Deepwater Horizon Oil Spill: Using Microcosms to Study Effects Of

DEEPWATER HORIZON OIL SPILL: USING MICROCOSMS TO STUDY EFFECTS OF

CRUDE OIL IN COASTAL SEDIMENTS

ERIKA KRISTINE RENTSCHLER

DR. RONA J. DONAHOE, COMMITTEE CHAIR

DR. YUEHAN LU DR. GEOFFREY TICK DR. PATRICIA SOBECKY

A THESIS

Submitted in partial fulfillment of the requirements for the degree of Master of Science in the Department of Geological Sciences in the Graduate School of The University of Alabama

TUSCALOOSA, ALABAMA

2013

ABSTRACT

Approximately 4.9 million gallons of crude oil traveled with ocean currents to reach the

Gulf coast after the Deepwater Horizon oil drilling rig explosion. Microcosm experiments were conducted to determine how oil contamination affects the concentration and distribution

(between solid and aqueous phases) of trace elements in a salt marsh environment. Sediment and seawater from a salt marsh at Bayou La Batre, Alabama, were measured into jars and spiked with

500 ppm MC-252 oil. The solid phase and aqueous samples were analyzed by ICP-OES, ICP-

MS, and IC. A second experiment was conducted using various concentrations (0 ppm, 10 ppm,

100 ppm, 500 ppm, 1000 ppm, 2500 ppm) of MC252 oil.

ICP-OES data show variations in aqueous elemental concentrations occurred over the 14 day experiment. The pH for the water in the experiments ranged from 6.93 to 8.06. Significant positive correlations (r>0.75) were found in the solid phase samples between iron and the following elements: aluminum, cobalt, chromium, and nickel. Aqueous iron concentrations were highly correlated (r>0.75) with solution pH.

The presence of iron oxide and clays in the salt marsh sediment indicates potential for adsorption of trace elements sourced from the environment and from crude oil contamination.

The release of aqueous Fe (II) observed between two and 14 days is likely caused by reductive dissolution of iron-bearing clays or iron oxide. All the samples that contained oil behaved in similar ways with respect to time, but the controls showed almost no changes in the concentrations of the trace elements. Although the levels of some trace elements in the solid phase changed during the experiments, their final concentrations were at the same levels as the control samples. With the exception of nickel, the 14 day samples contained lower trace metal concentrations than the sterile control which contained no oil. The reason for this is likely

attributable to the in situ oil-degrading bacteria, which were found to be present in the sediment.

The oil-degrading bacterial community increased in the presence of oil and decreased as the oil concentration decreased. Oil-degrading bacteria are capable of inducing reductive dissolution in

Fe (III) minerals.

iii

DEDICATION

This thesis is dedicated to everyone who helped and guided me through the trials and tribulations of this research. In particular, my husband, my family, and my “fur babies” (Angel,

Ninja, and Paytra). You all have been an amazing source of emotional support and encouragement.

ACKNOWLEDGMENTS

I would like to thank Dr. Rona Donahoe for her guidance and for sharing her knowledge of geochemistry. I would like to thank my committee members, Yuehan Lu, Geoff Tick, and

Patricia Sobecky for their invaluable input, inspiring questions, and support of both the thesis research and my academic progress. I thank Ghanashyam Neupane (Hari) for showing me how to perform an alkalinity titration, a microwave digestion, and for finding things in the lab for me when I had no idea where to look. I thank Sidhartha Bhattacharyya (Sid) for analyzing my oily samples on the ICP and for teaching me how to arrange the spreadsheets to make sense of all that data. Thank you to Jason Harvell, my husband, for going with me on sampling trips, helping me in the lab when nobody else could, and for bringing me cupcakes when I’m stressed out. Drew

Raulerson has been indispensable with his four-wheel drive truck, strong arms, and his willingness to help inside and outside the lab. I thank Whitney Harris for driving 400 miles with me, out of her own curiosity, and for grabbing me for balance in the water that one time, which led to disaster! I thank all of the undergraduate research assistants for the extra hands.

This research would not have been possible without the support of my friends and fellow graduate students. I am lucky to have such an amazing support system. Thank you to my family who never stopped encouraging me. You told me I could be whatever I wanted to be and I will always strive to prove you right.

CONTENTS

ABSTRACT ...... ii

DEDICATION ...... iv

ACKNOWLEDGMENTS ...... v

LIST OF TABLES ...... viii

LIST OF FIGURES ...... ix

1. INTRODUCTION ...... 1

2. STUDY AREA ...... 8

3. METHODOLOGY ...... 11

a. Field Sampling Methods and Materials ...... 11

b. Experimental Design ...... 11

c. Laboratory Methods ...... 14

4. RESULTS ...... 18

a. Time Series Major Elements (Aqueous) ...... 18

b. Time Series Minor Elements (Aqueous)...... 20

c. Time Series Trace Elements (Aqueous) ...... 22

d. Time Series Solid Phase Sample Composition ...... 31

e. Concentration Variation Major Elements (Aqueous) ...... 36

f. Concentration Variation Minor Elements (Aqueous) ...... 39

g. Concentration Variation Trace Elements (Aqueous) ...... 40

h. Concentration Variation Solid Phase Sample Composition ...... 42

i. Geochemical modeling ...... 45

5. DISCUSSION ...... 49

6. CONCLUSION ...... 55

REFERENCES ...... 57

APPENDIX I ...... 60

APPENDIX II ...... 72

vii

LIST OF TABLES

Table 1. Time Series Experiment Design ...... 10

Table 2. Concentration Variation Experiment Design ...... 11

Table 3. Time Series Major Element Chemistry (aqueous) ...... 17

Table 4. Time Series Major Element Chemistry (aqueous), cont...... 17

Table 5. Time Series Minor Element Chemistry (aqueous) ...... 18

Table 6. Time Series Trace Element Chemistry (aqueous) ...... 19

Table 7. Time Series Trace Element Chemistry (aqueous), cont...... 20

Table 8. Regression results of Time Series trace elements ...... 29

Table 9. Concentration Variation Major Element Chemistry (aqueous) ...35

Table 10. Concentration Variation Major Element Chemistry (aqueous), cont...... 36

Table 11. Concentration Variation Minor Element Chemistry (aqueous) .37

Table 12. Concentration Variation Trace Element Chemistry (aqueous) ..38

Table 13. Regression results of Concentration Variation trace elements ..40

Table 14. Trace metals found in salt marshes in South Carolina (Sanger et al., 1999) and trace metal averages for Time Series Experiment sediment ...... 50

viii

LIST OF FIGURES

Figure 1. Transformation of Fe (II, III) at an oxic-anoxic boundary in a water or sediment column ...... 4

Figure 2. Main principle of aerobic degradation of hydrocarbons by microorganisms ...... 5

Figure 3. Location of Bayou La Batre ...... 9

Figure 4. Aerial View of Sample Site Location ...... 10

Figure 5. Trend in iron concentration for Time Series Experiment aqueous samples...... 21

Figure 6. Trend in copper concentration for Time Series Experiment aqueous samples...... 24

Figure 7. Trend in nickel concentration for Time Series Experiment aqueous samples...... 25

Figure 8. Trend in zinc concentration for Time Series Experiment aqueous samples...... 26

Figure 9. Trend in vanadium concentration for Time Series Experiment aqueous samples...... 27

Figure 10. Trend in arsenic concentration for Time Series Experiment aqueous samples...... 28

Figure 11. Trend in Time Series minor and trace metals concentrations in aqueous samples...... 29

Figure 12. Trend in pH for Time Series aqueous samples ...... 30

Figure 13. Time Series Experiment solid phase trace element concentrations ...... 32

Figure 14. Time Series Experiment solid phase trace element concentration trends ...... 33

Figure 15. Time Series Experiment solid phase zinc concentration trend .34

Figure 16. Time Series Experiment solid phase iron concentration trend .35

Figure 17. Trend in aqueous iron concentration for Time Series Experiment Samples and the 21 day Concentration Variation Experiment sample containing 500 ppm oil (MC252-500) ...... 41

Figure 18. Trend in Concentration Variation solid phase trace elements ..43

Figure 19. Trend in Concentration Variation solid phase trace elements, cont...... 44

Figure 20. Calculated mineral saturation index values for Time Series Experiment 14 day sample (BLB-14d) and controls ...... 46

Figure 21. Saturation index trend for Time Series Iron Oxyhydroxides ...47

Figure 22. Mineral saturation indices for Concentration Variation Experiment aqueous samples ...... 48

CHAPTER 1

INTRODUCTION

On April 20th, 2010, the Deepwater Horizon oil drilling rig, located in the Gulf of

Mexico about 41 miles off the Louisiana coast (28.74°N, 88.39°W), exploded, burned for two days, and sank. The explosion killed 11 platform workers and injured 17, starting an underwater oil leak that would continue for 86 days. The vertical depth of the oil well is 10, 683m. The

Deepwater Horizon oil spill is now the largest offshore oil spill in U.S. history, surpassing the

1989 Exxon Valdez oil spill off the coast of Alaska. The estimated amount of crude oil that gushed into the Gulf of Mexico is about 4.9 million gallons. The oil slick, which was measured at roughly 75,000 square kilometers, traveled with ocean currents and reached the coasts of

Louisiana, Mississippi, Alabama, and Florida (Cleveland et al. 2011). A few weeks after the spill, the U.S. EPA and Coast Guard approved (somewhat controversially) the use of chemical dispersants to break the oil slick into small droplets, thus presumably making it more readily bioavailable.

The effects of the oil on wildlife and local economies were damaging. Up to 32 national wildlife refuges along the Gulf Coast were affected by the oil spill, according to the U.S. Fish and Wildlife Service (Cleveland et al. 2011). The immediate effects of the BP oil spill on wildlife were devastating. As of January 25 th , 2011, 2079 oiled birds, 456 oiled sea turtles, and

6,833 dead animals (birds, mammals, sea turtles and other reptiles) were reported by the U.S.

Fish and Wildlife Service and the National Oceanic and Atmospheric Administration (USFWS,

NOAA 2011).

A federal team of investigators found layers of oil buried beneath four to five inches of sand along the Bon Secour National Wildlife Refuge (Raines 2010). A group of scientists from

Auburn University found evidence that remnant oil exists in the form of tar balls and submerged, discontinuous tar mats buried a few centimeters to a meter below the surface slightly offshore of

Alabama beaches (Hayworth et al. 2011).

Storm surges, caused by hurricanes, are another potential threat to Gulf Coast ecosystems. Storm surge can cause mixing of coastal sediments and transfer of sediment from offshore to coastal or lagoonal areas (Mount 1984). Mixing could release trapped layers of oil from contaminated sediment and storm surge could bring weathered crude oil into shallower water to contaminate coastal wetland, estuary, and shelf sediments. This may be more likely to happen in the future due to the increase in hurricane frequency and intensity observed since 1995

(Webster et al. 2005).

The Macondo oil (Mississippi Canyon Block 252 or “MC-252”) discovered by

Deepwater Horizon is a light, sweet crude oil, which is a complex mix of hydrocarbons and other chemicals. MC-252 is low in sulfur content and high in alkanes (NOAA 2010). Alkanes are hydrocarbons, also known as paraffins, where the carbon atoms form single covalent bonds in chains that are easily bioavailable as food for microorganisms (Manahan 2001). Alkanes are generally the most readily degradable components in petroleum (Venosa and Zhu 2003).

Once crude oil is released into the ocean, the contaminants are immediately subject to weathering and to transport by currents and dispersive processes towards the shore (Urriza

2010). Weathering is an overall process involving evaporation of VOC’s, dissolution of the water soluble compounds, dispersion, photochemical oxidation, oil emulsification (which forms

“mousse”), absorption onto suspended particulate materials, and sinking/sedimentation (Maure

1995). In a study conducted after the Prudhoe Bay oil spill, it was found that more than 51% of the oil became attached to particulates after the first day of the experiment (Wong et al. 1984).

This study is focused on the oil that goes through the process of sedimentation, where oil near the shore adheres rapidly to particulate matter and is deposited with the sediment (Maure 1995).

There are several potential ways in which trace elements may be introduced into the environment after an oil spill: (1) adsorbed trace elements may be released from coastal sediments via reductive dissolution of Fe (III) solid phases; (2) trace elements associated directly with the crude oil may partition to the sediment/water interface; and (3) released drilling fluids and/or produced waters are common sources of trace element contamination around oil wells.

Iron oxyhydroxide solid phases readily form between Fe (III) and other trace metals, especially in aqueous semi-alkaline environments like the ocean (Krauskopf and Bird 2003). For example, hydrous ferric oxide (HFO) can form via the following reaction:

3+ - Fe (aq) + 3OH (aq) <==> Fe(OH) 3(s)

HFO and other iron oxyhydroxides have high specific surface areas and high adsorption capacities for trace elements (Manahan 2001). Adsorption on Fe (III) solid phases is important in the transport and sequestration of trace metals in the environment (Benjamin and Leckie

1980). The reduction of Fe (III) minerals in aquatic systems is part of the iron cycle:

+ 2+ Fe(OH) 3(s) + 3H + e- <==> Fe + 3H 2O

Reduction of ferric oxyhydroxides can be induced by the oxidation of organic compounds. These redox reactions can be mediated by several different processes, either

enzymatic or non-enzymatic. During microbial degradation, carbon dioxide is released into the water to form carbonic acid, which lowers the pH. This influences the mechanism of ion exchange by creating a greater net positive charge on the solid surfaces and enhancing desorption of trace elements. Most of the Fe (III) in sedimentary environments is reduced by microorganisms which gain energy for growth by oxidizing organic compounds, using Fe (III) as an electron acceptor (Lovley 1991). Figure 1 shows how Fe changes between the solid and aqueous phases through oxidation and reduction. Figure 2 shows the main aerobic pathway used when microbes degrade organic pollutants.

Figure 1. Transformation of Fe (II, III) at an oxic-anoxic boundary in a water or sediment column. Note: The Fe (III) peak lays on top of Fe (II) peak. (Stumm and Morgan 1996)

Figure 2. Main principle of aerobic degradation of hydrocarbons by microorganisms (Das 2011)

If oil cannot be visibly detected, the presence of certain metals in the water or sediment may be an indication of oil contamination. Trace metals often found in relation to crude oil include cadmium, chromium, copper, lead, manganese, nickel, vanadium, and zinc (Sneddon et al. 2006). Heavy metals have been found in sediment near offshore drilling sites (Newbury

1979). The presence of crude oil in coastal sediments could induce reductive dissolution of iron oxy-hydroxide phases present. Aromatic hydrocarbons, especially toluene, benzoate, phenol, and p-cresol have been found to stimulate oxidation by certain types of bacteria and induce the reduction of Fe (III) (Lovley 1991). Trace elements (along with phosphate) which are sorbed onto ferric oxyhydroxide phases will desorb as the iron is reduced and enter the water column

(Lovley 1991). This is of concern because benthic organisms can bio-accumulate trace elements, allowing them to enter the food cycle (Sneddon et al. 2006).

The hypothesis to be tested by the proposed research is: Contamination of coastal sediments by oil will affect the concentration and distribution (between solid and aqueous phases) of trace elements in coastal sediment/water systems. Contamination includes trace metals, which can bind to the sediment or be released in the water column. The objectives of this study are: (1) collect sediment and water samples from the Bayou La Batre salt marsh; (2) characterize the water and sediment chemistry; and (3) conduct microcosm experiments (as explained in Methods section) to determine the effect of crude oil on the concentration and mobility of metals in coastal sediments.

The purpose of this study is to gain a better understanding of how crude oil contamination affects the geochemistry of nearshore zones of the Gulf coast. This work contributes to ongoing research into the effects of the Deepwater Horizon oil spill on the environment. The geochemical aspects and the biological aspects of the oil spill contamination

must be examined in order to ensure the safety of the organisms living in the contaminated area, as well as organisms which may ingest the toxic substances and transport it up the food chain to humans. The microcosm experiments will serve as a baseline for oiled areas in the Gulf of

Mexico that are undergoing clean-up efforts. This study will help predict the effects oil transported into coastal sediments by hurricanes or other storm events could have on the distribution of trace elements between the sediment and the water column. This is important because released trace elements could bio-accumulate and negatively affect organisms living in the coastal environment. The goal of this study is to determine whether oil spill contamination can stimulate adsorption, desorption, or dissolution of trace elements in coastal sediment and the rates at which these processes take place.

CHAPTER 2

STUDY AREA

The study site is the Bayou La Batre salt marsh (30.38°N, 88.30°W). It lies on the

Alabama Gulf Coast in southern Mobile County about 10 miles west of Mobile Bay (Fig. 3 and

Fig. 4). The sample site is indicated by the yellow star on both figures. The study area is considered coastal lowlands, with generally flat, swampy areas underlain by alluvial, deltaic, estuarine, and coastal deposits. The sediment consists of inorganic clays of high plasticity, poorly-graded sands, silty-sand, and sandy-clay. The upper two to five feet of sediment consist of very soft, black to dark gray clay. This sediment contains a high percentage of water by weight and organic materials in concentrations of 8% to 24% by weight (Graham 1988).

Previous studies at Bayou La Batre have shown that sediments may contain unusually high concentrations of metals and other constituents. However, the concentrations of nutrients, heavy metals, high molecular weight hydrocarbons, and pesticides are highly variable. In a 1977 study by the Gulf South Research Institute, it was found that mercury, arsenic, copper, zinc, cadmium, and lead existed in concentrations greater than average crustal abundance. Highest metal concentrations exist in the fine-grained, organic-rich sediments (Graham 1988).

Figure 3. Location of Bayou La Batre

CHAPTER 3

METHODOLOGY

Field Sampling Methods and Materials

Coastal salt water marsh sediment and seawater samples were collected from the Bayou

La Batre sampling site prior to the two experiments, on March 17 th , 2011 and September 12 th ,

2011. Sediment and water samples were collected about 6-9 meters from the shoreline. The sediment samples were collected from the top 15-30 cm layer using a shovel (covered in duct tape to avoid contamination from the metal of the shovel head) and stored in an acid-washed bucket. The bucket of sediment was refrigerated upon return to the laboratory. Before the sampling event, 1 liter polypropylene bottles were rinsed with doubly deionized (DDI) water having >18 M Ω resistivity, then soaked in a 15% nitric acid bath for seven days, rinsed 5 times with DDI water, and soaked for three days in DDI water. At the sampling site, the DDI-filled acid-washed bottles were emptied away from the sampling location, rinsed three times with seawater, then filled and capped under the surface. The bottles were transported on ice and refrigerated upon return to the laboratory, about six hours after sample collection. A two-liter sample of MC-252 oil was obtained from BP’s Houston office.

Experimental Design

The collected sediment and seawater samples were used to set up two bench-top microcosm experiments (Time Series and Concentration Variation) in 500 ml glass jars with polypropylene lids. The glass jars were acid-washed and combusted at 450°C for four hours to remove remaining organics. The jar lids were also acid-washed and rinsed with methanol,

acetone, and hexane to remove organics. Six microcosms served as experimental controls. They contained either water and sediment, or water and sediment with oil. Four controls were sterilized and two controls were not sterilized to determine the influence of microbial processes.

The microcosm experiments consisted of: (1) a Time Series experiment using 500 ppm MC-252 oil, and (2) a Concentration Variation experiment using ranges (0 ppm, 10 ppm, 100 ppm, 500 ppm, 1000 ppm, 2500 ppm) of MC-255 oil, concentrations representative of those observed in coastal sediments after the Deepwater Horizon accident (USFWS 2011). All microcosms were set up in duplicate.

The sample labels are identified as Bayou La Batre (“BLB”) and the corresponding number represents the number of hours or days the jar was shaking on the shaker table. For example, “BLB-6” is the jar that was harvested after six hours. Samples from jar “BLB-7d” were harvested after seven days. The controls are labeled as non-sterile controls (NSC) or sterile controls (SC). The non-sterile control (NSC) and one sterile control (SC1) microcosm jars were free of oil.

Table 1. Time Series Experiment Design

Time Oil Sterile/ Sediment Seawater Sample Name (days) (ppm) Non-Sterile (g) (g) BLB-0 0 500 Non-Sterile 326.94 173.06 BLB-6 0.25 500 Non-Sterile 326.94 173.06 BLB-12 0.5 500 Non-Sterile 326.94 173.06 BLB-24 1 500 Non-Sterile 326.94 173.06 BLB-48 2 500 Non-Sterile 326.94 173.06 BLB-7d 7 500 Non-Sterile 326.94 173.06 BLB-14d 14 500 Non-Sterile 326.94 173.06 NSC 14 0 Non-Sterile 326.94 173.06 SC1 14 0 Sterile 326.94 173.06 SC2 14 500 Sterile 326.94 173.06

Table 2. Concentration Variation Experiment Design

Oil Sterile/ Sediment Seawater Sample Name (ppm) Non-Sterile (g) (g) NSC-0ppm 0 Non-Sterile 281.56 118.45 MC252-10 10 Non-Sterile 281.56 118.45 MC252-100 100 Non-Sterile 281.56 118.45 MC252-500 500 Non-Sterile 281.56 118.45 MC252-1000 1000 Non-Sterile 281.56 118.45 MC252-2500 2500 Non-Sterile 281.56 118.45 SC-0ppm 0 Sterile 281.56 118.45 SC-10ppm 10 Sterile 281.56 118.45 SC-100ppm 100 Sterile 281.56 118.45 SC-500ppm 500 Sterile 281.56 118.45 SC-1000ppm 1000 Sterile 281.56 118.45 SC-2500ppm 2500 Sterile 281.56 118.45

In the Time Series Experiment, 326.94g of wet sediment and 173.06g of seawater were measured into 500mL glass jars (with lids) and spiked with 500 ppm MC-252 oil. Twenty jars, including duplicates and both sterile (autoclaved) and non-sterile controls, were placed on a shaker table at 100 rpm for a total experiment time of 14 days. The jars were sacrificed at predetermined time intervals of zero hours, six hours, 12 hours, 24 hours, 48 hours, 7 days, and 14 days.

In the Concentration Variation Experiment, 281.56g of sediment and 118.45g of seawater were weighed in the 500mL glass jars (with lids) and spiked with a range of MC252 oil: 10 ppm,

100 ppm, 500 ppm, 1000 ppm, and 2500 ppm. Eighteen jars, including duplicates and both sterile (autoclaved) and non-sterile controls, were placed on a shaker table at 100rpm for a total experiment time of 21 days. The jars were sacrificed at the end of the 21 day experiment in the same manner as the Time Series Experiment.

Laboratory Methods

Each 500mL jar held 200g dry sediment and 200g of seawater. Before setting up the experiments, a Minus Water calculation was performed to determine the porosity of the sediment and the amount of sediment and seawater needed for each microcosm. A weighed amount of wet sample sediment was placed into a crucible and baked at 65-70 degrees Fahrenheit until reaching a consistent weight. The sediment was weighed at its dry weight. For the Time Series

Experiment, 27.43g of wet sediment was weighed and dried, resulting in a dry weight of 16.78g.

An algebraic expression was set up to determine that 326.94g of wet sediment is equivalent to

200g of dry sediment. Example: X/200g = 27.43g/16.78g. The seawater needed in the microcosm was determined by subtracting the amount of wet sediment needed (326.94g) from the total volume of the glass jar (500g), resulting in 173.06g of seawater. The same method was

used for the Concentration Variation Experiment, which needed 281.56g of wet sediment and

118.45g of seawater.

At the appropriate time points, microcosms were taken off the shaker table. After separation by filtering and centrifugation, the aqueous and solid phase samples were processed for analysis. The original seawater samples and the aqueous phase experiment samples were filtered through 0.45 micron nylon membrane filters and stored in 60mL PE bottles at 4°C. The aqueous samples for cation analysis were preserved by acidification with 70% w/w ultrapure

Optima Nitric Acid to 2% HNO 3 and refrigerated before analysis for major, minor, and trace element cations via inductively coupled plasma-optical emission spectroscopy (ICP-OES). The aqueous samples for anion analysis were frozen before analysis via ion chromatography (IC).

After removing the sediment from the glass jars, it was centrifuged for 20 minutes at

750rpm to extract more seawater. The wet sediment was spread onto glass watch glasses or plastic weighing trays, covered with paper towels, and allowed to air dry. The dry sediment was crushed using a mortar-and-pestle or a ceramic-lined SPEX 8510 Shatterbox to pass through a

400 mesh sieve. The sieved sediment was a bulk mount for X-Ray Diffraction (XRD) analysis.

The remaining crushed sediment was used to make clay smears. About 2g of crushed sediment was floated in a 1L glass beaker for approximately 45 minutes until the heavier minerals settled.

The top layer of water containing the suspended sediment was filtered using a 45 micron filter.

The filter was pressed onto a slide and analyzed via XRD.

Microwave-assisted partial acid digestion was performed using EPA Method 3051A (UA

EPA 1994). Approximately 0.5g of the uncrushed sediment and 10mL of 70% w/w Optima

Nitric Acid (HNO 3) were added into microwave digestion vessels. Samples were digested, six vessels at one time, inside a Milestone mls 1200 mega High Performance Microwave Digestion

Unit. Each run contained one “blank” vessel that only contained acid. Between each run, the vessels were cleaned with 10mL HNO3 and soaked in deionized water overnight. The sediment digestate solutions were refrigerated in 30mL PE bottles until analysis for major, minor, and trace element cations via inductively coupled plasma optical emission spectroscopy (ICP-OES).

Mineral saturation indices were calculated using the PHREEQC geochemical modeling program.

Immediately prior to analysis, instrument calibration standards were prepared by dilution with a 5000 ppm Na solution to matrix-match the experimental samples and thus obtain more accurate values for the major elements. The undiluted samples were analyzed for minor elements and trace elements. The ICP-MS was also used to analyze and obtain more accurate values for arsenic, cobalt, copper, selenium, titanium, and zinc in the Time Series aqueous samples.

Measurement of pH was taken with a VWR symphony SP90M5 meter. To calibrate, the pH electrode and ATC probe were rinsed with deionized water and placed in a buffer solutions with pH of 4, 7, and 10. When the pH icon stopped flashing, the meter displayed the actual pH value read by the electrode. The calibration button was pressed until the value of the flashing digit matched the actual value of the buffer solution. Each pH buffer solution was used in this manner until the meter was fully calibrated to all three buffer solutions.

Measurement of TDS (total dissolved solids) was performed using a gravimetric TDS measurement. The weight of a crucible was measured and a measured amount of unfiltered seawater was added. The crucible and water were baked in a small oven at around 21°C with a piece of aluminum foil lightly covering the crucible. After all water evaporated, the crucible was removed from the oven, allowed to cool, and weighed. The weight of the empty crucible was subtracted from the final weight of the crucible to determine TDS.

The unfiltered seawater was titrated to calculate alkalinity using methods outlined in

Standard Methods for the Examination of Water and Wastewater (APHA 2006). The seawater was titrated with a solution of 0.0213N HCl. The HCl acid solution was added to the seawater in a titration burette until the inflection point of 4.3pH was reached. During the addition of the acid, the solution was agitated using a magnetic stirrer. Duplicate titrations were performed for each sample. Alkalinity was calculated by multiplying the volume (mL) of HCl used, the normality of the HCL, and a factor of 50,000. This number was divided by the volume (mL) of the seawater sample. The resulting number is the total alkalinity measured in mg/L CaCO 3.

Geochemical modeling of the Time Series Experiment and Concentration Variation

Experiment aqueous samples was completed using PHREEQC (Parkhurst and Appelo 1999).

Specifically, this computer code was used to perform element speciation and mineral saturation index calculations to aid in the interpretation of chemical trends observed in aqueous and solid phase samples. Mineral saturation index (SI) values were calculated using PHREEQC for both experiments. The SI value is calculated by comparing the chemical activities of the dissolved ions of the mineral (ion activity product) with the mineral solubility product (K sp ):

SI = log(IAP/K sp )

A mineral saturation index shows whether water will tend to dissolve or precipitate a mineral. When the solution is undersaturated with respect to a mineral, the SI value will be negative. When the solution is supersaturated with respect to a mineral, the SI value will be positive.

CHAPTER 4

RESULTS

Time Series Experiment

Sediment and seawater, collected from a salt marsh at Bayou La Batre, Alabama, were measured into jars and spiked with 500 ppm MC-252 oil. Twenty microcosms, including duplicates and both sterile and non-sterile controls, were placed on a shaker table at 100rpm. At predetermined time points (0 hours, 6 hours, 12 hours, 24 hours, 48 hours, 7 days, and 14 days) the microcosms were taken down and harvested for analysis. The solid phase and aqueous samples were analyzed by ICP-OES, ICP-MS, and IC

Major Elements (Aqueous) - Major element concentrations in the seawater sample are in the 100-20,000 ppm range. Total dissolved solids (TDS) for the BLB seawater was 2916 ppm, which is appropriate for brackish water (Water Quality Association). The alkalinity of the

BLB seawater was 83.07mg CaCO 3/L. Chloride, sodium, calcium, potassium, and magnesium are present in levels normal for brackish water in the Time Series Experimental aqueous samples. Sulfate, bromide, silica, and nitrate (in most samples) concentrations are elevated even for pure ocean water (Pilson 1998). These results are shown in Table 1.A in Appendix I.

Major element chemistry and the corresponding pH for the Time Series Experiment aqueous samples are listed in Tables 3 and 4.

Table 3. Time Series Major Element Chemistry (aqueous)

Time Ca Mg Na Sample Name (days) pH (mg/L) (mg/L) (mg/L) BLB-0 0 7.96 207.92 654.82 5377.25 BLB-6 0.25 7.97 209.33 660.57 5377.51 BLB-12 0.5 7.76 209.51 655.72 5398.63 BLB-24 1 7.65 206.73 636.45 5234.75 BLB-48 2 7.96 209.06 659.89 5359.23 BLB-7d 7 7.48 217.95 660.22 5365.29 BLB-14d 14 7.57 211.92 652.17 5359.67 NSC (no oil) 14 7.70 213.64 640.75 5222.02 SC1 (no oil) 14 7.52 219.89 620.76 5529.32 14 7.54 223.34 632.95 5692.67 SC2 (500ppm)

Table 4. Time Series Major Element Chemistry (aqueous), cont.

K SO 4 Cl Br Sample Name (mg/L) (mg/L) (mg/L) (mg/L) BLB-0 213.68 1996.05 13738.03 319.20 BLB-6 212.67 1950.18 13956.20 302.40 BLB-12 215.83 1892.06 14305.12 269.15 BLB-24 215.33 2318.33 15224.33 400.27 BLB-48 208.86 1698.84 11265.23 243.82 BLB-7d 208.07 1790.99 12263.12 284.82 BLB-14d 211.79 1895.84 13574.35 301.65 NSC (no oil) 213.64 1253.50 8541.79 177.04 SC1 (no oil) 245.66 2540.58 17860.97 441.96 251.33 2073.07 14560.50 332.07 SC2 (500ppm)

Minor Elements (Aqueous) – Minor elements are defined as existing in the 1-100 ppm range. Minor element chemistry of the Time Series Experiment aqueous samples is shown in

Tables 5. Iron is elevated in samples BLB-7d and BLB-14d. Aluminum, boron, barium, iron, manganese, molybdenum, nickel, and strontium values were obtained by ICP-OES. Beryllium, cadmium, chromium, silver, Sn, Sb, Tl and lead concentrations were less than the limit of detection.

In all data plots, error bars were calculated for the y-axis using the standard deviation of the replicates (jar 1 and jar 2). For the microwave digestions, error bars were plotted using the standard deviation of the average of the triplicate samples. In the case of no replication, the error bars were plotted using the %RSD of the calibration standards used in ICP-OES analysis. So error is equivalent to the %RSD multiplied by the concentration of the calibration standard nearest to the actual sample concentration.

Table 5. Time Series Minor Element Chemistry (aqueous)

Time B Sr Fe Mn Si Sample Name (days) pH (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) BLB-0 0 7.96 2.29 3.77 0.0535 2.3905 3.73 BLB-6 0.25 7.97 2.32 3.89 0.0382 2.2743 3.71 BLB-12 0.5 7.76 2.25 3.80 0.0450 1.6589 3.52 BLB-24 1 7.65 2.24 3.80 0.0680 0.7759 3.39 BLB-48 2 7.96 2.20 3.84 0.0417 1.4464 4.06 BLB-7d 7 7.48 2.37 3.85 7.6312 4.8481 8.05 BLB-14d 14 7.57 2.51 3.84 6.1290 4.6048 9.88 NSC (no oil) 14 7.70 2.33 3.97 0.1208 2.3129 8.06 SC1 (no oil) 14 7.52 3.36 4.08 0.2552 3.6001 9.26 14 7.54 3.54 4.34 0.0998 3.5161 10.37 SC2 (500ppm)

Figure 5. Trend in iron concentration for Time Series Experiment aqueous sample s

The graph in Figure 5 tracks changes in iron concentration in the aqueous samples over time in the Time Series Experiment. The iron concentration remained approximately constant through day 2 of the experiment . After the second day, there was an increase in iron concentration from ~0 ppm to 8 ppm by day 8. After day 8, the concentration fell to ~6 ppm by the end of the 14 day experiment. The average concentration for the experimental duplicates is shown on the graph, but values for the two jars are also shown to illustrate the differences between the duplicate jars for the iron concentrations . Possible reasons for the large standard deviations will be discussed later. The non-sterile control and two sterile controls show ed lower concentrations of iron at the end of the experiment , compared to the iron content s of the experimental solutions.

Trace Elements (Aqueous) – Trace elements are defined as those having concentrations less than 1 ppm. Trace elements for the Time Series Experiment aqueous samples are shown in

Table 6 and Table 7. Zinc, nitrate, molybdenum, nickel, copper, aluminum, arsenic, cobalt, titanium, barium, selenium, and vanadium values were obtained by ICP-MS. The nitrate value for SC1 is high, but may be due to contamination by nitric acid that was used in preparing the jars for the experiment.

Table 6. Time Series Trace Element Chemistry (aqueous)

Time Zn NO 3 Mo Ni Cu Sample Name (days) pH (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) BLB-0 0 7.96 0.061 0.000 0.017 0.001 0.138 BLB-6 0.25 7.97 0.048 1.195 0.017 0.001 0.123 BLB-12 0.5 7.76 0.056 0.000 0.017 0.001 0.110 BLB-24 1 7.65 0.060 1.529 0.015 0.033 0.102 BLB-48 2 7.96 0.048 0.000 0.010 0.001 0.097 BLB-7d 7 7.48 0.043 0.000 0.008 0.001 0.091 BLB-14d 14 7.57 0.082 1.149 0.014 0.001 0.092 NSC (no oil) 14 7.70 0.074 0.714 0.011 0.001 0.089 SC1 (no oil) 14 7.52 0.048 10.979 0.054 0.005 0.117 SC2 (500ppm) 14 7.54 0.055 2.571 0.054 0.004 0.112

Table 7. Time Series Trace Element Chemistry (aqueous), cont.

Al As Co Ti Ba Se V Sample Name (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) BLB-0 0.042 0.030 0.045 0.847 0.069 0.073 0.095 BLB-6 0.036 0.024 0.043 0.831 0.063 0.075 0.095 BLB-12 0.041 0.041 0.611 0.832 0.058 0.054 0.095 BLB-24 0.042 0.073 0.209 0.828 0.039 0.072 0.095 BLB-48 0.038 0.019 0.027 0.827 0.036 0.066 0.095 BLB-7d 0.039 0.022 0.039 0.838 0.046 0.069 0.095 BLB-14d 0.044 0.026 0.024 0.812 0.044 0.060 0.095 NSC (no oil) 0.046 0.034 0.010 0.838 0.037 0.061 0.095 SC1 (no oil) 0.232 0.043 0.010 0.899 0.046 0.087 0.152 0.093 0.040 0.046 0.048 0.068 0.154 SC2 (500ppm) 0.909

Figure 6. Trend in copper concentration for Time Series Experiment aqueous samples

The average aqueous copper concentration (Figure 6) decreased rapidly during the first two days from ~0.14 to ~0.10 mg/L, then decreased more gradually between the second and seventh day. The average aqueous copper concentration slowly decreased between day 7 and day 14. The decreases in copper coincide with the decrease of iron shown in Figure 3 for the last week of the experiment. The non -sterile control (NSC) for the Time Series Experi ment matches the concentration of the copper in the jars at day fourteen, but the copper concentrations of the two seawater samples were slightly lower. However, the sterile controls (SC1 and SC2) were slightly higher in concentration at the 14 day point, but they were not as high as the starting point concentration for copper.

Figure 7. Trend in nickel concentration for Time Series Experiment aqueous samples

The average nickel concentration (Figure 7) peaked to 0.033pm at one day. The large error bar suggests this peak may not be real. It decreased between day one and day two to close to 0 ppm. The initial seawater sample nickel concentration was closer to the concentration at day

1. The Ni concentration of the non -sterile control closely matches the values for all samples with the exception of day 1. The Ni concentrations of the sterile controls were both slightly higher than that for the day 14 sample.

Figure 8. Trend in zinc concentration for Time Series Experiment aqueous samples

The average zinc concentration (Figure 8) decreased from ~0.06 ppm to ~0.045 ppm in the first six hours, then increased back up to 0.06 ppm at day one. Zinc concentration decreased to

~0.04 ppm between d ay one and day 7, then increased to a maximum on day 14 . For the last week, there was an increase to ~0.08 ppm. The trends between day two and fourteen is inverse to the trend in the iron graph during that time. Both of the non -sterile controls match the concentrations in the jar closely. T he seawater has a lower concentration than the jar.

Figure 9. Trend in vanadium concentration for Time Series Experiment aqueous samples

The average vanadium concentration (Figure 9) remained constant over time. The non- sterile control was higher than the starting point of the experiment. The non-sterile control matches the concentration for day 14. The seawater had a higher value than the starting concentration in the experiment. The sterile controls are both higher than the ending point at day

14 at ~0.15 ppm.

Figure 10. Trend in arsenic concentration for Time Series Experiment aqueous samples

The average arsenic concentration (Figure 10) decreased slightly in the first six hours, increased to a peak of ~0.07 ppm at day one, then decreased again until day three. There was a gradual increase in As concentration from ~0.02 ppm at day three until day 14 at ~0.024 ppm.

The As concentrations of the non -sterile control samples were approximately the same as the 14 day sample. The seaw ater sample had slightly lower As concentrations than the microcosm sample.

Figure 11. Trend in Time Series minor and trace metal concentrations in aqueous samples

The average aqueous concentrations of trace metals commonly associated with crude oil

(Cu, Ni, Zn, V, Cd, Pb) are shown in Fig. 11. As shown in the previous figures, vanadium, lead, and nickel remain fairly constant . Concentrations of zinc, vanadium, and copper are higher than concentrations of nickel and lead. Nickel, lead, copper, zinc, vanadium, and arsenic levels at the end of 14 days were the same or approximately the same as in the non -sterile controls (Figs. 6-

10).

Figure 12. Trend in pH for Time Series aqueous samples

The changes in pH in the aqueous samples are shown in Fig. 12. The pH show ed variability within the first two days of the Time Series Experiment , which coincide d with concentration trends observed in the trace element plots. Solution pH decreased between day 2-

7, then increased slightly during the second week of the experiment. The pH values of the microcosm samples were higher than those of the original seawater and the non -sterile controls for the first week of the experiment, but then lower. The pH in the two sterile control s was lower than the pH of the seawater sample and non-sterile controls.

Solid Phase Sample Composition - XRD data showed the presence of Quartz (SiO 2),

Kaolinite (Al 2Si 2O5(OH) 4), Saponite (Ca(Mg,Fe) 3((Si,Al) 4O10 )(OH) 2(H 2O), Microcline

(KAlSi 3O8), and Hematite (Fe 2O3). Microwave digestion data for the Time Series Experiment solid phase samples are given in Appendix I, Tables 1.D.1.–1.D.6. Nickel, lead, copper, zinc, vanadium, and arsenic levels at the end of 14 days were the same or approximately the same as in the non-sterile controls (Figure 14). With the exception of nickel, the 14 day samples contained lower trace metal concentrations than the sterile control which contained no oil.

Correlation coefficients were calculated for the Time Series solid phase samples between iron and trace elements (aluminum, cobalt, chromium, copper, lead, manganese, nickel, and zinc). Cobalt, chromium, nickel, and aluminum are significantly correlated with iron, indicated by the correlation coefficient (r) in Table 8. Bold numbers in the table indicate significant correlations (r>0.75, p=0.0002).

Table 8. Regression results of Time Series trace elements. Al As Co Cr Cu Fe Mn Ni Pb Zn 1.00 -0.20 0.62 0.98 0.04 0.79 0.30 0.87 0.30 0.02 Al 1.00 0.18 -0.23 0.31 -0.01 0.09 0.06 0.20 0.27 As 1.00 0.58 0.29 0.75 0.44 0.83 0.10 0.06 Co 1.00 0.04 0.79 0.31 0.84 0.36 0.07 Cr 1.00 0.14 0.69 0.19 0.30 0.00 Cu 1.00 0.44 0.88 0.39 0.02 Fe 1.00 0.45 0.50 0.19 Mn 1.00 0.38 0.09 Ni 1.00 -0.03 Pb 1.00 Zn

Figure 13. Time Series Experimen t solid phase trace element concentration trends.

Figure 13 shows Time Series Experiment solid phase concentrations of trace elements commonly associated with crude oil, as determined by microwave -assisted acid digestion. Zinc concentrations are higher than nickel, lead, and copper concentratio ns, in all four samples.

Vanadium and arsenic concentrations were below detection. There was no significant difference between the solid phase trace element concentrations of the non -sterile control, the sterile control that contained no oil, and the 14 d ay sample. However, the Zn, Pb and Ni concentrations of these three samples were higher than those of the sterile control that contained oil.

Figure 14. Time Series Experiment solid phase concentration trends.

Figure 15. Time Series Experiment solid phase zinc concentration trends.

Figure 14 and Figure 15 show changes in the solid phase concentrations of trace elements commonly associated with crude oil. The plots show a difference in concentration between zinc and the other four elements ( copp er, nickel, lead, and titanium ). For this reason, zinc was plotted separately. The concentrations of nickel and titanium remain constant throughout the experiment. Lead showed a very slight increase in the first three days, then a decrease of ~0.5 ppm between day 3 and day 7, then a slight increase of ~0.4 ppm at day 14. Copper peak ed at ~5 ppm at six hours, decreased after 12 hours to ~4.2 ppm, increased slightly by day 2, then decreased to ~4 ppm at day 14. Zinc concentration increased from ~40 ppm to ~55 ppm between

6-12 hours, decreased to ~37 ppm after twelve hours, increased again to ~50 ppm between day 2-

7, then decreased to ~37 ppm at day fourteen.

Figure 16. Time Series Experiment solid phase iron concentration trend.

The iron content of the solid phase (Figure 16) decreased during the six to twelve hour period. Iron increased between day one and day two, decrease d between day 2-7, and increased during the last week of the experiment. These solid phase concentration trends vary inversely with the iron concentrations trends in the aqueous samples. This is expected to occur as iron leaves the solid phase to enter the solution, and vice versa.

Concentration Variation Experiment

Sediment and seawater, collected from a salt marsh at Bayou La Batre, Alabama, were measured into jars and spiked with a range of concentrations (0 ppm, 10 ppm, 100 ppm, 500 ppm, 1000 ppm, and 2500 ppm) of MC-252 oil. Eighteen microcosms, including duplicates and both sterile and non-sterile controls, were placed on a shaker table at 100rpm. At the end of three weeks, the microcosms were taken down and harvested for analysis. The solid phase and aqueous samples were analyzed by ICP-OES, ICP-MS, and IC.

Major Elements (Aqueous) – Major elements of seawater exist in the 100-20,000 ppm range. Major element chemistry and the corresponding pH values are listed in Tables 9 and 10.

The sample labels are designated as Macondo Well Oil (“MC252”) with the concentration of oil added as a suffix. For example, “MC252-10” indicates that 10 ppm crude oil was added to the microcosm jar. The controls are labeled as non-sterile controls (NSC) or sterile controls (SC).

Total dissolved solids (TDS) for the MC252 seawater was 6509 ppm, which is in the range of brackish to highly brackish water. The alkalinity of the MC252 seawater was 95.85mg

CaCO 3/L. Detailed results are shown in Appendix II, Table 2.A.

Table 9. Concentration Variation Major Element Chemistry (aqueous)

DO Ca Mg Na Sample Name (mg/L) pH (mg/L) (mg/L) (mg/L) NSC-0ppm 8.16 7.35 351.96 1076.35 8335.02 MC252-10 7.52 7.38 362.10 1085.02 8510.14 MC252-100 7.67 7.34 347.11 1089.93 8445.83 MC252-500 6.04 7.49 339.55 1084.71 8547.44 MC252-1000 7.20 7.71 333.23 1073.39 8459.84 MC252-2500 6.48 7.49 316.76 980.28 8001.09 SC-0ppm 7.89 7.89 350.51 1096.73 8944.79 SC-10ppm 6.63 7.53 343.40 1025.85 8607.06 SC-100ppm 6.89 7.70 365.73 1106.93 8873.02 SC-500ppm 7.54 7.71 383.84 1150.26 9484.43 SC-1000ppm 7.20 7.56 369.97 1111.50 9275.96 SC-2500ppm 7.61 7.73 333.91 1082.20 8599.92 8.11 7.55 327.55 1071.25 8644.21 SW-2

Table 10. Concentration Variation Major Element Chemistry (aqueous), cont.

K Cl SO 4 Br Sample Name (mg/L) (mg/L) (mg/L) (mg/L) NSC-0ppm 325.23 7335.67 1258.42 225.43 MC252-10 321.74 13051.64 1951.03 416.02 MC252-100 327.44 12980.88 1720.82 376.46 MC252-500 334.36 5521.45 1698.52 448.66 MC252-1000 326.00 9495.25 1193.18 347.21 MC252-2500 335.47 13195.39 2048.45 436.26 SC-0ppm 360.63 14818.95 2287.99 524.04 SC-10ppm 387.63 16990.10 2800.96 537.66 SC-100ppm 372.28 15744.15 2205.43 495.42 SC-500ppm 426.89 12989.20 1644.13 417.20 SC-1000ppm 402.55 15120.22 2193.03 469.85 SC-2500ppm 326.75 14762.39 2072.37 520.52 331.50 14774.35 2309.52 441.56 SW-2

Minor Elements (Aqueous) - Minor element chemistry for the Concentration Variation experiment is shown in Table 11. All minor element values were obtained by ICP-OES.

Table 11. Concentration Variation Experiment Minor Element Chemistry (aqueous)

Fe Si Mn B NO 3 PO 4 Sr V Sample Name (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) NSC-0ppm 0.85 7.44 3.08 3.63 8.48 0.00 6.34 6.20 MC252-10 14.37 10.19 2.93 3.52 26.99 0.00 6.33 6.16 MC252-100 15.32 11.29 2.98 3.46 21.43 0.00 6.22 6.08 MC252-500 4.33 11.55 2.26 3.46 0.00 2.09 6.18 6.08 MC252-1000 0.18 11.18 2.02 3.28 7.49 9.14 6.13 5.96 MC252-2500 0.04 8.22 1.48 3.29 8.00 8.89 5.68 5.42 SC-0ppm 0.03 18.08 1.32 4.11 24.42 15.45 6.21 6.04 SC-10ppm 0.04 4.97 1.97 3.89 28.60 2.17 5.98 5.82 SC-100ppm 7.16 27.61 2.58 4.37 41.02 12.64 6.38 6.25 SC-500ppm 0.03 8.00 2.00 4.11 0.00 2.63 6.56 6.35 SC-1000ppm 0.06 6.39 1.91 3.91 0.00 1.53 6.50 5.98 SC-2500ppm 0.05 10.65 1.51 3.23 3.90 12.40 6.14 5.98 SW-2 0.00 0.01 0.00 3.41 17.96 0.00 6.11 6.00

Trace Elements (Aqueous) – Trace elements are present in the 1-100 ppm range. Trace element chemistry for the Concentration Variation experiment is shown in Table 12. All trace element values were obtained by ICP-MS. Cadmium, chromium, copper, lead, titanium, and zinc were below LOD. In general, trace element values were lower in the Concentration Variation Experiment than in the Time Series Experiment.

Table 12. Concentration Variation Experiment Trace Element Chemistry (aqueous)

Al As Mo Ni Se Ba Co Sample Name (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) NSC-0ppm 0.0386 0.0052 0.0099 0.0060 0.0026 0.0468 0.0209 MC252-10 0.0730 0.0039 0.0095 0.0002 0.0026 0.0464 0.4353 MC252-100 0.1349 0.0048 0.0072 0.0002 0.0038 0.0484 0.0148 MC252-500 0.2314 0.0090 0.0071 0.0002 0.0055 0.0469 0.0011 MC252-1000 0.0503 0.0104 0.0086 0.0002 0.0095 0.0441 0.0224 MC252-2500 0.0490 0.0217 0.0739 0.0027 0.0150 0.0414 0.0043 SC-0ppm 0.0421 0.0363 0.0048 0.0002 0.0082 0.0325 0.0115 SC-10ppm 0.0657 0.0244 0.1550 0.0125 0.0026 0.0389 0.0191 SC-100ppm 0.0514 0.0394 0.0063 0.0002 0.0162 0.0422 0.0056 SC-500ppm 0.0644 0.0304 0.1611 0.0125 0.0025 0.0445 0.0314 SC-1000ppm 0.0866 0.0235 0.1646 0.0109 0.0078 0.0462 0.0075 SC-2500ppm 0.0443 0.0024 0.0078 0.0002 0.0095 0.0434 0.0103 0.0391 0.0103 0.0130 0.0006 0.0026 0.0290 0.0106 SW-2

Figure 17. Trend in aqueous iron concentration for Time Series Experiment samples and the 21 day Concentration Variation Experiment sample containing 500 ppm oil (MC252 -500).

Figure 17 is identical to the aqueous iron concentration graph from the Time Series

Experiment (Figure 5), but t he twenty -one day time point has been added from the Concentration

Variation Experiment 500 ppm sample ( MC252-500). From day 7-21, the aqueous iron concentration steadily decreased . The non -sterile control (no oil) and the ster ile control s from the Concentration Variation Experiment show lower iron concentrations than sample MC252 -

500.

Concentration Variation Solid Phase Sample Composition The sediment contained more sand than the Time Series Experiment. Correlations were calculated for the Concentration Variation sediment samples between iron and trace metals aluminum, barium, chromium, copper, nickel, lead, titanium, and zinc (Table 13). Chromium, copper, lead, and aluminum are significantly correlated (>75%) with iron. Bold numbers in the table indicate significant correlations (r>0.75, p=0.0002).

Table 13. Regression results of Concentration Variation trace elements. Al Ba Cr Cu Fe Ni Pb Ti Zn 1.00 0.81 0.99 0.89 0.91 0.70 0.72 -0.27 0.44 Al 1.00 0.77 0.58 0.57 0.27 0.41 0.27 0.18 Ba 1.00 0.94 0.95 0.70 0.79 -0.31 0.44 Cr 1.00 0.96 0.69 0.89 -0.50 0.51 Cu 1.00 0.74 0.88 -0.55 0.56 Fe 1.00 0.51 -0.57 0.48 Ni 1.00 -0.57 0.48 Pb 1.00 -0.71 Ti 1.00 Zn

Figure 18. Trend in Concentration Variation solid phase trace elements

The bar graph in Figure 1 8 shows the solid phase concentrations (ppm) of four trace elements which are commonly associated with crude oil. The plot shows that the concentration of zinc increases as the crude oil concentration increases. Solid phase l ead stays constant fo r the

10 ppm, 100 ppm, 500 ppm, and 1000 ppm , but is considerable higher in the 2500 ppm oil sample. Nickel and copper concentrations don’t exhibit any apparent relationship to the concentration of MC-252 oil . The results shown on this bar graph appear s imilar to the results of the Time Series sediment trace elements in Figure 12 in terms of relative abundances of the elements.

Figure 19. Trend in Concentration Variation solid phase trace elements, cont.

The bar graph in Figure 1 9 shows the solid phase concentrations (ppm) of four trace elements found in the solid phase microwave digestions for the sterile controls and non -sterile controls. After twenty-one days, the concentrations in the trace elements shows zinc increases from ~22 ppm to ~52 ppm as the crude oil concentration increases , with the exception of the

MC252-SC-10 sample. Lead ranges from ~2.7 ppm to ~7.3 ppm and does not show a pattern with the amount of oil added to the microcosm . Nick el and copper are in similar ranges of concentra tion and also don’t show a significant pattern.

Geochemical Modeling (Time Series Experiments) A mineral saturation index is an index that shows whether water will tend to precipitate or dissolve a mineral. When the value is negative, the mineral is dissolving. When the value is positive, the mineral is precipitating. Mineral saturation index (SI) values were calculated using

PHREEQC for the Time Series Experiment aqueous samples. Figures 20, 21, and 22 show the saturation indices for minerals which are present in the salt marsh sediment or which may be expected to form as secondary phases. Most of the salt marsh sediment minerals predicted to dissolve into the water are typical of the coastal/estuary environment; clays, iron oxides, quartz, feldspars, evaporites, and some sulfate minerals. The fourteen day sample and the non-sterile control (no oil) showed similar SI values. The sterile controls were similar to each other, but had much lower sulfate minerals (Barite, Alunite) SI values than the fourteen day sample and the non-sterile control. Figure 19 shows that iron oxyhydroxide minerals are precipitating between days 2-7 and dissolving on days 7-14.

Figure 22 shows mineral saturation indices for the Concentration Variation Experiment aqueous samples. Most of the salt marsh sediment minerals predicted to dissolve into the water are typical of the coastal/estuary environment; (clays, iron oxides, quartz, feldspars), evaporites, and some sulfate minerals. They are the same minerals predicted to be dissolving in the Time

Series Experiment. All of the aqueous samples show similar SI values, with the exception of

MC252-500, which was more undersaturated with respect to sulfate minerals (barite, gypsum, anhydrite) than the other aqueous samples and controls.

Figure 20. Calculated mineral saturation index values for Time Series Experiment 14 day sample (BLB-14d) and controls

Figure 21 . Saturation Index trend for Time Series Iron Oxyhydroxides

Figure 22. Mineral saturation index values calculated for Concentration Variation Experiment aqueous samples

CHAPTER 5

DISCUSSION

Based on the experimental results obtained, oil contamination does affect the concentration and distribution (between aqueous and solid phases) of trace elements in the coastal environment. Some trace elements (Cu, Cr, Pb, Al, Ni), but not all, correlated highly with iron in the solid phase samples. The Time Series Experiment aqueous sample plots (Figures

7,8, and 10), show that trace elements (nickel, zinc, arsenic) were released into the water within the first 24 hours, which is earlier than the 2-7 day release of iron. This indicates that iron is not the controlling factor in their release and that the most likely explanation for early trace element release is ion exchange. During microbial degradation, carbon dioxide is released into the water to form carbonic acid, which lowers the pH. This influences the mechanism of ion exchange by created a more net positive charge on the solid surfaces and encourages desorption of trace elements. Time Series solid phase sample plots show a small increase after 24 hours, which is inversely proportional to the aqueous sample plots, as would be expected. Zinc is the only trace element that increases in concentration during the last 7 days. Zinc is not highly correlated with iron or aluminum, so it may be sourced by the oil.

Bacteria are responsible for the temporal changes observed in Fe and trace element concentrations for the Time Series Experiment. Dr. Patricia Sobecky’s research group in the

University of Alabama’s Department of Biological Sciences collaborated with this study and provided some information about the microbial communities present in the Bayou La Batre salt

marsh sediment. Their samples were taken from the same location as those collected for this study. They measured total hydrocarbons in the salt marsh sediment and found the highest levels in June and July of 2010. Total hydrocarbon concentrations decreased in September of 2010 and were no longer detectable by October of 2010 (Beazley 2012). No hydrocarbons were detected at the time near-surface sediment samples were collected for use in the Time Series and

Concentration Variation experiments in March and August of 2011.

Bacteria from the phylum Proteobacteria and class Deltaproteobacteria were found in the

UA biology study and in a study of Florida beach sediment (Kostka et al., 2011). Kostka et al.,

(2011) took oiled sediment from Pensacola beach, identified and quantified the oil-degrading bacteria present in the samples, and observed the in situ response of the oil-degrading bacterial population after the Deepwater Horizon oil spill. Dr. Asim Bej’s research group in the

Department of Biology at the University of Alabama, Birmingham, identified iron-reducing bacteria in the sediment from the BLB sample site that was used in this study.

The trace element plots for Time Series Experiment samples that contained oil showed different concentration trends with time, but the sterile control samples showed almost no changes in trace element concentrations. This suggests microbial activity was the primary control of trace element behavior in the samples that were not autoclaved. As discussed in the

Introduction, bacteria are capable of producing organic chelates which solubilize Fe (III) minerals (Luther et al., 1992). When crude oil was introduced into the microcosm, the most likely catalyst for change in the experimental jars was the oil-degrading bacteria. If inorganic processes were dominant, then there would have been similar changes observed in the experimental controls. The MC-252 oil created a reducing environment and provided a carbon source to facilitate Fe reduction by bacteria.

Figures 16 and 17 show that MC-252 oil is a direct source of trace elements to the coastal environment. Comparison of aqueous major ion concentrations between the Time Series

Experiment samples and the Concentration Variation Experiment sample that contained 500 ppm oil, shows higher concentrations of all elements (with the exception of chloride) in the

Concentration Variation Experiment sample. However, the Time Series Experiment aqueous samples had higher concentrations of most trace elements compared to the Concentration

Variation Experiment, with the exception of B, Sr, and V. These differences are most likely due to the heterogeneity of the sediment. In the solid phase samples, the major ion concentrations

(Ca, Mg, and K) were lower in the Concentration Variation Experiment than in the Time Series

Experiment solid phase samples. Most of the trace element concentrations in the Concentration

Variation Experiment solid phase samples were also lower than those in the Time Series

Experiment, with the exception of Sr and Cr concentrations, which were approximately the same.

Because the trace element concentrations were mostly lower for the Concentration Variation

Experiment solid phase and aqueous samples, this indicates the small differences in element concentrations are due to heterogeneity of the sediment.

Quartz, feldspars, and carbonates are found in coastal sediment, but these minerals do not typically contain many trace metals (Windom 1989). The salt marsh sediment collected at the

BLB site contained elevated trace element concentrations, which correlated with Fe. There are higher concentrations of trace metals detected in the Time Series Experiment solid phase samples, which contained higher percentages of fine-grained particles compared to samples from the Concentration Variation Experiments.

In a South Carolina estuary study in which the authors (Sanger et al., 1999) examined the distribution of trace metals in sediment, it was found that estuaries in tidal creeks contained

significantly higher concentrations of trace metals associated with urban activites. The data collected (Table 12) by Sanger et al. (1999) from non-impacted estuaries serves as a baseline level of trace elements in their study area. The natural levels of trace elements in non-impacted estuary sediment measured by Sanger et al. (1999) were significantly higher than those determined by this study for the Bayou La Batre sediment. This indicates that the data in this study could be expected to underestimate the potential release of trace elements from sediments having higher concentrations.

Table 14. Trace metals found in salt marshes in South Carolina (Sanger et al., 1999) and trace metal averages for Time Series Experiment sediment Sal t mar sh As Cd Cr Cu P b Mn Ni Zn unimpacted (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) BS-U 21.7 0.00 72.90 20.30 22.40 252.60 18.10 76.50 BS-L 18.8

Roeder et. al. (2011) hypothesized that trace metals can be used as potential indicators of oil contamination in marine sediments and beach sands. Their study focused on finding elevated concentrations of nickel and vanadium, even after degradation of the crude oil had taken place.

In marine sediments collected in DeSoto Canyon and Pensacola Beach, Roeder et al. (2011) found enrichment with nickel (3.02-3.37 ppm), vanadium (0.95-1.17 ppm), and cobalt (0.12 ppm) at different depths. Analysis of crude oil from the Deepwater Horizon spill found elevated concentrations of nickel (0.86 ppm), vanadium (2.76 ppm), and cobalt (0.084 ppm). Partial digestion of BLB sediment showed that it contained 3.60 ppm of nickel, 28.25 ppm of vanadium, and 1.96 ppm of cobalt, comparatively. Time Series solid phase samples contained higher nickel, lower vanadium, and higher cobalt concentrations than the Roeder study. This suggests that while studies of oil spill impacted areas are helpful, the heterogeneity of sediment trace element content may be a factor to consider when interpreting different levels of trace element concentrations, even at the same site.

One mechanism for the release of trace elements in Time Series Experiment samples within the first 24 hours could be oil degradation. The size of the oil-degrading bacterial populations increased significantly in the presence of hydrocarbons and then decreased when the hydrocarbons were no longer detected (Beazley 2012). Hydrocarbon degradation tests performed by Dr. Yuehan Lu, a Biogeochemist in the Department of Geological Sciences at the

University of Alabama, found that most biodegradation occurred within the first six hours. By the end of the 14 day Time Series Experiment, about 90% of the added oil was degraded by microbial processes (Beazley 2012).

A second potential mechanism for the release of Time Series trace elements within the first 24 hours is ion exchange. The drop in pH balances with the trace element charges released

within the first 24 hours, suggesting ion exchange. Also microbial oil degradation can provide a reducing environment, which can also cause a drop in pH.

CHAPTER 6

CONCLUSIONS

The objectives of the study were to determine the impact of oil spill contamination on the concentration and mobility of trace elements in the coastal environment, to determine the mechanisms for the release of trace elements into the water column, and to collect data about sediment and seawater that can serve as data to help with clean-up efforts in oil areas in the Gulf of Mexico. It was hypothesized that oil contamination would affect the concentration of trace elements in the environment and that it would influence bioaccumulation within the coastal ecosystem. It was also hypothesized that the resident oil-degrading bacterial community would stimulate reductive dissolution of the Fe (III) minerals.

Based on the experimental results obtained, oil contamination does affect the concentration and distribution (between aqueous and solid phases) of trace elements in the coastal environment. The trace element distribution alternated between the solid phase and the aqueous phase throughout the experiment. Trace elements released during the first 24 hours of the experiment were released via reductive dissolution. Although some trace elements (Cu, Cr,

Pb, Al, Ni) in the solid phase samples correlate highly with iron, iron is not the controlling factor in their release. This is indicated by the trace element release into the aqueous phase at least a day earlier than the release of iron into the aqueous phase. Ion exchange is a possible explanation for desorption of trace elements, evidenced by the drop in pH balances during the first 24 hours of the experiment.

Bacteria and oil degradation were responsible for the temporal changes observed in iron and trace element concentrations. Trace elements in samples that contained oil showed different concentration trends with time, but the experimental control samples showed almost no change in trace element concentrations with time. This suggests that microbial activity mobilized some trace elements in the samples that were not autoclaved. When crude oil was introduced into the microcosm, the most likely catalyst for trace element concentration changes in the experimental jars was the oil-degrading bacteria, which were found to be present by UA biologists. If equilibrium (inorganic) processes were dominant, then there would have been similar changes in the experimental controls.

The results of this study help to explain the mechanisms of trace element release in coastal sediments and can be used as a resource in the on-going efforts of tabulating the impacts of oil contamination in nearshore environments along the Gulf coast. Understanding the activities of the oil-degrading microbes and the role they play in cleaning up the coastline is important. Future stages of research are needed to understand how to minimize the impact of oil contamination for the health of organisms that live in estuaries and other impacted areas.

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APPENDIX I

Time Series Experiment Data

Table 1.A. General data for wet sediment and seawater in Time Series

Alkalinity pH (mg TDS Na K Ca Mg Si SO 4 NO 3 PO 4 Fl Al B Sample Name CaCO3/L)(mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) Br (mg/L) Cl(mg/L) (mg/L) As (mg/L)(mg/L)

BLB-BOTTLE-17.66 83.07 2915 5114.43 192.84 195.980000 623.05 0.00 0.004 0.00 550.94 49.93 0. 4035.48 0.044 0.015 2.32

BLB-BOTTLE-27.66 83.07 2915 5255.75 197.66 203.200000 646.86 0.00 0.004 0.00 971.35 129.06 0. 7341.02 0.049 0.017 2.25

BLB-0-1-MD1N/A N/A N/A 126.01 40.89 27.65 63.31 9.41 N/AA 203.666 N/A 0.226 N/A N/A

BLB-0-1-MD2N/A N/A N/A 164.45 51.96 27.41 76.42 5.44 N/AA 296.069 N/A

BLB-0-1-MD3N/A N/A N/A 141.03 50.64 35.83 75.26 6.13 N/AA 301.580 N/A

Ba Be Cd Co Cr Cu Fe Mn Mo Pb Ti V Porosi ty Ni (mg/L) Se (mg/L) Sr (mg/L) Zn (mg/L) Sample Name(mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (%)

BLB-BOTTLE-10.023 0.000 0.001 0.000 0.000 0.083 0.018.004 0.070 0.001 4.163 0.006 0.001 0.767 0 0.041 3.225 N/A

BLB-BOTTLE-20.028 0.010 0.001 0.001 0.000 0.081 0.008.004 0.067 0.009 4.150 0.011 0.005 0.756 0 0.037 3.138 N/A

BLB-0-1-MD10.434 0.015

BLB-0-1-MD20.610 0.013

BLB-0-1-MD30.613 0.014

Table 1.B.1. Major ion chemistry of Time Series Experiment aqueous samples. Cation concentrations measured by ICP-OES. Anion concentrations measured by IC.

Na K Ca Mg Br SO 4 Cl Sample Name (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) BLB-0-1 5343.43 210.89 206.63 650.52 265.26 1617.43 11596.59 BLB-0-2 5411.06 216.46 209.21 659.11 373.15 2374.68 15879.47 BLB-6-1 5474.94 219.15 215.42 679.89 268.01 1931.77 13673.56 BLB-6-2 5280.08 206.19 203.24 641.25 336.80 1968.60 14238.85 BLB-12-1 5298.71 204.08 215.83 655.55 269.15 1892.06 12042.14 BLB-12-2 5431.87 212.05 220.06 664.90 269.15 1892.06 16568.10 BLB-24-1 5487.27 212.50 213.25 661.54 341.73 1894.26 13416.73 BLB-24-2 5309.99 219.16 205.76 649.90 458.82 2742.39 17031.93 BLB-48-1 5520.89 220.59 218.62 669.20 181.65 1476.64 9515.14 BLB-48-2 5198.46 202.99 205.21 635.13 305.98 1921.05 13015.33 BLB-7D-1 5279.15 216.19 208.61 639.26 252.29 1496.97 9962.11 BLB-7D-2 5190.34 214.48 204.84 633.64 317.35 2085.01 14564.12 BLB-14D-1 5345.75 208.61 208.82 651.25 369.64 2227.58 16037.81 BLB-14D-2 5372.71 209.12 209.31 650.58 233.67 1564.10 11110.89 BLB-NSC-1 5187.01 205.10 214.32 637.48 214.58 1425.12 9882.36 BLB-NSC-2 5257.03 222.17 212.97 644.02 139.51 1081.89 7201.23 BLB-SC1-1 5499.87 233.21 219.38 619.29 446.02 2516.68 18141.31 BLB-SC1-2 5558.77 258.12 220.41 622.23 437.90 2564.48 17580.62 BLB-SC2-1 5647.64 250.94 222.53 633.95 385.81 2245.91 15375.33 BLB-SC2-2 5737.70 251.72 224.15 631.95 278.32 1900.22 13745.67 SW-1 Bottle 1 5114.43 192.84 195.98 623.05 49.93 550.94 4035.48 SW-1 Bottle 2 5255.75 197.66 203.20 646.86 129.06 971.35 7341.02 LOD 0.13 0.07 0.00 0.07 N/A N/A N/A

Table 1.B.2. Minor element chemistry of Time Series experiment aqueous samples. Cations measured by ICP-OES. B Fe Mn Sr Si Sample Name (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) BLB-0-1 2.27 0.03 2.30 3.74 3.77 BLB-0-2 2.31 0.07 2.48 3.79 3.69 BLB-6-1 2.32 0.06 2.06 3.91 4.02 BLB-6-2 2.32 0.02 2.49 3.87 3.39 BLB-12-1 2.22

Table 1.B.3. Trace element chemistry of Time Series Experiment aqueous samples. Cations measured by ICP-OES. Al As Ag Ba Be Cd Co Cr Cu Sample Name (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) BLB-0-1 0.042

Table 1.B.4. Trace element chemistry of Time Series Experiment aqueous samples. Cations measured by ICP-OES. Mo Ni Pb Se Sb Sn Tl Ti Zn V Sample Name (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) BLB-0-1 0.017

Table 1.D.1. Time Series Experiment solid phase sample microwave digestion data. Al As B Ba Be Cd Co Cr Cu Fe Mn Sample Name (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) BLB-0-1-MD1 4073.316 4.516

Table 1.D.2. Time Series Experiment solid phase sample microwave digestion data Mo Ni Pb Se Sb Ag Sn Sr Tl Ti Zn V Sample Name (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) BLB-0-1-MD1

Table 1.D.3. Time Series Experiment solid phase sample microwave digestion data .

Table 1.D.4. Time Series solid phase sample MD data averages and standard deviations.

Al As B Ba Be Ca Cd Co Cr Cu Fe Mg Sample Averages (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) BLB-0-1-MD 5342.100 2.838 24.295 11.053 0.282 605.950 0.356 1.960 9.138 4.239 7778.212 1433.285 BLB-6-1-MD 4724.647 4.366 38.616 9.823 0.340 524.854 6.674 2.049 8.216 4.904 7783.672 1352.665 BLB-12-1-MD 5229.786 2.863 24.295 11.067 0.314 2388.562 0.356 1.905 8.925 4.171 7547.006 1364.190 BLB-48-1-MD 5096.266 2.748 24.295 10.524 0.349 662.161 0.356 1.973 8.668 4.575 7793.578 1502.371 BLB-7d-1-MD 4818.757 1.999 24.295 9.931 0.336 542.894 0.356 1.951 8.413 4.527 7643.591 1434.016 BLB-14d-1-MD 5462.242 2.274 24.921 11.209 0.196 602.499 0.133 1.962 9.295 3.917 8021.953 1452.853 BLB-SC1-1-MD 4377.685 2.237 35.527 9.033 0.339 573.814 0.184 1.851 7.619 4.054 7608.967 1443.268 BLB-SC2-1-MD 6187.611 3.819 24.921 12.732 0.409 625.542 0.190 2.117 10.036 4.390 8642.484 1652.649 BLB-NSC-1-MD 4552.320 2.752 24.921 9.204 0.388 566.039 0.133 1.965 7.959 4.640 7696.805 1382.263 Standard Deviation BLB-0-1-MD 44.915 0.059 0.000 0.084 0.001 3.914 0.000 0.006 0.063 0.009 16.670 5.926 BLB-6-1-MD 17.070 0.014 1.013 0.037 0.001 1.553 0.447 0.009 0.022 0.011 10.820 2.988 BLB-12-1-MD 44.975 0.061 0.000 0.089 0.001 133.056 0.000 0.005 0.064 0.018 18.978 4.326 BLB-48-1-MD 7.209 0.053 0.000 0.008 0.001 1.316 0.000 0.003 0.013 0.010 4.225 2.587 BLB-7d-1-MD 35.234 0.000 0.000 0.063 0.001 1.803 0.000 0.007 0.052 0.012 9.647 6.381 BLB-14d-1-MD 6.165 0.046 0.000 0.014 0.004 6.157 0.000 0.003 0.023 0.001 8.089 2.187 BLB-SC1-1-MD 31.392 0.043 0.750 0.051 0.002 2.648 0.004 0.008 0.037 0.024 8.672 1.903 BLB-SC2-1-MD 7.527 0.007 0.000 0.026 0.001 2.903 0.004 0.002 0.009 0.014 20.881 5.814 BLB-NSC-1-MD 25.219 0.043 0.000 0.041 0.001 0.422 0.000 0.003 0.036 0.007 7.409 2.712

Table 1.D.5. Time Series solid phase sample MD data averages and standard deviations

Mn Mo Ni Pb Se Sb Ag Sn Sr Tl Ti V Sample Averages (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) BLB-0-1-MD 122.423 6.762 3.597 5.186 5.809 4.584 0.465 3.408 11.993 4.267 28.276 28.251 BLB-6-1-MD 123.359 11.112 3.562 5.357 5.809 7.277 3.639 9.598 10.771 10.621 15.307 28.251 BLB-12-1-MD 120.058 6.762 3.493 5.306 5.809 4.584 0.465 3.408 25.389 4.267 26.210 28.251 BLB-48-1-MD 126.062 6.762 3.536 5.419 5.809 4.584 0.465 3.408 12.386 4.267 19.819 28.251 BLB-7d-1-MD 123.974 6.762 3.314 5.086 5.809 4.584 0.465 3.408 11.231 4.267 17.393 28.251 BLB-14d-1-MD 118.570 1.664 3.593 5.245 0.969 0.823 0.109 0.591 10.983 1.587 24.869 15.630 BLB-SC1-1-MD 116.598 1.664 3.141 5.582 0.969 0.823 0.109 0.591 16.641 1.587 17.564 15.630 BLB-SC2-1-MD 132.618 1.664 4.121 6.010 0.969 0.823 0.109 0.591 12.882 1.587 31.074 15.630 BLB-NSC-1-MD 124.325 1.664 3.401 5.162 0.969 0.823 0.109 0.591 10.765 1.587 13.184 15.630 Standard Deviation BLB-0-1-MD 0.129 0.000 0.019 0.018 0.000 0.000 0.000 0.000 0.065 0.000 0.570 0.000 BLB-6-1-MD 0.071 0.308 0.018 0.004 0.000 0.190 0.224 0.438 0.022 0.449 0.122 0.000 BLB-12-1-MD 0.257 0.000 0.021 0.029 0.000 0.000 0.000 0.000 1.000 0.000 0.525 0.000 BLB-48-1-MD 0.071 0.000 0.002 0.017 0.000 0.000 0.000 0.000 0.005 0.000 0.037 0.000 BLB-7d-1-MD 0.109 0.000 0.012 0.005 0.000 0.000 0.000 0.000 0.044 0.000 0.346 0.000 BLB-14d-1-MD 0.142 0.000 0.005 0.021 0.000 0.000 0.000 0.000 0.034 0.000 0.055 0.000 BLB-SC1-1-MD 0.088 0.000 0.012 0.041 0.000 0.000 0.000 0.000 0.365 0.000 0.369 0.000 BLB-SC2-1-MD 0.271 0.000 0.004 0.016 0.000 0.000 0.000 0.000 0.063 0.000 0.193 0.000 BLB-NSC-1-MD 0.108 0.000 0.012 0.010 0.000 0.000 0.000 0.000 0.019 0.000 0.167 0.000

Table 1.D.6. Time Series solid phase sample MD data averages and standard deviations.

Zn Si K Na Sample Averages (mg/kg) (mg/kg) (mg/kg) (mg/kg) BLB-0-1-MD 39.568 139.834 956.565 2876.657 BLB-6-1-MD 38.148 137.445 894.076 2425.005 BLB-12-1-MD 53.716 165.169 912.596 2569.629 BLB-48-1-MD 36.608 173.583 934.903 3331.296 BLB-7d-1-MD 49.400 127.451 934.750 3201.843 BLB-14d-1-MD 37.367 200.504 982.399 2961.067 BLB-SC1-1-MD 41.621 174.136 878.646 3230.369 BLB-SC2-1-MD 58.382 174.172 1075.614 3268.384 BLB-NSC-1-MD 31.160 145.199 899.197 2992.972 Standard Deviation BLB-0-1-MD 0.489 1.731 4.937 15.819 BLB-6-1-MD 0.623 0.407 1.990 17.003 BLB-12-1-MD 1.300 1.402 5.156 4.121 BLB-48-1-MD 0.530 2.739 0.465 14.700 BLB-7d-1-MD 0.786 0.789 4.499 26.266 BLB-14d-1-MD 0.593 1.645 0.906 17.816 BLB-SC1-1-MD 0.349 3.605 1.881 5.473 BLB-SC2-1-MD 0.629 1.765 2.321 10.491 BLB-NSC-1-MD 0.082 1.535 2.163 5.529

APPENDIX II

Concentration Variation Experiment Data

Table 2.A. General data and amount of wet sediment and seawater in each jar for Concentration Variation Experiment

Alkalinity

(mg TDS Na K Ca Mg Si SO 4 NO 3 PO 4 Cl Br Al As B Ba Sample NameCaCO3/L) pH (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L)

SW-2 Bottle 17.6 95.85 6509 8579.78 329.35 326.6035 1067.46 0.00

SW-2 Bottle 27.6 95.85 6509 8708.63 333.64 328.51.58 1075.04 0.00

MC252-NSC-0-1N/A N/A N/A 658.63 89.15 71.61 161.50 6.7639.554 N/A 0.248 N/A1.667 1.350 N/A N/A N/A 4

MC252-NSC-0-2N/A N/A N/A 197.94 47.68 26.31 61.09 32.0039.148 N/A

Be Cd Co Cr Cu Fe Mn Mo Ni Pb Se Sr Ti Zn V Porosi ty

Sample Name(mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (%)

SW-2 Bottle

MC252-NSC-0-10.036

MC252-NSC-0-2

Table 2.B.1. Major ion chemistry of Concentration Variation Experiment aqueous samples. Cations measured by ICP-OES. Anion concentrations measured by IC.

Na K Ca M g SO 4 Br Cl Sample Name (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) MC252-10-1 8483.98 324.23 356.34 1079.33 2214.25 416.13 14231.17 MC252-10-2 8536.29 319.26 367.86 1090.72 1687.81 415.90 11872.11 MC252-100-1 8454.82 335.75 348.71 1096.72 1535.81 379.98 13116.85 MC252-100-2 8436.83 319.13 345.51 1083.14 1905.82 372.93 12844.91 MC252-500-1 8516.31 330.69 340.88 1080.48 2060.36 506.53 0.00 MC252-500-2 8578.57 338.03 338.23 1088.93 1336.67 390.80 11042.91 MC252-1000-1 8465.73 324.17 327.51 1067.51 1240.75 341.84 10632.24 MC252-1000-2 8453.94 327.82 338.95 1079.28 1145.62 352.59 8358.26 MC252-2500-1 9581.94 421.64 379.11 1151.83 2538.95 468.00 15147.20 MC252-2500-2 6420.25 249.31 254.40 808.72 1557.94 404.53 11243.58 SC-0ppm 8944.79 360.63 350.51 1096.73 2287.99 524.04 14818.95 SC-10ppm 8607.06 387.63 343.40 1025.85 2800.96 537.66 16990.10 SC-100ppm 8873.02 372.28 365.73 1106.93 2205.43 495.42 15744.15 SC-500ppm 9484.43 426.89 383.84 1150.26 1644.13 417.20 12989.20 SC-1000ppm 9275.96 402.55 369.97 1111.50 2193.03 469.85 15120.22 SC-2500ppm 8599.92 326.75 333.91 1082.20 2072.37 520.52 14762.39 NSC-0-1 8253.88 325.46 352.61 1068.01 2516.83 450.87 14671.34 NSC-0-2 8416.15 325.00 351.31 1084.68 SW-2 Bottle 1 8579.78 329.35 326.60 1067.46 2301.41 494.11 14952.49 SW-2 Bottle 2 8708.63 333.64 328.51 1075.04 2317.62 389.01 14596.22 LOD 38.17 35.98 2.10 2.85 N/A N/A N/A

Table 2.B.2. Minor element chemistry of Concentration Variation Experiment aqueous samples. Cations measured by ICP-OES.

Si Fe B Mn NO 3 PO 4 Sr V Sample Name (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) MC252-10-1 10.50 11.02 3.53 3.02 11.68 0.00 6.39 6.18 MC252-10-2 9.88 17.72 3.52 2.84 42.30 0.00 6.27 6.14 MC252-100-1 11.09 12.41 3.46 2.52 25.94 0.00 6.20 6.07 MC252-100-2 11.48 18.22 3.47 3.44 16.92 0.00 6.24 6.10 MC252-500-1 11.35 2.43 3.47 2.23 0.00 0.00 6.24 6.13 MC252-500-2 11.76 6.23 3.44 2.29 0.00 4.18 6.13 6.04 MC252-1000-1 11.11 0.17 3.26 2.08 14.99 10.12 6.10 5.92 MC252-1000-2 11.25 0.19 3.30 1.96 0.00 8.15 6.16 5.99 MC252-2500-1 8.28 0.04 4.11 1.75 16.01 5.48 6.62 6.32 MC252-2500-2 8.17 0.04 2.48 1.22 0.00 12.30 4.74 4.53 SC-0ppm 18.08 0.03 4.11 1.32 24.42 15.45 6.21 6.04 SC-10ppm 4.97 0.04 3.89 1.97 28.60 2.17 5.98 5.82 SC-100ppm 27.61 7.16 4.37 2.58 41.02 12.64 6.38 6.25 SC-500ppm 8.00 0.03 4.11 2.00 0.00 2.63 6.56 6.35 SC-1000ppm 6.39 0.06 3.91 1.91 0.00 1.53 6.50 5.98 SC-2500ppm 10.65 0.05 3.23 1.51 3.90 12.40 6.14 5.98 NSC-0-1 7.47 0.05 3.67 3.06 16.95 0.00 6.42 6.21 NSC-0-2 7.41 1.65 3.59 3.09 6.26 6.19 SW-2 Bottle 1

Table 2.B.3. Trace element chemistry of Concentration Variation Experiment aqueous samples. Cation concentrations measured by ICP-OES. Al As Ba Be Cd Co Cr Cu Sample Name (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) MC252-10-1 0.037

Table 2.B.4. Trace element chemistry of Concentration Variation Experiment aqueous samples. Cation concentrations measured by ICP-OES. Mo Ni Pb Se Sb Sn Tl Ti Zn Sample Name (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) MC252-10-1 0.010

Table 2.C.1. Concentration Variation Experiment solid phase sample microwave digestion data.

Al As B Ba Be Cd Co Cr Cu Fe Mn Sample Name (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) MC252-10-1-MD1 6219.97

Table 2.C.2. Concentration Variation Experiment solid phase sample microwave digestion data. Al As B Ba Be Cd Co Cr Cu Fe Mn Sample Name (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) MC252-SC-0-MD1 5740.65

Table 2.C.3. Concentration Variation Experiment solid phase sample microwave digestion data.

Mo Ni Pb Se Sb Ag Sn Sr Tl Ti Zn V Sample Name (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) MC252-10-1-MD1

Table 2.C.4. Concentration Variation Experiment solid phase sample microwave digestion data.

Mo Ni Pb Se Sb Ag Sn Sr Tl Ti Zn V Sample Name (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) MC252-SC-0-MD1

Table 2.C.5. Concentration Variation Experiment solid phase sample microwave digestion data. Na K Ca Mg Si Sample Name (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) MC252-10-1-MD1 2192.31 1081.56 434.37 1056.38 1861.54 MC252-10-1-MD2 2196.83 1060.14 426.01 1031.18 899.39 MC252-10-1-MD3 2213.46 1110.38 429.75 1068.43 1147.84 MC252-10-2-MD1 2838.74 1069.84 480.13 1150.40 1096.13 MC252-10-2-MD2 2739.11 982.62 447.54 1055.01 160.92 MC252-10-2-MD3 2909.82 1071.23 476.82 1142.71 206.05 MC252-100-1-MD1 4112.73 1042.37 619.62 1257.68 710.81 MC252-100-1-MD2 4019.18 1017.46 593.70 1219.96 848.06 MC252-100-1-MD3 4002.62 1081.19 597.01 1239.26 63.55 MC252-100-2-MD1 4410.03 1033.16 580.04 1270.58 159.89 MC252-100-2-MD2 4732.61 1134.05 635.12 1377.04 1341.86 MC252-100-2-MD3 5079.06 1230.37 680.82 1469.41 1346.03 MC252-500-1-MD1 3408.10 1086.02 552.86 1168.56 1646.44 MC252-500-1-MD2 2939.37 1069.02 434.03 1105.20 3736.05 MC252-500-1-MD3 2969.66 901.85 503.93 1023.90 173.39 MC252-500-2-MD1 3577.57 1099.23 591.95 1229.58 1178.61 MC252-500-2-MD2 2682.59 899.29 393.76 991.17 179.58 MC252-500-2-MD3 2732.30 959.78 424.25 1042.98 675.20 MC252-1000-1-MD1 2955.23 969.34 440.44 1068.10 208.62 MC252-1000-1-MD2 2942.00 946.98 441.92 1062.87 403.06 MC252-1000-1-MD3 2967.59 940.95 438.16 1059.38 72.85 MC252-1000-2-MD1 3088.07 1009.80 474.62 1120.06 1225.86 MC252-1000-2-MD2 3454.69 1158.44 524.49 1254.95 537.89 MC252-1000-2-MD3 3130.41 1023.51 481.98 1131.67 3616.25 LOD 93.65 85.02 6.87 26.93 17.58

Table 2.C.6. Concentration Variation Experiment solid phase sample microwave digestion data. Na K Ca Mg Si Sample Name (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) MC252-SC-0-MD1 4873.72 1151.79 557.00 1430.69 1857.13 MC252-SC-0-MD2 4726.07 1209.20 538.70 1388.47 36302.97 MC252-SC-0-MD3 5149.52 1214.98 581.32 1464.53 2484.66 MC252-SC-10-MD1 8696.76 2521.17 1027.75 3436.35 8350.22 MC252-SC-10-MD2 8633.16 2449.44 1022.66 3368.68 341.77 MC252-SC-10-MD3 8990.23 2592.48 1056.28 3470.37 88.23 MC252-SC-100-MD1 3634.61 1058.75 492.68 1188.94 4587.16 MC252-SC-100-MD2 3209.80 986.30 450.83 1068.06 9192.87 MC252-SC-100-MD3 3510.23 1072.07 487.89 1196.21 323.17 MC252-SC-500-MD1 2514.08 784.37 389.65 909.84 20.01 MC252-SC-500-MD2 3207.95 1000.61 500.32 1182.58 117.05 MC252-SC-500-MD3 3134.77 997.02 492.20 1154.77 239.82 MC252-SC-1000-MD1 3887.30 901.75 494.44 1128.22 3887.61 MC252-SC-1000-MD2 4011.98 905.02 498.92 1139.65 1185.36 MC252-SC-1000-MD3 3744.00 852.00 471.78 1070.93 224.14 MC252-SC-2500-MD1 6193.80 1110.31 616.43 1624.37 181.76 MC252-SC-2500-MD2 5824.00 995.10 567.51 1485.26 67.29 MC252-SC-2500-MD3 6014.69 1029.60 576.24 1513.08 48.18 MC252-NSC-0-1-MD1 12645.57 1702.31 1394.94 3120.96 111.03 MC252-NSC-0-1-MD2 12651.84 1778.25 1393.20 3164.92 115.52 MC252-NSC-0-1-MD3 14220.48 1868.58 1508.65 3404.40 178.91 MC252-NSC-0-2-MD1 3978.63 994.83 530.15 1233.00 1612.29 MC252-NSC-0-2-MD2 3938.97 839.06 526.14 1163.07 116.81 MC252-NSC-0-2-MD3 1026.91 522.49 1269.39 190.85 LOD 167.44 87.76 2.11 13.47 17.87

Table 2.D.1. Concentration Variation solid phase sample microwave digestion data averages and standard deviations. Al Co Cr Cu Ba Zn Sample Averages (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) MC252-10 MD 5920.60 1.70 8.67 3.49 38.53 26.50 MC252-100 MD 5375.94 1.60 7.98 3.90 37.99 31.16 MC252-500 MD 5380.12 1.91 8.38 4.11 38.75 32.95 MC252-1000 MD 5481.80 1.59 7.92 3.80 37.33 35.20 MC252-2500 MD 4739.76 1.58 7.20 3.07 35.01 40.60 MC252-SC-0 5890.58 1.77 9.08 3.71 40.77 21.66 MC252-SC-10 16332.38 3.65 20.94 9.12 58.29 47.76 MC252-SC-100 5130.20 1.77 8.02 3.89 39.60 25.70 MC252-SC-500 5173.01 1.44 7.52 3.17 34.57 26.17 MC252-SC-1000 4387.82 2.30 7.72 4.10 34.40 26.63 MC252-SC-2500 5301.38 1.67 7.51 3.26 36.06 52.38 MC252-NSC 6787.02 2.03 10.60 5.78 34.01 42.04 Standard Deviation MC252-10 MD 319.17 0.18 0.39 0.32 2.66 6.00 MC252-100 MD 484.45 0.09 0.57 0.63 2.06 3.33 MC252-500 MD 491.19 0.52 1.04 0.94 3.57 2.35 MC252-1000 MD 317.27 0.08 0.53 0.14 2.97 0.33 MC252-2500 MD 114.71 0.01 0.04 0.02 0.24 6.87 MC252-SC-0 190.08 0.20 0.43 0.39 1.22 3.45 MC252-SC-10 736.98 0.06 0.44 0.09 6.77 9.20 MC252-SC-100 334.73 0.22 0.36 0.05 1.63 2.50 MC252-SC-500 777.84 0.18 1.05 0.46 4.66 3.19 MC252-SC-1000 182.27 1.51 1.55 2.07 3.13 8.55 MC252-SC-2500 451.61 0.15 0.56 0.24 1.63 3.81 MC252-NSC 2266.78 0.96 3.64 2.37 3.34 10.35

Table 2.D.2. Concentration Variation solid phase sample microwave digestion data averages and standard deviations. Fe Mn Ni Pb Sr Ti Sample Averages (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) MC252-10 MD 4724.19 47.37 2.60 4.57 9.46 233.72 MC252-100 MD 4471.71 47.32 2.48 4.38 10.98 222.59 MC252-500 MD 4533.75 45.64 2.79 4.75 9.82 208.63 MC252-1000 MD 4408.81 46.52 2.42 4.54 9.56 206.24 MC252-2500 MD 4210.18 42.53 4.78 3.02 8.87 188.86 MC252-SC-0 4971.41 50.91 2.62 3.38 11.95 297.21 MC252-SC-10 12373.16 112.87 7.84 7.37 22.71 180.62 MC252-SC-100 4705.57 48.24 2.50 3.67 10.34 238.46 MC252-SC-500 4154.73 41.84 2.24 2.70 8.95 213.15 MC252-SC-1000 3742.93 39.95 2.83 3.66 9.41 228.61 MC252-SC-2500 4619.58 45.08 2.56 3.28 11.58 155.86 MC252-NSC 7929.45 72.14 4.35 6.45 17.62 89.92 Standard Deviation MC252-10 MD 258.24 2.54 0.06 0.07 0.50 21.34 MC252-100 MD 229.91 2.65 0.07 0.24 0.65 17.95 MC252-500 MD 320.00 3.04 0.19 0.04 0.89 26.49 MC252-1000 MD 320.49 2.11 0.03 0.35 0.68 16.75 MC252-2500 MD 264.20 2.53 3.50 0.55 1.13 51.83 MC252-SC-0 108.72 1.04 0.50 0.56 0.64 104.23 MC252-SC-10 226.86 1.80 0.07 0.24 0.44 43.78 MC252-SC-100 251.42 1.52 0.10 0.17 0.01 9.17 MC252-SC-500 552.21 5.12 0.42 0.66 1.25 19.86 MC252-SC-1000 138.62 3.09 1.49 1.55 0.08 19.38 MC252-SC-2500 231.83 2.17 0.18 0.12 0.54 13.20 MC252-NSC 3670.68 27.95 1.91 2.61 8.25 82.09

Table 2.D.3. Concentration Variation solid phase sample microwave digestion data averages and standard deviations. Na K Ca Mg Si Sample Averages (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) MC252-10 MD 2515.05 1062.63 449.10 1084.02 895.31 MC252-100 MD 4392.71 1089.77 617.72 1305.65 745.03 MC252-500 MD 3051.60 1002.53 483.46 1093.57 1264.88 MC252-1000 MD 3089.67 1008.17 466.93 1116.17 1010.75 MC252-2500 MD 2780.54 873.13 470.97 1008.03 2056.63 MC252-SC-0 4916.44 1191.99 559.01 1427.90 13548.25 MC252-SC-10 8773.38 2521.03 1035.56 3425.13 2926.74 MC252-SC-100 3451.55 1039.04 477.13 1151.07 4701.06 MC252-SC-500 2952.27 927.33 460.72 1082.40 125.63 MC252-SC-1000 3881.10 886.25 488.38 1112.93 1765.70 MC252-SC-2500 6010.83 1045.00 586.73 1540.90 99.08 MC252-NSC 9487.10 1436.61 1070.62 2417.27 426.91 Standard Deviation MC252-10 MD 348.48 42.83 23.91 49.98 640.64 MC252-100 MD 437.49 80.60 36.64 97.15 554.25 MC252-500 MD 363.64 93.13 78.74 91.82 1339.90 MC252-1000 MD 194.78 80.73 33.92 74.69 1337.97 MC252-2500 MD 385.71 117.67 54.81 87.36 1935.45 MC252-SC-0 214.93 34.93 21.38 38.11 19708.66 MC252-SC-10 190.47 71.52 18.12 51.77 4698.58 MC252-SC-100 218.40 46.16 22.90 71.98 4435.95 MC252-SC-500 381.24 123.82 61.68 150.09 110.15 MC252-SC-1000 134.10 29.71 14.55 36.83 1899.44 MC252-SC-2500 184.93 59.13 26.09 73.61 72.24 MC252-NSC 5087.27 481.18 497.41 1118.49 663.24