Superfund Research Program

Multidisciplinary Research to Develop Models to Study the Impacts of Exposure to Chlorpyrifos

Release Date: 06/04/2003

Until June 2000, when the US EPA restricted or eliminated some non-agricultural uses of chlorpyrifos, an estimated 20 million pounds of chlorpyrifos were applied in the United States every year - about half for agricultural uses, and half for residential uses. Chlorpyrifos exposure can cause neurological effects stemming from the inhibition of cholinesterase, an enzyme necessary for the proper transmission of nerve impulses. Symptoms include nausea, headaches, confusion and dizziness. Exposure to high doses can result in tremors, convulsions or respiratory paralysis, sometimes leading to coma and death. The potential impacts of pre-natal and peri-natal chlorpyrifos exposures are less well documented.

Animal models have been used to study chemicals like chlorpyrifos, with results demonstrating that exposure to high run-off levels can affect the development of the embryonic muscle, notochord and nervous system. Drs. Elwood Linney and Edward Levine are working with a team of researchers at Duke University to develop experimental systems to determine whether lower, environmentally relevant levels of exposure will also effect such changes. Their goal is to develop, refine and quantitate biological models that could function as sensitive biosensors for gene expression, developmental and reproductive effects of exposure to chlorpyrifos.


photos of zebrafish embryos


The applicability of mammalian models for the examination and detection of developmental effects of exposure to Superfund site chemicals is limited by the expense of working with sufficient numbers of animals, and the fact that embryonic examinations require sacrifice of the female carrying the embryos and sacrifice of the embryos. This constrains mammalian embryonic examinations to "snapshots" at specific developmental time points. The Duke researchers are using zebrafish (Danio rerio) as a model species because zebrafish develop rapidly, have a clear chorion (egg sack) so that the scientists have continuous visual access to the embryo throughout development, and the intact organism is available for behavioral studies after it hatches.


To begin an assessment of the molecular processes of chlorpyrifos exposure impacts on neurodevelopment, the Duke researchers are characterizing the behavioral phenotype of chlorpyrifos toxicity in zebrafish. While numerous validated methods are available to examine learning in rodents, learning in zebrafish is just beginning to be studied. In order to monitor the effect of early chlorpyrifos exposure on later choice accuracy performance, the Duke researchers developed a three-chambered tank and test methods that assess spatial discrimination learning in zebrafish, and can differentiate response latency (time elapsed between stimulus and response) from choice accuracy.

The Duke researchers exposed zebrafish embryos to low (10 ng/ml) or high (100 ng/ml) levels of chlorpyrifos from fertilization to hatching (5 days). The high dose caused a significant premature mortality in adult fish. Short-term low and high exposures during early development were found to cause disruptions in choice accuracy and motor function in zebrafish when they were tested as adults. Specifically:

  • Both low and high level chlorpyrifos exposure caused significant impairments in spatial discrimination in the adult fish.
  • The low dose caused an increase in response latency, while the high dose caused a decrease in response latency - both effects were readily apparent at the beginning of testing but became attenuated over the course of testing and by the last block of sessions the effects were no longer apparent. The biphasic nature of the effect on response latency suggests possible multiple mechanisms of chlorpyrifos toxicity.
  • Both doses caused significant impairments in choice accuracy. The high dose caused a more pronounced impairment than the low dose, and the high dose effect became more pronounced over time.

These findings have become the focus of additional research to determine the molecular/cellular changes affected by these early exposures. Dr. Linney is exploring the use of transgenic zebrafish to delineate changes in the development of the embryos upon chlorpyrifos exposure. These lines express green or yellow fluorescent protein in either all of their cells or in embryonic neuronal cells. The Duke scientists are just beginning to use a new laser scanning confocal microscope to investigate 3-dimensional changes in structures after chlorpyrifos exposure. They are trying to define critical windows of vulnerability to chlorpyrifos so that microarray analysis can be performed within this window using a 16,000 gene zebrafish microarray.

These studies represent significant progress towards the goal of development of fish models that could act as biosensors for Superfund chemical interactions - models that would allow continuous qualitative examination of developmental effects plus offer the possibility of quantitating effects of the chemicals.

For More Information Contact:

Edward D. Levin
Duke University
Neurobehavioral Research Laboratory
Duke University Medical Center
Durham, North Carolina 27710
Phone: 919-681-6273

Elwood A Linney
Duke University Medical Center
Duke Medical Center
Box 3020 Med Ctr.
Durham, North Carolina 27710
Phone: 919-684-6095

To learn more about this research, please refer to the following sources:

  • Levin ED, Chrysanthis E, Yacsin K, Linney EA. 2003. Chlorpyrifos exposure of developing zebrafish: effects on survival and long-term effects on response latency and spatial discrimination. Neurotoxicol Teratol 25(1):51-57. PMID:12633736
  • Udvadia AJ, Linney EA. 2003. Windows into development: historic, current, and future perspectives on transgenic zebrafish. Dev Biol 256(1):1-17. PMID:12654288

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