Salmon (Oncorhynchus ssp) and steelhead (Oncorhynchus mykiss) populations in the Pacific Northwest have declined drastically in recent years. Many runs have been listed under the U.S. Endangered Species Act (ESA). One contributing factor may be the degradation of water quality due to the presence of agricultural pesticides in spawning and rearing habitat. Previously, streams within an intensive agricultural region of Oregon were tested for a class of insecticides, the organophosphates (OPs), known to be extremely toxic to salmonids. Chlorpyrifos and Azinphos-methyl were detected at concentrations exceeding state water quality levels. Brain and plasma acetylcholinesterase (AChE) activity (the target enzyme of OP toxicity) in juvenile steelhead was decreased between 0-20% during 24 and 96 hour laboratory exposures within these environmental levels. This low inhibition is generally considered to be within safe limits.  

Recent studies, however, showed exposure to the OP, diazinon, also inhibits olfactory function of Atlantic salmon at these environmental levels. Olfactory mediated processes, such as reproduction, feeding, predator avoidance, and home stream imprinting, are critical for the survival of these fish.  

My research at Hokkaido University and the Toya Lake Station focused on evaluation techniques to assess the olfactory function of Rainbow trout and Masu salmon. These techniques were then used to test olfactory function after short exposures to the pesticide, Chlorpyrifos. 

Evaluation of fish olfaction function included electro-physiogram recordings, patch-clamp analysis, Y-maze tests and AChE enzyme assays. Other activities included pituitary cell imaging and continuous blood collection techniques. 

First, we evaluated the integrity of AChE activity in various regions of the brain and the olfactory organs. The AChE assay measures the activity of the neurological enzyme, acetylcholinesterase. This enzyme regulates the transmission of neurological signals within the nerve synapse. OP insecticides will inhibit this enzyme, causing uncontrolled synapse firing. 

Second, we recorded olfactory nerve impulses before, during and after exposures to low levels of Chlorpyrifos. A tungsten probe was directly inserted into the olfactory nerve, then the olfactory epithelium was perfused individually with two amino-acids, serine or alanine, or to a dilute saline solution. 

Third, patch-clamp analysis was performed on individual olfactory cells from a Rainbow trout and responses to a mixture of amino-acids was observed. Patch-clamping involves directly attaching a glass electrode to the either the surface of an individual cell (cell attachment) or to establish contact with the  inner cell (whole cell). No fish in this experiment were exposed to the OP, but future investigations will utilize this technique for possibly determining the site and mechanism of olfactory inhibition.  

Fourth, behavioral responses were tested with a Y-maze challenge. Fish were placed at the base of a Y-shaped tank and each arm of the tank treated with either control water or water treated with a mixture of amino-acids. Fish with intact, healthy olfactory functions typically prefer these stimulants in response to feeding behavior. Preference is determined by time spent within each side of the Y-maze, recorded by video-monitoring. No OP-exposed fish were evaluated at this time, but future experiments are planned for this test. 

Research over the next couple years will detail the effects of OP exposure to salmon and steelhead fish at current environmental levels. This will involve identifying concentrations at which olfactory function becomes impaired, time of inhibition and recovery, mechanism of action, and behavioral implications.