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Society of Toxicology 2013

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Holly Richendrfer, Ph.D.
Holly Richendrfer, Ph.D.

Chlorpyrifos, an organophosphate pesticide, affects zebrafish larval behavior differently depending on the developmental time period of exposure. The study found distinct effects on avoidance, thigmotaxis, and activity levels depending on whether larvae were treated from 0-6 days post fertilization, 4-6 days, or 6-7 days. Longer exposure periods and higher concentrations had more pronounced effects. Exposure late in development from 6-7 days predominantly affected time spent on the edge and sides of lanes. The results suggest chlorpyrifos impacts neurodevelopment in a stage-specific manner.

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Chlorpyrifos affects specific types of zebrafish larval behavior if administered during distinct developmental time periods Holly Richendrfer and Robbert Créton Department of Molecular Biology, Cell Biology and Biochemistry; Brown University, Providence, RI, USA. SUMMARY RESULTS 4-6 dpf treatment A large percentage of the population has been diagnosed with neurobehavioral disorders such as anxiety, ADHD, and autism. The role that environmental toxicants play on their development is concerning. We found that exposure to the organophosphate pesticide (OP), chlorpyrifos, during different periods of development had distinctive effects on A B zebrafish larval behavior when assessed at 7dpf. Figure 1 – Behavioral assay setup. A) 15 minutes of INTRODUCTION blank background and B) 15 minutes of a moving red •OPs are highly ubiquitous pesticides and are used on millions of bar acres of farm land in the US alone every year. 0-6 dpf treatment C D •The role that chronic low levels of OPs have on the development of Figure 4 – Results from larvae neurobehavioral disorders is still largely unknown. treated with CPF from 4-6 dpf •OPs have been implicated in the development of ADHD, autism that showed significant differences. A) Percent and anxiety in humans, rats and mice. down, B) Percent on edge of •Zebrafish larvae are ideally suited for high-throughput studies of lane, C) Percent on the Side, D) A B Swim speed, E) Percent rest. vertebrate behavior because large numbers of embryos can be E collected daily and directly exposed to chemicals. •A novel high-throughput assay was created in our lab for DISCUSSION behavioral analyses of zebrafish larvae to measure thigmotaxis •We found significant differences in behavioral measures such as and avoidance behavior a (common anxiety measures). avoidance, thigmotaxis (preference for the side, end or edge of the lane), C D Previously, our lab has verified that anxiety can be detected by and speed of movement depending upon the days of CPF treatment thigmotaxis behavior in our assay. during larval development. METHODS •There is an indication that the longer treatment period (0-6 dpf) affects larvae administered the two higher concentrations of CPF (fig 2 c, d, f, •Larvae were exposed to egg water (EW), DMSO and three and g) which was not found in shorter treatment periods (fig 3 and 4) concentrations of chlorpyrifos (CPF) (from 0.001- 0.1 µM) E F Figure 2 – Results from (which only affected larvae treated with 0.1 µM CPF). during different time points in development. On days larvae larvae treated with CPF •Larvae treated with 0.1 µM CPF from 6-7 dpf showed less of a response were not treated, all groups were housed in egg water. from 0-6 dpf. to the moving bar, and preferred to be on the edge less compared to all Behavioral analysis was always performed when larvae were at 7dpf. Both Acute (1-2 days) and sub-chronic (6 day) treatments other groups (figure 3) which could be attributed to lower swim speeds of pesticides were administered to the larvae. G (fig 3 C) or be indicative of anxiolytic properties of CPF. • Five larvae were placed in each lane in 5 lane plates which 6-7 dpf treatment •Measures of edge, side, and end seem to be most affected when larvae were positioned on top of a laptop screen. Behavioral assays are treated later in development (fig 4) indicating that CPF has different were all performed on the 7th day of development regardless of effects during restricted developmental days. treatment days. The assays were used to analyze •Speed and rest behavior were all significantly affected by 0.1µM CPF thigmotaxis, avoidance behavior, rest, and swim speed. The whether or not it was acute or sub-chronic and even when the larvae assay utilized a PowerPoint presentation shown to the larvae for A B were not in the pesticides during behavioral testing, indicating that CPF 30 minutes (five fish per well) (Fig 1). affects developmental processes. These results have implications in •For the first half of the assay there were no visual stimuli neurobehavioral disorders that affect activity levels. (1A), in the second half a red moving bar was shown to the •Follow up studies will be performed to image the developing brain of larvae (1B). larvae that are treated with pesticides in order to determine if there are •Time lapse imaging was used to capture larval placement within C D any neural structure abnormalities. the wells . Figure 3 – Results from larvae treated with CPF from 6-7 dpf that indicated significant differences. A) Percent down, B) Percent on edge of lane, C) Swim speed, D) Percent rest. There were no significant This work was supported by the National Institute of Child Health and Human differences in fish-fish distance, End or Side of lane Development, R01 HD060647 and the National Institute of Environmental Health Sciences and F32 ES021342.

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Society of Toxicology 2013

  • 1. Chlorpyrifos affects specific types of zebrafish larval behavior if administered during distinct developmental time periods Holly Richendrfer and Robbert Créton Department of Molecular Biology, Cell Biology and Biochemistry; Brown University, Providence, RI, USA. SUMMARY RESULTS 4-6 dpf treatment A large percentage of the population has been diagnosed with neurobehavioral disorders such as anxiety, ADHD, and autism. The role that environmental toxicants play on their development is concerning. We found that exposure to the organophosphate pesticide (OP), chlorpyrifos, during different periods of development had distinctive effects on A B zebrafish larval behavior when assessed at 7dpf. Figure 1 – Behavioral assay setup. A) 15 minutes of INTRODUCTION blank background and B) 15 minutes of a moving red •OPs are highly ubiquitous pesticides and are used on millions of bar acres of farm land in the US alone every year. 0-6 dpf treatment C D •The role that chronic low levels of OPs have on the development of Figure 4 – Results from larvae neurobehavioral disorders is still largely unknown. treated with CPF from 4-6 dpf •OPs have been implicated in the development of ADHD, autism that showed significant differences. A) Percent and anxiety in humans, rats and mice. down, B) Percent on edge of •Zebrafish larvae are ideally suited for high-throughput studies of lane, C) Percent on the Side, D) A B Swim speed, E) Percent rest. vertebrate behavior because large numbers of embryos can be E collected daily and directly exposed to chemicals. •A novel high-throughput assay was created in our lab for DISCUSSION behavioral analyses of zebrafish larvae to measure thigmotaxis •We found significant differences in behavioral measures such as and avoidance behavior a (common anxiety measures). avoidance, thigmotaxis (preference for the side, end or edge of the lane), C D Previously, our lab has verified that anxiety can be detected by and speed of movement depending upon the days of CPF treatment thigmotaxis behavior in our assay. during larval development. METHODS •There is an indication that the longer treatment period (0-6 dpf) affects larvae administered the two higher concentrations of CPF (fig 2 c, d, f, •Larvae were exposed to egg water (EW), DMSO and three and g) which was not found in shorter treatment periods (fig 3 and 4) concentrations of chlorpyrifos (CPF) (from 0.001- 0.1 µM) E F Figure 2 – Results from (which only affected larvae treated with 0.1 µM CPF). during different time points in development. On days larvae larvae treated with CPF •Larvae treated with 0.1 µM CPF from 6-7 dpf showed less of a response were not treated, all groups were housed in egg water. from 0-6 dpf. to the moving bar, and preferred to be on the edge less compared to all Behavioral analysis was always performed when larvae were at 7dpf. Both Acute (1-2 days) and sub-chronic (6 day) treatments other groups (figure 3) which could be attributed to lower swim speeds of pesticides were administered to the larvae. G (fig 3 C) or be indicative of anxiolytic properties of CPF. • Five larvae were placed in each lane in 5 lane plates which 6-7 dpf treatment •Measures of edge, side, and end seem to be most affected when larvae were positioned on top of a laptop screen. Behavioral assays are treated later in development (fig 4) indicating that CPF has different were all performed on the 7th day of development regardless of effects during restricted developmental days. treatment days. The assays were used to analyze •Speed and rest behavior were all significantly affected by 0.1µM CPF thigmotaxis, avoidance behavior, rest, and swim speed. The whether or not it was acute or sub-chronic and even when the larvae assay utilized a PowerPoint presentation shown to the larvae for A B were not in the pesticides during behavioral testing, indicating that CPF 30 minutes (five fish per well) (Fig 1). affects developmental processes. These results have implications in •For the first half of the assay there were no visual stimuli neurobehavioral disorders that affect activity levels. (1A), in the second half a red moving bar was shown to the •Follow up studies will be performed to image the developing brain of larvae (1B). larvae that are treated with pesticides in order to determine if there are •Time lapse imaging was used to capture larval placement within C D any neural structure abnormalities. the wells . Figure 3 – Results from larvae treated with CPF from 6-7 dpf that indicated significant differences. A) Percent down, B) Percent on edge of lane, C) Swim speed, D) Percent rest. There were no significant This work was supported by the National Institute of Child Health and Human differences in fish-fish distance, End or Side of lane Development, R01 HD060647 and the National Institute of Environmental Health Sciences and F32 ES021342.