PAH Degradation in Wetland Soils As Influenced by Redox Potential

Louisiana State University LSU Digital Commons

LSU Master's Theses Graduate School

2014 PAH degradation in wetland soils as influenced by redox potential Haoxuan Zhang Louisiana State University and Agricultural and Mechanical College, [email protected]

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Recommended Citation Zhang, Haoxuan, "PAH degradation in wetland soils as influenced by redox potential" (2014). LSU Master's Theses. 374. https://digitalcommons.lsu.edu/gradschool_theses/374

This Thesis is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Master's Theses by an authorized graduate school editor of LSU Digital Commons. For more information, please contact [email protected]. PAH DEGRADATION IN WETLAND SOILS AS INFLUENCED BY REDOX POTENTIAL

A Thesis Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Master of Science in The Department of Civil and Environmental Engineering

by Haoxuan Zhang B.S., Beijing University of Aeronautics and Astronautics, 2011 May 2014

ACKNOWLEDGEMENTS I would like to express my deep gratitude and sincere appreciation to my exceptional advisor, Dr. Malone (Department of Civil and Environmental Engineering) for his valuable guidance, encouragement, and commitments to complete this Thesis. His continuous support throughout my master studies is greatly appreciated, and he continually assisted me with my scientific writing. I also wish to thank my academic committee advisors, Dr. Adrian

(Department of Civil and Environmental Engineering), Dr. Gambrell (Department of

Oceanography and Coastal Sciences), Dr. Theegala (Department of Biological and Agricultural

Engineering) for their cooperation, guidance, and patience.

I also wish to thank Department of Civil and Environmental Engineering of Louisiana

State University for providing me wonderful research facilities. Thanks are due to Dr. Malone and other group personnel for their help and assistance in this study. Thanks to Martin S. Miles help me to collect the soils. And thanks to Batubara for making laboratory experiment which were of a great value to this research. Also thanks to Department of Ocean and Environment for helping me test the samples.

Finally, I would like to express my gratitude to my parents. I could not finish without their constant support, patience, and sacrifice both financially and mentally through these years.

I would like to thank my friends Batubara, Alt, Chen and Nguyen for their encouragement and helps during these years.

TABLE OF CONTENTS ACKNOWLEDGEMENTS ...... ii

LIST OF TABLES ...... iv

LIST OF FIGURES ...... v

ABSTRACT ...... vi

CHAPTER I INTRODUCTION ...... 1

CHAPTER II LITERATURE REVIEW ...... 4 2.1 Background Information ...... 4 2.2 Chemical and Physical Properties of PAHs ...... 7 2.3 PAHs Biodegradation ...... 12 2.3.1 Physical Factors Influences on Biodegradation ...... 13 2.3.2 Redox Potential Influences on Biodegradation ...... 15 2.3.3 PAH Concentration, Organic Content, Nutrient, pH Influences ...... 19

CHAPTER III METHODOLOGY AND MATERIALS ...... 21 3.1 Equipment System ...... 21 3.2 Soils Sample and PAHs Pollutants ...... 25 3.3 Redox Potential Measurement ...... 28 3.4 PAHs Sampling and Analysis ...... 30 CHAPTER IV RESULTS AND DISCUSSION ...... 32

CHAPTER V CONCLUSION ...... 43

REFERENCES ...... 44

APPENDIX A REDOX POTENTIALS DURING FIVE TIDES ...... 50

APPENDIX B PAH CONCENTRATIONS OVER 120 DAYS ...... 53

THE VITA ...... 56

iii

LIST OF TABLES TABLE PAGE

2.1 U.S. EPA’s Priority Pollutant PAHs and Selected Properties ……………………………. 8

2.2 Reference Potentials Varies With Temperature and Solution Concentration …………… 17

2.3 Reduction Reactions and Redox Potential Range in Soil ……………………………….. 17

3.1 Properties of Phenanthrene, Pyrene, Benzo[e]pyrene ………………………….………... 26

3.2 Representative Soils Characteristics ………………………………………….…….…… 27

4.1 First Order Biodegradation Reaction Rate Constants of PAHs ………………….……… 35

4.2 Summary Statistics for Redox Potentials …………………………………….………….. 40

4.3 Fisher's F-Test / Two-tailed Test of Redox Potentials …………………………………… 41

4.4 T-test for Two Independent Redox Potential Samples / Two-tailed Test ……….….……. 41

4.5 Z-test for Two Independent Redox Potential Samples / Two-tailed Test …….….………. 42

APPENDIX A. Redox Potentials during Five Diurnal Tides …………………….……….. 50

APPENDIX B. Concentration of PAHs Over 120 Days Impacted By Tidal Actions …..…. 53

LIST OF FIGURES FIGURE PAGE

2.1 Widespread Sources of PAHs ……………………………………………………………. 4

2.2 Structures of U.S. EPA’s 16 Priority Pollutant PAHs …………………………………...... 9

3.1 Flow Chart of Equipment System ………………………………………………………. 21

3.2 Simulating Tides in Experimental Tanks by Hydraulic Control Tank ………………….. 22

3.3 Design of Equipment System for Hydraulic Control ………………………….………… 23

3.4 Variation of Water Level in Experimental Tank during a Tidal Cycle …………….…….. 23

3.5 ORP Electrodes Placed in Periodically Emerged Tray and Submerged Tray ……….…... 24

3.6 Molecular Structures of Phenanthrene, Pyrene, Benzo[e]pyrene ………………….……. 26

3.7 Soils and Sand Layers in the Tray Seperated by Geotech Cloth …………….……….….. 27

3.8 Flow Chart of Transport and Consumption of Oxygen in Equipment System ………….. 29

3.9 Plastics Straws Were Used to Fill Holes Where Samples Were Taken………………...… 30

4.1 Tidal Action Greatly Impacted Biodegradation Rate of Phe in First 50 Days ……...…… 33

4.2 Tidal Action Obviously Impacted Biodegradation Rate of Pyr in Later Period ……..….. 34

4.3 Tidal Action Continuously Impacted the Biodegradation Rate of Bep ……..…….…….. 35 4.4 Comparison of Biodegradation Ability among Phe, Pyr, and BeP in Submerged Soils………………………………………………… 36

4.5 Comparison of Biodegradation among Phe, Pyr, and BeP in Periodically Emerged Soils ……………………………………………………………. 37

4.6 Redox Potentials Fluctuated during 5 Tidal Cycles in Periodically Emerged Soils (Electrode B) ……………………………………….……. 38

4.7 Comparison of Redox Potentials (Measured by Electrodes B, D, H) in Periodically Emerge Soils during Five Tidal Cycles …………………………….…….. 39

4.8 Comparison of Redox Potentials between Permanent Submerged and Periodically Emerged Soils ………………….……………. 40 v

ABSTRACT Polycyclic aromatic hydrocarbons (PAHs) are a common contaminant in wetland soils.

They are a group of compounds widely distributed in the environment and tend to accumulate in soils. Major contribution to removal of PAHs is biological degradation. For investigating the biodegradation potential of PAHs influenced by tidal actions, equipment was built for simulating the tidal actions, and concentrations of phenanthrene, pyrene, and benzo[e]pyrene were added to the soils samples which were collected from wetland. Experiments were then conducted over 120 days. Redox potentials and PAHs concentrations were measured and analyzed. Results are concluded: 1) influenced by tidal action, phenanthrene, pyrene, benzo[e]pyrene were rapidly biodegraded during the first 40 days followed by slow but continuous biodegradation in the next 80 days, 2) tidal action enhanced approximately 15.2%,

13.9%, 12.2% of the removal efficiencies of phenanthrene, pyrene, and benzo[e]pyrene in first

37 days, 3) redox potential can change rapidly and significantly in coastal wetland soils in response of flooding and draining, 4) redox potentials in submerged soils and periodically emerged soils were significantly different, which is 70 mV higher in the periodically emerged one.

CHAPTER I INTRODUCTION Polycyclic aromatic hydrocarbons (PAHs) are a group of over 100 different chemicals which consist of fused aromatic rings (Abbey, 2011; ATSDR, 2005). PAHs accumulated in wetland soil may come from two major sources: atmospheric deposition and oil spillage

(Greenberg et al., 1985). PAHs are the products of incomplete combustion released in the air from volcanoes, forest fires, burning coal, and automobile exhausts (ATSDR, 2005). They attach onto the particulate matters that are suspended in air, and eventually deposit with rainfall.

PAHs are also present in crude oil spill that have spent time in the ocean. Eventually, they pollute the shore (U.S. EPA, 2013).

Actually, spillage of crude oil is hard to avoid. The United States Environmental

Protection Agency (U.S. EPA) reported a number of large oil spills in the Gulf of Mexico over the last two decades. These spills have increased the concentration of PAHs in Louisiana wetland soils. Aromatic compounds are of major concern in wetland soils since they were listed as priority pollutants by the U.S. EPA. Many researchers have already demonstrated that these kinds of aromatic compounds are not only toxic to many marine coastal species, but also pose a high risk to human health because of their carcinogenic, mutagenic, and teratogenic effects

(Abbey, 2011; Ambrosoli, 2005; Catallo et al. 1995; Harvey, 1997). Therefore, studies on the removal of PAHs are becoming a main concern for environmental scientists.

The major pathway of PAHs loss in soil is biological degradation (Ambrosoli, 2005;

Lu, 2011). Due to their low water solubility and high hydrophobicity, PAHs are readily adsorbed onto particulate matter, and are also difficult to evaporate (Abbey, 2011; Lu, 2011).

Coastal and marine soils ultimately become the reservoir for PAHs (Hughes, Beckles et al.

1997). Once in the soils, they are slowly degraded by bacterium. The chemical and physical properties of the PAHs compounds affect biodegradation as well as environmental factors. In general, low molecular weight PAHs (which have 2-3 rings) are more easily degraded biologically (Stronguilo et al., 1994; Huesemann et al., 1995; Mackay, 2000 and Callcott, 1998).

On the other hand, environmental factors including pH, temperature, nutrient, soil, and redox conditions impact the PAH biodegradation rate.

Biological degradation of PAHs can occur aerobically and anaerobically (Heider et al.

1999; Head. 2006). Aerobic bacteria can utilize PAHs as carbon and energy source in the presence of oxygen (Eriksson et al. 2003; Quantin et al. 2005). However, under reducing conditions, the biodegradation of organic matters is the result of anaerobic bacterial respiration

(Ambrosoli, Petruzzelli et al. 2005). Generally, wetland soils are associated with low dissolved oxygen and reduced redox potentials (Gambrell, 1996). PAHs compounds often undergo slow biodegradation in anaerobic conditions (Alexander, 1999). In the past two decades, a number of various bacteria have been identified as “PAH degraders”, including the common genera of

Pseudomonas (Weissenfels et al. 1990), Sphingomonas (Desai et al. 2008), Cycloclasticus

(Geiselbrecht et al. 1998), some anoxic bacteria of Deltaproteobacteria (Musat et al. 2009) and

Alcaligenes (Weissenfels et al., 1990). Along with the disappearance of soil oxygen, the decrease in redox potential is the most striking change observed when a soil is flooded

(Gambrell, 1996). Therefore, soil redox potential measurements have been widely used to distinguish the biological processes occurring in flooded soils (Gambrell, 1996).

Tidal actions can change the redox potential in wetland soils, especially for the intertidal soil. The electron availability in intertidal soils is expected to be higher during low tides. By

simulating tidal action in a bench scale equipment, this study is aimed to 1) determine the tidal influence on redox potential in wetland soils and 2) quantify the biodegradation rate of phenanthrene, pyrene, and benzo[e]pyrene in wetland soils under changing redox conditions.

CHAPTER II LITERATURE REVIEW 2.1 Background Information

Polycyclic aromatic hydrocarbons are ubiquitous in natural environment (Abbey, 2011;

Lu, 2011; Rivas, 2006). As Figure 2.1 shows, PAHs are formed and released into the environment through both natural and anthropogenic sources (Abbey, 2011; Wang, 2001).

Natural sources include volcanoes and forest fires. Anthropogenic sources include (Harvey et al., 1997): 1) incomplete combustion of fossil fuels, wood, and municipal waste, 2) crude oil spill, 3) discharges from industrial plants and waste water treatment plants, 4) cigarette smoke,

5) charbroiled meat, 6) coal tar, coke, asphalt, creosote, asphalt roads, and roofing tar.

Figure 2.1 Widespread Sources of PAHs

Although most PAHs enter the environment via the atmosphere, sediments and soils are the primary environmental depository (Dabestani and Ivanov, 1999; Juhasz and Naidu, 2000).

But certain PAHs move through soil and eventually contaminate underground water (ATSDR,

2005). Forest fires and airborne pollution deposition are the main source of soil PAHs in remote areas. Background levels of PAHs in soil resulting from natural processes are estimated to be in the range of 1-10 mg/kg (MA DEP, 2002). However, because of growing industrial activities, the concentration of PAHs among the United States have increased in the past 100-150 years

(Abbey, 2011). In a long term research in Louisiana, Catallo et al (1995) report that most of the soils samples showing mutagenic activity were from soils deposited between 1955 and 1980, with virtually no activity in soils deposited before 1955 or after 1980. Catallo also indicated that 1955-1980 is a period when American industry was very active, which induced high pollution of PAHs. Wild and Jones (1995) also found that concentrations of PAHs in urban industrial soils can be 10-100 times higher than in remote soils. In soils at industrial sites, concentrations and type of PAHs found vary depending on the type of industry. For instance,

Juhasz and Naidu (2000) report total PAH concentrations of 5863 mg/kg at a creosote production site; 18,704 mg/kg at a wood preserving site; 821 mg/kg at a petrochemical site; and 451 mg/kg at a gas manufacturing plant site. Since thousands of industrial plants were built along Mississippi River, the wastewater and air pollution discharged from these industrial plants have definitely impacted the wetland soils.

The BP Spill (20 April, 2010) is considered as the largest accidental marine oil spill of the petroleum industry. The BP Spill source was located approximately 52 miles southeast of

Venice, Plaquemines Parish, Louisiana, (28.73667˚ N, 88.38722˚ W) (U.S. EPA, 2010). It

claimed eleven lives and cost billions of dollars for environmental remediation (Robertson,

2010). U.S. EPA has been sampling, analyzing, and assessing water and soils quality along the

Gulf Coast. Between May and June 2010, the coastal water contained 40 times more PAHs than before the spill (U.S. EPA, 2010). According to the satellite images, the spill directly impacted 68,000 square miles (180,000 km2) of ocean which is comparable to the size of

Missouri (Norse, 2010). As of July 2011, about 1,074 miles (1,728 km) of coastline in

Louisiana, Mississippi, Alabama and Florida had been oiled since the spill began (Polson,

2011). At present, PAHs in Louisiana wetland soils are believed to result mainly from the BP oil spill.

Laboratory mice that were fed with high levels of one PAH during pregnancy had difficulty reproducing and so did their offspring. These offspring also had higher rates of birth defects and lower body weights (ATSDR, 2005). Humans can directly be exposed to toxic compounds through ingestion, inhalation, and dermal exposure. Schneyer (2010) indicates that

PAHs enter the food chain through organisms such as plankton or fish. Human can indirectly be exposed to PAHs by taking contaminated fish. These compounds are hard to be degraded and difficult to purge from human body. Many studies have proved that PAHs pose a high risk to human health because of their carcinogenic, mutagenic, and teratogenic effects (Abbey, 2011;

Ambrosoli, 2005; Catallo, Schlenker et al. 1995; Harvey, 1997). They can impact the respiratory, reproductive, and neurological systems, cause birth defects and cancer (Abbey,

2011; Adonis, 2000).

In summary, PAHs are widespread in environment. Their potential harm to human beings is drawing more and more public attentions. In response to the increasing PAHs in the

soil, researchers are studying these aromatic compounds. Knowledge of their properties and biodegradation are essential.

2.2 Chemical and Physical Properties of PAHs

Molecular weight, molecular structure, water solubility, and vapor pressure of each

PAH compound decides the potential for its transportation and biodegradation (Jones et al.,

1996; Pierzynski et al., 2000; Reid et al., 2000a). Therefore, knowledge of chemical and physical properties of these PAHs are essential in determining the appropriate environmental remediation technique.

Polycyclic aromatic hydrocarbons can be classified by molecular weight: a low molecular weight (LMW) PAH has two or three fused rings; and a high molecular weight

(HMW) PAH has four or more fused rings (Harvey, 1997). Diesel combustion is the primary source of lighter molecular weight PAHs, while gasoline combustion is a main source of heavy molecular weight PAHs (Juhasz and Naidu, 2000). Table 2.1 gives U.S. EPA’s priority pollutant PAHs and their selected properties.

Smaller molecules, such as benzene, are not a polycyclic aromatic hydrocarbon.

Naphthalene is the simplest example of a PAH. Benzo[e]pyrene is not one of the 16 U.S. EPA priority pollutants, but it still chosen to be listed in Table 2.1 due to its carcinogenic, mutagenic, and teratogenic effects to human beings. In general, the PAHs resistance to oxidation, reduction, and evaporation increases with increasing molecular weight. Therefore, high molecular weight

(HMW) PAHs are more difficult to be removed, and thus, they pose a higher risk for human beings and other animals.

Table 2.1 U.S. EPA’s Priority Pollutant PAHs and Selected Properties (ATSDR, 2005)

PAH Name Number Molecular Solubility in Vapor Log of Rings Weight(g/mol) Water(mg/L) Pressure(mm Hg) Kow Naphthalene 2 128.17 31 8.89×10−2 3.37

Acenaphthene 3 154.21 3.8 3.75×10−3 3.92

Acenaphthylene 3 152.18 16.1 2.90×10−2 4.07

Anthracene 3 178.23 0.045 2.55×10−5 4.54

Phenanthrene 3 178.23 1.1 6.80×10−4 4.45

Fluorene 3 166.22 1.9 3.24×10−3 4.18

Fluoranthene 4 202.26 0.26 8.13×10−6 4.90

Benz[a]anthracene* 4 228.29 0.011 1.54×10−7 5.61

Chrysene* 4 228.29 0.0015 7.80×10−9 5.91

Pyrene 4 202.26 0.132 4.25×10−6 5.18

Benzo[a]pyrene* 5 252.32 0.0038 4.89×10−9 6.06

Benzo[b]fluoranthene* 5 252.32 0.0015 8.06×10−8 6.04

Benzo[e]pyrene 5 252.32 0.0063 7.60×10-7 6.44

Benzo[k]fluoranthene* 5 252.32 0.0008 9.59×10−11 6.06

Dibenz[a,h]anthracene* 6 278.35 0.0005 2.10×10−11 6.84

Benzo[g,h,i]perylene* 6 276.34 0.00026 1.00×10−10 6.50

Indeno[1,2,3-cd]pyrene* 6 276.34 0.062 1.40×10−10 6.58

U.S. EPA classified PAH in italics as possible human carcinogens (NTP, 2005)

Figure 2.2 Structures of U.S. EPA’s 16 Priority Pollutant PAHs (Lundstedt, 2003)

Symmetry, planarity, and the presence of substituents also affect PAH solubility, evaporability, and biodegradability. Planar PAHs are less reactive (i.e. less soluble) and biologically less toxic (Dabestani and Ivanov, 1999). Figure 2.2 shows the molecular structures of U.S. EPA’s 16 priority pollutant PAHs.

Alternant PAH compounds that are planar and symmetrical require a relatively high energy to dissolve because of their ability to fit closely in a lattice. Thus, they tend to be less soluble (Harvey, 1997). As the compounds deviate from planarity or symmetry they tend to be more soluble in organic solvents. For instance, as Figure 2.2 and Table 2.1 shows, benzo[a]anthracene and chrysene both have four rings and they are isomerism compounds (two or more compounds having the same molecular formula but a different arrangement of atoms within the molecule). But due to the centrosymmetric structure of chrysene, it is more difficult

to dissolve in water (0.0015 mg/L) than benzo(a)anthracene (0.011mg/L).

Most of the PAHs has a low solubility and do not dissolve easily in water at normal temperature. As the number of benzene rings in a PAH compound increases, solubility generally decreases (Wilson and Jones, 1993). Solubility is estimated by octanol-water partition coefficients (Kow). There are substantial amounts of data on the relationship between aqueous solubility and octanol-water partition coefficients (Kow) (Mackay and Callcott, 1998).

The Kow coefficients of U.S. EPA priority PAHs have been listed in Table 2.1. Following equation is an inverse relationship between Kow and solubilities:

푎푚표푢푛푡 표푓 표푟푔푎푛𝑖푐 푐ℎ푒푚𝑖푐푎푙 𝑖푛 표푐푎푡푎푛표푙 (푚푔/퐿) Kow= 푎푚표푢푛푡 표푓 표푟푔푎푛𝑖푐 푐ℎ푒푚𝑖푐푎푙 𝑖푛 푤푎푡푒푟 (푚푔/퐿)

These coefficients are a measure of the difference in solubility of the compound in these two phases. Hydrophobic compounds have high octanol/water partition coefficients. Due to their low solubility and high hydrophobicity in water, PAHs are most likely to stick tightly to particles, and thus ultimately accumulated in soils (Hughes, Beckles et al. 1997).

Another important property of PAHs is vapor pressure. Vapor pressure defines the point at which PAHs in the liquid state either evaporate into a gaseous state or condense back to a liquid state. A more volatile compound has a higher vapor pressure (Mackay and Callcott,

1998). Table 2.1 lists the vapor pressure of the PAHs. For instance, phenanthrene (vapor pressure=9.07×10-2 Pa) is more volatile and would evaporate more rapidly than pyrene (vapor pressure=3.33×10-4 Pa). Polycyclic aromatic hydrocarbons vapor pressures are important for determining the risks associated with dredging soils due to their carcinogenic, mutagenic, and teratogenic effects to researchers. Because of their transfer between two phases (soil and air), knowledge of vapor pressure of PAHs can keep field sampling and lab working in safe.

Volatilization is a major transfer process of PAHs in air, soil and water. And Wild and

Jones (1993) reported some abiotic loss of low molecular weight (LMW) PAHs from soil or water surface by volatilization. Vapor pressures controlling volatilization have been found depend upon the temperature, humidity and wind speed. Henry’s Law is used for estimating the transfer process of PAHs in both soil and water source (Eweis et al., 1998).

Henry’s Law:

푝 m = HRT = (T+273) 퐶1

Where,

m= Henry’s Law coefficient (atm·m3/mol)

H= dimensionless Henry’s Law coefficient

R= universal gas constant (atm m3/(mol·K))

T= temperature (oC)

p= partial pressure of the solute in the gas above the solution (atm)

3 Cl= the liquid-phase contaminant concentration (g/m )

Volatilization of PAHs from soil, atmospheric particles, water and vegetation increases with air temperature (Ausma et al., 2001; Sofuoglu et al., 2001). Wind above the surface enhance the volatilization of PAHs (Currado and Harrad, 2000; Sofuoglu et al., 2001). Some low molecular weight PAHs are semi-volatile organic compounds. If m more than 0.304 pa m3

/mol, then the chemical is volatile, but if m less than 0.304 pa m3/mol, then volatilization is not an important transfer (Eweis et al., 1998). For instance, the henry’s law constants of oxygen

3 3 and CO2 are 78020 pa m /mol and 2980 pa m /mol. Comparing to oxygen and CO2, henry’s law constants of pyrene and phenanthrene are 1.71 pa m3/mol and 4.29 pa m3/mol at 25 °C

respectively which is much smaller. Although their henry’s law constants are higher than 0.304 pa m3/mol, they are hard to evaporate. In general, most of the PAHs are likely to attach onto particulate matters in soil, has low solubility and difficult to evaporate.

2.3 PAHs Biodegradation

Biological degradation of PAHs is the process where PAH structures are altered from their original form (Abbey, 2011). The end product of biodegradation is the complete mineralization of PAHs to CO2, water, microbial carbon, and other inorganic compounds

(Lundstedt, 2003). The major pathway of PAHs in soils is degradation by microbial metabolism.

Other degradation pathways like photolysis, hydrolysis and chemical oxidation processes do not contribute measurably to loss of PAHs from soil (Sims, 1983).

In soil, PAH compounds generally undergo a two-phase loss process (Alexander, 2000;

Reid et al., 2000a; Semple et al., 2004; Niqui-Arroyo and Ortega-Calvo, 2006): 1) Rapid initial loss of low molecule weigh (LMW) PAHs to biodegradation. This removes the more labile fraction and results in a high molecular weight HMW PAH-dominated profile. 2) Slower loss as contact time increases (PAHs decay). For instance, Uyttebroek et al. (Uyttebroek, Spoden et al., 2007) reported a two phase loss upon contaminated soil with phenanthrene and pyrene.

There was rapid biodegradation of PAHs during the first 30 days, followed by slow but continuous biodegradation rates so that almost all the spiked amount was mineralized during the 140 day experimental period.

Bacterial, fungal and algal species have been found to degrade PAH compounds

(Cerniglia et al., 1992), and microbial degradation is believed to be one of the major processes to clean up PAHs contaminated soils (Hughes et al., 1997). Previous studies have reported that

indigenous microbial communities could have a considerable potential to remedy the oil- contaminated soils (Ramsay et al., 2000) and remove phenanthrene from aqueous solution

(Tam et al., 2002, 2003). Numerous bacteria genera and species can degrade 2- or 3-ring PAHs have been identified, but few genera have shown ability to degrade HMW PAHs. In the past two decades, a number of various bacteria have been identified as “PAH degraders”, including the common genera of Pseudomonas (Weissenfels et al. 1990), Sphingomonas (Desai et al.

2008), Cycloclasticus (Geiselbrecht et al. 1998), some anoxic bacteria of Deltaproteobacteria

(Musat et al. 2009) and Alcaligenes (Weissenfels et al. 1990).

The physical and chemical properties of the particular PAHs compounds being biodegraded impact this process, as well as environmental factors. Cerniglia (1992) summarized several factors that influence the biodegradation rate of PAHs: a) physical factors: salinity, temperature, and solubility; b) chemical influences: PAH molecular structures, PAH concentration, organic content, nutrient, pH, and redox potential; c) biological influences: bacteria type, population, distribution, previous exposure.

2.3.1 Physical Factors Influences on Biodegradation

PAH solubility, water salinity, temperature can influences the biodegradation of PAHs.

Most of the PAHs has a low solubility and do not dissolve easily in water at normal temperature

(Abbey, 2011; Lu, 2011). In wetland soils, PAHs are most likely to stick tightly to particles

(Hughes, Beckles et al. 1997). PAHs can only be biodegraded after they have been released

(e.g., by desorption) into an aqueous phase containing microorganisms that are capable of degrading them. (Reid, 2000; Johnsen, 2005; Rijnaarts et al., 1990; Volkering et al., 1992).

Mass transfer through diffusion to the aqueous phase is the rate limiting step in biodegradation

in soils because it often controls PAH availability to microorganisms (Volkering et al., 1992).

Yang et al (2011) also indicated that the biodegradations of low molecular weight PAHs were governed by desorption from soils; while for the higher molecular weight PAHs, microbial degradation potential was the controlling factor. High molecular weight PAHs (e.g. benzo[e]pyrene) are more recalcitrant because of their very low solubility (Cerniglia, 1992;

Wilson and Jones, 1993; Juhasz and Naidu, 2000).

Solubility of PAH compounds in water is dependent upon temperature, pH, ionic strength (concentration of soluble salts), and other organic chemicals (i.e. dissolved organic carbon) (Pierzynski et al., 2000). Biodegradation rates of PAHs vary depending on solubility of the PAH compounds (Alexander, Zhang et al. 1999). In general, low molecular weight PAHs are degraded more rapidly than high molecular weight PAHs (Jarabak, Harvey et al. 1997).

PAHs with lower solubility are more difficult to be degraded in soils.

Environmental conditions such as temperature have an important effect on the selection, survival, and growth of microorganisms (Alexander, 1999; Harmsen, 2007). In general, optimal growth for a particular microorganism occurs within a fairly narrow range of temperature although most microorganisms can survive within much broader limits (Alexander,

1999; Olson et al., 2003; Straube et al., 2003; Harmsen et al., 2007). The ideal soil temperature range for biodegradation is between 15 and 45°C, with maximum rates of biodegradation occurring from 25 to 35°C (Sims et al., 1990).

Salts, often present in dredge soils, have a negative impact on microorganisms. With increasing levels of salinity, rates of hydrocarbon metabolism decrease (Ward and Brock,

1978).

2.3.2 Redox Potential Influences on Biodegradation

Redox potential (also known as reduction potential, oxidation / reduction potential,

ORP, pE, ε, or Eh, mV) is a measure of the tendency of a chemical species to acquire electrons and thereby be reduced. The redox potential is positive or high in strongly oxidizing systems and negative or low in strongly reducing systems (Gambrell, 1996). Redox (reduction- oxidation) reactions include all chemical reactions in which molecular changed their oxidation state. Redox reactions involve the transfer of electrons between species.

Pankow (1991) described the negative logarithm of the electron activity (pe) as the master variable for describing the equilibrium position for all redox couples in a given system:

pe = - log (e-)

It can be shown that pe is related to Eh by

Eh = pe*(2.303*R*(T+273))/F Where: R = gas constant = 8.314 J/（K·mol）

T = temperature, oC

F = Faraday constant = 96.485*103 C/mol

At 25°C (298°K) this simplifies to

Eh = pe *0.05916 and pe =Eh / 0.05916

According to Faulkner et al. (1989), redox is a quantitative measure of electron availability and is indicative of the intensity of oxidation or reduction in both chemical and biological systems. When based on a hydrogen scale, redox (Eh) is derived from the Nernst

Equation (Stumm and Morgan 1981):

o ni nj Eh= Eh + 2.3 ·(R·T)/nF·log[ i·(ox) / j·(red) ] Where:

o Eh = potential of reference, mV

n = number of moles of electrons transferred

F = Faraday constant = 96.485 J/ (mol·mV)

(ox) and (red) = activity of the oxidants and reductants, respectively

A reference electrode is an electrode which has a stable and well-known electrode

o potential or Eh . The high stability of the electrode potential is usually reached by employing a redox system with constant (buffered or saturated) concentrations of each participants of the redox reaction (Bard, 2000; Gambrell, 1996). Potential of reference depends on the material of reference electrode. Two kinds of platinum usually applied in redox potential measurements:

1) Ag/AgCl, 2) Hg/Hg2Cl2. Equations used for Hg/Hg2Cl2 (Calomel) potential calculating are:

- - Hg2Cl2(s) + 2e = 2 Hg (liq) + 2Cl

2 3 E = E1 - d1 * (dT) - d2 * (dT ) - d3 * ( dT )

E, E1 in mV, dT = T - 25 for T in ºC .

Dean (1963) estimated that for saturated KCL solution, E1 = + 241.2, d1 = 0.661, d2 =

-3 -7 o 1.75x10 , d3 = 9.0x10 . For instance, when room temperature controlled at 20 C, the potential of calomel reference electrodes is 244.5 mV in saturated KCL solution. By applying this equation above, Table 2.2 shows the potential of calomel reference electrodes in different concentration of KCL solutions.

Table 2.2 Reference Potentials Varies With Temperature and Solution Concentration

Temperature (oC) 10 15 20 25 30 Saturation KCL 250.7 247.6 244.5 241.2 237.9 3.5 M KCL 256.0 254.0 252.0 250.0 248.0 1.0 M KCL 283.7 282.6 281.4 280.1 278.7

Handbook of Analytical Chemistry

M stands for molarity. 3.5 M KCL means 3.5 mol KCl in 1 liter of solution.

Generally, the redox potential of surface water and oxidized upland soils is better characterized by dissolved O2 and atmospheric O2 measurements, respectively. However, soil redox potential measurements have been widely used to characterize the intensity of redox potential and relate this to biological processes occurring in flooded soils. Along with the disappearance of soil O2, the decrease in redox potential is the most striking change caused by flooding a soil (Gambrell, 1996). Measured redox potentials of reduction reactions are listed in

Table 2.3.

Table 2.3 Reduction Reactions and Redox Potential Range in Soil

Reduction Reactions Measured Redox Potential (mV)

O2 → H20 400 ~ 600

NO3 → N2 200 ~ 400

2+ MnO2 →Mn 100 ~ 200

2+ Fe(OH)3 → Fe -50 ~ 100

2- 2- SO4 → S -200 ~ -100

CO2 → CH4 -300 ~ -220

Although PAHs are known to be most often biodegraded aerobically (Cerniglia, 1992;

Kastner et al., 1998; Yuan et al., 2000), evidence for anaerobic biodegradation is available for

some PAHs (Ambrosoli, 2005; Eriksson et al, 2003; Heider, 1999). In fact, microorganisms are known to have a strong potential for the biodegradation of a wide range of organic chemicals, either in oxic or in anoxic conditions (Heider et al. 1999; Head. 2006). The action is termed aerobic when oxygen is used for the electron acceptor, and reactions involving other electron accepters are considered anaerobic (Tchobanoglous, Burton et al. 2003). Aerobic bacteria can utilize PAHs as carbon and energy source in the presence of oxygen (Eriksson et al. 2003;

Quantin et al. 2005). However, under reducing conditions, the biodegradation of organic matters is the result of anaerobic bacterial respiration (Ambrosoli, Petruzzelli et al. 2005).

Eriksson et al (2003) have found aerobic biodegradation at 20°C removed 92% of the phenanthrene and 71% of pyrene in a Varta soil, but anaerobic biodegradation removed only

7% of phenanthrene and 1% of pyrene. The biodegradation kinetics was well described by first- order kinetic model or zero-order kinetic model (Bregnard et al. 1996; MacRae and Hall 1998).

In coastal marine soil, oxygen is less available in the subfacial layer. Normally, soils are associated with low dissolved oxygen and reduced redox potentials (Gambrell, 1996). PAH compounds often undergo slow biodegradation in anaerobic conditions (Alexander, 1999). The feasibility of anaerobic biodegradation of PAHs under sulfate-reducing condition has been demonstrated in a number of investigations (Coates et al. 1996; Zhang and Young 1997; Lei et al. 2005).

“The term anoxic is used to distinguish the use of nitrite or nitrate for electron acceptors from the others under anaerobic conditions” (Tchobanoglous, et al. 2003). Vitte et al. (2011) observed a similar removal efficiency of PAHs in anoxic/oxic switched condition and permanent oxic condition. Similarly, Abril et al. (2010) and Bastviken et al. (2004)

demonstrated that an anoxic period preceding an oxic period promotes the organic matter biodegradation. In addition, Hulthe et al. (1998) showed that re-exposure to oxygen of oxic coastal soils buried to anoxia stimulated the biodegradation of organic matter. Switching between anoxic/oxic conditions stimulates both anaerobic and aerobic metabolisms, enhancing the biodegradation of refractory PAH compounds (Aller, 1994; Hulthe et al. 1998). It is probably because it prepared a condition for a better degradation by aerobic metabolisms after the oxygen supply (Vitte et al., 2011).

2.3.3 PAH Concentration, Organic Content, Nutrient, pH Influences

Microbial activity usually functions optimally at a certain carbon: nitrogen: phosphorus ratio (Abbey, 2011). Lack of sufficient carbon and nutrient sources to sustain the growth of biodegrading microorganisms may affect the biodegradation success (Odokuma and Dickson,

2003; Ward and Singh, 2004). In contaminated sites, where organic carbon levels are often high due to the nature of the PAHs, available nutrients (N/P) can be rapidly depleted due to microbial utilization (Breedveld and Sparrevik 2000). Therefore, it is common practice to supplement contaminated soil with nutrients, generally nitrogen and phosphates, to stimulate the in situ microbial community and therefore enhance bioremediation (Breedveld and

Sparrevik 2000). The levels of nutrient addition required for PAH biodegradation are generally thought to be similar to those required for other organic pollutants. Lei et al. (2005) found that an amendment of inorganic N and P did enhance the level of PAH biodegradation. Also, it has been demonstrated that concentrations of phenanthrene did not change significantly without inorganic fertilizer (N, P), but decreased more than 25 times in soil amended with N and P

(Betancur-Galvis et al. 2006). The amount of nutrients present and the state of the nutrients

(organic, inorganic) is important for biodegradation. Addition of nutrients through fertilizers can enhance biodegradation.

Soil organic matter was an important factor influencing the biodegradation of PAHs.

Many studies have shown that the bioavailability of PAHs was controlled by the interaction between PAHs and soil organic matter, especially the sequestration in the carbonaceous materials (Luthy et al., 1997; Cornelissen et al., 2005; Ghosh, 2007)

Another important factor for the biodegradation activity is the pH of the soil or sediment as it may affect the solubility and bioavailability of the pollutants and nutrients. Soil pH has an important effect on the selection, survival, and growth of microorganisms. Since the vast majority of bacteria exhibit optimal growth at or near neutral pH values, most laboratory-based biodegradation studies have been carried out in pH range of 5.0–9.0 (Yuan et al. 2001; Kim et al. 2003; Chang et al. 2008; Lu et al. 2011). Biodegradation was enhanced after pH of the soil was adjusted from 5.2 to 7.0 by adding CaO. Soil pH also impacts microbial activity and can alter the community composition (i.e. fungal vs bacterial dominated). The ideal pH range for bacteria is generally between 6.0 and 8.0 (Eweis et al., 1998). Soil pH also influences the mobility of nutrients and metals. For example, phosphorous solubility is maximized at pH 6.5 and metal mobility minimized above pH 6.0 (Sims et al., 1990). Availability of metals, which are toxic to some microorganisms, can greatly reduce biodegradation of PAHs.

CHAPTER III METHODOLOGY AND MATERIALS The investigation was conducted in three phases. Section 3.1 consist of equipment building for simulating tidal actions. Section 3.2 shows the soils sample mixed with PAHs compounds and nutrients. Section 3.3 covers measuring and analyzing of the redox potentials.

Section 3.4 includes biodegradation studies of PAHs.

3.1 Equipment System

In this research, a diurnal tide (only one high and one low tide each day) was simulated in a bench scale equipment for measuring redox potentials in wetland soils. Equipment consisted of experimental tanks, dilution tanks, controlling tank, trays of soil, ORP measuring electrodes, ORP reference electrode, tubes, valves, timers, pumps, and reservoir of dilution water (Figure 3.1). Redox potentials were measured in one of the four experimental tanks.

Figure 3.1 Flow Chart of Equipment System

Figure 3.2 Simulating Tides in Experimental Tanks by Hydraulic Control Tank

When a tidal cycle begins, air is pumped into the air chamber into the hydraulic control tank (Figure 3.2). Water is slowly pushed out from the air chamber bottom by the air pressure increase. Therefore, the water level in hydraulic control tank increases. Tubes and valves are attached for connecting the air chambers in experimental tanks and controlling tank. Thus, the air pressure in hydraulic control chamber and experimental chamber are always equal. So as the air pressure in experimental chambers increases, water is pushed out from the bottom chamber and the water level in experiment tanks increases (Figure 3.3). Overflows pass through the water outlets and go to drain.

Figure 3.3 Design of Equipment System for Hydraulic Control

Valves were applied in a semi-open status to adjust the speed of air passing through. In order to control the rising or falling of water level, one air pump and two valves were connected in a line (Figure 3.1). The air inflow rate (q1) of hydraulic control chamber is adjusted by valve

1, and the air outflow rate (q2) of hydraulic control chamber is adjusted by valve 2 (Figure 3.1).

During pumping, q1 is fixed to be higher than q2 so that water level increases with the air pressure. When pump is turned off, inflow rate is zero but outflow rate is still equal to q2 so that the water level falls in experimental tanks.

80 70 60 50 40 30 20

Water Level WaterLevel (mm) 10 0 0 2 4 6 8 10 12 14 16 18 20 22 24

Time (hour)

Figure 3.4 Variation of Water Level in Experimental Tank during a Tidal Cycle

Figure 3.4 shows the variation of water levels in experiment tanks in a single tidal cycle.

The length of pumping time or flooding time was controlled by the timer. In this research, the air pump was on from 12:00 to 23:59 and was off at other times. As a result, all of the soils trays were submerged during 13:00 to 1:00 if we assumed that rising and falling of water needs

60 minutes. The highest water level is about 70 mm higher than the lowest water level which use around 60 minutes to 80 minutes to rise to raise the water.

Figure 3.5 ORP Electrodes Placed in Periodically Emerged Tray and Submerged Tray

Figure 3.5 shows a permanent submerged tray of soils and a periodically emerged tray of soils that were placed in one of four experiment tanks. Three ORP electrodes and the reference electrode was placed in periodically emerged tray, and one ORP electrode was placed in a submerged tray. Electrodes were fixed approximately 2 cm below the soil surface. Other reports indicated that about 10% of water in the bay, where we collected soils from, is lost to the ocean in a single tidal cycle. For this reason, dilution tanks and reservoir were used for simulating the tide in the bay.

3.2 Soils Sample and PAHs Pollutants

The soil samples used in the experiments were collected by Mr. Miles from wetlands at the mouth of Mississippi River (Empire, Louisiana; 29º23’55”, 89º36’31”W). These samples were a viscous soil composed of aged organic matter and soils subjected to contamination by discharges from petroleum related industry plant built along Mississippi River, as well as oil spills from the Gulf of Mexico. Soils sampling locations are not necessarily representative of widespread coastal conditions. Instead, they were selected to reflect soils with a history of PAH exposure.

For studying the tidal action influences on environmental remediation of wetland soils, phenanthrene (Phe),pyrene (Pyr), benzo[e]pyrene (BeP) were chosen as the compounds in this research since they exhibit intermediate toxicity (Nisbet and Lagoy, 1992), hydrophobicity, and environmental persistence (Mackay et al., 1992) relative to the 16 U.S. EPA priority PAHs.

Pyrene and phenanthrene are the dominant PAHs produced from incomplete combustion of oil and oil products (Prahl et al., 1984), and they also originate from oil spills and seeps. Pyrene has been shown to be toxic to a wide range of marine organisms, like copepods (Hjorth et al.,

2007) and microalgae (Long et al., 2001; Lei et al., 2003; Petersen et al., 2008). Although it is not as problematic as benzo[e]pyrene, animal studies have shown pyrene is toxic to kidneys and liver.

Initial concentrations of phenanthrene, pyrene, benzo[e]pyrene are 180.67 ug/kg,

172.67 ug/kg, 163.67 ug/kg respectively. Table 3.1 shows phenanthrene, pyrene and benzo[e]pyrene has relative low water solubility and vapor pressure comparing to other PAHs, their evaporation can be neglected. Soils were mixed with chemicals (KNO3+Na2HPO4·7H2O).

And the C: N: P at a ratio of 100:10:4 (only carbon of PAHs is calculated in the ratio) was in the nutrient. The initial Table 3.1 and Figure 3.6 show the chemical and physical properties of phenanthrene, pyrene, benzo[e]pyrene.

Table 3.1 Properties of Phenanthrene, Pyrene, Benzo[e]pyrene (ATSDR, 2005)

PAH Name Number Molecular Solubility in Log Vapor of Rings weight(g/mol) Water(mg/L) Kow Pressure(mmHg) Phenanthrene 3 178.23 1.10 4.45 6.80×10−4 Pyrene 4 202.26 0.132 5.18 4.25×10−6 Benzo[e]pyrene 5 252.32 0.0063 6.44 7.60×10-7

Figure 3.6 Molecular Structures of Phenanthrene, Pyrene, Benzo[e]pyrene (Lundstedt, 2003)

In wetland soils, PAHs are most likely to stick tightly to particles (Hughes, Beckles et al. 1997). But certain PAHs move through soil and eventually can contaminate the underground water (ATSDR, 2005). The rate of water movement in unsaturated soils is very slow. However, once the soil becomes saturated, water flow rates can increase dramatically (Letey and Oddson,

1972) and rates are controlled principally by soil texture and structural development. Hydraulic conductivity (K) describes the ease with which a fluid (usually water) can move through pore spaces or fractures. K dependents on the soil moisture content and pore size distribution. For clay, Hydraulic conductivity (K) ranges from 1.00×10−11 to 1.00 ×10−6. For fine sand, K ranges from 1.00×10−6 to 1.00×10−5 (Freeze and Cherry, 1979). The possible characteristics for the soils we measured are assumed and presented in Table 3.2. Each group of soils was then equally divided into 8 trays.

Table 3.2 Representative Soils Characteristics (Tchobanoglous, 2003)

D Range D K Range K(m/s) sandy clay 0.0039-0.06mm 7um 1.00×10−9 to 1.00×10−8 1.00×10−8 fine sand 0.006-2.0mm 1mm 1.00×10−6 to 1.00×10−5 1.00×10−5

Figure 3.7 Soils and Sand Layers in the Tray Seperated by Geotech Cloth

Soils diameters and porosity can influences the rate of water movement and the redox potentials in wetland soils. The characteristics of soils also impact the fate of PAHs (Abbey,

2011). As to the particle size of soil, the order of the biodegradation rates of PAHs in water– soil systems was fine silt > clay > coarse silt (Xia and Wang, 2008). In this research, 1 cm depth of fine sand were set under 3 cm depth of contaminated soils (sandy clay). Geo-tech cloths were used for separating the layers and maintaining the structures. The surface of soils was approximately 826 cm2. There are plenty of holes in the bottom of soil trays so that water can go through (Figure 3.7).

3.3 Redox Potential Measurement

The redox potential indicates the availability of free electrons and the oxidizing or reducing tendency of soils. The redox potentials of soils were measured in millivolts [mV] using ORP electrodes. For measuring redox potential, 3 electrodes were put in the periodically emerged soils, and 1 electrode was put in permanent submerged soils (Figure 3.8). The measuring points were selected randomly among the surface. Redox potentials were measured at approximately 25 mm depth below soils surface by connecting electrodes onto the ORP tester.

Aerobic biodegradation may only happen on the surface of soils which are not represented the redox measurements.

Mettler Toledo’s pH Meter (FiveEasy FiveGo FE20/FG2) was used for ORP measuring.

Calomel reference electrode was applied in experiments. For room temperature controlled at

20oC, the potential of calomel reference electrodes is 245 mV in saturated KCL solution.

Therefore, we corrected the raw data of redox potentials by adding 245 mV.

The transport and consumption of oxygen in this designed equipment system is illustrated in Figure 3.8. As figure shows, aerobic biodegradation occurs in the surface of soils and anaerobic biodegradation occurs at bottom layer. Microorganism consumed oxygen aerobically. Microorganism utilized oxygen for PAHs biodegradation. Impacted by tidal action, the moisture of soils fluctuate with water level. Atmospheric oxygen was absorbed into water phase and soils phase in order to supply biological degradation. Soils structure and properties affect the transport of oxygen between soils layers.

Figure 3.8 Flow Chart of Transport and Consumption of Oxygen in Equipment System

The redox potentials were designed to be measured every half an hour. The switching points between different redox conditions were important and measured. Measurements of redox potential in wetland soils were conducted over 120 days. Most of the data were measured only during the day and some of the data were measured during the whole day. Redox potentials were measured in full tidal cycles for investigating the tidal impact on redox potentials in soils.

For long term influence, the average redox potentials can represent the redox condition of soils.

3.4 PAHs Sampling and Analysis

The bench scale equipment was used for studying the biodegradation of phenanthrene, pyrene, and benzo[e]pyrene. Experiments were conducted over 120 days at 20oC. Soil pH was

7.90, and water pH was 8.10. Soil samples were taken by straws (D=5mm) from the trays every

7-10 days for PAH analysis. Figure 3.9 shows the straws we used to backfill the whole soils column. We use two kinds of straws for sampling: one was used to remove the soil core, another one was used to fill the hole for preventing the breakdown of the soil matrix. By this way, the sampled soils core in straws is the soil collected from surface to 25mm depth. Therefore, the concentration of PAHs taken by straws can stands for the average concentration of PAHs in soils.

Figure 3.9 Plastic Straws Were Used to Fill Holes Where Samples Were Taken

Instrumental analysis was conducted by Mr. Scott Miles (Department of Oceanography and Coastal Sciences) and Ms. Doorce Batubara (Department of Civil and Environmental

Engineering). According to U.S. EPA Method 8272 which is specifically for the determination of dissolved PAHs in interstitial water or pore water in soil samples only. GC-FID and GC-MS were used for analysis. PAH concentration was determined by the Hewlett-Packard (HP) 5890 series II gas chromatography (GC) equipped with a flame ionization detector (FID) and DB-5 capillary column (30 m × 0.52 mm i.d., with 1.5 µm film thickness). The initial oven temperature was maintained at 100◦C for 1 min, then increased to 280◦C at a rate of 6◦C/min, and held at this temperature for 10 min. Both, the injector and detector temperatures were maintained at 280◦C. Helium was used as a carrier and a make-up gas at rates of 3.5 and 30 mL/min, respectively. Hydro-gen gas and airflow rates were maintained at 30 mL/min and 300 mL/ min, respectively. Exactly 1 µL of extracted sample was injected automatically under split- less mode. Under these conditions, the retention times of phenanthrene, pyrene, and benzo[e]pyrene were 15.31, 17.24, and 21.02 min, respectively.

CHAPTER IV RESULTS AND DISCUSSION Tidal action has been simulated in the bench scale equipment by switching between aerobic soils being flooded and flooded soils being drained. Redox potentials were intermittently measured in one of the four experimental tanks over 120 days for analyzing their long-term impact on PAH biodegradation in wetland soils. Additionally, redox potentials were measured in full tidal cycles for investigating the tidal impact on redox potentials in soils. One of the aims is to study the effect of tidal action on bacterial decay of PAHs.

Plant absorption of PAHs, wave erosion of soils, and wind effect were not considered in this lab experiment. Temperature was controlled at 20oC. The major pathway of PAHs in soil was assumed as microbial degradation, and the evaporation of phenanthrene, pyrene, benzo[e]pyrene was negligible due to their low vapor pressure at 20oC and low concentrations of PAHs employed. Soils are assumed mixed well and equally separated in the four experimental tanks. Measuring points were selected randomly among the soils surface.

Electrodes for measuring redox potential were fixed at approximately 25 mm below the soils surface, and we assumed that the redox potential readings can stand for the average condition in soils of four experimental tanks. Similarly, the concentration of PAHs reflect the average concentration in soils along depth.

Figure 4.1 to Figure 4.3 give the biodegradation curves of phenanthrene, pyrene, and benzo[e]pyrene comparing between permanent submerged soils and periodically emerged soils, respectively (Batubara, 2014). As Figure 4.4 shows, in first 30 days, 49.1% and 78.5% of phenanthrene were removed from permanent submerged and periodically emerged soils, respectively. However, the final biodegradation efficiencies of phenanthrene in permanent

submerged and periodically emerged soils were 96.3% and 96.2% respectively which were similar (Batubara, 2014). The area under the shaded curves illustrated the difference between phenanthrene concentrations in permanent submerged soils and phenanthrene concentrations in periodically emerged soils. This area shows the contribution of tidal actions to PAHs biodegradation. As area indicates, tidal action greatly impacted the biodegradation of phenanthrene in the first 50 days. The biodegradation efficiencies of phenanthrene were similar after 120 days. Because of its biodegradability is limited by many factors including bacteria, temperature, redox conditions, and nutrient, phenanthrene and other PAHs cannot be completely biodegraded.

200.00 180.00 160.00 140.00 120.00 100.00 80.00

60.00 Concentration (ug/kg) Concentration 40.00 20.00 0.00 0 10 17 24 30 37 45 52 59 65 75 80 87 95 101 109 116 122 Time (Days) Deviation Submerged Sediments Periodic Emerged Sediments

Figure 4.1 Tidal Action Greatly Impacted Biodegradation Rate of Phe in First 50 Days

Figure 4.2 shows the biodegradation of pyrene in soils. Similar analysis was conducted.

In first 30 days, 63.1% and 69.9% of pyrene were removed respectively from permanent submerged and periodically emerged soils. Final biodegradation efficiencies of pyrene in

permanent submerged and periodically emerged soils were 82.3% compared to 90.8% respectively (Batubara, 2014). As the area indicates, tidal action continually impacted the biodegradation of pyrene, and impacted its final biodegradation efficiencies.

200 180 160 140 120 100 80 60

Concentration (ug/kg) Concentration 40 20 0 0 10 17 24 31 38 45 52 59 65 75 80 87 95 101 109 116 122 Time (Days)

Deviation Periodical Emerged Submerged

Figure 4.2 Tidal Action Impacted the Biodegradation Rate of Pyr in the Later Period

As Figure 4.3 shows, in first 30 days, 51.2% and 59.4% of benzo[e]pyrene were removed respectively from permanent submerged and periodically emerged soils. Final biodegradation efficiencies of benzo[e]pyrene in permanent submerged and periodically emerged soils were 74.6% compared to 82.2% respectively (Batubara, 2014). As the area indicates, tidal action continually impacted the biodegradation of benzo[e]pyrene, and impacted its final biodegradation efficiencies.

200 180 160 140 120 100 80 60 Concentration Concentration (ug/kg) 40 20 0 0 10 17 24 31 38 45 52 59 65 75 80 87 95 101 109 116 122 Time (Days)

Deviation Periodical Emerged Submerged

Figure 4.3 Tidal Action Continuously Impacted Biodegradation Rate of Bep

Table 4.1 shows the first order biodegradation reaction rate constant calculated by data from Appendix B (Batubara, 2014). The biodegradation rates of phenanthrene, pyrene, and benzo[e]pyrene were higher in periodically emerged soils where tidal actions switched redox condition and greatly impacted the biodegradation rate. As the analysis above indicate, phenanthrene, pyrene, and benzo[e]pyrene were biodegraded rapidly in first 30 days (Phe

49.1%, Pyr 63.1%, BeP 51.2% respectively) followed by continues but slowly decay in later days. Compared to pyrene and benzo[e]pyrene, the final removal efficiencies of phenanthrene were similar in periodically emerged soils (96.3%) and permanent emerged soils (96.2%) due to its higher solubility, bioavailability.

Table 4.1 First Order Biodegradation Reaction Rate Constants of PAHs (per day) Phenanthrene Pyrene Benzo[e]pyrene Permanent Submerged 2.57 2.12 1.29 Periodically Emerged 3.64 3.19 2.36

Figure 4.4 shows the biodegradation compared among phenanthrene, pyrene, and benzo[e]pyrene in permanent submerged soils, where Table 4.1 also supports, phenanthrene is the easiest one to be degraded, and benzo[e]pyrene is the hardest one to be degraded in permanent submerged soils. It may concluded as that the chemical properties of PAHs governs the biodegradation rates in redox conditions where has little impact from tidal actions. Higher solubility and biodegradability (Phe) result in higher biodegradation rate.

200.00 180.00 160.00 140.00 120.00 100.00 80.00 60.00 Concentration (ug/kg) Concentration 40.00 20.00 0.00 0 10 20 30 40 50 60 70 80 90 100 110 120 130 Time (Days)

phenanthrene pyrene benzo[e]pyrene

Figure 4.4 Comparison of Biodegradation Ability among Phe, Pyr, and BeP in Submerged Soils On the other hand, Figure 4.5 gives the results with tidal impact on PAHs biodegradation in periodically emerged soils. Similar biodegradation curve and high biodegradation efficiencies (Phe 96.3%, Pyr 90.8%, BeP 82.2%) were presented. Chemical properties of PAHs also govern the biodegradation. But comparing biodegradation efficiencies in submerged soils (Phe 96.3%, Pyr 82.3%, BeP 74.6%), tidal action enhanced the biodegradation. In first 37 days, tidal action enhanced approximately 15.2%, 13.9%, and 12.2%

of the phenanthrene, pyrene, and benzo[e]pyrene removal efficiencies.

200.00 180.00 160.00 140.00 120.00 100.00 80.00 60.00 Concentration (ug/kg) Concentration 40.00 20.00 0.00 0 10 20 30 40 50 60 70 80 90 100 110 120 130 Time (Days)

phenanthrene pyrene benzo[e]pyrene

Figure 4.5 Comparison of Biodegradation among Phe, Pyr, and BeP in Periodically Emerged Soils The relations between PAH biodegradation and redox potential will be also discussed in short timeframe by investigating the tidal impact on redox potentials in 5 full tidal cycles.

The electrodes fixed in periodically emerged soils were marked as “B, D, H”, and the submerged one was marked as “A”. Figure 4.6 shows the redox potentials in periodically emerged soils (C: N: P=100: 10: 4) measured by electrode “B” (See Appendix A).

Redox potentials of group “B” has an obvious and typical fluctuation. Therefore, the data was chosen and figure was shown for illustrating the tidal impact on redox potentials.

Redox potential measures the tendency of a substance to accept or donate electrons. The redox potential is positive or high in oxidizing systems and negative or low in reducing systems. In general, the data was collected during 5 days from where the soils experienced diurnal tides showed a similar diurnal fluctuation in redox potential: the lowest redox potential happens after

the ending of flooding a soil and before the beginning of draining a soil; the highest redox potential happens after the ending of draining a soil and before the beginning of flooding a soil.

However, the data measured (See Appendix A), which some researches also supported, indicated that pumping water may increase the redox potential in the beginning of flooding by bringing in dissolved oxygen. Redox potential in water was measured around 188 mV at 20oC.

85 80 75 70 65 60

55 Redox Potential 50 45 40 0.0 1.0 2.0 3.0 4.0 5.0 Time (Days)

Periodically Emergerd Sediments (B)

Figure 4.6 Redox Potentials Fluctuated during 5 Tidal Cycles in Periodically Emerged Soils (Electrode B) Overall conclusions from Figure 4.6: 1) redox potential of periodically emerged soils can be a dynamic nonlinear variable in coastal wetland soils over daily scales, and the design of biogeochemical experiments reflected this, 2) redox potential can change rapidly and significantly in coastal wetland soils in response of flooding and draining, 3) the data collected during 5 days from where the soils experienced diurnal tides showed a similar diurnal fluctuation in redox potential.

Although, electrodes “B, D, H” are all fixed in the same periodically emerged soils experienced 5 diurnal tides (Figure 3.8), the redox potentials measured by electrodes “B, D, H”

were different. Figure 4.2 shows the redox potentials in periodically emerged soils measured by electrodes “B, D, H”.

100

0 0 1 2 3 4 5 B D

-50 H Redox Potential Redox Potential (mV)

-100

Time (Day) -150

Figure 4.7 Comparison of Redox Potentials (Measured by Electrodes B, D, H) in Periodically Emerge Soils during Five Tidal Cycles

Generally, Figure 4.7 showed the soils impacted by tides experienced a similar fluctuation in the redox potentials. The redox potentials in periodically emerged soils fluctuated from -150 mV to 80 mV during these 5 days. Differences in measuring points perhaps come about by circuiting of water along the probes which contributed to the variation of redox.

From Figure 4.7 and Appendix A, conclusions can be summarized: 1) average redox potentials in periodically emerged soils is approximately at -9.34 mV during these 5 days which may stands for an overall redox condition of biological degradations in periodically emerged soils, 2) the redox potentials fluctuated obviously as the variance and STD number indicate.

Figure 4.8 shows the average redox potentials (B, D, H) in periodically emerged soils compared to the redox potentials (A) in permanent submerged soils.

100

0 0.0 1.0 2.0 3.0 4.0 5.0

-50 Redox Potential(mV)

-100

-150 Time (Day)

Average ORP in Periodic Emerged Sediments Permenant Submerged Sediments

Figure 4.8 Comparison of Redox Potentials between Permanent Submerged and Periodically Emerged Soils Figure 4.8 compares the redox potentials between permanent submerged soils and periodically emerged soils. The solid line in Figure 4.8 is the average oxidation-reduction potential in periodically emerged soils measured by electrodes “B, D, H”, and the dashed line is the ORP in permanent submerged soils measured by electrode “A”. Statistical analysis of the two groups of ORP is presented in Table 4.2 to Table 4.5. F-test T-test, and Z-test were calculated by EXCEL and XLSTAT.

Table 4.2 Summary Statistics for Redox Potentials

Variable Observations Minimum Maximum Mean Std. deviation -60 122 -105.000 -56.000 -83.795 12.591 7.33 122 -42.000 30.333 -9.481 17.590

Hypothesized difference (D): 0

Significance level (%): 5

Population variances for the t-test: Use an F-test

Table 4.3 Fisher's F-Test / Two-tailed Test of Redox Potentials Ratio 0.512 F (Observed value) 0.512 F (Critical value) 1.431 DF1 121 DF2 121 p-value (Two-tailed) 0.000276 alpha 0.05

Test interpretation:

95% confidence interval on the ratio of variances: [0.358, 0.733]

H0: The ratio between the variances is equal to 1.

Ha: The ratio between the variances is different from 1.

As the computed p-value is lower than the significance level alpha=0.05, one should reject the null hypothesis H0, and accept the alternative hypothesis Ha. The risk to reject the null hypothesis H0 while it is true is lower than 0.03%. The variances of two samples are significantly different.

Table 4.4 T-test for Two Independent Redox Potential Samples / Two-tailed Test Difference -74.314 t (Observed value) -37.945 |t| (Critical value) 1.971 DF 219 p-value (Two-tailed) < 0.0001 alpha 0.05 Test interpretation:

H0: The difference between the means is equal to 0.

Ha: The difference between the means is different from 0.

95% confidence interval on the difference between the means: [-78.174, -70.45]

The number of degrees of freedom is approximated by the Welch-Satterthwaite formula.

As the computed p-value is lower than the significance level alpha=0.05, one should reject the

null hypothesis H0, and accept the alternative hypothesis Ha. The risk to reject the null hypothesis H0 while it is true is lower than 0.01%. T-test indicates that the two samples are significantly different. Table 4.5 Z-test for Two Independent Redox Potential Samples / Two-tailed Test Difference -74.314 z (Observed value) -37.945 |z| (Critical value) 1.960 p-value (Two-tailed) < 0.0001 alpha 0.05

Test interpretation:

95% confidence interval on the difference between the means: [-78.153, -70.476]

H0: The difference between the means is equal to 0.

Ha: The difference between the means is different from 0.

As the computed p-value is lower than the significance level alpha=0.05, one should reject the null hypothesis H0, and accept the alternative hypothesis Ha. The risk to reject the null hypothesis H0 while it is true is lower than 0.01%. Z-test also indicates that the two samples are significantly different.

Conclusion from statistical analysis: 1) the average redox potentials are approximately

70 mV higher in periodically emerged soils than that in submerged soils, 2) as the F-test result tells, the variances of two samples are significantly different which means the redox potentials fluctuate in periodically emerged soils, 3) redox potentials of submerged soils (always 20-40 mm below the water surface in this research) also fluctuated but not as much as emerged soils did, 4) as the t-test and z-test indicated, the redox potentials in submerged soils and periodically emerged soils were significantly different.

CHAPTER V CONCLUSION Tidal action has been simulated in the bench scale equipment by switching between aerobic soils being flooded and flooded soils being drained. Redox potentials were measured in full tidal cycles over 120 days for investigating the tidal impact on redox potentials in soils.

Supported by cited references, removal of PAHs and fluctuation of redox potential were observed. Major conclusions can be summarized from Chapter IV: 1) phenanthrene, pyrene, benzo[e]pyrene were rapidly biodegraded during the first 30 to 40 days followed by slow but continuous biodegradation in later 90 days, 2) chemical and physical properties of PAHs govern their biodegradation fate, especially in permanent submerged soils with little impact from tidal actions, 3) redox potential of periodically emerged soils can be a dynamic nonlinear variable in coastal wetland soils over daily scales, and the design of biogeochemical experiments reflected this, 4) redox potential can change rapidly and significantly in coastal wetland soils in response of flooding and draining, 5) the reprehensive data collected during 5 days from where the soils experienced diurnal tides showed a similar diurnal fluctuation in redox potential in periodically emerged soils, 6) as statistical analysis indicates, the mean and variance of redox potentials in submerged soils and periodically emerged soils were significantly different, which is 70 mV higher in periodically one, 7) biodegradation of PAHs in periodically emerged soils was more likely influenced by tidal actions, 8) tidal action enhanced approximately 15.2%,

13.9%, and 12.2% of the removal efficiencies of phenanthrene, pyrene, and benzo[e]pyrene in first 37 days.

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APPENDIX A REDOX POTENTIALS DURING FIVE TIDES

SUBMERGED PERIODICALLY EMERGED AVERAGE DATE TIME A B D H B,D,H 1-JUL 9:30 -60 75 36 -89 7 10:30 -58 76 44 -100 7 11:30 -59 75 45 -100 7 12:30 -56 76 48 -99 8 13:30 -57 75 40 -133 -6 14:30 -58 73 41 -130 -5 15:30 -58 75 36 -132 -7 16:30 -57 70 38 -138 -10 17:30 -58 67 35 -145 -14 18:30 -88 60 33 -133 -13 19:30 -105 80 45 -67 19 20:30 -102 78 43 -74 16 21:30 -79 73 44 -87 10 2-JUL 8:30 -80 57 1 -85 -9 9:30 -92 61 11 -90 -6 10:30 -91 61 15 -83 -2 11:30 -76 65 16 -90 -3 12:30 -83 66 13 1 27 13:30 -90 68 19 4 30 14:30 -93 67 18 -30 18 15:30 -95 66 8 -43 10 16:30 -93 63 3 -55 4 17:30 -91 61 0 -65 -1 18:30 -93 57 -14 -69 -9 19:30 -94 56 -27 -67 -13 20:30 -95 54 -26 -70 -14 21:30 -96 51 -32 -72 -18 22:30 -97 43 -35 -74 -22 23:30 -98 40 -30 -77 -22 0:30 -94 42 -26 -60 -15 1:30 -91 46 -22 -52 -9 2:30 -88 50 -18 -44 -4 3:30 -87 54 -14 -36 1 4:30 -86 57 -10 -28 6 5:30 -85 62 -6 -20 12 6:30 -84 66 -2 -12 17 7:30 -82 70 2 -4 23 3-JUL 8:30 -80 68 1 0 23 9:30 -82 70 11 3 28

10:30 -81 68 15 1 28 11:30 -76 65 16 0 27 12:30 -83 66 13 1 27 13:30 -90 68 19 4 30 14:30 -93 67 18 -30 18 15:30 -95 66 8 -43 10 16:30 -93 63 3 -55 4 17:30 -91 61 0 -65 -1 18:30 -93 57 -14 -69 -9 19:30 -94 56 -27 -67 -13 20:30 -89 53 -37 -75 -20 21:30 -93 50 -49 -76 -25 22:30 -93 45 -59 -77 -30 23:30 -93 43 -64 -77 -33 0:30 -92 48 -69 -77 -33 1:30 -91 49 -59 -74 -28 2:30 -91 50 -49 -72 -24 3:30 -90 50 -40 -70 -20 4:30 -88 56 -35 -66 -15 5:30 -86 59 -31 -61 -11 6:30 -84 60 -28 -56 -8 7:30 -82 61 -25 -51 -5 4-JUL 8:30 -80 61 -23 -46 -3 9:30 -75 62 -19 -42 0 10:30 -67 63 -17 -29 6 11:30 -61 63 -15 -31 6 12:30 -73 61 -14 -30 6 13:30 -71 70 -8 -33 10 14:30 -66 73 -6 -43 8 15:30 -64 72 -10 -47 5 16:30 -69 68 -25 -54 -4 17:30 -74 64 -43 -61 -13 18:30 -80 59 -57 -68 -22 19:30 -85 58 -59 -70 -24 20:30 -90 57 -61 -67 -24 21:30 -90 56 -80 -73 -32 22:30 -89 55 -88 -78 -37 23:30 -87 53 -96 -82 -42 0:30 -73 56 -95 -81 -40 1:30 -68 59 -83 -80 -35 2:30 -70 62 -71 -79 -29 3:30 -68 65 -59 -78 -24 4:30 -65 68 -47 -77 -19 5:30 -67 71 -35 -76 -13

6:30 -66 74 -23 -75 -8 5-JUL 7:30 -63 77 -11 -86 -7 8:30 -61 75 1 -100 -8 9:30 -57 74 1 -110 -12 10:30 -58 74 1 -130 -18 11:30 -73 80 4 -129 -15 12:30 -76 74 3 -124 -16 13:30 -77 78 10 -90 -1 14:30 -76 76 10 -98 -4 15:30 -85 75 7 -120 -13 16:30 -88 72 -5 -119 -17 17:30 -94 67 -20 -118 -24 18:30 -97 63 -30 -116 -28 19:30 -102 62 -34 -125 -32 20:30 -100 61 -40 -123 -34 21:30 -102 59 -46 -124 -37 22:30 -103 59 -53 -123 -39 23:30 -101 58 -62 -122 -42 0:30 -99 60 -46 -120 -35 1:30 -98 62 -39 -118 -32 2:30 -96 64 -32 -117 -28 3:30 -95 66 -25 -116 -25 4:30 -98 68 -18 -115 -22 5:30 -91 70 -11 -114 -18 6:30 -88 72 -4 -113 -15 7:30 -85 74 3 -112 -12 8:30 -86 76 10 -114 -9 6-JUL 9:30 -85 77 12 -117 -9 10:30 -87 77 13 -118 -9 11:30 -88 79 14 -123 -10 12:30 -88 78 16 -120 -9 13:30 -89 80 21 -123 -7 14:30 -90 77 15 -119 -9 15:30 -90 70 14 -143 -20 16:30 -90 71 9 -146 -22 17:30 -91 70 6 -145 -23 18:30 -92 69 3 -142 -23 19:30 -93 69 2 -136 -22 20:30 -87 68 -3 -147 -27 21:30 -94 60 -10 -141 -30

APPENDIX B PAH CONCENTRATIONS OVER 120 DAYS

Time (day) Sediment Levels PAH compounds Average(µg/kg) STDV(µg/kg) 0 Submerged and Emerged Phe 180.67 3.51 Pyr 172.67 7.02 BeP 163.67 11.15 10 Submerged Phe 166.33 10.26 Pyr 131.33 17.62 BeP 156.67 11.85 Periodically Emerged Phe 134.33 5.03 Pyr 147.33 8.02 BeP 123.23 28.45 17 Submerged Phe 143.67 7.77 Pyr 87.00 6.69 BeP 114.57 21.21 Periodically Emerged Phe 113.33 5.13 Pyr 91.73 6.13 BeP 90.53 16.30 24 Submerged Phe 121.33 11.50 Pyr 57.37 8.08 BeP 86.40 4.61 Periodically Emerged Phe 56.83 4.32 Pyr 65.03 6.64 BeP 74.33 10.91 30 Submerged Phe 92.03 5.95 Pyr 63.67 9.37 BeP 79.87 9.25 Periodically Emerged Phe 38.80 2.85 Pyr 52.00 9.46 BeP 66.50 5.51 37 Submerged Phe 68.83 16.90 Pyr 59.23 8.82 BeP 69.57 11.76 PAH 197.63 37.48 Periodically Emerged Phe 41.33 10.76 Pyr 35.27 3.63 BeP 49.53 6.75 45 Submerged Phe 61.83 12.10 Pyr 41.60 5.45 BeP 71.60 12.05 Periodically Emerged Phe 24.63 8.21 Pyr 26.43 4.32

BeP 43.93 8.05 52 Submerged Phe 37.63 6.27 Pyr 49.23 9.90 BeP 64.97 11.30 Periodically Emerged Phe 18.27 4.45 Pyr 29.80 4.76 BeP 39.03 7.52 59 Submerged Phe 30.13 4.76 Pyr 41.30 6.35 BeP 53.50 5.09 Periodically Emerged Phe 18.40 7.21 Pyr 21.97 1.38 BeP 39.67 2.28 65 Submerged Phe 21.40 2.79 Pyr 39.20 6.13 BeP 56.50 5.62 Periodically Emerged Phe 15.53 3.76 Pyr 19.73 1.04 BeP 43.47 8.18 75 Submerged Phe 15.50 2.40 Pyr 39.77 2.25 BeP 67.07 2.47 Periodically Emerged Phe 14.61 4.70 Pyr 25.77 3.42 BeP 41.73 8.47 80 Submerged Phe 14.77 0.91 Pyr 36.37 6.40 BeP 54.80 8.23 Periodically Emerged Phe 12.67 4.14 Pyr 16.07 2.73 BeP 39.00 3.65 87 Submerged Phe 14.37 0.80 Pyr 33.37 8.95 BeP 54.47 5.15 Periodically Emerged Phe 9.07 3.81 Pyr 14.90 2.95 BeP 40.37 3.61 PAH 64.34 10.37 95 Submerged Phe 10.86 1.10 Pyr 39.00 3.80 BeP 53.00 2.63 Periodically Emerged Phe 10.37 1.76 Pyr 17.50 2.35

BeP 39.43 4.53 101 Submerged Phe 14.37 1.38 Pyr 32.10 4.39 BeP 52.27 1.64 Periodically Emerged Phe 7.09 1.12 Pyr 11.30 0.87 BeP 37.53 6.86 109 Submerged Phe 8.62 1.31 Pyr 33.77 4.25 BeP 62.73 1.72 Periodically Emerged Phe 6.66 0.60 Pyr 16.03 1.60 BeP 44.20 1.61 116 Submerged Phe 9.36 2.35 Pyr 34.43 6.10 BeP 50.80 2.93 Periodically Emerged Phe 7.06 2.43 Pyr 16.53 2.68 BeP 29.40 3.48 122 Submerged Phe 6.71 1.11 Pyr 30.63 4.61 BeP 41.57 4.63 Periodically Emerged Phe 6.69 0.74 Pyr 15.80 3.57 BeP 29.10 3.22

THE VITA Haoxuan Zhang, a native of Beijing, China, received his bachelor’s degree at the Beijing

University of Aeronautics and Astronautics in 2011. Thereafter, he made the decision to enter graduate school in the Department of Civil and Environmental Engineering at Louisiana State

University. He will receive his master’s degree in May 2014 and plans to begin work at Baton

Rouge, US.