UPTAKE AND ACCUMULATION OF TRICLOSAN AND TRICLOCARBAN BY FOOD
CROPS IN A HYDROPONIC SYSTEM
By
Shannon Lea Henderson

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE
Biosystems Engineering
2013

ABSTRACT
UPTAKE AND ACCUMULATION OF TRICLOSAN AND TRICLOCARBAN BY FOOD
CROPS IN A HYDROPONIC SYSTEM
By
Shannon Lea Henderson
Triclosan (TCS) and triclocarban (TCC) are two antimicrobial chemicals that are used in many
personal care products. In this study, triclosan and triclocarban were added to hydroponic
nutrient media that was used to grow different food crops. The initial triclocarban and triclosan
media concentration was 500 ppb and concentrates were measured weekly. The concentration of
antimicrobials in the media decreased over the 4 week testing period. After the end of the 4
weeks, the plant tissue was also tested for triclocarban and triclosan concentrations. Most of the
antimicrobials in each plant was found in the root tissue. From the root concentration factor
(RCF) and concentrations in the plant tissues, onion is best at taking up triclocarban, while chili
appears to take up the most triclosan. Chili and cucumber seem to have high potential to take up
both chemicals. Celery, broccoli, and asparagus appear to have the lowest concentrations and
RCFs of both chemicals. When the exposure of the crops was calculated using national produce
consumption data, the average exposure to TCC and TCS from eating contaminated crops was
still much less than the NOAEL.

ACKNOWLEDGEMENTS
Funding for this project was made possible by a grant from the National Institute of Food and
Agriculture (NIFA) within the United States Department of Agriculture (USDA), Award number
2011-67019-20026. Thank you to Dr. Thelen in the Crop and Soil Science Department for
allowing us to use his accelerated solvent extractor. Thank you to Niroj for answering all of our
questions about his previous work on the topic. And thank you to all the students in Dr.
Reinhold’s lab who helped us with watering the plants, making nutrient media, or getting things
set up.

iii

TABLE OF CONTENTS
LIST OF TABLES .......................................................................................................................... vi
LIST OF FIGURES........................................................................................................................ vii
CHAPTER 1: INTRODUCTION .............................................................................................................1
CHAPTER 2: LITERATURE REVIEW ..................................................................................................3
2.1

Description of chemicals .................................................................................................. 3
2.1.1 Type of chemical........................................................................................................ 3
2.1.2 Chemical Properties ................................................................................................... 4
2.2.3 Antimicrobials in Biosolids ....................................................................................... 5
2.2 Physiochemical Processes of TCC and TCS ........................................................................ 7
2.2.1 Sorption to Soils......................................................................................................... 7
2.2.2 Leaching ..................................................................................................................... 8
2.2.3 Photodegradation ....................................................................................................... 8
2.2.4 Volatilization.............................................................................................................. 9
2.2.5 Reactions with other chemicals ................................................................................. 9
2.3 Biological Processes of TCC and TCS .............................................................................. 10
2.3.1 Microbial degradation .............................................................................................. 10
2.3.2 Bioaccumulation ...................................................................................................... 10
2.4 Possible risks and effects of TCC and TCS ....................................................................... 13
2.4.1 Environmental effects and harm to wildlife............................................................. 13
2.4.2 Possible Health Hazards .......................................................................................... 14
2.5 Root Physiology and the Rhizosphere ............................................................................... 14
CHAPTER 3: MATERIALS AND METHODS ................................................................................... 16
3.1 Materials ............................................................................................................................. 16
3.2 Experimental Setup ............................................................................................................ 16
3.3 TCC/TCS degradation in the media ................................................................................... 18
3.4 Sample extraction and analysis .......................................................................................... 19
3.5 Media Statistical Analysis .................................................................................................. 22
CHAPTER 4: RESULTS AND DISCUSSION ..................................................................................... 23
4.1 Media TCC and TCS.......................................................................................................... 23
4.1.1. E-pure vs. nutrient media without plants ................................................................ 23
4.1.2. Concentration of TCC and TCS in the nutrient media with plants ......................... 24
4.2 Plant TCC and TCS............................................................................................................ 28
4.3 Toxicity of TCC and TCS .................................................................................................. 42
4.4 Potential Exposure to antimicrobials ................................................................................. 44

iv

CHAPTER 5: CONCLUSIONS .............................................................................................................. 48
5.1 Conclusions ........................................................................................................................ 48
5.2 Future Research .................................................................................................................. 49
LITERATURE CITED ............................................................................................................................ 50

v

LIST OF TABLES
TABLE 1. Chemical properties of triclosan and triclocarban .......................................................... 4
TABLE 2. Translocation Factors and Root Concentration Factors for various crops ................... 33
TABLE 3. Distribution of TCC and TCS after 4 weeks ................................................................ 34
TABLE 4. Bulb and flower TCC and TCS concentrations ............................................................ 36
TABLE 5. Lipid content and triclosan and triclocarban in multiple crops .................................... 39
TABLE 6. Observations on toxicity effects of TCC and TCS on plants compared to control
plants. ............................................................................................................................................ 42

vi

LIST OF FIGURES
FIGURE 1. Chemical structures of TCS (top) and TCC (bottom) .......................................... 3
FIGURE 2. Hydroponic system setup. (For interpretation of the references to color in this and
all other figures, the reader is referred to the electronic version of this thesis) ....................... 17
FIGURE 3. Dionex ASE 200 .................................................................................................. 19
FIGURE 4. Extraction drying setup ........................................................................................ 20
FIGURE 5. Shimadzu LC-MS ................................................................................................ 21
FIGURE 6. Filtering the samples ............................................................................................ 21
FIGURE 7. Concentration of TCC and TCS in E-pure water and Nutrient solution when
treated with 500ppb of TCC and TCS for 4 weeks .................................................................. 24
FIGURE 8. Concentration of TCC (top) and TCS (bottom) in the growth media of different
crops when treated with 500ppb of TCC and TCS for 4 weeks .............................................. 27
FIGURE 9. Concentration of TCC and TCS in the shoot of different crops when treated with
500ppb of TCC and TCS for 4 weeks ................................................................................... 29
FIGURE 10. Concentration of TCC and TCS in the root of different crops when treated with
500ppb of TCC and TCS for 4 weeks ..................................................................................... 30
FIGURE 11. Mass balances for TCS and TCC ...................................................................... 34
FIGURE 12. Concentrations of TCC and TCS in cucumber flowers, beet bulbs, onion bulbs,
and potato tubers ...................................................................................................................... 35
FIGURE 13. Transpiration Stream Concentration Factors for TCS and TCC ....................... 37
FIGURE 14. Cabbage toxicity comparison between the control and treated plants as shown by
the number of healthy and dried leaves ................................................................................... 43
FIGURE 15. Pictures of cabbage control and two treated cabbage plants ............................ 43
FIGURE 16. Potential exposure to antimicrobials from food crop consumption ................... 46
FIGURE 17. Potential exposure from crops compared to other routes ................................. 47

vii

CHAPTER 1: INTRODUCTION
Pharmaceuticals and personal care products have been used for decades, but have only
recently emerged as a concern for water resources. Triclocarban (TCC) and triclosan (TCS) are
common chlorinated aromatic compounds used as antimicrobial agents in products such as soaps
and toothpastes (Aryal and Reinhold 2011; Cha and Cupples 2010; USEPA 2002; USEPA 2010)
which end up in waste water treatment plants (WWTP) through water disposed of in sinks and
drains. In wastewater treatment plants, triclosan and triclocarban sorb with large particles that
settle out of solution into what is called “biosolids”. Biosolids are often applied to agricultural
fields because of the high nutrient content. While there are regulations regarding treatment of
biosolids before use as a fertilizer, triclosan and triclocarban are not on the list of chemicals that
are regulated in biosolids. Chemicals in biosolids can then be introduced into the soil and sorb to
soil particles, where most of the initial chemical in the biosolids will remain. Crops might then
be able to take up chemicals in the soil like triclosan and triclocarban.
Though the bioavailable fraction of these contaminants may be low due to low solubility
in water and sorption to soil and organic particles, accumulation of both TCC and TCS in plants
has been observed (Aryal and Reinhold 2011) and the fruits or vegetables from these plants may
be consumed by people. Triclocarban and triclosan are not considered health hazards by the EPA
or FDA, but animal studies have demonstrated that triclocarban and triclosan could result in
disruption of endocrine functions and the central nervous system respectively, and hence further
investigations are required (Paul and others 2010; USEPA 2010; USFDA 2010). Studies also
indicate the induction of antimicrobial resistance in bacteria in biosolid applied agricultural lands
(USFDA 2010). Several pathogens are known to be resistant to both TCC and TCS at high
concentrations. For example, E. coli and P. aeruginosa, are resistant to up to 1024 μg/mL of
1

triclosan (Coulborn Rebecca and others 2010; Coulborn and others 2010). However, information
on bacterial resistance to triclocarban is limited. Plants, including food crops, are capable of
taking up a number of organic contaminants into its biomass (Kim and others 2005b; Loffredo
and others 2010a; Murano and others 2009; Zhang and others 2009). The studies indicate higher
concentration of organic contaminants such as TNT, dieldrin, dibenzodioxin, dibenzofuran and
bisphenol in the roots of the plants compared to the shoot. Similarly, TCC and TCS
accumulation in pumpkin and zucchini were higher in the root than the shoot (Aryal and
Reinhold 2011) which emphasizes concern over the accumulation of organic contaminants in
edible root crops. Based on the hypothesis that plants are capable of accumulating both TCC and
TCS more in the root and shoot, the objectives of this study are to 1) screen various crops plants
on the uptake and accumulation of TCC and TCS using a hydroponic system 2) understand the
changes in TCC and TCS in the growth media.
The information gained from this research will help guide policy in matters concerning
these chemicals. For instance, it will help determine if the use of these chemicals should be
regulated, or if the use of biosolids application is safe, and whether it should be limited to certain
amounts of certain crops. It can also help determine if phytoremediation is a good method of
removing these chemicals from the soil without posing a threat to public health. This research is
very important since biosolids application is very common and has many benefits both to farmers
and the environment. The purpose of this study is to determine if crops can take up TCC and
TCS and how much.

2

CHAPTER 2: LITERATURE REVIEW

2.1

Description of chemicals

2.1.1 Type of chemical
Triclosan and triclocarban (TCS and TCC) are chemicals that are used as antimicrobial
agents in personal care products such as antibacterial soaps and lotions. Triclocarban and
triclosan are chlorinated aromatic compounds (USEPA 2002; USEPA 2010). The chemical
formula for triclocarban is C13H9Cl3N2O, and the formula for triclosan is C12H9Cl3O2 (Halden
and Paull 2005; USEPA 2002). Figure 1 shows the chemical structures of TCC and TCS (from
Wikipedia.org).

Triclosan

Triclocarban
FIGURE 1. Chemical structures of TCS (top) and TCC (bottom)(images from wikipedia.org).

3

Both chemicals are used as antimicrobial agents and preservatives in products such as
soaps and deodorants (USEPA 2002; USEPA 2010). As well as being used in antimicrobial
personal care products, triclosan is also used as a commercial pesticide and as a “material
preservative” in products such as adhesives, fabrics, and plastics (USEPA 2010).

2.1.2 Chemical Properties
Table 1 lists chemical properties of triclosan and triclocarban found in literature. The
solubility of both TCC and TCS in water is very low, especially for TCC, due to their organic
and nonpolar natures. The low vapor pressure indicates that TCC and TCS are not volatile. The
aerobic half-life will be discussed in another section.
TABLE 1. Chemical properties of triclosan and triclocarban
Property
Molecular weight
(g/mol)
Dispersion coefficient
(L kg)

Triclosan

Triclocarban

290

315.59

Reference(s)
(Chen and others 2009;
Halden and Paull 2005)

33-55

193-296

(Cha and Cupples 2010)

Log KOW

4.8

3.5

Aerobic half-life (d)
Solubility in water
(mg/L)

20-58

87-231

1.97-4.6

0.045

pKa

4.5

12.77

Vapor pressure (Pa)

0.00062

4.81 x 10^-7

Melting point (°C)

180

140

4

(Halden and Paull 2005;
Snyder and others 2010)
(Wu and others 2009)
(Halden and Paull 2005;
Snyder and others 2010)
(Chu and Metcalfe 2007;
Snyder and others 2010)
(Chen and others 2009;
Snyder and others 2010)
(Halden and Paull 2005)

2.2.3 Antimicrobials in Biosolids
Both triclosan and triclocarban are hydrophobic organic compounds and hence have high
affinity for organic matter (Kwon and others 2010a; Kwon and others 2010b). The products of
the WWTPs are biosolids and effluent water. Approximately 50% of biosolids are used as
fertilizers for agricultural fields as it reduces the dependence on chemical fertilizer (USEPA
2007). These bio-solids comprise of a high concentration of organic matter (80%), allowing
preferential partitioning of TCC and TCS (Cha and Cupples 2009; Kinney and others 2006).
Studies indicate that approximately 76 and 50% of TCC and TCS respectively are sorbed into the
organic fraction of biosolids from WWTPs (Kwon and others 2010b). Walters et al. (2010)
reported finding triclosan in biosolids/soil mixtures in an initial concentration of 1265 ppb and
triclocarban at 2715 ppb. Chu and Metcalfe (2007) reported the presence of triclosan and
triclocarban in biolsolids and activated sludge in ranges of 0.68-11.55 ppm for triclosan and
2.17-5.95 ppm for triclocarban (Chu and Metcalfe 2007).
According to EPA regulation 40 CFR part 503, there are varying qualities of biosolids
depending on the degree of treatment for pathogens, pollutants, and vector attraction. Treated
biosolids can be applied to agricultural fields, though there are restrictions on how they can be
applied and how soon crop scan be harvested depending on the quality of the biosolids. While
some states may have more strict regulations, the additional regulations in Michigan mainly
focus on applying at certain distances from water bodies and sources (Jacobs and others 2003).
There are four different quality classifications based on how much treatment the biosolids have
had. With regards to pathogens, biosolids can either be Class A or Class B. The goal of Class B
is to reduce the pathogens to a low enough number that there they do not pose a threat to public
health. The goal of Class A treatment is to reduce the pathogens to below detectable levels.

5

Class B biosolids have more restrictions on how long after application crops can be harvested.
Class A biosolids have more rigorous treatments against pathogens and are less regulated. For
pollutants or contaminants, biosolids are categorized as either following ceiling concentration
limits (CCL) or pollutant concentration limits (PCL). PCLs are lower concentrations than CCLs,
and if biosolids do not exceed the PCLs, then the cumulative pollutants throughout the lifetime
of the site do not need to be tracked. The CCLs are the maximum concentrations of each
pollutant that can be present in land-applied biosolids. Exceptional quality (EQ) biosolids are
Class A and meet all the PCLs and CCLs and can in general be used freely as any other type of
fertilizer. Pollutant concentration (PC) biosolids can be either Class A or Class B, but must meet
all PCLs for pollutants. There are more restrictions on where and how these biosolids can be
applied, for instance, they can only be applied in bulk and not on lawns or other lands easily
accessible by the public. Cumulative Pollutant Loading Rate (CPLR) biosolids can be either
Class A or Class B and can only be applied in bulk. They typically have at least one pollutant
over the PCL, but not the CCL, and the cumulative pollutants must be tracked. Annual Pollutant
Loading Rate (APLR) biosolids are sold in bags or other containers with instructions on safe use.
They are Class A and have at least on PCL exceeded, but no CCLs. The annual loading of the
pollutants must be tracked. TCS and TCC are not currently regulated under part 503, and
therefore do not have a PCL or CCL for biosolids, though TCS is under review by the EPA
(USEPA 2012).

6

2.2 Physiochemical Processes of TCC and TCS

2.2.1 Sorption to Soils
The most likely pathway for both TCC and TCS once introduced to an agricultural field
is to sorb to soils, and triclocarban sorbs more strongly than triclosan (C. Wu 2009; Cha and
Cupples 2010; Wu and others 2009). Wu et al. (2009) studied the adsorption and degradation of
triclosan and triclocarban in sandy loam soil and silt clay soil both with and without biosolids
amendments. Triclocarban had stronger sorption that triclosan in all soils tested, and sorption for
both compounds was higher in sandy loam than silt clay, possibly because the large amount of
clay in the silt clay inhibited interactions between the compounds and organic matter (Wu and
others 2009). The study found that adding biosolids to the soil increased pH, organic matter, and
cation exchange capacity; the effects were found to be able to last several years, and biosolids
amended soils sorbed more antimicrobials in all cases except triclocarban in silt clay, likely
because of the increased organic matter (Wu and others 2009). Sorption of triclosan decreased
slightly with increasing pH (from 4 to 8), but triclocarban sorption was not significantly affected
by pH; the decrease in sorption is likely due to the change in speciation for triclosan, as the study
found that the amount of the neutral form of triclosan changed from 100% to 39% with the
increase in pH from 4 to 8, but the sorption of the anionic form was still relatively high (Wu and
others 2009). The study also found that when the two compounds coexisted in the same solution,
the sorption of both compounds decreased at low concentrations, but co-solution had less of an
effect with higher concentrations (Wu and others 2009). The sorption of these chemicals can
change based on the properties of the soil, but regardless, triclocarban is more likely to sorb to

7

soils than triclosan. Cha and Cupples (2010) also found that triclocarban sorbs to soils more
strongly than does triclosan in fields receiving biosolid applications.

2.2.2 Leaching
The leaching of chemicals from soils to groundwater is a common concern with any
chemical that has the potential to pose health risks to the public. Triclosan typically is not
thought to leach out of soil systems, but rather becomes immobilized in the soil (USEPA 2010).
Cha and Cupples (2010) studied leaching potential of triclocarban and triclosan using a leaching
potential model. The study found that triclocarban had a groundwater ubiquity score (GUS) of
less than -0.5, and triclosan had a GUS of less than 0.7, which are both less than the 1.8 required
to be leachable or transitional under the Gustafson’s criteria (Cha and Cupples 2010). Therefore,
both chemicals are much more likely to sorb to soils than to leach into groundwater. However,
Aryal and Reinhold (2011) found in soil column studies that there were concentrations of
triclosan and triclocarban in the leachate from the columns in ranges of 460-1670 μg/L for
triclosan and 120-370 μg/L for triclocarban. Leaching was reduced in columns that contained
plants (Aryal and Reinhold 2011). Leaching can occur, but not often, and is not much of a
concern. Leaching may be a concern in certain circumstances, such as rain too soon after
biosolids application.

2.2.3 Photodegradation
Triclosan can undergo photodegradation through photo-oxidation which can result in
breakdown to many other products (Lawrence and others 2009; Ozaki and others 2011). Ozaki
et al. (2011) studied the photodegradation of triclosan on dried loamy sand under simulated
8

sunlight conditions and found that the half-life of triclosan in sunlight was 17 days and that the
data fit the light penetration-limited model best. Photodegradation of triclosan can form
dichlorodioxins and chlorophenols, both of which can be toxic and persistent (Lawrence and
others 2009). Triclosan can be degraded relatively quickly by light, but the rate of degradation is
limited by light availability, and therefore by how deep within the soil the chemical is.
Furthermore, even though light can cause triclosan to degrade, the products left behind could
potentially have worse effects for health and the environment than the original triclosan.
2.2.4 Volatilization
Triclosan is typically not thought to volatize out of most soil or water systems (USEPA
2010). Triclocarban is non-volatile (USEPA 2002). As shown in table 1, both chemicals have
very low vapor pressures, so volatilization is expected to be a very small portion of removal of
the chemicals. Chen et al. (2009) found volatilization to be an insignificant portion of the
removal of the chemical from soil.

2.2.5 Reactions with other chemicals
The most common degradation product of triclosan is methyl-triclosan, in which a methyl
group attaches to the phenol group of triclosan (Bester 2005). Ozone is also known to react
rapidly with the phenol group, either by adding oxygen or by opening the phenol ring (Suarez
and others 2007). TCC can also be degraded by ozone (Tizaoui and others 2011). The phenol
group of triclosan was found to be readily oxidized by manganese oxide (Zhang and Huang
2003). This reaction produced quinones of triclosan and 2,4-dichlorophenol, though the report
mentioned there are most likely other products that were not detected by the methods used in the
study.
9

2.3 Biological Processes of TCC and TCS

2.3.1 Microbial degradation
Wu et al. (2009) found that the two compounds can be degraded in soils under aerobic
conditions, but not under anaerobic conditions. Aerobic degradation of triclocarban can form
chloroanilines (Lawrence and others 2009). Triclocarban was degraded less than triclosan,
possibly due to less bioavailability to microbes due to stronger sorption to the soil (Wu and
others 2009). The half-life of triclocarban was higher than triclosan in all treatments (87-231
days and 20-58 days, respectively); half-lives were higher in silt clay soils than sandy loam soils,
while there was no significant difference in amended soils and not amended soils (Wu and others
2009). Methyl-triclosan is the most commonly reported product from triclosan and stays in the
environment longer than triclosan (Lawrence and others 2009). Triclocarban dechlorination
products (dichloro-, monochloro, and nonchlorinated carbanilides) were found in sediments in
the Chesapeake Bay area, but the investigators were unsure whether it was due to microbial
activity or chemical degradation, but claim that it is an anaerobic process (Miller and others
2008).

2.3.2 Bioaccumulation
Bioaccumulation is “the accumulation of a substance, such as a toxic chemical, in various
tissues of a living organism” (2002). The bioaccumulation of triclosan, methyl triclosan and
triclocarban has been documented in algae growing in a stream that receive effluents from a
wastewater treatment plant (Coogan and others 2007), so the chemicals are often present in
aquatic ecosystems. There is some possibility of bioaccumulation of triclosan and triclocarban in
10

aquatic systems (USEPA 2010), but the chance is relatively low that much TCC or TCS would
reach the trophic level containing fish; the bioconcentration factor (BCF) is 137 for the whole
fish, but only 13 for muscle tissue (USEPA 2002), which indicates that the concentration in
muscle tissue is 13 times greater than that in the water. However, since in most cases triclosan
and triclocarban remain in the soil, it is more likely that these chemicals would be taken up by
plants than be introduced into aquatic systems.
Plants can sometimes be used to take chemicals from contaminated soils, a practice called
phytoremediation, but this is not always advisable if the plants grown in the soil are meant for
human consumption, depending on how much the plant can uptake and where the contaminants
are stored in the plant. Therefore, there is concern that these chemicals could contaminate food
crops and cause a health risk. While there has not been an extensive amount of work done on the
effects on plants by these chemicals, one study found that pumpkin and zucchini take up
triclosan and triclocarban and accumulate the chemicals (Aryal and Reinhold 2011). Generally
the study found that there were higher concentrations of the antimicrobials in the roots than any
other part of the plants (Aryal and Reinhold 2011). For most crops that would indicate little
chance of consuming contaminated parts of plants, but holds a particular concern for root crops
such as carrots or onions. Another study found similar results with lettuce and radish (Pannu
2012). A study with carrots also found more TCS in the roots than the leaves, but also found that
more TCS was found in the peel, or outer layer, or the root than in the core (Macherius 2012).
The same study found that barley and meadow fescue accumulated TCS and other contaminants,
but did not differentiate between root and shoot for those crops. Another study found that algae
took up significant amounts of TCC, TCS, and methyl-triclosan from water (Coogan and others
2007). The uptake of antimicrobials by plants raised in soil was higher when applied via

11

irrigation water than when applied with biosolids (Wu and others 2010). When soybean plants
were treated with TCC and TCS, the antimicrobials accumulated in the roots and was
translocated to different parts of the plant including the bean.
Crops and other plants have been shown to take up several other organic contaminants.
For instance, onions have been shown to uptake TNT in both hydroponic systems and soil
systems (Kim and others 2005a; Kim and others 2004). Again, most of the contaminant was
found in the roots rather than the shoots or leaves. Five forage grasses and three horticultural
species (cucumber, marrow plant, and radish) were found to remove bisphenol A from water
after 16 days, likely with a combination of plant uptake, biotransformation, and microbial
degradation (Loffredo and others 2010b). Corn, lettuce, and potato were found to take up the
veterinary antibiotic sulfamethazine from manure-amended soil (Dolliver and others 2007).
Twelve different crops had dioxins in the roots and shoots after only a 4 day exposure (H. Zhang
2009). The crops tested included four from the genus Curcurbita, such as pumpkin and
cucumber, as well as sorghum, rice, wheat, maize, tomato, soybean, Chinese cabbage, and
garland chrysanthemum. Root uptake was mostly accomplished by lipophilic adsorption. The
curcurbits tested appeared to have better ability to translocate the dioxins from the root to the
shoot, though wheat and sorghum also had dioxins in the shoot tissue. The other crops tested had
shoot concentrations close to that of the respective controls. The curcurbits also had the highest
transpiration stream concentration factors (TSCF), which indicates that the main mechanism for
translocation to the shoot is transpiration (H. Zhang 2009). DDT is another chlorinated aromatic,
like TCC and TCS. It has been shown to be taken up by hairy root cultures of Cichorium intybus
and Brassica juncea (Suresh and others 2005). DDT was also taken up by pumpkin plants, and
the concentration in the stem and leaves decreased with distance from the roots, indicating that

12

transpiration may be a major mode of translocation for pumpkin (Aslund and others 2010). In
another study, pumpkin took up polychlorinated byphenyl (PCB), another chlorinated aromatic
(Aslund and others 2008). PCB-5 was taken up by pumpkin, corn, and soybean (Li and others
2011).

2.4 Possible risks and effects of TCC and TCS

2.4.1 Environmental effects and harm to wildlife
Agricultural fields are not the only places these chemicals can be introduced. They are
often detected in open water systems, usually deposited there after going through the wastewater
treatment plant. Triclosan has been found in 36 monitored streams in the United States that
receive effluent from wastewater treatment plants (USEPA 2010). When these chemicals get
into surface waters, they can have serious effects on the ecosystem. Lawrence et al. (2009)
studied the effect of triclocarban and triclosan on river biofilms and found that they significantly
affected the structure and functionality of the biofilms. The study found that the chemicals,
triclosan especially, were toxic to algae and other phototrophs, which affected the functional
groups of the biofilm to being more heterotrophic than autotrophic (Lawrence and others 2009).
That kind of shift can affect symbiotic activities in biofilms. The chemicals were also found to
“suppress carbon utilization” in microbes (Lawrence and others 2009). According to an EPA
study, typical triclosan levels in aquatic systems are not more than the levels of concern for fish,
but can be more than the levels of concern for aquatic plants, which can harm aquatic ecosystems
(USEPA 2010).

13

2.4.2 Possible Health Hazards
Triclocarban and triclosan are not considered health hazards by the EPA or FDA, but
animal studies on triclosan and triclocarban have demonstrated that they could be endocrine
disrupting chemicals at 0.3 μg/ml for TCC in the presence of native androgens (only 3 times the
human exposure from a whole-body shower) and 300 mg/kg/d for TCS, which the FDA and EPA
plan to investigate further when reviewing the chemicals again (Chen and others 2008; Paul and
others 2010; USEPA 2010; USFDA 2010). There have also been studies on bacteria indicating
that triclosan may be able to lead to antibiotic resistant bacteria after being exposed to 1-4 μg/ml
(Christensen and others 2011; Suller and Russell 2000; USFDA 2010). Furthermore, the FDA
(2010) finds no evidence that antibacterial handsoap containing triclosan is any more effective at
removing bacteria from hands than normal soap and water, which even further warrants more
research into possible health hazards and decreasing the concentrations of these chemicals in soil
and water or the concentration allowed in products.

2.5 Root Physiology and the Rhizosphere
Plants typically have either a taproot system or a fibrous root system. A taproot system
consists of one main root that begins as the embryonic root and continues to grow downwards as
well as smaller lateral roots that grow out from the taproot, whereas a fibrous system consists of
many small adventitious roots growing from the stem (Campbell and others 2008). In most
plants, a majority of plant uptake of water and nutrients is done near the tips of the roots, with
the aid of root hairs. Each root has a root cap at its end to protect the root as it grows through the
soil. There is also usually a layer of polysaccharide slime produced around the root cap to
lubricate the root as it moves through the soil. Water and nutrients are transported out of the root
with the xylem, and sugars and other storage materials are transported to the roots with the
14

phloem (Campbell and others 2008). Many plants have modified roots in order to better obtain
or store water and nutrients. For instance, potatoes and beets have such large parts of their roots
designed for storage. The bulbs of onions are actually not roots at all, but modified stems that
grow underground. The bulb actually contains a short stem with many layers of what are the
bases of the leaves that continue above ground; these leaf bases are used mainly for storage
(Campbell and others 2008).
Plant roots often have mutually beneficial relationships with bacteria and fungi in the
soil. About 80% of extant land plant species have some sort of symbiotic relationship with fungi
and roots, called a mycorrhiae (Campbell and others 2008). Interactions between bacteria, fungi,
and plants roots are often crucial to a plant’s growth. Roots exude materials such as
carbohydrates and other nutrients, which can attract or deter different kinds of bacteria, fungi,
and nematodes (Fogel 1988). Contaminants can sometimes affect the complex rhizospohere in
the soil. One study found that several pharmaceutical chemicals, including triclosan, can have a
negative impact on mycorrhizal communites; however, the concentrations needed to cause such
impacts were greater than is found in the environment (Hillis and others 2008). Organic
contaminants such as polyaromatic hydrocarbons (PAHs) and phenols have been shown to harm
mycorrhizal communities and decrease biodiversity in the rhizosphere (Cairney and Meharg
1999).

15

CHAPTER 3: MATERIALS AND METHODS
This study consisted of growing several different food crops in a hydroponic system
spiked with the two chemicals of interest, triclosan and triclocarban. They were exposed to the
chemicals for one month with weekly samples of the media taken. After the month, the plant
tissue was divided into above ground and below ground sections and tested for TCC and TCS
concentration.

3.1 Materials

Triclocarban [CAS 101-20-2], triclosan [CAS 3380-34-5] and C13- triclocarban were
purchased from Tokyo Chemical Industry and Calbiochem. Ammonium acetate (>99.99%),
acetone (>99.7%), methanol (99.8%), and methanol (>99.99% for LCMS) from were purchased
from VWR. Planting materials for the hydroponic study were procured from Garden Harvest
Supply, Burpee, and Tasteful Garden and plants of uniform age or size were used for the
experiment. When possible the plants were raised from seeds (cucumber, tomato, cabbage, okra,
chili), else tubers (potato, beet), bulbs (onion) or whole plants (celery) were used.

3.2 Experimental Setup

To avoid the effect of sorption by biosolids or soil and to understand the worst case
scenario of complete bioavailability, the current study was performed hydroponically. The
uptake and translocation of TCC and TCS were studied in cucumber, tomato, cabbage, okra,
chili, beet, celery, onion, broccoli, asparagus, and potato. The effect of plant moisture content,
16

fresh weight, transpiration and media pH on TCC and TCS uptake was also investigated.
Following uptake, the toxicity of these chemicals on plants was studied. The change in media
TCC and TCS concentrations in the presence of the plants were studied to understand the
difference in uptake.
Seeds were germinated or other propagating material were raised in a potting mix and
once the roots were well established the plants were transferred to a nutrient solution hydroponic
system with constant aeration in order to provide oxygen to the plant roots. After 1 week of
acclimatization, plants were transferred to 1L amber glass jars with 900mL test solutions that
were prepared in a nutrient medium made for aquatic plants (APHA 1999). Figure 2 shows an
example of the hydroponic setup.

FIGURE 2. Hydroponic system setup. (For interpretation of the references to color in
this and all other figures, the reader is referred to the electronic version of this thesis)

17

Standard solutions of both TCC and TCS mixtures were prepared in methanol from the
respective stocks. Each of the crops was raised for one month in the test solution prepared in
nutrient media having 500ppb of TCC and TCS. Each treatment had 5 replicates. No-plant
controls in the TCC/TCS media and plant control in the absence of TCC/TCS in the nutrient
media were also maintained. The crop plants were raised in these test solutions at constant
aeration with temperature maintained at constant temperature of 23±2°C and light supply. Two
mL of the media samples were collected weekly and analyzed for TCC/TCS immediately. Loss
of media by evapotranspiration was made up by adding nutrient solution every week. The total
amount of water lost from each bottle was calculated after adjusting the no-plant control media
loss (evaporation) to give the transpiration loss.
After the test period of one month, the plants were washed in tap water and rinsed in
distilled water. The plants were then separated into the above and below ground parts and flower,
tubers or bulbs when available. The fresh weights of the plant parts were taken before the plants
were dried in an incubator at 55°C for 2 days. The pH of the media was taken both before and
after the experiment.

3.3 TCC/TCS degradation in the media

Separate sets of experiments were performed to understand the behavior of TCC and TCS
under different aqueous systems without the presence of plants. Solutions of 500ppb of
triclocarban and triclosan were prepared in e-pure water, nutrient solution and different
components of the nutrient solution (A, B and C). Media pH was measured in each bottle both
before and after the experiments to understand the effect of pH on loss. All the systems were
maintained with continuous aeration. The experiments were performed in triplicates. There were
18

also sets of experiments conducted testing the behavior of TCC and TCS in media in the
presence and absence of both light and aeration.

3.4 Sample extraction and analysis

The dried plant samples were weighed and ground in a grinder and the samples extracted
using a Dionex ASE 200, as shown in figure 3. Cellulose thimbles were first placed in the
extractor cells and were half filled with sand, followed by the sample and then filled with sand.
One of the samples in each set of the plants was spiked with 1.2ppm of C-13 labeled triclocarban
(a stable isotope) to know the extraction and analysis efficiency. The extractor specifications
were as follows: temperature- 100oC, pressure-1500 psi, static time-5 min and flush volume 100%. The collected extracts in amber vials were then dried in N gas, as shown in figure 4, and
then reconstituted in 3mL of 1:1 methanol: acetone mixture. The mean recoveries calculated for
the method used in this study are 87.5% for triclocarban, 69.6% for triclosan, and 93.5% for the
C-13 triclocarban.

FIGURE 3. Dionex ASE 200
19

FIGURE 4. Extraction drying setup
The samples were analyzed for TCC and TCS concentrations using a Shimadzu LC-MS
2010 EV, as shown in figure 5, with an Allure biphenyl column (5 μm, 150 x 2.1 mm) from
Restek using a binary gradient of 75% methanol and 25% 5-mM. Qualitative analysis was done
in negative electrospray ionization with scan mode and quantification by selected ion monitoring
(SIM) mode. TCC and TCS were identified by retention time (tR ± 0.1 min), specific molecular
ions (m/z: 313 of triclocarban and 287 for triclosan), and reference ions (m/z 315 and 317 for
triclocarban and m/z 289 and 291 for triclosan (Aryal and Reinhold 2011). Mobile phases were 5
mM ammonium acetate and methanol, for which a binary gradient from 75% to 100% methanol
was used. Linear calibration for quantification of the analysis results was performed with
prepared samples of at least six concentrations. Detection limits were 0.00001µg/mL for media,
and 0.0001µg/g dry tissue for plants. Collected media samples from all the experiments were
diluted in 2 mL methanol and the resulting 4mL diluted sample was analyzed with the LC-MS.
Media samples and plant tissue extractions were both passed through a 0.2μm PTFE filter into an
amber glass vial for the LC-MS that were then placed into the autosampler to be analyzed using
the LC-MS.
20

FIGURE 5. Shimadzu LC-MS

FIGURE 6. Filtering the samples

21

3.5 Media Statistical Analysis

Statistical analysis was performed using SigmaPlot (version 11.0). One way-ANOVA
and Student-Newman-Keuls (SNK) tests were used to compare the data. Reported values are in
mean ± standard error of the mean. P-values of less than 0.05 were considered statistically
significant. While the data did not pass the automatic normality check for the ANOVA test,
when a more detailed normality check (in the appendix) was run, only one or two sets of data
failed, so the data was treated as normal.

22

CHAPTER 4: RESULTS AND DISCUSSION

4.1 Media TCC and TCS

4.1.1. E-pure vs. nutrient media without plants
Experiments with 500ppb of TCC and TCS mixture in e-pure and nutrient media
indicated a loss of TCC from both the e-pure and nutrient media. Loss of triclocarban from epure water was minimal compared to nutrient media, which accounts for ~ 43% loss in 30 d/ 4 w
(Figure 7). This indicates that nutrients play a major role in the loss of TCC. However, it is not
clear whether this loss is by a chemical or microbial degradation process. One study found that
triclosan can be oxidized by manganese oxide (Zhang and Huang 2003). While manganese
oxide is not used in the making of the nutrient solution used in this study (manganese chloride is
used for manganese), there are other metal oxides that could have a similar effect, and with the
multitude of chemicals in the nutrient solution, there may be reactions that result in the formation
of manganese oxide in the solution. Therefore, chemical degradation is a possibility, though
more testing would be needed to determine exactly what causes the loss in the media. In the case
of TCS, loss from both the e-pure (~30%) and nutrient media (~40%) in 30 d indicates the
possibility of degradation (figure 7). Here, loss may indicate a reduction in concentration in the
water soluble fraction of the media or degradation into other compounds.

23

Day 1

Media TCC mg/L

0.7

Day 4

Day 30

0.6
0.5
0.4
0.3
0.2
0.1
0

E-Pure

Nutrient

Media TCS (ppm)

0.6
0.5
0.4
0.3

0.2
0.1
0

E-Pure

Nutrient

FIGURE 7. Concentration of TCC and TCS in E-pure water and Nutrient solution when
treated with 500ppb of TCC and TCS for 4 weeks. Error bars represent standard error.
4.1.2. Concentration of TCC and TCS in the nutrient media with plants
The concentration of both TCC and TCS decreased with time for all the crop plants tested
over the 4 week experiment (figure 8). The concentrations in the control experiment
(hydroponic media with no plants) also decreased over time, showing that although the
chemicals are taken up by the plants (as discussed in the next section), it is not the only
mechanism for removal from solution for the chemicals.

24

The pH is an important determinant factor of the rate of degradation or loss of both TCC
and TCS. Triclocarban can undergo direct and indirect photolysis at pH 7 (Guerard and others
2009). Rapid degradation of triclosan to 2,8-dichlorodibenzo-p-dioxin is occurs at higher pH
(Sanches-Prado et al., 2006). Plant root exudates can alter the pH of the hydroponic system
which may enhance the degradation of antimicrobials. These exudates contain low molecular
weight organic acids which can house a number of microbes which may aid in antimicrobial
degradation in the growth media. The pH of the media before and after the experiments was
determined to understand the impact of pH on the change in concentration. The initial pH of the
nutrient media ranged from 7.4 to 7.7 and the final media pH ranged from 7.3 to 7.8 and 7.3 to
8.1 in the unplanted and planted media, indicating any impact of pH on both TCC and TCS
would be similar in unplanted and planted media.
With TCS, the majority of the chemical was still in the solution after 4 weeks, with the
exception of a few crops. With TCC the majority of the chemical was “unaccounted for,” which
means that it was neither in the solution nor in the plant tissue. Presumably unaccounted for
portions of the TCC and TCS were degraded, settled out of solution, reacted with one of the
compounds in the nutrient media, or attached to settled particles. In many of the experiments,
cloudiness was noticed in the media after the first or second week. If left without aeration after
removing the plants, the cloudy gelatinous precipitate would settle to the bottom of the bottle.
With the beet experiment, the bottles were left alone for several weeks after the plants were
removed in order to allow the gelatinous precipitate to settle to the bottom. The precipitate was
filtered out, dried, and weighed. It was then diluted and analyzed with the LC-MS. An average
of 17.7 mg/L TCC and 2.63 mg/L TCS was found in the precipitate, compared to the final media
concentration of 0.1-0.4 mg/L for TCC and TCS. Therefore, it was observed that a portion of the

25

TCC and TCS settled out of solution, though more testing would be needed to determine if this
precipitate is a product of interactions between nutrients or microbes. Any samples analyzed by
the LCMS were filtered, so the gelatinous precipitate and particulates would have been filtered
out and would not have affected the results found for the aqueous concentrations. Since the
concentrations found in the media were approximately constant after the first two weeks of
exposure, especially for triclocarban, it is possible that some of the TCC and TCS from the
precipitate would go back into solution whenever any was taken up by the plant, therefore
keeping the concentration in the media at a constant equilibrium concentration.

26

Change in TCC
Concentration

0.6
0.4
0.3

mg/L TCS

Control
Cucumber
Tomato
Okra

0.5
mg/L TCC

Change in TCS
Concentration

0.2
0.1

0.6

0

1

3

0.3
0.2
0.1

0.6
mg/L TCC

0.5

0

1

2
3
Weeks

0.2

0

1

2
Weeks

2
3
Weeks

3

Control
Celery
Asparagus

0

0.6

0.1
0

0

1

Cabbage
Broccoli

2
3
Weeks

4

Change in TCS
Concentration

Control
Beet
Onion
Potato

0.3

0

4

Change in TCC
Concentration

0.4

4

0.2

0.6
0.5
0.4
0.3
0.2
0.1
0

mg/L TCS

0

1

0.4

Change in TCS
Concentration

Control
Cabbage
Celery
Broccoli
Asparagus

0.4

0.6

Cucumber
Okra

4

Change in TCC
Concentration

0.5
mg/L TCC

2
Weeks

mg/L TCS

0

Control
Tomato

4

Control
Onion

Beet
Potato

0.4
0.2
0

0

1

2
3
Weeks

4

FIGURE 8. Concentration of TCC (top) and TCS (bottom) in the growth media of different

crops when treated with 500ppb of TCC and TCS for 4 weeks. Error bars indicate standard
error.
27

4.2 Plant TCC and TCS

The concentration of both TCC and TCS in the eleven plants studied was higher in the
roots than the shoots (figures 9 and 10). Broccoli, asparagus, and potato had the lowest
concentrations for the roots, and broccoli, asparagus, and onion had the lowest concentrations for
the shoots. Chili and cabbage had the highest shoot concentration for both TCC and TCS, while
root concentration varied more from the two chemicals and the different crops. Onion had the
highest root concentration of TCC, but not TCS. Cucumber had the highest root concentration of
TCS. Chili had relatively high concentrations for both TCC and TCS, while okra and cabbage
had relatively high concentrations for TCC. According to a one-way ANOVA StudentNewman-Keuls (SNK) test, TCS shoot concentrations were not significantly different and the
only value that was significantly different for the TCC shoot data was for chili. The power for
the TCS shoot test was 0.731, which is relatively close to the desired 0.8. The power for the
TCC shoot test was 0.758. However, while the pairwise comparison for TCS shoot data showed
no differences, the ANOVA test said that there were differences. The test was run again without
chili (the largest concentrations), and with that test not only was the normality test passed, but
the power increased to 0.989. With the new test, a couple different statistical groups were found.
For TCC root concentrations, chili and onion roots had significantly higher concentrations.
Cucumber had the highest TCS concentrations in the roots, and tomato and chili also had
significantly higher concentrations than the other plants. The power for the TCS root test was 1
with an alpha value of 0.05. The power for the TCC root test was 0.988. The high power values
indicate that with the data provided, the test would be very likely to detect any differences in the
data.

28

Shoot TCC and TCS Concentration (ppm)
b
4.00
AB

TCC
TCS

mg/kg DW

3.00
A

2.00

1.00

A

AB

a

AB
a

a

a
a

a

B
a

0.00

B

a

AB

B

ab

B

a B

FIGURE 9. Concentration of TCC and TCS in the shoot of different crops when treated with
500ppb of TCC and TCS for 4 weeks. Error bars indicate standard error.

29

Root TCC and TCS Concentration (ppm)

1,000

a

A

TCC

mg/kg DW

600

AB

ab

800
ab

a

ab

B
ab

400

200

C

b

C
b

0

C

b

C
C
b

C b C

C

FIGURE 10. Concentration of TCC and TCS in the root of different crops when treated with
500ppb of TCC and TCS for 4 weeks. Error bars indicate standard error.

Table 3 and figure 11 both show the mass balances of TCS and TCC. Most of the plants
had most of the TCC or TCS in either the solution or the “unaccounted for” portion, which
means that it is either settled out of solution, in the gelatinous precipitate found in the bottles of
media, degraded by light or microbes, or possibly metabolized by the plant (though more
research needs to be done to determine if this is a possibility). In general TCS has higher
percentages in the solution, while TCC has higher percentages unaccounted for. Since TCC is
less soluble than TCS, the unaccounted for portion is likely in large part the portion of the
chemical that has come out of solution.

30

The translocation factors (TF), ratio of the average concentration of TCC or TCS in the
shoot to the corresponding concentration in the root, and the root concentration factors (RCF),
the ratio of the concentration of TCC or TCS in the root to the final concentration in the
hydroponic media, are shown in Table 2. A low TF indicates a low amount of TCC or TCS
transported from the root to the shoot. The TF for TCC was less than that of TCS for every crop
except celery and beet, implying that the translocation from the root to the shoot is more limited
for TCC. There was no significant difference for the TFs for TCS of the different plants, but for
TCC celery was significantly different from cucumber, potato, okra, tomato, and cabbage. The
power for the test for TCC was 0.908, while the power for the test for TCS was 0.379. The low
power may be due to some of the data sets having smaller sample sizes. No high correlation was
found between TF and root or shoot concentration or TCC or TCS, even when potential outliers
were removed. The TF found here are similar to, but somewhat smaller than, those found for
pumpkin and zucchini (0.0013 for triclocarban, and 0.082 for triclosan) (Aryal 2010), but the
smaller numbers are not surprising since Aryal kept plants in the hydroponic system for two
months, while in this study the plants were only in the hydroponic system for one month. Higher
RCFs indicate a higher potential for accumulating TCC or TCS. Aryal (2010) found RCFs of
TCS for pumpkin and zucchini that were much smaller than those found in this study. Chili had
the highest RCF for TCS and onion had the highest RCF for TCC.
Judging by the concentrations of the antimicrobials in the roots of the crops and the RCF,
it would seem that onion is best at taking up triclocarban, while chili appears to take up the most
triclosan. Chili and cucumber seem to have high potential to take up both chemicals. Celery,
broccoli, and especially asparagus appear to have the lowest concentrations and RCFs of both

31

chemicals. From the concentrations in shoots, it appears that chili took up the highest
concentrations of triclosan and triclocarban.

32

TABLE 2. Translocation Factors and Root Concentration Factors for various crops
TCC
Shoot
SE
Root
SE
TF
SE
RCF
SE
TCS
Shoot
SE
Root
SE
TF
SE
RCF
SE

TCC and TCS Translocation Factor (TF) and Root Concentration Factor (RCF) in different crops
Cucumber Tomato Cabbage Okra
Chili Celery Beet
Onion Broccoli Asparagus
0.44
0.55
0.97
0.44
2.94
0.86
0.53
0.24
0.12
0.059
1.26
1.48
1.97
0.82
2.41
0.34
0.48
0.12
0.039
0.026
531
411
519
594
772
85
205
851
71
23
29
39
90
147
229
15
49
277
25
14
0.00082
0.0014 0.0022 0.0012 0.0054 0.015 0.0024 0.00049 0.0035
0.015
0.0003
0.0003 0.00092 0.00048 0.0028 0.0075 0.0012 0.00046 0.0021
0.0064
3288
2403
3233
6158
7573
576
1786
8610
674
38.73
407
203
551
1281
2395
178
431
2566
218
5.54
Cucumber Tomato Cabbage Okra
Chili Celery Beet
Onion Broccoli Asparagus
1.26
1.48
1.97
0.82
2.41
0.34
0.48
0.12
0.23
0.056
0.34
0.28
0.27
0.46
1.05
0.09
0.13
0.12
0.11
0.024
815
520
207
177
601
100
193
277
15
120
120
27
106
22
173
42
26
57
3
24
0.0016
0.0029
0.032
0.0042 0.0098 0.0053 0.0029 0.0012
0.032
0.037
0.0004
0.0006
0.013
0.0019 0.0079 0.0028 0.001 0.0012
0.025
0.017
2576
1538
732
1353
4896
327
783
1892
96
15.49
536
122
354
303
927
101
126
267
28
3.056

33

Potato
0.36
0.03
94
18
0.0014
0.00014
1275
258
Potato
0.24
0.13
26
3
0.0099
0.0055
304
45

TABLE 3. Distribution of TCC and TCS after 4 weeks

Crop

Solution
(%)

Cucumber
Tomato
Cabbage
Okra
Chili
Beet
Celery
Onion
Broccoli
Asparagus
Control

69.02
67.63
50.44
35.66
27.67
51.28
51.80
28.76
27.67
24.40
48.00

TCS
Plant
Plant (g)
(%)
3.80E-05
4.71E-05
1.75E-05
6.36E-05
2.34E-05
3.39E-05
9.44E-05
2.26E-05
0.000005
0.000009

Unaccounted
(%)

Solution
(%)

22.54
21.91
45.67
50.21
67.14
41.18
27.22
66.22
71.19
73.08
52.00

34.57
33.65
32.96
18.81
21.26
23.30
31.31
19.44
20.53
19.95
24.00

8.44
10.46
3.89
14.13
5.19
7.53
20.97
5.02
1.15
2.52

34

TCC
Plant
Plant (g)
(%)
2.46E-05
3.71E-05
4.06E-05
2.08E-04
3.00E-05
3.59E-05
8.21E-05
4.90E-05
0.000023
0.000018

5.47
8.25
9.01
46.25
6.66
7.97
18.25
10.89
5.01
5.20

Unaccounted
(%)
59.96
58.09
58.03
34.95
72.09
68.73
50.44
69.67
74.46
74.85
76.00

TCS Mass Balance
100
90

Percent TCS

80
70
60
50

Unaccounted

40

Plant tissue

30

Solution

20
0

Cucumber
Tomato
Cabbage
Okra
Chili
Beet
Celery
Onion
Broccoli
Asparagus
Potato
Control

10

100
90
80
70
60
50
40
30
20
10
0

Unaccounted
Plant tissue

Solution

Cucumber
Tomato
Cabbage
Okra
Chili
Beet
Celery
Onion
Broccoli
Asparagus
Potato
Control

Percent TCC

TCC Mass Balance

FIGURE 11. Mass balances for TCS and TCC. Error bars indicate standard error.

35

Table 4 and figure 12 show the data from the cucumber flowers, beet bulbs, onion bulbs,
and potato tubers, including three layers of the tubers. There was no significant difference found
between the miscellaneous parts of the different plants for TCC or TCS. However, the power of
the test for TCS was only 0.35 and the power for the test for TCC was only 0.548, which is less
than the desired power of 0.8. This may be due in part to the small sample size of some of the
data sets. For instance, both the cucumber flowers and beet bulbs only had enough biomass to
constitute one sample each. In general the flowers and bulbs had higher concentrations than the
shoots, but still much lower concentrations that those in the roots. The exception is the potato
tubers, whose concentrations were comparable to the shoot concentrations. With both onion
bulbs and potato tubers, effort was taken to make sure that the bulb or tuber was not submerged
in the media, though the onions were smaller and more prone to the bottom dipping in the media.
Both roots and bulbs or tubers were washed before analysis in order to remove as much surface
TCC and TCS as possible, but that might still be a factor in the higher concentrations. The
potato tubers were large enough to separate into layers. There was the peel, a middle section of
the tuber, and the core. The core had the highest concentration of the different layers. The peel
had one replicate out of five with very high TCC and TCS, but it was found to be a statistical
outlier and not included when making calculations.
TABLE 4. Bulb and flower TCC and TCS concentrations
Crop
Cucumber
Beet
Onion
Potato tuber peel
Potato tuber middle
Potato tuber core
Potato tuber total

Bulb or Flower
TCS (ppm)
3.80
4.81
16.37
0.10
0.05
0.32
0.14
36

SE
--9.75
0.01
0.01
0.13
0.05

Bulb or Flower
TCC (ppm)
1.89
5.31
25.60
0.10
0.10
0.24
0.11

SE
--13.16
0.02
0.01
0.05
0.01

TCC and TCS Concentration of Bulbs and Flowers

Concentration (ppm)

25

TCS

20

TCC

15
10
5
0

Cucumber Beet bulb
flower

Onion Cucumber
bulb
shoot

Beet
shoot

Onion
shoot

TCC and TCS Concentration of Tubers

Concentration (ppm)

0.4

0.3

TCS
TCC

0.2

0.1

0

Potato tuber Potato tuber Potato tuber Potato tuber
peel
middle
core
total

Potato
shoot

FIGURE 12. Concentrations of TCC and TCS in cucumber flowers, beet bulbs, onion bulbs,
and potato tubers. Error bars indicate standard error.

Moisture content of the crops ranged from 35.2-96.3%. Measured biomass (dry weight)
for the roots and shoots was highest for celery. The lowest shoot biomass was in tomato, and the

37

lowest root biomass was in onion (not including the weight of the bulb). When the root or shoot
biomass for each crop was graphed against the corresponding antimicrobial concentration, there
was not a very strong linear correlation in any case (R2 < 0.5), indicating that the different
concentrations are likely not merely due to larger plants having more tissue to dilute the
antimicrobials. There was also no correlation over 0.6 between TCS and TCC concentration in
either the shoot or the root. The fresh weights of the plants were also compared to the
antimicrobial concentrations, and also had no strong correlations. Moisture content also had no
strong correlation with antimicrobial concentration. When transpiration was graphed against
TCC and TCS concentration, there was no strong correlation, as well as when the transpiration as
graphed against the total mass of TCS and TCC found in the plant tissue. When the final pH was
graphed against antimicrobial concentration in the roots and shoots of the plants, the highest R2
value was 0.59 from shoot TCC concentration with a statistical outlier removed. With the outlier
2

included, the R value was only 0.19, and the next highest was only 0.37. Therefore, there is
likely not a significant effect on TCC or TCS from pH.

38

TABLE 5. Lipid content and triclosan and triclocarban in multiple crops
Plant

Lipid %
DW

Cucumber root

2.7

Cucumber leaves

0.7

Tomato root

0.12

Tomato stem

0.7

Cabbage root
Cabbage leaves

0.68
0.43

Okra root

1.7

Okra stem and leaf (av)

2.9

Pepper root

4.4

Celery root

1.45

Celery leaves

1.3

onion root

2.7

onion bulb

1

onion leaves

0.77

Reference
(Murano and others
2009; Murano and
others 2010)
(Bulder and others
1989)
(Gonzalez and others
2003)
(Gonzalez and others
2003)
(Gao and others 2005)
(Gao and others 2005)
(Gopalakrishnan and
others 1982)
(Gopalakrishnan and
others 1982)
(Lyons and Lippert
1966)
(Chiou and others
2001)
(Buttkus 1978)
(Cooper and Losel
1978)
(RodrÃ-guez Galdón
and others 2009)
(Novitskaya and others
2006)

TCC (ppm)

TCS (ppm)

531.41

815.73

0.44

1.26

411.89

520.80

0.55

1.48

519.43
0.97

207.03
1.97

594.98

177.09

0.44

0.82

772.60

601.80

72.01

84.56

0.86

0.34

851.02

277.12

25.60

16.37

0.24

0.12

Lipid content of plants was suggested as a possible factor in chemical accumulation in
plant tissue. A literature search provided several numbers for lipid content for some of the plants
tested in this study. Table 5 shows the lipid values found in literature and the TCC and TCS
values found for those crops on this study. No correlation was found with lipid content and
either TCC or TCS in shoots or roots. The highest R2 value found was 0.36 (root TCC vs. lipid
content). Lipid content is not likely to have a large factor in accumulation in plant tissue, at least
not for the crops tested in this study.
39

Phytoextraction is the ability of plants to remove metals and other contaminants from soil
and translocate them into above ground biomass (Robinson and others 2003). The
phytoextraction (the ratio of shoot concentration to final media concentration) ranged from
0.69% (Asparagus for TCS) to 61.03% (Chili for TCS). Only half of the phytoextraction values
were over 5%, and only 3 values are over 10%. Those values are the chili TCC and TCS values
(28.63% and 61.03%, respectively) and onion TCC (12.02%). Broccoli and asparagus had the
lowest values. A study with curcurbits and a different organic contaminant, p,p’-DDE, found
phytoextraction values ranging from 0.023% to 0.78% (White and others 2003). However, the
plants were grown in contaminated soil rather than hydroponic media, so it is not surprising that
the phytoextraction would be so much lower in a soil study since less of the chemicals would be
bioavailable. Aryal (2010) found values of 0.0014 for TCC and 0.024 for TCS in the hydroponic
study, and 15.87 for TCC and 3.72 for TCS in the soil column study
The transpiration stream concentration factor (TSCF) is the mass of chemical in the shoot
divided by the product of the total transpiration and the average of the initial and final media
concentration (H. Zhang 2009). It represents the ratio of concentration in the sap and the nutrient
media and can be used to quantify how easily chemicals can be taken up and translocated in a
plant through its transpiration stream. Figure 13 shows the TSCFs for both TCS and TCC.
Onion had by far the highest TSCF for both chemicals. Potato, okra, and cabbage also had high
values for both chemicals. Chili had a high value for TCC, but a mid-range value for TCS.
Asparagus had the lowest TSCFs for both TCC and TCS, followed closely by broccoli and
tomato. Celery had a low value for TCS, but a mid-range value for TCC.. The high values
indicate the plants such as onion use mainly transpiration to uptake TCC and TCS, whereas
plants with lower values likely use another mechanism more than transpiration.

40

TSCF

4.8E-07
4.6E-07
4.4E-07
4.2E-07
4E-07

Transpiration Stream Concentration Factor

5E-08

TSCF

4E-08

TCS

3E-08

2E-08
1E-08

0

TSCF

1.00E-06

Transpiration Stream Concentration Factor

9.00E-07
8.00E-07
4.00E-08
3.50E-08
3.00E-08

TCC

TSCF

2.50E-08

2.00E-08
1.50E-08
1.00E-08
5.00E-09
0.00E+00

FIGURE 13. Transpiration Stream Concentration Factors for TCS and TCC. Error bars indicate
standard error.

41

4.3 Toxicity of TCC and TCS

Table 6 shows the observations made on toxicity effects from the antimicrobials on the
plants compared to the plants grown in the control solution (not containing TCC or TCS). In this
study several plants had negative effects at 500ppb TCS when treated for 1 month. With okra
and onion, the plant in the control media also had negative effects, presumably due to the
methanol in the control media. Therefore, for those two plants the toxic effects may be due to the
methanol instead of the TCC or TCS. Figure 14 shows data on cabbage toxicity as an example.
The graph shows the number of dried and healthy leaves on both the control plant and the treated
plants. For cabbage the control had all healthy leaves, while most of the leaves on the treated
plants dried out. Figure 15 shows the comparison between the control cabbage and two of the
treated cabbage plants. The dry mass of the treated cabbage shoots ranged from 0.25 to 1.34,
indicating a range of health conditions. This range in health conditions possibly brought on by
TABLE 6. Observations on toxicity effects of TCC and TCS on plants compared to control
plants.
Plant

Treated with TCC/TCS

Control

Cucumber

Yellowing of leaf margins in older
leaves.
New leaves have yellow veins

No negative effect

Cabbage

Yellowing and drying of older
leaves
Slower new leaf formation

No negative effect

Okra

Yellowing of leaf margins in older
leaves.
New leaves have yellow veins
Blotching
Leaf fall

Yellowing of leaf margins in older
leaves.
New leaves have yellow veins
Blotching
Leaf fall

Onion

Drying of older leaves after 2
weeks

Drying of older leaves after 2 weeks
42

the toxicity of the chemicals could lead to a decrease in uptake. For instance, the chili plants,
some of which had a pest problem, had the widest variability in its data. Some of this variability
may have been caused by the health conditions of the plants, though more testing would need to
be done to make any solid conclusions.

12

Leaf number

10
8

Healthy

6

Dried

4
2
0

Control

Treated

FIGURE 14. Cabbage toxicity comparison between the control and treated plants as
shown by the number of healthy and dried leaves

FIGURE 15. Pictures of cabbage control (left) and two treated cabbage plants (middle and
right)

43

4.4 Potential Exposure to antimicrobials

Phytoremediation is a good way to remove contaminants from soil, but if the
contaminants are too concentrated in edible portions, food crops cannot be used for
phytoremediation. Furthermore, if contaminants are concentrated enough in edible portions of
food crops, the use of biosolids may need to be reconsidered. Since the hydroponic study
represents a scenario of maximum bioavailability of the chemicals, it can be considered a “worstcase scenario” of the accumulation of the chemicals by crops. Therefore, if there is no health
hazard from the amounts of chemicals taken up in the hydroponic study, there is not likely to be
a health hazard from the use of biosolids on agricultural fields. The value of no observable
adverse effect level (NOAEL) for TCS is 30 mg/kg (ppm) and the lowest observed adverse effect
level (LOAEL) is 100 mg/kg (ppm) (USEPA 2008). The NOAEL for TCC is 25 mg/kg (ppm)
and the LOAEL is 75 mg/kg (ppm) (USEPA 2002). However, those values do not consider the
possibility of endocrine disruption, which recent studies show could be a potential risk for TCS
and TCC (Chen and others 2008; USEPA 2010; USFDA 2010). Figure 16 shows the exposure
in ng TCC and TCS/kg crop/day based on consumption data from the Exposure Factors
Handbook from the National Center for Environmental Assessment (Assessment 2011)
(pumpkin and zucchini data came from Aryal and Reinhold, 2011). The exposure was calculated
by multiplying the concentration in the edible portion of the plant (shoot, root, or bulb) by the
average consumption by each age group by the average dry weight content of the crop. Note that
TCC has a much lower scale than TCS. Figure 16 shows that infants are exposed to higher
concentrations of both TCC and TCS. It also shows that the highest exposure to TCS comes
from zucchini and pumpkin, while the highest exposure to TCC comes from cabbage, potato, and

44

chili pepper. Figure 17 shows the estimated exposure from the mean of the crops compared to
3

other routes of exposure. The estimated exposure from vegetables is 10 times greater than that
from drinking water and 10

0.5

2

to 10 times less than that from product use, which refers to using

products containing TCC or TCS. The total estimated exposure is 10

2.9

3.3

to 10

times less than

the acute NOAEL. The total exposure from water, product use, and exposure from vegetables
(as estimated by this study) is still three orders of magnitude less than the NOAEL.

45

Potato
Broccoli
Pumpkin
Beet
Chili

Whole population
Middle age adult
1-2 years

Cabbage
Cucumber
0

400
800
Exposure (ng TCS/kg-d)

0

20

1200

1600

Potato
Asparagus
Broccoli
Zucchini
Pumpkin
Onion
Beet
Celery
Chili
Okra
Cabbage
Tomato
Cucumber
40
60
Exposure (ng TCC/kg-d)

80

FIGURE 16. Potential exposure to antimicrobials from food crop consumption

46

Total
exposure

NOAEL
Product
use
Mean
Vegetables
Sum Exp.
Vegetables

TCC
TCS

Water
0

2
4
6
Log Exposure (log ng/kg/d)

FIGURE 17. Potential exposure from crops compared to other routes

47

8

CHAPTER 5: CONCLUSIONS

5.1 Conclusions
This study found that the eleven plants tested took up TCC and TCS after one month of
exposure in a hydroponic system. In all crops tested the concentration of the chemicals in the
roots were much larger (two or three orders of magnitude) than the concentrations in the shoots.
The translocation factors were slightly lower than those found in pumpkin and zucchini in
previous research, which is likely due to the shorter exposure time. Chili and okra seem to have
high potential to take up both antimicrobials because of their high RCFs. Onion had a high RCF
for TCC. Celery, broccoli, and asparagus had the lowest RCFs for both TCC and TCS. Since
more TCC and TCS was removed from the media than was found in the plant tissue, plant uptake
was not the only mechanism responsible for removing these chemicals from solution, though
more testing is needed to determine what exactly caused the TCC and TCS to be removed from
the solution. TCC and TCS were found in precipitate collected from the bottom of the bottle
after the beet experiment, though it is uncertain if that is from settling, microbial activities, or
attachment to particulates. Based on the crops studied in this project, exposure to TCC and TCS
from consumption of plants exposed to contaminants would not pose a health risk, since the sum
of exposure from all the crops studied is less than estimated exposure from use of products
containing TCC and TCS. The total exposure from estimated water exposure, product use
exposure, and vegetable consumption exposure is still three orders of magnitude less than the
reported NOAEL. However, this is not considering recent findings that indicate that TCC and
TCS could both be endocrine disrupting chemicals. This study furthers research that shows that
crops can uptake organic contaminants found in soil, which aides in governmental reviews of
such chemicals.
48

5.2 Future Research
The next phase in this research involves testing crops that took up the most TCC and TCS
in the hydroponic study in a soil system to have a better understanding of the real-world system.
There will also be more investigation into what mechanisms are used to take up TCC and TCS
and whether they are passive or active. For that, the transpiration stream and enzyme activity will
need to be closely monitored during the exposure period. Furthermore, it would be beneficial to
test even more types of crops. Originally this study was going to include fruits and berries in
order to include several types of food crops, but the varieties selected did not thrive in a
laboratory environment and therefore were not used. Comparing fruits and vegetables would be
an interesting and more complete look into both phytoremediation possibilities as well as
exposure potential.

49

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