Biodegradability of Hydrocarbon Contaminants during Natural ...

BIODEGRADABILITY OF HYDROCARBON CONTAMINANTS DURING NATURAL ATTENUATION OF CONTAMINATED

GROUNDWATER DETERMINED USING BIOLOGICAL AND CHEMICAL OXYGEN DEMAND

A Master’s Thesis Presented to the Faculty of California Polytechnic State University

San Luis Obispo

In partial fulfil lment of the requirements for the degree of

Master of Science in Civil and Environmental Engineering

By

Evan B. Larson

February 2004

COPYRIGHT OF MASTER’S THESIS

I grant permission for the reproduction of this thesis in its entirety or any of its parts, without further authorization from me, provided it is referenced appropriately.

Evan Larson Date

i i

MASTER’S THESIS APPROVAL TITLE: BIODEGRADABILITY OF HYDROCARBON

CONTAMINANTS DURING NATURAL ATTENUATION OF CONTAMINATED GROUNDWATER DETERMINED USING BIOLOGICAL AND CHEMICAL OXYGEN DEMAND

AUTHOR:

EVAN B. LARSON

DATE SUBMITTED:

FEBRUARY 2004

THESIS COMMITTEE MEMBERS:

Dr. Yarrow Nelson Date

Dr. Nirupam Pal

Date

Dr. Christopher Kitts

Date

i i i

ABSTRACT

BIODEGRADABILITY OF HYDROCARBON CONTAMINANTS DURING NATURAL ATTENUATION OF CONTAMINATED GROUNDWATER

DETERMINED USING BIOLOGICAL AND CHEMICAL OXYGEN DEMAND

Evan Larson

Natural attenuation is being evaluated as a possible method of remediation of hydrocarbon contamination at the Guadalupe Restoration Project (GRP) at a former oil field on the Central Coast of California. The site is contaminated with hydrocarbons in the C10 to C30 range, which were used as a diluent to facilitate oil extraction. The GRP is located in an ecologically sensitive coastal area and thus it is important to remediate the hydrocarbon contamination with minimal disturbance. Natural attenuation is the microbial degradation and weathering of a contaminant, and interest has grown throughout the environmental community in its application over the past decade. To explore the feasibility of using natural attenuation at the GRP, a series of experiments were conducted to determine the biodegradation rates of total petroleum hydrocarbon (TPH) in groundwater from the site; and to evaluate the sustainability of biodegradation with weathering. In order for natural attenuation to be sustainable at this site, it is important that the hydrocarbons remain biodegradable as they are weathered. To test for this sustainability, biodegradability was determined for a series of groundwater samples, which had weathered differently. Biodegradability was measured as the ratio of biological oxygen demand (BOD) to chemical oxygen demand (COD). BOD/COD ratios were measured for diluent-contaminated groundwater from monitoring wells C8-39, G4-3, 206-C, 209-D, 209-E, H3-7, H2-1 and M4-4. The TPH concentrations ranged from 4.2 ppm to 29 ppm. Sampling was originally planned along the transect of a single plume to observe biodegradation patterns along the transect as the hydrocarbons presumably become more weathered down-gradient. Due to constraints concerning the nesting pattern of the Western Snowy Plover, this method of sampling was abandoned. As a surrogate method of collecting samples with varying degrees of hydrocarbon weathering, the series of monitoring wells listed above were used to provide a range of TPH concentrations, and wells with low TPH concentrations far from source zones were presumed to be more weathered. The range of BOD/COD values for these groundwater samples were 0.01 to 0.09, suggesting slow biodegradation. BOD/COD did not correlate with TPH concentration (R2 = 0.03). BOD/COD ratios did not significantly change with increasing TPH concentration, suggesting weathering did not significantly influence biodegradability. BOD/COD ratios decreased with distance from source, indicating the possibility of decreased biodegradability with increased weathering and a variation in diluent chemistry. COD correlated with TPH values fairly well with an R2 value of 0.74. BOD had a very weak correlation with TPH concentration (R2 = 0.41). The average COD/TPH value was 18.1. This COD/TPH ratio is approximately five times the expected theoretical oxygen demand (ThOD) of hydrocarbons of 3.5. This high value may be attributed to the presence of other oxidizable organics. BOD/COD ratios approaching the value of 0.4 have been reported for biodegradable material. However, the low BOD/COD ratios observed in this research were most likely because of slow biological degradation leading to low 5-day BOD values.

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

4.2.2 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

4.3 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 BOD/COD Ratio - Biodegradability.......................................................61

6 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 (BOD5) 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. , 2001a , 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. A BOD/COD ratio that

remains similar throughout the range of 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, 199b).

Biodegradation may reduce the surface contamination on the soil

particles, leading to a lack of nutrients. 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

Figure 3.3 Guadalupe plume map.

Not to scale

7

Unocal purchased the outstanding share of the GOF in 1953, and became

the operator (GRP, 2002). The former Guadalupe Oil Field consists of

over 2700 acres of land. 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, 1999b). 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 l ines 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 contaminants to be destroyed by

biodegradation (USEPA, 2003). 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 lit t le 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,

1999a).

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, 1999b). The

process of dilution and/or dispersion alone does not 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, 1999a).

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, 1999b).

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). I t

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, 1999b). Inhibitory compounds may

result from incomplete biodegradation, giving rise to by-products or

intermediates more toxic than the original compound (Alvares et al,

2001a). In addition to toxicity, the mobility of transformation products

may exceed that of the parent compound (USEPA, 1999b). 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,

1999b). Natural attenuation is not appropriate for high concentrations of

contaminant, due to toxicity factors (USEPA, 1999b). 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

BOD5 (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 BOD5/COD and BOD5/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, 2001b). Low BOD5/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 BOD5/COD was

more than 0.3 the wastewater had a better biodegradability. Similar

statements were made for a BOD5/COD ratio of 0.4 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 airt ight container and kept

in a controlled environment for a preselected period of t ime. In the

standard test, a 300-mL BOD bottle is used and the sample is incubated at

20°C for five days (Peavy et al. , 1985). The BOD is then calculated from

the initial and final dissolved oxygen (DO) concentration.

3.8 Chemical Oxygen Demand

The chemical oxygen demand (COD) test is used to measure the total

organic content of industrial wastes and municipal and natural

wastewaters. During the determination of COD, organic matter is

converted to carbon dioxide and water using a strong chemical oxidizing

agent (dichromate) in the presence of a catalyst and strong acid. In the

COD test, organic materials are oxidized regardless of the biological

assimilability of the substances. As a result, COD values are greater than

BOD values and may be much greater when significant amounts of

biologically resistant organic matter are present (Sawyer and McCarty,

1978).

3.9 Chemistry of Diluent Contamination at the Guadalupe Site

Diluent from the Guadalupe Restoration Project is a hydrocarbon

consortium with a carbon range of nC10 to nC3 0. Figure 3.3 shows

common fuel ranges with respect to carbon length. According to this

17

chart, the diluent at Guadalupe is essentially a diesel range oil (DRO).

Water solubility plays an important role when considering the fate of

diluent constituents. Constituents with low solubility exist as a separate-

product, whereas diluent chemicals with a high solubility generally are

dissolved in the groundwater. A majority of the diluent at the Guadalupe

Oil Field has low solubility. Diluent from Guadalupe has a reported

solubility of 30 mg/L (Haddad and Stout, 1996). The diluent composition

over the Guadalupe site is considerably variable. The difference in

diluent makeup can be explained by source oil variation and weathering

(Barron and Podrabsky, 1999).

Haddad and Stout made the following conclusions on the diluent

chemistry:

• The carbon length of the diluent ranges from <nC10 to >C3 0. About

70% of the diluent falls in the diesel range of nC1 0 to nC2 5.

• Saturated, aromatic, polar, and asphaltic fractions respectively make

up 60%, 17%, 8%, and 15% of the separate-phase diluent. The

dissolved-phase fractions were not available for review.

Haddad and Stout (1996) also reported the total petroleum hydrocarbon

(TPH) composition as well as the BTEX concentrations (Table 3.1 and

3.2).

18

C 3 6C 2 4

R E F E R E N CE : T P H I N S O I L P R I M E R , E L A I N E M . S C H W E R K O . D A T E D : 0 9 / 0 1 / 9 3 N o n - me a s u r a b l e T P H d u e to

v o l a t i l i z a t io n

1 70 C

3 40 F

1 70 C

3 40 F

C 1 0

D i e s e l Ra ng e

G a s o l i n e R a n g e

C 1 0

C 6

1 70 C

3 40 FB o i l i n g

P o i n t R a n g e

1 40 F

1 40 F

C 3 6

C 2 0

C 2 4 - 3 0

C 2 4

C 1 2

C 1 7

C 1 2

C 8

C 8

C 1 0

C 6

C 6

C 4

C 4

Lube Oi l s & heavier

Fuel Oi l s

Diese l

Semi-quant i f iable

Kerosene

Gasol ine

Measurable TPH

Table 3.1 Summary of separate-phase diluent analysis.

Constituent Concentration Range (mg/kg)

Benzene <2.0 to 120 Toluene <2.0 to 74 Ethylbenzene 2.2 to 200 Total Xylene 5.3 to 370 TPH: nC10 to nC32

910,000 to 990,000

Figure 3.4 Carbon ranges for common diluent constituents compared to common petroleum distillates (Elliot, 2002).

19

Table 3.2 Summary of dissolved-phase diluent analysis. Constituent Concentration Range (µg/L)

Benzene <0.5 to 9.1 Toluene <0.5 to 5.1 Ethylbenzene <0.5 to 7.4 Total Xylene <0.5 to 17 TPH: nC6 to nC10 nC10 to nC32

<50 to 400 870 to 16,000

(E l l io t 2002)

3.10 Cal Poly Natural Attenuation Project Unocal is currently funding remediation research at California

Polytechnic State University, San Luis Obispo through the Environmental

Biotechnology Institute (EBI). This project is part of a larger set of

experiments aimed at determining how the concentrations of petroleum-

derived hydrocarbons change due to remediation by natural or engineered

methods. Some of the other research projects are biosparging, steam

injection and phytoremediation. Anaerobic aspects of natural attenuation

of diluent were considered separately (Maloney, 2003), and further

natural attenuation research is currently underway.

20

CHAPTER 4

MATERIALS AND METHODS

4.1 Groundwater Sampling

Bob Pease collected groundwater samples from the Guadalupe site. Three

to five well volumes were purged before sample collection. Some

groundwater samples were used for BOD and COD method development,

while others were used for the natural attenuation experiment.

Monitoring wells sampled were G4-3, H2-1, H3-7, 206-C, 209-D, 209-E

and M4-4.

4.2 BOD Measurement

BOD measurements involve seeding a test sample, storing the sample for a

specified time and obtaining the initial and final dissolved oxygen (D.O.)

values.

In the BOD experiments, triplicates were run of the seed and dilution

water. These were respectively called seed control and dilution water

blank.

The BOD standard (glucose glutamic acid test, GGA) is intended to be a

reference point for evaluation of dilution water quality, seed

effectiveness, and analytical technique. GGA reagents were purchased

from Hach Co. USA.

21

BOD is computed using the following equation (Eqn. 1):

BOD = (D1 - D2) - (B1 - B2)f / P (1)

Where,

BOD = biochemical oxygen demand, mg/L

D1 = DO of diluted sample 15 minutes after preparation, mg/L

D2 = DO of diluted sample after incubation at 20°C, mg/L

B1 = DO of seeded dilution water blank before incubation, mg/L

B2 = DO of seeded dilution water blank after incubation, mg/L

f = ratio of seed in sample to seed in blank

= % seed in D1 / % seed in B1

P = decimal fraction of sample used

= mL of sample, Vs / 300 mL

4.2.1 BOD Inoculum

Inoculum for the BOD measurements must be of appropriate strength for

obtaining meaningful BOD data. Viable bacterial populations must be

present to have enough oxidation occurring to yield accurate BOD

measurements. However, the inoculum should not produce more than 10%

of the total oxygen consumption during the BOD analysis.

Preliminary Experiment Inoculum Preparation

The groundwater used for preparation of inoculum for preliminary experimentation had

an original TPH concentration of approximately 2 ppm. This sample was collected by

22

Ken Hoffman from C8-39 and had been sitting in the laboratory for a few months at room

temperature. The TPH concentration would have been depleted after a several month

storage period. This preliminary inoculum was formulated by mixing 1000 mL of the 2

ppm Guadalupe groundwater from C8-39 and 100 g of diluent-contaminated soil from the

Guadalupe site. This inoculum was continuously stirred for aeration at ambient

temperature for one-week prior to use. This will be labeled Ii.

Inoculum Preparation for Preliminary BOD Tests

Inoculum for the BOD2 0 and BOD6 experiments were prepared by using

one Polyseed® BOD Seed Inoculum capsule (Interbio, Woodland, TX),

about 100g diluent-contaminated soil and 100 mL of I i and 900 mL of

Guadalupe groundwater for a total volume of 1 liter. The groundwater for

this inoculum preparation is the depleted 2ppm TPH collected by Ken

Hoffman from monitoring well number C8-39 as described above. This

inoculum was labeled I i i . Continuous stirring following initial

preparation was used for aeration.

Inoculum Preparation for BOD5 Tests on Groundwater

For final BOD experiments, a third inoculum was prepared one month

following the preparation of I i i . This was used for the final BOD5

experiments using several groundwater sources. 100 mL of inoculum I i i

was added to 900 mL of Guadalupe groundwater, 100 g diluent-

contaminated soil and one Polyseed® capsule. The 900 mL of

23

groundwater used was a fresh sample from well number C8-39 at 8-12ppm

TPH, provided by Bob Pease. Continuous stirring was used for aeration

until use for BOD measurement four days later.

4.2.2 Dilution Water

Dilution water is used to provide trace elements to microbial populations

and to dilute samples to a measurable BOD range. Mineral deficiencies

and pH shifts can cause low BOD results. Thus, Hach BOD nutrient

buffer pillows (Hach Co., Loveland, CO) were used in the preparation of

dilution water for all BOD experiments. Each pillow contains buffer and

nutrients specified by the U.S. Environmental Protection Agency

(USEPA) and American Public Health Association (APHA) in the

Standard Methods for the Examination of Water and Wastewater (1999).

Dilution water was prepared by adding one Nutrient Buffer Pillow to 3 L

of deionized water.

Dilution water was bubbled with air, which passed through a 2-µm inline

filter, for at least twenty minutes to ensure maximum dissolved oxygen.

Air was filtered to avoid lubrication oils from the air pump and foreign

particulates in the ambient air from being introduced into the dilution

water. To ensure undiluted samples had the same final nutrient

concentration as the diluted samples, additional nutrients were also added

to full-strength (non-diluted) samples by adding 3 mL of 100x nutrient

24

stock to each BOD bottle. Similarly, a proportional amount of nutrient

stock was added to all 50% diluted BOD bottles to ensure nutrients were

not a limiting factor. The 100x solution was prepared by adding one

nutrient buffer pillow, to 30 mL deionized water.

4.2.3 Nitrification Inhibitor

Nitrogenous biochemical oxygen demand (NBOD) is the amount of

oxygen required for biological oxidation of ammonia to nitrate via

nitrification (Tchobanoglous and Schroeder, 1987). BOD determinations

may be inadequate for evaluating efficiency of treatment processes if

nitrifying bacteria are present. NBOD usually occurs after seven days of

incubation. NBOD was not of interest in this experiment since it would

erroneously indicate biological activity by overestimating the BOD,

resulting in an overestimation of the actual biological removal efficiency.

Nitrification inhibitor was therefore used throughout these experiments to

inhibit NBOD.

Hach Formula 2533™ nitrification inhibitor, 2-chloro-6-(trichloromethyl)

pyridine (TCMP), eliminates the nitrifying interference when testing

samples. Nitrification inhibitor can be used with the USEPA-accepted

BOD dilution method (HACH, 2003). Results of BOD tests completed

with inhibitor are referred to as carbonaceous BOD (CBOD).

This nitrification inhibitor is plated on an inert salt , which allows

inhibitor to dissolve quickly in samples. 0.16 g of nitrification inhibitor

25

were added to each 300 mL BOD bottle to make a final concentration of

10 mg/L TCMP. Hach Formula 2533™ nitrification inhibitor was used for

all BOD bottles.

4.2.4 Dissolved Oxygen Measurement

Dissolved oxygen concentrations were made with a YSI model 58

dissolved oxygen meter with YSI model 5905 Self-Stirring BOD Probe

(YSI Inc., Yellow Springs, Ohio). 300 mL BOD bottles with Vapor-

sealing caps were obtained from Wheaton Science Products (Millville,

NJ).

Figure 4.1 BOD analysis setup: DO meter, probe and BOD bottle.

Dissolved Oxygen Calibration

1) The YSI 58 DO meter was connected to the YSI 5905 probe

and the instrument was allowed to warm-up/stabilize for 15

minutes prior to use.

26

2) The probe was zeroed and calibrated at a temperature as close

as possible to the temperature of the sample to be measured

to obtain the highest accuracy of measurement. Setting the

function switch to ZERO and adjusting the display to read

00.0 with the O2 ZERO control zeroed the YSI 58.

3) Following zeroing of the DO meter, the function switch was

set to % Mode .

4) The BOD probe was placed in a BOD bottle containing about

one inch of water to provide a 100% relative humidity

calibration environment.

5) When the display reading had stabilized, the 02 CALIB

control locking ring was unlocked and the display was

adjusted to the CALIB VALUE obtained from the

pressure/altitude chart in Appendix F of the instruction

manual. The locking ring was then relocked to prevent

inadvertent changes.

4.2.5 BOD Incubation

All BOD bottles were placed in an incubator, in the absence of light, at

20°C ±1°C for five, six or twenty days. All bottles had a wet seal and had

a cap to act as a vapor seal over the top of the BOD bottle seal to ensure

no evaporation of the wet seal.

27

4.2.6 Fe Effects on BOD

To investigate possible effects of reduced iron on the measurements of

BOD, the BOD was measured for groundwater samples with and without

Fe2 + added. The concern was that abiotic Fe2 + oxidation to Fe3 + could

consume oxygen, leading to interference with the BOD test. Guadalupe

diluent-contaminated groundwater from C8-39 was used for this

experiment, and it contained approximately 2 ppm TPH. An iron

concentration of 40 mg/L was added to this groundwater, using FeSO4 and

used for two sets of samples. One set of samples was bubbled for 10

minutes to oxidize the Fe+ 2. Another set of samples was not bubbled.

This comparison was made to determine if bubbling could be used to

eliminate any biological oxygen demand due to Fe2+ oxidation in the event

that such oxygen demand was significant.

4.2.7 Dilution Effects on BOD

Single source samples from well number C8-39 and the series of

groundwater samples were tested to examine BOD changes due to dilution

and as a part of the general testing of the BOD series. Some samples were

diluted to bring final DO values to within a usable range and to verify

BOD values should remain constant. This would be expected when

looking at the definition of the P-value in Eqn. 1.

28

4.3 COD Measurement

COD was analyzed using the accu-TEST™ mercury-free micro-COD

system (Bioscience Inc., Bethlehem, PA). Potassium bipthalate (KHP)

(Spectrum Chemical Co., Redondo Beach, CA) was used as a COD

standard. Absorbances of KHP standards of known concentrations were

measured and the COD in mg O2/L was calculated using the stoichiometric

relation between KHP and oxygen (Eqn. 2).

KC8H5O4 + 29/4 O2 → 8 CO2 + 5/2 H2O + K+ (2)

A Hitachi U-3010 UV/Vis spectrophotometer was used to measure

absorbance for all COD analyses and calibrations. Bioscience 5-150 mg/L

low range COD vials were used for iron oxidation experiments using aged

2 ppm concentration groundwater. The COD of low range vials was

determined using a spectrophotometer at 440 nm by measuring the

decrease in concentration of the Cr (VI) ion. Vials were incubated for

120 minutes at 150°F ± 2°F. Before analysis, the vials were allowed to

cool in the dark to prevent further oxidation. In the COD experiments, a

DI water control blank was run in triplicate.

Bioscience 20-900 mg/L standard range COD vials were used for

experiments using fresh 8-12 ppm or refrigerated samples from the other

wells requiring a higher range COD. The COD of standard range vials

29

was determined using a spectrophotometer at 600 nm by measuring the

concentration of the produced Cr (III) ion.

4.3.1 COD Calibration

The COD method was calibrated by measuring the COD of KHP standards.

The standard curve was created for samples ranging from 5-150 mg/L

(Low Range) and used to convert the measured absorbance at 440 nm to

mg/L COD. Samples above 150 mg/L were tested using the 20-900 mg/L

(Standard Range) vials and calibrated at 600 nm. For both ranges, the

absorbance was plotted against COD concentration for duplicates of each

KHP concentration.

4.3.2 Measurement of Iron Oxidation Effects on COD

COD was measured for groundwater samples with and without added Fe2 +

to test for COD of dissolved iron. 2.5 mL of Guadalupe diluent

contaminated groundwater from C8-39 was used for this test with aged 2

ppm TPH. One triplicate of test samples contained Fe2 + at a concentration

of 40 mg/L FeSO4, while another triplicate of test samples did not have

Fe2 + added. COD was measured using Bioscience 5-150 mg/L COD vials

with absorbance measured at 440 nm. The KHP standard curve for this

COD range was developed using the following concentrations in

triplicate: 1, 2, 5, 10, 20 and 30 mg/L COD or mgO2/L. The resulting

30

trendline from the KHP standards used to convert absorbance to COD

yielded R2 = 0.96 (Figure 4.2).

4.3.3 COD Measurements of Groundwater Series

Low-range Bioscience COD vials (5-150 mg/L) were used for initial COD

measurements, until i t was realized that some of the samples were out of

this range. COD was measured in triplicate for this series of six

groundwater samples. For the final COD analysis using the 20-900 mg/L

Bioscience COD vials, the KHP standard curve was developed using the

following standard concentrations in triplicate: 25.5, 81.8, 204.5, 409.0,

613.4 and 817.9 mg/L COD or mgO2/L. The resulting trendline from the

KHP standards used to convert absorbance to COD yielded R2 = 0.99

(Figure 4.3).

4.3.4 UV/Vis Spectrophotometer Analysis

As mentioned previously, a Hitachi U-3010 UV/Vis spectrophotometer

was used for all COD analysis and calibration. Before each use, the

spectrophotometer was calibrated with DI water and a zero value was

recorded from an average value of the control blank triplicates.

31

y = -0.0129x + 0.6391R2 = 0.9556

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 5 10 15 20 25 30 35

COD (mg/L)

Abs

orba

nce

at λ

= 4

40 n

m

Figure 4.2 Absorbance vs. COD of KHP standards, 5-150 mg/L range. Measuring the decrease in concentration of the Cr (VI) ion.

32

y = 0.0003x + 0.0537R2 = 0.9985

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0 100 200 300 400 500 600 700 800 900

COD (mg/L)

Abs

orba

nce

at λ

= 6

00 n

m

Figure 4.3 Absorbance vs. COD of KHP standards, 20-900 mg/L range.

Measuring the concentration of the produced Cr (III) ion.

33

4.3.5 COD of Phenol Solutions

COD was measured for phenol, to establish COD vs. ThOD. Phenol is a

hydrocarbon compound with a known theoretical oxygen demand (ThOD)

of 2.38 mg/L. The stoichiometric relation for the ThOD of phenol is

given in Eqn. 3 below.

Calculation of ThOD for phenol:

Phenol: C6H5OH Molecular Weight: 94 g/mol

C6H5OH + 7O2 → 6CO2 + 3H2O (3)

(7 mol O2) * (32 g/mol O2) = 224 g O2

(224 g O2) / (94 g/mol)

ThOD = 2.38 g O2/ mol

34

CHAPTER 5

RESULTS

5.1 BOD Results

5.1.1 Preliminary BOD Measurements and Effect of Iron Oxidation on

BOD Measurement

The BOD method and possible effects of Fe on BOD measurements were

tested using a groundwater sample collected from well C8-39 with an

original TPH concentration of 2 ppm that was stored at room temperature

for two months. For the undiluted, full-strength (FS), groundwater

samples, the DO only decreased by 0.4 mg/L (Table 5.1). The same

groundwater samples diluted by 50% also showed a DO decrease of about

0.35 mg/L. These results suggest very minimal biodegradation of this

aged groundwater sample, probably because much of the TPH was

degraded before the experiment.

The DO depletion from the dilution water blank exhibits what should be

the minimal, with an average of 0.17 mg/L (Table 5.1). DO for the seed

controls actually increased slightly, buy by a negligible 0.25 mg/L

Glucose glutamic acid (GGA) BOD standards were run to ensure that the

appropriate amount of seed was used and as a measure of general

reliability of the BOD test. The measured BOD standard values of 450

mg/L were about 2% higher than the upper end of the expected range of

35

396 ± 61mg/L, based on the ThOD of GGA. The BOD of GGA standards

were nearly identical for the samples with 2 mL or 4 mL of standard

solution (Table 5.1), indicating good reliability of the BOD methods

employed. The slightly lower BOD of the sample with 6 mL of standard

solution was likely the result of oxygen depletion, to below 2 mg/L.

No appreciable effect of Fe2 + on measured BOD was observed. Undiluted

samples without Fe added exerted a BOD5 of 0.66 ±0.02 mg/L while

samples with Fe added (no sparging) exerted a BOD of 0.57 ±18 mg/L

(Table 5.1 and Figure 5.1). This difference is within the experimental

error of the BOD measurement and is negligible. Sparging the samples

with Fe added slightly reduced the observed BOD (Table 5.1 and Figure

5.1), but this difference was also within the experimental error of the

BOD measurements. These results suggest that reduced Fe will not

interfere with the BOD measurements for the groundwater samples.

0.00.10.20.30.40.50.60.70.8

Groundwater Samples With Iron [40 mg/L] error bars indicate ± 1 standard deviation

Figure 5.1 Iron effect on BOD5.

BO

D5 (m

g/L)

no Fe Fe w/o sparge

Fe w/ sparge

36

Table 5.1 BOD results and statistics for method development. Sample

description(C8-39 @ 2ppm)

mLof

100x

Bottle number

Dilution(%)

SeedVolume (mL)

Sample Volume

(mL)

Dilution(P-value)

DO initial (mg/L)

DO final

(mg/L)

DeltaDO

(mg/L

BOD5

values(mg/L)

Avg.BOD5

(mg/L)

BOD5

SD(mg/L)

FS, w/o Fe 3 1 -- 10 287 0.957 8.76 8.35 0.41 0.68 FS, w/o Fe 3 2 -- 10 287 0.957 8.72 8.35 0.37 0.64 0.66 0.02 FS, w/o Fe 3 3 -- 10 287 0.957 8.72 8.33 0.39 0.6650%, w/o Fe 3 4 50 10 143.5 0.478 8.75 8.34 0.41 1.1150%, w/o Fe 3 5 50 10 143.5 0.478 8.75 8.45 0.30 0.88 0.98 0.1250%, w/o Fe 3 6 50 10 143.5 0.478 8.75 8.42 0.33 0.94

FS, w/Fe, w/o sparge 3 7 -- 10 287 0.957 7.20 7.02 0.18 0.44FS, w/Fe, w/o sparge 3 8 -- 10 287 0.957 7.90 7.40 0.50 0.77 0.57 0.18FS, w/Fe, w/o sparge 3 9 -- 10 287 0.957 7.92 7.70 0.22 0.48FS, w/Fe, w/sparge 3 10 -- 10 287 0.957 8.20 8.09 0.11 0.37FS, w/Fe, w/sparge 3 11 -- 10 287 0.957 8.00 7.79 0.21 0.47 0.41 0.05FS, w/Fe, w/sparge 3 12 -- 10 287 0.957 7.92 7.78 0.14 0.40

seed control -- 13 -- 10 290 0.967 8.75 8.95 -0.20 -0.21seed control -- 14 -- 10 290 0.967 8.72 8.99 -0.27 -0.28 -0.25 0.04seed control -- 15 -- 10 290 0.967 8.77 9.03 -0.26 -0.27DW blank -- 16 -- -- 300 1.000 8.80 8.60 0.20 0.20DW blank -- 17 -- -- 300 1.000 8.97 8.90 0.07 0.07 0.17 0.09DW blank -- 18 -- -- 300 1.000 8.93 8.70 0.23 0.23

BOD Std., 2ml -- 19 -- 10 2 0.007 8.80 5.72 3.08 462BOD Std., 2ml -- 20 -- 10 2 0.007 8.81 5.78 3.03 455BOD Std., 4ml -- 21 -- 10 4 0.013 8.85 2.08 6.77 508BOD Std., 4ml -- 22 -- 10 4 0.013 8.80 3.15 5.65 424BOD Std.,6ml -- 23 -- 10 6 0.020 8.88 0.6 8.28 414BOD Std.,6ml -- 24 -- 10 6 0.020 8.88 0.5 8.38 419

458 5.3

417 3.5

466 59

37

5.1.2 Preliminary BOD Method Testing: Dilution Effects (6-day BOD)

This experiment tested for dilution effects using one fresh groundwater

sample from well C8-39, at 8-12 ppm TPH. The groundwater sample was

kept refrigerated prior to use, to maintain the high TPH concentration.

Full strength samples using 10 mL of seed had an average BOD6 of 4.31

mg/L ± 0.13 mg/L and with 20 mL of seed, had an average BOD6 of 4.00

mg/L ± 0.05 mg/L (Table 5.2 and Figure 5.2). This suggests 10 mL of

seed is sufficient for the BOD analysis. Similarly, 50% strength samples

using 10 mL of seed had an average BOD6 of 5.69 mg/L ± 0.24 mg/L and

with 20 mL of seed, had an average BOD6 of 5.59 mg/L ± 0.35 mg/L

accounting for dilution in the calculation of BOD (Table 5.2 and Figure

5.2). The seed control samples with 10 mL seed had an average BOD6 of

0.06 mg/L ± 0.01 mg/L and with 20 mL seed had an average BOD6 of 0.16

mg/L ± 0.05 mg/L (Table 5.2).

The calculated BOD exerted by the 50% diluted groundwater samples, at

5.69 ± 0.24 mg/L and 5.59 mg/L ± 0.35 mg/L, were approximately 30%

higher than those calculated for the full strength samples, at 4.31 mg/L ±

0.13 mg/L and 4.00 mg/L ± 0.05 mg/L (Table 5.2). They were expected to

be nearly equal. Therefore dilution experiments were conducted and an

evaluation of dilution effects based on these experiments is given below

in section 5.1.4.

38

The BOD standard samples (GGA) with 10 mL seed had an average BOD6

of 468.0 mg/L ± 12.7 mg/L and with 20 mL seed had an average BOD6 of

474.8 mg/L ± 3.2 mg/L (Table 5.2). These standards show excellent

agreement and are relatively close to the expected value of 396 ± 61

mg/L.

39

Table 5.2 BOD results for 6-day test with single groundwater sample (fresh C8-39, 8-12 ppm). Bottle numbers 29 and 31, with respective final DO concentrations of 3.09 and 6.29 mg/L, are outliers and not used.

Sample(C8-39)

mLof

100x

Bottle number

Dilution(%)

SeedVolume (mL)

Sample Volume (mL)

Diluiton (P-value)

DO initial (mg/L)

DO final

(mg/L)

BOD6

values(mg/L)

Avg.BOD6

(mg/L)

BOD6

SD(mg/L)

8-12 ppm 3 25 ---- 10 287 0.957 4.95 0.89 4.198-12 ppm 3 26 ---- 10 287 0.957 4.97 0.79 4.31 4.31 0.138-12 ppm 3 27 ---- 10 287 0.957 4.94 0.64 4.448-12 ppm 3 28 ---- 20 277 0.923 5.23 1.72 3.968-12 ppm 3 29 ---- 20 277 0.923 5.23 3.09 2.47 4.00 0.058-12 ppm 3 30 ---- 20 277 0.923 5.25 1.67 4.038-12 ppm 3 31 50 10 143.5 0.478 7.29 6.29 2.048-12 ppm 3 32 50 10 143.5 0.478 7.30 4.63 5.53 5.69 0.248-12 ppm 3 33 50 10 143.5 0.478 7.36 4.53 5.868-12 ppm 3 34 50 20 138.5 0.462 7.49 4.99 5.578-12 ppm 3 35 50 20 138.5 0.462 7.51 5.16 5.25 5.59 0.358-12 ppm 3 36 50 20 138.5 0.462 7.50 4.83 5.94seed ctrl. ---- 37 ---- 10 290 0.967 8.42 8.37 0.05seed ctrl. ---- 38 ---- 10 290 0.967 8.41 8.35 0.06 0.06 0.01seed ctrl. ---- 39 ---- 10 290 0.967 8.43 8.38 0.05seed ctrl. ---- 40 ---- 20 280 0.933 8.31 8.50 -0.20seed ctrl. ---- 41 ---- 20 280 0.933 8.36 8.52 -0.17 -0.16 0.05seed ctrl. ---- 42 ---- 20 280 0.933 8.49 8.58 -0.10BOD std. ---- 43 ---- 10 2 0.007 8.59 5.41 477.0BOD std. ---- 44 ---- 10 2 0.007 8.47 5.41 459.0BOD std. ---- 45 ---- 20 2 0.007 8.58 5.40 477.0BOD std. ---- 46 ---- 20 2 0.007 8.57 5.42 472.5DW blank ---- 47 ---- ---- 300 1.000 8.64 8.51 0.13DW blank ---- 48 ---- ---- 300 1.000 8.59 8.50 0.09 0.10 0.02DW blank ---- 135 ---- ---- 300 1.000 8.59 8.50 0.09

12.7468.0

3.2474.8

40

Figure 5.2 Effects of dilution and inoculum volume on the BOD6 of contaminated groundwater (C8-39, 8-12 ppm).

of dilution and inoculum volume on the BOD

Error bars indicate ± 1 standard deviation Error bars indicate ± 1 standard deviation

5.1.3 Preliminary 20-day BOD Test for BOD Method 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

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

6 of contaminated groundwater (C8-39, 8-12 ppm).

0

110 mL seed

20 mL seed

2

3

4

5

6

7B

OD

6 (m

g/L)

50% FS 20 mL seed

FS 10 mL seed

50%

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 BOD5 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)

mLof

100x

Dilution(%)

SeedVolume (mL)

Sample Volume

(mL)

Dilution(P -value)

DO initial (mg/L)

DO final

(mg/L)

BOD20

values(mg/L)

Ave.BOD20

(mg/L)

BOD20

SD(mg/L)

C8-39 3 -- 10 287 0.957 4.85 0.02 4.50C8-39 3 -- 10 287 0.957 4.68 0.03 4.31 4.38 0.10C8-39 3 -- 10 287 0.957 4.70 0.03 4.33C8-39 3 -- 20 277 0.923 4.97 0.10 4.58C8-39 3 -- 20 277 0.923 5.12 0.02 4.83 4.78 0.18C8-39 3 -- 20 277 0.923 5.23 0.03 4.94C8-39 3 50 10 143.5 0.478 7.02 0.03 14.06C8-39 3 50 10 143.5 0.478 7.11 0.03 14.25 14.11 0.12C8-39 3 50 10 143.5 0.478 7.00 0.03 14.02C8-39 3 50 20 138.5 0.462 6.65 0.03 13.64C8-39 3 50 20 138.5 0.462 6.79 0.03 13.95 13.85 0.18C8-39 3 50 20 138.5 0.462 6.80 0.03 13.97

Seed Ctrl. -- -- 10 290 0.967 8.30 7.76 0.56Seed Ctrl. -- -- 10 290 0.967 8.34 7.80 0.56 0.55 0.02Seed Ctrl. -- -- 10 290 0.967 8.17 7.66 0.53Seed Ctrl. -- -- 20 280 0.933 8.23 7.56 0.72Seed Ctrl. -- -- 20 280 0.933 8.30 7.65 0.70 0.70 0.02Seed Ctrl. -- -- 20 280 0.933 8.32 7.69 0.68BOD Std. -- -- 10 2 0.007 8.25 4.40 577.5BOD Std. -- -- 10 2 0.007 8.28 4.43 577.5BOD Std. -- -- 20 2 0.007 8.23 4.33 585.0BOD Std. -- -- 20 2 0.007 8.32 4.40 588.0DW Blank -- -- -- 300 1.000 8.35 7.94 0.41DW Blank -- -- -- 300 1.000 8.41 7.95 0.46 0.45 0.04DW Blank -- -- -- 300 1.000 8.42 7.93 0.49

577.5

586.5

0.00

2.12

>>>>>>>>>>>>

>

>

>

>

43

Figure 5.3 Guadalupe Restoration Project, Diluent Tanks(Lundegard, 2002).

44

Not to scale

area

Figure 5.4 Detail of sampled monitoring well locations (Lundegard, 2002).

45

BOD5 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 BOD5 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, BOD5 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 BOD5 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 BOD5 results within 15% of each

other. Similarly, 25% and 50% diluted H2-1 samples had average BOD5

values within 3% of each other and 10% and 25% diluted M4-4 samples

had average BOD5 values within 10% each other. These results show

consistently that the use of dilution does not significantly affect the

measured BOD5 of the groundwater samples (Figure 5.5).

46

Table 5.4 BOD5 data for BOD method testing on series of groundwater samples Wells 206-C, 209-E and M4-4 at 50% depleted all O2 and are repeated later.

SampleTPH

Conc.(ppm)

mLof

100x

Dilution(%)

SeedVol.

(mL)

Sample Vol. (mL)

Dilution (P-value)

D.O. initial (mg/L)

D.O. final

(mg/L)

BOD5

values(mg/L)

Ave.BOD5

(mg/L)

BOD5

SD(mg/L)

G4-3 4.2 3 -- 10 287 0.957 9.90 6.57 3.55G4-3 4.2 3 -- 10 287 0.957 9.92 7.06 3.06 3.43 0.33G4-3 4.2 3 -- 10 287 0.957 9.82 6.35 3.70206-C 5.7 3 -- 10 287 0.957 7.83 0.30 7.94206-C 5.7 3 -- 10 287 0.957 7.78 2.24 5.86 6.99 1.05206-C 5.7 3 -- 10 287 0.957 7.67 0.87 7.18209-D 7.5 3 -- 10 287 0.957 8.79 6.88 2.07209-D 7.5 3 -- 10 287 0.957 8.85 6.89 2.12 2.08 0.03209-D 7.5 3 -- 10 287 0.957 8.84 6.94 2.06209-E 8.2 3 -- 10 287 0.957 8.56 0.05 8.96209-E 8.2 3 -- 10 287 0.957 8.55 0.06 8.94 8.95 0.01209-E 8.2 3 -- 10 287 0.957 8.56 0.06 8.95H3-7 11.0 0.75 25 10 72.31 0.241 8.14 6.63 6.33H3-7 11.0 0.75 25 10 72.31 0.241 8.15 6.91 5.21 5.66 0.60H3-7 11.0 0.75 25 10 72.31 0.241 8.15 6.86 5.42H3-7 11.0 1.5 50 10 144.25 0.481 8.01 5.87 4.52H3-7 11.0 1.5 50 10 144.25 0.481 8.06 5.68 5.02 4.82 0.26H3-7 11.0 1.5 50 10 144.25 0.481 8.07 5.74 4.91H2-1 13.0 0.75 25 10 72.31 0.241 7.86 5.94 8.03H2-1 13.0 0.75 25 10 72.31 0.241 7.94 6.55 5.84 6.79 1.13H2-1 13.0 0.75 25 10 72.31 0.241 7.94 6.39 6.50H2-1 13.0 1.5 50 10 144.25 0.481 7.54 4.46 6.47H2-1 13.0 1.5 50 10 144.25 0.481 7.58 4.57 6.33 6.61 0.36H2-1 13.0 1.5 50 10 144.25 0.481 7.59 4.25 7.02M4-4 29.0 0.3 10 10 28.97 0.097 8.19 6.79 14.57M4-4 29.0 0.3 10 10 28.97 0.097 8.19 6.82 14.26 14.39 0.16M4-4 29.0 0.3 10 10 28.97 0.097 8.20 6.82 14.36M4-4 29.0 0.75 25 10 72.31 0.241 7.81 3.92 16.21M4-4 29.0 0.75 25 10 72.31 0.241 7.81 4.12 15.38 15.74 0.43M4-4 29.0 0.75 25 10 72.31 0.241 7.85 4.10 15.63M4-4 29.0 1.5 50 10 144.25 0.481 6.92 0.04 14.38M4-4 29.0 1.5 50 10 144.25 0.481 6.92 0.04 14.38 14.39 0.02M4-4 29.0 1.5 50 10 144.25 0.481 6.96 0.06 14.42

Seed Ctrl. -- -- -- 10 290 0.967 8.34 8.40 -0.06Seed Ctrl. -- -- -- 10 290 0.967 8.35 8.44 -0.09 -0.07 0.02Seed Ctrl. -- -- -- 10 290 0.967 8.35 8.40 -0.05BOD Std. -- -- -- 10 2 0.007 8.41 6.29 318.0BOD Std. -- -- -- 10 2 0.007 8.43 6.60 274.5DW Blank -- -- -- -- 300 1.000 8.70 8.59 0.11DW Blank -- -- -- -- 300 1.000 8.72 8.61 0.11 0.11 0.01DW Blank -- -- -- -- 300 1.000 8.72 8.60 0.12

296.3 30.8

>>>

>>>

>>>

>

>

>

>>>

47

***mL of 100x is for inoculum/nutr ient addit ion

G4-3 206-C 209-D 209-E H3-7 H3-7 H2-1 H2-1 M4-4 M4-4 M4-40

2

4

6

8

10

12

14

16

18

Ave

rage

BO

D5

(mg/

L)

FS

25%

FS

FS

FS

50%

25%

50%

10%

25%

50%

Figure 5.5 Effect of sample dilution on BOD5 determination for several groundwater samples.

Error bars indicate ± 1 standard deviaton.

48

49

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 BOD5 through dilution (Table

5.5). Seed control blanks and BOD5 standard samples (GGA) performed

as expected. The dilution water blanks had an average BOD5 of -0.09

mg/L ± 0.03 mg/L (Table 5.5).

SampleTPH Conc.(ppm)

mLof

100x

Dilution(%)

SeedVol.

(mL)

Sample Vol. (mL)

Dilution(P-value)

DO initial (mg/L)

DO final

(mg/L)

BOD5

values(mg/L)

Avg.BOD5

(mg/L)

BOD5

SD(mg/L)

209-E 8.2 1.5 50 10 144.25 0.481 8.32 1.95 12.14209-E 8.2 1.5 50 10 144.25 0.481 8.32 1.50 13.08 12.93 0.72209-E 8.2 1.5 50 10 144.25 0.481 8.28 1.23 13.56206-C 5.7 1.5 50 10 144.25 0.481 7.19 2.95 7.71206-C 5.7 1.5 50 10 144.25 0.481 8.03 2.88 9.61 9.71 1.16206-C 5.7 1.5 50 10 144.25 0.481 8.06 2.81 9.82

Seed Ctrl. ---- 1.5 ---- 10 290 0.967 8.34 7.26 1.12Seed Ctrl. ---- 1.5 ---- 10 290 0.967 8.37 7.24 1.17 1.10 0.07Seed Ctrl. ---- 1.5 ---- 10 290 0.967 8.35 7.36 1.02BOD Std. ---- 3 ---- 10 2 0.007 8.35 5.62 409.5BOD Std. ---- 3 ---- 10 2 0.007 8.34 5.63 406.5DW Blank ---- ---- ---- ---- 300 1.000 8.53 8.66 -0.13DW Blank ---- ---- ---- ---- 300 1.000 8.53 8.61 -0.08 -0.09 0.03DW Blank ---- ---- ---- ---- 300 1.000 8.53 8.60 -0.07

408 2

Table 5.5 BOD data for BOD5 repeat analysis of oxygen depleted samples 209-E and 206-C. For well 206-C, one BOD5 value was nearly two standard deviations off.

50

5.2 COD Results 5.2.1 Iron Oxidation Effects on COD Measurement and Dilution Effects

The aged 2 ppm TPH groundwater sample with Fe2 + added [40 mg/L FeSO4]

exhibited an average COD value of 34.50 ± 0.82 mg/L, while the same

groundwater sample without Fe2 + added had an average COD value of 35.98 ±

1.49 mg/L (Table 5.6 and Figure 5.6). These values are within experimental

error, suggesting COD exerted by the oxidation of Fe2 + to Fe3 + is negligible.

The 50% diluted samples had an average COD value of 15.28 mg/L, with a

standard deviation of 0.71 mg/L (Table 5.6). This is approximately half the

COD value of the full strength groundwater, as expected.

The calibration curve from the KHP standards used to convert absorbance to

COD yielded R2 = 0.96 (Figure 4.2).

52

Table 5.6 Iron oxidation COD data. The last value for a blank is an outlier.

Figure 5.6 Effect of iron oxidation on COD measured.

Sample Absorbanceat 345 nm

COD(mg/L)

Avg. CODmg/L

SD(mg/L)

FS w/Fe2+ 0.122 35.26FS w/Fe2+ 0.131 34.60 34.50 0.82FS w/Fe2+ 0.144 33.63FS no Fe2+ 0.131 34.60FS no Fe2+ 0.091 37.55 35.98 1.49FS no Fe2+ 0.115 35.7850% diluted 0.394 15.1650% diluted 0.401 14.64 15.28 0.7150% diluted 0.382 16.04

Blank 0.631 1.17Blank 0.637 0.73 0.95 0.31Blank 0.577 5.16

0

5

10

15

20

25

30

35

40

COD SamplesError bars indicate ± 1 standard deviation

CO

D (m

g/L)

w/ Fe2+ w/o Fe2+

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

COD(mg/L)

Avg. COD(mg/L)

COD SD(mg/L)

G4-3 4.2 61.8G4-3 4.2 63.5 62.6 0.85G4-3 4.2 62.6206-C 5.7 86.0206-C 5.7 89.4 89.1 2.98206-C 5.7 91.9209-D 7.5 129209-D 7.5 126 128 1.53209-D 7.5 128209-E 8.2 145209-E 8.2 144 146 2.18209-E 8.2 148H3-7 11.0 159H3-7 11.0 157 157 2.34H3-7 11.0 155H2-1 13.0 257H2-1 13.0 252 255 2.83H2-1 13.0 257M4-4 29.0 258M4-4 29.0 258 258 0.00M4-4 29.0 258

DI Blank ---- 6.48DI Blank ---- 6.48 7.19 1.23DI Blank ---- 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)

Avg.COD(mg/L)

COD SD(mg/L)

G4-3 4.2 75.9G4-3 4.2 66.0 70.4 5.0G4-3 4.2 69.3206-C 5.7 135.3206-C 5.7 82.5 110.0 26.5206-C 5.7 112.2209-D 7.5 138.5209-D 7.5 141.8 140.7 1.9209-D 7.5 141.8209-E 8.2 161.6209-E 8.2 171.5 163.8 6.9209-E 8.2 158.3H3-7 11.0 207.8H3-7 11.0 164.9 182.5 22.5H3-7 11.0 174.8H2-1 13.0 296.9H2-1 13.0 300.2 309.0 18.2H2-1 13.0 329.9M4-4 29.0 333.2M4-4 29.0 329.9 331.0 1.9M4-4 29.0 329.9

DI H2O ---- 21.5DI H2O ---- 18.2 18.2 3.3DI H2O ---- 14.9

55

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

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

Sample TPH(mg/L)

COD(20-900 mg/L)

COD(5-150mg/L)

G4-3 4.2 70.4 62.6206-C 5.7 110.0 89.1209-D 7.5 140.7 128.1209-E 8.2 163.8 146.0H3-7 11.0 182.5 157.2H2-1 13.0 309.0 255.4M4-4 29.0 331.0 257.7

out of range

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

http://www.cartage.org.lb/en/themes/Sciences/Chemistry/Miscellenous/Helpfile/Erroranalysis/Average/Average.htm

http://www.cartage.org.lb/en/themes/Sciences/Chemistry/Miscellenous/Helpfile/Erroranalysis/StandardDeviation/StandardDeviation.htm

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)

St o t /4.65 = sqrt(0.000896 + 0.000296 + 0.00139)

St o t /4.65 = 0.0508

St o t = 0.236

Since we only report error to 1 significant figure, the answer to this

problem would be 4.7+-0.2

58

Table 5.10 Final results of COD, BOD and calculated BOD/COD ratios for groundwater series. SD and RSD indicate standard deviation and relative standard deviation, respectively.

Sample TPH(mg/L)

Avg. COD(mg/L)

COD SD(mg/L)

COD RSD BOD(mg/L)

BOD SD(mg/L)

BOD RSD BODCOD

BODCODRSD

G4-3 4.2 70.4 5.04 0.07 3.4 0.33 0.10 0.049 0.121206-C 5.7 110.0 26.46 0.24 9.7 1.16 0.12 0.088 0.269209-D 7.5 140.7 1.90 0.01 2.1 0.03 0.02 0.015 0.021209-E 8.2 163.8 6.87 0.04 12.9 0.72 0.06 0.079 0.070H3-7 11.0 182.5 22.45 0.12 4.8 0.26 0.05 0.026 0.135H2-1 13.0 309.0 18.17 0.06 6.6 0.36 0.05 0.021 0.080M4-4 29.0 331.0 1.90 0.01 15.7 0.43 0.03 0.048 0.028

59

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 R2 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).

Figure 5.7 COD vs. TPH plot for series of groundwater sError bars indicate ± 1 standard deviation.

5.3.2 Correlation of BOD with TPH

Measured BOD5 of the groundwater samples did not correlate

TPH concentration. The slope for BOD vs. TPH has a value o

BOD per mg TPH, with an R2 value of only 0.41 (Figure 5

average BOD/TPH ratio is 0.73 ± 0.35. This ratio is much lowe

ThOD of 3.5 mg/L, but this is to be expected since the TP

y = 10.10x + 73R2 = 0.74

0

50

100

150

200

250

300

350

400

0 5 10 15 20 25TPH concentration (ppm)

CO

D (m

g/L)

60

M4-4

H2-1

H3-7
.33
209-D

209-E

G4-3
206-C
amples.

well with

f 0.39 mg

.8). The

r than the

H is only

30

partially biodegraded in 5 days. Well 209-D has a very low

biodegradation rate (Figure 5.8).

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 R2 value of only 0.03

(Figure 5.9). Biodegradability (BOD/COD values, Table 5.10) does not

y = 0.39x + 3.50R2 = 0.42

0

2

4

6

8

10

12

14

16

18

0 5 10 15 20 25 30TPH concentration (ppm)

BO

D (m

g/L)

G4-3

209-E

206-C

M4-4

H2-1

H3-7

209-D

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).

Figure 5.9 BOD

A trend of dec

source zone wa

Tanks (DT) area

ratio decreased

plume the BOD/

about 650 ft .

together and lit

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0

Bio

degr

adab

ility

(BO

D/C

OD

)

WEATHERED FRESH

y = -0.0006x + 0.0527R2 = 0.03

209-D

/COD vs. TPH plot for series of groundwater samplError bars indicate ± 1 standard deviation.

reasing BOD/COD ratio with increasing distance f

s observed for three separate plumes from the Dil

(Figure 5.10). For the northern DT plume the BOD/C

by a factor of two over 200 ft , and for the southern

COD ratio decreased by a factor of six over a distanc

The two wells in the central DT plume were very c

tle difference in biodegradability was observed (Fi

5 10 15 20 25

TPH concentration (ppm)

62

M4-4

H2-1

H3-7

209-E

G4-3

206-C

es.

rom

uent

OD

DT

e of

lose

gure

30

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

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.1

0 200 400 600Distance from sour

Bio

degr

adab

ility

(BO

D/C

OD

)

209-E, 8.2 ppm

206-C, 5.7 ppm

G4-3, 4.2 ppm

H3-7, 11 ppm

H2-1, 13 ppm

Northern DT

Central DT

Figure 5.10 BOD/COD vs. distance down plum series of groundwater samples gro number and TPH concentration ar point on the graph.

64

DT = Diluent Tanks

800 1000 1200ce (ft.)

209-D, 7.5 ppm

Southern DT

e from source plot for uped by location. Well e indicated for each

CHAPTER 6

DISCUSSION

6.1 Reliability of BOD Tests

O2 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 BOD5 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 BOD6 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 BOD5 increases with the dilution factor is

indicative of sample toxicity. Thus toxic samples with a high dilution

67

factor will give a high BOD5 value because of diluted toxicity effects,

while less dilute samples will give a lower BOD5 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 R2 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 Fe2 + [40 mg/L FeSO4] (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 l ikely 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 CO2 production from groundwater samples taken

from the biosparge unit at the GRP showed high CO2 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 O2/mg TPH (Table 5.10).

The ThOD of hydrocarbons is approximately 3.5 mg O2/mg TPH. The

stoichiometric relation for the ThOD of hexane is given in Eqn. 4.

Calculation of ThOD for hexane:

Generic hydrocarbon: C6H1 4 Molecular Weight: 86 g/mol

C6H14 + 19/2O2 → 6CO2 + 7H2O (4)

(9.5 mol O2) * (32 g/mol O2) = 304 g O2

70

(304g O2) / (86 g/mol)

ThOD = 3.53 g O2/ 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 litt le 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

fi ltration. 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 O2/mg TPH. This was also run to test

the COD method and calculations. The average COD value of 0.95 mg

O2/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 O2/mg phenol for all

samples analyzed, with an average of 0.95 mg O2/mg phenol (Table 6.1).

A COD vs. phenol bar graph is shown in Figure 6.1.

Table 6.1 COD of phenol solutions

Samplephenol conc.

(mg/L)

Absorbanceat 600 nm

COD(mg/L)

mg Phenol

DI Blank 0.016 ---- ----DI Blank 0.015 ---- ----DI Blank 0.018 ---- ----

150 0.06 144.05 0.960150 0.059 140.75 0.938150 0.058 137.45 0.916300 0.103 285.90 0.953300 0.104 289.19 0.964300 0.104 289.19 0.964

73

Figure 6.1 COD vs. phenol concentration for standard phenol solutions.

0

50

100

150

200

250

300

350

150Phenol concentration (mg/L)

error bars indicate ± one standard deviation

CO

D (m

g/L)

150 300

COD/phenol = 0.96

COD/phenol = 0.94

74

CHAPTER 7

CONCLUSIONS

The range of BOD/COD vales for this project were 0.01 to 0.09,

suggesting slow biodegradation. In 1987, Gilbert stated a

BOD/COD value below 0.4 suggests a low biodegradability.

BOD/COD ratios did not correlate with increasing TPH

concentration suggesting weathering did not significantly influence

biodegradability. This also may indicate the contaminant is not

becoming recalcitrant during biodegradation.

BOD/COD ratios suggest biodegradability may decrease with

distance down-plume from source. A limited number of wells were

used and more well should be used for further analysis.

Methods were successfully demonstrated for BOD and COD. Tests

did not have any significantly unusual occurrences.

COD/TPH values ranged from 11.4 to 23.8, with an average of 18.1.

This average value is approximately five times higher than the

expected value of 3.5 mg/L based on ThOD. This may mean TPH

values should be evaluated at the beginning and end of an

experiment.

COD values may have been high due to the presence other

oxidizable organics.

The biodegradability decreased with distance down-plume from

source, possibly signifying a decrease in biodegradability as the

hydrocarbons are weathered.

75

CHAPTER 8

RECOMMENDATIONS

The following are recommendations for future evaluation of the

biodegradability of hydrocarbon-contaminated groundwater.

It would be beneficial to run TPH analysis with more than one

sample, and having multiple independent lab analysis. This would

put to rest any uncertainty in the reported TPH values.

Measuring actual TPH degradation rates using duplicate initial and

final TPH analyses would be preferable to relying on O2

consumption or respiration rates.

Biodegradability should be estimated for a greater number of wells

with some actual plume transects.

Using TOC as a supplement to the testing methods would help in

obtaining a better understanding of the organic fraction of the

samples.

The use of a more precise method of measuring D.O. would be

beneficial in obtaining BOD values.

76

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