BIODEGRADABILITY OF HYDROCARBON CONTAMINANTS DURING NATURAL ATTENUATION OF CONTAMINATED GROUNDWATER DETERMINED USING BIOLOGICAL AND CHEMICAL OXYGEN DEMAND A Master’s Thesis Presented to the Faculty of California Polytechnic State University San Luis Obispo In partial fulfillment of the requirements for the degree of Master of Science in Civil and Environmental Engineering By Evan B. Larson February 2004
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BIODEGRADABILITY OF HYDROCARBON CONTAMINANTS DURING NATURAL ATTENUATION OF CONTAMINATED
GROUNDWATER DETERMINED USING BIOLOGICAL AND CHEMICAL OXYGEN DEMAND
A Master’s Thesis Presented to the Faculty of California Polytechnic State University
San Luis Obispo
In partial fulfil lment of the requirements for the degree of
Master of Science in Civil and Environmental Engineering
By
Evan B. Larson
February 2004
COPYRIGHT OF MASTER’S THESIS
I grant permission for the reproduction of this thesis in its entirety or any of its parts, without further authorization from me, provided it is referenced appropriately.
Evan Larson Date
i i
MASTER’S THESIS APPROVAL TITLE: BIODEGRADABILITY OF HYDROCARBON
CONTAMINANTS DURING NATURAL ATTENUATION OF CONTAMINATED GROUNDWATER DETERMINED USING BIOLOGICAL AND CHEMICAL OXYGEN DEMAND
AUTHOR:
EVAN B. LARSON
DATE SUBMITTED:
FEBRUARY 2004
THESIS COMMITTEE MEMBERS:
Dr. Yarrow Nelson Date
Dr. Nirupam Pal
Date
Dr. Christopher Kitts
Date
i i i
ABSTRACT
BIODEGRADABILITY OF HYDROCARBON CONTAMINANTS DURING NATURAL ATTENUATION OF CONTAMINATED GROUNDWATER
DETERMINED USING BIOLOGICAL AND CHEMICAL OXYGEN DEMAND
Evan Larson
Natural attenuation is being evaluated as a possible method of remediation of hydrocarbon contamination at the Guadalupe Restoration Project (GRP) at a former oil field on the Central Coast of California. The site is contaminated with hydrocarbons in the C10 to C30 range, which were used as a diluent to facilitate oil extraction. The GRP is located in an ecologically sensitive coastal area and thus it is important to remediate the hydrocarbon contamination with minimal disturbance. Natural attenuation is the microbial degradation and weathering of a contaminant, and interest has grown throughout the environmental community in its application over the past decade. To explore the feasibility of using natural attenuation at the GRP, a series of experiments were conducted to determine the biodegradation rates of total petroleum hydrocarbon (TPH) in groundwater from the site; and to evaluate the sustainability of biodegradation with weathering. In order for natural attenuation to be sustainable at this site, it is important that the hydrocarbons remain biodegradable as they are weathered. To test for this sustainability, biodegradability was determined for a series of groundwater samples, which had weathered differently. Biodegradability was measured as the ratio of biological oxygen demand (BOD) to chemical oxygen demand (COD). BOD/COD ratios were measured for diluent-contaminated groundwater from monitoring wells C8-39, G4-3, 206-C, 209-D, 209-E, H3-7, H2-1 and M4-4. The TPH concentrations ranged from 4.2 ppm to 29 ppm. Sampling was originally planned along the transect of a single plume to observe biodegradation patterns along the transect as the hydrocarbons presumably become more weathered down-gradient. Due to constraints concerning the nesting pattern of the Western Snowy Plover, this method of sampling was abandoned. As a surrogate method of collecting samples with varying degrees of hydrocarbon weathering, the series of monitoring wells listed above were used to provide a range of TPH concentrations, and wells with low TPH concentrations far from source zones were presumed to be more weathered. The range of BOD/COD values for these groundwater samples were 0.01 to 0.09, suggesting slow biodegradation. BOD/COD did not correlate with TPH concentration (R2 = 0.03). BOD/COD ratios did not significantly change with increasing TPH concentration, suggesting weathering did not significantly influence biodegradability. BOD/COD ratios decreased with distance from source, indicating the possibility of decreased biodegradability with increased weathering and a variation in diluent chemistry. COD correlated with TPH values fairly well with an R2 value of 0.74. BOD had a very weak correlation with TPH concentration (R2 = 0.41). The average COD/TPH value was 18.1. This COD/TPH ratio is approximately five times the expected theoretical oxygen demand (ThOD) of hydrocarbons of 3.5. This high value may be attributed to the presence of other oxidizable organics. BOD/COD ratios approaching the value of 0.4 have been reported for biodegradable material. However, the low BOD/COD ratios observed in this research were most likely because of slow biological degradation leading to low 5-day BOD values.
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ACKNOWLEDGEMENTS
I would like to thank my friends and family for their patience and support as they waited
for this day to arrive.
Also, I would like to give a special thanks to Unocal for their support and funding of
research at Cal Poly.
Finally, I must thank Dr. Yarrow Nelson for his guidance, patience and insight. It was a
pleasure to be around his upbeat attitude, refreshing sense of humor and great personality.
v
TABLE OF CONTENTS
List of Tables .......................................................................................................................x
List of Figures .................................................................................................................... xi
Biochemical oxygen demand (BOD) is defined as the amount of oxygen
required by bacteria while stabilizing decomposable organic matter under
aerobic conditions (Sawyer and McCarty, 1978). It is a test applied to
measure the amount of biologically oxidizable organic matter present and
determining the rates at which oxidation will occur or BOD will be
16
exerted (Sawyer and McCarty, 1978). In order to make the test
quantitative, the samples must be placed in an airt ight container and kept
in a controlled environment for a preselected period of t ime. In the
standard test, a 300-mL BOD bottle is used and the sample is incubated at
20°C for five days (Peavy et al. , 1985). The BOD is then calculated from
the initial and final dissolved oxygen (DO) concentration.
3.8 Chemical Oxygen Demand
The chemical oxygen demand (COD) test is used to measure the total
organic content of industrial wastes and municipal and natural
wastewaters. During the determination of COD, organic matter is
converted to carbon dioxide and water using a strong chemical oxidizing
agent (dichromate) in the presence of a catalyst and strong acid. In the
COD test, organic materials are oxidized regardless of the biological
assimilability of the substances. As a result, COD values are greater than
BOD values and may be much greater when significant amounts of
biologically resistant organic matter are present (Sawyer and McCarty,
1978).
3.9 Chemistry of Diluent Contamination at the Guadalupe Site
Diluent from the Guadalupe Restoration Project is a hydrocarbon
consortium with a carbon range of nC10 to nC3 0. Figure 3.3 shows
common fuel ranges with respect to carbon length. According to this
17
chart, the diluent at Guadalupe is essentially a diesel range oil (DRO).
Water solubility plays an important role when considering the fate of
diluent constituents. Constituents with low solubility exist as a separate-
product, whereas diluent chemicals with a high solubility generally are
dissolved in the groundwater. A majority of the diluent at the Guadalupe
Oil Field has low solubility. Diluent from Guadalupe has a reported
solubility of 30 mg/L (Haddad and Stout, 1996). The diluent composition
over the Guadalupe site is considerably variable. The difference in
diluent makeup can be explained by source oil variation and weathering
(Barron and Podrabsky, 1999).
Haddad and Stout made the following conclusions on the diluent
chemistry:
• The carbon length of the diluent ranges from <nC10 to >C3 0. About
70% of the diluent falls in the diesel range of nC1 0 to nC2 5.
• Saturated, aromatic, polar, and asphaltic fractions respectively make
up 60%, 17%, 8%, and 15% of the separate-phase diluent. The
dissolved-phase fractions were not available for review.
Haddad and Stout (1996) also reported the total petroleum hydrocarbon
(TPH) composition as well as the BTEX concentrations (Table 3.1 and
3.2).
18
C 3 6C 2 4
R E F E R E N CE : T P H I N S O I L P R I M E R , E L A I N E M . S C H W E R K O . D A T E D : 0 9 / 0 1 / 9 3 N o n - me a s u r a b l e T P H d u e to
v o l a t i l i z a t io n
1 70 C
3 40 F
1 70 C
3 40 F
C 1 0
D i e s e l Ra ng e
G a s o l i n e R a n g e
C 1 0
C 6
1 70 C
3 40 FB o i l i n g
P o i n t R a n g e
1 40 F
1 40 F
C 3 6
C 2 0
C 2 4 - 3 0
C 2 4
C 1 2
C 1 7
C 1 2
C 8
C 8
C 1 0
C 6
C 6
C 4
C 4
Lube Oi l s & heavier
Fuel Oi l s
Diese l
Semi-quant i f iable
Kerosene
Gasol ine
Measurable TPH
Table 3.1 Summary of separate-phase diluent analysis.
Constituent Concentration Range (mg/kg)
Benzene <2.0 to 120 Toluene <2.0 to 74 Ethylbenzene 2.2 to 200 Total Xylene 5.3 to 370 TPH: nC10 to nC32
910,000 to 990,000
Figure 3.4 Carbon ranges for common diluent constituents compared to common petroleum distillates (Elliot, 2002).
19
Table 3.2 Summary of dissolved-phase diluent analysis. Constituent Concentration Range (µg/L)
Benzene <0.5 to 9.1 Toluene <0.5 to 5.1 Ethylbenzene <0.5 to 7.4 Total Xylene <0.5 to 17 TPH: nC6 to nC10 nC10 to nC32
<50 to 400 870 to 16,000
(E l l io t 2002)
3.10 Cal Poly Natural Attenuation Project Unocal is currently funding remediation research at California
Polytechnic State University, San Luis Obispo through the Environmental
Biotechnology Institute (EBI). This project is part of a larger set of
experiments aimed at determining how the concentrations of petroleum-
derived hydrocarbons change due to remediation by natural or engineered
methods. Some of the other research projects are biosparging, steam
injection and phytoremediation. Anaerobic aspects of natural attenuation
of diluent were considered separately (Maloney, 2003), and further
natural attenuation research is currently underway.
20
CHAPTER 4
MATERIALS AND METHODS
4.1 Groundwater Sampling
Bob Pease collected groundwater samples from the Guadalupe site. Three
to five well volumes were purged before sample collection. Some
groundwater samples were used for BOD and COD method development,
while others were used for the natural attenuation experiment.
Monitoring wells sampled were G4-3, H2-1, H3-7, 206-C, 209-D, 209-E
and M4-4.
4.2 BOD Measurement
BOD measurements involve seeding a test sample, storing the sample for a
specified time and obtaining the initial and final dissolved oxygen (D.O.)
values.
In the BOD experiments, triplicates were run of the seed and dilution
water. These were respectively called seed control and dilution water
blank.
The BOD standard (glucose glutamic acid test, GGA) is intended to be a
reference point for evaluation of dilution water quality, seed
effectiveness, and analytical technique. GGA reagents were purchased
from Hach Co. USA.
21
BOD is computed using the following equation (Eqn. 1):
BOD = (D1 - D2) - (B1 - B2)f / P (1)
Where,
BOD = biochemical oxygen demand, mg/L
D1 = DO of diluted sample 15 minutes after preparation, mg/L
D2 = DO of diluted sample after incubation at 20°C, mg/L
B1 = DO of seeded dilution water blank before incubation, mg/L
B2 = DO of seeded dilution water blank after incubation, mg/L
f = ratio of seed in sample to seed in blank
= % seed in D1 / % seed in B1
P = decimal fraction of sample used
= mL of sample, Vs / 300 mL
4.2.1 BOD Inoculum
Inoculum for the BOD measurements must be of appropriate strength for
obtaining meaningful BOD data. Viable bacterial populations must be
present to have enough oxidation occurring to yield accurate BOD
measurements. However, the inoculum should not produce more than 10%
of the total oxygen consumption during the BOD analysis.
Preliminary Experiment Inoculum Preparation
The groundwater used for preparation of inoculum for preliminary experimentation had
an original TPH concentration of approximately 2 ppm. This sample was collected by
22
Ken Hoffman from C8-39 and had been sitting in the laboratory for a few months at room
temperature. The TPH concentration would have been depleted after a several month
storage period. This preliminary inoculum was formulated by mixing 1000 mL of the 2
ppm Guadalupe groundwater from C8-39 and 100 g of diluent-contaminated soil from the
Guadalupe site. This inoculum was continuously stirred for aeration at ambient
temperature for one-week prior to use. This will be labeled Ii.
Inoculum Preparation for Preliminary BOD Tests
Inoculum for the BOD2 0 and BOD6 experiments were prepared by using
one Polyseed® BOD Seed Inoculum capsule (Interbio, Woodland, TX),
about 100g diluent-contaminated soil and 100 mL of I i and 900 mL of
Guadalupe groundwater for a total volume of 1 liter. The groundwater for
this inoculum preparation is the depleted 2ppm TPH collected by Ken
Hoffman from monitoring well number C8-39 as described above. This
inoculum was labeled I i i . Continuous stirring following initial
preparation was used for aeration.
Inoculum Preparation for BOD5 Tests on Groundwater
For final BOD experiments, a third inoculum was prepared one month
following the preparation of I i i . This was used for the final BOD5
experiments using several groundwater sources. 100 mL of inoculum I i i
was added to 900 mL of Guadalupe groundwater, 100 g diluent-
contaminated soil and one Polyseed® capsule. The 900 mL of
23
groundwater used was a fresh sample from well number C8-39 at 8-12ppm
TPH, provided by Bob Pease. Continuous stirring was used for aeration
until use for BOD measurement four days later.
4.2.2 Dilution Water
Dilution water is used to provide trace elements to microbial populations
and to dilute samples to a measurable BOD range. Mineral deficiencies
and pH shifts can cause low BOD results. Thus, Hach BOD nutrient
buffer pillows (Hach Co., Loveland, CO) were used in the preparation of
dilution water for all BOD experiments. Each pillow contains buffer and
nutrients specified by the U.S. Environmental Protection Agency
(USEPA) and American Public Health Association (APHA) in the
Standard Methods for the Examination of Water and Wastewater (1999).
Dilution water was prepared by adding one Nutrient Buffer Pillow to 3 L
of deionized water.
Dilution water was bubbled with air, which passed through a 2-µm inline
filter, for at least twenty minutes to ensure maximum dissolved oxygen.
Air was filtered to avoid lubrication oils from the air pump and foreign
particulates in the ambient air from being introduced into the dilution
water. To ensure undiluted samples had the same final nutrient
concentration as the diluted samples, additional nutrients were also added
to full-strength (non-diluted) samples by adding 3 mL of 100x nutrient
24
stock to each BOD bottle. Similarly, a proportional amount of nutrient
stock was added to all 50% diluted BOD bottles to ensure nutrients were
not a limiting factor. The 100x solution was prepared by adding one
nutrient buffer pillow, to 30 mL deionized water.
4.2.3 Nitrification Inhibitor
Nitrogenous biochemical oxygen demand (NBOD) is the amount of
oxygen required for biological oxidation of ammonia to nitrate via
nitrification (Tchobanoglous and Schroeder, 1987). BOD determinations
may be inadequate for evaluating efficiency of treatment processes if
nitrifying bacteria are present. NBOD usually occurs after seven days of
incubation. NBOD was not of interest in this experiment since it would
erroneously indicate biological activity by overestimating the BOD,
resulting in an overestimation of the actual biological removal efficiency.
Nitrification inhibitor was therefore used throughout these experiments to
inhibit NBOD.
Hach Formula 2533™ nitrification inhibitor, 2-chloro-6-(trichloromethyl)
pyridine (TCMP), eliminates the nitrifying interference when testing
samples. Nitrification inhibitor can be used with the USEPA-accepted
BOD dilution method (HACH, 2003). Results of BOD tests completed
with inhibitor are referred to as carbonaceous BOD (CBOD).
This nitrification inhibitor is plated on an inert salt , which allows
inhibitor to dissolve quickly in samples. 0.16 g of nitrification inhibitor
25
were added to each 300 mL BOD bottle to make a final concentration of
10 mg/L TCMP. Hach Formula 2533™ nitrification inhibitor was used for
all BOD bottles.
4.2.4 Dissolved Oxygen Measurement
Dissolved oxygen concentrations were made with a YSI model 58
dissolved oxygen meter with YSI model 5905 Self-Stirring BOD Probe
(YSI Inc., Yellow Springs, Ohio). 300 mL BOD bottles with Vapor-
sealing caps were obtained from Wheaton Science Products (Millville,
NJ).
Figure 4.1 BOD analysis setup: DO meter, probe and BOD bottle.
Dissolved Oxygen Calibration
1) The YSI 58 DO meter was connected to the YSI 5905 probe
and the instrument was allowed to warm-up/stabilize for 15
minutes prior to use.
26
2) The probe was zeroed and calibrated at a temperature as close
as possible to the temperature of the sample to be measured
to obtain the highest accuracy of measurement. Setting the
function switch to ZERO and adjusting the display to read
00.0 with the O2 ZERO control zeroed the YSI 58.
3) Following zeroing of the DO meter, the function switch was
set to % Mode .
4) The BOD probe was placed in a BOD bottle containing about
one inch of water to provide a 100% relative humidity
calibration environment.
5) When the display reading had stabilized, the 02 CALIB
control locking ring was unlocked and the display was
adjusted to the CALIB VALUE obtained from the
pressure/altitude chart in Appendix F of the instruction
manual. The locking ring was then relocked to prevent
inadvertent changes.
4.2.5 BOD Incubation
All BOD bottles were placed in an incubator, in the absence of light, at
20°C ±1°C for five, six or twenty days. All bottles had a wet seal and had
a cap to act as a vapor seal over the top of the BOD bottle seal to ensure
no evaporation of the wet seal.
27
4.2.6 Fe Effects on BOD
To investigate possible effects of reduced iron on the measurements of
BOD, the BOD was measured for groundwater samples with and without
Fe2 + added. The concern was that abiotic Fe2 + oxidation to Fe3 + could
consume oxygen, leading to interference with the BOD test. Guadalupe
diluent-contaminated groundwater from C8-39 was used for this
experiment, and it contained approximately 2 ppm TPH. An iron
concentration of 40 mg/L was added to this groundwater, using FeSO4 and
used for two sets of samples. One set of samples was bubbled for 10
minutes to oxidize the Fe+ 2. Another set of samples was not bubbled.
This comparison was made to determine if bubbling could be used to
eliminate any biological oxygen demand due to Fe2+ oxidation in the event
that such oxygen demand was significant.
4.2.7 Dilution Effects on BOD
Single source samples from well number C8-39 and the series of
groundwater samples were tested to examine BOD changes due to dilution
and as a part of the general testing of the BOD series. Some samples were
diluted to bring final DO values to within a usable range and to verify
BOD values should remain constant. This would be expected when
looking at the definition of the P-value in Eqn. 1.
28
4.3 COD Measurement
COD was analyzed using the accu-TEST™ mercury-free micro-COD
system (Bioscience Inc., Bethlehem, PA). Potassium bipthalate (KHP)
(Spectrum Chemical Co., Redondo Beach, CA) was used as a COD
standard. Absorbances of KHP standards of known concentrations were
measured and the COD in mg O2/L was calculated using the stoichiometric
relation between KHP and oxygen (Eqn. 2).
KC8H5O4 + 29/4 O2 → 8 CO2 + 5/2 H2O + K+ (2)
A Hitachi U-3010 UV/Vis spectrophotometer was used to measure
absorbance for all COD analyses and calibrations. Bioscience 5-150 mg/L
low range COD vials were used for iron oxidation experiments using aged
2 ppm concentration groundwater. The COD of low range vials was
determined using a spectrophotometer at 440 nm by measuring the
decrease in concentration of the Cr (VI) ion. Vials were incubated for
120 minutes at 150°F ± 2°F. Before analysis, the vials were allowed to
cool in the dark to prevent further oxidation. In the COD experiments, a
DI water control blank was run in triplicate.
Bioscience 20-900 mg/L standard range COD vials were used for
experiments using fresh 8-12 ppm or refrigerated samples from the other
wells requiring a higher range COD. The COD of standard range vials
29
was determined using a spectrophotometer at 600 nm by measuring the
concentration of the produced Cr (III) ion.
4.3.1 COD Calibration
The COD method was calibrated by measuring the COD of KHP standards.
The standard curve was created for samples ranging from 5-150 mg/L
(Low Range) and used to convert the measured absorbance at 440 nm to
mg/L COD. Samples above 150 mg/L were tested using the 20-900 mg/L
(Standard Range) vials and calibrated at 600 nm. For both ranges, the
absorbance was plotted against COD concentration for duplicates of each
KHP concentration.
4.3.2 Measurement of Iron Oxidation Effects on COD
COD was measured for groundwater samples with and without added Fe2 +
to test for COD of dissolved iron. 2.5 mL of Guadalupe diluent
contaminated groundwater from C8-39 was used for this test with aged 2
ppm TPH. One triplicate of test samples contained Fe2 + at a concentration
of 40 mg/L FeSO4, while another triplicate of test samples did not have
Fe2 + added. COD was measured using Bioscience 5-150 mg/L COD vials
with absorbance measured at 440 nm. The KHP standard curve for this
COD range was developed using the following concentrations in
triplicate: 1, 2, 5, 10, 20 and 30 mg/L COD or mgO2/L. The resulting
30
trendline from the KHP standards used to convert absorbance to COD
yielded R2 = 0.96 (Figure 4.2).
4.3.3 COD Measurements of Groundwater Series
Low-range Bioscience COD vials (5-150 mg/L) were used for initial COD
measurements, until i t was realized that some of the samples were out of
this range. COD was measured in triplicate for this series of six
groundwater samples. For the final COD analysis using the 20-900 mg/L
Bioscience COD vials, the KHP standard curve was developed using the
following standard concentrations in triplicate: 25.5, 81.8, 204.5, 409.0,
613.4 and 817.9 mg/L COD or mgO2/L. The resulting trendline from the
KHP standards used to convert absorbance to COD yielded R2 = 0.99
(Figure 4.3).
4.3.4 UV/Vis Spectrophotometer Analysis
As mentioned previously, a Hitachi U-3010 UV/Vis spectrophotometer
was used for all COD analysis and calibration. Before each use, the
spectrophotometer was calibrated with DI water and a zero value was
recorded from an average value of the control blank triplicates.
31
y = -0.0129x + 0.6391R2 = 0.9556
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 5 10 15 20 25 30 35
COD (mg/L)
Abs
orba
nce
at λ
= 4
40 n
m
Figure 4.2 Absorbance vs. COD of KHP standards, 5-150 mg/L range. Measuring the decrease in concentration of the Cr (VI) ion.
32
y = 0.0003x + 0.0537R2 = 0.9985
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0 100 200 300 400 500 600 700 800 900
COD (mg/L)
Abs
orba
nce
at λ
= 6
00 n
m
Figure 4.3 Absorbance vs. COD of KHP standards, 20-900 mg/L range.
Measuring the concentration of the produced Cr (III) ion.
33
4.3.5 COD of Phenol Solutions
COD was measured for phenol, to establish COD vs. ThOD. Phenol is a
hydrocarbon compound with a known theoretical oxygen demand (ThOD)
of 2.38 mg/L. The stoichiometric relation for the ThOD of phenol is
given in Eqn. 3 below.
Calculation of ThOD for phenol:
Phenol: C6H5OH Molecular Weight: 94 g/mol
C6H5OH + 7O2 → 6CO2 + 3H2O (3)
(7 mol O2) * (32 g/mol O2) = 224 g O2
(224 g O2) / (94 g/mol)
ThOD = 2.38 g O2/ mol
34
CHAPTER 5
RESULTS
5.1 BOD Results
5.1.1 Preliminary BOD Measurements and Effect of Iron Oxidation on
BOD Measurement
The BOD method and possible effects of Fe on BOD measurements were
tested using a groundwater sample collected from well C8-39 with an
original TPH concentration of 2 ppm that was stored at room temperature
for two months. For the undiluted, full-strength (FS), groundwater
samples, the DO only decreased by 0.4 mg/L (Table 5.1). The same
groundwater samples diluted by 50% also showed a DO decrease of about
0.35 mg/L. These results suggest very minimal biodegradation of this
aged groundwater sample, probably because much of the TPH was
degraded before the experiment.
The DO depletion from the dilution water blank exhibits what should be
the minimal, with an average of 0.17 mg/L (Table 5.1). DO for the seed
controls actually increased slightly, buy by a negligible 0.25 mg/L
Glucose glutamic acid (GGA) BOD standards were run to ensure that the
appropriate amount of seed was used and as a measure of general
reliability of the BOD test. The measured BOD standard values of 450
mg/L were about 2% higher than the upper end of the expected range of
35
396 ± 61mg/L, based on the ThOD of GGA. The BOD of GGA standards
were nearly identical for the samples with 2 mL or 4 mL of standard
solution (Table 5.1), indicating good reliability of the BOD methods
employed. The slightly lower BOD of the sample with 6 mL of standard
solution was likely the result of oxygen depletion, to below 2 mg/L.
No appreciable effect of Fe2 + on measured BOD was observed. Undiluted
samples without Fe added exerted a BOD5 of 0.66 ±0.02 mg/L while
samples with Fe added (no sparging) exerted a BOD of 0.57 ±18 mg/L
(Table 5.1 and Figure 5.1). This difference is within the experimental
error of the BOD measurement and is negligible. Sparging the samples
with Fe added slightly reduced the observed BOD (Table 5.1 and Figure
5.1), but this difference was also within the experimental error of the
BOD measurements. These results suggest that reduced Fe will not
interfere with the BOD measurements for the groundwater samples.
0.00.10.20.30.40.50.60.70.8
Groundwater Samples With Iron [40 mg/L] error bars indicate ± 1 standard deviation
Figure 5.1 Iron effect on BOD5.
BO
D5 (m
g/L)
no Fe Fe w/o sparge
Fe w/ sparge
36
Table 5.1 BOD results and statistics for method development. Sample
5.1.2 Preliminary BOD Method Testing: Dilution Effects (6-day BOD)
This experiment tested for dilution effects using one fresh groundwater
sample from well C8-39, at 8-12 ppm TPH. The groundwater sample was
kept refrigerated prior to use, to maintain the high TPH concentration.
Full strength samples using 10 mL of seed had an average BOD6 of 4.31
mg/L ± 0.13 mg/L and with 20 mL of seed, had an average BOD6 of 4.00
mg/L ± 0.05 mg/L (Table 5.2 and Figure 5.2). This suggests 10 mL of
seed is sufficient for the BOD analysis. Similarly, 50% strength samples
using 10 mL of seed had an average BOD6 of 5.69 mg/L ± 0.24 mg/L and
with 20 mL of seed, had an average BOD6 of 5.59 mg/L ± 0.35 mg/L
accounting for dilution in the calculation of BOD (Table 5.2 and Figure
5.2). The seed control samples with 10 mL seed had an average BOD6 of
0.06 mg/L ± 0.01 mg/L and with 20 mL seed had an average BOD6 of 0.16
mg/L ± 0.05 mg/L (Table 5.2).
The calculated BOD exerted by the 50% diluted groundwater samples, at
5.69 ± 0.24 mg/L and 5.59 mg/L ± 0.35 mg/L, were approximately 30%
higher than those calculated for the full strength samples, at 4.31 mg/L ±
0.13 mg/L and 4.00 mg/L ± 0.05 mg/L (Table 5.2). They were expected to
be nearly equal. Therefore dilution experiments were conducted and an
evaluation of dilution effects based on these experiments is given below
in section 5.1.4.
38
The BOD standard samples (GGA) with 10 mL seed had an average BOD6
of 468.0 mg/L ± 12.7 mg/L and with 20 mL seed had an average BOD6 of
474.8 mg/L ± 3.2 mg/L (Table 5.2). These standards show excellent
agreement and are relatively close to the expected value of 396 ± 61
mg/L.
39
Table 5.2 BOD results for 6-day test with single groundwater sample (fresh C8-39, 8-12 ppm). Bottle numbers 29 and 31, with respective final DO concentrations of 3.09 and 6.29 mg/L, are outliers and not used.
Table 5.5 BOD data for BOD5 repeat analysis of oxygen depleted samples 209-E and 206-C. For well 206-C, one BOD5 value was nearly two standard deviations off.
50
5.2 COD Results 5.2.1 Iron Oxidation Effects on COD Measurement and Dilution Effects
The aged 2 ppm TPH groundwater sample with Fe2 + added [40 mg/L FeSO4]
exhibited an average COD value of 34.50 ± 0.82 mg/L, while the same
groundwater sample without Fe2 + added had an average COD value of 35.98 ±
1.49 mg/L (Table 5.6 and Figure 5.6). These values are within experimental
error, suggesting COD exerted by the oxidation of Fe2 + to Fe3 + is negligible.
The 50% diluted samples had an average COD value of 15.28 mg/L, with a
standard deviation of 0.71 mg/L (Table 5.6). This is approximately half the
COD value of the full strength groundwater, as expected.
The calibration curve from the KHP standards used to convert absorbance to
COD yielded R2 = 0.96 (Figure 4.2).
52
Table 5.6 Iron oxidation COD data. The last value for a blank is an outlier.
Figure 5.6 Effect of iron oxidation on COD measured.
St o t /4.65 = sqrt(0.000896 + 0.000296 + 0.00139)
St o t /4.65 = 0.0508
St o t = 0.236
Since we only report error to 1 significant figure, the answer to this
problem would be 4.7+-0.2
58
Table 5.10 Final results of COD, BOD and calculated BOD/COD ratios for groundwater series. SD and RSD indicate standard deviation and relative standard deviation, respectively.
Figure 6.1 COD vs. phenol concentration for standard phenol solutions.
0
50
100
150
200
250
300
350
150Phenol concentration (mg/L)
error bars indicate ± one standard deviation
CO
D (m
g/L)
150 300
COD/phenol = 0.96
COD/phenol = 0.94
74
CHAPTER 7
CONCLUSIONS
The range of BOD/COD vales for this project were 0.01 to 0.09,
suggesting slow biodegradation. In 1987, Gilbert stated a
BOD/COD value below 0.4 suggests a low biodegradability.
BOD/COD ratios did not correlate with increasing TPH
concentration suggesting weathering did not significantly influence
biodegradability. This also may indicate the contaminant is not
becoming recalcitrant during biodegradation.
BOD/COD ratios suggest biodegradability may decrease with
distance down-plume from source. A limited number of wells were
used and more well should be used for further analysis.
Methods were successfully demonstrated for BOD and COD. Tests
did not have any significantly unusual occurrences.
COD/TPH values ranged from 11.4 to 23.8, with an average of 18.1.
This average value is approximately five times higher than the
expected value of 3.5 mg/L based on ThOD. This may mean TPH
values should be evaluated at the beginning and end of an
experiment.
COD values may have been high due to the presence other
oxidizable organics.
The biodegradability decreased with distance down-plume from
source, possibly signifying a decrease in biodegradability as the
hydrocarbons are weathered.
75
CHAPTER 8
RECOMMENDATIONS
The following are recommendations for future evaluation of the
biodegradability of hydrocarbon-contaminated groundwater.
It would be beneficial to run TPH analysis with more than one
sample, and having multiple independent lab analysis. This would
put to rest any uncertainty in the reported TPH values.
Measuring actual TPH degradation rates using duplicate initial and
final TPH analyses would be preferable to relying on O2
consumption or respiration rates.
Biodegradability should be estimated for a greater number of wells
with some actual plume transects.
Using TOC as a supplement to the testing methods would help in
obtaining a better understanding of the organic fraction of the
samples.
The use of a more precise method of measuring D.O. would be
beneficial in obtaining BOD values.
76
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