Examining the Feasibility of Vermicomposting Biosolids Prior to Land Application to Remove Triclosan and Methyl Triclosan

Item

Title

Eng Examining the Feasibility of Vermicomposting Biosolids Prior to Land Application to Remove Triclosan and Methyl Triclosan

Date

2016

Creator

Eng Weibel, Whitney P

Subject

Eng Environmental Studies

extracted text

EXAMINING THE FEASIBILITY OF VERMICOMPOSTING
BIOSOLIDS PRIOR TO LAND APPLICATION
TO REMOVE TRICLOSAN AND METHYL TRICLOSAN

by
Whitney P. Weibel

A Thesis
Submitted in partial fulfillment
of the requirements for the degree
Master of Environmental Studies
The Evergreen State College
December 2016

©2016 by Whitney P. Weibel. All rights reserved.

This Thesis for the Master of Environmental Studies Degree
by
Whitney P. Weibel

has been approved for
The Evergreen State College
by

________________________
Erin Martin, Ph. D.
Member of the Faculty

________________________
Date

ABSTRACT
Examining the feasibility of vermicomposting municipal biosolids prior to land
application to remove triclosan and methyl triclosan
Whitney P. Weibel
More than 4 million dry tons of nutrient-rich biosolids are applied to land every year in
the United States. Current wastewater treatment plants (WWTPs) are ineffective in
removing all pharmaceuticals and personal care products (PPCPs) from biosolids prior to
land application. Triclosan (TCS) is a widely used broad-spectrum bacteriostatic. Both
TCS and its degraded form, methyl triclosan (Me-TCS), are hydrophobic and accumulate
in biosolids. As potential endocrine disruptors, removing TCS and Me-TCS from
biosolids is crucial. The feasibility of using earthworms (Eisenia fetida) to vermicompost
biosolids sourced from the City of Tacoma, Pierce County and the City of Lynden was
examined. Method development included determining the ratio of biosolids to paper
mulch that would allow for earthworm survival. Potassium, phosphorus, total organic
carbon, total Kjehldahl nitrogen (TKN), and pH were also evaluated in the biosolids
based on earthworm survival and reproduction. Total organic carbon appeared to be
positively associated with earthworm survival and TNK inversely so, which is believed to
be due to the presence of ammonia that is toxic to earthworms. Due to instrument
uncertainty and the lack of replication, the effect of earthworms on TCS concentrations
was inconclusive for all three biosolids sources. If earthworms were left in the substrate
for more time, perhaps there would have been a discernable difference in measured TCS
and Me-TCS concentrations. However, the presence of earthworms increased the
concentrations of Me-TCS, compared to a control, in the substrate composed of the City
of Tacoma’s biosolids. The difference observed in the City of Tacoma’s biosolids is
believed to be due to the initial concentration of TCS that then degraded into Me-TCS.
Based on past research (Domínguez, Aira, and Gómez-Brandón, 2010), it is believed
microbes excreted in E. fetida’s feces contributed to the increase in Me-TCS formation
the City of Tacoma’s biosolids.

Table of Contents
List of figures and tables ......................................................................................................v
List of acronyms ............................................................................................................... vii
Acknowledgments............................................................................................................ viii
1. Introduction ......................................................................................................................1
2. Literature review ..............................................................................................................4
2.1. Triclosan and methyl triclosan ................................................................................4
2.2. Sources of triclosan and other pharmaceuticals and personal care products ..........6
2.3. Triclosan’s impact on humans ................................................................................7
2.4. Pathways by which triclosan and PPCPs enter the environment ..........................10
2.5. Historical use of land-applied biosolids in the United States ...............................13
2.6. Environmental impacts of triclosan and methyl triclosan.....................................13
2.7. Factors affecting triclosan and methyl triclosan stability .....................................15
2.8. Current wastewater treatment processes ...............................................................18
2.9. Other wastewater treatment options......................................................................22
2.10. Bioremediation....................................................................................................23
2.11. Vermiremediation ...............................................................................................24
2.12. Earthworm anatomy and reproduction................................................................28
2.13. Current study.......................................................................................................29
3. Biosolids ........................................................................................................................31
4. Earthworms ....................................................................................................................33
5. Pilot study ......................................................................................................................33
6. Experiment I: 89 to 11 ...................................................................................................36
6.1. Parameters measured ............................................................................................37
6.2. Substrate preparation ............................................................................................37
6.3. Sample collection ..................................................................................................38
6.4. Results ...................................................................................................................38
6.5. Discussion .............................................................................................................39
7. Experiment II: 2 to 1 ......................................................................................................40
7.1. Parameters measured ............................................................................................41
7.2. Substrate preparation ............................................................................................41
7.3. Sample collection ..................................................................................................41
7.4. Results ...................................................................................................................41
7.5. Discussion .............................................................................................................42
8. Experiment III: Bulking material and concentration .....................................................46
8.1. Parameters measured ............................................................................................46
8.2. Substrate preparation ............................................................................................46
8.3. Sample collection ..................................................................................................49
8.4. Results ...................................................................................................................49
8.4.1. Triclosan and methyl triclosan concentrations ............................................52
8.5. Discussion .............................................................................................................53
9. Experiment IV: Three biosolids sources ........................................................................58

 

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9.0.1. Substrate preference .....................................................................................59
9.1. Parameters measured ............................................................................................60
9.1.1. Substrate preference .....................................................................................60
9.2. Substrate preparation ............................................................................................60
9.2.1. Substrate preference .....................................................................................63
9.3. Sample collection ..................................................................................................63
9.4. Results ...................................................................................................................64
9.4.1. Earthworm survival......................................................................................64
9.4.2. Reproduction ................................................................................................67
9.4.3. Triclosan and methyl triclosan concentrations ............................................69
9.4.4. Eisenia fetida substrate preference ..............................................................72
9.4.5. Nutrients.......................................................................................................73
9.5. Discussion .............................................................................................................75
9.5.1. Earthworm survival......................................................................................75
9.5.2. Reproduction ................................................................................................78
9.5.3. Triclosan and methyl triclosan concentration ..............................................80
9.5.4. Eisenia fetida substrate preference ..............................................................87
9.5.5. Nutrients.......................................................................................................88
10. Conclusion ...................................................................................................................89
References ..........................................................................................................................93
Appendix A – United States Environmental Protection Agency’s list of 88 synonyms for
triclosan ....................................................................................................117
Appendix B – Standard Operating Procedure Pharmaceutical and Personal Care Products
by EPA Method 8270D............................................................................118


 

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List of Figures
Figure 2.1. Concentrations of triclocarban, triclosan and methyl triclosan in anecic or
endogenic earthworms from fields amended with biosolids........................................29
Figure 8.1. Diagram of stacked containers used in Experiment III: Bulking material and
concentration ................................................................................................................48
Figure 8.2. Total weight (g) of earthworms (E. fetida) alive in each substrate after 30-day
experiment....................................................................................................................50
Figure 8.3. Amount of total Kjeldahl nitrogen, phosphorus, and potassium measured in
the four test substrates prior to the addition of earhtworms (E. fetida) .......................51
Figure 8.4. Total organic carbon (g/kg) measured in the four test substrates prior to the
addition of earhtworms (E. fetida) ...............................................................................52
Figure 8.5. Concentration of triclosan and methyl triclosan before and after
vermicomposting repeated pilot substrate ...................................................................53
Figure 9.1. Diagram of stacked containers used in Experiment IV: Three biosolids
sources..........................................................................................................................62
Figure 9.2. Diagram of divided containers used in Experiment IV: Three biosolids
sources..........................................................................................................................63
Figure 9.3. Number of earthworms added to each substrate at the beginning and after 35day experiment .............................................................................................................65
Figure 9.4. The weight (g) per earthworm in substrates composed of three parts paper
mulch and four parts biosolids sourced from the City of Lynden, Tacoma, and Pierce
County at the beginning and end of the 35-day experiment ........................................66
Figure 9.5. The weight (g) per earthworm in containers with divided substrates
composed of compost and biosolids sourced from the City of Lynden, Tacoma, and
Pierce County at the beginning and end of the 35-day experiment .............................67
Figure 9.6. Total number of cocoons counted within divided substrates composed of
biosolids from the City of Lynden, Tacoma, and Pierce County and compost ...........68
Figure 9.7. Total number of cocoons counted within each substrate consisting of
biosolids from the City of Lynden, Tacoma, and Pierce County.................................69
Figure 9.8. Triclosan concentrations before and after E. fetida exposure and control, for
substrates compose of biosolids and paper mulch from the City of Tacoma and Pierce
County ..........................................................................................................................71


 

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Figure 9.9. Methyl triclosan concentrations (µg/kg) in substrates composed of paper
mulch and biosolids from the City of Lynden, Tacoma, and Pierce County before and
after E. fetida exposure and control .............................................................................72
Figure 9.10. Percent of total earthworms at the end of the 35-day experiment in divided
containers found in the compost and either Pierce County, City of Tacoma, or City of
Lynden’s biosolids .......................................................................................................73
Figure 9.11. Total Kjeldahl nitrogen (g/kg) in the biosolids and substrates composed of
three parts paper mulch to four parts biosolids (by dry weight) from City of Lynden,
Tacoma, and Pierce County before E. fetida were added ............................................75
Figure 9.12. The difference in triclosan concentration that can be explained by the
formation of methyl triclosan in substrates composed of paper mulch and biosolids
sourced from the City of Tacoma and Pierce County after 35-day exposure to E.
fetida ............................................................................................................................84
Figure 9.13. Initial total Kjeldahl nitrogen (TNK) and percent of total worms added that
died in substrate made of three parts paper mulch and four parts biosolids from the
City of Tacoma, Lynden, and Pierce County ...............................................................89

List of Tables
Table 2.1. Physiochemical properties of triclosan and methyl triclosan .............................5
Table 2.2. Wastewater treatment cost and effectiveness of PPCP removal summary for
Washington State .........................................................................................................23
Table 3.1. Characteristics of the wastewater treatment plants from which biosolids were
obtained (Washington State) ........................................................................................33
Table 9.1. Mean (standard deviation) temperatures of substrate and ambient room
temperature ..................................................................................................................64
Table 9.2. Nutrients of substrates composed of paper mulch and biosolids from the City
of Lynden, Tacoma, and Pierce County.......................................................................74


 

vi
 

List of Acronyms
BAF

Bioaccumulation factor

C

Celsius

EQ

Exceptional quality

g

Gram

GC/MS/MS

Gas chromatograph double mass spectrometer

K

Potassium

kg

Kilogram

L

Liter

µg

Microgram

M

Mean

Me-TCS

Methyl triclosan

NH3

Ammonia

NH4+

Ammonium

P

Phosphorus

PPCP

Pharmaceuticals and personal care products

SD

Standard deviation

SE

Standard error

TCC

Triclocarban

TCS

Triclosan

TKN

Total Kjeldahl nitrogen

TOC

Total organic carbon

U. S. EPA

United States Environmental Protection Agency

U. S. FDA

United State Food and Drug Administration

WWTP

Wastewater treatment plant


 

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Acknowledgments
This project would not have been if it weren’t for the City of Tacoma, especially Daniel
Thompson, Stuart Magoon, Mark Bozlee and Nicole Riley.
My sincerest gratitude to my advisor and reader, Erin Martin, for her patience and
thoughtful direction. Kathleen Saul and Kevin Francis for their support and
encouragement throughout my time in the Masters of Environmental Studies program.
Thank you to my friends, cohort, and carpool. They all know more about earthworms and
biosolids than they ever wanted.
Special thanks to my husband and family for their unending love and support.
And to Peter Kareiva and Laura Hubbard, for their encouragement and confidence in my
abilities.
Do mischief.


 


 viii

1. Introduction
In the United States, wastewater treatment plants (WWTPs) generate over eight
million dry tons of biosolids annually, which are the end result of the wastewater
treatment process (U. S. Environmental Protection Agency, 1999). Biosolids are
generated from both residential and industrial wastewater treatment and can contain
heavy metals, pathogens, plus pharmaceuticals and personal care products (PPCPs) such
as hormones, steroids, antibiotics soaps, shampoos, and household cleaners (Xia,
Bhandari, Das, & Pillar, 2005). Rather than incinerating or dumping biosolids in landfills,
a number of cities around the world reuse the biosolids to amend soils because they have
plant-available nitrogen and phosphorus (Jacobs & McCreary, 2001). At least 55 percent
of biosolids produced in the U. S. are reused in a beneficial manner: applied to land for
restoration purposes, and in forestry and agricultural practices (North East Biosolids &
Residuals Association, 2007). Despite the contamination, the processed biosolids are a
nutrient-rich soil additive that homeowners, farmers, landscapers and forest managers can
use (Outwater, 1994).
While most pathogens and heavy metals are removed from the final product, per
government standards, anthropogenic chemical compounds have been found to persist in
the final product destined for land application (Kinney et al., 2006). For example, one
study found that one third to one half of the anthropogenic contaminants were not
removed and were consistently present in biosolids, suggesting current wastewater
treatment processes are not effective at removing PPCPs from biosolids (Kinney et al.,
2006). The ecosystems where biosolids are applied can become contaminated with the
chemicals, which in turn can be taken up by flora and fauna and leach into water sources

1

(Prosser & Sibley, 2015). Solutions are needed to remove these contaminants from the
biosolids before they are scattered throughout the environment.
This study focuses on using earthworms as a potential solution for removing
PPCPs from biosolids destined for land application. Earthworms have been used to
process biosolids, to enrich and aerate the soil and make nutrients, like phosphorus and
nitrogen, readily available for plant use (Rajpal, Bhargava, Chopra, & Kumar, 2014;
Sinha, Bharambe, & Chaudhari, 2008; Sinha, Herat, Bharambe, & Brahamhatt, 2009;
Singh & Suthar, 2012; Suthar & Singh, 2008). Earthworms have also been found to take
up, or bioaccumulate, chemical contaminants and heavy metals found in biosolidsamended soils (Higgins, Paesani, Chalew, Halden, & Hundal, 2011; Kinney et al., 2006,
2008, 2010, 2012; Macherius et al., 2014, Pannu, O'Connor, & Toor, 2012). However,
their ability to bioaccumulate PPCPs from biosolids derived from WWTPs for purposes
of removal remains largely unexplored.
Biosolids are a great alternative to chemical fertilizers to enrich soils, but if the
biosolids are polluted with anthropogenic chemical contaminants, application of biosolids
may be doing the environment a disservice. Furthermore, if the anthropogenic chemical
contaminants are antimicrobials they may render beneficial bacteria and other
microorganisms ineffective (World Health Organization, 2016). Triclosan (TCS) and its
degraded form, methyl triclosan (Me-TCS), are anthropogenic chemical contaminants,
which are also antimicrobials, used in common household and industry products.
Triclosan has endocrine disrupting properties seen in rats (Jung, An, Choi, & Jeung,
2012) as well as human tissue (Ahn et al., 2008; Gee, Charles, Taylor, & Darbre, 2008).
Additionally, researchers have observed positive associations between TCS exposure and

2

earlier onset of puberty (Wolff et al., 2010) and increased body mass index (Lankester,
Patel, Cullen, Ley & Parsonnet, 2013).
Triclosan and Me-TCS are also hydrophobic and therefore are more readily found
in biosolids rather than the processed wastewater (Chenxi, Spongberg, & Witter, 2008;
Lozano, Rice, Ramirez & Torrents, 2013; Ying & Kookana, 2007). If earthworms are
capable of bioaccumulating antimicrobial agents, like TCS and Me-TCS, perhaps they
can be used to remove them and other contaminants that are not being removed by
current WWTP processes in order to produce a cleaner product for land application and
soil amendment purposes. The possibility of incorporating earthworms into large scale
processing of biosolids, specifically for removal of TCS, and Me-TCS, has to the extent
of my knowledge yet to be evaluated and has significant potential for research
opportunities. Evaluating earthworms’ ability to bioaccumulate and subsequently remove
TCS and Me-TCS from biosolids prior to land application is an initial step to determining
the potential of PPCP removal from biosolids.
To understand how earthworms can be incorporated in the biosolids process we
must first understand TCS, Me-TCS, and other PPCPs, their source, impact on humans,
and route into the environment. The historical use of land-applied biosolids and impact
on the environment is discussed as well as the fate and prevalence of TCS and Me-TCS
in the environment. Next, to demonstrate why a solution is needed, the shortcomings of
current wastewater treatment processes are explored; specifically, the challenge of
removing anthropogenic chemical contaminants, which can enter the environment
through the land application of biosolids. A discussion of bioremediation, a mechanism
used to remove chemicals and other anthropogenic contaminants from soils, will follow.

3

Because there is an appreciable use for the biosolids waste and with future limits on the
disposal of biosolids in expensive and spatially limited landfills, a solution is needed to
reduce the amount of pollutants in the biosolids that we add to the environment. Finally,
this thesis is discussed and how it fills gaps in the literature and expands the possibility of
incorporating earthworms into processing of biosolids.
2.1. Triclosan and methyl triclosan
Triclosan (2,4,4'-trichloro-2'-hydroxydiphenyl ether, see Table 2.1) is a synthetic
broad-spectrum bactericide (Orhan, Kut, & Gunseoglu, 2007; Reiss, Lewis, & Griffin,
2009). Methyl triclosan is the biodegraded product of TCS and has been found to be more
persistent in soils with a half-life four times that of TCS (104 days versus 443 days;
Lozano, Rice, Ramirez & Torrents, 2012). While the exact process is not entirely known,
Me-TCS is most likely formed by microbial methylation within WWTPs (Boehmer,
Ruedel, Wenzel & Schroeter-Kermani, 2004) during aerobic digestion and in anoxic
conditions (Chen et al., 2011). The “cleaned” wastewater that leaves the treatment plant
is called the effluent and concentrations of Me-TCS, relative to TCS, were found to be
greater than three times that of the wastewater that comes into the treatment plant, which
is called the influent (Lindström et al., 2002). The half-life of TCS depends on the
medium in which it is found: air-borne TCS has a half-life of one day, 60 days in water,
120 days in soil, and 540 days in sediment; the researchers offered no explanation for
these differences in the TCS half-lives they calculated from models they created but is
perhaps due to oxygen availability (Halden & Paull, 2005). However, other research
indicates that TCS has a half-life of over one year in water (Ciba-Geugy Limited, 1990,
as cited in Ohron, 2014). In sewage sludge TCS has been observed to have a half-life of

4

315 to 770 days depending on its depth within a settling bed (Chen, Pauly, Rehfus &
Bester, 2009). This variation in half-life is most likely due to environmental factors such
as pH, temperature, and oxygen levels. The long half-life of TCS in sewage sludge
suggests the need for alternative methods for its removal.
Table 2.1. Physiochemical properties of triclosan and methyl triclosan
Triclosan

Chemical formula: C12H7Cl3O2
CAS number: 3380-34-5
Molecular mass: 289.54 g mol-1
log Kow: 4.2-4.8
Melting point: 55°C (131°F)
Boiling point: 120°C (248°F)
Water solubility: 4.621 mg L-1

Methyl triclosan

Chemical formula: C13H9Cl3O2
CAS number: 4640-01-1
Molecular mass: 303.56 g mol-1
log Kow: 5
Melting point: 43-45°C (109-113°F)
Boiling point: 358.7°C (678°F)
Water solubility: 0.4 mg L-1

Sources: Chen et al., 2011; National Center for Biotechnology Information (n.d.); Toronto Research
Chemicals (n.d.).
Note: Chemical Abstract Services is abbreviated as CAS. The partition coefficient is log Kow, which is the
ratio of a chemical’s concentration in octanol relative to the chemical’s concentration in water; it is an
indicator of a compound’s lipophilicity. Compounds can be considered highly lipophilic with
measurements up to 6 and 6.5 (Organization for Economic Cooperation and Development, 2002).

The European Commission’s Scientific Committee on Consumer Safety (SCCS)
(2010) stated that there is little known regarding the biochemistry of the biodegradation
of TCS. The SCCS states that the Minnesota Biocatalysis and Biodegradation Database,
which was inaccessible during the time of this writing, has nothing documented for TCS
yet the database claims to contain information regarding xenobiotic, or synthetic,
chemical compounds (University of Minnesota, 2016). Therefore, there is a gap in
knowledge of biodegradation of TCS and intermediary products.
5

2.2. Sources of triclosan and other pharmaceutical and personal care products
Triclosan was patented in 1964 and introduced as a surgical scrub in United States
in 1972 (Halden, 2014; Jones, Jampani, Newman, & Lee, 2000) and was quickly
incorporated into many home and personal care products, including toothpastes,
cosmetics, soaps and even plastics (Reiss et al., 2009). By 1978 it was detected in aquatic
environments and sediments (Hites & Lopez-Avila, 1979; Jungclaus, Avila, & Hites,
1978) and by 1981 Me-TCS was detected in fish (Miyazaki, Yamagishi, & Matsumoto,
1984).
During the writing of this thesis, the United States Food and Drug Administration
(U. S. FDA) enacted a rule banning the use of 19 antiseptic chemicals, including TCS, in
over-the-counter products intended for consumer use as a wash, which is to go into effect
September 6, 2017 (Safety and effectiveness of consumer antiseptics, 2016). It must be
stressed that this ban is only for over-the-counter consumer antiseptic washes; the ban
does not include products used in food service, hospitals, in products that are not rinsed
off after use, or its use in materials such as plastics and other items. Therefore, TCS and
Me-TCS will continue be used and enter WWTPs.
The U. S. Environmental Protection Agency (U. S. EPA) lists 88 synonyms for
triclosan. Some of the most common in consumer goods include Microban®, Biofresh®,
Irgasan® DP 300, Lexol 300, and Cloxifenolum (EPA, 2016; Glaser, 2004; entire list can
be found in Appendix A). According to Microban®’s website (2016), working in
multiple industries, products are infused with TCS during the manufacturing process.
This process of infusing materials with anti-microbial TCS creates a material that is
inhabitable for microbes. As one would expect it can be found in medical products such

6

as scrubs, masks, and polymer-based storage containers. However, it is also found in
everyday household materials such as air filters, kitchen and bathroom fixtures, carpets,
countertops, insulation, door hardware, paints, tile, and grout; not to mention clothing,
cutting boards, and children’s toys. Microban® also markets products for commercial use
from food prep and storage, baby changing stations and highchairs, elevator buttons and
paper towel dispensers.
In general, TCS can be found in hand and dish soaps, face wash, toothpaste and
toothbrushes, cosmetics, deodorant, household cleaners, as well as various other
household personal care products and wares (Glaser, 2004). About 96 percent of products
that contain TCS are ultimately washed down the drain to WWTPs (Reiss, Mackay,
Habig, & Griffin, 2002).
Antibacterial hand soaps, for consumer use, can contain TCS concentrations
ranging from 0.1% to 0.45% (weight/volume). This amount results in the total
concentration of TCS in your average bottle of hand soap to contain 0.221 g/mL to 0.994
g/mL, respectively.
2.3. Triclosan’s impact on humans
The direct impact of TCS on human health is not totally understood but it has
been determined that TCS-resistant microbes can become more abundant merely through
exposure to TCS (McNamara, LaPara, & Novak, 2014). The possibility of antibiotic
resistance prompted Minnesota’s Governor, Mark Dayton, to sign a bill that will go into
effect January 2017 banning all products containing TCS (Landau & Young, 2014). Also,
in an effort to “’protect our Great Lakes and water supplies’” New York State Senator,
Tim Kennedy, announced legislation that would prohibit the sale of household cleaning

7

products containing TCS, and derivatives such as triclocarban, in the state of New York
(Pignataro, 2015). In September 2006 Germany disallowed the use of TCS in plastics in
contact with food (European Commission, 2010). The United States Food and Drug
Administration (FDA) (2013) states that there is no evidence to support the idea that
using antibacterial soap is any better than using regular soap when washing hands. It has
even been argued that using antibacterial soap may lead to a false sense of security
leading individuals to not wash their hands as well because they believe antibacterial
soap does a better job than regular soap (Alliance for the Prudent Use of Antibiotics,
2011). On September 2, 2016, the FDA banned the use of TCS and triclocarban (TCC),
along with 17 other chemicals, from soaps but the U. S. EPA will still allow it in other
household textiles, such as cutting boards, toys, and hairbrushes (Safety and
Effectiveness of Consumer Antiseptics, 2016). Methyl triclosan is not included in the list
of banned chemicals because it is merely a byproduct of TCS, not a chemical that is
produced for a specific use, other than laboratory testing. These recent events regarding
the banning of TCS indicates a growing concern over the wide spread use in many
consumer products.
Researchers have evaluated the presence of TCS in humans, specifically testing
breast milk of nursing mothers, blood, and urine. Of 36 nursing mothers, those who used
toothpaste, deodorant or soap that contained TCS had higher concentrations of the
contaminant in their breast milk (0.022 to 0.95 µg/g) compared to mothers who did not
use PPCPs not containing TCS (<0.018 to 0.35 µg/g) (Allmyr, Adolfsson-Erici,
McLachlan, & Sandborgh-Englund, 2006). It should be noted that TCS concentrations
were also measured in the blood plasma of each mother and significantly more TCS was

8

found there than in the milk, indicating the infants were not exposed to TCS as much as
their mothers were. Additionally, every participant in the study had TCS present in their
body indicating that PPCPs are only one route of TCS exposure for the mothers since
some participants indicated not using products containing TCS.
The National Health and Nutrition Examination Survey, conducted annually by
the Centers for Disease Control and Prevention in the U. S., was the source of 2,517 urine
samples evaluated for TCS concentrations (Calafat, Ye, Wong, Reidy, & Needham,
2008). Triclosan was present in 74.6 percent of the samples with values ranging from 2.4
to 3,790 µg/L. The researchers found concentrations correlated to age and socioeconomic status. Specifically, concentrations of TCS were highest among individuals in
their thirties and those in higher-income households.
Triclosan is considered an endocrine disruptor, indicating it interferes with the
hormonal system in mammals (Chen et al., 2008). Exposure to TCS is also considered a
potential contributor to the development of breast cancer, and may otherwise be harmful
to the immune systems of mammals (Bertelsen et al., 2013; Clayton, Erin, Todd, Dowd &
Aiello, 2010). Studies evaluating the effect TCS has on humans are limited but there are
studies that utilize rats to test TCS endocrine disrupting properties. Jung (2012) studied
the effects TCS has on rats’ hormone receptors, finding TCS blocked specific estrogen
receptors. Jung’s results support the potential human health risks of TCS. In vitro
research found that TCS blocks estrogen and androgen receptors in tissue (Ahn et al.,
2008; Gee et al., 2008). Evaluating 2,058 male and 1,979 female human participants
(average age 49, 35-65), researchers found a positive association with TCS exposure,
measured from a urine sample, and an increased body mass index (Lankester et al.,

9

2013). In 2010, Wolff et al. followed 1,151 girls (ages six to eight) through puberty to
evaluate the relationship of breast and pubic hair development, in the U. S. Researchers
found a slightly inverse association with pubic hair development and TCS exposure
(measured from a urine sample). The results suggest a potential connection between TCS
exposure and a child’s development through puberty. Research on the impact TCS has on
humans is, by no means, conclusive or complete but there is evidence to suggest there are
negative consequences to TCS exposure whether the one being exposed is a rat or human.
2.4. Pathways by which triclosan and PPCPs enter the environment
The ecological risk of TCS and other PPCPs exposure through leaching from
land-applied biosolids or in the effluent water from WWTPs has been widely reviewed
(Coogan & Point, 2008; Fuchsman et al., 2010; Kreuzinger, Clara, Strenn, & Vogel,
2004; Reiss et al., 2002, 2009; Ying & Kookana, 2007; Xia et al., 2010). Researchers use
either modeling or laboratory simulations to evaluate risks of TCS. Reiss et al. (2002)
consider TCS concentrations in lotic, or flowing water aquatic systems, to be of little to
no concern for fish and vertebrates but that some algae, especially those close to WWTP
effluent discharge locations, have some risk. In 2009, Reiss et al. evaluated the TCS
concentrations and associated potential risk to earthworms; again concluding no
significant risk was indicated in their research. This is to be expected because TCS, even
amounts up to 300 mg/kg in soil, was not lethal to earthworms but did damage the DNA
of E. fetida (Lin, Zhou, Xie, & Liu, 2010).
Ying and Kookana (2007) conducted a preliminary risk assessment of a “worstcase scenario” of TCS concentrations in WWTP effluent (mean (M)=142 µg/L,
maximum=434 µg/L, minimum=23 µg/L) measured in Australia and the biosolids

10

(M=5.58 mg/kg, maximum=16.69 mg/kg, minimum=0.09 mg/kg). Through their analysis
of the limited available literature on the toxicity of TCS, they calculated a “risk quotient”
where values greater than one indicates risk and values less than one do not. Ying and
Kookana calculated the risk quotient for applying biosolids to land and in effluence from
WWTPs as 1.36 and 1.5, respectively. This indicates there is a risk of TCS effecting soils
when biosolids are applied to land and effecting aquatic organisms from WWTP
effluence.
Coogan and Point (2008) set up an experiment where aquatic snails and algae
were exposed to WWTP effluence in Texas to determine how much each would
bioaccumulate TCS and Me-TCS, among other PPCPs. After two weeks of exposure
snail tissue samples were tested and compared to tissue samples collected prior to
exposure. Algal tissue was compared to surrounding water, rather than a pre and postexposure comparison. The researchers found that prior to exposure, snails had TCS and
Me-TCS concentrations of 5.9 µg/kg and 0.8 µh/kg, respectively, and 58.7 µg/kg
(standard error (SE)=3.39) and 49.8 µg/kg (SE=2.49) after two weeks of exposure to
WWTP effluence. The water surrounding the alga was measured at 0.112 µg/kg of TCS
and 0.041 µg/kg of Me-TCS and after two weeks of exposure TCS and Me-TCS algal
concentrations measured 162 µg/kg (SE=17.6) and 50.4 µg/kg (SE=5.21). This research
indicates there is definitely bioaccumulation of TCS and its degraded form Me-TCS by
the snails and algae tested in this study.
Fair et al. (2009) evaluated the plasma of wild Atlantic bottlenose dolphins
(Tursiops truncatus) blood samples for the presence of TCS. Of 26 individual dolphins
sampled, 27% had detectable levels of TCS (≥0.033 µg/g) in blood samples. While the

11

levels detected in sampled dolphins does pose a lethal threat, the effects of chronic
exposure is unknown. The researchers express concern with the increasing human
population living in coastal communities leading to an increase in WWTP effluence into
waters inhabited by dolphins.
Through the land application of biosolids, PPCPs can leach into groundwater
(Kreuzinger et al., 2004; Xia et al., 2010). Triclosan and other PPCPs can potentially
accumulate in the food chain and travel to higher trophic levels through the uptake of
plants and animals living in soils amended with biosolids (Kelsey, Colino, & White,
2005; Kinney et al., 2010; Harris et al., 2000; Wild, Harrad, & Jones, 1994). Xia et al.
(2010) observed leaching of TCS in biosolids-amended soils in their evaluation of fieldapplied biosolids. By measuring TCS concentrations at various depths in a field that was
amended with biosolids, they determined that there is a potential for TCS leaching but
believe transformation of TCS was likely the cause for decreased concentration
measurements in soil samples.
Agyin-Birikorang, Miller, and O’Connor (2010) evaluated the difference between
soil, biosolids, and biosolids-amended soils and retention of TCS. Due to its hydrophobic
nature, TCS is more likely to be in biosolids rather than WWTPs’ effluent. Within the
biosolids tested, the TCS appeared to be retained whereas in the unamended sandy soils,
greater mobility, or leaching, of TCS was observed. Overall, they found that substrates
with high organic carbon resulted in lower mobility of the compound, compared to
substrates with higher organic carbon.

12

2.5. Historical use of land-applied biosolids in the United States
Regulations established back in the 1970’s focused mostly on pollutants common
during that time (heavy metals) and pathogen reduction. Pharmaceuticals and personal
care products were not addressed because they were not of concern, but as more PPCPs
are becoming more readily available and developed at accelerated rates, they are getting
more attention (Hildebrandt, 2007).
The processing of biosolids for consumer use results in a mostly odorless soil
with minimal pathogens; the amount of pathogens allowed in biosolids designated for
land-application use must meet specific U. S. Environmental Protection Agency (U. S.
EPA) standards to qualify as Class A or B Biosolids (U. S. EPA, 2016). Compared to
anaerobic digestion, the final product of aerobically digested sludge is considered to have
a higher fertilizer value, in terms of available nitrogen, phosphorus, potassium, pH, and
carbon-nitrogen ratio (Outwater, 1994).
2.6. Environmental impacts of triclosan and methyl triclosan
Triclosan has been found to be toxic in aquatic and terrestrial ecosystems. In a
survey of 139 U. S. streams TCS was among the seven most common anthropogenic
compounds detected. Effluent, or the processed wastewater, from WWTPs, and land
application of biosolids are the primary routes for TCS to enter the environment (Ying &
Kookana, 2007). In fish, TCS has been observed to cause lengthening of fins and changes
in sex ratios (la Farré, Pérez, Kantiani, & Barceló, 2008) and in frogs, disruption in
thyroid hormone and associated gene expression has been observed, affecting the
metamorphosis process from tadpole to frog (Veldhoen et al., 2006). While very few

13

studies have evaluated the effects of Me-TCS, one study did fine it to be toxic to blood
cells in abalone (Gaume et al., 2012).
Triclosan and Me-TCS have been found to be toxic to aquatic organisms by
fragmenting or causing irreversible damage to DNA (Binelli, Cogni, Parolini, Riva, &
Provini, 2009; DeLorenzo et al., 2008; la Farré et al., 2008). Both TCS and Me-TCS have
been found to bioaccumulate in fish, algae, earthworms, and snails (Balmer et al., 2004;
Boehmer, Ruedel, Wenzel, & Schroeter-Kermani, 2004; Coogan, Edziyie, La Point, &
Venables, 2007; Higgins et al., 2009/2011; Kinney et al., 2008; Snyder, O’Connor, %
McAvoy, 2011). Fish and aquatic invertebrates ingest triclosan by feeding on organisms
that live in contaminated soils. The concentration of Me-TCS was measured in fish in
various lakes in Switzerland WWTP effluence. The concentrations of Me-TCS measured
up to 35 µg/g (wet weight) compared to no detectable levels of Me-TCS in fish from a
remote lake receiving no WWTP effluence (Balmer et al., 2004). Although it has not
been studied directly, it is assumed that Me-TCS could have effects similar as TCS in the
environment and, consequently, organisms (Reiss et al., 2009).
Triclosan has also been shown to move through the food web affecting different
species of birds and mammals. Triclosan and Me-TCS are lipophilic, meaning they
readily pass through cells’ walls and accumulate in fats and lipids (Chedgzoy, Winckle &
Heard, 2002). The accumulation of TCS and Me-TCS allows for the contaminants to
move up through the food chain through consumption of plants or animals, like
earthworms or soil microorganisms, by birds and fish (Reiss et al., 2009).

14

2.7. Factors affecting triclosan and methyl triclosan stability
There are many processes, such as biodegradation, methylation, chlorination,
photolysis, and combustion that can transform TCS into other compounds. Biological
methylation of TCS results in Me-TCS (Bester, 2005; Boehmer et al., 2004). Photolysis,
or photodegradation, of TCS in aqueous solutions results in 2,8-dichlorodibenzodioxin
and other dioxin derivatives (Aranami & Readman, 2007; Latch, Packer, Arnold, &
McNeill, 2003; Mezcua et al., 2004; Lores, Llompart, Sanchez-Prado, Garcia-Jares, &
Cela, 2005; Sanchez-Prado et al., 2006) which the U. S. EPA (2015) states are persistent
organic pollutants, are highly toxic, and carcinogenic, and can accumulate in the food
chain. Combustion of TCS leads to another dioxin, the formation of di- and
trichlorodibenzo-p-dioxin (Kanetoshi, Ogawa, Katsura, Okui, & Kaneshima, 1988).
Chlorophenols, specifically 2,4,6-trichlorophenol and 2,4-dichlorophenol (Kanetoshi
Ogawa, Katsura, & Kaneshima, 1987; Rule, Ebbett, & Vikesland, 2005; Canosa et al.,
2005; Greyshock & Vikesland, 2006), are transformations of TCS that are on the U. S.
EPA’s “Priority Pollutant List” (Effluent limitations guidelines and standards, 2013).
Finally, when TCS reacts with chlorine, such as right before treated water is discharged
from a WWTP, chloroform is formed (Fiss, Rule, & Vikesland, 2007; Greyshock &
Vikesland, 2006; Rule et al., 2005). Chloroform was used as an anesthetic for nearly 100
years, from the 1847 to the beginning of its decline in 1932 (Wawersik, 1996). In
humans, chloroform was found to cause jaundice of the liver, depression of the central
nervous system decreasing respiratory rates, as well as effects on the heart and kidneys
(U. S. EPA, 2016). Chloroform is not believed to be a major concern in harming the
environment or human exposure to chloroform in the environment unless there is a spill

15

or some other occurrence of extremely high quantities being exposed to nature (Scottish
Environment Protection Agency, n.d.). However, the U. S. EPA has classified chloroform
as a probable carcinogen to humans (U. S. EPA, 2000).
The product of transformed and degraded TCS can result in toxic and non-toxic
compounds (National Industrial Chemicals Notifications and Assessment Scheme
[NICNAS], 2009). Under dark, anaerobic conditions, TCS is quite stable (McAvoy,
Schatowitz, Jacob, Hauk, & Eckhoff, 2002). However, in aerobic conditions TCS
degrades more readily into Me-TCS and, both being hydrophobic, tend to concentrate in
the solids and are removed from the water during the wastewater treatment process.
When TCS enters a WWTP most stays within the dewatered biosolids even after
extensive plant processes and treatments (Lozano et al, 2013; Chenxi et al., 2008; Ying &
Kookana, 2007). On average, 96 percent of TCS that entered WWTPs was “eliminated”
from the wastewater effluent but 30 percent was found in the sludge (Bester, 2003). The
researcher did not test for any other compounds that could have transformed from TCS in
the study, which may explain the 66 percent of unaccounted for TCS. Methyl triclosan
occurs at much lower concentrations than TCS. However, Me-TCS is more hydrophobic,
persistent in the environment because is more resistant to biodegradation and photolysis,
and more lipophilic making it more readily bioaccumulative than its parent form, (Dann
& Hontela, 2011; Mackay, & Barnthouse, 2010; NICNAS, 2009). Another factor that can
affect the transformation of TCS is whether oxygen is present or not.
To evaluate the effect oxygen has on the degradation of TCS and the formation of
Me-TCS, Chen et al. (2011) measured the change of TCS and Me-TCS concentrations in
sludge in three environments, aerobic, anaerobic and anoxic before and after an 80-hour

16

period. Adding a constant flow of oxygen through the substrate created the aerobic
condition, a constant flushing of nitrogen gas and potassium nitrate (KNO3) maintained
the anaerobic and anoxic conditions, respectively. They found, in the aerobic
environment, TCS concentrations decreased by 49 percent (30 µg/L to 15 µg/L) whereas
Me-TCS concentrations increased by 16 percent (4.5 µg/L to 5 µg/L). The anoxic
environment decreased TCS concentrations by 16 percent (32 µg/L to 29 µg/L) and a 17
percent increase in Me-TCS concentration (4.1 µg/L to 4.8 µg/L). Anaerobic
environment decreased TCS concentrations 11 percent (32 µg/L to 28 µg/L) but no
change in Me-TCS concentrations was detected. Overall, they determined that only one
percent of TCS degraded into Me-TCS in aerobic conditions, less in anoxic conditions,
and no TCS was observed to degrade into Me-TCS in anaerobic conditions.
In their evaluation of the effects of natural conditions on the degradation of TCS
and formation of Me-TCS from biosolids applied to fields, Butler, Whelan, Sakrabani
and Van Egmond (2012) observed a seasonal affect. They measured the greatest decrease
in TCS concentrations between July and October when soil moisture was low and
temperatures were warm. In loamy sand soil a 59 percent decrease in TCS was measured,
whereas in sandy clay loam and clay soils a 72 and 74 percent decrease in TCS
concentration was measured, respectively. They believe the temperature was a factor in
its impact on microbial activity affecting TCS biodegradation and moisture in soil was
believed to create a more anaerobic condition, in which TCS did not degraded into MeTCS. They also measured the formation of Me-TCS over the course of a year observing
66% percent of TCS degrade into Me-TCS in sandy clay loam, 64 percent in loamy sand,
and 39 percent in clay soils.

17

Biosolids are typically stored in piles or tanks prior to land application, such as on
a farm or in a garden. Chenxi et al. (2008) evaluated the persistence of seven
pharmaceuticals and one antibacterial compound, TCS, in stored biosolids over a 77-day
period. They tested the biological degradation of the compounds in the biosolids in
aerobic and anaerobic conditions, with and without light, at varied lengths of time.
Continuously pumping air through the biosolids in a bucket created the aerobic condition
and putting a lid on the bucket to restrict airflow created the anaerobic condition.
Triclosan, along with three other pharmaceuticals, showed no change in concentration in
any of the conditions tested. Considering previous research suggested a reduction of TCS,
Chenxi et al. attribute their results of no change on the “strong affiliation of TCS to the
organic-rich particles in biosolids and the resulting strong sorption might prevent TCS
from photo and biodegrading” (p. 516). Their reasoning for attributing the organic-rich
particles for TCS not degrading is the organic particles inhibit electron transfer from
TCS, thus restricting degradation (Reineke, 2001 – as cited in Chenxi et al. 2008). Given
that triclosan concentrations were not affected by aerobic or anaerobic storage conditions
or length of time stored in Chenxi et al.’s 2008 study, alternative mechanisms are needed
to remove this compound from biosolids, perhaps through the bioaccumulation of the
material in earthworms.
2.8. Current wastewater treatment processes
Anthropogenic contaminants are present in biosolids because wastewater
treatment processes are not 100 percent effective at removing said chemical compounds.
The primary process WWTPs utilize is called digestion. Similar to how the human
stomach breaks down the food we eat, wastewater digestion incorporates the natural

18

ability of microorganisms and bacteria to break down organic matter and pathogens
within the incoming wastewater, also called the influent. There two primary methods of
digestion is aerobic and anaerobic digestion. Anthropogenic chemical contaminants enter
WWTPs and are not all removed by current wastewater processes (Deegan et al., 2011).
Aerobic digestion requires the pumping of oxygen into processing tanks holding
the influent. Heat is naturally produced from the microorganisms breaking down the
plethora of organic matter within the influent converting it into carbon dioxide. Aerobic
digestion is typically utilized in smaller WWTPs, with capacities of less than five million
gallons per day. It is a faster process where the liquid influent is processed in the tanks
for 12-24 hours (Outwater, 1994; Thompson, D., personal communication, February 6,
2015). Aerobic is more costly than anaerobic digestion because of energy needed to
pump oxygen into the processing tanks but produces a mostly odorless and more
biologically stable product that also has higher fertilizer value than anaerobically digested
wastewater (Outwater, 1994).
Anaerobic digestion takes place in the absence of oxygen. This is to encourage
bacteria to convert fats, carbohydrates, proteins into organic acids and alcohols, which
are then converted into carbon dioxide and methane. In some cases the anaerobic
wastewater treatment process can result in net energy production because the methane
created is then used by the WWTP (Marchaim, 1992).
Following either anaerobic or aerobic digestion, sewage sludge is piped into pools
where the heavy solids sink to the bottom where they are removed from the pool from a
drain located at the bottom of the pool. The heavier solids at the bottom of the tank,
called biosolids, are dewatered, or pressed to remove excess water, before being used for

19

land-application purposes, incinerated, or taken to the landfill.
McAvoy et al. (2002) evaluated the concentration of TCS in the influent and
effluent wastewater from five WWTPs in Ohio and the digested sludge from three plants.
When it comes to aerobic versus anaerobic sludge digestion, McAvoy et al. found a
greater decrease in TCS concentrations in the two WWTPs that aerobically digested
sludge (14.7 and 12.2 µg/g of TCS in undigested sludge down to 4.2 and 1.5 µg/g in
digested sludge, respectively) compared to another plant, which utilized anaerobic
digestion, which saw no change in TCS concentrations. Interestingly, the WWTP that
anaerobically digests their sludge had the lowest concentration of TCS in the influent
wastewater yet showed the concentrations of TCS in the effluent to more than double,
from 7.5 µg/g to 15.6 µg/g (dry weight). The authors attribute a 50 percent reduction in
the overall amount of solids during the digestion process to this increase in TCS
concentration. Triclosan is synthetic and cannot be created except in a lab, by humans
(Orhan et al., 2007; Reiss et al., 2009). What is fascinating is Me-TCS was determined
present in the influent and effluent wastewater in all five of the WWTPs evaluated, but
accurate concentrations could not be determined because they were generally below
detectible levels. The exception was for the WWTP that anaerobically digests their
sludge, where Me-TCS was not detected after processing the wastewater. While the
authors did not discuss this finding, considering the hydrophobic nature of Me-TCS,
assuming it concentrated in the solids during the wastewater treatment process is not
unreasonable. Either way, the Me-TCS concentrations at the end of the digestion process
of the sludge did not greatly differ between the anaerobic (0.13 µg/g, dry weight) and two
aerobic (0.17 µg/g and 0.13 µg/g, dry weight) WWTPs.

20

There are more than 16,583 publicly owned wastewater treatment plants in the U.
S. (LeBlanc, Matthews, & Richard, 2009). In 2004, 51 percent of the WWTPs in the U.
S. used anaerobic digestion of wastewater and sewage sludge (EPA, 2006). Therefore,
based on McAvoy et al.’s (2002) findings, more than half of the WWTPs in the U. S. are
not removing as much TCS as WWTPs utilizing aerobic digestion for wastewater
treatment.
Wastewater treatment plants’ effluence is the primary route for PPCPs to enter the
environment. This indicates current wastewater processes are not removing the
contaminants (Gao, Ding, Li, & Xagoraraki, 2012; Miao, Bishay, Chen, & Metcalfe,
2004; Vienoa, Tuhkanen, & Kronberg, 2007). Furthermore, the majority of PPCPs are
hydrophobic and end up in the biosolids during the wastewater treatment process (Kinney
et at., 2006; Strachan, Nelson, & Sommers, 1983). Therefore, it is expected that TCS and
Me-TCS will behave similarly and become concentrated in the biosolids.
Kinney et al. (2006) investigated the presence of PPCPs and other contaminants
in biosolids destined for land application. Nine different WWTPs’ biosolids were
collected from seven states within the U. S. Of those nine WWTPs, 87 compounds were
screened for and every sample of biosolids contained at least 30 of these compounds,
some as many as 55 contaminants. Twenty-five contaminants were found in every
sample. Triclosan was one of the 87 contaminants tested for and was present in all of the
WWTP biosolids samples. The TCS concentration across the nine WWTPs ranged from
1.2 µg/g to 32.9 µg/g, by dry weight (mean and median concentration 10.5 µg/g and 10.2
µg/g, respectively). The most common contaminants were fairly consistent across all the
samples analyzed. This is surprising because the WWTPs, from which the biosolids

21

samples were obtained, did not have similar methods in the production of biosolids and
have varied population demographics from which the biosolids originated. This indicates
that regardless of the biosolids source’s population demographics and the treatment
processes utilized by WWTPs PPCPs persist and remain in the biosolids that are then
applied to land. Through the land application of biosolids the environment is being
polluted with anthropogenic contaminants, which can result in flora and fauna taking up
the contaminants and potentially moving through the food chain (Prosser & Sibley,
2015).
2.9. Other wastewater treatment options
Currently, there are a number of options for treating wastewater but many are cost
prohibitive or are not effective in the removal of PPCPs (see Table 2.2, below).
Especially in arid climates, reclaimed water (WWTP effluent) is pumped into constructed
wetlands called treatment wetlands. The purpose of treatment wetlands is to further
improve water quality after the primary wastewater treatment processes (U. S. EPA,
1993). An evaluation of contaminant concentrations of the inlet and outlet of a treatment
wetland, which receives two million gallons per day with average water retention time of
three to four days, showed a decrease in some contaminants but there was a six percent
increase in TCS during summer months. During winter months, however, researchers
measured TCS concentrations to decrease by 29 percent at the inlet and outlet. The
potential reason for the increase in concentration was not discussed in the published
report but researchers did collect fish living in the water. These fish were not measured
for TCS concentrations but perhaps the fish and other micro biota has something to do
with the differences observed (Barber, Keefe, Antweiler, Taylor, & Wass, 2006).

22

Table 2.2. Wastewater treatment cost and effectiveness of PPCP removal summary for
Washington State
Treatment
Primary
Secondary
Filtration
Activated Sludge
Microfiltration
Ultrafiltration
Nanofiltration
Granular Activated Carbon
Powdered Activated Carbon
Reverse Osmosis
Riverbank Filtration
Membrane Bioreactor
Electrodialysis Reversal
Ozonation
Flocculation

Percentage of
facilities using
treatment
100%
100%
20%
-0%
0%
0%
0%
0%
--15%
0%
Few
--

Relative Cost
----Very expensive
Very expensive
Very expensive
--Very expensive
-Very expensive
Expensive
--

Relative
Effectiveness of
PPCP removal
----Poor
Poor
Excellent
Excellent
Excellent
Excellent
Poor
--Excellent
Poor

Note: Jones, 2008, as cited in Lubliner, Redding, & Ragsdale, 2010

2.10. Bioremediation
Remediation is the process of removing pollutants or contaminants, such as heavy
metals (Barker & Bryson, 2002), petroleum (Atlas, 1995), waste from drilling (RojasAvelizapa, Roldan-Carrillo, Zegarra-Martinez, Munoz-Colunga, & Fernandez-Linares,
2007), and other hazardous materials (Sayara, Borràs, Caminal, Sarrà, & Sánchez, 2011)
from soils, sediments, ground water, or surface water. Bioremediation is the utilization of
natural processes for remediation treatments. There are various types of bioremediation.
To name a few: anaerobic and aerobic remediation utilize microbes in the absence and
presence (respectively) of oxygen to degrade the pollutants or contaminants (Russell et
al., 2011); phytoremediation utilizes plants to uptake contaminants in soils before being
harvested (Salt et al., 1995); mycoremediation involves mushrooms to sequester
contaminants in the fruit bodies which are then harvested and disposed of (Bhatt,
23

Cajthaml, & Šašek, 2002); vermiremediation incorporates earthworms to bioaccumulate
contaminants and heavy metals (Chachina, Voronkova, & Baklanova, 2015; Dabke,
2013). In addition, vermicomposted organic matter has been found to show an increase in
total nitrogen, available phosphorus and a desirable decrease in the carbon to nitrogen
ratio (Suthar & Singh, 2008).
Utilizing earthworms for the purposes of removing anthropogenic contaminants
from biosolids is the chosen form of bioremediation for a number of reasons. Sinha et al.
(2009) put it perfectly, “vermicomposting is a self-promoted, self-regulated, selfimproving, self-driven, self-powered and self-enhanced, low or no energy requiring zerowaste technology, easy to construct, operate and maintain” (p 880). Meaning that
earthworms self-regulate their population, where if there is enough food, they multiply
and if food is scarce, they do not (Edwards & Bohlen, 1996). Additionally, they improve
the substrate in which they find themselves in that they neutralize soil pH, aerate the soil
through tunneling and improve water retention (Ismail, 1998, as cited in Gajalakshmi,
Ramasamy & Abbasi, 2001)
2.11. Vermiremediation
Earthworms have been studied in soils that have been amended with biosolids and
have shown to bioaccumulate triclosan and other anthropogenic contaminants (Higgins et
al., 2011; Kinney et al., 2010; Lin et al., 2010; Pannu et al., 2012; Snyder et al., 2011).
To evaluate anthropogenic contaminants in soils treated with biosolids researchers
have looked at bioaccumulation of these contaminants in various species of earthworms
(Higgins et al., 2011; Kinney et al., 2006, 2008, 2010; Macherius et al., 2014). Kinney et
al. (2008) assessed the use of earthworms as a diagnostic tool for evaluating the presence

24

of 77 anthropogenic contaminants in soils amended with biosolids, manure, or land not
amended. Soil and earthworm samples were collected from the four commercial
agronomic production sites; two of three sites received an application of biosolids or
manure 31 days prior to sample collection; the third site had no known history of
biosolids or manure amendments. Prior to application, the manure and biosolids were
tested for contaminants so researchers knew what contaminants to expect in the soil or
earthworm samples to be collected in the future. It does not appear the soil was sampled
and measured for contaminants prior to the application of the swine manure or biosolids.
Triclosan was found in the biosolids (10.5 µg/g, dry weight), but not in the manure
collected for testing prior to land application. After 31 days, researchers measured TCS
concentrations in the soil and worms to be 160 µg/g and 1,740 µg/g, dry weight,
respectively in the biosolids-amended treatment. One hundred and fifty six days
following the application of biosolids, the researchers collected samples again and found
TCS concentrations to have decreased in the soil, to 96 µg/g, dry weight, and an increase
in TCS concentrations in the worms, 2,610 µg/g, dry weight. Contaminants, such as some
fragrances and detergent metabolites, bioaccumulated within earthworms at quantifiable
levels, yet the soils did not have any discernible measure. The biosolids had detectable
levels of some contaminants, so researchers knew the contaminants might be present in
the soil after the biosolids application. The findings from this study indicate earthworms
magnify anthropogenic contaminants present in soils and may be used as indicators for
contaminants that may be below detectible concentrations. Even though contaminants
may be below detectable levels, they can still accumulate in flora and fauna and work
their way up trophic levels of the food chain.

25

Kinney et al. (2008) also calculated the bioaccumulation factor (BAF), which is
the ratio of the mean concentration of contaminants found inside the earthworm to the
mean concentration of contaminants to the corresponding soil that had detectable levels.
Researchers use the BAF to determine the ratio by which earthworms are able to
bioaccumulate a contaminant, relative to the concentration of the contamination in the
soil (Organization for Economic Co-operation and Development, 2002). Kinney et al.
found that of all the contaminants detected in the worm and soil samples, TCS had the
highest BAF of 27; biogenic sterol cholesterol had the second-highest BAF of 21.4
(which has no known direct threat to environmental ecology but can biotransform into
testosterone (Fernandes, Cruz, Angelova, Pinheiro, & Cabral, 2003)). The results from
this study indicate the higher concentrations of TCS detected in the worms could only be
due to the application and presence of biosolids.
Snyder, O’Connor, and McAvoy (2011) evaluated bioaccumulation of
triclocarban (TCC) by earthworms (E. fetida). Related to TCS, TCC also has antibacterial
properties and is also a possible endocrine-disruptor (Chen et al., 2008; DiamantiKandarakis et al., 2009) and is found in personal care products that end up in biosolids
applied to land. Snyder et al. (2011) mixed biosolids that contained measureable amounts
of TCC with three types of soil: fine sand, silty clay loam, and artificial soil, and added
earthworms. Based on real-life application rates of biosolids in agricultural settings, the
TCC concentration in the amended test soils was estimated to be 6.9 µg/g, calculated
from the measured biosolids concentration and load rate. After 31 days the worms were
removed from the fine sand, silty clam loam and artificial soils and concentrations of
TCC in the earthworm tissue was recorded as 127±14 µg/g, 142±8.4 µg/g, and 36.5±0.89

26

µg/g, respectively. The difference in the concentrations of bioaccumulated TCC was
attributed to the difference the amount of organic matter, the earthworms’ main food
source, within each soil. In the artificial soil, the organic matter was 2.5 and 10 times
greater than the silty clay loam and fine sand, respectively, which provided a higher
volume of uncontaminated food for the worms to consume. Snyder et al. also calculated
the BAF for the fine sand, silty clam loam and artificial soils, 18±3.5, 20±2.1, and
5.2±0.22, respectively. The BAF values correspond with the concentration of TCC
bioaccumulated by the worms from the soil.
Looking exclusively at TCS, Pannu et al. (2012) evaluated earthworm
bioaccumulation from fine sand and silty loam clay soil in a laboratory. The soil samples
in this study were spiked with TCS of varying concentrations (0.05, 0.07, 0.1, 0.15, 0.55
and 1 µg/g) and earthworms remained in the soil for a period of 28 days.
Bioaccumulation was assessed through the calculation of BAF in which average values
for the fine sand and silty loam clay, regardless of the spiked TCS amount, was 6.5±0.84
µg/g and 12±3.08 µg/g, respectively. These values follow the trend seen in Snyder et al.’s
(2011) findings described above. Using the values from Snyder et al.’s research, Pannu et
al. determined the BAF values for TCC in the fine sand and silty loam clay soils were
greater but not significantly different than the BAF values calculated for TCS.
Higgins et al. (2011) evaluated bioaccumulation of TCS and TCC by E. fetida.
The researchers conducted their study evaluating the rate of bioaccumulation of the two
contaminants after earthworms were in contaminated and control soils for 1 day, 5, 7, 9,
14, and 21 days. They found rapid and consistent TCC accumulation by E. fetida but
inconclusive evidence for TCS, from biosolids-amended soils. Substrates were created
27

with “high” and “low” concentrations of TCS and TCC, which were established by
adjusting the amount of biosolids applied to each test soil to reach the desired
concentrations. The researchers concluded the lack of relationship between TCS exposure
and accumulation by earthworms was most likely due to TCS transforming into degraded
compounds, such as Me-TCS, once the worms had consumed the parent contaminant.
There have only been a few studies evaluating Me-TCS bioaccumulation in
earthworms. One of the studies investigated bioaccumulation of TCC, TCS, and Me-TCS
in the soil and earthworm four years after biosolids were applied to a plot (Macherius et
al, 2014). All three compounds were detected in the soil. Triclosan and TCC
concentrations in the soil (0.0015 µg/g and 0.013 µg/g, respectively) decreased 100 to
1,000 times compared to their concentrations in the biosolids (10.9 µg/g and 4.94 µg/g,
respectively) applied four years prior. Methyl triclosan had a concentration six times that
of TCS (0.009 µg/g and 0.0015 µg/g, respectively) in the soil four years following the
application of biosolids. Similar results were observed in earthworm bioaccumulation of
Me-TCS which was at least double that of TCS or triclocarban in endogenic earthworms
(Figure 2.1). This study supports the importance of studying a chemical compound as
well as its transformative or degraded form. Prior to this study, there was no published
research demonstrating bioaccumulation of Me-TCS in earthworms.
2.12. Earthworm anatomy and reproduction
Earthworms are made up of mostly water and fat (Washington State University
Whatcom Extension, 2016) making them ideal for lipid-bonding contaminants, like TCS
and Me-TCS. Eisenia fetida is more commonly known as red wiggler, redworm, tiger
worm, or the red Californian earthworm. They are in the kingdom Anamalia, phylum

28

Figure 2.1. Concentrations of triclocarban, triclosan, and methyl triclosan in anecic or
endogenic earthworms from fields amended with biosolids.
Concentration (ng/g dry weight)

Figure 2.1. Concentrations of triclocarban, triclosan, and methyl triclosan in samples of
anecic or endogenic earthworms from fields that were amended with biosolids four years
prior. Endogenic earthworms live in the upper layers of soil in the area surrounding
plants’ roots. Anecic earthworms create vertical burrows that can be up to six feet deep
(Macherius et al. 2014).
Annelida, class Clitellata, order Heplotaxida, family Lumbricidae, genus Eisenia, and
species fetida. They live primarily in leaf litter, mulch and manure. Like all earthworms,
E. fetida are hermaphroditic, but two earthworms are required for reproduction. After
copulation, each worm creates a cocoon from which 2-5 baby worms will hatch after
approximately 32-72 days. After hatching, E. fetida reach sexual maturity within 53-76
days (Edwards, 1988, as cited in Edwards & Bohlen, 1996).
2.13. Current study
There is a need for some way to effectively remove TCS and Me-TCS from
biosolids prior to land application because it is clear from the presented information that
current wastewater treatment processes are not effective; TCS and Me-TCS persist in the
biosolids produced. In a review of literature to evaluate the economic potential of
vermicomposting municipal solid waste researchers concluded that vermicomposting is a
29

great alternative to filling dumps and that by using vermicomposted municipal waste can
benefit the quality of soils to which the vermicast is applied (Singh, R., Singh, P., Araujo,
Ibrahim, & Sulaiman, 2011).
Taking into consideration the potential economic benefits of vermicomposting
municipal waste and the fact that earthworms can bioaccumulate contaminants that
persist in municipal biosolids following wastewater treatment processes, a solution may
be at hand. Combining these two theories to create a product, vermicomposted municipal
biosolids, that can improve soils, utilize our waste in a beneficial way, and reduce
polluting our environment seems to be a benefit all around.
Earthworms have shown to improve the nutrients available for plants, improve the
microbial community, and aerate soils (Edwards & Bohlen, 1996). In addition, they have
been observed bioaccumulating anthropogenic contaminants from soils amended with
biosolids suggesting the land applied biosolids did not have all the anthropogenic
contaminants removed during processing (Higgins et al., 2011; Kinney et al., 2006, 2008,
2010; Macherius et al., 2014). To my knowledge, there are no studies evaluating the
bioaccumulation of TCS and Me-TCS by earthworms for the purpose of removing or
filtering such contaminants out of biosolids prior to land application. Based on the fact
that WWTPs are unable to effectively remove TCS and Me-TCS, all known
anthropogenic contaminants, this research is clearly needed. If earthworms can remove
anthropogenic contaminants from biosolids, then incorporating vermiculture into the
wastewater treatment process may result in a more environmentally friendly product for
land application; one that may be economically feasible and possibly profitable.

30

The City of Tacoma, for example, sells biosolids as a product called TAGRO,
short for Tacoma Grown, to anyone who wants to use the material (City of Tacoma,
2013a). At times, they have even had to close their gates because they had run out of
product to sell (Cohen, 2015). TAGRO is sold by the truckload from the WWTP and in
bags at local Ace Hardware stores in addition to a number of other locally run garden
stores (City of Tacoma, 2013b). Customers can even fill a few buckets worth for free to
try it out or if they only need a small amount (personal observation). TAGRO is made
with biosolids classified as Class A EQ (Exceptional Quality), the U. S. EPA’s highest
rating for biosolids (City of Tacoma, 2013c). Class A EQ biosolids meet and exceed
Class A standards in pathogen and heavy metals reduction (U. S. EPA, 1999). So, while
the product is safe for humans to use the contaminants that are not removed in the
wastewater treatment process are being applied to land in the form of individuals’ lawns
and gardens and commercially on fields and forests.
Whether earthworms can be used to remove chemical contaminants from
biosolids prior to land application will be determined by comparing the concentrations of
TCS and Me-TCS in biosolids after vermicomposting to a control of substrate to which
earthworms are absent. The application of earthworms to biosolids for the distinct
purpose of removing anthropogenic contaminants has yet to be evaluated.
3. Biosolids
Biosolids were collected from three WWTPs in Western Washington (The City of
Tacoma, Lynden, and Pierce County), each utilizing a different process of biosolids
digestion (see Table 3.1). The sites were selected because they were willing to participate
in this study.

31

The City of Tacoma’s Central Wastewater Treatment Plant in Tacoma, WA,
utilizes dual-digestion in processing sludge. Influent is first digested aerobically for 8 to
12 hours before it is anaerobically digested for 30 days prior to settling out solids to be
dewatered. The process produces Class A Exceptional Quality biosolids, which are
combined with sawdust and sand to create TAGRO that is then sold to residents and local
businesses. The Plant serves a population of approximately 258,000 individuals (Morris
Pumps, 2008).
Pierce County’s Chambers Creek Regional Wastewater Treatment Plant, in
University Place, WA, utilizes anaerobic digestion to process the treatment plant’s
influent. Sludge is processed for 30-35 days before it is sent to settling tanks to separate
out the solids to be dewatered to create Class B biosolids. Biosolids were collected for
this study prior to the Plant’s final step of heating the biosolids to create their final Class
A biosolids fertilizer product because of access and ease of collecting enough material.
Additionally, following the final heating step the final product consists of small,
desiccated pellets and would have to be re-hydrated for the purposes of this study. The
Plant serves a population of nearly 288,000 (Tobin, A., personal communication, October
21, 2016).
The City of Lynden, located in Whatcom County, WA, utilizes aerobic digestion
to process the Plant’s influent. Influent is processed for 25-30 days before solids are
separated out and dewatered to create Class B biosolids. The Plant’s serves a population
of approximately 13,000 individuals (Goree, T., personal communication, October 25,
2016).

32

Table 3.1. Characteristics of the wastewater treatment plants from which biosolids were
obtained (Washington State).
Wastewater treatment
plant
City of Tacoma
Pierce County
City of Lynden

Wastewater treated
gal/per day

Population served

Primary treatment
process

38 million
17.4 millionc
1.2 milliond

258,000 b
288,000c
13,000d

Duala
Anaerobic
Aerobic

Note. aDual indicates that the biosolids are processed aerobically and anaerobically prior to biosolids
collection. cInformation obtained from Morris Pumps, 2008. cInformation obtained from Tobin, A.,
personal communication, 2016. dInformation obtained from Goree, T., personal communication, October
25, 2016. All other information obtained from Northwest Biosolids Management Association’s website
(http://www.nwbiosolids.org/membership_agencies.htm)

4. Earthworms
Eisenia fetida, or red wigglers, were purchased from three different suppliers in
Western Washington, depending on availability. Worms were purchased from Yelm
Earthworms and Castings located in Yelm, 3 in 1 Earthworms located in Poulsbo, and
Northwest Redworms located in Camas.
Upon receipt, worms were separated from the substrate in which they were
transported, by way of tabling. Tabling is a process where the worms and substrate are
placed on a table and a light is shined down upon the surface of the pile to encourage the
worms to travel to the bottom of the pile. Substrate is removed as the worms continue to
travel towards the table, away from the light. At the end of the tabling process, there are
only worms left at which point they were weighed and added to appropriate treatments.
5. Pilot study
An initial pilot study was conducted to ensure worms could survive in the
substrate mixture of biosolids, from the City of Tacoma, and paper mulch using a ratio of
four parts biosolids to three parts paper mulch by wet weight (Ndegwa, Thompson, &
Das, 2000). Unfortunately, values were mistaken and 20 percent moisture was used in the

33

calculations of the moisture content of the biosolids, rather than the actual 80 percent, and
considered the paper mulch dry or zero percent moisture rather than the seven percent
that it is. This mistake was not realized until later in the study; the assumed or correct
percent moisture used in each of the following sections in this study is identified in each
part. After the mistake of percent moisture was realized, the ratio of biosolids to paper
was recalculated and the actual ratio for this pilot study was closer to 1 to 4.
The substrate for the pilot study was prepared by adding biosolids and paper
mulch, at a ratio of four to three (wet weight), to the worm bin and mixed together by
hand. Distilled water was added to achieve the moisture content required for the
earthworms to survive, approximately 80 percent moisture (Ndegwa, Thompson, & Das,
2000), which was determined by look and feel. Earthworms were separated from the
substrate in which they were transported and placed in a pile on the surface of the
substrate the day after it was prepared.
The worm bin was comprised of one 68-liter polyethylene plastic containers (42
cm high, 61 cm wide, 41 cm deep) with a surface area of 0.24 m2. To create the ideal
stocking density for vermicomposting biosolids of 1.6 kg of worms/m2 (Ndegwa,
Thompson, & Das, 2000), 384 g of E. fetida were needed. In one pound (453 g) there are
approximately 1,000 sexually mature red wigglers; therefore 845 adult earthworms were
added to the worm bin (Yelm Earthworms and Castings, personal communication, 2015).
Paper mulch was added to the biosolids to provide bedding and a carbon
supplement for the worms. Premium Paper 100, a 100% hand sorted recycled newsprint
without added dye, was purchased from Applegate Mulch. The bin was filled in a singlebatch with enough substrate to equal 0.75 kg of the biosolids and paper mulch (wet

34

weight) mixture per kg of worms per day, for the anticipated duration of the experiment
(Ndegwa & Thompson, 2000). The depth of substrate did not exceed 0.3 m, suggesting
that material heating from microbial decomposition would not occur (Lindgern,
Pettersson, Kaspersson, Jonsson, & Lingvall, 1985). Distilled water was sprayed on the
surface of the substrate to maintain the moisture content earthworms require throughout
the experiment.
When checking the worms the morning after they were added to the substrate,
most of the worms were found crawling up the sides attempting to escape, or had
succeeded in escaping from the worm bin. Thinking the substrate appeared heavy on
paper mulch, two handfuls of 100 percent biosolids were applied to half of the bin’s
surface. Worms will not stay in a bin if there is not enough food, water, or oxygen (Fong
& Hewitt, 2016). Additionally, more water was added to the substrate, as it appeared on
the dry side. Earthworms are photophobic (Chengelis, 1990) so a light was kept on the lid
of the worm bin, which had half-inch holes drilled for air and a little light, to encourage
the worms to burrow into the substrate.
By the second day, most all of the worms had burrowed down into the substrate
that had not had additional biosolids applied to the surface. After noticing there were no
worms on the half of the bin to which the additional dually digested biosolids were
applied, the two handfuls of biosolids were removed because there was no other reason
for all the earthworms to be in the substrate on the opposite end of the bin. This
observation led to the idea there was something about the biosolids that was repulsive or
at least not appealing to the earthworms. Otherwise, it is believed the earthworms did not
try to escape after the second day because the initial moisture content was not sufficient

35

for a habitable environment.
In the literature, only a few studies indicate use of anaerobically digested
biosolids (Benitez, Sainz, & Nogales, 2005; Gaylor, Harvey, & Hale, 2013; Prosser,
Lissemore, Topp, & Sibley, 2014) whereas the majority of studies did not specify
whether the biosolids were aerobically or anaerobically digested. Further research was
conducted after (Experiment III: Bulking material and concentrations, described in
section 8 below), which suggested that anaerobically digested biosolids, similar to the
City of Tacoma and like that Pierce County’s biosolids, are toxic to earthworms
(Hartenstein et al., 1981).
The pilot study began October 26, 2015 and concluded November 30, 2015.
During the 35-day study, there were no further mass escapes and the earthworms thrived
and were even reproducing, as evident by the presence of cocoons, indicating favorable
environmental conditions.
6. Experiment I: 89 to 11
Upon successful completion of the pilot project, biosolids collected from the City
of Tacoma’s central WWTP were to be vermicomposted for a 45-day period. The
concentration of TCS and Me-TCS were to be measured every five days for the duration
of the experiment to track the change in concentration between two bins with worms and
one control that contained no worms. Preliminary analysis of the City of Tacoma’s
biosolids indicated sufficient concentrations of TCS to where additional contamination of
TCS to biosolids was not needed (M. Bozlee, personal communication, 2016).
To determine the total amount of paper mulch and biosolids necessary to feed the
worms for the duration of this study, values were used from Ndegwa, Thompson, and

36

Das (2000). They fed the worms a mixture of biosolids and paper mulch that consisted of
89 percent biosolids and 11 percent paper mulch, by dry weight, over the entire duration
of their study. These values were followed for this study, but again, the percent moisture
for the biosolids was calculated as 20 percent, rather than the actual 80 percent. This
miscalculation resulted in less biosolids overall than had the actual percent moisture been
used. This miscalculation was noticed and corrected in Experiments II and III.
Experiment I of this study began January 15, 2016 and concluded 15 days later on
January 29, 2016.
6.1. Parameters measured
If the experiment was successful, soil nutrients were to be measured (method in
parentheses) at the beginning and end of the experiment and consisted of total organic
carbon (EPA 9060A), phosphorus (EPA 365.4), potassium (EPA 6010C), total Kjeldahl
nitrogen (SM 4500-Norg B), pH (EPA 9045D), and percent solids (SM 2540 G).
Nutrients were to be measured because the final product is used for amending soil.
Earthworms change the nutrients available to plants (van Groenigen et al., 2014). By
measuring nutrient concentrations before and after the experiment, it could be determined
whether introducing earthworms into the biosolids had an effect on said nutrients.
6.2. Substrate preparation
The substrate was prepared in the same manner as in section 5 with the following
additional measures: The biosolids were allowed to off-gas for five days prior to mixing
with the paper mulch and room’s ambient temperature was monitored and averaged 16.8°
C (standard deviation (SD) = 1.11).

37

6.3. Sample collection
On scheduled collection days, five soil samples were taken from each of the three
bins (two treatments and one control). Samples were combined to make one compound
sample, from which 50 grams were transferred into Whirl-Pak bags to create one
compound sample for each of the two treatment bins and the one control. Samples were
collected every five days for 15 days with the first sample being only substrate. The U.S.
Environmental Protection Agency (1989) recommends samples of equal amounts to be
taken from multiple locations within each treatment container and thoroughly mixed
together to create a compound sample.
A randomly generated number table was used to pre-determine the location from
which each sample was taken, on a four by six grid. Samples were taken from the center
of each grid location and were stored at four degrees Celsius, or cooler, until they were
transported to the City of Tacoma’s Environmental Services’ laboratory for analysis
(U.S. EPA, 1989).
Each day, dead worms were removed and counted. The dead worms were only
counted and not weighed due to desiccation and decomposition. Individuals were counted
regardless of maturity; therefore a smaller, younger worm was counted the same as a
larger, more mature worm.
6.4. Results
Within a week of adding the worms to the substrate, each bin lost 343 and 477
worms, or 41 and 56 percent, respectively. The percentage of dead worms was
determined by converting the weight of the worms added, 384 g, to the approximate
number of individuals using the ratio of 1,000 individuals per pound of earthworms

38

(Knipple, D., personal communication, April 1, 2016).
Nine days into the experiment, additional earthworms were purchased and added
to the bins to replace the amount that had died in case the earthworms that were initially
put in the bins on day one were not healthy. After the additional earthworms were added,
worms continued to die, another 143 and 311 worms, or an additional 17 and 37 percent,
respectively, per bin. The experiment was terminated after 15 days.
Substrate temperatures were recorded on a daily basis to ensure temperature was
optimal for worm survival. The control bin’s mean substrate temperature was 21.7° C
(SD = 1.09) and the worm bins’ averaged 22.0° C (SD = 1.09) and 23.7° C (SD = 1.58).
The ambient temperature averaged 16.7° C (SD = 6.84) outside of the worm bins.
6.5. Discussion
This portion of the experiment strongly indicated there was something in their
environment that was killing the worms. Kaplan, Hartenstein, Neuhauser, and Malecki
(1980) determined the optimal substrate temperature for worm growth is between 20° and
29° Celsius and if pH is below five or above nine, earthworms die within a week. The
substrate temperatures in this study were within the optimal limits for earthworms and the
biosolids had a pH of 7.6, again, well within the range of earthworm survivability.
During the pilot study, additional biosolids were applied directly to half of the
substrate surface the morning after the earthworms were added to the substrate fearing
the substrate did not contain sufficient amount of feed. Upon further investigation the
following day, there were few, if any, earthworms in the substrate directly below the
additional biosolids. This observation suggests the earthworms did not want to be around
such high concentrations of biosolids.

39

With this in mind, perhaps the biosolids to paper mulch ratio (89:11), calculated
from Ndegwa, Thompson, and Das (2000) and subsequently used in this experiment, was
too biosolids heavy. On the other hand, mixing four parts biosolids to three parts paper
mulch does not seem practical if applied to a larger, possibly commercial, scale biosolidsvermicomposting process, due to required amount of paper mulch that would need to be
purchased. The ratio of 89:11, biosolids to paper mulch, perhaps results in too much
biosolids for healthy worm survival. It was not until after Experiment III (described in
section 8 below) that it was discovered that anaerobically digested biosolids, similar to
the City of Tacoma and like that of Pierce County’s biosolids, are toxic to earthworms
(Hartenstein et al., 1981). Without this knowledge, at the time, Experiment II of this
study evaluated earthworm survival in substrate composed of the City of Tacoma’s dually
digested biosolids and paper mulch mixed at a ratio of 2:1 with the hypothesis that the
previous experiment was too heavy in the amount of biosolids added.
7. Experiment II: 2 to 1
Considering the substrate created in Experiment I appeared to be biosolids-heavy
(even with the calculations including the mistaken 20 percent moisture) and the substrate
created for the pilot appeared paper mulch-heavy for practical purposes, the ratio of
biosolids to paper mulch was adjusted to two parts biosolids to one part paper mulch, by
dry weight. These calculations were completed using the correct percent moisture for the
biosolids of 80 percent and seven percent moisture for the paper mulch.
Experiment II of this study began February 13, 2016 and concluded 20 days later
on March 3, 2016.

40

7.1. Parameters measured
Parameters measured in this part of the experiment were the same as measured in
section 6.
7.2. Substrate preparation
The same worm bin setup was used in this portion of the study, as described in
section 5. The substrate was prepared the in the same manner as in section 6. The room’s
ambient temperature was monitored and averaged 16.8° C (SD = 1.11).
7.3. Sample collection
Samples were collected in the same manner as in section 6. Each day, individual
worms that were dead were removed and counted. This was done, as described in section
6. Substrate temperatures were recorded on a daily basis.
7.4. Results
Earthworms began to die quickly. By Day five, 224 and 225 worms were counted
as on the surface of the substrate, between the substrate and the side of the worm bins, or
had escaped and died from desiccation. Worms found dead between the substrate and the
sides of the worm bin were decomposed, making it difficult to accurately count the
number of individuals that died. As such, it was estimated to the best of the researcher’s
ability.
Considering 384 g of worms (or 845 individuals) were added to each worm bin,
the number of dead worms by Day 5 accounted for 26.5 and 26.6 percent, respectively, of
the total earthworms added. During sampling, live worms were seen deeper in the
substrate but dead and decomposed worms were also observed between the substrate and
the side of the bins but were not removed because to remove them would disturb the

41

substrate more than desired.
After Day 5, no more earthworms were found dead on the surface of the substrate.
No additional earthworms were purchased or added to the bins for this experiment. The
experiment was terminated after 20 days due to the total number of worms counted as
dead and the lack of live worms observed in the substrate during sampling.
Following termination of this portion of the study, the substrate from the worm
bins was sifted through by hand to count the number of worms that were still alive and if
any cocoons could be observed. Only seven worms were found alive in one bin and 13 in
the other; no cocoons were found in either bin. The 20 surviving earthworms were placed
into freshly mixed substrate that had a ratio of two to one, biosolids to paper mulch. The
following day, 12 worms were found dead in or on the substrate. Eight earthworms were
not accounted for in or around the container.
Substrate temperatures were recorded on a daily basis. The control bin’s average
substrate temperature was 22.55 (SD = 1.92) degrees Celsius and the worm bins’
averaged 23.04 (SD = 2.29) and 21.7 (SD = 2.08) degrees Celsius.
7.5. Discussion
Even with a biosolids to paper mulch ratio of two to one, earthworms continued to
quickly perish. The number of E. fetida counted as dead (224 and 225) and the number
found alive (7 and 13) does not equal the calculated total of individuals placed in each bin
(845). The totals actually account for only 27.3% and 28.2%, respectively. The
discrepancy in total calculated earthworms added and the total number counted as dead
and the final survivors is attributed to the fact that earthworms begin to decompose
quickly, after death. Earthworms are made up of 75 to 90 percent water and when they

42

die, they decompose very quickly (Washington State University Whatcom Extension,
2016).
The earthworms that survived this 20-day experiment were large and robust.
Perhaps age or size of the earthworms used contributes to their ability to survive in such
environments. Artuso, Kennedy, Connery, Grant, and Schmidt (2010) evaluated the
impact of soils amended with various concentrations biosolids on earthworm
survivability. They observed significantly fewer juvenile earthworms in the substrates
with the highest amount of biosolids but was not related to the presence of heavy metals,
the parameter measured in this study. Therefore, the researchers believed there is another
variable at play that was not measured in their study.
Kinney et al. (2012) also observed an increase in earthworm mortality and a
decrease in juveniles and cocoons in soils amended with the highest amounts of biosolids.
The researchers compared biosolids that had been aged for different time-periods because
previous studies have shown that ageing biosolids can decrease toxicity and
bioavailability, which is the ability for organisms to take-up the contaminant (Alexander,
R. & Alexander, M., 1999; White, Kelsey, Hatzinger, & Alexander, 1997).
The 20 surviving earthworms were collected from the two worm bins and placed
in another container of freshly mixed substrate of the exact same proportion of biosolids
to paper mulch, two to one. The substrate was made from biosolids collected for this
portion of the experiment so there was no difference in materials used, except they had
aged in a dark container for the duration of this part of the experiment. The following
day, 12 of the 20 earthworms were found dead on the surface of, or in the substrate. Eight
worms were unaccounted for and were presumed to have escaped but were not found in

43

the area surrounding the container.
In Experiment II it was made clear there was something in the environment or
substrate that was causing the earthworms to perish. Still being unaware of the toxicity of
anaerobically digested biosolids, it was learned that the City of Tacoma had recently
acquired a new dewatering system; they moved from a belt press to a screw press and
were still in the process of determining the correct amount of polymer to add to the
material. The addition of polymers is standard practice for most WWTPs that dewater
biosolids (Ross, R., personal communication, 2016) but was unknown to the author, at
the time, as to whether the polymer added to the biosolids during the dewatering process,
was potentially causing the earthworms to perish. During the dewatering process of
biosolids, polymer is added as a flocculant to improve the separation of water and solids
(Oleszkiewicz & Mavinic, 2002)
The addition of polymers is standard practice including a large-scale biosolidsvermicomposting operation in Granville, Pennsylvania (Weaver, P., personal
communication, 2016). Considering this facility was able to maintain healthy
earthworms, even with the addition of polymers during the dewatering of biosolids,
polymers were ruled out as a potential reason for the earthworms’ death. The Material
Safety Data Sheet for the polymer used by the City of Tacoma was obtained and no
previous research turned up that indicates it is toxic in any way to earthworms (Ross, R.,
personal communication, February 18, 2016). Moreover, Kaplan et al. (1980) determined
that, even at high concentration, inorganic additives used in the dewatering process to
better congeal the material was harmless to earthworms.
However, in the large-scale biosolids-vermicomposting facility in Granville,

44

Pennsylvania the bedding and carbon supplement supplied to the earthworms was wood
chips, rather than paper mulch. Paper mulch was used in this experiment because
previous studies evaluating earthworms’ ability to process biosolids also did as such
(Ndegwa & Thompson, 2002; Ndegwa, Thompson, & Das, 2002).
The City of Tacoma has a bountiful supply of wood shavings that would be
desirable for use of bedding if they were to start a large-scale vermicomposting operation
(Thompson, D., personal communication, 2016). Perhaps the paper mulch inhibited the
flow of oxygen through the substrate, because it can compress when saturated, possibly
creating an anaerobic environment that is not suitable for worm survival (Weaver, P.,
personal communication, 2016).
It is hard to say whether the earthworms did better or worst in this substrate,
compared to the substrate used in Experiment I. It was assumed that they would do better
because the ratio of biosolids to paper mulch was lighter on the biosolids, but the rate at
which they perished would not support that hypothesis. Therefore, in Experiment III that
follows, four smaller containers were filled with different substrates to evaluate whether
one substrate was more harmful or suitable for earthworm survival. Wood shavings,
supplied by the City of Tacoma, were used to create a substrate of proportions similar to
the substrate used in the large-scale biosolids-vermicomposting facility (Weaver, P.,
personal communication, 2016). To determine if there was a component of the substrate
that was causing the worms to perish, total organic carbon, total nitrogen, phosphorus,
potassium, and pH were measured when the worms were put into each container.

45

8. Experiment III: Bulking material and concentration
This portion of the study was completed in an attempt to determine which
parameters within the substrate resulted in earthworm survival. The following variables
were examined by The City of Tacoma’s Environmental Services’ Laboratory: pH,
potassium (K), phosphorus (P), total organic carbon (TOC), total Kjeldahl nitrogen
(TKN), which is the sum of organic nitrogen, ammonia (NH3) and ammonium (NH4+).
Experiment III began February 28, 2016 and concluded, 30 days later, on March
28, 2016.
8.1. Parameters measured
The parameters measured in section 6 and 7 were also measured in this section,
with the exclusion of substrate temperature because little fluctuation had been observed
in Experiments II and I. The detailed laboratory methods for testing TCS and Me-TCS,
which follow EPA Method 8270D for semi-volatile organics PPCP, and can be found in
Appendix B. Additionally, the weight of worms and number of cocoons produced was
measured at the end of the 30-day experiment.
8.2. Substrate preparation
Dually digested biosolids were spread onto plastic sheeting to a depth of a few
centimeters and allowed to off-gas for 10 days after being collected directly from
dewatering from the City of Tacoma’s Central Wastewater Treatment Plant. In
Experiments I and II, biosolids were allowed to off-gas for five days, however the
biosolids for Experiment III were allowed the extra time in an effort to allow more
ammonia to off-gas as earthworms are sensitive to ammonia (Edwards & Bohlen, 1996).

46

After off gas time period, the biosolids still had a strong ammonia odor (similar to
Experiments I and II).
Four mixtures were created for this part of the experiment to test worm survival in
biosolids substrates with the addition of the paper mulch or wood shavings, mixed with
different concentrations of biosolids. Further, the substrates and the biosolids themselves
were examined to assess if they were causing the earthworms to perish.
The substrate created for the Pilot (section 5) was replicated to see if it was an
initial fluke the worms survived and flourished in the material. The ratio of biosolids to
paper mulch by dry weight, using the correct percent moisture, for the initial pilot was
1:4. The second substrate consisted of two parts biosolids and one part wood shavings by
volume. This substrate was created based on the process by which a successful largescale vermicomposting of biosolids, in Granville, PA, operated (Weaver, P., personal
communication, 2016). The third substrate was the true ratio of 89:11, biosolids to paper
mulch by dry weight, based on correct and accurate percent moisture. The last substrate
was 100 percent biosolids to rule out any potential effect the addition of paper mulch or
wood shavings may have interacted with the biosolids creating an uninhabitable
environment for the earthworms. Distilled water was added to achieve approximately 80
percent moisture.
In addition, the possibility that the earthworms were shocked or overly stressed
when placed on the biosolids substrate, led to a new approach of stacking two containers
with the earthworms in a familiar substrate in the lower container and, with holes drilled
in the bottom of the upper container, so that earthworms could move up into the novel
substrate as they pleased (see Figure 8.1; Monroy, Aira, & Dominguez, 2009). Collecting

47

vermicompost from the source where the earthworms were purchased ensured the worms
would be in material with which they were familiar. This material contained the organic
matter in which the worms were raised, as well as their feces. The worm supplier utilizes
coconut coir as bedding for their worm-growing operation and some was obtained for the
experiment. One part coconut coir was mixed with two parts vermicompost. The coconut
coir was added to provide bedding and increase the volume of substrate in the compost.
Coconut coir’s nutrient value is relatively low (Richards, 2006) therefore its addition
would reduce the amount of feed available to the worms, encouraging them to utilize the
biosolids substrate while still providing habitable environment.
Figure 8.1. Diagram of stacked containers used in Experiment III: Bulking material and
concentration.
Ven8la8on-holesLidUpper-Container-

Biosolids/Paper-

Lower-ContainerCompost-

Earthworms-

Figure 8.1. Schematic of the preparation of stacked containers of substrate, compost, and
earthworms. Upper container is in contact with the substrate in the lower container. Two
thirds of the earthworms (by weight) were placed on the surface of the compost, prior to
stacking the upper container with the test substrate. One third of the earthworms (by
weight) were placed on top of the test substrate in the upper container).

48

Container size was reduced from Experiment I and II to 1.24-liter polypropylene
plastic containers (12 cm high, 18 cm wide, 18 cm deep) for a surface area of 0.03 m2
because smaller amounts of material were needed, and fewer earthworms were utilized.
Each container’s base was wrapped in foil (Kwon & Xia, 2012) because the containers
were clear and earthworms are photophobic (Phillips, Checkai, Chester, Wentsel, &
Major, 1994). Ventilation holes were drilled into each of the four containers’ lids and to
the sides of each container. The containers of earthworms and biosolids substrates were
not disturbed for the duration of this portion, Experiment III.
8.3. Sample collection
All substrate samples were collected to analyze pH, potassium (K), phosphorus
(P), total organic carbon (TOC), total Kjeldahl nitrogen (TKN), which is the sum of
organic nitrogen, ammonia (NH3) and ammonium (NH4+) prior to the addition of the
earthworms and at the end of the 30-day experiment. Additionally, a sample of the repeat
of the pilot study’s substrate was collected prior to the addition of worms and after the
30-day experiment ended to be tested for TCS and Me-TCS concentrations at the City of
Tacoma’s Environmental Services Laboratory. The laboratory methods for each test in
listed in section 6.1. Only the repeated pilot substrate was tested for TCS and Me-TCS
concentrations because it was the only substrate to have substantial earthworm survival at
the end of the 30 days.
8.4. Results
Of the 31 grams of worms added to each substrate, the pilot repeat saw an
increase in total weight of worms to 44 grams, suggesting the worms were actually
growing. The biosolids and wood shavings substrate resulted in a 20-gram decrease of

49

live worm weight, for a total of 11 grams at the end of the experiment. The 89 to 11,
biosolids to paper mulch, substrate and the biosolids-only substrate, had only five grams
and 0.7 grams, of live worms, respectively, at the end of the experiment (see Figure 8.2).
Cocoons were observed only in the repeated pilot substrate and the biosolids and wood
shavings test substrate where 51 cocoons were counted in the repeated pilot substrate and
only one cocoon found in the biosolids and wood shavings substrate.
Figure 8.2. Total weight (g) of earthworms (E. fetida) alive in each substrate after 30-day
experiment.

Figure 8.2. Weight (grams) of live worms in each substrate. The initial weight of worms
added to each container was 31 g, therefore values greater than 31 g indicate growth,
whereas values less than 31 g indicate death and decomposition of worms.
The biosolids-only substrate had the highest amount of TKN (48.7 g/kg) followed
by the biosolids and paper mulch (89:11), biosolids and wood shavings, and the repeat of
the pilot substrate (42.9 g/kg, 30.5 g/kg, and 19.5 g/kg, respectively; see Figure 8.4). The
biosolids-only substrate had the greatest amount of phosphorus (27.7 g/kg) followed by
50

biosolids and paper mulch (89:11), biosolids and wood shavings, and the repeated pilot
substrate (25.8 g/kg, 20.7 g/kg, and 13.4 g/kg, respectively). The highest amount of
potassium was measured in the biosolids-only substrate (2,310 mg/kg), followed by
biosolids and wood shavings, biosolids and paper mulch (89:11), and then the repeated
pilot substrate (2,190 mg/kg, 2,130 mg/kg, and 1,070 mg/kg, respectively).
Figure 8.3. Amount of total Kjeldahl nitrogen, phosphorus, and potassium measured in
the four test substrates prior to the addition of earhtworms (E. fetida).

Figure 8.3. Amount of total Kjeldahl nitrogen (TKN), phosphorus (P), and potassium (K)
in biosolids and paper mulch substrate (89:11, by dry weight), repeat of the pilot substrate
(1:4, by dry weight, biosolids to paper mulch), biosolids-only substrate, and biosolids
mixed with wood shavings (2:1, by volume).
Total organic carbon was highest in the pilot repeat (389 g/kg), followed closely
by biosolids and wood shavings, biosolids and paper mulch (89:11), and the biosolidsonly substrate (321 g/kg, 320 g/kg, and 309 g/kg, respectively; see Figure 8.4).

51

The pH of the four substrates ranged from 7.2 (biosolids and wood shavings) to
7.7 (biosolids and paper mulch, 89:11), which is well within earthworms’ pH tolerance of
five to nine (Kaplan et al., 1980).
Figure 8.4. Total organic carbon (g/kg) measured in the four test substrates prior to the
addition of earhtworms (E. fetida).

Figure 8.4. Amount of total organic carbon (TOC) in biosolids and paper mulch substrate
(89:11, by dry weight), repeat of the pilot substrate, biosolids-only substrate, and
biosolids mixed with wood shavings (2:1, by volume).
8.4.1 Triclosan and methyl triclosan concentrations
The TCS concentrations in the repeated pilot substrate decreased from 3,200
µg/kg to 880 µg/kg (75%) from before and after earthworm exposure. Methyl triclosan
concentrations increased from an undetectable level (minimum detection limit = 5 µg/kg)
in the repeated pilot substrate before earthworms were added to 29 µg/kg (480%) after 30
days of exposure to earthworms (see Figure 8.5).

52

Figure 8.5. Concentration of triclosan and methyl triclosan before and after
vermicomposting repeated pilot substrate.

Figure 8.5. Triclosan and methyl triclosan concentration (µg/kg dry) in repeated pilot
substrate (1:4 biosolids to paper mulch, by dry weight) prior to earthworms (E. fetida)
and after 30 days of vermicomposting.
8.5. Discussion
The largest amount of worm survival was seen in the repeated pilot substrate,
which actually showed an overall increase in live earthworm weight. This substrate
contained the lowest concentration of biosolids suggesting it may be potentially the cause
for the earthworms’ inability to survive. Interestingly, the biosolids-only substrate had the
least amount of surviving earthworms, only one individual (0.7 grams), and had the
highest amount of TKN while the repeated pilot had the lowest TNK but most surviving
earthworms. Wei and Liu (2005) found that high ammonia nitrogen concentration
inhibited growth and were initially toxic to earthworms. Unfortunately, the test available
in the current experiment, TKN, is the sum of organic nitrogen, ammonia, and
ammonium so it is impossible to determine which nitrogen compound concentration is
high and therefore the cause of the toxic environment. Edwards (1988, as cited in
Edwards & Bohlen, 1996) found that earthworms will leave a substrate once it becomes
anaerobic because they are very sensitive to ammonia and will not survive in substrates
containing high ammonia levels. The aerobic digestion of sludge creates ammonia but it
53

is typically released into the atmosphere (Maramba, 1978), like in an open tank similar to
the City of Lynden’s WWTP. Anaerobic digestion retains the ammonia that is produced
(Maramba, 1978), sometimes to levels that can actually become toxic and inhibit the
microbes from digesting and stabilizing the raw sewage sludge (Hansen, Angelidaki, &
Ahring, 1998).
The production of cocoons appeared to follow the trend of surviving earthworms,
which makes sense since there must be mature individuals in order to reproduce.
However, viability of cocoons was not evaluated. Reinecke, A., Reinecke, S., and
Maboeta, (2001) evaluated the effect metal toxicity on E. fetida reproduction and cocoon
viability. While they did not observe a difference in cocoon production of the worms in
contaminated substrate, compared to a control, they did observe a decrease in cocoon
viability in the soil contaminated with sublethal amounts of toxins.
Total organic carbon was measured highest in the repeated pilot substrate (389
g/kg) with the biosolids and wood shavings and 89:11 substrates second and third closest
(321 and 320 g/kg, respectively) and biosolids last with 309 g/kg of TOC. Interestingly,
the number of individual earthworms at the end of the 30-day experiment followed the
same pattern where the repeated pilot substrate had 44 individuals at the end, followed by
the biosolids and wood shavings and 89:11 substrates with 11, five, and one individual
(respectively). Additionally, the repeated pilot substrate had 51 cocoons where as the
only other substrate to have any cocoons was the biosolids and wood shavings where
only one cocoon was counted. Earthworms need carbohydrates, or carbon, and protein to
survive (Avis, 2011). Stachell (1967, as cited in Edwards & Bohlen, 1996) observed a
positive correlation between palatability and soluble carbohydrates in E. fetida. Perhaps

54

the earthworms in this experiment found the carbon-based paper mulch palatable in that
they are able to thrive in the repeated pilot substrate better than in the biosolids and wood
shavings substrate or the 89:11 substrate, that did not have nearly as much paper mulch to
biosolids as the repeated pilot substrate.
Knowing the TCS and Me-TCS concentrations before and after earthworm
exposure, the amount of expected amount of TCS degrading into Me-TCS can be
mathematically extrapolated. As mentioned earlier in section 2.7, Chen et al. (2011)
determined one percent of TCS transformed into Me-TCS in aerobic laboratory
conditions while Butler et al. (2012) measured up to 66 percent of TCS transforming into
Me-TCS in sandy loam clay soil during warm, dry months in a field setting. If the
minimum, one percent, of 3,200 µg/kg of TCS transformed into Me-TCS were applied
the total would be 32 µg/kg of Me-TCS (Chen et al., 2011). Whereas, if the maximum, 66
percent (Butler et al., 2012), of TCS transformed into Me-TCS, 2,112 µg/kg of Me-TCS
would be the expected amount observed.
Based on the expected percent of Me-TCS formation (one to 66) from TCS
degradation, the current study more closely aligns with Chen et al.’s (2011) findings of
one percent of TCS accounts for the formation of the measured Me-TCS. In the present
study, the formation of Me-TCS accounts for only one percent of the 75 percent decrease
in TCS concentration. The formation of Me-TCS cannot explain the total decrease in
TCS concentration, which would indicate there are other factors involved in the further
reduction of TCS observed. Something other than the formation of Me-TCS caused the
other 2,320 µg/kg of TCS to not be present in the substrate after exposure to earthworms.

55

To see Me-TCS concentrations below the level of detection is expected because it
is only formed through the process of TCS degradation. Therefore, with time, TCS would
degrade and Me-TCS concentration would be expected to increase, as seen in Figure 8.5.
The biosolids collected from the City of Tacoma were collected directly after dewatering,
the last stage of the wastewater treatment process. The plant does not age their biosolids;
they are used immediately and were collected as such.
The European Commission (2010) states TCS is degraded by photolysis
(exposure to light), chlorination, ozone treatment, and aerobic bacterial hydrolysis or the
breakdown of chemicals by bacteria in water. At The City of Tacoma’s WWTP where the
biosolids for this experiment were collected, chlorination does not occur until just prior to
release of effluent water back into the environment, after separation of the biosolids; so
no chlorine was introduced to cause degradation. The substrate was collected directly
following the dewatering process at the WWTP and was mostly kept in the dark
throughout the experiment, additionally, ozone treatment is not incorporated at The City
of Tacoma’s WWTP. Aerobic bacterial hydrolysis is the only factor the European
Commission lists as a primary degrader of TCS that cannot be ruled out in the current
experiment. However, the presence of earthworms may have an impact on the observed
decrease TCS concentration but further research will be needed to fully support this.
The consideration that earthworms may be responsible for the decrease in
measured TCS concentration is consistent with findings from previous research that
earthworms can bioaccumulate TCS (Higgins et al., 2011; Kinney et al., 2006, 2008,
2010; Macherius et al., 2014). Unfortunately, only the repeated pilot substrate had
earthworms survive in the material for evaluation of TCS and Me-TCS concentration

56

In search of the impact of potassium and phosphorus on earthworm survival, no
studies were found that would indicate there is any amount of either chemical that
inhibits earthworm survival or which causes death. However, phosphorus was positively
associated with earthworm survival.
It turns out that anaerobic sludge can be toxic to earthworms (Hartenstein, 1981).
Masciandaro, Ceccanti, and Garcia (2000) found that when anaerobically digested
biosolids were spread onto fields the amount of earthworms that left the area was
positively correlated with an increasing amount of biosolids. This is supported in the
current research in that very few worms survived in the biosolids-only substrate.
Additionally, when anaerobically digested biosolids were applied to the surface of half of
the substrate in the initial pilot study (section 5) the majority of earthworms appeared to
move to the area of substrate without added biosolids.
As the amount of material added to the biosolids was increased, from 89:11 (by
dry weight) biosolids to paper, 2:1 (by volume) biosolids and wood, and the repeated
pilot substrate (1:4, biosolids to paper, by dry weight) the amount of worms that survived
also increased. This suggests that while the anaerobically digested biosolids are toxic to
earthworms, an environment can be created in which the earthworms can survive by the
addition of other material or bedding.
Once Hartenstein’s publication was discovered, and subsequently confirmed here
in Experiment III, biosolids processed differently were sourced. In Experiment IV, that
follows, biosolids from the City of Lynden, that processes their incoming wastewater
aerobically, Pierce County that processes their incoming wastewater anaerobically, and
from the City of Tacoma that utilizes a dual digestion process of aerobic followed by

57

anaerobic digestion were obtained and earthworm survival and reproduction was. The
purpose for including Pierce County’s anaerobically digested sludge was to determine if
the earthworms had not survived in The City of Tacoma’s biosolids due to it being
anaerobically digested as the second step of the dual digestion process or perhaps another
unknown factor.
9. Experiment IV: Three biosolids sources
Through further discussion, Weaver, P. (2016) stated he had the most success
with vermicomposting aerobically digested sludge. Upon researching peer-reviewed
literature, it appears others have found anaerobically digested sludge to be toxic to
earthworms (Hartenstein, 1981; Masciandaro et al., 2000). There are two possibilities for
why anaerobically digested biosolids are toxic to earthworms, one being an oxygen
deficiency in the substrate because of limited compaction, and therefore minimal
aeration, and the other is the anaerobic process, utilized at WWTPs, results in toxic
compounds (Masciandaro et al., 2000). The biosolids used in Experiments I, II, and III
were all dually digested, initially aerobically followed by anaerobically digestion.
Therefore, aerobically digested, Class B biosolids were obtained from the City of
Lynden, Washington and anaerobically digested, Class B biosolids were obtained from
Pierce County’s Chambers Creek Wastewater Treatment Plant to compare worm
survival, reproduction, and ultimately contaminant concentrations to that of the City of
Tacoma’s Central Wastewater Treatment Plant’s Class A Exceptional Quality (EQ)
biosolids.
The primary difference between Class A and B biosolids is the amount of
pathogens allowed in the final product. In Class A biosolids, pathogen levels must be

58

nearly eliminated from the material whereas Class B biosolids can have pathogens to a
certain level. Class B biosolids tend to have more plant available nitrogen and are
therefore preferred by farmers but are more regulated and have more restrictions on usage
(Oregon Association of Clean Water Agencies, 2009). Class A EQ biosolids meet and
exceed Class A standards in pathogen and heavy metals reduction (U. S. EPA, 1999).
According to a 2004 survey, 23 percent of biosolids were processed to the Class A level
and 34 percent were processed to a Class B level (North East Biosolids and Residuals
Association, 2007).
9.0.1. Substrate preference
While anaerobically digested biosolids have been shown to be toxic to
earthworms (current study; Hartenstein, 1981), they were able to survive and thrive in the
Pilot study’s substrate (section 5). Therefore, at that concentration of biosolids and
carbon supplement, the biosolids were habitable but may not be preferred by earthworms.
An additional test was performed to determine whether aerobically or anaerobically
digested biosolids are more preferable to earthworms, compared to compost.
Experiment IV had a staggered start time because of biosolids availability.
Beginning April 4, 2016 and concluding on May 15, 2016 each treatment lasted 35 days.
The number of days was increased from Experiment III, which was 30 days, to ensure the
earthworms had enough time to process the material to test before and after
concentrations of TCS and Me-TCS.

59

9.1. Parameters measured
Parameters measured in Experiment III were the same as measured in section 8 of
this study, in addition to substrate temperatures. All samples, before, the control, and
after earthworm exposure, were measured for concentrations of TCS and Me-TCS.
9.1.1. Substrate preference
The parameters measured in testing the earthworms’ substrate preference was
number and weight of earthworms added initially, and again after 35 days. Cocoons were
also counted but the viability of cocoons was not tested or measured.
9.2. Substrate preparation
Biosolids were spread onto plastic sheeting to a depth of a few centimeters and
allowed to off-gas for 12 days after being collected from each WWTP and prior to being
mixed with paper mulch and distilled water. In Experiment III, the biosolids were
allowed to off-gas for 10 days. An additional two days was added here, in Experiment IV,
because the time was available and may have allowed for even more of the ammonia
smell to off-gas.
The substrate mixture was a ratio of four parts biosolids to three parts paper
mulch, by dry weight. The ratio of 4:3, biosolids to paper mulch, was chosen because a
ratio of 2:1, in Experiment II, resulted in major earthworm mortality and there was
success in the pilot study of which the substrate was prepared at a ratio of 4:3, biosolids
to paper mulch, but by wet weight, 1:4 by dry weight. In an effort to determine whether
the earthworms are capable of bioaccumulating TCS and Me-TCS from biosolids,
effectively removing the contaminants, the worms needed to survive and therefore a
substrate suitable for survival, rather than practical application, was chosen.

60

Three different substrates were created from biosolids from the three WWTPs, for
a total of nine unique substrates. No replicates were created due to limitations in funding.
These substrates were created to test whether the presence of earthworms had an impact
on TCS and Me-TCS concentrations in a biosolids and paper mulch substrate and the
substrate preference of E. fetida between biosolids and compost.
Each container’s base was wrapped in foil because earthworms prefer dark
conditions (Kwon & Xia, 2012). All containers were checked regularly for mold growing
on the surface of the substrate and sides of the container; any observed mold was
removed and the amount of material removed with the mold was weighed and recorded.
Each container had a lid in which ventilation holes had been drilled. Thermometers were
placed through a ventilation hole in the lid of each container into the substrates and
remained there for the duration of the experiment to obtain daily temperatures.
The same containers used in Experiment III were also used here. Additionally, the
stacked container approach (see Figure 9.1; Monroy et al., 2009) was used in the set up
evaluating the presence of earthworms’ influence on concentrations of TCS and Me-TCS
in the substrates; otherwise, all other treatments, including the control, were not stacked
containers.
For each biosolids and paper mulch treatment, a large batch was prepared for each
biosolids source and divided between two containers; one in which earthworms were
added and the other was the control, which was allowed to age the duration of the 35-day
experiment. The preparation of the three containers evaluating earthworms’ preference is
described below in section 9.2.1.

61

Compost was collected from Northwest Redworms, in Camas, WA at the time
earthworms were acquired. The compost consists of horse manure, grass clippings, and
sawdust pellets, which is turned and aged for more than one year. Northwest Redworms
grow their worms in this compost. For this experiment, four parts compost and one part
peat moss (wet weight) was mixed to create a substrate that would be familiar to the
earthworms, limiting the shock and stress of being placed in a novel substrate, while not
providing enough that they would be able to survive by simply consuming the familiar
compost. In this section of the experiment the ratio of compost to peat moss, a coconut
coir alternative, was increased from Experiment III (2:1) to allow enough bedding for
earthworms to utilize in the substrate preference sub-experiment.
Figure 9.1. Diagram of stacked containers used in Experiment IV: Three biosolids
sources.
Ven8la8on-holesLidUpper-Container-

Biosolids/Paper-

Lower-ContainerCompost-

Earthworms-

Figure 9.1. Schematic of the preparation of stacked containers of substrate, compost, and
earthworms. Upper container, with holes drilled into bottom to allow earthworms to
travel between substrates, is in contact with the substrate in the lower container. Two
thirds of the earthworms (by weight) were placed on the surface of the compost, prior to
stacking the upper container with the test substrate. One third of the earthworms (by
weight) were placed on top of the test substrate in the upper container.
62

9.2.1. Substrate preference
A sub-experiment was created to test earthworm preference between biosolids
from each of the three sources and a compost mixture. Layered on top of moistened paper
mulch, compost and peat moss mixture and biosolids each evenly covered half of the
paper mulch surface (see Figure 9.2). Distilled water was added to the biosolids and
paper mulch to achieve approximately 80 percent moisture (by weight). There was no
control substrate this substrate-preference sub-experiment.
Figure 9.2. Diagram of divided containers used in Experiment IV: Three biosolids
sources

Ven+la+on'holes'

Biosolids'
Paper'mulch'

Lid'

Compost'
Earthworms'

Figure 9.2. Schematic of the preparation of divided containers of biosolids, compost, and
earthworms. Paper mulch was evenly spread across the bottom of the container. Half of
the paper mulch was covered with biosolids while the other half was covered with
compost. All the worms were sandwiched between the paper mulch and compost at the
beginning of the experiment.
9.3. Sample collection
Samples were collected from all substrates for laboratory analysis prior to the
addition of the worms. After the 35-day experiment, each substrate was sorted by hand to
remove and count all earthworms and cocoons and each substrate was well mixed before
63

being packed into individual Whirl-Pak bags. Samples were frozen until they were
transported to the City of Tacoma’s Environmental Services’ laboratory. All biosolids
and paper mulch substrate samples were collected to analyze the percent solids, pH, TCS
and Me-TCS concentrations, pH, potassium (K), phosphorus (P), total organic carbon
(TOC), total Kjeldahl nitrogen (TKN), which is the sum of organic nitrogen, ammonia
(NH3) and ammonium (NH4+) prior to the addition of the worms and at the end of the 35day experiment. Laboratory methods used for each test are listed in section 6.1.
9.4. Results
Substrate and ambient room temperatures were recorded on a daily basis and are
presented in Table 9.1.
Table 9.1. Mean (standard deviation) temperatures of substrate and ambient room
temperature.
Divided
Biosolids Biosolids and
Biosolids
Ambient room
Source
substrate
and paper
paper with
control
temperature
with worms
control
worms
Lynden
18.4 (1.9)
18.0 (1.9)
18.4 (1.7)
17.8 (1.7)
17.3 (1.8)
Tacoma
17.3 (1.7)
18.8 (1.8)
17.5 (1.6)
17.7 (1.7)
17.6 (1.7)
Pierce County 19.1 (1.8)
18.1 (1.8)
18.0 (2.2)
18.5 (1.8)
17.8 (1.8)
Note. Mean (standard deviation) temperature, in Celsius, of ambient room temperature and substrates made
from the City of Lynden, Tacoma, and Pierce County’s biosolids and paper mulch.

9.4.1. Earthworm survival
Of the two substrates created using The City of Tacoma’s dually digested
biosolids, all the worms survived the duration of the 35-day experiment. The divided and
biosolids and paper mulch substrates created using the City of Lynden’s aerobically
digested biosolids saw a loss of one (4%) to three (12.5%) earthworms, respectively.
Substrates created using Pierce County’s anaerobically digested biosolids resulted in a
decrease of eight earthworms (45%) Interestingly, the total number of earthworms added

64

to the divided substrate composed of Pierce County biosolids and compost showed an
increase of one individual worm (5.9%; see Figure 9.3); this is assumed a counting error
and is discussed further in section 9.5.1).
Figure 9.3. Number of earthworms added to each substrate at the beginning and after 35day experiment.

Figure 9.3. The number of earthworms added to each substrate at the beginning (blue)
and the number of earthworms remaining at the end (red) of the 35-day experiment.
The total weight of earthworms added to each substrate at the beginning of this
experiment was 30 grams. The number of earthworms added to each container was
counted, as well, for both the biosolids and paper mulch substrates and divided
containers. The total weight and number of earthworms added to each container was used
to calculate the weight per earthworm at time they were added as well as at the end of the
35-day trial. Figure 9.4 illustrates the weight per earthworm in the substrates composed
of four parts biosolids to three parts paper mulch, by dry weight. Initially the City of
Lynden had the lowest weight per earthworm (1.25 g/earthworm), followed by the City
of Tacoma and Pierce County (1.5 g/earthworm and 1.63 g/earthworm, respectively). At
the end of the 35-day experiment, the earthworms in the substrate composed of Pierce
County’s biosolids weighed 0.94 grams per earthworm and had lost the most amount of
weight per earthworm (0.69 g/earthworm lost). Earthworms in the substrate composed of
the City of Tacoma’s biosolids weighed 1.17 grams per earthworm, which is an overall
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loss of 0.33 grams per earthworm. Earthworms in the substrate composed of the City of
Lynden’s biosolids weighed 1.08 grams per earthworm and had the smallest amount of
loss of weight per earthworm (0.17 g/earthworm lost).
Figure 9.4. The weight (g) per earthworm in substrates composed of three parts paper
mulch and four parts biosolids sourced from the City of Lynden, Tacoma,
and Pierce County at the beginning and end of the 35-day experiment.

Figure 9.4. The weight (g) per earthworm in substrates composed of three parts paper
mulch and four parts biosolids sourced from the City of Lynden, Tacoma, and Pierce
County at the beginning (blue) and end (red) of the 35-day experiment.
Similarly, the divided substrate containers followed the same pattern as the
biosolids and paper mulch substrates. When the earthworms were added to the substrate
the City of Lynden had the lowest weight per earthworm (1.3 g/earthworm; see Figure
9.5), followed by the City of Tacoma and Pierce County (1.36 g/earthworm and 1.76
g/earthworm, respectively). At the end of the 35-day trial, the earthworms in the divided
container with Pierce County’s biosolids weighed 1.13 grams per earthworm and had lost
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the most amount of weight per earthworm (0.63 g/earthworm lost). Earthworms in the
divided container with the City of Tacoma’s biosolids weighed 1.13 grams per
earthworm, which is an overall loss of 0.23 grams per earthworm. Earthworms in the
divided container with the City of Lynden’s biosolids weighed 1.24 grams per earthworm
and had the smallest amount of loss of weight per earthworm (0.06 g/earthworm lost).
Figure 9.5. The weight (g) per earthworm in containers with divided substrates
composed of compost and biosolids sourced from the City of Lynden,
Tacoma, and Pierce County at the beginning and end of the 35-day
experiment.

Figure 9.5. The weight (g) per earthworm in containers with divided substrates composed
of compost and biosolids sourced from the City of Lynden, Tacoma, and Pierce County at
the beginning (blue) and end (red) of the 35-day experiment.
9.4.2 Reproduction
Earthworm cocoons were counted at the conclusion of this 35-day experiment for
the preference sub-experiment (see Figure 9.6) and the biosolids and paper mulch
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substrates (see Figure 9.7). Cocoon production within the compost and biosolids
substrates in the divided containers sub-experiment showed similar tends across the three
biosolids sources. The majority of cocoons were found in the compost (248, 252, and
200) compared to the biosolids (53, 35, and 12) of the City of Lynden, Tacoma, and
Pierce County, respectively.
In the substrates composed of biosolids mixed with paper mulch the City of
Lynden had the most cocoons (303) followed by the City of Tacoma with 271 and then
Pierce County had the fewest cocoons with 114 counted at the end of the 35-day
experiment.
Figure 9.6. Total number of cocoons counted within divided substrates composed of
biosolids from the City of Lynden, Tacoma, and Pierce County and compost.

9.4
 and
 

Figure 9.6. The total number of cocoons counted within divided substrates composed of
biosolids from the City of Lynden, Tacoma and Pierce County (blue) and the number of
cocoons counted in the side composed of compost (red).

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Figure 9.7. Total number of cocoons counted within each substrate consisting of
biosolids from the City of Lynden, Tacoma, and Pierce County.

Figure 9.7. Total number of cocoons counted within each substrate consisting of four
parts biosolids, from the City of Lynden, Tacoma, and Pierce County, and three parts
paper mulch after 35 days of exposure to earthworms.
9.4.3. Triclosan and methyl triclosan concentration
Triclosan concentrations were measured in the biosolids and paper mulch
substrates for Pierce County, the City of Tacoma, and the City of Lynden before and after
earthworm exposure and the control (see Figure 9.8). The City of Tacoma’s
Environmental Services’ laboratory, that analyzed the samples, allows for 20 percent
uncertainty between soil sample duplicates, due to GC/MS/MS calibrations (Bozlee, M.,
personal communication, November 21, 2016). Therefore, a 20 percent uncertainty has
been applied to the measured TCS and Me-TCS values.

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The TCS concentration in the City of Lynden’s substrate prior to the addition of
earthworms was 48 µg/kg (± 9.6). The substrates, after earthworm exposure and the
control, which was allowed to simply age throughout the duration of the 35-day
experiment, both had TCS concentrations below the limit of detection (39 µg/kg). After
bring exposed to earthworms for 35 days the TCS concentration appeared to decrease in
the substrates created with Pierce County and City of Tacoma’s biosolids mixed with
paper mulch (17% and 16%, respectively) but the 20 percent instrument uncertainty
results in overlapping of error bars, indicating inconclusive change (see Figure 9.8). For
the control (where no worms were added), there was no difference in TCS concentration
between the substrates made with Pierce County’s biosolids before and after the
experiment (3,500 ± 700 µg/kg). The biosolids and paper mulch substrate made with the
City of Tacoma’s biosolids measured 4,300 ± 860 µg/kg before and 5,200 ± 1,040 in the
control after the 35-day experiment. This 20.9 percent increase is believed to be due to
the instrument uncertainty and is discussed further in section 9.5.3.
Methyl triclosan concentrations within the substrates composed of paper mulch
and biosolids from the City of Lynden, Tacoma and Pierce County varied greatly within
and between substrates (see Figure 9.9). Concentration of Me-TCS in the substrate made
with the City of Lynden’s aerobically digested biosolids appeared to decrease from the
beginning of the study (110 ± 22 µg/kg), in the control at the end of the 35-day
experiment (82 ± 16.4 µg/kg), and in the substrate exposed to earthworms (75 ± 15
µg/kg). However, the 20 percent instrument uncertainty results in overlap of the error
bars indicating and inconclusive difference in values. The substrate composed of

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Figure 9.8. Triclosan concentrations before and after E. fetida exposure and control, for
substrates compose of biosolids and paper mulch from the City of Tacoma
and Pierce County.

Figure 9.8. Triclosan concentrations (µg/kg) in substrates composed of paper mulch and
biosolids from the City of Lynden, Tacoma, and Pierce County at the beginning of the
experiment (blue), in the control after the experiment (containing no worms)(red), and
after exposure to E. fetida (green). Error bars represent 20 % instrument uncertainty. For
each WWTPs’ biosolids and paper mulch substrate, a large batch was prepared and
divided between two containers; one in which earthworms were added and the other was
the control, which was allowed to age the duration of the 35-day experiment.
the City of Tacoma’s dually digested biosolids saw the largest difference between the
substrate at the beginning of the study (5 ± 1 µg/kg), the control (59 ± 11.8 µg/hg) and
the substrate exposed to earthworms (160 ± 32 µg/kg). The Me-TCS concentrations in
the substrate made with Pierce County’s anaerobically digested biosolids appeared to
increase slightly between the samples at the beginning (14 ± 2.8 µg/kg), the control
substrate after (15 ± 3 µg/kg) and the substrate exposed to earthworms (16 ± 3.2 µg/kg).

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There is no discernable difference when the 20 percent uncertainty is applied to these
values.
Figure 9.9. Methyl triclosan concentrations (µg/kg) in substrates composed of paper
mulch and biosolids from the City of Lynden, Tacoma, and Pierce County
before and after E. fetida exposure and control.

Figure 9.9. Methyl triclosan concentrations (µg/kg) in substrates composed of paper
mulch and biosolids from the City of Lynden, Tacoma, and Pierce County at the
beginning of the experiment (blue), in the control after the experiment (containing no
worms)(red), and after exposure to E. fetida (green). Error bars represent 20 % instrument
uncertainty. For each WWTPs’ biosolids and paper mulch substrate, a large batch was
prepared and divided between two containers; one in which earthworms were added and
the other was the control, which was allowed to age the duration of the 35-day
experiment.
9.4.4. Eisenia fetida substrate preference
Substrate preference was determined by counting the number of earthworms and
cocoons within the biosolids or compost, and the paper mulch under each substrate, on
the final day of the experiment. After the 35-days the worms spent in the substrates, the

72

paper mulch was well incorporated into the corresponding substrate above it. In the
divided substrate composed of the City of Lynden’s biosolids and compost, 64 percent
(14 earthworms) of the earthworms at the end of the experiment (22 earthworms) were
found in the compost while only 36 percent (8 earthworms) were found in the biosolids
(see Figure 9.10). Similarly, for the City of Tacoma 36 percent (8 earthworms) of the
earthworms at the end of the experiment (22 earthworms) were found in the biosolids and
64 percent (14 earthworms) were found in the compost. Unexpectedly, the majority of the
earthworms 72 percent (13 earthworms) of the earthworms at the end of the experiment
(18 earthworms) were found in Pierce County’s biosolids while 27 percent (5
earthworms) were found in the compost.
Figure 9.10. Percent of total earthworms at the end of the 35-day experiment in divided
containers found in the compost and either Pierce County, City of Tacoma,
or City of Lynden’s biosolids.

Figure 9.10. Percent of total earthworms at the end of the 35-day experiment in the
divided containers found in the compost (blue) and either Pierce County, City of Tacoma,
or City of Lynden’s biosolids (red).
9.4.5. Nutrients
Potassium (K) and phosphorus (P) concentrations were measured for each paper
mulch and biosolids substrate from the City of Lynden, Tacoma, and Pierce County
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before worms were added, only, due to cost restrictions and because no literature could
be found suggesting it would affect the survival or growth of the earthworms (see Table
9.2). Total organic carbon (TOC), total Kjeldahl nitrogen (TKN, see Figure 9.12), and pH
were measured for each paper mulch and biosolids substrate before earthworms were
added, post exposure and for the control (no worms).
Table 9.2. Nutrients of substrates composed of paper mulch and biosolids from the City
of Lynden, Tacoma, and Pierce County.
TOC
TKN
K
P
Source
pH
(g/kg)
(g/kg)
(g/kg)
(g/kg)
Lynden
Before
331
34
6.5
3.3
17
After worms
332
30.6
5.9
Control
369
38.8
6.1
Tacoma
Before
395
21.7
7.7
1.39
13
After worms
339
25.3
7.3
Control
340
22.7
7.1
Pierce County
Before
393
36.4
8.2
1.36
15.5
After worms
418
39
6.3
Control
387
38.1
6.2
Note. Total organic carbon (TOC), total Kjeldahl nitrogen (TKN), pH, potassium (K) and phosphorus (P) of
substrates composed of three parts paper mulch to four parts biosolids (by dry weight) sourced from the
City of Lynden, Tacoma, and Pierce County. Substrates were sampled before and after exposure to E.
fetida and a control substrate (no earthworms added) which was allowed to age for the 35-day period.

The TKN (g/kg) was measured for the City of Lynden, Tacoma and Pierce
County’s biosolids and the substrate consisting of four parts biosolids and three parts
paper mulch (by dry weight). Of the three sources, Pierce County had the highest amount
of TKN in both the biosolids and the biosolids and paper mulch mixture (80 and 36.4
g/kg, respectively), the City of Lynden had the second highest amount of TKN in the
biosolids and biosolids and paper mulch mixture (68.3 and 34 g/kg, respectively), and the
City of Tacoma had the lowest amount of TKN in the biosolids and biosolids and paper
mulch mixture (46.3 and 21.7 g/kg, respectively).

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Figure 9.11. Total Kjeldahl nitrogen (g/kg) in the biosolids and substrates composed of
three parts paper mulch to four parts biosolids (by dry weight) from City of
Lynden, Tacoma, and Pierce County before E. fetida were added.

Figure 9.11. Total Kjeldahl nitrogen (g/kg) in the biosolids (dark grey) and substrates
composed of three parts paper mulch to four parts biosolids (by dry weight; light grey)
from City of Lynden, Tacoma, and Pierce County before earthworms were added.
While all substrates were prepared to be 80 percent moisture (or 20 percent
solids), some variation was observed when sampled at the laboratory but nothing to the
extent that would indicate conditions not favorable to earthworm survival (Edwards &
Bohlen, 1996).
9.5. Discussion
9.5.1. Earthworm survival
Within the substrates composed of four parts biosolids and three parts paper
mulch, the one made with Pierce County’s biosolids had nearly half of the earthworms
added, perish (see Figure 9.3). In the substrates made with the City of Lynden’s biosolids,
there was very little loss of earthworm life. The substrates composed of the City of

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Tacoma’s dually digested biosolids saw no loss of earthworms in either the biosolids and
paper mulch mixture or the divided substrate containers.
The greatest loss of earthworms was observed in the biosolids and paper mulch
substrates composed of the City of Lynden’s aerobically (12.5 % of total earthworms
added has perished) and Pierce County’s anaerobically (42.1% of total earthworms added
has perished) digested biosolids. The City of Lynden’s divided biosolids and compost
container was the only biosolids source that lost earthworms (4%). It is believed that
fewer earthworms perished in the divided containers, compared to the biosolids and paper
mulch substrates, because the earthworms had a more habitable option, the compost, that
was exposed to air other than the biosolids and paper mulch mixture, which had the
compost sandwiched between the lower and upper containers. However, the divided
substrate composed of Pierce County’s biosolids had 76 percent of the surviving
earthworms in the biosolids and experienced the greatest loss of earthworms compared to
substrates made with the City of Tacoma and Lynden’s biosolids. Considering most of
the earthworms within the divided containers with the City of Tacoma and Lynden’s
biosolids were found in the compost it was unexpected to find the majority of earthworms
in Pierce County’s biosolids. Because the source of biosolids was the only difference
between the containers and the divided container with Pierce County’s biosolids
experienced the most loss of the three sources the biosolids are believed to be reason for
the earthworms inability to thrive, but finding the majority in the biosolids was
unexpected and unexplainable.
The increase in the total number of earthworms in the divided substrate composed
of Pierce County’s biosolids is attributed to a counting error. The chance one worm left a

76

container, travelled between the two, and ended up in this container is possible but does
not seem probable. Additionally, it would be impossible for a cocoon to have hatched and
worm matured in the 35-day period of this study as it takes four months for E. fetida to
mature from fertilization (Tripathi & Bhardwaj, 2004). One other possibility is that there
was freshly hatched earthworm on one of the counted adult worms that was not seen
when placed into the substrate. However, every effort was made to ensure only the adult
worms were being added to test substrates. Therefore, a counting error is attributed to the
observed increase.
When comparing the weight per earthworm within each substrate before and after
the 35-day experiment, regardless of whether the substrate was the biosolids and paper
mulch mixture or the divided container with biosolids and compost, the greatest loss of
weight per earthworm was observed in the substrates composed of Pierce County’s
biosolids and the smallest weight loss was observed in the substrates composed of the
City of Lynden’s biosolids (see Figure 9.4 and 9.5). In their research evaluating survival
and growth of E. fetida Kaplan et al. (1980) found that anaerobically digested sludge did
not contain sufficient nutrients for growth in the form of weight gain, in addition to
various environmental factors that affected growth and survival. The environmental
factors that Kaplan et al. found as impacting growth and survival of E. fetida,
temperature, soil moisture, and pH, were maintained as consistent as possible between
containers or were within optimal parameters in this study. What Kaplan et al. noticed,
regarding the use of sludge or biosolids, is that if anaerobically digested material was
used, layering or mixing it with soil resulted in greater earthworm growth. Additionally,
Neuhauser, Kaplan, Malecki, and Hartenstein (1980) determined that the presence of soil

77

increases earthworm growth due to the inorganic matter in the soil. Neuhauser et al. also
observed that the particle size of the material was inversely related to earthworm weight
gain; the smaller the material the greater weight gain was observed.
In the current study, the biosolids were not sieved to ensure size but when
preparing the substrates there was an observable and tactile difference in the biosolids.
The City of Lynden’s biosolids were smooth, almost clay like, while the biosolids from
Pierce County were much more coarse and did not absorb water well. Based on the
findings by Neuhauser et al. (1980) it is possible the particulate size of the biosolids
resulted in the difference in weight per earthworm at the end of the 35-day study.
9.5.2. Reproduction
A large amount of cocoons were produced in all the divided compost-biosolids
substrates, with the majority of the cocoons found in the compost, regardless of the
source of the biosolids. While the same amount of compost was provided in the
substrates that were a mixture of biosolids and paper mulch and in the divided substrate
containers, the only difference is the compost in the divided container was in the upper
portion and may have increased the amount of available oxygen, which was not a
parameter measured in this experiment. This exposure to air may have created a more
ideal environment for the earthworms
Edwards and Bohlen (1996) found that temperature and moisture are correlated to
cocoon production and growth. However, there was no remarkable difference in
temperature or moisture in the present experiment. The Pierce County biosolids and
paper mulch substrate had the fewest amount of cocoons between the three biosolids
sources but that would be expected when the overall total of earthworms decreased by

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nearly half over the course of the experiment. Additionally, regardless of the substrate
treatment, those made with Pierce County’s biosolids had the lowest number of cocoons
compared to the other substrates made with the City of Tacoma or Lynden’s biosolids.
The only major difference in nutrients between the three biosolids sources is the TKN of
the substrate before earthworms were added (see Figure 9.12). The biosolids and paper
mulch substrate created with Pierce County’s biosolids had TKN of 36.4 g/kg. However,
this is not that much different from the substrate created with the City of Lynden’s
biosolids (34 g/kg). While the substrate created with the City of Tacoma’s biosolids had
TKN of (21.7 g/kg). Unfortunately, due to the limitations of the TKN test, it is not
possible to determine which, organic nitrogen, ammonia (NH3) and ammonium (NH4+), is
responsible for the impact the TKN may or may not have had on earthworm
reproduction.
Perhaps the particulate size of the biosolids contributed to the reproduction
success by earthworms. While cocoon production does not follow the trend of observed
tactile differences between the biosolids, Pierce County being the most coarse and the
City of Lynden being the least coarse (by personal observation), perhaps the size differed
just enough to result in sufficient versus insufficient nutrition for the earthworms.
Another factor that may be at play, which was not quantitatively measured, is that the
City of Tacoma’s WWTP utilizes a dual-digestion process. The wastewater that enters
the treatment plant is first aerobically digested before it is anaerobically digested. The
effect an initial aerobic digestion has on the biosolids does not stand out in the metrics
measured in this study.

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9.5.3. Triclosan and methyl triclosan concentration
The concentration of TCS in the substrate made with the City of Lynden’s
biosolids was above quantifiable limit (30 µg/kg) only in the sample tested at the
beginning of the experiment. Therefore only the substrates created with the City of
Tacoma and Pierce County’s biosolids are compared here regarding TCS concentration.
Overall, TCS concentrations decreased by 17 and 16 percent in the substrates made with
City of Tacoma and Pierce County’s biosolids, respectively, from before and after
exposure to earthworms. However, when the 20 percent uncertainty is applied to the TCS
values measured, the error bars overlap indicating inconclusive results (see Figure 9.8).
Triclosan is a synthetic chemical compound that does not exist in the natural
environment (U. S. EPA, 2010). That said, within the substrates made with the City of
Tacoma’s biosolids, TCS concentration increased by 20.9 percent in the control substrate
compared to the sample tested in the beginning (4,300 ± 860 to 5,200 ± 1,040 µg/kg).
The uncertainty in laboratory measurements of TCS and Me-TCS in soil samples is
typically about 20 percent (M. Bozlee, personal communication, November 21, 2016).
The error bars, for the before and control substrate made with the City of Tacoma’s
biosolids, overlap indicating they may be the same concentration and the difference in the
values measured is due to the instrument uncertainty. Overall, there is no discernable
difference within and across biosolids sources.
The substrate made with the City of Tacoma’s biosolids that was exposed to
earthworms had a TCS concentration of 3,600 ± 720 µg/kg, which is a 16 percent
decrease from the beginning of the 35-day study. Initially, the substrate made with Pierce
County’s biosolids had TCS concentration of 3,500 ± 700 µg/kg before exposure and

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2,900 ± 580 µg/kg after 35 days of exposure to earthworms; the difference between the
measured values of TCS is 600 µg/kg but with the 20 percent instrument uncertainty, the
values overlap indicating no discernable difference. No difference in TCS concentration
was observed in the control substrate that did not have earthworms added and was
allowed to age throughout the 35-day trial.
Approximately one to 66 percent of TCS degrades into Me-TCS through
biological mythelation (Butler et al., 2012; Chen et al., 2011). The difference in TCS
concentrations observed in the current study between the biosolids from the City of
Tacoma and Pierce County and paper mulch substrates before and after exposure to
earthworms is greater than one percent of the initial TCS concentrations but less than 66
percent. The City of Tacoma’s biosolids and paper mulch substrate had TCS
concentration of 4,300 ± 860 µg/kg before exposure and 3,600 ± 720 µg/kg after
exposure to earthworms; the difference between the measured values of TCS is 700
µg/kg, which is greater than one percent of the starting concentration (43 µg/kg) yet far
less than the possible 66 percent (2,838 µg/kg).
If the TCS degradation in this experiment is like that observed by Chen et al.
(2011) and Butler et al. (2012), one and 66 percent, respectively, we would expect there
to be 43 µg/kg to 2,838 µg/kg of Me-TCS formed in the substrates composed of the City
of Tacoma’s biosolids and 34 µg/kg to 2,310 µg/kg of Me-TCS formed in the substrates
composed of Pierce County’s biosolids. Again, the process by which TCS degrades into
Me-TCS is not entirely known, it is likely due to microbial methylation (Boehmer et al.,
2004).

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Interestingly, and unexpectedly, in the substrate composed of the City of
Tacoma’s biosolids 59 ± 11.8 µg/kg of Me-TCS was formed in the control substrate (no
earthworms added) and 160 ± 32 µg/kg of Me-TCS in the substrate with earthworms.
This does not align the results that had been anticipated that Me-TCS concentrations
would be less in the substrate with earthworms because past research has observed their
bioaccumulation of TCS and Me-TCS (Macherius et al., 2014). When comparing the
values with the 20 percent uncertainty the error bars do not overlap indicating a
discernable difference between Me-TCS concentrations between the substrate made with
the City of Tacoma’s biosolids before, after exposure to earthworms, and the control.
However, because there were no replicates to compare these numbers to, there is no way
to know whether these numbers are typical, therefore repeating this experiment, with
replicates, is necessary to draw founded conclusions.
As TCS concentrations decrease due to degradation, Me-TCS concentrations are
expected to increase. The Me-TCS concentrations in the substrates made with Pierce
County’s biosolids remained stable between the samples collected in the beginning of the
trial (14 ± 2.8 µg/kg) compared to that of the substrate exposed to earthworms (16 ± 3.2
µg/kg) and the control (15 ± 3 µg/kg). Methyl triclosan concentrations within the City of
Lynden’s biosolids and paper mulch substrate decreased more so in the substrate exposed
to E. fetida (110 ± 22 µg/kg initially, to 75 ± 15 µg/kg) compared to the control substrate
(110 ± 22 µg/kg initially to 82 ± 16.4 µg/kg; see Figure 9.7). This may support the
possibility that the earthworms’ bioaccumulated the TCS and Me-TCS (Macherius et al.,
2014) as the TCS concentrations decreased as well, but the 20 percent instrument
uncertainty, the values overlap indicating inconclusive results. However, it is unlikely the

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total amount of Me-TCS observed to decrease was only due to degraded because it is
known to persist longer in the environment than its parent compound, TCS (Lindström et
al., 2002). It did appear to decrease some in between the beginning and the control of the
biosolids and paper mulch substrate made with the City of Lynden’s biosolids, but not a
difference that is conclusive due to instrument uncertainty.
Interestingly, the substrates made with the City of Tacoma’s biosolids measured
the greatest increase in Me-TCS concentrations. At the beginning of the trial the
concentration of Me-TCS measured below the detectible limit of 5 µg/kg. At the end of
the 35-day experiment, the control substrate, not exposed to earthworms, measured 59 ±
11.8 µg/kg of Me-TCS and the substrate exposed to earthworms had 160 ± 32 µg/kg of
Me-TCS. The difference in Me-TCS concentration observed in the substrates made with
the City of Tacoma’s biosolids was greater than anticipated. The substrate exposed to the
earthworms was expected to have the lowest Me-TCS concentration because it was
hypothesized the earthworms would bioaccumulate the chemical compound, which was
clearly not the case (see Figure 9.7).
Putting aside the TCS concentration increase of 900 µg/kg observed in the control
substrate after the 35-day trial compared to the initial substrate, a 700 µg/kg difference
was observed in the TCS concentration from the initial substrate and the substrate
exposed to earthworms for 35 days. Twenty-two percent of TCS degrading into Me-TCS
is possible (Butler et al., 2012) but is nearly 100 times greater than what was measured in
the substrate made with Pierce County’s biosolids that was exposed to earthworms (see
Figure 9.12).

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Figure 9.12. The difference in triclosan concentration that can be explained by the
formation of methyl triclosan in substrates composed of paper mulch and
biosolids sourced from the City of Tacoma and Pierce County after 35-day
exposure to E. fetida.

Figure 9.12. The total difference of measured triclosan concentration from the initial
substrate compared to substrate exposed to earthworms for 35 days, which can be
explained by the formation of Me-TCS during the same time period for substrates
composed of biosolids from the City of Tacoma and Pierce County.
The City of Tacoma measured higher levels of Me-TCS in the substrates exposed
to earthworms compared to the control substrates. In the biosolids and paper mulch
substrate made with the City of Tacoma’s biosolids, there was a sharp increase in MeTCS concentration between the initial substrate tested, control substrate, and the substrate
exposed to earthworms. While Me-TCS concentrations were expected to increase as TCS
degraded the increase observed in the substrates made with the City of Tacoma’s
biosolids was greater than anticipated. The City of Lynden may not have followed suit
because there was half as much TCS in the substrate compared to Me-TCS. However, a
32 percent decrease in Me-TCS concentration was observed in the substrate exposed to
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earthworms while Me-TCS concentration decreased by 25 percent in the control
substrate, not exposed to earthworms; evidence that there is something affecting the MeTCS concentration that can not be attributed to the presence of earthworms.
The substrate composed of Pierce County’s biosolids had TCS concentration of
3,500 ± 700 µg/kg before exposure and 2,900 ± 580 µg/kg after exposure to earthworms;
the difference between the measured values of TCS is 600 µg/kg. The concentration of
Me-TCS at the beginning of this experiment in Pierce County’s biosolids measured 14 ±
2.8 µg/kg and at the end increased to 16 ± 3.2 µg/kg in the substrate that was exposed to
earthworms while TCS concentrations decreased from 3,500 ± 700 to 2,900 ± 580 µg/kg
in the same substrate. Triclosan concentrations decreasing by 600 µg/kg cannot not only
be due to degradation into Me-TCS because Me-TCS concentration only increased by 2
µg/kg. This suggests there are other factors affecting the decrease in overall TCS
concentration. While it is possible the earthworms bioaccumulated the additional TCS,
especially because past research supports this (Higgins et al., 2011; Kinney et al., 2006,
2008, 2010; Macherius et al., 2014), it cannot be ruled out that there were other factors
influencing the decrease in TCS. Therefore, either the earthworm’s bioaccumulated the
majority of the TCS that did not degrade into Me-TCS or it degraded into another
compound that was not measured in the current study. If the TCS was bioaccumulated by
the earthworms, one would expect to see increased concentrations of TCS in the
earthworm tissues and if the TCS degraded into another compound, we would expect to
see the concentrations of that degraded compound increase when comparing substrates
before and after earthworm exposure.

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A possible mechanism that was not measured in this study is the effect the
earthworms have the microbial community in the substrate. One factor that was not
measured that may have impacted the TCS and Me-TCS concentrations is the impact
earthworms have on the microbial community (European Commission, 2010).
Domínguez, Aira, and Gómez-Brandón (2010) evaluated microbial activity in the
presence and absence of earthworms. They found that when earthworms were present,
microbes in the soil were more effective at utilizing available energy compared to a
control, which was conducted without added earthworms. Over a four-week period,
microbial respiration increased nearly 90 percent simply through the process of
earthworms consuming and defecating soil (Scheu, 1987). Additionally, Gómez-Brandón,
Aira, Lores, and Domínguez (2011) looked at the microbes in manure and microbes
excreted within earthworm casts. Finding that while earthworms decreased the overall
biomass, or amount, of microbes in the substrate, yet the activity of the microbe
community did not change, even with fewer microbes present after earthworm digestion.
Turning back to the current study, perhaps the more active microbes excreted by
the earthworms in their casts were breaking down the TCS into Me-TCS, which is then
bioaccumulated by the earthworms. This may explain the increase in Me-TCS
concentrations seen in substrates that were exposed to earthworms created with the City
of Tacoma’s biosolids. While the exact process by which TCS degrades into Me-TCS is
not entirely known, Me-TCS is most likely formed by microbial methylation (Boehmer et
al., 2004). Perhaps the microbes excreted by earthworms assisted in the microbial
methylation that is believed to be the process by which Me-TCS is formed.

86

It would be interesting to see what would have happened had the earthworms
been kept in the substrates for a longer period of time. If given more time, they may
bioaccumulate more TCS and Me-TCS through consumption of the contaminated
substrate and through direct contact with the compounds in the substrate in which they
are living. The amount of TCS present in the substrates composed of the City of Tacoma
and Pierce County’s biosolids may have seen a reduction had the earthworms been
allowed more time to process the material. Additionally, if the microbes are indeed
increasing the rate at which TCS degrades into Me-TCS, increasing the time the
earthworms are in and consuming the substrate would further increase the microbes’
activity as well. Because Me-TCS is more lipophilic than TCS it would be more readily
bioaccumulated by the earthworms in the substrate.
9.5.4 Eisenia fetida substrate preference
The divided container approach was taken to determine whether earthworms
prefer aerobically digested sludge or anaerobically digested sludge over compost. While
more worms did survive, it did not appear as though there was a difference in the
preference of dually versus aerobically digested biosolids because substrates made with
the City of Tacoma and Lynden’s biosolids had healthy worm survival while
anaerobically digested Pierce County did not.
Dually digested City of Tacoma and aerobically digested City of Lynden had the
majority of the earthworms in their biosolids. Whereas, both the divided container and
the biosolids and paper mulch mixture created with anaerobically digested Pierce
County’s biosolids had the majority of the earthworms in the compost, indicating a
preference for the compost over the biosolids. The reason for this difference is not

87

understood and cannot be explained with the data from this thesis. Based on knowledge
gained and past research (Hartenstein, 1981; Masciandaro et al., 2000; Weaver, P.,
personal communication, 2016) it was expected that earthworms would mist likely be in
the aerobically digested biosolids and less likely to be in the anaerobically digested
biosolids, but that was not the case in this experiment.
9.5.5. Nutrients
Tacoma experienced no loss of earthworms and had lowest TKN (21.7 g/kg) in
divided and biosolids and paper mulch substrate followed by substrates composed of the
City of Lynden and Pierce County’s biosolids (34 g/kg and 36.4 g/kg, respectively) and
experienced loss of earthworms in a similar fashion (City of Lynden=12.5% and Pierce
County = 42.1%; see Figure 9.14). As for cocoon production, Pierce County had the
greatest amount of TKN and lowest number of cocoons (200) but the pattern does not
hold when it comes to the City of Tacoma and Lynden, in that Lynden had a higher TKN
value than Tacoma but more cocoons (Lynden = 252 and Tacoma = 248; see Figure
9.13).
The correlation of the TOC in the substrate to the survival of earthworms
observed in Experiment III was also seen here, in Experiment IV. As the TOC increased,
so did the total number of individual earthworms alive at the end of the 35-day
experiment.

88

Figure 9.13. Initial total Kjeldahl nitrogen (TNK) and percent of total worms added that
died in substrate made of three parts paper mulch and four parts biosolids
from the City of Tacoma, Lynden, and Pierce County.

Figure 9.13. Total Kjeldahl nitrogen (g/kg, blue) within substrate prior to the addition of
earthworms and percent of total earthworms added that died (purple) in substrate made of
three parts paper mulch and four parts biosolids sourced from the City of Tacoma,
Lynden, and Pierce County.
10. Conclusion
The question at the heart of this thesis is whether the presence of earthworms (E.
fetida) effects TCS and Me-TCS concentrations in biosolids destined for land application.
The answer is maybe yes and no. The “maybe” is because there were no replicates in
these experiments due to financial limitations and due to the unanticipated methods
development (Experiments I, II, and III). However, the results obtained from laboratory
analysis do suggest Me-TCS concentrations increased in the presence of earthworms in
the substrates composed of the City of It is unclear whether the repeated Pilot substrate
(in Experiment III) had an elevated Me-TCS concentration after earthworm exposure
because there was no control substrate that did not have earthworms with which to
compare.
89

As for the TCS and Me-TCS concentrations in the biosolids, it is possible the
presence of earthworms may have reduced the overall concentrations, more so than the
microbial degradation of TCS into Me-TCS alone. It is unclear whether TCS degraded
into compounds other than Me-TCS, as they were not tested in this study, or if the
earthworms bioaccumulated the contaminants, as they were not tested for TCS and MeTCS. It is indicative that there may be a relationship between the presence of E. fetida in
biosolids and an increase in Me-TCS concentrations, in the City of Tacoma’s biosolids.
This study established first steps in developing methods for the use of biosolids
from Pierce County, the City of Tacoma, and Lynden in the application of biosolidsvermicomposting. Future research can evaluate the TCS and Me-TCS concentrations in
the substrates as well as the earthworms. Due to the lack of replicates, it is impossible to
know whether the observations and results in this study are representative of trends one
could anticipate seeing if repeated. A simple study including replicates would allow for
stronger data and conclusions. However, there are some interesting findings that can shed
light down hallways of knowledge towards possible avenues of successful PPCP
removal. All PPCPs will not be banned as the U. S. and Germany have done with TCS
because some are necessary. Even though TCS was banned, we do not know the effects
of long-term exposure on wildlife and the environment to know how things will react;
much less how the degraded forms of TCS will affect us and the environment in the
future.
Overall, the initial steps of this study support the findings of Hartenstein et al.
(1981) that anaerobically digested biosolids create a toxic environment for earthworms.
Even though the biosolids obtained from the City of Tacoma are dually digested, initially

90

aerobic followed by anaerobic digestion, the earthworms were not able to survive in
biosolids-heavy substrates. However, with the addition of sufficient carbon-based
bedding, the earthworms were able to survive and thrive. By creating substrates
composed of aerobically digested biosolids from the City of Lynden and anaerobically
digested biosolids from Pierce County for comparison the results are not so
straightforward. More earthworms survived in substrate made with aerobically digested
biosolids compared to substrate made with anaerobically digested biosolids but the dually
processed biosolids from the City of Tacoma resulted in no loss of earthworms.
Something to note is that the initial steps in determining the appropriate biosolids
to paper mulch ratio was based on the City of Tacoma’s biosolids. The C to N ratios of
the substrates composed of Pierce County and the City of Lynden’s biosolids mixed with
the same ratio of paper mulch were 11 and 10, respectively. Compared to the City of
Tacoma, it is easy to see they have more nitrogen as the City of Tacoma’s C to N was 18
Therefore, perhaps with further testing appropriate C to N ratios can be accomplished
using biosolids from Pierce County and the City of Lynden. Additionally, simply
allowing the earthworms more time to process the biosolids and be in contact with the
substrate may result in clearer results.
If earthworms are indeed capable of bioaccumulating, and effectively removing,
TCS and Me-TCS from biosolids, they may be utilized in the removal of the
contaminants, and perhaps other PPCPs, prior to land application. Deegan et al. (2011)
reviewed a variety of wastewater treatment options and their ability and efficiency in
removing PPCPs. They evaluated published literature testing wastewater treatments that
have been added to traditional secondary sewage treatment: aerobic digestion, anaerobic

91

digestion and oxidation ditches, which are ditches with mechanized agitators to create an
aerobic environment. These additional treatments include membrane filtration, reverse
osmosis and activated carbon and can be costly and time consuming to incorporate into
an already existing WWTP. Unfortunately, they found that there is no solution for
removing all PPCPs that enter a WWTP.
Washington State’s Department of Ecology tested the influent, effluent and
biosolids of five WWTPs in the Pacific Northwest for 172 organic compounds, including
72 PPCPs, 27 hormones/steroids, and 73 semi-volatile organics (Lubliner et al., 2010). Of
the all samples collected and tested, every sample had detectible levels of PPCPs. Only
12 of the 172 compounds (7%) were not detected following secondary wastewater
treatment technologies, mentioned in the previous paragraph) and were not preset in the
biosolids. Triclosan was of detectable levels after the secondary treatment, and in the
biosolids, but not in wastewater after a tertiary treatment (defined by Lubliner et al. as a
chemical addition, filtration, or nutrient removal). Interestingly, approximately 20 percent
(mostly polycyclic aromatic hydrocarbons or PAHs) of the 172 compounds were detected
only in the biosolids further supporting the need to develop a method for removing these
contaminating compounds prior to the land-application of biosolids. Biosolids are rich in
nutrients and great for amending soils but their application on land with the
anthropogenic contaminants only pollutes our environment and puts organisms at risk.
This research illuminates one potential avenue for a potential solution, the utilization of
earthworms.

92

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Appendix A
United States Environmental Protection Agency’s list of 88 synonyms for triclosan:
1. TCL
45. MLS001066347
2. 72779
46. MLS001074876
3. T1872
47. MLS001335937
4. 524190
48. MLS001335938
5. C12059
49. SAM002554907
6. D06226
50. SMR000471847
7. DP-300
51. Irgasan DP300
8. IN1424
52. Microshield T
9. S00100
53. Oxy Skin Wash
10. Trisan
54. Irgasan DP 300
11. CH 3565
55. NCGC00159417-02
12. CH-3565
56. NCGC00159417-03
13. CID5564
57. NCGC00159417-04
14. D014260
58. UNII-4NM5039Y5X
15. DB08604
59. Stri-Dex Face Wash
16. Irgasan
60. Triclosan; Irgasan
17. AC-10667
61. MolPort-003-666-702
18. AC1L1KMN
62. Triclosan (USP/INN)
19. Aquasept
63. SSL Brand of Triclosan
20. I01-2897
64. Stri-Dex Cleansing Bar
21. LS-67854
65. Triclosan Reckitt Brand
22. Manusept
66. SterZac Bath Concentrate
23. Sapoderm
67. Clearasil Daily Face Wash
24. 222-182-2
68. Ster Zac Bath Concentrate
25. 3380-34-5
69. Ster-Zac Bath Concentrate
26. CHEMBL849
70. Dermtek Brand of Triclosan
27. CPD0-1227
71. Reckitt Brand of Triclosan
28. HSDB 7194
72. Triclosan Pharmachem Brand
29. Lexol 300
73. Stri-Dex cleansing bar (TN)
30. TL8002539
74. Pharmachem Brand of Triclosan
31. 88032-08-0
75. Trans Canaderm Brand of Triclosan
32. C12H7Cl3O2
76. GlaxoSmithKline Brand of Triclosan
33. CCRIS 9253
77. Procter & Gamble Brand of Triclosan
34. Cliniclean
78. Johnson & Johnson Brand of Triclosan
35. Cloxifenol
79. 5-CHLORO-2-(2,4-DICHLOROPHENOXY)PHENOL
36. HMS2093L17
80. 5-Chloro-2-(2,4-dichloro-phenoxy)-phenol
37. 112099-35-1
81. 2,4,4'-Trichloro-2'-hydroxydiphenyl ether
38. 164325-69-3
82. 2-Hydroxy-2',4,4'-trichlorodiphenyl Ether
39. 261921-78-2
83. Phenol, 5-chloro-2-(2,4-dichlorophenoxy)40. BRN 2057142
84. 2,4,4'-Trichloro-2'-hydroxy diphenyl ether
41. Triclosanum
85. Ether, 2'-hydroxy-2,4,4'-trichlorodiphenyl
42. CHEBI:164200
86. Phenyl ether, 2'-hydroxy-2,4,4'-trichloro43. CPD000471847
87. Irgasan DP-300
44. Cloxifenolum
88. 5-Chloro-2-(2,4-dichlorophenoxy)phenol
117

Appendix B

Standard Operating Procedure
Pharmaceutical and Personal Care Products by EPA Method 8270D

City of Tacoma
Environmental Services Laboratory

_______________________________________________

_____________

Stuart Magoon
Assistant Division Manager

Date

____________________________________________________
Greg Perez
Senior Environmental Lab Analyst – Organics Lead

______________
Date

____________________________________________________
Lori Zboralski
Senior Environmental Lab Analyst – QA Officer, LIMS Administrator

______________
Date


 

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Disclaimer:
Please note that the City of Tacoma’s Environmental Services Laboratory Standard Operating
Procedures (SOPs) are adapted from published methods. They are intended for internal use only
and are specific to the equipment, personnel, and samples analyzed at the Environmental
Services Laboratory. This SOP is not intended for use by other laboratories nor does it supplant
official published methods. Distribution of this SOP does not constitute an endorsement of a
particular procedure or method.
Any reference to specific equipment, manufacturer, or supplies is for descriptive purposes only
and does not constitute an endorsement of a particular product or service by the author or by the
City of Tacoma.
Although the lab follows the SOP in most instances, there may be instances in which the lab uses
an alternative methodology or procedure with quality assurance and management approval.
The method is for “research only”. It has not been vetted through our normal validation
process.
Currently, the SOP document review process is not complete for this first version and thus
unsigned.
SOP Revision History
Revision Date
1/8/2016


 

Rev Number
1.0

Summary of Changes
New SOP

Sections
all

Reviser(s)
Mark Bozlee

119
 

1.0

Scope and Application
1.1

This document is the Standard Operating Procedure (SOP) for the analysis
of Pharmaceutical and Personal Care Products by method SW846 8270D.
Refer to the Project and Sample Analysis Request Form in Element for
project specific compounds and reporting limits. The following compounds,
including typical MRLs, can be determined by this method:
S8270_BNA
Analyte
Triclosan
Methyl Triclosan

1.2

2.0

3.0


 

MRL
20
10

Units
ng/g
ng/g

The analysis portion of this method is to be used by, or under the direct
supervision of, analysts experienced in the use of Agilent gas
chromatography/mass spectrometry (GC/MS/MS) systems, MassHunter
software and in the interpretation of mass spectral data.

Summary of Procedure
2.1

The samples consisting of biosolids and paper mulch are milled using a
Cryomill without liquid nitrogen (SOP 1022 Cryomill Sample Processing)
followed by a vortex and sonication extraction.

2.2

The semivolatile compounds are introduced into the GC/MS/MS by injecting
the sample extract into a GC equipped with a narrow-bore fused-silica
capillary column. The GC column is temperature-programmed to separate
the analytes, which are then detected with a tandem MS connected to the
gas chromatograph. Analytes eluted from the capillary column are
introduced into the mass spectrometer via a direct connection.

2.3

A characteristic (precursor) m/z is further broken down into a characteristic
daughter (product) m/z for each compound and quantitated. An additional
daughter ion (qualifier ion) is also measured for even further identification.
An individual compound is identified by comparing the GC retention time, a
precursor ion, a product qualifier ion, and ratio of qualifier to quantifier ion to
an authentic standard. See Table 14.1 The concentration is determined by
using the response of the product quantitative ion and a multipoint
calibration of the target analytes with isotope dilution technique. Isotope
dilution provides automatic correction of the target analyte concentrations.

Interferences
3.1

Solvents, reagents, glassware, and other sample processing hardware may
yield artifacts and/or interferences to sample analysis. All of these materials
must be demonstrated to be free from interferences under the conditions of
the analysis by analyzing method blanks. Also refer to SW-846 Method
8000 for a discussion of interferences.

3.2

Raw GC/MS/MS data from all blanks, samples, and spikes must be
evaluated for interferences. Determine if the source of interference is in the
preparation of the samples and take corrective action to eliminate the
problem. Contamination by carryover can occur whenever high-

120
 

concentration and low-concentration samples are sequentially analyzed. To
reduce carryover, the sample syringe must be rinsed with solvent between
sample injections. Whenever an unusually concentrated sample is
encountered, it should be followed by the analysis of solvent to check for
cross contamination.
4.0

5.0

Safety
4.3

Refer to Chemical Hygiene and Laboratory Health and Safety Plan for
standard lab safety practices. See Section 13.0.

4.4

The toxicity or carcinogenicity of each reagent used in this method have not
been precisely defined; however, treat each chemical compound as a
potential health hazard. Reduce exposure to these chemicals to the lowest
possible level by whatever means available. Prepare primary standards of
these toxic compounds in a hood.

Equipment and Supplies
5.1

Vials – 4, 8 and 12 mL, amber glass, with polytetrafluoroethylene (PTFE)lined screw cap.

5.2

Gas tight syringes, various volumes

5.3

Hamilton Digital Dilutor - \\fspwes01\general\qa\sop\5019 Maintenance and
Operation of Hamilton Digital Diluter_v4.pdf

5.4

Metal Spatula

5.5

Vortex mixer

5.6

Sonic bath

5.7

Single use aluminum weighing pan

5.8

100 ml volumetric flask, ground glass joint with stopper

5.9

Balance accurate to 0.0001g (Mettler MS 3045 S/N B021037549). See SOP
..\Current\1015_Analytical Balance Calibration and Maintenance_v2.pdf

5.10 Syringe filters (0.45 micron) and self-filtering autosampler vials, (0.2 micron)
5.11 GC/MS/MS System
5.11.1 Agilent 7890 GC complete with all required accessories including
syringes, columns, and gases. The GC includes a front multi-mode
inlet (MMI) capable of large volume injection and a rear split/splitless
inlet.
5.11.2 Inlet Liners - The following liners are recommended. Any liner
yielding suitable chromatography may be substituted providing the
same type is used for the initial calibration and sample analysis.
5.11.2.1 Single-taper 2.3-mm i.d. focus liner with inert glass wool.
5.11.3 Analytical Column – 20m x 0.18mm x 0.18µm DB-5
5.11.4 Agilent 7000 GC/MS/MS - mass selective detector capable of
scanning from 35 to 1020 amu every 1 msec or less, and producing
a mass spectrum which meets all the criteria of


 

121
 

perfluorotributylamine (PFTBA) injected through the GC inlet. See
Section 8.1.
5.11.5 Agilent MassHunter data acquisition and analysis software
5.11.6 A Laboratory Information Management System (LIMS) capable of
computing and storing data acquired using Mass Hunter or
Chemstation software. This laboratory uses Promium® Element
DataSystem® (referred to in this SOP as LIMS or Element™).
6.0

Reagents and Standards
6.1

Standard Solutions: all standards are entered into LIMS. See an example
of a calibration standard entered into LIMS in Section 14.2 and a example of
a standard in Section 14.3.
6.1.1

Purchase commercially prepared certified stock solutions stock
solutions. Store, protected from light, at 4oC or as recommended by
the standard manufacturer. Check stock standard solutions
frequently for signs of degradation or evaporation, especially just
prior to preparing calibration standards from them. Replace stock
standard solutions by the manufacturer’s expiration date or sooner if
comparison with quality control check samples indicates a problem.

6.1.2

Pharmaceutical Mix #2 - Available from Restek. Contains Triclosan
in methanol at 200 µg/mL.

6.1.3

Methyl Triclosan – Neat analytical standard. Available from SigmaAldrich
6.1.3.1

6.2


 

Add 20 mg of Methyl Triclosan to 100 mls of acetone in a
100 ml volumetric. Mix well. Transfer solution to five 12 ml
amber vials. Store at -10°C. Prepare fresh every 6
months.

Internal standard solutions
6.2.1

(13C12) Triclosan in nonane 100 ug/mL - Purchased from
Cambridge Laboratories Inc. This is the working solution. The
Element standard type must be ‘Internal Std’. Spike each sample or
calibration extraction with 5 µL of the internal standard solution,
resulting in a concentration of 100 ng/ml.

6.2.2

(13C12) Methyl Triclosan in nonane 100 ug/mL - Purchased from
Cambridge Laboratories Inc. This is the working solution. The
Element standard type must be ‘Internal Std’. Spike each sample or
calibration extraction with 2 µL of the internal standard solution,
resulting in a concentration of 40 ng/ml.

6.3

Surrogates - Surrogated are not used in this method. The need for
surrogates is eliminated by the use of isotopic dilution. Isotopic internal
standard recovery correction eliminates the need for surrogates.
Surrogates may be added according to a QAPP or according to professional
judgement.

6.4

Laboratory Control Sample (LCS) - Use the same source as the initial
calibration standards to restrict the influence of standard accuracy on the
determination of recovery through preparation and analysis.

122
 

6.4.1

6.5

S8270_PPCP BS - Add 2.0 ul of the stock 200 ppm triclosan stock
(Pharmaceutical Mix #1) and 2.5 ul of the 200 ppm methyl triclosan
stock into a 8 ml amber vial with 0.15 g paper mulch. Add 4988.5 ul
of methanol and follow the extraction procedure from 10.3.5 to
10.3.12.

Matrix Spike (MS) - Use the same source as the initial calibration standards
to restrict the influence of standard accuracy on the determination of
recovery through preparation and analysis
6.5.1

S8270_PPCP MS - Add 2.0 ul of the stock 200 ppm triclosan stock
(Pharmaceutical Mix #1) and 2.5 ul of the 200 ppm methyl triclosan
stock into a 8 ml amber vial with 0.25 g of sample. Add 4988.5 ul of
methanol and follow the extraction procedure from 10.3.5 to 10.3.12.

6.6

Solvents – acetone, methylene chloride, and other appropriate solvents. All
solvents are pesticide quality or equivalent

6.7

Paper mulch – Premium Paper, 100% hand sorted recycled newsprint
without added dye, from Applegate Mulch. Milled (Section 10.2). Paper
mulch in calibration standards and samples increase the sensitivity of
methyl triclosan and triclosan. Components in the paper mulch act as
matrix enhancers by allowing more analyte to reach the detector, as shown

below.
6.8

6.9


 

Intermediate Standards (IMD Std) – Vortex to mix after all stock standard
additions. Store all intermediate solutions at -10°C. Prepare fresh every 6
months or sooner if degradation is detected.
6.8.1

S8270_PPCP: Triclosan/Methyl Triclosan Mix - 2000 ppb – Add 20
ul of each 200 ppb stock to 1960 mL of methylene chloride in a 4 mL
amber vial for a 2000 µg/mL solution.

6.8.2

S8270_PPCP: Triclosan/Methyl Triclosan Mix - 100 ppb – Add 50 ul
of 2000 ppb Triclosan/Methyl Triclosan mix to 950 mL of methylene
chloride in a 2 mL amber vial for a 2000 µg/mL solution.

Working Standards - For each calibration standard, add 0.15 g of paper
mulch and the specified amount of standard to a 8 mL screw cap amber vial
containing the methylene chloride (MeCl2). Add the appropriate amount of
IS (6.2) to each vial and vortex. Points may be added or subtracted to meet
project requirements. Store at -10 °C. Prepare fresh every 3 months or

123
 

sooner if degradation is detected. Enter the Element LIMS standard type as
‘Calibration’.
6.9.1

Std

Std Conc

CAL1
CAL2
CAL3
CAL4
CAL5
CAL6
CAL7
CAL8
CAL9
CAL10
CAL11
CAL12

200 mg/L Stocks
200 mg/L Stocks
200 mg/L Stocks
200 mg/L Stocks
2000 Int mix ng/ml
2000 Int mix ng/ml
2000 Int mix ng/ml
2000 Int mix ng/ml
2000 Int mix ng/ml
2000 Int mix ng/ml
2000 Int mix ng/ml

6.9.2

7.0

8.0

100 Int mix ng/mL

µL of Triclosan
Std

µL of MeCl2

Conc ng/mL

25 each
12.5 each
5 each
2.5 each
125
62.5
25
12.5
5
2.5
1.25
12.5

4943
4968
4983
4988
4868
4931
4968
4981
4988
4991
4992
4981

1000
500
200
100
50
25
10
5
2
1
0.5
0.25

S8270_PPCP continuing calibration standard – Add 0.15 g of paper
mulch to a 8 ml amber vial. Add 2.5 ul of the 200 ppm stock
solutions (pharmaceutical mix #2 and methyl tricolsan), 5 ul of
(13C12) triclosan and 2 ul of (13C12) methyl triclosan. Add 4988 mls
of methylene chloride. The target analytes are at a concentration of
100 ng/ml.

Sample Collection, Preservation and Handling
7.1

Collect samples in 8 oz Whirl-Pak container

7.2

Wrap with aluminum foil

7.3

Samples have a 14 day hold time but may be frozen at -18 °C (Freezer
Room 224) per Puget Sound Protocols in order to extend the holding time
from collection to extraction for up to 1 year.

7.4

Store extracts and milled samples when not being used for analyses at 18°C. Extracts are stored in 2 ml amber vials and milled samples are stored
in 40 ml amber VOA vials protected from light in screw cap vials equipped
with unpierced PTFE-lined septa. Complete analysis within 40 days of
extraction.

7.5

Qualify the results of any samples which exceed these limits as estimated
values.

Quality Control and Method Performance
8.3


 

S8270_PPCP Calibration Working Standards - A typical calibration
set is listed in the following table.

Tuning - Check tune by clicking the MS TUNE icon in the Instrument
Control panel to display the Tune dialog box. Then click on the Autotune’s

124
 

Check Tune tab. Click on “Check Tune”. Acceptable parameter limits are
as follows: See Section 14.4 for check tune.


 

8.3.1

Reanalyze any samples that are injected more than 12 hours after a
Autotune or Check Tune and mark the original analysis as not
reportable.

8.3.2

If Check Tune does not pass, check on Autotune tab. See Section
14.5. Make sure “EI high sensitivity autotune”, “Save tune file when
done” and “Default filename” are checked. Click on Autotune

8.4

Initial Demonstration of Performance (IDP) - Perform once by each analyst
prior to reporting sample results. Repeat the IDP when a major change is
made to the extraction, analysis method or equipment. IDP consists of the
analysis of four replicates of the laboratory control sample. The IDP is
acceptable if the average recovery of the four results is within the LCS
limits. IDP data is stored on \\fspwes01\Transfer\7000SV1\LLOQ and IDP
as a .pdf file named analyst initials_analysis name_IDP_date

8.5

Lower Limit of Quantitations (LLOQ) are determined the first time the
method is performed on the instrument and repeated annually, or if there is
a major change in the procedure or equipment. The LLOQ check is carried
through the same preparation and analytical procedures as environmental
samples and other QC samples.The verification is performed by the
extraction and analysis of an LCS (or matrix spike) at 0.5 – 2 times the
current LLOQ levels. Analyze in the same manner as samples. LLOQ data
is stored at \\fspwes01\Transfer\7000SV1\LLOQ and IDP

8.6

Method Blank - Prepare a method blank (Batch#-BLK#) of one per day or
one per 20 samples whichever is more frequent. Analyze the blank to
demonstrate that the system and extraction are free from contamination.
Use 0.15 g of milled paper mulch and extract as a sample (Section 10.2 and
10.3). If contaminated, evaluate if the GC system is the contamination
source by analyzing an instrument blank of methylene chloride. Clean the
inlet and the split vent line if GC is the source to remove higher molecular

125
 

weight target compounds that build up in the inlet as the system sits idle.
Reanalyze blanks with concentrations greater than or equal to ½ the Lowest
Level of Quantitation (LLOQ) after eliminating the GC as the contamination
source.

8.7

8.8


 

8.6.1

If the blank contains a concentration greater than or equal to the
LLOQ and the sample concentration is less than the LLOQ, report
the LLOQ value with a "U".

8.6.2

If the sample concentration is greater than the LLOQ and within 10x
the blank concentration qualify the sample concentration with a “UJ”.
Complete a QC Variance form and consult project manager to
determine if re-extraction is required to meet minimum project
reporting limits.

8.6.3

If the sample concentration is greater than 10x the blank
concentration, report the sample concentration without qualification.

8.6.4

If gross contamination exists in the blank (i.e. saturated peaks),
positive sample results may require rejection and be qualified as
unusable "R". Unusable data may require re-extraction. Complete a
QC Variance form and consult project manager to determine if reextraction is required.

Laboratory Control Sample - Prepare a blank spike (Batch#-BS#) the more
frequent of one per 14 days or one per 20 samples as per 6.4.
8.7.1

The recovery limits are specified by the project QAPP. If not
specified by the QAPP they are set by control chart of LCS
recoveries or default to 70-130% if data is insufficient for control
chart. Qualify the data as “J” for detects and “UJ” for non-detects if
the LCS recovery is less than the lower recovery limit. Data should
be qualified “R” if recoveries are below 20%. Complete a QC
Variance form. If data has been “R” qualified, initiate a Corrective
Action.

8.7.2

Reanalyze LCS if recovery is outside the criteria after evaluating
whether GC system maintenance could improve recovery and taking
any actions indicated. Consult with senior analyst if recovery is still
outside the criteria to determine whether re-extraction is possible
within sample holding times. Report data associated with the best
recovery. Delete results in Element for non-reported LCS leaving an
internal standard as an indication of the additional analysis.

8.7.3

Qualify the data with a “J” for detects and complete a QC Variance
form if the LCS recovery exceeds the upper recovery limit. Do not
qualify non-detects.

Matrix Spike/Matrix Spike Duplicate - Prepare one matrix spike (MS) and
matrix spike duplicate (MSD) the more frequent of one set per 14 days or
per 20 samples as per 6.5. Report results from the same dilution as
reported for the native sample unless the MS/MSD result is over the
calibration range. The recovery limits are specified by the project QAPP. If
not specified by the QAPP they are set by control chart of LCS recoveries or
default to 70-130% if data is insufficient for control chart.

126
 

8.9

9.0

8.8.1

Do not qualify data if the sample concentration exceeds the spike
concentration by a factor of four or more.

8.8.2

No qualification of the data is necessary on MS and MSD data
alone. In those instances where it can be determined that the results
of the MS and MSD affect only the sample spiked, limit qualification
to this sample only. However, it may be determined through the MS
and MSD results that a laboratory is having a systemic problem in
the analysis of one or more analytes, that affect all associated
samples.

8.8.3

Reanalyze MS or MSD if recovery is outside the criteria after
evaluating whether GC system maintenance could improve recovery
and taking any actions indicated. Consult with senior analyst if
recovery is still outside the criteria to determine whether reextraction is possible within sample holding times. Report data
associated with the best recovery. Delete results in Element for nonreported MS/MSD leaving an internal standard as an indication of
the additional analysis.

8.8.4

Qualify the data with a “J” for detects and a “UJ” for non-detects if
the LCS recovery is less than the lower recovery limit. Data should
be qualified “R” if recoveries are below 20%. Complete a QC
Variance form. If data has been “R” qualified, initiate a Corrective
Action.

Internal Standards - Add 5 µL of (13C12) Triclosan and 2 ul of (C13C12)
Methyl Triclosan Internal Standard Solutions prior to extraction. Compare
labelled internal standard responses to the latest continuing calibration
standard. The limits are retention time within 15 seconds and daughter SIM
ion area within a factor of two (-50% to +100%). The retention time of each
analyte and its corresponding isotope should be within +/- 6 seconds of
each other for each analysis.
8.9.1

Recovery limits for isotopic dilution methods may be overly
conservative (Section 11.4.4, EPA 8000D) since built in recovery
correction is one of the principle advantages of isotopic calibration.
Consult project manager and use professional judgement if
recoveries are outside of limits.

8.9.2

In general, qualify non-detects calculated with a internal standard
response <50 as estimated (J) and <20% as rejected (R). Do not
qualify non-detects calculated with a high internal standard
response. Consult project manager and use professional judgement.

Calibration and Standardization
9.1

Perform Initial calibration for each new instrument, and repeat when any
major changes or maintenance (ion source cleaning or repair, column
removal or replacement, etc.) are performed or when continuing calibration
fails. Enter into LIMS the calibration curve. An example is shown in Section
14.6.
9.1.1


 

Prepare calibration standards at a minimum of seven concentration
levels for each parameter of interest. See Section 6.9.

127
 

9.1.2

Allow standards to come to room temperature prior to analysis.
Inject 1 ul of filtered extract using splitless injector. Analyze each
calibration standard. Data analysis software calculates relative
response factors (RR) for each compound using the equation:

Calibrate the native compounds with a labeled analog using the following
equation:
RR = (An) (C1)
(A1) (Cn)
An = The area of the daughter m/z for the native compound
Al = The area of the daughter m/z for the labeled compound.
C1 = The concentration of the labeled compound in the calibration
standard(ng/mL).
Cn = The concentration of the native compound in the calibration standard
(ng/mL).
9.1.3

9.1.4

9.2

9.1.3.1

Qualify the associated detected results as estimated (“J”) if
calculated using an linear or quadratic coefficient that is
out of limits. Complete a QC Variance Form.

9.1.3.2

Perform percent recovery check on each calibration point
by re-fitting the response from each calibration point back
into the curve. If the recalculated concentration is not
within ± 20% of the standard’s true concentration or other
recovery criteria outlined in a project-specific QAPP. If
recoveries are failing, try different curve fits or redefine the
range of quantitation. Often quadratic curve that is
inversely weighted (1/x) helps accuracy at the lower
concentrations.

Retention Time - Recheck the integration and identification of a
target analyte if the retention time does not agree within +/- 15
seconds of that target analyte in the other calibration standards.

Calibration Verification
9.2.1


 

Use the RR for calculations. Evaluate the individual RRs compared
to the calibration curve to determine if there is a consistent high or
low bias indicating a problem with a particular point. Remake or
reanalyze the calibration standard if a problem standard or injection
is indicated. Evaluate chromatography to determine if system
maintenance could improve peak shape or response enough to
warrant maintenance and repeating the calibration. Evaluate linear
or quadratic RR curve plots of response ratios for best fit. Linear
curves must have correlation coefficients greater than 0.990 and
quadratic curves must be greater than 0.995

Calibration Check Standard (CCV) - Inject the mid-range standard at
the beginning of each 12-hour period after the tuning and column
performance test. Limit is the calculated result within plus/minus
20% of known value. Should this standard fail to meet those
parameters, repeat the test using a fresh calibration standard.

128
 

9.2.1.1

If the CCV fails the criteria for 20% or more of the
calibration analytes, or if a calculated result is not within
plus/minus 20% of known value, or if an estimated value is
not acceptable to the project manager prepare a new
calibration curve.

9.2.1.2

IS Retention Time – Evaluate the retention times of the IS
in the calibration verification standard immediately after or
during data acquisition. If the retention time for any internal
standard changes by more than 15 sec from that in the
mid-point standard level of the most recent initial
calibration sequence, then inspect the chromatographic
system for malfunctions and make corrections, as required.
When corrections are made, reanalyze samples analyzed
while the system was malfunctioning.

9.2.1.3

IS response - If the area for any of the IS in the calibration
verification standard changes by a factor of two (-50% to
+100%) from that in the mid-point standard level of the
most recent initial calibration sequence, inspect the mass
spectrometer for malfunctions and make corrections, as
appropriate. When corrections are made, reanalyze
samples analyzed while the system was malfunctioning.

10.0 Procedure
10.1 Preparation
10.1.1 Obtain sample(s) from sample freezer in room 224
10.1.2 Add sample to a single use aluminum weighing pan (may need to
thaw the sample a bit first)
10.1.3 Make a thin layer that covers the bottom of the pan. Two pans per
sample
10.1.4 Put excess sample back into the freezer
10.1.5 Place pans under hood using aluminum foil tents over the samples
to block light but allow air flow over sample. Leave hood lights off

10.1.6 Dry sample in hood at room temperature for 4 days. The
biosolids/paper mix tends to clump. Separate clumps into smaller
pieces after the first day and continue drying the full 4 days.
10.2 Milling


 

129
 

10.2.1 Using the Retsch Cryomill at room temperature (without liquid
nitrogen), add air dried sample (or paper mulch for calibration
standards) to a 50 ml steel grinding jar
10.2.2 Fill container roughly 2/3 full
10.2.3 Add three 10 mm steel balls
10.2.4 Follow the SOP L:\QA\SOP\Current\1022_Cryomill Sample
Processing_v1.pdf, except liquid nitrogen cooling is not used
10.2.5 Several cycles of adding dried sample to steel grinding jar may be
needed for each sample to have sufficient quantities
10.2.6 Combine milled samples into a 44 ml amber VOA vial using a clean
spatula
10.2.7 Store milled sample in sample freezer at -20°C (Rm 224).
10.2.8 Scrupulously clean grinding jar and steel balls. Wipe off excess
solids, wash with DI water and wipe clean with paper towel. Rinse
apparatus and steel balls 3 times with methylene chloride and dry
before next sample
10.3 Extraction
10.3.1 Add 0.25 g of milled sample or 0.15 g of milled paper mulch for
calibration standards to an 8 ml amber screw cap vial
10.3.2 Record the sample weight to 0.0001 grams on the printed LIMS
Bench Sheet and enter into LIMS.
10.3.3 Add appropriate amounts for calibration standards and matrix spikes
directly to sample according to 6.9.1
10.3.4 Add 5 ul of (C13C12) triclosan and 2.0 ul of (C13C12) methyl
triclosan to each calibration standard and sample
10.3.5 Add appropriate amounts of methylene chloride according to 6.9.1
10.3.6 Vortex 2 min
10.3.7 Sonicate 5 minutes
10.3.8 Vortex 1 min
10.3.9 Prefilter into an 4 ml amber vial using 0.45 syringe filter
10.3.10 Take 0.5 ml aliquot into self-filtering vials
10.3.11 Save extract in semi-voa freezer (Rm 231)
10.3.12 Filter using 0.2 micron self-filtering vials
10.4 Batch - In Element® LIMS select “Laboratory”and then select “Batch”.
Select the Vortex micro extraction from the drop down method under
“Preparation Method”. Select Semi Volatile organics from drop down menu
under “Batch Department”. Select Soil from drop down menu under “Batch
Matrix”. Select available methods Click on Department under “List Analyses
by” and select S8270_PPCP from available analysis. Save the Batch. See
example in Section 14.7.


 

130
 

10.5 Bench Sheet - After saving the Batch in Element® LIMS, a bench sheet is
created. Save the bench sheet. See example in Section 14.8. Print the
sequence using print format “seq_sxname.rpt”.
10.6 Sequence – In Element® LIMS select “Laboratory”and then select
“Sequence”. Set up the initial calibration or continuing calibration sequence.
Save the sequence. See example in Section 14.9. Print the sequence using
print format “seq_sxname.rpt”.
10.7 Turn on the LC\MS\MS PC.
10.8 Method Setup - Load the appropriate GC acquisition method
(8270_PPCP_Triclosan-MTS). Adjust the gas chromatographic operating
parameters to obtain suitable chromatography.
10.8.1 Adjust the ion groupings and dwell time such that it produces at least
15 to 20 scans per chromatographic peak for quantitative analysis
and 10 to 15 scans for qualitative analysis
10.8.2 Set up the Sequence in MassHunter, See Section 14.10 as an
example.
10.8.3 Sequence, Run Sequence to start from the first line of Sequence,
Position and Run Sequence to start sequence from a different line.
10.9 Tune Check – Perform a tune check each day to evaluate the instrument
status against the manufacturer’s requirements. Autotunes and tune checks
are automatically stored in the directory (Data)
D:\MassHunter\GCMS/1/7000/TuneReports.
10.9.1 Check Tune – Load the 8270_PPCP acquisition method.
10.9.2 If the check tune fails, perform instrument maintenance and/or a full
autotune. See Section 8.1.2 for the autotune procedure.
10.9.3 Print the autotune or checktune report. See Section 14.11 for
evaluation of tune report.
10.10 Condition system with five blank injections containing milled paper mulch
(Section 8.4) before calibration or continuing calibration sequence. This
ensures that matrix components have sufficiently masked the active sites in
the system (Section 2.4).
10.11 Calibration - Calibrate the system as described in Section 9.1. Save the
analysis method as S8270_PPCP_YYYYMMDD.m
10.12 Continuing Calibration - Perform a CCV check as described in Section 9.2.
Allow extracts to come to room temperature prior to analysis
10.13 Method Blank - Analyze a method blank prior to sample analyses in order to
ensure that the total system (introduction device, transfer lines and
GC/MS/MS system) is free of contaminants. If the method blank indicates
contamination, analyze a solvent blank to demonstrate that the
contamination is not a result of carryover from standards or samples.
10.14 Inject the same volume (1 ul) of the sample extract or QC extract into the
GC/MS/MS system as was used for the calibration standards.


 

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10.15 If the response for any compound exceeds the highest calibration standard,
dilute the extract and reanalyze or re extract using less sample. If using
diluted extract, add additional internal standard solution to the diluted
extract to maintain the same concentration as in the calibration standards.
Create a reshot (RE) in Element from the original sequence to import the
dilution result. Label dilutions with “#X” in the Misc. Info field where # is the
dilution factor and enter this # in the Sample Multiplier field. For example a 1
to 10 dilution of Outfall 230 would have Misc. Info: Outfall 230 10X and
Sample Multiplier:10.
10.16 Back-up Instrument Data - Copy the analysis subdirectory to
\Transfer\Instrument Backups\Instrument Name\DataYYYY.
11.0 Data analysis and Calculation
11.1 Open Agilent MassHunter quantitative analysis (‘QQQ quantitative analysis’
icon). Select the menu item “file” and then “new batch”, navigate to the data
subdirectory, double click on the data subdirectory and type the batch file
name in the “.batch.bin” file box as ‘S8270_PPCP_ICAL_YYYYMMDD’ for
an initial calibration.
11.2 Select the menu item “file” and then “add samples”, select the appropriate
files from the analysis subdirectory and click on “ok”.
11.3 Click on the box next to the “continuing calibration” file or the initial
calibration file at the CCAL level, select the menu item “method” and then
“open” and “open method from existing batch”. Navigate to the subdirectory
of the last previous batch, click on the previous “batch.bin” file and click on
“open”.
11.3.1 Click on “globals setup” and compare to the example in Section
14.12 for the appropriate entries to insure no multipliers are applied
to the on-column results.
11.3.2 Select the menu item “update” and then “update retention times”.
Select all and then “ok”
11.3.3 Select the menu item “update” and then “update qualifier ratios”.
Select all and then “ok”
11.3.4 Click on “exit” under “save/exit” and “yes” to apply this method to the
batch.
11.4 Select the menu item “analyze” and then select “analyze batch” if the batch
includes the initial calibration files. Select “quantitate batch” if the initial
calibration files are not included in the batch. Analyzing a batch without the
initial calibration files will overwrite the calibration, repeat Section 11.3 to
restore the initial calibration files. See Section 14.13 for example batch in
MassHunter quantitative analysis.
11.5 Qualitative Analysis – Evaluate the product quantitation and qualifier ions
listed in Section 14.1 for individual compound information. Use the following
criteria to make a qualitative identification:
11.5.1 The quantitation and qualifier ions of each parameter of interest
must maximize in the same or within one scan of each other.


 

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11.5.2 The retention time must fall within +/- 15 seconds of the authentic
compound. Evaluate retention time shifts against the surrogate and
internal standard for consistency.
11.5.3 The relative peak heights of the qualifier ions must fall within ±20%
of the relative intensities of these ions in the authentic compound
with relative intensities greater than 10%. Qualifier ion relative
intensities that are less than 10% should fall within ±30% of the
relative intensities of these ions in the authentic compound. Use
professional judgment in interpretation where interferences are
observed.
11.6

Quantitative Analysis – Quantitate identified parameters based on the
integrated abundance from the product quantitation ion

11.7

Compute the concentration of each compound in the extract using the RR from
the calibration data and following equation:
Cex (ng/ml) = (An) (C1)
(A1) (RR)
Cex = Concentration of the target analyte in the extract:other terms are defined
in Section 9.1.2
11.8 Avoid manual integrations unless necessary when the software does not
produce proper integrations
11.8.1 Investigate and evaluate any flags generated by the analysis
software for outliers to the calibration or qualifier criteria.
11.8.2 Select the menu item “view” and then “compounds-at-a-glance” to
view the standard and sample chromatograms side by side, if
necessary, to evaluate retention time shifts. See Section 14.14.
11.8.3 Select the menu item “analyze” and then select “analyze batch” if
any changes are made to the initial calibration files or the calibration
type. Select “quantitate batch” if any changes are made to the
continuing calibration or sample files.
11.8.4 Select the menu item “file” and then select “save batch”.
11.8.5 Generate the Element® LIMS import file. Select the menu item
“report” and then “generate” and navigate to the template file
“C:\MassHunter\Report Templates\Quant\en-US\Letter\ LIMS\lims
export files full.xltx”. Leave “report folder” on the default option which
will save the report to the batch subdirectory. Click on “ok”. The
Element® LIMS file will automatically be saved in that batch’s
subdirectory under “\QuantReports\batch name\lims export files
full.xltx”.
11.8.6 Generate a PDF file for each calibration or sample analysis
sequence. Select the menu item “report” and then “generate”,
navigate to the template file “C:\MassHunter\Report
Templates\Quant\en-US\Letter\
ISTD\Parts_Graphics\QuantReport_ISTD_Complete_B_05_01.xltx”.
Leave “report folder” on the default option which will save to the
batch subdirectory. Click in the box next to “output PDF to screen”.


 

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Click on “ok”. The PDF will automatically be saved in that Batch’s
subdirectory under \QuantReports\batch file name1\QuantReport_ISTD_Complete_B_05_01 .pdf.
11.9 Result Calculation
11.9.1 Sample concentration calculates in Element® LIMS using the
following equation:
Conc, (ug/kg or ng/g)  = (Cu ×V ×D)/(W ×S)  
Where
Cu = Concentration on column (ng/ml) = IResult
V = Sample Volume (mL) = Final (mL) = 5 mls
D = Dilution factor = Diln
W = Weight of sample (g)
S = Percent solids/100 = 100% solids is default for air dried
samples
11.10 Back-up Instrument Data – Copy the analysis subdirectory to
\\Transfer\Instrument Backups\7000SV1\DataYYYY. Use a data stick for the
transfer if quantitative analysis was performed on the QQQ computer.
11.11 Import into Element® LIMS
11.11.1 See Section 14.15 for a list of corresponding MassHunter and
DataTool fields
11.11.2 Use \Element\DataTool\CrossTables\SemiVolatiles as the cross
table. Set Units = 1 in Data Tool, units off the instrument are in
µg/L.
11.11.3 The DataTool file type for import is Agilent Mass Hunter LIMS
(*.xlsx). Check the box for “multiplier field” for the “take dilution
factor from” window in DataTool.
11.11.4 Import the file \\Transfer\Instrument Backups\7000Sv1\
DataYYYY\YYYYMMDD_COT\QuantReports\batch name\lims
export files full.xlsx.
11.11.5 Merge files. Edit and replace the acquisition method name in the
analysis column with “S8270_PPCP”, Instrument 1” in the
“instrument” column with “7000 SV1”, ZZZ” in the “analyst” column
with analyst initials if they did not import, and Element® LIMS export
files full-### in the “File_Name” column with the Data File
number.d as it appears in the pdf. Edit and replace the “sample
name” in the “Lab_Number” column if it does not match the name
as it appears in the Element® LIMS sequence.
11.12 Copy \\Transfer\Instrument Backups\7000
SV1\DataYYYY\YYYYMMDD_COT QuantReports\batch file name1\QuantReport_ISTD_Complete_B_05_01.pdf to the
\\Element\Data_PDF\Sequence\ subdirectory and rename as “TYMDD##”,
where TYMDD## is the Element® LIMS sequence number.


 

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11.13 Review Results – In Element® LIMS, review the imported “IResult” column in
“data review” table against the PDF.
11.13.1 Check that all dilution values are entered in the “diln” column.
11.13.2 Review any handwritten changes made to amounts or standards
on the “preparation log” sheet to ensure they have been updated in
Element® LIMS.
11.13.3 Investigate any red-flagged rows, correct if possible or generate an
Analysis QC Variance form to document and explain the variance.
11.13.4 For initial calibration: Review the Element® LIMS calibration
columns of cal type and LR COD or QR COD against the initial
calibration PDF. Uncheck standard points in Element® LIMS that
were not included in the calibration curve on the PDF to get the
Element® LIMS columns to match for each compoud
12.0 Pollution Prevention and Waste Management
12.1 Seal the vials for disposal containing sample extracts or expired standards
metal can waste container for this satellite area until disposed of by lab pack
in accordance with the laboratory’s waste disposal manual. Keep in use
waste disposal cans in the hood in room 231. This is the designated
satellite collection area for this waste stream. When waste container is full,
notify the Hazardous Waste Manager for removal to Hazardous Waste
Storage area.
12.2 Collect waste solvents in an appropriate waste container and dispose of in
accordance with the ES Laboratory Hazardous Waste Disposal Manual.
13.0 References
Test Methods for Evaluating Solid Waste, Physical/Chemical Methods SW-846
Method 8270D
Determinative Chromatographic Separation, EPA Method 8000D
City of Tacoma Environmental Services Chemical Hygiene Health and Safety Plan
2.0”, 2014
City of Tacoma Environmental Services Draft Hazardous Waste Disposal
Manual_v5, 2016
City of Tacoma Environmental Services 2015_Laboratory Quality Manual_v4,
2015


 

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14.0 Tables, Diagrams, Flowcharts and Validation Data
14.1 Characteristic Ions for Analytes and Internal Standard Isotope
Compound
Triclosan

CAS#
3380-34-5

Methyl Triclosan

4640-01-1

(13C12) Triclosan
(13C12) Methyl
Triclosan

Precursor Ion
287.6
217.8
301.7
251.7
302
302
313.9
313.9

Daughter Ions(s)
217.8 Quant
155.1 Qualifier
251.7 Quant
188.8 Qualifier
230 Quant
119.1 Qualifier
263.9 Quant
244.1 Qualifier

14.2 LIMS Calibration Standard


 

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14.3 LIMS Standard


 

137
 

14.4 Check Tune Screen Tab


 

138
 

14.5 Autotune Screen


 

139
 

14.6 LIMS Bench Sheet


 

140
 

14.7 LIMS Sequence

14.8 MassHunter Sequence


 

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14.9 Evaluate Tune Report


 

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143
 

14.10 Globals Setup

14.11 MassHunter Quantitative Analysis


 

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14.12 Compounds at a Glance

14.13 Corresponding Fields between MassHunter and DataTool
MassHunter Field
Data File

DataTool Instrument Data
File_Name

DataTool Merged Upload
FileID

Sample Name

Lab_Number

LabNumber

Aqu. Method File Name
Compound Name

Analysis
Analyte

Analysis
Analyte

Comment

Misc

(no upload match)

Dilution

Dilution

Result

InitialResult

Units

InitialUnits

Analyzed

Analyzed

RTime

RT

Response

RESP

Instrument

Instrument

Chemist

Analyst

1

1

4

Diln.

1,2

1,2

Final Conc.

7

Concentration Units
AcqTime
RT

7

7

Response

7

Instrument

6

Acq. Operator
1

3

5

Entered in Acquisition Worklist table
May be edited in QQQ Quantitative Analysis
3
Entered under Acquisition Worklist Run Parameters
4
Entered in QQQ Quantitative Analysis, MRM Compound Setup
5
Entered in QQQ Quantitative Analysis, Concentration Setup
6
Entered under Instrument Name during Agilent Configuration, the 6430 is Instrument 1
7
Generated field based on data acquisition for the individual file
2


 

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Media

Weibel_WPMESthesis2016.pdf