a 0.6 to 1 million kg/ year combined input into U.S. environment (EPA, 2003; Halden and Paull, 2005). The greatest discharge of triclocarban and triclosan into the environment is through municipal application of biosolids to fields, as more than 50% of biosolids are land applied (Heidler et al., 2006). Triclocarban is generally detected in U.S. surface waters at concentrations from 10 to 1550 ng/L (Halden and Paull, 2005; Sapkota et al., 2007), whereas triclosan is detected in U.S.

* Corresponding author. Tel.: þ1 517 432 7732; fax: þ1 517 432 2892. E-mail address: [email protected] (D.M. Reinhold). 0043-1354/$ e see front matter ª 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2011.08.027

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rivers and streams from 3.0 to 75 ng/L (Bester, 2003; Ying and Kookana, 2007). Additionally, agricultural soils previously amended with biosolids can accumulate 1.2e65 ng/kg triclocarban and 0.16e1.0 ng/kg triclosan (Cha and Cupples, 2009). Once introduced into the environment, triclocarban and triclosan sorb to soils or sediments and are not predicted to readily degrade (Halden and Paull, 2005; Ying et al., 2007). Experimental half-lives of triclocarban and triclosan range from 87 to 231 and 18e58 days respectively in aerobic soil, with longer half-lives in anaerobic soils (Wu et al., 2009; Ying et al., 2007). Triclocarban and triclosan are lowly soluble in water with solubilities of 45 mg/L for triclocarban (Snyder et al., 2010) and 462 mg/L for triclosan (EPI Suite 4.0). Triclocarban and triclosan have high octanolewater partitioning coefficients (KOW) with log KOW of 4.90 for triclocarban and log KOW of 4.76 for triclosan (EPI Suite 4.0). Henry Law constants of 4.52  1011 atm-m3/mole for triclocarban and 2.13  108 atmm3/mole for triclosan (EPI Suite 4.0) indicate that they are nonvolatile. Release of antimicrobials into the environment may result in bioaccumulation of antimicrobials in aquatic organisms and humans. For example, up to 58 mg/kg triclosan and 299 mg/kg triclocarban was measured in snails near wastewater treatment plant (WWTP) effluent (Coogan and La Point, 2008). Furthermore, concentrations of 2.4e3790 mg/L triclosan were measured in 74.6% of urine samples collected from the U.S. general population (Calafat et al., 2008). Triclosan, at the concentration of 0.01e19 mg/kg, was also detected in plasma of Swedish women who did not use personal care products containing triclosan, indicating unintentional systemic exposure to antimicrobials through sources other than personal care products (Allmyr et al., 2006). Antimicrobials have the potential to adversely impact human and ecosystem health. In humans, disruption of endocrine activity by triclosan and triclocarban is expected at concentrations of 29e3150 mg/L (Ahn et al., 2008). At much lower concentrations, triclocarban and triclosan disrupt critical ecological processes. In rivers, antimicrobials adversely affect biofilm structure and function (Lawrence et al., 2009). Freshwater microbial communities are sensitive to 2.9 mg/L triclosan (Johnson et al., 2009) and concentrations as low as 150 ng/L triclosan can have physiological effects on thyroid hormone, body weight, and hind limb development in frogs (Fraker and Smith, 2004; Veldhoen et al., 2007). Consequently, current environmental concentrations of antimicrobials have the potential to disrupt aquatic ecosystems. In terrestrial ecosystems, triclocarban and triclosan can inhibit soil respiration, nutrient recycling, and plant growth (Liu et al., 2009). Development of microbial and drug resistance has also been reported (Heath et al., 1998; Walsh et al., 2003). Studies have demonstrated that plants such as pumpkin and zucchini have potential to accumulate hydrophobic chlorinated organic pollutants. In field experiments of polychlorinated biphenyls (Aroclor 1254/1260), Cucurbita pepo ssp pepo cv. Howden (pumpkin) plants took up, translocated, and accumulated 7.6 mg/kg PCBs in plant shoots (Aslund et al., 2007). Studies have also demonstrated that C. pepo species (pumpkin and zucchini) can extract and translocate dichlorodiphenyltrichloroethane (DDT) and its metabolites (White et al., 2003) and polychlorinated dibenzo-p-dioxins and

dibenzofurans (Hu¨lster et al., 1994). Pumpkin and zucchini showed more potential for phytoaccumulation than other crops. For example, pumpkin and zucchini translocated 1.8e36 times more 2,4,8-trichlorodibenzo-p-dioxin and 2e4.3 times more 1,3,6,8-tetrachlorodibenzo-p-dioxin than 10 other food crops (Zhang et al., 2009). Additionally, the capability of switch grass (Panicum variegatum L.) to reduce PCB concentration in soils through unidentified mechanism has been demonstrated (Dzantor et al., 2000). Recent studies have also detected triclocarban and triclosan in shoot tissues and beans of soybean plants that were planted in biosolids-amended soils (Wu et al., 2010). The study presented herein evaluates the hypothesis that triclocarban and triclosan, being chlorinated aromatic organic pollutants similar to DDT and PCBs, can be taken up by plants to reduce antimicrobial concentrations in soil and the migration of antimicrobials to water resources. Understanding the fate of triclocarban and triclosan in vegetated fields to which biosolids have been applied is crucial to understanding the entrance of antimicrobials into water resources and quantifying the human health effects of consuming food grown on land to which biosolids are applied. The aims of this study were to: (i) assess the effects of plant growth on leaching of antimicrobials from land applied biosolids, (ii) evaluate accumulation of antimicrobials in pumpkin, zucchini and switch grass, and (iii) preliminarily evaluate relevance of triclocarban and triclosan phytoaccumulation to human health.

2.

Materials and methods

2.1.

Soil, seeds and biosolids

The plant varieties used for this study were pumpkin (C. pepo cultivar Howden), zucchini (C. pepo cultivar Gold Rush), and switch grass (P. variegatum). The specific varieties of C. pepo were chosen based on their reported accumulation potential for hydrophobic organic pollutants (Aslund et al., 2008; Lunney et al., 2004; Wang et al., 2004; White et al., 2003). Switch grass was selected as a non-vegetable plant that has potential to stimulate rhizosphere microbial degradation of hydrophobic aromatic pollutants like PCBs (Dzantor et al., 2000). Pumpkin and zucchini seeds were obtained from Johnny Seeds, Maine, whereas switch grass seedlings were obtained from the Department of Crop and Soil Sciences, MSU. Soil for the study, a screened sandy clay loam, was obtained from East Lansing, MI. Biosolids were collected from a nearby wastewater treatment plant and were analyzed for triclocarban and triclosan prior to use by previously developed methods (Cha and Cupples, 2009) using pressurized solvent extraction and tandem mass spectrometry. Triclocarban and triclosan were present at concentrations of 8.18  0.56 mg/kg and 0.18  0.01 mg/kg dry mass of biosolids, respectively.

2.2.

Chemicals

Triclocarban [CAS 101-20-2] (>98%) was obtained from Tokyo Chemical Industry and triclosan [CAS 3380-34-5] from

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Calbiochem. Mobile phase and extraction solvents were obtained from VWR, Inc. LCeMS solvents were of MS grade.

2.3.

Experimental columns

Plants were grown in experimental columns with diameters of 14.7 cm and lengths of 30 cm. The bottom 7.6 cm of the soil column was filled with soil without mixing biosolids. The next 15.2 cm of soil was thoroughly mixed with biosolids at the application rate of 0.73 dry Mg per 1000 m2 (Cha and Cupples, 2009). Solid content of biosolids was 4.8% (dry basis) and 200 g of wet biosolids were applied before seed sowing, with an additional 60 g of wet biosolids applied after 8 weeks to simulate a second field application. Experimental design included quadruple columns for pumpkin and zucchini and triplicate columns for switch grass and no plant controls. Seeds were sown at the rate of a seed per column except for switch grass, for which plants were transplanted directly. Plants were maintained at a constant temperature (23  2  C) using a light regime of 16 h light: 8 h dark.

2.4.

Sampling

For leachate sampling, columns were flooded with equal volumes of water so that a minimum of 100 mL water leached from each column. Leachate samples were collected in amber bottles under each soil column once every two weeks. Samples were stored immediately at 4  C until prepared for analysis. Plants were harvested at the end of 22 weeks. Plant tissues were separated into roots, leaves, and stems for pumpkin and zucchini and into roots and shoots for switch grass. Plant tissues were rinsed carefully to remove soil and dust particles, air-dried at room temperature (23  2  C), weighed and stored in amber bottles in the refrigerator until sample extraction. Soil samples from each column, at depths of 5 cm, 10 cm, 15 cm, and 20 cm, were collected in amber bottles, screened to remove plant roots, homogenized by mixing, and stored at 8  C until sample preparation.

2.5.

Sample preparation

Aqueous samples were prepared as published previously (Halden and Paull, 2004, 2005). Samples were passed through a solid phase extraction (SPE) cartridge (Oasis HLB 3 cc, Waters Corporation) and eluted with 4 ml of 50% methanol and 50% acetone containing 10 mM acetic acid. Elutes were dried under nitrogen, reconstituted in 1 ml of 50% methanol and 50% acetone, filtered through a 0.2 mm PTFE membrane, and analyzed by liquid chromatography mass spectrometry in negative electrospray ionization mode (LCeMS/ESI(e)). Soil samples were prepared and extracted as previously published (Cha and Cupples, 2009) by pressurized liquid extraction (PLE), using a Dionex ASE 200 accelerated solvent extractor. Triplicate subsamples of soil (5 dry g each) from each column sample were extracted. A fourth subsample was weighed and dried for at least 24 h at 105  C and again weighed for moisture determination. Extraction on the ASE utilized acetone with oven temperature of 100  C, extraction pressure of 1500 psi, static time of 5 min, and flush volume of 100%. The

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extracts were evaporated to dryness under nitrogen, reconstituted in 50% methanol and 50% acetone, spiked with 6 ppm triclosan and triclocarban, and filtered through a 0.2 mm filter before analysis by LCeMS. For quality assurance, all analytical runs included a blank, a control, and a sample triplicate. The recovery from the methods was 94.03  9.76% for triclocarban and 83.39  19.48% for triclosan. For plant samples, frozen plant tissues were oven dried, grounded in mortar and pestle, and extracted using the same method as for soil described above. All analytical runs included a blank, a control, and a sample triplicate except for roots, where sufficient biomass for replicate analysis was not available.

2.6.

LC/MS analysis

A Shimadzu LCeMS 2010 EV was used to analyze samples for triclocarban and triclosan. Samples were separated using Allure biphenyl column (5 mm, 150  2.1 mm) from Restek Cor. using a binary gradient of 75% methanol and 25% 5-mM ammonium acetate to 100% methanol. MS parameters were: curved desolvation line (CDL) of 1.5 V, block and CDL temperature of 30  C and nitrogen desolvation gas flow rate of 1.5 L/min. MS negative electrospray ionization mode with scan mode was used for method development and identification, whereas selected ion monitoring (SIM) mode was used for quantification. Retention time (tR  0.1 min), detection of characteristic molecular ions (m/z 313 for triclocarban and m/z 287 for triclosan), and detection of reference ions (m/z 315 and 317 for triclocarban and m/z 289 and 291 for triclosan) were used to identify the target molecules (Halden and Paull, 2005). Quantification was performed using external, linear calibration and a minimum of six calibration levels. The limits of detection were 10e100 ng/L for water and 0.1e1.0 ng/kg for soils and plants.

2.7.

Statistical analysis

All statistical analysis was performed in Sigma Plot (v 11.0). A one tailed t-test was used for all pair-wise comparisons and one-way ANOVA was used for all other comparisons. The reported values are in mean  standard error.

2.8. Relevance of plant accumulation of antimicrobials to human health Accumulation of antimicrobials by pumpkin and zucchini represents the potential for direct exposure of humans to antimicrobial through ingestion of vegetables. To quantify the potential impacts of accumulation of antimicrobials by pumpkin and zucchini on human health, doses of exposure for different routes were compared. The resulting dose of triclocarban and triclosan from consumption of pumpkin and zucchini exposed to biosolids was predicted by assuming fruit concentrations equal to the range of observed stems and leaves concentrations. This assumption is conservative in that it likely over predicts actual risk because: (i) leaves had lower concentrations of antimicrobials than that of stems in this study and another similar study with soybeans (Wu et al., 2010) and (ii) decreases in concentrations of PCB and DDE were observed in the stem of pumpkin and zucchini as the distance

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from root increased (Aslund et al., 2007; White et al., 2003). Predicted doses were compared to doses from other routes of exposure: product use (EPA, 2002), drinking water exposure (EPA, 2002) and exposure from eating soybean produced from fields that receive biosolids (Wu et al., 2010). The dose calculations were based on 5 g/day average soybean (Reinwald et al., 2010) and 11.5 g/day average pumpkin and zucchini consumption per capita in USA (USDA, 2011).

3.

Results and discussions

3.1.

Leaching of antimicrobials

Both triclocarban and triclosan were detected in leachate from the experimental soil columns. As shown in Fig. 1, the concentrations increased initially for two weeks and then decreased. The lag in peak concentration was likely due to sorption of antimicrobials to bottom 7.6 cm of soil where biosolids were not applied. In saturated soil systems, sorption and biodegradation were primary removal mechanisms for triclocarban (Drewes et al., 2003; Essandoh et al., 2010). The maximum triclosan concentration in leached water from pumpkin, zucchini, switch grass and control were 530  180 mg/L, 1670  540 mg/L, 460  280 mg/L, 710  450 mg/mL respectively, all observed in second week. Triclocarban concentrations also followed a similar trend with maximum concentrations of 210  160 mg/L, 190  110 mg/L, 120  40 mg/L, and 370  360 mg/L for pumpkin, zucchini and switch grass and

Fig. 1 e Triclocarban and triclosan concentrations in leached water (mg/ml) with time (weeks). Points represent mean and error bars represent standard error. Volume of water leached each week was statistically similar between columns.

control, respectively. An initial peak with maximum concentration of 110  81 mg/L triclosan and 3.4  2.2 mg/L triclocarban, followed by a rapid decrease in concentrations, has previously been observed in runoff from agricultural field after application of biosolids (Sabourin et al., 2009). Higher concentrations in this experiment were likely due to the relatively short columns. The second addition of biosolids increased antimicrobial concentrations immediately. For triclocarban, the observed second peak was not statistically different from the first peak (P ¼ 0.6). In contrast, triclosan was observed at lower concentrations after the second biosolids application (P ¼ 0.03). Triclosan, despite being present at a lower initial concentration in the applied biosolids, was collected at higher concentrations than triclocarban in leachate during the first four weeks. The higher concentrations of triclosan in the leachate were attributed to its higher solubility and decreased affinity for sorption when compared to triclocarban. The aqueous solubility of triclosan is approximately 10 times that of triclocarban. Sorption of triclocarban to soils is also much stronger than sorption of triclosan, with sorption coefficients (Kd) of 1029 L/kg and 231 L/kg (respectively) for sandy loam soils (Wu et al., 2009). Despite triclosan and triclocarban being weak acids with acid dissociation constants (Ka) of 107.9 and 1012.7 (respectively), the role of soil pH, as affected by addition of biosolids, was not expected to greatly impact sorption of triclosan or triclocarban. While sorption of the anionic form of triclosan is less than the sorption of the neutral form of triclosan, sorption of the anionic form of triclosan is still considerable, resulting in only a slight decrease in net triclosan sorption in soils when the pH increased from 4 to 8 (Wu et al., 2009). Triclocarban sorption was almost unaltered when pH of soil was increased from 4 to 8 (Wu et al., 2009). As measurements indicated the soil pH after addition of biosolids to the experimental columns were between 7 and 8, the increase in pH from the addition of biosolids to the soil columns was expected to only minimally decrease the sorption of triclosan to the soil and was not expected to affect triclocarban sorption to the soil. In contrast to the first four weeks, more triclocarban leached from the columns than triclosan over the remaining 18 weeks. Reduction in the relative leaching of triclosan was most likely due to increased microbial degradation of triclosan. In aerobic soils, degradation of triclosan was characterized by a half-life of 18e58 days, while degradation of triclocarban exhibited a half-life of 87e231 days (Wu et al., 2009; Ying et al., 2007). Additionally, the initial concentrations of triclocarban in the biosolids were much higher than were the initial concentrations of triclosan. The total masses of antimicrobials leached from the vegetated soil columns over 22 weeks were not significantly different from the total masses leached from the control columns. However, the highest concentrations of antimicrobials were leached in week 2, prior to full establishment of the plants. When only considering the total antimicrobials leached after the second addition of biosolids, there were significant differences between the masses of antimicrobials leached from the control and vegetated columns, with P values of <0.01 for pumpkin, 0.01 for zucchini and 0.01 for switch grass. These results suggest that established plants can

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reduce leaching of antimicrobials, prompting additional analysis of the fate of antimicrobials in vegetated soils.

3.2.

Soil concentration of antimicrobials

Soil concentrations of antimicrobials at the end of 22 weeks are summarized in Table 1. Soil concentrations of triclocarban in zucchini columns and of triclosan in zucchini and pumpkin columns were substantially lower than were soil concentrations in control columns (0.02 < P < 0.05). However, soil concentrations of triclocarban in pumpkin columns were similar to those in control columns (P ¼ 0.09). Soil concentrations of triclosan and triclocarban in switch grass columns were similar to or greater than soil concentrations in control columns. The observed difference in final soil concentration of antimicrobials between pumpkin, zucchini, and switch grass columns may have resulted from difference in plant growth in the column. Shoot mass of switch grass plants was 0.49  0.24 g as opposed to 3.74  0.93 g for pumpkin and 5.39  0.28 g for zucchini. Root masses were 0.91  0.40 g for switch grass, 0.08  0.03 g for pumpkin and 0.10  0.03 g for zucchini. Development of an extensive root mass in the absence of robust above ground growth did not appear to influence soil concentration of triclosan in switch grass columns. However, results indicated pumpkin and zucchini may decrease soil concentrations of triclocarban and/or triclosan after land application of biosolids.

3.3.

Concentrations of antimicrobials in plant tissues

Triclocarban and triclosan were detected in roots, stems, and leaves of pumpkin, zucchini, and switch grass. Antimicrobials concentrations in plant tissues (Figs. 2 and 3) ranged from 1.10 mg/kg in leaves to 39.5 mg/kg in roots. Root concentrations of antimicrobials were generally higher than concentrations in stems and leaves, with the exception of triclocarban concentrations in pumpkin tissues. However, plant tissue concentrations were highly variable and higher root concentrations were usually not statistically significant compared to shoot concentrations. No consistent trends in change in concentration of antimicrobials in stem and leaf tissues in pumpkin and zucchini were observed. A general decrease in concentration from root to stem to leaves to fruits has been previously observed for pumpkin and zucchini

Fig. 2 e Plant concentrations of triclocarban. Points represent mean and error bars represent standard error.

accumulation of PCBs (Aslund et al., 2007) and soybean accumulation of triclocarban and triclosan (Wu et al., 2010). A similar decrease from stem to leaf was observed for the accumulation of antimicrobials by pumpkin; however, the decrease was only significant for triclosan. In contrast, concentration of antimicrobials in zucchini increased from stem to leaf, with a significant increase observed for triclocarban. Further studies with higher number of replicates could clarify the significance of the observed trends in plant concentrations of antimicrobials. The hydrophobicities of triclocarban (log KOW ¼ 4.9) and triclosan (log KOW ¼ 4.76) indicate the potential for bioaccumulation. Soil concentrations of antimicrobials were significantly less than the concentrations of antimicrobials in stems, leaves, and water for all plant species (P values from

Table 1 e Soil antimicrobial concentrations and P values for comparison with controls. ND refers to values which were detected but less than quantitation limit (<0.001 mg/kg). Triclocarban, mg/kg Pumpkin Zucchini Switch grass Control

0.055  0.003 (P ¼ 0.091) 0.038  0.004 (P ¼ 0.040) 0.241  0.026 (P ¼ 0.045) 0.097  0.012

Triclosan, mg/kg

Sum, mg/kg

ND (P ¼ 0.021)

0.055  0.003 (P ¼ 0.073) 0.038  0.004 (P ¼ 0.032) 0.166  0.014 (P ¼ 0.155) 0.105  0.013

ND (P ¼ 0.021) 0.001  0.000 (P ¼ 0.457) 0.007  0.001

Fig. 3 e Plant concentrations of triclosan. Points represent mean and error bars represent standard error.

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<0.05 except for between soil and zucchini roots). Triclocarban root bioaccumulation factors, the ratio of root concentration to soil concentrations (g/g), were 11.01  5.06 for pumpkin, 40.27  46.34 for zucchini, and 30.92  9.41 for switch grass. For triclosan, the root bioaccumulation factors (g/g) were 972  398 for pumpkin, 1822  260 for zucchini, and 874  706 for switch grass respectively. These values are comparably higher than 1.0e3.3 for DDT bioaccumulation for the same plant varieties (Lunney et al., 2004) and higher than root bioaccumulation factors of 2.2e5.8 for triclosan and 1.7e2.0 for triclocarban observed for soybean (Wu et al., 2010). Triclocarban translocation factors (the ratios of shoot concentration to root concentration) were 0.78  0.55 g/g for pumpkin, 0.27  0.19 g/g for zucchini, and 0.52 g/g for switch grass. For triclosan, translocation factors were 0.30  0.21 g/g for pumpkin, 0.16  0.05 g/g for zucchini and 0.81 g/g for switch grass. Low translocation factor imply limited transport from root to shoot. These translocation factors are comparable to those reported in Aslund et al. (2007) and Lunney et al. (2004) for accumulation of DDT, DDD, and DDE and PCBs by the same species. Wu et al. (2010) reported translocation factors of 0.01e0.28 and 0.16e1.77 for triclocarban and triclosan, respectively, in soybean plants, indicating the translocation of antimicrobials is comparable for pumpkin, zucchini, and soybean. However, as root bioaccumulation factors were greater for pumpkin and zucchini than for soybeans, results indicated that pumpkin and zucchini have greater potential to accumulate antimicrobials than soybeans. A mass balance for each column was completed (Fig. 4). A significant mass fraction of triclocarban was not accounted for in most of the columns, indicating other mechanisms of antimicrobial loss, such as microbial degradation, phytostimulation, or phytodegradation. After eight weeks, the largest portion of triclocarban remained in the soil, indicating that sorption plays an important role in fate of triclocarban in

Fig. 4 e Mass balance analyses of triclosan and triclocarban in vegetated and control columns.

vegetated soils. For zucchini, total mass accumulation in leaves was highest followed by stems and roots accumulation. In contrast, stems accumulated the greatest mass, followed by leaves and then by roots, in pumpkin. Accumulation of antimicrobials was greater in roots than in shoots for switch grass owing to its limited shoot production. With the exception of triclosan in zucchini columns (where all mass was accounted for in leachate and plant samples), the unaccounted mass of antimicrobials was greater in planted columns than in unplanted controls, supporting the observation that presence of plants may promote removal of antimicrobials by microbial degradation or other unidentified mechanisms. Analysis of coefficients of variation showed that there was minimal variation between the samples in the same column, but rather large variation between the columns. Additionally, large variability was observed in plant masses between columns. Consequently, the observed variability in the experiment was most likely due to inherent biological variability within plantbased systems, rather than variability due to sampling or procedure.

3.4. Relevance of plant accumulation of antimicrobials to human health The doses calculated for multiple routes of exposure to antimicrobials, shown in Fig. 5, are substantially less than the noobservable adverse effect level (NOAEL) of 25 mg/kg bw/d for triclocarban (EPA, 2002). Therefore, none of the examined exposure routes present concerns, on an acute basis, to human health. Exposure of triclocarban from eating pumpkin and zucchini grown in fields receiving biosolids is two orders less than exposure from using products containing triclocarban, about 35 times greater than exposure from drinking water, and about 250 times greater than exposure from eating soybeans grown in fields receiving biosolids. Assuming a per capita consumption of 534 g/day fresh vegetables (USDA, 2001e2002) and that the triclocarban concentrations in pumpkin shoots are representative of all vegetables, the

Fig. 5 e Comparison of triclocarban dose associated with multiple routes of exposure for an adult. Error bars represent maximum and minimum doses. The NOAEL (noobservable adverse effect level) for triclocarban is 25 mg/ kg-bw/day.

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resulting antimicrobial exposure from vegetable consumption is (at worst case) similar to the exposure from using personal care products. Therefore, the acute health risk from antimicrobial accumulation by vegetables in fields receiving biosolids is likely minimal. However, additional studies, utilizing a more diverse range of vegetable and fruit crops, are needed to confirm this assessment. However, there is also the issue of antibiotic resistance due to antimicrobial exposure. While there is dearth of information about the development of microbial resistance from using products containing triclocarban, many reports indicate development of antibiotic resistance due to the triclosan. For example, the susceptibility of Escherichia coli, Proteus mirabilis, and Staphylococcus aureus to antibiotics was reduced by 40e400 times from exposure to triclosan (Saleh et al., 2011; Stickler and Jones, 2008; Suller and Russell, 2000). Though reduction of soil antimicrobial concentrations by plants may help to reduce antimicrobial resistance in soils, the ingestion of antimicrobials through food might increase drug and antimicrobial resistance in humans. More research is needed to explore this potential ramification of the accumulation of antimicrobials by food crops.

4.

Conclusions

Research indicated that established plants reduce the migration of antimicrobials from biosolids to water resources through phytoaccumulation and additional unidentified mechanisms. The soil concentrations of triclocarban and/or triclosan in pumpkin and zucchini columns were less than soil concentrations in unplanted columns. Additionally, the masses of antimicrobials for which the study was unable to account were generally greater in columns with plants than in columns without plants. Pumpkin, zucchini, and switch grass plants accumulated triclosan and triclocarban in mg per kg (dry weight) concentrations. In general, roots had higher concentrations of antimicrobials than shoots, but less total mass accumulation of antimicrobials due to high production of shoots. Consequently, results indicate that plants (i) reduce leaching of antimicrobials from fields to which biosolids have been applied, (ii) directly impact the fate of antimicrobials through phytoaccumulation, and (iii) decrease the persistence of antimicrobials in soil systems through additional, unidentified mechanisms. There was no acute human risk to humans due to eating pumpkins and zucchini produced from field to which biosolids have been applied. However, additional fieldscale studies with fruit production and more food crops are necessary to more clearly understand human exposure to antimicrobials through food crops. More research is also needed to identify and quantify mechanisms that dictate the fate of antimicrobials in vegetated fields receiving biosolids.

Acknowledgments The authors are grateful to Reinhold Research group members, Department of Biosystems and Agricultural Engineering, Michigan State University. Also, special thanks to the

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funding agencies: Center for Water Science, MSU, Michigan Agriculture Experiment Station and College of Engineering, MSU. We would like to thank Dr. Alison Cupples and Dr. Jong M. Cha for assistance with extractions and biosolids analysis.

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