Effects of Metam Sodium on Soil Microbial Communities: Numbers, Activity, and Diversity

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Authors Sederholm, Maya

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EFFECTS OF METAM SODIUM ON SOIL MICROBIAL COMMUNITIES: NUMBERS, ACTIVITY, AND DIVERSITY

By

Maya Sederholm

______

A Thesis Submitted to the Faculty of the

DEPARTMENT OF SOIL, WATER AND ENVIRONMENTAL SCIENCE

In Partial Fulfillment of the Requirements For the Degree of

MASTER OF SCIENCE

In the Graduate College

THE UNIVERSITY OF ARIZONA

2016

STATEMENT BY AUTHOR

The thesis titled Effects of Metam Sodium on Soil Microbial Communities: Numbers, Activity, and Diversity prepared by Maya Sederholm has been submitted in partial fulfillment of requirements for a Master’s degree at the University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library.

Brief quotations from this thesis are allowable without special permission, provided that an accurate acknowledgement of the source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author.

SIGNED: Maya Sederholm

APPROVAL BY THESIS DIRECTOR

This thesis has been approved on the date shown below:

Ian Pepper June 30, 2016 Professor, Soil, Water and Environmental Science

2 ACKNOWLEDGEMENTS

This study was supported by the National Science Foundation (NSF) Water and

Environmental Technology (WET) Center, The University of Arizona.

I would like to thank Dr. Bradley Schmitz, Dr. Luisa Ikner, Alexander Wassimi, and Maria

Campillo for their technical assistance in the laboratory. I would like to thank Dr. Reina Maier for help with stoichiometry and Dr. Walter Betancourt and Dr. Aditi Sengupta for assistance on molecular analyses. I would like to thank Mr. Richard Wagner, Mr. Jeffrey Bliznick, Mr. Stephen

Husman, Mr. Randy Norton, and Ms. Julia Rosen for helping with the construction, safety, and legal aspects of the project. We wish to acknowledge The University of Arizona Genetics Core for their sequencing services and Motzz Laboratory, Inc. for their soil analyses.

Lastly, I would like to thank my academic advisor, Dr. Ian Pepper, for providing the opportunity to conduct this research project and for guiding me through my academic career.

3 TABLE OF CONTENTS

LIST OF TABLES AND FIGURES ……………………………………………………………………………………………… 6 ABSTRACT ………………………………………………………………………………………………………………………...….. 7 CHAPTER 1: INTRODUCTION AND OBJECTIVES …………………………………………………….……………….. 8 CHAPTER 2: LITERATURE REVIEW ……………………………………..………………………………………………… 9 1. History of Fumigation ……………………………………..…………………………………………………………….…… 9 2. Metam Sodium ……………………………………..…………………………………………………………………..……… 10 3. Applications of MS ……………………………………..…………………………………………………………………..… 10 4. Epidemiological Studies ……………………………………..……………………………………………………..……… 11 5. Pesticide Regulation ……………………………………..……………………………………………………………..…… 13 6. Retention of MS and MITC …………………………………….……………………………………………………….…. 14 7. Combinations of Fumigants …………………………………………………………………………………………….... 15 8. Effects of MS on Microbial Communities …………………………………………………………………………… 15 REFERENCES ……………………………………………………………………………………………………………….…..…. 17 APPENDIX A …………………………………………………………………………………………………………………..…… 20 1. INTRODUCTION ………………………………………………………………………………………………………….…… 22 2. METHODS ……………………………………………………………………………………………………………………….. 25 2.1. Safety Requirements for the Project ……………………………………………………………………...……….. 25 2.2. Establishment of Field Plots …………………………………………………………………………………….…….. 25 2.3. Metam Sodium Application …………………………………………………………………………………..……….. 25 2.4. Field Sampling &Transport ………………………………………………………………………………………..….. 26 2.5. Soil Moisture Content ………………………………………………………………………………………………...….. 27 2.6. Heterotrophic Plate Counts ………………………………………………………………………………………….... 27 2.7. LuminUltra® ……………………………………………………………………………………………………………..….. 28 2.8. Dehydrogenase Activity Assay ……………………………………………………………………………………….. 28 2.9. DNA Extraction ………………………………………………………………………………………………………….….. 29 2.10. 16S Gene Amplification and Purification …………………………………………………………………….... 29 2.11. Sequencing and Analysis ……………………………………………………………………………………………... 29 2.12 Hazardous Waste Handling and Disposal …………………………………………………………………..….. 30 2.13. Statistical Analysis……………………………………………………………………………………………………….. 30 3. RESULTS ………………………………………………………………………………..……………………………………...… 31 3.1. Environmental Metadata ……………………………………………………………………………………………..… 31 3.2. Culturable Heterotrophic Numbers ……………………………………………………………………………...… 31 3.3. LuminUltra® Microbial Equivalents ……………………………………………………………………….……… 31 3.4. Dehydrogenase Activity Assay TPF Concentration ………………………………………………………..… 32 3.5. Molecular Results ………………………………………………………………………………………………………..… 32 3.6. Microbial Diversity: Richness ………………………………………………………………………………………… 33 3.7. Microbial Diversity: Community Composition …………………………………………………….………..… 33 4. DISCUSSION …………………………………………………………………………………………………………………….. 35 5. TABLES ………………………………………………………………………………………………………………………..….. 43 6. FIGURES ………………………………………………………………………………………………………………………….. 47 7. REFERENCES …………………………………………………………………………………………………………….…….. 54 APPENDIX B: SUPPLEMENTARY MATERIAL ………………………………………………………………………… 59 1. Motzz Laboratory, Inc. Soil Analysis Report ………………………………………………………………………. 59

4 2. Total Activity: LuminUltra® ………………………………………………………………………………………...…… 59 3. Statistical Analyses ………………………………………………………………………………………………………..… 60 4. DNA Extraction Quality ………………………………………………………………………………………………….… 67 5. Number of Sequences per Sample …………………………………………………………………………………..… 68 6. Standard Operating Procedure …………………………………………………………………………………….…… 69 7. Chemical Hygiene Plan ………………………………………………………………………………………………..…… 72 8. Respirator Certification ………………………………………………………………………………………….………… 79

5 LIST OF TABLES AND FIGURES

Chapter 1

Table 1. Occupational pesticide illnesses, California 1950-1988

Figure 1. MS decomposition in the environment

Chapter 2

Table 1. Soil Sampling Schedule

Table 2. Environmental Metadata

Table 3. Numbers: HPCs and LuminUltra®

Table 4. Total Activity: Dehydrogenase Activity Assay

Table 5. OTU Table Summary Statistics

Table 6. Relative Abundances of Top Phyla

Figure 1. Schematic of Plot and Drip Irrigation System

Figure 2. Carboy, Irrigation System, and Tarp Set-Up

Figure 3. Environmental Metadata

Figure 4. Numbers: HPCs

Figure 5. Numbers: LuminUltra®

Figure 6. Total Activity: Dehydrogenase Activity Assay

Figure 7. Number of OTUs per Sample

Figure 8. Community Richness

Figure 9. Community Composition- Phylum

Figure 10. Community Composition- Order

6 ABSTRACT

Metam sodium is a fumigant often used as a crop pretreatment in agriculture to control a wide array of pests that may inhibit plant yields. Previously, there have only been limited studies conducted on the effects of metam sodium on native soil microbial communities and plant pathogens, and results have been inconsistent. This present study utilized control and metam sodium-treated field plots to examine the effects of metam sodium on soil microbes in terms of numbers, activity, and diversity. Metam sodium did not cause significant changes in culturable heterotrophic numbers, as shown by heterotrophic plate counts, but may have adversely affected non-culturable microbes since metam sodium did affect microbial activity.

Specifically, the LuminUltra® and dehydrogenase activity assays both showed a significant decrease in total activity in treated plots one day after soil treatment, with a return to pre- application conditions within seven days. Illumina Next-Generation Sequencing of the 16S rRNA gene showed slight changes in richness and community composition throughout the 28-day study, but initial and final communities were similar in both control and treated soils. Overall, some soil microbes were adversely affected by metam sodium, but the resilience of the soil microbial community allowed for an apparent rapid recovery in terms of numbers, activity, and diversity.

7 CHAPTER 1: INTRODUCTION AND OBJECTIVES

Fumigation is a method to control a wide range of pests in a variety of disciplines using various gaseous pesticides. Initially discovered to kill insects that diminish crop yields or destroy stored grains, fumigants are now used for other applications such as treating sewer roots in pipe systems or removing pathogens from biosolids and manures.

Soil fumigants are often applied to soil as a solution through drip irrigation systems or shanks, where the solution degrades into the gaseous component. Metam sodium (MS), one of the most widely used soil fumigants throughout the US, is a methyl dithiocarbamate salt that is often sold dissolved in water as a commercial product, such as Vapam®.

Recently, MS was certified by the Environmental Protection Agency (EPA) as an approved method for converting Class B biosolids to Class A due to its ability to inactivate helminthes and viruses. This raised the question: What are the effects, in terms of numbers, activity, and diversity, of MS on soil microbial communities? To answer this question, a month-long study was conducted using a combination of culturable, biochemical, and molecular assays to evaluate the ecological impacts of MS on native soil microbes.

Of particular interest were the following: (a) Is there an initial decrease in numbers, activity, or diversity?; (b) If so, what is the duration of these adverse effects; (c) Does the soil ever return to pre-application conditions?; and (d) Is the soil quality diminished for future agricultural use?

8 CHAPTER 2: LITERATURE REVIEW

1. History of Fumigation

Early use of fumigants was first documented in the late 1800s when the sources of plant- causing diseases were being discovered (Iranzo, Olmstead, & Rhode, 2000). Powdery mildew

(Uncinula necator) in the eastern United States (US) was known to damage many crops, and in

1845 was discovered in England, its first non-native habitat. It spread throughout Europe with grave agricultural consequences, and a variety of sulfur-based solutions were used to attempt to combat the problem. Around the same time, Phylloxera aphid was also transported from the eastern US to Europe. Farmers tried various chemical and biological methods to control the aphids, all of which failed. Eventually, several scientists- A. M. Grison, Pierre Ducharte, J. H.

Léveillé, M. J. Berkeley, E. Tucker, and Giovanni Zanardini- successfully used the first chemical fumigant, carbon disulfide, for treatment (Iranzo et al., 2000).

By the 1930s, several more chemicals were available to farmers and the public, especially to control nematodes, which had become a problem for agriculture. Methyl bromide (MeBr) was first reported in France in 1932, where it was used during a quarantine period for plant, vegetable, and fruit protection against insects (Warnert, 2010). In 1941, USDA workers Al

Taylor and C.W. McBeth were the first to use MeBr in agricultural fields, where it successfully controlled a wide range of pests, including nematodes, fungi, and weeds, while increasing crop yields (Warnert, 2010). However, in the late 1990s, atmospheric scientists discovered that MeBr caused ozone depletion, and the 1997 Montreal Protocol on Substances that Deplete the Ozone

Layer determined that MeBr would be phased out of the US and other developed countries by

2005 (US EPA, 2016). Today, some of the most commonly used soil fumigants include metam

9 sodium, chloropicrin, dazomet, 1,3-dichloropropene (telone), and dimethyl disulfide (EPA,

2016).

2. Metam Sodium

Use of metam sodium (MS) as a soil fumigant has been registered in the US since 1954, and has replaced MeBr in agriculture as a pre-plant soil treatment. It is stable at a pH above 8.8 and unstable below a pH of 7. When dissolved in water, it quickly degrades into methyl isothiocyanate (MITC), hydrogen sulfide, carbon disulfide, and elemental sulfur (Duke & Jessen,

1996; Deguigine, Lagarce, Boels, & Harry, 2011). MITC released into the atmosphere is further broken down by rapid gas-phase photolysis into methyl isocyanate, methyl isocyanide, sulfur dioxide, hydrogen sulfide, carbonyl sulfide, N-methylformamide, and methylamine, with a half- life of about 10 hours to one day (Geddes, Miller, & Taylor, 1995). MS decomposition in both acidic and alkaline environments is represented in Figure 1:

Figure 1: MS decomposition in the environment (Cain, 2010)

3. Applications of MS

Fumigants have important implications outside of agriculture. They are used for root control in pipes and sewers, which can be problematic in wastewater systems. Intrusive roots

10 cause stoppages, leaks, overflows, and foundation damage, which can lead to septic pools, contamination of surface water, and human exposure to pathogens. Therefore, non-selective, non-systemic, contact herbicides such as MS are often used to kill the roots and prevent further damage. Currently, MS is applied in conjunction with dichlobenil, a root growth inhibitor with longer lasting herbicidal effects, as a foam. This process effectively kills roots within a few hours and any residual MS is diluted in wastewater treatment facilities, and rapidly broken down to harmless by-products. (Duke & Jessen, 1996).

MS is also used to treat sewage sludge, biosolids, and manure to inactivate pathogens.

MITC has the ability to infiltrate the ascaroside layer of Ascaris eggs and kill them. Helminth eggs are extremely persistent and capable of surviving in the environment for long periods of time.

This was demonstrated by a study thatspiked sewage sludge with Ascaris suum eggs and, following MS treatment, tested egg viability after 3 weeks of incubation. Data shows that MS was highly effective in inactivating A. suum eggs (Cain, 2010).

4. Epidemiological Studies

Because fumigants can be dangerous to animals and humans, epidemiological case studies and risk assessments have been conducted. Various laboratory studies with animals show that acute MS ingestion over several days can cause reproductive issues, including weight loss in pregnant females and abnormal development in their fetuses. It has also been shown to reduce immune system cells, reduce production of certain hormones, reduce strength of muscles, cause anemia, damage lungs and liver, and alter behaviors (Cox, 2006). EPA classifies MS and MITC as probable human carcinogenic and mutagenic agents, as they damage living tissue through amino acid inactivation (Lowit, 2004).

11 A human case study looked at the incidence of childhood cancer in conjunction with exposure to pesticides in California. Linking statistics from the California Cancer Registry and

California's Department of Pesticide Regulation, one study found that there was no clear trend between commercial pesticide exposure rates and incidence of childhood cancer (Reynolds et al.,

2002). This contradicts the positive associations found in case-control studies that often include exposure data in personal home and garden settings. For example, a study found associations between pesticides and leukemia, brain cancer, non-Hodgkin's lymphoma, Hodgkin's disease, and soft-tissue sarcoma in both children and adults, with a noticeably higher incidence observed in children, possibly due to greater exposure and susceptibility in younger individuals (Zahm &

Ward, 1998).

An observational study described some of the first cases where irritant-induced asthma was reported among adults working or living near the location of a metam sodium (MS) spill that occurred in 1991 in Mt. Shasta City and Shasta Lake. Following a tank car accident, over

71,922.83 liters (L) of MS was released into the Sacramento River. The breakdown product, airborne MITC fumes, traveled an estimated 40 miles, and residents and workers reported headaches, nausea, wheezing, burning eyes, skin rashes, and acute respiratory illnesses over the next several days. Average concentrations of MITC were 4-5 parts per billion (ppb) six days after the spill, with maximum concentrations reaching 1,600 ppb. Data such as location, age, sex, medical records, symptoms, and exposure were collected from people in the surrounding areas, and 48 patients out of 197 evaluated were identified as being negatively affected (with persistent respiratory health effects and asthma) by the MS spill (Cone et al., 1994). Many more were occupationally exposed during the public safety response and subsequent cleanup.

12 The California Department of Public Health has estimated that over 90% of agriculture occupational health issues are due to pesticide exposure. Daily acute exposure may lead to skin and eye irritations, burns, and inflammation, but many workers eventually develop chronic pulmonary diseases. The number of reported cases has drastically increased over the decades, as demonstrated below by the adapted table (Maddy, Edmiston, & Richmond, 1990).

Occupational pesticide illnesses, California 1950-1988

Year Cases Year Cases Year Cases Year Cases

1950 115 1960 424 1970 311 1982 628 1952 211 1962 270 1972 325 1984 490 1954 121 1964 280 1974 424 1986 562 1956 326 1966 290 1976 602 1987 744 1958 365 1968 259 1980 600 1988 903

Table 1: Number of reported cases of occupational pesticide illnesses with systemic or pulmonary symptoms or both in California 1950-1988 (Adapted from Maddy et al., 1990)

Although rare, severe incidents of pesticide exposure have been known to cause human death, with an average of one case per year in the US being attributed to accidental occupational exposure. A conservative World Health Organization risk assessment model in 1985 estimated up to 20,000 deaths worldwide each year due to pesticide exposure, and this model along with others have predicted increasing numbers of deaths throughout the years (Levine & Doull 1992).

In addition to an accidental or occupational overdose, occasional suicide attempts have been documented (Maddy et al., 1990).

5. Pesticide Regulation

Due to studies demonstrating the immense dangers of MS, many national regulations and laws have been put into place to protect applicators, farmers, and the general public from exposure. The Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) of 1972 states that handlers must be certified (40 CFR 171 Certification of Pesticide Applicators) to apply or

13 supervise application and that all pesticides must follow specific directions on product labels.

States may implement more conservative or specific regulations. The US Environmental

Protection Agency (US EPA) enforces FIFRA with fines and jail time if terms are not met. Along with FIFRA and state laws, the Endangered Species Act examines the potential fate and transport of fumigants through geospatial and ecological risk assessment models and to determine areas where fumigants are banned (US EPA, 2016). Occupational Safety and Health Administration

Hazard Communication Standards defines the hazard and its classification and labeling, which must be followed by all fumigant producers and users (Duke & Jessen, 1996; EPA, 2016; USDL,

2013).

6. Retention of MS and MITC

Although many occupational and public health studies have been conducted on soil fumigants, ecological data remains sparse and inconsistent. One of the main issues with fumigant application is the difficulty of effective application. Farmers face problems with uneven application, inconsistent lateral dispersion in the soil, and rapid volatilization. Efficacy depends largely on environmental factors: soil texture, soil structure, organic matter content, moisture content, and temperature (Meszka et al., 2010). Soils with low organic matter content exhibit quick MITC decomposition and release (within a few days), while soils high in organic matter and carbon retain over 33% of the initial MITC amount over a 15 day period (Riffaldi, Filippelli,

Levi-Minzi, & Saviozzi, 2000). However, organic amendments to soil accelerate MS degradation to MITC (Gan, Papiernik, & Crowley, 1998). Also, increasing moisture content results in decreasing MITC emission rates from the soil (Zheng, Yates, Papiernik, & Nunez, 2006).

Therefore, determination of best-management practices using soil amendments is required to increase the efficacy of MS and MITC.

14 Repeated applications of fumigants have been shown to alter soil microbial communities while decreasing the fumigant's retention time and ability to target soil organisms. Repeated applications of MS increase degradation rates to MITC., leading to losses in biocidal effects on microbes (Ibekwe, Papiernik, & Yang, 2004; Triky-Dotan et al., 2000). A study of soil samples collected upstream and downstream of the Sacramento River one year after the MS spill of 1991 also reflected this. Downstream soil microbial communities were less sensitive to MITC addition than upstream soils, with populations of fatty acid-rich microbes and other gram-positive microbes increasing in relative abundance (Taylor et al., 1996).

7. Combinations of Fumigants

MS application in conjunction with an additional fumigant may offer enhanced protection against pests (Ingham, Hamm, Baum, & Merrifield, 2007). MS in combination with 1,3- dichloropropene, aldicarb, or oxamyl proved effective at combating corky ringspot disease in potato crops, caused by the soil-borne fungi Paratrichodorous spp. and Trichodorus spp. (Ingham et al., 2007). Reduced rates of MS in combination with 1,3-dichloropropene was equally as effective at killing pathogenic plant fungi as higher rates of MS alone (Hamm, 2003). The rate of application was also important, with higher doses of MS leading to lower rates of respiration, as measured by CO2-C evolution (Riffaldi et al., 2000).

8. Effects of MS on Microbial Communities

Data on total and culturable numbers and microbial activity have been inconsistent among studies. Toyota et al., 1999 showed that 50% of total bacteria and 90% of culturable bacteria were killed from MS application, but numbers recovered 26 days after treatment. In

Meszka et al., 2010, total fungal numbers decreased by up to 3500-fold while bacteria increased.

Pseudomonads increased by 100-fold while most Bacillus spp. decreased or were unaffected

15 (Meszka et al., 2010). A microcosm study using soil slurries found that heterotrophic activity was reduced for at least 18 weeks after initial MS application (Macalady, Fuller, & Scow, 1998).

However, increasing doses of subsequent applications of MS on the same soil led to increasing relative abundances of actinomycetes and other gram-positive bacteria (Harwood & Russell,

1984; Macalady et al., 1998).

MS can suppress ammonia-oxidizing bacterial activity (Yan et al., 2013). Another paper observed that ammonium and nitrate oxidizers reduced by about 4 logs, with nitrate oxidizers recovering slightly after about 100 days (Toyota, Ritz, Kununaga, & Kimura, 1999). There can also be an overall decrease in microbial taxa with higher %GC contents (Toyota et al., 1999).

These shifts in community composition and function may alter the ability of soils to degrade pollutants, break down organic matter, and contribute to important biogeochemical cycles such as nitrification within the soil ecosystem.

16 REFERENCES

Cain, George D. "Sanitizing Sewage Sludge: The Intersection of Parasitology, Civil Engineering, and Public Health*." Journal of Parasitology 96.6 (2010): 1037-1040.

Cone, James E., Wugofski, Lee, Balmes, John R., Das, Rupali, Bowler, Rosemarie, Alexeeff, George, and Shusterman, Dennis. "Persistent respiratory health effects after a metam sodium pesticide spill." CHEST Journal 106.2 (1994): 500-508.

Cox, Caroline. "Metam sodium." Journal of Pesticide Reform 26 (2006): 12-16.

Deguigne, Marie B., Lagarce, Laurence, Boels, David, and Harry, Patrick. "Metam sodium intoxication: the specific role of degradation products–methyl isothiocyanate and carbon disulphide–as a function of exposure." Clinical Toxicology 49.5 (2011): 416-422.

Duke, Kevin and Jessen, Eric. "Sewer Line Root Control." US Environmental Protection Agency. Sewer Line Chemical Root Control. 1996.

Gan, J., Yates, S. R., Papiernik, S., and Crowley, D. "Application of organic amendments to reduce volatile pesticide emissions from soil." Environmental Science & Technology 32.20 (1998): 3094-3098.

Geddes, Jason D., Miller, Glenn C., and Taylor, George E. "Gas phase photolysis of methyl isothiocyanate." Environmental Science & Technology 29.10 (1995): 2590-2594.

Hamm, Philip B., Ingham, Russell E., Jaeger, Joy R., Swanson, William H., and Volker, Kurt C. "Soil fumigant effects on three genera of potential soilborne pathogenic fungi and their effect on potato yield in the Columbia Basin of Oregon." Plant Disease 87.12 (2003): 1449-1456.

Harwood, John L., and Russell, Nicholas J. "Distribution of lipids." Lipids in Plants and Microbes. Springer Netherlands, 1984. 35-70.

Ibekwe, A. Mark, Papiernik, Sharon K., and Yang, Ching-Hong. "Enrichment and molecular characterization of chloropicrin-and metam-sodium-degrading microbial communities." Applied Microbiology and Biotechnology 66.3 (2004): 325-332.

Ingham, R. E., Hamm, P. B., Baune, M., and Merrifield, K. J. "Control of Paratrichodorus allius and corky ringspot disease in potato with shank-injected metam sodium." Journal of Nematology 39.3 (2007): 258-262.

Iranzo, Susana, Olmstead, Alan L., and Rhode, Paul W. "Historical perspectives on exotic pests and diseases in California." Exotic Pests and Diseases: Biology and Economics for Biosecurity (2000): 55-67.

17 Levine, Robert S., and Doull, John. "Global estimates of acute pesticide morbidity and mortality." Reviews of Environmental Contamination and Toxicology. Springer New York, 1992. 29-50.

Lowit, Anna. "Quantification of carcinogenic potential for MITC with metam sodium cancer slope factor." Memorandum. US Environmental Protection Agency. Washington, D.C. 13 May 2004. Web.

Macalady, J. L., Fuller, M. E., and Scow, K. M. "Effects of metam sodium fumigation on soil microbial activity and community structure." Journal of Environmental Quality 27.1 (1998): 54-63.

Maddy, Keith T., Edminston, Susan, and Richmond, Donald. "Illness, injuries, and deaths from pesticide exposures in California 1949–1988." Reviews of Environmental Contamination and Toxicology. Springer New York, 1990. 57-123.

Meszka, B., Chałańska, A., Sobiczewski, P., Bryk, H., Malusa, E., and Slusarski, C. "Changes in microorganisms populations in the soil after fumigation." Communications In Agricultural and Applied Biological Sciences 76.4 (2010): 751-755.

US Department of Labor. "Occupational Safety and Health Standards: Toxic and Hazardous Substances." 8 Feb. 2013. Web. 20 Apr. 2016. .

Reynolds, Peggy, Von Behren, Julie, Gunier, Robert B., Goldberg, Debbie E., Hertz, Andrew, and Harnly, Martha E. "Childhood cancer and agricultural pesticide use: an ecologic study in California." Environmental Health Perspectives 110.3 (2002): 319.

Riffaldi, R., Filippelli, M., Levi-Minzi, R., & Saviozzi, A. "The influence of metam sodium on soil respiration." Journal of Environmental Science & Health Part B 35.4 (2000): 455-465.

Taylor, George E., Schaller, Kastli B., Geddes, Jason D., Gustin, Mae S., Lorson, Gwen B., and Miller, Glenn C. "Microbial ecology, toxicology and chemical fate of methyl isothiocyanate in riparian soils from the upper Sacramento river." Environmental Toxicology and Chemistry 15.10 (1996): 1694-1701.

Toyota, Koki, Ritz, Karl, Kuninaga, Shiro, and Kimura, Makoto. "Impact of fumigation with metam sodium upon soil microbial community structure in two Japanese soils." Soil Science and Plant Nutrition 45.1 (1999): 207-223.

Triky-Dotan, Shachaf, Ofek, Maya, Austerweil, Miriam, Steiner, Bracha, Minz, Dror, Katan, Jaacov, & Gamliel, Abraham. "Microbial aspects of accelerated degradation of metam sodium in soil." Phytopathology 100.4 (2010): 367-375.

18 US Environmental Protection Agency. "Assessing Pesticides under the Endangered Species Act." 29 March 2016. Web. 5 Apr 2016. .

US Environmental Protection Agency. "Soil Fumigant Chemicals." 7 Apr. 2016. Web. 11 Apr. 2016. .

U.S. Environmental Protection Agency. "The Phaseout of Methyl Bromide." Ozone Layer Protection- Regulatory Programs. 6 Oct. 2015. Web. 28 Jan. 2016. .fz

Warnert, Jeannette. "Soil Fumigant History." Western Farm Press. 28 May 2010. Wed. 31 Mar. 2016. .

Yan, D., Wang, Q., Mao, L., Li, W., Xie, H., Guo, M., and Cao, A. "Quantification of the effects of various soil fumigation treatments on nitrogen mineralization and nitrification in laboratory incubation and field studies." Chemosphere 90.3 (2013): 1210-1215.

Zahm, Shelia H. and Ward, Mary H. "Pesticides and childhood cancer." Environmental Health Perspectives 106.Suppl 3 (1998): 893.

Zheng, Wei, Yates, Scott R., Papiernik, Sharon K., and Nunez, Joe. "Conversion of metam sodium and emission of fumigant from soil columns." Atmospheric Environment 40.36 (2006): 7046- 7056.

19 APPENDIX A

EFFECTS OF METAM SODIUM ON SOIL MICROBIAL COMMUNITIES:

NUMBERS, ACTIVITY, AND DIVERSITY

Sederholm, Maya R.†*, Bradley W. Schmitz†, Ian L. Pepper†

Manuscript to be submitted to Soil Biology & Biochemisty

† Water & Environmental Sustainable Technology (WEST) Center, The University of Arizona, 2959

W. Calle Agua Nueva, Tucson, Arizona 85745, USA

† Department of Civil & Environmental Engineering, National University of Singapore, Block EIA,

#07-03, No. 1 Engineering Drive 2, Singapore, 117576

* Corresponding Author: Water & Environmental Sustainable Technology (WEST) Center, 2959 W.

Calle Agua Nueva, Tucson, AZ 85745

Email: [email protected]

Phone: (520) 626-3328

Fax: (520) 573-0852

20 KEYWORDS: Soil Fumigant; Metam Sodium; Soil Microbial Communities

ABBREVIATIONS: MeBr, methyl bromide; US EPA, United States Environmental Protection

Agency; MS, metam sodium; MITC, methyl isothiocyanate; rRNA, ribosomal ribonucleic acid;

RLSS, Research Laboratory and Safety Services; PPE, personal protective equipment; HPCs, heterotrophic plate counts; tATP, total adenosine triphosphate; CFUs, colony-forming units; MEs, microbial equivalents; TPF, triphenyl formazan; TTC, 2,3,5-triphenyltetrazolium chloride; DNA, deoxyribonucleic acid; QIIME, Quantitative Insights Into Microbial Ecology

21 1. INTRODUCTION

Fumigation is a common treatment for pest control in agricultural soils. Specifically, it is often used as a crop pretreatment for plant pathogenic insects, fungi, and nematodes, among others (US EPA, 2015). Soil fumigants are frequently applied into soil as liquid solutions via drip irrigation systems or shanks. Once incorporated into the soil, the fumigants degrade into their gaseous active components as well as other breakdown products. Methyl bromide (MeBr) was once the most common soil fumigant, but was phased out by 2005 following the Clean Air Act and Montreal Protocol on Substances that Deplete the Ozone Layer due to its immense contributions leading to stratospheric ozone depletion (US EPA, 2015). Following this, MeBr was subsequently replaced by metam sodium (MS), which is currently the most commonly used soil fumigant (Zheng et al., 2006) and the third most commonly used agricultural pesticide (Pruett et al., 2001) in the US. MS is registered for agricultural use on food and feed crops as well as turf grass.

In the presence of water, MS salt hydrolyzes into methyl isothiocyanate (MITC), a highly toxic and volatile gas with broad pesticidal activity (Zheng et al., 2006). Byproducts include carbon disulphate, dihydrogen sulfide, and elemental sulfur (Deguigne et al., 2011). MITC infiltrates cell walls and causes membrane denaturation (Cain, 2010), but the mechanisms by which organisms are affected have not yet been identified. Thus, MS can be used to adversely affect soil organisms. It is effective against pests when applied in sufficient concentrations, but is prone to uneven application rates due to soil structure (Candole, Csinos, & Wang, 2007). In the field, MS-treated soils are immediately covered with tarps or plastic mulch to retain more MITC for longer periods of time. The unreacted MITC in the soil eventually diffuses into the atmosphere, where it is broken down through photolysis (Cain et al., 2010).

22 Safety precautions must be considered when handling MS due to several acute adverse health effects on animals and humans including skin, eye, nose, throat, and lung irritation, urticaria, and moderate burns (Deguigne et al., 2011). MITC exposure through inhalation causes irritation, nausea, headaches, dizziness, and shortness of breath (Deguigne et al., 2011).

Recently, MS in conjunction with the MagnaGro® process became an EPA-approved technology for the conversion of Class B biosolids to Class A biosolids utilized for land application (Tamimi, 2012). A recent unpublished study evaluated the treatment of Class B biosolids with metam sodium for the potential to convert Class B biosolids to Class A. During the evaluation of the process, it was discovered that the product effectively killed Ascaris ova and inactivated viruses (Tamimi, 2012). This in turn raised questions about the effects of metam sodium on the rest of the microbial community, particularly bacteria.

The present study examines the effects of MS on soil microbial populations in terms of numbers, activity, and diversity over a four-week period. Previous studies suggest that the lowest levels of biomass and activity were reached within the first week following MS application

(Ibekwe et al., 2001), and complete recovery in numbers occurred after 26 days (Toyota et al.,

1999). To provide a thorough and unique evaluation of the effects of MS on soil bacterial communities, a series of assays was conducted on untreated (control) and MS-treated soils to evaluate the effects of this fumigant on soil microbial populations. Heterotrophic plate counts

(HPCs) on R2A media were used to provide the number of culturable heterotrophic bacteria, actinomycetes, and fungi. LuminUltra® and the dehydrogenase activity assay were used to measure total microbial activity. Prokaryotic diversity, in terms of richness and community composition, was quantified based on 16S rRNA gene amplicons identified through Illumina

Next-Generation Sequencing (NGS). Specifically, we evaluated: i) the initial adverse impacts of

23 MS on soil bacteria; ii) the rate of recovery of the community; and iii) whether or not the MS- treated soil regained its status as an ecologically healthy soil.

24 2. METHODS

2.1. Safety Requirements for the Project

Due to the potential for adverse health effects from exposure to MS, rigorous safety prerequisites were necessary prior to the initiation of the project.

i) A Standard Operating Procedure and Chemical Hygiene Plan (Appendix B) were created with oversight from The University of Arizona's Research Laboratory & Safety Services (RLSS).

ii) A preliminary medical surveillance was conducted before the start of the experiment on all personnel who would directly or indirectly work with proximity to MS. A pulmonary test, a chest x-ray, and blood tests were also conducted at The University of Arizona Campus Health.

Once cleared, respirator fitting and testing was conducted at The University of Arizona

Occupational Health Services: Risk Management.

iii) Finally, prior to project initiation, personal protective equipment (PPE) including a full-face respirator, organic vapor cartridges (3M™ Ammonia/Methylamine/Particulate P100

APR Cartridge, cat. no. 60924), a Tyvek® suit, a chemical-resistant apron, and chemical-resistant gloves were acquired.

2.2. Establishment of Field Plots

Plots were established at The University of Arizona Campus Agricultural Center in

Tucson, Arizona. Following raking and loosening of the topsoil, replicate plots were established.

Briefly, ~1x1 m triplicate control and treated plots were staked out. The control plots were spatially separated from the treated plots by a 6-m buffer zone.

2.3. Metam Sodium Application

A drip irrigation system was utilized to apply MS evenly within each plot. The drip irrigation system for a single plot consisted of five standard half-inch drip irrigation lines spaced

25 15.24 centimeters (cm) apart, with each line containing 7 emitters each spaced 10.16 cm apart

(Figure 1).

All lines were secured in place via plastic hooks inserted into the soil. The drip systems from each individual treated plot were connected to a main line attached to a 36-gallon (136.28-

L) dispensing carboy (Figure 2). The carboy was placed on a table 1 m above the ground. A

6.40x4.57 square m clear plastic tarp was placed over all of the treated plots and accompanying drip system (Figure 2) to prolong the retention time of the MITC gas. The perimeter of each tarp extended beyond the plots by 1.83 m in each direction and was weighed down by rocks and soil.

Tarps were used to cover all plots for 7 days. Control plots were set up identically.

For the treated plots, the carboy was filled with 136.28 L of tap water and 176 milliliters

(mL) of Vapam® HL (381.05 kilograms/cubic meter), which were mixed together quickly before replacing the carboy lid. The carboy for the control plots contained 136.28 L of tap water.

To apply the solution, the carboy valve was opened and the water gravity-fed through the lines and onto the plots, where the water infiltrated to a depth of 12.70 cm. Each plot received about a third (45.42 L) of the carboy solution, resulting in an application rate of 0.07 L per square meter (m). This rate was chosen because farmers typically add MS at 0.05 to 0.07 L per square m (Vapam® HL, 2015), as per manufacturers' recommendations.

2.4. Field Sampling & Transport

Due to safety requirements, the RLSS Chemical Safety Manager was present during the MS application in the field and subsequent sampling periods to monitor and document airborne

MITC concentrations and potential exposure.

A hand trowel pre-sterilized with 70% ethanol was used to collect soil samples down to a

10.16-cm depth at three randomly selected locations within each plot. Samples were added to a

26 sterile 1-L Nalgene bottle, mixed to create a composite sample, placed in a cooler, and transported to the laboratory for immediate processing. RLSS monitored MITC concentrations during MS application, 24 hours post-application, and during indoor laboratory processing.

Soil samples for analysis were taken at pre-application (0 days), 24 hours (1 day) after initial MS application, and then weekly for 28 days (Table 1).

2.5. Soil Moisture Content

The soil gravimetric moisture content was determined on a dry weight basis for each sampling period using the oven-drying method, as previously described (ASTM D2216, 2010;

Pepper & Gerba, 2004). Briefly, duplicate 10 grams of moist soil were placed in aluminum dishes, dried in an oven at 110°C for 24 hours, and then reweighed. The gravimetric moisture content (MC) was calculated as:

MC= (W- D)/ D (Eq 1) where W= wet weight of the soil and D= dry weight of the soil. The arithmetic average moisture content was calculated on a dry weight basis as the mean of the duplicates from one control plot.

2.6. Heterotrophic Plate Counts

Heterotrophic plate counts (HPCs) were determined for each soil sample using a culturable dilution and plating method, as previously described (ASTM D5465, 2012; Pepper and

Gerba, 2004). Briefly, 10 grams of moist soil were placed into 95 mL of autoclaved 0.85% NaCl solution, resulting in a 10E-1 dilution, from which subsequent 10-fold dilutions were created.

The samples were spread plated on R2A agar (Difco cat. no. 218261, Sparks, MD, USA) and incubated in the dark for 5 days at room temperature (22°C). All colonies (bacteria, actinomycetes, and fungi) were counted and ultimately reported as colony-forming units (CFUs) per gram of soil on a dry weight basis.

27 2.7. LuminUltra®

LuminUltra® Deposit and Surface Analysis (DSA) kit (prod. no. DSA-100, Fredericton, NB,

Canada) was used to measure total microbial activity, as described in the manufacturer's protocol. The kit uses a luciferin-luciferase protein-enzyme complex to measure adenosine triphosphate (ATP) concentrations in samples. Briefly, one gram of moist soil was mixed in the kit's UltraLyse 7™ tube containing the tATP extraction reagent. Subsequently, one mL of the resulting solution was placed into an UltraLute™ tube, inverted, and 100 µL of the mixture was aliquotted and combined with 2 drops of Luminase™, containing the protein-enzyme complex.

The tube was inserted into a Luminometer™ to detect the amount of light produced and provide a subsequent estimation of ATP concentration. Microbial equivalents (MEs) per gram of dry soil were calculated via the following calculation:

tATP (MEs/g) = tATP (pg ATP/g) * 1 ME/0.001 pg ATP (Eq 2) where tATP represents the ATP concentration measured.

2.8. Dehydrogenase Activity Assay

Dehydrogenase activity measured total heterotrophic activity based on the amount of triphenyl formazan (TPF) produced, as previously described (Thalmann 1968; Pepper and

Gerba, 2004; Casida, 1977). Briefly, six grams of moist soil were combined with 2.5 mL of autoclaved H2O and 1 mL of 3% 2,3,5-triphenyltetrazolium chloride (TTC) in quadruplicate 50- mL conical tubes. Two tubes were amended with 0.03 g of glucose to evaluate the activity response of the microbial community to added available substrate. The conical tubes were sealed, vortexed, and incubated at room temperature (22°C) for 5 days. 50 mL of 1M methanol was used to extract all TPF from each tube, and the solution was filtered through Whatman®

#42 filter paper. Eluent was collected in a tube and absorbance was measured with a

28 spectrophotometer and fitted to a standard curve at 485 nm to give the concentration of TPF per gram of dry soil.

2.9. DNA Extraction

MoBio Laboratories, Inc. PowerSoil® DNA Isolation Kit (cat. no. 12888-50, Carlsbad, CA,

USA) was used to extract community DNA from 8 grams of moist soil, as described via the manufacturer's protocol. Briefly, bead technology and centrifugation in conjunction with a series of kit solutions were used to lyse cells, extract DNA, and remove inorganic contaminants, humic acid, and other PCR-inhibitory substances. The final extracted DNA was held in Tris buffer

(EDTA-free), and was stored in a 1.5-mL DNA Lo-Bind Eppendorf tube at -20° Celsius.

2.10. 16S Gene Amplification and Purification

A custom Illumina 16S rRNA amplicon sequencing protocol (16S Metagenomic

Sequencing Library Preparation, 2016) was followed. 16S rRNA gene amplification was performed using specific primers targeting the V3 and V4 regions of the 16S rRNA gene. These primers were ligated to overhang adapter sequences. The full-length primer sequences were:

(forward) TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG and (reverse)

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC (Klindworth et al.,

2013).

2.11. Sequencing and Analysis

Samples were sequenced at The University of Arizona Genetics Core using a MiSeq sequencer (Illumina). For quality control, samples were minimally trimmed through

Trimmomatic software to remove Illumina adapter sequences and sequences which fit the following parameters: LEADING:3; TRAILING:3; SLIDINGWINDOW:4:15 (Bolger, Lohse, & Usadel,

2014). A quality check was conducted on MiSeq Reporter software, with default parameters

29 used to remove low quality reads. Bioinformatics software Quantitative Insights into Microbial

Ecology (QIIME) (Caporaso et al., 2010; Kuczynski et al., 2012) was used to process and analyze reads. First, paired-end reads were combined using the fastq-join method (Aronesty, 2011).

Next, the open-reference OTU-picking method (Rideout et al., 2014) was used to clump sequences based on 97% nucleotide similarity. Then, sequences were aligned and clustered for phylogenetic tree preparation through uclust (Edgar, 2010). GreenGenes reference database

(DeSantis et al., 2006) provided chimera screening/removal and alignment to sequences in the database. Taxonomic assignment at various levels was given by RDP Classifier (Wang et al.,

2007). All default parameters were used throughout the workflows. Output files were used to analyze OTU statistics, create alpha rarefaction curves, create stacked bar graphs of relative abundances of taxa per sample, and create beta ordination plots.

2.12. Hazardous Waste Handling and Disposal

Vapam®, methanol waste, and all media with potential MS and MITC contamination were collected in sealed, labeled plastic containers and picked up by Hazardous Waste Management at

Risk Management Services.

2.13. Statistical Analysis

One-way analysis of Variance (ANOVA) using Tukey-Kramer post-hoc test was run in

Statplus:mac LE 2016 (StatPlus, 2016) in order to compare replicate values between time periods for control and treated samples separately. An unpaired two-sample t-test assuming equal variances and normal distribution was conducted in Statplus:mac LE 2016 (StatPlus, 2016) to compare whether replicate control and treated values per time period differed. A cutoff value of p ≤ 0.05 was used for all tests.

30 3. RESULTS

3.1. Environmental Metadata

The soil at the study site was a Gila coarse-loamy, mixed, superactive, calcareous, thermic

Typic Torrifluvent (Soil Survey Staff, 2016) with good aeration and drainage. The soil organic matter content was low (1.2%). The pH was slightly alkaline, at 7.9, with an electrical conductivity of 1.3 deciSiemens per meter. Soil texture was comprised of 54% sand, 34% silt, and 12% clay, classified as a sandy loam. At the beginning of the study, the soil moisture content was 12.4%. However, it increased noticeably at 1 day when tap water was added to each plot, then gradually decreased over time. Both air and soil temperatures generally decreased over time (Table 2; Figure 3).

3.2. Culturable Heterotrophic Numbers

Initial HPCs in the soil were 1.52E7 CFUs in the control plots and 1.01E7 CFUs in the treated plots (Table 3). One day after MS application, numbers increased in both the control and treated plots, with a 0.41 log increase in the control plots and a 0.54 log increase in the treated plots. Within 7 days, HPC counts decreased to values similar to the 0-day numbers in both control and treated plots. From days 7 to 28, numbers stayed fairly constant. At 28 days, culturable numbers statistically increased (p = 0.005) in treated samples relative to control plots

(Table 3; Figure 4).

3.3. LuminUltra® Microbial Equivalents

LuminUltra® ME values were initially 7.03E7 in control plots and 1.30E8 in treated plots, but these values were not significantly different. At 1 day, the control plots increased significantly (p = 0.004) to 1.09E8 while the treated plots decreased to 1.24E7. At 7 days, MEs in the treated plots increased but values were not statistically different from the control plots.

Between 14 and 28 days, control and treated ME values increased (Figure 5). In addition, on

31 days 14 and 28, values were significantly higher in control plots related to treated plots, with p- values of 0.017 and 0.001, respectively (Table 3).

3.4. Dehydrogenase Activity Assay TPF Concentration

TPF concentrations in control and MS-treated soils were relatively low throughout the

28-day trial. At 1 day, both MS-treated soil and MS-treated soil amended with glucose had significantly lower dehydrogenase activity than in control plots, with p values of 0.005 and 0.000, respectively. At 14 and 21 days, the glucose-amended MS-treated plots had significantly higher

TPF values than corresponding control plots, with p values of 0.000 and 0.012, respectively. At

28 days, non-amended MS-treated samples had significantly higher (p = 0.014) values than those in control samples, but values in glucose-amended soils were similar (Table 4; Figure 6).

3.5. Molecular Results

Using a depth of 30,000 sequences per sample to normalize the data and 10 iterations to get means and standard deviations, the average minimum number of observed OTUs in a sample was 4,243.5 OTUs, which was for the 28 day treated sample, while the maximum number of

OTUs was 4,878.5 OTUs, which was for the 14 day control. Interestingly, at every time period except at 1 day, the MS-treated samples had lower numbers of observed OTUs. At one day, the treated sample had a slightly higher average number of OTUs (4,480.2) than the control (4,387.4) (Table 5;

Figure 7).

For all sequences, 98.15% were classified as bacterial, 0.17% were classified as archaeal, and 1.68% were unknown or unclassified. The largest contributors to the community in terms of phyla were: Proteobacteria (28.56% of the total community), Planctomycetes (15.13%),

Actinobacteria (14.82%), Acidobacteria (9.66%), Chloroflexi (9.06%), Bacteroidetes (6.46%),

Firmicutes (4.56%), Gemmatimonadetes (3.62%), "Unassigned" (1.68%), Verrucomicrobia

32 (1.63%), TM7 (1.61%), and Cyanobacteria (1.27%). The top 15 classes were:

Alphaproteobacteria (19.52%), Phycisphaera (10.66%), Actinobacteria (6.99%), Planctomycetes

(4.31%), Bacilli (4.17%), Anaerolineae (3.79%), Deltaproteobacteria (3.69%), Acidobacteria-6

(3.56%), Chloracidobacteria (3.35%), Gammaproteobacteria (3.23%), Acidimicrobiia (3.19%),

Cytophagia (2.73%), Saprospirae (2.31%), MB-A2-108 (2.15%), and Betaproteobacteria (2.06%)

(from QIIME data, not shown).

3.6. Microbial Diversity: Richness

Alpha rarefaction curves in QIIME showed that in all three metrics- Phylogenetic

Diversity, Chao1, and Observed OTUs (Faith and Baker, 2006; Chao and Lee 1992)- the control samples generally had higher numbers than the treated samples of OTUs (Figure 8).

For the Observed OTUs metric, the samples with the highest average number of OTUs per

30,000 sequences were the control samples at 14, 28, and 21 days, respectively (Table 5; Figure

8). The samples with the lowest number of OTUs were the treated sample at 28, 0, and 14 days, respectively (Table 5; Figure 8).

3.7. Microbial Diversity: Community Composition

With respect to phyla, Actinobacteria, Chloroflexi, and Firmicutes all increased noticeably in relative abundance in both control and treated samples throughout the project (Figure 9).

From 0 days to 28 days, Actinobacteria increased by 2.59% in control samples and 5.38% in treated samples (Table 6). This was due mostly to the notable increase in the order

Actinomycetales at 14 days (Figure 10). Chloroflexi increased by 0.74% in control samples and

2.00% in treated samples from 0 to 28 days (Table 6). Firmicutes remained constant in control samples, but increased by 1.69% in treated plots (Table 6). Firmicutes was comprised mostly of

Bacilli (91.51%), with much less Clostridia (8.47%) and no Mollicutes detected. The increase

33 from 0 to 28 days is driven mostly by Bacillales, which increased noticeably throughout the study

(Figure 10).

Acidobacteria and Bacteroidetes decreased in both control and treated samples throughout the project. Between 0 and 28 days, Acidobacteria decreased by 1.17% and 3.93% in control and treated soils, respectively. Bacteroidetes decreased by 1.83% and 1.72% in control and treated soils, respectively (Table 6), due mostly to the decrease of Cytophagales (Figure 9;

Figure 10).

Also of note is the increase in Proteobacteria in control samples at day 1, whereas

Planctomycetes decreased in treated samples at 1 day. Over all time periods, Proteobacteria was comprised mostly of Alphaproteobacteria (19.52%), with lower amounts of Deltaproteobacteria

(3.69%), Gammaproteobacteria (3.23%), and Betaproteobacteria (2.06%). Specifically,

Alphaproteobacteria represented a similar percent of the community at all time periods except at 1 day, where it increased to 23.53% of the community in control samples and decreased to

15.27% in treated samples (Table 6). The decrease in treated plots of Planctomycetes was driven mostly by WD2101, which decreased noticeably at 14 days in treated plots (Figure 9;

Figure 10).

Archaea made up a very small percentage of the total community, with only 0.125%

Crenarchaeota, 0.043% Eurarchaeota, and 0.003% Parvarchaeota.

34 4. DISCUSSION

The soil had low amounts of organic matter and clay, allowing for an aerated soil since it had been tilled frequently. These soil conditions likely resulted in high rates of gas flux to the atmosphere, which could have affected the ability of the soil to retain MITC. Soil moisture content notably increased after the application of Vapam® due to the significant addition of water to all plots. Soil and air temperatures steadily decreased throughout the study, except for a noticeable increase in both at 21 days.

Culturable counts increased in all plots at 1 day, but decreased by 7 days. This noticeable increase in HPC counts in both control and treated plots at 1 day was most likely in response to the addition of water, which increased soil moisture. Culturable methods show only viable microbes that can grow on the specific medium of choice. There is ample evidence that culturable methods may only account for 10% or less of the total microbial community in the sample, and frequently less than 1% (Maier, Pepper, and Gerba, 2009). Therefore, the effect of

MS on the entire microbial community may not be represented by HPC counts. In addition, dilution and plating methodology is known to be prone to significant errors, as indicated by the large standard deviations in HPC data. Soil dilutions also incur additional errors.

Overall, the number of culturable heterotrophic microbes on R2A media did not noticeably change throughout the study. However, at 28 days, the number of CFUs in treated plots was statistically larger (p = 0.005) than the number in control plots. Overall, gross numbers of culturable microbes were not dramatically affected by MS treatment. However, it is important to note that culturable counts reflect the net balance of both the death and growth of soil organisms. Thus, although the number of cells remained constant, the individual members of the community may have changed. Also, by growing bacteria, actinomycetes, and fungi

35 together without adding any inhibitory substances, their growth and distribution on the plates may have been affected by competition for space and resources.

LuminUltra®, a direct measure of ATP concentrations and total activity, gives an indirect measurement of total microbial load. In the LuminUltra® assay, the concentration of ATP is converted to microbial equivalents, which represent total viable microbial numbers. The equation used for this conversion assumes that all ATP comes from typical Escherichia coli-sized cells (LuminUltra®, 2013). Therefore, every cell is assumed to contain exactly 0.001 pg of ATP.

The t-test between treatment types shows at 1, 14, and 28 days that the ATP concentrations in control samples were significantly higher than values in treated samples

(Table 3). Although, control sample values are also higher at 7 and 21 days, they are significantly larger than values in treated samples due to the larger standard deviation (average standard deviation of 1.50E4) in ATP concentration. This data indicates that although MS did not reduce culturable counts, it did inactivate portions of the microbial community. Therefore,

LuminUltra® is a better indicator than HPCs of microbial health because it measures the real- time concentration of total biomass rather than a small subset of culturable heterotrophic microbes.

In the dehydrogenase assay, TPF concentrations in all soil samples were low throughout the 28-day trial, perhaps due to the low organic matter content of the soil. Concentrations of TPF in non-amended samples were always significantly lower than ones in glucose-amended samples

(Figure 6), reflecting the strong influence of glucose on the heterotrophic soil microbial community that was present in the soil prior to glucose addition. One day after the addition of

MS, glucose-amended treated soils showed significantly reduced values of dehydrogenase activity (Figure 6), presumably due to the adverse effects of MS. However, by 7 days, TPF

36 concentrations in glucose-amended MS-treated samples increased to levels similar to those in control samples. At 14 and 21 days, values in glucose-amended MS-treated samples were significantly larger than those in control samples (Table 4). Then, at 28 days, concentrations were once again noticeably (but not statistically due to large standard deviations) larger in glucose-amended control plots than in treated plots. Overall, the LuminUltra® and dehydrogenase assay data validate each other, both showing the short-term adverse effects of

MS on microbial activity, followed by a rapid rebound.

An evaluation of the total numbers of observed OTUs obtained from each soil sample show that the control soil usually had many more OTUs than the MS-treated sample did. At most time periods, it is observed that the treated soil yielded fewer OTUs than the control soil.

However, at 1 day only, the treated sample had a larger number of OTUs than the control sample.

This illustrates a possible inhibition of MS to soil DNA recovery or amplification but not on microbial richness. It is important to note that gross estimates of the microbial community in terms of culturable counts or activity tell only part of the story. The ability of soil microbial numbers or activity to rebound after an adverse effect may well be due to a few surviving members of the community increasing in numbers while other microbes decrease or die off. If this is the case, decreased microbial diversity due to MS addition would be expected, which is not observed.

For alpha diversity, the community was not sampled completely, as different samples yielded vastly different numbers of sequences. Therefore, all samples were sampled to an even depth (30,000 sequences). This way, all of the curves begin to plateau at around the same number of sequences, and can be directly compared.

37 In terms of community composition, there were noticeable changes in the relative abundances in some phyla and orders during the experiment. Alphaproteobacteria, which comprise more Proteobacteria than any other class, are known to rapidly consume and degrade some acidic herbicides, including 2-methyl-4-chlorophenoxyacetic acid and 2,4- dichloroprophenoxyacetic acid, in soil. Most of the degradation is conducted by

Sphinnogmoadaceae (Liu et al., 2011), a family found within the order Sphingomonadales, which was mainly responsible for the observed increase in Proteobacteria's relative abundance at 1 day in control samples relative to treated samples (Figure 9). Interestingly, Planctomycetes was observed to increase slightly at 1 day in treated soils, at the same period of time when

Proteobacteria decreased in relative abundance. This suggests that Planctomycetes in general may be more resistant to MS than Proteobacteria is. Planctomycetes are unique in their ability to carry out anammox reactions, which involve the anaerobic oxidation of ammonium to N2 using nitrite as an electron acceptor (Kuenen, 2008).

Actinobacteria increased in relative abundance in both control and treated samples after

7 days and were seemingly unaffected by MS. They are known for having high GC-contents, ranging from 46% to 73% (Zhao et al., 2007). A previous study found that gram-positive, non- spore-forming Actinobacteria were more resilient and persistent in the environment that other spore-formers (Willersley et al., 2004). However, this is inconsistent with the majority of studies that show that spores are the hardiest types of cells. The specific characteristics that make

Actinobacteria so resilient are still unknown, but the unusually high GC-content may contribute, making them more resistant to degradation and therefore more likely to be isolated and identified through sequencing work. An earlier study found that DNA isolated from various soil types after treatment with the herbicide 2-chloro-4-(ethylamino)-6-(isopropylamino)-1,3,5-

38 triazine (atrazine) were mostly from gram-positive microbes with high GC contents, and a large number of the isolates were specifically Actinomycetales (Rousseaux et al., 2001). Interestingly,

Actinomycetales is the main order of Actinobacteria, with noticeable increases in treated soils relative to control soils.

Bacteroidetes gradually decreased throughout the study. A previous study found that soil microbial communities often contain this phylum, which is strongly positively correlated with increasing pH (Lauber et al., 2009).

Some Firmicutes have relatively low GC contents while others have high GC contents

(Rocha and Danchin, 2002). Bacilli, which comprise the majority of the Firmicutes community, are among the taxa with low GC contents, but the fact that they increase in relative abundance in all samples throughout the project may be a reflection of the Bacilli being endospore-formers

(Onyenwoke et al., 2004).

Other changes in community composition to note are the gradual decrease in

Acidobacteria and increase in Chloroflexi throughout the project. Acidobacteria is significantly correlated with low pH (Lauber et al., 2009) while Chloroflexi is a diverse phylum physiologically but is often known as an anoxygenic phototroph (Bryant and Frigaard, 2006). Despite the slight changes in these phyla during the 28-day trial, initial and final microbial communities were not drastically different.

Molecular analyses indicate the presence of specific intact DNA sequences, regardless of whether they come from viable or non-viable microbes. Previously, it was assumed that DNA degrades quickly in the environment, however, a recent review addresses the issue of relic DNA, which is extracellular DNA that can last in soil for perhaps weeks or even years (Carini et al.,

2016). Therefore, 16S rRNA amplicon libraries of soil microbial communities should take relic

39 DNA into consideration, since it could affect perceived microbial diversity and composition.

There is evidence that an average of 40% of total prokaryotic and fungal DNA extracted from soil could be relic DNA (Carini et al., 2016). However, it is believed that soils lower in organic matter and higher in pH, electrical conductivity, and calcium content do not support large amounts of relic DNA (Carini et al., 2016). The soil utilized in this study had these characteristics (Arizona

Cooperative Extension, 1998), therefore, there may have been minimal misrepresentation due to relic DNA in our samples.

Low extraction efficiency in the DNA isolation could prevent the identification of rare taxa. A comparison of molecular culture-independent assays with culturable assays showed that the soil prokaryotes captured by culturing were in very low abundance, giving greater access to the "rare biosphere" (Shade et al., 2012). Contrarily, the molecular assays tend to identify the most abundant taxa and exclude rare species. The low extraction efficiency of community DNA from soil using the Powersoil® DNA Isolation kit was demonstrated to be 5.28% of the total DNA in the soil (Dineen et al., 2010) or an average final concentration of 22 ng of DNA/µL of solution

(Kennedy et al., 2014).

However, assuming all sequences are affected proportionally equally, this low yield would not affect the relative abundances of the dominating taxa. The lack of recovery could have been due to the presence of inhibitory substances, such as humic substances, or from the breaking up of DNA strands during vortexing or centrifugation. Bias in the DNA recovered could also come from organisms with higher GC contents, which stabilize their DNA sequences and allow them to persist for longer periods in soil, making them more likely to be detected in molecular work.

The V3 and V4 regions of the 16S rRNA gene were targeted for sequencing because they are hypervariable regions within this gene that are used to identify taxa down to the species level

40 and to supplement soil microbiological databases. The affinity of the primers used may have affected the identification of the sequence segments of interest. The taxonomic classifications of the sequences were determined by comparing the nucleotide sequences clustered by uclust

(Edgar, 2010) to those in the incomplete GreenGenes database (DeSantis et al., 2006).

Classification is difficult and biased due to the fact that only previously identified sequences are available to align and compare with sample reads. There were significant portions of each sample that resulted in the categories of "unknown" or "unclassified," which may mean that there are more types of taxa that remain unidentified.

The addition of Vapam® solution may have supplemented the soil with a significant amount of carbon and nitrogen. The soil was a Gila fine sandy loam with low organic matter, so the addition of these nutrients may have been a significant contribution for the microbes. The mass of a plot of soil to a depth of 12.7 cm is 164,591.67 grams. A typical Escherichia coli cell weighs 2.8E-13 grams in dry weight (Ingraham et al., 1983), with 1.34E-13 grams of carbon

(Bratbak et al., 1984) and 3.92E-14 grams of nitrogen. Assuming the MS infiltrated 12.7 cm deep into the soil and that the carbon and nitrogen inputs were used with 100% efficacy, the soil should have received enough carbon and nitrogen to increase by about one log: specifically, by

1.89E8 cells (based on carbon amounts) or 3.75E8 cells (based on nitrogen amounts) per gram of soil. The soil started out with about 7 logs of microbes, so this is a significant contribution if the microbes utilized the MS as a nutrient source. This may be why a slight increase in numbers is seen from 0 days to 1 day in the HPC assay in the treated plots. However, there is >1 log decrease in the treated plots in LuminUltra®.

This study is unique because it utilizes a variety of methods, each with their own merits and biases, to evaluate the microbial communities in different ways. The culturable technique

41 may represent only a very small subset of the total community, but it presents the live and viable cells. The biochemical assays represent the total active community, which may better represent the total live biomass. Molecular techniques identify all microbes whose sequences are intact, but it may not be possible to determine which populations the MS has adversely affected.

Overall, this project shows that MS does not have a lasting significant effect on the soil microbial communities. Both subtle and significant changes in numbers, activity, and diversity occurred during the 28-day study, but at the conclusion of the study, control and treated soils had similar microbial properties at the macro-level. Therefore, this study suggests that Vapam® may be fine to use on agricultural soils. A more extensive study that looks also at soil microbial function may conclude that this fumigant is safe in terms of soil microbial quality and ecological health.

42 5. TABLES

Table 1: Soil Sampling Schedule Time Period Date 0 days November 4 1 day November 5 7 days November 11 14 days November 18 21 days November 25 28 days December 2

Table 2: Environmental Metadata

Time Period Moisture Content Air Temp. (°C) Soil Temp. (°C)

0 days 11.095% 59.2 60.8

1 day 18.11% 54.5 58.1

7 days 9.64% 49.3 52.7

14 days 7.895% 46.2 49.1

21 days 5.71% 65.5 53.4

28 days 6.445% 44.2 43.5

Soil temperature taken from top 10.16 cm of soil.

43 Table 3: Numbers: HPCs and LuminUltra®

Time Period HPCs (Log10 CFUs/g dry soil) LuminUltra® (Log10 MEs/g dry soil) Control Treated p-value Control Treated p-value

0 days 7.17 ± 0.11 7.00 ± 0.04 0.063 7.82 ± 0.20 8.10 ± 0.12 0.106 a ab d 1 day 7.58 ± 0.08 7.54 ± 0.08 0.991 8.04 ± 0.03 7.03 ± 0.29 0.004 c b a 7 days 7.14 ± 0.07 6.96 ± 0.09 0.056 7.74 ± 0.06 7.61 ± 0.12 0.153 a ab bc 14 days 7.08 ± 0.06 7.09 ± 0.05 0.801 7.58 ± 0.03 7.34 ± 0.10 0.017 ab a ab 21 days 7.17 ± 0.03 7.26 ± 0.05 0.359 7.82 ± 0.22 7.82 ± 0.13 0.989 b ab cd 28 days 6.90 ± 0.03 7.11 ± 0.06 0.005 8.04 ± 0.03 7.73 ± 0.06 0.001 ab b bc

P ≤ 0.05. T-test results bolded if significant. P-values given by triplicate data. Tukey-Kramer post-hoc test done when there was a statistical difference in the data within groups. Results indicated by different letters (a, b, etc.). The same letter within a column indicates no significant difference between the groups; different letters within the same column indicates significant differences.

44 Table 4: Total Activity: Dehydrogenase Activity Assay

Time Period Dehydrogenase Assay (µg TPF/g dry soil) Control Treated p-value Control + G Treated + G p-value

0 days 1.37 ± 0.63 2.44 ± 2.00 0.442 24.65 ± 3.54 25.03 ± 2.68 0.837 ab a b b 1 day 3.13 ± 1.66 0.63 ± 0.32 0.005 12.25 ± 0.48 3.89 ± 3.12 0.000 b a a a 7 days 1.69 ± 0.88 3.45 ± 2.00 0.077 9.13 ± 0.40 8.37 ± 4.44 0.663 ab a a ab 14 days 1.40 ± 0.72 1.10 ± 0.47 0.310 9.64 ± 0.82 12.71 ± 0.41 0.000 ab a a ab 21 days 0.64 ± 0.54 0.27 ± 0.02 0.523 9.82 ± 0.53 11.59 ± 1.40 0.012 a a a ab 28 days 0.42 ± 0.04 0.69 ± 0.10 0.014 18.31 ± 6.63 12.73 ± 14.32 0.362 a a ab ab

P ≤ 0.05. T-test results bolded if significant. G = glucose-amended. P-values given by triplicate data. Tukey-Kramer post-hoc test done when there was a statistical difference in the data within groups. Results indicated by different letters (a, b, etc.). The same letter within a column indicates no significant difference between the groups; different letters within the same column indicates significant differences.

Table 5: OTU Table Summary Statistics

Time Period Number of OTUs Control Treated

0 days 4,545.4 ± 39.76 4,244.8 ± 23.93

1 day 4,387.4 ± 34.91 4,480.2 ± 27.79

7 days 4,466.3 ± 31.78 4,306.6 ± 7.38

14 days 4,878.5 ± 33.45 4,297.7 ± 24.82

21 days 4,799.8 ± 24.10 4,298.2 ± 24.79

28 days 4,810.5 ± 38.76 4,243.5 ± 25.71

Number of observed OTUs using 30,000 sequences per sample and 10 iterations.

45 Table 6: Relative Abundances of Top Phyla

Phylum Control Treated 0 days 28 days 0 days 28 days

Acidobacteria 13.60% 12.43% 9.19% 5.26%

Actinobacteria 7.76% 10.35% 17.64% 23.02%

Bacteroidetes 11.72% 9.89% 4.20% 2.48%

Chloroflexi 5.98% 6.72% 10.59% 12.59%

Cyanobacteria 1.34% 0.84% 2.09% 0.51%

Firmicutes 1.51% 1.51% 6.12% 7.81%

Gemmatimonadetes 4.13% 3.86% 2.47% 2.98%

Planctomycetes 15.32% 17.72% 14.32% 12.37%

Proteobacteria 28.55% 28.51% 27.43% 28.22%

TM7 1.90% 2.13% 1.15% 1.33%

Unassigned 1.91% 1.92% 1.52% 1.50%

Verrucomicrobia 3.24% 1.84% 1.56% 0.62%

46 6. FIGURES

Figure 1: Schematic of Plots and Drip Irrigation System

47 Figure 2: Carboy, Irrigation System, and Tarp Set-Up

Figure 3: Environmental Metadata

20 20% 18 16 15%

C) 14 ° 12 10 10% 8 6

5% Moisture Content

Temperature ( 4 2 0 0%

Time Period

48 Figure 4: Numbers: HPCs 8 20%

7.5 15%

7 10% CFUs/g dry soil 10 6.5 5% Moisture Content Log

6 0%

Time Period

CFUs: Colony-forming Units from HPCs.

Figure 5: Numbers: LuminUltra® 8.5 20%

8 15%

7.5 10% 7 MEs/g dry soil 10

5% Moisture Content Log 6.5

6 0%

Time Period

MEs: Microbial Equivalents from LU

49 Figure 6: Total Activity: Dehydrogenase Activity Assay 30

25

20

15

10 μg TPF/g dry soil

5

0

Time Period

Figure 7: Number of OTUs per Sample 5000

4800

4600

4400

4200

4000 Number of Observed OTUs

3800

Time Period

Number of observed OTUs using 30,000 sequences per sample and 10 iterations.

50 Figure 8: Community Richness

51 Figure 9: Community Composition- Phylum

Control Samples

0 days

1 day

7 days

14 days

21 days

28 days

0% 20% 40% 60% 80% 100%

Treated Samples

0 days

1 day

7 days

14 days

21 days

28 days

0% 20% 40% 60% 80% 100%

Include all taxa above 1% of the total community.

52 Figure 10: Community Composition- Order

Control Samples

0 days

1 day

7 days

14 days

21 days

28 days

0% 10% 20% 30% 40% 50% 60% 70%

Treated Samples

0 days

1 day

7 days

14 days

21 days

28 days

0% 10% 20% 30% 40% 50% 60% 70%

Include all taxa above 2% of the total community.

53 7. REFERENCES

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"AZ Master Gardener Manual: Soil pH." Arizona Cooperative Extension: College of Agriculture, The University of Arizona. 1998. Web. 15 Apr. 2016. .

Bolger, Anthony M., Lohse, Marc, and Usadel, Bjoern. "Trimmomatic: a flexible trimmer for Illumina sequence data." Bioinformatics (2014): btu170.

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Candole, Byron L., Csinos, Alexander S., and Wang, Dong. "Distribution and efficacy of drip-applied metam-sodium against the survival of Rhizoctonia solani and yellow nutsedge in plastic-mulched sandy soil beds." Pest Management Science 63.5 (2007): 468- 475.

Caporaso, J. Gregory, Bittinger, Kyle, Bushman, Frederic D., DeSantis, Todd Z., Andersen, Gary L., and Knight, Rob. "PyNAST: a flexible tool for aligning sequences to a template alignment." Bioinformatics 26.2 (2010): 266-267.

Caporaso, J. Gregory, Kuczynski, Justin, Stombaugh, Jesse, Bittinger, Kyle, Bushman, Frederic D., Costello, Elizabeth K., Fierer, Noah, Gonzalez Peña, Antonio, Goodrich, Julia K., Gordon, Jeffrey I., Huttley, Gavin A., Kelley, Scott T., Knights, Dan, Koenig, Jeremy E., Ley, Ruth E., Lozupone, Catherine A., McDonald, Daniel, Muegge, Brian D., Pirrung, Meg, Reeder, Jens, Sevinsky, Joel R., Turnbaugh, Peter J., Walters, William A., Widmann, Jeremy, Yatsunenko, Tanya, Zaneveld, Jesse, and Knight, Rob. "QIIME allows analysis of high-throughput community sequencing data." Nature Methods 7.5 (2010): 335-336.

Carini, Paul, Marsden, Patrick J., Leff, Jonathan W., Morgan, Emily E., Strickland, Michael S., and Fierer, Noah. "Relic DNA is abundant in soil and obscures estimates of soil microbial diversity." BioRxiv (2016): 043372.

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Casida, L. E. "Microbial metabolic activity in soil as measured by dehydrogenase determinations." Applied and Environmental Microbiology 34.6 (1977): 630-636.

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Deguigne, Marie B., Lagarce, Laurence, Boels, David, and Harry, Patrick. "Metam sodium intoxication: the specific role of degradation products–methyl isothiocyanate and carbon disulphide–as a function of exposure." Clinical Toxicology 49.5 (2011): 416-422.

DeSantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, Keller K, Huber T, Dalevi D, Hu P, and Andersen GL. "Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB." Applied and Environmental Microbiology 72.7 (2006): 5069-5072.

Dineen, S. M., Aranda, R. T., Anders, D. L., and Robertson, J. M. "An evaluation of commercial DNA extraction kits for the isolation of bacterial spore DNA from soil." Journal of Applied Microbiology 109.6 (2010): 1886-1896.

Edgar Robert C. 2010. Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26(19): 2460-2461.

Faith, Daniel P. and Baker, Andrew M. "Phylogenetic diversity (PD) and biodiversity conservation: some bioinformatics challenges." Evolutionary Bioinformatics 2 (2006).

Fetterer, R. H., Jenkins, M. C., Miska, K. B., and Cain, G. D. "Metam sodium reduces viability and infectivity of Eimeria oocysts." Journal of Parasitology 96.3 (2010): 632-637.

Ibekwe, A. Mark, Papiernik, Sharon K., Gan, Jianying, Yates, Scott R., Yang, Ching-Hong, and Crowley, David E. "Impact of fumigants on soil microbial communities." Applied and Environmental Microbiology 67.7 (2001): 3245-3257.

Ingraham, John L., Maaløe, Ole, and Neidhardt, Frederick C. Growth of the Bacterial Cell. Sinauer Associates, 1983.

Kennedy, Nicholas A., Walker, Alan W., Berry, Susan H., Duncan, Sylvia H., Farquarson, Freda M., Louis, Petra, and Thomson, John M. "The impact of different DNA extraction kits and laboratories upon the assessment of human gut microbiota composition by 16S rRNA gene sequencing." PloS One 9.2 (2014): e88982.

55 Klindworth, Anna, Pruesse, Elmar, Schweer, Timmy, Peplies, Jörg, Quast, Christian, Horn, Matthias, and Glöckner, Frank Oliver. "Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies." Nucleic Acids Research (2012): gks808.

Kuczynski, Justin, Stombaugh, Jesse, Walters, William Anton, González, Antonio, Caporaso, J. Gregory, and Knight, Rob. "Using QIIME to analyze 16S rRNA gene sequences from microbial communities." Current Protocols in Microbiology (2012): 1E-5.

Kuenen, J. Gijs. "Anammox bacteria: from discovery to application." Nature Reviews Microbiology 6.4 (2008): 320-326.

Lauber, Christian L., Hamady, Micah, Knight, Rob, and Fierer, Noah. "Pyrosequencing-based assessment of soil pH as a predictor of soil bacterial community structure at the continental scale." Applied and Environmental Microbiology 75.15 (2009): 5111-5120.

Liu, Ya-Jun, Liu, Shuang-Jiang, Drake, Harold L., and Horn, Marcus A. "Alphaproteobacteria dominate active 2-methyl-4-chlorophenoxyacetic acid herbicide degraders in agricultural soil and drilosphere." Environmental Microbiology 13.4 (2011): 991-1009.

Littell, Ramon C. SAS. John Wiley & Sons, Ltd, 1996.

"LuminUltra® Test Kit Instructions: Deposit & Surface Analysis." 5 Apr. 2013. Web. 10 Feb. 2016. .

Maier, Raina M., Pepper, Ian L., and Gerba, Charles P. Environmental Microbiology. Vol. 397. Academic press, 2009.

Neidhardt, Frederick Carl, Ingraham, John L., and Schaechter, Moselio. "Physiology of the bacterial cell: a molecular approach." (1990): 30.

Onyenwoke, Rob U., Brill, Julia A., Farahi, Kamyar, and Wiegel, Juergen. "Sporulation genes in members of the low G+ C Gram-type-positive phylogenetic branch (Firmicutes)." Archives of Microbiology 182.2-3 (2004): 182-192.

Pepper, Ian L. and Gerba, Chuck P. Environmental Microbiology: A Laboratory Manual. Second Edition, Elsevier Academic Press. 2004.

Pruett, Stephen B., Myers, L. Peyton, and Keil, Deborah E. "Toxicology of metam sodium." Journal of Toxicology and Environmental Health Part B: Critical Reviews 4.2 (2001): 207-222.

Rideout, J. R., He, Y., Navas-Molina, J. A., Walters, W. A., Ursell, L. K., Gibbons, S. M., Chase, J., McDonald, D., Gonzalez, A., Robbins-Pianka, A.,Clemente, J. C., Gilbert, J.A., Huse, S.M., Zhou, H.W., Knight, R., and Caporaso, J.G. "Subsampled open-reference clustering creates

56 consistent, comprehensive OTU definitions and scales to billions of sequences." PeerJ 2 (2014): e545.

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58 APPENDIX B: SUPPLEMENTARY MATERIAL

1. Motzz Laboratory, Inc. Soil Analysis Report Electrical Sand Silt Clay Organic pH Conductivity, EC Classification (%) (%) (%) Matter (%) (dS/m) 7.9 1.3 54 34 12 SANDY LOAM 1.2

2: Total Activity: LuminUltra®

Time Period LuminUltra® pg ATP/g dry soil Control Treated p-value

0 days 4.82 ± 0.20 5.10 ± 0.12 0.106

1 day 5.04 ± 0.03 4.03 ± 0.29 0.004

7 days 4.74 ± 0.06 4.61 ± 0.12 0.153

14 days 4.58 ± 0.03 4.34 ± 0.10 0.017

21 days 4.82 ± 0.22 4.82 ± 0.13 0.989

28 days 5.04 ± 0.03 4.73 ± 0.06 0.001

P-value rejected at rejected at ≤ 0.05. Bolded if significant. By treatment type, p = 0.19675. By time period, p = 0.61440.

59 3) Statistical Analyses a) HPC Control

b) HPC Treated

60 c) LuminUltra® Control

61 d) LuminUltra® Treated

62 e) Dehydrogenase Non-Amended Control

63 f) Dehydrogenase Non-Amended Treated

64 g) Dehydrogenase Glucose-Amended Control

65 h) Dehyodrogenase Glucose-Amended Treated

66 4. DNA Extraction Quality

Read 1 Sample 260 Raw 280 Raw 320 Raw 260 280 260/280 ng/µL 0-C 0.058 0.053 0.045 0.01 0.006 1.65 9.916 0-T 0.048 0.044 0.039 0.006 0.003 2.083 5.751 1-C 0.05 0.046 0.04 0.008 0.004 1.775 7.832 1-T 0.047 0.043 0.038 0.006 0.003 2.168 5.53 7-C 0.078 0.069 0.056 0.02 0.012 1.664 20.279 7-T 0.046 0.042 0.037 0.005 0.003 2.089 5.437 14-C 0.053 0.048 0.041 0.01 0.005 1.829 9.693 14-T 0.045 0.042 0.038 0.004 0.002 2.117 4.224 21-C 0.051 0.047 0.041 0.007 0.004 1.797 7.123 21-T 0.055 0.051 0.042 0.011 0.007 1.505 10.665 28-C 0.052 0.048 0.042 0.008 0.004 1.775 7.96 28-T 0.082 0.08 0.071 0.008 0.007 1.11 7.866

Read 2 Sample 260 Raw 280 Raw 320 Raw 260 280 260/280 ng/µL 0-C 0.061 0.055 0.045 0.013 0.007 1.785 13.263 0-T 0.053 0.049 0.043 0.007 0.004 1.691 7.068 1-C 0.056 0.05 0.043 0.011 0.006 1.802 10.893 1-T 0.049 0.045 0.041 0.005 0.003 1.899 5.227 7-C 0.077 0.069 0.056 0.019 0.011 1.667 18.939 7-T 0.058 0.052 0.045 0.01 0.006 1.725 10.178 14-C 0.061 0.054 0.046 0.013 0.007 1.747 12.691 14-T 0.054 0.05 0.044 0.007 0.004 1.715 7.333 21-C 0.05 0.046 0.041 0.006 0.003 1.822 6.087 21-T 0.048 0.044 0.04 0.005 0.003 1.826 5.164 28-C 0.05 0.047 0.043 0.004 0.002 2.233 4.226 28-T 0.099 0.097 0.093 0.003 0.002 1.37 2.882

67 5. Number of Sequences per Sample

Time Period Number of Sequences Control Treated

0 days 97,537 51,231

1 day 189,324 72,719

7 days 217,726 30,728

14 days 90,157 68,120

21 days 167,766 68,467

28 days 120,614 53,209

Number of sequences read by QIIME after quality control.

68 6. Standard Operating Procedure

University of Arizona Laboratory Standard Operating Procedure

Title: Metam Sodium Effects on Microbial Activity and Populations in Soil and Land-Applied Biosolids

Approval Holder (AH): Ian Pepper Approval #: 20246

Approval Holder Phone Number(s): (520) 626-3328

Approval Safety Coordinator (ASC): Ian Pepper

Approval Safety Coordinator Phone Number(s): (520) 626-3328, (520) 307-4396

Department: Soil, Water and Environmental Science

1. Purpose This standard operating procedure (SOP) is intended to provide guidance on how to safely apply metam sodium and handle contaminated soil and supplies in Ian Pepper’s laboratory. Laboratory personnel should review this SOP, as well as the appropriate Safety Data Sheet(s) (SDSs), before handling the metam sodium. If you have questions concerning the requirements within this SOP, contact your Approval Holder (AH) or Approval Safety Coordinator (ASC).

2. Scope This SOP applies when the research team is handling metam sodium out in the field and when samples are being taken, transported, and handled. It applies to all workers under Ian Pepper's Hazardous Chemical Approval- the project coordinator (Ian Pepper), the graduate researcher (Maya Sederholm), and the staff technician (Jeff Bliznick). Any researchers that may come into contact with contaminated samples and supplies (Brad Schmitz, Alex Wassimi, Maria Campillo, Luisa Ikner, etc.) should also review this SOP.

3. Hazard Description Metam sodium (MS) is a soil fumigant that is an acute toxin and probable carcinogen. It is irritating and corrosive to the skin, eyes, and respiratory tract. It rapidly degrades into methyl isothiocyanate (MITC), which is a lachrymator (tear gas) that targets the nervous system and causes pain, crying, and vomiting.

4. Setup All handlers must wear the specific PPE at all times in the field. Once the samples have been transported to the laboratory, they must be handled/diluted under a chemical fume hood and kept there until sealed in a hazard bag.

69

5. Personal Protective Equipment

All handlers in the field must wear specific PPE: a Tyvek suit with hood, full face respirator with organic vapor cartridges, 28 mil Viton gloves, close-toed shoes, and boot covers. It is recommended that the mixer/applicator (Bliznick) also wears an impermeable apron. All researchers in the laboratory must wear a lab coat, long pants, gloves, and close-toed shoes at all times and may choose to wear goggles when diluting the samples in water.

6. Process

The applicators and sample collectors will put on all of the PPE before starting the field work. The mixer/applicator will load a carboy with the solution and hook it up to the injector. As the solution gravity-feeds into the soil, all personnel will stand at least 10 feet away. Once the carboy is empty, cores will be taken immediately and then the plastic tarp will be placed over the entire plot.

The samples (in closed nalgene bottles) will be transported to the laboratory in a University-registered vehicle, and will be placed immediately under a chemical fume hood. From there, the samples can be handled by any researcher wearing the appropriate PPE.

7. Cleanup

Waste (MS solution, field samples, biosolids, diluted samples, etc.) will be disposed of in sealed hazard bags and picked up by the hazardous waste team (Jeff Christensen) in a timely manner.

8. Handling & Storage of MS and Contaminated Supplies The MS solution will be stored in a closed container (carboy) in a shed at the Campus Agricultural Center. Once the injector, soil augers, and PPE have been used, they will be rinsed thoroughly with water and left outside to completely dry before being put in the shed. If the MS contaminates any other items at any point (ex: University vehicle, road), these items will also be rinsed with water and left to dry out in the open.

9. Warnings and Recordkeeping The contaminated plots will each have three 8.5x11'' laminated signs (shown below) staked at waist- height. The date of fumigation will be written on the sign and all people not in full PPE must stay outside the buffer zone (10 feet) for a minimum of 5 days after application.

70

The MS applicators and handlers will keep a copy of the Fumigation Management Plan (FMP) and post-application summary for 2 years from the date(s) of application. For each application, the date, weather conditions, and MITC air concentrations (taken by a Research Laboratory & Safety Services worker) will be recorded.

71 7. Chemical Hygiene Plan

University of Arizona Laboratory Chemical Hygiene Plan

Approval Holder (AH): Ian Pepper Approval #: 20246

Approval Holder Phone Number(s): (520) 626-3328

Approval Safety Coordinator (ASC): Richard Wagner

Approval Safety Coordinator Phone Number(s): (520) 626-5467

Department: Soil, Water and Environmental Science

Laboratory Locations with Hazardous Chemical Use/Storage: Building Room Room Type Veterinary Science & 404 Incubator Microbiology Veterinary Science & 405 Refrigerator Microbiology Veterinary Science & 407 Cultural lab Microbiology Veterinary Science & 409 Cultural lab Microbiology Veterinary Science & 412 Centrifuge room Microbiology Veterinary Science & 414A PCR lab Microbiology Veterinary Science & 410 Molecular lab Microbiology Veterinary Science & 416 Molecular lab Microbiology Veterinary Science & 418 Autoclave room Microbiology Campus Agricultural Center ? Storage shed WEST Center 1145 Laboratories

72 Summary of Changes: N/A

RLSS Use Only: Amendment #: Click here to enter text. Amendment Date: Click here to enter a date.

AH Electronic Signature: Sign by entering full name. Date: Click here to enter the date.

1 Introduction

Purpose

This Laboratory Chemical Hygiene Plan (LCHP) addresses the specific hazards and available control measures associated with the chemicals within Ian Pepper’s inventory for his/her University of Arizona (UA) laboratories. The LCHP has been created to comply with the requirements of the Code of Federal Regulations, Title 29 Section 1910.1450.

Scope

This LCHP provides information that is specific to Ian Pepper’s laboratory and is not covered in the University Chemical Hygiene Plan (UCHP). The LCHP is an addition to the UCHP, and shall not contradict the UCHP. In any instance where the LCHP contradicts the UCHP, either the UCHP shall be upheld or approval for variance from the UCHP will be provided by the Research Laboratory & Safety Services (RLSS).

The Approval Holder must review this plan for completeness and accuracy at least annually. All laboratory workers under Approval Number 20246 must read and affirm to this LCHP through the Research Laboratory and Safety Services (RLSS) User Dashboard (rlss.arizona.edu/services) upon amendment. The AH or ASC must also perform laboratory-specific training based off of this LCHP to all laboratory workers. A template for laboratory-specific training can be found on the RLSS website. Worker affirmation to this LCHP through the RLSS User Dashboard also includes an affirmation that the worker has received adequate laboratory-specific training and had the opportunity to have all questions answered by the AH or ASC.

Standard Operating Procedures

All laboratory workers under Ian Pepper’s Hazardous Chemical Approval must comply with all University Standard Operating Procedures (USOPs) found within the UCHP.

In addition to the USOPs, laboratory workers under this Approval must also adhere to the Laboratory Standard Operating Procedures (LSOPs) included in Appendix A of this plan. Emergency Plans/Procedures

73

IN CASE OF AN EMERGENCY: CALL 911 to contact The University of Arizona Police Department (UAPD)

Emergency Preparedness

The following emergency equipment is available in the laboratory for laboratory workers to use if they are appropriately trained.

Emergency Equipment Location(s) Fire Extinguisher VSCM inside each lab, also in hallway outside Room 418 First Aid Kit VSCM Room 410, also brought along to the CAC during MS application and sample collection and transport Chemical Spill Kit VSCM Room 410, also brought along to the CAC during MS application and sample collection and transport Emergency Eye Wash VSCM Room 410, also brought along to the CAC during MS application and sample collection and transport

Chemical Spill

Make sure the handler is wearing the appropriate PPE while cleaning up the spill. If the spill occurs outside (at the CAC or in the back of the University truck), flush the area with water and let dry. If a sample spills in the laboratory, mop up and place the towels in a double sealed hazard bag to be picked up by the hazardous waste team.

Chemical Exposure

Call 911 if a laboratory worker is exposed to a hazardous chemical and requires immediate medical attention. Perform first aid assistance described in Section 8.4 of the UCHP if you’ve been appropriately trained, and it is safe to do so. Notify Ian Pepper at (520) 626-3328 as soon as is practical. Inform the Research Laboratory & Safety Services and Risk Management Services of all chemical exposures.

If the chemical exposure does not require immediate medical attention, but the laboratory worker feels unwell, he/she should call the Arizona Poison & Drug Information Center at 520- 626-6016 for further information and recommendations.

Chemical Exposure First Aid Assistance

If metam sodium (MS) or a contaminated sample splashes in the face or eyes, use the eye-wash bottles to flush the chemical off/out. If the MS spills on the skin, rinse off thoroughly in the sink with running water.

Fire/Explosion

74 In the case of an explosion or fire in the laboratory, assist any person in immediate danger if it can be accomplished without risk to you. Immediately evacuate the area and call 911 from a campus phone, or call 911 from a non-campus phone and mention the incident is on the UA campus. If an alarm is not yet sounding, activate the fire alarm system by pulling a manual fire alarm pull station and meet your fellow laboratory workers at the pre-determined destination.

If the fire is relatively small and contained, and a laboratory worker has been appropriately trained on the use of a fire extinguisher, he/she may attempt to extinguish the fire, following the instructions in Section 8.3 of the UCHP.

Turn off any heat sources, including Bunsen burners and autoclaves, if the fire is small and contained. If the fire is large or uncontrolled, immediately leave the building via stairwells and find a secure location outside and away from the building.

Chemical Hazards and Controls

The following chemical hazard classes represent the chemicals that may be used or stored in Ian Pepper’s laboratories according to the RLSS User Dashboard. • Delayed Health Hazard • Inhalation Hazard • Contact (Eye & Skin) Hazard • Ingestion Hazard • Corrosive • Flammable

Control measures specific to Ian Pepper’s laboratories to address these hazards are detailed in the following sections.

Engineering Controls

All metam sodium-applied samples and contaminated supplies (particularly hazardous chemicals) in the laboratory will be handled within a certified chemical fume hood. Metam sodium wastes must be stored in double sealed hazard bags until picked up by the hazardous waste team.

Work Practices

All required PPE must be worn at all times while working in the field or laboratory:

The injector, soil augers, and other contaminated supplies must be rinsed with water thoroughly and dry completely outside before being stored.

Personal Protective Equipment

All handlers in the field must wear specific PPE: a Tyvek suit with hood, full face respirator with organic vapor cartridges, 28 mil Viton gloves, close-toed shoes, and boot covers. It is

75 recommended that the mixer/applicator (Bliznick) also wears an impermeable apron. All researchers in the laboratory must wear a lab coat, long pants, gloves, and close-toed shoes and may choose to wear goggles when diluting the samples in water.

Designated Areas

All particularly hazardous chemicals (i.e. select carcinogens, developmental/reproductive toxins, or chemicals that are fatal if inhaled, ingested, or come in contact with the skin) must be used and stored in areas designated for that purpose. Designated areas can be a piece of equipment (e.g. chemical fume hood), and area of a lab (e.g. a lab bench where ethidium bromide is used), or an entire lab itself (e.g. a dark room where particularly hazardous chemicals are used).

The following table describes all of the designated areas available for the use and storage of particularly hazardous chemicals in Ian Pepper’s laboratories.

Building Room Description Campus Agricultural Center Shed Metam Sodium solution, stored in carboy in shed

Laboratory Procedures Requiring Prior Approval

Laboratory workers must obtain prior approval from Ian Pepper before working with metam sodium or isothiocyanate, or before performing any of the following procedures: applying MS to soil or biosolids, measuring metadata in situ, collecting samples, transporting samples, diluting, plating, or performing any of the assays involved in the experiment.

Hazardous Gases

Laboratory workers must review all relevant safety information (i.e. UCHP, USOPs, LSOPs, etc.) and discuss experiments and procedures involving methyl isothiocyanate before beginning any such experiments. The hazards presented by these gases, as well as the control measures in place to decrease the likelihood of exposure to these gases, will be evaluated by the RLSS.

7 Waste Disposal

Waste (MS solution, field samples, biosolids, diluted samples, etc.) will be disposed of in double sealed hazard bags and picked up by the hazardous waste team (Jeff Christensen) in a timely manner.

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Appendix A: Laboratory Standard Operating Procedures

10. Purpose This standard operating procedure (SOP) is intended to provide guidance on how to safely apply metam sodium and handle contaminated soil and supplies in Ian Pepper’s laboratory. Laboratory personnel should review this SOP, as well as the appropriate Safety Data Sheet(s) (SDSs), before handling the metam sodium. If you have questions concerning the requirements within this SOP, contact your Approval Holder (AH) or Approval Safety Coordinator (ASC).

11. Scope This SOP applies when the research team is handling metam sodium out in the field and when samples are being taken, transported, and handled. It applies to all workers under Ian Pepper's Hazardous Chemical Approval- the project coordinator (Ian Pepper), the graduate researcher (Maya Sederholm), and the staff technician (Jeff Bliznick). Any researchers that may come into contact with contaminated samples and supplies (Brad Schmitz, Alex Wassimi, Maria Campillo, Luisa Ikner, etc.) should also review this SOP.

12. Hazard Description Metam sodium (MS) is a soil fumigant that is an acute toxin and probable carcinogen. It is irritating and corrosive to the skin, eyes, and respiratory tract. It rapidly degrades into methyl isothiocyanate (MITC), which is a lachrymator (tear gas) that targets the nervous system and causes pain, crying, and vomiting.

13. Setup All handlers must wear the specific PPE at all times in the field. Once the samples have been transported to the laboratory, they must be handled/diluted under a fume hood and kept there until sealed in a hazard bag. 4a. The plots must have three laminated 8.5x11'' hazard signs (shown below) staked at waist level and should be placed at least 10 feet away from the fumigation zone. The date of fumigation will be written on the sign and all people not in full PPE must stay outside the buffer zone (10 feet) for a minimum of 5 days after application.

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14. Personal Protective Equipment

All handlers in the field must wear specific PPE: a Tyvek suit with hood, full face respirator with organic vapor cartridges, 28 mil Viton gloves, close-toed shoes, and boot covers. It is recommended that the mixer/applicator (Bliznick) also wears an impermeable apron. All researchers in the laboratory must wear a lab coat, long pants, gloves, and close-toed shoes at all times and may choose to wear goggles when diluting the samples in water.

15. Process

The applicators and sample collectors will put on all of the PPE before starting the field work. The mixer/applicator will load a carboy with the solution and hook it up to the injector. As the solution gravity-feeds into the soil, all personnel will stand at least 10 feet away. Once the carboy is empty, cores will be taken immediately and then the plastic tarp will be placed over the entire plot.

The samples (in closed nalgene bottles) will be transported to the laboratory in a University- registered vehicle, and will be placed immediately under a fume hood. From there, the samples can be handled by any researcher wearing the appropriate PPE.

16. Cleanup

Waste will be disposed of in double sealed hazard bags and picked up by the hazardous waste team (Jeff Christensen) in a timely manner.

17. Handling & Storage of MS and Contaminated Supplies The MS solution will be stored in a closed container (carboy) in a shed at the Campus Agricultural Center. Once the injector, soil augers, and PPE have been used, they will be rinsed thoroughly with water and left outside to completely dry before being put in the shed. If the MS contaminates any other items at any point (ex: University vehicle, road), these items will also be rinsed with water and left to dry out in the open.

78 8. Respirator Certification

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