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ACCUMULATION OF ANTIMICROBIALS BY PLANTS:
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NIROJ ARYAL

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5108 K:IProj/Acc&Pres/CIRCIDateDue.indd

ACCUMULATION OF ANTIMICROBIALS BY PLANTS: IMPACTS ON
ANTIMICROBIAL FATE AND RISK
BY

NIROJ ARYAL

A THESIS
Submitted to
Michigan State University

in Partial Fulfillment of the Requirements
for the Degree of

MASTER OF SCIENCE
Biosystems Engineering

2010

ABSTRACT

ACCUMULATION OF ANTIMICROBIALS BY PLANTS: IMPACTS ON
ANTIMICROBIAL FATE AND RISK

BY
NIROJ ARYAL

Triclocarban and triclosan, two widely used antimicrobials in consumer use
products, adversely affect ecosystems and potentially human health. The application of
biosolids to agricultural fields introduces triclocarban and triclosan to soil and water
resources through runoff. This research examined the effects of plant grth on the fate
and migration of antimicrobials to water resources, focusing on plant accumulation of
antimicrobials and a risk characterization following plant accumulation of antimicrobials.

Pumpkin, zucchini, and switch grass were grown in soil columns to which
biosolids were applied. Leachate from soil columns was assessed every other week for
concentration of triclocarban and triclosan. At the end of trial, concentration of
triclocarban and triclosan was determined for soils, roots, stems, and leaves using liquid
chromatography mass spectrometry. In addition, pumpkin, and zucchini were grown
hydroponically in medium spiked with triclocarban and triclosan to study the
phytoaccumulation potential of plants for antimicrobials.

Results indicated that plant growth reduced the leaching of antimicrobials from
columns. Plants accumulated triclocarban and triclosan, but not at high enough
concentrations to be considered hyperaccumulators. There was negligible risk of
antimicrobials from eating pumpkin and zucchini fruits produced from fields to which

biosolids have been applied.

Dedication

To my Late Grandmother

Mishri Maya Aryal

iii

ACKNOWLEDGEMENTS

First and foremost, I would like to express my sincere gratitude to my major
advisor Dr. Dawn Reinhold for putting faith in me, and for providing continuous support
and exceptional guidance. Her encouragement, sound ideas, inspiration, and immense
knowledge helped me throughout the research and also in thesis writing.

I would also like to thank rest of my thesis members: Dr. Steve Safferman and
Dr. Hui Li for their encouragement, insightful comments, and suggestions.

My thanks to Dr. Alison Cupples, Civil and Environmental Engineering
department, for letting use her lab and Dr. Jong M. Cha for guiding me in ASE.

I am grateful to lab technicians, Ms. Kristine Van Winkle and Louis Faivor,
graduate secretary Barb Delong and other staffs and faculties in BAE.

I am indebted to Reinhold Research Group Members: Natalie, Brad,
Catherine, Kevin, Shannon (11.), Patrick, Shannon (M.), Sunirat, Devin, Alice, Ben ,
and Umesh and Ramkrishna for their help and creating workable fun environment.

My funding agencies: Center for Water Science (CWS), MSU and Michigan
Agriculture Experiment Station, and College of Engineering deserve special mention.

I thank my entire family for providing a loving environment for me: my wife
Ishara, my brothers Yuroj and Suroj, my sister-in-law Shanti and my nephew Oscar.

Lastly, and most importantly, I wish to thank my parents, Surya Bahadur Aryal

and Mohan Maya Aryal, for their love, care, and support.

iv

TABLE OF CONTENTS

LIST OF TABLES .................................................................................. vii

LIST OF FIGURES ................................................................................ ix

CHAPTER 1 INTRODUCTION

1.1. Rationale ................................................................................. 1
1.2. Objectives ............................................................................... 3
CHAPTER 2 LITERATURE REVIEW
2.1. Uses of Triclocarban and Triclosan .................................................. 5
2.2. Occurrence and Fate of Triclocarban and Triclosan ................................ 6
2.3. Impacts of Triclocarban and Triclosan ............................................. 12
2.4. Plant Interaction with Chemicals and Phytoremediation of Chlorinated
Organics ................................................................................. 14
2.4.1. Green Liver Model ....................................................... 17
2.5. Phytoremediation and Hydroponics ................................................ 18
2.6. Extraction Procedures for Antimicrobial Analysis ................................ 19

CHAPTER 3 MATERIALS AND METHODS

3.1. Soil Column Experiment ............................................................ 22
3.1.1. Soil, Seeds and Biosolids ................................................ 22
3.1.2. Chemicals .................................................................. 23
3.1.3. Experimental Columns ................................................... 24
3.1.4. Sampling .................................................................. 26
3.1.5. Sample Preparation ....................................................... 27
3.1.6. LC-MS analysis ........................................................... 30
3.1.7. Statistical Analysis ....................................................... 32
3.1.8. Risk Characterization .................................................... 32

3.2. Hydroponic Experiment .............................................................. 34
3.2.1. Plants and Experimental Setup ......................................... 34
3.2.2. Plant Culture in Media ................................................... 35
3.2.3. Sampling .................................................................. 37
3.2.4. Sample Preparation ...................................................... 37
3.2.5. LC - MS analysis .......................................................... 38

CHAPTER 4 RESULTS AND DISCUSSIONS
4.1. Soil Column Experiment ............................................................. 39
4.1.1. Leaching of Antimicrobials ............................................. 39
4.1.2. Soil Concentrations of Antimicrobials ................................ 42

4.1.3. Plant Concentrations of Antimicrobials ............................... 43
4.1.4. Risk of Land-applied Antimicrobials in Vegetated Systems ...... 48

4.2. Hydroponic Experiment ............................................................. 52
4.2.1. Aqueous Concentrations of Antimicrobials ........................... 52
4.2.2. Plant Concentrations of Antimicrobials ................................ 54

4.2.3. Comparison of Soil Column and Hydroponics Antimicrobial
Concentrations .................................................................... 59
CHAPTER 5 CONCLUSIONS AND FUTURE RESEARCH ............................ 64

APPENDICES

A.l Review of Triclocarban and Triclosan Extraction ................................ 66

A2 Threshold Toxicity data of Triclocarban and Triclosan from Different
Research .................................................................................................... 68

REFERENCES ...................................................................................... 76

vi

LIST OF TABLES

Table 2.1 Structure and Properties of Triclocarban and Triclosan .............................. 6
Table 2.2 Occurrence of Triclocarban and Triclosan in the Environment ................... 10
Table 3.1 Chemicals Used in the Experiment ................................................... 23
Table 3.2 Nutrient Solution Composition ........................................................ 36

Table 4.1 Occurrence of Maximum Concentration of Triclocarban and Triclosan in

Leachate Water ...................................................................................... 39
Table 4.2 Soil Antimicrobial Concentrations and P-values for Comparison with

Controls ............................................................................................... 42
Table 4.3 Biomass Produced in Soil Column Experiment and Root to Shoot Ratio ....... 43

Table 4.4 P-Values for Comparison of Antimicrobial Concentrations between Plant
Parts .................................................................................................... 45

Table 4.5 P-Values for Comparison of Soil Antimicrobial Concentrations and Plant
Antimicrobial Concentrations ...................................................................... 45

Table 4.6 Bioaccumulation and Translocation Factors for Triclocarban and
Triclosan ............................................................................................. 46

Table 4.7 Risk Characterization of Triclocarban and Triclosan .............................. 51

Table 4.8 Comparison of Biomass Produced and Root to Shoot Ratio in Hydroponic
Experiment with Soil Column Experiment ...................................................... 56

Table 4.9 P-Values for Comparison of Hydroponic Experiment with Soil Column
Experiment ........................................................................................... 60

Table 4.10 Comparison of Translocation Factors and Bioaccumulation Factors for Two
Experiments .......................................................................................... 61

Table 4.11 Coefficients of Variation in Different Units in the Experiment ................. 62

Table A.l Review of Extraction of Triclosan and Triclocarban from Different Matrices in
Different Literatures ................................................................................ 66

Table A2 (a) Microbial Resistance (Aqueous and Soil) ....................................... 68

vii

Table A2 (b) Ecological Health (Aqueous and Soil) ............................................ 68

Table A2 (c) Human Health ........................................................................ 76

viii

LIST OF FIGURES

Figure 1.1 Comparison of Chemical Structure of DDT, PCBs, TCS, and TCC.. . . . . . ...2

Figure 2.1 Comparison and Occurrences of Threshold Toxicity Values of Triclosan and
Triclocarban to Microorganisms, Animals and Plants in Both Soil and Aqueous

Ecosystem ............................................................. , .............................. 12
Figure 3.1 Experimental Setup Showing Six Columns and Amber Bottles to Collect

Leachate Water ...................................................................................... 25
Figure 3.2 Plants Growing in Columns under Constant Regime Lights ..................... 26
Figure 3.3 Solid Phase Extraction ................................................................. 28
Figure 3.4 Accelerated Solvent Extractor Used in the Experiment ........................... 29
Figure 3.5 LC-MS Used in the Experiment ...................................................... 30
Figure 3.6 A sample Chromatogram along with Calibration Curve 31
Figure 3.7 Vases before Wrapping in Foil, Afier Adding Antimicrobial Solution and

Transplantation of Pumpkin and Zucchini Seedlings .......................................... 35
Figure 3.8 Experimental Vases after Some Weeks of Plants Growth ........................ 36

Figure 4.1(a) Triclocarban Concentrations in Leached water with Time. . . . . . . . .41

Figure 4.1(b) Triclosan Concentrations in leached water with Time .......................... 41
Figure 4.2 Plant Concentrations of Triclocarban. ............................................... 44
Figure 4.3 Plant Concentrations of Triclosan ................................................ I ...... 44
Figure 4.4 Triclocarban Mass Balance Analysis ................................................ 47
Figure 4.5 Triclosan Mass Balance Analysis .................................................... 48

Figure 4.6 Box Plots of Triclocarban Concentrations Available in Different Literatures
and Research Data ................................................................................... 49

Figure 4.7 Box Plots of Triclosan Concentrations Available in Different Literatures and
Research Data ....................................................................................... 50

ix

Figure 4.8 Triclocarban Concentrations with Time .......................................... '....53
Figure 4.9 Triclosan Concentrations with Time ................................................. 53

Figure 4.10 Triclocarban Concentrations in Pumpkin Roots and Shoots, loglo scale for
concentration ......................................................................................... 55

Figure 4.11 Triclosan Concentrations in Shoots and Roots of Pumpkin and Zucchini ..... 57

Figure 4.12 Mass Balance Analysis of Triclocarban in Hydroponic Experiment... . . . . . ...58
Figure 4.13 Mass Balance Analysis of Triclosan ................................................ 59
Figure 4.14 Comparisons of Soil Experiment and Hydroponic Experiment Data ......... 6O

Chapter 1: Introduction

1.1 Rationale

Triclocarban and triclosan are two very widely used antimicrobials worldwide.
Added to daily use products like hand soaps, lotions, toothpastes, cosmetics and
deodorants, annual per capita usage in the US. is 1,130 mg triclocarban and 1,030 mg
triclosan. Consequently, the combined input of antimicrobials into the US. environment
is 0.6 - 1 million kg (Halden and Paul] 2005, EPA 2003). Triclocarban and triclosan are
not readily biodegradable, persistent and hydrophobic (EPA 2002b, EPA 2008b). These
“down the drain” contaminants are not readily degraded and strongly sorb to solids in
wastewater treatment plants (Heidler, Sapkota and Halden 2006, Heidler and Halden
2007). However, a fraction of antimicrobial does migrate to water resources. The
widespread practice of applying digested sludge from wastewater treatment plant to
agricultural fields introduces triclocarban and triclosan into agricultural soils (Kinney et
a1. 2008, Cha and Cupples 2009). Once in agricultural soils, triclocarban and triclosan
can contaminate runoff water and then water resources (Topp et a1. 2008, Sabourin et a1.
2009). Triclocarban is ranked top 10 in occurrence and top 20 in concentration among 96
organic pollutants in the United States water resources (Halden and Paull 2005).

Moreover, triclocarban and triclosan are toxic to animals, plants and potentially
humans. For example, algae growth is affected at current environmental concentrations
(Johnson et al. 2009). Antimicrobials also affect soil and aqueous ecological health. More
details about the fate and effects of antimicrobials are discussed in Chapter 2: Literature

Review.

Continuous release of antimicrobials to water resources and soils necessitates
exploration of sustainable management strategies. Phytoremediation could be a
sustainable approach to managing these types of continuously released organic pollutants.
Phytoremediation is inexpensive, environmentally friendly, and requires low maintenance.
Polychlorinated biphenyls (PCBs), dichlorodiphenyltrichloroethane (DDT) and their
metabolites, which have two chlorinated benzene rings similar to triclocarban and
triclosan (Figure 1.1), have been shown to be phytoremediated (Aslund et al. 2007,
Lunney, Zeeb and Reimer 2004). Specifically, pumpkin and zucchini can phyto or
hyperaccumulate compounds like PCBs, and DDT and its metabolites (Dzantor, Chekol

and Vough 2000, Lunney et al. 2004, Wang et al. 2004, Aslund et al. 2008, White et al.

2003)
CI CI CI OH CI
0‘
or O O or Cl Cl
Dichlorodiphenyltrichloroethane (DDT) Triclosan (TC S)
it
0. .Q
Clx Clx C' 0'
Cl
Polychlorinated biphenyls (PCBs) Triclocarban (TCC)

Figure 1.1 Comparison of Chemical Structure of DDT, PCBs, TCS, and TCC

Given the widespread use, high production volume and established risks to

humans and the environment, there is a pressing need to evaluate antimicrobials with

2

regards to occurrence, fate and effects in broad system-based scenario. Most research on
triclocarban and triclosan has focused on occurrence and fate in water resources and
wastewater treatment plants (Coogan et al. 2007, Halden and Paull 2004, Halden and
Paull 2005, Heidler et al. 2006, Heidler and Halden 2007, Sapkota, Heldler and Halden
2007, Young et al. 2008, Chu and Metcalfe 2007, Yu and Chu 2009, Miller et al. 2008b,
Hua, Bennett and Letcher 2005, Bester 2003, Vikesland, Rule and Greyshock 2004, Wu
et al. 2007, Poiger et al. 2003, Nishi, Kawakami and Onodera 2008, Bester 2005,
Thompson et a1. 2005, Morrall et al. 2004, Sabaliunas et a1. 2003, Montes et al. 2009) and
very few on agricultural soils (Cha and Cupples 2009, Kinney et al. 2008, Chu and
Metcalfe 2007). The fate of antimicrobials in agricultural fields with plants is unknown,
especially with regards to the role of plant growth in migration of antimicrobials from
fields to water resources afier land application of biosolids from wastewater treatment
plants. Understanding the interactions between plants and antimicrobials is crucial to both
(i) quantify the phytoremediation potential of plants for these compounds and (ii) to

assess accumulation of antimicrobials in vegetables grown in biosolid-applied field.

1.2 Objectives

The thesis presented herein evaluates the hypothesis that triclocarban and
triclosan, being similarly structured to other chlorinated aromatic organic contaminants
like DDT, DDE and PCB (Figure 1.1), can be phytoremediated to reduce antimicrobial
concentrations in soil and the migration of antimicrobials to water resources. Pumpkin
and zucchini were selected due to their reported potential of PCBs and DDT

phytoremediation. This study also aims to quantify the human health effects of

consuming food grown on land to which biosolids are applied. Switch grass was selected
as a non-vegetable plant that is a potential bioenergy crop and has potential to
phytoremediate hydrophobic aromatic compound like PCBs (Dzantor et al. 2000).

The specific aims of this study were to:

(i) Assess the effects of growth of pumpkin, zucchini and switch grass on
leaching and soil accumulation of triclocarban and triclosan from land
applied biosolids,

(ii) Evaluate phytoaccumulation of triclocarban and triclosan in pumpkin,
zucchini and switch grass,

(iii) Evaluate phytoaccumulation of triclocarban and triclosan in pumpkin and
zucchini grown hydroponically, and '

(iv) Characterize health risk associated with observed accumulation of

triclocarban and triclosan by pumpkin and zucchini to humans.

Chapter 2: Literature Review

2.1 Uses of Triclocarban and T riclosan

Antimicrobials such as triclocarban (3,4,4’-trichlorocarbanilide) and triclosan (5-
chloro-2-(2,4-dichlorophenoxy)-phenol) are widely used in personal care products like
toothpaste, soaps, deodorants, cosmetics and lotions (Ying, Yu and Kookana 2007). A
retail survey of national brand liquid and bar soaps in 1999 to 2000 reported that
antibacterial agents TCC and TCS were present in 76% of liquid soaps and 29% of bar
soaps available nationally (Perencevich, Wong and Harris 2001). Added to soaps at levels
of 0.5 to 5% (w/w), production of TCC exceeds one million pound per year. Triclosan is
used in consumer products at concentrations of 0.69 — > 99 % (Heidler et al. 2006, EPA
2002b). Some personal consumer applications of triclosan include hand soaps, toothpaste,
deodorants, laundry detergent, fabric softeners, facial tissues, antiseptics for wound care, and
medical devices. Triclosan is used for commercial, residential and industrial applications to
minimize bacterial growth on equipment and instruments. Industrial uses include conveyer
belts, fire hoses, dye bath vats, ice making instruments, and HVAC coils. Triclosan is also
used as a material preservative in toys, paints, mattresses, clothing, brooms, mulch, floors,
shower curtains, awnings, tents, toilet bowls, urinals, garbage cans, refuse container liners,
insulation, concrete mixtures, grouts, and upholstery fabrics adhesives, fabrics, vinyl, plastics,
polyethylene, polyurethane, polypropylene, floor wax emulsions, textiles (footwear, clothing),
caulking compounds, sealants, rubber, and latex paints (EPA 2008b).

The structure and properties of triclocarban and triclosan are shown in Table 2.1.
Both are chlorinated aromatic organic compounds with low solubility in water and high

octanol - water partition coefficient. Both are similarly- structured to poly- chlorinated

biphenyls (PCBs) and dichlorodiphenyltrichloroethane (DDT) (Figure 1.1).

Table 2.1 Structure and Properties of Triclocarban and Triclosan

 

 

 

 

 

 

 

 

 

 

Triclosan Triclocarban
Molecular Structure 0H CI 0
6" We
on on
Cl ' Cl °'
CAS registry no. 3380-34-5 00101-20-2
Molecular Weight 289.55 315.59
Dissociation Constant, 8.14 at 20°C 12.7
13Ka
Water Solubility 10 mg/L at 20°C 0.02366 mg/L at 25°C
Octanol -Water 4.76 at 25°C, pH 7 4.90 at 25°C, pH 7
Partition Constant
(Log KOW)

 

Reference: (Halden and Paull 2005) and EPI SUITE 4.0

2.2 Occurrence and Fate of Triclocarban and Triclosan

Diverse uses of triclocarban and triclosan lead to high volume use with an annual
per capita usage of 1,130 mg triclocarban and 1,030 mg triclosan nationwide, resulting in
annual disposal of greater than 330,000 kg triclocarban and 300,000 kg triclosan in the
US. (Halden and Paull 2005). The primary route through which triclocarban and
triclosan enter the environment is domestic sewage discharge to wastewater treatment
plants (WWTP) (Chu and Metcalfe 2007, Heidler et al. 2006, Sapkota et al. 2007).

Removal of antimicrobials in WWTP predominantly results from sorption to
wastewater particulate matter (78 i 11 % for triclocarban and 80 :t 22 % for triclosan)
(Heidler et al. 2006, Heidler and Halden 2007). Consequently, dewatered municipal
sludge accumulates 51 i 15 mg triclocarban and 30 :l: 11 mg triclosan per kg of sludge
(Heidler and Halden 2007, Heidler et al. 2006). Approximately 50% of US biosolids are

land applied (EPA 2007). Heidler et al. (2006) estimated that major part of approximately

 

three-quarters of triclocarban used by consumers is ultimately released into the
environment through land application of municipal sludge as biosolids for agriculture
(Heidler et al. 2006). Combined input of triclosan and triclocarban into US. environment
exceeds 0.6 to 1 million kg/year (EPA 2003), with only 5,800 kg of triclocarban and
2,600—10,400 kg of triclosan resulting from discharge of effluent of activated sludge
treatment plants into US. water resources nationwide (Halden and Paull 2005).
Agricultural soils previously amended with biosolids contains triclocarban and triclosan
in the range of 1.20 - 65.10 ug/kg triclocarban and 0.16 - 1.02 ug/kg triclosan (Cha and
Cupples 2009). Marine sediment samples at the outflow of urban wastewater treatment
plant contained triclosan from 0.27 - 130.7 jig/kg (Aguera et al. 2003).

Triclocarban ranks in the top 10 in occurrence and top 20 in maximum
concentration among 96 organic pollutants in US. water resources (Halden and Paull
2005). A summary of the occurrence of antimicrobials in the environment is shown in
Table 2.2. Triclocarban is present in the environment at broader range than triclosan.
Triclocarban is detected in US. rivers and streams from 0.09 — 1.55 ug/L (Halden and
Paull 2005, Sapkota et al. 2007), in sediment pore water from 1.77-10.78 ug/L (Chalew
and Halden 2009, Miller et al. 2008a), in WWTP influents from 4.1-8.1 ug/L (Heidler et
al. 2006, Halden and Paull 2005) and in WWTP effluents from 0.11-0.17 pg/L (Heidler
et al. 2006, Halden and Paull 2005). Triclosan is detected in rivers from <0.003-0.075
ug/L (Bester 2003, Ying and Kookana 2007), in WWTP influents from 3.8 to 16.6 ug/L
(McAvoy et al. 2002) and in WWTP effluents from 0.800 — 37.8 ug/L (Aguera et al.

2003). Annual loadings of antimicrobials to water resources are attributed to activated

sludge treatment plants (3 9-67%), trickling filter plants (31-34%) and combined and
sanitary sewer overflows (2-7% and <0.2% respectively) (Halden and Paull 2005).

Once introduced into the environment, triclocarban and triclosan tend to sorb to
soil or sediment (Heidler and Halden 2007). Miller et al. (2008) indicated the persistence
of triclocarban in agricultural soils (Miller et al. 2008a). Additionally, triclocarban and
triclosan computer fate modeling for biodegradability using Quantitative Structure-
Activity Relationship (QSAR) indicate that these compounds do not degrade quickly with
primary biodegradation half-lives of weeks and ultimate biodegradation half-lives of
months (Halden and Paull 2005, Ying et al. 2007). Experimental half-lives of triclocarban
and triclosan were 108 and 18 days respectively, in aerobic soil, with longer half lives in
anaerobic soils (Ying et al. 2007). Triclosan is reported to form even more persistent and
toxic degradation products like 2,8-dichlorodibenzo-p—dioxin (DCDD) (Gledhill 1975,
Aranami and Readman 2007).

Accumulation of triclocarban and triclosan has been reported in aquatic organisms
and humans. Computer modeling using EPIWIN PBT (persistence, bioaccumulation and
toxicity) profiler, developed by USEPA, estimated that triclocarban and triclosan are
potentially bio-accumulative (Ying et a1. 2007). Triclocarban and triclosan tend to adsorb
to particulate matter in waters, which are eaten by aquatic organism like fish, crustaceans,
clams, oysters, snails leading to detectable concentration of antimicrobials in their tissues
(Balmer et al. 2004, Ramirez et al. 2009). For example, maximums of 58.7 ug/kg
triclosan and 299 ug/kg triclocarban was detected in snails near WWTP effluent (Coogan

and La Point 2008). Triclocarban can also bioaccumulate rapidly in filamentous algae

(Cladophora spp.) and in snail at three orders of magnitude greater than ambient water
concentrations (Coogan et al. 2007, Coogan and La Point 2008).

Bioaccumulation of antimicrobials has been also observed in humans. A survey of
the US. general population in 2003 - 2004 detected concentrations of triclosan in 74.6%
of urine samples at the concentration of 2.4 - 3,790 ug/L (Calafat et al. 2008). Also,
triclosan was detected in plasma and breast milk of Swedish mothers who did not use any
personal care products containing triclosan, indicating systemic exposure (Allmyr et al.

2006)

Table 2.2 Occurrence of Triclocarban and Triclosan in the Environment. Concentrations in mean :1:
standard deviation. WWTP = Wastewater treatment plant

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Triclocarban, Triclosan,
Medium (pg/L or (pg/L or Location References Remarks
ugflg) fig/kg)
Aqueous 6.7 a 0.1 6.1 a 1.6 MD, USA (“amen and Paull
2005)
‘55 East Coast, .
é Aqueous 6.1 :t 2.0 USA (He1d1er et al. 2006)
,5 Aqueous 0.4 - 9.4 (Farre et al. 2008)
& Aqueous 1.1 - 1.3 Germany (Bester 2003)
Mid-Atlantic, (Heidler and Halden
E Aqueous 0.8 - 10.8 USA 2007)
Aqueous 3.8-16.6 (McAvoy et al. 2002)
Aqueous 0.01-4.0 ON, Canada (Lishman et al. 2006)
I; Aqueous 0.19 0.12 TX, USA (Coogan et al. 2007) At
a.
3 Algae 109.0 150 .0 TX, USA (Coogan et al. 2007) WWTP
Aqueous 0.08 0.06 TX, USA (Coogan et al. 2007)
Algae 401.0 146.0 TX, USA (Coogan et al. 2007)
. 5.9 - 58.7 (Coogan and La Point
Snail 9.8 - 299.0 wet wt TX, USA 2008)
0.03 :h (Halden and Paull
... Aqueous 0.11 :1: 0.0 1 0.02 MD, USA 2005)
G
0 East Coast, .
é Aqueous 0.17 :1: 0.03 USA (Heidler et al. 2006)
0 Aqueous 0.8 -37.8 Spain (Aguera et al. 2003)
E Ageous 0.04-0.06 Germany (Bester 2003)
0.023- . (Ying and Kookana l9
Aquews 0.434 Ausual‘a 2007) effluents
A ueous 0.001- Mid-Atlantic, (Heidler and Halden Tertiary
9 0.240 USA 20% effluent
Aqueous 0.2 - 2.7 (McAvoy et al. 2002)
Aqueous 3252- ON, Canada (Lishman et al. 2006)
5 1,0003: 15,000 East Coast, .
Sludge ((19,) US A (Heidler et al. 2006)
2 170-5 970 620'0- (Chu and Metcalfe
Sludge (:11?) ’ 11,550 ON, Canada 2007)
dry wt
_ 90.00- (Cha and Cupples
% Sludge 4,890 9,280 7060 MI, USA 2009)
=‘- I 000- WWTP at
o 9
.3 Sludge 1,300 Germany (Bester 2003)
m -
Biosolid 10,500 53““ (Kinney et a1. 2008)
90.00- .
Biosolid 16,790 Australia (Ymg and K°°kana 1? .
(db) 2007) biosol1ds
SI d e 20,000- Mid-Atlantic, (Heidler and I-Ialden Digested,
“ g 55,000 USA 2007) dewatered

 

10

 

Table 2.2 Cont’d

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Triclocarba Triclosan,
Medium 11, (pg/L or (pg/L or Location References Remarks
rig/kg) rig/Kg)
Dried, primary; co-
Sludge 19,300 i 7,100 USA (58%?“ et al. contaminant DCC,
m tetraCC also found
3 2,170- Samples of activated
g Sludge (6:0)0'11’550 5,970 gynada SEEESZOM) sludge and treated
35 ’3’ (dry) biosolids
500.0-
Sludge 15,600 $31;ng et al. Digested
(dry)
. _ 0.16 - (Cha and Previously biosolid
f—g Sod 1'2 65'10 1.02 MI’ USA Cupples 2009) amended
5 . Midwest , (Kinney et al. Previously biosolid
E S011 833'0 USA 2008) amended
.50 . Midwest , (Kinney et al. Minimally affected
3 8011 160'0 USA 2008) by manures
75, Soil 69 0 Midwest, (Kinney et al. Previously Swine
m ' USA 2008) manure applied
. _ (Halden and Known input or
River 5'6 6'7 Pau112004) wastewater
River 3%?83' Germany (Bester 2005)
. Up to . (Ying and Outfall, upstream,
RIVCI'S 0.075 Australia Kookana 2007) downstream
Downstream of
River 0.084z+:O.1 10 USA (Sap kota et al. sewage treatment
2007)
plants
. (Sapkota et al. Upstream of sewage
1.. River 0.012zt0.015 USA 2007) treatment plants
8 . Runoff from
«.1
B Runoff 0.003 t 0.002 3'33? i Canada gihggmm et al. dewatered municipal
é ' sludge applied field
0.258 (Topp et al. Runoff from biosolid
3
m Runoff :t0.039 Canada 2008) applied field
1.770 :t 0.670 (Miller et al Mono -, di -, and
8 Aqueous (avg); 3.96 USA 2008a) ' nonchlorinated
8. (max) carbanilide found
‘5 4.05 3:3.38; (Miller et al. DCC=15.5iI .8
“g ,3 ““6"“ 10.78 @ax) USA 2008a) mm
:0 "' 0.26- (Chalew and .
a) “I _
m 3 0.24 32.90 382.8 Hal den 2009) Estlmated
700.0-I,600 30.0 a (Miller et al. MCC=_4"*2'4
dry wt 10 0 USA 2008a) mg/kg’
' NCC=0.5iO.l mfig1
<20.00 — 8,200 USA (EPA 20023)
‘55 0.270- S ain (Aguera et al. Sediments at outflow
§ 130.7 p 2003) of WWTP
'3 0 006 S ain (Morales-Munoz Sediment at WWTP
m ' p et al. 2005) outflow

 

 

 

11

2.3 Impacts of Triclocarban and Triclosan

Release of antimicrobials into the environment and subsequent accumulation in
organisms raises a number of concerns about human health and ecosystem health. Figure
2.1 shows threshold toxicity values and reported occurrences of TCS and TCC in

different environmental matrix (Appendix A] and A2) published in different literatures.

 

 

 

 

 

 

 

 

12 ~
11 e xMicroorganisms +Animals X
10 - 0 Plants OTCS occurrence aqueous
__ 9 __ I TCS soil occurence A TCC occurence
g i
8 i- l x
E“ E I . . ‘ l
o g 7 Q l l l . ‘ i
< l x . l A '
ch—_ 6 P | I g . l
. 1 1 ’ i
C ~ 0
.3 .o S E I F o f i
I! 3 4 ~ g ' 1 § 1
E I I l
s—- 3 - l - ; :
c I 1
8 2 ~ 1 I ' I I 8 I
1—0 H I I I
go o. 1 + 1
— Q 1 T : I . E
I
Toxicity I Occurrence Toxicity Occurrence ' Toxicity Occurrence'
TCS : TCS TCC
Ecological Health (Soil) Ecological Health (Aqueous)

 

 

 

Figure 2.1 Comparison of Occurences and Threshold Toxicity Values of Triclosan and Triclocarban
to Microorganisms, Animals and Plants in Both Soil and Aqueous Ecosystem. References are given at
Appendices AJ and A.2

Human Health. Multiple studies have documented adverse human health effects
from antimicrobials. Triclocarban significantly reduced mammalian reproduction in rats
at 0.25 % of diet and caused methemboglobeminema in humans (1971, Ponte et al. 1974,
Johnson, Navone and Larson 1963). Methemboglobeminema was caused by the use of

enema solutions made from soaps containing triclocarban. Margin of exposure (MOE),

12

which is ratio of no-observable adverse effect level (N OAEL) to the estimated exposure
dose, is used to quantify risk. Exposure of triclosan or its residues can be from dietary
(i.e., food and drinking water), residential, and other non-occupational sources like hand
soaps and toothpaste, and from all known or plausible exposure routes (oral, dermal, and
inhalation). MOE for triclosan for children from 6 years to adults ranged from 4,700-
19,000 and was 290 for infants (EPA 2008b). Nolen and Dierckman (1979) found
significant decrease in rat conception rate, in the number of pups, and in the number of
pups that survived until weaning at 0.25% of diet (Nolen and Dierckman 1979). Maternal
toxicity, including weight losses, abortions, and deaths were observed in rats at 50
mg/kg/day (Nolen and Dierckman 1979). Additionally, triclocarban and triclosan disrupt
endocrine activity at concentrations of 29-3,150 ug/L potentially affecting human and
animal health (Ahn et al. 2008). Endocrine disruption resulted from bioactivity

amplification of endogenous hormones (Chen et al. 2008).

Ecosystem Health. Triclocarban and triclosan adversely impact both aqueous and
soil ecosystems. Triclocarban and triclosan are toxic to microorganisms and hence can
disrupt critical ecological processes like nitrification in WWTP and waste recycling
(N eumegen, F ernandez-Alba and Chisti 2005, lDokianakis, Komaros and Lyberatos 2004).
In aqueous ecosystems, antimicrobials can disrupt river biofilm community structure and
function, microbial community composition, algal biomass, algal architecture and algal
activity (Lawrence et al. 2009). River biofilms are thin layer of microorganisms on solid
surfaces like rocks, sediments, and plants which are different from their suspended or free

floating counterparts (Beer 2006). River biofilms are responsible for majority of

13

microbial processes in aquatic ecosystem. Freshwater microbial communities are
sensitive to 2.895 ug/L triclosan, which is within the range of observed freshwater
triclosan concentrations (Johnson et al. 2009). Additionally, triclocarban at concentration
of 17.04 ug/l and triclosan at 3.400 - 5.210 ug/l inhibit algal growth, adversely affecting
primary productivity (Yang et al. 2008, Tatarazako et a1. 2004). Concentrations of
triclosan as low as 0.150 ug/L can cause both behavioral and physiological effects on
thyroid hormone, body weight, and hind limb development in frogs (Fraker and Smith
2004, Veldhoen et al. 2007). Genotoxic and cytotoxic effects in Zebra mussel (Dreissena
polymorpha) hematocytes by triclosan may be potentially dangerous to entire aquatic
biocenosis (Binelli et al. 2009). In terrestrial ecosystems, triclocarban and triclosan
inhibited soil respiration and nutrient recycling and plant growth (Liu et a1. 2009).
Development of microbial and drug resistance has also been reported (Heath et al. 1999,
Heath et al. 2000, Heath et al. 1998, Hoang and Schweizer 1999, McMurry, Oethinger

and Levy 1998, Walsh et a1. 2003).

2.4 Plant Interaction with Chemicals and Phytoremediation of Chlorinated
Organics
Phytoremediation is the use of green plants to remove, contain or detoxify
pollutants in the environment. Phytoremediation processes include:
o rhizodegradation or pfihvtostimulation (degradation of organic
contaminants in the rhizosphere);

o phytostabilization (immobilization of contaminants in the rhizosphere);

l4

0 phytoaccumulation or hyperaccumulation (translocating and concentrating
contaminants into plant tissues);

0 thtodegradation or phytotransformation (uptake of organic contaminants
with subsequent transformation, conjugation and sequestration); and

0 phflovolatilization (uptake and release of same contaminant dming
transpiration (ITRC 2001).

Characteristics that influence the uptake of organic pollutants by plants are
hydrophobicity, polarity, sorption properties, and solubility (ITRC 2001) .
Hydrophobicity, measured by a chemical’s octanol - water partitioning coefficient (Kow),
was initially used to characterize uptake and subsequent translocation of an organic
molecule with an optimum range of 1 S log Kow S 3 (Dzantor 2007). At log Kow values
2 3, the pollutant is tightly sorbed onto roots, limiting translocation to shoots. At log Kow
values less than 1, the specific affinity of the pollutant to be sorbed onto plant roots is
limited (Dzantor 2007).

The previous notion of plant uptake of chemicals only occurring in intermediate
hydrophobicity range is not universally applicable (Dettenmaier, Doucette and Bugbee
2009). Lipid content of plant species and Kow were related to partitioning and uptake of
lipopholic organic chemicals with log Kow values > 3 in root and water systems (Gao et
a1. 2008). Sites contaminated with organic hydrophobic pollutants with log Kow >3 have
been remediated by direct plant uptake (ITRC 2001). Additionally, the uptake of
halogenated phenols by Lemna minor was affected primarily by subsequent positioning,
but not by hydrophobicity as munber of halogen substituents did not affect uptake rate

(Tront et al. 2007, Reinhold and Saunders 2006). Pollutant uptake rate was controlled by

15

rate of enzymatic transformation internal to plants, not by rate of partitioning into plants,
and was not related to log Kow or dissociation constant (pKa) (Tront et al. 2007,

Reinhold and Saunders 2006).

After a pollutant is uptaken and translocated by plants, it can be metabolized to
reduce phytotoxicity associated with the pollutant. Plant metabolism of organic pollutants,
vital for phytoremediation, appears to follow detoxification or elimination metabolic
processes collectively known as ‘green liver model’ described in separate section below
(Burken 2003). The extensive number of substrates with which plant enzymes react
allows plants to transform many xenbiotic organic pollutants (Burken 2003).

Studies have demonstrated that plants such as pumpkin, zucchini and switch grass
can be used as phytoaccumulators, hyperaccumulators, and phytostimulators for
hydrophobic chlorinated organic pollutants. Hyperaccumulators are distinguished from
phytoaccumulators by the ability to accumulate at least 106 ug/kg (dry weight) of a
chemical (McCutcheon and Schnoor 2003, ITRC 2001). In situ field experiments on the
phytoextraction of polychlorinated biphenyls (Aroclor 1254/ 1260), C ucurbita pepo spp
pepo cv. Howden (pumpkin) plants took up, translocated, and hyperaccurnulated 7,600
ug/kg PCBs in plant shoots, indicating that pumpkin is a potential PCB phytoextractor
(Aslund et al. 2007). An exponential decrease was observed in PCB concentration in the
stern of pumpkin as the distance from root increased (Aslund et al. 2007). Shoot
bioaccumulation factor (BAFshoot), defined as ratio of concentration in shoot to that in soil,
was 0.15 in pumpkin plants (Aslund et al. 2008). Cucurbita pepo species extracted and
translocated dichlorodiphenyltrichloroethane (DDT) and its metabolites,

dichlorodiphenyldichloroethhane (DDD) and dichlorodiphenyldichloroethylene (DDE),

16

with translocation factors (ratio of Shoot concentration to root concentration) of 1.8 and
1.2 and bioaccumulation factors (ratio of plant concentration to soil concentration) of 3.3
and 2.0 for zucchini and pumpkin, respectively (Lunney et al. 2004). The authors
concluded that extraction was related to high transpiration rates, large above-ground
biomass, and composition of root exudates. Zucchini (Cucurbita pepo spp prop cv Black
Beauty) phytoextracted 1.3% of weathered p, p’-DDE with 98% in aerial tissues under
field conditions (Wang et al. 2004). Subspecies variation in phytoextraction of p, p’-DDE
was observed in 21 cultivars of C. pepo spp taxana and C. pepo spp pepo (White et al.
2003). Average bioaccumulation factor of 0.283 was observed in zucchini fruit in
phytoextraction study of p, p’- DDE compared to 0.87 for leaves, 5.40 for stems and 7.22
for roots, indicating decreasing concentration along the stem from root (White et al.
2003). Hulster et al. (1994) found that polychlorinated dibenzo-p-dioxins and
dibenzofurans were uptaken through roots and translocated to shoots and fruits by
pumpkin and zucchini (Hulster, Muller and Marschner 1994). Green house scale
experiment on the evaluation of ability of switch grass (Panicum variegatum L.) to
stimulate dissipation of PCB in soil in the laboratory confirmed the potential of switch

grass to do so (Dzantor et a1. 2000).

2.4.1 Green liver-model.
The “green liver” model first appeared in 1977 based on metabolic processes
observed in enzyme studies from plant cell cultures and the metabolism of nonpolar
compounds like DDT and benzo(a)pyrene (Sandermann 1994). Burken (2003) has

described green-liver model in three common phases: transformation, conjugation, and

17

elimination or storage. After pollutants enter the plant tissue, the first phase is the
transformation of the initial substrate, including oxidation, reduction, or hydroxylation
(Burken 2003). Plant enzymes like Cytochrome P- 450 and peroxidases are common to
phase I metabolism (Burken 2003).

Conjugation (phase II) follows transformation to generally produce more water-
soluble and less-toxic compounds that can be deposited in vacuoles or incorporated into
bound residues through sequestration in phase III (Burken 2003). Exceptions include
organic pollutants with easily conjugated functional groups such as phenols, which are
directly conjugated through phase II metabolism without undergoing phase I
transformation. Phase II metabolism includes conjugation with malonic acid, D-glucose,
glutathione, cysteine, and other amino acids and carbohydrates utilizing the functional
groups produced from phase I transformations (Burken 2003).

Phase III, sequestration, follows conjugation and includes isolation of phase 11
products to three terminal fates, specifically the vacuole, the apoplast, or cell walls of the
plant (Burken 2003). In phase 11, active transportation of conjugates is facilitated and
controlled by a glutathione pump (Burken 2003). Some residues are bound to stem or
leaves or lignified, for example, residues of DDT in experiments with Canadian

waterweed (Elodea canadensis) (Garrison et al. 2000).

2.5 Phytoremediation and Hydroponics
Hydroponics is a technology for growing plants in nutrients solution or water
without use of soil. Practiced for centuries to produce food and vegetables, hydroponics

research evolved to space applications, and waste recycling, and phytoremediation (Jones

18

1997). Screening of plants suitable for removing particular type of pollutant and
identifying exact removal mechanisms of pollutants are eased by hydroponic systems
(Nzengung 2007). Additionally, better control of the environment like light, temperature,
humidity, nutrients input, root zone, air, bioavailability of targeted contaminant and less
complexity of soil-related processes due to absence of soil make hydroponics system
ideal for phytoremediation studies (N zengung 2007). Identifying fate of any pollutants in
vegetated systems using hydroponic systems gives freedom of fewer variables due to
controlled environment.

However, hydroponics research might exaggerate the results as doses are higher
than environmental concentrations. Interactions between pollutants and environmental
matrices, like soil, can affect plants response (Zabudowska et al. 2009). Root biology
may differ in hydroponics than in soil systems and selection of species for field site based
on hydroponic experimentation might mislead (Zabudowska et al. 2009). Hydroponic

research is difficult for water insoluble contaminants and for larger trees and shrubs.

2.6 Extraction Procedures for Antimicrobial Analysis

The goal of solid liquid extraction is to remove the analyte from a solid matrix
(e. g., soil, biosolid, plant) to a liquid solvent with minimal loss, degradation, or
contamination. While slight variations in protocol are numerous, there are only a few
basic techniques to extract organic chemicals from plant tissues (Anderson et al. 1997).

Among several clean-up extracts including solid phase extraction (SPE), liquid-
liquid extraction (LLE), gel permeation chromatography (GPC) and semi-preparative

HPLC, SPE has been preferred in most instances because it is fast with low organic

19

solvent use, presents low contamination risk and can be used in-line (Diaz-Cruz, de Aida
and Barcelo 2003).

Pretreatment techniques used to solubilize and remove organic chemicals from
plant tissues include digestion (treatment of sample with strong alkaline or acidic
solutions before extracting with an organic solvent), sonication (using high-intensity
ultrasonic vibrations to lyse cells rapidly and increase solvent contact with the sample),
homogenization (disrupting plant cells before solvent extraction), and solvent extraction
(using affinity of a compound for a particular solvent) (Anderson et al. 1997). Personal
care products from solid matrices have been extracted using ultrasonication and by
simple stirring of the sample with polar organic solvents or mixtures of solvents, or with
aqueous solutions containing additives or buffers (Diaz-Cruz et al. 2003).
Homogenization or high intensity mechanical extraction methods are simple, quick, and
efficient in removing chemicals from solid matrices, including plant tissues and employ
tissue gn'nders or homogenizers to disrupt the sample and increase contact with the
solvent (Anderson et al. 1997). A disadvantage of sonication and homogenization,
however, is the increase in temperature from the sonication probe that can lead to
chemical losses from volatilization and sample degradation (Anderson et al. 1997). Thus,
it is extremely important to establish the percent recovery by using spiked controls or
internal standards. Solvent extraction of plant tissues with an organic solvent has also
been carried out in a separatory funnel of a soxhlet apparatus (Anderson et al. 1997).

With advancements in extraction techniques, the use of more advanced extraction
techniques, such as pressurized liquid extraction (PLE) and microwave-assisted

extraction (MAE) are increasingly being utilized in extraction of triclocarban and

20

triclosan. A review of methods used to extract and clean-up triclocarban and/or triclosan

from solid matrix is given in the appendix A.1 (Table Al).

21

Chapter 3: Materials and Methods

Research consisted of two sections: soil column experiments and hydroponic
experiments. In soil column experiment, four pumpkin, four zucchini, three switch grass
and three controls (no plant) were grown in biosolids applied soil in columns to meet
objective I and II. To meet objective III, five pumpkin, five zucchini and five controls
were grown hydroponically in nutrient solution spiked with triclocarban and triclosan.
The results from soil column experiment were used to characterize risk mentioned in

objective IV.

3.1 Soil Column Experiment
In soil column experiments, plants were grown in soil columns inside laboratory.
Leachate samples were analyzed for 22 weeks for concentration of triclocarban and

triclosan and soil and plant samples were analyzed at the end of the experiment.

3.1.1 Soil, Seeds and Biosolids

The crops used in this study were pumpkin (C ucurbita pepo Howden cultivar),
zucchini (Cucurbita pepo cultivar Gold Rush) and switch grass (Panicum variegatum).
Pumpkin and zucchini seeds were obtained from Johny Seeds, Winslow, Maine, and
switch grass seedlings were obtained from the Department of Crop and Soil Sciences,
Michigan State University. Soil used in this study was collected from Michigan State
University to represent locally available soil. The field had not been previously amended
with biosolids. The texture of soil was sandy clay loam (sand 50.8 %, silt 28.4 % and

clay 20.8 %). The collected soil was screened through a 2 mm sieve prior to use.

22

Biosolids were collected from Delhi Charter Township wastewater treatment plant in
Michigan in February 2009. The biosolids was analyzed for triclocarban and triclosan
prior to use by previously developed methods using liquid chromatography tandem mass
spectrometer (LC-MS-MS) (Cha and Cupples 2009). Triclocarban and triclosan were
present at concentrations of 8.18 :1: 0.56 mg/kg and 0.18 d: 0.01 mg/kg dry mass of

biosolids, respectively.

3.1.2 Chemicals
A list of chemicals used in the experiment, along with the manufacturer and the

purity, are given in Table 3.1.

Table 3.1 Chemicals Used in the Emriment

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Chemicals Company Purity
Triclocarban [CAS 101-20-2] Tokyo Chemical Industry Co. Ltd. >98 %
Triclosan [ CAS 3380-34-5] Calbiochem >98%
Ammonium acetate Sigma Aldrich >99.7 %
Acetic acid Columbus Chemical Industry Inc. >99.7%
Acetone J .T. Baker >99.7%
Methanol J .T.Baker >99.8%
Methanol EMD Chemical Inc. >99.99%
Water (MS solvents, for nutrient
preparation, standards Millipore system 18.2 M!)
reparation)
Hoagland basal salt Sigma Aldrich

 

Combined stock solution for both chemicals was prepared using methanol and
stored at 40C. Working standard solutions were prepared for every batch analysis by

diluting stock solution.

23

3.1.3 Experimental Columns.

Plants were grown in experimental columns with diameter of 14.7 cm and length
of 30 cm. The column size was selected to give enough depth and width to the pumpkin
roots while being not too large. Plants were grown in the selected columns to test grth
before deciding on the size of the columns. The experimental columns featured leachate
collection at the bottom through a tightly held fimnel (Figure 3.1). The bottom 7.6 cm of
the soil column was filled with soil without mixing biosolids. To replicate actual field
practice, the next 15.2 cm of soil was thoroughly mixed with biosolids at the application
rate of 0.73 dry Mg per 1000 m2 (3.25 dry tons per acre) (Cha and Cupples 2009). A
depth of 15.2 cm is equivalent to plough depth. Additional triclocarban and triclosan
were not added. Solid content of biosolids was 4.8 % (dry basis) and 200 g of biosolids
were applied before seed sowing, with an additional 60 g of biosolids applied after 8
weeks of seed sowing to simulate a second field application. The second application of
biosolids was done to study migration behavior of triclocarban and triclosan and once

plants were established to provide nutrients to plants.

24

 

 

 

 

Figure 3.1 Experimental Setup Showing Six Columns and Amber Bottles to Collect Leachate Water

Experimental design included quadruple columns for pumpkin, zucchini, and
switch grass and triplicate for no plant controls. The quadruple columns were selected to
guarantee three replicates if any of the four replicates were to die. One switch grass plant
died during the experimental period. Seeds were sown except for switch grass, for which
plants were transplanted directly fi'om the greenhouse, Department of Soil and Crop
Sciences, MSU. Soil was kept moist during plant establishment. Plants were maintained
at a constant temperature (23 :t 2 0 C) under plant growth lights using a light regime of 16
h light: 8 h dark (Figure 3.2). The temperature and light duration were selected based on
the average temperature and light duration during the months in which pumpkin are

grown around East Lansing.

25

 

Figure 3.2 Plants Growing in Columns under Constant Regime Lights

3.1.4 Sampling

For leachate sampling, columns were flooded with equal volumes of water so that _
a minimum of 100 mL of water leached from each column every two weeks. Water
samples were collected in amber bottles under each soil column. Samples were stored
immediately at 4°C until sample preparation.

Plants were harvested at the end of 22 weeks when plants started to senescence.
Plant tissues were separated into roots, leaves, and stems for pumpkin and zucchini and
into shoots and roots for switch grass. Plant tissues were rinsed carefully with deionized
water to remove soil and dust particles, air-dried, weighed and stored in amber bottles in
refrigerator at 4°C until sample extraction. Leaves and stems samples of each plant were
stored in an amber bottle without sub-sampling. Before preparation of samples for

extraction, leaves and stems were sampled to triplicate sub-samples each.

26

Soil samples representative of different depths (0-15.2 cm) of soil around the root
zone were collected from each column. Plant residues were removed and soil was
homogenized by mixing and stored at 4°C until sample preparation. Soil from each
column was stored in an amber bottle. Before extraction, soil from each column was

sampled to triplicate sub-samples.

3.1.5 Sample Preparation

Aqueous samples were prepared as published previously (Halden and Paull 2005,
Halden and Paull 2004). The procedure began with filtering the samples through
Whatrnan qualitative glass filter paper (7.0 cm) without applying vacuum to remove soil
particles. Exactly 100 m1 of filtrate was 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 (Figure 3.3). Elutes were dried under a
gentle stream of nitrogen, reconstituted in 1 ml of 50:50 methanol acetone, filtered
through 13 mm syringe filter with 0.2 pm PTFE membrane to amber autosarnpler vials
and analyzed by liquid chromatography mass spectrometer in negative electrospray
ionization mode (LC/ESI(-)/MS). Stock solutions for analyzing water samples were
prepared by spiking known concentrations following similar procedures to water sample

preparation.

27

 

Figure 3.3 Solid Phase Extraction

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 (Figure 3.4). Triplicate subsamples of soil from each
column sample were extracted and a fourth subsample was weighed and dried for at least
24 h at 105°C and again weighed for moisture determination. A glass fiber filter was
inserted at the outlet of stainless steel extraction cell (11 ml) body, a thin layer of Ottawa
sand added from top followed by 5 g of thawed soil sample and a thin layer of Ottawa
sand, and sealed on both sides using fiits, rings and caps. Extraction on ASE (accelerated
solvent extractor) was carried out in acetone with conditions: oven temperature of 100°C,
extraction pressure of 1500 psi, static time of 5 min and flush volume of 100%. The
extracts were evaporated to dryness under nitrogen gas, reconstituted in 940 pl of 50%

methanol and 50% acetone mixture, spiked with 6 rig/ml triclosan and triclocarban in

28

50% methanol and 50% acetone, and filtered to auto-sampler vials through 0.2 pm filter
before analysis by LC-MS. For quality assurance, all analytical runs included a blank, a
control and three sub-samples from a column. The recovery from the methods was 94.03

:h 9.76 % for triclocarban and 83.39 :t 19.48 % for triclosan.

 

Figure 3.4 Accelerated Solvent Extractor Used in the Experiment

For plant samples, frozen plant tissues were oven dried at 105°C, grounded in
mortar and pestle and extracted using the same method as for soil described above. For
quality assurance, cutting of plant samples was done with metal blades rinsed with
methanol between uses to minimize cross contamination. All analytical runs included a
blank, a control and three sub-samples except for roots, where sufficient biomass for
replicate analysis was not available. A blank run, control run and a sample re—run was

performed once for each 15 samples to check the accuracy and precision of LC-MS.

29

3.1.6 LC-MS Analysis

A Shimadzu LC-MS 2010 EV was used to analyze samples for triclocarban and
triclosan (Figure 3.5). Samples were injected by auto-inj ector in the autosampler
controlled by Shimadzu lab solutions software (v 3). Triclocarban and triclosan were
separated using Allure biphenyl column (5 um, 150 X 2.1 mm) from Restek Cor. MS
(Mass Spectrometer) 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 with scan mode was used for method development and

identification whereas selected ion monitoring (SIM) mode was used for quantification.

 

Figure 3.5 LC- MS Used in the Experiment

Retention time (tR i 0.1 min), detection of characteristics molecular ions (de-
protonated molecular mass [M-H']): 313 of triclocarban and 287 for triclosan, and
detection of reference ions, m/z (molecular mass) 315 and 317 for triclocarban and m/z

289 and 291 for triclosan were used to identify the target molecules (Halden and Paull

30

2005). Reference ions were present due to naturally occurring 37Cl atoms. Mobile phases
were 5 mM ammonium acetate as phase A and methanol as phase B. The total flow rate
was 0.2 mL min]. A binary gradient 20 min method with LC flow time program of 75%
B for 0-2 minutes, increased linearly to 100% B in 13 min, held to 14 min, decreased
linearly to 75% B in 15 min and held up to 20 min for re-equilibration, was used.
Quantification was performed using external, linear calibration and a minimum of five
calibration levels (Figure 3.6) (Halden and Paull 2005). The regression coemcient (R2) of
the curves was greater than 99.5 %. The detection limits were 10-100 ng/L for water, and

100-1000 ng/kg for plants and soils.

7
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Quantitation View for Triclocarban

31

3.1.7 Statistical Analysis

All statistical analysis was performed in Sigma Stat (version 11.0). A two tailed t-
test was used for all comparison purposes. The reported values are in mean :1: standard
error of mean.

Being a biological system, variability was observed in the experimental data. An
effort was made to identify the sources of variability. Coefficients of variation (CVs)
were calculated to quantify sources of data variability. CVS were calculated for LC-MS
analysis, accelerated solvent extraction, experimental sample replicates and field
replicates. The values are reported as mean CV :t standard error of mean of CV. For
experimental sample replicates (single column or reactor), CV of sub-samples for each
column was calculated and averaged over a plant variety or control. For field replicates

(multiple replicates), CVs of each plant variety and control were calculated and averaged.

3.1.8 Risk Characterization

To evaluate the relevance of this study to human health, a risk characterization for
triclocarban was completed. Exposure assessment was done using the worst case scenario.
The concentration of leaf or stem was assumed equal to pumpkin and zucchini fruit
concentration. This is assumed to be the worst case scenario because (i) leaves had lower
concentrations of antimicrobials than that of stems in this study, and both leaves and fruit
get water from the stem, (ii) an exponential decrease in concentration of PCB was
observed in the stem of pumpkin as the distance from root increased (Aslund et a1. 2007),
and (iii) an average bioaccumulation factor of 0.283 was observed in zucchini fruit in

phytoextraction of p, p’- DDE compared to 0.87 for leaves, 5.40 for stems and 7.22 for

32

roots (White et al. 2003). Humans are also exposed to antimicrobials through contact
while using liquid soaps, bars and body wash, and through ingestion while drinking water.
Dietary, residential and non-occupational sources and their exposure were considered for
the worst case scenario. Accordingly, maximum triclocarban used in bar soap (5 %), in
liquid soaps (5 %) and in body wash (0.5 %) were used to calculate margin of exposure.
An absorption value of 0.39% (EPA 2002a), maximum daily fruit consumption of 200 g
pumpkin and/or zucchini fresh weight per person (Cook 2004), solid content of 10% in
pumpkin and/or zucchini, maximum daily drinking water of 3 L and maximum leaf/stem
concentration in this study to represent the fruit concentration was used to characterize
risk. A no-observable adverse effect level (N OAEL) of 25 mg/kg bw/d for triclocarban
and 30 mg/kg bw/d for triclosan was used to calculate margin of exposure (EPA 2002a).

Aggregate margin of exposure was calculated using following equation (EPA 2001).

l

 

MOE, =

 

 

1 1 1
+ 4. ........ +
MOEl MOE 2 MOE”
Where, MOET = Aggregate margin of exposure

MOELZMn = Margin of exposure for individual exposure route

33

3.2 Hydroponic Experiment

Plants were grown in hydroponic medium to expose them to higher
concentrations of antimicrobials and quantify maximum accumulation of antimicrobials.
Plants were grown for two months in nutrient solution spiked with triclocarban and
triclosan. The concentration of antimicrobial in nutrient solutions was monitored and
plant samples analyzed for presence of triclocarban and triclosan at the end of the

experiment.

3.2.1 Plants and Experimental setup

The plants selected for the hydroponic study were pumpkin (Cucurbita pepo Howden
cultivar) and zucchini (Cucurbita pepo cultivar Gold Rush) for the reasons mentioned in
section 1.2 and successful test grth in nutrient media. Switch grass was not selected
due to poor growth in preliminary hydroponic studies.

The experiment setup consisted of glass vases filled with glass marbles to support
plants (Figure 3.7). Vases had 10 cm top and bottom diameter, 19.5 cm height and 7.5 cm
diameter at the mid-height section. Vases were covered with aluminium foil to minimize
photodegradation of antimicrobials. There were total of fifteen vases, five each for

pumpkin and zucchini and controls without plants.

34

.......

 

Figure 3.7 Vases before Wrapping in Foil, After Adding Antimicrobial Solution and Transplantation
of Pumpkin and Zucchini Seedlings

3.2.2 Plants Culture in Nutrient Media

Pumpkin and zucchini seeds were germinated in antimicrobial-flee soil, grown for
a week and transplanted to vases filled with marbles (Figure 3.7). Triclocarban and
triclosan, at 1 11M concentration each, was prepared in 280 ml nutrient solution, added to
each vase and initial level of the solution was marked (Figure 3.7). The solubility of
triclocarban in nutrient solution necessitated a 1 11M concentration of triclocarban.
Triclosan was all spiked at 1 11M. Each week, reactors were supplemented with basal salt
nutrients, the composition of which is given in Table 3.2 (American Public Health
Association, American Water Works Association. and Water Pollution Control
Federation. 1998). Nutrients were added so that the weekly addition was equivalent to the
weekly requirements for plant growth (Rouphael and Colla 2005). The basal salt nutrient

solution was selected instead of Hoagland’s nutrient solution because of extremely low

35

triclocarban solubility in Hoagland’s and to decrease microbial growth in the non-sterile

systems. Plants after some weeks of growth are Shown in Figure 3.8.

 

Figure 3.8 Experimental Vases after Some Weeks of Plants Growth

Table 3.2 Nutrient Solution Composition

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Nutrient Salt Stock Solution Element Concentration,
Solution Concentrationng. mg/L
25.5 N 42.0
NaNO3 Na 110.0
A NaHCO; 15.0 C 21.4
1.04 K 4.69
K2HPO4 P 1.86
CaC12.2H20 4.41 Ca 12.0
MgClz 5.7 Mg 29.0
B Fer 0.096 Fe 0.33
NazEDTA.2H20 0.3
MnClz 0.264 Mn 1.15
MgSO4.7H20 14.7 S 19.1
H3B03 0.186 B 325
C NazMoO4.2H20 7.26 Mo 28.8
ZnClz 3 .27 Zn 15 .7
CoClz 0.78 Co 3.54
CuClz 0.009 Cu 0.04

 

 

 

 

 

 

Reference: (American Public Health Association., American Water Works Association. and Water
Pollution Control Federation.) 1 ml of stock solution A, B and C was added to make 100 ml of
nutrient solution.

Evaporation from top surface was greater than nutrient solution to be added each
week. Thus, reagent water was added each week to bring the solution level in the vase to
the original level. Therefore, after adding nutrient solution and reagent water, reactor

solutions were allowed to diffuse for five hours. Five hours duration was based on a prior

36

food dye test. In food dye test, added dye diffused homogeneously within the solution in

less than an hour.

3.2.3 Sampling
Weekly sampling was done for each vase. Prior to sampling, a syringe needle (60 ml)
was placed at the center of the reactor and the nutrient solution was mixed. For sampling,
0.5 mL of the solution in the vase was pipetted out and stored in an amber vial. Plants
were harvested at the end of two months when pumpkin and zucchini showed slowed
growth and the beginning of senescence. Plants were washed with reagent water, air dried,
separated into shoots and roots, weighed and stored at 4°C until analysis. Low plant

biomass did not allow replications of samples during extraction.

3.2.4 Sample Preparation

Aqueous samples were prepared immediately after sampling on weekly basis.
Samples were diluted with 1.5 ml of methanol to increase detection of triclocarban. The
resulting 2 ml solution was filtered through 0.2 pm PTFE membrane filter to amber
autosampler vials and analyzed in LC-MS.

For plant samples, samples at 4°C were taken out of refrigerator, oven dried,
grounded using mortar and pestle, weighed, and extracted using a Dionex ASE 200
accelerated solvent extractor. The method used was same as described in section 3.1.5
excluding the addition of triclocarban and triclosan. Triclocarban and triclosan was

spiked at concentrations of 0.5 uM after extraction to make final volume to 1 mL.

37

3.2.5 LC- MS Analysis:
The method used for analysis in LC-MS was same as described in soil column LC-
MS analysis section. External linear calibration with at least five calibration levels from

0.1 uM to 1.2 1.1M was used for this experiment.

38

Chapter 4: Results and Discussions

4.1 Soil Column Experiment

- Both triclocarban and triclosan were detected in leachate from the experimental

soil columns. As shown in Figure 4.1, concentrations of antimicrobials increased initially

4.1.1 Leaching of Antimicrobials

for two weeks and then decreased. The lag in peak concentration was likely due to

sorption of antimicrobials to the bottom 7.6 cm of soil where biosolids were not applied.

Previous studies suggest strong sorption of antimicrobial to soil. For example, in

saturated soil systems, sorption was the primary removal mechanism for triclocarban

(Drewes et al. 2003, Essandoh et al. 2010). The maximum triclocarban and triclosan

concentration in leached water from pumpkin, zucchini, switch grass and control are

given in Table 4.1.

Table 4.1 Occurrence of Maximum Concentration of Triclocarban and Triclosan in Leachate Water.

Values are presented in mean :1: standard error.

 

 

 

 

 

 

 

 

 

 

Plants Triclocarban Time for Time for Triclosan
maximum maximum maximum maximum
concentration, concentration concentration concentration,
ng/ml for for triclosan, rig/ml

triclocarban, weeks
weeks

Pumpkin 2 0.21 :1: 0.16 2 0.53 d: 0.18

Zucchini 2 0.19 :I: 0.11 2 1.67 i 0.54

Switch 2 0.12 :t 0.04 2 0.46 :I: 0.28

Grass

Control 10 0.37 :I: 0 .37 2 0.71 :I: 0.45

 

Triclosan, despite having a lower initial concentration in the applied biosolids,

was collected at higher concentrations than triclocarban in leachate water during 0-22

weeks. This phenomenon was consistent with the greater hydrophobicity and lower

solubility of triclocarban than triclosan. For weeks 4-22, total triclocarban that leached

39

 

was greater than that of triclosan. Reduction in the relative leaching of triclosan was most
likely due to more rapid microbial degradation of triclosan as compared to triclocarban.
Degradation of triclosan in aerobic soils was more rapid with a half life of 18 days, than
was degradation of triclocarban, with a half life of 108 days (Ying et a1. 2007). Observed
concentrations of triclosan were 1.9-8.8 times higher than those in surface runoff
following land application of biosolids previously reported as 0.26 :t 0.04 11ng (Topp et
al. 2008).

Leachate concentrations of triclocarban were 40-123 times greater than observed
maximum concentrations of 0.003 i 0.002 11ng triclocarban following application of
dewatered municipal biosolid (Sabourin et al. 2009). Higher concentration was likely due
to collection of leachate water from relatively short columns indicating a limitation to
these types of studies. The second addition of biosolids, replicating field practices,
increased antimicrobial concentration immediately. For triclocarban, the observed second
peak was greater than first peak concentration although the increase was not statistically
significant (P= 0.6). In contrast, triclosan was observed at lower concentrations after the

second biosolids application (P=0.03).

40

 

1' ------9 Pumpkin
0‘7 ° Biosolid
addition --I-- Zucchini
0.6 n
- A- Switch Grass
0.5 a
1r —-x— Control

 

Triclocarban Concentration, [.13] ml

 

 

 

 

Weeks

 

 

 

Figure 4.1(a) Triclocarban Concentrations in Leached Water with Time. Points represent mean and
error bars represent standard error.

 

2.5 — . pumpkin
--I--Zucchini

2.0 ~ - t— Switch Grass
—X—Control

1.5

    

1.0 Biosolid addition

0.5

Triclosan Concentration, ug/ ml

 

 

0.0

 

 

 

Figure 4.1(b) Triclosan Concentrations in Leached Water with Time

41

The total antimicrobials leached from the soil columns over 22 weeks were not
significantly different than the control. However, plants were not established until week
four. The total antimicrobials leached from weeks 4-22 were significantly different than
that of the control, with p values of <0.01 for pumpkin, 0.01 for zucchini and 0.01 for
switch grass. Consequently, the results indicated that plants play a role in reducing

leaching of antimicrobials to water resources.

4.1.2 Soil Concentrations of Antimicrobials
Soil concentrations of antimicrobials at the end of 22 weeks are summarized in Table
4.2. Soil concentrations of both triclocarban and triclosan in pumpkin and zucchini
columns were less than that of control columns with p-values of 0.06-0.07. However,
antimicrobial concentrations in switch grass columns did not differ from that of control (p
= 0.88). The observed difference in final soil concentration of antimicrobials may have

resulted from difference in plant growth in the column.

Table 4.2: Soil Antimicrobial Concentrations and P-values for Comparison with Controls

 

 

 

 

 

Triclocarban, jig/g Triclosan, rig/g Sum, pg/g
Pumpkin 0.047i0.007 (P=0.06) 0.000:I:0.000 (P=0.07) 0.047i0.008 (P=0.07)
Zucchini 0.042i0.019 (P=0.07) 0.00:t0.00 (P=0.07) 0.042:I:0.019 (P=0.07)
Switch Grass 0.080i0.045 (P=0.52) 0.008i0. 002(P=0.20) 0.088i0.033 (P=0.88)
Control 0. 175:1:0.063 0.021d:0.009 0.1 96:20.070

 

 

 

 

 

 

Table 4.3 Shows root and Shoot biomass produced in soil column experiment along
with ratio of shoot to root. Shoot mass of switch grass was 7.6 and 11 times smaller than
that of pumpkin and zucchini, respectively. Root mass of switch grass was 11.3 and
9.1times greater than that of pumpkin and zucchini, respectively. Table 4.3 also shows

that growths of plants were variable. Results indicated pumpkin and zucchini do

42

influence soil concentrations of antimicrobials after land application of biosolids.

However, poor growth of switch grass did not yield conclusive result.

Table 4.3 Biomass Produced in Soil Column Experiment and Root to Shoot Ratio
Plants Soil Column Experiment

Root, g Shoot, & Shoot/ Root
Pumpkin 0.08 :1: 0.03 3.74 :t 0.93 1339.8 :1: 1309.5
Zucchini 0.10 :I: 0.03 5.39 :L- 0.28 69.49 :E 15.86
Switch
Grass

 

 

 

 

 

0.91 :I: 0.4 0.49 :l: 0.24 1.426 :t 1.19

 

 

 

 

 

 

Residual triclocarban concentration in soil was higher than residual triclosan
concentration. For example, the soil concentration of triclocarban was approximately 8.3
times greater than that of triclosan in control columns. This could be due to triclocarban
being i) more immobile of the two based on Koc values (Table 1.1), ii) more resistant to

microbial degradation, and iii) being the less soluble of the two chemicals.

4.1.3 Plant Concentrations of Antimicrobials

Both triclocarban and triclosan were observed in plant roots, stems and leaves.
Antimicrobials concentrations in plant tissues (Figure 4.2 and Figure 4.3) ranged from
1.1 ug/ g in leaves to 39.5 ug/g in roots. Antimicrobial concentrations in root tissues were
higher than those in leaves, stern, and soil; however, they were not significantly different
(p > 0.05). As previously observed for pumpkin and zucchini accumulation of PCBs
(Aslund et al. 2007), concentration generally decreased from roots to stems to leaves.
Though there were antimicrobial concentration differences, concentrations of
antimicrobials in stem, leaves and root were typically not statistically different (p values

from 007-098) (Table 4.4).

43

 

 

25.0

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Pumpkin

 

 

 

 

Zucchini

 

El Root
20.0
E Stem
\
it
a 15.0
3 Leaf
H 4
1!
our
1: ..
en
E
O 10.0
u
u
u
I-
5.0
0.0
Pumpkin Zucchini Switch Grass
Figure 4.2 Plant Concentrations of Triclocarban
60.0 a
Cl Root
50.0 — Stem
Leaf
40.0 r
E
\
DD
:1.
. 30.0 r
C
o
'5
E
‘E
3 20.0 -
C
o
u
U)
u
I- 10.0 a
0.0

 

Switch Grass

 

Figure 4.3 Plant Concentrations of Triclosan

44

 

 

Table 4.4 P-Values for Comparison of Antimicrobial Concentrations between Plant Parts
Plants Triclocarban Triclosan
Stem Leaf Stem Leaf
Pumpkin Root 0.93 0.929 0.181 0.066

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Stem - 0.981 - 0.025
Zucchini Root 0.196 0.24 0.095 0.082

Stem - 0.068 - 0.119
Switch Grass Root 0.266 0.674

 

A one way ANOVA analysis for comparison of different plant parts using Sigma Plot
indicated that soil concentration of antimicrobials were significantly different (p values
from <0.01 to 0.06 except for between soil and zucchini roots) from concentrations of
stems, leaves, and water for all plant species (Table 4.5). Triclocarban bioaccumulation
factors, the ratio of plant concentration to soil concentration, are given in Table 4.6.
These values are comparably higher than those for DDT bioaccumulation by Lunney et al.
(2004) for the same varieties of zucchini (Cucurbita pepo L. cv. Senator hybrid) and
pumpkin (Cucurbita pepo cv. Howden), which were 3.0 and 2.0, respectively. PBT
profiler modeling developed by EPA suggests that TCC and TCS are potentially
bioaccumulative (log BCF 3.074 for triclocarban and 2.565 for triclosan) (Ying et al.
2007)

Table 4.5 P-Values for Comparison of Soil Antimicrobial Concentrations and Plant Antimicrobial
Concentrations

 

 

 

 

 

 

 

 

 

 

Plants Plant Parts Triclocarban Triclosan
Root 0.068 0.041
Pumpkin Stem 0.037 0.003
Leaf 0.004 0.003
Root 0.13 0.061
Zucchini Stem 0.002 0.001
Leaf <0.001 <0.001
Switch Grass Root 0.014 0.011
Stem 0.027 0.024

 

 

 

 

 

45

Table 4.6 Bioaccumulation and Translocation Factors for Triclocarban and Triclosan

 

 

 

 

 

 

Plants Bioaccumulation Factor Translocation Factor
Triclocarban Triclosan Triclocarban Triclosan
Pumpkin 11.0 i 5.06 972 d: 398 0.81 :t 0.63 0.46 :l: 0.08
Zucchini 40.3 i 46.3 1822 i 260 1.91 :l: 1.83 0.35 i 0.21
Switch Grass 30.92 :1: 9.41 874 i 706 0.82 i 0.47 1.23 i 0.80

 

 

 

 

 

 

Translocation factors, the ratio of shoot concentration to the root concentration are
given in Table 4.6. Low translocation factor imply that the antimicrobials transport from
root to shoot was limited. Triclosan gets uptaken into plant roots easier than triclocarban,
but was only limitedly transported from roots to shoots. The translocation factors are
comparable to those for DDT, DDD, and DDE and PCBs reported in Aslund et al. (2007)
and Lunney et al. (2004) for the same plant varieties. None of the plants were
triclocarban or triclosan hyperaccumulators as the accumulated concentrations were not
greater than 106 ug/kg (dry weight) (McCutcheon and Schnoor 2003, ITRC 2001).

Mass balance for each column was assessed (Figure 4.4 and 4.5). Significant mass
fraction of triclocarban remained unaccounted for most of the columns indicating other
mechanisms of antimicrobial loss, such as microbial degradation, phytostimulation, or
phytodegradation. The largest portion of triclocarban remained in the soil suggesting that
triclocarban was sorbed to soil particles. Results are similar to previously published
studies. Essandoh et al. (2010) reported that sorption and biodegradation were two
primary mechanism of triclocarban removal in saturated aquifer system and attributed
5 6 % of total loss of triclocarban to biodegradation for the first nine days which
decreased to 7 % by 16 days (Essandoh et al. 2010). For zucchini, though root
concentrations were greater than shoot concentrations, total accumulation in leaves was

highest followed by total stem accumulation and root accumulation. In contrast, stems

46

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
resulting from less shoot production. Unaccounted mass for triclosan was relatively less
compared to triclocarban. This is likely due to the greater loss of soluble TCS in first four

weeks of studies.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

   

 

IUnaccounted DSoil ILeaves mStem BRoot IWater Leached
1 r-
7 7/
C
o 0.7 ' / /
(B i—
t 0.6 A
§ 0.5 ~
2
5 0.4 -
'2
g 0.3 ~
.2
,2 0.2 -
o.1 ~
0 . l l 1
Pumpkin Zucchini Switch Grass Control

 

 

 

Figure 4.4: Triclocarban Mass Balance Analysis

47

 

Unaccounted C] Soil I Leaves [11 Stem 8 Root Water Leached

1.0
0.9
0.8
0.7
0.6
0.5
0.4

0.3

Triclosan Mass Fraction

0.2

0.1

 

0.0

Pumpkin Zucchini Switch Grass Control

 

 

 

Figure 4.5: Triclosan Mass Balance Analysis

4.1.4 Risk of Land-applied Antimicrobials in Vegetated Systems
Concentrations of triclocarban and triclosan known to promote adverse impacts in
soil and aquatic ecosystems, and environmental occurrences in soil and water are
summarized in box plots in Figure 4.6 and 4.7.

In figure 4.6, leachate triclocarban concentrations of planted columns in this
experiment (box plots 5 and 6) were higher by about an order of magnitude than water
concentration of triclocarban at ambient environment (box plot 1). In contrast, soil
concentrations of triclocarban in the experimental columns (box plots 9 and 10) were

lower by about an order magnitude than that in environment (box plot 8).

48

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

3’

g 12

3

_l

a 10 ‘

C

E 8-

E i 1:!
c 4- Li?— : .v _.I_ ‘ ‘1 .

s we i

E 2 .J

“If

I:

m 0 ' ' ' ' ' ' ' ' ' fl
0

..l

01 2 3 4 5 6 7 8 91011

Figure 4.6 Box Plots of Triclocarban Concentrations Available in Different Literatures and Research
Data 1) Occurrence in Water, 2), 3) and 4) Threshold Toxicity Values for Microorganisms,
Invertebrates and Plants, 5) Experimental Water Concentrations in Planted Columns, 6)
Experimental Water Concentrations in Control, 7) and 8) Occurrence in Biosolids and Soils, 9)
Experimental Soil Concentration in Planted Columns and 10) Experimental Soil Concentrations in
controls. Concentrations are in Logarithmic Scale in parts per trillion, or ng/L for Liquid and ng/kg
for Solid Matrices. References are given in the Table in Appendix A.l and A.2.

For triclosan, as shown in Figure 4.7, triclosan concentrations in planted columns
leachate (box plots 5 and 6) had higher concentration range than environmental water
concentrations (box plot 1). In contrast, concentration of triclosan in soil in the
experimental columns (box plots 10 and 11) were lower than that in environment (box

plot 8).

49

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

310
<-

m

c

'5 3"

:1

U7

:6" . Tm

.2 m

t: 2-

8 +

C O

3

2 0‘ .

.9

ti I T I I I I I I I I I I
3 012 3 4 5 6 7 8 9101112

Figure 4.7 Box Plot of Triclosan Concentrations Available in Different Literatures and Research
Data 1) Occurrence in Water, 2), 3) and 4) Threshold Toxicity Values for Microorganisms,
Invertebrates and Plants, 5) Experimental Water Concentrations in Planted Columns, 6)
Experimental Water Concentrations in Control, 7) and 8) Occurrence in Biosolids and Soils, 9)
Threshold Toxicity Values for Soil Health, 10) Experimental Soil Concentration in Planted Columns
and 11) Experimental Soil Concentrations in controls. Concentrations are in Logarithmic Scale in
parts per trillion, or ng/L for Liquid and ng/kg for Solid Matrices. The limits of detection were 10-
100 ng/L for water, 100-1000 ng/kg for plants. References are given in a Table in Appendix A.l and
A.2.

Concentration of triclosan and triclocarban detected in all matrices (plants, soil
and water) in this experiment ranged from 0.00087 to 5.34 ug/ml triclocarban and
0.00072 to 6.17 ug/ml triclosan in water, 0.5 to 28.92 ug/g triclocarban and 6.73 to 81.21
ug/g triclosan in root, 0.8 to 6.6 ug/g triclocarban and 1.21 to 21.70 ug/g triclosan in
shoot. The concentrations in soil and water are within the range of environmental

concentrations reported in different literatures (Table 2). Concentration in plant parts

50

could not be directly compared to other studies due to lack of relevant studies.
Comparing with the values in Figure 4.6 and 4.7, there is high risk for both soil and
aquatic ecosystem health including soil respiration, enzymatic activity and
microorganisms from concentrations observed in this study. Therefore, land application
of biosolids is likely to adversely impact the ecosystem functioning and health of both
land and water.

Uptake of antimicrobials by pumpkin and zucchini represents the first
documentation of exposure of humans to antimicrobial through ingestion of vegetables.
Margin of exposure (MOE), which is the ratio of no-observable adverse effect level to
exposure dose, are given in Table 4.7. MOE values for pumpkin and zucchini were
comparable to that of using bar or liquid soap containing triclocarban and were
substantially less than that of drinking water contaminated with antimicrobials. In other
words, the health risk from eating pumpkin and zucchini grown on biosolid-applied fields
is likely greater than that from drinking water and is similar to that of using antimicrobial

products.

Table 4.7 Risk Characterization of Triclocarban and Triclosan

 

 

 

 

 

 

 

 

 

 

 

 

Exposure Resulting dose* NOAEL’

Chemical Route mg/kg bw / d mg/kg bw/d :1ng MOE
Dermal-bar soap 0.10 0.005 25 5000
Dermal-liquid soap 0.11 0.006 25 4167

TCC Dermal body wash 0.07 0.0004 25 62,500
Oral-drinking water 1.38x 10$ 25 18,1 l5,942
Pumpkin/zucchini“ 200 g (max) 0.011498 25 2175
Aggregate 1092
Oral-drinking water 56 ng/L 2413-06 30 12500000
Pumpkin,/zucchini 0.010896 30 2754

TCS .

Aggregate (excluding

. . . 30 4700
pumpkin and zucchini)
Aggfigate 1736

 

 

 

 

 

 

 

References: (EPA 2002a, EPA 2008a, Cook 2004)
Resulting dose = estimated 0.39% dermal absorption of TCC (EPA 2002a)
MOE = margin of exposure; NOAEL= No-observable adverse effect level

51

 

4.2 Hydroponic Experiment

4.2.1 Aqueous Concentrations of Antimicrobials

Triclocarban concentration normalized to the initial concentration with time is
shown in Figure 4.8. Triclocarban concentration rapidly decreased with time for up to
two weeks and then decreased gradually for all vases. The concentration of triclocarban
at the end of experiment was 0.73 i 0.01 1.1M, 0.55 i 0.01 uM and 0.61 i 0.03 uM for
pumpkin, zucchini and control, respectively. The decrease in triclocarban concentration
in vases was probably due to photodegradation or chemical degradation. Sorption to
marbles was unlikely as sorption of triclocarban to glass has not been reported.

Previous studies have documented phytodegradation of triclocarban. Triclocarban
was removed from surface waters with direct and sensitized phytolysis with measured
half life of 24 hours in sunlight (Trouts 2008). Additionally, in planted vases, the
concentrations could have decreased due to phytodegradation, rhizodegradation and/or
phytoaccumulation. Concentrations in pumpkin vases did not decrease as much as
controls, possibly due to shading of the reactors by well-developed foliage resulting in
decreased photodegradation. Concentration in zucchini reactors were less than that in the
control. Group comparison between pumpkin, zucchini and control concentrations using
Sigma Plot indicated that both pumpkin and zucchini were not statistically different from
control (P values of 0.52-0.54). However, pumpkin plant significantly differed from

zucchini (P value of 0.013).

52

 

 

1.2

 

 

 

 

 

 

 

 

 

 

Weeks

~4--Pumpkin
-- -Zucchini
: 1 +Control
‘2
a: 0.8 ~
0
c
o
0
I: 0.6 ~
cu
in
.2
’5
I— 0.4 -
'U
c»
g
a:
E 0.2 ~
0
z
0 L
0 2 4 6 8 10
Weeks
Figure 4.8 Triclocarban Concentrations with Time
1.2 r
--O--Pumpkin
c 1 -I-Zucchini
é , +control
.b
5
o 0.8 -
c
o
0
5 0.6 -
.a
I-
cc
0
8
': 0.4 ~
I-
'u
31
E 0.2 -
2
o l l l l J
0 2 4 6 8 10

 

Figure 4.9 Triclosan Concentrations with Time

53

 

The plot of concentration of triclosan normalized to the initial concentration with
time is shown in Figure 4.9. Concentration of triclosan gradually decreased with time for
all vases. The concentration of triclosan at the end of experiment was 0.34 i 0.01 uM,
0.39 :1: 0.01 uM and 0.36 d: 0.07 uM for pumpkin, zucchini and control (respectively).
The decrease in triclosan concentration in all vases was probably due to photodegradation
or chemical degradation. Sorption to marbles was unlikely as no other studies have
reported sorption of triclosan to glass. Previous literature on photodegradation of
triclosan reported that triclosan photodegraded in fresh water with half life of 8 days
(Aranami and Readman 2007). In planted vases, plants could have accumulated, or
phytodegraded triclosan or have stimulated rhizodegradation. The sharp decrease in
concentration of triclosan towards the end of experiment is interesting. A longer
experimental period could have made it clear about whether concentration of triclosan
would continue to decrease, remain the same or was an error. Group comparison between
pumpkin, zucchini and control concentrations using Sigma Plot indicated that both
pumpkin and zucchini were not statistically different from control (P values of 0.94 and

0.309).

4.2.2 Plant Concentrations of Antimicrobials

Though triclocarban concentrations in nutrient solutions were not significantly
lower than that in control, concentration of triclocarban was detected in the plant tissues,
both roots and shoots. The triclocarban concentrations in log scale in ng/kg for both
pumpkin and zucchini shoots and roots are shown in Figure 4.10. Triclocarban

concentration ranged from 133 - 530 ug/g in roots and from 0.08 - 0.60 ug/g in shoots.

54

As was observed in soil column experiment with triclocarban and triclosan, root
concentration of triclocarban was higher than that of shoot. While concentration of
triclocarban in pumpkin root was lower than that in zucchini roots, pumpkin shoots had
higher triclocarban concentration than zucchini shoot. Since roots were well developed in
hydroponic experiment, roots played a major role in total accumulation. Triclocarban
concentration in nutrient solution was higher at the end of the experiment in pumpkin
than in zucchini. Reduced concentration in solution of zucchini vases could have been
due to more root concentration and accumulation. A statistical comparison of the
concentration of triclocarban between pumpkin and zucchini roots and shoots showed
that the concentrations of pumpkin are statistically different than concentrations of
zucchini (p < 0.001). But, root did not differ statistically from shoot for both pumpkin

and zucchini (p = 0.871 and 0.997).

 

 

 

 

 

 

 

 

 

 

   

 

 

14 —

12 r T T URoot
3 IShoot
é
2’ 1o »
c“
o
3
S 8 *
C
o
0
5 a -
0
U
U .L
l:- 4
a
2

2 .—

o 1

Pumpkin Zucchini

 

 

 

Figure 4.10 Triclocarban Concentrations in Pumpkin Roots and Shoots, log“. Scale for
Concentration

55

Both roots and shoots contained detectable concentrations of triclosan (Figure

4.11). Triclosan concentration ranged from 12.07 - 130.61 ug/g in roots and from 0.56 -

25.43 ug/ g in shoots. Pumpkin had lower root and shoot concentration than zucchini. As

was observed in the soil column experiment with triclocarban and triclosan,

concentrations of triclosan in roots were higher than that in the shoots. Since roots were

well developed in hydroponic experiment, roots played major role in distribution of

triclosan (Table 4.8). This is consistent with the fact that triclosan concentration in

nutrient solution in pumpkin was higher than in zucchini.

Table 4.8 shows the comparison of shoot to root ratio for both soil column

experiment and hydroponic experiment. Shoot to root ratio of soil column experiments is

higher than that of hydroponic experiments by a factor of 224 for pumpkin and 12 for

zucchini. Pumpkin shoot in soil column experiment has most grth in all. A high

variability in biomass growth between columns is seen in both soil column experiment

and hydroponic experiment.

Table 4.8 Comparison of Biomass Produced and Root to Shoot Ratio in Hydroponic Experiment with
‘ Soil Column Experiment

 

 

 

 

 

 

 

 

 

 

Plants Hydroponics Soil Column Ex oeriment
Root, g Shoot, g Shoot/Root Root, g Shoot, Shoot/Root
Pumpkin 0.07 i 0.02 0.43 :t 0.10 5.98 :t 0.47 0.08 :1: 0.03 3.74 :1: 0.93 1339.8 a 1309.5
Zucchini 0.07 d: 0.01 0.39 d: 0.06 5.72 i 0.82 0.10 i 0.03 5.39 i 0.28 69.49 :1: 15.86
Switch Grass 0.91 i 0.4 0.49 :t 0.24 1.426 :1: 1.19

 

 

 

56

 

 

1:1 Root
IShoot

 

70’

 

 

 

TCS Concentration, uglml
S

20*

 

 

 

 

 

 

o i .

Pumpkin Zucchini

 

 

 

 

 

Figure 4.11 Triclosan Concentrations in Shoots and Roots of Pumpkin and Zucchini

A mass balance analysis of triclocarban in both pumpkin and zucchini is shown in
Figure 4.12. A large portion of triclocarban remained in the nutrient solution and sorbed
to the roots. Roots accumulated more triclocarban than shoots in both pumpkin and
zucchini in contrast to soil column experiments where root had lower accumulation. This
is likely due to the well developed root system in hydroponics. Moreover, difficulty in
retrieving all roots in soil column also added to the discrepancy. A significant mass
remained was unaccounted for in both pumpkin and zucchini indicating another
mechanism of loss. The unaccounted amount is less than expected based on decrease in
the concentration of triclocarban in control vases. This might have been possibly due to
preferential mechanism of triclocarban to accumulate to plants over photodegradation or
chemical degradation and prevention of photodegradation from shading of reactors by

plant leaves.

57

 

1.0

  
 
 
 
 
 
  

Z Unaccounted
0.9
Water

0.8
I Shoot

0.7
B Root
0.6
0.5

0.4

0.3

Triclocarban Mass Fraction

0.2

0.1

0.0

 

 

Pumpkin Zucchini Control

 

Figure 4.12 Mass Balance Analysis of Triclocarban in Hydroponic Experiment

Triclosan mass analysis was completed at the end of the experiment (fig 4.13). As
opposed to triclocarban, the largest fraction was unaccounted in both pumpkin and
zucchini. As was found in triclocarban mass analysis, triclosan also remained in water in
substantial amount. Root accumulation was higher than shoot accumulation for both

pumpkin and zucchini, though not as substantially as for triclocarban.

58

 

1.0
D Unaccounted

 
   
  
  

0.9 Water
1]] Shoot

B Root

0.8

0.7

0.6

0.5

0.4

Triclosan Mass Fraction

0.3

0.2

0.1

0.0

Pumpkin Zucchini Control

 

 

 

Figure 4.13 Mass Balance Analysis of Triclosan

4.2.3 Comparison of Soil Column and Hydroponics Antimicrobial
Concentrations

Box plots in Figure 4.14 shows the log concentrations of all matrices in both soil
column and hydroponic experiments. Box plot 3, triclocarban concentration of
hydroponic solution at the end of experiment, is clearly higher than concentrations of
triclocarban in water and soil in the column experiments (box plots 1 and 2). This was
expected since the hydroponic solution was spiked with higher concentrations of
triclocarban. The case of triclosan, shown in box plots 6, 7 and 9, was similar. Plant

59

triclocarban. The case of triclosan, shown in box plots 6, 7 and 9, was similar. Plant
triclocarban concentration in hydroponic experiment (box plot 5) is on average is higher
than that for soil column experiment (box plot 4). The same phenomenon is observed for
triclosan (box plots 9 and 10). Concentration of triclocarban and triclosan in water in
hydroponics was statistically different from that in soil and water in soil column studies
(Table 4.9). However, plant concentrations of triclocarban and triclosan in soil column
experiment were not always statistically different than that in hydroponics experiment
(Table 4.9). When comparing between the soil and water concentrations with plant
concentration of antimicrobials, plants in the soil column experiment were clearly more
efficient in extracting antimicrobials. Translocation factors and translocation factors for

both experiments are summarized in below Table 4.10.

Table 4.9 P-Values for Comparison of Iiydromnic Experiment with Soil Column Experiment

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Soil Column Experiment
0 ,_, Soil Plant Water
g g Triclocarban Water <00] <0.01
3: Plant 0.233
“a :9} Triclosan Water <0.001 <0.001
I W Plant <0.001
10000 3
g mo 5
E *- T , g
or O o i
3 ‘° E T. ’ Q 3
: g :
s 3 '
0.1 : .!. j. T g
1; _ g
0.001 > r r I I f ' ' ' ' '— :
0 1 2 3 4 5 6 7 8 0 10 11

Figure 4.14 Comparisons of Soil Experiment and Hydroponic Experiment Data. Box plot
Concentrations 1), 2) Water and Soil TCC in Soil Column Experiment, 3) Hydroponic Water TCC,
4), 5) Plant TCC in Soil Column and Hydroponics, 6), 7), Water and Soil TCS in Soil Column
Experiment, 8) Hydroponic Water TCS, 9), 10) Plant TCS in Soil Column and Hydroponics.

60

Table 4.10 Comparison of Translocation Factors and Bioaccumulation Factors for Two Experiments

 

 

 

 

 

 

 

 

 

 

 

. . . . Translocation Bioaccumulation factor
Antimicrobial Experiment Factor Root Shoot
TCC Soil Column 0.52 i 0.33 45.44 :1: 38.02 15.87 :1: 8.27
Hydroponics 0.0013 d: 0.0010 1.78 i 0.54 0.0014 :t 0.0010
TCS Soil Column 0.35 :1: 0.28 1242 i 561 372 i 208
Hydroponics 0.082 i 0.079 0.64 i 0.38 0.024 :1: 0.02

 

 

Roots extracted triclocarban and triclosan more efficiently from soil columns
than from nutrient solutions in the hydroponic study by factors of 25.5 and 1941
respectively. Translocation of antimicrobials from root to shoot is more in soil column
than in hydroponic studies by a factor of 4.3 for triclosan and 400 for triclocarban. This
indicates that triclocarban is more difficult to uptake to plant roots but easier to
translocate to aerial plant parts than triclosan. More liphophilicity of triclocarban might
have made it easier to translocate up to shoots. The results above indicate that
hydroponics can be a tool to screen if plants are phytoaccumulators or not. However,
hydroponics might not represent the actual case for phytoaccumulation of antimicrobials
triclosan and triclocarban by pumpkin and zucchini. Though plants in soil column
experiment were efficient in removing antimicrobials from soil rather than soil, screening
plants is easier in hydroponic experiment owing to its ease of experimentation with
regards to cost and time.

Table 4.11 contains coefficients of variation for both hydroponic and soil column
experiment. Coefficients of variation were calculated as described in Section 3.1.7. As
expected, LC-MS contributed the least amount of variability. For the soil column
experiment, variability of LC-MS data for all matrices was comparable. However, for
hydroponic samples, variability in observed aqueous concentrations was greater than that

for plant samples most likely due to directly analyzing the aqueous samples as opposed to

61

extracting solid phase. The lowest variability and small coefficient of variation in LC-MS
data suggested that LC-MS produced good quality data for analysis of triclocarban and
triclosan. As LC-MS analysis is cheaper than LC-MS-MS analysis, LC-MS analysis is an
economical option for analyzing triclocarban and triclosan while producing good data
quality.

Table 4.11 Coefficients of Variation in Different Units in the Experiments

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Soil Column Experiment Hydroponics
Unit Experiment
Soil Stem Leaves Roots Water Shoot Root Water
001$ 0.02 $ 0.19 d:
LC-MS 0.00 0.03 $ 0.05 0.00 0.01 $ 0.01 003
ASE 0.10 $ 0.00 - 0.10 $ 0.00 -
5 Single 0.60 i 0.38 :t 095$ _ _ _ _ _
fi Column 0.00 0.12 0.25
0
if, Multiple 0.59 $ 0.53 :t 1.26 085$ 061$ 0.64 $ 0:8 0.011.
; Columns 0.12 0.12 $0.44 0.17 0.12 0.12 016 0.00
0.00 $ 0.00 $ 0.15 $
LC-MS 0.02 0.01 $0.02 000 0.00 $0.01 0.00
ASE 0.22 $0.01 - 0.22 $0.01 -
Single 0.12 $ 0.17 :1: 0.28 _ - _ _ _
g Reactor 0.00 0.12 $0.06
:8 Multiple 144$ 0.47 $ 0.92 0.71 $ 0.68 $ 1.32 $ 0:2 0.08 $
5 Reactors 0.53 0.08 $0.03 0.10 0.07 0.21 028 0.06

 

 

 

 

 

 

 

 

 

 

 

ASE also contributed to variability of data. Variability in ASE data included
variability due to LC-MS as ASE extracted samples were analyzed in LC-MS for
quantification of triclocarban and triclosan. Data variability in triclocarban concentration
was less than that for triclosan concentration.

Single column or reactor variability stands for variability seen across the sub-
samples of the same column or reactor. Single reactor variability for hydroponic
experiment could not be calculated as the sample mass was not sufficient enough to sub-

sample. Variability within a single column was due to variability in analytical methods,

62

i.e. LC-MS and ASE, and heterogeneity observed within the column. For example,
previous literature has shown exponential decrease in concentration of PCBs in shoot as
the distance from root increase (Aslund et al. 2007). Since stems were sampled as a unit,
the variability would have been increased as compared to sampling stems on the basis of
the length from root. The single column or reactor variability was higher than ASE
variability by a factor of 4 to 9 for triclocarban and a factor of 0.5 to 1.3 for triclosan.
Unsurprisingly, coefficient of variation is largest for multiple columns for both
hydroponic and soil column experiments. Variability in plant varieties or control is as
high as 6.4 times the variability due to ASE. In addition to variability sources mentioned
above, this also accounts for difference in variation among the reactors or columns.
Variation of mass of plants in different columns or reactors are given in Table 4.8.
Moreover, multiple column variability could also be by local micro-environment. The
greatest source of variation in the experiments is due to heterogeneity within a column
and variability among multiple reactors or columns. Soil and shoot had the greatest
variability. LC-MS contributed the greatest variability in aqueous samples of hydroponic
experiment. However, variability in multiple reactors in hydroponic experiment is less
- than that in soil column experiment for both aqueous and plant samples. For example,
coefficient of variation of triclocarban concentration in pumpkin stem in soil column
experiment is four times greater than that in hydroponic experiment. Thus, hydroponic
experiment can be more accurate option to screen plants for phytoremediation studies,

particularly if a more precise LC-MS method is developed.

63

Chapter 5 Conclusions and Future Research

Research indicated that plants influence the migration of antimicrobials from
biosolids to water resources through a yet unidentified mechanism. Though pumpkin,
zucchini and switch grass up took antimicrobials, they did not accumulate high enough
concentrations to be considered as hyperaccumulators of triclocarban and triclosan. Roots
had higher concentration than shoot, but less total mass accumulation of antimicrobials
than shoots. Pumpkin and zucchini significantly reduced soil concentrations of
antimicrobials. There was no or very minimal risk of antimicrobials to human due to
application of biosolids to pumpkin and/or zucchini field. However, there is low to high
risk to aquatic plants, microorganisms and invertebrates and soil ecosystem health. More
research is needed to identify and quantify the exact mechanism that dictates the fate of
antimicrobials in biosolid- applied vegetated systems. Field scale research is needed to
fully represent field conditions. Additionally, study of hyperaccumulation in fruits and
those plant parts which human consume is recommended along with risk of eating those
fruits and vegetables.

The hydroponic experiment indicated that while there was accumulation of
triclocarban and triclosan, the planted reactors did not differ significantly from the
control. Root concentrations were higher than shoot concentrations for both pumpkin and
zucchini in both the hydroponic and the soil column experiments. However, root mass
accumulation of antimicrobials was higher than shoot accumulation for both pumpkin
' and zucchini, in contrast, to soil column study. Variability in both the experiments was
mainly due to multiple reactors rather than analytical error. Large number of columns

with longer duration could make the results better by providing more data points per

64

sampling event and more number of sampling events. While hydroponic experiments
could be a good option for selecting the aquatic species, it might not be the best
representative for the terrestrial plants as was observed in this experiment. Development
of very good root system in hydroponics and a relatively undeveloped shoot system may
not be representative of terrestrial plants. Further research on screening more species,
terrestrial in soil column and aqueous in water column is recommended. Also, triclosan

could be spiked higher due to higher solubility than triclocarban if conducted separately.

65

 

 

 

 

 

 

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