Progress 03/30/00 to 12/31/03
One goal of this project was to develop and refine procedures of Toxicity Identification Evaluation (TIE) procedures for several invertebrate species focusing on species resident in the Sacramento-San Joaquin Delta, which are important food organisms for juvenile Chinook salmon and possibly other at-risk fish species. Toxicity tests were developed at the Aquatic Toxicology Laboratory (ATL) at UC Davis. Two commonly used pesticides, the organophosphate diazinon and the pyrethroid esfenvalerate were chosen as model test compounds, and 96-hour LC50s were determined for each species. LC50s were also determined for the P450 enzyme inhibitor piperonyl-butoxide (PBO). Sublethal effects of two pesticides, chlorpyrifos and esfenvalerate, were determined in juvenile Chinook salmon. Four to five month old salmon were exposed to a range of pesticide concentrations, and tissue samples of surviving fish were analyzed for stress protein expression, cytokine transcription, and
acetylcholinesterase (AChE) activity. At the highest concentrations, both pesticides led to complete mortality, whereas medium and low concentrations resulted in high survival rates. Significant differences in stress proteins, cytokines and AchE activity were found between control and surviving chlorpyrifos-exposed fish. Elevated stress protein expression was the only detectable response to esfenvalerate. To test the hypothesis that pesticide exposure enhances susceptibility to disease, we performed a 3-month experiment in which we exposed juvenile chinook salmon to nominal concentrations of 5 ug/L chlorpyrifos and 0.1 ug/L esfenvalerate for 4 days. These concentrations did not cause mortality within this time period. The fish were subsequently exposed to infectious concentrations of Infectious Hematopoietic Necrosis Virus (IHNV), and maintained in flow-through conditions for 7 weeks. Mortality was recorded and samples for biomarker analysis taken after 1) pesticide exposure, 2)
pesticide and virus exposure and 3) at the end of the experimental period. Samples have been analyzed for acetylcholine esterase activity, and results indicate that enzyme activity was reduced to 54% after the 4-day exposure to chlorpyrifos, and remained low (79%) after 16 days of recovery. Enzyme activity was equal to controls after 56 days. Significantly higher levels of stress proteins were found in muscle and gills of fish exposed to the combined stressors (pesticides and pathogen) than in fish exposed to individual stressors. Cytokine analysis showed that interleukin-1 m-RNA was significantly higher in esfenvalerate-exposed fish than in all other groups. Mx protein mRNA was elevated in virus-infected fish. To test if pesticide exposure and associated changes in cytokine transcription result in significant impairment of immune cell function, fish were exposed to sublethal concentrations of esfenvalerate and chlorpyrifos, and blood samples taken after 6, 8, 10 weeks to perform
quantification of fish antibody to a specific antigen (dinitrophenylated keyhole limpet hemocyanin). This immune-system endpoint is being related to cytokine transcription and other biomarker endpoints.
This project enhances our ability to identify pesticide impacts in the watershed. The use of resident species in toxicity testing and toxicity identification procedures strengthens the link between toxicity quantified by standard laboratory tests and the ecosystem of the Sacramento-San Joaquin watershed. Our studies on the sublethal effects of dormant-spray insecticides in juvenile salmon will enable decision makers to better evaluate chronic effects of common pesticides and help in determining water quality standards and TMDLs.
- Eder K.J., Leutenegger C.M., Koehler H.R., Wheelock C.E., Hammock B.D., Wilson B.W., Werner I. 2003. Molecular and cellular biomarker responses to pesticide exposure in juvenile Chinook salmon (O. tshawytscha). Abstract, 12th International Symposium on Pollutant Responses in Marine Organisms (PRIMO), Safety Harbor, Florida, 8-13 May 2003.
- Werner I., Deanovic L.A., Kuivila K., Orlando J., Pedersen T. 2003. Concentrations of organophosphate pesticides and corresponding bioassay toxicity in the Sacramento-San Joaquin Delta. Abstract, Calfed Science Conference, Sacramento, California, January 14-16, 2003.
- Eder K.J., Clifford, M.A., Hedrick R.P., Werner I. 2003. Cellular effects of exposure to pesticides and virus in juvenile Chinook salmon (O. tshawytscha). Abstract, 24th Annual Meeting of the Society of Environmental Toxicology and Chemistry North America, November 9-13, 2003, Austin, Texas.
- Eder K.J., Leutenegger C.M., Wilson B.W. and Werner I. 2004. Molecular and cellular biomarker responses to pesticide exposure in juvenile Chinook salmon (Oncorhynchus tshawytscha). Marine Environmental Research.
- Rosenkranz, P., Deanovic, L. and Werner, I. 2001.Using the estuarine amphipod Gammarus daiberi in toxicity testing. Abstract, State of the Estuary Conference, San Francisco, CA, October 9-11, 2001.
- Eder, K., Leutenegger, C., Koehler, H.-R. and Werner, I. 2002. Stress protein expression and immunological responses in juvenile chinook salmon (Oncorhynchus tshawytscha) exposed to pesticides. Abstract, Society of Environmental Toxicology and Chemistry, Vienna, Austria, May 12-16, 2002.
- Eder, K., Leutenegger, C., Koehler, H.-R. and Werner, I. 2002. Stress protein expression and immunological responses in juvenile chinook salmon (Oncorhynchus tshawytscha) exposed to pesticides. Abstract, Regional Chapter, Society of Environmental Toxicology and Chemistry, Davis, CA, May 16-17, 2002.
- Eder, K.J., Leutenegger, C.M., Koehler, H.R., Wheelock, C.E., Hammock, B.D., Wilson, B.W. and Werner, I. 2002. Molecular and cellular biomarker responses to pesticide exposure in juvenile Chinook salmon (O. tshawytscha). Abstract, Society of Environmental Toxicology and Chemistry, Salt Lake City, Utah, November 16-20, 2002.
- Werner I.2003. Using biomarkers to detect sublethal effects of pesticides in juvenile Chinook salmon (Oncorhynchus tshawytscha) and medaka (Oryzias latipes). Abstract, NorCal SETAC Annual Meeting, May 6-7, 2003, Berkeley, CA. _
Progress 01/01/02 to 12/31/02
Identification of pesticides of concern: In collaboration with UC Berkeley and USGS co-investigators we conducted an initial data review to identify pesticides of concern and field sites: We reviewed existing information from the Central Valley Regional Water Quality Control Board (CVRWQCB), USGS NAWQA and Toxics Programs, DPR and other agencies and watershed projects such as the Sacramento River Watershed Project to compile data on pesticide concentrations in the Sacramento/San Joaquin Rivers and Delta. To supplement the above concentration data we compiled literature data on pesticide toxicity and reviewed results of past Toxicity Identification Evaluations (TIE) in the Rivers and Delta to establish probable agents of toxicity. Resident species test development: Testing protocols have been established for the cladocera Simocephalus vetelus, the midge larva Chironomus riparius and the amphipod Gammarus daiberi. We are presently running a series of tests comparing
pesticide sensitivities of resident (Delta) Hyalella azteca (Amphipoda) with cultured animals obtained from a commercial supplier. Effects of pesticide exposure in juvenile Chinook salmon: Analysis of biomarkers from juvenile chinook exposed to chlorpyrifos and esfenvalerate in the laboratory continues. All biomarkers analyzed so far (acetylcholine esterase and carboxyl esterase activities, stress protein levels and cytokine m-RNA changes) were altered by chlorpyrifos at a concentration of 1 ug/L (nominal), whereas only stress proteins were induced by esfenvalerate at a concentration of 0.1 ug/L (nominal). To test the hypothesis that pesticide exposure enhances susceptibility to disease, we performed a 3-month experiment, where we exposed juvenile Chinook salmon to 5 ug/L chlorpyrifos and 0.1 ug/L esfenvalerate for 4 days. These concentrations did not cause mortality within this time frame. The fish were subsequently exposed to infectious concentrations of Hematopoietic Virus, and
maintained in flow-through conditions for 7 weeks. Mortality was recorded and samples for biomarker analysis taken after 1) pesticide exposure, 2) pesticide and virus exposure and 3) at the end of the experimental period. Samples will be analyzed in 2003. Development of Toxicity Identification Evaluation (TIE) procedures: We have determined LC50 values for S. vetelus, C. tentans, C. riparius, Hyalella azteca and G. daiberi for the P450 inhibitor piperonyl-butoxide (PBO) used in TIE procedures, and the pesticides diazinon and esfenvalerate. Field studies: During the winter of 2002 we regularly sampled five sites (Ulatis Creek, Vernalis, Knight's Landing, mouth of the Tuolomne, and Yuba City). Only one water sample was toxic in the 2001/2002 storm season: Ulatis Creed collected 03/20/02. Mortality of C. dubia was 100% within 48 hrs and TIEs indicated that toxicity was due to an organophosphate pesticide.
- No publications reported this period