Antibiotic Uptake by Vegetable Crops from Manure-Applied Soils

Final Report for LNC08-294

Project Type: Research and Education
Funds awarded in 2008: $139,420.00
Projected End Date: 12/31/2010
Region: North Central
State: Minnesota
Project Coordinator:
Dr. Satish Gupta
University of Minneota
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Project Information

Summary:

Since their discovery, antibiotics have been instrumental in treating infectious diseases that were previously known to kill humans and animals. However, their widespread use as a feed additive in food animal production has raised concerns on the possibility of antibiotics appearance in the food supply particularly organically grown foods where animal manure is a major source of plant nutrients. This study characterized the extent of antibiotic uptake by vegetable crops from soils that have been fertilized with antibiotics mixed turkey manure and hog manure, both fresh and after composting. The study involved composting hog and turkey manures in the field, characterizing antibiotic degradation during composting, setting up the field plots at two sites representing clay loam and sandy loam soils, growing 11 different vegetable crops, and characterizing antibiotic presence in various plant tissues and soil at harvest. Nutrient source treatments were fresh and composted hog manure, fresh and composted turkey manure, and inorganic fertilizer. Antibiotics were mixed into the manure as a concentrated feed or in a powder form. Targeted antibiotic concentrations were 10 mg L-1 for liquid hog manure or 10 mg kg-1 for solid turkey manure on dry weight basis. Composting of liquid hog manure was done by mixing wood shavings with manure containing antibiotics and then composting the mixture in cement bins. Vegetable crops tested for antibiotic uptake were spinach, lettuce, cabbage, carrots, radish, onion, garlic, tomatoes, green pepper, sweet corn and potatoes. Antibiotics analyzed in manure, soil, and plant tissues were chlortetracycline, tylosin, monensin, virginiamycin, and sulfamethazine. All analysis was done with ELISA kits.

The results showed that except for chlortetracycline, antibiotic degradation was slower during composting of turkey manure than composting of hog manure. During composting of hog manure, tylosin had the shortest half-life (5 days) followed by chlortetracycline (46 days), sulfamethazine (74 days) and monensin (128 days). Comparatively during composting of turkey manure, tylosin had a half-life of 16 days, followed by chlortetracycline (17 days), virginiamycin (25 days), monensin (257 days) and sulfamethazine (462 days). Except for sulfamethazine and one case of monensin, soil antibiotic concentrations were below the limits of quantification (LOQ) at both planting and at harvest for all antibiotics. Monensin concentration in raw hog manure treatment at planting at both sites and concentration of sulfamethazine at planting and sometimes at harvest for all manure treatments at both sites were above the LOQ. Between planting and harvest, there was greater degradation of sulfamethazine in the Staples plots (49% to 95%) than in the Waseca plots (21% to 99%). Except for some isolated treatments, concentrations of monensin, tylosin, chlortetracycline, and virginamycin in plant tissues of all vegetable crops were below the LOQ at both sites. For some manure treatments, sulfamethazine concentrations were higher than the LOQ for almost all vegetable crops. However, there was no consistent pattern between plant sulfamethazine concentration and its corresponding soil concentrations. In general, sulfamethazine concentrations were higher in radish skin than radish root, carrot skin than carrot root, and potato skin than potato tuber. These differences may be due to direct contact of the skin of these below ground vegetables with soil containing antibiotics and possibly due to lower water content of the skin than inner tissue. Sulfamethazine concentration in most vegetable tissues was less than 10 µg kg-1.

The second objective of this study was to document the presence or absence of antibiotics in certified organic vs. conventional commercial vegetables that have been grown in manure applied soils. For this objective we surveyed a group of five certified organic vegetable growers and two conventional vegetable growers who use either fresh or composted manure on their farm. We analyzed their manure, soil, and vegetable samples for presence of antibiotics. We also analyzed the vegetable produce from two grocery stores who sell certified organic produce. Concentrations of antibiotics found in 5 farms (where samples were available) were much lower than the concentration of antibiotics used in our controlled field study at Waseca and Staples. Soil samples from these farms also did not exceed the LOQ of tetracycline, virginiamycin, and monensin. There were two farms where sulfamethazine concentrations in soil samples were higher than the LOQ. Except for a few cases, concentrations of all antibiotics in vegetables were below the LOQ. Highest sulfamethazine concentration (27.34 µg kg-1) occurred in lettuce harvested in one certified organic farm. In two other certified organic farms, 18% and 54% of vegetables had higher sulfamethazine concentration greater than the LOQ. The conventional turkey manure farm had the highest number (78%) of vegetables containing sulfamethazine. Although some of the concentrations detected are higher than the concentration we detected in vegetables from our experimental plots, they are still relatively low concentrations. There was no specific trend among vegetables in terms of sulfamethazine presence above the LOQ. Except for few cases, antibiotic concentrations in vegetables from two grocery stores were generally below the LOQ. Since vegetables from producers and grocery stores were not analyzed in replicates, we are uncertain whether these concentrations above the LOQ are real or false positives. Overall, various antibiotic concentrations detected in vegetables from our field experiments, farmers’ fields, and from grocery stores are relatively low.

Several organizations have established acceptable daily intake (ADI) values for some of the veterinary pharmaceuticals. The ADI values indicate levels that can be ingested daily over a lifetime without any health risks. Most ADI values given for many veterinary pharmaceuticals are less than 10 µg kg-1 body weight per day. Assuming body weights varying from 50-75 kg, total permissible intake will be equivalent to 500-750 µg of pharmaceutical per day. The maximum concentration of antibiotics in vegetable produce in this study is less than 30 µg kg-1 wet weight. This means, one would need to consume 17-25 kg of produce every day to reach the ADI values suggested by various organizations. These simple calculations suggest that antibiotic uptake by various vegetable crops analyzed in this report is not of a major health concern to most adults unless one is allergic to that particular pharmaceutical. In this study, we only characterized the potential uptake of five antibiotics. Because there are other pharmaceuticals that are also administrated to food animals and are excreted in manure, additional studies will be needed to address the potential of those pharmaceuticals to appear in vegetable crops from manure applied soils.

Since our study shows that most antibiotics degrade over time, a practical guideline for organic producers who use conventional manure for plant nutrient source will be to (1) apply composted manure rather than fresh manure, and/or (2) let the manure sit in the soil for as long as possible before planting vegetables in the spring. This extra time will help degrade antibiotics and thus lower the concentration available for plant uptake.

A pdf copy of the full report is available at the following url:
https://netfiles.umn.edu/xythoswfs/webui/_xy-22381669_1-t_adwst0WX

Acknowledgements

The authors gratefully acknowledge the funding for this project from the North Central Region Sustainable Agriculture Research and Education (NCR-SARE) program. Earlier work on antibiotic uptake by vegetables by Dr. Kuldip Kumar and subsequently by Dr. Holly Dolliver and Dr. Kumar form the basis for this project. The senior PIs are grateful to Drs. Kumar and Dolliver for laying the foundation for this project. There were several individuals who helped the PIs in their field and laboratory work. We are thankful to Josh Worely for his help in vegetable harvest and in laboratory processing of vegetable produce; Dr. Charlie Rohwer, Curt Miller, Scott Coy, Sue Schoenfeld, Kraig Deling, and Gabriel Malima for their help with composting of manure, field set-up and maintenance of vegetable plots at Southern Research and Outreach Center at Waseca; and Bob Schaefer, Michelle Johnson, Matt McNearney, Anina Christensen, Brendan Ward and Anubha Garg for their help with plot set-up and other field work at Central Lakes Irrigation Center at Staples. The senior PI also acknowledges helpful discussions with Mr. Tom Halbach on manure composting. The authors are grateful to both certified organic and conventional vegetable growers for their participation in the survey part of the study.

Introduction:

Since their discovery, antibiotics have been instrumental in treating infectious diseases that were previously known to kill humans and animals. However, their widespread use as a feed additive in food animal production has raised concerns on (1) the development of antibiotic resistance bacteria in the environment and (2) the appearance of antibiotics in food and water supplies. The main pathway for these impacts is when manure containing antibiotics is land applied (Kumar et al., 2005b).

According to the Institute of Medicine about 22.7 million kilograms of antibiotics are used annually in the United States out of which 60% is used in human medicine, and 32% and 8% for non-therapeutic and therapeutic use in livestock agriculture (Shea, 2003). On the other hand, the Union of Concerned Scientists estimate that 15.9 million kilograms of antibiotics are used annually in the United States out of which 13% is used in human medicine, 78% and 6% for non-therapeutic and therapeutic use in livestock agriculture, and 3% in pets (Shea, 2003). Non-therapeutic use of antibiotics in animal agriculture is not only to increase the animal’s ability to absorb nutrients from feed and reach market weight on time, but also to counteract the effects of crowded living conditions and poor hygiene in intensive animal agriculture. Although the antibiotic dose varies from 1 to 100 g per ton of feed depending upon type and size of the animal and the type of antibiotic, as much as 80% of the antibiotics administered orally may pass through the animal unchanged in urine and manure (Levy, 1992).

There is very little literature on antibiotic uptake by plants from manure applied soils. Most of the earlier research was directed towards identifying stimulation or toxicity of antibiotics to plants (Norman, 1955; Batchelder, 1981 & 1982). Milgiore et al. (1995) tested the toxicity of sulphadimethoxine and found that 300 mg/L of sulphadimethoxine in agar plate altered the normal development and growth of roots, hypocotyls, and leaves of millet, peas, and maize. Final bioaccumulation amounts in root and stalk/leaves were: 2070 µg/g and 110 µg/g for millet over an 8 day period, 178 µg/g and 60 µg/g for peas over an 18 day period, and 268 and 12.5 µg/g for maize over an 8 day period. Later, Milgiore et al. (1996) showed that sulphadimethoxine bioaccumulation varied in barley roots and foliage from 48 to 79 µg g-1 and 12 to 19 µg g-1. Jjemba (2002) summarized the literature on potential impact of veterinary and human drugs in manure and biosolids on plants and concluded that phytotoxicity of these drugs varies between different plant species and depended upon soil and biosolids characteristics. However, many of the studies in the literature like those above were done in cultures, agar, or soils that were artificially spiked with antibiotics.

The earlier research on antibiotic uptake by vegetable crops was reported by Bewick (1979) who used tylosin and terramycin (oxytetracycline) fermentation wastes as sources of fertilizer for tomatoes. The authors reported that there was no antibiotic detected in any of the tomatoes when these wastes were mixed with compost containing peat. However, two recent greenhouse studies by the associates of the investigators showed that onions, cabbage, and corn take up chlortetracycline and corn, lettuce, and potato take up sulfamethazine from soils mixed with antibiotic containing fresh (non-composted) hog manure (Kumar et al., 2005a; Dolliver et al., 2007). The chlortetracycline treatments included manure where antibiotics had passed through the animal gut as well as manure that was artificially spiked. The chlortetracycline uptake amount increased from 0 to 0.017 µg/g of fresh weight with an increase in antibiotic concentration in soil from 0 to 1600 ?g/pot. However, there was no uptake of tylosin by corn, cabbage, and lettuce possibly due to the larger size of its molecule. The soil used in this experiment was Hubbard loamy sand.

Dolliver et al. (2007) showed a significant difference in sulfamethazine uptake between the control (soil amended with manure with no antibiotics) and the sulfamethazine treatments (soil amended with manure containing sulfamethazine). However, there was no difference in sulfamethazine uptake between 50 mg/L (5 mg pot-1) and 100 mg/L (10 mg pot-1) treatments. The soil used in this experiment was a Waukegan silt loam mixed with sand, peat and compost. Both studies were done for six weeks and antibiotics were only measured in the leaf tissue. From these studies it is not apparent whether or not antibiotic concentrations remains the same in leaves if allowed to grow for full maturity (growing season) and also if antibiotic concentrations in fruits (edible part) are at the same level as in leaves.

A recent study in UK reported the uptake of 10 veterinary medicines by lettuce and carrots from soils that had been artificially spiked with these drugs (Boxall et al., 2006). Seven antibacterial drugs included amoxicillin, enrofloxacin, florfenicol, oxytetracycline, sulfadiazine, trimethoprim, and tylosin. The results showed that after 5 weeks of plant growth, florfenicol, and trimethoprim were present in lettuce at concentrations of 15 µg/kg and 6 ?g/kg whereas enrofloxacin, florfenicol, trimethoprim were present in carrot roots at concentrations of 2.8 µg/kg, 5 µg/kg, and 5.3 µg/kg. The nominal antibiotic concentration was 1.5 mg of a given antibiotic in each container of light loamy sand with low organic matter content. The authors also reported that enrofloxacin (8.5 µg/kg) and florfenicol (38 µg/kg) were higher in carrot peel than the carrot root. However, oxytetracycline, tylosin and sulfadiazine concentrations in lettuce and carrot were below the detection limits. Using the acceptable daily intake (ADI) values reported in the literature for antibiotics in meat, both Boxall et al. (2006) and Dolliver et al. (2007) concluded that antibiotic concentrations in plants did not pose any appreciable risk to a majority of the population. However, both authors concluded that the prolonged effect of continued exposure may be important in some populations which have a weak immune system.

Recently, Herklotz et al. (2010) used cabbage (Brassica rapa var. pekinensis) and Wisconsin Fast Plants (Brassica rapa) to test potential uptake and accumulation of carbamazepine, salbutamol, sulfamethoxazole, and trimethoprim at 232.5 µg L-1 concentrations under hydroponic conditions. All four chemicals were detected in the root and leaves of cabbage. Concentration in roots were 98.9 ?g kg-1 carbamazepine, 114.7 µg kg-1 salbutamol, 138.3 µg kg-1 sulfamethoxazole, and 91.3 µg kg-1 trimethoprim. While all four pharmaceuticals were detected in leaf, stem, and root of Wisconsin Fast Plant, only carbamazepine and salbutamol were detected in the seedpods. In an ecological risk assessment study of tricolsan effects on cucumber, corn, ryegrass, soybean, tomato, and wheat in quartz sand, Reiss et al. (2009) found significant reduction in mean shoot length at the two highest concentrations tested (280 and 930 µg kg-1). In this study, shoot and root dry weight also decreased significantly at the highest concentration relative to the control.

With greater emphasis on sustainable farming, there is even greater demand for organic foods, where manure is the primary source of nutrients. At this time, there are no guidelines on the presence of antibiotics in manure used for certified organic farming.

Furthermore, most of the studies in the literature have been done on a few vegetable crops and only for a short time. Also, the previous research does not indicate the extent of antibiotic uptake under natural soil conditions. Furthermore, there are hardly any data on antibiotic uptake by vegetable plants grown in soils mixed with composted manures.

Approved Antibiotic for use in Agriculture

Agricultural producers use antibiotics in feed and water not only to treat, prevent, and control disease, but also to encourage growth and feed efficiency. McEweon and Fedorka-Cray (2002) observed that about 83% of food-producing animals are administered at least one antibiotic for disease prevention or growth promotion. According to Shea (2003), roughly 90% of the antibiotics used in agriculture are administered for non-therapeutic uses such as growth promotion. Table 1 lists FDA approved antibiotics for growth promotion as well as for treatment of food animals. In this study, we selected five antibiotics for plant uptake from manure applied soils. The antibiotics were: chlortetracycline, monensin, tylosin, virginiamycin, and sulfamethazine. The following text briefly describes the properties of these antibiotics.

Chlortetracycline: Tetracyclines are widely used in livestock production for therapeutic treatment of intestinal and respiratory infections and also for growth promotion. Chlortetracycline, the first member of tetracycline group, was discovered in the late 1940s by Dr. Benjamin Duggar. Tetracycline inhibits cell growth by inhibiting protein synthesis. Tetracyclines are excreted as active compounds and enter the soil environment when manure is land applied as a source of nutrients for crops. De Liguoro et al. (2003) applied manure to soil from calves treated with oxytetracycline (820 ?g/kg) and found that oxytetracycline was present in soil even after 5 months but it could not be detected in water. Hamscher et al. (2002) reported that tetracycline and chlortetracycline could be measured in a soil treated with liquid manure at soil depths down to 30 cm and are known to persist and accumulate in soil. However, persistence of chlortetracycline depends upon the soil temperature. Gavalchin and Katz (1994) showed that there was no degradation of chlortetracycline applied at 5.6 mg kg-1 of soil at 4°C after 30 days. However, only about 44% and 88% of the applied chlortetracycline remained in the soil at 30°C and 20°C, respectively. Recommended dosage for turkeys is 25 mg of tetracycline per pound body weight (FDA Center for Veterinary Medicine). Figure 1 shows the molecular structure and Table 2 lists the molecular properties of tetracycline.

Monensin: Monensin belongs to the ionophore (ion-carrier) group of antimicrobials mainly used in veterinary medicine (Haney and Hoehn, 1967). Among several identified types, monensin A is derived from the fermentation of the fungus Streptomyces cinnemonensis and is a sodium-selective carboxylic ionophore. Monensin is approved as a growth promoter for cattle and poultry but not for swine. Monensin is the only FDA permitted feed additive for lactating cows to increase milk production. Monensin enhances growth and feed efficiency in cattle by altering ruminal fermentation and in poultry by preventing disease (Tedeschi et al., 2003).

Approximately 1.5 million kilograms of monensin is used subtherapeutically in the cattle and poultry industry in the USA (Mellon et al., 2001). Monensin prevents and controls coccidiosis in cattle and calves (Mellon et al., 2001). However, more than 90% of monensin is excreted and concentrations greater than 1 mg L-1 have been detected in manure (Kumar et al., 2005b). Unlike many antibiotics used in veterinary medicine, monensin is not used in human medicine because it is highly toxic (Dolliver et al., 2008a). Monensin has also been shown to adversely affect plant growth at concentrations >1 mg kg?1 (Brain et al., 2004). Although there is potential for monensin to persist in the environment due to lack of hydrolysis and slow photolysis, the biodegradation rates suggest a rapid biological attenuation (Tedeschi et al., 2003). Monensin half-life has been reported from less than 2 days (Sassman and Lee, 2007) to 13.5 days (Carlson and Mabury, 2006). Sassman and Lee (2007) reported the partition coefficient of monensin varies from 0.92 to 78.6 (L kg-1) for various soils at an aqueous phase concentration of 0.05 ?mol L-1. Figure 2 and Table 3 show the molecular structure of monensin and its properties.

Tylosin: Tylosin was identified as the top ten most frequently detected antibiotics in surface water in the United States from 1999 to 2000 (Kolpin et al., 2002). Tylosin is one of many macrolide antibiotics that is used exclusively in veterinary applications. When used as a therapeutic drug, tylosin inhibited the ability of bacteria to synthesize its proteins. Tylosin is used as a growth promoter in swine, cattle, and poultry production (Rabølle and Spliid, 2000). For growth promotion, tylosin concentrations in swine feed ranges from 10 to 100 g tylosin/ton feed (Elanco Animal Health Tylan® Premix product label). Figure 3 shows the molecular structure of tylosin and Table 4 lists it molecular properties.

Sulfamethazine: Sulfamethazine belongs to sulfonamides class of antibacterial. Sulfonamides were discovered in the 1930s and have been widely used in aquaculture, agriculture, and in veterinary and human medicine. Sulfamethazine is used for treatment of respiratory disease and for growth promotion in cattle, sheep, swine, and poultry. Daily recommended dose of sulfamethazine sodium in drinking water per pound body weight varies from 56 to 113 mg for swine and 53 to 130 mg for turkey (FDA Center for Veterinary Medicine). Sorption of sulfamethazine decreases with an increase in pH (Boxall et al., 2002). Sukul and Spiteller (2006) reported sulfamethazine concentration as high as 40 mg kg-1 in liquid manure from swine and calves. Figure 4 and Table 5 show the molecular structure of sulfamethazine as well as its properties, respectively.

Virginiamycin: Virginiamycin belongs to the streptogramin class of antimicrobials and is known to promote livestock growth by increasing ileal amino acid digestibility (Stewart et al., 2010). Virginiamycin has also been used to prevent and control diseases in poultry, swine, and cattle. It consists of two principal components designated as macrolactone virginiamycin M1 and the hexadepsipeptide virginiamycin S1. The commercial product contains about 75% of the M1 factor, 20% of the S1 factor and 5% of other minor analogues. The presence of the S component markedly enhances the antimicrobial activity of the M component. Virginiamycin is also used in the ethanol (biofuel) industry to prevent microbial contamination. Figure 5 shows the molecular structure of virginiamycin M1 and S1 factors and Table 6 lists their molecular properties.

Except for monensin, all other antibiotics discussed above can be used in swine and poultry production. Monensin cannot be used in swine as subtherapeutic agent. Table 7 lists the residue limits in various meat tissues for all five antibiotics used in our study. Antibiotic residues in animal products are regulated by the United States Department of Agriculture, Food Safety and Inspection Service. According to Van Dresser and Wilcke (1989), penicillin, oxytetracycline, and sulfamethazine are the most frequently detected residues in food products.

Figures and Tables for Introduction Section

Project Objectives:

The goal of this study was to determine the extent of antibiotic uptake by vegetable crops from soils that have been fertilized with antibiotic laden turkey manure and hog manure, both fresh and after composting. In this paper, we refer to fresh manure as manure that is just taken out of the turkey barn or from a hog lagoon and has not gone through the composting or extensive curing process.

Specific objectives of the study were: (1) to test the extent of antibiotic (chlortetracycline, tylosin, monensin, virginiamycin, and sulfamethazine) uptake by eleven vegetable crops (spinach, lettuce, cabbage, carrots, radish, onion, garlic, tomatoes, green pepper, sweet corn and potatoes) from two different textured soils that have been fertilized with antibiotic-containing turkey manure and hog manure, (2) to document the differences in antibiotic uptake by vegetables that have been fertilized with fresh vs. composted turkey and hog manures, and (3) to document the presence or absence of antibiotics in certified organic vs. conventional commercial vegetables that have been grown in manure applied soils.

Since sulfamethazine has been shown to be taken up by vegetable plants and since it is also slow in degrading during composting (Dolliver et al., 2007), we were further interested in studying the adsorption behavior of sulfamethazine in our two soils. Recently, Kahle and Stamm (2007) suggested that presence of phosphorus may have some effect on sulfamethazine adsorption on soils. Since manure contains significant amount of phosphorus, an additional objective of this study was to test the adsorption potential of sulfamethazine in our two soils both with and without the presence of phosphorus.

Cooperators

Click linked name(s) to expand
  • Yogesh Chander
  • Vince Fritz
  • Dong Hee Kang
  • Helene Murray
  • Carl Rosen
  • Ashok Singh

Research

Materials and methods:

Field Study:

The field study was conducted at two locations, the Southern Research and Outreach Center (SROC) in Waseca, MN and the Central Lakes College Agricultural Station in Staples, MN. The soil at the Waseca site is Webster clay loam whereas the soil at the Staple site is a Verndale sandy loam. Eleven vegetables representing leafy greens, root crops, and fruit were grown in soils amended with four manure treatments and a control (no manure). The four manure treatments were: 1) fresh turkey manure, 2) composted turkey manure, 3) fresh hog manure, and 4) composted hog manure. Hog manure was taken from the gestation barn at SROC hog facility at Waseca, MN whereas the turkey manure was obtained from a conventional farm where antibiotics are used per industry standard. Limited antibiotics had been used at the hog facility at SROC. Composting of both manures was done at Waseca.

Hog Manure Composting: Hog manure composting involved mixing liquid hog manure with wood shavings made from pellets that have not been treated with any chemical. A total of 70 cubic yards of wood shavings were mixed with 19,021 gallons of liquid hog manure containing antibiotics (Fig. 6). Liquid manure was applied to wood shaving in three batches on 7 July 2008. The first batch of liquid manure (6,330 gallons) contained antibiotics. Table 8 lists the amount of antibiotic feed or powder that was mixed with manure prior to its mixing with wood shavings. Mixing of antibiotics involved adding known amount of antibiotic feeds containing chlortetracycline, tylosin, and monensin and powder sulfamethazine in 250 gallons of manure from the pig nursery. Mixing was done by circulating manure in the container using a small sump pump. Antibiotics were mixed with pig nursery manure on 2 July 2008 which was then added to the first batch of liquid manure in a large tank. Manure in the large tank (>6,000 gallons) was thoroughly mixed by running the pump. After mixing, the manure was applied to the wood shavings. The water content of wood shavings was 11.63 ± 0.65 g per 100 g.

Wood shavings and liquid hog manure mixing was done with a front end loader on a cement slab with walls on three sides. The floor had some vents that were thought to be closed. However, one of the vents was inadvertently left open and as a result there was some leakage of manure from the third batch of manure. We estimated we lost about 1000-4000 gallons of the last batch. Since the first batch of manure contained all antibiotics and this manure was soaked up by the wood shavings, we assumed no antibiotics were lost from the leakage of the third batch of manure. On 10 July 2008, we moved the manure mixed shavings to two cement bins for composting.

Every two weeks the compost pile was mixed by moving the composting material from one bin to another bin (Fig. 7). The composting material was moved with a front end loader. Each bucket of composting material was thinly spread in the new bin and small amount of water was sprayed on the material with a hose. This process was repeated until the compost pile was fully rebuilt again. Table 9 lists weekly average water contents and temperatures of the compost pile before mixing. Figure 8 shows a plot of daily temperature variation in the composting piles.

Turkey Manure Composting: Turkey manure compost was started on 12 August 2008. Twenty three tons of turkey manure was hauled from a conventional turkey growers farm to SROC. The grower stated that he had used neomycin in this flock. In his nursey flock, he had also used BMD (Bacitracin Methylene Disalicylate). He also mentioned that he uses coccidiostats such as coban and avatech depending upon the feed mill but some flocks receive Coccivac vaccine instead. Water based antibiotics were used for treatment only and these included primarily SDM/Albon (sulfamethazine) or chloratetracycline (CTC) and on rare occasions it included penicillin.

Turkey composting was done on a cement slab enclosed with three walls. Turkey manure was unloaded on a cement pad and mixed in with antibiotic feed or powder using a front end loader (Fig. 9). Water was also sprayed on the turkey manure pile as it was mixed in with antibiotics. A total of 1025 gallons of water was added to the manure pile before composting. The antibiotics added included CTC, Tylosin, monenesin, sulfamethazine, and virginiamycin (Table 8). Just like hog manure compost pile, turkey manure compost pile was also inverted/mixed every two weeks. To ensure efficient composting, water was added periodically to the turkey manure pile. All mixing was done with a front end loader. Table 10 lists the water content of the turkey manure composting pile. Figure 10 shows the variation in temperature of the turkey manure pile duirng composting.

Samples of the hog and turkey manure compost piles were taken on 18 September 2008 for TKN analysis. Another set of samples were taken on 9 October 2008 for NH4-N and percent ash content. Table 11 summarizes the results of these analyses.

Antibiotic Mixing in Fresh Hog and Turkey Manures: Fresh hog and turkey manure were mixed with the same antibiotics that we used in composted manure. We aim to have antibiotic concentration of 10 mg L-1 of each antibiotic in liquid hog manure and 10 mg kg-1 dry turkey manure in fresh turkey manure. For fresh hog manure, antibiotics were added @ 686.5 g of CTC-50, 858g of tylan-40, 429 g of rumensin-80 in 2000 gallons of liquid hog manure. Again the liquid hog manure was obtained from SROC.

For turkey manure, antibiotics were added in proportion to what was added to turkey manure before compost. We added 362 g of CTC-50, 451 g of tylan-40, 226 g of rumensin-80, 39.8 g of sulfamethazine and 637.2 g of Stefac-20 to 5.7 metric tons of wet turkey manure. This was in proportion to the amount that was added in 20.88 metric tons of turkey manure before composting. Fresh turkey manure (5.7 metric tons) was again obtained from the same farm on 23 October 2008. Antibiotics were mixed in the fresh turkey manure in the driveway at SROC. Feed CTC, tylan, rumensin, and stefac were hand broadcast over the turkey manure. Sulfamethazine was first dissolved in 5 gallon of water and then solution was sprayed with a cup on the turkey manure. Turkey manure was mixed with a front end loader and then covered up with a tarp and allowed to cure for 2 days. Table 12 lists some of the nutrient analysis in the fresh turkey manure sample.

Application Rate: Composted and fresh hog and turkey manures were applied based on N availability. For composted manure, it was assumed that only 10% of the organic-N and all of the NH4-N was available first year. For both hog and fresh turkey manures, N calculations were solely based on TKN analysis. For fresh turkey manure, we assumed 55% of TKN was available (Blanchet and Schmitt, 2007). For fresh hog manure, we assumed 50% of the TKN was available in first year (Schmitt, and Rehm, 1998). Based on our analysis, we calculated applications of 175 kg of composted hog manure, 14.9 kg of composted turkey manure, 87 liters of fresh hog manure, and 7.72 kg of fresh turkey manure for each 3.1 m x 3.1 m (10 x 10 feet) plot.

Staples Plots: All manure application rates were based on application of 112 kg-N ha-1. The fertilizer (control) treatment was also supplied with an equivalent amount of N with urea. Based on the initial analysis of fresh and composted manure, we estimated following applications of manure to 3.1m x 3.1 m plot:
Composted hog manure= 175 kg; Fresh hog manure=87 liters
Composted turkey manure=14.9 kg; Fresh turkey manure=7.7 kg
Fertilizer treatment =226.6 g of urea, 28 kg/ha of P2O5 and 56 kg/ha of K2O

Composted hog and turkey manure were first applied to plots at Staples on 21 October, 2008. Composted hog manure was applied with a front end loader whereas composted turkey manure was applied with 5 gallon buckets. Front end loader weight was calibrated to 166 kg of composted hog manure by loading it with weighed number of 5 gallon buckets of composted hog manure. This weight is slightly different than the target weight stated above because not enough composted hog manure was delivered to Staples. Composted turkey manure was directly weighed in 5 gallons buckets and hand applied. After application of composted manures, plots were roto-tilled to incorporate the composted manure in each plot.

Fresh hog and turkey manures were applied to Staples plots starting on 29 October 2008. Fresh hog manure was transported from Waseca to Staples in 120 gallon tanks whereas fresh turkey manure was transported in 5 gallon buckets. Fresh hog manure was first applied to 5 garlic plots @ 87 liters per plot.

Except for the liquid hog manure plots, garlic was planted at Staples on 30 October 2008. Garlic was not planted in fresh hog manure plots because they were too wet from manure application. In fresh hog manure plots, garlic was planted on 10 November 2008. Eighty cloves were planted in each plot. Their weight varied from 0.6 to 0.8 kg. Smaller cloves were planted on the outside of each plot.

Fresh turkey and hog manure was again transported to Staple and applied to additional plots on 4 November 2008. From here on antibiotics were mixed in each fresh hog manure tank. Mixing was done by stirring the tank with a stick. Another load of fresh hog manure was taken to Staples on 11 November 2008. At the end of this year, there were still 6 plots that needed fresh hog manure. Manure on these plots was applied on 21 and 22 April 2009. Manure was roto-tilled in the plots next day. We also broadcast applied the fertilizers to the control treatment and then mixed in with the soil with the tillage equipment. On 2 June 2009, it appeared that plants in composted hog manure plots were under N stress. This may be because of high C:N ratio of wood shavings. We decided to add 50 lbs of N as urea to composted hog manure plots.

The eleven vegetables that were grown were: leaf lettuce (‘Glossy Green’ or ‘Green Wave’), spinach (‘Bloomsdale Long Standing’), cabbage (‘Quisto’), carrot (‘Legend’), radish (‘Crimson Giant’), onion (‘Norstar’ or ‘Copra’), garlic (‘Music’), tomato (‘Sunshine’), pepper (’Alliance’), sweet corn (‘Ambrosia’ ), and potato (‘Yukon Gold’ ). Garlic was planted in the fall following fresh and composted manure application and the other vegetables were planted at appropriate times the following spring. Before planting in the spring, all plots except garlic plots) were tilled twice with a disc to create fine seed bed conditions. During this mixing it is likely that raw and composted manure were nearly uniformly mixed in with soil. Lettuce, spinach, radish, carrot, and sweet corn were direct seeded. Cabbage, tomato, and pepper were seeded into plug trays in the greenhouse and then transplanted at appropriate times. Potato was planted from B size tubers. Population densities and plant spacing were in accordance with the established production methods for each crop (Table 13). Both Waseca and Staples sites were irrigated overhead as needed. Peppers and tomatoes were planted in the greenhouse on 3 April and 9 April 2009, respectively. Table 13 lists the planting of various vegetable crops at Staples and Waseca. All crops were planted or transplanted in 30 inch rows.

We used a planter wheel to plant all crops except cabbage, pepper, and tomatoes. Tomatoes were planted 15-23 cm deep hole whereas pepper and cabbage were planted in 10-15 cm and 8-10 cm deep holes, respectively. One cup of fertilizer solution of Millers Sol-u-Gro (12-48-8 fertilizer) mixed at the rate of ½ cup in 5 gallons water was also applied to each transplant and then covered with soil.

Waseca Plots: Fresh hog manure was injected on 26 October 2008 @525 gallons per pass with a total of 4 passes on all the plots. The application rate was 11,438 gallons per acre. Fresh and composted turkey manure and composted hog manure was surface applied on 30 October 2008. Fresh and composted turkey manure and composted hog manure were weighed separately for each plot. Rates of their application were same as at the Staple site. After manure application, all plots were chisel plowed to mix the manure into the soil. A planter was used to build up ridges in the plot for garlic planting. Garlic was planted on 3 November 2008. The planter was lifted up between plots to prevent cross contamination of plots. The application rate for urea was the same as for the Staples plots. Fertilizer treatment also received 28 kg/ha of P2O5 and 56 kg/ha of K2O. Vegetable types, varieties, and spacing were same as at the Staples site.

Plot Design

Plots were laid out as randomized complete block design. Three blocks represented the three replications. Figures 11 and 12 show the layout of the plots at Staples and Waseca. Within each block, nutrient source (manure and fertilizer) treatments were randomized. Within each nutrient source treatment, the sequence of crop layout was same for all replications. This arrangement helped facilitate the field operations.

Plots were established with fertilizer (Fer), raw hog (RH) manure, composted hog (CH) manure, raw turkey (RT) manure, composted turkey (CT) manure treatments as the main plot and vegetable as the subplot. Each treatment was replicated three times. Weeds were controlled by hand hoeing. Each subplot was 3.1 m x 3.1 m with a separation of 1.53 m (5 feet) in between plots, both horizontally as well as vertically. Vegetables were hand harvested at peak maturity and a sub sample was analyzed within a day.

Vegetable Harvest and Processing

Vegetables were hand harvested at peak maturity and a sub sample of the produce was processed within a day. Processing involved carefully washing the vegetables with distilled water, wiping the extra water of the vegetables with paper towels, chopping the vegetables in coarse chunks, taking a subsample of chopped vegetable for water content, and then taking another subsample (20 g) of chopped vegetables and grinding it with 40 mL of peptone water in a blender. The blended mixture was then mixed in a rotating mixer (20 rpm) for 30 minutes in a cold room (4°C) and then centrifuged at 6000 rpm for 15 minutes. The supernant of the centrifuged mixture was then decanted and filtered through a 0.45-?m nonsorptive filter, frozen and stored for future analysis. Moisture content of the vegetable was measured by oven drying the subsample of chopped vegetable at 60°C.

Soil Sample Collection and Processing

Processing of soil samples was somewhat similar to the vegetable extraction procedures. Surface soil (0-15 cm) was collected just before planting (22 April at Staples and 29 May at Waseca) and after vegetable harvest to evaluate the fate of manure applied antibiotics in soils. Since timing of vegetable harvest was different for different vegetables, timing of soil sampling at harvest also varied. Fertilizer soil samples were used as a control. The antibiotic extraction procedure involved mixing 5 g of moist soil with 20 mL of peptone water in centrifuge tubes, shaking the suspension for 30 minutes in a rotating mixer at 20 rpm in a cold room (4°C), and then centrifuging the suspension at 6000 rpm for 15 minutes. The supernatant was decanted and saved. To the remaining soil, an additional 20 mL of peptone water was added, the mixture was shaken and then centrifuged again as outlined above. The supernatant was decanted and added to the earlier saved supernatant. The combined supernatant was then filtered through a 0.45-?m non-sorptive filter and filtrate frozen until the time of antibiotic analysis. Water content of the soil samples was measured by oven drying the soil at 105°C.

Survey of Vegetable Producers and Organic Grocery Stores

For the second objective, a group of five certified organic and two conventional vegetable growers who use either fresh or composted manure were selected for this study. The original plan was to enlist 10 growers representing each group. However, there was much reluctance on the part of conventional (non-certified organic) vegetable growers to participate in our study. For the growers participating in our study, we analyzed their produce, manure, and soil for presence of antibiotics. The procedure for antibiotic analysis of manure, soil, and plant samples was similar to that of our plot studies. Because of a lack of enough participants in each group, we were unable to run a statistical analysis. Instead we used the limits of detection (LOD) as the criteria to evaluate whether or not antibiotics in vegetable produce were present or absent in samples from each farm. The LOD is defined as three standard deviations of percent inhibition for 0 µL-1 standard and the LOQ is defined as six standard deviations of inhibition for 0 µgL-1 standard.

In addition to the survey of certified organic and conventional growers, we also bought vegetables from two organic grocery stores to assess the presence or absence of five antibiotics tested in this study. One of the stores generally sells locally grown organic produce (Store#1) whereas the second store (Store#2) is a grocery chain that sells organic vegetables that are grown in other parts of the United States as well as in Mexico and Canada. We analyzed the produce samples from these stores and compared them with limit of quantification (LOQ) and the antibiotic concentrations that we measured in vegetables from our plots at Staples and Waseca.

Antibiotic Analysis

Antibiotic analysis was done using the Enzyme-Linked Immunosorbent Assay (ELISA) kits for all five antibiotics used in this study. These kits were used primarily because they can measure small quantities of antibiotics in samples, which was the case for vegetable samples. Some of these kits have been tested thoroughly in the investigators laboratory for various matrices and cross-reactivity (Kumar et al., 2004, 2005a; Dolliver et al., 2007, 2008).

The procedures for antibiotic analysis followed ELISA kit manufactures instructions and measured optical density of sample extracts after incubation. The details of these procedures have been outlined by Kumar et al. (2005a) and Dolliver et al. (2008). Optical density was converted to percent inhibition which was then regressed against standard antibiotic concentration to develop a standard curve. A standard curve was developed each time the ELISA test was run.

% Inhibition=100-(ODsample or standard/OD0mg/L) [1]

Quantification of antibiotics in samples of shoots, leaves, roots, and fruit first involved extracting chemicals with buffered peptone water (BPW, pH 7.0). Peels of carrots, radishes, and potatoes were analyzed separately to determine if there was any difference in antibiotic concentrations between the skin and the root. Some of the positive samples from ELISA tests were further tested with HPLC to confirm the presence of these antibiotics. The procedure was similar to that of Migliore et al. (1996) and adopted by Dolliver et al. (2007).

Bioassay:

Plant samples were also bioassayed to assess whether or not antibiotics found in plant materials are biologically active. Since sulfamethazine concentrations were higher than LOQ in many vegetables, efforts were directed to bioassay the vegetable extracts for sulfamethazine only. Furthermore, only those samples that showed presence of sulfamethazine in HPLC analysis were bioassayed. Antimicrobial activity of sulfamethazine was determined against Actinobacillus suis. This procedure is similar to that of Chander et al. (2005). For the bioassay, overnight grown culture of A. suis was taken and its turbidity was adjusted to 0.5 McFarland units. The first step involved development of a standard curve. For preparation of the standard curve, 2-fold serial dilutions of sulfamethazine stock solution (10 mg ml-1) were made in 3% tryptic soy broth (TSB) to obtain an antibiotic concentration ranging from 4 µg ml-1 to 2048 µg ml-1. Each dilution was inoculated with 100 µl of 0.5 McFarland adjusted inoculums of A. suis. The inoculated samples were then incubated at 37°C overnight, after which five-fold dilutions of the samples were made in phosphate buffered saline (PBS, pH 7.0), mixing it well by vortexing, and then recording the OD at 650 nm.

For bioassay of vegetable extracts, the samples were first filtered using 0.45 micron filters, mixed with 3% TSB in 1:1 ratio, followed by inoculation using 100 µl of 0.5 McFarland adjusted inoculums of A. suis. For use as a positive control, 3% TSB without antibiotic was inoculated with 100 µl of A. suis culture. All tubes containing test samples and positive control were then incubated at 37°C. After overnight incubation, five-fold dilutions of all samples were made in phosphate buffered saline (PBS, pH 7.0), mixed well by vortexing, and then recorded the OD at 650 nm.

Sulfamethazine Sorption Tests

Although many researchers have run adsorption tests of sulfamethazine on soil, the adsorptive behavior in binary solution has not been evaluated yet. Since phosphate anions present in manure can form strong surface complexes with iron oxide via ligand exchange, Kahle and Stam (2007) suggested that these anions may compete for sorption places with antibiotics. Since manure contains significant amount of phosphorus, we were interested in testing this hypothesis for sulfamethazine. Although we did not test, clay content and soil pH are two other important factors in sulfamethazine adsorption in soils (Gao and Pedersen, 2005).

To assess adsorption behavior of sulfamethazine in presence of phosphorus, batch equilibrium experiments were performed with two types of soils (Verndale sandy loam from Staples, MN, and Webster clay loam from Waseca, MN) using single and binary solutions. Adsorption data were fitted with the Freundlich isotherm model: Cs=Kf Ceq1/n, where Cs (?mol kg-1) is the concentration of sorbate associated with the sorbent and Ceq (?mol kg-1) is the equilibrium chemical concentration in the aqueous phase, Kf (mmol1?1/nL1/n kg?1) is the nonlinear Freundlich isotherm coefficient, and 1/n (unitless) is a measure of the isotherm nonlinearity. The sorbate concentration (Cs) was calculated by taking the difference between the initial concentration (Ci, ?mol L-1) of antibiotic in solution and the equilibrium antibiotic concentration in solution; Cs=(Ci?Ceq)×v/m, where v is the solution volume (L) and m is the sorbent mass (kg).

Soil Preparation: For adsorption studies, Webster clay loam and Verndale sandy loam soils were collected from the control plots at SROC in Waseca, and the Central Lakes College Agricultural Station at Staples, MN, respectively. The Webster clay loam soil contained approximately 5.8% of organic matter, 34% of clay, 28% of sand, 38% of silt and a pH of 6.0. The bulk density of the soil was 1.22 Mg m-3 for 0-15 cm depth and 1.32 Mg m-3 for 15-30 cm depth. In addition, it contained 72 mg kg-1 and 258 mg kg-1 of phosphorus and potassium, respectively. The Verndale sandy loam soil contained 1.8% of organic matter, 69.4% sand, 24.5% silt, 6.1% clay and a pH of 7.0. The bulk density of the soil was 1.52 Mg m-3 from 0-26 cm and 1.73 Mg m-3 from 26-40 cm. Approximately 36 mg kg-1, 134 mg kg-1, 4 mg kg-1, 4.8 mg kg-1, 1492 mg kg-1, and 203 mg kg-1 of phosphorus, potassium, sulfur, zinc, calcium, and magnesium were present in this soil.

Time effect: Initial tests were run to estimate the equilibrium time for sorption studies. These tests were run using a solution that contained 1?molar (1 ?mole L-1) sulfamethazine and 10 mmolar (10 mmole L-1) of potassium chloride. Potassium chloride was used to adjust the ionic strength of the solution. Both sulfamethazine sodium salt (C12H13N4O2SNa) and potassium chloride (KCl) were obtained from Sigma-Aldrich Co. The adsorption test involved mixing of 2 g of air dried soil with 20 mL of antibiotic solution in 50 mL centrifuge tube for various times. Mixing times varied from 0.5 to 96 hours. Mixing was done in a rotating mixer (20 rpm) at room temperature. After mixing, the supernatant was separated out by centrifuging the suspension at 6000 rpm for 15 minutes, and then filtering it through a 0.45 µm non-sorptive filter. This test was run in duplicate for both soils.

Dilution effect: An additional test was also run to identify the best soil: solution ratio for adsorption studies. For this test, we also used the solution containing 1?molar sulfamethazine solution made form sodium salt (C12H13N4O2SNa) and 10 mmolar potassium chloride (KCl) solution. Again, the test involved mixing 2 g of air-dried soil with various amounts of antibiotic solution. Solid and liquid ratios varied from 1:1 to 1:100. For this experiment, mixing was done for 48 hrs. Previous tests showed that 48 hours was sufficient time to equilibrate sulfamethazine with Webster clay loam and Verndale sandy loam soils. After mixing, the supernatant was separated out by centrifuging the suspension at 6000 rpm for 15 minutes, and then filtering it through a 0.45 µm non-sorptive filter. This test was also run for both soils in duplicate.

Adsorption Experiment Design: The competitive effects of two chemicals (sulfamethazine and phosphorus) were tested by running the adsorption test on both soils at five antibiotic concentrations. The antibiotic concentrations were 0.09, 0.45, 0.9, 2.7, and 9 ?molar whereas phosphorus concentrations were 0, 1, and 10 mmolar. Each solution was adjusted for ionic strength with 0.01 mmolar KCl. The adsorption test was run by mixing 2 g of soil with 20 mL of the antibiotic solution in a rotating mixer for 18 hrs at room temperature. The supernatant was separated out by centrifuging the suspension at 6000 rpm for 15 minutes and then passing the supernatant through a 0.45 µm non-sorptive filter. This test was also run in duplicate for both soils.

The sorption concentrations (Cs) were plotted against the equilibrium solution concentration (Ceq) and the data were fitted with the Freundlich adsorption isotherm.

Cs=Kf(Ceq)1/n [2]

Sulfamethazine Analysis for Adsorption Test: Sulfamethazine concentrations for adsorption studies were analyzed with HPLC using a Dionex 600 Ion Chromatograph and an ED40 detector (Sunnyvale, CA). The working range was 30 to 1000 ?g L-1 with 10 ?g L-1 as a detection limit.

Research results and discussion:

ELISA Analytical Procedure

The standard curve of percent inhibition vs. antibiotic concentration for all antibiotics tested in this study was log-linear with a high coefficient of determination (Table 14). Standard deviation of the percent inhibition for 0 ?gL-1 standard was used to calculate the limit of detection (LOD) and the limit of quantification (LOQ) (Table 14). Limit of detection is defined as three standard deviations of percent inhibition for 0 ?gL-1 standard whereas limit of quantification (LOQ) is defined as six standard deviations of inhibition for 0 ?gL-1 standard.

Average percent inhibition ranged from 22.32% for 1 ?gL-1 standard to 71.98% for 25 ?gL-1 standard in case of monensin, 13.33% for 0.05 ?gL-1 standard to 82.76% for 5 ?gL-1 standard in case of sulfamethazine, 44.38% for 0.1 ?gL-1 standard to 88.84% for 100 ?gL-1 standard in case of tylosin, 39.03% for 0.39 ?gL-1 standard to 85.63% for 12.5 ?gL-1 standard in case of viginiamycin, and 20.71% for 0.15 ?gL-1 standard to 86.89% for 12.15 ?gL-1 standard in case of tetracycline.

Antibiotic Degradation during Composting

Figures 13 to 21 show the degradation of various antibiotics during composting of both hog and turkey manures. The data were fitted to the first order rate reaction (Eq. 3).

Ct=C0e-?t [3]

where Ct is the concentration of a given antibiotic at a given time (t), C0 is the initial concentration of the same antibiotic, and ? is the rate of degradation (d-1).

Table 15 lists the degradation rates and half life of four antibiotics during composting of hog manure and 5 antibiotics during composting of turkey manure. Except for tetracycline, antibiotic degradation was slower during composting of turkey manure than composting of hog manure. During composting of hog manure, tylosin had the shortest half-life (5 days) followed by chlortetracycline (46 days), sulfamethazine (74 days) and monensin (128 days). Comparatively during composting of turkey manure, tylosin had a half-life of 16 days, followed by chlortetracycline (17 days), virginiamycin (25 days), monensin (257 days) and sulfamethazine (462 days). Table 16 lists the concentration of various antibiotics in manure just at the time of their application to plots.

Water Content of Vegetables and Soil at Harvest

Water contents of the vegetables and the soil at the time of harvest are listed in Table 17. As expected vegetable water content varied with the type of vegetable and also for a given vegetable it varied between leaves, skin and bulb. Generally, water content of vegetables from Waseca was slightly higher than that from Staples.

Water contents of the soil samples at the time of harvest ranged from 23.4% to 38.9 % for the Waseca site and from 6.1 %to 13.6% for the Staples site. Higher water content at the Waseca site is due to high clay and organic matter contents of the Webster clay loam soil compared to Verndale sandy loam soil at Staples.

Antibiotic Concentration in Soil at Planting and Harvest

Figures 22 to 31 show the concentration of various antibiotics in soil just before planting and at the time of harvest. Each bar in these figures is an average of 33 (11 crops and 3 replications) measurements. Since different vegetables were harvested at different times, concentration after harvest represents an average over all harvest times. The figures also show the LOQ for each antibiotic. The results show that except for sulfamethazine and one case of monensin (raw hog manure treatment), soil antibiotic concentrations were below the limits of quantification at both planting and at harvest for all antibiotics. These results also show that for most part the antibiotics present in composted and raw manures at the time of their application either degraded or were tied up in the soil. Concentrations of monensin in raw hog manure treatment at planting at both sites (Figs. 22 and 23) and concentrations of sulfamethazine both at planting and harvest for all manure treatments at both sites (Figs. 24 and 25) were above the limits of quantification.

Monensin: Except for raw hog manure, monensin concentrations in soil at planting were below the LOQ (10.47 ?g kg-1 or 1.18 ?g L-1). It appears that high monensin concentrations in soil at planting for raw hog manure treatment are due to its high concentration in manure at the time of its application (Table 16). Monensin concentration in raw hog was 3240 ?g L-1 as compared to 1205 to 1432 ?g kg-1(dry weight basis) for raw turkey, composted hog, and composted turkey. Monensin concentrations in soils after raw hog manure application were 17.95 ± 25.15 ?g kg-1 at the Waseca site (Fig. 22) and 27.05 ± 15.15 ?g kg-1 at the Staples site (Fig. 23). Higher concentrations at the Staple site compared to the Waseca site are likely because raw hog manure to all plots at Waseca was applied at one time in the fall whereas at the Staples site raw hog manure application occurred in the fall in some plots and next spring in the remaining plots. Delayed application of raw hog manure at Staples occurred because of poor weather conditions including soil freezing in the fall. Even in spring, raw manure application at Staples was staggered because of limiting transport capacity to haul manure from Waseca to Staples. Monensin concentration in the soil at the time of harvest represented the residual concentration and this concentration was below the limits of quantification for all treatments (Figs. 22 and 23).

Sulfamethazine: Figures 24 and 25 show the concentration of sulfamethazine in soil at planting and after harvest. Sulfamethazine concentration at planting in all treatments was higher than the limits of quantification (2.98 ?g kg-1 or 0.33 ?g L-1). Initial sulfamethazine concentrations in manure were 2547 ?g kg-1, 4344 ?g kg-1, and 3128 ?g kg-1 (dry weight basis) for raw hog, raw turkey, and composted turkey manure treatments, respectively. Comparatively, sulfamethazine concentration in composted hog manure was much lower (314 ?g kg-1 dry weight basis) than for other manure treatments. Sulfamethazine concentrations in soil for the raw hog manure treatment was the highest at planting at both sites. These concentrations were 56.23 ± 15.81 ?g kg-1 and 72.61 ± 14.65 ?g kg-1 for Waseca and Staples plots, respectively. Except for the raw turkey manure treatment at both sites and composted turkey manure treatment at Staples, residual sulfamethazine concentration in soil was still higher than the LOQ. Highest residual sulfamethazine concentrations detected in the soil at the Waseca and the Staples sites were for the raw hog manure treatment and these concentrations corresponded to 20.98 ± 12.80 ?g kg-1 and 22.96 ± 5.26 ?g kg-1, respectively.

Tylosin, Chlortetracycline, and Virginiamycin: Figures 26 to 31 are plots of tylosin, chlortetracycline, and virginiamycin concentrations in soil both at the time of planting and at harvest. Concentrations for all three antibiotics in soil at both sites were below the limits of quantification i.e. 11.31 ?g kg-1 (or 1.27 ?g L-1), 3.1 ?g kg-1 (or 0.35 ?g L-1), and 5.94 ?g kg-1 (or 0.67 ?g L-1) for tylosin, chlortetracycline, and virginiamycin, respectively. These lower concentrations in soil were partially due to lower concentrations (14 to 172 ?g kg-1 dry weight basis) of these antibiotics in manure at the time of planting (Table 16).

Assessment of Matrix Effects in Plant Sample Analysis

A matrix effect can occur when a substance or substances in the matrix interfere with the ELISA assay, thus producing inaccurate results. In general, the more complex the matrix, the more likely a matrix effect will be encountered during ELISA analysis. In this study, matrix effect for various ELISA assays was assessed for samples extracted from vegetables. This assessment was made with pepper extractant. Pepper extractant was selected because it had a dark green color and we envisioned this could result in matrix interference. The procedure involved blending 20 g of pepper from the inorganic fertilizer treatment with 20 mL of peptone water in a blender, transferring the mixture to a 50 mL centrifuge tube, and then shaking the mixture for 30 minutes on a rotary mixer at 20 rpm in a cold room (4°C). After shaking, the mixture was centrifuged at 6000 rpm for 15 minute and then filtered through a 0.45-?m non-sorptive filter. To evaluate matrix effect from plant materials, a set of antibiotic and pepper extractant mixtures were prepared by mixing 0.3 mL of antibiotic solution of varying concentrations and 0.3 mL of pepper extractant. Antibiotics concentrations ranged from 1 to 25 ?gL-1 for monensin, 0.5 to 5 ?gL-1 for sulfamethazine, 0.1 to 10 ?gL-1 for tylosin, 1.35 to 12.15 ?gL-1 for chlortetracycline and 0.156 to 15.2 ?gL-1 for virginiamycin. These mixtures were then analyzed for various antibiotic concentrations using different antibiotic ELISA kits.

Figures 32 to 36 show the 1:1 plots of measured concentration vs. added antibiotic concentration for five different antibiotics tested in this study. The coefficient of determination (r2) varied from 0.97 to 1.0 for all five antibiotic tested. The test showed that except for chlortetracycline, the matrix effect from pepper extractant was statistically absent (slope not different than 1 and intercept not different than 0). The slope of line for chlortetracycline in Fig. 35 was statistically (at 5% significance) different than 1.0. Since concentrations of chlortetracycline in vegetables were always less than the quantification limits, it appears that ELISA test was still sufficient for characterization of antibiotics in vegetable produce.

Antibiotic Concentration in Vegetables and Soil

Figures 37 to 67 show the concentration of various antibiotics in vegetable and soil samples. Since there were 3 replications for each treatment, each antibiotic concentration is an average of 3 numbers. The following text describes uptake of each individual antibiotic by various vegetables.

Monensin: Figures 37 and 38 show the concentration of monensin in 11 different vegetable crops for various nutrient source treatments. For almost all treatments, monensin concentration in various vegetable crops was less than the limits of quantification (3.44 ?g kg-1). The exceptions were radish skin in composted hog manure treatment, carrot root in raw turkey manure treatment, and garlic scape and bulb in composted turkey manure treatment at the Waseca site and garlic bulb in raw turkey manure treatment at the Staples site. However, considering the large variation in concentrations between replications for each of these treatments, these concentrations are unlikely to be significantly different than the limits of quantification. Even though monensin concentration in soil for the raw hog manure treatment was the highest at planting at both the Waseca and Staples sites, monensin concentration in vegetables was less than LOQ. This is most likely due to its degradation in soil since its concentrations in soil at harvest were below the level of quantification; similar to other treatments (Figs. 13-14).

Sulfamethazine: Figures 39 and 40 show the concentration of sulfamethazine in various vegetables at the Waseca and Staples sites. Since sulfamethazine concentration in several vegetables was higher than the limits of quantification for various nutrient source treatments, we discuss the uptake of sulfamethazine by each vegetable type.

Radish: Figure 41 shows sulfamethazine concentration in radish skin and bulb for both Waseca and Staples sites. Generally, there was higher concentration of sulfamethzine in radish skin (0.48 ~4.80 ?g kg-1 at Staples, 0.06 ~2.84 ?g kg-1 at Waseca) than in radish root (0.13 ~0.67 ?g kg-1 Staples, 0.18 ~1.19 ?g kg-1 at Waseca) at both sites. Lower sulfamethazine concentrations in root may partially be due to its higher water contents. The moisture contents of harvested radish root varied from 95.1% to 96.3% as compared to 93.7% to 94.7% for radish skin. The LOQ for root and skin were 0.65 ?g kg-1 and 0.98 ?g kg-1, respectively. Concentrations of sulfamethazine were higher than the limits of quantification for radish skin from composted hog and composted turkey manure treatments at the Waseca site and raw and composted turkey manure treatments at the Staples site.

Figure 42 shows the sulfamethazine concentration in radish plots both at planting and harvest at Staples. Soil samples were not collected from the radish plots at Waseca. Approximately 78 to 83% of sulfamethazine in soil degraded between planting and harvest. Except for raw hog manure, sulfamethazine concentration in all plots was lower than or closed to LOQ (2.99 ?g kg-1). Sulfamethazine concentration in soil of raw hog treatment at harvest was 43.18 ± 7.63 ?g kg-1. It appears that there is no relationship between the sulfamethazine concentration in soil and its corresponding concentrations either in the radish skin or in the root.

Spinach: Sulfamethazine was also taken up by spinach from soils that have been amended with manure containing sulfamethazine (Fig. 43). For most treatments, sulfamethazine concentration in spinach was close the limits of its quantification (0.98 ?g kg-1). The exception was the spinach from raw hog manure treatment at Staples (4.82 ± 3.25 ?g kg-1). This high uptake appears to be related to high concentration of sulfamethazine in soil at the Staples site (Fig. 44). Other than the raw hog treatment, sulfamethazine concentration in the soil at the time of harvest for other treatments was lower than or close to LOQ (2.99 ?g kg-1). On an average, sulfamethazine concentration in soil decreased from 63% to 78% from planting to spinach harvest.

Lettuce: Sulfamethazine concentration in lettuce at both sites is plotted in Fig. 45. For the Staples site, sulfamethazine concentrations in lettuce for all treatments were near or below the LOQ (0.65 ?g kg-1). However, sulfamethazine concentrations in lettuce at the Waseca site were above the limits of quantification for both raw (1.77 ± 1.04 ?g kg-1) and composted hog (1.32 ± 1.82 ?g kg-1) manure treatments. Higher concentrations in lettuce for raw and composted hog manure treatments appear to be related to higher sulfamethazine concentration in soil for these treatments at the Waseca site (Fig. 46).

Soil analysis shows that sulfamethazine concentration was higher at both the Waseca and the Staples sites for both raw (41.32 ± 29.79 ?g kg-1) and composted (29.79 ± 25.36 ?g kg-1) hog manure treatments (Fig. 46). However, these higher concentrations at the Staples site did result in higher uptake of sulfamethazine by lettuce. There was slightly higher degradation in Staples plots than in Waseca plots. Approximately 26 to 81% and 78 to 91% of sulfamethazine in soil degraded between planting and lettuce harvest at Waseca and Staples, MN, respectively.

Carrots: Sulfamethazine concentrations in both skin and root of the carrots are plotted in Fig. 47. Except for composted hog and composted turkey manure treatments at Waseca, sulfamethazine concentration in carrot skin and carrot root was below the LOQ. The limits of quantification for carrot skins and roots were 0.64 and 0.97 ?g kg-1, respectively. Sulfamethazine concentrations in carrot skin from the composted hog and turkey manure treatments at Waseca were 1.47 ± 1.04 ?g kg-1 and 1.15 ± 0.60 ?g kg-1, respectively.

Soil analysis showed that raw hog manure treatment at both sites had the highest sulfamethazine concentrations at planting (Fig. 48). However, these high concentrations did not translate to high uptake of sulafmethazine by carrot from these treatments. Sulfamethazine concentration in soils from composted hog manure treatments at Waseca was higher than its corresponding concentrations at the Staples site. However, sulfamethazine concentration in composted turkey manure treatment at planting was higher at the Staple than the Waseca site. At both sites, there was significant degradation of sulfamethazine from raw hog manure treatment. Approximately 67 to 79% and 79 to 97% of sulfamethazine degraded in soil between carrot planting and harvest at the Waseca and the Staple sites, respectively.

Garlic: Sulfamethazine concentration in garlic and garlic scapes are plotted in Fig. 49. Except for garlic scapes from composted turkey manure treatment at Waseca, sulfamethazine concentrations in garlic and garlic scapes from all other treatments at both sites were lower than the LOQ (0.92 ?g kg-1). Although sulfamethazine concentration in garlic scapes from composted turkey manure treatment at Waseca (1.25 ± 1.16 ?g kg-1) was higher than the LOQ, the error bar indicates it may not be significantly different than LOQ.

Figure 50 shows sulfamethazine concentration in garlic plots at both sites. At harvest, raw hog manure treatment (28.93 ± 5.19 ?g kg-1) at Staples and composted hog manure treatment (9.12 ± 3.97 ?g kg-1) at Waseca had the highest sulfamethazine concentrations in soil. However, there does not appear to be any correlation between sulfamethazine concentration in soil and sulfamethazine concentration in garlic. Approximately 65 to 99% (Waseca, MN) and 78 to 90% (Staples, MN) of sulfamethazine in soil degraded between planting and harvest of garlic.

Cabbage and Pepper: Sulfamethazine concentrations in cabbage and pepper are plotted in Figs. 51 and 52. Except for pepper from composted hog manure treatment at Staples, sulfamethazine concentrations in both cabbage and pepper for all other treatments at both sites were lower than the LOQ. The LOQ for cabbage and pepper were 0.64 ?g kg-1 and 0.48 ?g kg-1, respectively. Considering large error bar in sulfamethazine concentration of pepper for composted hog manure treatments at Staples (1.79 ± 2.80 ?g kg-1), it would appear that it may not be significantly different than LOQ.

No soil samples were collected from the cabbage plots at Waseca or from the pepper plots at either site. Soil samples collected from Staples after cabbage harvest were analyzed for sulfamethazine (Fig. 53). Approximately 84 to 95% of sulfamethazine in soil degraded from planting to harvest in the cabbage plots at Staples. Raw hog manure treated soils at Staples showed the highest sulfamethazine concentration (22.2 ± 3.65 ?g kg-1) at cabbage harvest.

Onions: Sulfamethazine concentrations in onions are plotted in Fig. 54. Except for composted turkey manure plots at both sites, sulfamethazine concentrations in onion for all other treatments were lower than the limits of quantification (0.43 ?g kg-1). Large error bars in sulfamethazine concentration in composted turkey manure treatment suggest that these slightly higher concentrations may not be statistically significantly different than LOQ.

Sulfamethazine concentrations in soil from onion plots is plotted in Fig. 55. At harvest, raw hog manure treatment had the highest sulfamethazine concentration in soil at both sites (17.07 ± 2.62 ?g kg-1 at Waseca and 15.90 ± 8.45 ?g kg-1 at Staples). Between planting and onion harvest, 63 to 96% and 78 to 94% of sulfamethazine in soil degraded at the Waseca and Staples sites, respectively. There does not appear to be any correlation between sulfamethazine concentrations in soil and onions.

Potatoes: Sulfamethazine concentrations in potato are plotted in Fig. 56. Sulfamethazine concentrations in potato tuber from both sites were lower than LOQ (0.62 ?g kg-1). However, sulfamethazine concentrations in potato skins appear to be generally higher than LOQ. Sulfamethazine concentration in potato skin from raw hog, composted hog, and raw turkey manure treatments at Staples were higher than the corresponding values from Waseca. Comparatively, sulfamethazine concentration in potato skins from composted turkey manure treatment at Waseca was higher than Staples.

Figure 57 shows sulfamethazine concentrations in soil from potato plots. Composted hog manure at Waseca (20.92 ± 3.44 ?g kg-1) and raw hog manure at Staples (23.07 ± 10.48 ?g kg-1) had the highest sulfamethazine concentration in soil at harvest. From 21 to 91% and 62 to 92% of sulfamethazine degraded in soil from planting to harvest in potato plots at Waseca and Staples, respectively.

Tomatoes: Sulfamethazine concentrations in tomatoes are plotted in Fig. 58. Sulfamethazine concentrations in tomatoes from composted hog manure and composted turkey manure treatments at Waseca and composted hog manure treatment at Staples were higher than the LOQ (0.32 ?g kg-1). However, the error bars are quiet large for composted hog treatment thus suggesting that differences may not be very different than the LOQ. For other treatments, sulfamethazine concentrations in tomato from both sites were lower than the LOQ.

Sulfamethazine concentrations in soil from tomato plots are shown in Fig. 59. Sulfamethazine concentrations in all manure treatments were higher the limits of quantification (2.99 ?g kg-1). Raw hog manure treated soils showed the highest sulfamethazine concentration at both sites (30.44 ± 33.70 ?g kg-1 at Waseca and 10.07 ± 7.18 ?g kg-1 at Staples). Between planting and harvest, approximately 35 to 82% and 83 to 94% (Staples, MN) of sulfamethazine in soil degraded for various treatments at Waseca and Staples, respectively.

Sweet Corn: Sulfamethazine concentrations in sweet corn kernels are plotted in Fig. 60. Sulfamethazine concentrations in sweet corn for all treatments at both sites were lower than LOQ (0.59 ?g kg-1).

Figure 61 shows sulfamethazine concentrations in soils from sweet corn plots at two sites. Approximately 67 to 80% (Waseca, MN) and 49 to 96% (Staples, MN) of sulfamethazine in soil degraded between corn planting and harvest. At harvest, raw hog manure treated soils had the highest sulfamethazine concentration at Waseca (36.87 ± 15.54 ?g kg-1) followed by composted hog at Staples (13.59 ± 4.15 ?g kg-1). Raw hog and composted hog manure at Staples and Waseca and raw hog manure treatments at Waseca had concentrations above the LOQ. There does not appear to be any relationship between soil and sweet corn kernels sulfamethazine concentrations. It is possible that sweet corn likely has lower antibiotic concentration because of the large biomass of the corn plant.

Tylosin, Tetracycline, and Virginiamycin:

Tylosin, tetracycline, and virginiamycin concentrations in plant samples are shown in Figs. 62 to 67. For almost all manure treatments, concentrations of these antibiotics in plant samples were less than the limits of quantification (4.47 ?g kg-1 for tylosin, 1.02 ?g kg-1 for tetracycline, and 2.18 ?g kg-1 for virginiamycin). Exceptions were tetracycline concentration in garlic bulb from the composted turkey manure treatment at Waseca (Fig. 64) and virginiamycin concentration in garlic bulb at Waseca (Fig. 67) and Staples (Fig. 67). These concentrations were higher than the LOQ.

For both sites, tylosin, tetracycline, and virginiamycin concentrations in soils both at planting and harvest for all manure treatments did not exceed their corresponding LOQ (Figs. 26 to 31). The limits of quantification for tylosin, tetracycline, and virginiamycin at harvest were 13.67 ?g kg-1 (Figs. 26 and 27), 3.12 ?g kg-1 (Fig. 28 and 29), and 6.68 ?g kg-1 (Figs. 30 and 31), respectively.

Survey of Organic and Conventional Producers

The “Organic” label implies that products were produced according to strict legal and regulated organic standards. According to the United States Department of Agriculture (USDA) “Organic food is produced without using most conventional pesticide, fertilizer, bioengineering, or ionizing radiation. Before a product can be labeled ‘organic,’ a Government-approved certifier inspects the farm where the food is grown to make sure the farmer is following all the rules necessary to meet USDA organic standards.” For animal products, livestock must be raised without use of hormones or antibiotics. In case of organic crops, vegetable corps must be grown without the use of chemical pesticides and inorganic fertilizers. Use of animal manure on organic farms is allowed and furthermore there is no restriction on the use of animal manure from conventional farms where antibiotics may have been administered for disease prevention or for growth promotion. The only rule governing the use of manure is in terms of pathogens i.e. vegetables grown in manure applied soils should not be sold for 90 days after manure application if they are touching the soil and for 60 days after manure application if they are not touching the soil.

In this study, we also analyzed the produce samples from five vegetable growers who are certified organic growers and may or may not have used manure on their land, two non-certified organic growers who have used manure from conventional farms, and two grocery stores who sell certified organic produce. The following text describes our result from these surveys.

Survey of Vegetable Producers: Most of the produce sold at farmer’s markets is grown at a smaller scale and many of these farmers use composted or fresh manure as a source of nutrients for their crops. To further test the potential of antibiotic uptake by vegetable plants under grower’s field conditions, a set of certified organic and non-certified organic vegetable producers who had used either fresh or composted manure were also solicited to participate in our study on antibiotic uptake by plants. Their produce, manure, and soil were analyzed for presence of antibiotics. The characteristics of various farms that participated in our antibiotic uptake by vegetable crops survey are listed in Table 18.

Table 19 summarizes the concentration of monensin, sulfamethazine, tetracycline, and virginiamycin in manure from 5 farms (A,B,D,E, and G). Manure samples were not available from farms C and F. Concentrations of antibiotics found in 5 farms are much lower than the concentration of antibiotics used in our controlled field study at Waseca and Staples. The highest monensin and virginamycin were detected in farm D which had used turkey manure from a conventional farm. The highest sulfamethazine concentration was detected in manure from farm B and this is 10-200 times less than the concentration measured in manure samples at the time of its application in our experimental field study. Highest tetracycline concentration was detected in farm A which was nearly similar or 15 times less than the maximum concentration in manure samples at the time of its application in our study.

Soil samples were also collected from each farm to evaluate the presence of antibiotics in soils. Figure 68 shows the plot of tetracycline, monensin, sulfamethazine, and virginiamycin, concentrations in soil samples obtained from vegetable growers fields participating in our survey. Tetracycline, virginiamycin and monensin concentrations in these soil samples did not exceed LOQ. Sulfamethazine concentrations in the soil collected from farms A (9.76 ?g kg-1) and B (12.66 ?g kg-1) were higher than LOQ (2.73 ?g kg-1) but less than or nearly equal to the concentration at the time of planting in our field experiment at Waseca and Staples (Figs. 24 and 25).

Figures 69 through 72 show the concentration of various antibiotics in vegetables collected from farms participating in our survey. Except for a few cases, tetracycline (Fig. 69), monensin (Fig. 70), and virginiamycin (Fig. 71) concentrations in most produce samples were less than the LOQ (0.83 ?g/kg for tetracycline, 2.17 ?g/kg for monensin, and 1.58 ?g/kg for virginiamycin). However, sulfamethazine concentration (Fig. 72) in many produce samples was greater than the LOQ (0.78 ?g kg-1). Highest sulfamethazine concentration (27.34 ?g kg-1) occurred in lettuce harvested in farm C. Conventional turkey manure farm (Farm D) had the highest number (78%) of plants containing sulfamethazine. Farm E (organic farm) showed 54% of vegetables had higher sulfamethazine concentration than the LOQ (0.78 ?g kg-1). Only 18% of the vegetables at farm F (organic farm) were over the LOQ. Although some of the concentrations are higher than the concentration we detected in vegetables from our experimental plots, they are still relatively low concentrations. Figure 73 shows the concentration of sulfamethazine in various vegetable parts from different farms. As stated above, conventional farm D showed the most presence of sulfamethazine in various vegetables of any other farm in our survey. Monensin, tetracycline, and virginiamycin concentration in various vegetable parts are presented in the Appendix A.

Survey of Organic Grocery Stores: Besides the comparisons of organic vs. conventional producers, we also analyzed the organic produce from two grocery stores. One of the stores (store#1) mostly sold locally produced organic vegetables whereas the other grocery store (store#2) was part of a national chain and sold organic produce grown in other parts of the United States. Table 20 lists the produce that we analyzed from these two organic grocery stores.

Figures 74 thru 77 show the plots of monensin, sulfamethazine, tetracycline, and virginiamycin concentrations in vegetable samples from the two grocery stores. Monensin concentrations in many of the vegetables were lower or close to the LOQ (2.17 ?g kg-1). The exceptions were cabbage from both stores, and onions, carrot peels, and tomatoes from grocery store #2. Highest monensin concentration (4.26 ?g kg-1) was detected in cabbage sample collected from store #1.

In general, sulfamethazine concentration was less than or close to the LOQ (0.78 ?g kg-1) in various vegetables from both stores (Fig. 75). The exception was sulfamethazine concentration in onions (7.28 ?g kg-1) from store#2. All vegetables from both stores had tetracycline concentrations less than the LOQ (0.83 ?g kg-1) (Fig. 76). Virgniamycin concentrations in potato skin and onion collected from store #2 and onion collected from both stores exceeded the LOQ (1.58 ?g kg-1, Fig. 77). Since these vegetables were not analyzed in replicates, we are uncertain whether or not these differences are significantly different. Furthermore, we do not know whether these vegetables were grown in soils where manure containing these antibiotics had been applied. Overall, the concentrations detected are very low.

Bioassay:

We also ran the bioassay test to quantify the biological activity of sulfamethazine in vegetable extracts from our field study. Figure 78 shows the standard curve for growth of A. suis at different sulfamethazine concentrations. For this test, we used the vegetable extracts that showed higher concentration of sulfamethazine. However, the optical density of the suspension after incubation ranged from 0.7 - 1.16 which suggested that antibiotic concentrations in the plant extracts were too low to have any inhibitory effect on the growth of A. suis. In other words, the minimum inhibitory concentrations of A. suis were too high to be detect the presence of sulfamethazine in the vegetable extracts.

Implications

Several organizations have established acceptable daily intake (ADI) values for some of the veterinary pharmaceuticals. These include the United Nations’ Food and Agriculture Organization in collaboration with World Health Organization (FAO-WHO), Australian Health authority, and the Drinking Water Authority in Germany (Webb et al., 2003). The ADI values indicate those levels that can be ingested daily over a lifetime without any health risks (Dolliver et al., 2007). For regulatory purposes, maximum residue levels (MRLs) have also been established and these values are less than 1.2 mg per kilogram of fresh weight of animal tissue. The minimum ADI value given is 10 ?g kg-1 of body weight per day (Table 21). Assuming body weights of most adults vary from 50-75 kg, the total permissible intake will be equivalent to 500-750 ?g of pharmaceutical per day. The maximum concentration of antibiotics in vegetable produce in this study is less than 30 ?g kg-1 wet weight. This means, one would need to consume 17-25 kg of produce every day to reach the recommended ADI values. These simple calculations suggest that antibiotic uptake by various vegetable crops analyzed in this report is not a major health concern for most adults unless one is allergic to that particular pharmaceutical.

Adsorption Potential of Sulfamethazine

Environmental impact of antibiotics is greatly influenced by their mobility in soil. Mobility of these chemicals, in turn, is strongly influenced by their sorption potential to soils. Batt et al. (2006) reported that concentration of sulfamethazine (SMZ) in the groundwater near an animal feeding operation ranged from 0.076 to 0.22 ?g L?1. Thiele-Bruhn et al. (2004) reported that sulfonamides have relatively high polarity and high water solubility which results in their weak affinity for soil particles thus leading to their increased mobility in soil. A field study showed that sulfonamides were rapidly transported in surface runoff from a clay soil that had been recently fertilized with pig manure slurry (Boxall et al., 2002).

Since adsorption reduces the bioavailability of chemicals, it can also contribute to a lack of apparent biodegradation (Bushey and Dzombak, 2004). Between pH 6 and 7, most of the sulfamethazine is uncharged and phosphorus exists as negatively charged ion (H2PO4-) in solution. Adsorption of anions or uncharged chemical species on soils depends on the positive charge of soil surface. In many cases, adsorption of sulfamethazine and phosphate are limited because most soil particles are negatively charged. In this study, we evaluated the adsorption potential of sulfamethzine on two soils in presence of different phosphorus concentrations in solution. Before undertaking the adsorption experiment, experiments were done to define the optimum equilibrium time for adsorption studies as well as the optimum solid: solution ratio for adsorption. All sulfamethazine concentrations were analyzed using HPLC.

Equilibrium Time: Equilibrium time is an important parameter in adsorption studies since it determines the adsorption kinetics of an adsorbate. Figure 79 shows the variation in sulfamethazine adsorption as a function of equilibration time varying from 0.5 to 96 hours. The results show that sulfamethazine adsorption is quite rapid (approximately 1 hr) in both Webster clay loam and Verndale sandy loam soils. To avoid the variation in adsorption reaction during initial stages, we selected 18 hours of reaction time for equilibration in our study.

Dilution Effect: Dilution effects were examined at three solid: liquid (S/L) ratios (1:100, 1:10, 1:1). Other operational parameters such as room temperature, rate of soil and sulfamethazine solution mixing (20 rpm), equilibration time (18hrs), pH (natural), and sulfamethazine concentration in solution (1?molar sulfamethazine plus10 mmolar potassium chloride) were kept constant.

The results show that sulfamethazine removal (adsorption) increased with an increase in solid:liquid ratio (Fig. 80). This means, increasing solid proportion increases the number of active sites available for adsorption. The results also show that for a given solid: liquid ratio, sulfamethazine removal (adsorption) is higher in a Webster clay loam than Verndale sandy loam soil. This is expected considering Webster clay loam soil has higher clay and organic matter contents and thus more adsorption sites than the Verndale sandy loam.

Adsorption Behavior: Figures 81 and 82 show sulfamethazine adsorption by Webster clay loam and Verndale sandy loam soils with and without the presence of phosphate in solution. The results show that sulfamethazine concentration was higher in Webster clay loam than Verndale sandy loam soil. This is expected considering Webster clay loam has higher clay (34%) and organic matter (5.8%) content than the Verndale sandy loam (6.1% and 1.8%, respectively). The results show that for a given sulfamethazine concentration in solution, its adsorption on Webster clay loam soil increased in the presence of phosphorus in solution. Comparatively, sulfamethzaine adsorption on Verndale sandy loam soil was lower in the presence of phosphorus in solution. This suggests that there was some competition for adsorption sites between sulfamethazine and phosphate ion in Verndale sandy loam due to limited adsorption capacity (clay content=6.1%, organic matter content=1.8%). Phosphorus forms a very strong surface complex with iron oxide via ligand and then competes for adsorption sites (Gu et al., 1995) with sulfamethazine. Comparatively, there was no competition between sulfamethazine and phosphate ions in Webster clay loam. Lack of competition for adsorption sites in Webster clay loam soil may be due to high organic matter content (5.8%) followed by high clay content (34%). Kahle and Stamm (2007) showed that adsorption capacity of organic sorbents for sulfonamides was a magnitude to 100-fold higher than that of clay minerals.

The above difference in adsorption behavior of sulfamethazine in Webster clay loam and Verndale sandy loam is also well illustrated in Fig. 83. This graph shows sulfamethazine concentration remaining in solution when 10 &?molar sulfamethazine solution containing 0, 1, and 10 mmolar phosphorus solution was brought in equilibrium with two different soils. The results show that there was more sulfamethazine remaining in solution for Verndale loamy sand (less adsorption) than for Webster clay loam soil (more adsorption). For a given soil, there was not much difference in sulfamethazine concentration remaining in solution (similar adsorption) from 0 to 1 mmolar P solution but sulfamethazine remaining in solution increased (less adsorption) for Verndale sandy loam and decreased (more adsorption) for Webster clay loam when P concentration increased from 1 to 10 mM.

Parameters (Kf and n) values for the Frendluich adsorption isotherms as function of phosphate concentration and soil types are listed in Table 22. Kf values at low phosphate concentration are nearly same as that for 0 phosphorus concentration. Comparatively, both Kf and n change at higher P concentrations. The Kf value for Webster clay loam is also higher than that of Verndale sandy loam.

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Research conclusions:

Findings from our NCR-SARE funded research on antibiotic uptake by vegetable plants from manure-applied soils led to a call for proposals from USDA-AFRI program on "Bioaccumulation of antibiotics in plants receiving recycled water". Because of his expertise in this area, Dr. Gupta was invited and then Chaired the panel that subsequently funded several projects around the United States on various aspects of pharmaceutical uptake by plants from recycled or grey waters.

Participation Summary

Educational & Outreach Activities

Participation Summary:

Education/outreach description:
Radio Interviews and Presentations
  1. Interview with Greg Cima of the American Veterinary Medical Association (AVMA) “Researchers study antimicrobial uptake in crops” AVMA Vol 236, No. 5, 1 March 2010.
  2. Interview with Stephanie Domet on Main Street Program of CBC radio in Nova Scotia. The interview was on our research on the presence of antibiotics in vegetables. (1/15/09). Interview with Mathew Cimitile, December 18. The article appeared in 2009 Environmental Heath News (Jan 6, 2009) under the heading “Crops absorb livestock antibiotics, science shows” and subsequently in Scientific American (6 Jan 2009) under the heading “Worried about antibiotics in your beef? Vegetables may be no better”. http://www.environmentalhealthnews.org/ehs/news/antibiotics-in-crops http://www.sciam.com/article.cfm?id=vegetables-contain-antibiotics
  3. Interview with Ron Frisen of the Manitoba Cooperator, Crops can absorb livestock antibiotics (Jan 22, 2009). http://digital.manitobacooperator.ca/xta-asp/pageview1.asp?tpl=iframe_main&pc=mc
  4. Kang, D., Gupta, S.C., C. Rosen, V. Fritz, H. Murray., A. Singh, Y. Chander. 2011. Antibiotic uptake by vegetable crops from manure-applied soils. Agronomy Abstract. 158-14.
  5. Kuldip, Kumar, Lakhwinder Hundal, Satish Gupta, Albert E. Cox and Thomas C. Granato. 2009. Uptake of pharmaceutical and personal care products by plants – Potential mechanisms. Agronomy Abstract. 246-3.
  6. Presentation on “Antibiotic use in livestock and its consequences on human health” to the Minnesota Fruit & Vegetable Growers Association, St. Cloud, MN. 20 January 2011.
  7. Presentation on “Does Antibiotic Use in Livestock Affect Human Health?” at the Minnesota Fruit & Vegetable Growers Association, St. Cloud, MN. 21 January 2010.
  8. Presentation on “Antibiotic Feeding in Livestock and Its Consequences on the Environment” to the University of Minnesota Women’s Club, St. Paul, MN. 24 March 2010.
  9. Presentation on “Antibiotic Feeding in Livestock and Its Consequences on the Environment at Fifth Annual Regional Bioscience Conference, Worthington, MN 2 April 2009.
  10. Poster Presentation on Antibiotic Uptake by Vegetable Crops from Manure-Amended Soils at a field day at the Southern Research and Outreach Center, Waseca, MN. 10 September 2009.
  11. Organized a session on “Emerging Contaminants: Interactions with Mineral Surfaces, Environmental Transport, and Bioavailability” at the 2009 Soil Science Society meetings in Pittsburgh, PA.
  12. Presentation on Antibiotic Feeding in Livestock and Its Consequences on the Environment in the Department of Applied Economics, University of Minnesota. 7 April 2008.
  13. Presentation on “Antibiotic Feeding in Livestock and Its Consequences on the Environment” in the Dept. of Soil, Water, & Climate. 30 Jan. 2008. A pdf copy of the full report is available at the following url: https://netfiles.umn.edu/xythoswfs/webui/_xy-22381669_1-t_adwst0WX

Project Outcomes

Recommendations:

Areas needing additional study

Since this study only characterized the uptake of 5 antibiotics from manure applied soils, there is further need to characterize uptake of other additives (other antibiotics and pharmaceuticals) in various livestock feeds that can potentially appear in animal manure.

Information Products

Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.