phytoremediation potential of vetiver grass

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PHYTOREMEDIATION POTENTIAL OF VETIVER GRASS [CHRYSOPOGON ZIZANIOIDES (L.)] FOR TETRACYCLINE a

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Rupali Datta , Padmini Das , Stephanie Smith , Pravin Punamiya b

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, Dil M. Ramanathan , Ramana Reddy & Dibyendu Sarkar

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Department of Biological Sciences, Michigan Technological University, Houghton, Michigan, USA b

Department of Earth and Environmental Studies, Montclair State University, Upper Montclair, New Jersey, USA c

New Jersey Center for Science, Technology and Mathematics, Kean University, Union, New Jersey, USA Accepted author version posted online: 05 Jul 2012.

To cite this article: Rupali Datta, Padmini Das, Stephanie Smith, Pravin Punamiya, Dil M. Ramanathan, Ramana Reddy & Dibyendu Sarkar (2013): PHYTOREMEDIATION POTENTIAL OF VETIVER GRASS [CHRYSOPOGON ZIZANIOIDES (L.)] FOR TETRACYCLINE, International Journal of Phytoremediation, 15:4, 343-351 To link to this article: http://dx.doi.org/10.1080/15226514.2012.702803

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International Journal of Phytoremediation, 15:343–351, 2013 C Taylor & Francis Group, LLC Copyright ISSN: 1522-6514 print / 1549-7879 online DOI: 10.1080/15226514.2012.702803

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PHYTOREMEDIATION POTENTIAL OF VETIVER GRASS [CHRYSOPOGON ZIZANIOIDES (L.)] FOR TETRACYCLINE Rupali Datta,1 Padmini Das,2 Stephanie Smith,1 Pravin Punamiya,2 Dil M. Ramanathan,3 Ramana Reddy,1 and Dibyendu Sarkar2 1

Department of Biological Sciences, Michigan Technological University, Houghton, Michigan, USA 2 Department of Earth and Environmental Studies, Montclair State University, Upper Montclair, New Jersey, USA 3 New Jersey Center for Science, Technology and Mathematics, Kean University, Union, New Jersey, USA The presence of veterinary and human antibiotics in soil and surface water is an emerging environmental concern. The current study was aimed at evaluating the potential of using vetiver grass as a phytoremediation agent in removing Tetracycline (TC) from aqueous media. The study determined uptake, translocation, and transformation of TC in vetiver grass as function of initial antibiotic concentrations and exposure time. Vetiver plants were grown for 60 days in a greenhouse in TC contaminated hydroponic system. Preliminary results show that complete removal of tetracycline occurred within 40 days in all TC treatments. Initial concentrations of TC had significant effect (p < 0.0001) on the kinetics of removal. Tetracycline was detected in the root as well as shoot tissues, confirming uptake and rootto-shoot translocation. Liquid-chromatography-tandem-mass-spectrometry analysis of plant tissue samples suggest presence of metabolites of TC in both root and shoot tissues of vetiver grass. The current data is encouraging and is expected to aid in developing a cost-effective, in-situ phytoremediation technique to remove TC group of antibiotics from wastewater. KEY WORDS: tetracycline, antibiotics, phytoremediation, vetiver grass

INTRODUCTION Soil and surface water contamination with human and veterinary antibiotics (VAs) is an emerging environmental concern. Four compounds namely tetracycline (TC), oxytetracycline (OTC), chlortetracycline (CTC), and doxycycline (DC) belong to a class of antibiotics which are commonly called as tetracyclines (TCs). The TCs are broad-spectrum antibacterials which are widely used as human antibiotics (TC, OTC) as well as in animal feed (CTC, OTC, TC, and DC) to maintain health and improve growth efficiency (Kemper 2008). Capelton et al. (2006) prioritized 83 veterinary medicines for detailed risk assessments based on their usage, potential to reach the environment, and toxicity profile. CTC, OTC, and TC ranked 16, 28, and 31 respectively in their priority list with high usage and potential to reach the environment and priority for the risk assessment (Capelton

Address correspondence to Rupali Datta, Department of Biological Sciences, Michigan Technological University, 1400 Townsend Drive, Houghton, MI, USA. E-mail: [email protected] 343


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et al. 2006). Primary usage of CTC is restricted to cattle and pigs, OTC is mostly used in veterinary medicines (cattle, sheep, and pigs), but also used as a human drug; whereas TC is one of the most widely used human antibiotics, that is also used in horse, sheep and pigs (Kemper 2008). In community hospitals, TC along with penicillin and ampicillin are the most used antibiotics, which account for about half of the antibiotics given to patients (Sheckler and Bennett 1970). Being bioactive substances, antibiotics are highly effective at low doses and cannot be completely eliminated by the animal or human organisms, and thus excreted after short time of retention (Kemper 2008). Excretion rates of TCs vary between 40–90% depending on the mode of application, the excreting species, and time after administration (Berger, Petersen, and Buning-Pfau 1986; Haller et al. 2001; Halling-Sørensen et al. 2001). Intracorporal degradation of the antibiotics often takes place in the feces, but after excretion these metabolites can be transformed back to their parent compounds (Langhammer 1989). In last decade, many studies from several countries found occurrences of TCs in sewage influent and effluent, in surface water, groundwater and even drinking water (Kemper 2008; Sarmah, Meyer, and Boxall 2006). For instance, presence of TCs in river water (1µg L−1) was first reported in England (Watts et al. 1982). Tetracyclines (ranged from 1000 µg L−1) were most frequently detected in the wastewater lagoons at swine and poultry animal feeding operations from six states of United States (Meyer et al. 2003). A U.S. Geological Survey study found large proportions of TCs ( 70% of the total TC in the plant tissues were located in the shoot which is in contrast with the findings of Migliore, Cozzolino, and Fiori (2003) and Kong et al. (2007) who suggested that the antibiotics mainly accumulate in the root tissues of the plants. Root to shoot translocation factors (TF) (calculated as the TC in shoot/TC in root) of TC in vetiver grass significantly (p = 0.0003) increased with increasing initial TC concentrations in solution (Table 1). The high TF (> 1) across higher TC treatments (10 and 15 mg L−1) indicates the potential of vetiver grass as an effective phytoremediation agent. Unknown peaks other than TC found in the HPLC chromatogram of all root and shoot samples led us to further analyze these samples using liquid chromatography-tandem mass spectrometry. Figure 2 shows the LC-MS/MS chromatograms of root (2A) and shoot (2B) samples. In all root samples, major peaks were found at higher m/z than that of TC (m/z = 445). In contrast, in the shoot samples, major peaks were at lower m/z as compared to the TC. This suggests presence of TC conjugates in the root samples, whereas in shoot, the degradation products of tetracycline prevail. Absence of these peaks in the hydroponic solution and in the control plants not treated with TC suggests that these unknown peaks are plant metabolites of TC. Calculations were also performed to check for the presence of solvents conjugate, mainly ACN-TC and MeOH-TC and salt conjugate, Na-TC and K-TC. Analysis by mass spec revealed absence of both the solvent and salt conjugate with TC. Ongoing studies involve performing liquid chromatography–tandem mass spectrometry to identify the newly formed daughter compounds and metabolites via Meta ID (metabolite identification) in the plant tissue. LTQ-Orbitrap will be used to determine and identify and plant conjugate /non-conjugate metabolites using ion trap (both positive and negative mode simultaneously). Future studies aims towards developing solid phase extraction (SPE) techniques for extraction of compounds from plant tissue and robust and sensitive mass spectrometry method to identify and quantify TC and its metabolites. Our ultimate goal is to develop an in situ biological treatment system to remove TCs from manure stockpiles at lagoons and wastewater using constructed wetlands. This study is the first step towards our long-term goal of remediating TCs contaminated soil and water


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Figure 2 LC-MS-MS chromatograms of the vetiver root (2A) and shoot (2B) suggesting the presence of TC metabolites.

using vetiver grass. Results demonstrated the ability of vetiver grass to uptake, translocate, and biotransform tetracycline. Given the huge biomass of vetiver grass compared to any similar species and it’s tolerance of a wide variety of environmental conditions and matrices indicate its potential as a phytoremediation agent in aqueous systems. The effectiveness of vetiver grass in removing organic contaminants has been reported in various previous reports (Makris et al. 2007; Das et al 2010; Marcacci et al. 2006). Tetracycline from the aqueous media and structural similarity between tetracycline group of antibiotics led us speculate that vetiver grass would also be able to remediate OTC and CTC. We are in process of designing a greenhouse experiment to study the phytoremediation potential (uptake and metabolism of detoxification) of vetiver grass in remediating TC, OTC, and CTC from contaminated municipal wastewater and manure lagoons.

ABBREVIATIONS TC OTC CTC HPLC LC-MS/MS

Tetracycline Oxytetracycline Chlortetracycline High performance liquid chromatography Liquid chromatography tandem mass spectrometry.

ACKNOWLEDGMENTS Student author SS acknowledges the Summer Undergraduate Research Fellowship provided by Michigan Technological University. Student authors PD, PP and RR acknowledge their respective universities for Graduate Assistantships.

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