THE HEALTH EFFECTS AND A BIOREMEDIATION PROCESS FOR VINYL
CHLORIDE
by
Meng-Chen Lee
B.S. M.S., University of Iowa, 2010, 2012
Submitted to the Graduate Faculty of
Department of Environmental and Occupational Health
Graduate School of Public Health in partial fulfillment
of the requirements for the degree of
Master of Public Health
University of Pittsburgh
2014
UNIVERSITY OF PITTSBURGH
GRADUATE SCHOOL OF PUBLIC HEALTH
This essay is submitted
by
Meng-Chen Lee
on
April 28 , 2014
and approved by
Essay Advisor:
James Peterson, PhD
______________________________________
Associate Professor
Department of Environmental and Occupational Health
Graduate School of Public Health
University of Pittsburgh
Essay Reader:
Na Wei, PhD
______________________________________
Assistant Professor
Department of Civil and Environmental Engineering
Swanson School of Engineering
University of Pittsburgh
ii
Copyright © by Meng-Chen Lee
2014
iii
James Peterson, PhD
THE HEALTH EFFECTS AND A BIOREMEDIATION PROCESS FOR VINYL
CHLORIDE
Meng-Chen Lee, MPH
University of Pittsburgh, 2014
ABSTRACT
Vinyl chloride is a colorless organic gas with high volatility. The carcinogen is the secondary
pollutant from chlorinated ethenes that have been extensively used in plastic industries and
facilities during manufacturing. Since 1970s numerous reports have appeared regarding a distinct
relationship between high level of exposure to vinyl chloride and angiosarcomas of the liver
(ASL), in which occupational exposure is of high concern for potential risk of public health.
Improved bioremediation for dense non-aqueous phase liquid (DNAPL) contamination has been
utilized to reduce vinyl chloride concentration in groundwater. To understand the factors
controlling VC degradation in the presence of both substrates (methane and ethene) and both
microbial groups (methanotrophs and etheneotrophs), cometabolic activities of methanotrophs,
etheneotrophs, and both microbial groups are discussed respectively in this essay.
iv
TABLE OF CONTENTS
1.1
DNAPL AND VINYL CHLORIDE APPLICATIONS .................................... 1
1.2
VINYL CHLORIDE ENVIRONMENTAL CONTAMINATION AND
REMEDIATION................................................................................................................... 2
1.2.1
Physical characteristics of vinyl chloride ...................................................... 2
1.2.2
Regulation of vinyl chloride ............................................................................ 4
2.1
VINYL CHLORIDE EXPOSURES ................................................................... 6
2.1.1
General population exposure .......................................................................... 6
2.1.2
Occupational exposure .................................................................................... 8
(Source: Kielhorn, Melber et al. 2000) ..................................................................... 10
2.2
ADVERSE HEALTH EFFECTS OF VINYL CHLORIDE .......................... 11
2.2.1
Effects of short-term exposure ..................................................................... 11
2.2.2
Liver cancer observed from long-term exposure to vinyl chloride ........... 12
2.2.3
Angiosarcomas of the liver observed from long-term exposure to vinyl
chloride ........................................................................................................................ 14
2.2.4
Hepatocellular carcinoma observed from long-term exposure to vinyl
chloride ........................................................................................................................ 16
2.2.5
Other malignant neoplasms observed from long-term exposure to vinyl
chloride ........................................................................................................................ 16
v
3.1
VC COMETABOLISM BY METHANOTROPHS ....................................... 18
3.2
COMPETITIVE INHIBITION AND PRODUCT TOXICITY OF CAHS TO
METHANOTROPHS......................................................................................................... 20
3.3
VC COMETABOLISM BY ETHENEOTROPHS ......................................... 21
3.4
VC COMETABOLISM IN THE PRESENCE OF BOTH MICROBIAL
GROUPS ............................................................................................................................. 22
3.4.1
Microbial behavior with VC and methane mixtures .................................. 25
3.4.2
Microbial behavior with VC and ethene mixtures ..................................... 25
3.4.3
Microbial behavior with methane and ethene mixtures without VC ....... 26
3.4.4
Microbial behavior with mixtures of methane, ethene, and VC ............... 26
BIBLIOGRAPHY ....................................................................................................................... 31
vi
LIST OF TABLES
Table 1. Occupational exposure: reported levels of vinyl chloride in workplace air samples in
VC/PVC production plants ........................................................................................................... 10
Table 2. Results of Poisson Regression Analysis of Liver Cancer, Angiosarcoma, and
Hepatocellular Carcinoma by Time Since First Employment, Duration of Employment, and
Cumulative Exposure .................................................................................................................... 13
vii
LIST OF FIGURES
Figure 1. Formation of vinyl chloride from higher chlorinated ethenes by microorganisms ......... 4
Figure 2. Log relative risk and 95% confidence interval of all liver cancer (N=71), by cumulative
exposure to vinyl chloride (ppm-years) ........................................................................................ 14
Figure 3. Log relative risk and 95% confidence interval of liver angiosarcoma (N=37), by
cumulative exposure to vinyl chloride (ppm-years) ..................................................................... 15
viii
PREFACE
I would like to thank my family for their selfless support of my education in the past nine years.
The path from studying Microbiology to Environmental Science, and to Environmental &
Occupational Health has been the journey to discover what my heart truly desires. It is their
love that has motivated me to keep on going when times were rough, it is the faith they have in
me so that I always know that I can do it.
I would like to thank the professors who have given me opportunities to learn. Without the
training you have given me, I would never know what I am capable of, and what I am lack of.
I am also grateful that I was never alone on this journey. I have always had great friends
surrounded me to keep me warmed and accompanied. I shall always cherish the time we spent
together and the laughter we shared with each other. I would like to thank each one of you who
helped me when I was down, encouraged me when I was low, and gave me a push when I was
weak. I know I could not make it to this far without all of my dear friends.
Finally, I thank god. Thank you for giving me your blessing so that I was never left alone. Thank
you for leading me to the path that enriches me. Thank you for always giving me what is the best
for me.
I know I should do a lot better and I promise you I will. I know I still have much to learn on this
path to pursue my goal, and with your love and faith I shall keep on going and never stop.
ix
1.0
1.1
INTRODUCTION
DNAPL AND VINYL CHLORIDE APPLICATIONS
Chlorinated ethenes are the most common source of dense non-aqueous phase liquid (DNAPL)
contamination in groundwater, including tetrachloroethene (perchloroethene or PCE),
trichloroethene (TCE), cis-1,2-dichloroethene (cis-DCE), trans-1,2-dichloroethene (trans-DCE),
1,1-dichloroethene (DCE), and vinyl chloride (VC). All of these compounds, with the exception
of VC, have densities greater than water and therefore can create a distinct sinking layer of the
compound.
Large quantities of chlorinated ethenes are still used in numerous industries and facilities
during manufacturing. The advantage of being non-flammable and highly evaporable had made
tetrachloroethene and trichloroethene popular dry cleaning solvents after the petroleum shortages
caused by World War II in the 1940s (Ballard and Baer 1989). Polymerization of VC to produce
polyvinyl chloride (PVC) homopolymer/copolymer resins was also developed early in the 20th
century, and its production capacity has almost doubled since the 1980s for a wide variety of
applications. At a global level, demand for PVC exceeds 35 million tonnes per year. PVC
manufacture represents 99% of the total vinyl chloride applications. The production of Western
Europe is estimated for 1996 to be 5,209 thousand tonnes with an annual growth of 2%
compared to 4% in North America and 9.5% in Asia (WHO, 1999).
1
PVC is one of the most commonly used thermoplastic materials in daily short-life products, such
as PVC packaging materials used in food, cleaning materials, textiles, beverage packaging
bottles, and medical devices. It is also extensively utilized in long-life products such as pipes,
window frames, cable insulation, floor coverings, roofing sheet, etc (Sadat-Shojai and
Bakhshandeh 2011).
1.2
VINYL CHLORIDE ENVIRONMENTAL CONTAMINATION AND
REMEDIATION
1.2.1 Physical characteristics of vinyl chloride
Vinyl chloride is a colorless organic gas with high volatility, this characteristic acts as a
significant transport mechanism in the environment. The high volatility and low solubility (1.1
g/litre) at 25 °C in water of VC has made the removal of VC from surface water to be considered
the most rapid route. Therefore, the concentrations measured in water and sediment generally do
not exceed 10g/L. However, volatilization of VC is unlikely to occur in groundwater, where
there’s no direct exposure to air. As a result, much higher levels of up to 56000g/L have been
found in groundwater samples from contaminated sites. The half-lives of VC measured in
models for disturbed or quiescent water range from <1h to 5h and from 2.5 to 43h for natural
water bodies. Conversely, VC may remain for months or years in groundwater, which poses a
threat to the health of animals.
2
When DNAPLs other than vinyl chloride are released into the subsurface, they migrate
downwards below the level of the groundwater table. Due to the slow dissolution rate of
DNAPLs, areas in the subsurface containing DNAPL serve as lasting sources of groundwater
contamination (Wymore and Akladiss 2009).
Under anaerobic conditions the reductive dechlorination biodegradation process carried
out by bacteria such as Dehalococcoides can converts PCE (perchloroethene ) to TCE
(trichloroethene) ,TCE to c-1,2-DCE (dichloroethene), c-1,2-DCE to VC, and VC to ethene
(Neumann, Scholz-Muramatsu et al. 1994, Maymó-Gatell, Chien et al. 1997, Smidt and De Vos
2004, Sung, Fletcher et al. 2006) (Figure. 1). However, when these dominant anaerobic
processes fail to go to completion, the more toxic secondary pollutant VC will accumulate in
groundwater zones, where significant amount of methane and ethene may also be generated from
methanogenesis and reductive dechlorination of chlorinated ethenes. In some situations, the
groundwater returns to aerobic conditions down gradient of the source with methane, ethene,
VC, and possibly cis-DCE still present. Natural attenuation of VC could occur via oxidation by
aerobic microorganisms as a VC plume intersects with groundwater containing molecular
oxygen (Freedman, Danko et al. 2001, Broholm, Ludvigsen et al. 2005).
3
Figure 1. Formation of vinyl chloride from higher chlorinated ethenes by microorganisms (Source:
(Kielhorn, Melber et al. 2000)
1.2.2 Regulation of vinyl chloride
The evidence showing vinyl chloride to be carcinogenic in both animal studies and in human
case reports first came out in the early 1970s. In 1974, Congress passed the Safe Drinking Water
Act and vinyl chloride is one of the Hazardous Air Pollutant (HAP) that is regulated by the
Environmental Protection Agency (EPA) in order to ensure protection of public health and the
environment. The environmental statutes that are associated with VC include the Clean Air Act
4
(CAA), the Safe Drinking Water Act, the Resource Conservation and Recovery Act. The
regulation of vinyl chloride became effective in 1989. Under Section 112 of the Clean Air Act,
The EPA requires that VC emissions from VC/PVC production facilities not exceed an average
of 10 ppm over a 3-hour period. Under the Safe Drinking Water Act, the EPA has set an
enforceable regulation for VC, called a maximum contaminant level (MCL), at 0.002 mg/L or 2
ppb. Furthermore, EPA determined that the zero maximum contaminant level goals (MCLG) and
0.002 mg/L or 2 ppb MCL for vinyl chloride are still considered adequately protective of human
health in the review of VC as part of the Six Year Review (OECD SIDS 2001).
Overall, vinyl chloride contamination of groundwater, on which 50% of Americans
developed for their potable supply, may cause prolonged human and environmental health
problems since it is very difficult to locate and treat DNAPL source areas (Barbash and Roberts
1986). Chlorinated solvent remediating technologies have shifted over the last 30 years.
Currently, aggressive source zone in situ bioremediation (ISB) of chlorinated ethenes has proven
to be successful for degrading chlorinated ethenes to the levels that are ideal for natural
attenuation to occur. This paper summarizes the epidemiologic studies on adverse health effects
associated with vinyl chloride exposure, as well as the limitation and progress of technologies in
DNALP source zone remediation (West and Kueper 2012).
5
2.0
REVIEW ARTICLES
In 1999, Toxic Release Inventory (TRI) data reported 848,576 lb of vinyl chloride was emitted to
the air, 106 lb to water and 405 lb to land or underground injection in the US. The partitioning
tendency of VC in different environmental compartments based upon the Mackay Level 1 model
is as followed: 99.99% air, 0.01% water, <0.01% soil and ,<0.01% sediment. However, high
levels of vinyl chloride contamination was reported to present in soil, groundwater, aquifers, and
wells near landfill and industrial waste disposal sites from several countries (Euro Chlor 1999). It
is crucial to understand the potential exposure routes to vinyl chloride and the adverse health
effects results from contacting the carcinogen.
2.1
VINYL CHLORIDE EXPOSURES
2.1.1 General population exposure
Inhalation of air polluted with VC, mainly in the vicinity of VC/PVC plants or waste disposal
sites, is of greater concern than atmospheric concentration of vinyl chloride in ambient air. As
the ambient concentration of vinyl chloride is usually less than 3 µg/m3, much higher
concentration- up to 8000 µg/m3 and 100 µg/m3 of vinyl chloride had been observed near
6
industrial plants and waste disposal sites, respectively in the past. Indoor air concentration in the
residential house near to landfills could reach a maximal concentration of 1000 µg/m3. However,
such exposure is likely to be transient, so there is very little exposure of the general population to
vinyl chloride in the air (IPCS, 1999).
The contaminating concentrations of vinyl chloride in surface waters vary from 10 µg/L
to 200,000 µg/L due to its high volatility. One study reported high concentrations, up to 200 000
µg/litre of vinyl chloride were detected in well water in the vicinity of a PVC plant in Finland 10
years after leakage (Salkinoja-Salonen, Uotila et al. 1995). Areas contaminated with chlorinated
hydrocarbons contained approximately 60 000 µg/litre of vinyl chloride in the leachate (Brown
and Donnelly 1988) and 56 000 µg/litre in a contaminated sand aquifer (Semprini, Kitanidis et
al. 1995) . In 2000, a vinyl chloride concentration of 27 000 µg/litre was detected in site
groundwater above a drinking-water aquifer (Peterson et al., 2000).
Unplasticized polyvinyl chloride (uPVC) tubing and bottles are possible sources of vinyl
chloride in drinking-water. Malck (1999) studied the migration of vinyl chloride from uPVC
pipies into water under different parameters. The study showed that after 30 days of exposure
exposure of double distilled water at 45 oC, the uPVC pipes released more than 2.5 ppb of VC
into the contact water, which is just above the permissible level in drinking water set by the U.S.
Environmental Protection Agency, 2.0 ppb (Al-Malack, Sheikheldin et al. 2000). Other studies
into PVC-bottled drinking-water have identified vinyl chloride at levels of 0.06–0.18 µg/litre
(Benfenati, Natangelo et al. 1991) and <0.6 µg/litre (Fayad, Sheikheldin et al. 1997). In 1975,
vinyl chloride has been identified in peanut oil samples packaged in PVC bottles at levels
ranging from 0.3 to 3.3 ppm (Williams and Miles 1975). Overall, the frequency of occurrence of
7
vinyl chloride is expected to be higher in such water than in tap water since bottled-water is
always in contact with new PVC packaging material.
2.1.2 Occupational exposure
Before vinyl chloride was recognized as a human carcinogen, no regular workplace monitoring
and precautions were taken against contact with the toxic gas while the autoclaves or reactor
vessel cleaners emptied the PVC reactors manually with spatulas and chisels. In the 1950s and
earlier, the VC workers were estimated to be exposed to as much as 1000 ppm (2600 mg/m3). By
the mid-1970s, the exposure level was reduced to one-tenth of this, the levels were generally
lower than 1-5 ppm (2.6-13 mg/m3) after 1975 in many countries (Kielhorn, Melber et al. 2000).
In the United States, the number of workers exposed to vinyl chloride has been estimated
to be approximately 80,000 during 1981-1983 and more than 5000 in Sweden during 1975-1980.
Factories in many countries that failed to reduce the emission of vinyl chloride below the
regulated level in the early 1970s were forced to close. However, in other countries where the
VC polymerization plants were not forced to close for socioeconomic reasons, the workers
continued to be exposed to high levels of vinyl chloride emitted from the old-fashioned
technologies (Hozo, Andelinović et al. 1996).
Table 1 summarizes the occupational exposure of vinyl chloride in VC/PVC production
plants in the following countries: China, Croatia, Egypt, Finland, Italy, Poland, Russia, and
Taiwan. Thirty years (1949-1987) of the reported levels of vinyl chloride in VC/PVC plants
from Croatia revealed that employees were exposed to an average vinyl chloride concentration
of 543 mg/m3 with an occasional peak concentration of 1300 mg/m3. Vinyl chloride exposure
8
levels reported from Italy from 1950 to 1985 showed a wide range of concentrations from 131300 mg/m3. The data collected after 1980 from countries like Finland, Poland, and Russia
showed significant reduced vinyl chloride exposure levels, where the mean concentrations
ranged from 0.2 to 400 mg/m3. The VC/PVC production plants from Asian countries such as
China and Taiwan also showed high levels of vinyl chloride exposure levels ranging from 0.13 to
1009 mg/m3 within the specified investigation years (Kielhorn, Melber et al. 2000).
9
Table 1. Occupational exposure: reported levels of vinyl chloride in workplace air samples in VC/PVC
production plants
(Source: Kielhorn, Melber et al. 2000)
10
2.2
ADVERSE HEALTH EFFECTS OF VINYL CHLORIDE
After vinyl chloride was extensively produced in the late 1930, various effects caused by
exposure to vinyl chloride were noticed. The reported symptoms include hepatic abnormalities in
vinyl chloride workers, acroosteolysis, Raynaud-type-phenomenon and sclerodermoid skin
lesions (Lelbach & Marsteller, 1981). Starting from 1974, numerous reports have appeared
regarding a distinct relationship between high level of exposure to vinyl chloride (as much as
2600 mg/m3, 1000 ppm) and angiosarcomas of the liver (ASL), a type of rare tumor that
constitutes only 2% of all primary tumors of the liver in the general population. (Makk, Creech et
al. 1974, Tamburro 1984). Furthermore, studies have shown that the average latent period
between starting work in an occupation involving VC exposure and ASL diagnosis/death for 99
cases was 22 years (Purchase, Stafford et al. 1987).
Several further cases series and small epidemiologic studies also indicated that vinyl
chloride may also be connected with other (non-ASL) cancer sites and types, including
hepatocellular carcinoma, respiratory system, digestive system other than the liver,
lymphopoietic and hematopoietic tissue, brain and other central nervous system and malignant
melanoma (Kielhorn, Melber et al. 2000).
2.2.1 Effects of short-term exposure
For the workers who exposed to high level concentration of vinyl chloride (about 1000 pm) for
more than 1 month to several years prior to 1974, several specific pathological syndromes were
reported and defined as “vinyl chloride illness”.
Symptoms described were earache and
headache, dizziness, unclear vision, fatigue and lack of appetite, nausea, sleeplessness,
11
breathlessness, stomachache, pain in the liver/spleen area, pain and tingling sensation in the
arms/legs, cold sensation at the extremities, loss of libido and weight loss (Thiess & Versn,
1974). Clinical findings includes scleroderma-like changes in the fingers with subsequent bony
changes in the tips of the fingers described as acroosteolysis, peripheral circulatory changes
similar to Raynaud’s diseases, and enlargement of the liver and spleen with a specific
histological appearance, and repiratory manifestations (Lange et al., 1974, Veltmann et al.,
1975).
2.2.2 Liver cancer observed from long-term exposure to vinyl chloride
Two large cohort studies of male factory workers who had been exposed to vinyl chloride for
more than a year from the United States and Europe were conducted to determine the adverse
health effects associated with long-term vinyl chloride exposure. Wong et al. conducted a cohort
study (covered 1942-1982) included 10,173 male who had been exposed with vinyl chlorides in
37 plants for at least 1 year in United Sates before January 1973. On the other hand, the update
study (Ward et al. 2001) on a major European cohort study of mortality and cancer incidence
among VC/PVC workers (Simonato et al. 1991) was published in 2001. The observation period
was different for different factories, starting for most in1955, extended until 1986, then updated
from 1993 through 1997. The multicentric study comprised a total of 12,700 male workers with
at least 1 year of employment in 13 factories in three countries (Norway, Sweden, and the United
Kingdom). These two studies provide the most informative data on the health effects associated
with exposure to VC.
The increase in standardized morality ratio (SMR) for liver cancer reported from the two
European cohort studies showed similar results: 2.86 (95% CI= 1.86-4.25) in the original
12
analysis by Simonato (1991) and 2.40 (95% CI= 1.80-3.14) in the updated investigation by Ward
in 2001. The data from the international programme on chemical safety (IPCS) task group
indicated a 5-fold excess risk for liver cancer observed among workers exposed to vinyl chloride
(WHO, 1999).
A strong association between liver cancer and time since first exposure to vinyl chloride,
duration of employment, and estimated ranked and quantitative exposures was confirmed in both
original and follow-up investigation (Simonato, Labbe et al. 1991). Poisson regression analysis
of liver cancer performed by Ward et al. showed strong positive trends for “time since first
employment”, duration of employment, and cumulative exposure (Table2).
Table 2. Results of Poisson Regression Analysis of Liver Cancer, Angiosarcoma, and
Hepatocellular Carcinoma by Time Since First Employment, Duration of Employment, and
Cumulative Exposure (Ward, Boffetta et al. 2001).
Table 2. Results of Poisson Regression Analysis of Liver Cancer, Angiosarcoma, and Hepatocellular
Carcinoma by Time Since First Employment, Duration of Employment, and Cumulative Exposure
(Source: Ward, Beffetta et al. 2001)
13
In this study, the cumulative dose was subdivided into 13 categories, and found a fairly
monotonic increase in rate ratios, with a rate ratio (RR) of 82 (95% CI= 21-323) in the highestdose category of > 18,300 ppm-years. The (natural) logarithmic transformation of cumulative
exposure provided the best fit when the midpoints of the dose categories was used to model dose
as a continuous variable. The RR for one logarithmic unit of cumulative exposure was 2.0 (95%
CI= 1.7-2.4). The estimated categorical rate ratios for cumulative exposure and liver cancer
along with the continuous curve and CIs were shown in Figure 2.
Figure 2. Log relative risk and 95% confidence interval of all liver cancer (N=71), by cumulative
exposure to vinyl chloride (ppm-years) (Source: Ward, Boffetta et al. 2001)
2.2.3 Angiosarcomas of the liver observed from long-term exposure to vinyl chloride
In the USA study, a total of 15 angiosarcomas were recorded on the death certificate. Although
no expected numbers were calculated, this must be a very marked excess, since the annual
incidence of liver angiosarcoma is approximately 2 per 10 million, which would in this cohort
lead to an expected number of approximately 0.05. The authors noted that when these 15
angiosarcomas were excluded, there remained 22 other liver and biliary tract tumours; this
14
represents an excess (5.7 expected, SMR 3.86, p<0.02). In addition, this result is likely to be an
overestimate, since some of these other liver cancers were likely to be angiosarcomas(Wong,
Whorton et al. 1991).
Simonato et al. reported a 45-fold excess risk for ASL in workers exposed to VC >
10,000 ppm-years, and the updated study (Ward et al., 2001) estimated an approximately 200
times greater risk of ASL than the general population (Table 2).
Exposure response was estimated for 37 angiosarcomas in a model including 12 dose
categories in the updated European study. Compared to the exposure-response relation for liver
cancer, a sharper relation is observed for ASL, where the RR for a logarithmic unit of cumulative
exposure was 2.9 (95% CI= 2.2-3.9). Figure 3 shows the estimated categorical rate ratios for
cumulative exposure and liver angiosarcomas along with the continuous curve and CIs(Ward,
Boffetta et al. 2001).
Figure 3. Log relative risk and 95% confidence interval of liver angiosarcoma (N=37), by cumulative
exposure to vinyl chloride (ppm-years) (Source: Ward, Beoffetta et al. 2001)
15
2.2.4 Hepatocellular carcinoma observed from long-term exposure to vinyl chloride
A noticeable exposure-response trend with both duration of employment and cumulative vinyl
chloride exposure was present for the ten known cases of hepatocellular carcinoma, suggesting
an association between vinyl chloride and this tumor. Two updates of parts of the Italian cohort
(Pirastu, Comba et al. 1990) have shown that an elevated mortality of liver cancer (11 observed,
5.7 expected cases, SMR 1.93) was observed in Marghera. In an extended survey for hepatic
cancers, four further cases were uncovered. There were 5 angiosarcomas, 5 hepatocellular
carcinomas, 3 cirrhotic carcinomas, and in two cases the histology was not known (Ietri, Belli et
al. 1997). The studies have shown that the risk for hepatocellular carcinoma is not as great as the
risk for ASL.
2.2.5 Other malignant neoplasms observed from long-term exposure to vinyl chloride
Statistically significant increase of brain and CMS tumours after occupational exposure to vinyl
chloride were reported in USA surveys by Waxwiler et al. (1997), as well as in Sweden (Byron
et al., 1976) and Germany (Greiser et al., 1982).
In the cohort study conducted by Wong et al. (1991) in USA showed that the relative risk
was elevated for cancers of the brain and other CNS cancers (SMR 1.80, CI 1.14–2.70).
Mortality was highest in the group with the longest duration of exposure (SMR 1.64, 1.20 and
3.85, for workers with the duration of exposure < 10 years, 10–20 years and > 20 years,
respectively), while no trend with latency was observed (SMR 1.83, 1.58 and 2.10, for latencies
of < 20, 20–30 and > 30 years).
16
An update of the study by Wong et al. was completed in 1999, in which specific cancers
reported to be associated with vinyl chloride exposure both through 1982 and through 1985 had
shown no excess values, including lung cancer, cancers of lymphatic and hematopoieic tissue,
emphysema and pneumoconiosis. (Kielhorn, Melber et al. 2000) (Mundt, Dell et al. 2000). The
same conclusion was confirmed by the European study. The statistical analysis suggested no
clear pattern with duration of employment, ranked level of exposure, or cumulative exposure to
vinyl chloride; also, vinyl chloride exposure was not strongly related to other cause of death, or
incident cancer, including brain cancer, lung cancer,
soft-tissue sarcoma, non-Hodgkin’s
lymphoma and malignant melanoma (Ward, Boffetta et al. 2001).
17
3.0
COMETABOLIC BIODEGRADATION OF VINYL CHLORIDE IN THE
PRESENCE OF METHANE AND ETHENE
Cometabolic biodegradation processes have been extensively studied in the past thirty years
because of their potential usefulness in biotransforming many of the chlorinated aliphatic
hydrocarbons (CAHs) found in naturalsystems. However, cometabolic activity is also an
unavoidable side reaction that may be harmful to the microorganisms. To understand the factors
controlling VC degradation in the presence of both substrates (methane and ethene) and both
microbial groups (methanotrophs and etheneotrophs), cometabolic activities of methanotrophs,
etheneotrophs, and both microbial groups are discussed respectively. In addition, competitive
inhibition between the primary (carbon and energy source) and secondary substrates (pollutants),
and the toxic effects from the intermediate compounds on methanotophs and etheneotophs are
reviewed in this chapter.
3.1
VC COMETABOLISM BY METHANOTROPHS
Several reports showed that chlorinated ethenes are not degraded in the absence of methane in
groundwater (Wilson et al., 1983). In the presence of oxygen and methane, methanotrophs can
cometabolize chlorinated aliphatic compounds very rapidly, on the order of days.
18
In small-inoculum experiments, a particulate methane monooxygenase (pMMO)
expressing methanotrophic mixed culture was added to the sample bottles, in which 34% of
methane and 94 to 99% of the chlorinated aliphatic hydrocarbons (CAHs) (t-DCE, c-DCE, and
VC) were in solution. The transformation yield ((Ty), defined as the mass of CAH degraded per
unit mass of growth substrate consumed, is useful in characterizing the finite value of CAH
cometabolism that occurs in a culture system). The highest observed Ty expressed as moles of
CAH per mole of methane for VC was 0.20 mol of VC/ mol of methane when the equilibrium
aqueous concentration of VC and methane were 17 µM and 30 µM respectively. However,
MMO failed to oxidize both VC and methane when the VC concentrations were high at 86 µM
and 340 µM. This can be due to competitive inhibition, the availability of reducing power and/or
the toxic effect of the transformation product (Anderson et al., 1997).
Large-inoculum experiments were done under different growth condition in order to
confirm the high yields for VC found in the small-inoculation experiments. The maximum Ty
values for VC was 0.25 mol of VC/mol of methane, which is very similar to the result obtained
from small-inoculum experiments. However, when comparing the two experiments, maximum
Ty
value of small-inoculums was found in the bottle containing the lowest methane
concentrations (30 µM) and the lowest VC concentration (17 µM), while with the large
inoculums, much higher methane concentration (180 µM) together with highest VC
concentration (260 µM) gave the maximum transformation yield. The results suggested that
factors such as reducing-energy availability, competitive inhibition, and transformation toxicity
need to be corrected to reach its balance, so that the maximum values of Ty could be obtained
(Anderson et al., 1997).
19
Fogel et al. utilized a mixed culture containing methane-oxidizing bacteria (CL-M)
isolated from lake sediment to demonstrate its ability to degrade TCE and other chlorinated
ethenes to CO2. It is observed that acetylene inhibits 100 ng/ml of TCE degradation completely,
suggesting that it is methanotrophs that are responsible for TCE oxidation among the methaneutilizing culture since acetylene is a specific inhibitor of methane monooxygenase. The fact that
12mM methanol can cause 50% inhibition, indicating that methanotrophs are very sensitive to
inhibition by methanol. This mixed culture of methanotrophs is observed to degrade VC from
600 ng/ml to less than 0.2 ng/ml in 1 day. Obligate methanotrophs are typically the major
component in the mixed culture, although one or more heterotrophic organisms could be present
as well.
Therefore, methane-oxidizing bacteria are benefited by the presence of these
heterotrophic organisms since they can consume the potential inhibiting organic compounds
produced by the methanotrophs (Fogel et al., 1986). The authors conclude that mixed cultures
are more stable than pure cultures in terms of environmental remediation efforts.
3.2
COMPETITIVE INHIBITION AND PRODUCT TOXICITY OF CAHS TO
METHANOTROPHS
Transformation capacity (Tc) is a defined as the amount of cometabolic compound degraded
(dSc,µmol/L) divided by the amount of cells that are inactivated during this process(dx, mg/L).
Chang et al. (1996) conducted a study in which four methane-oxidizing cultures (a mixed
methanotrophs culture grown under both chemostat and batch conditions, a pure culture of
CAC1, and a pure culture of M. trichosporium OB3B grown under batch conditions) and 18
individual chlorinated and nonchlorinated aliphatics compounds were utilized to test the
20
hypothesis that “the product toxicity of chlorinated aliphatic hydrocarbons to methane-oxidizing
cells decreases in proportion to their chlorine content” (Chang et al. 1996).
In an effort to accurately reflect the product toxicity associated with cometabolic
degradation reactions, formate (20 mM) was amended into the cultures to secure the availability
of reducing energy without exerting competitive inhibition effects that would occur if methane is
added during the reaction. Similar results observed with the four different cultures supported the
hypothesis, indicating that within similar carbon structure groups (methane, ethanes, and
ethenes), the highest Tc values were found in nonchlorinated compounds. However, 1,1-DCE
appeared to have lower Tc values /higher toxicity than TCE, cDCE and tDCE. In addition, the
results of degradation kinetics for TCE, cDCE, and VC showed no direct relationship between
the degradation rate and chlorine content (Chang et al. 1996).
3.3
VC COMETABOLISM BY ETHENEOTROPHS
Numerous organisms are known to grow on ethene according to previous research, including
several strains of Mycobacterium (Hartmans et al, 1985, Hartmans et al. 1992). To date,
Mycobacterium, Nocardioides, and Pseudomonas strains are reported to grow on VC and
assimilate ethene with the same enzymes (Jin and Mattes, et al. 2008). In one study, an ethene
enrichment culture was used to evaluate aerobic biodegradation of VC in the presence of ethene,
ethane and VC. VC removal was most extensive in the bottles containing both ethene +VC,
followed by ethane+ethene+VC, and only VC. Ethene biodegradation was fastest when it was
the sole substrate, and the highest rate of ethane oxidation occurred in bottles that contained both
ethene and ethane. The results indicated that ethene is essential for ethene-utilizing organisms to
21
induce the alkene monooxygenase in order to cometabolize VC and catabolize ethane (Jin and
Mattes, et al. 2008). This work also confirmed that ethane culture was able to degrade both
ethene and VC since alkane monooxygenase has a broader substrate range than AkMO. VC
biodegradation by the ethene and ethane grown cultures were reported to be roughly equivalent.
Interestingly, ethene was still consumed first by ethane-grown culture fed with ethane, ethene,
and VC. This result is consistent with the fact that alkane monooxygenases have broader
substrate range than AkMO reported in the literature (Jin and Mattes, et al. 2008).
The effect of varying ratios of VC to ethene on the rate of removal of each compound
was investigation as follows: (1) VC only; (2) ethene only; (3) VC: ethene = 1:3; (4) VC: ethene
= 1:1; (5) VC : ethene = 3:1. Initially, VC removal rates were uniformly high, and started to
diverge as the experiment progressed. By day 64, the bottles that received equimolar amounts of
VC and ethene displayed the highest VC removal rate. In contrast,the rate of VC degradation in
the bottles that contained unequal amount of VC and ethene are significantly reduced;
approximately 90% reduction occurred in the bottle that contained only VC. The results
suggested that at ethene to VC levels greater than a 1:1 molar ratio, VC degradation was
competitively inhibited by primary substrate that have higher affinity to AkMO (Jin and Mattes,
et al. 2008).
3.4
VC COMETABOLISM IN THE PRESENCE OF BOTH MICROBIAL GROUPS
The first microcosm studies comparing methane and ethene as primary substrates for
cometabolism of VC by native methanotrophs and etheneotrophs were done by Freedman et al in
2001 (Freedman et al., 2001). The sediment and groundwater were taken from monitoring well
22
#3037 at a Superfund site in California. The initial concentration of methane, ethene, and VC
were equivalent to what was measured in the anaerobic portion of the groundwater that was
closest to the interface with the aerobic zone, where ethane and methane were an order of
magnitude lower than that of the VC: 200µg/l methane, 112µg/l ethene, and 2600 µg/l VC.
The whole period of microcosm operation lasted 150 days, during the first 76 days of
microcosm operation a noticeably slower rate of VC consumption occurred when just CH4 was
present. Beyond day 75, VC biodegradation was fastest with just ethene present in the bottle. The
results indicated that ethene may be a more effective primary substrate for alkene
monooxygenase(s). The results of cumulative amounts of VC, ethene and methane consumed in
the microcosm bottles indicated the presence of methane alone with ethene inhibited ethene
biodegradation, thereby inhibiting VC consumption. The fact that VC alone did not inhibit
ethene consumption, but did inhibit methane uptake suggests that methanotrophs are more
susceptible to the presence of VC than are etheneotrophs. The results from these microcosms
confirm that ethene and methane promoted cometabolism of VC even when VC aqueous phase
concentration was an order of magnitude higher than that of the ethene and methane (Freedman
et al., 2001).
A series of microcosm studies evaluating ethene’s role in VC cometabolism were done
by Begley in 2011. The purpose of this study was to establish the feasibility of a full-scale
aerobic biostimulation treatment system at a demolition debris landfill in Carver, Massachusetts.
By 2002, the site was assessed to have a detached VC plume 3000’ long, 40’ wide and 30’ thick,
located 50’ below the water table with persistent low levels of VC (2 to 27 μg/L) extending
4600’ downgradient from the landfill.
23
A mixed site groundwater sample was used to generate a series of microcosms to test for
aerobic VC degrading activity. The groundwater contained 50 µM of methane and was amended
with 1 ml of 100% O2, MSM, 13µM ethene and 0.5 µM VC. VC degradation was complete in 21
days when groundwater was amended with minerals and 13µM ethene, compared to 9% VC
degradation from unamended groundwater, and 25% VC degradation from the groundwater that
was amended with mineral nitrogen, phosphorus, and trace elements after 21 days. In this
ethene-supplemented microcosm study, VC degradation lagged in the first 12 days, then the
concentration of VC started to decrease to nondetectable levels by day 21 after ethene
consumption. The result indicated that VC- degrading activity could be simulated by the addition
of ethene, and VC is consumed slower than ethene in this groundwater sample. However, rapid
degradation of methane was observed in both unamended and amended groundwater samples,
but the cometabolism of VC was limited. The results suggested that the addition of oxygen did
not stimulate the native methanotrophic bacteria to degrade VC in the site groundwater (Begley
et al., 2012).
Another microcosm experiment was done with the groundwater from well 63-I at the
Carver site in MA. The concentrations of methane, ethene and VC contained in the groundwater
are 195, 0.35 and <2 µg/L respectively. The relative abundance of etheneotroph and
methanotroph were estimated by qPCR analysis of functional genes, showing that methanotophs
and etheneotrophs are both present in the samples, and the methanotrophs are more than
etheneotrophs by 2-3 orders of magnitude. The screening also showed that methanotrophs,
etheneotrophs and VC-assimilators were all present in the groundwater sample (Findlay et al.)
Relevant observations from this study are described below.
24
3.4.1
Microbial behavior with VC and methane mixtures
The lag time for VC utilization decreased from 14 days to 10 days, shorter than that of VC as
sole substrate (13days). This suggested that cometabolic oxidation of VC (0.47 µM) was carried
out by methanotrophs rather than by VC-assimilators in the presence of 1µM methane (Findlay
et al.).
The lag time for initial methane utilization and the time for 50% methane degradation
both increased when 0.47µM VC and 0.45µM of methane were in the aqueous phase of the
sample, suggesting that methane utilization by methanotrophs was inhibited by similar aqueous
concentration of VC. This phenomenon was overcome if increasing amount of methane (0.45
µM, 1.28 µM, 3.6 µM) was fed to the microcosms. However, VC degradation is enhanced in the
presence of higher methane concentration, which supports the idea that VC is primarily oxidized
cometabolically (Findlay et al.).
3.4.2
Microbial behavior with VC and ethene mixtures
The initiation of VC consumption was about 16 days for microcosm experiments with VC (0.47
µM) alone and with both VC and ethene (3.0 µM). However, etheneotrophs started to oxidize
intensively after day 32, resulting in 77% of VC degradation by day 43, which was the time
required for VC-assimilator to oxidize just 50% of VC. Increasing concentration of ethene (1.3
µM, 3.8 µM,10.7 µM) did not affect the lag time for VC use significantly, but did increase the
time for 50% VC degradation. These results suggest that there is competition between VC and
ethene for the alkene monooxygenase binding site. However, the presence of ethene enhanced
25
VC cometabolic oxidation because in each test VC was degraded faster in the presence of ethene
than when alone (Findlay et al.).
3.4.3
Microbial behavior with methane and ethene mixtures without VC
The time required for methanotophs in the groundwater sample to degrade 50% of 1 µM
methane was 20 days, after lagging for about 9 days. When 3 µM of ethene was fed into the
bottle contained 1 µM of methane, both lagging period and times for 50% degradation increased
to 12 days and 26 days respectively. On the contrary, the times for 50% of 3 µM of ethene
decreased significantly from 39 days to 20 days when 1 µM of methane was fed into the
groundwater samples. The results are expected because methanotrophs can cometabloize ethene
using MMO, which impacts its ability to oxidize methane since methane and ethene are
competing for the active site. Another reason for faster ethene loss could be due to the inducing
of AkMO gene expression by epoxyethane generated by methanotrophs (Findlay et al.).
3.4.4 Microbial behavior with mixtures of methane, ethene, and VC
In 2010 experiments, VC degradation took only 19 days to reach 50% degradation when tested
with 1.3 µM methane and 3.8 µM ethene, it is faster than with only 1.3 µM methane (32 days) or
with only 3.8 µM ethene (23 days). It took 44 days to obtain 50% VC degradation with VC
alone. VC and ethene oxidation are shown to be a synchronized event, whilst great variability
were shown in terms of times to reach 50% methane degradation. For example, it took more than
70 days for methnotrophs to oxidize 50% of methane in the microcosm containing only 0.5 µM
26
methane and 3.8 µM ethene, which indicated that low concentration made methantophs
susceptible to VC inhibition (Findlay et al.).
Similar time frames for VC degradation were shown in 2011 experiments, where VC
degradation took only 20 days to reach 50% degradation when tested with 1 µM methane and 3
µM ethene, it is faster than with only 1.3 µM methane (31 days) or with only 3.8 µM ethene (38
days). It also confirmed that VC degradation did occur simultaneously with ethene use (Findlay
et al.).
From these microcosm results, groundwater scenarios were created with relatively low
concentrations of methane, ethene and VC. It is shown that methane utilization by
methanotrophs was inhibited when similar or low concentrations of VC and methane were
contained in the groundwater samples. In addition, the presence of ethene also impacted the
methane utilization by MMO. Nevertheless, ethene degradation is enhanced by the presence of
methane. VC degradation is observed to be proceeded via cometabolism by methanotrophs and
ethenenotrophs, and it has the highest potential to be fully degraded when methane and ethene
are both present (Findlay et al.). This work concluded that VC cometabolic activity in
groundwater system is shaped by the relative abundance of native methanotrophs and
etheneotrophs as well as the aqueous concentrations of methane, ethene, and VC at the specific
site.
27
4.0
CONCLUSION
U.S. EPA classified vinyl chloride as a known human carcinogen by inhalation exposure (EPA
2000). The carcinogen is one of the 188 hazardous air pollutants (HAPs) listed under Section 112(b)
of the 1990 Clean Air Act Amendments, and its emissions are regulated from more than 170
industrial air pollutant source categories. The primary target organ for vinyl chloride exposure is
the liver. The association between angiosarcoma of the liver and vinyl chloride exposure is well
documented for occupational exposures. Noncancer liver pathologies have also been associated
with vinyl chloride exposure, including liver necrosis and cysts.
General population are at risk for exposure to vinyl chloride from ambient air contaminated
with vinyl chloride by emissions released from polyvinyl chloride plastics production and
manufacturing facilities, as well as some incinerators. Contamination of groundwater and drinking
water with vinyl chloride-contaminated run-off from such manufacturing facilities is also a concern
for public health. Reports of effects of chronic exposure to vinyl chloride focus primarily on
occupational adult exposures and experimental animal studies. Evidences of chronic vinyl chloride
exposure causing angiosarcoma of the liver in occupationally-exposed adults have been shown from
cohort studies worldwide. In addition, other cancers of the liver, as well as lung and brain cancer,
have been reported in occupational exposure studies and experimental animal studies (Kielhorn,
Melber et al. 2000, Ward, Boffetta et al. 2001).
28
In Situ bioremediation is a destruction technology that takes advantage of
microorganisms natural metabolism to break down contaminants to less toxic end products.
Under anaerobic condition higher chlorinated ethenes like PCE and TCE will be converted to
less chlorinated ethenes by microorganisms through reductive dechlorination process. When the
groundwater returned to aerobic condition downgradient of the source with methane, ethene, VC,
cis-DCE and several other compounds still present, natural attenuation of VC could occur more
readily via oxidation by aerobic microorganisms as a VC plume intersects with groundwater
containing molecular oxygen (Mattes, Alexander et al. 2010). As the results, a complete
degradation of chlorinated ethenes shall be achieved if a two-stage treatment system is proposed,
which consisting anaerobic as first stage, and aerobic as the second.
Biostimulation is the process of stimulating active bioremediation through the addition of
substrates that encourage microbial growth. Aerobic bioremediation of chlorinated ethenes can
be enhanced by injecting methane or ethene and oxygen into a dilute VC plume to stimulate
growth of the ethenotrophs/methanotrophs and subsequent cometabolic oxidation of VC
(Semprini 1997, Eguchi, Kitagawa et al. 2001, Wendlandt, Stottmeister et al. 2010). However,
one of the disadvantage of growth-linked bioremediation process is that the target bacteria may
not be populous enough for biodegradation to proceed at an acceptable rate, and thus
bioaugmentation would be needed at most sites. In addition to suitable condition s for bacteria
growth (i.e., redox conditions, pH range, supply of electron donors, and presence of nutrients), it
is equally critical to measure the presence or absence of methanotrophs and etheneotrophs.
Even more promising than cometabolism, future research can be focusing on
investigating the role of VC-assimilators, the bacteria are widespread and readily isolated from
contaminated and uncontaminated environments, for example Nocardioides sp. strain JS614
29
(ATCC BAA-499) that can directly degrade VC and ethene as a growth substrate (Coleman,
Mattes et al. 2002, Mattes, Coleman et al. 2005).
30
BIBLIOGRAPHY
Anderson, J.E. et al. (1997). “Transformation yields of chlorinated ethenes by a methanotrophic
mixed culture expressing particulate methane monooxygenase.” Appl. Environ.
Microbiol 63: 687-693.
Al-Malack, M. H., et al. (2000). "Effect of water quality parameters on the migration of vinyl
chloride monomer from unplasticized PVC pipes." Water Air and Soil Pollution 120(12): 195-208.
Ballard, M. and N. S. Baer (1989). "Conservation I: Halogenated hydrocarbon dry-cleaning
solvents." International Journal of Museum Management and Curatorship 8(3): 336-341.
Barbash, J. and P. V. Roberts (1986). "Volatile organic-chemical contamination of groundwater
resources in the united-states." Journal Water Pollution Control Federation 58(5): 343348.
Benfenati, E., et al. (1991). "Migration of vinyl-chloride into pvc-bottled drinking-water assessed
by gas-chromatography mass-spectrometry." Food and Chemical Toxicology 29(2): 131134.
Begley, J.F. et al. (2012). "Oxygen and ethene biostimulation for a persistent dilute vinyl
chloride plume." Groud Water Monit. Remediat. 32: 99-105.
Broholm, K., et al. (2005). "Aerobic biodegradation of vinyl chloride and cis-1,2dichloroethylene in aquifer sediments." Chemosphere 60(11): 1555-1564.
Brown, K. W. and K. C. Donnelly (1988). "An estimation of the risk associated with the organicconstituents of hazardous and municipal waste landfill leachates." Hazardous Waste &
Hazardous Materials 5(1): 1-30.
Byron D. et al. (1976). "Mortality and cancer morbidity in a group of Swedish VCM and PVC
production wrokers." Environ Health Perspect 17: 167-170
Chang, H.L. et al. (1996). "Biodegradation of individual and multiple chlorinated aliphatic
hydrocarbons by methane-oxidazing cultures." Appl. Environ. Microbiol 62: 3371-3377.
31
Coleman, N. V., et al. (2002). "Phylogenetic and kinetic diversity of aerobic vinyl chlorideassimilating bacteria from contaminated sites." Applied and Environmental Microbiology
68(12): 6162-6171.
Eguchi, M., et al. (2001). "A field evaluation of in situ biodegradation of trichloroethylene
through methane injection." Water Research 35(9): 2145-2152.
Environmental Protection Agency (EPA) (2000). "Integrated risk information system:Vinyl
chloride"
U.S.
Environmental
Protection
Agency.
http://www.epa.gov/iris/subst/1001.htm
Euro Chlor (1999). Euro Chlor risk assessment for the marine environment, OSPARCOM
Region- North Sea: Vinylchloride. Brussels, Euro Chlor pp 1-25.
Fayad, N. M., et al. (1997). "Migration of vinyl chloride monomer (VCM) and additives into
PVC bottled drinking water." Journal of Environmental Science and Health Part aEnvironmental Science and Engineering & Toxic and Hazardous Substance Control
32(4): 1065-1083.
Findlay, M. et al."Aerobic vinyl chloride metabolism in groudwater microcosms by a
methanotrophic and etheneotrophic consortium." Bioremediation Consulting, Inc.
Freedman, D. L., et al. (2001). Substrate interactions during aerobic biodegradation of methane,
ethene, vinyl chloride and 1,2-dichloroethenes. 43: 333-340.
Fogel, M.M., et al. (1986). "Biodegradation of chlorinated ethenes by a methane-utilizing mixed
culture." Appl. Environ. Microbiol. 51: 720-724.
Hartmans, S. et al. (1985). "Bacterial-degradation of vinyl chloride." Biotechnol. Lett. 7: 383388.
Hartmans, S. et al. (1992). "Aerobic vinyl chloride metabolism in Mycobacterium Aurum L1."
Appl. Environ. Microbiol. 58: 1220-1226.
Hozo, I., et al. (1996). "Vinylchloride monomer exposure by the plastic industry workers basic
condition for liver angiosarcoma appearance." Medicinski arhiv 50(1-2): 9-14.
Greiser E. et al. (1982). "Vinyl chloride exposure and mortality of German chemical workers in
comparison to comparison of non-exposed chemical workers and PVC workers." Zent. bl
Arbeitsmed Arbeitsschutz Prophyl 32: 44-62 (in German).
Ietri, E., et al. (1997). "Cohort mortality study of rubber and plastics product makers in Italy."
Occupational Medicine-Oxford 47(7): 417-422.
32
International Programme on Chemical Safety (1989). "Environmental health criteria 215: Vinyl
chloride."
World
Health
Organization.
http://www.inchem.org/documents/ehc/ehc/ehc215.htm.
Jin, Y.O. & Mattes, T.E. (2008). "Adaptation of aerobic, ethene-assimilating Mycobacterium
strains to vinyl chloride as a growth substrate. Environ Sci Technol 42(13): 4784-4789.
Kielhorn, J., et al. (2000). "Vinyl chloride: Still a cause for concern." Environmental Health
Perspectives 108(7): 579-588.
Lange CE, Juhe S., Stein G.& Veltman G. (1974) "The so-called vinyl chloride disease: An
occupationally caused systemic sclerosis." Int Arch Arbeitsmed 32: 1-32 (in German).
Lelbach WK & Marsteller HJ (1981). "Vinyl chloride- associated disease." Adv Intern Med
Pediatr 47: 1-110.
Makk, L., et al. (1974). "Liver-damage and angiosarcoma in vinyl-chloride workers - systematic
detection program." Jama-Journal of the American Medical Association 230(1): 64-68.
Mattes, T. E., et al. (2010). "Aerobic biodegradation of the chloroethenes: pathways, enzymes,
ecology, and evolution." Fems Microbiology Reviews 34(4): 445-475.
Mattes, T. E., et al. (2005). "Physiological and molecular genetic analyses of vinyl chloride and
ethene biodegradation in Nocardioides sp. strain JS614." Archives of Microbiology
183(2): 95-106.
Maymó-Gatell, X., et al. (1997). "Isolation of a bacterium that reductively dechlorinates
tetrachloroethene to ethene." Science 276(5318): 1568-1571.
Mundt, K. A., et al. (2000). "Historical cohort study of 10 109 men in the North American vinyl
chloride industry, 1942-72: update of cancer mortality to 31 December 1995."
Occupational and Environmental Medicine 57(11): 774-781.
Neumann, A., et al. (1994). "Tetrachloroethene metabolism of Dehalospirillum multivorans."
Archives of Microbiology 162(4): 295-301.
Peterson M.L. et al. (2000). "Characterization of chlorinated hydrocarbons above a drinking
water aquifer." In: Wickramanayake GB, ed. Risk, regulatory and monitoring
considerations. Columbus, OH, Battelle Press, pp. 79-86.
Pirastu, R., et al. (1990). "Mortality from liver-disease among italian vinyl-chloride monomer
polyvinyl-chloride manufacturers." American Journal of Industrial Medicine 17(2): 155161.
Purchase, I. F. H., et al. (1987). "Vinyl-chloride - an assessment of the risk of occupational
exposure." Food and Chemical Toxicology 25(2): 187-202.
33
Sadat-Shojai, M. and G.-R. Bakhshandeh (2011). "Recycling of PVC wastes." Polymer
Degradation and Stability 96(4): 404-415.
Salkinoja-Salonen, M., et al. (1995). "Organic halogens in the environment: studies of
environmental biodegradability and human exposure." Environmental Health
Perspectives 103: 63-69.
Semprini, L. (1997). "Strategies for the aerobic co-metabolism of chlorinated solvents." Current
Opinion in Biotechnology 8(3): 296-308.
Semprini, L., et al. (1995). "Anaerobic transformation of chlorinated aliphatic-hydrocarbons in a
sand aquifer based on spatial chemical-distributions." Water Resources Research 31(4):
1051-1062.
Simonato, L., et al. (1991). "A collaborative study of cancer incidence and mortality among
vinyl-chloride workers." Scandinavian Journal of Work Environment & Health 17(3):
159-169.
Smidt, H. and W. M. De Vos (2004). Anaerobic microbial dehalogenation. 58: 43-73.
Sung, Y., et al. (2006). "Geobacter lovleyi sp. nov. strain SZ, a novel metal-reducing and
tetrachloroethene-dechlorinating bacterium." Applied and Environmental Microbiology
72(4): 2775-2782.
Tamburro, C. H. (1984). "Relationship of vinyl monomers and liver cancers - angiosarcoma and
hepatocellular-carcinoma." Seminars in Liver Disease 4(2): 158-169.
Thiese AM & Versen P (1974). "Occupational medical thoughts on the so-called 'vinyl chloride
disease'." Arbeitsmed Sozialmed Praventivmed 7: 146-148 (in German).
The Organization for Economic Co-operation and Development (2001). " SIDS Initial
assessment
report
for
SIAM
13."
http://www.inchem.org/documents/sids/sids/VINYLCHL.pdf.
Veltman G, Lange CE., Juhe S., Stein G. & Bachner U. (1975). " Clinical manifestations and
course of vinyl chloride disease." Ann NY Acad Sci 246: 6-17
Ward, E., et al. (2001). "Update of the follow-up of mortality and cancer incidence among
European workers employed in the vinyl chloride industry." Epidemiology 12(6): 710718.
Wendlandt, K. D., et al. (2010). "The potential of methane-oxidizing bacteria for applications in
environmental biotechnology." Engineering in Life Sciences 10(2): 87-102.
34
West, M. R. and B. H. Kueper (2012). "Numerical simulation of DNAPL source zone
remediation with in situ chemical oxidation (ISCO)." Advances in Water Resources
44(0): 126-139.
Williams, D. T. and W. F. Miles (1975). "Gas-liquid-chromatographic determination of vinylchloride in alcoholic beverages, vegetable oils, and vinegars." Journal of the Association
of Official Analytical Chemists 58(2): 272-275.
Wilson, J.T. et al. (1983). "Biotransformation of selected oraganic pollutants in groundwater."
Dev. Ind. Microbiol 24: 225-233.
Wong, O., et al. (1991). "An industry-wide epidemiologic-study of vinyl-chloride workers, 19421982." American Journal of Industrial Medicine 20(3): 317-334.
Word Health Organization (1999). "Environmental health criteria 215 Vinyl chloride."
http://whqlibdoc.who.int/ehc/WHO_EHC_215.pdf.
Wymore, R. and N. Akladiss (2009). In situ bioremediation of chlorinated ethenes: DNAPL
source zones.
35