MICROBIAL PERSISTENCE IN THE PRESENCE OF TRICLOSAN ...

The Pennsylvania State University

The Graduate School

College of Science, Engineering and Technology

MICROBIAL PERSISTENCE IN THE PRESENCE OF TRICLOSAN:

EVALUATION OF GREEN ROOF SUBSTRATES IRRIGATED WITH

ARTIFICIAL GREYWATER

A Thesis in

Environmental Pollution Control

by

Heather C. Bloss

© 2015 Heather C. Bloss

Submitted in Partial Fulfillment of the Requirements

for the degree of

Master of Science

May 2015

ii

The thesis of Heather C. Bloss was reviewed and approved* by the following:

Katherine H. Baker Associate Professor of Environmental Microbiology Thesis Adviser Shirley E. Clark Associate Professor of Environmental Engineering Program Coordinator Daniel M. Borsch Senior Lecturer *Signatures are on file in the Graduate School

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ABSTRACT

The installation of green roofs is an increasing trend to reduce the urban heat

index, control stormwater runoff, and provide wildlife habitat. Lightweight soil substrate

can reduce the modifications required for existing structures to support a green roof.

Recycled crumb rubber offers an alternative to conventional green roof medium that both

reduces the weight of a green roof and repurposes tires entering the waste stream.

Vegetation can be maintained on such roofs through the reuse of household greywater,

significantly reducing freshwater demand. Analysis of the microbial quality of

conventional and recycled soil irrigated with artificial greywater was evaluated via

culturable heterotrophs and most probable number assays for coliforms. Samples from

both soil types irrigated with greywater supplemented with triclosan contained organisms

resistant to triclosan. Triclosan resistant Escherichia coli were detected on two occasions

in commercial media but did not persist. At no point were resistant E. coli detected in

recycled substrate. The presence of triclosan caused fluctuations in the concentration of

total coliforms present in commercial soil. Replicate plating further underscored the

effect low-level, consistent biocide exposure has on the ability of microorganisms to

develop resistance.

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TABLE OF CONTENTS List of Tables ..................................................................................................................... vi

List of Figures ................................................................................................................... vii

Acknowledgements ............................................................................................................ ix

Dedication ........................................................................................................................... x CHAPTER 1: INTRODUCTION .................................................................................... 1

1.1 Green Roofs ....................................................................................................................... 1

1.2 Greywater .......................................................................................................................... 3

1.3 Triclosan ............................................................................................................................ 7

1.4 Resistance ........................................................................................................................ 11

CHAPTER 2: EXPERIMENTAL ................................................................................. 14

2.1 Model Green Roof Systems ................................................................................................. 14

2.1.1 Media Selection ............................................................................................................ 14

2.1.2 Planting ......................................................................................................................... 14

2.1.3 Vegetation Control ....................................................................................................... 15

2.1.4 Greywater ..................................................................................................................... 16

2.1.5 Sampling ....................................................................................................................... 16

2.2 Soil Nutrient Quality ............................................................................................................ 17

2.3 Heterotrophic Plate Counts .................................................................................................. 17

2.4 Most Probable Numbers ...................................................................................................... 17

2.5 Antibiotic and Triclosan Resistance .................................................................................... 18

CHAPTER 3: RESULTS ............................................................................................... 20


3.2 Soil Nutrient Quality ............................................................................................................ 20

v

3.3 Heterotrophs ......................................................................................................................... 23



CHAPTER 4: DISCUSSION ......................................................................................... 38


4.2. Soil Nutrient Quality ........................................................................................................... 38

4.3 Heterotrophic Plate Counts .................................................................................................. 40



4.6 Conclusion ........................................................................................................................... 43

WORKS CITED ............................................................................................................. 45

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LIST OF TABLES

Table 1: Grass Seed Composition ..................................................................................... 15

Table 2: Replica Plate Preparations .................................................................................. 18

Table 3: Potential Leachate Comparison to Water Quality Standards ............................. 39

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LIST OF FIGURES

Figure 1: Layers of a Conventional Green Roof ................................................................. 2

Figure 2: Chemical Structure of Triclosan .......................................................................... 7

Figure 3: Total Nitrogen ................................................................................................... 21

Figure 4: Total Ammonia-Nitrogen .................................................................................. 22

Figure 5: Chemical Oxygen Demand ............................................................................... 22

Figure 6: Total Phosphorus ............................................................................................... 23

Figure 7: Heterotrophs in Greywater Irrigated Recycled Soil .......................................... 24

Figure 8: Heterotrophs in Greywater with Triclosan Irrigated Recycled Soil .................. 24

Figure 9: Heterotrophs in Greywater Irrigated Commercial Soil ..................................... 25

Figure 10: Heterotrophs in Greywater with Triclosan Irrigated Commercial Soil ........... 25

Figure 11: Coliforms in Greywater Irrigated Recycled Soil ............................................. 26

Figure 12: Coliforms in Greywater with Triclosan Irrigated Recycled Soil .................... 27

Figure 13: Coliforms in Greywater Irrigated Commercial Soil ........................................ 27

Figure 14: Coliforms in Greywater with Triclosan Irrigated Commercial Soil ................ 28

Figure 15: Antibiotic and Triclosan Replicates of Greywater Irrigated Recycled Soil

Heterotrophs .............................................................................................................. 30

Figure 16: Replica Plate Averages of Greywater Irrigated Recycled Soil Heterotrophs . 31

Figure 17: Antibiotic and Triclosan Replicates of Greywater with Triclosan Irrigated

Recycled Soil Heterotrophs ...................................................................................... 32

Figure 18: Replica Plate Averages of Greywater with Triclosan Irrigated Recycled Soil

Heterotrophs .............................................................................................................. 33

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Figure 19: Antibiotic and Triclosan Replicates of Greywater Irrigated Commercial Soil

Heterotrophs .............................................................................................................. 34

Figure 20: Replica Plate Averages of Greywater Irrigated Commercial Soil Heterotrophs

................................................................................................................................... 35

Figure 21: Antibiotic and Triclosan Replicates of Greywater with Triclosan Irrigated

Commercial Soil Heterotrophs ................................................................................. 36

Figure 22: Replica Plate Averages of Greywater with Triclosan Irrigated Commercial

Soil Heterotrophs ...................................................................................................... 37

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ACKNOWLEDGEMENTS

I would like to thank the following people for their contributions to this work:

A.R. Bowers, J.L. Reed, T.M. Kell, L.K. Mehalik, J.M. Felker, and A.S. Mickey

In addition, words cannot express enough gratitude to Dr. Katherine H. Baker, Dr.

Shirley Clark, and Dr. Daniel Borsch for their academic support, guidance, and words of

encouragement throughout my time spent researching at Penn State.

I would like to thank my family and friends for their support through this process.

Specifically, I would like to thank my husband for his extraordinary support through

early mornings, late nights, and everything in between.

Finally, I must thank Lily Bloss for inspiration, motivation, and contribution to this work.

x

DEDICATION

This work is lovingly dedicated to my daughter, Lily, who has inspired me in ways I never

knew existed prior to meeting her.

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CHAPTER 1 INTRODUCTION

1.1 Green Roofs

Green roofs date back centuries as aesthetically appealing rooftop gardens.

Beyond appearance, green roofs offer benefits such as stormwater runoff control,

reduction to the urban heat index, and providing wildlife habitats. One disadvantage to

green roof installation is the structural modifications necessary to support the load of soil

on the roof. For this reason, there is a demand for a lightweight soil substitute that still

supports plant growth. Commercially available green roof growth substrate typically

consists of a combination of peat and expanded shale. A more lightweight option is to

utilize recycled tires in the form of shredded or crumb rubber in combination with

compost material to create an artificial soil suitable for growing plants. This reduces the

number of tires entering the waste stream as well as reduces the modifications required

for an existing structure to support a green roof.

Conventional green roof setup involves several layers of materials (Figure 1).

The top layer, generally the thickest layer, is the substrate layer. This is followed by a

filter layer to keep small soil particles from clogging the third layer, the drainage layer.

There are several other layers beneath this that are designed to protect the roof from

water and potential root damage. Vila et al. (2012) examined rubber crumbs as a drainage

layer. They determined that the use of rubber crumb did not present any issues with

regards to water retention capacity, plant health, or temperature of the growing medium

(Vila et al, 2012).

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Figure 1: Layers of a Conventional Green Roof

Image Source: http://greenerheights.wordpress.com/

Standard soil supports the growth of plants and microbial species including both

bacteria and fungi. In the recycled material, the organic matter provides nutrients for

growth while the rubber component provides structure to support a root matrix. Mickey et

al. (2013) illustrated the growth capabilities of the both recycled tire soil and commercial

growth medium by monitoring growth on two green roof plots on a roof in Harrisburg,

PA, USA. Microbial soil quality was also monitored. Culturable heterotrophs decreased

in commercial growth substrate and increased in the recycled tire substrate over time.

Initial sampling indicated that the commercial plot contained more coliforms than the

recycled plot. Over time, coliforms declined to below detectable levels on both plots

(Mickey et al., 2013).

Crampton et al. (2014) observed that rainwater causes leaching of materials from

crumb rubber that are inhibitory to the growth of bacteria. When the concentration of

rubber leachate was diluted to concentrations more characteristic of green roof

application, bacterial survival significantly increased. Upon investigating the effects of

zinc leachate on bacterial growth, it was observed that a strain of Salmonella enterica

serovar Typhimurium less susceptible to high concentrations of zinc expressed an up-

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regulation in the zinc efflux gene zntA and a down-regulation in the zinc influx gene

znuA. The authors suggest that washing crumb rubber in an acidic solution prior to

application may be beneficial (Crampton et al., 2014).

1.2 Greywater

Greywater reuse is increasingly considered to be a sensible option for irrigation in

areas where fresh water supply is limited (Pinto et al, 2010). Greywater includes the

water collected from households, office buildings, schools, laundry facilities, etc. that

was used for bathing, dishwashing, laundering clothing and hand washing (Eriksson et al,

2002). It differs in composition from blackwater, which is the water used for toileting.

Greywater composition will vary from location to location and will be affected by

individual household lifestyle (Eriksson et al., 2002). Greywater collected from kitchens

can include soaps from dishwashing manually or in a machine and food wastes such as

fats, oils, grease, sugars, artificial sweeteners, coffee, tea, starches, and pathogenic

microorganisms from raw meat and egg preparations. Greywater collected from

bathrooms and laundry machines can contain shampoos, soaps, perfumes, dyes, hair, hair

styling products, bleaches and other cleaning agents, surfactants, fabric softeners,

preservatives, toothpaste, cosmetics and microorganisms associated with skin, respiratory

and intestinal flora (Eriksson et al., 2002; Ghaitidak and Yadav, 2013; Maimon et al,

2010). The volume contributed from kitchen, bathroom, and laundry can also vary by

location and will affect the ratio of these products to water as well as the temperature of

the collected water. Pinto et al. (2010) showed that laundry detergents could have a

significant effect on the pH of water. They created artificial greywater using laundry

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detergent only and observed an increase from 7.0 in plain water to 10.5 in greywater

using standard potable water (Pinto et al., 2010).

Surfactants are concentrated when greywater is separated from the total

wastewater stream. Untreated greywater retains soaps and detergents and carries them on

to soil during irrigation. These detergents can alter soil properties and potentially affect

vegetation. Travis et al. (2010) observed significantly greater amounts of surfactants, oil,

and grease in soil irrigated with untreated raw greywater than in soil irrigated with either

treated greywater or freshwater. The untreated greywater also promoted water repellency

in sand and loam soils. Based on the effects of short-term irrigation, they hypothesize that

long-term irrigation with untreated greywater will result in more exaggerated effects as

lipids accumulate in the soil. Treated greywater irrigation produced results similar to

using freshwater, highlighting the importance of treatment before use (Travis et al.,

2010).

Since greywater is diverted from toilet water, greywater appears to be a suitable

choice for reuse in applications that do not necessitate fresh water. While the microbial

load of greywater is less than in standard wastewater, opportunistic pathogens still

present a health risk upon exposure during use. Furthermore, organisms could potentially

persist following application to soil. The persistence of high numbers of coliforms in soil

could present problems if the soil is to be used for growing crops, as a recreational space,

or in a green roof setting where runoff could be contaminated. While avian and animal

defecation intermittently adds pathogens to the soil, consistent irrigation with greywater

has the potential to periodically dose the overall soil area with pathogenic organisms.

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Pathogen removal is crucial for the reuse of greywater. Several methods have

been investigated for home treatment of greywater including coagulation and

flocculation, constructed wetland treatment, filtration, rotating biological contactor,

membrane bioreactor, sequencing batch reactor, and up-flow anaerobic sludge blanket

reactor (Ghaitidak and Yadav, 2013). Most treatments are designed to address wastewater

and are not appropriate for greywater, particularly on the household scale. Gross et al.

(2007) highlight the issues of varying volume and composition over time for household

greywater collection and designed a recycled vertical flow bioreactor (RVFB) that

maintains steady flow for treatment. The RVFB reduced chemical contaminants to levels

that are acceptable for recreation or irrigation and reduced E. coli concentration to meet

US EPA recreational water quality criteria (Gross et al., 2007).

Winward et al. (2008) developed three constructed wetland treatments, vertical

and horizontal flow reed beds, and a green roof water recycling system. All treatments

were able to meet Germany’s standards for greywater reuse for total coliforms and

Pseudomonas aeruginosa using low strength greywater. However, no treatment could

produce useable water when high strength greywater was treated. Only the vertical flow

reed bed met California State’s total coliform standard in 44% of samples tested.

Additionally, they observed an increase in concentrations of total coliforms, E. coli, and

P. aeruginosa in greywater supplemented with additional shampoo (Winward et al.,

2008). This indicates that the storage of greywater could provide conditions favorable to

bacterial growth.

The presence of the fecal coliform, E. coli, is the best-known efficient indicator of

fecal contamination in water and soil. Fecal contamination may not be the only issue the

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reuse of greywater presents. As noted by Gross et al. (2007), P. aeruginosa and

Staphylococcus aureus may also be of interest when evaluating the safe reuse of

greywater. These organisms are known colonizers of humans. Both are capable of

causing opportunistic infections of the skin and respiratory tract leaving the elderly,

young, and immune-compromised most vulnerable (Winward et al., 2008).

In 2013, Benami et al. evaluated soil treated with freshwater and soil treated with

greywater, excluding kitchen waste, treated by a recirculating vertical flow constructed

wetland. They found that both freshwater and greywater irrigated soils contained the

same types of pathogens indicating that treated greywater reuse may not pose any

potential health risk. However, they were unable to differentiate viable from nonviable

pathogens and suggest further research is necessary (Benami et al., 2013).

Literature is limited on the evaluation of the removal of chemical contaminants in

soaps and surfactants from greywater following treatment. Eriksson et al. (2003)

identified nearly 200 xenobiotic organic compounds in household greywater collected

from bathrooms only. If greywater is to be reused for irrigation purposes, the fate of these

chemicals and whether they are removed during treatment should be investigated prior to

ground application. One of these compounds, triclosan, is being increasingly studied.

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1.3 Triclosan

Figure 2: Chemical Structure of Triclosan

Image Source: http://pubchem.ncbi.nlm.nih.gov/image/imagefly.cgi?cid=5564&width=300&height=300

Triclosan (5-chloro-2-(2,4-dichlorophenoxy)phenol, TCS) is a chlorinated,

residue-forming antimicrobial agent that is used in various commercial products. TCS is

included in body care products such as hand soaps, toothpaste, and deodorants, as well as

laundry detergent, cleaning agents, plastics, paints, fabrics, clothing, pillows, mattresses,

carpets, toys, pacifiers, toothbrushes, etc. (Levy, 2001; Lozano et al., 2011; Halden,

2014). TCS has become almost ubiquitous in products advertised as “antibacterial.”

Liquid hand soaps generally contain TCS in concentrations of 0.1% to 0.45%

weight/volume (Aiello et al., 2007).

An increasing amount of data indicates that including TCS in household products

is not beneficial to consumers or the environment and in fact may be harmful. In

December 2013, the U.S. Food and Drug Administration proposed new legislature that

would require companies to “provide more substantial data to demonstrate the safety and

effectiveness of antibacterial soaps” (FDA, 2013). In response to this, some companies

such as Crest toothpaste now advertise their products as “100% triclosan-free” (Crest,

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2015). S. Levy (2001) found that TCS alone may be capable of killing E. coli but that

adding the TCS to soap appeared to reduce it’s effectiveness. Furthermore, TCS cannot

reach its potential efficacy when most individuals do not wash their hands for the

recommended 30 seconds (Levy, 2001; Aiello, 2007). Contact time is critical to a

biocide’s antibacterial activity.

Human exposure is greatest through application to the body and through the use

of TCS treated toothpastes. Infant exposure can be attributed to house dust and hand-to-

mouth activity (Bedoux et al., 2012). Studies on the health effects upon human exposure

are limited. Endocrine disruption due to TCS exposure was observed in rats and frogs

(Halden, 2014; Bedoux, 2012). Mean human urine concentrations were found to be

approximately 12 µg/L detected in roughly 70% of tested samples (Bedoux et al., 2012).

In 2007-2009 TCS was detected in 100% of urine samples and 51% of provided cord

blood samples from 181 expectant mother/infant pairs (Pycke et al, 2014).

Following the use of body care products, TCS enters the household wastewater

flow and is discharged to the treatment plant. Influent wastewater concentrations in the

U.S. have been reported to range from 600 – 86,200 ng/L (Bedoux et al., 2012). TCS is a

lipophilic compound with a high octanol/water partitioning coefficient of 4.8 (Halden and

Paull, 2005), which makes the majority of TCS partition to sludge during wastewater

treatment. Consistent low dosing of surface waters occurs from the remaining TCS in

treated water. This can add up to as much as 5200 – 18,824 kg/yr deposited in to U.S.

waterways (Drury et al, 2013).

Microbial communities play an important role in the health of aquatic ecosystems.

The addition of an antimicrobial to the biome has the potential to induce change at the

9

lowest levels and in time affect larger organisms as well. Drury et al. (2013) investigated

the effects TCS has on benthic bacterial communities. They found that exposure to TCS

in both the field and laboratory settings resulted in increased TCS resistance.

Additionally, TCS reduced sediment bacterial diversity and caused shifts in taxonomic

composition of sediment communities (Drury et al, 2013).

Bacterial biofilms participate in the removal of inorganic nutrients from water

systems. Proia et al. (2011) found that TCS exposure lead to immediate effects on biofilm

viability. Biofilms recovered within two weeks following exposure, however, the authors

indicate that frequent pulse doses of TCS to biofilm communities could give rise to long-

term effects on structure and function (Proia et al., 2011).

TCS has the potential to be methylated by biological processes during wastewater

treatment (Ying et al., 2007). Methyltriclosan (MTCS) is a more persistent pollutant that

is more lipophilic than it’s parent compound TCS. MTCS has an octanol/water

partitioning coefficient equal to 5.0 (Lozano et al, 2011). Lipophilic compounds tend to

bioaccumulate in fish and other aquatic organisms and MTCS is no exception. MTCS

was detected in fish up to 600 ng/g (Bedoux et al., 2012) as well as in fish in Switzerland

(Ying et al., 2007). No studies at this time associate any linkage between human

exposure and fish consumption.

Biosolid application to soil, most commonly on farmland, introduces TCS to soil.

In 2007, 55% of the 7 million dry tons of biosolids generated were land applied (Lozano

et al., 2012). Cha and Cupples (2010) determined that the leaching of TCS into

groundwater is unlikely due to the compounds high affinity for soil. Lozano et al. (2012)

observed that all TCS detected one year following application was within the first 10 cm

10

of soil. Kwon and Xia (2012) investigated a field that had biosolids applied for 33

consecutive years and detected TCS in the top 15 cm of soil only. Volatilization is not of

concern as the vapor pressure coefficient is low (4 x 10-6 mm Hg at 20°C) (Lozano et al,

2012).

TCS can also undergo methylation in soil. Degradation of TCS occurs under

aerobic conditions. Reports of half-life range from 7.5 – 104 days (Lozano et al., 2012;

Ying et al., 2007; Park et al., 2013). Degradation is significantly slower in biosolid-

amended soil than in soil without biosolids (Park et al., 2013; Kwon and Xia, 2012).

Lozano et al. (2012) and Kwon and Xia (2012) report that 12.7% and 12.1%,

respectively, of degraded TCS was converted to MTCS. Lozano et al. (2012) report that

84% of TCS applied with biosolids degraded within one year of application and soil

returned to background levels after two years. MTCS increased in concentration over

year one, then declined through year two and three but never reached background levels

in the three consecutive years tested, illustrating the persistence of MTCS in soil. The

estimated half-life for MTCS was 443 days (Lozano et al., 2012).

Information is lacking on whether MTCS has more significant endocrine

disrupting properties than TCS. TCS shares structural similarity with the known

persistent environmental pollutants, dioxins, and has been labeled as a predioxin (Halden,

2014). TCS carries the potential for conversion to a dioxin following photodegradation

(Bedoux et al., 2012; Halden, 2014).

Data on the effect of TCS exposure to soil microbial communities are limited.

Stream sediment data, as previously mentioned, indicates negative effects on community

diversity (Drury et al., 2013). Previous research in this laboratory, (Harrow et al., 2011),

11

also showed a reduction in microbial community diversity in soil irrigated with greywater

containing TCS. The occurrence of antibiotic resistant bacteria was also promoted by the

presence of TCS in the irrigation water (Harrow et al, 2011).

1.4 Resistance

McMurry et al. (1998) identified the specific gene, FabI, acted upon by TCS.

This confirmed that TCS acts upon a specific bacterial target and is not a general biocide,

as once thought (McMurry et al., 1998). The FabI gene is responsible for the production

of enoyl-acyl carrier protein reductase, which allows for the synthesis of fatty acids. A

mutation in FabI significantly disables TCS from causing cell lysis (McMurry et al.,

1998). Mutant strains of E. coli were uninhibited by up to 100 times more TCS than wild-

type strains (Levy, 2001). Furthermore, the mutant E. coli were capable of surviving in

soap containing TCS diluted with only 3 parts water (Levy, 2001). A related gene, FabK,

provides natural resistance to TCS in other bacterial species including E. faecalis and S.

pneumonaie (Levy, 2001). Widespread environmental exposure to TCS allows for the

potential for naturally resistant organisms to flourish, as well as for the promotion of the

expression of resistance genes in wild-type bacteria.

Resistance to TCS may also confer resistance to antibiotics to the organism. A

second mechanism by which bacteria exert resistance to TCS is through the expression of

efflux pumps (Levy, 2001). Efflux pumps vary across bacterial species by what each

pump is capable of pumping out of a particular cell. The AcrAB pump in E. coli can

pump “pine oils, organic solvents, triclosan, quaternary ammonium compounds,

chloroxanol, and chlorhexidine” out of the cell (Levy, 2001).

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Multi-drug efflux pumps are also largely implicated as responsible for clinical

antibiotic resistance. Chuanchuen et al. (2001) demonstrated that TCS is also a substrate

for the nfxB gene in P. aeruginosa, selecting for multi-drug resistance and a 94-fold

increase in the minimum inhibitory concentration (MIC) of ciprofloxacin. Birosova and

Mikulasova (2008) also illustrated that repeated exposure to 0.25 and 0.5 MIC of TCS

promoted ciprofloxacin resistance in Salmonella enterica serovar Typhimurium. All

tested doses (0.125, 0.25, and 0.5 MIC TCS) increased the resistance of Salmonella

enterica serovar Typhimurium to TCS and repeated low-level exposure to 0.25 MIC TCS

conferred resistance to tetracycline and chloramphenicol as well (Birosova and

Mikulasova, 2008). Overproduction of the efflux pump SmeDEF in Stenotrophomonas

maltophilia is responsible for resistance to tetracycline, chloramphenicol and

ciprofloxacin (Sanchez et al., 2005).

Low-level environmental exposure may promote the expression of multi-drug

pumps in natural habitats. Birosova and Mikulasova (2008) demonstrated under

laboratory conditions that exposure of Salmonella enterica serovar Typhimurium to low-

level TCS concentrations helped to retain the antibiotic resistant strains in the population,

but did not increase the total amount of strains present (Birosova and Mikulasova, 2008).

Surface waters accepting contribution from wastewater treatment plant (WWTP) outflow

were examined in New Jersey, USA. Middleton and Salierno (2013) found that the level

of TCS resistance was dependent upon the sampling location in relation to the WWTP

discharge site. Isolates from effluent were significantly more resistant to TCS than

upstream isolates. 78.8% of fecal coliform isolates were resistant to TCS expressing a

MIC of 43.2 µg/mL. 89.6% of Citrobacter freundii isolates were resistant to four classes

13

of antibiotics (Middleton and Salierno, 2013). A second sampling site in New Jersey

revealed another relationship between TCS resistance and resistance up to three classes of

antibiotics. Fecal coliform species that exhibited high-level resistance to TCS included

Escherichia, Enterobacter, Serratia, and Citrobacter. TCS resistant bacteria were

significantly more resistant to chloramphenicol and nitrofurantoin (Middleton and

Salierno, 2013).

14

CHAPTER 2 EXPERIMENTAL

2.1 Model Green Roof Systems

2.1.1 Media Selection

Two types of green roof systems were constructed using commercial media and

shredded recycled tires obtained from several tire recyclers. The compost for the media

and formulated commercial media were supplied by the Penn State Recycling Center.

The recycled media component was made from a combination of 85 percent by weight

rubber and 15 percent by weight compost material. The rubber was mixed in a 50/50

proportion of shredded rubber and rubber crumbs.

2.1.2 Planting

Each type of soil was used to fill 18 individual pots on each of eight separate

trays. Each pot was filled with the same volume of media. Given the differences in the

bulk density between the media, this resulted in each type of pot having a different

weight of media added to it. 180 ± 0.5 grams of soil were added to each commercial pot

and 80 ± 0.5 grams of rubber mixture were added to each recycled pot. All pots were

contained within a larger tray to collect leachate. The trays were divided into four

categories, Recycled Greywater (RG), Recycled Triclosan (RT), Commercial Greywater

(CG), and Commercial Triclosan (CT) allowing for one replicate tray (18 pots) of each

treatment type. Each tray was planted with 0.5 ± 0.1 grams of grass seed by sprinkling

seed over each pot using a 1-teaspoon measuring spoon. The seed used was a

15

Landscaper’s Sun and Shade Mix of Premium Grass seed. A detailed list of the seed

types in the mixture can be found in Table 1. The prominent type of seed in the mixture

was ryegrass. Trays were placed on individual shelves keeping all RT and CT trays on

the lowest shelves to prevent contamination of TCS-free trays in the event of a leak.

Light was provided by a fluorescent light above each individual shelf that was controlled

by a timer set to adjust with the local sunrise and sunset in the United States Eastern

Standard Time zone.

Table 1: Grass Seed Composition: Stauffers of Kissel Hill Landscaper’s Sun and Shade

Mixture

29.75% Protégé GLR Perennial Ryegrass

29.51% Manhattan 6 Perennial Rye Grass

13.88% Kentucky Bluegrass

13.66% Creeping Red Fescue

5.99% Appalachian Kentucky Bluegrass

5.89% Brooklawn Kentucky Bluegrass

1.32% Inert Matter

2.1.3 Vegetation Control

Plant growth was trimmed twice during the study. The grass was cut to a height of

one half inch above the rim of each pot using standard scissors. Grass clippings were

dried according to treatment category.

16

2.1.4 Greywater

Trays were watered according to their classification. Synthetic greywater (GW)

and greywater supplemented with triclosan (GWT) were created following the methods

used by Harrow et al. (2011). The dosage of 20mg/L of TCS in the artificial greywater

simulated a 100-fold dilution of 0.2% (2,000 mg/L) soap into water. The concentration of

TCS is diluted in water during the use of TCS-containing products.

Greywater was made immediately prior to use each week with standard tap water.

In addition to the recipe used, researchers making the greywater simulated hand washing

in the container to add organisms to the water typically found colonizing human skin.

Several different researchers prepared the greywater throughout the course of the

research and no pattern was recorded to identify any person making the water in relation

to any sample. Trays were watered one day per week with 500mL GW or GWT divided

evenly among each pot in the tray. Two additional days per week each tray was watered

with 500mL of tap water. Leachate collection trays were rinsed with tap water once per

week.

2.1.5 Sampling

Samples were taken five times over the course of the study. One pot was

sacrificed per tray at each sampling. Any vegetation was removed from the pot and

discarded. Ten grams of the remaining soil was transferred aseptically to a sterile bottle

containing 95mL of sterile dilution water. The resultant mixture was utilized as the initial

dilution of soil and any further dilutions were made using this bottle. Six serial dilutions

of the initial soil mixture were made to complete the testing.

17

2.2 Soil Nutrient Quality

Fifteen to twenty grams of soil were transferred to an aluminum tray and an initial

weight was recorded immediately after removing the grass from the pot. Remaining soil

was immediately frozen. Moist soil was dried for one week in a 105°C oven after which a

dry weight was recorded. Moisture content of the soil was calculated.

Nutrient testing was conducted to determine the Chemical Oxygen Demand

(COD), Total Nitrogen, Nitrogen as Ammonia and Total Phosphorus of each soil sample.

Test kits were ordered from Hach for each nutrient test and the tests were conducted

following the instructions provided.

2.3 Heterotrophic Plate Counts

Soil dilutions (103, 105, and 107) from each sample were plated onto regular

strength Tripticase Soy Agar (Difco). 100 microliters of each soil dilution was spread

onto the agar, plates were flipped when dry, and stored at 25°C contained in a humidity

chamber. Colonies were counted after five days of incubation.

2.4 Most Probable Numbers

Most Probable Number (MPN) tests were conducted following an adaptation of

EPA Method 1680. A presumptive test was conducted by inoculating five Hach tubes

containing 10mL of Laurel Tryptose Broth (LTB) with the initial soil dilution. The next

two serial dilutions were used to inoculate two additional sets of five tubes. This process

was repeated for all eight samples. Tubes were incubated at 35°C for 24 ± 2 hours.

18

Confirmation of the presence of E. coli was completed by transferring the culture

in each positive LTB tube to a tube containing 10mL of EC media. Negative LTB tubes

were allowed an additional 24 ± 2 hours of incubation at 35°C. EC tubes were incubated

in a 44.5°C water bath for 24 ± 2 hours. Negative tubes were placed back into the water

bath and checked after an additional 24 ± 2 hours. All LTB or EC tubes were incubated

for a maximum of 52 hours.

2.5 Antibiotic and Triclosan Resistance

Countable plates of both TSB and TSB + TCS agar were replicated using

sterilized velveteen and a replica-plating tool. All plates were replicated on to TS agar

supplemented with an antibiotic or combination of an antibiotic plus TCS. The nine

categories and antibiotic dosages can be found in Table 2.

Table 2: Replica Plate Preparations

Treatment Antibiotic (µg/mL) Triclosan (mg/L) Trypticase Soy Broth 0 0

Ciprofloxacin 10 0 Ciprofloxacin + Triclosan 10 20

Chloramphenicol 12.5 0 Chloramphenicol + Triclosan 12.5 20

Tetracycline 10 0 Tetracycline + Triclosan 10 20

Vancomycin 3 0 Vancomycin + Triclosan 3 20

The initial plate was pressed onto the velveteen and removed. The non-TCS plates

were then stamped onto the velveteen. The original plate was pressed onto the velveteen

19

again followed by the four plates containing TCS. The TSB plate was the last plate

stamped onto the velveteen.

All plates were incubated inside of plastic sleeves at 25°C for five days. On the

fifth day, all plates were moved to 4°C incubators. Antibiotic plates were counted and

compared to the original respective plates.

20

CHAPTER 3 RESULTS


Vegetation slowly died off towards the end of the study, which occurred over a

time in excess of a typical growing season. Thus, the final sampling was conducted on

pots in which the grass had completely died-off. The vegetation die-off was gradual and

began at the August sampling. The grass growing in commercial media died before the

grass growing in recycled media. Grass in commercial media appeared to have a more

dense root structure and growth filled in quicker. Small insects were observed living in

the trays beneath the commercial media pots. Although it is unclear when they were first

observed, the insects were rinsed away once each week during watering. They were

observed in one tray beneath one recycled sample towards the end of the study.

Dry weight of the grass was recorded following the trimming in August. There

was no visible difference between GW and GWT trays so the grass was divided by

substrate type. Recycled grass weighed 5.6 grams dry. Commercial grass weighted 7.83

grams dry. Commercial soil yielded approximately 1.4 times more vegetation than

recycled soil.

3.2 Soil Nutrient Quality

Nutrient testing revealed that recycled soil treated with greywater contained the

highest levels of all nutrients tested in all samples. There was no apparent effect on

vegetation or increased numbers of heterotrophs in the recycled media. Recycled soil

21

samples consistently contained higher amounts of all tested nutrients than the

complimentary commercial soil samples receiving identical irrigation (Figure 3; Figure

4; Figure 5; Figure 6). On average, soil treated with GW retained more overall nutrients

than soil treated with GWT. The presence of TCS in the soil correlates with reduced total

nitrogen, ammonia nitrogen, total phosphorus, and COD. While nutrients in commercial

soil were reduced in the presence of TCS, they were more similar to commercial GW

than recycled media GW and GWT treatments are to one another. Commercial soil

nutrient content was less affected by GWT treatment than recycled soil nutrients.

Figure 3: Total Nitrogen

0

2

4

6

8

10

12

14

16

18

GW - Recycled GW - Commercial GWT - Recycled GWT - Commercial

mg/

L

Total Nitrogen

22

Figure 4: Total Ammonia-Nitrogen

Figure 5: Chemical Oxygen Demand

0

0.2

0.4

0.6

0.8

1

1.2

1.4


mg/

L Total Ammonia-Nitrogen

0

50

100

150

200

250

300

350


mg/

L

COD

23

Figure 6: Total Phosphorus

3.3 Heterotrophs

The number of culturable heterotrophs increased over time in the recycled

medium for the GW treatment (Figure 7). Commercial soil irrigated with GW

experienced an increase and decrease in detectable heterotrophs that matched the growth

and decline in vegetation (Figure 9). Heterotrophs maintained more stable numbers in

GWT irrigated soils but these numbers were lower than GW irrigated soils (Figure 8;

Figure 10). TCS resistant organisms were not consistently detected in GW samples

throughout the course of the study for either substrate but were more frequently detected

in recycled soil. GWT treated samples contained TCS resistant organisms in both

commercial and recycled media in all samples. Overall, the numbers of TCS resistant

heterotrophs cultured were significantly greater in both substrates irrigated with GWT

than when irrigated with GW. The August sampling of commercial soil showed that

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35


mg/

L Total Phosphorus

24

almost all culturable GWT heterotrophs were triclosan-resistant organisms. The August

sampling was the last sampling with well-established vegetation.

Figure 7: Heterotrophs in Greywater Irrigated Recycled Soil

Figure 8: Heterotrophs in Greywater with Triclosan Irrigated Recycled Soil

0 1 2 3 4 5 6 7 8 9

10

6/23/14 7/21/14 8/11/14 9/15/14 9/29/14

log

CFU

/gra

m d

ry so

il

Sample Date

Heterotrophs in GW Irrigated Recycled Soil

Soy Agar

Soy Agar + Triclosan

0 1 2 3 4 5 6 7 8 9

10

6/23/14 7/21/14 8/11/14 9/15/14 9/29/14

log

CFU

/gra

m d

ry so

ul

Sample Date

Heterotrophs in GWT Irrigated Recycled Soil

Soy Agar


25

Figure 9: Heterotrophs in Greywater Irrigated Commercial Soil

Figure 10: Heterotrophs in Greywater with Triclosan Irrigated Commercial Soil

0 1 2 3 4 5 6 7 8 9

10

6/23/14 7/21/14 8/11/14 9/15/14 9/29/14

log

CFU

/gra

m d

ry so

il

Sample Date

Heterotrophs in GW Irrigated Commercial Soil

Soy Agar


0 1 2 3 4 5 6 7 8 9

10

6/23/14 7/21/14 8/11/14 9/15/14 9/29/14

log

CFU

/gra

m d

ry so

il

Sample Date

Heterotrophs in GWT Irrigated Commercial Soil

Soy Agar


26


Total coliforms increased with vegetation growth and declined when vegetation

decreased in all treatments except GWT irrigated commercial soil. Total coliforms

decreased faster in GWT treated soil than GW treated soil upon the loss of vegetation.

The number of total coliforms cultured from GWT treated commercial samples fluctuated

throughout the study. E. coli was not present in all GW samples but was detected on more

dates in commercial media than recycled media (Figure 11; Figure 13). TCS resistant E.

coli remained below detectable levels for all recycled media samples (Figure 12). TCS

resistant E. coli were cultured from two commercial media samples (Figure 14).

Figure 11: Coliforms in Greywater Irrigated Recycled Soil

0 1 2 3 4 5 6 7 8 9

10

6/23/14 7/21/14 8/11/14 9/15/14 9/29/14

log

MPN

/gra

m d

ry so

il

Sample Date

Coliforms in GW Irrigated Recycled Soil

Total

E. coli

27

Figure 12: Coliforms in Greywater with Triclosan Irrigated Recycled Soil

Figure 13: Coliforms in Greywater Irrigated Commercial Soil

0 1 2 3 4 5 6 7 8 9

10

6/23/14 7/21/14 8/11/14 9/15/14 9/29/14

log

MPN

/gra

m d

ry so

il

Sample Date

Coliforms in GWT Irrigated Recycled Soil

Total

E. coli

0 1 2 3 4 5 6 7 8 9

10

6/23/14 7/21/14 8/11/14 9/15/14 9/29/14

log

MPN

/gra

m d

ry si

l

Sample Date

Coliforms in GW Irrigated Commercial Soil

Total

E. coli

28

Figure 14: Coliforms in Greywater with Triclosan Irrigated Commercial Soil


Antibiotics unexpectedly appeared to have no effect on the growth of organisms

in commercial or recycled samples. Due to the fact that all four tested antibiotics did not

inhibit growth, the results were averaged (Figure 16; Figure 18; Figure 20; Figure 22).

There are no significant differences between the TSA replicate plate and the antibiotic

replicate plate for plates initially dosed with TCS or TCS-free plates in recycled media

(Figure 15; Figure 17). The average colony forming units (CFUs) on antibiotic plates

from commercial soils are lower than the average CFUs on TSA (Figure 20; Figure 22).

Figure 19 shows a slight sensitivity to ciprofloxacin in GW treated commercial soil that

reduces the average. Figure 21 shows that GWT treated commercial soil was more

sensitive to the combination treatment of ciprofloxacin and TCS than any other treatment.

0 1 2 3 4 5 6 7 8 9

10

6/23/14 7/21/14 8/11/14 9/15/14 9/29/14

log

MPN

/gra

m d

ry so

il

Sample Date

Coliforms in GWT Irrigated Commercial Soil

Total

E. coli

29

Recycled GW samples that were never exposed to TCS were sensitive to

treatment in TCS containing agar. Samples originally plated on TCS agar developed

resistance to TCS after just one exposure. Soil samples from both types of media clearly

show the development of triclosan resistance following low-level exposure. Samples of

GW irrigated soil plated onto low dose TCS plates developed resistance to triclosan

following that single exposure while samples irrigated with GWT showed resistance

upon initial plating. While there were organisms intrinsically resistant to TCS present in

both types of soil, low level TCS exposure appears to promote selection for this

resistance.

30

Figure 15: Antibiotic and Triclosan Replicates of Greywater Irrigated Recycled Soil Heterotrophs

0

1

2

3

4

5

6

7

8

9

10

log

CFU

/gra

m d

ry so

il

Replica Plate Agar Treatment

Replicates of GW Irrigated Recycled Soil Heterotrophs

Soy Agar


31

Figure 16: Replica Plate Averages of Greywater Irrigated Recycled Soil Heterotrophs

0

1

2

3

4

5

6

7

8

9

10

Replicated Original

TSA Replicate Antibiotic Antibiotic + Triclosan

log

CFU

/gra

m d

ry so

il


Replicate Averages of GW Irrigated Recycled Soil Heterotrophs

Soy Agar


32

Figure 17: Antibiotic and Triclosan Replicates of Greywater with Triclosan Irrigated Recycled Soil Heterotrophs

*There were no replicate Tetracycline or Tet + Triclosan plates created for the Soy Agar

0

1

2

3

4

5

6

7

8

9

10

log

CFU

/gra

m d

ry so

il


Replicates of GWT Irrigated Recycled Soil Heterotrophs*

Soy Agar


33

Figure 18: Replica Plate Averages of Greywater with Triclosan Irrigated Recycled Soil Heterotrophs

0

1

2

3

4

5

6

7

8

9

10

Replicated Original


log

CFU

/gra

m d

ry so

il


Replicate Averages of GWT Irrigated Recycled Soil Heterotrophs

Soy Agar


34

Figure 19: Antibiotic and Triclosan Replicates of Greywater Irrigated Commercial Soil Heterotrophs

*There were no replicate Tetracycline or Tet + Triclosan plates created for the Soy Agar + Triclosan.

0 1 2 3 4 5 6 7 8 9

10

log

CFU

/gra

m d

ry so

il


Replicates of GW Irrigated Commercial Soil Heterotrophs*

Soy Agar


35

Figure 20: Replica Plate Averages of Greywater Irrigated Commercial Soil Heterotrophs

0

1

2

3

4

5

6

7

8

9

10

Replicated Original


log

CFU

/gra

m d

ry so

il


Replicate Averages of GW Irrigated Commercial Soil Heterotrophs

Soy Agar


36

Figure 21: Antibiotic and Triclosan Replicates of Greywater with Triclosan Irrigated Commercial Soil Heterotrophs

*There was no replicate Tet + Triclosan plate created for the Soy Agar.

0 1 2 3 4 5 6 7 8 9

10

log

CFU

/gra

m d

ry so

il


Replicates of GWT Irrigated Commercial Soil Heterotrophs*

Soy Agar


37

Figure 22: Replica Plate Averages of Greywater with Triclosan Irrigated Commercial Soil Heterotrophs

0

1

2

3

4

5

6

7

8

9

10

Replicated Original


log

CFU

/gra

m d

ry so

il


Replicate Averages of GWT Irrigated Commercial Soil Heterotrophs

Soy Agar


38

CHAPTER 4 DISCUSSION


The final sampling was conducted on extinct grass. Two possible reasons were

considered in explaining why the grass died. First, the grass completed a full growth

cycle; second, the root structures became ‘pot-bound’ in the small amount of soil

available and ceased growth. While the second scenario is a possibility, it is less likely

due to the fact that vegetation in both types of media stopped growing. The root

structures in the commercial media were much denser and more established than the root

structures in the recycled media. This would indicate that the commercial grass should

have died long before the recycled grass. While the commercial grass did die first, the

root structures in the recycled media never reached the same thickness that the

commercial grass did. The best explanation for the loss of vegetation at this time is that

this type of grass completed its growth cycle. TCS treatment is not considered to be a

factor in vegetation loss since all samples ceased growth regardless of whether they were

irrigated with TCS or not.

4.2. Soil Nutrient Quality

Nutrient levels were higher in recycled soil than commercial soil for both GW and

GWT treatments. The most similar results were COD in GWT treated soils. The

increased nutrient levels in the recycled soil may reflect increased sorption to the rubber

particles. Although there was more nutrients present in recycled GW treated soil, there

was not an increase in vegetation or heterotrophic organisms.

39

High nutrient levels are of concern with regards to nonpoint source pollution to

waterways. Excess nitrogen and phosphorus cause eutrophication and excess ammonia is

toxic to aquatic species. Leachate from these model green roof systems was not tested for

nutrient content. Assuming ten percent of the nutrient content of the soils leaches from

the soil to runoff, the impact of a green roof is compared to 2013 EPA water quality

criteria in Table 3. The nitrogen and phosphorus standards are based on EPA Aggregate

Nutrient Ecoregion IX Reference Conditions, 25th Percentile (US EPA(a), 2014) which

includes the Chesapeake Bay Region. Ammonia concentrations are based on EPA

Aquatic Life Ambient Water Quality Criteria For Ammonia Freshwater 2013 (US

EPA(b), 2013).

Table 3: Potential Leachate Comparison to Water Quality Standards

10% Soil Values Recycled

GW Recycled

GWT Commercial

GW Commercial

GWT EPA

Guidelines Nitrogen (mg/L) 1.57 1.14 .86 .83 .69

Ammonia (mg/L) .12 .07 .05 .04 1.9

Phosphorus (µg/L) 29 22 26 16 36.56

Based on this ten percent assumption, the leachate levels of ammonia and

phosphorus would meet the guidelines for EPA Ecoregion IX water quality. Nitrogen

levels in the leachate would exceed water quality standards. Pre-washing the crumb

rubber, as recommended by Crampton et al., 2014, may potentially reduce the nutrient

load in the recycled soil. Further investigation is necessary to determine this.

40

4.3 Heterotrophic Plate Counts

It is unknown what amount of TCS was retained in the soil after watering the

grass or whether the TCS collected on soil particles over time. The amount of TCS

present did not kill heterotrophs but did promote the development of TCS resistant

organisms. It appears that the TCS may have prevented the non-resistant heterotrophs

from multiplying. Heterotrophs in the GW samples may have continued increasing if the

grass had kept growing. In the commercial soil GW samples there was a clear reduction

in heterotrophs as vegetation declined. Further investigation would be necessary to draw

any conclusions relating vegetation to heterotroph viability.

The presence of TCS in irrigation water clearly promotes the resistance of

heterotrophic organisms to TCS. Resistant organisms were consistently cultured from all

samples of GWT irrigated soil. This supports previous work that also implicates low-

level TCS exposure as a causative agent in the development or persistence of TCS

resistance in environmental settings (Harrow et al., 2011; Middleton and Salierno, 2013;

Drury et al, 2013).


Low doses of TCS appear to be effective in reducing the number of E. coli to

below detectable levels in recycled media. Low-level resistant E. coli were observed in

commercial soil. It is unclear whether something in commercial substrate aids in the

promotion of TCS resistance or in the growth of E. coli. There were slightly more E. coli

present in commercial soil irrigated with GW than recycled soil irrigated with GW.

41

All soils experienced a reduction in total coliforms towards the end of the study.

This decline is consistent with the loss of vegetation. Total coliforms were less affected

by TCS treatment than E. coli but the commercial soil treated with GWT experienced an

unexplainable fluctuation in detectable total coliforms throughout the course of the study.

TCS may cause stress on total coliforms and something in the commercial media may aid

in that stress. The presence of TCS resistant E. coli and total coliforms support previous

work that isolated resistant coliforms from surface waters dosed with low-levels of TCS

(Middleton and Salierno, 2013). In this research low doses of TCS supported the

selection for TCS resistant pathogens in soil. This indicates that there is potential for soil

receiving untreated greywater application to develop resistant E. coli provided that the

greywater contains TCS. There is no literature at this time that describes any available

GW treatment that removes TCS. Therefore, GW reuse for soil applications should be

done with caution.


Replicate plates grew unexpectedly high numbers of colonies on antibiotic-

containing plates. It is apparent that the minimum inhibitory concentrations (MICs) for

these antibiotics were not reached on this set of plates. Previous literature was used in

determining these concentrations but upon further evaluation, more recent literature

indicates the MICs have increased. It is possible that the soil microorganisms are highly

resistant to these four classes of antibiotics, but is unlikely. This is justified by the use of

vancomycin. This level of resistance to such a potent antibiotic seems far too unlikely to

accept these results and further investigation is required.

42

It is, however, much more apparent that TCS resistance is being selected for in

these microorganisms. Species present in the soil samples may possess the genes required

for TCS resistance but are not actively expressing those genes until necessary. This is

evident from the development of resistance from single exposure and highlights the

importance of reducing environmental dosing of TCS so as to not encourage the

expression of genes that may potentially enhance antibiotic resistance as well. The

antibiotic and TCS resistance plates may not adequately address antibiotic resistance but

do reaffirm the promotion of TCS resistance following low-level exposure.

By removing TCS from commercial products and reducing or eliminating the

dosing of waterways, it may be possible to eliminate the promotion of such species.

Serafini and Matthews (2009) demonstrated that upon removal from triclosan exposure,

resistant bacteria reverted to wild-type strains. Additionally, of the two bacterial types

used, E. coli took nearly twice as long as S. aureus to regain susceptibility to triclosan

indicating that some bacterial species may require longer periods without exposure to

once again be vulnerable to triclosan (Serafini and Matthews, 2009). While the genes that

code for resistance may still be present in the environment, the absence of TCS will

reduce the selection for the expression of these genes.

43

4.6 Conclusion

The use of recycled crumb rubber appears to be a suitable option to repurpose

tires entering the waste stream and reduce the weight a conventional green roof places on

a structure. The artificial soil did not promote excess growth or hinder the growth of

heterotrophs or coliforms. Resistant organisms were not increased by the recycled

substrate and resistant coliforms may in fact be less likely to persist. Greywater reuse is

best suited when water has been treated. Reuse for household purposes where the water

will ultimately reach the wastewater stream, i.e. toilet flushing, appears to be acceptable

at this time as triclosan would be entering the wastewater regardless. Diverting the water

temporarily will not increase the concentration of TCS entering wastewater flow. Upon

wastewater treatment, most of the triclosan will partition to sludge as in standard

blackwater. Soil application should still be done with caution, including biosolid

application. Using greywater for irrigation purposes can enhance triclosan resistance in

microbial soil communities and may also promote antibiotic resistance. The only way at

this time to know treated greywater is free of triclosan is to avoid the use of products

containing triclosan.

Triclosan should promptly be removed from household products. There is

insufficient evidence that the addition of triclosan to soaps helps to avoid illness or aids

in the removal of more bacteria from hands than traditional soap (Aiello et al., 2007).

There is increasing evidence that environmental harm may outweigh any perceived

benefit from the use of triclosan. Aside from the development of resistant organisms,

changes in microbial communities radiate to higher trophic levels. Widespread

dissemination of a biocide could have repercussions we are not able to predict yet.

44

Cautious use of antimicrobials when necessary helps to safeguard the benefits we reap

from them as humans. It also aids in the conservation of microbial processes we benefit

from without realizing.

The speed with which bacteria evolve requires that we minimize the exposure

microorganisms receive from our best means of protecting ourselves. If we wish to

continue benefitting from the use of antimicrobials we must only use them when

absolutely necessary. Reserving the use of triclosan for hospital settings reserves an

antimicrobial for a setting where it is far more necessary than in each individual

household or the nearest waterway.

45

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