Degradation of Di(2-ethylhexyl) phthalate

(DEHP) by Rhodococcus – Treatment Design

Rita Chen, Audrey Pottschmidt, John Schlichting

The Ohio State University

Bioremediation of Groundwater and Soil
Dr. Natalie Hull
November 25, 2019
Introduction

Nearly any item we encounter everyday has some form of plastic in it. In fact, a study in the
journal of Environmental Science and Technology found that humans may be consuming 39,000
to 52,000 microplastic particles each year (Gibbens, 2019). Plastic compounds can be found in
anything ranging from children’s toys to medical devices, clothing, personal care products, and
even household dust. Various plastic compounds are now gaining attention in the public eye as
emerging contaminants in the environment and water systems.

An example of these emerging contaminants is phthalates. Phthalates are a plastic additive that
are typically used to make plastic more malleable and flexible. They are used on a wide variety
of products and have been very popular in the media. The following paper gives an in-depth
study of a specific phthalate, di(2-ethylhexyl) phthalate or DEHP and potential remediation
pathways found to degrade it.

Compound Background (Structure, Production, Uses, Problems)

Di(2-ethylhexyl) phthalate, also known as DEHP, is a commonly manufactured chemical added

to polyvinyl chloride (PVC) to make the material more flexible. DEHP can be found in ordinary
household products such as wall covering, tablecloths, floor tiles, shower curtains, swimming
pool liners, toys, shoes, and more. In the medical industry, it is used in medical tubing, blood
storage bags, and other plastic applications (ATSDR, 2002). Approximately 2.7 million metric
tons of phthalates are produced a year, a quarter of which is DEHP. The low melting point and
high boiling point of phthalates make them useful as heat transfer fluids and carriers (van Wezel
et al., 2000).

DEHP is an organic compound, with an empirical formula of C24H38O4 (ASTDR, 2002). When
mixed with plastic, DEHP does not bind with the polymer. Instead, it mixes with the polymer
and enables the polymer strands to move flexibly. Since the DEHP is not bound, it slowly
leaches out of the plastic into the surrounding environment. In conjunction with its high volume,
DEPH has become ubiquitous in the environment. Human exposure can occur from DEHP
transferred to food from PVC film and food containers, and from disposable medical equipment.
Additionally, DEHP is found at low concentrations in the air, water, soil, and sediments
(OEHHA, 2009). DEHP can attach to suspended particles in the water or bind to dust particles
in the air.

For humans, the most probable route of exposure is through food; DEHP migrates into food from
plastics during processing and storage. DEHP can also be found in drinking water and the
ambient atmosphere. However, DEHP levels in the environment are generally too low to cause
adverse human health effects. Other routes of exposure include medical procedures such as
blood transfusions, kidney dialysis, and procedures that use respirators. For people that work in
manufacturing or factory environments, occupational exposure to DEHP may be possible if their
work involves large amounts of DEHP (USEPA, 2000).

Due to the omnipresence of DEHP in the human and natural environment, it is difficult to
quantify the average exposure of humans to the compound. A study conducted by the Institute
and Outpatient Clinic of Occupational, Social and Environmental Medicine at the University of
Erlangen in Erlangen, German aimed to estimate the daily intake of DEHP by the general public.
The research results showed that the median daily intake was 13.8 µg/kg body weight/day and
the 95th percentile intake was 52.1 µg/kg body weight/day (Koch et al, 2003). The European
Union Scientific Committee on Toxicity, Ecotoxicity, and the Environment has set a tolerable
daily intake (TDI) of 37 µg/kg body weight/day; similarly, the United State Environmental
Protection Agency (USEPA) has set a reference dose (RfD) of 20 µg/kg body weight/day. While
the median daily intake from the study is below both standards, there are considerable amounts
of people that exceed the standards (Koch et al, 2003).

Little studies and theories concerning DEHP’s effects on human health have been established.
DEHP’s status as an emerging contaminant and ubiquitous nature make research difficult;
additionally, direct administration of DEHP to humans would be unethical. Acute health effects
of large doses of DEHP (5-10g) have shown to cause gastrointestinal distress in humans.
Chronic effects have yet to be fully determined. However, studies using laboratory mice
demonstrated that chronic exposure to DEHP can cause formation of liver tumors (ATSDR,
2002). The United States EPA has listed DEHP has a probable carcinogen to humans (USEPA,
2000). Additionally, California’s Office of Environmental Health Hazard Assessment has listed
DEHP as a chemical under Proposition 65. Proposition 65 requires businesses to provide
warnings to Californians about significant exposures to the listed chemicals, which are suspected
to cause cancer, birth defects or other reproductive harm (OEHHA, 2009).

A study conducted by Environmental Health Perspective has confirmed that DEHP can be
transferred from pregnant mothers to babies. Blood samples were drawn from newborns to test
for core levels of DEHP. Infants with positive results of DEHP were positively correlated to
have shorter gestation periods and were born earlier than infants with no detectable levels of
DEHP (Latini et al, 2003). Even though it is unknown how the presence of DEHP will affect the
newborns’ development, the study confirmed transfer from mother as a possible route of
exposure for babies.

Microbial Taxa

As an emerging contaminant, DEHP degradation pathways are being studied in labs around the
world. Many microbial taxa have been isolated and found to degrade DEHP. The taxa that seems
to degrade DEHP most efficiently and effectively is rhodococcus pyridinivorans XB. The r.
pyridinivorans XB isolates were collected from the activated sludge of a sewage plant in
Guangzhou, China (Zhao et al 2018). This strain was found to be facultative anaerobic, gram
positive, non-flagellated, short rod bacteria with opaque, salmon pink, rounded colonies, see
Figure 1 below (Zhao et al 2018).
 Figure 1: Cellular Morphology or R. pryidinivorans XB (Zhao et al. 2018)

R. pyridinivorans XB uses DEHP as its sole carbon and energy source under optimal conditions
(Zhao et al 2018). The optimal degradation conditions for r. pyridinivorans XB were found to be
pH 7.08, 30.4 degrees Celsius, and an inoculum size of 0.6 (OD600nm) with an average
degradation rate of 99.1% (Zhao et al 2018). R. pyridinivorans XB, was however, able to grow at
a range of pH (5-9) and temperatures (20-40 degrees Celsius) suggesting that the microbe could
be used in a variety of environments (Zhao et al 2018). One competitive advantage that r.
pyridinivorans XB has over other microbes living in a DEHP contaminated environment is the
production of special extracellular polymeric substances (EPS). R. pyridinivorans XB was shown
to excrete these EPS’s in response to stress caused by high concentrations of DEHP in the
environment as a way of detoxifying the cell (Zhao et al 2018). These EPS’s can be seen in
Figure 1 (B-D) above as the morphology of the cell shifts based on exposure to DEHP. In Figure
1 B, a normal rod-shaped bacterium is shown compared to C-D where the cellular morphology
changes as EPS is produced due to exposure to 200mg/L of DEHP.

Another strain of rhodococcus that has been found to degrade DEHP is r. ruber YC-YT1. This
strain is also gram positive and rod shaped and was originally isolated from marine plastic debris
in coastal seawater in China (Yang et al. 2018). Similar to r. pyridinivorans XB, r. ruber YC-
YT1 is thought to use DEHP as its carbon source (Yang et al. 2018). R. ruber YC-YT1 had the
highest degradation rates in slightly alkaline solutions (pH of 6-10) at an optimal temperature of
about 30 degrees Celsius (Yang et al. 2018). Under optimal conditions, the lag phase was a total
of 10 hours and DEHP was completely degraded after 72 hours (Yang et al. 2018). A major
barrier to the degradation of DEHP by r. ruber YC-YT1, particularly in industrial wastewaters, is
salinity. The isolated r. pyridinivorans XB could survive a maximum NaCl concentration of 120
g/L while having a degradation rate of 80% or higher (Yang et al. 2018).
Other microbes that have been studied and found to degrade DEHP in laboratory conditions
include agromyces, fusarium culmorum, and pseudomonas but all at much lower rates than
rhodococcus. A future application of bioremediation of DEHP could involve mixed communities
of microbes working together to degrade the contaminants. As an example, a consortium of
microbes was found to degrade 93.46% of 1000mg/L DEHP after 48 hours (Li et al. 2018). This
consortium was made up of seven different species and families of microbes but mainly made up
of Gordonia, Rhodococcus, and Achromobacter and provides potential efficiencies to degrading
high concentrations of DEHP.

Biodegradation Pathways

Phthalates enter the environment due to the production, usage, and disposal of plastic products.
In 2015, the world produced 381 million tons of plastic which was almost 200 times the amount
produced in 1950. Plastic recycling is more prevalent now than in the 1950s, plastic incineration
and disposal have been consistently chosen (Greyer, 2017). While incineration can introduce
local air emission problems, disposal is a primary source of plastic and plastic related chemicals
in the environment. Phthalates have been detected in various environments including air
(Wensing et al. 2005), soil (Xu et al. 2008), sewage (Gavala et al. 2003), wastewater (Roslev et
al. 2007), and natural waters. Phthalates are not chemically bonded to the plastics polymer and
can eventually migrate from the plastics into the environment. DEHP has low-water solubility
and concentrates in soils and river sediments. Industrial production of DEHP is generally carried
out following the reaction of phthalic anhydride with 2-ethylhexanol. Esterification is a reaction
of an alcohol and an acid to produce an ester and H2O. Esterification of phthalic anhydride by
alcohol generally takes place in two stages. The first stage is fast and can be carried out in the
absence of catalyst. However, esterification of the second carboxylic group is slow and must be
facilitated by an acid catalyst with the resulting water being removed from the reaction mixture
to achieve DEHP.

Because DEHP and phthalates are common additives in plastics, the globalization of plastic has
led to the globalization of phthalates in all types of environments prompting phthalate exposure.
These substances are endocrine disrupting compounds (EDC) that can affect environmental
fauna development and human development depending upon the time and type of exposure.
DEHP joins polychlorinated biphenyls (PCBs), dichlorodiphenyltrichloroethane (DDT), and
bisphenol A (BPA) as chemicals commonly used in production that are classified as EDCs.
Properly remediating DEHP from the environment or building would greatly benefit
environmental and public health. The recommended drinking water levels of DEHP in the US is
6 ug/L (WHO, 2004). Any initial reduction of DEHP would reduce later costs of removal.
DEHP is recalcitrant due to the steric effect of phthalates side ester chains, which hinders the
hydrolytic enzymes from binding to the phthalates and thereby inhibits hydrolysis. (Liang et al.
2008). The steric effect reduces the rate of chemical interactions slowing the overall rate. This
effect is caused by overlapping atoms and electrons occupying the same space, favorable
collision paths or binding is not favored. Studies have demonstrated that phthalates with shorter
ester chains like DMP, DEP, DBP, DPP, DPrP, and BBP are more readily biodegraded and
mineralized (Chang et al. 2007).
Challenges of bioremediation with microbes involve sustaining growth in the medium by
providing enough nutrients and stimulating the growth of the desired microbes. Additionally, this
microbial growth could increase the environment’s chemical oxygen demand or alter the
environment causing and biodegradation to be incomplete or hindered. Bioremediation of
polluted sites using enzymes may be quite advantageous compared to classical methods of
inoculating and sustaining microbes on polluted sites. Generally, most microbial capabilities are
observed ex situ under laboratory conditions and actually inoculating a biological site can vary
due to unknown factors. Enzymes offer a lower activation energy for degradation mechanisms to
occur and can provide cells with energy to grow (Peterson and Staples 2003). Common
microbial PAE enzymes are phthalate oxygenase, phthalate dioxygenase, phthalate
dehydrogenase, phthalate decarboxylase, lipase, and esterase. Under aerobic and anaerobic
conditions, microbial and enzymatic degradation pathways generally occur in 2 main steps:
primary degradation and ultimate degradation. Primary degradation usually converts DEHP to
MEHP and PA. Ultimate degradation tends to convert PA to CO2 and CH4 (Juvancz et al. 2008).

Figure 2: Proposed DEHP degradation pathway by R. ruber YC-YT1 (Yang et al. 2018)
Figure 2 demonstrates the proposed pathway of DEHP degradation by Rhodococcus ruber YC-
YT1 (Yang et al. 2018). The study used high-performance liquid chromatography - mass
spectrometry (HPLC-MS) to identify the intermediate metabolites formed during this pathway.
For primary degradation, this microbe utilizes selective hydrolysis on two of DEHP’s ester
bonds. The breaking the first bond produces MEHP and an alcohol. Breaking the second ester
bond produces PA and an alcohol. For ultimate degradation, this microbe uses decarboxylation
to form benzoic acid which can be degraded in the benzoate metabolic pathway producing CO2
and CH4. The other microbe investigated, Rhodococcus pyridinivorans XB, utilizes a similar two
stage degradation pathway, however several important intermediates are generated (Zhao et al.
2018). This study used HPLC-MS to identify six main intermediates as dibutyl phthalate (DBP),
MEHP, 2-Ethylhexanol, and PA during primary degradation and 3,4 - dihydroxyphthalate and
protocatechuate (PCA). The KEGG database outlined in the pathway tutorial has limited entries
for DEHP degradation. However, the entry found relating to DEHP described a similar
degradation verifying the degradation intermediates like 2-Ethylhexanol. The XB strain first
hydrolyzes the DEHP with phthalate hydrolase to form DBP, MEHP, and 2-Ethylhexanol. The
MEHP is hydrolyzed to form PA. The ultimate degradation pathway involves β-oxidation and
requires further investigation. However, the steps proposed involved phthalic acid dioxygenase
forming PCA which can then be cleaved and utilized in the tricarboxylic acid cycle (TCA cycle).
The 2-Ethylhexanol could not be utilized by XB therefore this byproduct could accumulate in the
environment causing toxic effects and incomplete degradation of DEHP. It is suggested that
these microbes be coupled with other phthalate reducing microbes in order to reduce any toxic
effects of byproducts formed during the degradation and emergence of DEHP.
DEHP Treatment & Bioremediation Applicability
DEHP has proven to be a ubiquitous compound in the environment. Plastic waste pollution is a
major problem across various ecosystems, and it will be important to develop a technology of
bioremediating DEHP efficiently in a wide range of environmental conditions. As previously
discussed, Rhodococcus ruber YC-YT1 is capable of degrading DEHP quickly in 72 hours.
Expanding on Yang et al’s research, YC-YT1 can be applied to bioremediating other
contaminated sites.

Overview of Case Study

The case study that was screened for degradation of DEHP was based on a study conducted in
China on biodegradation of DEHP in contaminated water and soil (Yang et al. 2018). In this
study, Rhodococcus ruber YC-YT1, a bacterial strain with super salt intolerance, was isolated
from marine plastic debris sludge. This strain was chosen specifically for DEHP degradation
because plastic debris heavily pollutes the marine ecosystem. Furthermore, most DEHP-
degraders can utilize no more than eight kinds of substrates. Finding one strain that is capable of
degrading DEHP and its intermediates in various environments is important for remediating a
ubiquitous contaminant such as DEHP.

To determine the optimal environmental conditions for DEHP degradation, single-factor

optimization experiments were conducted in batches. The environmental factors tested were pH,
temperature, salinity, and glucose concentration. Additionally, degradation efficiency was tested
by performing tests at minimum and maximum concentrations of DEHP. Residual DEHP
concentration was measured using a gas chromatograph (GC).

Results from the single-factor optimization batch tests showed that the optimal conditions for
DEHP degradation are pH ranges between 6.0-10.0 and temperatures greater than 30 degrees
Celsius. YC-YT1 can also withstand up to 120 g/L of NaCl and a glucose concentration of up to
5 g/L. Growth outside of these ranges decreases significantly but is not completely inhibited
(Yang et al 2018).

It is also important to factor in various metabolites produced by YC-YT1 when screening for
design parameters. Some key metabolites that form when YC-YT1 degrades DEHP are MEHP,
BA, and PA. BA is a central intermediate found in the anaerobic phthalate metabolism and is
then oxidized to form phenol to be metabolized through the TCA cycle (Yang et al. 2018). Other
metabolites that have been found in DEHP degradation applications using different sets of
microbes include EHPP, BEHP, and MBP (Li et al. 2018). The biodegradation of DEHP, is,
therefore, a very complicated process and still highly misunderstood by microbiologists. Because
of the complex nature of this degradation pathway, it is important to take advantage of
indigenous microbes when possible, in order to encourage full degradation of DEHP. If the
bioremediation site in question has been contaminated for a long period of time, it can be
assumed that microbes able to degrade both DEHP and these metabolites could be present
naturally. If they are not present, inserting a consortium of bacteria that are known to degrade
DEHP is the most effective way of fully remediating a highly concentrated spill of DEHP.

Some key bacterial strains to consider for the treatment consortium include Mycobacterium sp.
YC-RL4, Gordonia sp., and Achromobacter sp. Mycobacterium sp. YC-RL4 is known to have
hydrophobic properties which helps the microbe remediate DEHP and other phthalates that are
otherwise biologically unavailable for most microbes (Yang et al. 2018). Gordonia sp. and
Achromobacter sp. have been studied as useful microbial partners when paired with
Rhodococcus sp. to degrade DEHP in a high salt environment (Li et al. 2018). The consortium
used in Li’s study was able to degrade DEHP concentrations as high as 2000 mg/L by 92.80%
within only three days (Li et al. 2018). This degradation rate supersedes that of the isolated YC-
YT1 strain from the above case study as this consortium could degrade double the concentration
of DEHP in the same timeframe. The degradation kinetics of this consortium was found to fit to
a first-order model with R^2 confidence ranging from 0.905 to 0.964 and a degradation half-life
of 3.61637-4.35519 days. This suggests that a consortium bacterium, therefore is a better
application for our site because the consortium is a very effective and efficient solution for
extremely high concentrations of DEHP.

Suggested Treatability Study

While literature review served as remedy selection, careful and deliberate analysis of the
bioremediation site in question must be conducted. To assess the applicability of utilizing YC-
YT1 at a specific site, several steps are suggested for remedy selection. The environmental
conditions of the site should be determined in order to replicate the microcosm in batch testing;
this would aid in determining the site characteristics fall within the optimal conditions for YC-
YT1 growth. Additionally, metagenomics testing should be conducted to determine the
microorganisms already present at the site. Rates of primary DEHP degradation should also be
determined to evaluate the timeframe for a full-scale remediation project.

For remedy design/action, a pilot study will be conducted in-situ. The specific design parameters
are discussed below. This phase will apply the bioremediation technology from the case study
onto another contaminated site.

Treatment Design

Based on bacterial strains outlined in Part 1 and Part 2’s case study, this treatment design uses a
consortium of known DEHP-degrading species to remediate an idealized site. A watershed
monitoring well near the Great Miami River in Hamilton County Ohio observed MEHP during
routine sampling. Historical records showed a plastic manufacturing plant operated from 1986 to
2005. The plant improperly disposed of its PVC by-products, primarily scrap linoleum flooring
and piping, using a simple compacted clay liner. This liner was found to have a life expectancy
of ~10 years (TCS, 2008). The disposal site has been excavated, but adsorbed DEHP remains in
the subsurface. Based on these factors, DEHP concentrations are assumed to be 1000 mg/L.
Additionally, bioremediation via biosparging can be an effective method to degrade this
endocrine disruptor. The treatment process involves: background wells, consortium inoculation,
enzyme and nutrient supply, injection wells, extraction wells, activated carbon, monitoring and
verification. Background wells determine the presence of other DEHP sources. Consortium is
aerated and inoculated with groundwater and soil mix from background wells then re-injected.
Enzymes, including peroxidase and lipase, and nutrients, depleted by surrounding agricultural
activities, are aerated, then injected with the consortium. Because of utility pipelines and the
county road, the treatment is split, and the permission of local farmers is necessary. The
extraction wells uptake treated groundwater and filter using granular activated carbon. These
GAC filters can be used to remediate further and monitor contaminant concentrations. Additional
monitoring wells are created along the direction of subsurface flow. The concentrations of DEHP
and its metabolites are observed, and degradation is verified using high-performance liquid
chromatography or GC. These are idealized conditions; injection rates would be adjusted based
on monitoring well results and in-field samples. Further investigation of the subsurface
hydrology, geochemistry, and composition would be needed to size the injection and extraction
well depths. Due to DEHP’s environmental presence and chemical properties, sampling
procedures must involve method blanks and field blanks to eliminate lab error (Connelly, 2002).

This proposed treatment design selects microbes that are known to degrade DEHP following the
pathway described in Figure 2. However, engineered consortia unlock the collective benefits of
microbial growth with gene editing, quorum sensing, extracellular polymeric substances, and
other operations focused on contaminant degradation (Desai, 2010). Soil concentrations of
DEHP at plastic processing sites are expected to range from 30 to 354 mg/kg (TCS, 2008). Given
the assumptions of the idealized treatment design, this proposed system could fail in the
environmental conditions via natural competition, redox variations, and pH fluctuations.
Therefore, biotechnologies should be implemented to enhance degradation. A direct-push
delivery of the injection solution and nutrients was found to increase efficient delivery within
soils with clay compositions (Christiansen, 2010). This delivery system could benefit more
complex ground subsurface because the pressurized push introduces fissures spanning
impermeable zones. Additionally, this could increase microbial access to adsorbed DEHP
compounds. Another biotechnology could be applied to this contaminated site. The addition of
maize mucilage to soils was observed to increase microbial carbon by 23% (Benizri, 2007). This
demonstrates phytodegradation and bioaugmentation that could be implemented to treat surface
deposited DEHP. Introducing plants and rhizodeposits like mucilage helps stabilize the native
microbial community especially within agricultural developments (Lebeau, 2011).
Conclusion

Utilizing a microbial consortium containing the novel bacteria Rhodococcus ruber YC-YT1, a
plastic manufacturing site (Figure 3) was analyzed for bioremediation applications, the group
selected biosparging (Figure 5). Biosparging adds oxygen to the saturated zone and is often
combined with biostimulation and bioventing. Generally, aerobic degradation occurs at faster
rates than anaerobic. The soil and redox characteristics were found using the National
Conservation Soil Survey database (Figure 4). The soil near the site was found to support
microbial growth. From HPLC and GC results, environmental factors were observed performing
the primary degradation step to MEHP. The group performed metagenomic analyses of site
samples and determined native microbial species were capable of degrading DEHP.

Key design features were implemented based on the Part 1 review and the selected case study.
Given the site location being farmland, the group realizes the invasiveness of the injection and
extraction wells. Therefore, selecting optimal conditions to reduce overall disruption while
removing DEHP is preferred. Because many of the by-products of DEHP degradation still
exhibit carcinogenic effects, the complete mineralization of these phthalates is essential. To
achieve ultimate degradation of DEHP, enzymes known to contribute to primary degradation,
including peroxidases and lipases, were added to increase native and consortia degradation rates.
While YC-YT1 has been observed to completely degraded DEHP, an engineered consortium, as
previously described, increases co-metabolism effects and overall removal of DEHP. While this
increases costs of inoculating additional microbes and creating batch enzymes, the degradation
of DEHP will be completer and more thorough over a shorter amount of time. Further
observations of the GAC filters and monitoring wells will be required to ensure successful
bioremediation.
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Appendix

Figure 3 - Overview of Proposed Biosparging Treatment at the closed PVC plant

Figure 4 - Hamilton County soil data from National Resource Conservation Survey

Figure 5 - Process overview of biosparging, treating groundwater