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
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
ii
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
iii
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 diluentcontaminated groundwater from monitoring wells C8-39, G4-3, 206-C, 209-D, 209-E, H37, 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 downgradient. 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.
iv
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
1
INTRODUCTION .........................................................................................................1
2
PROJECT SCOPE .........................................................................................................4
3 BACKGROUND ...........................................................................................................5
3.1
Former Guadalupe Oil Field .................................................................................5
3.2
Natural Attenuation as a Treatment Technology ..................................................9
3.2.1
Biodegradation........................................................................................11
3.2.2
Dilution/ Dispersion................................................................................12
3.2.3
Volatilization...........................................................................................12
3.2.4
Adsorption...............................................................................................13
3.3
Advantages of Natural Attenuation ....................................................................13
3.4
Limitations of Natural Attenuation.....................................................................14
3.5
Site Criteria .........................................................................................................15
3.6
Determination of Contaminant Biodegradability................................................15
3.7
Biochemical Oxygen Demand ............................................................................16
3.8
Chemical Oxygen Demand .................................................................................17
3.9
Chemistry of Diluent Contamination at the Guadalupe Site ..............................17
3.10 Cal Poly Natural Attenuation Project .................................................................20
4 MATERIALS AND METHODS.................................................................................21
4.1
Groundwater Sampling .......................................................................................21
4.2
BOD Measurement .............................................................................................21
4.2.1
BOD Inoculum........................................................................................22
4.3
4.2.2
Dilution Water ........................................................................................24
4.2.3
Nitrification Inhibitor..............................................................................25
4.2.4
Dissolved Oxygen Measurement ............................................................26
4.2.5
BOD Incubation ......................................................................................27
4.2.6
Fe Effects on BOD..................................................................................28
4.2.7
Dilution Effects on BOD ........................................................................28
COD Measurement .............................................................................................29
4.3.1
COD Calibration ....................................................................................30
4.3.2
Measurement of Iron Oxidation Effects on COD ...................................30
4.3.3
COD Measurements of Groundwater Series...........................................31
4.3.4
UV/Vis Spectrophotometer Analysis......................................................31
4.3.5 COD of Phenol Solutions........................................................................34
5
RESULTS ....................................................................................................................35
5.1 BOD Results .......................................................................................................35
5.1.1
Preliminary BOD Measurements and Effect of
Iron Oxidation on BOD Measurements ..................................................35
5.1.2
Preliminary BOD Method Testing: Dilution Effects (6-day BOD)........38
5.1.3
Preliminary 20-day BOD Test for BOD Method....................................41
5.1.4
Dilution Effects and BOD Analysis of Several Groundwater Samples..42
5.1.5
Repeat BOD Analysis of Diluent-Contaminated
Groundwater Samples.............................................................................49
5.2 COD Results .......................................................................................................52
5.2.1
Iron Oxidation Effects on COD Measurement and Dilution Effects......52
5.2.2
COD of Groundwater Series...................................................................54
vii
5.2.3
Repeat COD Series Testing ....................................................................55
5.3 Final Compilation of BOD, COD and TPH Data ...............................................56
5.3.1 Correlation of COD with TPH................................................................59
5.3.2 Correlation of BOD with TPH................................................................59
5.3.3
6
BOD/COD Ratio - Biodegradability.......................................................61
DISCUSSION ..............................................................................................................65
6.1
Reliability of BOD Tests ....................................................................................65
6.2
Effect of Iron on BOD ........................................................................................66
6.3
Effect of Dilution ................................................................................................67
6.3.1 Effect of Dilution on BOD Measurement...............................................67
6.3.2 Effect of Dilution on COD Measurement...............................................68
6.4
Reliability of COD Tests ....................................................................................68
6.5
Biodegradability..................................................................................................69
6.6
ThOD of Hydrocarbons ......................................................................................70
7 CONCLUSIONS..........................................................................................................75
8
RECOMMEDATIONS................................................................................................76
REFERENCES ..................................................................................................................77
viii
LIST OF TABLES
Table 3.1 Summary of separate-phase diluent analysis .......................................................19
Table 3.2 Summary of dissolved-phase diluent analysis .....................................................20
Table 5.1 BOD results and statistics for method development............................................37
Table 5.2 BOD results for 6-day test with single groundwater sample
(fresh C8-39, 8-12 ppm) ......................................................................................40
Table 5.3 BOD results for 20-day test with single groundwater sample
(fresh C8-39, 8-12 ppm) ......................................................................................43
Table 5.4 BOD5 data for BOD method testing on series of groundwater samples..............47
Table 5.5 BOD data for BOD5 repeat analysis of oxygen depleted samples.......................50
Table 5.6 Iron oxidation COD data......................................................................................53
Table 5.7 Data for COD of groundwater series ...................................................................54
Table 5.8 Repeat COD analysis of groundwater series using 20-900 mg/L vials ...............55
Table 5.9 Comparison of high and low range COD tests ....................................................56
Table 5.10 Final results of COD, BOD and calculated BOD/COD
ratios for groundwater series................................................................................59
Table 6.1 COD of phenol solutions .....................................................................................73
ix
LIST OF FIGURES
Figure 3.1 Guadalupe site map ............................................................................................5
Figure 3.2 Aerial view of Guadalupe site ............................................................................6
Figure 3.3 Guadalupe plume map........................................................................................7
Figure 3.4 Carbon ranges for common diluent constituents................................................19
Figure 4.1 BOD analysis setup: DO meter, probe and BOD bottle.....................................26
Figure 4.2 Absorbance vs. COD of KHP standards, 5-150 mg/L range..............................32
Figure 4.3 Absorbance vs. COD of KHP standards, 20-900 mg/L range............................33
Figure 5.1 Iron effect on BOD5 ...........................................................................................36
Figure 5.2 Effects of dilution and inoculum volume on the BOD6
of contaminated groundwater (C8-39, 8-12 ppm).............................................41
Figure 5.3 Guadalupe Restoration Project, Diluent Tanks area ..........................................44
Figure 5.4 Detail of sampled monitoring well locations .....................................................45
Figure 5.5 Effect of sample dilution on BOD5 determination for several
Groundwater samples .........................................................................................48
Figure 5.6 Effect of iron oxidation on COD measured........................................................53
Figure 5.7 COD vs. TPH plot for series of groundwater samples .......................................59
Figure 5.8 BOD vs. TPH plot for series of groundwater samples .......................................61
Figure 5.9 BOD/COD vs. TPH plot for series of groundwater samples .............................62
Figure 5.10 BOD/COD vs. distance down plume from source plot for
series of groundwater samples grouped by location ..........................................64
Figure 6.1 COD vs. phenol concentration for standard phenol solutions............................74
x
CHAPTER 1
INTRODUCTION
A grant from Unocal has enabled Cal Poly to perform research on various
remediation methods for the former Guadalupe Oil Field, now known as
the Guadalupe Restoration Project (GRP). A kerosene-like substance was
previously used as a diluent for facilitating the extraction of crude oil at
this site.
Leaky pipes and tanks caused significant hydrocarbon
contamination.
The research goal is to find better ways of treating the
diluent contaminated soil and groundwater.
Natural attenuation is the microbial degradation and weathering of a
contaminant and its application at the Guadalupe Site is the focus of this
research. The goal of this project is to determine if hydrocarbons become
recalcitrant after a certain amount of biodegradation and to find any trend
in biodegradability with weathering of hydrocarbons in the groundwater.
These questions were addressed by evaluating the current and long-term
biodegradability of DPD (dissolved phase diluent) in the groundwater at
the
Guadalupe
site.
Biodegradability
of
DPD
was
measured
by
determining the ratio of 5-day biological oxygen demand (BOD 5 ) to
chemical oxygen demand (COD). This BOD/COD ratio has been reported
as a measure of biodegradability in a number of projects (Gilbert 1987,
Alvares et al., 2001 a , b , Imai et al., 1998, Mantzavinos et al., 1996 & 2001,
1
Koch et al., 2002, Kumar et al., 1998, Geenens et al., 2000, Chun and
Yizhong, 1999).
The Guadalupe Restoration Project has to consider the presence of several
endangered species, which makes the use of natural attenuation attractive.
The presence of ecological receptors such as the Red Legged Frog and the
Western Snowy Plover raises some concern.
Samples of groundwater taken with different DPD concentrations serve as
surrogates for the aging of DPD over time, since low concentrations are
probably farther from the source zone.
The contaminant concentration
should decrease as the contaminant is broken down by various means over
time, discussed in further detail in Section 3.2.
remains
similar
throughout
the
range
of
A BOD/COD ratio that
concentrations
would
be
considered good evidence for sustained biodegradability.
There
are
several
factors
that
could
ultimately
limit
long-term
biodegradation, including nutrient and/or electron acceptor availability
and changes in chemical composition of the hydrocarbon mixture.
Biodegradation patterns and nutrient availability can fluctuate over the
long term.
Sometimes levels of contaminant can appear to fall within
desired limits for a period, then suddenly spike again (Leeson and
Hinchee, 1997).
Contaminants can sorb to particles in the geo-matrix,
2
which can limit the bioavailability of the contaminants (USEPA, 199 b ).
Biodegradation
may
reduce
the
surface
particles, leading to a lack of nutrients.
contamination on
the
soil
The microbial population may
then subside (Leeson and Hinchee, 1997). Once the surface contamination
is gone, the contaminant from interstitial spaces and pores may migrate
out of the matrix, leading to a new spike in measurable contaminant
concentration.
Preliminary tests on the BOD and COD methods were performed to insure
iron oxidation was not a source of significant interference, and dilution
was varied to test the use of appropriate strength inoculum and the
possibility of dilution affecting BOD measurements.
As a control,
dilution water blanks containing no hydrocarbons were used.
After preliminary testing of the BOD and COD methods, BOD and COD
were measured for groundwater samples from a series of 7 monitoring
wells.
BOD/COD ratios were calculated to examine any trends in
biodegradability with TPH concentration.
3
CHAPTER 2
PROJECT SCOPE
The specific objectives of this project included:
1. Measure the BOD/COD ratio as an indication of biodegradability of TPH
in groundwater samples taken from a transect down-plume of diluent
contamination.
2. Determine the sensitivity of the BOD analysis.
3. Conduct preliminary tests to optimize the BOD and COD analyses.
4. Determine the suitability of the inoculum for BOD tests in terms of
kinetics of BOD exertion (i.e. is sufficient oxygen consumed in 5days
from the samples).
5. Determine if Fe(II) will significantly contribute to BOD or COD.
4
CHAPTER 3
BACKGROUND
3.1 Former Guadalupe Oil Field
The Guadalupe Restoration Project (GRP) is located on the Central Coast
of California, northwest of the city of Guadalupe and along the southern
edge of San Luis Obispo County (Figure 3.1).
The site was previously
called the Guadalupe Oil Field (GOF) and was in operation from the late
1940's through the mid 1990's.
Figure 3.1: Guadalupe site map.
5
Figure 3.2 Aerial view of Guadalupe site.
6
Not to scale
Figure 3.3 Guadalupe plume map.
7
Unocal purchased the outstanding share of the GOF in 1953, and became
the operator (GRP, 2002).
over 2700 acres of land.
The former Guadalupe Oil Field consists of
The dunes lie between the Pacific Ocean, the
Santa Maria River, and privately owned agricultural land.
The majority
of the oil field lies in San Luis Obispo County but a southeastern portion
lies in northern Santa Barbara County.
It is one of the last intact dune
ecosystems in the state of California and is home to a variety of
threatened and endangered species (GRP, 2002).
Diluent (a diesel or kerosene-like substance) was used as a thinning agent
during active production, to help the viscous crude oil flow through the
pipes and aid in the on-site crude oil extraction.
The soil and
groundwater became significantly contaminated with diluent from a series
of spills and leaks from the storage tanks and transmission pipes that went
unreported and untreated. As a result, contaminant plumes developed in
over eighty sites at the Guadalupe Oil Field.
For a few days in 1988 and again in early 1990, following storm events,
diluent began to appear on the beach and it became clear that the diluent
was a threat to surrounding waters and the surrounding ecosystem (GRP,
2002).
The last use of diluent was in 1990.
In the following years the
Guadalupe Oil Field ceased operation and the Guadalupe Restoration
Project arose.
8
3.2 Natural Attenuation as a Treatment Technology
Natural attenuation processes include a variety of physical, chemical, or
biological processes that, under favorable conditions, act without human
intervention
to
reduce
the
mass,
toxicity,
mobility,
volume
or
concentration of contaminants in soil or groundwater (USEPA, 1999 b ). It
can be more aesthetically attractive than having above ground treatment
systems and could be less disruptive to the terrestrial ecosystem. Natural
attenuation
can
be
a
cost
effective
alternative
as
long
as
the
contamination is in low to moderate levels. Highly contaminated sources
should be removed by some method such as free product recovery,
bioventing, or soil vapor extraction prior to use of natural attenuation
(USEPA, 2003).
To take advantage of using natural attenuation, the effectiveness must be
confirmed in order to validate its use for remediation. Criteria for the site
must be evaluated for suitability for natural attenuation.
If the site is
suitable, methods of tracking the progress of natural attenuation must be
implemented.
Also, such criteria as the ability to remove or neutralize
contaminants and time effectiveness need to be evaluated.
To document natural processes reducing contaminant concentrations,
several lines of evidence may be required. Historical trends of decreasing
contaminant concentrations are one line of evidence. Another is to have a
9
retreating or stable plume, possibly indicating microorganisms are
removing dissolved contaminants from groundwater at a rate greater than
or equal to the rate at which the source is adding them.
Natural
biodegradation can leave chemical indicators, also known as footprints.
Documenting such chemical indicators is another way to exemplify the
occurrence of natural attenuation.
Indicators include changes in water
chemistry left by the attenuation reactions as well as intermediates.
Biodegradation
of
contaminants
is
directly
related
to
changes
in
groundwater chemistry such as the biological consumption of natural
levels of oxygen, nitrate, and sulfate and the creation of byproducts such
as dissolved iron (II), manganese (II), and methane (USEPA, 2003). For
example, biodegradation of toluene by aerobic bacteria consumes oxygen
from the groundwater and adds inorganic carbon as the toluene is
converted
to
carbon
dioxide.
Once
such
reactions
are
postulated,
monitoring is necessary to show that the attenuation processes continue
(Macdonald, 2000). Geochemical indicators can also be used to estimate
the
site-specific
potential
for
biodegradation (USEPA, 2003).
contaminants
to
be
destroyed
by
Formation of intermediates (eg. TCE,
DCE, vinyl chloride) and laboratory treatability tests are other means of
proving biodegradation (Macdonald, 2000).
Most contaminants can degrade or transform by a number of different
mechanisms, depending on site conditions, and often many different
10
mechanisms act in concert.
For example, the initial stages of benzene
biodegradation often consume all of the oxygen in the groundwater; so
later stages proceed by different biotransformation pathways. As a
consequence, the search for footprints of natural attenuation must
consider the unique conditions of the site (Macdonald, 2000).
Depending on the contaminant type and site-specific characteristics,
breakdown or removal of contaminants through natural attenuation occurs
by different mechanisms and biotransformation pathways. The four main
processes of natural attenuation include biodegradation, dilution, sorption
and volatilization (USEPA, 2001).
A description of each mechanism is
given in the following sections.
3.2.1 Biodegradation
One
of
the
most
important
components
of
natural
attenuation
is
biodegradation, the change in form of compounds carried out by living
creatures
such
as
microorganisms.
Biodegradation
of
petroleum
compounds occurs when they serve as the primary source of food and
energy to naturally occurring soil and groundwater bacteria (USEPA, 1999b).
Under the right conditions, microorganisms can cause or assist chemical
reactions that change the form of the contaminants so that little or no
health risk remains. Biodegradation is important because many important
components of petroleum hydrocarbon contamination can be destroyed by
11
biodegradation,
biodegrading
microorganisms
are
found
almost
everywhere, and biodegradation can be very safe and effective (USEPA,
1999 a ).
3.2.2 Dilution/ Dispersion
Contaminants will mix with soil and groundwater as seasons change and
groundwater levels rise and fall. As the dissolved contaminants mix and
move farther away from the source area, the contaminants are dispersed
and diluted to lower and lower concentrations over time. Eventually the
contaminant concentrations may be reduced so much that the risk to
human and environmental health will be minimal (USEPA, 1999 b ).
process
of
dilution
and/or
dispersion
alone
does
not
The
destroy
a
contaminant.
3.2.3 Volatilization
Many petroleum hydrocarbons evaporate readily into the atmosphere,
where air currents disperse the contaminants, reducing the concentration.
In some cases, this means of natural attenuation may be useful, since
some contaminants can be broken down by sunlight (USEPA, 1999b).
Vapors in contact with soil microorganisms may also be biodegraded in
the vadose zone (USEPA, 1999 a ).
12
3.2.4 Adsorption
Many contaminants are prevented from entering the groundwater and
migrating off-site, due to adsorption onto soil particles (USEPA, 2003).
The soil and sediment particles through which the groundwater and
dissolved contaminants move can sorb the contaminant molecules onto the
particle surfaces, and hold bulk liquids in the pores in and between the
particles, thereby slowing or stopping the movement of the contaminants.
This process can reduce the likelihood that the contaminants will reach a
location where they would directly affect human or environmental health
(USEPA, 1999 b ).
3.3 Advantages of Natural Attenuation
Some of the inherent advantages of natural attenuation include generation
of lesser volume of remediation wastes, reduced potential for cross-media
transfer of contaminants commonly associated with ex situ treatment, and
reduced risk of human exposure to contaminants, contaminated media, and
other hazards, and reduced disturbances to ecological receptors (USEPA,
1999a). Natural attenuation can be used in conjunction with or as a follow
up to more active remedial measures (USEPA, 1999a).
Some other
advantages include having a lower degree of intrusion with fewer surface
structures, a potential for the application to all or part of a given site,
depending on site conditions and remediation objectives (USEPA, 1999b). It
13
also offers potentially lower overall remediation costs than those
associated with active remediation (USEPA, 1999b).
3.4 Limitations of Natural Attenuation
The use of natural attenuation may require more time than active methods
to achieve cleanup goals, and thus may require a long-term commitment to
monitoring and associated costs (USEPA, 2003). In some cases, if natural
attenuation rates are too slow, the plume could continue to migrate, which
can lead to the required use of land and groundwater controls (USEPA,
2003).
Site characterization is expected to be more complex and costly
than other active methods (USEPA, 1999 b ).
Inhibitory compounds may
result from incomplete biodegradation, giving rise to by-products or
intermediates more toxic than the original compound (Alvares et al,
2001 a ).
In addition to toxicity, the mobility of transformation products
may exceed that of the parent compound (USEPA, 1999 b ).
Hydrologic
and geochemical conditions amenable to natural attenuation may change
over time and could result in renewed mobility of previously stabilized
contaminants, adversely influencing remedial effectiveness (USEPA,
1999 b ). Natural attenuation is not appropriate for high concentrations of
contaminant, due to toxicity factors (USEPA, 1999 b ).
If there is a high
contaminant concentration, natural attenuation is commonly used in
conjunction with an active method.
14
3.5 Site Criteria
Considering environmental receptors is only one of the many factors
involved in determining if a petroleum hydrocarbon contaminated site is a
candidate for using natural attenuation as a remedial method.
Location
should be of primary concern and be in an area with little risk to human
health or the environment from direct contact with contaminated soil or
groundwater.
The contaminated soil and groundwater should also be
located an adequate distance from potential receptors.
This exemplifies
the importance of having a good conceptual model of the site. Every site
needs at least a simple model showing groundwater flow, contaminant
locations and concentrations, and possible natural attenuation reactions.
(Macdonald, 2000)
3.6 Determination of Contaminant Biodegradability
The evaluation of biodegradability of organic compounds in aqueous
medium can be performed by many options including shake-flask batch
tests
measuring
biogas
production,
activated
sludge
simulation,
biochemical oxygen demand (BOD), static test (Zahn-Wellens method),
respirometry, dissolved organic carbon (DOC), total organic carbon
(TOC), chemical oxygen demand (COD), metabolism and identification of
transformation products (ISO, 2003).
The ratio of BOD/COD has also
been used as a measure of biodegradability. The BOD/COD ratio gives a
gross index of the proportion of the organic materials present which are
aerobically degradable within a certain period of time, e.g. 5 days for
15
BOD 5 (Mantzavinos et al., 1996).
BOD is a measure of the oxidation
occurring due to microbial activity while COD measures the highest
extent of oxidation a material may undergo. Details of BOD and COD are
given in the following two sections.
The BOD 5 /COD and BOD 5 /TOC ratios are commonly used indicators of
biodegradability
improvement,
where
a
value
of
zero
indicates
nonbiodegradability and an increase in the ratio reflects biodegradability
improvement (Alvares et al, 2001 b ). Low BOD 5 /COD values (usually less
than 0.1) indicate their resistance to conventional biological treatment
(Koch et al., 2002, Imai et al., 1998).
Chun and Yizhong studied
photocatalytically treated wastewater contaminated with azo dyes from
the processing of wool.
They found when the ratio of BOD 5 /COD was
more than 0.3 the wastewater had a better biodegradability.
statements
were
made
for
a
BOD 5 /COD
ratio
of
0.4
Similar
using
nonbiodegradable substituted aromatic compounds (Gilbert, 1987).
3.7 Biochemical Oxygen Demand
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 airtight container and kept
in a controlled environment for a preselected period of time.
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
wastewaters.
of
industrial
wastes
and
municipal
and
natural
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 nC 1 0 to nC 3 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 separateproduct, 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 <nC 1 0 to >C 3 0 .
About
70% of the diluent falls in the diesel range of nC 1 0 to nC 2 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
C6
C10
C4
Measurable TPH
Semi-quantifiable
C24
C6
C4
Gasoline
C8
C12
Kerosene
C8
C36
C17
Diesel
C24
C12
C24-30
Fuel Oils
C20
Lube Oils & heavier
C36
Boiling
Point
Range
140 F
340 F
340 F
340 F
140 F
170 C
170 C
170 C
Gasoline
Range
C6
C10
Diesel Range
C10
REFERENCE:
TPH IN SOIL PRIMER, ELAINE M.
SCHWERKO.
DATED: 09/01/93
Non-measurable TPH due to
volatilization
Figure 3.4 Carbon ranges for common diluent constituents compared to
common petroleum distillates (Elliot, 2002).
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
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
<50 to 400
870 to 16,000
nC10 to nC32
(Elliot 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 petroleumderived 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
effectiveness, and analytical technique.
from Hach Co. USA.
21
dilution
water
quality,
seed
GGA reagents were purchased
BOD is computed using the following equation (Eqn. 1):
BOD = (D 1 - D 2 ) - (B 1 - B 2 )f / P
(1)
Where,
BOD = biochemical oxygen demand, mg/L
D 1 = DO of diluted sample 15 minutes after preparation, mg/L
D 2 = DO of diluted sample after incubation at 20°C, mg/L
B 1 = DO of seeded dilution water blank before incubation, mg/L
B 2 = DO of seeded dilution water blank after incubation, mg/L
f = ratio of seed in sample to seed in blank
= % seed in D 1 / % seed in B 1
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 BOD 2 0 and BOD 6 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.
inoculum
was
labeled
Iii.
Continuous
stirring
following
This
initial
preparation was used for aeration.
Inoculum Preparation for BOD 5 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 BOD 5
experiments using several groundwater sources. 100 mL of inoculum I i i
was added to 900 mL of Guadalupe groundwater, 100 g diluentcontaminated
soil
and
one
Polyseed®
23
capsule.
The
900
mL
of
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.
and pH shifts can cause low BOD results.
Mineral deficiencies
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 O 2 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 0 2 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
Fe 2 + added.
The concern was that abiotic Fe 2 + oxidation to Fe 3 + could
consume oxygen, leading to interference with the BOD test.
diluent-contaminated
groundwater
from
C8-39
was
used
experiment, and it contained approximately 2 ppm TPH.
Guadalupe
for
this
An iron
concentration of 40 mg/L was added to this groundwater, using FeSO 4 and
used for two sets of samples.
minutes to oxidize the Fe + 2 .
One set of samples was bubbled for 10
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 Fe 2 + 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 O 2 /L was calculated using the stoichiometric
relation between KHP and oxygen (Eqn. 2).
KC 8 H 5 O 4 + 29/4 O 2 → 8 CO 2 + 5/2 H 2 O + 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.
29
The COD of standard range vials
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 Fe 2 +
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 Fe 2 + at a concentration
of 40 mg/L FeSO 4 , while another triplicate of test samples did not have
Fe 2 + added. COD was measured using Bioscience 5-150 mg/L COD vials
with absorbance measured at 440 nm.
COD
range
was
developed
using
The KHP standard curve for this
the
following
concentrations
triplicate: 1, 2, 5, 10, 20 and 30 mg/L COD or mgO 2 /L.
30
in
The resulting
trendline from the KHP standards used to convert absorbance to COD
yielded R 2 = 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 it 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 mgO 2 /L. The resulting trendline from the
KHP standards used to convert absorbance to COD yielded R 2 = 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
0.7
Absorbance at λ = 440 nm
0.6
0.5
0.4
0.3
y = -0.0129x + 0.6391
R2 = 0.9556
0.2
0.1
0
0
5
10
15
20
25
30
COD (mg/L)
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
35
0.35
Absorbance at λ = 600 nm
0.30
0.25
0.20
0.15
y = 0.0003x + 0.0537
R2 = 0.9985
0.10
0.05
0.00
0
100
200
300
400
500
600
700
800
COD (mg/L)
Figure 4.3 Absorbance vs. COD of KHP standards, 20-900 mg/L range.
Measuring the concentration of the produced Cr (III) ion.
33
900
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: C 6 H 5 OH
Molecular Weight: 94 g/mol
C 6 H 5 OH + 7O 2 → 6CO 2 + 3H 2 O
(7 mol O 2 ) * (32 g/mol O 2 ) = 224 g O 2
(224 g O 2 ) / (94 g/mol)
ThOD = 2.38 g O 2 / mol
34
(3)
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 Fe 2 + on measured BOD was observed. Undiluted
samples without Fe added exerted a BOD 5 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.
0.8
BOD5 (mg/L)
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
no Fe
Fe w/
sparge
Fe w/o
sparge
Groundwater Samples With Iron [40 mg/L]
error bars indicate ± 1 standard deviation
Figure 5.1 Iron effect on BOD5.
36
Table 5.1 BOD results and statistics for method development.
Sample
description
(C8-39 @ 2ppm)
FS, w/o Fe
FS, w/o Fe
FS, w/o Fe
50%, w/o Fe
50%, w/o Fe
50%, w/o Fe
FS, w/Fe, w/o sparge
FS, w/Fe, w/o sparge
FS, w/Fe, w/o sparge
FS, w/Fe, w/sparge
FS, w/Fe, w/sparge
FS, w/Fe, w/sparge
seed control
seed control
seed control
DW blank
DW blank
DW blank
BOD Std., 2ml
BOD Std., 2ml
BOD Std., 4ml
BOD Std., 4ml
BOD Std.,6ml
BOD Std.,6ml
mL
of
100x
3
3
3
3
3
3
3
3
3
3
3
3
-------------
Seed Sample Dilution
DO
Bottle Dilution
Volume Volume (P-value) initial
number
(%)
(mL)
(mL)
(mg/L)
1
-10
287
0.957
8.76
2
-10
287
0.957
8.72
3
-10
287
0.957
8.72
4
50
10
143.5
0.478
8.75
5
50
10
143.5
0.478
8.75
6
50
10
143.5
0.478
8.75
7
-10
287
0.957
7.20
8
-10
287
0.957
7.90
9
-10
287
0.957
7.92
10
-10
287
0.957
8.20
11
-10
287
0.957
8.00
12
-10
287
0.957
7.92
13
-10
290
0.967
8.75
14
-10
290
0.967
8.72
15
-10
290
0.967
8.77
16
--300
1.000
8.80
17
--300
1.000
8.97
18
--300
1.000
8.93
19
-10
2
0.007
8.80
20
-10
2
0.007
8.81
21
-10
4
0.013
8.85
22
-10
4
0.013
8.80
23
-10
6
0.020
8.88
24
-10
6
0.020
8.88
37
DO
final
(mg/L)
8.35
8.35
8.33
8.34
8.45
8.42
7.02
7.40
7.70
8.09
7.79
7.78
8.95
8.99
9.03
8.60
8.90
8.70
5.72
5.78
2.08
3.15
0.6
0.5
Delta
DO
(mg/L
0.41
0.37
0.39
0.41
0.30
0.33
0.18
0.50
0.22
0.11
0.21
0.14
-0.20
-0.27
-0.26
0.20
0.07
0.23
3.08
3.03
6.77
5.65
8.28
8.38
BOD5 Avg. BOD5
values BOD5
SD
(mg/L) (mg/L) (mg/L)
0.68
0.64
0.66
0.02
0.66
1.11
0.88
0.98
0.12
0.94
0.44
0.77
0.57
0.18
0.48
0.37
0.47
0.41
0.05
0.40
-0.21
-0.28 -0.25
0.04
-0.27
0.20
0.07
0.17
0.09
0.23
462
458
5.3
455
508
466
59
424
414
417
3.5
419
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 BOD 6 of 4.31
mg/L ± 0.13 mg/L and with 20 mL of seed, had an average BOD 6 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 BOD 6 of 5.69 mg/L ± 0.24 mg/L and
with 20 mL of seed, had an average BOD 6 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 BOD 6 of
0.06 mg/L ± 0.01 mg/L and with 20 mL seed had an average BOD 6 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 BOD 6
of 468.0 mg/L ± 12.7 mg/L and with 20 mL seed had an average BOD 6 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.
Sample
(C8-39)
8-12 ppm
8-12 ppm
8-12 ppm
8-12 ppm
8-12 ppm
8-12 ppm
8-12 ppm
8-12 ppm
8-12 ppm
8-12 ppm
8-12 ppm
8-12 ppm
seed ctrl.
seed ctrl.
seed ctrl.
seed ctrl.
seed ctrl.
seed ctrl.
BOD std.
BOD std.
BOD std.
BOD std.
DW blank
DW blank
DW blank
mL
of
100x
3
3
3
3
3
3
3
3
3
3
3
3
----------------------------------------
Bottle
number
Dilution
(%)
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
135
------------------50
50
50
50
50
50
----------------------------------------
Seed
Volume
(mL)
10
10
10
20
20
20
10
10
10
20
20
20
10
10
10
20
20
20
10
10
20
20
----------
Sample
Volume
(mL)
287
287
287
277
277
277
143.5
143.5
143.5
138.5
138.5
138.5
290
290
290
280
280
280
2
2
2
2
300
300
300
Diluiton
(P-value)
0.957
0.957
0.957
0.923
0.923
0.923
0.478
0.478
0.478
0.462
0.462
0.462
0.967
0.967
0.967
0.933
0.933
0.933
0.007
0.007
0.007
0.007
1.000
1.000
1.000
40
DO
initial
(mg/L)
4.95
4.97
4.94
5.23
5.23
5.25
7.29
7.30
7.36
7.49
7.51
7.50
8.42
8.41
8.43
8.31
8.36
8.49
8.59
8.47
8.58
8.57
8.64
8.59
8.59
DO
final
(mg/L)
0.89
0.79
0.64
1.72
3.09
1.67
6.29
4.63
4.53
4.99
5.16
4.83
8.37
8.35
8.38
8.50
8.52
8.58
5.41
5.41
5.40
5.42
8.51
8.50
8.50
BOD6
values
(mg/L)
4.19
4.31
4.44
3.96
2.47
4.03
2.04
5.53
5.86
5.57
5.25
5.94
0.05
0.06
0.05
-0.20
-0.17
-0.10
477.0
459.0
477.0
472.5
0.13
0.09
0.09
Avg.
BOD6
(mg/L)
BOD6
SD
(mg/L)
4.31
0.13
4.00
0.05
5.69
0.24
5.59
0.35
0.06
0.01
-0.16
0.05
468.0
12.7
474.8
3.2
0.10
0.02
7
6
BOD6 (mg/L)
5
4
3
2
1
50%
20 mL
seed
50%
10 mL
seed
FS
20 mL
seed
FS
10 mL
seed
0
Figure 5.2 Effects of dilution and inoculum volume on the BOD 6 of contaminated
groundwater (C8-39, 8-12 ppm).
Error bars indicate ± 1 standard deviation
5.1.3 Preliminary 20-day BOD Test for BOD Method
Because of the low oxygen depletion rates observed i the preliminary experiments
described above, a 20-day BOD test was run. All oxygen was depleted in the 20-day
test and therefore, BOD could not be calculated (Table 5.3). Dissolved oxygen
levels essentially went to zero for all groundwater samples. The seed control using
20 ml seed showed a 0.70 mg/L uptake with a standard deviation of 0.08 mg/L. The
seed control using 10 ml seed only showed 0.55 mg/L uptake with a standard
deviation of 0.11 mg/L (Table 5.3). These are within the recommended range of 0.6
and 1.0 mg/L. The dilution water blank had an average D.O. depletion of 0.45 mg/L
with a standard deviation of 0.04 mg/L (Table 5.3). According to Standard Methods,
the recommended depletion for dilution water blanks in a 20-day test should be less
than 0.5 mg/L (APHA). The test performed was within the specified limit. The
41
rapid DO depletion for these samples in 20-days was encouraging because it
indicates that biodegradation rates are producing measurable DO shifts. Because of
this rapid DO depletion for the fresh groundwater sample, subsequent tests were run
as standard 5-day BOD tests.
5.1.4 Dilution Effects and BOD Analysis of Several Groundwater Samples
Additional 5-day BOD tests were run using several groundwater samples to make
sure dilution does not alter the BOD 5 result, since the previous 6-day test was not
convincing. The series of groundwater samples used for all further experiments were
taken from near the Diluent Tanks area (Figure 5.3).
A closer version illustrates
where each of the seven wells sampled are located (Figure 5.4).
42
Table 5.3 BOD results for 20-day test with single groundwater sample (fresh C8-39, 8-12 ppm).
Sample
(8-12 ppm)
mL
of
100x
Dilution
(%)
Seed
Volume
(mL)
Sample
Volume
(mL)
Dilution
(P -value)
DO
initial
(mg/L)
C8-39
C8-39
C8-39
C8-39
C8-39
C8-39
C8-39
C8-39
C8-39
C8-39
C8-39
C8-39
Seed Ctrl.
Seed Ctrl.
Seed Ctrl.
Seed Ctrl.
Seed Ctrl.
Seed Ctrl.
BOD Std.
BOD Std.
BOD Std.
BOD Std.
DW Blank
DW Blank
DW Blank
3
3
3
3
3
3
3
3
3
3
3
3
--------------
------50
50
50
50
50
50
--------------
10
10
10
20
20
20
10
10
10
20
20
20
10
10
10
20
20
20
10
10
20
20
----
287
287
287
277
277
277
143.5
143.5
143.5
138.5
138.5
138.5
290
290
290
280
280
280
2
2
2
2
300
300
300
0.957
0.957
0.957
0.923
0.923
0.923
0.478
0.478
0.478
0.462
0.462
0.462
0.967
0.967
0.967
0.933
0.933
0.933
0.007
0.007
0.007
0.007
1.000
1.000
1.000
4.85
4.68
4.70
4.97
5.12
5.23
7.02
7.11
7.00
6.65
6.79
6.80
8.30
8.34
8.17
8.23
8.30
8.32
8.25
8.28
8.23
8.32
8.35
8.41
8.42
43
BOD20
DO
final values
(mg/L) (mg/L)
0.02
0.03
0.03
0.10
0.02
0.03
0.03
0.03
0.03
0.03
0.03
0.03
7.76
7.80
7.66
7.56
7.65
7.69
4.40
4.43
4.33
4.40
7.94
7.95
7.93
> 4.50
> 4.31
> 4.33
> 4.58
> 4.83
> 4.94
> 14.06
> 14.25
> 14.02
> 13.64
> 13.95
> 13.97
0.56
0.56
0.53
0.72
0.70
0.68
577.5
577.5
585.0
588.0
0.41
0.46
0.49
Ave.
BOD20
(mg/L)
BOD20
SD
(mg/L)
> 4.38
0.10
> 4.78
0.18
> 14.11
0.12
> 13.85
0.18
0.55
0.02
0.70
0.02
577.5
0.00
586.5
2.12
0.45
0.04
Not to scale
Figure 5.3 Guadalupe Restoration Project, Diluent Tanks area
(Lundegard, 2002).
44
Figure 5.4 Detail of sampled monitoring well locations
(Lundegard, 2002).
45
BOD 5 values were obtained for the seven groundwater samples, with some
diluted samples (Table 5.4). Virtually all oxygen was depleted in samples
from wells 206-C, 209-E and M4-4 50%.
The BOD analyses for the
samples from wells 206-C and 209-E were therefore repeated, and results
are given in Section 5.1.5. BOD measurements were not repeated for well
M4-4, since two other dilutions from this testing event provided useful
data. The seed control blanks had an average BOD 5 of 0.07 mg/L ± 0.02
mg/L, while the BOD standard (GGA) average was 296.3 mg/L ± 30.8
mg/L and the dilution water blanks average was 0.11 mg/L ± 0.01 mg/L.
Effects of dilution were examined using groundwater samples H3-7, H2-1
and M4-4. In all cases, BOD 5 values determined were similar regardless
of dilution (Figure 5.5 and Table 5.4). H3-7 samples with a TPH
concentration of 11.0 ppm had an average BOD 5 of 5.59 mg/L ± 0.60
mg/L at 25% strength and 4.75 mg/L ± 0.26 mg/L at 50% strength. The
dilutions for these samples provided BOD 5 results within 15% of each
other. Similarly, 25% and 50% diluted H2-1 samples had average BOD 5
values within 3% of each other and 10% and 25% diluted M4-4 samples
had average BOD 5 values within 10% each other. These results show
consistently that the use of dilution does not significantly affect the
measured BOD 5 of the groundwater samples (Figure 5.5).
46
Table 5.4 BOD 5 data for BOD method testing on series of groundwater samples
Wells 206-C, 209-E and M4-4 at 50% depleted all O 2 and are repeated later.
Sample
G4-3
G4-3
G4-3
206-C
206-C
206-C
209-D
209-D
209-D
209-E
209-E
209-E
H3-7
H3-7
H3-7
H3-7
H3-7
H3-7
H2-1
H2-1
H2-1
H2-1
H2-1
H2-1
M4-4
M4-4
M4-4
M4-4
M4-4
M4-4
M4-4
M4-4
M4-4
Seed Ctrl.
Seed Ctrl.
Seed Ctrl.
BOD Std.
BOD Std.
DW Blank
DW Blank
DW Blank
TPH
mL
Seed Sample
Dilution
Conc.
of
Vol.
Vol.
(%)
(ppm) 100x
(mL) (mL)
4.2
3
-10
287
4.2
3
-10
287
4.2
3
-10
287
5.7
3
-10
287
5.7
3
-10
287
5.7
3
-10
287
7.5
3
-10
287
7.5
3
-10
287
7.5
3
-10
287
8.2
3
-10
287
8.2
3
-10
287
8.2
3
-10
287
11.0 0.75
25
10
72.31
11.0 0.75
25
10
72.31
11.0 0.75
25
10
72.31
11.0
1.5
50
10
144.25
11.0
1.5
50
10
144.25
11.0
1.5
50
10
144.25
13.0 0.75
25
10
72.31
13.0 0.75
25
10
72.31
13.0 0.75
25
10
72.31
13.0
1.5
50
10
144.25
13.0
1.5
50
10
144.25
13.0
1.5
50
10
144.25
29.0
0.3
10
10
28.97
29.0
0.3
10
10
28.97
29.0
0.3
10
10
28.97
29.0 0.75
25
10
72.31
29.0 0.75
25
10
72.31
29.0 0.75
25
10
72.31
29.0
1.5
50
10
144.25
29.0
1.5
50
10
144.25
29.0
1.5
50
10
144.25
---10
290
---10
290
---10
290
---10
2
---10
2
----300
----300
----300
Dilution
(P-value)
0.957
0.957
0.957
0.957
0.957
0.957
0.957
0.957
0.957
0.957
0.957
0.957
0.241
0.241
0.241
0.481
0.481
0.481
0.241
0.241
0.241
0.481
0.481
0.481
0.097
0.097
0.097
0.241
0.241
0.241
0.481
0.481
0.481
0.967
0.967
0.967
0.007
0.007
1.000
1.000
1.000
BOD5
D.O.
D.O.
initial
final values
(mg/L) (mg/L) (mg/L)
9.90
6.57
3.55
9.92
7.06
3.06
9.82
6.35
3.70
7.83
0.30 > 7.94
7.78
2.24 > 5.86
7.67
0.87 > 7.18
8.79
6.88
2.07
8.85
6.89
2.12
8.84
6.94
2.06
8.56
0.05 > 8.96
8.55
0.06 > 8.94
8.56
0.06 > 8.95
8.14
6.63 > 6.33
8.15
6.91 > 5.21
8.15
6.86 > 5.42
8.01
5.87
4.52
8.06
5.68
5.02
8.07
5.74
4.91
7.86
5.94
8.03
7.94
6.55
5.84
7.94
6.39
6.50
7.54
4.46
6.47
7.58
4.57
6.33
7.59
4.25
7.02
8.19
6.79
14.57
8.19
6.82
14.26
8.20
6.82
14.36
7.81
3.92
16.21
7.81
4.12
15.38
7.85
4.10
15.63
6.92
0.04 > 14.38
6.92
0.04 > 14.38
6.96
0.06 > 14.42
8.34
8.40
-0.06
8.35
8.44
-0.09
8.35
8.40
-0.05
8.41
6.29
318.0
8.43
6.60
274.5
8.70
8.59
0.11
8.72
8.61
0.11
8.72
8.60
0.12
Ave.
BOD5
(mg/L)
BOD5
SD
(mg/L)
3.43
0.33
> 6.99
1.05
2.08
0.03
> 8.95
0.01
5.66
0.60
4.82
0.26
6.79
1.13
6.61
0.36
14.39
0.16
15.74
0.43
> 14.39
0.02
-0.07
0.02
296.3
30.8
0.11
0.01
*** mL of 100x is for
ino cu lu m/nu tr ien t add ition
47
18
25%
16
10%
50%
14
Average BOD5 (mg/L)
12
10
FS
FS
25%
8
50%
25%
6
50%
FS
4
FS
2
0
G4-3
206-C
209-D
209-E
H3-7
H3-7
H2-1
H2-1
M4-4
M4-4
M4-4
Figure 5.5 Effect of sample dilution on BOD 5 determination for several groundwater samples.
Error bars indicate ± 1 standard deviaton.
48
5.1.5 Repeat BOD Analysis of Diluent-Contaminated Groundwater
Samples
Some bottles previously ran out of oxygen and testing was repeated here,
with more dilution. The groundwater samples retested were 209-E and
206-C. This test obtained better values of BOD 5 through dilution (Table
5.5). Seed control blanks and BOD 5 standard samples (GGA) performed
as expected. The dilution water blanks had an average BOD 5 of -0.09
mg/L ± 0.03 mg/L (Table 5.5).
49
Table 5.5 BOD data for BOD 5 repeat analysis of oxygen depleted samples 209-E and 206-C.
For well 206-C, one BOD 5 value was nearly two standard deviations off.
Sample
TPH
Conc.
(ppm)
mL
of
100x
Dilution
(%)
209-E
209-E
209-E
206-C
206-C
206-C
Seed Ctrl.
Seed Ctrl.
Seed Ctrl.
BOD Std.
BOD Std.
DW Blank
DW Blank
DW Blank
8.2
8.2
8.2
5.7
5.7
5.7
-------------------------
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
3
3
----------
50
50
50
50
50
50
-------------------------
BOD5
Seed Sample
DO
DO
Dilution
Vol.
Vol.
initial
final values
(P-value)
(mL) (mL)
(mg/L) (mg/L) (mg/L)
10
144.25
0.481
8.32
1.95
12.14
10
144.25
0.481
8.32
1.50
13.08
10
144.25
0.481
8.28
1.23
13.56
10
144.25
0.481
7.19
2.95
7.71
10
144.25
0.481
8.03
2.88
9.61
10
144.25
0.481
8.06
2.81
9.82
10
290
0.967
8.34
7.26
1.12
10
290
0.967
8.37
7.24
1.17
10
290
0.967
8.35
7.36
1.02
10
2
0.007
8.35
5.62
409.5
10
2
0.007
8.34
5.63
406.5
---300
1.000
8.53
8.66
-0.13
---300
1.000
8.53
8.61
-0.08
---300
1.000
8.53
8.60
-0.07
50
Avg. BOD5
BOD5
SD
(mg/L) (mg/L)
12.93
0.72
9.71
1.16
1.10
0.07
408
2
-0.09
0.03
5.2 COD Results
5.2.1 Iron Oxidation Effects on COD Measurement and Dilution Effects
The aged 2 ppm TPH groundwater sample with Fe 2 + added [40 mg/L FeSO 4 ]
exhibited an average COD value of 34.50 ± 0.82 mg/L, while the same
groundwater sample without Fe 2 + 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 Fe 2 + to Fe 3 + 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 R 2 = 0.96 (Figure 4.2).
52
Table 5.6 Iron oxidation COD data.
The last value for a blank is an outlier.
Sample
2+
FS w/Fe
2+
FS w/Fe
2+
FS w/Fe
2+
FS no Fe
2+
FS no Fe
2+
FS no Fe
50% diluted
50% diluted
50% diluted
Blank
Blank
Blank
Absorbance
at 345 nm
COD
(mg/L)
0.122
0.131
0.144
0.131
0.091
0.115
0.394
0.401
0.382
0.631
0.637
0.577
35.26
34.60
33.63
34.60
37.55
35.78
15.16
14.64
16.04
1.17
0.73
5.16
Avg. COD
mg/L
SD
(mg/L)
34.50
0.82
35.98
1.49
15.28
0.71
0.95
0.31
40
35
COD (mg/L)
30
25
20
15
10
5
w/ Fe2+
w/o Fe2+
0
COD Samples
Error bars indicate ± 1 standard deviation
Figure 5.6 Effect of iron oxidation on COD measured.
53
5.2.2 COD of Groundwater Series
The measured COD of the seven groundwater samples are listed in Table
5.7. COD calculations were made based on the calibration curve from
KHP (Figure 4.2). The blanks had an average COD value of 7.19 mg/L ±
1.23 mg/L (Table 5.7). The COD values for wells G4-3, 206-C, 209-D
and 209-E were within the range of the COD vials used (5-150 mg/L).
However wells H3-7, H2-1 and M4-4 exhibited COD above this range, and
so COD analyses of all samples were repeated (see next section).
Table 5.7 Data for COD of groundwater series
Sample
TPH Conc.
ppm
G4-3
4.2
G4-3
4.2
G4-3
4.2
206-C
5.7
206-C
5.7
206-C
5.7
209-D
7.5
209-D
7.5
209-D
7.5
209-E
8.2
209-E
8.2
209-E
8.2
H3-7
11.0
H3-7
11.0
H3-7
11.0
H2-1
13.0
H2-1
13.0
H2-1
13.0
M4-4
29.0
M4-4
29.0
M4-4
29.0
DI Blank
---DI Blank
---DI Blank
----
COD Avg. COD COD SD
(mg/L)
(mg/L)
(mg/L)
61.8
63.5
62.6
0.85
62.6
86.0
89.4
89.1
2.98
91.9
129
126
128
1.53
128
145
144
146
2.18
148
159
157
157
2.34
155
257
252
255
2.83
257
258
258
258
0.00
258
6.48
6.48
7.19
1.23
8.60
54
5.2.3 Repeat COD Series Testing
Repeat analyses for the COD of seven groundwater samples are given in
Table 5.8. COD calculations were based on the calibration curve from
KHP (Figure 4.3). The COD values were all within the range of the COD
vials used (20-900 mg/L).
Table 5.8 Repeat COD analysis of groundwater series using 20-900
mg/L vials.
Sample
TPH
(mg/L)
COD
(mg/L)
G4-3
G4-3
G4-3
206-C
206-C
206-C
209-D
209-D
209-D
209-E
209-E
209-E
H3-7
H3-7
H3-7
H2-1
H2-1
H2-1
M4-4
M4-4
M4-4
DI H2O
DI H2O
DI H2O
4.2
4.2
4.2
5.7
5.7
5.7
7.5
7.5
7.5
8.2
8.2
8.2
11.0
11.0
11.0
13.0
13.0
13.0
29.0
29.0
29.0
----------
75.9
66.0
69.3
135.3
82.5
112.2
138.5
141.8
141.8
161.6
171.5
158.3
207.8
164.9
174.8
296.9
300.2
329.9
333.2
329.9
329.9
21.5
18.2
14.9
55
Avg.COD
(mg/L)
COD SD
(mg/L)
70.4
5.0
110.0
26.5
140.7
1.9
163.8
6.9
182.5
22.5
309.0
18.2
331.0
1.9
18.2
3.3
All COD data used with BOD and TPH comparisons will come from these
results for consistency. These results agree well with the previous COD
measurements using the 5-150 mg/L vials. Some data from samples tested
with 5-150 mg/L COD vials were out of range; therefore this set of data
obtained using 20-900 mg/L COD vials will be used.
Results are fairly
consistent between the separate COD testing events, except for the
measurements made out of range (Table 5.9).
Table 5.9 Comparison of high and low range COD tests
Sample
TPH
(mg/L)
COD
(20-900 mg/L)
COD
(5-150mg/L)
G4-3
206-C
209-D
209-E
H3-7
H2-1
M4-4
4.2
5.7
7.5
8.2
11.0
13.0
29.0
70.4
110.0
140.7
163.8
182.5
309.0
331.0
62.6
89.1
128.1
146.0
157.2
255.4
257.7
out
of
range
5.3 Final Compilation of BOD, COD & TPH Data
A comprehensive compilation of all BOD, COD and TPH data and
calculations is given in Table 5.10.
The BOD values used in the final
analysis and calculation of biodegradability were taken from the two BOD
analysis runs, which used the series of groundwater samples (Table 5.4
and Table 5.5). Some of the data from each sampling event proved to be
unusable because of DO depletion and so a composite was used to
56
represent the BOD final results presented in Table 5.10.
The most
representative samples, most closely matching the guidelines specified by
the APHA for BOD analysis, were used.
The COD data was obtained
using the same groundwater samples and values used for final compilation
are taken from Table 5.8.
To get the standard deviation (SD) and relative standard deviation (RSD)
for final BOD/COD ratios, I followed a basic procedure. I first obtained
the relative standard deviations, and then multiplied the relative standard
deviation by the average BOD/COD value to obtain the reported standard
deviation.
When I multiplied or divided average values with their
standard deviations, I didn't simply add the standard deviations to produce
the final standard deviation.
Instead, I squared the fractional standard
deviations, added them, and then took the square root of the sum to get
the fractional total deviation. If I had values A +- dA, B +- dB, ... and
wanted to compute X = A*B*..., the total error dX is then
dX/X = sqrt( (dA/A) 2 + (dB/B) 2 +...)
Note that I added the squares of the errors even when dividing the actual
values.
An example should make this clearer.
Assume we have the following
three values with their standard deviations
•
A = 1.67 +- 0.05
•
B = 5.23 +- 0.09
•
C= 1.88 +- 0.07
57
and we want to compute X = A*B/C. The actual problem is trivial:
(1.67 * 5.23)/1.88 = 4.65
To compute the standard deviation of the result, we must sum the squares
of the relative errors and then take the square root.
dX/4.65 = sqrt( (0.05/1.67) 2 + (0.09/5.23) 2 + (0.07/1.88) 2 )
S t o t /4.65 = sqrt(0.000896 + 0.000296 + 0.00139)
S t o t /4.65 = 0.0508
S t 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.
TPH Avg. COD COD SD COD RSD BOD BOD SD BOD RSD BOD
Sample
(mg/L) (mg/L)
(mg/L)
(mg/L) (mg/L)
COD
G4-3 4.2
206-C 5.7
209-D 7.5
209-E 8.2
H3-7 11.0
H2-1 13.0
M4-4 29.0
70.4
110.0
140.7
163.8
182.5
309.0
331.0
5.04
26.46
1.90
6.87
22.45
18.17
1.90
0.07
0.24
0.01
0.04
0.12
0.06
0.01
3.4
9.7
2.1
12.9
4.8
6.6
15.7
59
0.33
1.16
0.03
0.72
0.26
0.36
0.43
0.10
0.12
0.02
0.06
0.05
0.05
0.03
0.049
0.088
0.015
0.079
0.026
0.021
0.048
BOD
COD
RSD
0.121
0.269
0.021
0.070
0.135
0.080
0.028
5.3.1 Correlation of COD with TPH
The slope for COD vs. TPH has a value of 10.1 mg/L COD per mg/L TPH,
with an R 2 value of 0.74 (Figure 5.7). This is three times the expected
slope when using the approximate hydrocarbon ThOD value of 3.5 mg/L
(see section 6.6).
400
H2-1
350
COD (mg/L)
300
M4-4
250
200
209-E
H3-7
150
y = 10.10x + 73.33
R2 = 0.74
209-D
100
50
G4-3
206-C
0
0
5
10
15
20
25
30
TPH concentration (ppm)
Figure 5.7 COD vs. TPH plot for series of groundwater samples.
Error bars indicate ± 1 standard deviation.
5.3.2 Correlation of BOD with TPH
Measured BOD 5 of the groundwater samples did not correlate well with
TPH concentration. The slope for BOD vs. TPH has a value of 0.39 mg
BOD per mg TPH, with an R 2 value of only 0.41 (Figure 5.8).
The
average BOD/TPH ratio is 0.73 ± 0.35. This ratio is much lower than the
ThOD of 3.5 mg/L, but this is to be expected since the TPH is only
60
partially
biodegraded
in
5
days.
Well
209-D
has
a
very
low
biodegradation rate (Figure 5.8).
18
16
M4-4
14
209-E
BOD (mg/L)
12
10
206-C
8
H2-1
6
y = 0.39x + 3.50
2
R = 0.42
H3-7
4
G4-3
2
209-D
0
0
5
10
15
TPH concentration (ppm)
20
25
30
Figure 5.8 BOD vs. TPH plot for series of groundwater samples.
Error bars indicate ± 1 standard deviation.
5.3.3 BOD/COD Ratio - Biodegradability
The BOD/COD ratios were below 0.10 for all seven groundwater samples
(Table 5.10 and Figure 5.9).
The slope for BOD/COD vs. TPH is
essentially flat (Figure 5.9). BOD/COD vs. TPH was plotted and has no
observable correlation and a linear regression R 2 value of only 0.03
(Figure 5.9).
Biodegradability (BOD/COD values, Table 5.10) does not
61
decrease at low concentrations, corresponding to weathered material, as
would be expected if partially degraded diluent was more recalcitrant than
fresh diluent. Diluent does not appear to become more recalcitrant with
biodegradation and/or aging (weathering).
WEATHERED
FRESH
Biodegradability (BOD/COD)
0.12
0.10
206-C
y = -0.0006x + 0.0527
2
R = 0.03
209-E
0.08
0.06
G4-3
0.04
H3-7
0.02
M4-4
209-D
H2-1
0.00
0
5
10
15
20
25
30
TPH concentration (ppm)
Figure 5.9 BOD/COD vs. TPH plot for series of groundwater samples.
Error bars indicate ± 1 standard deviation.
A trend of decreasing BOD/COD ratio with increasing distance from
source zone was observed for three separate plumes from the Diluent
Tanks (DT) area (Figure 5.10). For the northern DT plume the BOD/COD
ratio decreased by a factor of two over 200 ft, and for the southern DT
plume the BOD/COD ratio decreased by a factor of six over a distance of
about 650 ft.
The two wells in the central DT plume were very close
together and little difference in biodegradability was observed (Figure
62
5.10).
Although there were only two samples per plume, these results
indicate the possibility of decreased biodegradability with increased
weathering.
Another interesting observation is that BOD/COD is quite different for the
different plumes.
For example, the observed biodegradability was much
lower for the wells from the central DT plume than the wells from the
northern and southern plumes even though both wells in the central DT
area were near the source zone and had high TPH concentrations (Figure
5.10).
This suggests that chemical composition differences between the
different plumes may have a very large impact on biodegradability.
63
0.1
DT = Diluent Tanks
206-C, 5.7 ppm
0.09
209-E, 8.2 ppm
Biodegradability (BOD/COD)
0.08
0.07
Northern DT
0.06
0.05
G4-3, 4.2 ppm
0.04
0.03
Southern DT
H3-7, 11 ppm
0.02
H2-1, 13 ppm Central DT
0.01
209-D, 7.5 ppm
0
0
200
400
600
800
1000
1200
Distance from source (ft.)
Figure 5.10 BOD/COD vs. distance down plume from source plot for
series of groundwater samples grouped by location. Well
number and TPH concentration are indicated for each
point on the graph.
64
CHAPTER 6
DISCUSSION
6.1 Reliability of BOD Tests
O 2 depletion adequate in fresh groundwater samples
The 6-day BOD test using diluent-contaminated groundwater and prepared
seed inoculum showed significant oxygen depletion in all sample bottles
with diluent-contaminated groundwater (Table 5.2), indicating sufficient
biodegradation occurred. The results from the 6-day experiment showed
oxygen uptake was adequate using fresh groundwater sample. The 5-day
BOD re-test using diluent-contaminated groundwater showed sufficient
oxygen depletion for all final samples used (Table 5.5). The blanks and
seeded blanks showed negligible oxygen consumption for all experiments.
Inoculum sufficient
Most BOD samples had appropriate oxygen consumption and residual
D.O. values.
The BOD standards were in the appropriate range most of
the time, which test for inoculum viability among other parameters, also
suggesting that appropriate amounts of inoculum were used.
Since
doubling the inoculum from 10 mL to 20 mL did not significantly increase
the oxygen uptake, 10 mL inoculum was used. Using 10 and 20 mL seed,
results were 2% and 4% above the upper limit of 457 mg/L.
Slightly
greater BOD exertion was observed for the 6-day samples with only 10
mL of seed compared to those with 20 mL of seed, but is not statistically
65
significant (Table 5.2). This indicates sufficient seed was used in these
tests.
Most GGA standards were within the appropriate range
The BOD standards for the 6-day experiment worked as expected. 10 and
20 ml of seed was added to BOD bottles with 2 mL of GGA stock solution
and dilution water.
The resulting BOD exerted by these standards was
468 and 475 mg/L for the 10 and 20 mL of seed added, respectively
(Table 5.2).
The BOD of the GGA in the 5-day test was slightly lower
than expected.
The resulting average BOD 5 exerted by these standards
was 296 mg/L (Table 5.4), 12% below the lower limit of 335 mg/L
(APHA, 1999). The resulting average BOD exerted by the standards for
the 5-day re-test was 408 mg/L for the 10 mL of seed added (Table 5.5)
and performed as expected.
6.2 Effect of Iron on BOD
The effect of iron on BOD was minimal. The difference in BOD of
samples with iron and without iron was very small and not statistically
significant (Table 5.1). The BOD of the non-sparged samples was slightly
greater than the sparged samples, but the difference between sparged and
not sparged is very small and not statistically significant. This test
confirmed no iron effect on BOD.
66
6.3 Effect of Dilution
6.3.1 Effect of Dilution on BOD Measurement
The calculated BOD 6 exerted by the 50% diluted groundwater samples was
somewhat higher than that calculated for the full strength samples (Table
5.2). They should have been nearly equal, but differed by approximately
30%.
This may have been partly caused by the nearly complete oxygen
depletion in the full strength samples (Table 5.2). Full strength samples
started with lower initial dissolved oxygen concentrations from not being
given oxygen-saturated dilution water. Because of this oxygen depletion,
the dilution experiment was repeated.
In the 5-day test, diluted samples are expected to have similar values and
were all relatively close. The average BOD exerted by the 25% and 50%
diluted samples of H2-1 differed by approximately 3%, while the 25% and
50% diluted samples of H3-7 differed by approximately 15%. The various
dilutions of M4-4 samples exhibited varying degrees of D.O. depletion.
The 10% dilution showed a small D.O. depletion while the 50% dilution
essentially depleted all oxygen.
The 25% diluted samples provided the
most desirable results, having at least a 2 mg/L D.O. reduction and having
a minimum D.O. residual of 1 mg/L (Table 5.4).
A 'sliding' effect whereby the BOD 5 increases with the dilution factor is
indicative of sample toxicity.
Thus toxic samples with a high dilution
67
factor will give a high BOD 5 value because of diluted toxicity effects,
while less dilute samples will give a lower BOD 5 due to more intensified
toxicity effects (Alvares et al (2), 2001).
This effect was not observed
with Guadalupe groundwater.
6.3.2 Effect of Dilution on COD Measurement
The diluted samples were run as a check on the effect of dilution on COD
measurement. The diluted sample values exhibited approximately half the
COD value of the full strength samples, as expected (Table 5.6).
This
concludes that dilution does not interfere with the analysis, and COD can
be reliably used for the groundwater samples with different TPH
concentrations.
6.4 Reliability of COD Tests
Good KHP calibration curves
Both calibration runs, having R 2 values of above 0.95, exemplify the high
reliability of the KHP calibration curves.
Important to use proper range
The importance of using proper range COD vials became apparent when
COD results were above 150 mg/L using the 5-150 mg/L (low range)
vials. These low range vials would not be suitable for all samples. A
68
switch to 20-900 mg/L vials was made, ensuring consistent results for the
range of concentrations for all groundwater samples.
No effect from Iron
There appears to be no significant chemical oxygen demand exerted by the
presence of Fe 2 + [40 mg/L FeSO 4 ] (Table 5.1).
The difference between
the samples, with and without iron, falls well within the limits of one
standard deviation (Table 5.1).
6.5 Biodegradability
BOD/COD ratios were only 0.01 to 0.09 (Table 5.10), suggesting either
the diluent isn’t very biodegradable, or biodegradation is very slow
(Gilbert, 1987).
Gilbert suggested that a BOD/COD ratio of 0.4 to be
considered biodegradable.
The low BOD/COD ratios observed in this
experiment are most likely the result of slow diluent biodegradation rates,
rather than low diluent biodegradability.
These BOD tests were
conducted for only five days, yet the time scale for diluent bioremediation
is much longer than this. Longer BOD experiments or employing the use
of long-term respirometry may be worthwhile to observe long-term
biodegradability.
Alternatively, rate constants for biodegradation could
be determined for each sample, and these rate constants could be used to
estimate the ultimate BOD from the observed five-day BOD values.
69
A project evaluating the CO 2 production from groundwater samples taken
from the biosparge unit at the GRP showed high CO 2 production without
nutrient addition (Waudby, 2003).
Short-term experiments by Waudby
showed no benefit from inorganic nutrient addition while it may prove to
be necessary for sustained biodegradation.
TPH degradation slowed
considerably after collecting 6 days of respirometry data.
Long-term
biodegradation was sustained but rates were slow and no minimum TPH
concentration was observed.
More long-term experiments in this area
were suggested to determine nutrient limitations.
6.6 ThOD of Hydrocarbons
COD/TPH ratio higher than expected based on ThOD
The COD/TPH ratios in Table 5.10 range from 11.4 to 23.8 with an
average of 18.1 mg COD/mg TPH. The average COD/TPH ratio of 18.1 is
approximately five times higher than the expected hydrocarbon ThOD
value of 3.5 mg O 2 /mg TPH (Table 5.10).
The ThOD of hydrocarbons is approximately 3.5 mg O 2 /mg TPH.
The
stoichiometric relation for the ThOD of hexane is given in Eqn. 4.
Calculation of ThOD for hexane:
Generic hydrocarbon: C 6 H 1 4
Molecular Weight: 86 g/mol
C 6 H 1 4 + 19/2O 2 → 6CO 2 + 7H 2 O
(9.5 mol O 2 ) * (32 g/mol O 2 ) = 304 g O 2
70
(4)
(304g O 2 ) / (86 g/mol)
ThOD = 3.53 g O 2 / mol
There are a number of possible reasons why the observed COD of the
groundwater samples were higher than the ThOD for TPH, including:
1. Other organic material contributes to COD
2. Error in COD measurement/ calculation
3. Error in TPH measurements
Each of these possibilities is discussed below.
Other oxidizable organics may be present in groundwater
Other oxidizable organics present in the groundwater would increase the
measured COD/TPH values. Other organic compounds may be associated
with TPH that might not show up using GC analysis. COD correlates with
TPH, but the TPH value of 29 ppm for well M4-4 seemed a little high,
considering the geometric mean of TPH for well M4-4 in previous
sampling events was 17.74 ppm (Lundegard 2002).
The previous
maximum value obtained was 22 ppm. Considering TPH values could be
lower, COD/TPH may have higher values.
This would support the
possible theory of other oxidizable organics being present. A comparison
of the COD of non-contaminated groundwater to contaminated samples
may be useful, also the use of total organic carbon (TOC) analysis in
conjunction with TPH analysis could be useful. There also could be more
71
bacteria present or their by-products when TPH is high, and these bacteria
or by-products could exert COD.
Errors in COD measurement?
Errors in measuring COD are unlikely.
COD is a simple test only
involving pipetting, heating and measuring light absorbance. Calculations
of COD involve creating and using a calibration curve and accounting for
blanks.
Comparisons to other COD calculations yielded similar results.
To test our COD method and calculations, the COD was measured using
the accu-TEST™ kits for solutions of phenol to compare to the ThOD of
phenol.
Measured TPH values may be below actual values
It is unlikely that the GC/MS method used by Zymax is measuring TPH
incorrectly.
Zymax is an EPA certified lab, so they are probably
following the appropriate guidelines.
They only filter samples when
specifically requested by the customer, so product would not be lost in
filtration.
They agitate samples during extraction, homogenizing the
sample and reducing the chance for an analytical error.
The first
paragraph of Section 6.6 also addressed the possibility of a measured TPH
value falling outside the expected range.
72
Comparison of COD to ThOD of phenol
A COD run with phenol was made to try to understand why the oxygen
demand of diluent contaminated groundwater was above the calculated
theoretical ThOD value of 3.5 mg O 2 /mg TPH. This was also run to test
the COD method and calculations.
The average COD value of 0.95 mg
O 2 /mg phenol was approximately 40% of the 2.38 mg/L ThOD of phenol
(Table 6.1).
The ThOD value was not expected to be attained due to
incomplete reaction, but the reported COD of phenol was expected to be
closer to the ThOD.
This experiment does show that our COD method
does not result in extraneously high COD in all cases.
The oxygen
demand of phenol was between 0.92 and 0.96 mg O 2 /mg phenol for all
samples analyzed, with an average of 0.95 mg O 2 /mg phenol (Table 6.1).
A COD vs. phenol bar graph is shown in Figure 6.1.
Table 6.1 COD of phenol solutions
Sample
Absorbance COD
mg
phenol conc. at 600 nm
(mg/L) Phenol
(mg/L)
DI Blank
0.016
------DI Blank
0.015
------DI Blank
0.018
------150
0.06
144.05
0.960
150
0.059
140.75
0.938
150
0.058
137.45
0.916
300
0.103
285.90
0.953
300
0.104
289.19
0.964
300
0.104
289.19
0.964
73
350
COD/phenol = 0.94
COD (mg/L)
300
250
200
COD/phenol = 0.96
150
100
50
0
150
300
150
Phenol concentration (mg/L)
error bars indicate ± one standard deviation
Figure 6.1 COD vs. phenol concentration for standard phenol
solutions.
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
REFERENCES
1. Alvares, A.B.C., C. Diaper and S.A. Parsons. "Partial oxidation by
ozone to remove recalcitrance from wastewaters - a review."
Environmental Technology 22 (2001): 409-427.
2. Alvares, A.B.C., C. Diaper and S.A. Parsons. "Partial oxidation of
hydrolysed textile azo dyes by ozone and the effect on
biodegradability." Trans IChemE 79.B (2001): 103-108.
3. APHA, Standard Methods for the Examination of Water and
Wastewater, 20 t h edition. American Public Health Association,
Washington, D.C. (1999): Section 5210.
4. Barron, M., and T. Podrabsky. “Guadalupe Oil Field NRDA:
Analytical Chemistry of Free-Phase and Dissolved-Phase Diluent.”
Trans. Boulder, CO: Stratus Consulting, Inc., 1999.
5. Chun, H., W. Yizhong. "Decolorization and biodegradability of
photocatalytic treated azo dyes and wool wastewater." Chemosphere
39.12 (1999): 2107-2115.
6. Elliot, K. 2002 "VOC Emissions From Willow Trees Grown on
Hydrocarbon-Contaminated Groundwater in a Two-Compartment
Growth Chamber." M.S. Thesis, California Polytechnic State
University, San Luis Obispo, CA.
7. Geenens, D., B. Bixio and C. Thoeye. "Combined ozone-activated
sludge treatment of landfill leachate." Water Science and
Technology 44.2 (2000): 359-365.
8. Gilbert, E. "Biodegradability of ozonation products as a function of
COD and DOC elimination by example of substituted aromatic
substances." Water Research 21.10 (1987): 1273-1278.
9. Green, C., et al. “Enhanced Biodegradation of Hydrocarbon
Contaminants Using Photocatalytic Pretreatment.” In Situ Aeration
and Aerobic Remediation Columbus, Ohio: Batelle Press 2001.
10.(GRP) Guadalupe Oil Field/ Guadalupe Restoration Project, 2003.
URL: http://www.guaddunes.com/
Last updated in October 2002
Pages viewed on Friday, December 3 r d , 2003
77
11.HACH, 2003
BOD Reagents: Nitrification Inhibitors
URL: http://www.hach.com/Spec/BOD_nitrificationInhib.htm
Last updated on November 1 s t , 2001
Pages viewed on Monday, November 3 r d , 2003
12.Haddad, B. and S. Stout. Diluent Geochemistry Report.
Corporation, 1996.
Unocal
13.Imai, A., et al. "Effects of pre-ozonation in refractory leachate
treatment by the biological activated carbon fluidized bed process."
Environmental Technology 19 (1998): 213-221.
14.ISO, 2003. URL:
http://www.iso.ch/iso/en/stdsdevelopment/tc/tclist/TechnicalCommi
tteeStandardsListPage.TechnicalCommitteeStandardsList?COMMID
=3729
Last updated on Monday, March 10 t h , 2003
Pages viewed on Monday, January 12 t h , 2004
15.Koch, M., et al. "Ozonation of hydrolyzed azo dye reactive yellow
84 (CI)." Chemosphere 46 (2002): 109-113.
16.Kumar, A., et al. "Biodegradable organic matter in industrial wastewaters as determined by formulated microbial mixture." Journal of
Environmental Biology 19.1 (1998): 67-72.
17.Leeson, A. and Hinchee, R. Soil bioventing : principles and
practice. Boca Raton, FL: CRC/Lewis Publishers, 1997.
18.Lundegard, P., 2002 "Empirical analysis of total petroleum
hydrocarbons in groundwater versus distance from diluent source
areas (Version 2.1)." Former Guadalupe Oil Field, San Luis Obispo
County, California: Unocal Corp. internal document.
19.Macdonald, J., "Evaluating Natural Attenuation for Groundwater
Cleanup." Environmental Science & Technology 34.15 (2000): 346
A-353 A.
20.Maloney, L., 2003 "Characterization of aerobic and anaerobic
microbial activity in hydrocarbon-contaminated soil." M.S. Thesis,
California Polytechnic State University, San Luis Obispo, CA.
78
21.Mantzavinos, D., et al. "Chemical treatment of an anionic surfactant
wastewater: electrospray-ms studies of intermediates and effect of
aerobic biodegradability." Water Research 35.14 (2001): 33373344.
22.Mantzavinos, D., et al. "Wet air oxidation of polyethylene glycols;
mechanisms, intermediates and implications for integrated
chemical-biological wastewater treatment." Water Research 51.18
(1996): 4219-4235.
23.Peavy, H., D. Rowe and G. Tchobanoglous. Environmental
Engineering. New York, New York: McGraw Hill, 1985.
24.Sawyer, C. and P. McCarty. Chemistry for Environmental
Engineering. New York, New York: McGraw Hill, 1978.
25.Tchobanoglous, G. and E. Schroeder. Water Quality. Reading,
Massachusetts: Addison-Wesley, 1987.
26.USEPA, 2001. Citizen's Guide to Monitored Natural Attenuation.
EPA/542/F-01/004.
27.USEPA, 2003.
URL: http://www.epa.gov/swerffrr/documents/petrol.htm
Commonly Asked Questions Regarding the use of Natural
Attenuation for Petroleum Contaminated Sites at Federal Facilities
Last updated on Monday, March 10 t h , 2003
Pages viewed on Friday, October 3 r d , 2003
28.USEPA, 1999 a . Monitored Natural
Hydrocarbons. EPA/600/F-98/021.
Attenuation
of
Petroleum
29.USEPA, 1999 b . Use of Monitored Natural Attenuation at Superfund,
RCRA Corrective Action and Underground Storage Tank Sites.
Office of Solid Waste and Emergency Response, Directive Number
9200-4-17P.
30.USEPA, 1994. How to evaluate Alternative Clean-up Technologies
for Underground Storage Tank Sites. EPA/10/B-94/003.
31.Waudby, J., 2003. "An investigation of factors limiting the
biosparge-mediated
in-situ
bioremediation
of
hydrocarbon
contaminated groundwater". M.S. Thesis, California Polytechnic
State University, San Luis Obispo, CA.
79