Final Report for GNE12-032
Veterinary anticoccidials, biochemically known as ionophores are used in therapeutic and sub- therapeutic levels in animal feed, for prophylaxis and growth promotion. A significant amount of ionophores have been detected in animal manure like poultry, and surrounding soil and waters as reported in various literature. In our previous study on poultry manure, we quantified four ionophores, namely monensin, salinomycin, narasin and lasalocid at the range of 20-100 µg kg-1. Ionophores have been found to be toxic to different species of flora and fauna including humans. Case studies on poultry farm workers, report, ionophore toxicity at the range of 0.5-5 mg kg-1. Ionophores can also contribute to clinical antibiotic resistance in pathogens by different mechanisms including horizontal gene transfer. As ionophores have been identified as emerging contaminant from manure, contaminating agricultural soils and risking farmers’ health, it is important to study its mobility in soil for further risk assessment. A batch equilibrium study has been performed with a very commonly found ionophore in the mid-atlantic region, called monensin. We found monensin to be strongly sorbed to soil particles containing more clay and organic matter. Soil factors such as pH and CEC were strongly co-related with sorption. Distinct desorption pattern was also observed.
In the United States (USA) more than 11 X 106 kg year-1 of antibiotics are used at sub-therapeutic levels (Hansen et al., 2009c; Mellon et al., 2001). Macrolides, ionophores, and antibiotics are the most commonly used antimicrobials in poultry, dairy and swine production. Animal manure is commonly used as fertilizer in agriculture throughout the world, providing a beneficial reuse for waste from livestock production, which otherwise would be costly to dispose of. Manure can be beneficial to farmers as it helps to increase crop production, improving soil quality and fertility when the manure is land-applied for agricultural purposes. But other undesired constituents such as antimicrobials can also enter the environment with manure application. The United States Department of Agriculture – National Agricultural Statistics Service (USDA-NASS) estimated the amount of manure that was applied to hectares of cropland and pastureland in the USA, based on 2002 and 2007 Censuses of Agriculture. Data shows that poultry manure was applied to 8,94,2061 hectares of land, with more than 50 % of the application occurring in atleast twenty-three States. In the Mid-Atlantic States of Pennsylvania, Virginia, Maryland, and Delaware it is estimated that poultry manure was applied at the manure application rate of 5.71 Mg per hectare to more than 10,00,00 hectares. Broiler chicken (chickens raised for meat) production has increased from 11 billion kg in 1990 to close to 22 billion kg in year 2009. Proportionately, the value of production also increased for the poultry industry. For broilers, value of production increased from 15 billion dollars in 1999 to above 20 billion dollars in 2009 and for layers it increased from 5 million dollars in 1999 to 7 million dollars in 2008 as per the 2002 and 2007 Censuses of Agriculture by USDA-NASS data as illustrated in the Ag-Atlas maps. Thus huge amount of poultry are produced every year in the US and millions of dollars are involved in this poultry industry with increasing production rate. This is directly co-related to huge amount of poultry manure production that is used in different ways. The most popular use of poultry manure is land application as fertilizers. Significant quantities of antimicrobials have been found in manure-amended soils, plants grown in the soils, and associated groundwater, surface water, and sediments (Kim and Carlson, 2006; Kumar et al., 2005a). This implies that antimicrobials from the manure do get transported into various components of the agro-ecosystem and hence have a high potential of entering the food web (Chee-Sanford et al., 2009). Antibiotics are known to cause antibiotic resistance in bacteria including pathogenic bacteria that present a human health concern. As the potential risks of using antibiotics in animal feed are becoming well known the use of alternative antimicrobials such as ionophores are increasing. Poultry companies classify them as ‘non-antibiotic’ compounds, as they are not used as clinical drugs. This has led to some confusion as ionophores have been found to be toxic to non-target species, including soil dwelling flora and fauna, higher animals and even human beings (Dowling, 1992; Hansen et al., 2009c). Ionophores have been popularly used for many decades as anticoccidant in poultry feed, coccidia being a major parasitic disease. They are also used as feed additives in cattle as they increase efficiency in post ruminal digestion by altering rumen microbial population through ion transfers across cell membranes and also known to decrease methane emission from them, methane being a severe greenhouse gas (Russell 2002). Significant levels of ionophores ranging from 0.005 µgml-1 to 50.0 mgml-1 have been found in public waterways, sediments, and soils near confined animal feeding operations (Paginini 2005). There is acute lack of data regarding the use of ionophores like monensin in animal feed, along with, knowledge on their occurrence, fate and transport. There is little published data on the sorption and desorption characteristics of ionophores relative to soil properties. It is important to study the mobility and persistence of these ionophores in the agricultural soils so as to better assess the environmental risks associated with them. This will enable the farmers to improve the feed management practices, so that these compounds are not allowed to accumulate and persist in the agricultural environment. It will not only lead to reduction of environmental and health risks for farm workers exposed to ionophores, but also improve soil and water quality and conserve natural resources which in turn will improve the lives of farm communities. This project deals with determination of sorption and desorption characteristics of the most commonly found ionophore named monensin, in coastal plain soils; that will give mechanistic information about its mobility in the agricultural soil systems, where poultry manure is typically land applied. References: 1. Hansen, M., Krogh, K.A., Bjorklund, E., Halling-Sorensen, B. and Brandt, A. Environmental risk assessment of ionophores. TrAC Trends in Analytical Chemistry, 2009, 28, 534-542. 2.Mellon, M., Benbrook, C. and Benbrook, K.L. Hogging it- Estimates of antimicrobial abuse in livestock. Union of Concerned Scientists Publication, Cambridge, MA, USA, 2001. 3.Kim, S.C. and Carlson, K. Occurrence of ionophore antibiotics in water and sediments of a mixed-landscape watershed. Water Research, 2006, 40, 2549-2560. 4.Kumar, K., C. Gupta, S., Chander, Y., Singh, A.K. and Donald, L.S., Antibiotic Use in Agriculture and Its Impact on the Terrestrial Environment. In Advances in Agronomy; Academic Press; 2005; 1-54. 5.Chee-Sanford, J.C., Mackie, R.I., Koike, S., Krapac, I.G., Lin, Y.-F., Yannarell, A.C., Maxwell, S. and Aminov, R.I. Fate and Transport of Antibiotic Residues and Antibiotic Resistance Genes following Land Application of Manure Waste. J Environ Qual, 2009, 38, 1086-1108. 6.Dowling, L. Ionophore toxicity in chickens: a review of pathology and diagnosis. Avian Pathology, 1992, 21, 355-368. 7.Russell, J.B. and Houlihan, A.J. Ionophore resistance of ruminal bacteria and its potential impact on human health. FEMS Microbiology Reviews, 2003, 27, 65-74. 8.Paginini, European Food Safety Authority. In ESFA J; 2005; 77.
The main objective of the proposed project was to determine sorption and desorption of the most commonly found ionophore in coastal plain soils that will give mechanistic information about its mobility in the agricultural soil systems, where poultry manure is typically land applied. This project determined the effect of soil texture, pH, CEC and EC on sorption-desorption in both the A and B-horizons of all the different soils that represent common textures on the Delmarva Peninsula.
Soil Collection and Preparation Soil samples were collected from all the 37 different soil map units of 5 farms from the Delmarva Peninsula. Both A and B-horizons were sampled. Giddings probe having 3.81 cm diameter and 1 m length has been used. The soil cores were sampled using the probe in triplicates and split at the A-B interface to get the A and B horizons. The 3 cores of each sample for both A and B horizons, were field sieved through 7 mm sieve and mixed together to form a composite sample. This lead to total of 74 soil samples to be analyzed (34 map units X 2 for A and B horizons). The samples were transported back to the lab, air-dried, ground and sieved through a 2-mm sieve. Physico-chemical parameters: The soil samples were tested for background ionophores using LC/MS/MS method used for poultry manure (Biswas et al 2012). Soil physical and chemical parameters were analyzed using standardized lab tests. The following tests were performed: soil texture (% sand, silt and clay), by hydrometer method -organic matter content, by loss on ignition -cation exchange capacity, by ammonium acetate method followed by colorimetric measurements using salicylate and sodium nitroprusside for color complexing, measured at 660 nm on a colorimeter. -pH and EC, using pH meter and EC electrodes. Ionophore Sorption Experiment Monensin has been selected as the ionophore to be used in the sorption and desorption studies, as it is the ionophore found in the greatest concentration in poultry litter of our previous study. Monensin sodium salt was purchased from Sigma Aldrich Co. (St. Louis, MO). Lasalocid used as internal standard for the LC/MS/MS runs. Preliminary study: Since no standardized method has been established for monensin batch equilibrium, a pilot study was done to determine the following in our system. - soil solution ratio (trial ratios are 1:5, 1:10, 1:20, 1:50, 1:100) - equilibration time for sorption ( 1hr, 6 hrs, 12 hours, 18hours, 24 hours, 48 hours, 72 hours) - initial concentration of monensin for the sorption study ( 1 µg/ml, 25 µg/ml, 50 µg/mL) - time required for complete desorption (1hr, 6 hrs, 12 hours, 18hours, 24 hours, 48 hours, 72 hours) Trial and error method has been used to determine the above parameters with references drawn from the literature review and EPA2008 guidelines for performing batch equilibrium. Equilibration time was estimated as when increase in shake time lead to less than 5% change in monensin concentration in the solution (EPA-OPPTS 835.1230, 2008). Based on preliminary study, 1 µg mL -1 initial concentration of monensin has been used with 18 hours of shake time and 1:20 soil solution ratio. As monensin is sparingly soluble in water, it is initially dissolved in methanol and diluted further to necessary concentration in aqueous solution. Dilution scheme used: Taken 1 g monensin and dissolve in 20 mL = 1000mg/20mL == 50mg/mL (Stock A) Take 2 ml of Stock A and add 98mL water = (2mL*50mg/mL) / 100mL = 1mg/mL Made 100x dilution in water, step dilute 10x each time, to make 1 µg/mL of initial concentration to be used. Thus aqueous solution of monensin was used for the batch equilibrium to represent soil-water system in the natural environment. With the optimized shake time of 18 hours, soil samples were shaken, supernatant transferred into centrifuge tubes, centrifuged for 15mins at 1000 rpm followed by filtration through 0.45 µm filter paper. The filtrates were stored at 40C until they were analyzed. For Quality Assurance/Quality Control (QA/QC) in the batch equilibrium study, controls including monensin solution+ no soil, aq. DI water (no monensin) + soil and aq. DI water (no monensin)+no soil, i.e. double blank were used. LC/MS/MS analyses: 1 mL samples of filtrate of each of the 74 soil samples were transferred into amber HPLC vials, and 10 µL of 2 µg mL-1 internal standard lasalocid were added. The solutions were analyzed using the LC-MS/MS. QA/QC samples were run along with unknowns. HPLC-MS/MS parameters were optimized to measure monensin in the aliquots. Optimization parameters included time program modification of mobile phases (acetonitrile and 0.1% aq. formic acid solution), Declustering potential (optimized to 50eV), focusing potential (optimized to 150 eV), cell exit potential (optimized to 24 eV) and collision energy (optimized to 55 eV) of the analyte to determine parent-daughter ion (m/z ratio of 693.7 for parent ion and 675.6 for daughter ion were selected from our optimized method. Ionophore desorption study: Following the sorption study, a desorption study was conducted. Solids were retained from the sorption experiment. The solids from the sorption study were desorbed using 20 mL of de-ionized water. Shake time as pre-determined in the preliminary study of 24 hours was used such that no further desorption takes place. This was followed by the same method of filtration, centrifugation and LC/MS/MS analysis as above. This generated a desorption curve and allowed us to determine if any of our treatment factors contributed in hysteresis between the sorption and desorption trends with Monensin.
Results from the physicochemical tests of soil and sorption and desorption by batch equilibrium, were studied using X-Y scatter plots to see if there were relationships and trends between the soil factors (pH, texture, EC, CEC) and mobility factors (sorption and desorption) in both A and B horizons of soil. Sorption, desorption, hysteresis and partition co-effecient data for all the 74 soil samples from A and B horizons have been presented from Table 1 to Table 4. Sorption of monensin was found to range from 4.78 µg g-1 to 16.23 µg g-1 soil for A horizon and 7.89 µg g-1 to 18.9 µg g-1 soil for B horizon. Desorption of monensin was found to range from 3.22 µg g-1 to 15.72 µg g-1 of soil for A horizon and 4.82 µg g-1 to 16.49 µg g-1 of soil for B horizons. Partition co-effecients or Kd values for each soil sample were calculated as followed: Kd (mL g-1) = Concentration of monensin in soil (µg g-1)/ Concentration of monensin in water (µg mL-1). Kd values ranged from 6.28-85.82 mL/g for A horizon and 12.39-124.56 mL/g for B horizon. We found Cation exchange capacity- the measure of soil activity, % organic matter and % clay content to favor sorption in both A and B horizons. In many of these cases this was followed by an increased desorption as well. Soils containing very high percentages of sand (>90%) had lower sorption and hence desorption. Though the rate of desorption in some of them were pretty high. pH of the soil samples ranged from 4-6.5 with majority of soils having pH ranging between 4.5-5.5. This is understandable as our soil samples were collected from non-agricultural lands where there were no history of manure application in the past decade and the soils weren’t limed. Since we saw pretty high Kd values that are the partition distribution co-effecients, we can say that monensin has affinity to sorb to soils in such pH ranges. Hysteresis, is the inability of desorption to mirror sorption causing retention or loss of monensin sorped to the soil. Hysteresis was seen in all soil samples. In some soils hysteresis was more pronounced than others as shown in table 4. Tables 5-11, show scatter plots of effects of % organic matter, % sand, silt, clay, cation exchange capacity, pH and Electrical conductivity on sorption, desorption, hysteresis and partition co-effecients (Kd) in both A and B horizons of soils. Table 12 consists of means and standard deviations (in parenthesis) of soil physicochemical parameters by sample sites and soil horizon
- Fig 1: Sorption of Monensin in the soil samples of A and B horizon
- Fig 2: Desorption of Monensin in the soil samples of A and B horizon
- Fig 3: Hysteresis observed in the soil samples of A and B horizon
- Fig 4: Partition Co-effecient distribution (Kd) values for A and B horizon soil samples
- Table 11: Effect of CEC on sorption-desorption of monensin
- Table 6: Effect of EC on sorption-desorption of monensin in soils
- Table 7: Effect of % clay on sorption-desorption of monensin
- Table 8 : Effect of % silt on sorption-desorption of monensin
- Table 10: Effect of % sand on sorption-desorption of monensin in soils
- Table 12: Means and standard deviation(in parenthesis) of physicochemical soil parameters by sample sites and soil horizons
- Table 5 : Effect of pH on sorption-desorption of monensin
- Table9 : Effect of % Organic matter on sorption-desorption of monensin
Our batch study consisted of 74 soils samples; all collected from 5 different farms in the mid-atlantic region of the US where there is a high concentration of poultry production. As monensin is used a poultry feed-additive and have been detected in poultry manure and associated soil-water system, it was important for us to study how monensin behave in soil and if the dominant soil factors affect the behavior. In our batch equilibrium study, we found that indeed monensin had a high affinity for soil and the sorption process was more pronounced in soils that had higher organic matter, cation exchange capacity and % clay. Since higher sorption was followed by higher desorption, it also indicates that monensin bound to the soils can also enter the associated water system through run-off or ground water through leaching. This is also validated by the literature studies, where monensin was found in soil-water systems around manure storage and application areas (Watanabe et al. 2008, Dolliver et al. 2008). This has a significant impact on farmers who work and live in farm areas where manure is either stored or applied to soil as fertilizer. Since this is a huge data-set, we are still processing the statistical analyses of our data using SAS 9.2 to study statistically the effects and co-effects of each soil factor on sorption and desorption and also if there are any compounded effect of the factors, that is hard to visualize from the XY scatter plots or simple statistical charts. We are also researching the mechanisms by which monensin can be sorbed to the soil particles and to what extend mechanisms such as adsorption, absorption, vander-waal’s effect, ionic bonding and clay minerology influence the sorption process. References: 1. Watanabe, N., Harter, T.H. and Bergamaschi, B.A. Environmental occurrence and shallow ground water detection of the antibiotic monensin from dairy farms. J. Environ. Qual., 2008, 37, S78-S85. 2.Dolliver, H.A.S. and Gupta, S.C. Antibiotic Losses from Unprotected Manure Stockpiles. J Environ Qual, 2008, 37, 1238-1244.
Education & Outreach Activities and Participation Summary
Several field technicians, soil scientists and general public gave positive feedback on our poster presentation at the International Annual ASA-CSSA-SSSA Meeting 2012, Cincinnati, Ohio. Many of them took a leaflet version of the poster. Everyone agreed that awareness should be created on use of antimicrobials in animal rearing like poultry and its health and environmental implications on the society. A leaflet version of the 2012 poster has been attached. After completion of the study, we plan to publish our work in peer reviewed international scientific journal. Along with that, spread awareness through public outreach materials, on the use of this class of antimicrobial called ionophores that are emerging contaminants for farm workers and the agro-ecosystem. We will be presenting another poster shortly from this project at the International Annual ASA-CSSA-SSSA Meeting 2013, Tampa, Florida.
A farmer from Ohio, at the ASA-CSSA-SSSA 2012 conference in Cincinnati, Ohio, was very impressed with this study. He himself has poultry farms but no idea about ionophores being added to feed and that it may be toxic at high concentrations. He took a leaflet version of our poster presentation and said that he wanted to share it with his fellow farmers at their meeting. We hope, the outreach material we publish on use of ionophores in animal feed and its environmental implications will be educational for all farm workers. Leaflet version of the 2012 conference poster is attached under publication section.
Areas needing additional study
We are currently working with statistical techniques using SAS 9.2 to statistically understand the effects of the physicochemical soil parameters on the sorption-desorption processes. We are using concepts of principle component analyses for the same. In future we want to expand on our batch equilibrium study and see the effect of microbial degradation on sorption-desorption by sterilizing soil. We also want to know how pH changes on a particular soil, will affect its mobility as we already found pH to influence sorption-desorption processes. We would also like to do adsorption kinetic study using multiple time points for individual soil type as well as multi-point isotherm, including different monensin concentrations for each soil. All of these studies will help us to get an in-depth knowledge of the sorption-desorption mechanisms and hence mobility, fate and transport of monensin in mid-atlantic soils.