Final report for FNE17-867
Project Information
Faraway Farm Alpacas located in Yorktown Heights, NY is home to 18 alpacas. We treat the alpacas monthly with subcutaneously injected doramectin prophylactically for meningeal worm disease. This drug class is known to be toxic to soil invertebrates and residues in manure can contaminate the soil environment. Using the manure from our alpacas, we implemented a well-controlled study to: 1) determine maximum concentration of doramectin in raw alpaca manure after subcutaneous injection – these concentrations were determined from raw manure samples taken daily for 7 days and then weekly up to 28 days after injection; 2) determine doramectin concentrations in turned and unturned piles of composted alpaca manure over the course of 18 months – samples were taken periodically from the composting piles; 3) compare temperature, and physical and chemical characteristics of composted alpaca manure between turned and unturned piles.
After the study’s subcutaneous injections, doramectin concentration in raw manure peaked at day 3 (565 ppb) and then decreased and was still detectable at day 28 (87.1 ppb). Overall, the curvilinear relationship between time and raw manure doramectin levels was best described by a significant S-curve function. Analysis of compost piles revealed that doramectin levels peaked at 14 days for turned (440.0 ppb) and unturned (487.0 ppb) compost piles. Curve estimation regression models indicated that significant curvilinear S-curve functions best fit the increase and then decrease in doramectin levels during the first month for both piles. There was a statistically significant difference in mean doramectin levels (p = .002) between the turned (M = 101.87) and unturned (M = 161.00) from month 1 to month 18 with turned pile doramectin levels consistently lower than the unturned pile.
There also was a statistically significant difference (p < .001) in pile mean temperatures between the turned (M = 95.09 °F) and unturned pile (M = 86.68 °F) with the turned pile consistently hotter. The peak temperature for the turned pile was 124 °F and was 110 °F for the unturned pile. The turned pile temperatures were in the thermophilic range (>50°C [122°F]) for 7 days while the unturned pile temperatures never reached the thermophilic threshold. Our baseline sample raw manure C:N ratio = 18.65. At 135 days (4.8 months) post baseline, the unturned pile C:N = 15.56, while the turned pile C:N = 11.42. The turned compost looked and felt like it was ready as a soil amendment. By 6 months after baseline both turned and unturned compost piles were teeming with earthworms with at least several hundred worms per pile.
We have ascertained the decay rate of subcutaneously administered doramectin in alpaca raw manure and in alpaca manure compost. Though an acceptable manure level of doramectin was beyond the scope of this experiment, we observed a plethora of earth worms at month 6 in both treatment piles which was encouraging.
Our outreach has included educational tours for such organizations as the Environmental Defense Fund and the NYC Watershed Agricultural Council. Preliminary results were presented at the Westchester County NY HS Science and Education Fair 2017-18 by our high school research assistant coauthor for which he won a conservation award. Planned outreach includes submitting a manuscript to a relevant peer-reviewed scientific journal and presentations at alpaca show workshops.
Faraway Farm Alpacas, located in Yorktown Heights, NY, is home to 18 alpacas. We treat the alpacas monthly with subcutaneously injected doramectin 1.0% (10 mg/ml, Dectomax®, Zoetis Animal Health, Kalamazoo, MI). Using the manure from our alpacas, we proposed a well-controlled study to: 1) determine maximum concentration of doramectin in alpaca manure after subcutaneous injection – these concentrations are determined from raw manure samples taken daily for 7 days and at 14 days after injection; 2) determine doramectin concentrations in turned and unturned piles of composted alpaca manure over the course of 6 months – samples are taken periodically from the composting piles; 3) compare temperature, and physical and chemical characteristics of composted alpaca manure between turned and unturned piles. The study provided data to determine decay rates of doramectin in raw manure, and in unturned and turned compost piles. It was expected that composting manure from alpacas that have been dewormed with doramectin decreases the concentration of doramectin in the resulting compost product and that the rate of decay will be greater for the optimally composted pile.
Most alpacas and llamas in eastern U.S. are treated with ivermectin (trade name Ivomec®) or doramectin (Dectomax®) via subcutaneous injection as part of a monthly regimen to prevent meningeal worm disease. Meningeal worm (Parelaphostrongylus tenuis), also known as deer worm or brain worm, is a roundworm parasite hosted by white-tailed deer. Snails and slugs are intermediate hosts. When alpacas or llamas (or other unnatural hosts such as goats, sheep, elk or moose) ingest the infective larvae, migration through the body occurs. Migration in the central nervous system can cause muscle weakness, incoordination, paralysis and death. No commercial vaccine is available therefore prevention is based on management principles such as reduction of exposure to deer, snails, etc., and with deworming every four weeks. Deworming every four weeks is efficacious in killing the infective larvae before the central nervous system is penetrated.
Doramectin and ivermectin are chemical formulations used to control external parasites such as mange mites, lice, and grubs, and also gastrointestinal and lung worms. More specifically, they are macrocyclic lactones produced as fermentation products of a soil microorganism and have been approved for subcutaneous injection in cattle and intramuscular injection in swine (subcutaneous injection only for ivermectin in swine). Veterinary studies have demonstrated that ivermectin can persist in manure and soil for months and that ivermectin can be excreted in manure in concentrations that can be highly toxic to organisms in the ecosystem. For example, Boxall et al. (2003; Are veterinary medicines causing environmental risks?; Environmental Science & Technology, 286-294) reported that macrocyclic lactones affect the mortality of dung invertebrate larvae at very low concentrations with 50% lethal concentration (LC50) values less than 36 parts per billion (ppb). In one of the few studies of doramectin, Kolar et al. (2008; Toxicity of abamectin and doramectin to soil invertebrates; Environmental Pollution, 151, 182-189) studied its effects on soil invertebrates in spiked soil and in feces from recently treated sheep. They found LC50 for springtail hexapods and white potworm at > 300,000 ppb in soil and 2,500 ppb in feces. LC50 for earthworms was > 228,000 ppb for soil and > 2,500 for feces. While there was no significant effect on the reproduction of earthworms, there was significant weight loss for surviving earthworms. In another study of doramectin in sheep manure, Kosuh et al. (2005; Degradation of abamectin and doramectin on sheep grazed pasture; Exotoxicology, 14, 627-635) found high concentrations of doramectin for numerous weeks but field conditions can play a role in its degradation. Kahn et al. (2008; Chemical contaminants in feedlot wastes: Concentrations, effects, and attenuation; Environment International, 34, 839-859) though found that macrocyclic lactones are susceptible to aerobic biodegradation in soils under suitable conditions and thus would be expected to degrade in the composting process.
Since alpacas are ruminants (actually modified ruminants with their fore-stomachs made up of three compartments) and the doramectin is administered via subcutaneous injection, trajectories of doramectin may be unique to them and other camelids such as llamas. A recent SARE-funded study (FNE12-750), though, explored ivermectin use in alpacas. In the exploratory study, "Ivermectin residue in vegetable plants and compost," Kemp (2015) found that ivermectin was present in an unturned alpaca manure pile approximately 5 years old (limit of detection set at 0.8 ppb). Results were inconclusive as to the specific time frame of ivermectin degradation. However, a well-designed study of horses was recently published online by the Cornell Waste Management Institute. Published as a research summary, Schwarz and Bonhotal (May 2016; The fate of ivermectin in manure composting) reported that there was exponential decay of ivermectin in manure from horses that had been treated orally with ivermectin paste. Thermophilic temperatures were reached in the compost pile at the first temperature recording. According to the Cornell Waste Management Institute (compost.css.cornell.edu/microorg.htm), under optimal conditions, composting proceeds through three phases: 1) the mesophilic, or moderate-temperature phase (>40°C [104°F]) which lasts for a couple of days, 2) the thermophilic, or high-temperature phase (>50°C [122°F]), which can last from a few days to several months during which high temperatures accelerate the breakdown of proteins, fats, and complex carbohydrates like cellulose and hemicellulose, the major structural molecules in plants, and finally 3) a several-month cooling and maturation phase. Temperatures over about 65°C (149°F) kill many forms of microbes and limit the rate of decomposition. In the Schwarz and Bonhotal study, after 175 days of composting, ivermectin concentration decreased from 1,590 ppb to 60 ppb in the composted material. It should be noted, though, that the research subjects were horses which are not ruminants (horses have single-chambered stomachs) and that the de-worming medication was administered orally. As far as we know, no well-controlled studies have investigated the decay rate of subcutaneously administered doramectin in alpaca raw manure or in alpaca manure compost.
The period of time following drug administration when environmentally significant concentrations are present in manure depend upon the physiochemistry of the drug administered, the species to which it is administered, and the formulation or delivery system in which it is administered (McKellar [1997], Ecotoxicology and residues of anthelmintic compounds, Veterinary Parasitology, 72, 413-435; Steel, [1993], Pharmacokinetics and metabolism of avermectins in livestock, Veterinary Parasitology, 48, 45-57). McKellar reported that for subcutaneous injection in cattle, ivermectin had persistent activity against gastrointestinal parasites for 21 days and doramectin for 21-28 days. McKellar's review included raw manure concentrations of ivermectin after subcutaneous administration in cattle: ivermectin was detected in raw manure for up to 13 days for grain fed cattle and up to 14 days for pasture fed cattle. We are not aware of such analogous studies for alpacas or llamas.
The 2012 U.S. Census of Agriculture reported 9,353 alpaca farms in the U.S. with 140,601 alpacas, and 15,296 llama farms with 76,086 llamas. The Alpaca Owners Association currently reports that there are 138,710 registered alpacas east of the Mississippi River. With many of these animals in the eastern U.S. being treated with doramectin or ivermectin, and evidence that these pharmaceuticals can be present in the animals’ manure, the fate of these medications in alpaca manure is of deep concern since they may pose environmental and health risks. In fact, without proper care, raw or improperly composted manure from these animals may contribute to agricultural pollution. With appropriate study, though, it can be determined when composted manure from de-wormed alpacas can be used without negative effect in the environment, whether on a farmer’s own fields or gardens, or sold as a compost product.
Cooperators
- - Technical Advisor (Educator and Researcher)
- (Educator and Researcher)
- (Educator and Researcher)
- (Researcher)
- (Researcher)
Research
As part of our farm's monthly regimen, 18 female and male alpacas were treated on May 31, 2017 with doramectin 1.0% (10 mg/ml) injectable. Recommended dosage is 1 cc/70 lb. administered via subcutaneous injection. This is extralabel use of the drug. Our alpaca weights ranged from 140 to 220 lbs. The day that the doramectin injection was given is considered Day 0. Raw manure was collected daily in the mornings from the alpaca housing areas and pastures and put into a composite pile. A baseline raw manure sample (500 ml) was collected from the initial composite pile the morning of Day 0 prior to doramectin injections. For Day 1 (1st day post injection), a raw manure sample (500 ml) was collected from that morning's composite pile. Half of the remaining manure then was placed on a pad that became the ‘turned’ pile and the remaining half was placed on another pad that become the ‘unturned’ pile. The same procedure was conducted on Days 2 to 7. On Days 14, 21, and 28 additional raw manure sample were taken. On Days 14, 21, 28, 56 (month 2), 112 (month 4), and 168 (month 6) samples (500 ml each) were drawn independently from each of the turned and unturned piles. The turned pile was thoroughly mixed daily for the first week and weekly for the remainder of the study while the unturned pile was not mixed at all. Hay was added to the turned pile to provide a source of carbon and maximize decomposition (On-Farm Composting Handbook, 1992; Natural Resource, Agriculture, and Engineering Service [NRARES] Cooperative Extension). The piles were adjacent to one another so that they received the same amount of sunlight and rain. Drainage trenches surrounded each pile to prevent contamination between piles.
Grab samples of 1,000 ml were taken from 5 locations around each pile at varying depths to insure that samples were representative of all 18 alpacas. The grab samples were thoroughly mixed in a bucket. Samples of 500 ml then were drawn from each bucket, placed in a sterile container, and frozen. The samples were stored at -80°C (-112°F) to prevent any further degradation of doramectin so as to determine doramectin levels at the time of sample collection. On September 11, 2017, 33 frozen samples were overnighted to the New Bolton Center-Toxicology Laboratory, School of Veterinary Medicine, University of Pennsylvania, Kennett Square PA. A sensitive method was developed using an API 4000, a triple quad LC/MS/MS (AB Sciex, Foster City, CA) analyzer equipped with a Prominence LC (Shimadzu, Columbia, MD). Manure used as a blank control was taken from a composite pile from alpacas at Tufts University School of Veterinary Medicine, North Grafton, MA that had not been treated prophylactically for meningeal worm disease with any medications. All reagents used were of analytical grade and deionized water was obtained from an in-house water purification system (EMD Millipore, Billerica, MA). The control sample was tested for the absence of doramectin prior to use in the study. A set of doramectin fortified compost samples from 0-1000 ppb (ng/g) were prepared and analyzed to obtain a calibration curve. The concentration of doramectin in study samples was measured by interpolating from the linear trendline of the calibration curve. The limit of detection was 10 ppb and all results were reported in ppb. On December 12, 2017, the remaining frozen samples were overnighted to the Toxicology Lab.
In addition to the samples to be assessed for doramectin, a baseline sample of manure was sent to Agro-One, Ithaca, NY to be analyzed for components such as pH, nitrogen, carbon, phosphorous, potassium, and carbon:nitrogen ratio. Four months later, composted manure samples were taken from the turned and unturned piles and sent to Agro-One for the same component analyses that were performed for the baseline sample.
The location of the manure piles ensured that they could get full sun all day. Each pile was placed on hard soil and was surrounded by an untreated wood and wire mesh frame 8’ wide by 8’ long and 4’ high. Pile temperatures were measured daily with a Reotemp Compost Thermometer Model No. 3LPU9, 36" x 1/4" diameter stem, ½" NPT mount, with a 0 to 200°F range. Amount of sunlight and rain, ambient temperatures and other weather data were collected daily with a Davis 6250 Weather Station with a Davis 6450 Solar Radiation Sensor.
Results
Weather data. During the initial 6-month study period, average daily temperatures ranged from 55 to 92 °F (M = 72.89 °F) and the average monthly rainfall ranged from 0.84 to 4.95 in. (M = 2.51 in.) with a total rainfall of 15.08 in. This was a relatively dry period of time with our 'normal' 6-month total of 27.56 in. (https://www.usclimatedata.com/climate/yorktown-heights/new-york/united-states/usny1632). We have on-site sunshine data for the first 3 months of the study. According to the World Meteorological Organization (2003; Manual on the Global Observing System; WMO-No. 544, Geneva), bright sunshine during a given period of time is defined as the sum of that sub-period which the direct solar irradiance exceeds 120 W/m2. According to that criterion, for 81 of the 90 days (90.0%) the compost piles received at least 8 hours of bright sunshine, and for 57 days (63.3%) there was at least 10 hours of bright sunshine.
Raw manure. Raw manure samples were collected prior to doramectin injection (baseline) and at days 1 to 7, 14, 21, 28. Samples were stored at -80 deg C (-112 deg F) and sent to the University of Pennsylvania Toxicology Lab in batches for analysis. The one month of raw manure data are displayed in Figure 1. At baseline, doramectin still was detectable in raw manure from the prior administration approximately 30 days before the start of the study. Doramectin levels in raw manure increased until day 3 (565 ppb) and then decreased and were still detectable at day 28 (87.1 ppb). Curve estimation regression models were used described the curvilinear relationship between time and doramectin raw manure levels. An S-curve function best fit the raw manure data from baseline to day 28, R2 = .29, with a trend-level p = .087. From peak concentration at day 3 to day 28, an exponential decay function significantly fit, R2 = .88, p = .001.
Compost piles. Turned and unturned compost pile samples were collected at days 1 to 7, 14, 21, 28, 56, 112, 168 (6 months), 168 (12 months), and 504 (18 months). Analysis of the first month of compost pile doramectin levels in Figure 2 reveals that for the turned pile doramectin level peaked at day 14 while the unturned pile doramectin had peaked at days 4 and 14. Of the 10 samples collected in the first 28 days of the study, doramectin levels for the turned pile were lower than the unturned pile for 8 (80%) of the collections.
Data for the 15 doramectin analysis results from day 1 through 18 months for both compost piles are displayed in Figure 3. For the 8 data points from peak levels at day 14 and through 18 months, curve estimation regression indicated that S-curve functions best fit the turned pile data, R2 = .73, p = .007, and the unturned pile data, R2 = .81, p = .002. With doramectin levels after the 1st month demonstrating linearity for the turned (R2 = .71, p = .036) and unturned piles (R2 = .54, p = .098), t tests for independent samples were conducted to assess differences in doramectin levels between turned and unturned piles. For the 6 data points from month 1 to month 18, the mean doramectin concentration for the turned pile (M = 101.87, sd = 20.52) was significantly lower than for the unturned pile (M = 161.00, sd = 26.75), p = .002.
Compost Pile Components. Component analyses revealed differences from baseline and between the turned and unturned piles. For example, a key index of the composting process is the C:N ratio, the ratio of the weight of carbon (C) to that of total nitrogen (N) in an organic material. The C:N ratio decreases during the composting process, with the ratio of finished compost typically close to 10:1, or 10. Our baseline raw manure sample C:N ratio = 18.65. At 135 days (4.8 months) post baseline, the unturned pile C:N = 15.56, while the turned pile C:N = 11.42. The turned compost looked and felt like it was ready as a soil amendment.
Compost Pile Temperatures. A paired-sample t test indicated that mean compost pile temperature (°F) was statistically significantly higher for the turned pile (M = 95.09, sd = 19.57) than the unturned pile (M = 86.68, sd = 13.51), p < .001. The peak temperature for the turned pile was 124 °F and was 110 °F for the unturned pile. The turned pile temperatures were in the thermophilic range (>50°C [122°F]) for 7 days while the unturned pile temperatures never reached the thermophilic threshold.
Earthworms. Although the presence of earthworms was not a primary outcome measure, it should be reported that by 6 months after baseline both turned and unturned compost piles were teeming with earthworms with at least several hundred worms per pile.
We proposed a well-controlled study to: 1) determine maximum concentration of doramectin in alpaca raw manure after subcutaneous injection; 2) determine doramectin concentrations in turned and unturned piles of composted alpaca manure over the course of 6 months; 3) compare temperature, and physical and chemical characteristics of composted alpaca manure between turned and unturned piles. It was expected that composting manure from alpacas that have been dewormed with doramectin decreases the concentration of doramectin in the resulting compost product and that the rate of decay will be greater for the optimally composted pile. Study data support these hypothesized results.
1). Raw Manure:
After the study's subcutaneous injections, doramectin concentration in raw manure peaked at day 3 (565 ppb) and then decreased and was still detectable at day 28 (87.1 ppb). Overall, the curvilinear relationship between time and raw manure doramectin levels was best described by a significant S-curve function. These results appear to be the first reported for raw manure doramectin levels for alpacas or llamas.
2). Composted Manure:
Doramectin levels peaked at 14 days for turned (440.0 ppb) and unturned (487.0 ppb) compost piles. Curve estimation regression models indicated that significant curvilinear S-curve functions best fit the increase and then decrease in doramectin levels during the first month for both piles. There was a statistically significant difference in mean doramectin levels (p = .002) between the turned (M = 101.87) and unturned (M = 161.00) from month 1 to month 18 with turned pile doramectin levels consistently lower than the unturned pile. Overall, curvilinear patterns of doramectin degradation were consistent with those reported by Schwarz and Bonhotal (2016) for oral administration of ivermectin in horses.
3). Compost Pile Characteristics:
Temperature. There was a statistically significant difference (p < .001) in pile mean temperatures between the turned (M = 95.09 °F) and unturned pile (M = 86.68 °F) with the turned pile consistently hotter. The peak temperature for the turned pile was 124 °F and was 110 °F for the unturned pile. The turned pile temperatures were in the thermophilic range (>50°C [122°F]) for 7 days while the unturned pile temperatures never reached the thermophilic threshold. Overall, curvilinear patterns of pile temperature changes were consistent with those reported by Schwarz and Bonhotal (2016) for oral administration of ivermectin in horses.
Components. We also analyzed the C:N ratio, a key index of the composting process. The C:N ratio decreases during the composting process, with the ratio of finished compost typically close to 10:1, or 10. Our baseline sample raw manure C:N ratio = 18.65. At 135 days (4.8 months) post baseline, the unturned pile C:N = 15.56, while the turned pile C:N = 11.42. The turned compost looked and felt like it was ready as a soil amendment.
Earthworms. Anecdotally, we found that both piles had a plethora of earthworms 6 months into the study. Although reporting on the environmental safety levels of doramectin in manure and compost is beyond the scope of this study, seeing earthworms was a good sign.
In conclusion, using a well-controlled study, we ascertained the decay rate of subcutaneously administered doramectin in alpaca raw manure and in alpaca manure compost, and have demonstrated some advantages of turned and hay-augmented compost piles. Hopefully this unique study can be replicated with a larger sample of alpacas.
Education & Outreach Activities and Participation Summary
Participation Summary:
-Environmental Defense Fund economists toured project as part of their annual meeting (October 2017).
-NYC Watershed Agricultural Council members have toured farm and project multiple times to educate other farmers.
-Westchester Counter HS Science and Education Fair 2017-18: Our high school intern has been invited to speak at WESEF based on an abstract of our study's preliminary findings; our intern was awarded the WESEF Conservation Award based on our research proposal.
-Study results will be presented to an East of Hudson NYC Watershed Agricultural Council Committee meeting in 2019.
-A manuscript will be submitted to a relevant peer-reviewed scientific journal such as Veterinary Parasitology in 2019.
Learning Outcomes
Environmental risks of veterinary medicines; optimal composting techniques
Project Outcomes
Using a well-controlled study, we have ascertained the decay rate of subcutaneously administered doramectin in alpaca raw manure and in alpaca manure compost. And we have provided quantitative and qualitative support for turning composted manure piles and adding sources of carbon to optimize doramectin degradation and enhance the composting process.
In retrospect, we feel that the study methodology was appropriate for the research questions. One possible limitation which was indicated in our SARE proposal review was the size of herd. Although the results are relevant to many small to moderate size alpaca farms, replicating the study with a larger herd is of interest. It was important to demonstrate that even with a herd of 18 alpacas, optimal composting techniques generated compost pile temperatures in the thermophilic range and significantly lower levels of residual doramectin. Future research will be needed to determine the environmental safety of the residual doramectin.