draft · 2018. 2. 12. · 32 review explores behavior of the tetracycline class of antibiotics from...

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Fate of Tetracycline Antibiotics in Dairy Manure-Amended

Soils

Journal: Environmental Reviews

Manuscript ID er-2017-0041.R1

Manuscript Type: Review

Date Submitted by the Author: 22-Sep-2017

Complete List of Authors: Pollard, Anne; Washington State University, Department of Crop and Soil Sciences Morra, Matthew; University of Idaho, Department of Soil & Water Systems

Keyword: Antibiotic, Tetracycline, Soil, Dairy, Manure

https://mc06.manuscriptcentral.com/er-pubs

Environmental Reviews

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1

Fate of Tetracycline Antibiotics in Dairy Manure-Amended Soils 1

2

Anne T. Pollard1 and Matthew J. Morra

a. Department of Soil & Water Systems, University of 3

Idaho, 875 Perimeter Drive MS 2340, Moscow, ID 83844-2340, USA 4

5

a Department of Soil & Water Systems, University of Idaho, 875 Perimeter Drive MS 2340, 6

Moscow, ID 83844-2340, USA. [email protected] 7

8

Corresponding author: 9

Anne T. Pollard 10

Department of Crop and Soil Sciences, Washington State University, Johnson Hall 233E, PO 11

Box 646420, Pullman, WA 99164-6420, USA 12

Phone: 208-301-8469 13

Email: [email protected] 14

15

16

17

Word count: 12,176 (including references and figures) 18

1 Present address: Department of Crop and Soil Sciences, Washington State University, Johnson

Hall 233E, PO Box 646420, Pullman, WA 99164-6420, USA. [email protected]

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Abstract: The U.S. dairy industry has changed significantly during the past 20 years. The 19

number of dairies declined 63% from 1997 to 2012 due to the rise in concentrated animal feeding 20

operations (CAFOs) and the concomitant decline of small dairy farms. Efficient and cost-21

effective dairies adhering to the CAFO business design are praised for their high milk 22

production. However, with a per capita daily manure production of 55 kg, storage and disposal of 23

manure at these large operations pose significant management challenges and environmental 24

risks. Application to surrounding agricultural fields is a common practice for disposing of 25

manure, but the fate and consequences of antibiotics present in dairy waste are issues of great 26

concern. Although antibiotics in the environment promote microbial resistance, their risks to 27

humans and the environment are not completely known. Understanding and predicting the fate of 28

antibiotics from dairy manure in soils is complicated by the variability and complex interactions 29

of soil factors in addition to the diversity of chemicals of emerging concern (CECs), their 30

amphoteric structures, and potential antagonistic and synergistic interactions among CECs. This 31

review explores behavior of the tetracycline class of antibiotics from dairy manure in the soil 32

environment. Tetracycline fate in soils depends significantly on soil pH, ionic strength, and soil 33

organic matter. Molecular charge and physicochemical properties of tetracyclines at typical soil 34

pHs encourage strong sorption to soils; however, this interaction is complicated by organic 35

matter and metals, and may also encourage development of antibiotic resistance. Furthermore, 36

tetracycline degradation products exhibit distinct properties from their parent compounds that 37

also must be considered. Increased knowledge of the behavior of tetracycline antibiotics in soil is 38

needed to enable mitigation of their potential risks. 39

Key words: Dairy manure; Antibiotic; Tetracycline; Soil 40

41

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1. Introduction 42

The transition from smallholder dairies to CAFO-style dairy operations has resulted in 43

massive generation of animal waste in a physically restricted location. While spreading manure 44

on surrounding farmland is a centuries-old practice of waste removal, the extensive amount of 45

waste that is being applied near CAFO dairies has triggered mounting concerns about the 46

potential human and environmental risks posed by CECs (chemicals of emerging concern) in 47

manure. Veterinary pharmaceuticals in concentrations of micrograms to nanograms per liter have 48

been found in surface and groundwaters located proximate to manure-applied soils, illustrating 49

the mobility of these CECs in soil and therefore their potential threat to the environment (Daghrir 50

and Drogui 2013). Given their low cost, minimal adverse side effects, and broad spectrum 51

application, compounds within the tetracycline family are among the most highly used 52

antibiotics for livestock and humans (Chopra and Roberts 2001; USDA 2008a; USFDA 2015). 53

Daghrir and Drogui (2013) reviewed tetracycline occurrence and toxicity, with an emphasis on 54

mitigating water treatment processes. Additional reviews exist on the presence and behaviors of 55

veterinary pharmaceuticals in soils (Tolls 2001; Thiele-Bruhn 2003); however, the fate of 56

tetracycline antibiotics in soils following dairy manure application has not been comprehensively 57

assessed. The variety of chemical compounds in dairy manure, the diversity of soil types to 58

which dairy waste is applied, and the various waste application practices complicate such 59

evaluation. This review addresses the occurrence, properties, and fate, including sorption, 60

transport, and degradation, of tetracycline (TC), chlortetracycline (CTC), and oxytetracycline 61

(OTC) antibiotics in dairy manure-amended soils. 62

2. Antibiotics in the dairy industry 63

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The source of detected pharmaceuticals in surface and groundwater has historically been 64

attributed to human use and the consequent release of effluents from sewage treatment plants 65

(Kolpin et al. 2002). However, the dominant consumer of pharmaceuticals in the United States is 66

livestock, encompassing swine, poultry, beef, and dairy cattle (USEPA 2013). Antibiotics are 67

used in the dairy industry to treat existing disease, to prevent disease, and for growth promotion, 68

though the latter is used only in pre-lactating calves (USDA 2008b; Chee-Sanford et al. 2009; 69

USEPA 2013). The use of antibiotics for growth promotion in livestock is banned in the 70

European Union, but remains a common practice in the USA and China, albeit not frequently for 71

dairy cattle (Sarmah et al. 2006; USEPA 2013; Zhou et al. 2013). Tetracycline antibiotics are 72

used in dairy operations to treat respiratory infections, diarrhea, navel infections, reproductive 73

disorders, mastitis, lameness, and other ailments (USDA 2009). In 2007, TC was given to 25% 74

of heifers to treat respiratory infections, down from 34.3% in 2002; 55% of heifers to treat 75

diarrhea and digestive disorders, a significant increase from 11.8% in 2002; and 42% of heifers 76

to treat lameness, unchanged from 2002 (USDA 2008a, 2009). 77

Antibiotics are administered to dairy cattle orally through feed and water, by injection, 78

topically, or via intramammary or intrauterine infusion (Zwald et al. 2004; Zhou et al. 2013). In a 79

survey of 99 conventional dairies from the states of New York, Wisconsin, Michigan, and 80

Minnesota, 98% reportedly used intramammary antibiotic infusions prophylactically in non-81

lactating cows (Zwald et al. 2004). In 2007, 49.5% of all US dairy operations fed milk replacer 82

medicated with a combination of OTC and neomycin to preweaned heifers (USDA 2008a). 83

Additionally, a survey of 80% of all US dairies indicated that 82% of all cows were given 84

antibiotics at dry-off, or the period between milk stasis and subsequent calving (USDA 2008a). 85

These results are similar to a survey of 201 dairies in The Netherlands in which 82.8% were 86

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found to administer antibiotics on dry cows (Barkema et al. 1998). From 2002 to 2007, the 87

percent of US dairy operation that add CTC or OTC to weaned heifer rations decreased from 88

62.4% to 14.4% for CTC and from 21.5% to 10.9% for OTC (USDA 2009). 89

It is difficult to accurately assess antibiotic use in dairies because 1) operations are not 90

required to report data; 2) data on which studies have been based result from voluntary 91

participation, which introduces a bias since non-responder dairy operations may differ from the 92

responders; 3) the majority of studies group beef and dairy cattle into a single category; 4) 93

standardized record keeping systems do not exist; 5) most respondents rely on memory regarding 94

antibiotic use, frequency, duration, and prevalence; 6) it is not possible to verify the accuracy of 95

reported findings; and 7) extra-label drug usage occurs, meaning antibiotics are used on animals 96

or for conditions that the drug is not specified (Zwald et al. 2004; Van Boeckel et al. 2015). For 97

example, 55% of conventional dairies surveyed in Wisconsin indicated extra-label use of 98

intramammary antibiotics (Pol and Ruegg 2007). An estimated 15.4 million kg of antimicrobials 99

were sold and distributed in 2014 specifically for domestic food-producing animals with 100

tetracyclines accounting for the greatest share overall at 43% (USFDA 2015). Furthermore, from 101

2009 to 2014, sales and distribution of tetracyclines for domestic food-producing animals 102

increased 25% (USFDA 2015). However, the report does not specify the amount distributed per 103

livestock species. Antibiotics are poorly metabolized in vivo and estimates of 30-90% of the 104

parent compound and its active metabolites are excreted in animal urine and feces (Chee-Sanford 105

et al. 2009; Daghrir and Drogui 2013). Moreover, because antibiotics are given for prophylactic, 106

metaphylactic, and growth-promoting purposes, the antibiotic load present in manure far 107

surpasses historic quantities when more conservative practices were employed. Through land-108

application of antibiotic-rich animal waste in concentrated geographical regions, the soil serves 109

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as a repository for pharmaceuticals from which tetracyclines may leach into groundwater, enter 110

surface waters via overland flow, or persist in soil (Kwon 2011; Zhou et al. 2013; Pan and Chu 111

2017). 112

Although a thorough investigation of the association between land-application of dairy 113

manure and development of antibiotic resistance in soil bacteria is beyond the scope of this 114

review, its great importance warrants a brief explanation. In addition to large quantities of 115

residual antibiotics in animal manure, commensal bacteria present in animal guts are likewise 116

present in high concentrations in animal waste (Chee-Sanford et al. 2009). Due to frequent 117

ingestion of antibiotics, commensal bacteria contain a high concentration of antibiotic-resistant 118

genes (ARG), which confer resistance to antibiotics in microorganisms (Wichmann et al. 2014). 119

In their metagenomic analysis of dairy cow manure to assess ARG present in dairy cow gut 120

bacteria, Wichmann et al. (2014) identified 80 unique ARG, including three that confer 121

tetracycline resistance: tetW, tetO, and an unspecified gene. Chambers et al. (2015) likewise 122

conducted a similar metagenomics analysis of ARG in dairy cow manure and 75% of detected 123

ARG were of the tetracycline class. Antibiotics present in animal waste, in addition to excreted 124

commensal bacteria, create a conducive environment for proliferation of ARG (Storteboom et al. 125

2007; Kyselková et al. 2015a,b; Peng et al. 2016). Manure samples from antibiotic-treated dairy 126

cows contained nearly the same quantity and distribution of ARG as manure samples from dairy 127

cows with no previous antibiotic exposure, illustrating the ease with which ARG transfer to other 128

bacteria by mobile genetic elements (Wichmann et al. 2014). In a survey of tetracycline resistant 129

genes in cow waste, water, and soil samples at six grassland-based dairy farms, 14 unique 130

tetracycline ARG were detected among the animal and environmental samples, with tetW and 131

tetQ detected in nearly every sample at each of the farms (Santamaría et al. 2011). It appears that 132

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tetW and tetQ arrived in soil and water samples as a result of horizontal gene transfer since those 133

ARG have the exact genetic sequences as the tetW and tetQ detected in animal waste samples. 134

Though research frequently focuses on the introduction of pathogenic microbes to the 135

environment from animal manure (Salgado et al. 2011), data indicate that manure application is 136

directly associated with increasing numbers of antibiotic-resistant bacteria in soil (Sengeløv et al. 137

2003; Peng et al. 2014). Residual antibiotics in land-applied manure exert a selective pressure on 138

resident soil microbes, causing a shift in the microbial community toward more resistant 139

microorganisms (Boxall et al. 2003; Pruden et al. 2006; Chessa et al. 2016). Recent studies show 140

that very low antibiotic concentrations in the environment can induce selection for resistance 141

(Gullberg et al. 2011; Liu et al. 2011). The soil environment may serve as a reservoir for ARG, 142

thereby contributing to the inefficacy of antibiotics in humans, as many of the drugs commonly 143

administered to livestock are also routinely used by humans (Storteboom et al. 2007). In addition 144

to widespread concern for development of antibiotic resistance, some antibiotics have also 145

demonstrated an ability to behave as hormones, thus having endocrine disrupting effects on 146

organisms (Hamscher et al. 2005). 147

2.1. Physical and chemical properties of tetracyclines 148

The tetracycline class of antibiotics contains some of the most widely used broad-spectrum 149

antibiotics for humans and animals because of their low cost and antimicrobial efficacy (Daghrir 150

and Drogui 2013; Chang et al. 2015). Tetracyclines function as antibacterial agents by attaching 151

to a transfer-RNA binding site on the 30S ribosomal subunit in bacteria, thereby inhibiting 152

protein synthesis (Carter et al. 2000). Numerous tetracycline antibiotics are used in the livestock 153

industry, with tetracycline (TC), chlortetracycline (CTC), and oxytetracycline (OTC) the most 154

commonly used antibiotics worldwide (Granados-Chinchilla and Rodríguez 2017). Among three 155

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dairy farms sampled in China, CTC constituted 86% of total antibiotics excreted by cows, 156

equating to 3.66 mg d-1

cow-1

(Zhou et al. 2013). In addition to therapeutic treatment of mastitis, 157

foot infections, respiratory disease, and metritis, they are also used prophylactically for disease 158

prevention in dry cows and for growth promotion in pre-lactating cows (Zwald et al. 2004; Pol 159

and Ruegg 2007; USEPA 2013). Chlortetracycline and OTC are among the top ten antibiotics 160

licensed for use as growth promoters in the United States (Hao et al. 2014). Tetracyclines are 161

poorly absorbed in the digestive tract, with an estimated 25% excreted in feces and an additional 162

50-60% excreted as the parent molecule or an active metabolite in urine (Feinman and Matheson 163

1978; Boothe 2015). The concentration and frequency of tetracyclines reported in dairy manure 164

vary. Oxytetracycline has been found in high concentrations in fresh dairy calf manure, 165

illustrating its poor intestinal absorption (De Liguoro et al. 2003). From three dairy farms 166

sampled in China, tetracyclines were present in fresh feces at only one farm, with 1450 µg kg-1

167

CTC and 17 µg kg-1

TC (Zhou et al. 2013). 168

Tetracyclines are large molecules with molecular weights ranging from 480.9 g mol-1

for TC 169

to 528 g mol-1

for CTC. They share a naphthalene ring structure with three ionizable functional 170

groups: tricarbonyl (pKa 3.2 to 3.3), phenolic β-diketone (7.46 to 7.78), and dimethylamine (8.9 171

to 9.6) (Teixido et al. 2012) (Fig. 1). The multiple polar functional groups give the molecules 172

amphoteric characteristics, whereby they may be a cation, zwitterion, or anion (Sassman and Lee 173

2005) (Table 1). At soil pH values between approximately 4 and 8, tetracyclines will exist as 174

zwitterions with a positive charge on the amine and a negative charge on the hydroxyl group in 175

the tricarbonyl (Sassman and Lee 2005; Li et al. 2010). 176

The distinctive physical structures of the tetracycline molecules attribute unique chemical 177

properties to the individual antibiotics. Tetracycline displays the basic structure of the class, with 178

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hydrogen at C10 of the D-ring replaced by a chlorine atom to produce CTC, and a hydroxyl 179

group at C6 of the B-ring substituted for the hydrogen to produce OTC (Teixido et al. 2012) 180

(Fig. 1). In addition to the structures of the parent molecules, tetracyclines can undergo 181

transformations to form numerous bioactive epimers (Kwon 2011). Due to substitutions at C10 182

and C6, CTC and OTC are more reactive in the environment than TC (Loftin et al. 2008). CTC 183

has greater solubility and mobility than TC or OTC (Kwon 2011; Teixido et al. 2012). In terms 184

of degradability, CTC is the most easily degraded, followed by OTC, then TC (Loftin et al. 185

2008). Increased reactivity, solubility, and degradability of CTC may be attributed to its reported 186

instability at alkaline pH (Figueroa et al. 2004). Avisar et al. (2010) reported lower sorption of 187

OTC to montmorillonite clay than TC as a result of molecular structure differences. The 188

presence of a hydroxyl adjacent to the cationic amine residue on OTC can cause steric hindrance, 189

decreasing its ability to sorb to clay surfaces. Tetracyclines are essentially non-volatile, as 190

indicated by their low Henry’s constants (3.91 x 10-26

to 3.45 x 10-24

atm m3 mol

-1) (Daghrir and 191

Drogui 2013). Their high water solubilities (0.008-0.062 mol L-1

) and low log Kow values (-1.25 192

to -0.62) suggest a hydrophilic nature (Daghrir and Drogui 2013). Furthermore, a negative log 193

Kow suggests that any tetracycline partitioning to soils is a result of electrostatic interactions, ion 194

exchange, and surface complexation (Jones et al. 2005). 195

2.2. Occurrence of manure-borne tetracyclines in soils 196

Predicting sorption, transport, and bioavailability of tetracycline antibiotics following land-197

application of dairy manure is challenging. Relatively few field studies have characterized the 198

fate of antibiotics in soil following land-application of manure, and even fewer specifically 199

address dairy manure. The potential environmental risks from tetracycline introduction to the 200

environment from manure application include development and spread of antibiotic resistance 201

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and introduction of antibiotic resistance to the food chain (Storteboom et al. 2007). Moreover, 202

antibiotic-resistant organisms exhibit unique physiological structures and processes compared to 203

their non-resistant counterparts (Walczak et al. 2011), resulting in unique interactions with the 204

soil environment. For example, in packed sand column studies comparing movement of 205

tetracycline-resistant (tetR) and tetracycline-susceptible (tet

S) E. coli isolated from dairy manure, 206

the tetR strains had greater mobility than the tet

S. The lower zeta potentials of the tet

R resulted in 207

greater repulsive forces between the tetR and negatively charged sand surfaces, thus facilitating 208

increased transport. The composition of outer membrane proteins between the resistant and 209

susceptible strains was also very different, which may have contributed to the increased mobility 210

of the tetR strains (Walczak et al. 2011). Drastic alteration of microbial physiology and behavior 211

due to concentrated and prolonged exposure to environmental antibiotics will necessitate 212

reevaluation of the current microbiological knowledge base. 213

Due to their widespread use and high sorption capacity, tetracyclines have been detected in 214

substantial concentrations in soils. In an analysis of antibiotics present in fresh cattle, swine, and 215

chicken manure, the tetracycline class of antibiotics was detected most frequently (81-91%) and 216

in the greatest concentrations, with CTC predominating at an average concentration of 8060 ± 217

23080 µg kg-1

(Hou et al. 2015). The concentration of CTC in cattle manure was far less than for 218

swine or chicken manure, averaging approximately 300 µg kg-1

. Moreover, the concentration of 219

CTC and TC detected in adjacent manure-amended soils was very similar to the concentrations 220

in the fresh manure. For example, the CTC concentration in fresh manure was approximately 221

1550 µg kg-1

while it was detected at ~ 1500 µg kg-1

in the adjacent field (Hou et al. 2015). In a 222

study from Germany of tetracycline behavior in a sandy soil (91.5% sand) repeatedly fertilized 223

with liquid pig manure containing initial concentrations of TC and CTC of 14.1-41.2 mg kg-1

and 224

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0.9-1.0 mg kg-1

, respectively, the average concentrations detected in soil were 158 µg kg-1

for TC 225

and 8.3 µg kg-1

for CTC (Hamscher et al. 2005). Hamscher et al. (2005) additionally found 226

consistently high concentrations of tetracyclines in the top 0-30 cm of soil for 2 years, illustrating 227

their persistence and consequent accumulation in soils with repeated manure application 228

(Hamscher et al. 2002, 2005), as was similarly determined in more recent studies examining 229

tetracycline occurrence in swine manure (Martinez-Carballo et al. 2007; Qiao et al. 2012). 230

In a study from northern China comparing antibiotic concentrations in soils after either 231

swine or dairy wastewater application (initial concentrations not specified), the levels of 232

tetracyclines differed significantly between the animal operations. No tetracyclines were detected 233

in the dairy field soil, but between 0 and 667 µg kg-1

were detected in the swine fields (depth of 234

soil sampling not specified) (Zhou et al. 2013). Tetracyclines were also not detected in manure 235

lagoon samples or soil leachates (sampling details not provided) from two conventional and two 236

organic German dairies, leading Kemper et al. (2008) to conclude that dairies are an insignificant 237

source of antibiotics to the environment. However, their negative results may also be influenced 238

by the tendency of tetracyclines to sorb strongly to soil particles and resist leaching. Contrasting 239

results from different animal manure studies can be attributed to the amount of antibiotics 240

originally administered; different animals’ physiologies and resultant metabolism of 241

tetracyclines; number of animals contributing to manure output, thus drug concentrations in the 242

manure; and overall different operational systems and methods utilized at swine and dairy farms. 243

Moreover, studies show that composition and concentration of antibiotics in animal manure are 244

affected by animal type, life stage, and husbandry practices (Hou et al. 2015). For example, the 245

concentration of CTC in fresh piglet manure was far greater (~ 35000 µg kg-1

) than in sow 246

manure (~14000 µg kg-1

) likely from high dosages of CTC administered to piglets for disease 247

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prevention (Hou et al. 2015). Furthermore, antibiotics at the dairies in Kemper et al.’s study 248

(2008) were only given for treatment of disease and typically to individual dairy cows, whereas 249

common practice on pig farms is to routinely administer antibiotics to entire herds for 250

prophylactic and metaphylactic purposes (Zhou et al. 2013). Similarly, Zhou et al. (2013) stated 251

that antibiotic use on dairy farms in China was primarily for treatment and prevention of disease, 252

and not for growth promotion, a common purpose of antibiotic administration on swine farms. 253

Quantities of antibiotics, including tetracyclines, in manure from a beef feedlot were 254

significantly greater than levels in dairy manure (specific values were not reported), presumably 255

resulting from high doses given to beef for growth promotion (Storteboom et al. 2007). These 256

studies highlight the variability in reported concentrations of tetracyclines in animal waste as a 257

result of diverse animal husbandry methods, such as administered amounts of drugs and 258

operating procedures. 259

2.3. Tetracycline sorption in soils 260

Aside from minor differences in their chemical properties, sorption behavior of TC, OTC, 261

and CTC on clay surfaces is extremely similar due to their structural resemblance (Figueroa et al. 262

2004). The main determinants of tetracycline sorption to soils in order of significance are pH, 263

which controls speciation and Coulombic or electrostatic interactions; CEC; metal content 264

(especially Fe2O3, K2O and to a lesser extent Al2O3), which influences surface complexation 265

with metal oxides and cation bridging; and soil texture, such as clay content (Jones et al. 2005; 266

Sassman and Lee 2005; Teixido et al. 2012; Peng et al. 2014) (Fig. 2). Depending on the soil 267

physicochemical properties, sorption coefficient Kd values for tetracyclines spiked in soils have 268

been measured ranging from < 200 to over 5,000 L Kg-1

(Table 2) (Rabølle and Spliid 2000; 269

Sassman and Lee 2005; Teixido et al. 2012; Chessa et al. 2016). Through batch-equilibration 270

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sorption studies in which 30 unique soils devoid of historical animal usage were spiked with 271

OTC, Jones et al. (2005) found that texture, oxide content, and cation exchange capacity 272

significantly influence OTC sorption in soils with ≤ 4% organic carbon (Table 2). Pearson 273

correlation and a principle components analysis correlating OTC sorption to 16 physicochemical 274

properties of the 30 soils showed OTC sorption coefficients (Kd) most positively correlated to 275

CEC, percent clay, surface area, Fe-oxide content, and exchangeable Mg2+

, Ca2+

, and Na

+. 276

Sorption was negatively correlated to percent sand. In general, tetracyclines sorb quickly and 277

strongly to soils, especially at low pH and in soils with high clay content, as seen in the batch 278

sorption studies of Sassman and Lee (2005) (Table 2) where they spiked variable soils with OTC 279

concentrations comparable to those found at livestock operations. Sorption of tetracyclines onto 280

‘Toronto’ soil (pH 4.18) averaged approximately 30 times greater than sorption onto ‘Drummer’ 281

soil (pH 7.49), though they had equal 21% clay contents. Chessa et al. (2016) drew similar 282

conclusions from batch equilibration studies where alkaline and acid soils were 1) spiked with 283

TC, or 2) incorporated with TC-spiked cattle manure (Table 2). The Kd value was five times 284

greater in a pH 5.77 soil with 41% clay compared to a pH 7.6 soil with 16.6% clay (Chessa et al. 285

2016). Moreover, the slight change in TC sorption after manure addition further suggests that 286

clay content and pH, rather than organic matter content, strongly influence TC sorption to soils. 287

A wide variability of Kd values among 30 soil samples from five represented soil orders 288

indicates that OTC sorption to soils cannot be determined solely based on soil order (Jones et al 289

2005). Nevertheless, Spodosol samples displayed the lowest attraction for OTC, which may be 290

due to low surface area of the coarse-textured soils (Jones et al. 2005). Other studies have also 291

determined that finer-textured soils display greater capacity for OTC sorption (Rabølle and 292

Spliid 2000; Loke et al. 2002) (Table 2). In a batch-equilibration study analyzing OTC-spiked 293

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soils, between 95 – 99% of OTC was sorbed to loamy sand and sandy loam soils, with Kd values 294

of 417 and 1026 mL g-1

, respectively (Rabølle and Spliid 2000) (Table 2). Despite this range in 295

Kd values, infrequent detection of tetracyclines in groundwater is consistent with their high Kd 296

values. In a comparison of CTC sorption kinetics onto two soils with similar pH and organic 297

matter content, but divergent particle size distribution, CTC rapidly sorbed onto both the sandy 298

loam and heavy clay soil, further support that tetracyclines have low probability of leaching 299

through the soil profile (Allaire et al. 2006). 300

Sorption of tetracyclines to montmorillonite and kaolinite clays depends on solution pH, 301

ionic strength, and organic matter content (Figueroa et al. 2004; Avisar et al. 2010; Zhao et al. 302

2011 and 2015). Estimating tetracycline sorption in soils based on Kd values attained from 303

research with pure clay minerals may be misleading, since clay surfaces in actual soils may be 304

coated with organic matter or metal oxides, thus affecting antibiotic sorption to the clay surface. 305

Furthermore, presence of ions in soil solution could impact antibiotic sorption also influencing 306

antibiotic bioavailability and mobility in soils (Figueroa et al. 2004). However, since limited 307

research has been conducted on the behavior of these chemicals in situ, results of pure clay 308

studies provide insight into tetracycline behavior in soils. 309

In addition to sorption to clay surfaces, tetracyclines intercalate the interlayer of 2:1 swelling 310

clays (Kumar et al. 2005). For example, Chang et al. (2009a,b) show through X-ray diffraction 311

(XRD) and Fourier transform-infrared (FTIR) analyses that tetracycline intercalates rectorite 312

between pH 1.5 – 8.7. Similarly, OTC intercalates montmorillonite between pH 1.5 and 11.0, as 313

evidenced by increased d-spacing observed through XRD analyses (Kulshrestha et al. 2004; 314

Aristilde et al. 2013). In a study of OTC desorption in30soils with variable properties, the 315

amounts desorbed in the Iredell Alfisol and the Sharkey Vertisol (37 and 53%, respectively) 316

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were noticeably less than the average 75 ± 14% desorbed from the larger set of soils (Jones et al. 317

2005). The high proportion of shrink-swell clays in these soils, coupled with high Ca2+

and Mg2+

318

contents, supports OTC intercalation, thereby yielding low recoveries (Jones et al. 2005; 319

Aristilde et al. 2016). Aristilde et al. (2016) found that when Ca2+

or Mg2+

were present in 320

solution, OTC adsorption to montmorillonite doubled. Furthermore, they detected a 120% 321

increase in adsorbed OTC when introduced with Mg2+

, accompanied by a 200% increase in OTC 322

intercalation of montmorillonite layers. Intercalation of OTC into montmorillonite can be 323

affected by ionic strength (Figueroa et al. 2004). Sorption edge experiments using three salt 324

concentrations showed that while the OTC cationic species interacted more strongly with 325

montmorillonite than the zwitterion, the cation was more sensitive to high ionic strength, as seen 326

in a near 13-fold decrease in Kd when ionic strength increased from 10 mM to 510 mM 327

(Figueroa et al. 2004). Although the adsorption capacity of kaolinite is far less than for swelling 328

clays due to a lack of interlayer accessibility, the rate of adsorption is much higher since 329

tetracycline is interacting only with the external surface and not intercalating (Li et al. 2010). 330

Tetracycline adsorption to clay has been characterized by two kinetically different processes: a 331

fast initial adsorption to outer surfaces of clays, followed by slower intercalation of swelling 332

clays and into micropores (Sithole and Guy 1987a). 333

The strong correlation between tetracycline sorption and effective cation exchange capacity 334

(CEC) could result from electrostatic attraction between the tertiary amine group that is 335

positively charged at environmentally relevant soil pHs and the negatively charged clay surface 336

(Jones et al. 2005; Zhao et al. 2015). As this study also found a strong positive correlation 337

between Kd and Mg2+

, Ca2+

, and Na+, cation bridging between negatively charged functional 338

groups and base cations may contribute to the strong interaction between tetracyclines and 339

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smectite clays (Figueroa et al. 2004). Tetracyclines also bind to proteins and anionic silanol 340

groups common on phyllosilicate surfaces (Sithole and Guy 1987a; Loke et al. 2002). Although 341

kaolinite has a low CEC compared to 2:1 swelling clays, sorption of tetracyclines on kaolinite 342

needs to be considered because it may contribute significantly to tetracycline sorption when 343

present in high concentrations in soils. Furthermore, the pH-dependent surface charge on 344

kaolinite influences tetracycline interactions with kaolinite, with maximum sorption on kaolinite 345

at pH 7 when it is a zwitterion (Li et al. 2010). Although the net charge of the tetracycline 346

zwitterion is zero, this species contributes significantly to total tetracycline sorption to soil clays 347

because it is the dominant tetracycline species at the common soil pH values of between 348

approximately 3.3 and 8. In addition to the dominant sorption mechanism of cation exchange 349

which predominates on kaolinite surface sites (Zhao et al. 2015), surface complexation via H-350

bonding is responsible for tetracycline sorption to kaolinite edge sites (Li et al. 2010; Zhao et al. 351

2015). The size and physical conformation of tetracycline aligned with the kaolinite surface does 352

not appear to influence sorption, but rather charge density shows the greatest effect (Li et al. 353

2010). For example, Zhao et al. (2011) concluded that TC adsorption on kaolinite decreased with 354

the increasing atomic radius and valence of metal cations in solution, suggesting that cations 355

interfere with outer-sphere complexation between TC and kaolinite. 356

As pH increases, tetracycline molecules become increasingly deprotonated, thus exhibiting a 357

greater net negative charge and correspondingly weaker sorption to soils (Sassman and Lee 358

2005; Peng et al. 2014; Zhao et al. 2015). This behavior is illustrated by decreasing Kd values 359

with increasing pH (Figueroa et al. 2004; Sassman and Lee 2005). For example, the Kd of OTC 360

sorption to Na-montmorillonite was approximately 7500 L eq-1

at pH 5, declining to 361

approximately 800 L eq-1

by pH 8 (Figueroa et al. 2004). This decrease in sorption from pH 5 to 362

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8 reflects the decrease in zwitterion abundance in that pH range and the significance of the 363

zwitterion species on total OTC sorption. Furthermore, total sorption of tetracyclines at any pH is 364

a sum of the sorption of cationic species and zwitterionic species (Figueroa et al. 2004). 365

Figureroa et al. (2004) concluded that although cationic OTC species comprise a small portion of 366

total OTC at pH 5.5, the force of attraction between the cationic species and montmorillonite is 367

20 times stronger than that of the zwitterion. Accordingly, 19% of total sorption was ascribed to 368

the cation and 81% to the zwitterion. 369

Tetracyclines can adsorb to soils via highly stable chelate complexes and ionic bridging with 370

metals, including divalent cations Cu2+

, Ca2+

, and Mg2+

(Loke et al. 2002; MacKay and 371

Canterbury 2005; Sassman and Lee 2005; Avisar et al. 2010; Zhang et al. 2011). While 372

tetracycline sorption decreases with increasing pH due to its deprotonation, sorption to alkaline 373

soils is enhanced when those soils contain high Ca2+

and Mg2+

concentrations (Arias et al. 2007). 374

Above pH 7 when anionic tetracycline species gain prominence, the presence of Ca2+

in solution 375

increases Kd values compared to tetracycline sorption in the absence of cations (Figueroa et al. 376

2004). As mentioned in numerous studies, this enhanced tetracycline sorption at alkaline pH is 377

attributed to cation bridging between the carboxylic residue of the tetracycline molecule and 378

negatively charged clay surfaces (Avisar et al. 2010). However, the presence of Ca2+

or Na+ in 379

solution below pH 7 results in significantly less sorption of OTC to montmorillonite as a result of 380

competition between positively charged OTC species and cations for sorption sites on the clay 381

surface. In contrast, the presence of Cu2+

at low soil pH (

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18

though the latter may be the dominant mechanism (Gu and Karthikeyan 2008) (Fig. 3). Sorption 386

of tetracyclines on SOM is highly pH-dependent between pH 2.5 and 10 due to variable charges 387

on the three ionizable functional groups, with maximum tetracycline sorption at pH 4.3 when the 388

zwitterionic form comprises 90% of total species. This species interacts primarily with 389

carboxylic acid functional groups on SOM via cation exchange (Avisar et al. 2010). Below pH 390

4.3, zwitterionic and cationic tetracycline species compete with H+

for binding on humic acids 391

and above 4.3, sorption decreases due to charge repulsion between the predominant anionic 392

tetracycline species and deprotonated SOM (Gu et al. 2007). Tetracycline sorption to SOM is 393

strongly influenced by the presence of multivalent cations (Fig. 3). For example, divalent cations 394

such as Ca2+

and Mg2+

exert a significant effect on tetracycline sorption to SOM at pHs >5, 395

leading to formation of ternary organic matter-M2+

- tetracycline complexes (Gu et al. 2007). 396

Bridging with multivalent cations has likewise been determined as a means of OTC sorption to 397

manure in soil (Sithole and Guy 1987b; Loke et al. 2002). The Kd values of tetracyclines to 398

manure thus cannot be determined by extrapolation from Kow or the fraction of organic carbon in 399

soil, for Kd values are much higher than expected based on the negative log Kow values (Sithole 400

and Guy 1987b; Loke et al. 2002; Jones et al. 2005). 401

While sorption of tetracyclines on clay surfaces decreases with increasing pH, a greater 402

overall decline in sorption was seen with increasing ionic strength (Sithole and Guy 1987a; 403

Figueroa et al. 2004; Li et al. 2010). Tetracycline sorption is strongly influenced by the 404

concentration of metals in solution, as evidenced by a more than 50% decrease in sorption to 405

SOM when ionic strength increased 10-fold (Gu and Karthikeyan 2008). This correlation was 406

especially strong for cationic tetracycline species, suggesting that cation exchange is the most 407

significant sorption mechanism at low pH (Figueroa et al. 2004). However, surface complexation 408

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19

is a significant sorption mechanism for zwitterionic tetracycline species that predominate 409

between pHs of approximately 3.3 and 8, indicated by their low sensitivity to ionic strength. 410

Hysteretic behavior has been noted for humic acid-tetracycline complexes, whereby a large 411

concentration of tetracycline bound irreversibly to humic acids via physical containment or 412

changes in humic acid conformation (Gu et al. 2007). Hysteresis may decrease bioavailability, 413

degradation, and mobility of tetracyclines in soils. 414

Organic matter is not a primary influence on tetracycline sorption to soils, which must be 415

taken into account when assessing the effect of land-application of manure (Teixido et al. 2012). 416

This conclusion is illustrated in Chessa et al.’s (2016) study in which TC Kd values were nearly 417

identical whether cow manure was applied to soil or not; this trend held for two distinct soils 418

with moderate and high levels of manure application. Humic substances, either dissolved or 419

bound to clay minerals, decrease tetracycline sorption, thus increasing its mobility (Avisar et al. 420

2010). For example, a concentration of 10 mg/L humic acid reduced sorption of OTC to 421

montmorillonite (Kulshrestha et al. 2004). Consequently, the potential exists for tetracyclines to 422

migrate via surface and groundwaters in soils high in organic matter (Gu and Karthikeyan 2008). 423

Jones et al. (2005) likewise saw a strong negative correlation between OTC sorption to 29 424

different soils (≤ 4% organic carbon) and percent soil organic carbon. In contrast, strong OTC 425

sorption to soils is sometimes attributed to high OM content. In a study of OTC sorption in three 426

distinct soils, Peng et al. (2014) concluded that strong adsorption to soil B was due to its high 427

OM content (42.7 g Kg-1

), in contrast to soil A and soil C with 10.4 and 13.4 g Kg-1

, 428

respectively. However, the low pH values (3.5 and 4.3, respectively) of soil B and C were 429

perhaps greater determinants of OTC sorption, considering their comparable Kf values of 3590 430

and 3640, respectively, in comparison to the low Kf (108) of soil A with a pH of 7.6. Similarly, 431

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Jones et al. (2005) suggest strong OTC sorption to the Burton soil may resulted in part from its 432

high organic carbon content (8.93%) The low 3.2 pH of the Burton soil likely contributed to its 433

strong OTC sorption. 434

Interaction with Al, Fe, and Mn Oxides 435

Interactions with metal oxides influence tetracycline behavior in soils. Carbonyl and 436

tricarbonyl functional groups on tetracyclines complex strongly with Al and Fe atoms on edges 437

of aluminum and iron hydrous oxides (HAO and HFO, respectively) (Gu and Karthikeyan 2005). 438

However, tetracyclines compete for edge sites with SOM since the latter likewise strongly 439

associates with HAO. Gu and Karthikeyan (2008) found that TC sorption onto HAO 440

significantly declined with increasing humic acid content, with an increase from 0.81% to 1.52% 441

organic carbon resulting in about a 40% decrease in TC sorption. Pils and Laird (2007) 442

concluded that TC and CTC adsorb in decreasing order to clay minerals > humics > clay-humic 443

complexes. Reduction in TC sorption in the presence of SOM may be due to competition for 444

sorption sites, and also reversal in aluminum oxide surface charge resulting from SOM sorption, 445

which would lead to repulsion between the oxide surface and deprotonated TC functional groups 446

(Gu and Karthikeyan 2008). Tetracycline-SOM-HAO interactions were not obviously influenced 447

by ionic strength, suggesting formation of inner sphere complexes (Gu and Karthikeyan 2008). 448

Chen and Huang (2009, 2010, 2011) show that Mn2+

, Cu2+

, MnO2, and Al2O3 catalyze 449

tetracycline transformations in soil including oxidations, dehydrations, isomerizations, and 450

epimerizations. The pathways by which these changes occur vary according to the metal, 451

resulting in various end products. A range of environmental fates for the tetracycline 452

transformation products ensues. 453

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2.4. Tetracycline transport through soils 454

Lysimeter-based studies are commonly used for examining leaching of tetracyclines through 455

soil. In a study where fresh pig manure slurry amended with 0.87 kg ha-1

OTC was added to 456

lysimeter cores (60 cm long x 24 cm diameter) of a sandy loam soil, HPLC analyses did not 457

detect OTC in any of the leachate samples (Blackwell et al. 2009). OTC was detected in the top 458

5 cm of soil in two lysimeters at concentrations of 27 and 94 µg kg-1

when manure was 459

incorporated into the soil, and at concentrations of 19 and 41 µg kg-1

in lysimeters where manure 460

had been applied to the soil surface (Blackwell et al. 2009). Detection of OTC in soil, but not in 461

leachate, is due to its strong sorption coefficient (though the authors did not provide a specific 462

Kd) and persistence in soil, as similarly concluded in numerous other studies (Rabølle and Spliid 463

2000; Hamscher et al. 2002, 2005; Aga et al. 2005; Kay et al. 2005). In a study involving 464

repeated field applications of pig manure containing tetracycline (4 mg L-1

) and chlortetracycline 465

(0.1 mg L-1

), TC and CTC were consistently detected in the top 30 cm of the soil, but there was 466

no evidence of TC or CTC in the 30-90 cm depth, nor were any tetracyclines found in 467

groundwater (detection limit was 50 ng L-1

) (Hamscher et al. 2002). 468

Despite large attractive forces between tetracyclines and soils, they may be transported 469

through the profile into groundwater via preferential flow processes (Kay et al. 2004; Aust et al. 470

2008). Increasingly alkaline soil pH may also contribute to tetracycline leaching since the 471

predominantly anionic species will not sorb as strongly to soils. In column studies investigating 472

the effect of acid rain on TC mobility in soil following surface application of TC-spiked chicken 473

manure (2000 µg TC kg soil-1

), TC was found in the top 20 cm at a concentration of 17.1 µg kg-1

474

after pH 3 rain, but was found at 0-40 cm depth at 6.57 µg kg-1

after pH 7 rain, demonstrating its 475

mobility in soil at higher pH (Pan and Chu 2017). In an extensive survey of three dairy farms in 476

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China, Zhou et al. (2013) analyzed the concentrations of 50 antibiotics in the fecal waste and 477

wastewater. Tetracycline and CTC were detected in the fecal samples at concentrations of 16.7 478

µg kg-1

and 1450 µg kg-1

, respectively. Tetracyclines (TC, OTC, and CTC) were consistently 479

found in association with suspended particles in wastewater at all three dairy farms at 480

concentrations ranging from 0 to 1710 µg kg-1

, with CTC usually occurring in the highest 481

concentrations (Zhou et al. 2013). Conversely, tetracyclines were rarely detected in the aqueous 482

phase (Zhou et al. 2013). 483

2.5. Influence of environmental conditions on tetracycline sorption and degradation 484

Environmental conditions also influence tetracycline sorption and degradation in soils. 485

Photolysis, heat, and microbial degradation have been implicated in tetracycline depletion in 486

manure piles (Storteboom et al. 2007). Degradation of tetracyclines correlates to temperature, 487

with greater persistence and accumulation in soils at low temperature (Hamscher et al. 2005; Li 488

et al. 2010), and higher temperatures stimulating increased molecular reactivity and greater 489

degradation (Loftin et al. 2008). Based on a high 86 ± 11% recovery of desorbed OTC from 490

diverse soils, Jones et al. (2005) conclude that removal of OTC from solution is due to sorption 491

mechanisms and not degradation. 492

Concentration of OTC in manured dairy calf bedding declined rapidly in the initial 10 days, 493

with a degradation half-life of < 10 d (De Liguoro et al. 2003). Moreover, OTC concentrations in 494

30-d and 105-d dry-piled manure differed according to superficial, intermediate, or deep position 495

in the pile. The superficial layer contained the highest concentrations, with amounts roughly 496

twice those found in the deep layer. Concentrations in the intermediate layer were roughly four 497

times less than the deep layer (De Liguoro et al. 2003). Potential factors responsible for the 498

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23

discrepancies between the positions in the manure pile include pH, antibiotic concentration, 499

temperature, and moisture. The strong attraction of tetracyclines to soils at low temperatures 500

implies that in those conditions, tetracyclines will be less available to degradation via abiotic or 501

biological processes. The higher temperature in the intermediate layer likely facilitated 502

tetracycline desorption and increased biological activity, thereby increasing its rate of 503

degradation (De Liguoro et al. 2003). In a comparison of high- and low-intensity manure 504

management, degradation rates of tetracyclines were greater in high-intensity management 505

systems in which manure piles were watered regularly, turned weekly, and amended with equal 506

parts dry leaves and fresh alfalfa (Storteboom et al. 2007). The increased degradation rate may 507

result from a greater moisture level that would support an active microbial community, or from 508

the increased oxygen level, supporting more efficient aerobic microbial degradation pathways. 509

Following incorporation of piglet manure, which initially contained approximately 100-300 510

mg CTC per kg dry wt manure, into the top 20 cm of two livestock field soils [a loamy sand soil 511

(A) and a sandy soil (B)], CTC dissipation was assessed and its half-life was 25 d in soil A and 512

34 d in soil B (Halling-Sørensen et al. 2005). Carlson and Mabury (2006) conducted a similar 513

CTC dissipation study, but using dairy manure spiked with an initial CTC concentration of 514

approximately 725 µg kg-1

and incorporated into the upper 12 cm of a sandy loam soil. Despite 515

the more than 100-fold less initial CTC concentration compared to the previous study, the 516

average half-life was 24 d in the manure-amended plots and 21 d in the non-manure plots. 517

Dissipation kinetics of OTC spiked in soils that were sterilized or non-sterilized, and under 518

aerobic or anoxic conditions (Yang et al. 2009) yielded significant differences between 519

treatments, and contrasting results from Carlson and Mabury (2006). For example, t1/2 of OTC in 520

aerobic, non-sterile soil A (initial concentration of 10 mg kg-1

) was 37 d, compared to 99 d when 521

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24

the soil was sterilized, or 62 d was non-sterile soil A was under anoxic conditions. Increasing the 522

initial OTC concentration in aerobic non-sterile soil A from 5 mg kg-1

to 30 mg kg-1

increased t1/2 523

from 29 d to 56 d (Yang et al. 2009).The contrasting half-life figures found in different studies 524

are due in part to compound structure, initial concentration, varying pH, soil redox status, 525

temperature, and microbial community. , The half-life of CTC measured in spiked soil interstitial 526

water in the dark at pH 3.0 and 4.1 was approximately 40 d, but dropped to about 2 d when pH 527

was increased to 8.5 (Søeborg et al. 2004). This pH variability could explain the high dissipation 528

rate of CTC (initial concentration 0.328 mg kg-1

) in spiked horse manure (pH 8.2-8.4), which had 529

t1/2 of 5.1 to 8.4 days (Storteboom et al. 2007). The initial concentration of tetracyclines in beef 530

feedlot manure of approximately 2 mg kg-1

declined to below the 0.01 mg kg-1

dry manure 531

detection limit within 6 months, while initial tetracycline concentrations in dairy manure of 532

approximately 0.4 mg kg-1

declined to below the detection limit within 4 months (Storteboom et 533

al. 2007). The discrepancy in initial tetracycline concentrations is expected since the beef cattle 534

are routinely administered subtherapeutic levels of tetracyclines, while they are given to dairy 535

cattle only for therapeutic purposes (Storteboom et al. 2007). 536

2.6. Tetracycline degradation products 537

Sparse information exists on the presence and fate of antibiotic degradation products in soils 538

following manure application. In addition to the parent molecule, four epimers of tetracyclines 539

can be excreted by livestock (Brambilla et al. 2007). Accordingly, Qiao et al. (2012) reported 540

that epimers 4-epitetracycline (ETC), 4-epi-chortetracycline (ECTC), and 4-epioxytetracycline 541

(EOTC) were the predominant degradation products detected in fresh and composted swine 542

manure samples. Though present in fresh swine manure, none of the anhydrous tetracycline 543

degradation products were detected in composted manure or treated soil samples, potentially 544

Page 24 of 53



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25

because dehydrated products only form in very acidic conditions (Qiao et al. 2012). Similar 545

results were achieved by Wu et al. (2011) who did not detect any of the 4-546

epianhydrotetracyclines in composted swine manure. In leachate samples taken from manure-547

amended soil columns, Kwon (2011) detected consistently elevated levels of CTC (0.075 ng mL-

548

1), almost continual release of ECTC, and in contrast to other reports, 4-epi-549

anhydrochlotetracycline (EACTC), albeit at low and infrequently occurring levels. Kwon 550

suggests detection of CTC and OTC parent molecules was due to increases in soil pH from 551

manure application, leading to increased mobility of the compounds. The minor presence of 552

epimers could be due to their formation requirement of acidic conditions. Considering that 553

anhydrous products exhibit a greater biological toxicity than the parent molecules, longer 554

composting times may mitigate potential biological risks from applying antibiotic-laden manure 555

to soils (Halling-Sørensen et al. 2002; Qiao et al. 2012). In a study of California dairy farms, 556

Watanabe et al. (2010) detected OTC, TC, and epi-tetracycline in surface soils following dairy 557

manure application at concentrations of 25 µg kg-1

, 8.8-105 µg kg-1

, and 163 µg kg-1

, 558

respectively. 559

Although CTC has a half-life of 5-84 days, half-lives of its degradation products can be as 560

high as 400 d in soil interstitial water (Søeborg et al. 2004). These recalcitrant metabolites 561

remain bioactive, thus able to continually exert selective pressure on soil microbes, which may 562

be responsible for persistence of ARG in soils past the time when parent compounds have been 563

depleted (Thiele-Bruhn 2003; Storteboom et al. 2007). Walczak and Xu (2011) likewise reported 564

persistence of antibiotic resistance in dairy manure with time, raising concern for the increased 565

distribution of ARG to the environment from manure application. 566

2.7. Biological risk of tetracyclines 567

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26

Assessing the biological risk of tetracyclines in soils is not simple. Their strong sorption and 568

persistence in soil would suggest low bioavailability (Tolls 2001), yet long residence times in 569

soil have been linked to development of antibiotic resistance (Kümmerer 2009, 2010; Kyselková 570

et al. 2015b). Allaire et al. (2006) saw no difference in CTC sorption between sterilized and non-571

sterilized sandy loam or heavy clay soils, illustrating a possible lack of biological influence on 572

tetracyclines during short residence times. In a study of plant uptake of various antibiotics from 573

soils, Boxall et al. (2006) noted decreased growth of carrots and lettuce when grown in soils 574

containing OTC residues. Modeling antibiotic transport in soils has been proposed as a method 575

for assessing potential environmental risks of antibiotics, but Blackwell et al. (2009) found that 576

predicted values produced from a commonly used pesticide fate model (FOCUS-PEARL v3.3.3) 577

highly underestimated antibiotic transport detected in field lysimeters. A multitude of factors 578

may contribute to the discrepancy, including interactions of manure colloids with antibiotics or 579

fluctuating temperature and pH, underscoring the complexity of predicting antibiotic fate in the 580

environment. Research suggests biological activity and toxicity of tetracyclines may be affected 581

when they are chelated with metals (Loke et al. 2002); however, metal type, complex speciation, 582

and organism tested all influence biological toxicity of chelated tetracyclines (Pulicharla et al. 583

2017). 584

In addition to the probable upsurge in antibiotic resistance of soil microbes, routine 585

application of manure to agricultural soils has raised concerns about potential risks to soil 586

microbial health, functioning, and community structure (Toth et al. 2011). For example, 587

chlortetracycline has been found to negatively impact microbial iron reduction in soil, with an 588

ED50 (effective dose with 50% inhibition of Fe-reduction) of 2.54 x 104 µg kg

-1 (Thiele-Bruhn 589

2005). However, when using lower concentrations of antibiotics, chlortetracycline demonstrated 590

Page 26 of 53



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no influence on iron reduction (Toth et al. 2011). Other microbial ecosystem functions adversely 591

influenced by antibiotics in soil include methanogenesis, nitrogen cycling, and sulfate reduction 592

(Ding and He 2010). Microbial community diversity as determined by Shannon’s diversity 593

index, as well as microbial substrate utilization, was significantly reduced with additions of OTC 594

to soil, even at OTC concentrations as low as 1 µM (Kong et al. 2006). 595

3. Conclusion 596

Tetracycline antibiotics enter agricultural soils from land-application of dairy manure. 597

Numerous factors affect their behavior in soils including soil pH, soil mineralogy, organic 598

matter, and the microbial community. Complex interactions make it difficult to readily predict 599

the fate of these CECs in soil. Soil solution pH, ionic strength, and soil organic matter content 600

especially influence tetracycline fate in soils. Due to their amphoteric structures, the behavior of 601

tetracyclines in soil is fundamentally governed by pH, which dictates the charge and physical 602

properties of the compounds. In typical soil pHs, these compounds bind to soil, yet soil organic 603

matter and metals greatly impact that association, and given the likelihood of high SOM and 604

metal content in manure, these variables must be seriously considered. In general, tetracyclines 605

are sorbed by soils at typical soil pHs. While this may be advantageous to waterways, its 606

consequential promotion of antibiotic resistance in microbes poses a serious threat to human 607

health. Moreover, tetracyclines degrade to form secondary products that exhibit yet additional 608

properties distinct from their parent molecules. Given the variable interacting factors that affect 609

tetracycline behavior in soil, additional research is needed to make well-informed management 610

decisions regarding land-application of dairy manure. 611

612

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4. Acknowledgements 613

This work was supported by the Agriculture and Food Research Initiative competitive grant 614

2013-67019-21375 from the USDA National Institute of Food and Agriculture. Financial support 615

was also provided by the University of Idaho, Department of Plant, Soil, and Entomological 616

Sciences. 617

618

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