Sustainability of Land-Application of Class B Biosolids on an Arid Soil

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Authors Zerzghi, Huruy Ghebrehiwet

Publisher The University of Arizona.

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SUSTAINABILITY OF LAND-APPLICATION OF CLASS B BIOSOLIDS ON AN ARID SOIL

Huruy Ghebrehiwet Zerzghi

______

A Dissertation Submitted to the Faculty of the

DEPARTMENT OF SOIL, WATER AND ENVIRONMENTAL SCIENCE

In Partial Fulfillment of the Requirements For the Degree of

DOCTOR OF PHILOSOPHY

In the Graduate College

THE UNIVERSITY OF ARIZONA

2008

THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE

As members of the Dissertation Committee, we certify that we have read the dissertation prepared by Huruy Ghebrehiwet Zerzghi entitled “SUSTAINABILTY OF LAND- APPLICATION OF CLASS B BIOSOLIDS ON AN ARID SOIL” and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of Doctor of Philosophy.

______Date: May, 2008 Dr. Ian L. Pepper

______Date: May, 2008 Dr. Charles P. Gerba

______Date: May, 2008 Dr. Raina M. Maier

______Date: May, 2008 Dr. Kelly A. Reynolds

Final approval and acceptance of this dissertation is contingent upon the candidate’s submission of the final copies of the dissertation to the Graduate College.

I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement.

______Date: May, 2008. Dissertation Director: Dr. Ian L. Pepper 3

STATEMENT BY AUTHOR

This dissertation has been submitted in partial fulfillment of requirements for an advanced 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 dissertation are allowable without special permission, provided that accurate acknowledgment of 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 interest of scholarship. In all other instances, however, permission must be obtained from the author.

SIGNED: Huruy Ghebrehiwet Zerzghi

ACKNOWLEDGEMENTS

I would like to thank and appreciate my major advisor Dr. Ian L. Pepper for his generous time and commitment. Throughout my doctoral work he encouraged me to develop independent thinking and research skills and greatly assisted me with scientific writing. I am also very grateful for having an exceptional doctoral committee and would like to thank Dr. Charles P. Gerba, Dr. Raina M. Maier, and Dr. Kelly A. Reynolds for their assistance, ideas, and time throughout these years. In addition, thanks to Dr. John P. Brooks for his assistance, teaching, and guidance in my study for being always available to answer my questions on the research. I am grateful to many persons who shared me their experiences, especially Karen Josephson, who generously shared her expertise and insights that supported and expanded my research. Thanks to Al Agellon, Dr. Atasi Ray- Maitra, Susan J. Miller, and Jaime Naranjo who helped in my project in sample collection, running experiments, and data analysis. I am pleased to thank my friend Dr. Desale B. Zerai, for his assistance in statistical analysis, formatting and reviewing the dissertation. I extend many thanks to my colleagues from Dr. Pepper lab and Dr. Gerba lab, Elizabeth A. Scott, Syreeta L. Miles who helped in my project and experiments, especially, Monisha J. Banerjee, I appreciate her input and assistance into the project including field sampling and sample analysis. Also I would like to thank the staff at the ERL especially, Elenor and Jennifer and SWES office that have been always helpful throughout my graduate career.

Lastly ,but not least I would like to thank my family and friends who have been a source of continuous encouragement, support, love and care in my graduate study and life.

This work was funded by the National Science Foundation Water Quality Center located at the University of Arizona.

TABLE OF CONTENTS

LIST OF FIGURES ……………………………………………………………………....7

LIST OF TABLES………………………………………………………………………...8

ABSTRACT………………………………………………………………….....………..11

INTRODUCTION ……………………………………………………………………....13

I. LITERATURE REVIEW: LONG-TERM IMPACTS OF BIOSOLIDS LAND APPLICATIONS ON MICROBIAL AND CHEMICAL PROPERTIES OF AN ARID SOIL……………………………………………………………………………………...15

Wastewater Treatment and Biosolids Production in the United States…...... ……...……15 Assessment of Health Risks Associated with Land Application of Biosolids…………...17 Pathogen Risks and Beneficial Use of Biosolids…………………………..……….18 Public Health Risks from Chemicals Found in Land-applied Biosolids…………...21 Effect of Long-term Land Application of Biosolids on Soil Microbial Community…....24 Effect of Long-term Land Application of Biosolids on Soil Chemical Properties……....26 Sustainability of Long-term Land Application of Biosolids…….…….…………………28 References………………………………………………………………………………..31

II. THE LONG TERM EFFECTS OF LAND APPLICATION OF CLASS B BIOSOLIDS ON THE SOIL MICROBIAL COMMUNITY……………………………38

Abstract………………………………………………………………………….…….…38 Introduction………………………..………………………………………...……..…….39 Materials and Methods……………….……………….………………….………………40 Study Site…………………………………………………………………………...40 Indigenous Soil Microbial Numbers…………………………………...…………..41 Microbial Pathogens, Indicators and Antibiotic Resistant Bacteria………….…...42 Microbial Activity…………………………………………………...………...... 45 Results……………………………………………..….………..……….……………...... 46 Discussion and Summary………………………………………………………………...50 References……………………………………………………………..…………………54 Tables and Figures …………………………………………..…………………………..58

III. INFLUENCE OF LAND APPLICATION OF CLASS B BIOSOLIDS ON SOIL BACTERIAL DIVERSITY……………………………………………………….62

Abstract…………………………………………………….……………..……..……….62 Introduction………………………………………………….….……………………..…63 Materials and Methods…………………………………………………………………...65 Study Area…...... 65 Soil Bacterial Community DNA Extraction and PCR Amplification of 16S rRNA....65 Cloning, Colony PCR and Sequencing of 16S rRNA …………………………..…..67 16S rRNA Sequence Analysis …………………………………….……………..….68 Results and Discussion ……………………………………..……..…………..………...69 rRNA Clone Sequencing and Phylogenetic Analyses………………..….…….…....69 Conclusion……………………………………………………………………………….78 References…………………………………………………….….……………………....79 Tables and Figures ….……………………………………………………………..…….83

IV. LONG-TERM EFFECTS OF LAND APPLICATION OF CLASS B BIOSOLIDS ON SOIL CHEMICAL PROPERTIES………………………………………………….85

Abstract………………………………………………………………………….……….85 Introduction………………………………………………………………………………86 Materials and Methods…………………………………………………………………...87 Results and Discussion ………………………………………………...... 90 Influence of Land Application of Biosolids on Soil Macro Elements …...... 90 Effect of Land Application of Biosolids on Soil Heavy Metals ……………………95 Summary………………………………………………………………..…...... 99 References………………………………………………………………...…………….102 Tables and Figures ……………………………………………………………………..106

REFERENCES…………………………………………………………………………111

LIST OF FIGURES

Figure 2.1 Land applications of liquid biosolids on Marana, Arizona agricultural field via biosolids injector. Photo courtesy H.G. Zerzghi………………………………………....41

Fig.2.2 NO3 concentrations in soils incubated from 0 to 72 hours……………...... 49

Figure 3.1. Neighbor-joining phylogenetic tree of 16S rRNA bacterial gene sequences from control (no biosolids amended) soil; the number in parenthesis indicates number of rRNA sequences. Bacterial genera are derived from sequences analysis result obtained from NCBI, GenBank at > 96 % identity……………………………………………….72

Figure 3.2 Neighbor-joining phylogenetic tree of 16S rRNA bacterial gene sequences from soils amended with the high biosolids rate of application; the number in parenthesis indicates number of rRNA sequences. Bacterial genera are derived from sequences analysis result obtained from NCBI, GenBank at > 96 % identity………………………73

Fig.4.1. Total nitrogen (%) and total carbon (%) levels measured in soil cores collected in 2005.Values are the average of all soil samples from 0-150cm and values with different letters are significant at the 95 % confidence level………………………………………92

Fig. 4.2. The influence of biosolids on soil P. Values are the mean of all soil analysis from 0-150cm and values with different letters are significant at the 95 % confidence level………………………………………………………………………………………94

Fig.4.3. Effect of biosolids on heavy metals concentrations. (Total and available concentrations of the diethylene triamine penta-acetic acid (DTPA) metals). Data represent mean values of soil samples taken in December 2005 averaged over 150 cm soil core samples; values with different letters are significant at the 95 % confidence level...96

LIST OF TABLES

Table 1.1 Table 1.1 Known principal pathogens of concern in Class B biosolids in the U.S. ……………………………………………………………………………...………20

Table 2.1 Microbiological assays used to detect microbial pathogens and antibiotic resistant bacteria………………………………………………………………………….43

Table 2.2 Mean microbial numbers based on cultural heterotrophic plate counts and acridine orange direct counts. Data represent mean values of 10 months of analyses from March to December of 2005……………………………………………………………..47

Table 2.3 Typical Pathogen and Indicator Loads in Tucson Sewage and Biosolids (Pima County); biosolids utilized for land application…………………………………………48

Table 2.4 Soil analyses for potential microbial hazards…………………………………48

Table 2.5 Influence of land application on sulfur oxidation (µgSO4 -S/g dry soil) and Dehydrogenase activity (µg TPF/ g dry soil). Data represents mean values of 16 soil samples (4 replicates x 4 treatments) analyzed on December of 2005……………………………50

Table 2.6 Soil gravimetric moisture contents (%) of the soils from the experimental plots. Data represents mean values of 10 months of analyses from March to December of 2005………………………………………………………………………………………58

Table 2.7 Cultural Heterotrophic Plate Counts (cfu/ g dry soil). Data represents mean values of 10 months of analyses from March to December of 2005…………………….59

Table 2.8 Total Direct Counts (number of cells /g dry soil). Data represents mean values of 16 soil samples (4 replicates x 4 treatments) analyzed on December of 2005…………………………………………………………………………….………...60

Table 2.9 Potential biological hazards…………………………………………………...60

Table 2.10 Influence of land application of Class B biosolids on sulfur oxidation (µgSO4 - S/g dry soil) and dehydrogenase activity (µgTPF/g dry soil). Data represents mean values of 16 soil samples (4 replicates x 4 treatments) analyzed on December of 2005…………..61

LIST OF TABLES- continued

Table 3.1. Comparison of identified bacterial species composition in the control soils (unamended) and the high biosolid-treated soils………………………………………...71

Table 3.2 Grouping and comparison of bacterial sequence clone isolates % distribution per sample using the Ribosomal Database Project II (classified at > 95 % percent identity) and library comparison were considered significant at the 95% confidence level, Marana, AZ………………………………………………………………………………75

Table 3.3. Summary of phylogenetic assignments of 16S rRNA gene clones from (a) unamended soil and (b) biosolid-treated soil, Marana, AZ……………………...…...... 83

Table 4.1. Soil chemical analyses used to determine soil pH, electrical conductivity (EC), CaCO3, NO3, total organic carbon (TOC), total nitrogen (TN), total phosphorus, available phosphorus, total metals, and diethylene triamine penta-acetic acid (DTPA) metal concentrations……………………………………………………………………………89

Table 4.2. Representative heavy metals (Inorganic pollutants) in biosolids…………….96

Table 4.3. Representative heavy metals in agricultural soils compared with metal concentration in high biosolid treated soils……………………………………………...98

Table 4.4 Chemical analyses conducted on all soil samples; values (averaged over the 0- 150 cm depth) with different letters are significant at the 95% confidence level………106

Table 4.5 N, P and TOC chemical analyses conducted on all soil samples. Mean values (µg/g) were considered significant at the 95% confidence level and are indicated with different letters………………………………………………………………………….107

Table 4.6 Total metal analyses conducted on all soil samples. Mean values (µg/g) were considered significant at the 95% confidence level and are indicated with different letters…………………………………………………………………………………..108

Table 4.7 Available metal analyses conducted on all of soil samples and available metal concentrations, DTPA metals (µg/g) were considered significant at the 95% confidence level (indicated with letters) except for Cd and Pb. are indicated with different letters..109

Table 4.8 Total metal analyses conducted on all soil samples; values (µg/g) averaged over 0-150 (cm) depth of total metals concentrations were considered insignificant at the 95% confidence level…………………………………………………………………...110

LIST OF TABLES- continued

Table 4.9 Typical C and N content of anaerobically digested biosolids. Means and standard deviations from biosolids samples collected from 1984 to 1990. TOC, total organic C; TN, total N……………………………………………….…………………110

ABSTRACT

This study evaluated the influence of annual land applications of Class B biosolids on the soil microbial and chemical properties monitored over 20 year period. The study was initiated in

1986 at the University of Arizona Marana Agricultural Center, Tucson, Arizona. The final application of biosolids was in March 2005, followed by growth of cotton from April through

November 2005. Surface soil samples (0-30 cm) were collected monthly from March 2005 through December 2005, and analyzed for soil microbial properties. Soil cores (0-150 cm) were also collected in December and analyzed for various soil chemical properties. The study showed that land application of Class B biosolids had no significant effect on the number of indigenous soil microbial numbers including bacteria, actinomycetes, and fungi (no bacterial or viral pathogens were present in soil samples collected in December) but enhanced microbial activity in the biosolid amended plots. Bacterial diversity was not impacted after 20 years of land application when evaluated through cloning and sequence analysis of bacterial 16S rRNA. Both soils had a broad phylogenetic diversity comprising more than five major phyla including:

Proteobacteria, Acidobacteria, Actinobacteria, Bacteroidetes, and Firmicutes. Chemical analyses showed that land application of biosolids significantly increased soil pH but did not affect soil salinity and CaCO3 values as compared with the control plots. However, this lack of increase in salinity was likely due to the leaching of soluble salts through the soil profile since irrigation rates. Land application significantly increased soil macro-nutrients including C, N and P and caution should be taken with respect to phosphate loadings to prevent nutrient contamination of surface waters. The biosolid amended soil concentrations of available and total metals were low

(compared to the typical background soil metal concentrations). Metal concentrations attenuated rapidly with increasing soil depth, and were generally similar to values found in control soils at a depth of 150cm. Increases in available metal concentrations were modest. It is important to note 12

that there are differences between these studies with respect to different cropping systems, biosolids type, climate and soil type, as well as irrigation rates in the arid southwest.

INTRODUCTION

Biosolids contain significant amounts of nitrogen (N), phosphorus (P), and complex organic matter that can benefit crop production. Biosolids are also beneficial for maintaining and improving soil structure and water-holding capacity (Giroux and Tabi,

1990).The amendment of to nonfood agricultural production land through land application of biosolids is an attractive environmental alternative for both soil conservation and residue disposal, but potential risks from the accumulation of heavy metals and organic pollutants, as well as pathogen contamination, must be considered.

In 1986, a study began at the University of Arizona Marana Agricultural Center,

21 miles north of Tucson to determine the long-term effects of continued annual application of biosolids to an irrigated field planted with cotton. The applied biosolids consisted anaerobically digested sewage sludge (biosolids) produced by the city of

Tucson. The aim of the investigation was to evaluate the benefits and hazards of land application of Class B biosolids on agricultural land. During this time, Brendecke et al.

(1993) conducted a research study to evaluate the effect of land application of biosolids on soil’s physical and chemical properties, and also on the soil microflora at the Marana study site. That study showed that 4 years of land application of biosolids to an agricultural desert soil in the southwest USA did not adversely affect microbial populations or activity. The current study evaluates the influence of 20 years of continuous land applications of Class B biosolids on microbial and chemical properties.

Biosolids have been annually land applied to the Marana study site for 20 years.

Monitoring soil quality by means of biological indices can be useful in managing and 14 maintaining the sustainability of soils that receive biosolids application (Fernades et al.,

2005). Having an agricultural field with a 20 year history of biosolids application provides a unique opportunity for evaluation of the long-term effects of biosolids on various biological and chemical parameters and its sustainability. It is the objective of this work to evaluate the effect of long-term land application of Class B biosolids on Soil microbial numbers, activity, diversity as well as soil chemical properties.

I. LITERATURE REVIEW: LONG-TERM IMPACTS OF BIOSOLIDS LAND

APPLICATIONS ON MICROBIAL AND CHEMICAL PROPERTIES OF AN ARID

SOIL

I. Wastewater Treatment and Biosolids Production in the United Sates

The growth of the world population and the requirements of today’s modern

society create large amounts of industrial and municipal waste or sewage (Pepper et al,

2006). Construction of waste collection systems date back to Roman times, where wastes

were normally discharged into the nearest body of water, including, lakes, streams, or the

ocean (Maier et al., 2000). However, this was simply a transfer of waste material from

one location to another (Pepper et al., 2006). Sewage treatment is a relatively modern practice that evolved from earlier practices that centered on cheap disposal with less

regard for environmental protection (Bastian, 2005). The main purpose of a modern-day

sewage treatment is to protect the environment and public health by treating sewage prior to discharge into the environment.

The wastewater treatment comprises three major steps: primary, secondary, and tertiary treatments. Primary wastewater treatment is a physical process involving the separation of large solids from waste stream and then subsequent sedimentation creating a primary sludge. Secondary treatment serves as a biological degradation in which the number of potential pathogens in the sludge is reduced (Pepper et al., 2006). The sludge produced from primary and secondary treatment (and sometimes tertiary) must be treated further through stabilization techniques in order to reduce the organic matter, pathogens and water content present in raw sludge (Maier et al., 2000, (Zaleski et al., 2005). 16

In the United States, the term biosolids implies treatment to produce Class A or

Class B biosolids that meet the land-application standards in the Part 503 Environmental

Protection Agency regulations (USEPA, 1994). Biosolids are a complex mixture of

organic and inorganic compounds of biological and mineral origin resulting from

treatment of sewage at wastewater treatment plants (Hurst, 1988) and approximately 5.6

millions of dry tons are produced every year (NRC, 2002).

Sludge stabilization techniques include: aerobic digestion, anaerobic digestion,

and composting. Aerobic digestion involves reducing odor, pathogen, and organic

content by adding air or oxygen in an open tank system with a moderate or mesophilic

(30-400C) digestion temperatures and converts organic solids to carbon dioxide, water,

and nitrogen. Increasing the oxygen content, temperature and retention time can enhance

inactivation of pathogens, in which pathogen concentrations determine the degree of

treatment level of product, i.e. Class B versus Class A biosolids (Pepper et. al., 2006).

On the other hand, anaerobic digestion occurs under low redox conditions and

microorganisms use carbon dioxide as a terminal electron acceptor to convert organic

substrate to methane and carbon dioxide. This process is the most common methods of

sludge stabilization that reduces the volatile solids by 30-60% and results in the

production of Class B biosolids. Class B biosolids are normally produced as a “cake” (≃

20% solids) or as liquid biosolids (≃ 5-8% solids) (Pepper et. al., 2006). Class B material

can be further treated to Class A standards following more stringent and enhanced

treatment (Pepper et. al., 2006). According to the U.S.EPA (1994), as long as treatment

facilities comply with the treatment and discharge regulations set by the 40 CFR Part 503 17

Biosolids Rule and the methods for the further treatment of biosolids are followed, and then land application of anaerobically digested biosolids is considered to be a safe practice.

II. Assessment of Health Risks Associated with Land application of Biosolids

It has been more than two centuries since a large variety of industrial and municipal waste residuals were first land applied as soil amendments to supplement and improve soil. Nutrients and organic solids from sewage were shown to increase crop production. Therefore, sewage farming was promoted as being a profitable as well as effective technology to reduce waterborne pollution of surface waters. In fact, soil interactions with pollutants were considered to be purifying processes, however, systems frequently failed as a result of unregulated loading of too much waste and other pollutants into the soil (Bastian, 2005).

U.S. approaches to develop these regulations for land applications of biosolids have changed over the years as more scientific information on the fate and effects of land applied biosolids were gathered. The 1970s were a period of intense searching for environmental risks related to land application of biosolids (Chaney et al., 1996). As a result, a considerable amount of data on the use of biosolids in the agricultural area was collected towards the latter part of the 20th century, which led to promulgation of strict regulations aimed at safe and economically feasible biosolids management practices

(Mantovi et al., 2004). 18

Initially, ocean disposal of treated sewage sludge or biosolids was the preferred

method of disposing of biosolids in the world's coastal cites (NRC, 2002). Forty years

ago, many American costal cities disposed wastewater solids directly into the ocean and

freshwater aquatic systems (Elliott and O’Connor, 2006). However, in the late 1980s , the

U.S. Congress banned ocean dumping of municipal wastes (Ocean Disposal Ban Act of

1988), and as an alternative (to ocean dumping, landfilling or incineration), the U.S.EPA promulgated standards for the use or disposal of sewage sludge (Code of Federal

Regulations Title 40, Part 503), which allows for and regulates land application of biosolids. The ban of ocean dumping led to increased land application of biosolids for soil-amendment and land reclamation purposes. In 2000, it was estimated that approximately 5.6 million tons of dry sewage sludge were used or disposed of annually in the United States (Pepper et al., 2006) and currently about 60% of all biosolids are land applied with most of the land receiving Class B biosolids (NRC, 2002).

Pathogen risks and beneficial use of biosolids

Biosolids from waste water treatment have been defined by U.S.EPA (1995) as primarily organic solid product yielded by municipal wastewater treatment processes that can be beneficiary recycled. Unlike the EPA regulations for chemical standards, the section of the U.S.EPA rule known as the 503 rule (40 CFR Part 503) for pathogens was promulgated without addressing pathogen risk assessment because methodologies had not been developed (Eisenberg et al., 2004 , NRC 1996); rather they were based on performance or technology-based standards, management and record-keeping practices 19

(Eisenberg et al.,2004). Most of the limits were based on risk assessment that includes a

number of assumptions. However, there have been disagreements in the scientific community over these assumptions used for the risk assessment (Gaskin et al, 2003). The goal of EPA regulations was to minimize the risk of adverse health effects from pathogens in biosolids applied to land and there are operational standards to reduce the potential for human exposure (NRC, 2002).

The 503 Rule permits Classes A and B biosolids for agricultural use. Class A is treated to ensure the pathogens are below detection limits and are applied without any crop restrictions. Class B biosolids are also treated to reduce pathogens but still contain

detectable levels of them. The known pathogens of concern in Class B biosolids are listed

in Table 1.1. Most public concerns are centered on the possible presence of these pathogens in biosolids. Opponents of land application of biosolids put the blame on

biosolids for the reported illness and deaths from or people who work at wastewater

treatments plant or those residents living near land application sites who are exposed to

dust from fields treated with biosolids (Zaleski et al., 2005).

Regardless of the continuing debate over the safety of land application of biosolids and the hazards they may impose on public health and the environment, the

U.S.EPA still supports land application of biosolids. A number of court cases have been

reported in relation to biosolids public health effects, but in all cases, but there has been no documentation of the link between the reported deaths or illness and biosolids exposure (Zaleski et al., 2005). Regardless of numerous studies that have showed low risks to human health posed by pathogens in biosolids, uncertainty and unease in the 20 public’s mind will likely to continue (Lewis et al., 2002, Rusin et al., 2003, Elliott and

O’Connor, 2006, Pepper et al., 2008).

Table 1.1.Known principal pathogens of concern in Class B biosolids in the U.S.

(Adapted from Pepper et al., 2008).

BACTERIA PROTOZOA Salmonella sp. Cryptosporidium Shigella sp. Yersinia Giardia lamblia Vibrio cholerae Campylobacter jejuni Toxoplasma gondii Escherichia coli

ENTERIC VIRUSES HELMINTH WORMS Hepatitis A virus Ascaris lumbricoides Adenovirus Ascaris suum Norovirus Trichuris trichiura Sapporovirus Toxocara canis Rotavirus Taenia saginata Enterovirus Taenia solium - Poliovirus Necator americanus - Coxsackievirus Hymenolepisnana - Echovirus - Enterovirus 68-91 Reovirus Astrovirus Hepatitis E virus Picobirnavirus

Of these pathogens, pathogenic antibiotic resistant strains may be of most concern

because these organisms have shown resistance to many commonly prescribed antibiotics

(Pepper et al., 2008). Kim and Aga (2007) reported that “sorbed antibiotics in land- applied biosolids pose a special concern because antibiotics that remain biologically active may potentially influence selection of antibiotic resistant bacteria.” However, the results of a study by Brooks et al. (2007) found that the influence of biosolids on the incidence of soil-borne antibiotic resistant bacteria was negligible. Overall, most studies tend to agree that public health risks from treatment or applying Class B biosolids are very low, but more studies on land application practices are needed for a broader public support, with reliable risk assessments and monitoring systems in place.

Public health risks from chemicals found in land-applied biosolids

Today many chemical compounds are used in the modern industrialized world that may end up in wastewater effluents or biosolids (Krester et al., 2005). There have been public health concerns with regard to risks of exposure to hazardous chemical components found in bioaerosols from biosolids (Lewis et al., 2002). Several days or weeks after land application of biosolids, degradation of biological and chemical components can produce various volatile irritants (organic and inorganic) , volatile fatty acids, alkyl amines and ammonia that winds may carry for miles (Dowd et al., 2000).

Biosolids can also contain other organic compounds and depending on its origin, biosolids may contain variable amounts of trace metals (Parat et al., 2007). Many industries have banned discharging or utilized strict pretreatment guidelines of unwanted 22

organic compounds (e.g. PCBs, DDT, heptachlor, etc) because of the health problems

that can result from dispersing or bioaccumulation in aquatic food chains (Chaney et al.,

1996). Other chemicals of concern and chemicals that have been the target of EPA investigation are “emerging chemicals of concern” including: endocrine disrupting

compounds (EDCs), polybrominated diphenyl ethers (PBDEs), pharmaceuticals, personal

care products (PPCPs), and endotoxins. EDCs are chemicals that interfere with endocrine

glands or their hormones. PBDEs are compounds utilized as flame retardants for

everyday household items including carpets and cushions. PBDEs are a known class of

EDCs that are typically present at ppm levels in municipal biosolids produced in the

USA. In a study by Lorenzen et al. (2006), EDCs levels following land application were very low because a wide range of them were rapidly degraded or mineralized in soil,

Roberts et al. (2006) found similar trends with respect to EDC degradation. A study conducted by Quanrud et. al. (2007) indicated that current practices of land application

are likely to be sustainable with respect to EDCs. However, given the increasing number

of medicines administrated for pain relief, control of cholesterol levels, and enhancement

hormones, then more exposure of humans to these endocrine disruptors is apparent,

increasing concern with respect to the overall health hazards.

Endotoxin, a lipoplysaccharide derived from breakdown of cell wall of gram-

negative bacteria, is a highly immunogenic class of molecules that can cause a broad

range of health effects including: fever, asthma, and shock (Pepper et al., 2008) and it is

present in everyday environments (mostly dust). Brooks et al. (2006) determined the extent of endotoxin attributed to land-application by collecting aerosol samples: during 23

land application of biosolids, during tractor operations on an agricultural field, and from

an aeration basin located within an open-air wastewater treatment plant. The study demonstrated that the greatest source of the endotoxin was from a wastewater treatment plant aeration basin. In addition, more endotoxin was aerosolized during tractor operations without biosolids than with biosolids. Similarly, other studies results have also

suggested that the majority of endotoxin may in fact be of soil origin rather than biosolid

treated soil (Paez-Rubio et al., 2006; Paez-Rubio et al., 2007, Brooks et al., 2006).

Current debates and concerns of public health risks from trace metals as a result

of land application of biosolids focus on two hypotheses known as the sludge protection

hypothesis and sludge time-bomb hypothesis. Advocates of the biosolids protection

hypothesis predict that trace metals bound within biosolids will remain absorbed to biosolids as long as trace metals of concern persist in soils. On the contrary, the biosolids time-bomb hypothesis predicts the release of trace metals as more biosolids-derived organic matter becomes degraded (Parat et al., 2007). However, several studies showed

that the potential public health hazard from trace metals is very low for two major

reasons. First, trace metals bioavailability tends to decrease over time. The second factor

is because the metal concentrations found within biosolids have decreased over the past

20 years due to improved point-source control or enhanced pretreatment technologies.

Thus, the potential for metal contamination of soil or surface waters from land

application of biosolids has decreased and studies in the southwest have indicated that

metals from land application are below toxic levels (Gaskin et al., 2003). However,

monitoring of long-term application of biosolids is important to protect human health. 24

III. Effect of long-term land-application of biosolids on Soil Microbial Community

Along with pathogenic and indicator organisms, land application of Class B biosolids may result in a higher numbers of heterotrophic bacteria, fungi and actinomycetes in response to additions of nutrients and plant productivity (Sullivan et al.,

2006, Pepper et al.,2008). The decay and survival of these organisms is governed by complex soil and environmental variables, including soil texture, organic matter content, pH value, temperature and moisture content (Land and Smith, 2007). Numerous studies have reported that land-application resulted in an initial increase of microbial populations but within relatively short periods of time, populations of the biosolid-treated soils become similar to non-amended (Brendecke et al., 1993, Lang and Smith, 2007; Lang et al., 2007; Lang et al., 2003; Pepper et al., 1993). In contrast, other studies have reported that the survival of microorganisms (especially indicators and pathogens) in biosolid- treated soil varies widely between 24 h to > 2 years (Lang et al., 2007).

In most studies, land application of Class B biosolids has been shown to enhance microbial activity (Sullivan et al., 2006, Pepper et al., 2008). The concept of using dehydrogenase activity or other physiological assays as an index for measuring general microbial activity is a useful technique and results can be used to predict soil fertility or changes in yield (Brendecke et al., 1993). There are other approaches that have been used to determine microbial activity including common microbial transformations such as nitrification and sulfur oxidation. A recent study evaluated the effect of long-term land application of Class B biosolids using the common microbial transformations: 25 nitrification or sulfur oxidation, where rates of both microbial transformations increased in soils from the biosolid amended plots, and increased with increased rate of biosolids application. Dehydrogenase activity also increased with increased biosolid amendment

(Pepper et al., 2008).

Functional diversity can also be an important mechanism that allows for soil microbial communities to successfully respond to changes in the soil environment.

Having such functional diversity of soil microorganisms is vital to maintain a healthy biosphere (Roane and Pepper, 2000). Land application of biosolids may result in significant changes in the structure, or richness of microbial communities at a given site

(USEPA, 1991). Several studies have reported the changes in microbial community structure in response to land application (Sullivan et al., 2006, Dennis and Fresquez,

1989). Responses of microbial communities to biosolids may differ depending on the length of the study, microbial community characterization methods, and the rate or method of application used. For instance, in the study by Sullivan et al., (2006), higher application rates of biosolids resulted in significant decline of the arbuscular mycorrhizal fungi (AMF) which are the critical components of soil microbial communities. Shifts or changes in soil microbial community can be attributed to increased soil fertility due to the land application of biosolids (Sullivan et al., 2006), or due to naturalized populations from biosolids (Ishii et al., 2006). Overall, there is a need for more long-term studies of the complex microbial communities and their interactions to determine resulting ecosystem dynamics (Sullivan et al., 2006).

IV. Effect of Long-term Land Application of Biosolids on Soil Chemical Properties

As discussed earlier there are many reasons for assessing the impact of land

application of biosolids on soil microbial ecology. Several long-term land application

studies have shown enhanced microbial activity and few adverse toxicity effects on soil

microbial communities. However, other land application issues arise from nutrient

additions, which are often defended as a major benefit of land recycling of biosolids

(Elliott and O’Connor, 2007). For instance, the recent study by, Parat et al. (2007)

reported that repeated applications of biosolids induced accumulation of biosolid-derived

organic carbon. Biosolids are good sources of C, N, P, K and other micronutrients for

crop production (Artiola and Pepper, 1992), but there are concerns related to N and P

over-loading that results from the mismatch between the biosolids nutrient content and

crop nutrient requirements.

Wang et al., 2003 reported on the potential for nitrate pollution of groundwater regardless of whether biosolids or inorganic fertilizer was applied to the soil. They showed that N mineralization was affected by soil type and temperature indicating that further research on these fields may help to determine appropriate N loading rates in order to avoid potential nitrate leaching to groundwater. In general, land-based recycling of biosolids were typically applied at rates designed to satisfy N requirements (Brandt et

al., 2004) but the issue of high phosphate concentrations of biosolids may limit land

application of biosolids. Elliott and O’Connor (2007) have stated that P is at “the

forefront of biosolids-related issues that may adversely affect long-term sustainability of

land-based recycling programs in the U.S.” Similarly, Tian et al., (2006) found that long- 27

term application of biosolids at high rate levels increased P in surface waters. Therefore,

P-based nutrient management is needed in order to minimize the potential of

contamination of receiving waters from biosolids-derived P (Wang et al., 2003).

In the current study, at Marana, no increases in soil salinity were observed following the 20 years of land application, no doubt in part because irrigation rates were in excess of consumptive water use rates for cotton, resulting in the leaching of salts through the soil profile. Ours results are similar to those of several other researchers who reported that long-term application of biosolids did not increase soil salinity, commonly measured by the electrical conductivity (EC) of a solution extracted from soil solution

(Cogger et al., 2001; Schroder et al., 2008).

Even though utilizing land application of biosolids benefits agriculture both by its fertilization and irrigation value, there are potential hazards because of the accumulation of metals including the arsenic and mercury (Gaskin et al., 2003). Accumulation of heavy metals on the top soil could have long-term negative effects on soil fertility and microbial

activity (Gibbs et al., 2006). Thus, the USEPA Part 503 regulations dealt with the

problem of accumulation by setting regulatory rules which determine the upper and lower

thresholds for safe land application. In states like Arizona, the potential accumulation of

heavy metals from biosolids was thought to be a limiting factor for biosolids use as a

nutrient source (Brendecke, 1993). In addition, several studies on metals from land

application of biosolids have found increased metals concentrations in biosolid-treated

soils. However, the increase of soil heavy metal concentrations was not significantly

higher above the background soil concentrations (Gaskin et al., 2003, Tiffany et al., 2000, 28

Pepper et al., 2008). Overall, concentrations of metals in biosolids have been decreasing

over the past several decades due to effective production land use bans, industrial pre-

treatment programs, and wastewater treatments (point-source control practices) especially

after the promulgation of the 40 CFR Part 503 regulations.

V. Sustainability of Long-term Land Application of Biosolids

Biosolids are a product of modern wastewater treatment and contain nutrients and

organic matter which can be used for agricultural use and reclamation of degraded soils

(Sanchez-Monedero, et al., 2004). Today, the major challenge for scientists and

regulators is the question of establishing a sustainable land application practice under a wide range of conditions (Bastian, 2005). Soil reactions with contaminants or nutrients in biosolids represent the key to sustainable land application systems. The soil environment contains numerous microorganisms and vegetations that react to the specific contaminants in land-applied residuals and modify the contaminants through biogeochemical cycling such as direct oxidation-reduction reactions, adsorption- desorption, biodegradation and plant uptake (Bastian, 2005). The initial basis for questioning the sustainability of land-application has been due to public concern over potential hazards and often linked to the production of odors, flies, or dust associated with land-application (Bastin, 2005).

“Sustainability of any process implies that the process can be maintained indefinitely and it necessitates addressing the needs of the present without compromising the ability of future generations to meet their needs” (Pepper et al., 2008). An evaluation 29 of land application of biosolid’s sustainability must be performed over long time periods, and the influence of repeated annual applications of biosolids on soil properties must be documented (Pepper et al., 2008). In essence, soil quality is maintained through the integration of soil microbial, chemical, and physical properties. Much research has shown long-term land application of biosolids can have a significant impact on these parameters.

Some of these studies may have differing results because of different cropping systems, biosolids type or rate, climate and soil type, irrigation rates and environmental conditions.

Researchers have shown that long-term land application of biosolids can have significant positive effects on the microbial and chemical properties of the soil (Artiola and Pepper, 1992, Brendecke et al., 1993, Elliott and O’Connor, 2007). In addition, there was no evidence of adverse toxicity effects on the soil microbial community with respect to numbers, activity, or diversity. In contrast, there were previous studies indicated that accumulation of metals following long-term applications of biosolids had reduced levels of microbial biomass and was shown to reduce nitrogen fixation (Brookes et al., 1986).

Long term land-application also increased soil macro-nutrients including C, N, and P (Elliott and O’Connor, 2007). After the promulgation, metals in the soils from fields that had received biosolids for a long period of time (13-20) years were similar to background levels reported for agricultural soils in the mid-west and southwest parts of

U.S and impacts on surface water quality were minimal (Gaskin et al., 2003, Pepper et al. , 2008). “To date there does not appear to be any chemical entity likely to limit land- application other than phosphate loadings in areas sensitive to surface water contamination (Pepper et al., 2008). Of course, vigilance is always necessary as new 30 pathogens and contaminants continue to emerge, and their presence and fate in biosolids need to be assessed to ensure safe and sustainable land application of Class B biosolids.

Soil physical properties are shaped by soil texture, which is intrinsic to any given soil, and soil structure, which is key to the functioning of the soil and highly variable

(Pepper et al., 2008). Several studies have shown that long-term additions of biosolids improved the physical properties of soils from semi-arid climate and Mediterranean conditions (Aggelides and Londra, 2000, Tsadilas et al., 2005). They have documented enhanced soil physical properties including: increased water retention, reduced soil bulk density, increased porosity, aggregation and aggregate instability, hydraulic conductivity and infiltrations. Most of these effects are due to an increase of soil organic matter from biosolids (Tsadilas et al., 2005). There have also been reports documenting decreased water infiltration due to soil compaction following land application of liquid municipal sewage sludge in eastern Nebraska (Stamatiadis et al. 1999). Soil compaction hardpan formations were reduced by developing appropriate methods of land application of municipal solid waste (Khalilian et al, 2002). However, provided that land application is carefully managed i.e., avoidance of traffic over saturated soils, then land application is likely to be sustainable with respect to soil physical properties (Pepper et al., 2008).

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II. THE LONG TERM EFFECTS OF LAND-APPLICATION OF CLASS B BIOSOLIDS ON THE SOIL MICROBIAL COMMUNITY

Abstract

This study evaluated the influence of 20 annual land applications of Class B biosolids on the soil microbial community. Specifically, the potential benefits and hazards of land application were evaluated by analysis of surface soil samples collected following the 20th land application of

biosolids. The study was initiated in 1986 at the University of Arizona Marana Agricultural

Center, 21 miles north of Tucson, Arizona. The final application of biosolids was in March 2005,

followed by growth of cotton from April through November 2005. Surface soil samples (0–1')

were collected monthly from March 2005, two weeks after the final biosolids application, through

December 2005, and analyzed for soil microbial properties. In the current study, the data show

that land application of Class B biosolids had no significant effect on indigenous soil microbial

numbers including bacteria, actinomycetes, and fungi compared to control plots. No bacterial or

viral pathogens were detected in soil samples collected from biosolid amended plots in December

(nine months after the last land application). However, plots that received biosolids had

significantly higher microbial activity or potential for microbial transformations, including

nitrification, sulfur oxidation, and dehydrogenase activity than control plots and plots receiving

inorganic fertilizers. In general, this study documents that land application of biosolids at this

particular site was sustainable throughout the 20 year period, with respect to soil microbial

properties.

Introduction

In 1986, a study was initiated at the University of Arizona Marana Agricultural

Center, 21 miles north of Tucson to determine the effects of long term annual

applications of biosolids to an irrigated agricultural field planted with cotton. The applied

biosolids consisted of anaerobically digested biosolids following wastewater treatment of

sewage from the City of Tucson. The aim of this part of the study was to evaluate the

influences of 20 annual land applications of Class B biosolids on the soil microbial community.

Earlier in this study, Brendecke et al. (1993) also evaluated the effect of land

application of biosolids on the soil microflora at the Marana study site. That study

showed that 4 years of land application of biosolids to an agricultural desert soil in the

southwest USA did not adversely affect microbial populations or activity. The current

study evaluates the influence of 20 years of continuous land applications of Class B

biosolids on microbial and chemical properties. The soil at this site is classified as a Pima clay loam. Biosolids were annually land applied to the Marana study site for 20 years.

Monitoring soil quality by means of biological indices can be useful in managing and maintaining the sustainability of soils that receive biosolid applications (Fernandes et al.,

2005).

Having an agricultural field with a 20 year history of biosolids application provides a unique opportunity for evaluation of the long-term effects of biosolids on various biological parameters. The objectives of this work were: i) to evaluate the effect of long-term land application of Class B biosolids on microbial numbers (including 40

bacteria, fungi, and actinomycetes); ii) to determine if microbial indicator organisms and pathogens found in the original biosolids persisted in the field soil following land application; and iii) to assess the potential for microbial activity using the common microbial transformations: nitrification, sulfur oxidation and dehydrogenase production.

Materials and Methods

Study Site

The biosolids throughout this study were anaerobically digested liquid Class B biosolids (approximately 8% solids) from the Ina Road Wastewater Treatment Plant in

Tucson, Arizona. The design of the experiment was a randomized block design, with four replicates of four treatments. The four treatments were: control plots; (no amendment), plots treated with inorganic fertilizer; plots treated with an agronomic (N based) rate of biosolids (1x, 8.0 t ha-1 yr-1); and plots treated with a high biosolids rate (3x, 24.0 t ha-1 yr-1). Land application of biosolids commenced in the spring of 1986 and annual

applications continued for twenty years. Each plot consisted of 6 rows and the

experimental field was 5m wide and 101m long. Each year, cotton (Gossypium hirsutum

L.) was planted in April and harvested in early November. The land applied biosolids

contained 8% solids and were injected into the soil using a semi-trailer applicator (6350

BALZER, WI) (Fig. 2.1).

Figure 2.1 Land application of liquid biosolids on Marana, Arizona agricultural field via biosolids injector . Photo courtesy H.G. Zerzghi.

Indigenous Soil Microbial Populations

The twentieth annual land application of biosolids occurred in March 2005,

followed by growth of cotton from April through November 2005. Soils were

conditioned via disking after the injection of biosolids into the surface (0-30cm) soils.

Surface soil samples (0-30cm) were collected monthly from March 2005, two weeks after

the final biosolids application, through December 2005, and analyzed for soil microbial

properties. Each soil sample was the result of composite sampling in which four sub-

samples were taken on a transect along each plot and thoroughly mixed prior to taking a

sample for microbial analysis. After each sampling, soils were sieved through a 2 mm 42

sieve and analyzed for; moisture content determination (%); culturable heterotrophic

plate counts (HPC) (cfu g-1 dry soil) and total bacterial counts (cells g-1 dry soil). In

December 2005, following the harvest of cotton, additional surface soil samples were

taken and analyzed for indigenous bacteria, fungi, and actinomycetes. In addition,

samples were analyzed for indicator organisms, pathogens, phage, and antibiotic resistant

bacteria.

Heterotrophic plate counts bacteria (HPCs) were determined on soil samples

using 10-fold serial dilutions of 0.85% saline (EMD Chemicals Inc. Gibbstown, NJ)

followed by plating on R2A agar ( Difco Co., Sparks, MD). Appropriate dilutions were

also prepared for total direct count analysis (Hobbie et al., 1977). Total direct count assays were done every two months using acridine orange staining and counting eight fields of view per replicate (Schmidt and Paul 1982).

Cultural heterotrophic counts for fungi were made using a spread plate technique on Rose Bengal-streptomycin agar from 10-fold serial dilutions. Culturable heterotrophic plate counts for actinomycetes were also determined using Glycerol-Casein agar (Pepper

and Gerba, 2004) and plates were incubated at 270C for 7 days.

Microbial Pathogens, Indicators, and Antibiotic Resistant Bacteria

Additional analyses of the last soil sample (December 2005) included: phage

(viruses that infect bacteria); enteroviruses; total coliforms; fecal coliforms; Salmonella;

and antibiotic resistant bacteria. This was important, as one goal of the study was to

determine the potential for survival indicator microorganisms and pathogens after 20 43 years of land application of biosolids. The methods used to detect these organisms are summarized in Table 2.1.

Table 2.1 Microbiological assays used to detect microbial pathogens and antibiotic resistant bacteria.

Organisms Assay or culture media used References Coliphage Bacteriophage assay 1 Pepper and Gerba, 2004. Enteroviruses Cell culture ASTM Method D499-89., 1993. Total coliforms MPN 1 APHA, Standard Method 9221, 1998. Fecal coliforms MPN 1 APHA, Standard Method 9221, 1998. Salmonella HE, XLD media 2 Cooper and Danielson, 1997. Antibiotic Resistant Ampicillin + R2A agar Jorgensen et al., 1999 Bacteria

Cephalothin + R2A agar Jorgensen et al., 1999.

Ciprofloxacin + R2A agar Jorgensen et al., 1999.

Tetracycline + R2A agar Jorgensen et al., 1999. 1 = Assay repeated twice, 2 = Assay repeated three times

The assay for coliphage (MS-2 15597-B1) was performed twice using variable amounts of aliquots from dilutions, using the double overlay procedure derived from

Pepper and Gerba (2004). The host bacterium, E. coli 15597, was grown to mid-log phase and the soft agar was then amended with broth culture of the host and coliphage suspension. The contents of the tube were vortexed and poured onto an agar plate containing an appropriate growth medium. Plates were incubated at 370C and examined for plaque formations (clear zones) after 12 hours.

Assays for entroviruses were performed by The University of Arizona

Department of Soil, Water, and Environmental Science, Environmental Virology Lab 44

(Tucson, AZ) according to the Recovery of Virus from Biosolids or Wastewater Sludge:

ASTM Method D4994-89).

Soil samples were evaluated for total and fecal coliforms using a three-tube ‘most probable number (MPN) method adapted form EPA Standard Method 9221 (American

Public Health Assoc., 1998). Ten grams of soil were added to a 95 ml of 0.85% saline

(EMD Chemicals Inc. Gibbstown, NJ) and placed on a shaker for 10 minutes. Ten fold dilutions were made and1 ml of each dilution was added to each of three tubes containing

10 ml of a lauryl tryptose broth, LTB (Difco, Co., Detroit, MI) and fermentation tubes.

Inoculated tubes were stored for 24 to 48 hours in a circulating water bath at 350C, and

were monitored for gas production. Positive and negative controls were also included in

the inoculation procedure. After 48 hours, the LTB tubes were scored positive or negative

for gas production and 0.1 ml of each positive tube of LTB was added to a tube

containing 10 ml brilliant green bile (BGB) broth and EC broth (Difco, co., Detroit, MI),

and both tubes containing fermentation tubes. The BGB and EC tubes were incubated at

350C and 44.5 0C for 48 hours respectively. BGB is utilized for enumerating total

coliform populations, while the EC medium confirms the presence of fecal coliforms. An

MPN index table was used to calculate numbers of total and fecal coliforms.

Soil samples were analyzed for Salmonella using a presence-absence assay.

Enrichment tubes containing 10 ml of XLD broth (Becton, Sparks, MD) were incubated

in a circulating water bath at 370C for 18-24 hours. A loopful of the enrichment was streaked onto Hekton Enteric (HE) agar (Difco, Co., Detroit, MI) for the selection of

Salmonella. 45

Soil samples were also analyzed for antibiotic resistant bacteria. This was done by

exposing samples to four antibiotics: ampicillin (32 µg ml-1) cephalothin (32 µg ml-1),

ciprofloxacin (4 µg ml-1), and tetracycline (16 µg ml-1) (Sigma Aldrich; St Louis, MO).

Each antibiotic was amended into R2A agar media (Difco). A 10-fold serial dilution was

prepared and aliquots of 0.1 ml sample were spread plate onto the antibiotic amended

agar. The plates were incubated for 5 days at 270C and antibiotic resistance counts were

made to determine the concentrations of antibiotic resistant bacteria (cfu g-1 dry soil).

Microbial Activity

The influence of 20 years of land application on microbial activity was evaluated

through studies involving nitrification, sulfur oxidation, and dehydrogenase production.

The nitrification potential was determined using a method which assessed the maximum

rate of nitrification within soil samples (Hart et al., 1994). The process involved the

oxidation of ammonium to nitrate in a two-step transformation by nitrifying organisms.

Fifteen grams of the sieved field moist soil were incubated under conditions specified in

the “shaken soil-slurry method for assessing the nitrification potential in soil”.

The rate of sulfur oxidation was determined by measuring the amount of sulfate

produced when soil is incubated with S0 under specific conditions. Sieved field moist

soils (240 gms) were amended with 0.5g S0. Sulfate was extracted using NaCl extraction

solution which aids in dispersing soil particles. The rate of sulfur oxidation was

determined by measuring changes in the soil sulfate concentration over a period of three 46

weeks by measuring turbidity on a spectrophotometer at 470nm (modified from Pepper

and Gerba, 2004).

Dehydrogenases are enzymes from soils found in all living organisms (Maier et

al., 2000). Studies on enzyme activities in soils have been used to measure the rates of

enzyme-catalyzed reactions in soil samples. Dehydrogenase activity was determined

using a procedure modified from Pepper and Gerba (2004) involving methanol extraction

and quantification using a spectrophotometer at 485nm. All statistics were run using the

statistical software package (SYSTAT Software, Inc, San Jose, CA). Results of all tests

were considered significant at the 95% confidence level.

Results

Heterotrophic plate count bacteria (HPC) on biosolids treated soils were

frequently higher than the counts on unamended (control) soil samples but overall

differences were not significant at the 95% level (Table 2.2). Direct counts from all soils

were higher than culturable counts (approximately 0.5 to 1.0 logs). Heterotrophic plate counts for actinomycetes were about one log lower than bacteria plate counts but an order

of magnitude higher than fungi plate counts (Table 2.2).

Table 2.2 Mean microbial numbers based on cultural heterotrophic plate counts and acridine orange direct counts. Data represent mean values of 10 months of analyses from March to December of 2005.

Unamended Inorganic Low High Variable (control) Fertilizer Biosolids Biosolids

Bacteria(cultural count ; cfu g-1) 5.4 x 107 5.3 x 107 6.6 x 107 7.4 x 107 Bacteria ( direct count; cell g-1) 1.6 x 108 1.5 x 108 1.8 x 108 2.1 x 108 Actinomycetes ( cfu g-1) 4.1 x 107 7.7 x 107 4.4 x 107 9.0 x 107 Fungi ( cfu g-1) 8.5 x 105 9.9 x 105 1.3 x 106 1.8 x 106

Table 2.3 shows representative values of pathogen and indicator loads present in the class

B biosolids utilized for land application. However soil concentrations of phage, enteroviruses,

and Salmonella in December 2005 show that there were no detectable numbers of these

organisms even though assays were repeated more than once (Table 2.4). There were

however very low counts of total and fecal coliforms present in the biosolid amended

soils. The fact that no pathogens were detected in the land-applied soil at the conclusion

of the study is important because of community concerns with respect to the persistence

of pathogens (Pepper et al., 2008). Antibiotic resistant bacterial (ABR) concentrations in

biosolids treated soils were one log higher than the unamended soils but did not

statistically differ in all these four antibiotics.

Table 2.3 Typical Pathogen and Indicator Loads in Tucson Sewage and Biosolids (Pima County); biosolids utilized for land application.

Bacteria (MPN/KG)1 Sample Date Raw Sludge Mesophilic (Biosolids)

Fecal Streptococci 1988 -1993 9.6 x109 1.2 x109 1994 -1999 1.2 x 1010 1.9 x109 2000 - 2006 3.1 x 1011 2.0 x109

Total Coliforms 1988 -1993 1.2 x 1012 2.7 x 1010 1994 -1999 7.5 x 1011 1.0 x 1010 2000 - 2006 7.5 x1011 1.0 x 1010

Fecal Coliforms 1988 -1993 2.7 x 1011 8.4 x109 1994 -1999 1.6 x 1011 2.9 x109 2000- 2006 1.7 x 1011 1.8 x109

Salmonella 1995 -1999 3.7 x 106 2.6 x 105 2000 - 2006 4.3 x 106 5.6 x 104

Enteroviruses (PFU/ 4g dry wt)2 1989 -1994 427 10 1995 -1999 78 34 2002 - 2005 21 1 1 Geometric means; 2 Arithmetric means Table 2.4 Soil analyses for potential microbial hazards. Inorganic Organisms Control Low Biosolids High Biosolids Fertilizer Phage 1 nd nd nd nd

Enteroviruses nd nd nd nd

Total coliforms 1 nd nd 4 MPN g-1soil 6 MPN g-1soil

Fecal coliforms 1 nd nd 4 MPN g-1soil nd

Salmonella 2 nd nd nd nd

Antibiotic 3.93 x 106 3.98 x 106 1.01 x 107 1.23 x 107 resistant bacteria

nd = Not detected; 1 = Assay repeated 2x; 2 = Assay repeated 3x

The microbial activity assays showed significant increases in activity in the biosolid amended soils. The high biosolids treated plots had higher nitrification activity indicated by the higher nitrate concentration in the soils as a function of time, then followed by the low biosolids treated plots (Fig.2.2). In the sulfur oxidation assays, soils

without sulfur amendment had similar sulfate levels throughout the assay with a range of

9 - 25 µg SO4-S per g dry soil. Activity increased in all soils samples after the addition of

0.5g elemental sulfur. The high biosolids treated soils showed significant elevations of

extracted sulfate concentrations indicating higher sulfur oxidation rates throughout the 72 hours of incubation (Table 2.5). Trends were similar in the dehydrogenase assay with the formation of TPF significantly higher in the high biosolids treated soils than the control, and low biosolids treated soils (Table 2.5). Interestingly, soils from the inorganic fertilizer plots showed the lowest levels of activities for both the sulfur oxidation and dehydrogenase assays.

Unamended Soil Inorganic Fertilizer Soil Low Biosolids 60 High Biosolids Soil

40 (ppm) 3 NO 20

0 20406080 Time (hr) Figure 2.2 NO3 concentrations in soils incubated from 0 to 72 hours. 50

Unamended Inorganic Variable Low Biosolids High Biosolids Fertilizer (control) Sulfur oxidation 260 205 337 430

Dehydrogenase activity 291 148 164 660

Discussion and Summary

Land application of biosolids has been problem considered to be a beneficial

fertilizer and soil conditioner for crop production (Lang and Smith 2007). In addition,

long term additions of biosolids have been shown to improve the physical properties of

amended soils in several studies (Aggelides and Londra, 2000; Tsadilas et al., 2005) but

there are concerns about its long-term sustainability and other environmental effects

(Love et al. 2001). In this study, soils analysed after 20 years of land application of Class

B biosolids (collected ten months after last land application) were not significantly

different than control soils, with respect to soil moisture content and the number of viable

heterotrophic plate counts (HPC) for bacteria, fungi and actinomycetes. The total direct

counts for bacteria were an order of magnitude higher than the HPC counts but again

treatment differences were not significant. The measured microbial counts are similar to

those expected in a normal, fertile soil (Pepper et al., 2006). Earlier studies at this site

(Marana) also reported similar numbers of heterotrophic bacteria, fungi, and 51 actinomycetes in both biosolids treated and control unamended soils (Brendecke et al.

1993).

Understanding the environmental persistence and fate of enteric pathogens introduced into the soil during land application is important to provide a sound scientific basis for sustainable land application of biosolids (Topp et al. 2003). Class B routinely contain bacterial pathogens and therefore site restrictions are imposed to allow for pathogen die off (Gerba and Smith, 2005) .

In this current study, Class B biosolids, which contain bacterial and viral pathogens were land applied and normally some pathogens and microbial indicators survive transiently after land application (Pepper et al., 1993). Ten months after the last biosolid application Salmonella and enteroviruses were not detected in any of the plots

.Total coliforms and fecal coliforms counts were < 6 MPN/g in plots that received Class

B biosolids. Other studies have also reported the presence of coliforms without the presence of Salmonella (Zaleski et al. 2005). Additionally, soil temperature, moisture, organic matter, pH, and antagonistic indigenous biota all have a major role in pathogen reduction processes in biosolid amended soil (Lang and Smith, 2007). A number of studies have reported inactivation of pathogens and indicators following land application

(Lang et al., 2007; Lang and Smith, 2007; Pepper et al., 1993). For example, in a recent study numbers of E. coli declined rapidly and survival was limited to three months (Lang and Smith, 2007). Also the survival of E. coli and other enteric microorganisms in biosolids amended agricultural soil have been evaluated in laboratory and field studies

(Lang and Smith 2007; Lang et al. 2007; Zaleski et al. 2005). The data reported here are 52

in agreement with other studies on the survival of microbial indicators and pathogens

introduced into soil during land application.

Today, a wide range of naturally occurring and synthetic antibiotics are frequently

used for therapy of infectious disease since the antibiotic era began following Alexander

Fleming’s discovery of penicillin nearly 80 years ago. In addition, antibiotics are

frequently utilized in animal feeds. Antibiotics are designed to act very effectively at low

doses and to be excreted from the body after a short residence time (Thiele-Bruhn, 2003).

Overuse of antibiotics has led to many resistant bacterial strains (Monroe and Polk 2000;

Lieberman 2003).

Recent concerns with land application of biosolids have focused on the potential

for enhanced level of antibiotic resistant bacteria. Data from this present study show that biosolid amended soils had one log higher antibiotic resistant bacteria (ARB) concentrations than the unamended soils, but there were no significant differences between treatments with respect to the ARB bacteria concentrations. Therefore, land application of Class B biosolids did not significantly affect soil ARB concentrations. This is likely to be due to inactivation of the introduced ARB following land application. In addition, the loss of plasmids carrying genes that code for resistance may have occurred due to the lack of selective pressure for antibiotic (Smith and Bidochka, 1998). The present data agree with a recent study by Brooks et al. (2007) and (Pepper et al. 2008) on

ARB concentrations in soil following land application.

Gibbs et al. (2006) also showed low metal biosolids generally have a beneficial effect on microbial activity and soil fertility while biosolids with high metal 53

concentration subsequently decrease both. In an earlier study conducted on the same

plots, Brendecke et al. (1993) concluded that because of the relatively low concentration of inhibitory substances in biosoolids, and due to high mineralization activity of carbon substrates, the long- term effect of biosolids on microbial activity is neither inhibitory nor

stimulatory. However, in this present study significant increases in the potential for

microbial activity were detected when activity was assessed using the common microbial

transformations: nitrification and sulfur oxidation. The rates of both microbial

transformations increased in soils from the biosolids amended plots, and increased with

increased rate of biosolids application. In addition to nitrification and sulfur oxidation,

dehydrogenase activity also increased with increased biosolids amendment.

In conclusion, the soil microbial community did not appear to be adversely affect

by 20 years of land application of Class B biosolids. In fact, land application to an arid

land appeared to be beneficial as evidenced by enhanced microbial activity. In addition,

no known pathogens were detected in the soil sampled 10 months after the last biosolids

application. Based on this study, land application with respect to its influence on the soil

microbial community would appear to be beneficial.

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Tables and Figures

Table 2.6 Soil gravimetric moisture contents (%) of the soils from the experimental plots. Data represents mean values of 10 months of analyses from March to December of 2005.

Unamended Inorganic Low High Time (Month) (control) Fertilizer Biosolids Biosolids

March 11 9 9 12 April 14 13 16 17 May 6 6 7 8 June 9 11 11 9 July 12 12 15 15 August 14 15 14 16 September 10 9 9 10 October 7 7 7 8 November 5 6 5 5 December 4 5 4 5

9 9 10 11 Mean

Table 2.7 Cultural Heterotrophic Plate Counts (cfu/ g dry soil). Data represents mean values of 10 months of analyses from March to December of 2005.

Unamended Inorganic Time (Month) Low Biosolids High Biosolids (control) Fertilizer

7 March 6.8 x 10 8.9 x 107 8.3 x 107 6.3 x 107

April 6.9 x 107 5.1 x 107 6.1 x 107 1.2 x 108

May 4.8 x 107 3.1 x 107 6.9 x 107 9.0 x 107

June 5.3 x 107 5.7 x 107 4.3 x 107 6.7 x 107

July 3.4 x 107 3.9 x 107 4.5 x 107 5.1 x 107

August 5.5 x 107 4.2 x 107 5.7 x 107 6.0 x 107

September 4.0 x 107 3.7 x 107 5.0 x 107 3.9 x 107

October 5.3 x 107 7.3 x 107 9.3 x 107 1.2 x 108

November 5.0 x 107 4.8 x 107 7.3 x 107 4.8 x 107

December 6.5 x 107 5.9 x 107 8.5 x 107 8.3 x 107

Mean 5.4 x 107 5.3 x 107 6.6 x 107 7.4 x 107

Table 2.8 Total Direct Counts (number of cells /g dry soil). Data represents mean values of 16 soil samples (4 replicates x 4 treatments) analyzed on December of 2005.

Inorganic Low High Time (Month) Control Fertilizer Biosolids Biosolids March 2.3 x 108 2.2 x 108 2.1 x 108 2.8 x 108

May 1.1 x 108 9.7 x 108 1.3 x 108 1.7 x 108

July 1.5 x 108 1.5 x 108 1.8 x 108 2.1 x 108

September 1.6 x 108 1.6 x 108 1.8 x 108 2.0 x 108

November 1.6 x 108 1.5 x 108 1.8 x 108 2.0 x 108

Mean 1.6 x 108 1.5 x 108 1.8 x 108 2.1 x 108

Table 2.9 Potential biological hazards.

Inorganic Organisms Control Low Biosolids High Biosolids Fertilizer Phage 1 ( per 10 g -1soil) nd nd nd nd Enteroviruses ( per 4 g -1 nd nd nd nd soil ) Total coliforms 1 nd nd 4 MPN g -1soil 6 MPN g -1soil

Fecal coliforms 1 nd nd 4 MPN g -1soil nd

Salmonella 2 nd nd nd nd ( cfuc g -1soil) ARB3-Ampicilin 3.6 x 106 4.1 x 106 7.1 x 106 1.3 x 107

ARB-Cephalothin 4.4 x 106 4.7 x 106 1.1 x 107 2.5 x 107

ARB-Ciprofloxacin 5.5 x 106 4.8 x 106 1.9 x 107 7.8 x 106

ARB-Tetracycline 2.3 x 106 2.4 x 106 3.0 x 106 3.5 x 106

Mean 3.9 x 106 4.0 x 106 1.0 x 107 1.2 x 107

nd = Not detected; 1 = Assay repeated 2x; 2 = Assay repeated 3x; 3 = Antibiotic resistant bacteria (cfu g -1soil) 61

Unamended Inorganic Low High Time (week) (control) Fertilizer Biosolids Biosolids 1 154 88 190 307 2 365 322 483 552 Mean of SO4-S levels 260 205 337 430 0 282 156 170 788 1 300 139 158 532 Mean of TPF levels 291 148 164 660

III. INFLUENCE OF LAND APPLICATION OF CLASS B BIOSOLIDS ON SOIL BACTERIAL DIVERSITY

Abstract

This project evaluated the influence of annual land applications of Class B biosolids on soil bacterial diversity monitored over a 20 year period. The study was initiated in 1986 at the

University of Arizona Marana Agricultural Center, 21 miles north of Tucson, Arizona. The final application of biosolids was in March 2005, followed by growth of cotton from April through

November 2005. Surface soil samples (0-30cm) were collected on December 2005, nine months after the final biosolids application, and analyzed for soil bacterial diversity. Replicated plots included control (unamended) soil and biosolid amended plots. The influence of land application on soil bacterial diversity was evaluated using 16S rRNA clone libraries. In the current study, bacterial diversity was not adversely impacted after 20 years of land application when evaluated through cloning and sequence analysis of bacterial 16S rRNA. In fact, the results showed that the known total number of identifiable species increased in the high rate biosolid plots, when compared to control (no biosolid) plots. The results from the Ribosomal Database Project II (RDP

II) 16S rRNA gene sequences analysis showed that both soils had a broad phylogenetic diversity comprising more than four major phyla including: Proteobacteria, Acidobacteria, Actinobacteria,

Bacteroidetes, and Firmicutes. Soil bacterial community members were typical of those found in arid sandy loam soil and southwest arid soil. Overall, the data show that bacterial diversity was either un impacted or enhanced following 20 years of land application of Class B biosolids. 63

Introduction

In 1986, a study was initiated at the University of Arizona Marana Agricultural

Center, 21 miles north of Tucson to determine the long-term effects of continued annual

land application of biosolids to an irrigated field planted with cotton. The applied

biosolids consisted of anaerobically digested sewage sludge (Class B biosolids) produced

by the City of Tucson. The aim of the investigation was to evaluate the benefits and

hazards of land application of Class B biosolids on agricultural land. Initial investigations

(Brendecke et al., 1993) showed that 4 years of land application of biosolids to an agricultural desert soil in the southwest USA did not adversely affect microbial populations evaluated via culturable numbers or microbial activity.

Monitoring soil quality by means of biological indices can be useful in managing and maintaining the sustainability of soils that receive biosolids application (Fernandes et

al., 2005). Having an agricultural field with a 20 year history of biosolids application

provides a unique opportunity for evaluation of the long-term effects of biosolids on

various biological parameters including the diversity of soil microbial community. Thus,

understanding microbial community structure shifts or any changes following land application of biosolids could help to develop best soil management practices (Sun et al.,

2004). Soil microbial diversity can be affected by soil health and quality, which in turn

can be affected by soil management. On the other hand, sustainable land use depends on

the health of soil microbial diversity because they play a critical role in maintaining soil

processes such as soil structure formation; decomposition of organic matter; toxin

removal; and nutrient recycling of C, N, P, and S (Garbeva et. al., 2004). 64

Assessing total soil microbial diversity is one of the primary challenges in modern microbial ecology (Holben et al., 2004) and little is known (only 0.1 to 1% of total species diversity) about the most complex and diverse microorganisms in the ecosystem, due to limitations of culture- based studies (Garbeva et. al., 2004). Recent progress in molecular microbial ecology using PCR-based techniques for community composition analysis have proven to be powerful and have been widely applied to generate much information on microbial diversity (Holben et al., 2004). In addition, these technologies enabled researchers to detect viable but non culturable (VBNC) bacteria (Brooks et al.,

2007). In this present study, culture-based results showed that there were no culturable bacterial or viral pathogens present in soil samples collected from biosolid amended plots in December (nine months after the last land application) (Zerzghi et al., 2008a).

Therefore, the purpose of this study was to evaluate the effect of long-term land application of Class B biosolids on the soil bacterial diversity of a Pima clay loam soil.

Bacterial diversity was determined through PCR amplification of small subunit 16S rRNA gene sequences that allowed identification of dominant bacterial species or genera.

Materials and Methods

Study Area

Class B biosolids have been used as soil amendments for 20 years at the

University of Arizona Marana Agricultural Center (1986-2005). The biosolids throughout

this study were mesophilic digested liquid Class B biosolids (approximately 8% solids)

from the Ina Road Wastewater Treatment Plant in Tucson, Arizona. The design of the

experiment was a randomized block design, four replicates of four treatments. The four

treatments were: control plots (no amendment), plots treated with inorganic fertilizer,

plots treated with a low application rate of biosolids (1x) 8.0 t ha-1 dry solids, biosolids applied at agronomic rates), and plots treated with a high biosolids rate (3x) 24.0 t ha-1dry solids. Land application of biosolids commenced in the spring of 1986 and annual applications continued for twenty years. Each year, cotton was planted around late April and harvested in early November. The land applied biosolids contained 8% solids and were injected into the soil using a semi-trailer applicator (6350 BALZER, WI) (Fig. 2.1).

Soil surface samples were collected in December 2005 to determine the long-term effects of Class B biosolids land application on soil bacterial diversity. Composite soil samples from each plot (plot size was 5 x 100 (m)) were homogenized in the field and transported to the lab, where soils were sieved through a 2.0 mm sieve.

Soil Bacterial Community DNA Extraction and PCR Amplification of 16S rRNA

Soil community DNA was extracted using MO BIO Ultra Clean Soil DNA

Isolation kit (MoBio Laboratories; Carlsbad, CA). A total of 1.0g soil per replicate or 66

4.0g soils (4 replicates per treatment extracted separately and combined) per treatment was used for community DNA extraction. Extracted DNA from each replicate was then composited into a single bulk sample of 200µl DNA solution. The composite extracted

DNA was repurified and concentrated to a 50 µl DNA solution using a Qiagen PCR purification kit (Qiagen Inc.; Valencia, CA). Analysis focused on soils from the control plots (no biosolids amendment) and high biosolids rate amended plots (3x).

Pre-sequencing PCR amplification of the 16S rRNA with the community DNA was performed in an Applied-Biosystems GeneAmp PCR system 2700 (Applied

Biosystems ; Foster City, CA) under the following conditions : 1X PCR Buffer , 2.5 mM

MgCl2, 0.2 mM dNTP (equimolar each of dNTPs), 1.2 U of Taq Gold DNA Polymerase

(Applied-Biosystems; Foster,City,CA),primers 341F (5’- CTCCTACGGAGGCAGCAG-

3’) and 1492R (5’-GGTTACCTTGTTACGACTT-3’) (0.4 µM each), dH2O (Integrated

DNA Technologies; Coralville, IA ), and 0.25 ng/µl template DNA concentration per

25µl volume. PCR profile: 10 min at 950C (initial denaturation), then cycled 35 times at:

950C (denaturation) 40 s, 510C (annealing) 45s, 720C (extension) 1min 10s, followed by a final extension at 720C for 5minutes and hold at 40C. The correct PCR products

(approximately 1151bp) were purified with a Qiagen PCR purification kit and eluted with

50µl elution buffer. The entire volume was then electrophoresed through a 1.5% (w/v) agarose and 1X Tris Borate gel at 120 V for 60 minutes. The gels were stained with ethidium bromide and visualized under an ultra violet (UV) light box operated at approximately 365nm. The PCR products were excised from the gel then purified using a

Qiagen Gel Purification kit according to the manufacture’s protocol. 67

Cloning, Colony PCR and Sequencing of 16S rRNA

The purified PCR was then ligated into a transport plasmid vector (TOPO TA

Cloning® kit, containing pCR®2.1-TOPO). Direct cloning of PCR product using these

commercially available kits is often difficult because proofreading polymerase removes the 3’ Adenine (A)-overhang necessary for TA cloning. Therefore, the purified PCR product was incubated with additional A (give DNTP concentration) for 60 min at 720C

for the addition of 3’A-overhang. After the addition of 3’-overhangs, 4 µl of the post

amplification product was used for ligation for 30 min at room temperature (22-230C) and then tubes were placed on ice(TOPO TA Cloning® vectors , Carlsbad ,CA). Once

ligated, the vectors were then transformed into Invirtrogen TM TOPO10 One Shot TM

Escherichia coli, chemically competent cells (each 50µl) following the manufacturer’s recommended manual. Protocols for the positive (TOPO TA® vector + 750bp DNA

template) and negative (TOPO TA® vector only) controls were performed according to the manufacturer’s protocol (Invitrogen; Carlsbad, CA).

A total of 760 white colonies (vector + PCR insert); more than 350 colonies per

treatment were selected for sequence analysis. Individual colonies were isolated with a

sterile pipet tips and placed into 100µl sterile PCR water and kept frozen at -80 0F ; a

colony PCR was performed to evaluate the cloning efficiency and confirm the insertion

of the 16S rRNA PCR product into the vector. PCR amplifications were performed using

a 1µl template from the colony solution and M13F (5’-CTGGCCGTTTTAC-3’) (and

M13R (5’- GTCATAGCTGTTTCCTG-3’) primers (Operon; Huntsville, AL). A 1400 bp

PCR product was visualized by agarose gel electrophoresis on an ethidium bromide 68

stained 1.2 % (w/v) gel. A total of 747 transformants with the correct insert were sent to

the University of Arizona sequencing lab (Biotechnology Computing Facility; Tucson,

AZ). The 16S rRNA products were purified and quantified prior sequencing using 341F primer and A3730XL DNA analyzer (Applied Biosystems; Foster, CA).

16S rRNA Sequence Analysis

Sequences were trimmed at fixed intervals (< 30 bp and > 750 bp using called

FAKtory (Biotechnology Computing Facility, University of Arizona) (Miller and Myers,

1999) and only sequences with at least 600 bp of readable sequence were used for further

analysis. Of these sequences, 711 16S rRNA gene sequences were initially analyzed by

using BLASTn (National Center for Biotechnology Information) which searches

sequence similarity against an updated GenBank database found at the University of

Arizona Center for Computing and Information Technology’s High Performance

Computer.

The isolates were identified by 16S rRNA sequence analysis and compared to

known sequences in GenBank. BLASTn results were then viewed and organized using

Sequence Comparison Output Organizing Tool (SCOOTaR) (Arizona Research

Biotechnology Computing Facility, University of Arizona). Sequences which showed at

least 96% homology were used for identification at the genus and species level.

Phylogenetic dendrograms were prepared using MEGA 3.1 integrated software for

molecular evolutionary genetics analysis and sequence alignment. The sequences were 69

aligned by using clustal x 1.83 with default settings (Kimura 2-parameter model for

substitutions) (Kumar et al., 2004).

Further analysis on all the sequences screened suspected chimeras or artifacts

using a new computer program, called Mallard (Ashelford et al., 2006). In addition, 16S rRNA gene sequences were also analyzed using Ribosomal Database Project II (RDP); including a program called “Classifier” and used to assign 16S rRNA gene sequences to the new phylogenetically consistent higher-order bacterial taxonomy which allows classification of both bacterial and archaeal 16S rRNA sequences (Wang et al., 2007).

The RDP naïve Bayesian classifier was designed to provide rapid classification of library sequences into the new phylogenetically consistent higher-order bacterial taxonomy and estimates the probability of observed difference in a given taxon using a statistical test.

Both 16S rRNA sequences libraries were also compared using the RDP Library Compare

Tool designed to compare microbial communities and results were considered significant

at the 95% confidence level (Wang et al., 2007)..

Results and Discussion

rRNA Clone Sequencing and Phylogenetic Analyses

In the current study, biosolid-treated soil was previously shown to have

increased microbial activity, microbial numbers, and the concentration of some chemicals

(Zerzghi et al., 2008a, Zerzghi et al., 2008b). Often, such changes are observed to cause

both changes in qualitative and quantitative composition of the soil microbial

communities (Wintzingerode et al., 1997). Out of the 711 16S rRNA sequences only 123 70 clones (17%) displayed greater than 96 % identity to the known GenBank sequences

(Table 3.1 and 3.2). Unique clone sequences from both soils are presented in phylogram

(Fig. 3.1a and 3.2). This may indicate that a considerable proportion (83%) of uncultured organisms present on soils represent unknown organisms (Table 3.1). Similarly, Cotta et al. (2003) found that approximately half of the sequences identified exhibited < 95% identity with sequences present in GenBank. Based on first (determined by using NCBI

Blastn) 16S rRNA sequence analysis of the two soil samples (control and biosolid-treated soils), the overall pattern of bacterial species composition appears to be similar.

However, the high biosolids amended soils had greater number of known genera. In addition the biosolid amended soil two which are known to be of biosolids origin:

Shigella sonnei and Escherichia coli. These are human pathogenic bacteria, however, we do not know whether or not these organisms were viable.

Table 3.1. Comparison of identified bacterial species composition in the control soils (unamended) and the high biosolid-treated soils.

Control soils (untreated soil) High biosolids rate amended soils Acidovorax avenae (2) Acidobacteria Agrobacterium tumefaciens (2) Agrobacterium tumefaciens Arthrobacter aurescens (3) Arthrobacter aurescens (4) Arthrobacter sp. (25) Arthrobacter sp.(21) Azoarcus sp. (2) Azoarcus sp. Bacillus licheniformis (10) Bacillus cereus Bacillus thuringiensis (4) Bacillus licheniformis (8) Bordetella parapertussis Bacillus subtilis Erythrobacter litoralis Bacillus thuringiensis (3) Leifsonia xyli Bradyrhizobium japonicum Myxococcus xanthus (4) Caulobacter crescentus (2) Polaromonas naphthalenivorans (2) Erythrobacter litoralis Sinorhizobium meliloti Escherichia coli Streptomyces avermitilis (2) Leifsonia xyli (3) Mesorhizobium sp. (2) Myxococcus xanthus Nitrosospira multiformis Nocardioides sp. Pseudomonas putida Shigella sonnei Sinorhizobium meliloti (3) Streptomyces avermitilis (2) Streptomyces coelicolor Xanthomonas campestris

Myxococcus xanthus (4)

Arthrobacter aurescens (3)

Arthrobacter sp. (25)

Leifsonia xyli

Streptomyces avermitilis (20)

Bacillus licheniformis (10)

Bacillus thuringiensis (4)

Erythrobacter litoralis

Agrobacterium tumefaciens (2)

Sinorhizobium meliloti

Bordetella parapertussis

Azoarcus sp. (2)

Acidovorax avenae (2)

Polaromonas naphthalenivorans

0.02

Agrobacterium tumefaciens Caulobacter crescentus (2) Bradyrhizobium japonicum Sinorhizobium meliloti (3) Mesorhizobium sp. (2) Erythrobacter litoralis Xanthomonas campestris Azoarcus sp. Nitrosospira multiformis Pseudomonas putida Escherichia coli Shigella sonnei Acidobacteria (2) Myxococcus xanthus Bacillus licheniformis (8) Bacillus subtilis Bacillus cereus Bacillus thuringiensis (3) Streptomyces avermitilis (2) Streptomyces coelicolor Nocardioides sp. Leifsonia xyli (3) Arthrobacter aurescens (4) Arthrobacter sp. (21)

0.02

Sequences were also screened due to recent report regarding numerous corrupt

16S rRNA gene sequences that can have substantial anomalies as a result of chimeras

(Ashelford et al., 2006; 2005; Cole et al., 2003; Head et al., 1998). Chimeras are PCR products produced when two or more phylogenetically different sequences become fused during PCR amplification process (Head et al., 1998). Mallard program was designed to minimize further database contamination with chimeras and anomalies. In the present study, 24% (90/369) of the submitted 16S rRNA gene sequences were seen as suspected chimeras from the unamended soils and 25 % of the 340 sequences from the biosolid amended soil. Based on this all suspected chimeric sequences were excluded from the final analysis. Earlier studies have shown that 30% of PCR products generated during amplification were chimeric (Wang and Wang, 1996). In another study (Asheford et al.,

2005) one library alone was found to contain up to 45.8% chimeras. Mallard program helps to indicate the presence of chimeric sequence but it was reported that it detect chimeras correctly with an average of 73.1% accuracy and there are 0.5 to 5.6% of library falsely identified as chimeric (Asheford et al., 2006).

The results from the Ribosomal Database Project II (RDP II) 16S rRNA gene sequences analysis are summarized in Table 3.2 with most sequences identified to known taxonomical levels. The results constituted soil organisms similar to bacterial community members similar to those found in another arid sandy loam soil (Brooks et al., 2007). All sequences were of bacterial origin and approximately 83% of these sequences obtained from both soil samples were classified to previously recognized bacterial phyla or divisions at > 95 % percent identity. Other studies have reported that 32% of sequences 75 obtained from Northern Arizona soils were grouped with previously recognized bacterial divisions (Kuske et al., 1997).

Before Mallard screening After Mallard screening

Bacterial group Control Soil Biosolid- Control Soil Biosolid-

( untreated) treated Soil ( untreated) treated Soil % % % distribution % distribution distribution distribution P < 0.05 Phylum (class) (279 clones) (253 clones) (369 clones) (340 clones) Acidobacteria 17.7 18.5 20.7 20.9 0.78 Actinobacteria 14.9 16.5 15.7 17.7 0.33 Bacteroidetes 5.2 7.1 6.1 9.1 0.17 Chloroflexi 1.1 0.3 1.4 0.4 0.27 Cyanobacteria 1.1 0 1.4 0 0.08 Firmicutes 6.3 7.4 7.1 7.5 0.44 Gemmatimonadetes 0.8 0.6 1.1 0.4 0.05 Nitrospira 0 1.2 0 1.6 0.78 Planctomycetes 0.8 0.6 1.1 0.8 0.46 Proteobacteria 31.8 29.1 29.3 31.5 na Proteobacteria (Alpha) 10.3 42.4 46.3 45 na Proteobacteria (Beta) 43.6 22.2 30.5 20 na Proteobacteria (Delta) 26.5 11.1 6.1 13.8 na Proteobacteria na (Gamma) 6.8 14.1 9.8 12.5 Proteobacteria na (Unclassified) 12.8 10.1 7.3 8.8 Verrucomicrobia 2.7 0.3 3.6 0.4 0.01* OP10 0.3 0 0.4 0 0.55 TM7 0.8 0 1.1 0 0.15 Unclassified Bacteria 16.6 18.5 11.1 9.8 na

na = not determined, * = significant value

There is considerable overlap between the two bacterial phyla from control soil

with no biosolids and the high rate biosolid amended soils. Statistical comparison of both

16S rRNA sequences libraries showed no significant differences between the two

bacterial communities composition, except for the phylum Verrucomicrobia which were

significantly higher in the unamended soils. Recently, Buckely et al. (2001) showed that

Verrucomicrobia composed 1.9 to 2.1 % of the microbial community rRNA present in

the soil samples. In addition, in this study, verrucomicrobial rRNA abundance was

significantly affected by environmental conditions that changed in relation to time, soil

history and soil depth, specifically numbers tended to be lower in fields with a history of cultivation. However, it is difficult to conclude why there were significantly higher numbers in the unameneded soil compared to the biosolid amended soil since assessing their variation of abundance requires in depth data on the biotic and abiotic characteristics of the soil (Buckley et al., 2001). The phyla Chloroflexi were reported to

be found exclusively in biosolid-aerosols (Brooks et al., 2007) and Baertsch et al. (2007)

found the phyla Chloroflexi to be the most abundant in bulk biosolids. However, in this

study members of phyla Chloroflexi found in biosolid amended soil were less than the

unamedned soil (Table 3.2). Similarly, Chloroflexi sp. have not been observed in all

microbial ecology studies related to anaerobic digester and biosolids (Moletta et al.,

2007, Baertsch et al. , 2007)

Overall, the analyses showed a broad phylogenetic diversity that comprised more

than five major phyla including:Proteobacteria, Acidobacteria, Actinobacteria,

Bacteroidetes, and Firmicutes.Sequences belonging to the bacterial phylum

Proteobacteria were the dominant group, which is consistent with other several studies

related to agroecosystems (Brooks et al., 2007, Kuske et al., 1997, Sun et al., 2004).

Further analysis to the Class and Genus level was possible for many of the phyla

especially for the phylum Proteobacteria (Table 3.3). Other studies have reported that

Proteobacteria comprised 35 to 37% of the bacterial community in agroecosystems (Sun

et al., 2004, Smit et al., 2001). In contrast, there have been other studies that reported a

lower percentage of Proteobacteria (16-18%). These differences may be due to the fact

that these agroecosystems were different (Sun et al., 2004). Results from the current

study suggest that Proteobacteria bacterial clones from both soils samples were closely

related to each other indicating that long-term biosolids land application did not affect

bacterial diversity.

Acidobacteria were the second most dominant group (Table 3.2). In the survey

study by Barns et al. (1999), where 43 environmental samples were surveyed by PCR amplification, Acidobacteria were reported to be as genetically and metabolically diverse, environmentally widespread, and ecologically as important as the well-known

Proteobacteria and gram-positive bacterial. Overall, the current study demonstrated that

members of the major phyla and genera of proteobacteria are widespread in the both soil

samples. Although a number of potential sources of bias may be present in 16S rRNA

gene sequences-based analysis of soil microbial communities (Kuske et al., 1997, Sun et 78

al. 2004), our results still suggest that comparisons of sequences at the genus level is

possible, sequence analysis of these sequences from the two soils suggested that bacterial

diversity in agricultural soils was not affected by long-term land application of Class B

biosolids.

Conclusion

In conclusion, results from this study suggest that bacterial community diversity

was not significantly affected by land application of Class B biosolids. Further, high rate

biosolids amended soil had a greater number of known bacterial at the genera level. Two

pathogenic genera from the biosolid amended soil were suspected to have biosolids origin

(Shigella sonnei and Escherichia coli) but it is not known whether these were actually

viable within the soil. The percentage of unclassified bacteria from each clone library was

reduced after Mallard screening and shows that Mallard was effective to detect PCR-

generated chimeras. Members of the major phyla including: Proteobacteria,

Acidobacteria, Actinobacteria, Bacteroidetes, and Firmicutes were widespread in both soil samples. Sequences belonging to the bacterial phylum Proteobacteria were the dominant group followed by Acidobacteria, which is consistent with other several studies related to agroecosystems. Overall, soil bacterial community members were typical of those found in arid sandy loam soils of the southwest.

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Tables and Figures Table 3.3. Summary of phylogenetic assignments of 16S rRNA gene clones from (a) unamended soil and (b) biosolid-treated soil, Marana, AZ. a. Control soil (unamended) Bacterial group % Number % Class Genus (at > 95% identity level) Confidence of Clones Distribution Alphaproteobacteria Phenylobacterium 98-100 2 1.7% Rhizobium 100 1 0.9% Ensifer 100 1 0.9% Methylobacterium 100 1 0.9% Unclassified Methylobacteriaceae 98 1 0.9% Devosia 98-100 3 2.6% Unclassified_Hyphomicrobiaceae 98 1 0.9% nd 95-100 16 13.7% Unclassified Sphingomonadaceae 99-100 5 4.3% Unclassified Rhodospirillaceae 100 5 4.3% nd 100 3 2.6% nd 96-100 12 10.3% Betaproteobacteria Unclassified Methylophilaceae 98 1 0.9% Azoarcus 100 2 1.7% Unclassified Oxalobacteraceae 100 1 0.9% Unclassified Alcaligenaceae 100 1 0.9% Ramlibacter 97 1 0.9% Unclassified Comamonadaceae 97-100 3 2.6% Azohydromonas 100 2 1.7% Unclassified Incertae sedis 5 100 1 0.9% nd 100 6 5.1% nd 98-100 13 11.1% Deltaproteobacteria Bdellovibrio 100 1 0.9% Geobacter 100 1 0.9% Unclassified Geobacteraceae 95 1 0.9% Unclassified Nannocystineae 95 1 0.9% Unclassified Polyangiaceae 100 2 1.7% Cystobacter 96 1 0.9% nd 100 4 3.4% nd 100 1 0.9% Gammaproteobacteria Pseudomonas 100 1 0.9% Lysobacter 98 1 0.9% nd 97-100 6 5.1% Proteobacteria (Unclassified) nd 95-100 15 12.8% 117 100.0% nd = Taxa uncertain at this level.

b. Biosolid amended soil % Number Bacterial group Confidenc of % Class Genus (at > 95% identity level) e Clones Distribution Alphaproteobacteria Unclassified Rhodobacteraceae 100 1 1.0% Phenylobacterium 100 2 2.0% Unclassified Caulobacteraceae 100 1 1.0% Sphingobium 100 1 1.0% Sphingomonas 100 2 2.0% Sphingosinicella 96-100 2 2.0% Unclassified Sphingomonadaceae 96-100 5 5.1% Bradyrhizobium 100 1 1.0% Microvirga 99 1 1.0% Devosia 100 1 1.0% Ensifer 100 3 3.0% Rhizobium 100 1 1.0% Unclassified Phyllobacteriaceae 100 1 1.0% nd 98-100 5 5.1% Unclassified_Rhodospirillaceae 95-100 8 8.1% nd 99 1 1.0% nd 95-100 6 6.1% Betaproteobacteria Nitrosospira 97 2 2.0% Unclassified Oxalobacteraceae 100 1 1.0% Unclassified Incertae sedis 5 98-100 3 3.0% nd 99-100 4 4.0% Azoarcus 100 1 1.0% nd 97-100 11 11.1% Deltaproteobacteria Bdellovibrio 95 1 1.0% Unclassified Polyangiaceae 98-100 3 3.0% Unclassified Cystobacterineae 100 1 1.0% nd 99-100 6 6.1% Gammaproteobacter ia Pseudomonas 99 1 1.0% Lysobacter 99-100 2 2.0% Unclassified_Xanthomonadaceae 99-100 3 3.0% Unclassified Enterobacteriaceae 100 2 2.0% nd 96-100 6 6.1% Proteobacteria (Unclassified) nd 97-100 10 10.1% Total 99 100.0%

nd = Taxa uncertain at this level.

IV. LONG-TERM EFFECTS OF LAND-APPLICATION OF CLASS B BIOSOLIDS ON SOIL CHEMICAL PROPERTIES

Abstract

This study evaluated the influence of 20 annual land applications of Class B biosolids on the soil chemical properties. Specifically, the potential benefits and hazards of land application were evaluated by analysis of depth (0-150cm) soil samples collected 9 months after the 20th land application of biosolids. The study was initiated in 1986 at the University of Arizona Marana

Agricultural Center, 21 miles north of Tucson, Arizona. The final application of biosolids was in

March 2005, followed by growth of cotton from April through November each year. Land application of biosolids significantly increased soil pH but did not affect soil salinity (measured by electrical conductivity, EC) and CaCO3 values as compared with the control (no amendment) plots. However, this lack of salinity was likely due to the leaching of soluble salts through the soil profile since irrigation rates (100 cm per growing season) were almost twice the evapotranspiration requirements for cotton. Long-term land-application significantly increased soil macro-nutrients including C N and P. Of all the macronutrients, caution with respect to phosphate loadings in some regions needs to be observed to prevent nutrient contamination of surface waters. The biosolid-amended soil concentrations of available and total metals were low

(compared to the typical background soil metal concentrations) and not hazardous. Metal concentrations attenuated rapidly with increasing soil depth, and were generally similar to values found in control soils at a depth of 150cm. Increases in available metal concentrations were modest. It should be also noted, that this study was conducted in the arid southwest and as such results were site specific. It is important to note that there are differences between these studies with respect to different cropping systems, biosolids type, climate and soil type, as well as irrigation rates in the arid southwest. 86

Introduction

In 1986, a study was initiated at the University of Arizona Marana Agricultural

Center, 21 miles north of Tucson to determine the effects of long-term annual applications of biosolids to an irrigated agricultural field annually planted with cotton.

The term biosolids implies treatment to produce Class A or Class B biosolids that meet the land-application standards in the Part 503 Environmental Protection Agency regulations (EPA, 1994). Class B biosolids are normally produced as a “cake” (≃ 20% solids) or as liquid biosolids (≃ 5-8% solids). Currently about 60% of all biosolids are land applied in the United States with most of the land receiving Class B biosolids (NRC,

2002). The applied biosolids consisted of anaerobically digested Class B biosolids (≃ 5-

8% solids) that resulted from wastewater treatment of sewage from the city of Tucson.

The goal of this study was to evaluate the influence of 20 annual land applications of

Class B biosolids on the soil chemical properties.

Earlier in this same study, Brendecke et al. (1993) also evaluated the effect of land application of biosolids on various chemical soil properties at the Marana study site.

That study showed that 4 years of land application of biosolids to an agricultural desert

soil in the southwest USA did not adversely affect soil chemical properties. The current

study evaluates the influence of 20 years of continuous land applications of Class B

biosolids on soil chemical properties. The soil at this site is classified as a Pima clay

loam. 87

Monitoring soil quality by measuring soil chemical properties can be useful in

managing and maintaining the sustainability of soils that receive biosolids application

(Fernandes et. al., 2005). Thus, having an agricultural field with a 20 year history of

biosolids application provided a unique opportunity for evaluation of the long-term

effects of biosolids on various chemical parameters and metal levels in soils. To date, a great deal of research has focused on anthropogenic activities, including the land application of biosolids and contaminant additions of metals to soil (Basta et al., 2005).

Materials and Methods

Municipal Class B biosolids have been used as a soil amendment for 20 years at

the University of Arizona Marana Agricultural Center (1986-2005). This replicated field

plot study had 4 treatments: i) control (no amendment); ii) inorganic fertilizer control; iii)

biosolids at an agronomic rate (1x) based on the nitrogen requirements for the growth of

cotton (160 kg N/ha), and iv) biosolids at a high rate (3x). Representative data on the C

and N contents of the biosolids applied to the field are presented in Table 4. 6. The land

applied biosolids contained 8% solids and were injected or applied by spray application

into the soil. All plots were utilized for the growth of cotton and furrow irrigated as

necessary for optimum plant growth. Water supplied as irrigation was approximately 100

cm per growing season, since the consumptive water use requirements for cotton are 92

cm per season.

Five sets of soil core samples were collected from 0 -150 (cm) depths in 30cm

intervals. Soil samples were taken from each of the treatment plots across a transect line 88 to determine the soil chemical properties as a function of depth. The samples were taken in December 2005, nine months after the 20th land application in March 2005. Soils were sieved through a 2 mm sieve, air dried and sent for analysis to the University of Arizona,

Water Quality Center Lab (WQCL). These soil core subsurface samples allowed for an evaluation not only of the influence of long - term land-application of biosolids on soil chemical properties, but also the influence of land application on various parameters as a function of soil depth.

Analysed soil chemicals clustered into two main groups: soil macro elements (N,

P, and C) and heavy metals. Specifically, soil samples were analyzed for : soil pH ; electrical conductivity (EC) ; CaCO3; NO3; total organic carbon (TOC) ; total nitrogen

(TN) ; total phosphorus ; available phosphorus; total metals; and diethylene triamine penta-acetic acid (DTPA) metal concentrations (Table 1). Analysis of variance was performed using the statistical software package (SYSTAT Software, Inc, San Jose, CA).

Results of all tests were considered significant at the 95% confidence level.

Variable Method used Instrument used References

1:1 soil to deionized Accument pH 50, Fisher pH Page et al., 1982 water ratio Scientific, Pittsburgh, PA

Markson model 15/16, 1:1 soil to deionized EC Amber Science, Sandiego, Page et al., 1982 water ratio CA

CaCO H SO4 digestion and - 3 2 Black, 1965 back titration Dionex ICS-100 Ion NO Ion Chromatography U.S.EPA. 1984 3 Chromatography,

Sunnyvale, CA Concentrations NA 1500, Nitrogen, TOC and TN determined using CNS Carbon/Sulfur analyzer, Artiola, 1990 analyzer Carlo Erba Instruments , Milan, Italy Ascorbic acid HITACH U-2000 Available P colorimetric method on Page et al., 1982 Spectrophotometer, San soil extracts Jose, CA

Dry soil samples digested Digester CEM and analyzed with Corporation MDS-2100, Total P and inductively coupled Snoqualmie, WA U.S. EPA 1986 total metals plasma-mass ICP-MS Elan 6100/ ICP-

spectrometry and optical OES optima 5300 DV, emission spectrometry PERKELMER Instruments, Shelton, CT Inductively coupled ICP-OES optima 5300 DTPA metals plasma-optical emission DV, PERKELMER Page et al., 1982 spectrometry Instruments, Shelton, CT

Results and Discussion

Influence of Land Application of Biosolids on Macro Elements

All biosolid treated plots had significantly higher soil pH values but land

application of biosolids did not significantly affect salinity (measured by electrical

conductivity, EC) or CaCO3 values as compared to the control (no amendment) plots (see

Table 1). Other researchers have also reported that the long-term application of biosolids did not significantly affect soil pH (Cogger et al., 2001; Schroder et al., 2008). However, previous studies at this same Marana site reported that there were significantly elevated

EC levels in the high biosolid treated soils immediately after biosolids application

(Brendecke et al., 1993). In this present study, all plots were utilized for the growth of cotton and furrow irrigated as necessary for optimum plant growth. Water supplied as irrigation was approximately 100 cm per growing season, since the consumptive water use requirements for cotton are 92 cm per season. Therefore the lack of salinity 9 months after the final biosolids application was likely due to the leaching of soluble salts through the soil profile since irrigation rates were almost twice the evapotranspiration requirements for cotton. A recent study by Schroder et al., (2008) showed that increased levels of EC in soils were due to high application rates of biosolids as compared to untreated controls. It is important to note that there are differences between these studies with respect to different cropping systems, biosolids type, climate and soil type, as well as irrigation rates in the arid southwest.

Long-term land-application significantly increased soil macro-nutrients including soil nitrate (see Table 2), total nitrogen (N) (Fig.4.1), total carbon (C) (Fig.4.1), and both 91

available and total phosphorous (P) concentrations (Fig.4.3a and b). Soil nitrate

concentrations in both biosolid amended soils and soils that received inorganic fertilizers

for 20 years were significantly higher than control (no amendment) plots when averaged

over all soil depths (0-150 cm), and total nitrogen increased significantly in biosolid-

amended soils. Plots which received the high biosolids rate had the highest concentration

of soil nitrate (Table 4.5). Nitrate values in both biosolid and fertilizer treated plots

exceeded 10 ppm NO3-N at most soil depths down to 150 cm. These data indicate the

potential for nitrate pollution of groundwater regardless of whether biosolids or inorganic

fertilizer is applied to the soil. Other studies have shown that N mineralization was

affected by soil type and temperature (Wang et al., 2003) and further research on these

fields may help to determine N loading rates in order to minimize potential leaching loss

of biosolid-derived N.

Previous studies from Marana experimental field indicated that, after five annual

applications of biosolids and inorganic fertilizer, plots showed no significant differences of total N concentrations (Artiola and Pepper, 1992). In the current study, the data

showed that in surface soil (0-60 cm), the high biosolids plots had significantly higher

levels of total N than the other treatments. However, analyses on the data from the low biosolids, inorganic fertilizer, and control plots showed no treatment differences in terms of total N content (Fig.4.1 and Table 4.5). Our results are similar to other studies of long- term land applications of biosolids which showed that high application rates of biosolids increased total N concentration (Schroder et al., 2008).

Total Nitrogen Total Carbon 0.7 0.12

b 0.6 c 0.10 a a ab a 0.5 0.08 a 0.4 b

0.06 0.3

0.04 0.2

0.02 (%) concentration C Total 0.1

Total N concentration (%) N concentration Total 0.00 0.0 Control Fertilizer Low BS High BS Control Fertilizer Low BS High BS Treatment Treatment

Total organic carbon significantly increased in biosolid amended (3x rate) soil after 20 years of land application (U of A study) (Fig.4.1). This is in contrast to analyses of the same plots following 5 land applications. Data from core soils samples collected in

1990 showed no differences in the soil total organic carbon when compared control versus biosolid amended soils (Artiola and Pepper, 1992). However, dissolved organic carbon increases were detected in the 1990 biosolid-amended soil samples. These data illustrate how difficult it is to increase soil organic matter in soils of the arid southwest

USA, due to high mineralization rates (Artiola and Pepper, 1992). But it is important to note that even the modest increases in total organic carbon found here are important to soil fertility in soils which are traditionally low in soil organic matter (0.5-1%) (Fuller, 93

1991). Land application of biosolids has also been shown to increase soil organic carbon

in other studies (Gibbs et al., 2006b; Mantovi et al., 2005).

In our study , data showed that plots which had received high rates of biosolids

had a mean total organic C level of 1.3% in the surface soils (0-30cm) which was slightly

higher than the total organic C levels in desert soils reported by Artiola and Pepper, 1992

(Table 4.5). Note that control values of total carbon in the surface (0-30cm) soil was

0.8%. However, the effect was of land applications on total organic C diminished at the lower (120-150 cm) depths of the soil profile. This is consistent with other reports by

Peterjohn and Schlesinger (1990), who found the mean organic levels were 0.72 % at the surface (0-10cm) and 0.42 % at a depth of 20-30cm 0.12 % at depths of 120-150 cm. In addition, our data also showed that that control plots had significantly higher levels of total organic C than plots which received inorganic fertilizer.

Land application (3x rate) of biosolids also significantly increased total and available soil phosphate concentrations, particularly in the surface (0-30 cm) soil (Fig.

4.2; Table 4.5). Increases in soil phosphate concentrations of biosolid amended soil were already evident in soil samples collected from the same study after 4 years of land application (Brendecke et al., 1993). These data are not unexpected since several other studies have documented phosphate increases following land application (Mantovi et al.,

2005). In addition, Elliott and O’Connor (2007) recently reported that long-term land application of biosolids resulted in accumulation of P in surface (0-15cm) soils and that P concentrations were greater than that needed for optimum crop yields. They also stated that P was at the forefront of biosolids-related issues that may adversely affect the long - 94 term sustainability of land-based recycling programs in the U.S. Such concern is based on the potential for water quality deterioration that can occur in surface waters due to eutrophication following phosphate accumulations in surface water runoff.

Fig. 4.2. The influence of biosolids on soil P. Values are the mean of all soil analysis from 0-150cm and values with different letters are significant at the 95 % confidence level.

Total Phosphorus Available Phosphorus 1200 40

1000 b c a 30 800 a a

600 20

400 b 10 200 a a

Available P concentration(%) Total P concentration (mg/Kg) 0 0 Control Fertilizer Low BS High BS Control Fertilizer Low BS High BS Treatment Treatment

Biosolids are good sources of P for crop productions in soils (Peterson et. al.,

1994) but are typically applied at rates designed to satisfy crop N requirements mainly to avoid nitrate leaching to groundwater. Several studies have reported that there is a mismatch between the biosolids N nutrient content and crop requirements of P, such that

P is supplied in excess of the optimum levels needed for crop growth and because application of biosolids is based on the N requirement of crops(Peterson et. al., 1994,

Schroder et al., 2008). While this surplus of soil P from biosolids is not harmful to crops 95

(Brandt, et. al., 2004), there is a potential for off-site migration that can cause

eutrophication in most P limited surface waters (Carpenter et. al., 1998). However, it appears that these issues are more important in the eastern USA, since in the arid southwest, surface waters are rare. That notwithstanding, phosphorus management will continue to be important for sustainable biosolids recycling in the U.S.

Effect of Land Application of Biosolids on Soil Heavy Metals

The biosolids applied in the University of Arizona 20 year land-application study

contained relatively low levels of heavy metals (Table 4.7). After 20 annual land

applications, soil data showed that significantly higher concentrations of available

diethylene triamine penta-acetic acid (DTPA) metals including: Cu, Zn and Ni were

detected within the biosolids treated plots (Fig. 4.3a, b, c and d; Table 4.7). On the other

hand, total Hg, Se, Cr and B values were significantly higher in land applied plots (Fig.

4.3e,f ; Table 4.6) and values decreased with increasing soil depth over all treatments. In

addition, the total and available metals levels were well correlated with biosolids

application rates.

Overall, data showed that low biosolids land application rates (1x) did not

significantly increase metal concentrations with the exception of a few metals (DTPA Zn,

Cu, and total Se). The biosolid-amended soil concentrations of other available and total

metals (Cu, Cd, Pb, Ni, As and Mo) were not significantly different between plots that

received biosolids and control plots (Table 4.8). Application of biosolids did not affect

significantly Cd soil concentrations but values were higher in the biosolid treated plots as 96

compared to plots that did not receive biosolids (control and inorganic fertilizer (Fig.

4.3b).

Table 4.2. Representative heavy metals (Inorganic pollutants) in biosolids.

Metal Mean 1 Minimum 1 Maximum1 Range 2 (mg/kg) Arsenic 12.4 0.3 316 3 – 23 Cadmium 65.5 0.7 8,220 7 - 15 Chromium 258 2.0 3,750 32 - 95 Copper 665 6.8 3,120 568 - 957 Lead 195 9.4 1,670 89 - 221 Mercury 4.1 0.2 47 0.1 - 6 Molybdenum 13.1 2 67.9 5 - 83 Nickel 77 2 976 26-51 Selenium 6.2 0.5 70.0 2 - 32 Zinc 1,693 37.8 68,000 800 - 1590 Silver NA NA 3 - 60

1 National biosolids survey (209 randomly selected wastewater plants from all regions of the U.S. Adapted from Kuchenrither and Carr, 1991. 2 Pima County biosolids used in current study. Adapted from Pepper et al., 2008. NA = Data not available

a. DTPA Cu b. DTPA Cd 5 0.06 c 0.05 a 4 a

b 0.04 3 a a ab a 0.03

2 0.02

1 0.01 DTPA Cd concentration (%) DTPA Cu concentration (%) 0 0.00 Control Fertilizer Low BS High BS Control Fertilizer Low BS High BS Treatment Treatment 97

c. DTPA Zn d. DTPA Ni 3.0 0.10

2.5 c 0.08 b 2.0 0.06 a a a 1.5 0.04 1.0

0.5 a a 0.02 DTPA Zn concentration (%) DTPA Ni concentration (%) 0.0 0.00 Control Fertilizer Low BS High BS Control Fertilizer Low BS High BS Treatment Treatment

e. Total Hg f. Total Se 1.0 0.07 0.06 b d 0.8 acd

0.05 ab b

0.6 0.04 a a a 0.03 0.4

0.02 0.2

0.01 (%) Se concentration Total Total Hg concentration (%) 0.00 0.0 Control Fertilizer Low BS High BS Control Fertilizer Low BS High BS Treatment Treatment

Other studies have indicated that although metal concentrations can increase in

soil due to biosolids application, these increases are frequently below the U.S. EPA

cumulative limits (Gaskin et al., 2003). In addition, metal concentrations in biosolids

have been decreasing over the past several decades due to improved point-source 98 controls. Finally, metal concentrations attenuated rapidly with increasing soil depth, and were generally similar to values found in control soils at a depth of 150 cm (Table 4.6 and 4.7).

Adriano (2001) reported that annual application of biosolids over a 13 yr period did not affect metal concentrations in soils as compared to the normal level found in agricultural soils of the United States. The current study has shown that the long-term annual applications of biosolids did not increase soil heavy metal concentrations above normal background concentrations or typical soil metal concentrations except for Cu and

Pb which had slightly higher levels than normal background ranges (Table 4.3).

Table 4.3. Representative heavy metals in agricultural soils compared with metal concentration in high biosolid treated soils.

Biosolid Typical treated Geometric Background Metal Minimum1 Maximum1 concentration Soil 3 mean1 Range4 in soil2 (current (mg kg-1) study) As - - - 5.4 6.4 0.10 – 55 Cd 0.18 < 0.01 2.0 0.1 0.4 0.03 – 0.94 Cr - - - 89.3 14.6 10–150 Cu 18.0 1.4 495.0 17.9 73.0 1.0 – 50 Pb 10.6 3.0 135.0 13.4 22.4 2 – 300 Hg - - - 0.03 0.2 0.20 – 5 Mo - - - - < 0.5 - Ni 16.5 2.2 269.0 35.7 12.1 5 – 500 Se - - - 0.2 1.0 - Zn 42.9 3.2 264.0 44.6 10.4 1.0 – 900

1 Summary statistics for concentrations of microelements in 3045 surface soil from major agricultural production areas of the USA. Adapted from Holmgren et. al., 1993. 2 Typical soil concentrations of heavy metals. Adapted from Adapted from Kuchenrither and Carr, 1991. 99

3 Mean metal concentrations in surface soils (0-30 cm) which received repeated high rate (24 t ha-1) land applications of Class B biosolids (Current Arizona study). 4 Adapted from Schroder et al., 2008.

Overall, this study shows that metal concentrations of soil from long-term land

application of biosolids were not hazardous. Similarly, Tian et al. (2006) concluded that

application of biosolids for land reclamation at high loading rates from 1972-2002 only

impacted surface water quality prior to the promulgation of the 40 CFR Part 503

regulations. After the promulgation, metal impacts on surface water quality were

minimal. In addition to reduced concentrations of metals in biosolids, new understanding

of trace element chemistry in biosolid-amended soil has shown that following termination

of land application, available metal concentrations essentially remain constant provided

the soil pH remains constant, or even decreases (Basta et al., 2005). Decreases may be

due to sorption to inorganic oxide surfaces or very recalcitrant organics present in soil of

non-biosolid origin (Basta et al., 2005).

Summary

In this study, the effect of long- term land applications of Class B biosolids on soil

chemical properties were variable with respect to parameters analysed. Long-term application of biosolids increased soil pH but not soil EC (salinity) or CaCO3. The lack of

salinity was probably due to the leaching of soluble salts through the soil profile since

irrigation rates (100 cm per growing season) were almost twice the evapotranspiration

requirements for cotton. Long-term land-application significantly increased soil macro- nutrients including soil nitrate, total nitrogen (N), total carbon (C), and both available and total phosphorous (P) concentrations. 100

Soil nitrate concentrations in both biosolid amended soils and soils that received inorganic fertilizers for 20 years were significantly higher than control (no amendment) plots when averaged over all soil depths (0-150 cm) and total nitrogen increased significantly in biosolid-amended soils. Plots which received the high biosolids rate of application had the highest concentration levels of soil nitrate. One possible negative effect was the potential for nitrate pollution of groundwater regardless of whether biosolids or inorganic fertilizer was applied to the soil. Therefore, further research on controlled irrigation rates for cotton may help to determine N loading rates in order to minimize potential leaching loss of biosolid or fertilizer-derived N. Total organic carbon significantly increased in biosolid amended (3x rate) soil after 20 years of land application (U of A study) to values of 1.3 % in the surface 0- 30 cm soil which is higher than the normal reported total organic C in desert soils. However, the effect of biosolids application on total organic C diminished at the lower (120-150 cm) depths of the soil profile.

Land application (3x rates) of biosolids also significantly increased total and available soil phosphate concentrations, particularly in the surface (0-30 cm) soil. These results are not unexpected as several other studies have also reported phosphate increases following land application. Long-term land application of biosolids resulted in the accumulation of P in surface (0-15cm) soils, and concentrations were greater than needed for optimum crop yields. Therefore P may be at the forefront of biosolids-related issues that could adversely affect the long-term sustainability of land-based recycling programs in the U.S. Such concern is based on the potential for water quality deterioration that can 101 occur in surface waters due to eutrophication following phosphate accumulations in surface water runoff. However, it appears that these issues are more important in the eastern USA, since in the arid southwest, surface waters are rare. That notwithstanding, phosphorus management will continue to be important for sustainable biosolids recycling in the U.S.

Long-term land application of biosolids increased the concentration of some soil heavy metals. However, this study shows that long-term annual applications of biosolids did not increase soil heavy metal concentrations above normal or background soil concentrations except for Cu and Pb, which showed slightly enhanced levels. Overall, concentrations of metals in biosolids have been decreasing over the past several decades due to effective point-source control practices especially after the promulgation of the 40

CFR Part 503 regulations (Pepper et al., 2008). In addition, increases in available soil levels were modest most likely due to high soil pH values. Finally, metal concentrations attenuated rapidly with increasing soil depth, and were generally similar to values found in control soils at a depth of 150 cm. Overall; this study shows minimal adverse effects on soil chemical properties indicating that land application with respect to chemical entities is sustainable in the arid southwest.

102

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106

Tables and Figures

Table 4.4 Chemical analyses conducted on all soil samples; values (averaged over the 0- 150 cm depth) with different letters are significant at the 95% confidence level.

Control Inorganic Low Biosolids High Biosolids Depth (cm) Fertilizer

pH 0 –30 7.7 a 8.0 a 8.2 b 7.9 b 30 – 60 7.7 8.1 8.0 7.9 60 – 90 7.6 8.0 7.8 7.9 90 – 120 7.4 8.1 7.9 7.9 120 – 150 7.6 8.0 8.0 8.1

EC ( mmhos /cm) 0 –30 0.6 a 0.6 a 0.9 a 0.7 a 30 – 60 0.5 0.6 0.8 0.6 60 – 90 0.7 0.9 1.0 0.7 90 – 120 1.1 1.0 1.1 0.6 120 – 150 0.9 0.8 1.0 0.4

CaCO3 (%) 0 –30 5.3 a 5.0 a 4.7 a 4.2 a 30 – 60 5.0 4.3 3.6 3.0 60 – 90 5.0 4.3 3.6 3.0 90 – 120 4.0 4.5 4.8 3.9 120 – 150 2.9 3.2 4.1 4.1

Table 4.5 N, P and TOC chemical analyses conducted on all soil samples. Mean values (µg/g) were considered significant at the 95% confidence level and are indicated with different letters. 107

Depth Control Inorganic Low Biosolids High Biosolids (cm) Fertilizer

NO3 (µg/g) 0 –30 7.5 a 19.4 b 16.4 c 57.6 c 30 – 60 8.2 17.2 26.9 93.6 60 – 90 5.2 6.0 41.4 7.8 90 – 120 4.1 35.8 70.4 36.8 120 – 150 11.9 26.5 62.1 15.4 Total Nitrogen (%) 0 –30 0.08 a 0.08 ab 0.10 a 0.14 b 30 – 60 0.06 0.05 0.06 0.06 60 – 90 0.05 0.09 0.05 0.05 90 – 120 <0.01 <0.01 0.04 <0.01

120 – 150 <0.01 <0.01 <0.01 <0.01 Total Organic Carbon (TOC) 0 –30 0.8 a 0.7 b 0.9 a 1.3 c 30 – 60 0.6 0.4 0.6 0.7 60 – 90 0.4 0.2 0.4 0.4 90 – 120 0.3 0.1 0.3 0.3 120 – 150 0.2 0.1 0.3 0.2 Total Phosphorous (µg/g) 0 –30 803 a 864 a 1037 a 1978 b 30 – 60 670 710 734 943 60 – 90 585 605 582 602 90 – 120 548 607 643 579 120 – 150 523 540 612 603 Available Phosphorous (µg/g) 0 –30 9.9 a 10.4 a 46.0 b 94.9 c 30 – 60 2.6 1.2 10.2 44.2 60 – 90 2.1 0.5 1.6 3.1 90 – 120 1.9 0.7 2.0 2.0 120 – 150 1.9 0.4 1.0 2.0

108 Table 4.6 Total metal analyses conducted on all soil samples. Mean values (µg/g) were considered significant at the 95% confidence level and are indicated with different

Depth (cm) Control Inorganic Low Biosolids High Biosolids Fertilizer Total Zn 0 –30 6.8 ab 6.8 b 7.6 ab 10.4 ac 30 – 60 6.4 6.5 6.7 7.2 60 – 90 5.4 5.4 5.3 4.9 90 – 120 4.6 4.6 4.9 4.1 120 – 150 5.5 3.5 4.3 4.2 Total Hg 0 –30 0.04 a 0.04 a 0.06 a 0.15 b 30 – 60 0.03 0.03 0.04 0.06 60 – 90 0.03 0.02 0.02 0.03 90 – 120 0.02 0.03 0.02 0.02 120 – 150 0.01 0.01 0.02 0.02 Total Se 0 –30 0.96 ab 0.80 b 0.89 acd 1.03 d 30 – 60 0.78 0.75 0.77 0.96 60 – 90 0.67 0.69 0.74 0.77 90 – 120 0.59 0.63 0.70 0.64

120 – 150 0.52 0.53 0.64 0.66 Total Cr 0 –30 13.4 a 13.9 bc 13.5 ac 14.6 ac 30 – 60 13.2 14.1 13.5 13.2 60 – 90 11.6 13.0 11.9 11.1 90 – 120 10.7 11.7 11.8 10.4 120 – 150 9.5 11.6 12.2 10.8 Total B 0 –30 7.0 ac 8.5 ac 7.9 ab 5.4 c 30 – 60 7.3 7.6 7.8 5.2 60 – 90 5.3 6.7 6.4 4.4 90 – 120 4.2 5.4 6.1 3.9 120 – 150 3.7 4.2 5.5 3.8

Table 4.7 Available metal analyses conducted on all of soil samples and available metal 109 concentrations, DTPA metals (µg/g) were considered significant at the 95% confidence level (indicated with letters) except for Cd and Pb. are indicated with different letters.

Control Inorganic Low Biosolids High Biosolids Depth (cm) Fertilizer Cu 0 –30 3.49 ab 3.46 a 5.24 b 11.19 c 30 – 60 2.95 2.87 3.36 4.95 60 – 90 2.24 2.21 2.41 2.08 90 – 120 1.51 1.53 1.87 1.57 120 – 150 0.94 0.87 1.26 1.37 Cd 0 –30 0.06 a 0.01 a 0.06 a 0.01 a 30 – 60 0.04 0.07 0.06 0.13 60 – 90 0.03 0.05 0.04 0.07 90 – 120 0.01 0.03 0.02 0.03 120 – 150 0.01 0.02 0.01 0.01 Zn 0 –30 0.90 a 1.02 a 2.83 b 8.58 c 30 – 60 0.41 0.47 0.89 2.37 60 – 90 0.17 0.26 0.21 0.26 90 – 120 0.16 0.16 0.25 0.26 120 – 150 0.18 0.21 0.24 0.23 Pb 0 –30 2.48 a 2.36 a 2.69 a 3.02 a 30 – 60 1.97 1.98 2.00 2.35 60 – 90 1.58 1.72 1.65 1.45 90 – 120 1.20 1.42 1.50 1.30 120 – 150 0.89 0.80 1.01 1.13 Ni 0 –30 0.08 a 0.09 a 0.11 a 0.17 b 30 – 60 0.05 0.05 0.05 0.07 60 – 90 0.04 0.04 0.04 0.04 90 – 120 0.03 0.03 0.04 0.04 120 – 150 0.03 0.02 0.03 0.04

110

Table 4.8 Total metal analyses conducted on all soil samples; values (µg/g) averaged over 0-150 (cm) depth of total metals concentrations were considered insignificant at the 95% confidence level.

a a a Control Inorganic Low Biosolids High Biosolids Metal Fertilizer a

Cu 37.7 38.4 41.4 43.4 Cd 0.22 0.22 0.22 0.24 Pb 17.4 17.4 17.2 17.2 Ni 9.2 9.6 10.0 9.6 As 5.4 5.2 5.5 5.7 Mo < 0.45 < 0.45 < 0.45 < 0.45

Standard Number of Parameter Amount deviation samples Total N (solids) 4.4 % ± 0.48 13 Total organic C (solids) 23.2 % ± 6.96 10 TOC/ TN ratio (solids) 5.26 :1 ± 0.92 15 Total solids 1.82% ± 0.90 20 Soluble TOC (supernatant) 1.270 mgl-1 ± 100 3 - -1 NH 4 N (supernatant) 1.000 mgl ± 177 6 - -1 NO 3-N (supernatant) < 1 mg l ± 120 3 TOC / TN ratio (supernatant) 1.3: 1 ± 0.15 3

111

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