Grantee Research Project Results
Final Report: Behavioral Toxicology and Pathology of Early Exposure to Toxic PCOs
EPA Grant Number: R828224Title: Behavioral Toxicology and Pathology of Early Exposure to Toxic PCOs
Investigators: Kane, Andrew S. , Salierno, James D.
Institution: University of Maryland - College Park , University of Maryland School of Medicine
EPA Project Officer: Packard, Benjamin H
Project Period: September 1, 2000 through August 30, 2003 (Extended to April 30, 2005)
Project Amount: $423,264
RFA: Ecology and Oceanography of Harmful Algal Blooms (1999) RFA Text | Recipients Lists
Research Category: Water , Aquatic Ecosystems
Objective:
Harmful algal blooms (HABs) in the Mid-Atlantic States, and associated fish morbidity and mortality events, have increased in frequency and severity over the past several decades. The ability to predict and characterize environmental effects of different HAB species is essential to HAB remediation and control. This study was designed to investigate the effects of low level (sublethal) HAB stressor exposure on fish swimming behavior and pathology. The hypotheses, as originally proposed, included: (1) identification of behavioral indices that are related to HAB and other toxins; (2) quantification of behavioral endpoints related to low-level HAB exposure; and (3) evaluation of pathology associated with Pfiesteria exposure to experimental fish, including killifish (mummichog, Fundulus heteroclitus) and menhaden (Brevoortia tyrannus). However, work with menhaden was discontinued after the first year, since complicating variables prevented behavioral exposures of this species, even after prolonged laboratory acclimation. Also, there was limited access to Pfiesteria cultures for exposure of intact fish. Therefore, the third hypothesis was modified to include molecular alterations in the brain of HAB-exposed fish, specifically changes in the expression of c-Fos, “an immediate-early repair gene.” Gill and skin pathology of exposed fish was also conducted in association with exposure to varying cell densities of Pfiesteria shumwayae and Chaetocero s concavicornis. Therefore, the final goal of this project was to investigate and quantify possible alterations in mummichog swimming behavior, neuronal activity, and pathology resulting from HAB exposure, demonstrating that fish can be affected by low level HAB stress, which may then alter individual and population dynamics in the environment.
In order to accomplish this goal, a software program was developed for the analysis of schooling, shoaling, and individual behaviors in fish. The software system was then coupled to an existing videography system which allowed the capture of 30 -minute video segments of schools and shoals of mummichog before, during, and after exposure to toxins. Additionally, predator avoidance and startle response behaviors were analyzed by subjecting fish before, during, and after exposures to a model predator (bird) and an auditory/vibratory stimulus.
An acclimation study was conducted in addition to an exposure to tricaine methanesulfonate (MS-222), a common fish anesthetic and reference toxicant, in order to validate the behavioral quantification system. The acclimation study consisted of measuring mummichog behavior over a 3-day period to obtain baseline activities and changes in behaviors over time. Exposure of fish to MS-222 was then conducted as an exposure validation and for comparison to HAB neurotoxins. Once the system was validated and data from the acclimation and MS-222 experiments were analyzed, mummichog were then exposed to known HAB neurotoxins, brevetoxin (PbTx-2), or saxitoxin (STX).
Investigations into possible alterations in brain activity resulting from HAB sublethal stressor exposure in fish were also conducted. c-fos, an immediate early gene and its protein product, c-Fos, are indicators of neuronal activity and stress in mammals. The procedure used to quantify c-Fos protein in mammals was adapted for use in mummichog brains. Initially, a transport stress experiment was conducted with mummichog to test the ability to quantify changes in c-Fos protein expression in physically stressed fish. Once changes in c-Fos expression were quantified in different regions of mummichog brains, mummichog were exposed to HAB neurotoxins PbTx-2, STX, domoic acid (DA), and the HAB species, P. shumwayae and C. concavicornis. Different regions of the brain were examined in an attempt to discern if stress-specific regional expression in c-Fos occurred.
Summary/Accomplishments (Outputs/Outcomes):
Exposure to sublethal HAB stressors in the laboratory resulted in quantifiable alterations in both behavior and neuronal activity in groups of mummichog. Mummichog exposed to HAB toxins and the anesthetic, MS-222, displayed significant alterations in schooling and shoaling behaviors and neuronal activity. Exposure to STX and MS-222 significantly decreased interactions and schooling and shoaling behaviors, whereas exposure to PbTx-2 increased these behaviors. The composition of schools, shoals, and sedentary groups of exposed fish significantly decreased from groups of five to groups of three fish after exposure. Additionally, exposure to all three toxins altered startle response and predator response behaviors. Twenty-four hours after exposure, groups of fish exposed to all three toxins had significant decreases in activity and frequency of behaviors compared with pre exposure activity. These results suggest that sublethal exposure to HAB toxins can have demonstrable effects on swimming and social behaviors, as well as on startle response and predator avoidance behaviors. These alterations can last for at least 24 hours, which can negatively affect groups of mummichog by reducing their ability to maintain schooling, shoaling, and avoidance behaviors in the laboratory. This is the first study to quantify alterations in social behavior of fish exposed to HAB toxins. These data will provide a better understanding of how environmentally realistic concentrations of HAB stressors affect fish behavior. Empirical behavioral observations were also recorded from fish exposed for the purpose of evaluating changes in exposure-related c-Fos expression. There were notable changes in swimming behavior when fish were exposed to Pfiesteria cultures, saxitoxin, and domoic acid, but not Chaetoceros cultures or brevetoxin.
Exposure of mummichog to DA, STX, PbTx-2, P. shumwayae, C. concavicornis, and transport stress resulted in significant alterations in c-Fos expression, an indicator of neuronal activity. The excitatory neurotoxins, DA and PbTx-2, and transport stress, increased c-Fos expression, while STX significantly inhibited c-Fos expression. In addition, exposure to P. shumwayae and C. concavicornis increased c-Fos expression in a density-dependent relationship. c-Fos expression in these fish may be associated with neuronal stress and alteration in signaling. Stressor-specific c-Fos expression was observed, with P. shumwayae and C. concavicornis increasing c-Fos expression in the telencephalon and optic lobes, and physical transport and chemical stressors increasing expression in the optic lobes. This is the first study to quantify alterations in c-Fos expression in the telencephalon and mesencephalon of fish. These alterations in brain activity may ultimately be linked to behavioral changes such as those quantified in the laboratory.
There were no histopathological alterations in the skin or gills of fish exposed to cultures of P. shumwayae or C. concavicornis for the short (120- minute) exposure times in this study. This does not obviate the behavioral or central nervous system changes that were observed in the c-Fos experimental exposures, rather it illustrates the early and sensitive detection of the alterations in c-Fos expression.
This study indicates that alterations in behavior and CNS activity can serve as sensitive indices of HAB exposure. Endpoints of greatest sensitivity to low-level HAB exposure were changes group schooling and shoaling dynamics, as well as changes in startle response dynamics. These endpoints have the potential to be streamlined and optimized to permit higher throughput assays for field water samples. The quantification of alterations in behavior and neuronal activity resulting from sublethal HAB stressor exposure in the laboratory may ultimately elucidate changes in individual and population dynamics of fish exposed to HAB events in the field.
Journal Articles on this Report : 4 Displayed | Download in RIS Format
Other project views: | All 9 publications | 5 publications in selected types | All 4 journal articles |
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Choich J, Salierno JD, Silbergeld EK, Kane AS. Altered brain activity in brevetoxin-exposed bluegill, Lepomis macrochirus, visualized using in vivo14 C 2-deoxyglucose labeling. Environmental Research 2004;94(2):192-197. |
R828224 (Final) |
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Kane AS, Salierno JD, Gipson GT, Molteno TCA, Hunter C. A video-based movement analysis system to quantify behavioral stress responses of fish. Water Research 2004;38(18):3993-4001. |
R828224 (Final) |
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Salierno JD, Snyder NS, Murphy AZ, Poli M, Hall S, Baden D, Kane AS. Harmful algal bloom toxins alter c-Fos protein expression in the brain of killifish, Fundulus heteroclitus. Aquatic Toxicology 2006;78(4):350-357. |
R828224 (Final) |
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Stine CB, Baya AM, Salierno JD, Kollner M, Kane AS. Mycobacterial infection in laboratory-maintained Atlantic menhaden. Journal of Aquatic Animal Health 2005;17(4):380-385. |
R828224 (Final) |
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Supplemental Keywords:
RFA, Scientific Discipline, Geographic Area, Water, Waste, Ecosystem Protection/Environmental Exposure & Risk, Limnology, Ecosystem/Assessment/Indicators, Ecosystem Protection, exploratory research environmental biology, Chemical Mixtures - Environmental Exposure & Risk, Contaminated Sediments, State, Chemistry, Ecological Effects - Environmental Exposure & Risk, Southeast, algal blooms, Ecological Effects - Human Health, Biology, Ecological Indicators, East Coast, ecological exposure, pfiesteria piscicida, North Carolina, agricultural runoff, ecology, bloom dynamics, fish kills, Shephard's Crook, whole-fish bioassay, suburban watersheds, economic assessments, fish lesions, Gyrodiniums, harmful algal blooms, ligand sensitive hosts, Everglades, environmental chemistry, pfiesteria, toxins, bioassay, dinoflagellate, Glenodriniums, Tilapia, Florida, fluorescence sensing, Virginia, ScripsiellasProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.