Embed Size (px)
Citation preview
\BIDDEGRADATION OF METHANOL AND TERTIARY BUTYL ALCDHOLIN PREVIOUSLY UNCONTAMINATED SUBSURFACE SYSTEMS,
by
Charles DouglasQG0ldsmitn, Jr.
Dissertation submitted to the Faculty of the
Virginia Polytechnic Institute and State University
in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
in
Environmental Sciences and Engineering
APPROVED: -. ,,„//7
J. T. Novak, Chairman
' 4
C!q g gpp'., _
Z ¢kt lll -
R. E. Benoit R. C. Hoehn/*7 l g
L}.; _. ........,. „*-{J
G. D. Boardman P. F. Scanlon
March, l985Blacksburg, VA
3/ BIODEGRADATION OF METHANOL AND TERTIARY BUTYL ALCOHDLE IN PREVIOUSLY UNCONTAMINATED SUBSURFACE SYSTEMS
äé byTx
Charles Douglas Goldsmith, Jr.
Committee Chairman: John T. Novak
Environmental Sciences and Engineering
(ABSTRACT)
The objective of this study was to determine the potential for
biodegradation in subsurface soils and groundwater from sites in
Milliamsport, PA, Nayland, NY, and Dumfries, VA. These subsurface
systems were characterized both physically, chemically and biologically.
Bacterial populations were substantial in all systems and ranged from
l03 to l08 colony forming units per gram. Soil sampling was done in a
quality-controlled aseptic manner using conventional drilling end
sampling equipment. A matrix of test—tube microcosms was used to
determine hiodegradation rates of methanol and t—hutyl alcohol at
concentrations ranging from l to 1000 mg/L. Methanol degraded readily
at all sites ranging from 0.8 mg/L/day to 20.4 mg/L/day and rates were
generally greater in the saturated zone. TBA biodegraded at all sites,
but was refractory in nature. Biodegradation rates for TBA in anaerobic
subsurface systems were found to increase directly with initial
concentration froml0”4
mg/L/day for l mg/L tol0”1
mg/L/day for 80
mg/L. TBA biodegradation in the aerobic system was essentially constant
over all concentrations. Biokinetic coefficients were determined for
methanol and TBA at each site based on plots of utilization rates versus
suhstrate concentration and reciprocal plots of these values. Ihg_§
vg1ggs_jgggd_ggggest-Lhat_ae;gbjg_subsurface«systems can utilize
9_]g9_!l9_]5„„§„„.a„gxߤtex„J‘e=;.te„t.*1a.n~„.a„e<2.>$_ip.„§;1usedmfor¤£gmpa;ati1e„puxp¤3es.
The KS of anoxic subsurface systems were
found to be large due to the low temperature (l0°C) found in aquifers.
The results indicate that methanol contamination in groundwater has much
less associated risk
tobiodegradation.However, TBA poses a much greater risk due to the very
slow removal rates at low concentrations, which could result in a
residual level for over a decade in some cases.
ACKNONLEDGEMENTS
iv
TABLE OF CONTENTS
Page
ABSTRACT
ACKNOWLEDGEMENTS................... iiii
LIST OF TABLES.................... vii
LIST OF FIGURES ................... ix
INTRODUCTION.......................... T
Background and Review of the Literature.......... 3Subsurface System .................. 3
Structure ..................... 3Environment .................... 4Biology ...................... 5
Groundwater ................... 5Soil....................... 5Biodegradation Studies.............. 7
Solutions to Aquifer Contamination.......... TTPhysical...................... ll
. Theoretical .................... TTStimulation of Natural Biodegradation ....... T2
Studies with Pure and Mixed Cultures......... T3Alcohols...................... T4Methanol..................... T4TBA ....................... T8
Aromatics ..................... 2TBenzene ..................... 22Toluene ..................... 22Xylene...................... 22Anaerobic Biodegradation............. 22
METHODS AND MATERIALS .......A.............. 27
Study Areas........................ 27Selection ...................... 27Descriptions..................... 27
Sampling ......................... 29Soil Extrusion Apparatus............... 34Groundwater ..................... 36
Microcosms ........................ 36Static sacrificial microcosms ............ 36Static continuous sampling microcosms ........ 37Circulating microcosms................ 39
v
METHODS AND MATERIALS (Cont'd)
Bacterial Enumeration................... 39Analytical Methods .................... 39
Gas Chromatography.................. 40Low Level Methanol Detection............ 40ARC0 Alternative Methods.............. 42
Standards Preparation ................ 45Alcohols...................... 45Aromatics ..................... 45
Lithium Analysis................... 47Anion Analysis.................... 47Microcosm Dosing................... 47
RESULTS AND DISCUSSION..................... 49
Soil Sampling....................... 49Extrusion Apparatus.................... 50Lithium Tracer ...................... 50Subsurface Bacterial Populations ............. 51Microcosm Dosing ..................... 61Groundwater Parameters .................. 64Biodegradation ...................... 64
Pennsylvania..................... 69Methanol...................... 69Circulating Microcosms............... 77TBA ........................ 77Control Microcosms................. 91
New York ....................... 96Methanol...................... 96TBA ........................ 100Groundwater .................... 108Control Microcosms................. 108
Virginia ....................... 108Methanol.............. ‘........ 108TBA ........................ 122Control Microcosms................. 122
Risk Assessment.................... 125
SUMMARY AND CDNCLUSIONS .................... 133
LITERATURE CITED........................ 134
APPENDIX A........................... 140
APPENDIX B........................... 167
APPENDIX C........................... 171
VITA.............................. 199
vi
LIST OF TABLES (Conti'd)
Table Page
l6 Residual tertiary butyl alcohol concentrations incontrols from the Pennsylvania unsaturated andsaturated zones autoclaved for 45 minutes ....... 93
l7 Determination of optimum sterilization of controlsby various treatments................. 94
l8 Saturated zone utilzation rates for New York ..... 99
l9 Biodegradation in New York groundwater ........ l09
20 Methanol, TBA, benzene, toluene and m-xylene controlmicrocosms for New York................ llO
2l Utilization rates (mg/L/day) for Virginia....... ll2
22 Methanol, TBA, benzene, toluene and m-xylene controlmicrocosms for Virginia................ l24
23 Summary of kinetic data from Pennsylvania, New Yorkand Virginia at lO°C. ................ l28
24 Time and distance requirements for the complete bio-degradation of l000 mg/L methanol and TBA in thesaturated zone moving a one m/day........... l30
viii
LIST OF FIGURES
Figure Page1 The serine pathway for methanol assimilation...... 17
2 Benzene biodegradation pathway............. 23
3 Toluene biodegradation pathway............. 24
4 Two pathways for meta—xy1ene biodegradation ...... 25
5 Location of study areas ................ 28
6 Soil extrusion apparatus................ 35
7 The separation of methanol, TBA, benzene, toluene, andm—xylene on a CN l500 column.............. 41
8 The separation of 50 ppb methanol and water on aPorapak QS column ................... 43
9 The separation of a benzene, toluene, and m-xylenestandard in methanol on a SP 1200/bentone 34 column . . 46
10 Bacteria found with depth by Acridine-Orange (A0)direct counts and soil extract (SE), SE plus formate,and SE plus methyl amine (MA) agar plate counts ofPennsylvania soil ................... 56
ll Bacteria found with depth by Acridine·0range (AO)direct counts and soil extract (SE), SE plus formate,and SE plus methyl amine (MA) agar plate counts ofNew York soil ..................... 57
12 Bacteria found with depth by Acridine-Orange (A0)direct counts and soil extract (SE), SE plus formate,and SE plus methyl amine (MA) agar plate counts ofVirginia soil ..................... 58
13 Methanol biodegradation in the Pennsylvania unsaturatedzone.......................... 70
14 Methanol biodegradation in the Pennsylvania saturatedzone.......................... 71
15 Methanol biodegradation in the Pennsylvania unsaturatedzone.......................... 72
ix
LIST OF FIGURES (Cont'd.)
Figure Page
l6 Methanol biodegradation in the Pennsylvania saturatedzone.......................... 73
l7 Determination of the kinetic constant K for methanolin the Pennsylvania soil................ 76
l8 Mean value for methanol utilization rates inPennsylvania soil ................... 78
l9 Comparison of rates obtained with tube and circulatingmicrocosms....................... 79
20 TBA biodegradation in the Pennsylvania unsaturatedzone.......................... 80
2l TBA biodegradation in the Pennsylvania saturated zone . 8l
22 TBA biodegradation in the Pennsylvania unsaturatedzone.......................... 83
23 TBA biodegradation in the Pennsylvania saturated zone . 84
24 TBA biodegradation in the presence of methanol in thePennsylvania unsaturated zone ............. 86
25 TBA biodegradation in the presence of methanol in thePennsylvania saturated zone .............. 87
26 TBA biodegradation in the presence of 700 mg/Lmethanol in the Pennsylvania saturated zone ...... 88
27 Determination of TBA kinetic constants using Lineweaver—Berk plot for the Pennsylvania soil .......... 89
28 Determination of rate order for TBA utilization inthe Pennsylvania soil ................. 90
29 BTX removal in sterile and non-sterile Pennsylvaniamicrocosms............b........... 95
30 Methanol biodegradation in the New York saturated zone. 97
3l Methanol biodegradation in the New York saturated zone. 98
32 Determination of the kinetic constants k KS and K formethanol in saturated New York soil .......... l0l
x
LIST OF FIGURES (Cont'd.)
Figure Page
33 Determination of the kinetic constant K for methano1in saturated New York soi1............... 102
34 TBA biodegradation in the New York saturated zone . . . 103
35 TBA biodegradation in the New York saturated zone . . . 104
36 Determination of rate order for TBA uti1ization in theNew York and Virginia soi1s .............. 105
37 Determination of kinetic constants k, KS, and K for TBAin New York and Virginia soi1s............. 106
38 Determination of the kinetic constant K for TBA inNew York and Virginia soi1s .............. 107
39 BTX remova1 in steri1e and non-steri1e New Yorkmicrocosms....................... 111
40 Methano1 biodegradation at various depths in theVirginia subsurface .................. 115
41 Methano1 biodegradation at various depths in theVirginia subsurface .................. 116
42 Determination of the kinetic constants k, KS and K formethano1 in the Virginia subsurface .......... 117
43 Determination of the kinetic constant K for methano1in the Virginia subsurface...........,... 118
44 The effect of TBA and TBA + BTX on methano1 bio-degradation in Virginia soi1s at 3.4 M (11 ft.) .... 119
45 The effect of TBA and TBA + BTX on methano1 bio-degradation in Virginia soi1s at 17 M (57 ft.)..... 120
46 The effect of TBA and TBA + BTX on methano1 bio-degradation in Virginia soi1s at 31 M (102 ft.) .... 121
47 TBA biodegradation at various depths in the Virginiasoi1s ......................... 123
48 BTX remova1 in steri1e versus non-steri1e Virginiamicrocosms....................... 126
xi
INTRODUCTION
The potential for and occurrence of groundwater contamination due
to gasoline leakage from pipelines and storage tanks is a major concern
of the petroleum industry, as well as the general public. Gasoline
spills of major proportions have been reported in the past (l,2) in
addition to the small quantities that are continually being lost.
Groundwater comprises more than 95 percent of all available
freshwater in the United States, and approximately BO percent of all
public water suppliers rely on this source. Groundwater has in the past
been considered a pristine liquid. However, a survey of almost 35,000
water supplies revealed a widespread problem of contamination (3).
Of specific concern in this study is gasoline containing alcohol
‘additives (gasohol) such as methanol and tertiary butyl alcohol (TBA).
These alcohols present a unique situation after entry into a subsurface
system. As gasohol travels through a groundwater aquifer, changes inI
component concentrations may occur. The mechanisms of disperson,
sorption, and biodegradation are responsible for these changes.
Dispersion is a function of hydraulics, while sorption is more complex.
Predicted retention times of several organic compounds in the soil
matrix of aquifers based on organic content of the matrix and the
octanol-water partition coefficient were found to agree well with
retention times in field studies (4). Sorption is responsible for
separating the compounds of gasohol and producing a component wave
effect as groundwater moves through the aquifer, Alcohols, particularly
2
methanol, are of concern since their presence in water is not easily
detected by taste and odor until levels potentially detrimental to human
health may be reached. Gasoline in general is not detectable until a
concentration of 0.5 mg/L is attained. Due to the solubility of
alcohols in water they would move ahead of the remaining gasoline
components which are more easily detected. Dispersion and sorption
result only in separation and concentration changes, but do not resolve
the mode of removal of these toxics from the groundwater system.
Biodegradation would seem to be a mechanism of great importance in
a groundwater aquifer, and little information of this kind is available.
Therefore, the objectives of this investigation were to l) quantify
bacterial numbers as they vary with depth, 2) determine the potential
for biodegradation by these microbes using an experimental microcosm
matrix for several uncontaminated sites as it relates to depth and
aquifer parameters, 3) determine the effect of benzene, toluene, and
m-xylene (BTX) on alcohol biodegradation rates and 4) obtain a general
risk assessment for groundwater contamination by gasohol by integrating
the collected data.
BACKGROUND AND REVIEW OF LITERATURE
This study encompasses several scientific fields that impact the
removal of subsurface contaminants. These are engineering,
microbiology, geology, and biochemistry. An understanding of the macro-and micro—subsurface, both physically and biologically, is important.
Therefore, the potential for natural assimilation of alcohols found ingasoline and the effects of multiple subsurface parameters will be
discussed.
Subsurface System
Structure
Great heterogeneity can exist within the subsurface, hut basically
these systems consist of two major entities. There is an unsaturated
and a saturated zone. The unsaturated zone is subject to influence by
vadose water and lies above the water table, which may rise and fall
within the unsaturated zone. The saturated zone, where water is held,
may be homogeneous or consist of several layers, some of which can cause
partial (aquitard or aquiclude) or complete (aquifuge) confinemert of
vertical water movement within the aquifer. These layers are currently
referred to as leaky confining or confining aquifers. Groundwater flow
may range from millimeters to meters in distance per year, but can
3
4
generally be said to be quite slow. Most aquifers are at some point
underlain by bedrock and can be recharged by downward seepage through
the unsaturated zone. Recharge can also occur by lateral flow or by
upward seepage from underlying strata (5).
Environment
The nutrient status of an aquifer can be said to be ogliotrophic.
Groundwater is generally low in organic carbon, low in temperature,
anaerobic and of low pH. There are, of course, exceptions to these
conditions at a given site. A major question with regard to subsurface
biodegradability is; can organisms existing under such conditions cope
with increased organic loading due to contamination? Ogliotrophic
organisms can live under very low nutrient conditions, but can adapt to
high nutrient conditions. The reverse adaptation is not thought to be
as easily done. These organisms usually have a high surface to volume
ratio and may have lower minimum substrate requirements to attain
measurable growth, but the maximum growth rate is also lower.
Subsurface bacteria prefer attachment and can often use multiple
substrates (6). These factors along with increased pressure, spatial
limitations, temperature, macro and micro nutrient limits, and others
were evaluated.
A parameter considered to be of major significance was temperature.
Soil temperature increases approximately 30°C/l000 meters in depth.
This would preclude microbial growth at depths greater than 2500m if
only thermophiles are considered. The average groundwater temperature,
however, to depths of about 60 ft usually approximates the mean annual
5
air temperature. The conclusion was that organisms in the subsurface
would take part in the assimilation of contaminants (7).
Biology
Until recently only the uppermost soil layers were believed to have
significant bacterial populations. This in part was probably due to the
expense and lack of methodology for obtaining aseptic samples from deep
soil regions. Groundwater was considered to be relatively pure and
bacteria-free.
Groundwater. Bacteria have been identified in groundwater and soils in
several areas where spills of contamirating organics have occurred (2,
8-l0) as well as uncontaminated areas (ll). These are presented in
Table l along with the type of contaminant for each case. It is
probably safe to assume, therefore, that the groundwater offers a
variety of microhabitats.
§gjl„ Several methods have been used to enumerate subsurface soil
bacterial populations. Wilson et al. made direct measurements by
utilizing epifluorescence microscopy with acridine orange staining (l2).
Counts were also obtained by culturing on soil extract agar. In
addition transmission electron microscopy (TEM) was used to study
organism morphology. Both gram—negative and positive cells were found.
Most frequent were small rods (<lpm) and cocci. This is consistent with
the findings cf Ghiorse and Balkwill (I3). TEM revealed that bacterial
cells were most always coated with a dense clay-like material and an
extracellular polymer matrix was found to be connecting cells to soil
particles. Most bacteria were small in size relative to nutrient rich
6
Tab1e 1. Microbes found in groundwater of contaminated anduncontaminated sites.
0rganism(s) Contaminant Reference
Pseudomonas spp. Gas01ine 2Arthrobacter
Pseudomonas Gaso1ine 8NocardiaAcinetobacterF1avobacteriumMicrococcus
Pseudomonas Gas oi1 9
Pseudomonas spp. Creosote 10Pseudomonas putidaPseudomonas aeruginosaPseudomonas stutzeriA1ca1igenes spp.A1ca1igenes denitrificans
Microcyc1us None 11ProsthecomicrobiumGa11iore11aCau1obacter -AgrobacteriumHyphomicrobiumP1anctomycesC1ostridiumNocardiaProtozoaYeasts
7
bacteria. This was not found to be true of bacteria observed in
groundwater by Hirsch and Rades-Rohkoh1 (11). They did find, in
agreement with Ni1son et a1., that groundwater bacteria carried
hair-1ike surface po1ymers. The differences in reported size cou1d be a
function of 1iving space, i.e., free f1oating in groundwater we11s
versus confinement in interstitia1 spaces.
whi1e Ni1son et a1. (12) be1ieved no organisms other than bacteria
existed in the subsurface soi1s, Ghiorse and Ba1kwi11 found e11ipsoid
ce11s with pointed ends, narrow fi1aments, and some ovoid forms that
were be1ieved to have been funga1 spores or yeast ce11s (13).
Supporting this contention were ninety different organisms found in we11
water from Northern Germany. Seventy—two were bacteria, ten were
protozoa, whi1e eight were fungi, a11 of which grew we11 at 9°C(11).
The depths to which bacteria can exist seems to be great (7). At a
site near Pensaco1a, F1orida, bacteria were found to 410 m by estimates
of biomass using five biochemica1 assays of fatty acids (14).
Biodegradation Studies .
Many researchers have tried to estimate bioaccumu1atior of
hazardous organic compounds using octano1/water partition coefficients
(15,16) or estimates of microbia1 degradation rates by eva1uation of
chemica1 structure (17). Recent review papers have done a commendab1e
job with respect to eva1uating the potentia1 for remova1 of hazardous
compounds from the environment by biodegradation (6, 18), but these
assessments cannot a1ways be accurate1y compi1ed to predict actua1
remova1 rates for a given system.M
8
No studies were found to exist dealing with the subsurface
biodegradation of alcohols. However, several dealing with various other
compounds in subsurface soils were available, as were investigations of
the biodegradation in well waters of gasoline, gas oil, and creosote.
Some of the original investigations in suhsurface soil biodegradation
can be subject to criticism. However, the work was done with no prior
experiences upon which to draw. Therefore, considerable knowledge was
gained and time saved by examination of these works.
The biodegradation of toluene, chlorobenzene, bromodichlormethane,
l,2-dichloroethane, l,l,2—trichloroethane, trichloroethylene, and
tetrachlorethylene were measured in subsurface soils from l.2, 3.0, and
5.0 m at Lula, Oklahoma (l2). This material was made into a slurry with
distilled water and a sterile solution of the listed compounds was added
and mixed before addition to 35 mL test tube microcosms. The slurry was
assumed to be uniform. At given time intervals duplicate microcosms
would he sacrificed for measurements of biodegradation. The initial
concentrations were found to range from 85 to 666_pg/L indicating very
poor mixing. All concentrations were normalized to the compound thought
to be closer to the expected value. After incubation at 20°C for l6
weeks toluene was found to degrade rapidly at all three depths. The
remaining compounds either degraded very slowly or not at all. The lack
of degradation of these other compounds could have been due to the need
for an extended incubation period, the use of sterile distilled water
versus sterile groundwater, which may contain essential inorganic
9
nutrients, or the presence of toluene, which is easily degraded and may
have been utilized preferentially.
A follow-up study of subsurface soils from Pickett, Oklahoma and
Fort Polk, Louisiana used similar techniques to evaluate biodegradation
(19). This time, however, the dose solution censisted of sterile well
water containing the contaminants, and known amounts were added to the
microcosm tubes containing the slurry to give a more reliable initial
concentration of approximately 1 mg/L. Chloroform, 1,1-dichloroethane,
1,1,1-trichloroethane, trichloroethylene, tetrachloroethylene, toluene,
chlorobenzene, and styrene were the compounds tested. Toluene and
styrene showed some evidence of biodegradation at both sites under
incubation at l7°C, while the others were utilized very slowly or not at
all.
The biodegradation rates of 47 water-soluble components of gas oil
were determined in groundwater at lO°C (9). The substrate was obtained
by shaking 1 ml of gas oil with 1 L of groundwater for 10 minutes to
achieve a total aromatic hydrocarbon concentration of 2 mg/L in the
water. All were identified by GC/MS. No compounds remained after 288
days„ Of particular interest were ortho, meta, and para-xylene and
toluene biodegradation. All were subject to complete degradation, but
meta and para-xylene were degraded much faster than ortho—xy1ene. Of 30
isolates it was found that just 12 strains actually affected the
composition of the hydrocarbons. Using GC/MS only four different
degradation spectra were found implying that only four metabolically
different strains existed. Studies with these four groups verified that
l0
the ortho substitution was most resistant to degradation. Intermediate
compounds that were identified agreed well with existing literature on
aromatic metabolism, and suggested, to some extent, that pure culture
studies are of predictive use.
At a creosote contaminated site in St. Louis Park, Minnesota, well
studies down gradient from the source revealed that phenolic compounds
and naphthalene were disappearing much faster than expected due to
dispersion and sorption (20). Methane was found in the water from the
contaminated area at 2-20 mg/L. Racterial counts of l08-109 per l00 mL
and isolates on creosote substrates confirmed the belief that bacterial
action was occurring (l0).
A pipeline loss of 100,000-250,000 gallons of gasoline occurred at
Glendale, California (2). As a result, one of the first studies to
suggest that bacterial degradation could be helpful in the natural
cleanup of well waters was conducted at this site. The wells that had
no gasoline taste or odor averaged 200/mL bacteria, while those
exhibiting taste and odor averaged50,000/mL.As
the result of another gasoline pipeline break a similar study
was performed on groundwater in Ambler, Pennsylvania (l). 0f the 3,l86
barrels lost to the soil almost half remained after all means of
physical extraction had been exhausted. To investigate the possibility
of bacterial action, populations were enumerated by growth on minimal
media salts agar incubated at l3°C in a dessicator containing gasoline
(Sunoco 260) vapors and by nutrient agar plate counts. Counts in all
11
we11 waters ranged from 103-104 per mL. Enumerations done with to1uene
and n—paraffin (gaso1ine constituents) p1ates were found to yie1d 106
bacteria/mL we11 water.
So1utions to Aquifer Contamination
Physica1
Genera11y on1y physica1 means have been considered and tested as a
means of "c1eaning up" an aquifer after a contaminant spi11. Most of
these methods are expensive and usua11y invo1ve pumping water from the
aquifer with subsequent rem0va1 or treatment and rep1acement. Treatment
schemes consist of air stripping (20), granu1ar activated carbon (3), or
b1ending contaminated groundwater with other c1ean water sources (22).
In some instances purging a11 the water from the aquifer, whi1e simu1-
taneous1y stopping the contaminating source has been successfu1 (23).
Theoretica1 ·Predictive mode1s have been deve1oped to aid in physica1 treatment
schemes by estimating the size of the area to be affected by a
contaminant p1ume. This is done by uti1izing measurab1e groundwater and
aquifer parameters. Soi1 profi1es, hydrau1ic conductivity, groundwater
ve1ocity, and adsorptive factors of the contaminant are a few of the
important inputs. Newer mode1s have inc1uded first order biochemica1
decay in addition to these and other parameters (24,25). Mode1s have
been shown to he very sensitive to degradation rates when these are
inc1uded (25), therefore, more recent attempts have recognized the
importance of biodegradation by emp1oying biofi1m kinetics (4).
12
However, very little real world data of this kind exists. In addition,
biodegradation rates may be site specific, but with enough data some
generalizations on these rates could be assumed to hold true for
aquifers that are similar in nature, i.e., soil type, anaerobic versus
aerobic, etc. for modeling applications. Models may be of importance in
the future for determining safe distances for spills near public water
supplies.
Stimulation of Natural Biodegradation
Biodegradation, it seems, is a potent force in the eventual removal
of unwanted compounds from the subsurface environment. There have been
attempts to enhance biodegradation rates in well water studies.
It was found that by supplementing gasoline minimal media agar with
0.2 percent ammonium nitrate or sodium nitrate, sodium or potassium
phosphates and magnesium sulfate that counts increased from 103-104 to
105-107 based on an increased concentration of electron acceptors for
enhanced anaerobic respiration (1). It was decided to amend well waters
with aerators as well as ammonium sulfate and mono and disodium
phosphates (8). After addition, oxygen was believed to be utilized and
the phosphate and nitrogen decreased, while microorganism counts
increased to 107 at the spill site and decreased towards the perimeter
to 104. Six months after nutrient treatment no gasoline was detectable
in well waters by ultra-violet spectroscopy. However, it cannot be
assumed that all gasoline was removed since each well only affects a
given area. It is evident, though, that of the 36 wells treated
bacterial action was accelerated.
13
In support of this contention lab studies on well-water gave
similar results (9). when natural groundwater was given 2 mg/L of mixed
aromatics from gas oil degradation proceeded, but stopped after 10 days
resulting in an increase of 102 to 106/mL. The groundwater was found to
be nitrogen deficient. The amount of inorganic nitrogen originally in
the water stoichiometrically corresponded to the amount of hydrocarbon
utilized. Upon the addition of ammonium chloride further degradation
resulted.
Studies with Pure and Mixed Cultures
To thoroughly understand the biological processes that take place
in the subsurface a review of research conducted with pure cultures is
of utmost importance. The effects of pH, temperature, oxygen, and
groundwater composition can be more effectively predicted when the
routes by which biodegradation occur are known.
To obtain energy biological systems can utilize various sources.
Both organic and inorganic molecules can serve as_these sources
(electron donors) and they can also serve as electron acceptors. In
this study we are concerned with the utilization of the alcohols,
methanol and tertiary butyl alcohol, as energy sources by suhsurface
organisms. Three routes of utilization should be considered. Aerobic
respiration occurs in the presence of oxygen. Anaerobic respiration
uses NOS, $0ä- or CO§- as electron acceptors. Fermentation occurs
without any such electron acceptors. The energy yielded to the organism
declines from the highest with aerobic respiration to the lowest with
l4
fermentation. The thrust of all these reactions, of course, is toconserve energy in ATP.
From this general information it could be expected that a subsur-
face system would be subject to varying efficiencies of contaminant
assimulation depending upon its oxygen and inorganic anion status.
Studies using pure or mixed cultures are valuable for determining
the biochemical pathways for the diverse types of organisms that have
already been shown to exist in the subsurface environment. There exists
extensive literature, in most cases, to aid in defining the fate of
biological contaminant molecules.Alcohols ‘Methanol. The utilization of C] compounds by microbes at one time was
considered to be obscure. It is now known that organisms capable of
such metabolism are ubiquitous. Studies done on aerobic and anaerobic
biological waste treatment have demonstrated the presence and usefulness
of C] utilizers for the removal of methanol and formaldehyde (26-29).
Several excellent review papers exist on the_utilization of C]
compounds (30-33). These will be used to explain how methanol may be
metabolized.
Generally, C1 metabolizing microorganisms are recognized by their
ability to utilize organic compounds that contain no carbon to carbon
bonds and are more reduced than carbon dioxide. Three pathways of C1
assimilation are known. These are the ribulose diphosphate pathway, the
ribulose monophosphate pathway (RMP) and the serine pathway.
l5
The general scheme of respiratory methanol utilization is as
follows (30).
methanol formaldehyde formatedehydrogenase dehydrogenase dehydrogenase
H„0
2H 2H NAD+ NADH + H+Assimilation
In the ribulose diphosphate pathway of CO? assimilation the CO?
formed from methanol combines with ribulose diphosphate to yield
phosphoglyceric acid (PGA) which in turn is converted to
glyceraldehyde-3-phosphate. This pathway is used by only a few bacteria.
C] bacteria are presumed to assimilate most of their carbon by
either the RMP or serine pathways. From studies of methane utilizing
bacteria the RMP pathway has been designated as Type I, while the serine
pathway has been designated Type II.
The RMP pathway found in Type I crganisms is more efficient than
the serine pathway in that it does not possess a complete tricarboxylic
acid (TCA) cycle. All of the carbon atoms for cell material are derived
from formaldehyde and there is no detectable 2-ketoglutarate dehydro-
genase (30). In condensed form the reactions are as follows (34).
a) 3Formaldehyde + 3Ribulose-5-P + 3hexulose-6+P + 3Fructose-6-Psynthase isomerase
b) 3Fructose-6-P + ATP +·3Ribulose-5-P + ADP + 3-phosphoglyceraldehydecell material
l6
c) Overall: 3Formaldehyde + ATP + 3-Phosphogylceraldehyde + ADP
Variants of this cycle have been demonstrated (33).
The serine pathway is somewhat longer but can be briefly described.
Formaldehyde is assimilated into cell material by the formation of
Acetyl—CoA, then three, four, five and six carbon compounds are
synthesized by the glyoxylate cycle (which regenerates oxaloacetate) and
the reversal of glycolysis (34). The cyclic reactions in Figure l
essentially demonstrate the serine pathway (30,32,34).
The first studies on the fermentation of methanol were conducted
over thirty years ago. work with Methosarcina explained the reacticn
involved for methanol utilization by 14C labeling (35).
l) CH3OH + H20 + CO2 + 6H
2) 3CH30H + 6H + 3CH4 + 3H20
3) 4CH30H + 3CH4 + CO2 + 2H20
Subsequent work has served to support these findings with this and
other organisms (36-38), but in addition concluded that growth yields
must be limited by nutrients or some factor other than the energy
source. A study on the anaerobic treatment of fuel oil censisting of 50
percent methanol and 50 percent higher alcohols, supportively found
indications that trace elements were of great importance in preventing
upset of the process.
Studies involving pure cultures have served to demonstrate the
biochemical diversity of methylotrophic bacteria (39-4l). A large
17
.1*9<1:..1>-x0Z1g 0
IU
.1
g g¤-RT
Öä 111
6 <DIO
g' <U *"E
1 <
Ü**T
m << 9}*2
14
1- U tu [-j
1-M
<t 1u0
nu1-
<t U
M0
<1:
Z>—
+ .1
M—'
<x
.
.J(D
ZE
1:
>—0
_3
I
I+IÄ|
+-1
m3,
*3
E 1,. 2 -ZM-< <~1
ME
Img
O
gg
ug<I LU
O
g
1-1-
ä
<[<
XE
Eg *6
.3
0>-
8*->IE
I+ —*
0 ua· tz?
QI (D
0.
>'•I•II
>.s
<Ü U.]
D
1..
Of4 1-
Q;
SA
]‘—‘Z (
>.,
u.1>
2
1-D
E]
1-
-1-E
Q
_
ungi
z
-
9Q
*62
1G
1S,
*6¤¤
3,E
J<
Qg
2
u.g
11
u.
l8
number of compounds can be oxidized, while not serving as a carbon or
energy source (cometabolism). The compounds listed in Table 2 can be
found in gasoline (Table 3) and are oxidizable by methylotrophs. From
this partial list it is obvious that these organisms are important .
operating factors in the environment and could be of commercial
importance due to their ability to create new products.
TBA. Tertiary butyl alcohol seems to be a compound that has been
neglected in microbial research. No information is available concerning
the metabolic pathways and very little exists for compounds of similar
structure. They have been described as strongly resistant to
biodegradation.
The quaternary alcohols, 2,2-dimethyl—l,3-propanediol and
pentaerythritol, were not metabolized by acclimated activated sludge
while quaternary dicarboxylic acids were easily metabolized (42).
A pathway for the degradation of 2,2-dimethylheptane to pivalic
acid and tertiary butylbenzene to the diol by Achromobacter was reported
(43) showing evidence for potential degradation of TBA based on similar
refractory molecular structures.
Attack on the alcohol group of TBA may be hindered by the molecular
structure, but it is feasible for enzymes to convert it to the
corresponding aldehyde, ketone or acid.
A list of compounds that may be amenable to fermentation was
proposed (44). These followed the conventional scheme of complex
organics being converted to a higher organic acid or acetic acid which
ultimately yields methane. TBA was found on this list based on the fact
. l9
Table 2. Compounds found in gasoline that are oxidizable byvarious methylotrophs and the resultant products.
Compound Product
Hexane l-hexanol2-hexanoln-hexanol
2-butanol 2-butanone
Benzene phenolhydroquinone
Toluene p-cresolbenzyl alcoholbenzoic acid
Ethylbenzene o—hydroxyethylbenzenep-hydroxybethylbenzenephenylethanol
Naphthalene Y-(l,6)—naphtholB-naphthol
20
Table 3. Gasohol composition.*
Constituent Volume %
Methanol 5
T—butyl Alcohol 5
Benzene** 2
Ethyl Benzene** 2
P-Xylene 2
0-Xylene 3
M-Xylene 4
C9-C10 Aromatics 9
N-Hexane _ 3
Total Paraffins, Olefins & Maphthalenes 57
Toluene** 8
*Furnished by Atlantic Richfield Co.**Priority pollutants
2l
that it is present in industrial wastewaters that are treated by
anaerobic processes. However, this is not conclusive proof of
degradation.
Aromatics.
As listed in Table 3 gasohol contains several aromatic compounds,
two of which are on the EPA priority pollutant list. Aromatics have
long been considered hazardous to human health. They are also
associated with "shock" loadings to wastewater treatment plants. For
this reason water soluble concentrations of benzene, toluene, and
m-xylene were investigated to determine their fate and impact on the
biodegradation rates of methanol and TBA in the subsurface.
Similar work with mixed substrates of benzene and aliphatic
hydrocarbons was done in the Chesapeake Bay giving site specific effects
(45). Early investigators documented the decomposition of benzene and
toluene in the soil (46), as well as the then known aromatic respiratory
degradation mechanisms (47-49). These organisms actually use aromatics
as a carbon source and therefore do more than just change the compound
as was the case with several methylotrophs. It is believed that a
substituted aromatic nucleus only serves to present an organism with a
choice as to the mode of attack (50). The literature on the metabolism
of aromatics is extensive and several papers will be utilized to
illustrate aromatic biodegradation pathways. In general it appears as
though there is a lack of substrate specificity by aromatic utilizing
organisms.
· 22
Benzene. The biodegradation of benzene is illustrated in Figure 2.
Most information obtained on this pathway was obtained from studies
utilizing Pseudomonas sp. (50-53).
Toluene. Toluene also is utilized by a defined pathway of reactions
very similar to benzene (51,54,55) as shown in Figure 3.
Äylggg. The identification of the degradation products of meta and
para-xylene was a difficult task, due to their instability, even with
the aid of ultraviolet, infrared and proton magnetic resonance spectra
(56,57). However, pathways have been discovered and are depicted in
Figure 4.
Anaerobic Biodeqradation. The method of fermentative ring fission is
quite different from the aerobic pathway. It was realized that some
hydrogen acceptor other than oxygen was used to rupture the benzene ring
(58). The common requirement found for successful anaerobic utilization
was the presence of nitrate (59,60). Degradation work done with
Pseudomonas PN-1 grown anaerobically on benzoate was expressed as
follows (60).
C7H602 + GKNO3 + 7C02 + 3N2 + 6KOH
The anaerobic metabolism of several aromatics was studied using
soil microorganisms in the presence of KN1803 substantiated that nitrate
was the electron acceptor (59).
The liberation of CO2 as 60-70 percent of the carbon of benzoate
indicated that the benzene ring had been disrupted.
23Qjg
·•-OO
E';°$U
.
=·&’ua
‘D
‘öO-5
E
.2 6as
¤
S 3Q
3
2: ä S Qg
°C •
°°m
U
Q OÜ)
§I
.2 •J;
. . I.°
2 ‘<?‘ö
lg O.
an E
I
•*
*0*S
\o° '
¤
’
*-
:
.
‘°'
—
0
·§ ‘—
E
0
0
E
EE
‘6¤
0 *‘
¤·
9
ET ä =‘=’2
I°"'
¤8 :%:1:
ÖQ
I0 g
0 ‘
2g
N
°-°
0)
2‘”
¤¤ ä'
0(D
' °
Q-
__
LI.ENZ'ä
24
‘ö
I I·c
O Q §
I..
O E§
4 A6I IO O-
_O
I·— ¤=
/0 I ¤ 0
ro ~"6‘
II
O.·¤
5
,_..Ä_i\=
O—Q E
6
Fo ..·E§6
00
S
ääé
%’
IQ
ga6‘ E
6
IOLIJ
L:
Iz
E
U u.:3Ǥ
. .J
E
0I-LL.
C25
M-xYL Ncaa EE4,
CH34 3
H cwöHO
cH3 HZOH-·€
H1; K;HO cn cu cwH Diol 3 3 34, 2,4-Dimethylphenol m—Me1WIbenzyI olcoholH3 C O 4/HO H
ÜHO CH3 · CH33,5·DIme1hyIpy1·ocofech0I m—T0Iu0Idehydc
coow 4
iii BH1 m—T0Iuic och?
00+1 HO 0001+ J,ou ,H
E owCH3 cH3
3-Nlethylsolicylic ocid D10! ocidH ion
cwöPrmetnglcmecholRing Flssion
Figure 4. Two pathways for meta-xylene biodegradation.
26
Under strict anaerobic conditions eleven aromatic lignin
derivatives indicated that 80 percent of the carbon was converted to CO2and CH4 (6l).
Employing 14C labeling of the number one and seven carbon atoms of
benzoate it was shown that the carboxyl of benzoate is only partly
reduced to methane with the CH4:CO2 ratio being l:5. The number onecarbon of benzoate goes mostly to methane while very little is oxidized
to CO2 with the CH4:CO2 ratio being 25:l. It is clear that the benzenering can be ruptured, but the mechanism is not certain (62).
Again working with 44C benzoate in a sewage sludge enrichment 44CH4
and 44CO2 were produced (63). Without utilization of other substrates
the stoichiometry would be:
444c6H6602 + 18H20 + 16446+14 + 944602 + 4602
The results proved to be within 3.5 percent for produced labeled
methane.
METHODS AND MATERIALS
Study Areas
Selection
Potential sites were limited to Atlantic Richfield Company (ARCO)
terminals in Virginia, Pennsylvania and New York. Basic geological
conditions at these sites were obtained from general documents by
Groundwater Technology, Inc. to aid in the selection of sites with
principally sandy soils and a relatively high water table. Preliminary
drilling was done in selected areas by local drillers under the
supervision of James 0'Brien of ARCO Petroleum Products using a small
diameter, split—spo0n sampler. At times, large gravels may not be
returned to the surface by this method, even when present in subsoils,
so some of the problems that could be encountered (i.e., gravelly sands)
were not totally known. The number of potential sites was narrowed from
these data and from discussions between ARCO personnel and Dr. G.N.
Clough of Virginia Tech. Based upon all available information, the
final selection process was made hy ARCO. The four sites chosen to be
most likely to permit successful sampling were Nilliamsport, PA;
Corning, NY; wayland, NY; and Dumfries, VA as shown in Figure 5.
Descriptions
williamsport, PA was chosen as the first study area. The
distribution terminal was located within 100 yards of the Susquehanna
River on level terrain at the base of a mountain range. The water table
was at approximately 14 feet and roughly corresponded te the level of
27
28
NEWU YORK
•_ •
/ , vmommPENNSYLVANIA
Figure 5. Location of study areas.
29
the river. Subsoils consisted of a loamy silt to a depth of 12 feet
followed by a medium-dense, clean sand from 15-l6 feet. Dense gravelly
sand was found to 30 feet. At 36-38 feet, a relatively lean, dense
sand, underlain by rock, was present .
The Corning, NY site proved to be an unsatisfactory sampling area
due to large cobbles throughout the subsoil that prevented efficient
drilling and sampling collection.
The wayland, NY site was marginally more successful. Only a
limited amount of material was sampled because of problems similar to
those at the Corning area. Drilling was conducted within 400 yards of a
small lake on flat terrain. The upper two feet of soil was dark and
interspersed with marl. Below this, a continuous layer of marl (E 6 in)° existed. The water table was at three to four feet.
The Dumfries, VA site was located adjacent to the Potomac River.
Excellent samples were obtained here to 102 ft. This was due to the
easily obtained alternating layers of sand and silty clay where iron
deposits were observed in the denser layers. The water table was at
43 feet. However some samples above this level appeared saturated.
Sampling
Samples of subsurface material at each site were to be obtained
from:
l) the unsaturated zone above the water table
2) the saturated zone below the water table
30
It was not necessary to maintain the ig situ soil structure or density
closely, an objective usually of primary concern in a geotechnical
investigation. For the sampling work the following goals were
applicable:
l) Apply minimum shock to the soil
2) Avoid introduction of any non-native agent into the soil being
sampled.
3) Extrude the samples at the site by a rapid procedure during which
only sterilized surfaces would he in contact with the soil.
4) Capture the samples in sterilized containers which could be sealed
immediately.
To define the proper techniques for sampling, Professors G.W.
Clough and J.T. Novak of VPI & SU traveled with Mr. J. 0'Brien of ARC0
Chemical to the EPA in Ada, Oklahoma to meet with researchers who had
developed sterile, soil sampling procedures. The EPA methods are
described by Wilson gt gl; (l2). Basically, the sampling apparatus
consisted of a heavy bronze cutting shoe fitted to a thick-walled sample
tube. The shoe has a sharp edge at the penetration end and flares to a
solid cylindrical shape into which the tube is inserted. The entire
apparatus was designed to be pushed into the ground. Because the bronze
cylindrical head is one inch larger than the tube, a gap is created
around the tube as the unit is advance into the soil. To keep the
specimen from falling out of the tube, a six-finger sample "catcher" is
inserted between the bottom of the sample tube and the flared section of
the cutting shoe. The particular sample catcher employed is used
3l
conventionally to retain rock core during oil well drilling. EPA
personnel indicated good success with their sampling apparatus.
However, they had only used it in local clean river bed sands at
relatively shallow depths.
For the ARCO sites, the EPA technique was felt to be inappropriate
on the basis that:
l) The soils were dense, and simple pushing procedures would likely
not penetrate enough distance for sampling.
2) Some of the soils were gravelly, and this would likely damage the
sharp edge of the bronze cutting shoe.
3) Below the water table, the Nilliamsport soils would likely collapse
into the gap created between the cutting shoe and the tube causing
withdrawal problems and possible loss of the cutting shoe.
Alternative sampling techniques were investigated.
A conventional sampling procedure was adopted to fit the project
requirements. The Narren George, Inc. drilling firm of Jersey City, NJ
offered four alternatives: l) an Osterberg hydraulic sampler; 2) a
Denison sampler; 3) a Pitcher Barrel sampler, and, 4) a Dames and Moore
sampler. A split spoon sampler was used in preliminary drilling.
The split spoon sampler is also known as the split barrel or split
tube sampler. There are several modified versions of this sampler but
basically it has a sample retainer located immediately above the barrel
shoe and the barrel in which the sample is held is split longitudinally
for easy sample removal. Split spoon sampling is accomplished by
driving the sampler with a l40-300 lb hammer. It is common practice to
32
record the number of blows for each six inches of sampler penetration.
The number of blows required for a l.5 inch sampler to driven l2 inches
with a given hammer weight can be used to determine the Standard
Penetration Resistance (SPR) of the soil. Split—spoon sampling is
generally used where it is necessary to determine stratification,
identification, consistency, and density of scils at a site (64).
The Osterberg piston sampler (hydraulic sampler) consists of an
actuating piston and a pressure cylinder. Introduction of fluid
pressure on top of the actuating piston executes the sampling by pushing
the Shelby tube into the soil (65).
A Denison sampler relies on a combination of jacking and coring to
obtain the sample. It is made of an outer rotating core barrel with a
bit, an inner stationary sample barrel with a cutting shoe, inner and
outer barrel heads, an inner barrel liner and an optional core retainer
(64).
The Pitcher sampler is basically a Denison sampler in which the
inner barrel is spring loaded to provide automatic adjustment of the
distance by which the cutting edge of the barrel leads the coring bit
(64).
Shelby tubes were the sampling tubes employed and were made of
brass or steel. Several types of samplers were used. The hydraulic
sampler is pushed into place, the Denison and Pitcher Barrel samplers
use rotary drilling and the Dames and Moore is driven. Table 4 lists the
advantages and disadvantages of each sampling method. The Dames and
Moore sampler was not used, however.
° 33
Table 4. Samplers considered for program.
lnsertionSampler Method Advantages Disadvantages
Hydraulic Hydraulic Minimal dis- Limited to looserPush turbance sands, no gravel
Denison Rotary Can drill into Must use drillingDrilling hard materials mud
Pitcher Rotary Drilling Can drill into Must use drillingBarrel or Spring Push hard soil, push mud
into soft soil
Dames and Driven Can penetrate Must be drivenMoore hard soils by hamering
34
Drilling mud is used to maintain hole stability, especially in
sands below the groundwater table, and is required for rotary drilling,
as in the case of the Denison and Pitcher Barrel samplers, to return
cuttings to the surface. while the mud serves to enhance the basic
drilling process, a primary concern was that it might contaminate the
soils. This was thought to be remote because the mud is very viscous
and unlikely to flow freely. As a quality control measure, lithium
chloride was added as a tracer to the mud.
Careful observations were also made for drilling mud penetration
into the samples. No visible evidence of drilling was found inside any
of the soil cores.
Soil Extrusion Apparatus
The extrusion device consisted of a hydraulically powered piston
capable of generating in excess of l000 psi, a dual clamp bed for
holding the sampling tube, and a paring device (Figure 6). As the soil
core was extruded from the sample tube, the first few centimeters were
cut away with a flame sterilized spatula to remove any possibly
contaminated material. The core was then forced through the sterilized,
stainless steel, paring device, which trimmed away the outer one
centimeter so that soil in contact with the sample tube walls was
discarded. The final portion of the core sample was also discarded.
The samples were extruded into acid·washed, sterile, one quart
containers with Teflon-lined lids, and transported in iced coolers to
the Virginia Tech labs for refrigeration.
35 /
— om Azo ‘=.."'
¤<:mmlO _ :-/ ä'°'* 2¤- •-
gt >-_} (IJQ .Jm\ 1m
=*· IEu:0g gr2.2%/X.][UO.
SSS-FUD.
SB}.: E2° Sml =—mz ¤*IQ g-¤-O : E
mg 6-.J |-I an
-*:1:EA
:.*0¢:>-DI
36
Groundwater
Several methods of groundwater collection from wells are available.
All of these are concerned with removing the water without contact with
any adsorptive surface and maintaining sterility. Most employ either
glass or Teflon tubing and a water lifting source, such as a peristaltic
or vacuum pump. This necessitates having a power source and involves
the transport of much equipment into the field. Problems were
encountered with lifting water from significant depths, leaks, and the
continual dismantling of the apparatus to empty the vacuum flask
contents.
The most successful method was the most simple. Acid-washed,
sterilized BOD bottles tied to a monofilament line were lowered down the
well. The bottles were self—filling and could be removed and capped
immediately constituting a sterile undisturbed 300 mL sample.
Field measurements were made on the temperature, dissolved oxygen
level, and pH of the groundwater. Samples were shipped to Virginia Tech
labs in ice-filled coolers. ,
Microcosms
Static Sacrificial Microcosms
A modification was made from the EPA procedure in that smaller test
tubes were used (l3 x l00 mm). This allowed more microcosms to be
prepared from less subsurface material. The sacrificing of duplicate
tubes with time as described by Wilson et al. (l2) was the procedure
followed with the exception that the organic concentration was measured
37
at zero time rather than calculated. The tube microcosms were filled
with soil with a sterile, stainless steel spatula, then dosed with
sterile, substrate-containing groundwater to simulate nutrient levels
present in the subsurface system but, at the same time, avoid the
introduction of additional bacteria. Soil and water were mixed in the
tube microcosm with a vortex mixer so that approximately l—l.5 mL of
water remained at the surface for sampling by direct injection into a
gas chromatograph.
The proposed Nilliamsport, PA test matrix for sole and mixed
substrates is given in Table 5. Fifteen sacrificial-tube microcosms
would be needed at each concentration, ten non—sterile and five sterile
controls. This would provide for duplicate microcosms plus one control
for five time intervals. By completing this matrix, 330 microcosms
would need to be prepared. For this reason alternative microcosms were
investigated.
Static Continuous Sampling Microcosms
The continuous microcosms were identical to the static microcosms
except that the screw-top contained a Teflon-coated, silicone septum. A
sterile syringe was used to remove a small sample (2-5 ut) from the
liquid on the surface of the tube, so each microcosm unit could he
monitored with time. This modification reduces the number of microcosms
and permits a superior assessment of degradation rates.
U <|‘ <|‘<i‘C •
••·-U O..I r—· r— ••¤·· \ <|'\ <'\C)4-* Lhw •—®\fü ><E N
• N •@E N~/ Xlr- xr-?Ö N |*N f"N 'LGDN NN NNO(4-*cQ N •
N •\M ON4-) •
¤—v7 •O
OLJU.C3COC/TOU <.>r~ LO
<E N LDLO LOI-OONN NNNE oo co--
-« C)r— ©:—JZ r- •—
LLIZ
X "U
L (IJ+-* •··—·
•fü UE: s:
•—GJOG) C)4-*L)+> Lw N LDfü CL 3 C) C) •L U4—*®\ I-OLD LOLDO
V) {ONEOO.C13I-» Or- (Du-17 (OO I-' r-vn Lu
Efü••—·4-*•r—C•,-
Q}4.) ••
'U fü •GJ LU«~ LDvr -0-*C..I •O (DON Ol-Dr- Ol-DCPQ „D(.) LO II)o :s EL U) C1
Q. _CJ<L}"BJ• I-
LO
G.r-.D GJfü 4-*
" r-l- fü •
OLU .C-I—*C/~ OWO-I OOP- C)£3•- U
Or- •——3 U
•— •— füu>1E
Ca r—·IIILIJ >, •OE 4-*LIJ 3 O.)E .¤ :
$5 GJr- r-OLQJOJ>>
0} CMCCXC ¤J·r—<1J<DIO (U •-C4-*N3fü
INI C -|·*$-Ce--}-*E EO wruwocvo·¤ oe-: E-•—».¤-•->ELG) L'4-4-* *4-"U II II II II II
E fU GJan •-—L :-4-* I<E¢DI-—><O G3 fUf¤ (DU)U •r·4-* •¤-L GJ}-0 Lw L3 EL (Dm Q)-•-JU 4-*C 4-*fü·•— fü; MU)Z Z E1
39
Circulating Microcosms
These microcosms consisted of l00 grams of subsurface soil
sandwiched between two layers of acid—washed, sterile sand. The
microcosm was filled with water from a bottom septum with a sterile
syringe. Sampling was possible from this port as well. Circulation of
the groundwater was achieved by inverting the flask daily, thus creating
capillary action through the soil and sand.
Bacterial Enumeration
Bacterial populations were enumerated hy Dr. R. E. Benoit and
G. Allen of the VPI&SU Microbiology Department. Several methods were
used. The direct counting method involved epifluorescence microscopy
with acridine—orange staining (l3). When excited by blue light, the dye
fluoresces bright green when present on an organism and dim orange when
on abiotic material. Plate counts were made using soil extract agar.
Plate counts were also made with select media, i.e., formate and methyl
amine, to encourage growth and isolation of C]-utilizing organisms.
Analytical Methods
A method was needed to detect 50 ppb methanol to meet certain state
standards for water. The problem encountered most often at low methanol
concentrations was the difficulty in separating methanol from water
because they are similarly polar. Purge and trap gas chromatography was
thought to be the best method as had been shown by previous alcohol and
water separations (65). This method is time—consuming and would limit
40
the number of microcosms that could be analyzed. Several months were
spent by ARC0 and Virginia Tech personnel in the development of methods
that could measure the 50 pph before actual microcosm studies could
begin. Several options for the analysis of alcohols, as well as
aromatics in water were found to be useful for the investigation.
Gas chromatggrgphy
The compounds to be measured by FID (Flame Ionization Detector);
i.e. methanol, TBA, benzene, toluene and m-xylene; could be separated in
the most efficient manner on a 7 ft x l/8 inch stainless steel column
packed with 0.2 percent Carbowax l500 on 80/l00 mesh Carbopak C. The
instrument operating conditions as listed below:
Injection port - l00°C
FID - 250°C
N2 carrier — 25 cc/min
Sample size - 2 pL
0ven temperature for separation of:
Methanol and TBA - l20°C isothermal
Alcohols + BTX - l20°C for 4 min. to l70°C at 20°C/min.
and hold 35 min.
The separation of all five compounds, as it was performed on a
Hewlett-Packard 5880A gas chromatograph, is shown in Figure 7. The
detection limits with this column were 0.l mg/L methanol, 0.05 mg/L TBA,
and 0.03 mg/L for benzene, toluene, and m-xylene by direct injection.
Low Level Methanol Detection. A 6 ft x l/8 inch stainless steel column
packed with l50-200 mesh Porapak QS at an oven temperature of l80°C was
41
— METHANOL 0.85 min.
TBA 2.8 min.
BENZENE 6.7 min.
. TOLUENE |2.8min.
m-XYLENE 33.5 min.
Figure 7. The separation of methano1, TBA, benzene, to1uene, andm-xylene on a CN 1500 co1umn.
42
used for the complete separation of methanol from water. This column is
capable of handling water-sample volumes of l0 uL. Nhen 5 uL samples
were used, concentrations of 25 pph could be detected but not
consistently. At lOO ppb, reproducability was j 2.7 percent and j l0.9
percent at 50 ppb. Care must be taken to prevent “ghosting" after the
injection of a high concentration sample. It can be eliminated by
purging the column with pure water. To decrease analysis time, the
column was shortened to 5.5 feet and the oven temperature was lowered to
l20°C isothermal. Concentrations of 50 ppb in as little as 2 uL volumes
could still be detected at the most sensitive atteruation setting. A
tracing of the Hewlett—Packard 588OA gas chromatogram is shown in Figure
8. Instrument conditions were the same as with the previous mixture
analysis, with the exception that the N2 flowrate was 30cc/min.
ARCO Alternative Methods. Alcohols analysis can be performed as well on
a 8 ft x l/8 inch stainless steel column packed with 3% SP l5OO on
80/l20 Carbopak B. Operating conditions were:
Injection port - l80°C _
FID - 230°C
N2 Carrier - 2l cc/min.
Sample size - l uL (direct injection)
5 ml (purge and trap)
Oven temperature for separations by:
l) Direct injection of:
Alcohols - 40°C to l60°C at 20°/min and hold 2 min.
Alcohols and BTX - 40°C to 220°C at 20 min and hold l0 min.
43
' éE3|“'
OU
U')O'
ä¢ ¤.{ff E; S
GU
COLGJ4-*ru3'¤„
Cru
I"°OC
2+->9.*
6 1:°‘
.¤E ¤.
¤¤
N oJ 1.0O 11-Z O .
§ 2'17 +§ 92 L
GJua
-2I·-—
1:6GJL3O3LI
44
2) Purge and trap - start program at "Desorb" on the LSC-2 model:
Alcohols - 2°C isothermal for 4 min to l80°C at 20°/min
and hold 5 min.
Alcohols and BTX - 2°C isothermal for 4 min to 220°C at
20°/min d hold l0 min.
A l0 ft x l/8 inch stainless-steel column packod with 5% SP
l200/l.75% Bentone 34 on l00/200 Supelcoport could he used for the
aromatic compound separations using either direct injection or purge and
trap. The instrument operating conditions are listed below.
Injection port - l50°C
FID — l50°C
N2 carrier — 30 cc/min.
Sample size - l uL direct injection
5 mL purge and trap
Oven temperature for separation by:
l) Direct injection — 80°C isothermal for 5 min. to l20°C at
5°/min. and hold 7 min.
2) Purge and Trap - start program at "Desorb“ on LSC-2 50°C .
isothermal for 5 min. to l20°C at 5°/min.
Purge and Trap Operating Conditions for Tekmar Model LSC-2 Automatic
Sample Concentrator:
Trap - l2 inch x l/8 inch 0D (0.0l0 inch wall)
stainless steel, filled with 6 inch Tenax and
3-l/4" silica gel
Purging vessel - 5 mL firt type
45
Purging temperature -60°C (used Tekmar jacketed 5 mL sampler and”
Brinkman/Lauda RM3-S recirculating water bath)
Purge gas - N2 at 40 cc/min.
Purge time - 12 min.
Desorb preheat - l80°C
Desorb - l80°C for 4 min.
Bake - 220°C for 7 min.
This same column was used for alternative direct injection BTX analysis
at the Virginia Tech labs but the oven temperature program was modified
to 70°C isothermal for 3 min. to l00°C at 6°/min and held for 3 min.
The resulting chromatogram is shown in Figure 9.
Standards Preparation
Alcohols. Methanol and TBA stock primary standards were made by
adding each to volumetric flasks by weight and diluting to volume with
water. Dilutions to any concentration could then be made. Alcohols
were found to be linear in area response from 0.1 to over 1000 mg/L when
analyzed by gas chromatography. _
Argmgtjgs. Benzene, toluene, and m-xylene are all insoluble in
water (66); thus, it is very difficult, if not impossible, to prepare
accurate standards of these aromatics in water. All three, however, are
infinitely soluble in each other and methanol.- Since methanol elutes
early in the chromatogram of these compounds it was used as the media to
contain the matrix standard. Benzene, toluene, and m-xylene were
desired in the concentrations of 7, 18 and 14 mg/L, respectively, in
microcosms due to their extractability in water. In a small septum
46
_:‘¤L«¤·¤c:eu~•->ua7-“2c GJ
E E-O 1E.1
ag< $6I :0!··· _ :3 _ E4LuE
E 14 E gg02 **0 °'° E?cu u.1 0* ggg;111 Ü ä'
QBE 3 w CE-1 ..] on.*2* G >- cmL;] P )f ’_c>¢¤CD -1::E 2:
¤.oäiäwä1'ZE
o4cuL.ä
LZ
47
sea1ed via1 a mixture of 0.7, 1.8, and 1.4 grams of BTX was added byU
syringe. If 0.39 g of this mixture is added to 100 mL methano1 in a
septum stoppered vo1umetric f1ask then 0.07 g benzene, 0.18 g te1uene
and 0.14 g m-xy1ene has been added or 700, 1800, and 1400 mg/L.
Di1utions can be made to any desired concentration. Linear area
response was found as with a1coho1 standards.
Lithium Ana1ysis
Lithium ch1oride was used as a tracer in dri11ing mud to monitor
potentia1 contamination of soi1 core samp1es. Soi1 samp1es were dried
in an oven at 150°C for 3 hours then p1aced in a dessicator unti1 coo1.
1ithium was extracted from the soi1 by digesting approximate1y one gram
of each dried samp1e in 5 mL of concentrated nitric acid for three hours
then mixing with 5 mL of disti11ed water. Each samp1e was fi1tered with
a 10 mL Buchner funne1 containing a g1ass—fiber fi1ter to remove
particu1ate matter. A b1ank was prepared with acid and water on1y.
Samp1es were ana1yzed by atomic adsorption spectrophotometry.
Anion Ana1ysis ,
Phosphates, ch1orides, nitrates and su1fates in the groundwater
were ana1yzed by ion chromatography in the Virginia Tech Bio1ogica1
Laboratories.
Microcosm Dosing
The weight of soi1 p1aced in each tube microcosm was known, as was
the soi1 moisture content. After the microcosm was dosed the weight
(m1) of added water was a1so known. The soi1 moisture (percent moisture
x weight soi1) p1us the added (dose) water equa1s the tota1 water
48 I
present in the microcosm. Then CIV] = C2V2, where C = concentration andV = volume, can be used to determine the concentration of substrate
needed in the dose water. For example, if the dose water added is l.5
mL and soil water is 2.7 mL (i.e. l8% soil moisture x 15 g soil per tube
microcosm) then total water is 2.7 + l.5 = 4.2 ml. If the desired,
final concentration in the microcosm water is l00 mg/L then solve the
equation for C], or dosing water concentration.
Known parameters:
C2 = l00 mg/L (desired ccncentration)
V2 = 4.2 mL (total water in microcosm to be at l00 mg/L)
V1 = l.5 mL (dose water added)
Solving:F = CgVg = (l00 mg/L)(4.2 mL) = 280 mg/L'l V] l.5 ml
Therefore, the concentration in the stock solution should be 280 mg/L so
that when it is diluted by the soil mixture, the final concentration
will be l00 mg/L.
RESULTS AND DISCUSSION
Soil Sampling
A hydraulic sampler was used to push a thin—wall, Shelby tube into
the soil. Because a vacuum was generated at the head of the sample
during the insertion of the tube using the hydraulic sampler, no
retainer was necessary to hold the sample in the tube. Samples of sand
were readily obtained by this method.
Sampling in the gravelly sands proved to be difficult, because the
"gravels" ranged from small pebbles to sizes exceeding the diameter of
the Shelby tube. These conditions ruled out the use of the hydraulic
sampler, and made sample recovery, even with either the Denison or
Pitcher Barrel, which work well for very dense sand and clays,
impossible in some cases. In many areas, the sand component turned out
to be very coarse and loosely packed, and even when using a retainer
ring at the bottom of the sampler, the soil would flow out of the tubes
resulting in sample loss. Only very limited success was achieved at
sampling gravelly sand. Generally the best results were obtained where
a sandy layer was encountered within the gravel or gravelly sand.
The field operations showed that all three of the samplers included
in the work could be used to obtain suitable specimens for the research.
The hydraulic sampler proved to be the best device because of its ease
of use. However, it is limited to sands which are essentially free of
gravel, and it may not be applicable in deeper holes were confining
pressures make the sand too stiff for it to penetrate.
49
50
Extrusion Apparatus
Generally, the system developed by Warren George Co. performed
well. However, two problems were encountered. First, because of
frictional resistance of soil in the tube, the clamps were unable to
hold the tube in place. Once the tube slipped, the extrusion device
—would no longer work. It appears that a retaining device should be
added to this system.
A second problem occurred when the Pitcher Barrel sampler was used.
This sampler uses a steel tube instead of brass. This tube is about 3/4
in. longer than the brass tube and would not fit into the bed of the
extrusion device. Therefore, all tubes required hand cutting of the
lower inch with a hacksaw before extrusion. Movement of the cutting
device will correct this problem.
Lithium Tracer
It was found that lithium concentrations in the same sample could
vary based on the extraction method employed. The tracer lithium in the
drilling mud should have been adsorbed by the soil if exposure occurred.
Lithium was added to the drilling mud at lO mg/L at PA and 45-50 mg/L at
VA and NY, respectively. However, naturally occurring lithium could be
_ extracted to varying concentrations based on the contact time
(digestion) in concentrated nitric acid causing some variation in the
resultant concentration. The samples either had to be completely
digested which requires extensive digestion time and the use of more
hazardous acids or all extracted for an equal time. An equal extraction
51
time procedure was selected. Analyses indicated that no lithium
contamination had occurred based on comparisons of split spoon samples
where no mud was used, and center core and pared core samples taken with
lithium dosed drilling mud. To further insure these results, samples
were analyzed from various depths of dosed and non-dosed drillings. The
results are shown in Table 6. The New York soil presented a unique
problem. During acid digestion the high concentrations of marl in the
soil reacted violently resulting in the loss of several samples.
Minimal non—dosed soil was collected at New York and the remaining
samples were required for microcosm preparation. Lithium was present in
the ppb (< l mg/kg) range in most samples and no drilling mud
contamination was evident.
Subsurface Bacterial Populations
Significant bacterial populations were found to be present in the
subsurface systems of all sites. The results of acridine-orange direct
counts and plate counts are listed in Tables 7-9. A general observation
can be made for all sites based on Figures lO-l2. Acridine-orange
direct counts were consistently higher than the soil extract and soil
extract plus formate and methylamine plate counts. A slight decrease in
bacterial numbers is evident in the unsaturated zone by A-0 direct
counts for Pennsylvania, while a decrease of four orders of magnitude is
shown for all plate counting methods in Figure l0. A similar response
is seen for New York with one order of magnitude decrease shown for all
counting methods from the surface through the saturated zone. The
52
Tab1e 6. Lithium ana1yses for Ni11iamsp0rt, PA, Nayland, NYand Dumfries, VA s0i1s.
Y Lithium Dosed Non-dosedH01eN0. Üeth(ft‘ C0r~c.1m 7l? 1 0 e No. se th ft
PA 1 7 0.31 PA 2 6.0 0.240.21 0.21
PA 1 8.5 0.22 PA 2 8.0 0.160.21 0.16
PA 1 9.0 0.33 PA 2 10.0 0.180.40 0.17
PA 4 9.0 0.19 PA 2 12.0 0.320.29 0.35
PA 3 36.0 1.171.08
PA 5 8.0 0.180.17
NY 1 4.0 1.06 NY 1 0.5 1.051.29
NY 1 4.0 1.06NY 1 6.0 1.98
1.86NY 2 3.0 0.77 .
0.94
VA 1 17.0 0.14 VA 1 12.0 0.230.11 0.29
VA 1 17.0 0.14VA 2 54.0 < 0.04 0.11
< 0.04 VA 2 13.0 0.120.10
VA 2 32.0 0.100.11
VA 2 57.0 < 0.04< 0.04
VA 2 102.0 < 0.04< 0.04
53
N ¢\| ÜO O Gn- •—— N
X X X<2] (\| I-D U1
+ CJ
N1.1.1+1 +1 +1• (/)<[ N N kOQ_ • • •
¢\| O1 C')••
+-*LOO-VIE {O (\l (*7cu • CD O O·•—
> N N r-N GiN X X X•¤-
2 · L1. N xo ao'U
• • •
4-* -6-* + O O<t‘
fü V7 ¤.« +1 +1 +IN + V7•r- N N 00 RDO
•,- • • •
w O GD ON ROU)
GJUfü N
'+·- 3L- '·6- N O') LD3 U C) G GU'! N N N-D3 X X XU) L
0*) N ä füQ • • • U)
•r·— LLJ G ¢\| E fü1/)
c; +1 +| +1 S-mO -6-* IOC•«- O LO Ü C U'l·•··+4
• • •3 ggg
fd (*7 (V7 N EG füN fOU GJE L -I-*ND. C7-6-* fO>~,
Q. IDG) L--6-*N N N -6-*L-,CJGJ
N G O CD ·•··••— ‘+—E
(U N N N C°OL-••- 3 fO++L- X X X GJCDGJ U7U7fO-6-*-6-*
-6-* O'! <' N CC UUU
• • • ·¤—fO-6-*fOfUeu CQ 1- •— N Es.us.s.
Q3 <L S-OfU·6-*-6-*+1 +1 +1 cn:-><><
*6- GJ+-*GJGJ• LO 05 KD CX|\ • • • >$•r—·GJNN
LO C") Ü C'O •r···•—
GJO•r—•—-OO
N L••-meh.Q OUOILO Ufüvll I
N N ||| 1N I\ ¤-- U-EE
• • Q')N' (V7 O3 N ++-|-J C1 x.1 3'·6— '~|•—©l-A-|I.|.IUJ
Q N QN (V)
-6-*
GJQ
1
. 54
Table 8. Bacterial popu1at1on 1n subsurface $011 at Wayland, NY.
cfu/g $011 (+ SD)
Degth ft(mQ A0 SE SE + F SE + MA
0 1.0 X 108 1.0 X 107 9.5 X 105 8.3 X 106
I 4.1 X 107I 4.0 X 106
I 2.7 X 106I 6.5 X 106
6 (1.8) 7.6 X 107 9.3 X 105 9.0 X 105 8.5 X 105
I 3.8 X 107 I 1.1 X 105 I 8.4 X 104 I 3.0 X 104
12 (3.7) 8.0 X 107 1.1 X 106 3.1 X 105 1.2 X 106
I 6.4 X 107 I 8.5 X 104 I 1.0 X 105 I 2.1 X 104
55
LO LD Ü' LD C'S S S S S S
( NO N LO N LD r- {D N RO N <I' N
2 O oo O O O O O• r-X S NX r—··>< NX NX NX
<‘|Z+ N
> Lu XS VXS XN XN XN XC)
•• U) L0<t' Nw Nu- <!'<t‘ Sw <'<\I
U1• • • • • •
aa ·=r+| N+| m+| m+| •-+| r~+|•r-
L*4-E3S
4-* >I5 GJ
'U (*0 (*7 ü' LO I-K7 C')
N S S C) S S S·•- • (*0 N <' N LO r- kO N LO N ü' N
O 'D LI- S (O S S S S S
V7 JJ NX S r-X NX NX NX NXV} + N
0) XI-D XEO XLD X¢\l XN XUN
Q + |_;_|•
V• • • • •
EV7 N(*7 ¢"’)(*) I-Dw (*0N NN NÜ
-6
<|‘+| •-+| <*>+I <'+Ir—+I N+I
UI VI-¤3 U
V7 X3
C W-•r·· ULO LD (*7 LO <I' Q'
C S S S S S S
O RO N RD N LD N LO N LD N LO N
·r·S(*7JJNX S NX NX NX NX NX
AU Nr- UJ ><N ><S
><•—- ><(\I Xt") ><w
=(_/) •
V• • • • •
g-NLI7 (*0¢' KON
€\|UN SLO NCJ
¤. ow+| c·o+| m+| ¤.r>+| cx+| -0-|+|
N(U•r-
S-Q14-‘
U„
(Udh N N N N N N N
S S S S S S SCO N N r- N N
I‘~N N N N N N
• O O O O O O O
UN NX NX NX NX NX NX NX
GJ XN XN X(\l X¢' X'<f' X§O XI-D.D SC (*'7(*7 •—·(\1 ON00 NED (*0LO w<2'gg
• • • • • • •
|--·•-+| r\+| m+| m+| •-+| r~+| <r+|
z- ¢·~ rxN ¢·~ rx d' d' g-
§' (V) g- • • •
E• • • |\ qg- r-
-/ (*0¤‘ Ch
•— t\I C")
-|J S S S S S S
'+-S r- <' S N S CQ
C N N (*7LO4-* N
GJS
56
C) ¤ A9 •
1•
$2 • l\()Z: 0 SIE
+I',_ A0.uJC3 2<D
3() 8IO' ,02 105 IO4 105 IOS IO7 IO
BACTERIA/9
Figure l0. Bacteria found with depth by Acridine-Orange (A0) directcounts and soil extract (SE), SE plus formate and SE plus
· methyl amine (MA) agar plate counts of Pennsylvania soil.
57
O 0/o •„ /
f'\4-w-
N-!
1 IOI-O. 0 •UJC3 •AO
¤SE¤SE+F^SE+MA
2OI
5 6 7 8IO IO IO IO
BACTERIA/g
Figure ll. Bacteria found with depth by Acridine-Orange (A0)direct counts and soil extract (SE), SE plus formate,and SE plus methyl amine (MA) agar plate counts ofNew York Soil.
0/
m
.D
¤
9gi
9
E
Ä
:^
§
#:5
{[1]+2
n
O
Sw
0
g°g
0
(D
·
QM
9
w
S;
cx:
—
gg
0
3*
G12
an
°‘¤_
9
<
S
0
GE
$1%'
Q-°-° I1‘j
EE
0
nn
(
ao
0
m
Ö3
1
.z~„,2
Hw-
..Lda
¤C$
0
$35
¤
—
Z5-?
4-FU);
0~
;Z.°}°
O
O
O
CI
07
'CiCU‘”
ä
ii
59
Virginia site consisted of an upper unsaturated zone to 45 feet followed
by a layered aquifer, i.e., alternating saturated and unsaturated soils.
Figure 12 reflects this by all counting methods. The plate counts
exhibited a dramatic decrease in the upper zone and this may be a
reflection of the unnatural fill material present through this depth or
an indication of the effect of dehydration upon bacterial numbers.
Acridine-orange counts seem to more accurately reflect the
microbial populations of the subsurface. Plate counts may not provide
the proper conditions for the growth of organisms that are adapted to a
subsurface environment. This is shown by Figures 10-12 where plate
counts follow the same trends as the direct counts except at a lower
number.
Methylamine and formate were added to the soil extract agar to
encourage the growth of C1 utilizing organisms. This seemed to make
little difference with the exception of the New York site where the
deepest formate count was significantly lower.
Past incubation counts made on Pennsylvania microcosms that had
been dosed with 100 mg/L methanol and 10 mg/L TBA demonstrated a 10 fold
increase in bacteria numbers confirming the substrate loss in the
microcosms as shown in Table 10. Had this not been done with saturated
zone samples there exists the possibility that increases in bacteria
numbers could be a result of rehydration.
Generally, it can be seen that in the Pennsylvania subsurface 106-
107 bacteria exist per gram of soil by the A—0 direct count throughout.
In New york 108 bacteria per gram were present on the surface while only
60
Table l0. Bacterial population in williamsport, PA saturated zonemicrocosms after a 30 day incubation.
Uncorrected cfu/gSamgle AO Direct Count + SD SE Medium + SD
Original 4.6 i 2.7 x l07 l.4 j 0.8 x l05
Microcosm + l00 mg/L 9 7Methanol l.6 j 8.2 x
l0’2.4 j 0.3 x l0
Microcosm + l0 mg/L 8 6TBA l.9 j 9.5 x l0 l.3 j 0.5 x lO
61
106 were found at Tower depths. In Virginia, as in New York, the
surface was 108, however, the subsequent profi1e was between 107- 108.
The types and numbers of bacteria present a1ong with the aquifer
characteristics can be used as a qua1itative predictive too1 for
decontamination of an aquifer. There exists the future possibi1ity of
integrating microcosm data with direct counts and an aquifer
characterization so that an approximate remova1 rate for various
compounds can be more rapid1y obtained.
Microcosm Dosing
Dosing experiments were performed using Wi11iamsp0rt, PA soi1 to
determine if the theoretica1 concentration by the CIV1 = CZVZ formu1a
w0u1d prove correct. The soi1 moisture was found to be approximate1y 18
percent. Triplicate tubes were dosed with s01utions containing methano1
and TBA to give theoretica1 concentrations based on soi1 weight and
added water. Theoretica1 versus actua1 measured concentrations after
mixing are 1isted a1ong with margin of error (Tab1e 11).
The experiment was repeated, based on the amount of water that
sh0u1d have been in the microcosm tubes to yie1d the measured
concentrations. The dose so1ution was physica11y added, so the error
must have been in the soi1 moisture va1ue. The dose s01uti0n
concentration was 1owered to agree with a soi1 moisture content of 8-10
percent. The resu1ts are given in Tab1e 12.
The measured and ca1cu1ated concentrations agree we11 with an
average difference between the two of 3.7 and 6.2 percent for methano1
62
Tab1e 11. Theoret1ca1 and measured concentrations of Methano1 and TBAin wi111amsport, PA unsaturated zone soi1.
Methano1/TBA (mg/L)Tube No. Ca1cu ated Conc. Measured Conc. Percent Error
1 98.9/99.1 142.1/146.3 43.7/47.6
2 87.6/87.8 131.8/135.1 50.4/54.0
3 97.8/98.0 -- /160.5 -- /63.7
63
Table 12. Theoretical and measured concentrations of Methanoland TBA in williamsport, PA unsaturated zone soilafter lnwering dose concentration.
Methanol/TBA (mg/LQTube No. Calcu ated Conc. Measured Conc. Percent Error
1 100.6/104.3 101.4/93.3 0.8/10.6
2 119.6/124.0 120.5/116.9 0.8/5.7
3 102.3/106.1 109.1/109.3 6.7/3.0
4 108.2/112.1 105.9/108.5 ?.l/3.2
5 114.5/118.7 104.7/110.8 8.6/6.7
6 98.6/102.2 102.2/110.6 3.7/8.2
64
and TBA, respectively. Sandy soils from the saturated zone had a
moisture content of approximately 20 percent, and calculated
concentrations using actual soil moisture agreed well with measured
concentrations as seen in the degradation data of dosed microcosms in
the Appendix. It appeared that some moisture in the soil from the
unsaturated zone must be "bound" in the silts and unavailable for
mixing, while water in the sandy, saturated soil must be free. These
results reaffirmed the belief that each microcosm must be analyzed
initially to obtain a true zero time substrate concentration.
Groundwater Parameters
Groundwater parameters for both Nilliamsport, PA and the wayland,
NY and Dumfries, VA sites are listed in Table l3. The sites are quite
different in most respects, which is useful for degradation rate
comparisons.
Biodegradation
The subsurface systems investigated here are of varying nature as
seen in Table l3 groundwater data. The Pennsylvania aquifer was of an
aerobic nature and contained relatively high nitrate and sulfate
concentrations. The New York and Virginia sites were anaerobic by
comparison with only the New York aquifer containing significant sulfate
levels. In considering the potential for degradation the aforementioned
conditions are of importance since an aerobic reaction yields more
energy in comparison to anaerobic respiration and fermentation. During
66
preparation and mixing of microcosms some air is inevitably introduced.
However, because the average groundwater dose is 4 mL the total amount
of oxygen in solution is minimal even if considered to be at saturation. 4
The anions, nitrate and sulfate, then are important electron acceptors
for the promotion of anaerobic respiration. Stoichiometric equations
for the utilization of methanol-by aerobic and anaerobic respiration,
and fermentative pathways can be written if nitrate and sulfate are
considered to be taken to nitrogen and sulfide, respectively.
The equations describing the metabolism of methanol are:
aerobicHigh Energy CH30H + l.402-————-—-» CO2 + 2 H20
_ anaerobicCH30H + NO3 · CO? + 0.5 N2 + 2 H?0
=anaerobic =CH3OH + 0.75 S04-———-————» CO? + 0.75 S + 2H?0
fermentationLow Energy 2CH30H —————————-~ l.5 CH4 + CO? + H20
From the groundwater data and assuming saturation of the dose
groundwater with oxygen at time zero the amount of methanol that can be
utilized by aerobic and anaerobic respiration can be calculated. The
Pennsylvania aquifer electron acceptors could stoichiometrically account
for 40 mg/L of methanol utilization, while New York and Virginia could
utilize 23 and 4 mg/L, respectively. The rate at which these reactions
proceed can be quantified by the determination of the associated
65
Table 13. Chemical analyses of groundwater at the Pennsylvania}New York and Virginia sites.
LocationParametersCl',
mg/L 9.94 8.00 12.24
BR”, mg/L -— 0.06 0.32
NOS, mg/L 53.40 1.54 0.11
NOE, mg/L —- 1.20 0.56
S0ä”,mg/L 27.64 52.00 8.10
P0ä”,mg/L ND ND ND
Fe2+,mg/L 0.44 0.05 4.23
Ca2+,mg/L 22.60 73.80 2.88
Mg2+,mg/L 4.65 14.40 3.88
Na+ ,m97L 2.78 33.40 13.50
K+ „m9/L 1.09 V3.50 4.54
TOC, mg/L 1.0 1.7 1.3
TOX none none 50.0 ppb
Alkalinity, mg/L as CaC03 none · 180 none
Dissolved Oxygen, mg/L 6.7 0.7 0.2
Temperature (°C) 11.0 10.0 10.0
pH 4.73 7.81 4.50
67
biokinetic constants. The Michaelis-Menten equation is the basis for
defining enzyme kinetics for reactions involving an enzyme and
substrate. To describe an enzymatic reaction the coefficients Km and
Vmax are required. These represent the Michaelis constant and the
maximum reaction velocity, respectively. Km and Vmax can be determined
from a plot of the velocity versus substrate concentration. Km, also
known as KS, is equal to that concentration of substrate that will allow
the reaction to proceed at one-half of its maximal velocity. where [S]
represents the substrate concentration, the velocity, v, is determined
by the Michaelis-Menten equation.
V =Vmax[S]
[S]+Km
The reciprocal of both sides of this equation yields the Lineweaver—Burk
modification.
1 . li1VVmax SVmaxA
plot of l/v versus l/s will permit a graphical determination of Km and
Vmax and can verify the results of Michaelis-Menten plots when dealing
with scattered data. An equation of the same form as the Michaelis-
Menten equation was proposed by Monod to explain specific growth rate
where p and umax were substituted for the velocity terms and the KS gave
the substrate concentration that would result in one-half the maximum
growth rate. This equation is not necessarily related to a specific
. 68
biochemical mechanism but represents an observed response for either
mixed substrates or mixed biological populations or both.
The modified Monod equation, commonly used to descrihe wastewater
treatment kinetics describes the specific substrate utilization rate as
a function of the limiting nutrient concentration (70). The general
equation form of the equation is:
_ k [SVQTE?1
where:
[S] = substrate concentrationg mg/L
k = maximum utilization rate; mg/L/day
KS = half-saturation constant
The value of q may be calculated for steady state systems as
<=,A&IX
where:
AS = substrate utilized;
mg/LAT= time; days
X = biomass concentration; mg/L
In this study, an X value specific for the substrates being studied
could not be determined so values of q are taken as AS/AT are from batch
microcosm data. For cases where a lag period occurred, this portion of
time was not included in the rate determinations. It should he
recognized that variations in q may reflect differences not only in
69
substrate responses but also in substrate specific organism numbers and
these are considered in the discussion of results.
EgmsylywMethanol. Methanol as a sole substrate at l and l0 mg/L was completely
degraded within one week in both the saturated and unsaturated zones.
At lO0 mg/L, methanol was also readily degraded in both zones as shown
in Figures T3 and T4. The biodegradation of methanol does not appear to
be inhibited by the addition of l, 5, and TO mg/L TBA nor by TO mg/L TBA
with 7, T8, T4 mg/L BTX. It has been shown that several methylotrophic
organisms are capable of oxidizing aromatic compounds. This may
contribute to the fact that no inhibition of alcohol biodegradation was
seen in the presence of the aromatics. The levels of benzene, toluene
and m-xylene were chosen based on their extractability from gasoline by
water as found by Atlantic Richfield. At 500-TOOO mg/L methanol was
biodegraded as shown in Figures T5 and T6. The addition of 50 mg/L TBA
showed no inhibitory effect on methanol biodegradation.
Both low and high methanol concentrations using sacrificed versus
septum-capped microcosms gave similar biodegradation results as seen in
the previously discussed figures and by examination of the rates as
listed in Table T4. Therefore, the remaining sites were studied by
septum-capped microcosms for several reasons. These are: less soil was
required which permitted the use of soil from sites where only small
quantities of soil were obtained, and more consistent data were provided
by continuously monitoring the same microcosms over time. Also, the
addition of BTX was difficult with sacrificial microcosms due to the
70
I2O
'OO• METHANOL (Socrificiol mlcrocosmsl¤ + Imq/L TBA (Soc.)v +IOmg/L TBA ($epium capped)A +IO mq/L TBA + BTX ( Cap.)
BO ·I
^ 60 '(I O
E ' °
ö' 40 _,Z •( .II-UJ "E 20 '
'
0 = ‘ ;O IO 20 30 40
TIME (days)
Figure I3. Methanol biodegradation in the Pennsylvania unsaturatedZONE.
7]
‘
aiCoN
O ·¤an m+>
4
CU*5
Q',}+-•
E ,„
3
gE
td
*.0
8 ¤¢2 E
6 8***
·¤
Eug
Z
-s"
Ü
.2^.=z§
g
53ä%<¤
E
8*;;'°
E-E
¤V
—••-*V"
E
•-J.!
^ s:
.<(.I\\
en O
\¤‘U‘
>‘ °•·
I°‘E6° +*
4P-E
• ()‘¤ cu
mmgg
NVZ+++ul *6,
.
*" o• E
OBü°•‘
• P
·•->E
0 O
é
<rO g
bw
3
LI.
72
E”T^
3'öggg cu•.
GJ
-*..1 S@O_|-J NZz\\ . E<<U*¤° O UI:1:EE *2 EÜ"}- O :Wtuggr)
c•¤><I
”gf!}
E
O.
LU 4%° 2rg .. E
" 6
I QrO
I': 4
·
C
GJ
U 46 . O U;
Ö O Q··‘
O O gg
LI.
(-]/6***) -lONVH.LHl/NI
73
6- 0A6 8 O ‘,_;¤.„„ N
=·8l,. HI
..1..1 I00 Q
„;
<< E
N
fiii Q O E
gw no E EE + 3
OU PulE
E
EO r~Q il §
GJ§ 6•
Lu .,5
E .§" E
(U¤• > CA ä,• • *0 'g
E
P•
¤••CQ
ga
S g <> 29 ¤¤ E
LL
('I/bw) “|ONVH.L3W
74
Table 14. Utilization rates (mg/L/day) for the Pennsylvania subsurface
Methanol
Unsaturateä Initiai Saturated InitialConcentration (mg/L) Rate Concentration (mg/L) Rate
106.2 4.19 115.6 4.51112.0 + lTBA 6.01116.1 + 5TBA 1.32 111.0 + 5 TBA 3.87111.1 + 10TBA 11.88* 157.9 + 15 TBA 20.38*92.9 + 10TBA + BTX 4.34* 118.5 + 10 TBA + 5.62*
BTX
577.0 2.11 1212.0 9.41929.0 2.63* 843.0 5.61*738.C + 50TBA 7.49717.0 + 50TBA 4.54* 683 + 50 TBA 6.44*
TBA
1.14 0.21 0.97 0.042.15 0.383.36 0.583.71 0.384.42 0.394.86 0.485.12 0.525.55 0.476.23 0.45 5.39 0.11
10.17 0.27
1.01 + 100 Me0H 0.016.20 + 100 Me0H 0.01 5.14 + 100 Me0H 0.119.61 + 100 Me0H 0.27* 15.1 + 100 Me0H 0.14*7.46 + 100 Me0H + BTA 0.20* 7.75 + 100 Me0H 0.02*
+ BTX70.8 0.26 100.8 1.7780.1 2.22* 128.6 0.67*49.4 + 700 Me0H 0.3148.3 + 700 Me0H 0.21* 45.3 + 700 Me0H 0.28*
*septem-cappeö microcosms
75
volatility and low solubility of these compounds in water. However,
they could be injected directly using a septum cap minimizing any chance
for loss due to volatilization.
There are several observations that can be made concerning the
plotted biological utilization rates. Methanol biodegradation begins
immediately in both the unsaturated and saturated zones and the data can
be represented as a zero order reaction rate or as a first order decay.
There is also considerable scatter in the rate data and this may reflect
organism variations, particularly since some organism clumping was
observed microscopically. In order to resolve some of the variations in
reaction rate and data scatter, a kinetic analysis was conducted.
A plot of q versus S (Figure l7) was used to evaluate the kinetic
response. Both unsaturated and saturated data are indicated. No
obvious rate order could be seen from the data in Figure l7. For
aerobic systems the maximum substrate utilization rate, k and KS, are
depressed at lower temperatures (70). This indicates that methanol
would be removed at a much greater rate from aquifers with a higher
temperature since these values approach those for municipal activated
sludge. The data shown in Figure l7 exhibits some scatter. This could
be a reflection of differences in organism populations or changes that
occurred in the microcosm over time. Initially, aerobic conditions
prevailed. A slow conversion to an anaerobic state then took place as
the oxygen was depleted and nitrate and sulfate were used as electron
acceptors. If any microcosms contained an undetected air space,
continuing aerobic conditions may have existed. This is a possible
76
c
I"
2 ° 2 2•¤lu O
'“f,
6 N äN 3 fs
o xS 'E“'
D•Q10:
F or ß 2D F5 E 22
‘€iZ ° O -¤ 8
U) D • O U• 00tuM ·
E M-:ooUIc
.°.‘,ESCE';6 EEBE
° SS°
0* cp 00 .
N
*0 O IO O anQO
S?(\1 N .. ... g)
‘ II(KOP/"I/ÖUJ) 3.LV¢‘:i NO|.LVZ|“||.L0
· 77
explanation for the outlying data. Evidence of anaerobic conditions was
seen in most microcosms towards the complete utilization of the
substrate in that black deposits formed throughout the soil. Based on
the sulfate and iron analysis of the groundwater these were most likely
iron sulfide. It would be logical to assume that if the microcosms were
maintained aerobically for the duration of the study that the time
involved for complete degradation would have been much less.
The rate order for methanol utilization was determined by a plot of
log rate versus log concentration and was found to be zero order as seen
in Figure l8. This is in agreement with the general slopes exhibited in
the arithmetic plots.
Circulating Microcosms. The Pennsylvania continuous sampling sand/soil
microcosms were incubated for one month. As can be seen in Figure l9
the degradation rates here were much slower than in the static
microcosms. These units were to be used originally as a monitor for
utilization rates to determine when static microcosms should be
sacrificed. Their slower rates could be attributed to two facts. The
ratio of substrate to soil was much greater than in the tube microcosms,
and the unsaturated zone soil was very compact and may have allowed
limited water percclation. Therefore, the use of these microcosms was
discontinued.
I§A„
Tertiary butyl alcohol biodegradation was complete within 50 days
at concentrations from l-l0 mg/L as shown in Figures 20 and 2l. At
concentrations from approximately 50-l30 mg/L TBA, complete biodegrad-
78
o SATURATED _E
• UNSATURATED\ I5E? 0L3 IO •tf 0
Z 5•
S2 O<3• 0SE
•P4:3 0I: 0Z3
0
4 IIOO 4 IOOO
METHANOL (mg/L)
Figure I8. Mean value for methanol utilization rates inPennsylvania soil.
79
'ZO \ CIRCULATING0&ICROCOSMSI00
80j VE. Vé 60-¤ V0z VVä 40n- V VV§ V F Tuer;
2Q V Ä; MICROCOSMS
&Q VI0 I0 20 30 40
T|ME(d0ys)
Figure 19. Cqmparison of rates obtained with tube and circu1ating
80
Oco
. 0°‘
.*5-0-*
¤B°öSSS
‘·¢=’
EEE¤>..1..1.1 /4'$’
x\\ ·;,¤*¤•¤ /
Oeg
EEE000 ß
gc
ti/ __ EE
/ . '“§/ · Q .2
/ — .0
J · E/ c g 2
/ d2- 22OCD (Q ¢ N Q in
°—°LI.
(1/¤w>v81
81°
IO
o TBA '8 • +|OO mq/L Methanol
6
A< \UtE 4 \Ä \1-
2 \
~ OO .0 20 gg 40 50
Time <¤¤vS1
Figure 21. TBA biodegradation in the Pennsylvania saturated zone.
82
ation required more than 200 days in the unsaturated zone and approxi-
mately 180 days in the saturated zone as shown in Figures 22 and 23.
The utilization rates (mg/L/day) exhibited variability in a few cases
(Table 14). This may be due to the infrequent occurrence of TBA
degraders in soil or a reflection of their acclimation time for this
particular site.
At the Pennsylvania study area, both methanol and TBA are
degradable to levels below the analytical detection limit. The
biological removal of methanol and TBA from the groundwater in this area
appears to be quite likely if given enough time and continuing
contamination does not occur. TBA, however, is more refractory than
methanol and would therefore persist for a longer time.
In the Pennsylvania unsaturated and saturated zones TBA
biodegradation sometimes exhibited a retarded rate when added with 100
mg/L methanol. At the 1 mg/L TBA concentration a definitive change in
the degradation rate occurred in the unsaturated zone in the presence of
methanol as compared to TBA alone. However, in the saturated zone a
similar rate was noted for 1 mg/L TBA only. A 20 day lag occurred for
the degradation of 5 mg/L TBA in the presence of 100 mg/L methanol in
the saturated zone supporting the initial observation at 1 mg/L in the
unsaturated zone that the presence of methanol may retard TBA
degradation. This phenomenon was also observed to occur at the higher
levels of TBA. At approximately 50 mg/L TBA with 700 mg/L methanol an
extended lag of greater than 140 days was seen for TBA biodegradation.
83
O<I'N
Il
l
O.. Q ·2 I?E S~•- II 3
.| Q TE}\ an 2Q - 3
E .2Ö C
ä ,9 ^ EÄ
" + z— E• ¤ O 3«§‘.’
¤ <¤ Lu §E; E
V S•G -„-• p
/”¤
Ö‘~
. <r ·· 3*•• °glf
E4 /• n gJ F-
••|$|¤ •¤O
Q g2 O <v
2 ¤¤ <¤Er)
3 °. ä.
EI(1/bw) va.;
84
00anII
/ <>E 22'U
ä g S-,5 E•v B2 I O 2
_J Q! E\ ¢¤
§ E(O Ümy, °·
•¤I
mo C'U -«—Q,] 2
• E .§{Z 2
/ Q 2X <r .9.D
CD$<(I-
6• N
,4
rl Q)
O G: O
L
X <.~! 3 3 3 $ 3 °§‘
('I/Ö"J)V9.L
85
Plots of both methanol and TBA biodegradation when simultaneously
added to the same microcosm are shown in Figures 24-26. It appears in
some cases that TBA biodegradation begins only after the removal of most
of the methanol. This is demonstrated at both high and low concentra-
tions of each compound. Sequential growth seem to most often occur at
higher concentrations of TBA and methanol. This is demonstrated in
Figure 26 where TBA biodegradation does not begin until methanol levels
are significantly lowered. Several mechanisms have been used to
describe the type of reaction shown in Figures 24-26. The kinetics of
enzymatic reactions having two or more substrates are generally more
complex than single substrate reactions since there may be several
enzyme-substrate complexes. Since more than one enzyme is also being
dealt with here it would be mere speculation to suggest which type of
sequential substrate utilization reaction is occurring.
Determination of kinetic coefficients was made using the
utilization rate data for TBA as shown in Figure 27. The response was
as expected for aerobic systems at low temperatures. The data shown
suggest that Monod kinetics may be used to describe saturated zone
kinetics but not the unsaturated response. The k, KS and K for the
saturated zone were found to be 0.6 mg/L/day, 23.5 mg/L and 0.026 day"],
respectively. A log-log plot of rate versus concentration suggests a
zero order rate for the unsaturated zone as shown in Figure 28 with a k
of 0.4 mg/L/day. Since bacterial numbers were found to be constant by
A-0 direct counts with depth it may be postulated that there is some
difference in activity of the bacterial populations in the unsaturated
86
|2O
-
\ IOC
.l\ OCDE O 2_J 6 xC!2 go é<i <II (DI- 4Q METI-IANOL 4 I-I.u 0 O
IOO O 2
‘
O Q• • ) O •
· Q O
TIME (days)
Figure 24. TBA biodegradation in the presence of methanol inthe Pennsylvania unsaturated zone.
87
IO
8I20
,:1
f\B 6 {li —— -0 ¤• „
A X4 ODät P
F-
E 40 METHAN01. A O 2ZS
\\\\\
O 0
0 I0 20 30 40 50TIME(doys) ·
Figure 25. TBA biodegradation in the presence of methanolin the Pennsylvania saturated zone.
— 88
TBA 1mg/ 1.)O O(IJ '<I‘ O
OO
0 N
<I.5
Ü 5IU1
(X E
E75
‘°6
0 Ü 80 EL1J
522 $.3—• 'Ul,. .5%
0Ei00 ,*23
/00
00OO
Q LO
(“l/ÖUJ) "|ONVH.L3 W
89
IO
•
8 SATURATED•
r•O·896
k•O' 59Ks•23·5
K-0-0 2-6
U 4E •
°
2I/k
‘I/Ks0
UNSATURATED4
oV 02”
. °O Oo2 000
0
-20 0 20 40 60 ‘· BO IO02
I/S X IO
Figure 27. Determination of TBA kinetic constants usingLineweaver-Burk plot for the Pennsylvania soil.
I90
IO’,§~ o SATURATEDE
¢ UNSATURATED\cn
OM •L?(IZI
9I—· O .4 O.*2* @6zg —-—-— C)——-C)C)——————————··—·—·—··—·———·*—·"*"*
P- C):> 0
‘0
C) C)
O
4
· I IC) NBC)
TBA (mg/L)I
Figure 28. Determination of rate order for TBA utilization inPennsylvania soil.
91
and saturated zones towards TBA. There is further evidence of this in
the arithmetic plots of concentration against time in Figures 22 and 23.
Qggtrol Microcosms
Problems were encountered initially with substrate loss in the
control static microcosms. Experimental results with Pennsylvania soil
are shown in Tables 15 and 16. It was determined by actual cell counts
that the controls were not rendered sterile by 45 minutes of
autoclaving. A study was conducted to determine the optimum
sterilization time. The various treatments are shown in Table 17.
Extensive autoclaving was required to maintain sterile controls for any
length of time. Even soil placed in a muffle furnace for 3 hours showed
some substrate loss after an extended period of time. This most likely
was due to contamination by the needle introduced through the septum cap
to obtain samples. This problem was corrected by using a heat
sterilizer for the gas chromatographic syringe after solvent rinsiog,
and by wiping the microcosm sampling surface with isopropanol. From the
controls, it can be seen, however, that in the case of alcohols that
disappearance is due to biodeoradation and not to adsorption.
A plot of BTX removal in sterile versus non—steri1e microcosms is
shown in Figure 29. It can be seen that when equally dosed microcosms
are followed with time that there is essentially no difference in the
rates. It is conceivable that BTX in the percentage found in gasoline
could be rapidly adsorbed allowing the more soluble constituents to move
ahead with the water front. The adsorption rate would be expected to
92
Table 15. Residual methanol concentrations in controls from thePennslyvania saturated and unsaturated zones autoclavedfor 45 minutes.
Cnncentration (mg/L)
Time (Days} 0 7 9 11 14 23
Unsaturated 10.4 11.610.0 9.112.7 9.512.8 11.111.7 10.0
111.8 108.4100.2 82.783.0 73.7
124.2 87.2101.9 67.5
Saturated 13.5 14.413.3 16.513.8 ND .8.9 MD6.0 ND
121.7 104.5172.4 ‘ 64.1104.5 73.5150.4 63.086.6 80.2
93
Table l6. Residual tertiary butyl alcohol concentrations in controlsfrom the Pennsylvania unsaturated and saturated zonesautoclaved for 45 minutes.
Concentration (mg/L)
Time (Days} 0 5 ll l9 32 52
Unsaturated 0.9 0.80.9 0.90.8 0.70.6 0.50.7 0.8
5.2 5.24.8 4.75.4 5.45.2 5.35.5 5.6
78.9 70.667.7 53.96l.8 47.865.4 52.9
Time (Days} 0 7 l3 2l 34 54
Saturated l.2 l.20.6 0.8l.l 0.7
l5.0 l2.7l0.4 7.ll4.4 l0.08.0 6.7
l4.5 l0.8
l46.l ll5.5l49.l ll3.5l92.7 l23.9l7l.0 l29.ll5l.3 96.9
94
Table 17. Determination of optimum sterilization of controlsby various treatments.
Methanol Concentration (mg/L)
Time (Days) O 11 26 47
Autoclaving time(min.)
45 70.3 7].999.9 99.389.5 89.089.9 7.9
114.2 124.6
120 99.3 99.2110.6 110.892.4 91.588.1 88.9
115.1 49.6
120 + 180 135.9 136.4 V104.6 104.9170.5 153.5143.8 137.9106.2 83.1
Muffle Furnace 229.4 229.7 231.3 162.5(septum caps) 226.8 227.7 228.3 217.7
95
2
LuJ-2
EE
LIJ
‘”
Lu
ä
J';)O 3
E•N E
u.IZ-
g
I—·Ovl
(DZ,6 g
X ¤ nÄ
I- < 4Lg *9
CU O •E‘§ E
·2 II
g‘Z
§
lO zä
· äS.
unG
E
¤/¤·¤> mg
96
vary from site to site based on the chemical characteristics of the
soil.
The adsorption of aromatic hydrocarbons has been investigated by
others. While one study demonstrated minimal adsorption (67), others
showed that sorption could occur and is related to soil properties,
particularly organic content. Xylene has been found to be sorbed more
readily than toluene and toluene more readily than benzene (68,69). 0ne
investigator demonstrated that the moisture content of the soil aided in
the formation of a pellicular film of gasoline on soil.
llävlexthBiodegradation was studied only in the saturated zone at New York
due to a shallow water table and a soil structure which made sample
collection difficult.
Methanol. Figures 30 and 31 show methanol utilization at initial
concentrations of approximately 80 and 500 mg/L, respectively. The
utilization rates (Table 18) were lower than at Pennsylvania, and
initially exhibited a lag period. The utilization rate beyond the lag
was quite rapid. A lag of approximately 20 days occurred with methanol
as a sole substrate at 80 mg/L. At the same concentration, however,
only a 10 day lag occurred when TBA or TBA and BTX were present. It is
interesting to note that complete biodegradation was accomplished in 35
days when TBA and BTX were added, while 60-75 days were required for
methanol or methanol and TBA. This appears to he an enhancement effect
which could again be related to the methyletrophs ability to oxidize
aromatics. Similarly, for 500 mg/L initial concentrations, methanol
97
OQ)
I1
cn}n.Q cn 8 §
¤ < < §4-»
.: "' l- ÜE Q •¤ =„:
. E + + $• ¤ 4 O ä„ ~* G E/ ä *“‘
'D .1:I *"
F
/u.• _ä
• ¤ E E0 *— EN S
Sr,· _OE'E
EL GJ
9 0 0 0 0 OO E
Q w Q <T N OO")
('I/BW) 'IONVHLBWO'!ni
98
OE
• O .*2 2
ON
gg Efü'2
¤¤ <> Zo G- E 25E c> Zev 07 gE + ° 8
ZO Q " lg
O'-
lgQ (0
. ¤¤ ä E'° GS
O y ¤¤ ·¤• „a/ Ü LU Q(D g. g- .2/ ¤ O ig/0/ ·
<t'.C77 EO O E
m° ä
Qg
Ü Q O Ö O O '-·—*9 so <r *9 cu —
('I/9*-9) 'IONVI-LLEIW
99
Table 18. Saturated zone utilization rates (mg/L/day)for New York.
Initial Concentration, mg/L Rate, mg/L/dag
Methanol
79.2 1.5880.2 + 10 TBA 1.1579.1 + 5 TBA + BTX 2.08
531 2.84512 + 90 TBA 6.92
IM-31.3 1.90 x 10_2
10.5 1.65 x 10_177.8 2.23 x 10
11.8 + 100 Me0H 5.05 x 10:g5.7 + 100 Me0H + BTX 6.52Vx 10_T
88.9 + 500 Me0H 1.40 x 10
100
degradation was great1y increased by the presence of TBA. The presence
of 90 mg/L TBA resu1ted in comp1ete biodegradation in 70 days compared
to 180 days for methano1 a1one. Enzyme inducement is accomp1ished by
the presence of a mo1ecu1e simi1ar in structure to the substrate being
degraded. Since TBA is an a1coho1, but more refractory than methano1,
enzyme inducement cou1d be occurring.
Kinetic p1ots were made for these data and are shown in Figures 32
and 33. The k, KS, and K were 5.4 mg/L/day, 200.3 mg/L, and 0.027
days-], respective1y. The 1arge KS is indicative of the increase in KS
associated with 1ow temperatures and anaerobic systems, as is the 1ow K
va1ue (70). Limited data were avai1ab1e due to minima1 soi1 sampies.
TBA. TBA biodegradation from the New York site as shown in Figures 34
and 35 is s1ower than that observed for the Pennsy1vania site. The
specific rates are 1isted in Tab1e 18. 0nce again there is an indica-
tion of enhanced biodegradation due to the presence of methano1 and BTX.
There is no indication of methano1 preference over TBA as was seen in
Pennsy1vania. At higher TBA concentrations the uti1ization rates were
near1y equa1 with and without methano1 and as a second substrate. A
1og-10g p1ot in Figure 36 of rate versus concentration shows that the
degradation rate is approximate1y first order. Due to the 1ow
temperature (10°C) and anaerobic conditions it wou1d be expected that
the KS wou1d be Targe (71). The KS va1ue of 463 mg/L (Figure 37)
supports this contention. This wou1d make extreme1y 1ow 1eve1s of TBA
very difficu1t to remove. Kinetic p1ots support this. Figure 38 shows
the K va1ue to be 0.0018 day-]. This is much sma11er than the K va1ue
IO]
IOO
•
1'=0·79k=·5·¢I2Ks=2003
•
NC)__
Q
X
Cf\
•
•
· 5 0”
IO 201/6 x I03
Figure 32. Determination of the kinetic constants k, KSand K for methanol in saturated New Yorks0i1.
102
_¢:
'3cE-•-3
OQ ä(O ~4—
·>c-•-•
• g
¢c
. oo
3 *63Eé
E 2ux
Ü öl ~•- äO>-
Q c 31 .9;P ät:
3 -;3l cu ä
+>+>S 8
66oo
• • ‘Q,s.ä„
O {I
O IÜ O
(ADP/7/Bw) 3iVH NO|.LVZI'IIJ.f1
102
0Q
1 0*.9
/ 2Q-~äé / " E
m T5.._•;QSg— 2S° 2•-E • 1:1 0 E”•v
O >‘25 ** g
<tOQ‘ Z
¤=.*29_ f O 2"** ¤¤ 7;;•¤<! >• ‘·‘
G O 3g2O')"‘
3 ·-.Q; <Z
¤ • Q O 1°-E•€Q
9. **9 0 <‘ N 0§’
('I/Ö*“)V8.|.
104
o99
Ü •G
/ 2o 2/ 1-* 1
2 Fi.9 <> ES}! BIDth
gg „ •8 E·__l0 / ..+ ä•|] AZ
GJ1l Ü-EÜ
Q}
, , o•u.§/|-ru
lI„. 9 <r §
DÄ ‘°<
I'1N
¤ U;(V')
1 O gG c> c> o c Q _g»Q ao co <s· cu ·—
V1/ßwxvai
IO5
I
• VIRGINIA• NEW YORK •/°•—| •°
IO•
•• ,ħ ••..I °•EIOE( •[ O
Z.9
9•
'ZiO
.J
EI ° E
IÖ4
INITIAL CONCENTRATION (mg/L)
Figure 36. Determination of rate order for TBA utilizationin the New York and Virginia soils.
106
700 /0
500 .
0
U'\—
0
3000
r = 0-76 Ok= 0-70Ks=463K=O-OO|5
. oo -I00 0:,00
0/ 6
O00-2 0-4 0-6 0-B I·0
I/S F
Figure 37. Determination of kinetic constants k, KS, and K~ for TBA in New York and Virginia soi1s.
107
0-25
9.§ • New YORK_, <> VIRGINIA
B ° /V O- I 5 I- = O.gI o
·I
u1 K= O·OOI8E 0 OzQ1- •
OG O ·
I: O
° 2;.O O
0 20 40 60 80 100METHANOL (mg/L)
Figure 38. Betermination of the kinetic constant K for TBA_ 10 New York and Virginia soils.
108
found for methanol. TBA appears to be much less biodegradable in anaerobic
systems than methanol.
Groundwater. Because of questions relating to the relative distribution
of organisms in soil versus free floating in groundwater, microcosms
consisting only of groundwater were dosed with methanol and TBA.
Tubes containing groundwater only, when dosed with concentrations of
methanol and TBA equivalent to those studied in soil microcosms,
exhibited very slow degradation rates. Results are given in Table 19
for a 180 day period. Minimal loss was found in sterile controls. This
suggests that biodegradation in the water matrix is insignificant.
Qontrol Microcosms. New York controls were autoclaved every four to
five days for 25 minutes each of five autoclavings. Table 20 shows that
these microcosms were effectively sterilized, since no appreciable loss
of methanol or TBA was found. BTX, however, once again was lost due to
adsorption as shown in Figure 39.
Virginia
The Virginia site provided a large quantity of data since drilling
was successful to 31 m. Five depths were investigated because the
aquifer seemed to be a layered one. Utilization rates are given in
Table 21. The data have been presented in several ways. Plots of the
effects of depth upon methanol and depth upon TBA biodegradation are
presented. Treatment effects, that is, single and multiple substrates
at individual depths are also presented.
Methanol. Methanol biodegradation rates in the saturated zone are
similar to the New York site except no lag occurs. It is evident in
109
Tab1e 19. Biodegradation in New York groundwater.
Concentration (mg/L) at time (days)
Comgounds 0 92 180
Methano1 103.1 72.2 50.9
997 757 685
TBA 0.6 0.4 0.1
8.1 8.1 7.6
73.7 75.1 73.5
Methano1/TBA 88.6/7.9 69.7/7.9 50.8/7.9
900/100.8 745/79.6 697/77.9
Methano1 Contro1s 83.4 83.6 76.8
TBA Contr01s 10.0 10.1 9.762.4 64.7 61.0
110
Tab]e 20. Methano], TBA, benzene, to1uene, andm—xy1ene contro1 microcosms for New York.
Concentration (mg/L) at time (days)
Comgounds 0 14 25 69
Methano]/TBA 9].5/1].8 88.8/11.9 86.5/11.6 88.3/11.7BTX 3.],4.],3.3 0.7,].],0.5 ND,ND,ND
101.9/88 100.6/8.6 92.9/8.4 9].7/8.73.2,5.0,4.2 0.3,0.3,0.Z ND,ND,ND
II]
20B T X0 A 0 STERILE
SI5·
* l NON··STER|LE\¤•
EXI- I0LD
5 .
0 5 I0 I5 20 25TIME (days)
Figure 39. BTX removal in sterile and non-sterile New Yorkmicrocosms.
112
Table 21. Utilization rates (mg/L/day) for Virginia.
Degth (ft)
Initial Concentration 11 30 57 80 102
Methanol (m /L)95.1 0.9387.3 + 10 TBA 0.8681.7 + 7 TBA + BTX 0.88
724.0 1.89
79.7 0.84878.0 2.12
68.5 1.0368.2 + 5 TBA 0.9780.2 + 7 TBA + BTX 0.79
691.0 2.36
87.5 2.36996.0 7.89
86.6A
2.3499.9 + 8 TBA _ 2.6794.1 + 8 TBA + BTX 1.47
799.0 3.57
(Continued)
114
Figure 40 that at 24 and 31 m methano1 uti1ization is greater than in
the unsaturated zone. Biodegradation of approximate1y 800 mg/L
methano1, however, on1y showed an increased rate at 24 m as shown in
Figure 41. Since bacteria1 counts showed no significant1y greater
popu1ation at 24 or 31 m than on the surface the exp1anation must be
e1sewhere. The groundwater ana1yses for Virginia did not revea1 a
reason for the increased rates in the saturated zone. It must be that
the bacteria1 popu1ation is more active since it is not subject to
dehydration from the rise and fa11 of the groundwater tab1e or there
exists a greater proportion of the popu1ation that is amenab1e to
methano1 biodegradation. This postu1ation is supported to some degree
by soi1 extract p1us formate or methyiamine agar p1ate counts. An
increase in the popu1ation is seen through this area. However, it is
interesting to note the increased rate when 24 and 31 meters are
considered the saturated zone. Kinetic data for methano1 further
substantiates the activity of the saturated zone. As seen in Figures 42
and 43 the kinetics are different for the two aquifer zones. The k, KS
and K for the unsaturated zone were 2.3 mg/L/day, 118.9 mg/L and 0.019
days-], whi1e they were 5.7 mg/L/day, 158.3 mg/L and 0.036day-]
for the
saturated zone.
Treatment effects as seen in Figures 44 and 45 at individua1 depths
showed no effect at 3.4 and 17 m on methano1 uti1ization rates.
However, Figure 46 demonstrates at 34 M it required approximate1y 62
days to degrade 100 mg/L methano1 in the presence of TBA and BTX.
115
IO
3 .29
0 -1->GJ„-„ / 5
E 1 4 <· CI 9 enI6:
§ Q- ..,°
8 ZE.gu IO m
- N IO ( %’
Q // 2O4 0 <> • ¤ ·
Ü?E
0 OU>
"·•->
/ 2 ä ‘°{l
-3_g _
I ·· :° Q E/ auE Lo ¤/ —- °’
<> i, '·i=‘,,·’
ow-'I CS-
IUD
° äé4 ,1 O
EV!1|•.
=» 6 O QO 0 0 0. Q g co <r cuO("I/Bw)'IONVHLEIW
116
O18°°I*1E
Ga. 0 I>O cu
I lo co Sc
I . Eqj 40,.
m·0
I 1 ¢ '§~ EI 3
'$’
II M 1;;
fä E Qo• E — .9
I E ‘* ¤ E· r~ ·~1· —- cu
S] '°m-NII
IO‘
° °‘
° "61:‘ eu.1:-•->0 ä
Q O O OQ . g O O O O „;·Q Q) (O *4* N ·=r"' zu
L('I/ÖH!) 'IONVH.L3W
117
Illr = C)·E)I
Ok- 2·26ks=n6·9 °/
IO K- O·O|9 000 (PUNSAT¤·
>_O
5 0°°
° ' k 670SAT V = 076 Ks= |58·3, K-0·O36
_IO _5 O 5 IO3
I/S X IO
Figure 42. Determination of kinetic constants k, K , and K formethanol in Virginia soil. S
118
0eä
O E• •- O .5ä E>
9 I- ° 5*-¤- .-E • g A 2<t <l E
O ° cn EÜ (0
E ä*5 S V ““
>£
E 5 O ö' 5cn 0: O Z IZ5 2 *°én.
I-
oc ä E¤' GJ
.C
*5,* IO
V:
E• |ä
E\ 2G 28cm <1· G
E-LI.VH NO|.LVZ|'l|.L0 · ääEI
U9
O¤> 0 „„'6m
•) .9>
O E
D D W
XS
..FOZ
{
ow
§ " E6
F- 9D
• E
‘*' N 0 *6
• >_) P P.,„ ä2 6
DD• 'D F-
»» E
<F' 5- Uä
'A
P)•éé
/“6‘“
> •„E0
O9• N $(6
if?
•0s.
¥giE
O O QO
9 °° <¤ 3 3 0
(-F/ßU·')'|ONVHJ.3|N
120
OQ
'E‘S
3 .2> E
4-*
Sä
» %O
O Egp 'S§
4-*
Xg
_, s- ¤>• » ¤ S0 Q) P. ,;<
( ( P V UJ °°I m cn Q E +
LU ID N ;E + + , 2• V V > Ef;
*46:-,4-*:;UU
O $2
mf"
E13
» ¤• si
O E0 ¤ 0 0 0 0
- Q (D (0 Q- N
2 ('1/Öl") "|ONVH.L3IN
T2]
OO..
4-•«¤
¢¤
E.s.
as 3c.l ID
""
O *
g
Z 4 4ggru
El··•l··•w
w Q §
E + + O .2
• b P(O Eo
U, 4-*
>>• 2§ 2
><lu
»—<> E T
>_ ';
EEE
D,•—
·.
EEQ
·•-•cu QS
· P •u-Ü;zugz»—Ä
>•, zu
O Q an O 0O E
Ü/Bw) '|ONVH.|.3W
l22
Methanol alone or methanol and TBA required only 35 days. This could be
a reflection of population dynamics, i.e., mostly alcohol degraders or
may reflect microcosm differences since the same differences are
demonstrated in Figure 40.
TBA. TBA biodegradation at the Virginia site was very slow. Figure 36
demonstrates that Virginia and New York had the same utilization rates
for TBA and exhibited a first-order reaction rate. The exact rate
values are given in Table 2l. The biodegradation of TBA at 60-80 mg/L
for all depths studied is shown in Figure 47. Lower concentrations of
TBA exhibit the same line slope as shown in Figure 47. Examination of
the utilization rates reveals no treatment effects as was found in New
York. The kinetic data for TBA was shown to be the same as New York in
Figures 37 and 38.
Similarities in kinetic data for both methanol and TBA at the New
York and Virginia sites are excellent indications that anaerobic
aquifers may react similarly to the same compounds. A change from
optimum pH reduces bacterial activity. Based on the mechanism of
non-competitive inhibition, the KS, though, does not change. The
maximum rate is reduced at non-optimum pH. .The pH of the New York site
was 7.8, while at Virginia it was 4.5. Only if these were the optimum
pH for these systems would no difference in the maximum rates be
expected. Temperatures were equal and do not need to be considered in
the comparison.
Control Microcosms. Virginia controls were autoclaved in the same
manner as New York. Table 22 shows that these microcosms were
123
O
b3
PC1O
ui
P•
Qev
°°E
¤ä
I8
og E
E
I
(0
;
.„ K<·‘
ä.5
°"<><>.°,;,°
ä‘ ä0.
M8
¤
Ebo
O
I- E4-*IO
•r>O
Q
¤
Oä
0
Ngg
l> O
E
°¤
ä
_•-
O
.
Q8 O
s
(OO
E
OOJ.
l24
Table 22. Methanol, TBA, benzene, toluene, andm-xylene control microcosms for Virginia.
Concentration (mg/L) at time (days)
Comgounds 0 l4 25 69
Methanol/TBA 74.4/7.8 72.4/7.8 70.0/7.6 73.2/7.8
BTX 7.4,l6.5,7.0 l.l,2.3,l.0 0.6,l.0,0.3 ND,ND,ND
82.2/7.4 67.2/7.5 66.2/7.4 66.5/7.25.5,l6.4,l3.5 l.5,3.5,3.5 0.7,l.0,0.5 ND,ND,ND
l25
effectively sterilized. BTX was lost due to adsorption as shown in
Figure 48.
Risk Assessment
Models are based upon proven scientific principles and are
generally expanded to include various correction factors to give a more
accurate prediction. This is the case in aquifer predictive equations
also.
Several mathematical models have been published which describe the
movement of contaminant compounds with groundwater through aquifers.
These generally deal with the factors of dispersion, adsorption and
biodegradation. These factors can only be accurately utilized when
specific data are available. For example, the organic content of the
soil has been demonstrated to directly correlate with the retention of
many compounds. The heterogeneity of aquifer material then complexes
the determination of adsorption factors. The total adsorption would be
the weighted sum of each aquifer layer down to the aquifer base. More
importantly, the values for hiodegradation rates_are not readily
available and may be site specific.
Even the simplest of several equations presented by Hoeks requires
that extensive data for an aquifer be available. Hoeks used the
equation
L = Q-Yt/(l+R)Co
126
20B T XO A ¤ STERILE
„„I5• A ¤ NON-STERILE
<'U!
EXIOI-m
'*———
O 5 IO I5 20 25
TIME(d¤ys) gFigure 48. BTX remova1 in steri1e vs. non-steri1e microcosms for
Virginia soi1.
l27
as a means of predicting the concentration of a degradable contaminant
at a given time (distance). Where if first order, Y = decay coefficient
(yr”]), t = time, R = Kd/e (Kd = adsorption coefficient; e = effective
water filled pore volume) or distribution factor and directly affects
the retardation term (l+R). If no retardation is assumed
Q. . E-YtCo
This is simply the expression for first-order reactions.
Simplifying assumptions, therefore, can be stated to aid in the
solution of contaminant transport and biodegradation. Groundwater flow
can be assumed to be unidirectional, whether it be horizontal, downward
or upward, and to have some flow velocity which can be expressed in
terms of distance per time. Alcohols are infinitely soluble in water
and since they are not readily adsorbed, the velocity of the groundwater
and the alcohol contaminants can be assumed equal. Dispersion must be
either gotten from field observations or estimated from empirical
relationships. Recent aquifer models have considered dispersion to be
not fully understood and consistently present approaches that do not
include dispersion as a factor in modeling. Deletion of dispersion
would only seem to make predictions liberal in nature.
It is reasonable to use basic plug flow reactor equations to
predict the time required to remove a substrate since reaction order and
biokinetic constants are known and summarized in Table 23. For zero
order reactions:
l28
Table 23. Sunmary of kinetic data from Pennsylvania, New York andVirginia at l0°C.
Parameter
Location Substrate k(mg/L/day} Ks(mg/LQ K(day—]}
Pennsylvania Me0H 6.3 -- —-
TBA saturated 0.6 23.5 0.026unsaturated 0.4 —- —-
New York Me0H 5.4 200.3 0.027TBA 0.7 463.0 0.00l8
Virginia Me0H saturated 5.7 l58.3 0.036unsaturated 2.3 ll8.9 0.0l9
TBA 0.7 463.0 0.00l8
129
ter = %(°„·°«Q
For first-order reactions:
1 (Col)'CPF =171The
amount of time ca1cu1ated can be direct1y converted into
distance from a point spi11 where no contamination wi11 remain, if the
groundwater ve1ocity is known. Assume the effective water-fi11ed pore
vo1ume in the aquifer to be 35%. An initia1 di1ution of the contaminant
can be ca1cu1ated as the concentration at CO based on an estimate of
aquifer vo1ume initia11y affected. For examp1e, if a surface area of 25
x 25 meters had 5000 L of a1coho1 spi11ed and the aquifer extended from
the surface to 30 meters there wou1d be 18750 m3 affected or (18750 x
0.35) 6562.5 m3 of groundwater avai1ab1e for di1ution at time zero. The
initia1 concentration wou1d be (5.0 x 103 L/6.56 x 106 L) 762 ppm by
vo1ume. The 1ength of time required for remova1 is obvious1y contro11ed
by the magnitude and nature of the spi11ed compound, percentage of water
in the aquifer, and the bacteria1 popu1ation.
To compare the effects of spi11s at the three sites investigated
the time required to remove 1000 mg/L a1coho1 was ca1cu1ated for each
saturated zone and is reported in Tab1e 24. It is evident from these
data that methano1 is readi1y biodegradab1e at a11 sites. 0n1y a very
high groundwater ve1ocity wou1d present prob1ems. TBA, however, is
biodegraded quite s1ow1y and cou1d exist in an anaerobic aquifer for
many years. The aerobic Pennsy1vania aquifer required approximate1y 0.4
130
Table 24. Time and distance requirements for the completebiodegradation of 1000 mg/L methanol and TBA inthe saturated zone moving at one m/day.
Time(yrs)/Distance(m)
Location Methanol TBA
Pennsylvania 0.430/158.7 1.21/ 442.0
New York 1.171/426.4 17.52]/6396.0
Virginia 0.88 /319.8 17.52]/6396.0
? - zero order- first order
131
years for TBA biodegradation. In either case for TBA a very s1ow
groundwater ve1ocity wou1d be desired.
The impact of groundwater ve1ocity upon the contaminated distance
is a1arming1y evident when a ve1ocity of 1 M/day is assumed. An initia1
spi11 of on1y 1000 mg/L methano1 wou1d impact 159-427 M. An equa1
amount of TBA wou1d affect 443 M in the best case and 6396 M in the
worst case. The distances were derived, of course, assuming that these
compounds are comp1ete1y degradab1e. A very important consideration is
whether or not a minimum contaminant 1eve1 exists at which bacteria1
uti1ization can no Tonger occur. This certain1y appears to be the case
with TBA at anaerobic sites. The 1ow temperature (10°C) increases the
KS, i.e., the amount of substrate needed to achieve ha1f the maximum
uti1ization rate. For examp1e, the TBA KS for New York and Virginia is
463 mg/L. Therefore, the 1ower the concentration, the Tower the
uti1ization rate becomes. At 1 mg/L TBA the uti1ization rate was
genera11y in the 2x10”3 mg/L/day range. For 0.1 mg/L TBA (100 ppb) the
rate wou1d be 2x10”4 mg/L/day, at 0.01 mg/L TBA the resu1ting rate is
2x10”5 and so on. A minimum biodegradab1e concentration may or may not
be a 1eve1 fit for human consumption. These 1eve1s have yet to be
determined.
SUMMARY AND CONCLUSIONS
Subsurface systems at Nilliamsport, PA, Wayland, NY, and Dumfries,
VA were characterized physically, chemically and biologically. Physical
parameters of the groundwater were determined in the field and
laboratory. Subsurface soil sampling was done in a quality-controlled
aseptic fashion using conventional drilling equipment and samplers. The
quantification of bacterial numbers at all depths sampled to 31 m was
done by acridine-orange stain direct counts, soil extract agar plate
counts and C] agar plate counts to determine if the potential for
biodegradation existed in subsurface systems. The biodegradation rates
of the gasoline additives, methanol and TBA, were studied using test
tube microcosms as was the effect of the gasoline components benzene,
toluene, and m-xylene upon the alcohol biodegradation. These rates were
used to estimate the residence times of the two alcohols in the three
systems investigated.
The results described in this dissertation warrant the following
conclusions:I
1. Bacterial populations exist in substantial numbers to 3l m
with minimal depth effects on these numbers by
Acridine—orange direct counts, however, soil extract agar
plate counts exhibited some population decrease in the
unsaturated zone.
2. Methanol was readily biodegradable in all subsurface soils
examined both aerobic and anoxic.
132 _
133
3. Methanol biodegradation rates were greater in the saturatedI
zones of anoxic systems..‘
4. In aerobic aquifer systems TBA was biodegraded more rapidly,.
5. Methanol exhibited a retardation of TBA biodegradation in some
cases. However, TBA demonstrated no such effect on methanol.
6. Benzene, toluene and m-xylene exhibited no adverse effect on
methanol or TBA biodegradation.
7. Kinetic data were found to be similar for anoxic aquifers and
both aerobic and anoxic aquifers followed established
temperature-substrate utilization reactions.
8. Groundwater contamination by TBA may persist because of its
recalcitrant nature, while methanol can be rapidly assimilated_
by subsurface microorganisms.
LITERATURE CITED
1. Jamison, V. N., Raymond, R. L. and Hudson, J. O., Jr.,"Biodegradation of High-octane Gaso1ine in Groundwater."0eve1opments in Industria1 Microbio1ogy, lg, 305-312 (1975).
2. McKee, J. E., Laverty, F. B. and Herte1, R. M., "Gaso1ine inGroundwater." Journa1 of The water Po11uti0n Contro1 Federation,"gg, 293-302 (1972).
3. Dyksen, J. E. and Hess, A. F., III., "A1ternatives for Contro11ingOrganics in Groundwater Supp1ies." Journa1 of the American waterworks Association, gg, 394-403 (1982).
4. McCarty, P. L., Rheinhard, M., and Rittman, B. E., "Trace Organicsin Groundwater." Environmenta1 Science and Techno1ogv, lg, 40-51(1981). .
5. Fetter, C. N., Jr., A 1ied H dro eo1o . Char1es F. Merri11Pub1ishing Co., Co1umbus, OH, 488 p. (1981).
6. Kobayashi, H. and Rittman, B. E., "Microbia1 Remova1 of HazardousOrganic Compounds." Environmenta1 Science and Techno1ogy, lg,170A-183A (1982).
7. McNabb, J. R. and Dun1ap, N. J., "Subsurface bio1ogica1 activity inre1ation to groundwater po11ution." Ground water, gg, 33-44(1975).
8. Raymond, R. L., Jamison, V. N. and Hudson, J. O., "Beneficia1Stimu1ation of Bacteria1 Activity in Groundwaters ContainingPetro1eum Products." American Institute of Chemica1 Engineers, gg,390-404 (1977).
9. Kappe1er, T. and Nuhrmann, K., "Microbia1 Degradation of thewater-So1ub1e Fraction of Gas 0i1. II. Bioassays with PureStrains." water Research, lg, 335-342 (1978).
10. Ehr1ich, G. G., Godsy, E. M., Goer1itz, D. F. and Hu1t, M. F.,"Microbia1 Eco1ogy of Creosote-Contaminatod Aquifer at St. LouisPark, Hinnesota." Deve1opments in Industria1 Microbio1ogy, gg,235-245 (1983).
11. Hirsch, P. and Rades-Rohkoh1, E., "Microbia1 Diversity in aGroundwater Aquifer in Northern Germany." Deve1opments inIndustria1 Microbio1ogy, gg, 183-200 (1983).
134 _
135
12. Wilson, J. T., McNabb, J. F., Balkwill, D. L. and Ghiorse, W. C.,"Enumeration and Characterization of Bacteria Indigenous to aShallow Water-Table Aquifer." Ground Water, 24, 134-142 (1983).
13. Ghiorse, W. C. and Balkwill, D. L., "Enumeration and MorphologicalCharacterization of Bacteria Indigenous to SubsurfaceEnvironments." Develogments in Industrial Microbiology, 24,213-224 (1983).
14. White, D. C., Frederickson, H. F., Gehron, M. J., Smith, G. A.,Parker, J. H., Nickels, J. S. and Findlay, R. H., "The ground wateraquifer microbiota: Biomass, community composition and nutritionalstatus." Develogments in Industrial Microbiology, 24, 201-211(1983).
15. Chiou, C. T., Fred, V. H., Schmedding, D. W. and Kohnert, R. L.,"Partition Coefficient and Bioaccumulation of Selected OrganicChemicals." Environmental Science and Technology, 44, 475-478(1977).
16. Neely, W. B., Branson, D. E. and Blau, G. E., "PartitionCoefficient to Measure Bioconcentration of Chemicals in Fish."Environmental Science and Technology, 2, 1113-1115 (1974).
17. Wolfe, N. L., Parris, D. F., Steen, W. C. and Baughman,"Correlation of Microbial Oegradation Rates with ChemicalStructure," Environmental Science and Technology, 44 1143-1144(1980.
18. Alexander, M., "Biodegradation of chemicals of environmentalconcern." Science, 244, 132-138 (1981).
19. Wilson, J. T., McNabb, J. F., Wilson, B. H., and Noonan, M. J.,"Biotransformation of selected organic pollutants in ground water."Develogments in Industrial Microbiology, 24, 225-233 (1983).
20. Ehrlich, C. G., Corelitz, 0. F., Godsey, E. M., and Hult, M. F.,"Degradation of phenolic contaminants in ground water hy anaerobicbacteria St.-Louis Park Minnesota USA." Ground Water, 22, 703-710 _(1982).
21. Althoff, W. F., Cleary, R. W., and Roux, P. H., "AquiferDecontamination for Volatile Organics: A Case History." GroungWateg, 42, 495-505 (1981).
22. Westerhoff, G. P. and Uhl, V. W., Jr., "Control Measures forGroundwater VOCS." Water Engineering and Management, 422, 30-33
((1982).
136
23. Bordeau, L. E. and Fulton, G. P., "Purge Aquifer to Remove VolatileOrganic Contamination." Public works, ll3, 72-73 (1982).
24. Hoeks, J., "Analytical Solutions for Transport of Conservative andNonconservative Contaminants in Groundwater Systems.“ Mater, Air,and Soil Pollution, lQ, 339-350 (1981).
25. Enfield, C. G., Carsel, R. F., Cohen, S. Z., Phan, T., and Walters,D. M., "Approximating Pollutant Transport to Ground Water." QgoundWater, 2Q, 711-722 (1982).
26. Grabinska-Loniewska, A., "Activated sludge bacteria participatingin the biodegradation of methanol, formaldehyde, and ethyleneglycol. I. Isolation and Identification." Acta Microbiologia «Polonica, Q, 75-81 (1974).
27. Grabinska-Loniewska, A., "Activated sludge bacteria participatingin the biodegradation of methanol, formaldehyde, and ethyleneglycol. II. Utilization of Various carbon and Nitrogencompounds." Acta Microbiologia Polonica, Q, 83-88 (1974).
28. Kim, N. J., Humenick, M. J. and Armstrong, N. E., "A Comprehensivestudy on the biological treatabilities of phenol and methanol-IAnalysis of Bacterial Growth and substrate removal kinetics by astatistical Method.“ Water Research, lQ, 1221-1231 (1981).
29. Lettinga, G., De Zeeuw, N. and Ouborg, E., "Anaerobic Treatment ofMastes Containing Methanol and Higher A1coho1s." Mater Resaerch,lQ, 171-182 (1981).
30. Colby, J., Dalton, H. and Nhittenbury, R., "ßiological andBiochemical Aspects of Microbial Growth on C1 Compounds." AnnualReview Microbiology, Q}, 481-517 (1979). .
31. Hanson, R. S., "Ecology and Diversity of Methylotrophic Organisms."Advances in Applied Microbiology, QQ, 3-39 (1980).
32. Higgins, I. J., Best, D. J., Hammond, R. C. and Scott, D.,"Methane-Oxidizing Microorganisms.“ Microbiological Review, MQ,556-590 (1981).
33. Quayle, J. R. and Ferenci, J., "Evolutionary Aspects ofAutotrophy." Microbiology Review, Q2, 251-273 (1978).
34. Brock, T. D., Biolo of Microor anisms. Prentice-Fall, Inc.,Englewood Cliffs, NJ, 802 p. (1979).
137
35. Stadtman, T. C. and Barker, H. A., "Studies on Methanefermentation-IX. The origin nf methane in the acetate and methano1fermentations by Methanosarcina." Journa1 of Bacterio1ogv, 61,81-86 (1951).
36. Smith, M. R. and Mah, R. A., “Growth and Methanogenesis byMethanosarcina Strain 227 on Acetate and Methan01." App1iedEnvironmenta1 Micr0bio1ogy, 36, 870-879 (1978).
37. Mah, R. A., Smith, M. R. and Baresi, L., "Studies on an acetate-fermenting strain of Methanosarcina." App1ied Environmenta1Microbio1ogy, 36, 1174-1184 (1978).
38. Zehnder, A. J. B. and Brock, T. D., "Methane formation and methaneoxidation by methanogenic bacteria." Journa1 of Bacterio1ogy, 132,420-432 (1979).
39. Haber, C. L., A11en, L. N., Zhao, S. and Hansen, R. S.,"Methy1otrophic Bacteria: Biochemica1 Diversity and Genetics."Science, 221, 1147-1153 (1983).
40. Da1ton, H., "Oxidation of Hydrocarbons by Methane Monooxygenasesfrom a Variety of Microbes." Advances in App1ied Microbio1ogy, 26,71-87 (1980).
41. Higgins, I. J., Best, D. J., and Hammond, R. C., "New findings inmethane-uti1izing bacteria high1ight their importance in thebiosphere and their commerica1 importance.“ Ngtggg, 266, 561-564(1980).
42. Mohanrao, G. J. and McKinney, R. E., "Activated S1udge Metabo1ismof Certain Quaternary Carbon Compounds." Advances in waterPo11ution Research, 2, 245-259 (1964).
43. Cate1ani, D., Co1ombi, A., Sor1ini, C. and Trecchni, V.,“Metabo1ism of Quaternary Carbon Compounds 2,2-dimethy1 heptane andtert-buty1 benzene." Apg1ied Envircnmenta1 Micr0bi01ogX• §Ä•351-354 (1977).
44. Speece, R. E., "Anaerobic biotechno1ogy for Industria1 wastewaterTreatment." Environmenta1 Science and Techno1ogy, 12, 410A-427A(1983).
‘
45. wa1ker, J. D. and Co1we11, R. R., "Degradation of Hydrocarbons andMixed Hydrocarbon Substrate by Microorganisms from Chesapeake Bay."Progress in water Techn010gy, 2, 783-791 (1975).
46. C1aus, D. and Ha1ker, N., "The Decomposition of To1uene by Sci1Bacteria." Journa1 of Genera1 Microbio1ogy, 36, 107-122 (1964).
138
47. Dag1ey, S., Evans, N. C., and Ribbons, D. W., "New pathways in theoxidative metabo1ism of aromatic compounds by microorganisms."Nature, 199, 560-566 (1960).
48. Evans, N. C., "The Microbia1 degradation of aromatic compounds."Journa1 of Genera1 Microbio1ogy, 99, 177-184 (1963).
49. Marr, E. K. and Stone, R. W., "Bacteria1 oxidation of benzene."Journa1 of Bacterio1ogy, 91, 425-430 (1961).
50. Gibson, D. T., "Microbia1 Degeradation of Aromatic Compounds."Science, 191, 1093-1097 (1968).
51. Gibson, D. T., "The microbiai oxidation of aromatic hydrocarhons."9ritica1 Reviews in Microbio1ogy, 1, 199-223 (1971).
52. Gibson, D. T., Koch, J. R. and Ka11io, R. E., "OxidativeDegradation of Aromatic Hydrocarbons by Microorganisms. I.Enzymatic Formation of Catech01 from Benzene." Biochemistry, 1,2653-2662 (1968).
53. Gibson, O. T., Cardini, G. E., Mase1es, F. C. and Ka11io, R. E.,"Incorporation of Oxygen-18 into Benzene by Pseudomonas gutida."
_ Biochemistry, 9, 1631-1635 (1970).
54. Gibson, D. T., Hensky, M., Yoshioka, H. and Mabry, T. J.,"Formation of +-cis-2,3-Dihydoxy-1-methy1cyc1o hexa-4,6-diene fromTo1uene by Psuedomonas putida." Biochemistry, 9, 1626-1630 (1970).
55. Kanemitsu, H., Fukada, M.and Yano, K., "P1asmid-BorneBiodegradation of To1uene and Ethy1benzene in a Pseudomonad."Journa1 Fermentation Techno1ogy, 99, 175-171 (1980).
56. Davey, J. F. and Givson, D. T., "Bacteria1 metabo1ism ofpara-xy1ene and meta-xy1ene -- Oxidation of a methy1 substituent."Journa1 of Bacterio1ogy, 119, 923-929 (1974).
57. Gibson, D. T., Mahadeva, V., and Davey, J. F., "Bacteria1Metabo1ism of para-xy1ene and meta-xy1ene - Oxidation of aromaticring." Journa1 of Bacterioiogy, 119, 930-936 (1974).
58. C1ark, F. M. and Fina, L. R., "The anaerohic decomposition ofbenzoic acid during methane fermentation.." Archives Biochemistryand Bioghysics, 99, 26-32 (1952).
59. Oshima, T., "On the anaerobic metabo1ism of aromatic compounds inthe presence of nitrate by soi1 microorganisms." 1gjtschrift furA11gemeine Mikrobio1ogie., 9. 386-394 (1965).
139
60. Taylor, B. F., Campbell, N. L. and Chinoy, I., "AnaerobicDegradation of the Benzene Nucleus by a Facultative Anaerobic
_ Microorganism." Journal of Bacteriology, 188, 430-437 (1970).
61. Healy, J. B., Jr. and Young, L. Y., "Anaerobic Bio Degradation ofll Aromatic compounds to Methane." Applied EnvironmentalMicrobiology, 88, 84-89 (1979).
62. Fina, L. R. and Fiskin, A. M., "The anaerobic decomposition ofbenzoic acid during methane fermentation. II. Fate of carbons oneand seven." Archives Biochemistrv Biophysics, 81, 163-165 (1960).
63. Nottingham, P. M. and Hungate, R. E., "Methanogenic fermentation ofben2oate." Journal of Bacteriology, 88, 1170-1172 (1969).
64. Hinterkorn, H. F. and Fang, H. Y., eds., Foundation EnoineerinHandbook. Van Nostrand Reinhold Company, New York, NY (1975).
65. Ramstad, T. and Nestrick, T. J., "Determination of polar volatilesin water by volatile organics analysis.“ water Research, 18,375-381 (1981).
66. Neast, R. C., ed., ßandbook of Chemistr and Ph sics. The ChemicalRubber Co., Cleveland, OH (1971).
67. Kanatharana, P., and Grob, R. L., "Gas chromatographic study ofHydrocarbons adsorbed on soils." Journal Environmental Science andHealth, 18, 59-77 (1983).
68. Rodgers, R. D., McFarlane, J. C. and Cross, A. J., "Adsorption andDesorption of Benzene in two soils and montmorillonite clay."Environmental Science and Technology, 14, 457-460 (1980).
69. Nathwani, J. S. and Phillips, C. R., "Adsorption-Desorption ofSelected Hydrocarbons in Crude Oil on Soils." Qhemosphere, 8,157-162 (1977).
70. Novak, J. T., "Temperature-substrate interactions in hiologicaltreatment." Journal of the water Pollution Control Federation, 48,1984-1994 (1974).
71. Lawrence, A.H., "Application of Process Kinetics to Design ofAnaerobic Processes." In: Anaerobic Biological TreatmentProcesses, F. G. Pohland, Symposium Chairman. American ChemicalSociety, Cleveland, OH (1971).
APPENDIX A
wi11iamsport, PA Biodegradation Data
140
l4l
Table A-l. Methanol biodegradation in the unsaturatedzone at l mg/L.
Concentration (mg/L)
Tjme (days) 0 7 24
l.3 ND*l.O ND‘ l.2 NDl.2 NDl.0 NDl.0 NDl.2 NDl.l NDl.3 NDl.D ND
*NÜ = non-detectahle (less than Ö.0F mg7L)
l42
Table A-2. Methanol biodegradation in the unsaturatedzone at l mg/L.
Concentration (mg/L)Time (days) 0 5 2l
l.4 NDl.0 NDl.6 NDl.4 NDl.4 NDl.2 NDl.2 NDl.6 NDl.l ND
_ l43
Table A-3. Methanol biodegradation in the unsaturatedzone at l0 mg/L.
Concentration (mg/L)Time (days} 0 7 24
ll.9 NDl4.2 NDll.0 NDl0.l NDl0.5 NDll.7 ND9.7 ND
l3.4 ND9.6 ND
l0.4 ND
144
Tab1e A-4. Methano1 biodegradation in the unsaturatedzone at 10 mg/L.
Concentration (mg/L)Time (days) 0 5 21
9.2 ND10.9 ND11.6 ND10.1 ND11.8 ND7.4 ND7.8 ND
12.1 ND14.3 ND8.3 ND
l45
Table A-5. Methanol biodegradation in the unsaturatedzone at l00 mg/L.
Concentration (mg/L)Time (days) 0 6 9 ll l4 23
l03.9 73.4l0l.4 67.9ll8.6 5l.2l06.2 5l.6l08.6 55.2l09.4 52.0l02.2 22.587.7 ND
l08.4 33.8l06.2 9.8
146
Table A—6. Methanol biodegradation in the unsaturatedzone at 100 mg/L.
Concentration (mg/L)Time (days) 0 5 7 9 12 21
99.3 57.594.5 74.081.7 28.5
100.6 16.494.2 53.192.5 7.489.7 33.0
103.5 28.1113.6 20.6117.5 21.2
l47
Table A-7. Methanol biodegradation in the unsaturatedzone at 700 mg/L.
Concentration (mg/L)Time (days) 0 l6 20 32 46 54 82
69l 620778 499705 525657 444603 442594 448680 456673 45l622 204577 403
l48
Table A-8. Methanol biodegradation in the unsaturatedzone at l000 mg/L.
Concentration (mg/L)Time (days) O l6 20 32 46 54 82
ll00 732ll24 777l386 730926 508
l225 609l495 6l6l209 400ll85 486ll79 463l282 440
¢\l IDCÜÜF7 (\l!~U7 I I IN F7F7¢\|
F7 Ih F7 QQQQ G1 G ZZZr- <f ¢
OJ UiIB C
UI I-
2 EO UN VI
G QQG ¢\|¢\Ir-'D VI <|' <|'¢Ll7 •
- ~U D r- IDNDC e-G\¢OJ 0 ¢\lQ\DI5 5L C= °*'OJ OJI5 CVi OC U3
L'D UC *4-I5 N Qüv- *9
ve F7 IDIDIN •
'U E r- ¤D\D<f $0ua an NOJ OI5 UL OD L
*‘~+J U —J
gn ,'“
E 6E
OJ Q C wdßr- <rwGQ OID QIDN GI-Q
C I5 ·r·Il7 www Qlh¤‘••— U OJ
CO OJ••- OJ COJ Q UI5 ID U'U th CI5 O*6, S" °GJ ·r Q‘#'F7 <'F7G·¤ m Q I17¢Q Ne-wQ : N NNN www
Q -l\
'3 EC
E 8OJ Oé’
'F d’\•-th Iflhlb VQ «—0¤m m~¤·O• N QNN www
T<U
-DIBI-
QIDUI LDIDOIw F'H~Q wu-<'c•QN r~Nw
F7F7\h F5¢\|¢G F7F7¤- (\I\D¢
Q¤~Q chQv~
‘U
Ih GJOJ Ü
I5 I5 U'U S- OJ
3 IBOJ L
E I5 3th OJ ·•e—C I5
I- 2 th
150
Table A-10. Tertiary butyl alcohol biodegradation in the unsaturatedzone at l mg/L.
Concentration (mg/L)Time (days) 0 5 ll 19 32 52
0.8 ND*0.7 ND0.6 ND2.0 ND0.7 ND1.0 ND0.8 ND0.7 ND0.6 ND0.7 ND
*ND = non-Hetectable (less than O.] mg7L)
l5l
Table A-ll. Tertiary butyl alcohol biodegradation in the unsaturatedzone at l mg/L.
Concentration (mg/L) .Time (days) 0 7 l3 2l 34 54
0.8 0.7l.l 0.9l.O 0.4l.O 0.4l.2 NDl.O NDl.3 NDl.O NDl.l ND0.9 ND
152
Table A-12. Tertiary butyl alcohol biodegradation in the unsaturatedzone at 5 mg/L.
Concentration (mg/L)Time (days) 0 5 11 19 32 52
5.0 5.04.7 4.85.0 5.05.4 5.35.2 6.05.3 5.55.1 5.54.8 5.15.3 ND5.7 ND
l53
Table A-l3. Tertiary butyl alcohol biodegradationin the unsaturated zone at l-6 mg/L.
Concentration (mg/L)Time (days) 0 2 5 l3
l.l 0.9 NDl.l 0.6 NDl.0 NDl.l ND2.3 l.9 0.42.l l.8 ND2.8 ND2.3 0.63.4 2.8 0.8 ND3.4 0.8 ND3.7 3.l l.9 ND3.7 l.8 ND4.4 2.5 ND4.9 3.9 2.5 ND5.l 4.l 2.8 ND5.l 2.2 ND5.2 4.7 2.6 ND5.5 3.0 ND5.6 4.7 3.4 ND6.2 4.8. ND
l54
Table A—l4. Tertiary butyl alcohol biodegradation in the unsaturatedzone at l0 mg/L.
Concentration (mg/L)Time (days} 0 7 l3 2l 34 54
ll.0 8.89.9 7.3
l0.9 6.36.5 2.5
ll.5 0.67.9 2.38.0 l.7
l2.3 ND5.7 ND7.2 ND
]55
Table A-l5. Tertiary butyl alcohol biodegradation in the unsaturatedzone at 70 mg/L.
Concentration (mg/L)
Time (days) 0 5 ll l9 32 52 56
70.9 65.770.7 60.]7].9 52.96].3 56.872.2 57.072.8 57.574.2 64.]7].4 66.569.5 55.572.7 56.]
*N0 = non-detectable (less than 0.4 mg7L)
156
Tab1e A-16. Tertiary buty1 a1coho1 biodegradation in the unsaturatedzone at 100 mg/L.
Concentration (mg/L)Time (days) 0 6 12 20 33 53 57
126.6 104.078.2 57.4
113.2 71.971.0 33.3
103.7 58.095.7 56.4
112.3 27.7114.9 14.5121.2 36.571.3 0.4
157
Table A-17. Tertiary butyl alcohol biodegradationin the saturated zone at 100 mg/L.
Concentration (mg/L)Time (days) 0 6 20 28 55 137 183 232
Unsaturated 74.7 69.7 5.6 ND - -85.2 80.9 10.0 ND - -80.3 77.4 31.0 11.4 0.5 ND
Saturated 176.1 135.5 121.7 112.4 96.3 35.7 ND -123.7 101.2 80.3 82.4 78.6 65.2 10.2 ND86.1 78.9 71.5 69.9 63.3 9.3 ND
158
Table A-18. Methanol/tertiary butyl alcohol biodegradation in theunsaturated zone at 100 mg/L.
Concentration (mg/L)Time (days) 0 11 15 27 41 49 76
106.0/1.0 ND/0.6122.8/1.1 37.3/0.8109.2/0.9 22.9/0.7114.9/0.9 20.8/0.8115.8/1.0 ND/ND122.0/1.2 ND/ND113.5/0.9 ND/ND79.1/0.7 ND/ND82.2/0.9 ND92.0/0.9 ND
159
Table A-19. Methanol/tertiary butyl alcohol biodegradation in theunsaturated zone at 100/5 mg/L.
Concentration (mg/L)Time (davs) 0 15 27 41 49 76
117.1/6.1 63.5/6.8125.6/6.9 62.0/7.4118.4/6.2 40.9/3.6111.1/5.8 65.3/3.1114.1/6.1 52.7/5.7115.5/6.2 24.4/5.7107.7/6.0 30.8/5.2118.2/6.2 16.2/5.3
l60
Table A—20. Methanol/tertiary butyl alcohol biodegradation in theunsaturated zone at l00/5 mg/L.
Concentration (mg/L)Time (days} 0 ll l5 27 4l 49 76
l25.7/6.8 63.6/5.693.7/4.6 68.5/4.799.7/5.0 32.9/4.5
ll2.2/5.9 62.9/6.2lll.0/6.8 ll.9/6.3 .69.4/3.2 ND/3.0
l03.5/5.3 ND/4.393.5/4.7 ND/0.l
l03.6/5.4 ND/0.ll05.5/5.7 ND/ND
161
Tab1e A-21. Methano1/tertiary buty1 a1coho1 biodegradation inthe saturated and unsaturated zone at 100/10 mg/Lusing septum—capped microcosms for continuoussamp1ing.
Concentration (mg/L)Time (days) 0 6 20 28 55
Unsaturated 119.4/9.8 44.0/7.0 ND/5.1 ND/0.9 ND/0.2126.7/11.8 53.8/7.5 ND/6.6 ND/5.4 ND/ND87.3/17.3 21.7/5.0 ND/ND - -
Saturated 152.8/12.3 17.7/8.3 ND/7.0 ND/6,6 ND/6.9153.1/13.0 35.0/8.3 ND/8.3 ND/7.9 ND/7.1167.8/20.0 54.1/9.1 ND/9.1 ND/8.6 ND/7.7
O'!
LOSO C\IW Ä
®LD•—-
'UGJ4-*füL34-*fü r-
O
C ON t\ID "$' f")
(D LO.C LD4-* (\|
C•P
CQ Loc:4-* .-I GSCfü \r-' ¢¢''U
¤‘•\]'ii
fü E •:t'LOS- <2'(\|O') '*
<l‘I-O
GJ C'O OO ·r—•P 4-*.Q fü
S-r- 4-)OC..CGJ
• •
O U|\ ®LOU C(\4 0")¤'P- O \\fü LJ (OLD
CDI\r- LO<T>~•
· '4-*-I3\‘°E’>~, <1·•.¤gi • •
füLO LO GWLO·P\ r- GOLD+-*@S-G
CNLOQ)|\ OGG4)) SWW
P-O4-*Cfüfü ©<I'
..C:¢U• •
4-JC ¢'LO(DU r- <fI-DEN P" \\
SO|\<|'t\I• LOI\
{\|(U
I<£¢D IN:-CO*~DO3f*)I\®®®r-
• • • • • • • • • •
.D (\|LO('f)f•\|§'¤’uI\®LO®fü G <'LO<f‘LO<|‘¤‘LOLD<"¤'
I-¤'f"')G\O\LO©U'•t\ILOlO<I'(\|kO®\.O<I'LO©C‘IO’l
P l·Dx\Dl'\§OI\¤)O‘•kOI.DII)>~•fü'U
G)E·•—I-
OOON ZZZG')N
I I I
Ihth
Fl 60*O OOOQ FINZ ZZZ
I 1-1-NQ
rxmus6 vvm mmm•— ID Q • • •
• ZON
II0II 60|") OFDFIL
• • • • 1 •
5 O1-O 600l0-* 6 I"IIf|"$ |")I'$f"IIIO PIr* 1- Ol OOO
-
• ZZZUO- QIFYNKU
0•\0
0-JU 1-
5E•-0 QON QONCQ
• I • • « •
OU OFI1- ¢)O•-•-wll)OJ
Ih•-I 6Ih<I 6506'UC x 661- Qlhüdv- EI FINCW NI'1I')LwUID
•-I COX
• O
-1-UI
-
IDOQ QOIDQ C OJ ¤ • « I • •
~•- I OIDÜ 1-Nr"1-Or- L
CN ÜNN IOQQOO! U 6fÜO 6NG00m u vww vmw•-6 CG w
0*3 8v-GO>• 30JwC Olälh Ililhlli
BCO-* r-OND NQN
. OCXNOL
U N IOQIO QOOEU NOIUI IWQO~•-III- Ihlhth IIIIDID4-IO-JOLlß-UL93w\•-IE
1-dw NFIO O0\N‘
OwO• • • • • •
CCUK
Ihißo- Qlhlh{cw Ovxm 1-OC
¤¤E www www
P')N num mvxvI
• • •-
• I
< vom wmv
0 6 xxx xxxr- r--O O3?).0 *0*0N Ol 6Q ID66 QDQO
I-
¢~ 1}vl IIX OJ ܤ ¤ I)T! L OJO 3 Q•J L3
III 0-*v•¤
7* Ih
164
2 2 2 2 2Z Z CJZ :-Z MZ
Q Q 0 Q 0 • 0 • 0 • 0
O n- Z2 Z2 LOS LO2 LOC.)•r- N NZ NZ I I NZ NZ NZ-:9 Q •• Q •· Q •~ Q •• D •·
rd Z2 ZC: Z2 Z2 ZZ'U Z Z Z Z ZfüL07GJ'UO•:-
•-OZ 2
0.: Z <r •· Q ZC G ^ ÖKO 2 GC) ZZ I--
••
0.: -C'J •OGZ
•Z • •· •:—
:- NZ G •Z '* LO
'• LOS KD •
Z-) Ü N"
NÖ NZ NZ NZ NG>< M Or- D •· DZ DZ Q •• C: ••
I Z • ZM Z•· Z •· ZOO ZM
E• Q 2
• •
• r- Z Z O G'UG7CCIü·:·- -
:-•·¤. .J(UE NCfü Uiwu: E Q Q QIJ Z Z Z 2 E E:—m •· w •· :—- •• N •• NZ <I‘Z NZQ; g
•|_Q •qj •Q· • 0 • 0 • 0
+-*0 Or- LO •I-DCD O
• Nm LOLO wm3
••—N NZ N• NZ N • N • N •
•~¢: +9 D •~ LOG 0 •• GO GO NOmq- (5 Z'-
• 0 •gQ Z 0 Z 0 • 0C4-* L
• ZG :-G FZ N :—·:-—·
wg: +J Q «• • • •
NO Si I-· CIGCU(U
0) U.DL E
O OMI- 0
:-OU).::5 no N OOOm Q • Q • C: •
UO•·—Z :-O MZ MN LOZ LO:-
'-•U • B • B • B • 0 • B • B
füO LON LOGO LOLD N<f' LON LDLD;_ px \ • \ • \ • \ • \ • \ •r—U GOG N:-— ZG GOr— GG CDLO>’•,- • 0 • 0 • 0 • 0 • 0 • 0
+9E #2 LOCO :-LO ZN :-GO :-N3 MZ M • GO • LD • GO • GO •
>5O.LO.füfü•:—K)+9l ·S-EGJ3+9+-*G\¤.M m O • •
:-0.: N • CO • G3 • NN ®m N:-om •gv) •(~_| •g·v) •,- • • •,-
C KO ••m
•• LD•·
m•~ LO:—- N
•·
fUU°l NI" NLO NN NOW N "NLD
Lg :·-• N • LO • <|' • ®<I"
•
4.:-,- •|\ •gQ •|_g) •@ • • •g·:)
man m •• m •· O •· mr- mM w:-E: mw oo~=r mN :—- •• O •• N •·
• • • :—N :-ID :-NQ- Q- gv) «
• •
• N N CX)d'N
I<0.: N 'U:—· U) GJ.0 >; +9 'Ufü fü fü <E GJI- '0 L CU +9
-9 3 I- ¢¤{J N>< L-
0} fü SEP" 3E In ON 4-*•r- C G! füI·- Q E an
G G G G G GZ Z Z Z Z Z
0 0 0 0 0 0
•— G G G G C3 C)N Z Z Z Z 21 Z
'Q 0 0 0 0 0 0GJ G G G G G G4.; Z Z Z Z Z Z
3L4-*CUUICD
G G G G'U G G Z Z Z ZL 0 0 0 0
ru•· •· Q Q Q Q
Q Q Z Z Z Z'Q
<Q‘E 0 0 0 0
<D C') •~ •~ LD (0 00 Q
ä
•·— r- G G G Z
L G G G G G34-)ruVI
..I(U N.C U)4-* E
G G G G G GE
•~ z Z z z z zQ E 0 0 0 0 0 0
L Or- N N N N Q ®q..• •|-(\| • • 6 • • •
vz -6-* C2 Q 0 Q Q Og_qE gg 0 0 0 0 0 0WV) L- G G N (\I r- •—QQ 4.> • • 6 • • ••-U C G •—- G G G G
O GJCJL UCU CQ;•r-· Or-·E LJ>~•><'OIG) N •·- r- r- NEg_ Q • • • • •
O- Z G G G G G
CU N KD OS G Cxl K) \g5| |\
• • 6 • 6 • Q')
E G G CD G •— n- E0; 0 0 0 0 0 0
GJ4-J Ü Ü G 00 Ü 07 C')gg,
• • • • • • Q(DCD
•·- G r- •—· r- G•
ZV) G•—OU) · C4-*C CU
•r- .C•m
4-*CJDC G V!(DU'! G3 07 Ü O3 • LO 07NQ;
• • • • Q1 • Q;CC
€\I¢\I Ü LO '• Ü r—· ·
QQ 0 0 0 0 Q- 0
GN O• d) N
• QG • N
• • Ü• CU
LC) Ü N Q r-· N ·• 0 0 0 0 0 0_Q
m cs O cn Q cx N ns1,\|
• • • • • • 4.>
Ir—· (\I Ü LC) LC Ü U
<C G)4-*
GJ GJ•—· 'O.¤ uNS CP' O
es 1; g:V) GJ:3 4-* °¤ IIIG fd CU ‘
'O L 4-* G-« 3 ¢¤ Z
4-* LG) ru Z3E um -6-*••-
C CUI‘* G U)
l66
Table A-26. Biodegradation of methanol and tertiary-butylalcohol in the unsaturated zone in duplicatecontinuous sampling sand/soil microcosms.
Concentration (mg/L)Time (days) 0 7 18 32
DoseMe0H ll8.6 ll2.0 l05.l 90.9Me0H ll8.0 l08.3 94.5 85.7TBA 5.l 5.0 5.l 3.2TBA 5.l 5.l 5.8 3.9TBA 5l.2 49.9 47.9 44.7TBA 50.5 50.5 46.5 43.2
Me0H/TBA l08.2/54.2 ll0.0/56.l 84.0/5l.2 68.9/49.8
Me0H/TBA l02.9/54.8 l06.5/55.0 87.l/5l.9 82.6/47.9
APPENDIX B
Nay1and, NY Biodegradation Data
167
PNNCBQ • • •
_ •-
• ,-5;QIDQ UIÖÖIDSDIBl'*
@@2 \DB\D• • • • • • • •
.NDZZZ |·D§'(V7 ©•··••— QBQ ß|\D(V7 IBQQ GOG•- QQQ ·zm zzn¢ • ¢N G
• •-
<Y®¢¤Qm QQQ me-o mmc nnm mmm mnn
IB(V7IBNNr- QQQ
•Z·
Z-•—
N SD r-LO(V7 (V7 IB
0CON G th
'U ® ~G•Q®
gp•@ • ,- • •
H IDBI-0 QMQ Qv-O OQM NQQ Gr-G OKDQ|¤
B•••gQ(*)•- ••• ••• •••\\\,-ggg
S- (*0 LOGF7 U7l-GQ •-•-•r- •-QGY U7Nf'7 QQr- BBB:¤ n¤Qm •- Qmm ·z
·nnQ
4-* 6D lblflv-ru M Q mmm$0
>·Z
‘U
CN•— A Q Q Q N N
Q1 g'! VB I- O1
-6-*C
c O4-*N
C S- Nr- (V)0 J-* B
•G •(V7Q•p Q •@ • ,-• • •
4-*gg QQ •••g,n|,r)Q ••• •«• •••\\\,.-gpy\
'U CU) LD(\|\D LDIFIÜ •••-•- •-IBU\ ¢‘(V7P· CIQIII BBB•¤ o nmm-
Qmc •z-
m•-roL U
•— I"! G FHDQ
3Q LD Q|V7Lh
°¤G„D
<NM uh
m Q •·
•mmI-
•NQ B •·QQm nmm ov-o Qmm mQn mr-- OQQ
Q ¢\|-••|\|,n\,p••• ••• •••\\\;-g°y\
E N r-•-(*7 Il')!-|’7{' •-r•!- •-QQ IDQSD \DN@ BBBG QBB r- QIDQ • • • F)!-N•— Q•-0 Qnn
nö IOQIB<’('V7|•O
¢ID{4-*é,
GUIID U!• • • •€\B
GPQ Q • •• GBI'! BQID GG ¢¢\D QGQ •—•-•— •-QO
p- R)•••t*)\QQ|•• ••• •«•QQQ,-QQ
I r- •-QG lhL(7Q' r-Q NQG •D•-ID NB•- \\\Qu- (V7lD¢ NBQU6D\DYB-D
IBF-
QGID r-¢mBQU-
•-(GQ Q • •• • • G¢G BNG BIB?) BBQ
•-•-r- r-QNQ QQQ (WBB • •
~•
-• • • • ,-Em
‘
OSOND IDIDQ •-QG NQO\D\D•-··
I-Dv-G BBB•- •-•·· QNDN
• • • QDI-hr- B¢'$
Q®•-•nQn mmm
<I
us I-B
· Ilr• I-‘UO ÜQ Ceu G
I) -C EE H < H·•· 0 Q 0I" I- Z
GPM
ID DED USOUS f'Sf'I•~ WQIN ·¤E Q--·O ZZZ
••· ••· •··Q22 nhtbtbN O¤—O cnxrx SO; xxx xxx
QQPpn gp n • •
ID QEE USOUS Or-IN OUSP •E • QODgg yxzz
•«• ••• •••gQZ;— nggpgg
P P EPG USI\I~ USNW \\\ \\\fü I-D IED DDD
ZZ Z22
I'OI'*)PQ} ig
• • •
PQ DEQ US Q IDQP GSIQQ •E • SDI-DQ..JO r-ZQ •
· ·•
- ~ ·• |~ZI~ !\\D\D
xr- e O O US|~Q Nenn xxx xxxES QGIID DDD DDDZZZ ZZZ
CO4J0S-OJC U1U\US FSEDIN Qcw e-~ coo oxw N" "' "°Q P Q(J
•-BP$@1
• • •
INIDBI IDE IÖIDUT USIWP • • • EDFPOS•n•rx
mmm O•-·OONQ Nv-rx xxx xxxP DDE
ZZZ 222
UIUSN
·• • I~\DU’S
PO? U•OU• MIDID IDNQ QQUS QKDSDygP-
W I•D¢'¢'°I Du-E OPQ f*‘)QO USED EDI")• P QQQ -22 22fü
OJ ID ¢\I
Q PO
<Q• an I-P \
I ID Pr·•
D 'U O OC C
U I5 IBP 0 •C .C.D E 4-* < 4-*
_
Il•r·CJI-I- Z I- Z
#Q •
O! Z (\I\D \ \
E QZ Z
P (*7
(V # ID# \ \
D5
Q •• II7
(*7II7(*7(*7
Q IZ 5
—I\
ECr r- 5
: an InCON \ \••-(\I(*7I5
O IhL P PJJCUUC
8MQ QD•Z •Z
Q)• Q) •~
an xc \¤. GJ @2 #2
(UC 52BIZ (UZ
(NI UI
IDC Nr-
LDO Ov-# 5 • \ •P #@ GIID '
QM OOgp • Q) •
¢\I
5gg •
• O(GQ
•-•-
IDKD \DUI• •
P U7@ ED?«•J P •~
O•
c NO QGI
L! ID ID
<Q• an I- _•- \
¤S·€
EUP U •CI--¤ E
•-*¤gg ~¢- UI- I- Z-I-
APPENDIX C
Dumfries, VA Biodegradation Data
171
INOQ
SDFIID RUNDR
• S SOIIDQ Pr-f")¢\|f'°)f"7 • S S
QSDSD
IRCDSD•S•SS<rOQ- somm •—•-O ~¤<t QON ONNMQ? SORR • • S
6-*UU GIQQI;. rl) S S S(*5PS-
B-
O ¢QS¤
A mwN MRQ O:-QSJ0 --~¢\If*)P ••• •S
SDSDSD •--0 RSD Qdu- r-RG)O) QUSD SDSDR • • •
•E OO-?)
nn ~¢ AhtbmU••- CL 9
B- JJGJ
JJ S0 -0G G -0 •••-
U PPP PPP •-•-Q (*70 QIDF) R•-RQu') S S • (\,||,[)(\| S n S • • S • S
OISIÖID SDSDSO I--0R¢OO SD¢SD SDRR • • •
-•-S.) OmQ
JJ SDRSDU'9ULUI
'S LO9 0 •<f••- -0 •Q RSNISD r-UISD P:-Q Qu-R
SSS••PQRSD SDRR Pr-0 RSD ¢\IS!IU\ GIQ0
-R¢\D RRR •
-•
P NSD0I RQSB
U
SO
F•SQ
QSDSB•0 •
S S S S9Q9• •-•-R 9\N QQSD QPRdßßyp- n S S S S SSO<rR
•¤r~r~ •-•-O Run NSDQ Qr~~¤P RRR S S S
QP Sg;
l\I¢¢\ -0·• •
-NMR •-v-Q R0 0¢9$ Q!-R
S S S S S S SSDRR •-•-0 R10 RR0 SDSDR•-· RQQ • . •
ChSD\D
QV! I-
R
· '°’5
'S*9
° SUan .:: -¤E JJ JJ
GJI- I- Z
LOOMO O!-'lß NIPG On-Q QN NGO
- - -@Z••(*)(ü|'V) •••.•I-
(NILD LDIITUW000ONOZ •Z
OGLO MQN OOQ <f'N 0GN 0N0•.
LON Il7L[)I«D r-•-O 0<f <f®N (DI-OOv- @00 • •Z
ON
¢(\ILO
NQG LDQ0Q;N
¢\l\D(b I—U'l!")¢\-IOIF)
-|NGE
0 GOM 0<f¤- Oe-0 NN RING 0@0•,-(V') •••;Qgp© •••••4JN
IDNCDI-DLDLDeg•- Lpgpßp • • .
L M!-N4* ¢\|l"')(\|CUUCOL)
0 QNLO 00G)
OJMMM<fM
GFILO
Nl\I•- 0¤)0
ND0<fLOMGM
GQQ
MMG INQI-O•*® O0 MN0
..es
LD OGO UÜUÜLÜ •-•r-C) 0< NGG LO¤—•—• NNM 000
-• •
4.)Or-•-
; <fI.OQG(.3
an I-•- \ ** I G I' I-
(.3 'U O OC C
U G G•- S .C,Q 4-* 4-V
_ G•v- (D E GJ‘
I- I- I I- Z
O• Q gbQ
• •
I- ID NN \ \ \Q v~ Q Q
M 1D •-·Q Q Q
N Q ID
Ol S0 NQ N E NQ Q I~ Q
Q Q NQ Q Q
r-O-2 OO Ih
Q•¤ ez •
rs r·O NG•
N-|O \Z \O \xt'! QD •· Q2 ODU} •¢\|
•e
•
E O·
QO NQ IDO Q2 ID€
G
IDL O • •J-! •O GIO r-: Q
• • JI •
mI-•-· s¤•- Q
uu! \ • \·\
CN INO QO QQ
• •~ « • •
Q MM Qv-•··
LD • ID·
QO O
G'! QQ)
• •
•O O•— IDQ
• • • •-«— r~¤n Q•••-
QO- I'- N
\D\D ¢\¢I SDN ~ LO • Q _
O O
Qv- Q•~ p-Q •·—
Q•~ • • •
I-\D NU! Q•••-M
Gir- tb
- NG! 1*001 ON • Q • Ui
O O
Q•
• QQUY I')!— r•GIID ODQ lb ·• Q .QON QQ O
J-! lb• ¤'• • Os
E so NO QO Q $0
<Q• II! I-
I- \I OU
!—
U‘U
OC
G! |U><r- (II LI-.D E -!-!Qru ·•·
UI- O-- E+
ID ¢Q Q • „G \ Q KD
Q Z Z
¢\|• LD (Üci • •
N \ ID NBQ an x x• Q QW7 Z Z
¢ \D• • [QQ LD ~Q \ \ WD
N •— I") \• • Q<!' N Z
•-J
E~¢ LD ¢ Gr
C Q QD Qom x \ \ZN Q Ih IDID Q GJ IDL ¢\| (NI•J
CUUCO(J
I') Q
Q ND¢ \ \\D C) Y~
O QQ •-
(0 Q lb
Q I-0 NDQ \ \ \KD Gl Q U'!
•- Q ¢\IP') ¢\I (\I
I~ N OQ QD N
\D \ \ \¤·~ ID Q ND r~JJ lb O QC Q Q (UOL)
<Q•vl I-
n- .I ru
•-
(J 'D OC°’E5
L GQN •¤-
U|—D- 2+
OOIDAIqg + • •
¤' \D<'B•-\0\D
Q'!-¢ Of')- •-f')¢\I IDG- GGNGI(g . • • •-QB 6 • + • • + • + +<f (\|¢O\ BBSD r-r-•-— IDDOS Bf'701
CULDID•-•- lD\b\D
+30 O-<|'q; gn • • •Q- F7 GIB-
<'\0BG07
LQ-
"6Ih ¤- USNLD
•-O•- GM- OOSN MNN_g(\; .••(v),-5 ••+ •+• •••
< \f'§ ¤\B•-•BBM) -·r-•·· IDOIUIu-Ih
(D C••— CL
••-
Q- 4-*
E E° EC 0 FIEID •-\DE Chäir- OIUI-·· EFIOS•,- (J5 •••(~;Q(¤ ••- ••• •••
CGI ¢\It.Dr- BB\D Gu-r-· IDOSGI B¢\IBC O LDQDB v-
•D\D•D
O Q
4-*G'UGLU'!G!
E••- •-Qlh Ih¢•- dvfür- -O¢\-I ¢\I¢\I•-gv) I • • r—\h°\ • • 6 • • • • • 6r- GGE EB\D G•-r- QDGGI GFIE
BBB•-r- Blbtb
¢\II
(J
+-QGI-
0$<fE Ihr-G GFIGI ¢O•- l")l\-IOIQ
• 6 • • • • ¤ • • « • •
BGYG EBB •-r-•- \DG¢§ COBBBQ r-r- Btbßß
•-IDQ Nmno •-nn-: nnsommv~¤•
·Q • • • f*7<'I-0 • • • « • • + • •
BG:-· GQB •-r-•- IDOYGI WEBBEE
•" v- BNDB
Ih
au•-
‘U0¢ .
IDE6-: <·•- U E
I- Z I-
¢\I $$2 QQLH Qu-# @(\IU7 0'I@•-$ ZZZ Iblünn • • • • . „ „ • •r- 000
•-r·-•-QGSQ $#LI7FZ IDIDID
GQ $$äQ ZZ •
Q
QNOQ #(*7
•-I\UiE
C $(*7I- QPQ $•-•- $@0 (*7('7QQ|„ßZ••;gq\|,n ••. ••• •••••·I~ GN @@0 •··•-•— I-DOIQ QN00; Q!\| •·- @00L
4-*EG!U¢0U
$¢\I0 QID00 Z
• •|\QI.h
ND (UI") 000LON'!
$¢\$N Z
• •\D IDND
LDU7
QQQ Bilhu'7 $•-•— Qt\|I~ Q(*7QQ Z ~
• QQUS « ¤ • • • • • • •
¤~ an QN IDNDIDID0 •—
0004-*CO
•VI
¢\,| .I IQ #
U 'U OC
0 I5I- U LB E J-! <I5 •v—
0 QI- !· Z I-
Qu-M')
Ow wmvQ1 I
••Q
EDFÜ QEQQQ
• • •
ENO("IQINI
Own
USED (NEED UDO \D\DE UIQID EDEDQ'Q@••QQ(\| •• ••• ••-ggg
Q ZOUI IDNDEDOv'EDQ EDEDED • • •
QEDF')QQC'7
• F5!-Eh4J (6 • • •
GJ F'! EEDF')U P1!-DEDM-
EED
S-M- QOED
·•-QEID NQQ Q0 Ehfßln IDOI~ |nk¤<I'
QQ«••¢\,|Q(*) •• ••• •••\\\
th P') QOQ EDSDED Dv- CWEED $¢\IE r-(GEOQ’EDED EDEDED
• •~
< v- kDNm> -I wmv
\•^D5
an EQ)S-
CM- FSGJE
O 6 EOUSKNI OQO U\O EDSDE OOGS EDEDQgn'; •••r-GEO••C
+-*¢\I GNIVED \D\D\DO•·• €')E\D G1¢\|E \D\DlD
••• C Qtbth Ehtblb•
- ~G9 $Ih•-
C U EDI-DEDO C-•-• Oé-3 L}6"U6L QFSE
'U EDQID IDOOI ED\bQQ Q
• • • EDEOQ• • • • • • • • ggg
••— I- Ehe-·¢\I IDIDED OO FSQE •--Ehr-O•¤$•-·
Q EDEED \D\DlD •-
¤ED-O@6DED
F)I
U
dl
I- EGON •Dr·l\I $0 $NM Ih•-th EDEDQQ
• • • Qßißß• n • • • • • • ggg
EDQ'|"‘) EDEDEO Or- f')®E ¢\IEDO•—®f*‘I
EhE\D \0&DE0• • •
r•¢\I•-·EEDQD
\DEIh
¢\(\I¢'7 @0Q $•- EEQ •-EO! EDEDQ_
IQ•••EDf")® •• ••- •••\\\
r-¤3Eh EDEIO Gl- MOE $0- \h\OQ\DE\D IDEO ~
• •¢\I|l)\OEEDED
{Q
V! I-E
Q•- •-
'U O O
S ' S-C
•C•-•
< +*·•·· U Q UI- Z I- Z
GCNDN •-—•—m mw Nwr- vnr-
-• •
Q ©vQ • • • • • • • • mmq•— vvm OO mww www xxxLhthhh
GQ
¢QmQG NN? ¢IDG
-• •,_
• • • • • • • « [QQQQQGGLhth—|
\UIE „-g P •<P In
QOl!) IQ. ählln Ix \D-•-In Z <l-DI-0 •
xes O Qng ZL
-69EG)UCQLJ
"’uäqié'N' xxxDAI!Z
•
Ix¢\|
OßbP \D¢
N
·
•Qnx
gp vz mQ ·P „ZNF')
OGEDGQ f'7lh\D QGgnggq«— gn nn mmm QO mnw m•—$ xxx• l\|•-• \h\D Q!-•-•
g QLJ
Q•m
QT *= 6 6 ‘LJ
‘U
,- q; L .C_QE 4-* < 4-*
Q -1- GJ Q GJ•— •—2 I- Z
¢\ R
R OG! R RQ O O
LD OQ N
O Q• •
O•
Q R R I-D<r w wr x• • Q
Q th ZQ N
füfü
QOO OZ OO•Z «
• •z
R• Or- Ih
•·
O \O \ • ROfü NZ OO OZ
»·~ QQ NO NQ.J QZ füz füZRU1E
C Q O NQ
• • ••«- NO RO OO4) • •· • • • •~
I¤ OO OO IDQLO \ • R • R
•
um wc OO RO:
• • • • • •
GJ Ofü IDQ ONU O
• fü • O•
E O O OGLJ
ID O R
OO Otv- OOOG'! OO OLG• • •
•- GO mr- moNO Ihdi r-QO
• O • O•
O O O '
r- Ib ID
ON OQ NQ
O¢\ Or- OOQ
• « •
t\Ifü RO •-O
RO Ne- I-OSO
• R•
O•
r- fü fü
R N Gt
Chfü füO OOOn- I-DO ON ·Q
• $ • •
¢~ QO NQ OOU OQ fü
• n-•
G O • RO! O¢Q H) • •
O R Q
<O_• III I-fü \I N r-
U 'D 0C
U l¤><r- U Sb-.¤ E PO•¤ ·•-
UD- I- Z4-
O
N R ID IhQ g • •
1- v-• ND II)
RG \® ®
fhC\I
C• ¢ Ih•—
\ lb IhQ M x x• Q Q
N z zF')
-1RGE
sa Ln .• RC R •OBD R W·PR ID \6-* • Ql¤ N ZL fü6-*CUUCO(J
U) lb• • @R lb •
lb R \ Iblb O O T¢ RUS •-
N #
R lDN \ \lb R G
I") u-
R BO• • lsR W •
Q \ x Ihes LD R F7 B6-* G N ZC U'! (NIQL}
<E. vs I-
F} R·I IB r-
U 'U OC
G1 |¤><n- GJ •-CI-.0 E Umlu ·•· UI- I- 2+
]8"A-
mmer OQQ mmmA A A A A A A
AB A-BQF7 r--Q GSBB CANI-hw
wBw Or-G BQw A-ww DDD www A A A A A A A AQ ZZZ wmv
•-•-Q OACDGB Fishww
IB• I-D QD
·+3 F7 ZzwQ} u-U*••
GG
L*4-•·- 4- I-B r--N
Q•-G Qu-w GPAA-
_;mgg A mmgp A A A A A A A A
O \F> ZZ67 www -r-Q ¤\¤)¤> F7BIh EI F7 ww< Q>
C• Ovr
·••(D
-A-I"C E"' ‘é‘GJ r- BABB Q-G1 Qu-B wm
Q UB QQ A • • • A A A A
CN ZZQ wBw•-•-G GQQ F'IQ
C G LD ww•r· LJ
CO
OB'D6
*6-U NwG Qwdl r-GQ GPQ NQ-5 m A A A Qwm A A A A A A A AO A- QQIB
v~r~•~A-A-0 .c¤¤c¤ wm
-•-wwB A- ww
QI
(J
U
Z6 BQA- BF7Q Gr-Q IADQID •-G|.A Q A A A A A A A A A A A
QNQ BQB Gr-O GQQ wBQQB ww
FHBB wr-GA NNQ ABQA- BBg A A A mmm A A A A A A A A ·
BFH- QQA- r--G F7(\-IO! NLBGQQ r-•- •-•- BB
III•¤ A-'U 0
CIII
0 S
E *6-' äI- 2 I-
gucsm mmm v~~r~ c¤«- <-<rc GOG mr~r~ nn-91 ¤n\¤
(O Y~Ui @$0*7
•-I\U!E
G l\I®¢Or-4*
G5L
4-*GUUGOQ •
G$|~•- U\¢G\ |~\0\D #I~•— P2
$$°‘6 °6 ¤SrÄv§ ¤Z««i
IQ •—I\I-D• •p \h("'>¢\|
4*COL)
• UI¢
J; 2 '5C°’
« 2.¤ E 4-* <15
•r-• 0 EI- I- Z I--
UT
Y')<f¢QQU') (\|P(\| •-•Q•(\,| Ihr-- • • •Q Q Qg°@ . •-
• . •
-
•
-ggixgpV Z Wtßsh PPP NQQ GQR! \\\PP LDWW QQQZZZ
- r~¢¤>4-)Q Q • • •dl N •
ZWNWU Y') Q\\\Il-QQC)Z2 •R!
R-IQ
LM-
PGWG·•- P QQN PRIQ GGR! ¢R!Q •
-•O —|¢ •Z•$bQ¢\| -•• •.•
«-•gg|\g°vs \Y') W Q WUTW PPP WQQ QGP \\\> sa
IDQ* Q
vs QU •v-·•- 4-)L FB
U- LE
4-)ZIP
= • • •Q ID WN? ¢GY') PRIRI RIWY') YOQN NNNQ QR! <rRlG Wthtb PPP NQQ GQP NN¤'••-
O (\l¢\I•-PP I-h\B© • • •IJ
WUGQPNN0
4-)N'UNL3) NFTQ'U PVQ W!-Q R!R!Q Q<f(\! GY')Y') WNWQ
P QRJN QLDN PPP NPQ QQP QQY')Q WNN •—•— mspgp . • •NDUTPQNQU)
I(-)
.2 NQ-·I- RIVQ IDQN Y")Y')P WYNIU) NMW NNNQWN PPP Nr-Q Rlv-Y') QNIDWGQ PP WWW • •
~Q!-<‘
GQQ
GY')Q
@•••@(V)@ -•• ••. •••°5¢q•)
'GPQ QNN •-•-P
QRIP WQP· · ~WGG PP WNN PY')Q‘
PGG
-<QvrI-\' l¤ PP'U 0OSSN
E .Q .Q4-)< 4-)·•- (D Q G)I- Z I- E
¢’Q@@5 • • • • • • « • • • • •
r- f*)<'<' OOP LDIDLD 55GI IDIDQIDLDLD 555
UIQID •—•®•-<'5® •-AIF7P') @55 • « • • • • • • •
w <r<r<.r •—•—•-- www wnw
\DO¥0i5 r•5@5 Lh¢I„ß
MmwQ ynqgßj • • • • • • • • • |p55pID 550r \\\
u- IISLDIO I | I
erID
QD IDLDID F·I-r- BDGGIr- LDLDID QQQJJ
ZZZEOL)
<Q• v•I- _I!) 5I M l- r-LD 'U O DC CU ¢¤ ns•-•
.C .:„D4-*
{ 4-*U ••- 0 Q ID _D- I-- Z I- Z
Q N
N 0-·· \ \ll) N N
!* QLI')
M Q
N 0 •N N KD
Q M v- \• •Q
G Q 2Ih Q
0 Q
N 0N N NF) N I.!)
·N
(Y) •
ID I-
IDG IDG MG-2 -2 -2
ix n gg n gg ••
.IQ \G \¤ ¤G\M M2 N2 GI2Q) • • • • • •E QG OIG r-G-r IDZ Q2 N2EO
4-*
E Q M •-4-* IDG NG NGQ • an • •· • •
0 NO! 00 000U0 \ • \
-N •
CN r-·G IDG MGQ
• • • • • ••
(J GIID NQ QNID • UI • N •
G G G
0 ID I")
GG MG MGNAD \DI¤D EDI-
0 \ •\ • \ •
•- ID•-I")•-
•—••-
Duv mm mm _Q • Ch • N •
G G G
G N G• (Q • •
NID •M IDN• •pg • • •
NM \r- IDIDID \
-N • \
-M0 •QIDN
IDN GQ Gr--Q • 1- • N •
¢\I r- 1-
•- M• •\ß
GI \D¢\I •GI- ••—
NN• •N
• • •mm xw wo 'G \-
r--\ •
¢~ QN •m QN• ••-• Nr- ••-4-* Ch •·
G•
ID •~
Ewm •—m QGS
(J 0 ID Ü
ä•Ih I-
ID N ”I IU r-
CJ 'U OC
0 I¤><•-0 .C|·-
-D E PQG ••-
Q)I- I- 2+
Q N 1- GQ
• • •
r- I\ ID Ih\ \ \
Q FI O
U) 7* \D \°\ \Q Q DZ Z Z
_
—lI\\I\GE
C0
L4-*C LD (V) I-
UG I~ G GCIO \ \ \Q I I IGJ
Ih
Q |\*0Z
IO• Q G• •
G \ ND G»~ G G \ \• • Q Q4-* O Z Z
~C QOQ
<Q• Ih I*‘|,ß g
I III v-u '¤ O
C0 •¤><•— 0 -CI-.¤ E +*¤vu
••-U
.
I- I- E+
O Or-Lh OOOO Z • •
ZZZr- (\I|·l7
OQIDNOQ
QGIO°\
• • •Pv~mom Q •—M
QON ONPN • • • Ih Z •
-Ib OOO
N IDNUI N ON N ZZZP QN
Il; GIQID GIID ONO[Q Z
•z
P ID QGIID W NN W Nä. r-(\I¢\l IDLD'U
IZ mo c:•·—¤gg
•Z
W WNID ID IDW W QL PNN IDW r-O‘•-
•• ..JID OQO LD WN LD NNes NNN mw Nr-$, s-r
*4- MmN m gccsO
-¤—<r Osmw Q WQN Q WNGINN PIDID QQll
|¤ L·•-*
C C Q Q Q OQLOwg 8Q ID •'-:·
Q Z•
·W OOO O OOOp :<r oco•— Q ~Qc·s Q cm Q zzz Q zzzIII GIIDID |¤O<’
'UIOLUI
N Ih OON IDOO*8 (Y) ll)P)O
N I•D<fGI N QNP N NWN N ID N O<'IDN Nthth P
LDWGI NNID <'NID OOIO NONgg
N Q!-OQ N QNP N QQQ N Q N ID OZ; QQID <f‘IDN QLDID NZ
• Pr-P NOW WQN OOP- WNQQ
NZZ• Q•.•
I N QWU7 N GIWP N QNID N Q GI QNQL} IDWN UID!-DLD„¤
eu NNID •-WW WQID NWO PQN.,.. Q
• • • (*) • • •Q
• • •H')•-ONI-O P OWN P Inv-GI P <'®I„D P QNNNQW NNN I-DNID WIDN IDNN
QQW QQO NNN
NQP <f NQO N IDQN Q QNN IhIDNW
QNN NWN ®#Ih
O NO O WI-D O NN O
Qih•JJ
gg-PO N O N
I- M Ih co Q
CIO-)En.••-G)I-O
O N#Ui N #@Ih ¢\I r-•-0 (NI GIGQID @ ("IO•¢\|G
|"‘)f")€'7G LDIOLD G O@G G R • • G @¢\JRr- Lhthtb r- £D@@ ¤- ##LD •- @#2 1- f")@@
f'7@N f'7@RR \'0¢'1f'7 U!
LDLDLD•—
cc•<rMII;
@@r- @•-@ @#•- 0Re- @00E P7 @@\D Ih NDRII) I-0 #NR Ih r-@0 R
•-RO46*. R LOIDIJÖ R |D\D\9 R #l-DI!) R thßlr- R LOQLDÜ°O
uu vom mmc <r·<rmlb R@I-D IDr-Rthlö\D @@@ 59 IDl")l\l KD I-D@I-0
*4-¢-> <* U'! N3!-• Oilhlh f'7lh@ IDN@ RIDN
th Elß Ui 9DNRO!Ü~.¢t.ß LOIDID ID @@@ th lbthth Ih Ih@f*) IJTLDU7
L O3 4-*G IB
L @#0 Of')? NRID VDRK) @O|·Ü4-* 4-*<f
©O’|@IDr•GROU
C# ßbthlh @ RRID @ *D@\D @ I-0ID@' # SDI-OlhG)
E UO
€np- Q4; {J'U |ÜR@
•-O•-N@@
•-•-¢\Ir-#@
FB O 6-|f">•·•N 00P*R O NOF) N f*7G)\D # |D@¢\I
la, (6 @ID@ 4*) RR@ K') \DID\D (*7 lbtßl-0 I") \Dth@Ü'UO
Z5 ¤••-•- r-@@ @#Q RKDR @0l*)n- th NLDN R \"')O*·D I-D V-GID R #r·ID *0@@*0 (\| RR@ N \O\D@N|¤„C
4-*éä •-0@ \„h¢•-RMR @@0
NI \DRO\ F') #@04 CD N@r-
'; •— IDRR r- @R@ •- \D@@ n- RRR 1- @\0RI
L)
Ü
.¤ mmm Ihn-O m•-NIh \DO't¢\|
f- @RR @RR *D\D\D R@R @\DR
NCÜR {\|I•h\D @O# @•••G\ f'$¢\lf')O I„h\D¢-h O f*’J@lD Q IDf'i@ O NFÖN O @!")@
\bRR Q@R \0R\D ®©r- @RR
th•
4-*10%-U •- D R Q N•-• FiI!)Q
-r-Ü 'D"
O OFQ N OFF N GQ N QGG Q GGOQ
• • • Q • • • Q• • Q • • • Q • • •
F FFO F FFF F OO F OOO F OOF
OOQ5 • • •Q FFO
QG FOFF • • 01
• • •
Q OO Q FFF
V; OFQO•"!‘° GOG FOF_;
0) • • • Ln • • • M1• • « Q
• • •
Ja F FFQ F FFF F QFQ LQ FFF0'U
I6 OFQ OFF OG OOQ FFFFQ • • • Q
• • •Q
• •Q
• • • Q
.IIß •-FO Ih FFF In OO lb FFO LD FFF5‘°'2
< O> C•· O OFQ FGSN GO OFG NFNgg •,-Q • • « Lp • • • Q
• • gp • • « Q
FFQ Q FFF Q OF Q FFO Q FFF
E LQ- J-!
O UC OFQ OQF QO OFG FNO
gn QQ• • • ¢\,| • • • Q
• • 51• • • Q • • •
1¤ Qt') FFO F) FFF l") OF F) FFO 0*7 FFF
¢O
'D FFQ GQF GO OFU\ FNN15 Ln
• • « 5• • •
Ln• •
5• • • Lg • • •
gi N FFO N OFF N OF N FFO N FFFGJ'UO
E FFQ GCUF GG FOQ NNOR) • • • Q
• • 93• . • Lp
F •-FO F OFF F OO F FFO F FFF
·=éQ FFF OCÜN GO GFQ l'Üf'7•-
Q• • • Q
• • •Q
• •Q
• • • gn ¤• •
Q) FFQ FFF OF OFO FFF
3OUF
O•-FQ FIDQ GF NNQ NQQQ
• • • Q • • • Q ~• Q • • • Q • • •
Fe-O FFF OF FFO 1-FF
4-IU9-'D F O F O N ·
F (*7 ID Q O
ED-·•·lIJ!-O ·
Gۀ
hl lßhllß ¢\l ¢\IQF"hlQ• • • • • Q • • • Q • • •Q • • •
v- \D¢ r- QGQ •- F200 r- Q|~|\ r- 000
¢Ä[L • •
Q IDQ
r- • • • (V) • • •
Q F200 Q IDGQ
IllE ÄÄ GIDID Ä\D\D QFIU4.) (V) « • gn • • • gn • • • Q
• • •
Q. Ä RD? Ä IDGG Ä GÄIN {D IDGUIQ: •- •—-'D
GGND GhlÄ ¢GF) F)IDO\ Ghllß
5- gp • •Q
• • • Q• « •
Q • « •Q
• • •
O eil) ND? Il) IDGG U') F')Ä\B ll) UIÄÄ SD \OG¢\I•·4 r- r-\
>an E GQ Il'2G•- 00Y~ NG0 •-GNgg Qqv • • gp • « • gg • • • gp • • • @ • • •
••• ••-G G¢’ <’IDGU) Ü YWÄND Ü' GQQ ¢ ÄGG
L 4-I n-•— •-•-\•~
G
E LJJ
° 5JJ U l-G \DGihl °’IO$hlpg gß • • [\| • « •
Q • • « {Q • • • Q • • •GF) INI!) f') LOOIG F) FYÄIO F') F7 IDGG
E L) r- •-•-OJJ
SG (GUI GG- IDIDÄ G-Ä hl\DF)g gn • • • • • gp « • • (L • • • [Q • • •
U2 hl Ä? ¢\l l•DO\Ui hl FIÄG hl GGG hI NGOGJ r- •-••-‘¤G
Q
GN •-Ghl Qu-F5 Gr-Q ¢¢hl ‘g @ • • (V) • • « Q
• • • (I') • • • Q • • •
p- •— nm ¤— mom «- mms •- cww•-— r~•-¤
r-•- •-• I-!-
aiJUlhlgg
Q . • Q • • • Q • • • { • • • gn • • ••- nm ~0Oc~ mwß cnww rx-C„Q !-!- •-•-
GI-
BG IDG!-H ÄÄG Il)§'r- GIDÄQ
• • Q• • •
Q • « • Q • • • Q • • •
ÄI0 GDCDUI F)QÄ F)hIG\ Ghlr-•- •—•- 0-I'-
VI •JJ
~
'U I- G Ä G hl1- F)ll)Eä
I- .
O RQO N N)\DP- N QRP N OO Q NNN)Q • • •Q • « •
Q • • « Q • •Q
-• •
P QOR P OPU7 P Qwlb P LOP- P RRQOOSD OOO LDLDLD LOO I-DUÜLB
OQR• • •
w ~:rwr~\D\D‘D
QOO
O OOOI•l’7I„l'|I-I')
IIIL füRO N)N)Q QR für-O9 (Y) • • •
Lh • • •LQ • •
Q • • •Q. R RIR R QNO R QN O RRQ_g OOO OOO Lhtb Lhthnf)
QQN) ON ONO[_
gp . • •Q
• I •Q • • •
Q • « Q • • •O ¢'\LD RQOI LO QCVID LD U\r••® LD U$¢\\ lb RRQ\•-
<OGDO OOO Ihtßl-I') LDO I!)u’)I„D
1 U)> E
vn C PIBQ OQN QOLD P-O |.hP¤-Q QQ • • • IQ • • •Q ¤ • • gp • •
Q • • ••P •PQQON Q RfüQ Q QNQ Q fülh Q QONlt O': ORR ONDKD LDOU7 ORD IDOO
LAJ
° ä-6-v u ms- MNN •„n0r~ wu- QNO45 QQ « • • ¢\| • • •Q • • • (\| • •
Q • « •ON) OQP N) Rfüw fü QNR fü N)R fü OQPL} KDWDR NDSOND NONDLD SDND Olhlb
AJ
8OU Ol-DN) ONIQ QOQ IDN f")ORg gn • • •
R « • • |„f) • • •R • •
Q • • •Q N QON) N RNR N QNR N MO N QO•-ORR OOO IDQDI-D OO LDOOO
QON) NNP IDOQ NO däfül")• • • H') • • •Q • • I (Y; ¤ •
{Q • • •|·— •— ¢\|L¤0s •-Ofüw •— •-LD•- P- OO P- QOPRRR R\DlD \D\D\D BDO LD\D\D
ciI
(.3 Nlfülb N)NQ \h•-Lt') PQ RIÜOQ • • •Q • • •
Q • • •Q • • gn • • •
U NOO OOR NOO OR (\I•-N!E RRR ROO OOO OO OOOIVO-
OQQ IDRQ RQ RR FONQ • « • Q • • • Q • •Q • •
Q • •'1RRO OOR OOP NLG OOPROO ROR LOOQD RR ORR
(I1•
AJ
·¤ •— c n c NP N) U) I Q
EK·•-UI-Q
mmm com svsomo mov mso
W xxx W xxx G xxxIn Ines- Ih 20-e WGes- GOF)
RIGW G•-In OOQQ
smo mov osmIx xxx xxx xxxQ •-¤—\"1 Q Q'xQ
QmmmsNm• QLDQ f')QRIVIE4-,D-GJÜ
s- ¢"I|*'I|x Güifx IxQ®
IB sU1W Ih!-OQ WlxßbQ xxx Q xxx fx xxx
L Q GRIN QGIDI-9-
GR-IO! QIDFI RIIDWDÜIII
GJ QGWGOxxx o xxx v xxx
4-: mmm owm moIB I-hlbln InInQ v-
C
7; 4-*·¤
•¤N•¤ I- xo ·o mus o¤¤•—-
Lé-’ •@ • • • • • • •
m E s•—s mmv sssGJ UID xxx In xxx W xxxRIQQ RI WWW RI |xIxQ
••- O Qmm mmv- ovm.¤ 9 msmmmmI-
S+* UT••- G •Q Qläfx BMGI
•@ 4 • • • • • •
Gem IDWQ Wlxßne G xxx G xxx W xxxs- RIWG r- 0101s- I- GQU1au Nmc m•·-m om•-,5 swo mmm msm
GJ
I- RIs- r- •Q Qu-Ix R|Gr-| •@
¤ • • • • • •
g) Pxlxlxxxx xxx Ih xxx
GJ
•-RII- G•'*¢eu mms smm mmmP-
Qs-60.:7 Ibsln 0H")GGes Int-DQxxx xxx xxxmms mov mvmGII-DID RIIDID s-MQIxGIx 7xWWIx
RIIn O
IQgd)D.
I-
mom OGGW¢\j • • • §- •
-
•
G WQW O I-¤u’>Q O LDWQ
Z ·ZQ
\h¢\¢\I
WNWxxx
ÜCNIID
<1 xxx O- IDIIIQ (Ü \Dlx\Dmx •-mm Q Q xxxE mmm zzz
OJJ
E QWQ SQ} mmm snCGÜ xxx LD xovx QNm N O
• • •Z‘-’
m•-N(\If"7l°\|
Qvxm O~m mmm
~¤Qs¤ LDWQ mrxsoxxx \D xxx xxx
W WWQ W•N•
W
INI
65f•'Hh OW
WQW WQA O xxx N xx
-W WNQ W IDQ
¢\I0 MQM P')L!
r- Ui ~!— JJG GUM-
U '¤•· N N•- U') Q
0 S r-r• JJ.¤ gtiGU •r•llb- I-D
‘
|\ N Ö N OD '\¤•Q v~ r~ Q •
xn \ \ x mx x unan w Q N xn r~M\DG Ui G! (U
¢\I F') ¢\I
O•® Ih Ut N
Q• • • •
I- SD N f\ \DN \ x x 0 x xQ N <r ¢¤ <r O O
F) \D I- LD $9• Q ¢ Q Q NIh
«C+9D-U'U•- N F') Ih O ¢g- • • • ••IB
¢$ Q N ® KD ~Q \ \ \ ¢ \ \ IO
ä Q ® N ® ¢ ®¢Ü—Q |\ Q ID ZQ
€¢ ¢ ¢\|
<>
V)U·•-
r-Q QL
•Z GIS ID IZ IOZ xßtg. Q » •z • .2 • • •Z¤•~ •-Q \b • N N •• \¤r— an •·
.JO \Z \Q \ Q \Q N-
\Q@ \f'7 \D
•IZ \D (Ü BIZ ®@ GZ
gg •(\,| • •~ • • ••
-
• • •-
u E G·
QD Ix DIG NQ NCQ sa LOG IDZ LD ÜZ (*72 FTZ
C CO O
44 JJ•¤ •¤ \D r- Q \D N·¤ L Q
• • • • ••¤
U ·O QD •-D NQ NG IDC;_g •„ • • •z • •• « • • •
UI 0•-¤-
\D¤-· G3 ••wßb SDR) \h<t
G) UI!] N • N • \@ UT N·
N • N•
U CN NO #0 Q2 N QD \D¢ NDQ Q • • • • • •· • • • • . •
·¤-U (Of') Qu- •-Q Of"! lb<f GN
.¤ Ih • ID ~ $2 ND • I"1 • tb •D D D D D
>< .I-Q‘D
Ui Ü GI LH Ö Ng gp) • • • « • •
M•O Or- LDO QO ¤••- DD
Q•~ • an • • • •¤ • • • ••( -—•-· nm mv wm row om
g „ g • • Q , • •
• \D© UIUI KDF) (VCIhG\•-N • I-O • Q • QD • LD • \b
-2 D D D D D DßE·¢-'gp •- Q N •- nn nnZ q· • • • • • •
•¢\| !*@ |"'* @l\l @¢ (UG'Q
• • • • n • n • • • •
gg Q g • g • • • „ ••- •'|"'> 01r- \D¢\| Gif') NQ r-®I
• • • • • •~
-
• • • • •
(J ING! N70!€€
Y\Ih (Ur- •-GiN • E ~ GS
-®
• N • Q •
U Ö Ö F- r- C'! F')
-DP3Q r-• • ND N GJ UIQ5 Q « • • •
•· ¢"}•·· •-<' Gif'? P700 I-DED•|„|1 • • • • • •• • • • •
Ui • \D<' QON) tbr- IDG IDA!_
ggg • • Q • • g .¤*r·•mw
O¤•<tQ NO OG
\D\D G'! • O\I\ \D¢ F7 •· r- •r~
-no ¢¤ •
¤> • nc: GQ<f • C') I") • •
*0 Y\ ¢
II! •4-)
IDW- l\U r- thr•
8gl-!G-
|—g
C') Ih ¢ G I!) I.!)
O 0 0 0 Nus no nnO \ x \ Ox x \N N G G NN• •
G Q
N LC) C') N
G O O NN N N N GNG th Q D ww• •
I") C')(\l
¢ O Il) G ü I.!)
N G O O us O Il).IG \ N \ N\ \ N\N N 0) Q Qt') Q Q
« •z
•Q
N Ner N C')
EO
8IBL8 I!) ¢ G Il) N¤
• • • • •
U G ID O N OUC') \ N N I|)\ NCN G I!) L!) NI.!) GQ
• • ••LJ
C') N I.!) NN N C')
C') I!). Ib I.!)
G O N O Il)¢ \ N IDN N \0 O us 0O Q •
c 0 ~¤· UsC') N C') N
C') G O N ¢
G I.!) O N IDN G \ \ \ C\I\ \
J 0 N 0 In nous 0ß N G N C') G¢ C') N N C') NL)
(Y UI'•- •-I
I IGN- NQ '¤ N th
0N N8.o EQ-l¤ vg ~I- I-
‘Ü IN
NPN N
Ih N N
P Q[D
M Q P
N W *0Q \ N NQ M P 9
9 QID Q
W Q
WN N NI") N Ih• N
(V) •
ID P
W9 W9 M9•z •z •z
P [Q •• gp •• gg •-IQ‘ N9 N9 N9NM WZ NZ 0\Z
E @9 99 P9‘ P WZ WZ NZC
· O
4-*
-0-* [D9 N9 N9, Z
• •- • • • •
U NG! WW WMUW N
• N-
N•
QN P9 W9 M9Q
• • • • • ••
9 GNU) NQ QNIh • 01 ~ N •
9 9 9
W LD M
99 M9 M9 '
NU'! WW Wr-W N • N
-N
•P WP MP PP
99 WO! lhlhW
• Oh • N •
. 9 9 9
9 N 9
NU'! NC'! WN
NM NP Wu')Ih N • N • N •
MW NQ WN
IDN PW ¢\•--Q « Q • N
•
N PP P
|* FI
N N•
¤~P sb- NN
WM NW W9P Q
• • • .• WN PLD QN
·4-* ·P~•- •P
Q cg • N • LD •·
O WC') 9W WQg} • P
• •
sa W ID Q
N VI-P 4-*
IL %"'NIQU
So
P gä3 QM
•r·QJI- I-9
-N •-· w
¢ IR C Ih„ Ö \ R R
« ¢ I") G• • •
.-JF! R C C. gw \ x x'
sa_
C·
9
4-*. OU
L-^ 4-*
C Ih (6 •-
Q • • • I
· uw r~ so nnCC \ R \_ 0(J
¢ RC \
' DZ
‘ •¤ ~¢ oR C C
-O x \ \• C C D Q
4-*• Z ZO
ÜUsf
° Ih•
r- 4-*I I5
L) 'U N
04-*
Ti g== —I- kg
The vita has been removed fromthe scanned document
![Chapter 4shodhganga.inflibnet.ac.in/bitstream/10603/35843/12... · ), tertiary butyl hydrogen peroxide (TBHP) and Cumene hydroperoxide (CHP). Besides these, aluminum [43, 44], copper](https://img.pdfslide.net/doc/110x75/5f318d4c4479b6276c09aff3/chapter-tertiary-butyl-hydrogen-peroxide-tbhp-and-cumene-hydroperoxide-chp.jpg)
