chlordane and dieldrin contamination and ......in the lab. also, i would like to thank my...
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CHLORDANE AND DIELDRIN CONTAMINATION AND BIODEGRADATION IN
ALA WAI CANAL SEDIMENT
A THESIS SUBMITTED TO THE GRADUATE DIVISION OF THE
UNIVERSITY OF HAWAIʻI AT MĀNOA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE
IN
CIVIL ENGINEERING
MAY 2012
By
Cindy M.Y. Lee
Thesis Committee:
Tao Yan, Chairperson
Roger Babcock
Michelle Teng
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ACKNOWLEDGEMENTS
This thesis would have not been possible without the encouragement, guidance, advice,
and assistance of several individuals. First, I would like to thank my advisor, Dr. Tao
Yan for his mentorship and involvement with this thesis. I would like to thank my
Graduate Committee members, Dr. Roger Babcock and Dr. Michelle Teng for their
valuable advice about my thesis. I would like to thank Mr. Joseph Lichwa and Ms.
Bunnie Yoneyama for their assistance with the laboratory equipment. I would like to
thank the current and previous members of Dr. Yan’s research group for their assistance
in the lab. Also, I would like to thank my supervisors and co-workers at Pearl Harbor
Naval Shipyard & IMF for their encouragement and advice. Finally, I would like to
thank my parents and brothers for their assistance and constant support.
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TABLE OF CONTENTS
Acknowledgements…………………………………………………………………… i
List of Tables…………………………………………………………………………. iv
List of Figures………………………………………………………………………… v
CHAPTER 1 INTRODUCTION……………………………………………………... 1
1.1 Chlordane and Dieldrin Contamination………………………………………. 1
1.2 Urbanization and Stream Modifications……………………………………… 2
1.3 Evaluating Potential Effects Associated with Locations Contaminated with
Chlordane and Dieldrin……………………………………………………….. 3
1.4 Treatment Efforts for Locations Contaminated with Chlordane and Dieldrin.. 4
1.5 Objectives……………………………………………………………………... 5
CHAPTER 2 HORIZONTAL AND VERTICAL DISTRIBUTION OF
CHLORDANE AND DIELDRIN IN ALA WAI CANAL SEDIMENT…………….. 6
2.1 Introduction…………………………………………………………………… 6
2.2 Methods……………………………………………………………………….. 8
2.2.1 Sample site and Collection……………………………………………… 8
2.2.2 Laboratory Analysis…………………………………………………….. 10
2.2.2.1 Sample Preparation……………………………………………... 10
2.2.2.2 Extraction……………………………………………………….. 11
2.2.2.3 Cleanup and Concentration……………………………………... 12
2.2.2.4 Calibration and Analysis………………………………………... 13
2.3 Results………………………………………………………………………… 16
2.3.1 Concentration Profiles of Sediment Cores……………………………… 16
2.3.2 Comparison of the Concentration of Surface Sediments to Aquatic Life
Protection Guidelines…………………………………………………… 18
2.4 Discussion…………………………………………………………………….. 20
CHAPTER 3 DETERMINE WHETHER OR NOT CHLORDANE CAN BE
BIODEGRADED IN ALA WAI CANAL SEDIMENT……………………………... 25
3.1.Introduction…………………………………………………………………… 25
3.2.Methods……………………………………………………………………….. 27
3.2.1 Sample Site and Collection……………………………………………... 27
3.2.2 Culture Preparation and Sampling……………………………………… 28
3.2.3 Extraction and Cleanup…………………………………………………. 29
3.2.4 Calibration and Analysis………………………………………………... 30
3.3 Results………………………………………………………………………... 33
3.4 Discussion…………………………………………………………………….. 34
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CHAPTER 4 CONCLUSIONS AND RECOMMENDATIONS…………………… 35
4.1 Conclusions…………………………………………………………………... 35
4.2 Recommendations……………………………………………………………. 36
Appendix A…………………………………………………………………………... 39
Appendix B…………………………………………………………………………... 43
Appendix C…………………………………………………………………………... 44
References……………………………………………………………………………. 67
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LIST OF TABLES
Table 1.1: Types of Stream Modifications…………………………………………... 3
Table 2.1: Summary of Analytical Methods………………………………………… 10
Table 3.1: Set Up of the Laboratory Microcosm Experiments………………………. 28
Table A.1: Dry Weights of Sediment Collected for Set A…………………………... 39
Table A.2: Dry Weights of Sediment Collected for Set B…………………………... 40
Table A.3: Concentration, % Moisture, and % Recovery of Sediment Collected for
Set A…………………………………………………………………………………. 41
Table A.4: Concentration, % Moisture, and % Recovery of Sediment Collected for
Set B…………………………………………………………………………………. 42
Table B.1: Years Structures Built in the Pālolo, Mānoa, McCully/Mōʻiliʻili, and
Makiki Areas………………………………………………………………………… 43
Table B.2: Number of Units in Structures in the Pālolo, Mānoa, McCully/Mōʻiliʻili,
and Makiki Areas…………………………………………………………………….. 43
Table C.1: Chlordane Concentration of Samples Taken from Microcosms…………. 44
Table C.2: pH of Samples Taken from Microcosms………………………………… 55
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LIST OF FIGURES
Figure 2.1: Areas Surrounding the Ala Wai Canal…………………………………... 9
Figure 2.2: Sediment Core Collection Sites…………………………………………. 10
Figure 2.4: Site 1 Concentration Profile……………………………………………... 16
Figure 2.5: Site 2 Concentration Profile……………………………………………... 16
Figure 2.6: Site 3 Concentration Profile……………………………………………... 17
Figure 2.7: Site 4 Concentration Profile……………………………………………... 17
Figure 2.8: Site 5 Concentration Profile……………………………………………... 17
Figure 2.9: Chlordane Concentration at the Surface for Each Site and the Probable
Effect Level (PEL) of Chlordane…………………………………………………….. 19
Figure 2.10: Dieldrin Concentration at the Surface for Each Site and the Probable
Effect Level (PEL) of Dieldrin………………………………………………………. 19
Figure 3.1: Sediment Core Collection Site…………………………………………... 27
Figure 3.2: Decachlorobipheyl Calibration Curve…………………………………... 31
Figure 3.3: Chlordane Calibration Curve……………………………………………. 32
Figure 3.4: Chlordane Concentration of Samples Taken from Microcosms………… 33
Figure C.1: pH of Samples Taken from Microcosms………………………………... 46
Figure C.2: Chromatographs of Samples Taken on January 14, 2012………………. 47
Figure C.3: Chromatographs of Samples Taken on February 4, 2012………………. 52
Figure C.4: Chromatographs of Samples Taken on March 10, 2012………………... 57
Figure C.5: Chromatographs of Samples Taken on March 31, 2012………………... 62
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CHAPTER 1 INTRODUCTION
1.1 Chlordane and Dieldrin Contamination
Chlordane and dieldrin are organochlorine pesticides, chlorine-containing
compounds, that were used worldwide for residential and agriculture applications to
control termites and other insects. Chlordane was produced in 1947 and was sold as an
insecticide for agricultural applications and termiticide for underground treatment around
the foundation of structures [30, 33]. Dieldrin was produced in 1948 and was also sold as
an insecticide and termiticide [27, 32].
Chlordane and dieldrin were banned in the 1970s for agricultural applications and
1980s for all other uses due to the harmful effects on aquatic life, wildlife, and humans
[4]. Chlordane and dieldrin are persistent, toxic, and can bioaccumulate in the food chain
[27, 30]. Even when chlordane and dieldrin are no longer used, bottom sediments and
marine biota display significant concentrations of organochlorine compounds since
organochlorine compounds take years to deplete from the environment [4].
Organochlorine compounds are commonly transported to water sources through soil
erosion and runoff [4]. Organochlorine compounds are hydrophobic, do not dissolve well
in water and bind strongly to sediment, making the compounds hard to deplete from the
environment [4].
Sediments are analyzed for organochlorine compounds since “sediment serves as
both a source and a removal mechanism for contaminants to and from the stream, and as
a means of contaminant transport downstream” [4]. Sediment also provides a habitat for
benthos [4]. Benthos ingest the organochlorine compounds in bottom sediments and fish
and birds then eat the benthos, which leads to the accumulation of organochlorine
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compounds in the fatty tissues of species in the food chain [4]. Also, marine biota are
analyzed to determine the presence of organochlorine compounds and contamination
levels since the accumulation of organochlorine compounds are harmful to aquatic life
and to those who consume them [4]. Bottom sediments and marine biota contain higher
concentrations of organochlorine compounds than the surrounding water since
organochlorine compounds are hydrophobic and bioaccumulative, which increases the
chances of detecting organochlorine compounds when analyzing sediment and tissue [4].
1.2 Urbanization and Stream Modifications
As the population increases more land is used to make room for residential,
industrial, commercial, and recreation areas. Also, land is needed to built roads,
freeways, and highways for these areas. In order to have more land for these
development projects, streams are modified for these projects. Stream modifications
include lined channels, blocked or filled in channels, elevated culverts, extended culverts,
and revetments [18, 38]. Also, vegetation near the stream maybe removed for these
stream modifications. Clearing of land for construction near streams or removal of
streamside vegetation can promote soil erosion and increase runoff to streams, which
contributes to chlordane and dieldrin contamination.
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Table 1.1: Types of Stream Modifications [18, 38]
Type of Stream Modification Description
Lined Channel Artificial channel where the natural banks and stream
bed have been replaced, usually with concrete.
Blocked or Filled In Channel Part of the original channel is blocked.
Elevated Culvert Relatively short (usually less than 60 meters) conduit
structures that are usually found under highways. An
artificial waterfall is created with these structures
since they are built above the water level
immediately downstream.
Extended Culvert Similar to an elevated culvert but longer and usually
found in residential areas.
Revetment Either one or both sides of the banks are reinforced
but the channel isn’t reinforced.
Vegetation Removed –
Channel Realigned
The stream is realigned and the vegetation on the
banks is removed.
1.3 Evaluating Potential Effects Associated with Locations Contaminated with
Chlordane and Dieldrin
Guidelines have been established for chlordane and dieldrin to determine what
concentration of chlordane and dieldrin will cause adverse biological effects [4]. The
Canadian Sediment Quality Guidelines (CSQG) for aquatic life is used to determine if
aquatic life expose to chlordane and dieldrin in sediment are affected by these
contaminants [4, 31]. The CSQG for aquatic life were established by conducting
sediment toxicity tests on various aquatic life such as mollusks, shrimp, and sea urchin
and oyster larvae to assess the biological significance of contamination levels found in
sediments and comparing the tests to contamination levels found in sediments and data
pertaining to the characteristics of the sediments [31]. The probable effect level (PEL) is
the “level above which adverse effects are expected to occur frequently” meaning “more
than 50% adverse effects occurs” when aquatic life is exposed to sediment above this
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level [31]. The PEL is 8.87 µg/kg dry weight for chlordane and 6.67 µg/kg dry weight
for dieldrin [4, 31].
1.4 Treatment Efforts for Locations Contaminated with Chlordane and Dieldrin
Because chlordane and dieldrin are still found in the environment and a potential
health hazard, studies have been done to find ways to deplete chlordane and dieldrin.
Chlordane and dieldrin undergo aerobic or anaerobic biodegradation in the water column
and in the sediment, respectively. Aerobic biodegradation involves the breakdown of
chlordane and dieldrin by microbes when oxygen is present [25], whereas anaerobic
biodegradation occurs when non-oxygen electron acceptors are utilized [28]. Chlordane
and dieldrin are hydrophobic and have low solubility levels, making it hard for microbes
to get to these in the sediment so that they can be degraded. Aerobic degradation has
been reported by Cuozzo et al. (2012) for chlordane and Matsumoto et al. (2009) for
dieldrin [8, 13]. Anaerobic degradation has been reported by Hirano et al. (2007) for
chlordane and Chiu et al. for dieldrin (2005) [7, 10]. From the studies reported, suitable
conditions are needed for degradation of chlordane and dieldrin to occur, which depends
on the contamination levels, temperature, and carbon and energy source [2, 7, 8, 10, 13,
14, 20]. Therefore, by determining the degradation microbes that are available in the
environment and discovering the right conditions and then optimizing the conditions for
the microbes will help the microbes to degrade chlordane and dieldrin.
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1.5 Objectives
The objectives are (1) investigate the contamination levels of chlordane and
dieldrin both horizontally (i.e. at different locations of the Ala Wai Canal) and vertically
(i.e. at different depth in the sediment) in Ala Wai Canal sediment, and (2) determine
whether or not chlordane in Ala Wai Canal sediment can be biodegraded. Objective 1
was accomplished by collecting sediment core samples at various locations along the Ala
Wai Canal and the concentrations of chlordane and dieldrin were quantified for each
sampling location to formulate a concentration profile. Objective 2 was accomplished by
performing laboratory microcosm experiments with Ala Wai Canal sediment spiked with
chlordane.
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CHAPTER 2 HORIZONTAL AND VERTICAL DISTRIBUTION OF
CHLORDANE AND DIELDRIN IN ALA WAI CANAL SEDIMENT
2.1 Introduction
The Ala Wai Canal was constructed in 1921-1928 by Walter F. Dillingham’s
Hawaiian Dredging Company to drain the “mosquito infested wetlands” and to “create
land suitable for development into commercial and residential real estate” [15].
Apartments and condominiums surround the Ala Wai Canal and the banks of the Ala Wai
Canal are landscaped with coconut palms and flowers. The Ala Wai area is used for
recreation with bike paths, canoes, kayaks, a golf course, and an illuminated sidewalk for
running and walking. However, there are signs posted near the Ala Wai Canal informing
people not to swim in the Ala Wai Canal and that the fish and shellfish in the Ala Wai
Canal are contaminated. Three streams, Pālolo, Mānoa, and Makiki, were altered to
make room for residential, commercial, and recreation areas and transports trash and
contaminates that drains into the Ala Wai Canal. Due to a low flow rate, the Ala Wai
Canal cannot naturally filter and clean the water so pesticides, toxic chemicals, heavy
metals, and trash accumulate in the Ala Wai Canal [17].
As part of the U.S. Geological Survey’s National Water-Quality Assessment
(NAWQA) program, stream bed sediment and fish samples were collected during the fall
of 1998 from six streams on Oahu to determine the occurrence of organochlorine
pesticides [4]. One of the sampling sites was Mānoa Stream. The chlordane
concentration in the sediment was 300 µg/kg dry weight [4]. The chlordane
concentration in the fish tissue was 950 µg/kg wet weight and 700 µg/kg wet weight [4].
The dieldrin concentration in the sediment was 150 µg/kg dry weight [4]. The dieldrin
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concentration in the fish tissue was 850 µg/kg wet weight and 1250 µg/kg wet weight [4].
The concentration of chlordane and dieldrin in the sediment exceeded the Canadian
Sediment Quality Guidelines (CSQG) for the protection of aquatic life, 8.87 µg/kg for
chlordane and 6.67 µg/kg for dieldrin [4, 31]. The concentration of chlordane and
dieldrin in the fish tissue exceeded the New York State Department of Environmental
Conservation (NYSDEC) guidelines for the protection of fish eating birds and mammals,
500 µg/kg for chlordane and 120 µg/kg for dieldrin [4, 31]. Since the Mānoa Stream is
contaminated with chlordane and dieldrin, the Ala Wai Canal will also be contaminated
since the contents of the Mānoa Stream are transported to the Mānoa-Pālolo Drainage
Canal and then to the Ala Wai Canal.
There is a concern about chlordane and dieldrin because of the harmful effects to
humans, other species, and the environment. The fish and the shellfish in the Ala Wai
Canal are unsafe to eat and may have developed deformities due to living in a
contaminated environment and ingesting chlordane and dieldrin or eating benthos that
ingest chlordane and dieldrin in the sediment. When the Ala Wai Canal was dredged, a
Mantis shrimp (Odontodactylus Scyllarus) that was much larger than the normal size was
pulled from the sediment and weighted in at “1.35 pounds and 15 inches” [9].
Before the Ala Wai Canal was contaminated, people used to fish and catch
shellfish in the Ala Wai Canal. Today, most people know not to consume the fish and
shellfish in the Ala Wai Canal, but first-generation immigrants who may not speak or
speak little English, go to the Ala Wai Canal to catch crabs since they may not know the
hazards of consuming contaminated crabs from the Ala Wai Canal [9]. People continue
to use the canal for recreational purposes, in spite of the presence of contaminants.
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Therefore, it is important the concentration levels of harmful contaminants, such as
chlordane and dieldrin, are accurately determined. This can be accomplished by
analyzing sediment since from sediment, the sources of contamination and where these
contaminants are being transported can be determined and trends can be discovered on
how these contaminants are being degraded naturally. With the up-to-date concentration
data, especially if the levels are high and exceeds the aquatic life guideline, sound
regulatory actions and warning mechanisms can be put in place to warn people about the
harmful health effects of consuming contaminated fish and shellfish and the hazardous
potentials associated with paddling in the Ala Wai Canal.
This study is focused on investigating the contamination levels of chlordane and
dieldrin both horizontally (i.e. at different locations of the Ala Wai Canal) and vertically
(i.e. at different depth in the sediment) in Ala Wai Canal sediment. This was
accomplished by collecting sediment cores along and near the Ala Wai Canal and
analyzing the layers of the sediment core for chlordane and dieldrin.
2.2 Methods
2.2.1 Sample Site and Collection
The Ala Wai Canal is approximately 2 miles (3.22 km) long, that receives water
and sediment from the Mānoa-Pālolo Drainage Canal and Makiki Stream and empties to
the Ala Wai Harbor. Besides, collecting runoff from the Pālolo, Mānoa, and Makiki
areas, the Ala Wai Canal also collects storm water runoff from the Kapahulu,
McCully/Mōʻiliʻili, Ala Moana, and Waikīkī areas (Figure 2.1).
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Figure 2.1: Areas Surrounding the Ala Wai Canal [34]
Sediment samples were collected on November 24, 2010 and November 25, 2010
at five transects along or near the Ala Wai Canal (Figure 2.2). Each transect had two
sampling sites where a sediment core was collected. At each sample site there was a
floating dock used by paddlers. A sediment core was collected at opposite ends of the
floating dock, one on the right side and the other on the left side. Sediment cores 2
inches (5.08 cm) in diameter and 8 inches to 20 inches (20.32 cm – 50.8 cm) in length
were collected using an AMS multi-stage sludge and sediment core sampler (American
Falls, Idaho, U.S.A). The sediment cores were capped and placed in an ice cooler with
ice packs. The sediment cores were taken to the laboratory and were frozen in the freezer
at -20 °C.
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Figure 2.2: Sediment Core Collection Sites [34]
2.2.2 Laboratory Analysis
The samples were prepared, extracted, cleaned, concentrated, and analyzed per
Environmental Protection Agency (EPA) Methods (Table 2.1).
Table 2.1: Summary of Analytical Methods
Type of Analytical Method Method
Pressurized Fluid Extraction SW-846 Method 3545A
Florisil Cleanup SW-846 Method 3620C
Fraction Concentration Appendix A to Part 136 Method 608
Gas Chromatography SW-846 Method 8081B
2.2.2.1 Sample Preparation
The samples were prepared using SW-846 Method 3545A [37]. The frozen
sediment cores were thawed and then removed from the plastic liner. The sediment cores
were sectioned at 2 inch (5.08 cm) intervals using a clean stainless steel knife. Any water
ALA WAI CANAL
Mānoa-Pālolo Drainage Canal
Drainage Canal
Makiki Stream
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found was removed from the sediment sample and discarded. The sediment samples
were dried in a hood for 48 hours to 72 hours on hexane-rinsed aluminum foil. Foreign
objects such as twigs, rocks, rope, and fish bones were removed from the sediment
samples and discarded. The sediment samples were grinded using a ceramic mortar and
pestle and then strained through a strainer to remove any remaining foreign objects.
Approximately 5 grams of ground sediment was weighed for extraction. Each sample
that will be extracted was mixed with the surrogate, 2 mL of decachlorobiphenyl at 250
µg/L, and was set aside so that the spiking solvent, acetone, would evaporate. An
additional 5 grams of ground sediment was weighed into a tared aluminum crucible and
dried overnight at 103 °C to 105 °C to calculate the percent dry weight.
2.2.2.2 Extraction
The samples were extracted using SW-846 Method 3545A [37]. An extraction
cell was prepared for each spiked sediment sample. A 20 mm disposable glass fiber filter
was placed in the bottom of a 5 mL cell and then weighed. The spiked sediment sample
was added to the cell and then weighted. The difference of the cell with sample and the
empty cell was used to calculate the weight of spiked sediment sample. Drying agent,
diatomaceous earth, was added to fill up the cell and was mixed with the spiked sediment
sample with a clean stainless steel spatula.
The spiked sediment samples were extracted with acetone and hexane (1:1, v/v)
using a DIONEX ASE 200 Accelerated Solvent Extractor (Sunnyvale, California, U.S.A)
with the following operating conditions. The oven temperature was set to 100 °C and the
pressure was set to 1500 psi to 2000 psi. The static time was set to 5 minutes. The
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flushing volume was set to 60% of the cell volume. The cell was purged with nitrogen
and the purge time and pressure was set to 60 seconds and 150 psi.
After extraction, the volume of each extract was measured. The extracts were
stored in the freezer at -20 °C until cleanup.
2.2.2.3 Cleanup and Concentration
The samples were cleaned using SW-846 Method 3620C [37]. A column
containing 20 grams of activated Florisil and 20 grams of anhydrous sodium sulfate on
top of the Florisil was prepared for each cleanup. One-tenth (1/10) of the total extract
volume was used for cleanup. The columns were pre-eluted with 60 mL of hexane and
the elutes were discarded. The extract was added to the column and 1 mL of hexane was
added twice to rinse the column after the extract was added. The columns were eluted
with two fractions containing ethyl ether and hexane. Fraction 1 contained 200 mL of
ethyl ether and hexane (6:94, v/v). Fraction 2 contained 200 mL of ethyl ether and
hexane.
The factions were concentrated using Method 608 [24]. The fractions were
concentrated in a hood using a Kuderna-Danish (K-D) apparatus on a hot water bath and
then further concentrated in a hood using a nitrogen evaporator. Three or four boiling
chips were placed in the 500 mL K-D flask with a 10 mL concentrator tube. The fraction
was poured into the K-D flask with a concentrator tube and a three-ball Snyder column
was attached to the K-D flask. The Snyder column was pre-wetted with 1 mL of hexane.
The K-D apparatus was placed on a water bath set to 85 °C. The water temperature was
increased and the placement of K-D apparatus in the water bath was adjusted to complete
the concentration. When the volume reached to approximately 5 mL, the K-D apparatus
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was removed from the water bath. When the K-D apparatus was cooled, the Snyder
column was removed and the K-D flask was rinsed with hexane. The fraction was placed
in a 5 mL vial for further concentration. The fraction was concentrated to 1 mL using an
N-EVAP™
III Nitrogen Evaporator with an OA-SYS™
Heating System (Berlin,
Massachusetts, U.S.A). The 1 mL of fraction was placed in a 2 mL gas chromatography
(GC) vial containing a cap with silicone/PTFE septa. The volume of each fraction was
adjusted with 1 mL of hexane. The fractions were stored in the freezer at -20 °C until
analysis.
2.2.2.4 Calibration and Analysis
The concentration of chlordane and dieldrin in the samples were determined using
SW-846 Method 8081B [37]. Chlordane and dieldrin in sediment was analyzed using a
Hewlett Packard 5890 Series II Gas Chromatograph equipped with an electron capture
detector (GC-ECD) (Palo Alto, California, U.S.A) with the following operating
conditions. The oven temperature was initially held at 100 °C for 2 minutes, ramped
from 100 °C to 160 °C at 15 °C/min, and then ramped from 160 °C to 270 °C at 5
°C/min. The carrier gas was helium (ultra high purity) and the pressure was set to 60 psi.
The makeup gas for the detector was nitrogen and the pressure was set to 25 psi. The
detector temperature was set to 300 °C. The GC-ECD also had an Agilent GC
AutoSampler Controller and 6890 Series Injector (Santa Clara, California, U.S.A) to
inject 2 µL of sample. The injector temperature was set to 225 °C. Also, a J&W DB-
XLB column of 30 m × 0.533 mm × 150 µm size was used (Folsom, California, U.S.A).
Chlordane and dieldrin concentrations were quantified using calibration standards
and were corrected by surrogate recoveries, ranging from 0.45% to 84.62%, and percent
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moisture, ranging from 7.87% to 30.65%. Five calibration standards at concentrations of
50 µg/L, 150 µg/L, 250 µg/L, 350 µg/L, and 500 µg/L were prepared for both chlordane
and dieldrin. Also, six calibration standards at concentrations of 200 µg/L, 250 µg/L, 300
µg/L, 350 µg/L, 400 µg/L, and 450 µg/L were prepared for the surrogate,
decachlorobiphenyl. The retention time windows were established for each compound.
Chlordane had four peaks and had a retention time of approximately 26.4 minutes for
peak 1, 26.6 minutes for peak 2, 26.8 minutes for peak 3, and 27.0 minutes for peak 4.
For calculations, the sum of the area of the chlordane peaks was used. Dieldrin had one
peak and had a retention time of approximately 28.0 minutes. Decachlorobiphenyl had
one peak and had retention time of approximately 32.8 minutes. The calibration factor
(CF), mean calibration factor (mean CF), standard deviation (SD), and relative standard
deviation (RSD) were calculated for each compound using the equations presented in
equation (1) to (4).
If the RSD was greater than 20%, a new set of calibration standards were prepared. The
concentration of each compound was calculated using the equation presented in equation
(1)
(2)
(3)
(4)
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(5), where Ax is the area of the peak, Vt is the total volume of the concentrated extract
(µL), D is the dilution factor, is the mean calibration factor (area/ng), Vi is the volume
of the extract injected (µL), and Ws is the weight of sample extracted (g).
The surrogate recovery was calculated using the equation presented in equation (6).
The percent moisture was calculated using the equation presented in equation (7), where
Ws is the weight of sample (g) and Wds is the weight of dry sample (g).
The concentration of chlordane and dieldrin was corrected using the surrogate recovery
and percent moisture. The corrected concentration was calculated using the equation
presented in equation (8).
(5)
(6)
(7)
(8)
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2.3 Results
2.3.1 Concentration Profiles of Sediment Cores
The sediment cores were analyzed to determine the concentration of chlordane
and dieldrin. The concentration profiles for each sediment collection site are shown
below (Figure 2.4 – 2.8).
Figure 2.4: Site 1 Concentration Profile
Figure 2.5: Site 2 Concentration Profile
0
10
20
30
40
50
60
0 50 100 150 200
Sed
ime
nt
De
pth
(cm
)
Concentration (µg/kg dw)
Site 1 Chlordane Site 1 Dieldrin
0
10
20
30
40
50
60
0 50 100 150 200
Sed
ime
nt
De
pth
(cm
)
Concentration (µg/kg dw)
Site 2 Chlordane Site 2 Dieldrin
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Figure 2.6: Site 3 Concentration Profile
Figure 2.7: Site 4 Concentration Profile
Figure 2.8: Site 5 Concentration Profile
0
10
20
30
40
50
60
0 50 100 150 200
Sed
ime
nt
De
pth
(cm
)
Concentration (µg/kg dw)
Site 3 Chlordane Site 3 Dieldrin
0
10
20
30
40
50
60
0 50 100 150 200
Sed
ime
nt
De
pth
(cm
)
Concentration (µg/kg dw)
Site 4 Chlordane Site 4 Dieldrin
0
10
20
30
40
50
60
0 50 100 150 200
Sed
ime
nt
De
pth
(cm
)
Concentration (µg/kg dw)
Site 5 Chlordane Site 5 Dieldrin
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The concentration profiles showed two different trends. For sediment cores collected
along Mānoa-Pālolo Drainage Canal and between Mānoa-Pālolo Drainage Canal and the
Drainage Canal near Isenberg Street, site 1, 2, and 3, the concentration decreases with
depth with fluctuations. For sediment cores collected between the Drainage Canal near
Isenberg Street and the McCully Street Bridge, site 4 and 5, the concentration increases
with depth with fluctuations.
The concentration profiles are different due to the location and how sediment is
transported to these locations. For site 1, 2, and 3, the concentration decreases with
depth, so this could mean that chlordane and dieldrin is depleting from the environment
but is still entering the environment through soil erosion and runoff. For site 4 and 5, the
concentration increases with depth, so this could mean that degradation of chlordane and
dieldrin by microbes might be occurring on the surface or that sediment containing
chlordane and dieldrin have not been settling in that area recently.
2.3.2 Comparison of the Concentration of Surface Sediments to Aquatic Life Protection
Guidelines
The concentrations of chlordane and dieldrin were compared with the Canadian
Sediment Quality Guidelines (CSQG) for aquatic life to determine if chlordane and
dieldrin in Ala Wai Canal sediment are affecting aquatic life in the Ala Wai Canal. The
probable effect level (PEL) was 8.87 µg/kg dry weight for chlordane and 6.67 µg/kg dry
weight for dieldrin [4, 31].
The concentration of the sediments at the surface ranged from 19.06 µg/kg dry
weight to 169.26 µg/kg dry weight for chlordane and 2.13 µg/kg dry weight to 35.63
µg/kg dry weight for dieldrin. Plots comparing the concentration of chlordane and
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dieldrin in surface sediment to the probable effect level are shown below (Figure 2.9 and
2.10). At all the sites, the concentration of chlordane and dieldrin in surface sediment
exceeded the probable effect level, except for dieldrin at site 5.
Figure 2.9: Chlordane Concentration at the Surface for Each Site and the Probable
Effect Level (PEL) of Chlordane
Figure 2.10: Dieldrin Concentration at the Surface for Each Site and the Probable
Effect Level (PEL) of Dieldrin
0
20
40
60
80
100
120
140
160
180
200
Site 1 Site 2 Site 3 Site 4 Site 5
Co
nce
ntr
atio
n (
µg/
kg d
w)
Chlordane Conc.
Chlordane PEL
0
5
10
15
20
25
30
35
40
Site 1 Site 2 Site 3 Site 4 Site 5
Co
nce
ntr
atio
n (
µg/
kg d
w)
Dieldrin Conc.
Dieldrin PEL
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2.4 Discussion
The Ala Wai Canal is a basin where sediment is deposited. The sediment is
transported from streams and drainage canals that discharge into the Ala Wai Canal. The
Ala Wai Canal reduces the amount of sediment transported into the nearshore coastal
waters of Waikīkī, which serves as a recreation area for tourists and residents. Large
quantities of sediment are transported to the Ala Wai Canal through storm water runoff
from surrounding areas, particularly during periods of high intensity rainfall [3].
The concentration profiles showed two different trends. The concentration of
chlordane and dieldrin either decreases or increases with depth. When the concentration
decreases with depth, this could mean that chlordane and dieldrin that was introduced in
the environment before the 1970s has been depleting but is still entering the environment
through soil erosion and runoff after the 1970s. When the concentration increases with
depth, this could mean that degradation of chlordane and dieldrin by microbes might be
occurring on the surface or that sediment containing chlordane and dieldrin have not been
settling in that area recently. Where the sediment is transported from, how the sediment
is transported, and disturbance near or at the sampling site can contribute to this
difference.
Higher concentrations of chlordane and dieldrin at the surface where found at site
1, 2, and 3, along the Mānoa-Pālolo Drainage Canal and near the outlet of the Mānoa-
Pālolo Drainage Canal. Chlordane and dieldrin were both used as a termiticide before
being banned in 1988 for chlordane and in 1985 for dieldrin. In Hawaii, 50,000 pounds
of chlordane and approximately 9000 pounds of aldrin (degrades to dieldrin) was used in
1977 for pest control [4]. Chlordane and dieldrin are transported through soil erosion and
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runoff, so the areas that were treated for termites contain chlordane and dieldrin that can
be transported to water sources. Higher concentrations of chlordane and dieldrin were
found at these sites since the soil and sediment from the Pālolo and Mānoa areas contains
more chlordane and dieldrin than the McCully/Mōʻiliʻili and Makiki areas. The areas of
Pālolo and Mānoa contain many older homes that were treated for termites while the
areas of McCully/Mōʻiliʻili and Makiki contain apartments and businesses that were not
treated as often for termites. Based on a census conducted in 2000 on selected housing
characteristics, around 80% to 84% of the structures in the Pālolo, Mānoa,
McCully/Mōʻiliʻili, and Makiki areas were built in 1979 or earlier and 80% of the
structures in the McCully/Mōʻiliʻili area and 84% of the structures in the Makiki area
contained 5 or more units while 19% of the structures in the Pālolo area and 23% of the
structures in the Mānoa area contained 5 or more units [36].
Most of the sediment deposited in the Ala Wai Canal comes from the Mānoa-
Pālolo Drainage Canal. Sediment is transported to the Ala Wai Canal through water
containing suspended sediment particles that flow to Ala Wai Canal and then eventually
settle to the bottom of the Ala Wai Canal depending on the flow rate and the size of the
sediment particles. More sediment particles settle to the bottom if the flow rate of the
water is slow and the water contains large suspended particles [3]. At the outlet of the
Mānoa-Pālolo Drainage Canal the flow rate decreases since the water coming from the
Mānoa-Pālolo Drainage Canal is entering a wider and deeper area [3]. This reduces the
ability to carry more suspended sediment particles, which eventually settles out overtime
and results in an area accumulated with sediment [3]. Sediment particles, around the size
of a sand particle, were found at areas near the outlet of the Mānoa-Pālolo Drainage
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Canal [3]. The proportion of slit to sand also increased with distance away from the
outlet of the Mānoa-Pālolo Drainage Canal [3]. Also, a lot of slit was found in the
unlined section of the Mānoa-Pālolo Drainage Canal [3]. The Mānoa-Pālolo Drainage
Canal, site 2, and the areas near the outlet of the Mānoa-Pālolo Drainage Canal, site 1 and
3, contained higher concentrations of chlordane and dieldrin than the areas further away
from the outlet of the Mānoa-Pālolo Drainage Canal, site 4 and 5. This can be due to the
amount of sediment transported and how the sediment is transported to the Ala Wai
Canal. The most amount of sediment was collected at the Mānoa-Pālolo Drainage Canal,
site 2, and the least amount of sediment was collected at the area furthest away from the
outlet of the Mānoa-Pālolo Drainage Canal, site 5. The sediment at the Mānoa-Pālolo
Drainage Canal, site 2, was composed mainly of slit, while the sediment near the outlet of
the Mānoa-Pālolo Drainage Canal, site 1 and 3, was composed of more sand than slit
when compared to the areas further away from the outlet of the Mānoa-Pālolo Drainage
Canal, site 4 and 5.
Construction can contribute to the contamination of chlordane and dieldrin
through soil erosion or runoff since construction areas may be dug up to construction a
new structure, building, or house. Also, the sediment layers may be disturbed due to
construction being done at or near the waterway. Site 4 and 5 is near the McCully Street
Bridge. There was a construction zone located at site 5. The construction occurring at
site 5 could have disturbed the sediment layers since the least amount of sediment was
collected at this site. Site 4 was near site 5 and had the same concentration profile trend.
The concentration profile at these sites was different from site 1, 2, and 3 and could have
been due the construction near these sites.
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Sediment was analyzed to determine the concentration of chlordane and dieldrin.
Chlordane and dieldrin are persistent, toxic, and can bioaccumulate in the food chain [27,
30]. There is concern about the harmful effects chlordane and dieldrin has on humans
since chlordane and dieldrin effects the nervous, immune, and reproductive system and
may cause cancer [32, 33]. The concentrations of chlordane and dieldrin for the
sediments at the surface exceeded the Canadian Sediment Quality Guidelines (CSQG) for
aquatic life at all sites, except for dieldrin at site 5. Therefore, residents and state and city
officials should be warned about the chlordane and dieldrin levels in the Ala Wai Canal
and especially in the input streams and drainage canals since there are no warnings about
the potentially contaminated fish or shellfish that resident might catch and consume in
those sites.
Through this investigation of the horizontal and vertical distribution of chlordane
and dieldrin in Ala Wai Canal sediment, the contaminations levels for chlordane and
dieldrin were determined for the sampling sites. The highest levels of contamination
come from the Pālolo and Mānoa areas, which contain older homes that were treated
before the 1980s with chlordane or dieldrin to control termites. The soil from these older
homes still contains chlordane and dieldrin that enter water sources through soil erosion
and runoff. Also, the concentration of chlordane and dieldrin in Ala Wai Canal sediment
are affecting aquatic life in the Ala Wai Canal. Plans need to be developed and
implemented to restore stream and channel banks which can help control erosion and to
educate residents about these contaminants that can be harmful to their health if the
contaminated fish and shellfish are consumed. Also, periodic testing is needed to
determine the presence of chlordane and dieldrin and the contamination levels at sites
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contributing to the transportation of chlordane and dieldrin down to the Ala Wai Canal
and at sites at the Ala Wai Canal and continuing monitoring of the contaminations levels
for chlordane and dieldrin is needed to identify trends.
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CHAPTER 3 DETERMINE WHETHER OR NOT CHLORDANE CAN BE
BIODEGRADED IN ALA WAI CANAL SEDIMENT
3.1 Introduction
Chlordane and dieldrin were both detected in Ala Wai Canal sediment. However,
higher concentrations were detected for chlordane. In this study, chlordane was used to
determine whether or not chlordane can be biodegraded in Ala Wai Canal sediment since
high concentrations of chlordane was detected in Ala Wai Canal sediment.
Chlordane was produced in 1947 and was sold as an insecticide for agricultural
applications and termiticide for protecting structures from termites [30, 33]. The United
States of America (U.S.A) produced 9.5 million kg of chlordane in 1974 [33]. In 1978,
the EPA banned the use of chlordane on food crops and phased out other applications of
chlordane used above the ground over the following 5 years [30]. From 1983 and 1988,
chlordane was only approved to be used as a termiticide for underground treatment
around home foundations [30]. In 1988, all uses of chlordane were banned [30].
Chlordane is no longer used today, but is still being detected in environmental
samples worldwide [4, 5, 7, 10, 11, 13, 14, 16, 20, 23, 27, 30, 32, 33, 39]. Chlordane has
been listed by the EPA’s Great Waters Program and the Stockholm Convention as a
pollutant of concern [8, 10, 20, 30, 35]. Chlordane can remain intact in the environment
for long periods, be widely distributed throughout the environment as a result of natural
processes through soil and water, accumulate in the fatty tissue of living organisms and
therefore becoming more concentrated higher up the food chain, and is toxic to aquatic
life, wildlife, and humans [10, 30, 39]. Therefore, data of contamination levels are
needed for regulation purposes, to pin point sources, to determine the risks involved with
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chlordane remaining in the environment, and to discover trends on how chlordane is
being degraded naturally. This information can be useful in determining how chlordane
can be degraded.
Microbial degradation can be used to remove pollutants from sediments.
However, not much research has been done showing chlordane degradation from
sediments and about the degradation pathways or degradation products of chlordane [8,
10, 20]. Chlordane refers to a mixture of chlordane isomers (cis-chlordane and trans-
chlordane), other chlorinated hydrocarbons, and by-products such as heptachlor and
nonachlor [8, 10, 20, 33]. Since chlordane contains a mixture of various compounds and
each compound degrades independently, this makes chlordane hard to degrade and in
determining the degradation pathways or degradation products of chlordane [10, 20, 33].
Anaerobic microbial degradation has been reported by Hirano et al. [10].
Degradation of chlordane occurred after 4 weeks for sediment collected at one sampling
site [10]. At another sampling site degradation of chlordane occurred immediately after
the experiment started while no degradation of chlordane was also observed at another
sampling site [10]. This was due to the different contamination levels and amount of
organic carbon found at the various sampling sites where sediment was collected [10]. If
the sediment is highly contaminated with chlordane and contains a lot of organic carbon,
the microbial population is sufficient enough to induce degradation of chlordane, while
for sediment less contaminated with chlordane and containing little organic carbon, the
microbial population needs time to grow before degradation of chlordane will occur [10].
This study is focused on determining whether or not chlordane in Ala Wai Canal
sediment can be biodegraded. This was accomplished by conducting laboratory
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microcosm experiments and analyzing the samples taken from the microcosms
approximately once a month to determine the percentage of chlordane remaining in the
microcosms and observing the chromatographs for any dechlorinating peaks.
3.2 Methods
3.2.1 Sample Site and Collection
Two sediment cores were collected at site 1 (Figure 3.1) on November 24, 2011
using an AMS multi-stage sludge and sediment core sampler (American Falls, Idaho,
U.S.A). The sediment below the surface (anaerobic layer) was kept and placed in a glass
jar. The sediment sample was stored in an ice cooler with ice packs and was immediately
taken to the laboratory and stored in a COY Laboratories anaerobic chamber (Grass Lake,
Michigan, U.S.A).
Figure 3.1: Sediment Core Collection Site [34]
ALA WAI CANAL
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3.2.2 Culture Preparation and Sampling
Microcosms were prepared (in triplicate) using 125 mL serum bottles (Figure
3.2). Each bottle contained approximately 20 g of wet sediment, 100 mL of culture
medium, 39.3 µL of chlordane at 3.05 M, and 1 mL of sodium propionate at 20 mM
(electron donor). The culture medium contained (per liter of deionized (DI) water) 0.001
g of resazurin, 0.1 g of MgCl2·6H2O, 0.1 g of CaCl2·6H2O, 3.0 g of Na2CO3, 0.5 g of
NH4Cl, 0.6 g of Na2HPO4, 8.7 g of K2HPO4, 10 mL of vitamin solution, and 10 mL of
mineral solution [6, 19, 21]. The bottles were capped with Teflon stoppers and aluminum
crimps. The bottles then were vigorously shaken and kept in a COY Laboratories
anaerobic chamber (Grass Lake, Michigan, U.S.A). Microcosms were also prepared (in
triplicate) as described above, except sodium sulfate was also added to the bottles (Figure
3.2). Sterilized control microcosms were also prepared (in triplicate) as described above
and then the bottles were wrapped in aluminum foil and autoclaved overnight at 103 °C
to 105 °C (Figure 3.2).
Table 3.1: Set Up of the Laboratory Microcosm Experiments
Set Components
A Ala Wai Canal Sediment, Chlordane, and Culture Medium
B Components from Set A and Sulfate
C Components from Set A and Autoclaved
Samples were taken from each microcosm approximately every 1 month for
chlordane analysis. The bottles were vigorously hand-shaken for 2 minutes and
approximately 2 mL of completely mixed sample was removed with a glass pipette and
placed in a 22.2 mL glass vial for extraction. An additional 2 mL of completely mixed
sample was removed and placed in a 2 mL centrifuge tube for measuring pH. The
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sample in the vial was measured to determine the weight of the completely mixed
sample. Each sample that will be extracted was mixed with the surrogate, 40 µL of
decachlorobiphenyl at 250 µg/L. The pH was determined by centrifuging the additional
sample taken for 10 minutes at 13,000 rpm. After centrifuging the sample, 1 mL of
supernatant was removed and placed in a glass tube with 2 mL of deionized (DI) water.
The pH was then measured using a Denver Instruments pH meter (Bohemia, New York,
U.S.A). The remaining supernatant was removed and discarded and the cells remaining
in the centrifuge tube was kept and stored in the freezer at -20 °C.
3.2.3 Extraction and Cleanup
The samples were extracted in a similar manner to the methods described by
Hirano et al. (2007) [10]. The samples were extracted twice with 6.25 mL of acetone.
The vials were shaken for 2.5 minutes on a shaker and placed in an ultrasonic cleaner and
then the acetone layer was transferred to a 125 mL serum bottle. The samples were then
extracted with 31.25 mL of deionized (DI) water containing 5% (by volume) NaCl and
12.5 mL of hexane. The bottles were shaken for 2.5 minutes on a shaker and the hexane
layer was transferred to a 22.2 mL glass vial and stored in the freezer at -20 °C.
The extracts were cleaned using SW-846 Method 3665A and 3660B [37]. Only 2
mL of extract was used for cleanup. First, 2 mL of the hexane layer from the extraction
was transferred to a 15 mL centrifuge tube containing 5 mL of deionized (DI) water
containing 50% (by volume) sulfuric acid. The extracts were then vortex for a minute
and set aside for a minute to allow the phases to separate. The hexane layer was
transferred to a glass tube and 1 mL of hexane was added to the centrifuge tube and then
hand-shaken. The hexane layer was removed and combined with the hexane layer in the
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glass tube. The hexane layer from the sulfuric acid cleanup was then cleaned using
copper powder to remove sulfur. Copper powder was filled up approximately to the 0.5
mL mark (2 g of copper powder) and 1 mL of hexane layer from the sulfuric acid cleanup
was transferred to a 2 mL centrifuge tube containing copper powder. The tubes were
shaken for a minute on a shaker and set aside to allow the phases to separate. The hexane
layer was transferred to a 2 mL gas chromatography (GC) vial. The remaining hexane
layer from the sulfuric acid cleanup was cleaned with copper powder and combined with
the hexane layer in the GC vial as stated above. The GC vials were stored in the freezer
at -20 °C until analysis.
3.2.4 Calibration and Analysis
The concentration of chlordane in the samples was determined using SW-846
Method 8081B [37]. Chlordane in the samples were analyzed using a Hewlett Packard
5890 Series II Gas Chromatograph equipped with an electron capture detector (GC-ECD)
(Palo Alto, California, U.S.A) with the following operating conditions. The oven
temperature was initially held at 100 °C for 2 minutes, ramped from 100 °C to 160 °C at
15 °C/min, and then ramped from 160 °C to 270 °C at 5 °C/min. The carrier gas was
helium (ultra high purity) and the pressure was set to 60 psi. The makeup gas for the
detector was nitrogen and the pressure was set to 25 psi. The detector temperature was
set to 300 °C. The GC-ECD also had an Agilent GC AutoSampler Controller and 6890
Series Injector (Santa Clara, California, U.S.A) to inject 2 µL of sample. The injector
temperature was set to 225 °C. Also, a J&W DB-XLB column of 30 m × 0.533 mm ×
150 µm size was used (Folsom, California, U.S.A).
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Chlordane concentrations were quantified using calibration standards and were
corrected by surrogate recoveries that were greater than 100%. Five calibration standards
at concentrations of 50 µg/L, 150 µg/L, 250 µg/L, 350 µg/L, and 500 µg/L were prepared
for chlordane. Also, five calibration standards at concentrations of 250 µg/L, 300 µg/L,
350 µg/L, 400 µg/L, and 450 µg/L were prepared for the surrogate, decachlorobiphenyl.
The retention time windows were established during the analysis of chlordane and
dieldrin in the sediment samples. Standard curves (Figure 3.2 and 3.3) were used to
determine the concentration of chlordane and decachlorobiphenyl from peak areas and
then adjusted based on the amount of hexane used for extraction and the amount of
completely mixed sample removed from the microcosms. The concentration of
chlordane was also corrected using surrogate recoveries.
Figure 3.2: Decachlorobipheyl Calibration Curve
y = 75685x R² = 0.9629
0
5000000
10000000
15000000
20000000
25000000
30000000
35000000
40000000
0 100 200 300 400 500
Are
a
Concentration (µg/L)
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Figure 3.3: Chlordane Calibration Curve
The concentration of decachlorobiphenyl was calculated using the equation presented in
equation (9).
The surrogate recovery was calculated using the equation presented in equation (10).
The concentration of chlordane was calculated using the equation presented in equation
(11).
y = 81767x R² = 0.9976
0
5000000
10000000
15000000
20000000
25000000
30000000
35000000
40000000
45000000
0 100 200 300 400 500 600
Are
a
Concentration (µg/L)
(9)
(10)
(11)
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3.3 Results
Laboratory microcosm experiments were performed to determine whether or not
chlordane in Ala Wai Canal sediment can be depleted. For a period of 81 days
(approximately 11 weeks), the microcosm experiments did not show any indications of
degradation of chlordane (Figure 3.4). After the initial sampling, each set of microcosm
experiments still had 97% or more chlordane remaining, except for the samples taken on
day 54 (approximately 8 weeks). The samples taken on day 54 had more chlordane than
the initial samples taken. This could have probably been due to the culture not being
mixed well and then a sample with a high spike of chlordane was removed from the
culture.
Figure 3.4: Chlordane Concentration of Samples Taken from Microcosms
(each point represents the average plus and minus one standard deviation of the mean)
-50
0
50
100
150
200
250
300
350
400
0 20 40 60 80 100
Ch
lord
ane
Co
nce
ntr
atio
n (
µg/
kg w
w)
Time (day)
Set A
Set B
Set C
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The chromatographs for the microcosm experiments also did not show any dechlorinating
peaks since the chromatographs for the chlordane containing microcosms (set A) and the
chlordane and sulfate containing microcosms (set B) contained the same peaks as the
sterilized control microcosms (set C).
3.4 Discussion
Degradation of chlordane did not occur during a period of 81 days (approximately
11 weeks). This means that degradation of chlordane did not start or that chlordane
cannot be depleted from Ala Wai Canal sediment. For degradation of chlordane to occur,
the microbial population needs time to grow to a sufficient size, known as the acclimation
period, before microbes could start degrading chlordane [10, 12]. During the acclimation
period, slight or no degradation occurs and is shown as a lag period on degradation
profiles. Suitable conditions are needed for degradation to occur. Sediment
contaminated with high levels of chlordane and contain a high percentage of organic
carbon can provide suitable conditions for the microbial population to grow [10]. Hirano
et al. (2007) reported that degradation of chlordane occurred immediately for sediment
contaminated with chlordane and containing the highest percentage of organic carbon
while no degradation occurred for sediment not contaminated with chlordane and
containing the least percentage of organic carbon [10]. A lag period was noticed for all
microcosm experiments containing Ala Wai Canal sediment. The Ala Wai Canal
sediment used for the microcosm experiments were contaminated with chlordane so the
sediment probably did not have enough microbes and needs more time for the microbial
population to increase before degradation will occur or the Ala Wai Canal sediment did
not have the right conditions to entice microbial degradation.
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CHAPTER 4 CONCLUSIONS AND RECOMMENDATIONS
4.1 Conclusions
Through this study about the horizontal and vertical distribution of chlordane and
dieldrin in Ala Wai Canal sediment and whether or not chlordane in Ala Wai Canal
sediment can be biodegraded, a chlordane and dieldrin concentration survey was obtained
and it was determined that no degradation of chlordane in Ala Wai Canal sediment
occurred for a period of 81 days (approximately 11 week).
The concentration of chlordane and dieldrin in Ala Wai Canal sediment are
affecting aquatic life in the Ala Wai Canal. This is concerning since chlordane and
dieldrin are persistent, toxic, and can bioaccumulate in the food chain [27, 30]. The
highest levels of contamination come from the Pālolo and Mānoa areas, which contain
older homes that were treated before the 1980s with chlordane or dieldrin to control
termites. The soil from these older homes still contains chlordane and dieldrin that enter
water sources through soil erosion and runoff. Therefore, plans need to be developed and
implemented to restore stream and channel banks which can help control erosion.
Periodic testing and continuing monitoring is also needed to determine the presence of
chlordane and dieldrin and the contamination levels at sites contributing to the
transportation of chlordane and dieldrin down to the Ala Wai Canal and at sites at the Ala
Wai Canal. Also, residents and immigrants that speak no or little English need be
educated about pollution and the harmful effects of consuming contaminated fish and
shellfish and drinking contaminated water.
Degradation of chlordane in Ala Wai Canal sediment was not observed for a
period of 81 days (approximately 11 weeks). The microbial population in the sediment
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needs to be large enough before degradation will occur, which depends on the
contamination levels, temperature, and carbon and energy source [2, 7, 8, 10, 13, 14, 20].
The Ala Wai Canal sediment used for the microcosm experiments were contaminated
with chlordane so the sediment probably did not have enough microbes and needs more
time for the microbial population to increase before degradation will occur or the right
conditions to entice microbial degradation.
4.2 Recommendations
The Ala Wai Canal is contaminated and the City and County of Honolulu and the
State of Hawaiʻi are seeking ways to clean up the Ala Wai Canal [18]. First, the sources
contributing to the contamination in the Ala Wai Canal needs to be determined. By
determining the source of contamination, these areas can be continuously monitored to
discover trends in the contamination levels, which can be used to determine how the
contamination is transported and what is contributing to the high levels of contamination.
Since the Ala Wai Canal contains contaminated fish and shellfish, periodic testing of the
fish and shellfish is also needed to determine the contamination levels in the fish tissue
and in the shellfish. Secondly, plans need to be developed and implemented to control
soil erosion, which contributes to contamination entering water sources, such as streams
and drainage canals. This can be done by restoring stream and channel banks and
preventing soil from entering nearby water sources when land is cleared or during
construction. By having these plans and monitoring the contamination levels, then state
and city officials can determine whether or not the soil erosion reduction practices are
effective. Lastly, residents and immigrants that speak no or little English need to be
educated about the contamination found in water sources and harmful effects of
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consuming contaminated fish and shellfish and drinking contaminated water. The Ala
Wai Canal has signs warning people not to swim and catch and consume the fish and
shellfish in the Ala Wai Canal. However, the contamination found in Ala Wai Canal is
also found at streams and drainage canals that discharge water and sediment to the Ala
Wai Canal [4, 5, 9, 17]. Therefore, signs are also needed at those contaminated sites to
prevent consumption of contaminated fish and shellfish and awareness and ways
residents can help out with the cleaning efforts are promoted through education.
Microbial degradation is one method that can be used to deplete contaminants
from the environment. The laboratory microcosm experiments performed in anaerobic
conditions did not show any signs of chlordane being depleted from Ala Wai Canal
sediment, however this can be due to the sediment probably not having enough microbes
and more time is needed for the microbial population to increase before degradation will
occur or the right conditions to entice microbial degradation. Therefore, future research
is needed to determine the conditions needed to promote microbial degradation. Other
techniques can be used to determine whether or not contaminants can be degraded from
the environment such as microbial degradation under aerobic conditions and
photodegradation [8, 13, 14, 20]. Future research, using these techniques could be used
to determine if these techniques are more suitable for degrading the contaminants found
in the Ala Wai Canal. Data pertaining to the characteristics of the sediment found in the
Ala Wai Canal was not gather for this study, however future research involving
degradation should include sediment characteristics, which can help determine what is
inhibiting and stimulating degradation of contaminants. If the containments in the Ala
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Wai Canal can be degraded, this can lead to more information that can be used to develop
and find ways to detect and eventually deplete the containments from the Ala Wai Canal.
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APPENDIX A
Table A.1: Dry Weights of Sediment Collected for Set A
Site Foil (g) Hood Dry (g) 1st Oven Dry + Foil (g) 2nd Oven Dry + Foil (g) 1st Oven Dry (g) 2nd Oven Dry (g) Dry Weight - 1st Dry Weight - 2nd RPD (< 4%)
1.1.1 1.7910 4.7169 5.6939 5.5978 3.9029 3.8068 82.74% 80.71% 2.49%
1.1.2 1.5527 5.1261 5.2874 5.2092 3.7347 3.6565 72.86% 71.33% 2.12%
1.1.3 1.4610 4.9233 4.9746 4.9024 3.5136 3.4414 71.37% 69.90% 2.08%
1.1.4 1.6546 5.1270 5.6772 5.5910 4.0226 3.9364 78.46% 76.78% 2.17%
1.1.5 1.3982 5.2896 5.4369 5.3418 4.0387 3.9436 76.35% 74.55% 2.38%
1.2.1 1.6762 5.1079 5.896 5.8139 4.2198 4.1377 82.61% 81.01% 1.96%
1.2.2 1.6100 4.6539 5.4731 5.4166 3.8631 3.8066 83.01% 81.79% 1.47%
2.1.1 1.3466 4.8947 5.2369 5.1233 3.8903 3.7767 79.48% 77.16% 2.96%
2.1.2 1.5912 4.7586 5.7939 5.6841 4.2027 4.0929 88.32% 86.01% 2.65%
2.1.3 1.3348 4.5704 5.2826 5.1612 3.9478 3.8264 86.38% 83.72% 3.12%
2.1.4 1.3742 4.7137 5.2663 5.1395 3.8921 3.7653 82.57% 79.88% 3.31%
2.1.5 1.3190 4.6444 4.9124 4.7903 3.5934 3.4713 77.37% 74.74% 3.46%
3.1.1 1.3664 4.7321 5.1247 5.0067 3.7583 3.6403 79.42% 76.93% 3.19%
3.1.2 1.3749 4.5855 5.1028 4.9896 3.7279 3.6147 81.30% 78.83% 3.08%
3.1.3 1.4703 5.3477 5.4254 5.3365 3.9551 3.8662 73.96% 72.30% 2.27%
3.1.4 1.4926 5.5514 5.5727 5.4639 4.0801 3.9713 73.50% 71.54% 2.70%
3.1.5 1.3229 4.8586 4.9578 4.8666 3.6349 3.5437 74.81% 72.94% 2.54%
4.1.1 1.3978 4.7994 5.1542 5.0373 3.7564 3.6395 78.27% 75.83% 3.16%
4.1.2 1.3602 4.9525 5.2626 5.1450 3.9024 3.7848 78.80% 76.42% 3.06%
4.1.3 1.3733 4.8288 5.1419 5.0302 3.7686 3.6569 78.04% 75.73% 3.01%
4.1.4 1.3848 4.8338 4.9446 4.8295 3.5598 3.4447 73.64% 71.26% 3.29%
4.1.5 1.3296 4.6529 5.2768 5.1546 3.9472 3.825 84.83% 82.21% 3.14%
4.2.1 1.4029 5.0559 5.5284 5.4215 4.1255 4.0186 81.60% 79.48% 2.63%
4.2.2 1.3451 4.9167 4.9153 4.8126 3.5702 3.4675 72.61% 70.52% 2.92%
5.1.1 1.4144 4.7803 5.2850 5.1801 3.8706 3.7657 80.97% 78.78% 2.75%
5.1.3 1.7077 4.8922 5.6334 5.6270 3.9257 3.9193 80.24% 80.11% 0.16%
5.1.4 1.7134 5.2300 5.6029 5.5004 3.8895 3.787 74.37% 72.41% 2.67%
5.2.2 1.5198 5.0714 5.5345 5.4134 4.0147 3.8936 79.16% 76.78% 3.06%
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Table A.2: Dry Weights of Sediment Collected for Set B
Site Foil (g) Hood Dry (g) 1st Oven Dry + Foil (g) 2nd Oven Dry + Foil (g) 1st Oven Dry (g) 2nd Oven Dry (g) Dry Weight - 1st Dry Weight - 2nd RPD (< 4%)
1.1.1 1.4243 5.4325 6.2115 6.1348 4.7872 4.7105 88.12% 86.71% 1.62%
1.1.2 1.5141 6.6205 7.6137 7.5286 6.0996 6.0145 92.13% 90.85% 1.40%
1.1.3 1.4799 5.6688 6.5145 6.4380 5.0346 4.9581 88.81% 87.46% 1.53%
1.1.4 1.4682 6.0164 6.6566 6.5673 5.1884 5.0991 86.24% 84.75% 1.74%
1.1.5 1.7829 5.0461 6.2452 6.1791 4.4623 4.3962 88.43% 87.12% 1.49%
2.1.1 1.4359 4.5471 5.1231 5.0473 3.6872 3.6114 81.09% 79.42% 2.08%
2.1.2 1.4707 4.6910 4.8753 4.7747 3.4046 3.3040 72.58% 70.43% 3.00%
2.1.3 1.7621 4.7167 5.2983 5.1861 3.5362 3.4240 74.97% 72.59% 3.22%
2.1.4 1.4622 5.5374 5.7530 5.6512 4.2908 4.1890 77.49% 75.65% 2.40%
2.1.5 1.5107 4.8700 5.1758 5.0934 3.6651 3.5827 75.26% 73.57% 2.27%
2.2.1 1.5496 5.9345 6.1425 6.0435 4.5929 4.4939 77.39% 75.72% 2.18%
2.2.2 1.7018 5.0640 5.3456 5.2502 3.6438 3.5484 71.95% 70.07% 2.65%
2.2.3 1.4302 6.4875 6.0840 5.9971 4.6538 4.5669 71.73% 70.40% 1.88%
2.2.4 1.6243 5.9217 5.8238 5.7222 4.1995 4.0979 70.92% 69.20% 2.45%
2.2.5 1.4525 4.7742 4.7634 4.6761 3.3109 3.2236 69.35% 67.52% 2.67%
3.1.1 1.5947 5.8309 5.8785 5.7950 4.2838 4.2003 73.47% 72.04% 1.97%
3.1.2 1.5821 5.8743 6.3072 6.2148 4.7251 4.6327 80.44% 78.86% 1.97%
3.1.3 1.6388 7.1634 6.9458 6.8397 5.3070 5.2009 74.08% 72.60% 2.02%
3.1.4 1.5298 5.0132 5.2234 5.1459 3.6936 3.6161 73.68% 72.13% 2.12%
3.1.5 1.4116 5.9844 6.1571 6.0720 4.7455 4.6604 79.30% 77.88% 1.81%
3.2.1 1.4978 6.1198 6.6975 6.6043 5.1997 5.1065 84.97% 83.44% 1.81%
3.2.2 1.3995 5.3476 5.5567 5.5481 4.1572 4.1486 77.74% 77.58% 0.21%
4.1.1 1.4500 5.6815 5.7455 5.6526 4.2955 4.2026 75.61% 73.97% 2.19%
4.1.2 1.8342 5.6015 6.3167 6.2238 4.4825 4.3896 80.02% 78.36% 2.09%
4.1.3 1.6638 6.8588 6.7731 6.6828 5.1093 5.0190 74.49% 73.18% 1.78%
4.1.4 1.6542 5.4424 5.7558 5.6678 4.1016 4.0136 75.36% 73.75% 2.17%
5.1.1 1.7177 6.4005 6.4712 6.3661 4.7535 4.6484 74.27% 72.63% 2.24%
5.1.2 1.4650 5.9795 6.3039 6.2152 4.8389 4.7502 80.92% 79.44% 1.85%
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Table A.3: Concentration, % Moisture, and % Recovery of Sediment Collected for Set A
Site % Moisture Decachlorobiphenyl Chlordane Dieldrin
Concentration (µg/kg) % Recovery Corrected Concentration (µg/kg) Corrected Concentration (µg/kg)
1.1.1 17.2571 38.6005 38.6005 4.6335 ND
1.1.2 27.1434 13.5235 13.5235 55.6066 12.8850
1.1.3 28.6332 24.6492 24.6492 29.5375 21.5654
1.1.4 21.5409 32.9427 32.9427 13.2811 8.0632
1.1.5 23.6483 28.7887 28.7887 50.0547 26.4352
1.2.1 17.3868 36.4656 36.4656 46.4633 103.1289
1.2.2 16.9922 7.0042 7.0042 149.7152 26.7666
2.1.1 20.5202 15.1120 15.1120 166.8800 36.9672
2.1.2 11.6820 19.7568 19.7568 94.8871 ND
2.1.3 13.6224 6.0449 6.0449 239.7678 37.8866
2.1.4 17.4300 15.2658 15.2658 248.0344 43.2297
2.1.5 22.6294 19.4735 19.4735 198.3966 29.1487
3.1.1 20.5786 9.3815 9.3815 98.2920 34.2479
3.1.2 18.7024 18.6311 18.6311 85.2549 26.8242
3.1.3 26.0411 32.6484 32.6484 12.3294 ND
3.1.4 26.5032 5.9299 5.9299 97.2823 6.9805
3.1.5 25.1863 13.1496 13.1496 329.7045 67.9782
4.1.1 21.7319 22.6872 22.6872 117.2141 ND
4.1.2 21.2034 15.8907 15.8907 186.8065 ND
4.1.3 21.9558 14.8456 14.8456 95.2276 ND
4.1.4 26.3561 15.6488 15.6488 174.1233 ND
4.1.5 15.1669 19.5134 19.5134 224.5136 11.5480
4.2.1 18.4023 84.6204 84.6204 5.9259 3.2471
4.2.2 27.3863 41.0242 41.0242 38.4477 16.6358
5.1.1 19.0302 20.1114 20.1114 42.4497 68.0089
5.1.3 19.7559 4.4452 4.4452 79.5071 23.7331
5.1.4 25.6310 3.7704 3.7704 80.0446 51.4833
5.2.2 20.8365 4.5594 4.5594 38.1209 3.4332
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Table A.4: Concentration, % Moisture, and % Recovery of Sediment Collected for Set B
Site % Moisture Decachlorobiphenyl Chlordane Dieldrin
Concentration (µg/kg) % Recovery Corrected Concentration (µg/kg) Corrected Concentration (µg/kg)
1.1.1 11.8785 1.6058 1.6058 272.6451 215.9787
1.1.2 7.8680 18.7285 18.7285 20.9477 4.7021
1.1.3 11.1876 5.4508 5.4508 2.8008 18.4174
1.1.4 13.7624 2.0673 2.0673 26.5557 20.9703
1.1.5 11.5693 2.0546 2.0546 61.0219 25.9825
2.1.1 18.9110 2.3487 2.3487 18.2532 ND
2.1.2 27.4227 2.2599 2.2599 29.5816 ND
2.1.3 25.0281 1.6988 1.6988 17.3368 ND
2.1.4 22.5124 25.4988 25.4988 2.8129 ND
2.1.5 24.7413 12.1535 12.1535 8.1374 ND
2.2.1 22.6068 17.9397 17.9397 9.2735 1.7002
2.2.2 28.0450 23.3906 23.3906 16.2134 3.4918
2.2.3 28.2651 4.1196 4.1196 33.6085 1.6746
2.2.4 29.0829 15.9097 15.9097 35.3632 4.2993
2.2.5 30.6502 10.2810 10.2810 46.9967 4.8068
3.1.1 26.5328 17.1221 17.1221 3.7832 2.6617
3.1.2 19.5632 0.5296 0.5296 5.3565 7.6469
3.1.3 25.9151 12.0620 12.0620 3.4210 0.7448
3.1.4 26.3225 0.4502 0.4502 133.5747 38.5814
3.1.5 20.7022 20.2479 20.2479 21.0320 2.8413
3.2.1 15.0348 15.9929 15.9929 11.2492 2.0478
3.2.2 22.2605 33.4727 33.4727 8.8245 3.2870
4.1.1 24.3950 15.2549 15.2549 5.5754 1.4339
4.1.2 19.9768 23.2928 23.2928 17.0707 3.9741
4.1.3 25.5074 1.4729 1.4729 110.5043 ND
4.1.4 24.6362 18.7748 18.7748 7.9908 3.9870
5.1.1 25.7324 22.1639 22.1639 10.2112 2.9091
5.1.2 19.0752 3.4479 3.4479 ND 0.8294
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43
APPENDIX B
Table B.1: Years Structures Built in the Pālolo, Mānoa, McCully/Mōʻiliʻili, and Makiki Areas [36]
Year Structure Built Pālolo Mānoa McCully/Mōʻiliʻili Makiki
1999 - March 2000 30 59 9 215
1995 - 1998 126 260 149 854
1990 - 1994 143 377 353 431
1980 - 1989 475 568 1698 1746
1970 - 1979 587 1216 5021 5497
1960 - 1969 823 1692 3416 4193
1940 - 1959 2054 2275 2864 2481
1939 or earlier 486 1106 588 859
Table B.2: Number of Units in Structures in the Pālolo, Mānoa, McCully/Mōʻiliʻili, and Makiki Areas [36]
Units in Structure Pālolo Mānoa McCully/Mōʻiliʻili Makiki
1 unit, detached 2870 4788 831 1383
1 unit, attached 587 587 370 321
2 units 176 175 447 300
3 or 4 units 157 247 1187 552
5 - 9 units 311 379 2212 1262
10 - 19 units 216 429 1786 2214
20 or more units 402 941 7241 10235
other 5 7 24 9
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44
APPENDIX C
Table C.1: Chlordane Concentration of Samples Taken from Microcosms
Date 1/10/2012 2/4/2012 3/10/2012 3/31/2012
Microcosm
#
Chlordane Conc.
(μg/kg)
Chlordane Conc.
(μg/kg)
Chlordane Conc.
(μg/kg)
Chlordane Conc.
(μg/kg)
Set A
C1 4.7538
44.8350
1.8411
14.9072
5.9514
47.9261
0.5149
4.3744 C2 15.4045 3.3444 8.9018 0.8492
C3 114.3467 39.5362 128.9250 11.7591
Set B
C4 113.6751
86.3150
48.4190
32.1544
144.3607
91.9541
13.1589
8.6294 C5 59.1320 23.5465 62.1205 5.0001
C6 86.1378 24.4977 69.3813 7.7293
Set C
C7 256.2968
231.3326
88.5691
85.0907
378.8256
320.7271
29.6408
24.2284 C8 211.8748 43.1056 319.4936 25.2333
C9 225.8261 123.5973 263.8621 17.8111
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Table C.2: pH of Samples Taken from Microcosms
Date 1/10/2012 2/4/2012 3/10/2012 3/31/2012
Microcosm # pH pH pH pH
Set A
C1 7.09
6.98
7.46
7.36
7.71
7.62
7.84
7.74 C2 6.96 7.35 7.61 7.73
C3 6.89 7.28 7.54 7.65
Set B
C4 6.97
6.98
7.34
7.34
7.91
7.90
8.03
8.03 C5 6.98 7.34 7.83 7.99
C6 6.99 7.35 7.97 8.07
Set C
C7 6.82
6.82
7.39
7.32
7.46
7.44
7.53
7.52 C8 6.83 7.31 7.47 7.55
C9 6.81 7.26 7.40 7.48
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Figure C.1: pH of Samples Taken from Microcosms
6.6
6.8
7
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7.4
7.6
7.8
8
8.2
0 20 40 60 80 100
pH
Time (day)
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Set B
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47
Figure C.2: Chromatographs of Samples Taken on January 14, 2012
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20
.51
20
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.21
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Chrom Perfect Chromatogram Report
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Chrom Perfect Chromatogram Report
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Chrom Perfect Chromatogram Report
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Chrom Perfect Chromatogram Report
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Figure C.3: Chromatographs of Samples Taken on February 4, 2012
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Chrom Perfect Chromatogram Report
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54
Chrom Perfect Chromatogram Report
Printed on 2/5/2012 12:49:20 PM Page 1 of 1
20
.10
20
.93
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.15
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.67
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.03
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.31
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.67
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.91
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.25
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.76
2
3.9
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.13
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.88
25
.20
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.60
25
.82
26
.26
2
6.4
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.60
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.80
27
.32
27
.74
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8.0
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.34
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.74
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.84
29
.16
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Printed on 2/5/2012 12:50:08 PM Page 1 of 1
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.39
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.87
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.15
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.67
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.02
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.31
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.67
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.90
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.24
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.56
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.76
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55
Chrom Perfect Chromatogram Report
Printed on 2/5/2012 1:09:23 PM Page 1 of 1
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.10
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.44
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.16
21
.51
22
.03
22
.32
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.84
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.25
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.77
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3.9
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.14
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.46
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.65
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.91
25
.17
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.56
25
.79
26
.26
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.60
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.79
27
.29
27
.73
28
.03
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.28
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.50
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.84
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.15
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Printed on 2/5/2012 1:10:14 PM Page 1 of 1
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.85
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.50
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.03
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.64
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.88
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.25
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.49
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.76
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.13
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.68
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.91
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.16
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.57
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.26
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.03
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56
Chrom Perfect Chromatogram Report
Printed on 2/5/2012 1:11:02 PM Page 1 of 1
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.08
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.44
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.14
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.50
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.02
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.31
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.64
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.48
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.45
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.91
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.16
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.56
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.79
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.26
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.60
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.79
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.73
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.02
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.23
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.38
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.50
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.83
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57
Figure C.4: Chromatographs of Samples Taken on March 10, 2012
Chrom Perfect Chromatogram Report
Printed on 3/11/2012 11:00:20 AM Page 1 of 1
20
.17
20
.95
21
.17
21
.68
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.04
22
.32
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.67
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2.8
4 23
.25
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.77
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3.9
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.14
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.42
24
.62
24
.88
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5.0
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.21
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.60
26
.26
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.60
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.80
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.30
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.62
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.90
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8.0
9
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.35
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.55
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4 2
8.8
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.16
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Chrom Perfect Chromatogram Report
Printed on 3/11/2012 11:01:48 AM Page 1 of 1
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.21
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.72
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.03
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.25
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.85
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58
Chrom Perfect Chromatogram Report
Printed on 3/11/2012 11:04:56 AM Page 1 of 1
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.10
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.48
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.16
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.67
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.03
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.32
22
.66
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.24
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.77
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.14
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.42
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.62
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.87
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4
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.20
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.60
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.26
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.60
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.80
27
.30
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.62
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.89
28
.35
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.54
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.74
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.84
29
.16
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Chrom Perfect Chromatogram Report
Printed on 3/11/2012 11:07:31 AM Page 1 of 1
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.12
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.99
2
1.1
7
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.67
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.03
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.32
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.68
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.25
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.77
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.14
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.42
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.63
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.88
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.21
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.60
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.26
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.60
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.80
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.30
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.62
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.90
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59
Chrom Perfect Chromatogram Report
Printed on 3/11/2012 11:09:29 AM Page 1 of 1
20
.10
20
.89
21
.16
21
.68
22
.03
22
.32
22
.68
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.91
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.25
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.58
2
3.7
7
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.93
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.14
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.87
25
.21
2
5.2
7
25
.60
25
.80
26
.26
2
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.60
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.80
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.31
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.72
28
.36
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.75
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.84
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.17
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Chrom Perfect Chromatogram Report
Printed on 3/11/2012 11:08:32 AM Page 1 of 1
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.13
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.17
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.68
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.03
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.32
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.68
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.00
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.25
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.77
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3.9
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.14
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.89
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.21
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.60
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.83
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.26
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.60
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.80
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.31
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.36
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.75
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.84
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60
Chrom Perfect Chromatogram Report
Printed on 3/11/2012 11:12:31 AM Page 1 of 1
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.10
20
.45
20
.92
21
.16
21
.52
22
.03
22
.32
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.64
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23
.25
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.77
2
3.9
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24
.14
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.43
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.63
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.91
25
.17
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.57
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.79
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.26
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6.4
1
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.60
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.80
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.23
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.61
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.72
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.03
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.36
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.50
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.84
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.15
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Chrom Perfect Chromatogram Report
Printed on 3/11/2012 11:13:17 AM Page 1 of 1
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.91
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.16
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.52
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.03
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.64
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.24
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.60
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.93
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.14
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.44
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.63
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.91
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.17
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.58
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.79
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.26
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.60
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.79
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.24
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61
Chrom Perfect Chromatogram Report
Printed on 3/11/2012 11:14:10 AM Page 1 of 1
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.09
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.44
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.90
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.15
21
.51
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.03
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.32
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.65
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.24
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.76
2
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.13
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.42
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.63
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.91
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.17
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.58
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.79
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.25
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.60
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.79
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.23
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.61
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.72
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.03
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.35
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.83
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62
Figure C.5: Chromatographs of Samples Taken on March 31, 2012
Chrom Perfect Chromatogram Report
Printed on 4/8/2012 11:47:16 AM Page 1 of 1
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.10
20
.43
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.92
21
.16
21
.67
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.03
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.32
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.66
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3.2
4
23
.76
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3.9
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.14
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.41
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.62
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.87
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5.0
3
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.20
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.27
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.59
2
5.7
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.25
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.60
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.79
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.29
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.61
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.88
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4
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.34
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.54
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.74
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.83
29
.15
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Chrom Perfect Chromatogram Report
Printed on 4/8/2012 11:49:05 AM Page 1 of 1
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.11
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.43
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.91
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.15
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.02
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.31
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.66
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.24
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.76
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.13
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.44
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.64
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.88
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.03
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.59
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.25
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.60
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.79
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.30
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.55
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63
Chrom Perfect Chromatogram Report
Printed on 4/8/2012 11:48:08 AM Page 1 of 1
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.08
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.76
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.14
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.66
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.76
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.13
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.40
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.62
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.87
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.19
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.26
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.58
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8
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.25
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.59
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.79
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.21
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.29
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.60
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.86
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.34
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.54
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.74
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.82
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.14
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