AGROCHEMICAL IMPACTS ON HUMAN AND ENVIRONMENTAL HEALTH: MECHANISMS AND MITIGATION

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0227662
• Project No.: NEV00744
• Multistate No.: W-2045
• Project Start Date: Jul 1, 2011
• Project End Date: Sep 30, 2015

Project Director
Pritsos, C.

Recipient Organization
UNIVERSITY OF NEVADA
(N/A)
RENO,NV 89557

Performing Department
Ag Nutrition and Vet Sciences

Non Technical Summary
There exist several sources of environmental contaminations which can impact wildlife species. Agrochemicals are routinely used to control weeds and insects on crops and gardens throughout the U.S. and Nevada. During the application process and subsequent to it, non-target organisms can come into contact with these agrochemicals either through direct spraying, or ingestion of the chemicals through food and or water. Another important source of environmental contamination is from anthropogenic origins such as mining. Acute toxicities resulting in death of the animals are easily identifiable and highly regulated. Sub-lethal exposures, however, are more difficult to identify and although these exposures may not cause immediate death to the organisms, they could cause long term effects which might impact the organism's ability to survive. Birds, and in particular migratory birds, may be exposed to these environmental contaminants repeatedly. Migratory birds are protected during migration by an international treaty, The Migratory Bird Treaty Act. As Nevada is part of an important flyway for several migratory bird species, this is a particularly important issue for Nevada particularly in light of the current and historic gold and silver mining conducted in the state. We have developed a model which we believe is useful in testing whether low-dose exposure to environmental and agrochemical contaminants has an adverse effect on migratory birds. We use the homing pigeon (Columba livia) as our avian model to determine the impact of various environmental and agrochemical exposures to migratory birds. Homing pigeons are trained to "home" from a distance of 60 miles and their time-of flight is monitored from this distance following two week exposures to the compounds of interest. Previous studies have tested cyanide and arsenic compounds as well as neurotoxic agrochemicals widely used in California on various crops. In most cases, a dose-dependent increase in the time required to return to the "homing" roost occurred. These results show that low-dose exposure to these compounds results in a decreased ability to fly back to the roost which would imply a decreased ability for migration. Many of these compounds have also shown effects on these birds ability to successfully reproduce. For the purposes of this study we want to test this model with other neurotoxic compounds which are persistent in the environment such as mercury and lead compounds. Mercury contamination is particularly of interest in Nevada, because of the mining conducted in the 1800's which resulted in widespread mercury contamination in the rivers in and around Virginia City, Nevada. We wish to test whether these compounds will have the same effect as the carbamate and organophosphate compounds had that we tested. We also wish to expand upon our understanding of the longer term effect of these exposures by testing the offspring of these birds for both learning (to home) and reproductive abilities. These studies will allow us to assess potential short and long term population level effects due to these exposures.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
133	0820	1000	25%
133	0820	1070	25%
133	5220	1150	25%
133	5220	1000	25%

Knowledge Area
133 - Pollution Prevention and Mitigation;

Subject Of Investigation
5220 - Pesticides; 0820 - Wild birds;

Field Of Science
1070 - Ecology; 1000 - Biochemistry and biophysics; 1150 - Toxicology;

Keywords

mercury exposure

lead exposure

neurotoxicity

homing pigeon

migratory birds

agrochemical exposure

avian model

reproductive toxicity

migratory effects

Goals / Objectives
3.Determine adverse impacts from agrochemical exposure to cells, organisms, and ecosystems. Agrochemicals are routinely used to protect crops from a wide variety of pests. The application of these pesticides however often results in the exposure of non-target organisms to these compounds. Migratory birds are particularly susceptible to these exposures as they may travel over long distances stopping to eat and drink at sites where these chemicals have been applied. This is particularly important from the standpoint that these birds are protected by an international treaty known as the Migratory Bird Treaty Act and that Nevada serves as a flyway for many migratory birds. Previous work from this laboratory has established the homing pigeon (Columba livia), as a model for assessing avian exposure to agrochemicals. Most recently, we have assessed the impact of the cholinesterase inhibiting agrochemicals (carbamates and organophosphates) on these birds. We have demonstrated that these neurotoxic compounds have a negative effect on the pigeons' homing and reproductive ability. Other neurotoxic compounds, used historically in agrochemicals, include mercury and lead which remain persistent in the environment. Mercury and lead contamination of ecosystems is a major concern as they bio-accumulate in avian species. While their high dose toxicity is well documented it is not clear whether low-dose exposure to these neurotoxins could effect migration in exposed birds. It is also unclear as to whether parental exposure to these contaminants has any affect on their offspring, particularly in terms of their learning abilities. In these studies, we propose to test the effect of mercury and lead compounds on the homing and reproductive abilities in our homing pigeon model. We will also test to see whether parental exposures to these compounds affect the learning abilities of the offspring in subsequent generations. We will compare the neurotoxicity of these compounds with that of the carbamates and organophosphates. This project is a Nevada contribution to the multi-state project W-1045:Agrochemical Impacts on Human and Environmental Health: Mechanisms and Mitigation. It contributes to objective 3 of the multi-state project: Determine Adverse Impacts from Agrochemical Exposure to Cells, Organisms and Ecosystems. The specific objective of this project is to determine the adverse impact of acute non-lethal neurotoxic agrochemical exposure on non-target migratory bird species. The three fundamental questions being asked by this research are: What is the effect of low-dose mercury or lead exposure on migratory bird flight, using the homing pigeon as a model: What is the effect of low-dose mercury or lead exposure on migratory bird reproduction, using the homing pigeon as a model: and What is the effect of low-dose mercury or lead exposure on the offspring of migratory birds exposed to these compounds, using the homing pigeon as a model

Project Methods
All pigeons utilized in the study will be housed and trained at the homing pigeon research facility located at the Agricultural Experiment Station at the University of Nevada, Reno. Over the course of several weeks the birds will be trained to return to the loft from a final distance of 60 miles. Once consistent flight times are established three control flights will be conducted. Return, times will be recorded by the electronic clock at the home loft entrance. Following the control flights the birds will be divided into groups and provided with deionized water containing either mercury (methylmercury chloride), lead (lead acetate) or nothing (control) at specified concentrations. The birds will then be exposed for two weeks to their source of water during which they will complete at least 3 dosed experimental flights. Following these initial exposures, lead and mercury concentrations will be increased every two weeks in a prescribed dosage scale-up manner until either behavior or toxicological effects warrant termination of the exposure. Data will be analyzed for differences in flight times for each group by ANOVA. At the conclusion of the exposure regimens and flight-time studies, birds from each of the groups will be selected for the collection of tissue samples including blood, brain and liver. Reproductive studies will be conducted in the early spring of year two. The birds will be separated into their respective groups as previously determined from the dosed flight studies (mercury, lead, control feed) and placed into the breeding loft. Each group will then be provided with water containing the appropriate substance at a concentration previously determined from the dosed flight studies. The birds will then be allowed to pair up with others in their respective group for breeding. Reproductive studies including fertility and offspring viability and post hatch weight measurements will be conducted. Offspring from each of the groups will begin training as previously described after reaching adulthood, approximately, 35 days of age. Each group will be provided water similar to that of their parents during the exposure period for two weeks prior to initiating the training and will remain with that water source throughout the training period. Each group will be trained separately in order to monitor their progress in training and to see whether differences occur in their ability to learn how to home from various distances. Differences in the number of flight attempts from varying distances and the length of time required for the birds to "home" will be used to determine whether their parental exposure had any impact on their learning ability to home. These birds will also be allowed to pair-up and mate during the third year of the study and reproductive and training of their offspring will follow as previously described. All statistics will be performed using Prism 3.0 statistical software. Differences in eggshell thickness and weights will be assessed with One-Way Analysis of Variance (ANOVA) and t-tests as appropriate. Fisher's Exact tests will be used to assess differences in fertility and offspring viability.

Progress 07/01/11 to 09/30/15

Outputs
Target Audience:Target audiences during this reporting period include research scientists, government agency representatives and regulators. This work was presented at a National Meeting of the Society of Environmental Toxicology and Chemistry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The work described in this study has provided training and professional development. A graduate student and several undergraduate students haveworked on this study and havereceivedtraining on working with birds, handling toxic chemicals and in research design and studies. This training was provided both by the P.I. of the project, Dr. Pritsos, and the research associate Mr. John Moye. Professional development was provided for to the undergraduate andgraduate students, research associate and P.I. through attendance of SETAC meetings to present the findings of these studies. How have the results been disseminated to communities of interest?The results of these studies have been dissmentated to the scientific community both through publication of the results in refereed journals and through presentations at national meetings. The results have also been disseminated to the local community through presentations of the work at the Nevada Agricultural Experiment Station field days held each year in Reno, NV. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Mercury is a toxic heavy metal commonly found in many ecosystems, particularly in US states with a complex mining and agriculture industries such as those found in Nevada and California. Much of its presence and persistence in the environment is credited to the use of organomercurials as fungicidal seed treatments or from mining activities. Avian species are often exposed to these mercury compounds. In avian species, reproduction is particularly sensitive to its toxic effects. Although a wide-ranging number of studies have detailed bioaccumulation and adverse effects on avian physiology and reproduction, the effect of non-lethal, low-dose exposures has not conversely been studied at length. While sensitivity may vary among species, previous studies suggest that methylmercury exposure at concentrations relevant to the environment can detrimentally impact breeding tendency and reproductive success, lower egg laying and hatching success, increase developmental abnormality frequency, and influence behavior of parent and chick. Prior work in the Pritsos laboratory has shown that at low methylmercury concentrations, flight times of homing pigeons from a particular distance are greatly affected. We will assess effects on reproduction and development at levels lower than those that may cause behavioral abnormalities in adults. Homing pigeons have been established in the Pritsos laboratory as a surrogate model for migratory birds and will be used in these studies. Mercury is one of the most common metals found in contaminated ecosystems. It occurs naturally, but high levels found in contaminated areas derive from human use practices. Among the most vulnerable species to exposure are birds that live, nest, or feed in or near these contaminated ecosystems. Because of the known neurological effects of mercury on birds, it is hypothesized that effects upon migratory ability would be evident after exposure to low levels of this metal, and effects may be exacerbated in young birds. Difficulties in following mercury exposed birds once they migrate away from contaminated areas have left investigators with insufficient data to establish exposure levels causing injury of migratory species due to migration disruption. Breeding pigeons were exposed to ~1.0 mg/kg/day methylmercury via the drinking water, and first round offspring were trained to home after fledging, while also continually exposed to methylmercury. The young pigeons were released individually for three flights, and flight times were assessed and compared to control young pigeon flight times from 3.5, 9, 21, 53, 65, and 98 air miles as well as two individual flights at ~50 air miles from multiple directions. Results indicate that methylmercury exposed birds exhibit slower flight times than controls during the initial flight, and generally improve on successive flights at each distance and direction. This may suggest orientation impairment and allude to migration disruption in migratory species.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: J.K. Moye C.R. Perez and C.A. Pritsos, Effects of parental and direct methylmercury exposure on flight activity in young homing pigeons (Columba livia). Environ. Pollut. 5:23-30 (2016).

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Target audiences during this reporting period include research scientists,government agency representatives and regulators. This work was presented at a National Meeting of the Society of EnvironmentalToxicology and Chemistry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Training activities have been provided to two undergraduate students who asist in the project. Both undergraduates participate in the collection and analyses of data. As part of the research team, they attend weekly laboratory meetings and are exposed to readng and evaluating scientific literature as well as discussions related to the researc project. Traning has also been provided to one graduate student who works closely with the P.I. on the project. How have the results been disseminated to communities of interest? Data was presented as part of a poster session at the Society of Environmental Toxicology and Chemistry annual meeting in Vancouver, BC Canada in November 2014. This is an international meeting. What do you plan to do during the next reporting period to accomplish the goals? Plans for the remainder of the project period, through June 2015 is to write the data up as a manuscript. Additional analyses of the data will be conducted.

Impacts
What was accomplished under these goals? Mercury is a toxic heavy metal commonly found in many ecosystems, particularly in US states with a complex mining and agriculture industries such as those found in Nevada and California. Much of its presence and persistence in the environment is credited to the use of organomercurials as fungicidal seed treatments or from mining activities. Avian species are often exposed to these mercury compounds. In avian species, reproduction is particularly sensitive to its toxic effects. Although a wide-ranging number of studies have detailed bioaccumulation and adverse effects on avian physiology and reproduction, the effect of non-lethal, low-dose exposures has not conversely been studied at length. While sensitivity may vary among species, previous studies suggest that methylmercury exposure at concentrations relevant to the environment can detrimentally impact breeding tendency and reproductive success, lower egg laying and hatching success, increase developmental abnormality frequency, and influence behavior of parent and chick. Prior work in the Pritsos laboratory has shown that at low methylmercury concentrations, flight times of homing pigeons from a particular distance are greatly affected. We will assess effects on reproduction and development at levels lower than those that may cause behavioral abnormalities in adults. Homing pigeons have been established in the Pritsos laboratory as a surrogate model for migratory birds and will be used in these studies. Last year we initiated studies to access the impact of methyl mercury on avian reproduction using the homing pigeon as our model. Findings were reported for exposure at 1.0 mg/kg body wt/day. The goal for this year was to conduct similar studies at lower dosages in order to assess a dose-dependent response. We conducted reproductive studies at 0.5 mg MeHG/kg body wt/day, or ½ the dosage tested the previous year. Pigeons were observed for two weeks to determine pairs of birds exhibiting mating behaviors. Band numbers of paired birds were recorded and the pairs assigned to one of two groups (control and Methyl mercury exposure). The control and the exposed group were each assigned five breeding pairs. Control and exposed breeding pairs were then separated into different breeding lofts outfitted with nest boxes, nest bowls, and nesting material to initiate breeding. Nest boxes in each breeding loft were numbered and as the boxes were occupied by a breeding pair, the bird's band number was recorded. The MeHg exposed groups received a concentration of approximately 1.0 mg/kg body weight methylmercury (II) hydroxide in deionized water, which served as their drinking water. Nest boxes were checked daily for the presence of eggs. After the beginning of dosing, all eggs laid within the following two week period were culled to ensure that all breeding occurred after parental exposure (8 day gestational period). After the two weeks, all eggs laid were recorded and monitored for progress in development. A number of reproductive parameters were collected including: date of egg laying, fertility rate of egg via candling, date of hatch, weight at hatching, weight at 7, 14, 21, 28, 35 and 49 days post-hatch, and date of fledging. Any deformities observed in the offspring were recorded. Survival rate of chicks were also observed and recorded for 10 weeks. Data was recorded for control and both groups of treated birds throughthree breeding cycles.The results of these studies showed a decrease in egg fertility and a decrease in overall reproductive success (proportion of actual number of offspring surviving to 10 weeks as compared to optimum number of offspring surviving to 10 weeks). The overall reproductive success after 3 successive rounds of parental exposure to MeHg at 0.5 mg/kg body wt/day was 75% of optimum reproductive success. For comparison, reproductive success for the 1.0 mg/kg body wt/day study was 37% and for the controls 94%. These 3 sets of reproductive success data show a dose dependent response to MeHg exposure. The number of days to fledge was determined as well in the offspring of the control and treated birds. For all three rounds the offspring of the MeHg exposed birds took statistically longer to fledge than the offspring of the control birds. MeHg exposed chicks tended to weigh more than their control counterparts. These results suggest that low-level exposure to MeHg results in negative effects on reproductive success and chick development in homing pigeons.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2014 Citation: McGlammery, K, Moye, J, Perez, C and Pritsos, C, Low Dose Methylmercury Effects on Reproductive and Development in Breeding Homing Pigeons (Columba livia), SETAC Annual Meeting, Vancouver, BC Canada, November 9-13, 2014

Progress 01/01/13 to 09/30/13

Outputs
Target Audience: Target audiences included other research scientists and regulators whosework involvesenvironmental exposure of avian species to contaminants. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Training activities have been provided to two undergraduate students who assist in the project. Both undergraduates participate in the collection and analyses of data. As part of the research team, they attendweekly laboratory meetings and are exposed to reading and evaluating scientific literature as well as discussions related to the research project. Training has also been provided to one graduate student who works closely with the P.I. on the project. How have the results been disseminated to communities of interest? Data is currently being collected and analyzed. Once this has been completed, the investigators will begin dissemination to the communities of interest. What do you plan to do during the next reporting period to accomplish the goals? Over the next reporting period we plan to expand upon the work conducted in the previous year by testing the effect of methylmercury on reproductive success in the homing pigeons at lower dosages than previously tested in order to get a doseresponseeffect for these expsoures. We would like to be able to determine a threshold level for reproductive effects from methylmercury exposure. These data would be very valuablefordetermining acceptable levels of methylmercury contamination in water.

Impacts
What was accomplished under these goals? Mercury is a toxic heavy metal commonly found in many ecosystems, particularly in US states with a complex mining and agriculture industries such as those found in Nevada and California. Much of its presence and persistence in the environment is credited to the use of organomercurials as fungicidal seed treatments or from mining activities. Avian species are often exposed to these mercury compounds. In avian species, reproduction is particularly sensitive to its toxic effects. Although a wide-ranging number of studies have detailed bioaccumulation and adverse effects on avian physiology and reproduction, the effect of non-lethal, low-dose exposures has not conversely been studied at length. While sensitivity may vary among species, previous studies suggest that methylmercury exposure at concentrations relevant to the environment can detrimentally impact breeding tendency and reproductive success, lower egg laying and hatching success, increase developmental abnormality frequency, and influence behavior of parent and chick. Prior work in the Pritsos laboratory has shown that at low methylmercury concentrations, flight times of homing pigeons from a particular distance are greatly affected. We will assess effects on reproduction and development at levels lower than those that may cause behavioral abnormalities in adults. Homing pigeons have been established in the Pritsos laboratory as a surrogate model for migratory birds and will be used in these studies. This past year, we initiated studies to access the impact of Methyl mercury on avian reproduction using the homing pigeon as our model. Pigeons were observed for two weeks to determine pairs of birds exhibiting mating behaviors. Band numbers of paired birds were recorded and the pairs assigned to one of two groups (control andMethyl mercuryexposure). The control and the exposed group were eachassigned five breeding pairs. Control and exposed breeding pairs were then separated into different breeding lofts outfitted with nest boxes, nest bowls, and nesting material to initiate breeding. Nest boxes in each breeding loftwere numbered and as the boxeswere occupied by a breeding pair, the bird’s band number was recorded. The MeHg exposed groups received a concentration of approximately 1.0 mg/kg body weight methylmercury (II) hydroxide in deionized water, which served as their drinking water. Nest boxes were checked daily for the presence of eggs. After the beginning of dosing, all eggs laid within the following two week period were culled to ensure that all breeding occurred after parental exposure (8 day gestational period). After the two weeks, all eggs laid were recorded and monitored for progress in development. A number of reproductive parameterswere collected including: date of egg laying, fertility rate of egg via candling, date of hatch, weight at hatching, weight at 7, 14, 21, 28, 35 and 49 days post-hatch, and date of fledging. Any deformities observed in the offspring were recorded. Survival rate of chicks were also observed and recorded for 10 weeks. Data was recorded for control and both groups of treated birds throughfour breeding cycles. The results of these studies showed a decrease in egg fertility rates, a decrease in survival rates (%of fertile eggs surviving to 10weeks, and a decrease in reproductive success (determined as the proportion of acutual # of offspring surviving to 10 weeks as compared to the ideal# ofoffspring surviving to 10 weeks) in the exposed group. With each round of breeding, the reproductive success in the exposed group decreased from 50% in round one to 17% in round four. Reproductive success in the control group was 90% in round one and 100%for round four.Based upon these results we have initiated similar studies at one half the methylmercury dose,0.5 mg/kg body weight methylmercury (II) hydroxide.

Publications

• Type: Journal Articles Status: Submitted Year Published: 2014 Citation: Estimating the Surface Area of Birds: Using the Homing Pigeon (Columba livia) As A Model.

Progress 07/01/12 to 12/31/12

Outputs
OUTPUTS: The first six months of this project have been used to complete all of the various regulatory requirements associated with the projects including development of MOU and IACUC forms associated with the project and in working with Methylmercury. We have also undertaken to expand our homing pigeon research facility to accommodate this and other research projects which have come out of our work on developing the homing pigeon model for Natural Resource Damage Assessment work. We have modified one of our existing lofts in order to allow for greater separation of birds within the loft for the purposes of keeping different experimental groups of birds separated from each other. We have also built a new loft at the another Nevada Agricultural Experiment Station facility in order to maintain birds at a separate facility (approximately 7 miles distance from the original loft site) in the cases where it could potentially confound studies if birds returning to the loft (at one location with several lofts) entered the wrong loft and were exposed to the wrong conditions. We have also paired up birds for breeding and are beginning studies to test the effect of environment exposures to methylmercury on reproduction in these birds. PARTICIPANTS: Chris Pritsos is the P.I. of the project and directly responsible for all aspects of the project. John Moye serves as the project manager for all research activities at the Nevada Avian Toxicity Facilities. Thivanka Muthumalage is a graduate student helping out with the experimentals. Cristina Perez is a graduate student working on the project Dr. Michael Hooper, USGS consults on this project USGS Environmental Research Center, Columbia, MO TARGET AUDIENCES: Target audiences include other research scientists, regulators and industry persons whose operations may contribute to the contamination of environmentally sensitive areas. PROJECT MODIFICATIONS: No modifications to the study at this time.

Impacts
The study has only commenced 6 months ago at the time of this report. The outcomes/impacts thus far are primarily related to the successful conversion of the loft at the Valley Road Field Station in order to accommodate more experimental groups at the loft and construction of the new loft at the Main Station Field laboratory. An important outcome/impact which has come out of the several years of related work on developing our homing pigeon model for use in Natural Resource Damage Assessment work as it relates to migratory birds is that the U.S. Dept of Interior, U.S. Fish and Wildlife Service has included our model in their NRDA related work on Avian Toxicology related to the Deepwater Horizon Oil Spill. We are also continuing to work with the USGS on methylmercury exposure to avian species.

Publications

• No publications reported this period