Kelly Luedke and Matt Bartley, Physiological effects of diazinon on fathead minnow fry (Pimephales promelas)

Abstract

The purpose of this experiment was to determine the physiological effects of diazinon on fathead minnow fry.  Diazinon is a general insecticide with an anticholinesterase effect.  Two day old fry were tested with four treatments of varying concentrations of diazinon in water (12.5, 25, 37.5, and 50 parts per billion).  Behavioral changes in swim movements and number of swim movements per minute were recorded.  At all concentrations of diazinon, fish showed significant decreases in the swim movements per minute over an hour interval (paired t-test).  Behavior of fish in water only (control) consisted of a constant, rhythmic pattern of zigzag darts in a forward direction, with an average of 84 swim movements per minute (mpm).  Initial behavior after exposure to diazinon included sporadic, explosive, long bursts of movements by the fish (0-10 minutes after exposure).  Thirty minutes after exposure, the fish could be seen twitching, and after one hour, there was little or no movement at all.  Only the fish exposed to 12.5 ppb diazinon survived after 24 hours, and these did not fully recover.  Based on the results, diazinon used for agricultural and residential purposes could have a devastating effect on the small aquatic organisms, such as Pimephales promelas, found in micro-environments exposed to runoff. 
 

Introduction

Diazinon is a common ingredient in many commercial insecticide products.  It is an organophosphorus compound that disrupts the nervous system of an organism by reducing the activity of acetylcholinesterase at nicotinic and muscarinic receptors.  Reducing the activity of this enzyme allows a prolonged effect of acetylcholine, a neurotransmitter, on the receptors.  Acetylcholine is the principle neurotransmitter used in insect sensory and motor systems and the only neurotransmitter used in vertebrate motor endplates (Girard, 2004).  The disabling of acetylcholinesterase can have a toxic effect leading to detrimental physiological effects or even death at certain concentrations.  Diazinon and other organophosphorus compounds have been used increasingly since the 1970’s when organochlorine pesticide use (such as DDT) was banned in the United States.  Substances like DDT are not readily biodegradable and last for years resulting in bioaccumulation in wildlife.  Diazinon is short-lived (an average lifespan of fourteen days, depending on temperature or soil and water pH) and does not bioaccumulate in organisms (Eisler, 1986).  Seemingly, this is an ideal pesticide with little environmental damage.  However, with conditions of low moisture and low temperatures, diazinon can stay biologically active for six months in contaminated soil (EXTOXNET, 2001).

It is possible for serious environmental damage to occur from use under certain conditions (Friend & Franson, 1998).  Direct exposure has been shown to kill birds, especially on golf courses. Larger animals in the food chain have died from secondary poisoning from consuming contaminated organisms (Friend & Franson, 1998).  Most recent research has been done on larger animals such as birds, mammals, or adult fish, but little research has been done on invertebrates or developing fish, which may become contaminated through residential and agricultural runoff.  Only very small concentrations are required to cause detrimental effects or death to these organisms (Eisler, 1986).  For example, EXTOXNET (2001) reported that the LC-50 for rainbow trout is 90-140 ppb, while warm water fish like fathead minnows are more resistant (LC-50 of 0.5 ppm-15 ppm).  Our experiment was motivated by earlier studies that tested the toxicity of diazinon on brook trout, rainbow trout, and (adult) fathead minnows (Allison and Hermanutz, 1977; S. Beauvais, S. Jones, S. Brewer, and E. Little, 2000). 

In 2000 over 13 million pounds of the insecticide diazinon were applied annually across the United States (EPA, 2000).  The Environmental Protection Agency (2000) also reported that almost 80 percent of this usage was for residential insect control, both indoors and outdoors.  The EPA (2000) reported that diazinon is highly toxic to freshwater fish and invertebrates following acute exposure.  Improper storage, usage, and disposal techniques for this chemical can lead to waste runoff, water contamination, and harm for many aquatic organisms.   

The purpose of our study was to test the behavioral effects of small concentrations of diazinon on developing fathead minnows, Pimephales promelas, and relate that to the physiological effects of the chemical on the organism’s nervous system.  This is directly related to the effects of water contamination due to runoff or irrigation.  Testing the immediate effects of diazinon on aquatic organisms as it enters a water supply is relevant, considering the relatively rapid degradation of the chemical in the environment.  The effects of this chemical can cause a decrease in survival mechanisms or even death in the exposed fish (S. Beauvais, S. Jones, S. Brewer, and E. Little, 2000).  We chose to observe the number of swim movements per minute for the fish and also the qualitative behavioral changes after exposure to different concentrations of diazinon.  Both sexes of fathead minnow fry have a constant rhythmic pattern of swimming, and so any changes in the organism can easily be detected by a change in this pattern.

Diazinon inhibits acetylcholinesterase, and consequently prolongs the activity of acetylcholine on the nicotinic and muscarinic receptors throughout the organism.  Our hypothesis was that the initial reaction of the fry to diazinon exposure would be an increase of swimming movements for the first hour.  After 1 hour of treatment, we predicted that the swimming movements per minute would decrease due to exhaustion of the energy supply.  The four test concentrations that were tested were 12.5, 25, 37.5, and 50 ppb of diazinon.  Note here also that the commercial diazinon product Spectracide is diluted to 25 percent, so we were careful to make the proper measurements for concentrations.  The concentrations of diazinon are lower than the LC-50 (the concentration of a substance that will kill 50 percent of the test subjects), so we predict that none of the exposures will kill the organisms.  The lowest concentration of treatment (12.5 ppb diazinon) would have a minimal effect on swimming movements, while the highest concentration (50 ppb diazinon) would significantly change normal swimming activity.

Methods

We used a common diazinon product called Spectracide (Spectrum Brands Div., United Industries, St. Louis, MO) as the model insecticide chemical product.  Our test subjects were fathead minnows (Pimephales promelas) in the fry stage, two days after hatching, approximately 1 cm or less in length, gender undeveloped.  Fish were obtained from the UW--Stevens Point College of Natural Resources Water Lab.  Four fish were exposed to each of four treatments (12.5, 25, 37.5, 50 ppb diazinon) for a total of sixteen fish.  The control and treatment water were kept at a constant room temperature.  Because minnows in the fry stage are still receiving nutrients from their yolk sac, they did not need to be fed. 

To transport the fry, we used a large-bore pipette with the tip cut off.  Before subjecting the fry to the diazinon, the fry’s swimming movement rate was observed and also the behavior of the fry was qualitatively noted.  These observations were used for the control and provided a baseline with which to compare treatments.  A 9 cm diameter glass circular dish was used to view the fry, and a video camera was mounted above the dish.  Three minutes of the fry’s actions were recorded, and then an average swim rate and behavior pattern was noted over this time period.  After the recording, the fry were transferred to the prepared dish with the appropriate diazinon solution.  When the last fry was transferred, the three minute video recording period began.  Observations were recorded 10 minutes, 30 minutes, 1 hour, and 24 hours after exposure to diazinon treatment.

This process was repeated for all remaining concentrations, such that each fish was first recorded under control conditions to provide a baseline of comparison, and then was exposed to one of the four solutions.  The qualitative data included notes from observing the first 5 minutes of the 37.5 ppb diazinon, demonstrating the initial reaction to diazinon.   We counted how many movements the fish made each minute for a total of three minutes, and then recorded the average number of swim movements per minute (mpm). The method of statistical analysis was to compare the control mpm to the four treatments at the progressive time intervals using paired t-tests. 

Results

Movements per minute

Paired t-tests were used to compare the average control swim movements per minute (mpm) and treatment mpm for at each of the time intervals (10 min, 30 min, 1 hour, 24 hours).  The mean swim movements per minute and standard deviations for the four concentrations at varying times of exposure can be found in Table 1.  The average (± SD) control mpm for all sixteen fish tested was 84 ± 21 mpm.  Figure 1 shows the general trend of swim movements per minute during diazinon exposure decreasing over time in comparison with the control values.  The fish exposed to 37.5 ppb diazinon showed a slightly different pattern of decreased movements, but overall each treatment group showed a decrease of swim movements to zero or almost zero after one hour of exposure. 

Effects of the lowest concentration treatment (12.5 ppb diazinon), were varied.  At 10 minutes, the mpm increased but was not significantly different than the control mpm (paired t-test, P=0.138).  After 30 minutes and one hour there was a significant decrease in mpm for the fish compared to the control (paired t-tests, P=0.003 and P=0.000, respectively).  After twenty four hours the fish had recovered in the sense that they had almost the same mpm, with an average of four movements per minute more as the control, so there was no significant difference (paired t-test P=0.656). 

Exposure to 25 ppb diazinon resulted in a more profound effect on the minnows in a shorter period of time.  There was no significant difference after 10 minutes of treatment (paired t-test, P=0.360), but after 30 minutes and one hour there were significant decreases in the mpm compared to the control for the minnows (paired t-tests, P=0.000 and P=0.000, respectively).  The twenty four hour test was discontinued because the fish tested at 25, 37.5, and 50 ppb diazinon died before twenty four hours.  As a result, we believe that we found the LC-50 for the organisms at this developmental stage (fathead minnows, two days old) to be between 12.5 ppb and 25 ppb diazinon.

The fish tested at 37.5 and 50 ppb diazinon showed significant decreases in mpm at 10 minutes, 30 minutes, and one hour relative to control movements (paired t-tests, P≤ 0.004 for all tests).  Both fish groups tested at 37.5 and 50 ppb diazinon for one hour did not exhibit any movements during the observation time but they were observed to still be living.

The largest decrease of mpm occurred during the 10 to 30 minute interval.   There was a significant decrease in mpm between 10 and 30 minutes of exposure for fish treated with 12.5, 25, and 50 ppb diazinon (paired t-tests, P=0.000, P=0.002, and P=0.000, respectively).  There was no statistically significant decrease between 10 and 30 minutes for the fish treated with 37.5 ppb diazinon, although there was an average 7 mpm decrease between the two intervals. 

Behavioral swimming patterns

Qualitative observations for the swimming behavior were recorded as well.  Control behavior consisted of a constant, rhythmic pattern of zigzag darts in a forward direction, with an average of 84 mpm (swim movements per minute).  Initial behavior after exposure to diazinon included sporadic, explosive, long bursts of movements by the fish (0-10 minutes after exposure).  Thirty minutes after exposure the fish could be seen twitching, and after one hour, there was little or no movement at all.  Only the fish exposed to 12.5 ppb diazinon survived after 24 hours, and these did not fully recover.  Although the fish regained the same number of swim movements per minute as observed in the control group, their swimming movements failed to produce forward motion.  The pattern was similar to the control group because they were constant rhythmic movements, but they typically stayed in one position and gained little forward distance. 

Discussion

Our original hypothesis that the swimming movement rate would increase over the first hour exposed to diazinon was not supported.  The only increase in swimming movement rate was observed within the first ten minutes exposed to treatment.  The second hypothesis stating that the swimming movement rate would decrease after one hour of exposure was supported by the data.  The reason the only increased mpm was observed within the first ten minutes was because the fry were very small and more susceptible to the diazinon in the water.  The diazinon was absorbed by the fry quickly and produced immediate effects.  Diazinon is converted to diazoxon in animals, which is a strong enzyme inhibitor (EXTOXNET, 2001).  The insecticide caused a breakdown of acetylcholinesterase (AChE) in the nervous system, thus causing prolonged effect of acetylcholine (ACh) on the nicotinic and muscarinic receptors.  This process caused the sporadic, explosive, long bursts of muscle spasms in the fry. 

The nicotinic receptors that receive ACh are involved in the somatic nervous system functions such as muscle contractions used for locomotion.  Consequently, diazinon allowed prolonged binding of ACh to the sarcolemma of the muscle fibers.  The muscarinic receptors that receive ACh are involved in the autonomic nervous system, specifically, the parasympathetic pathway.  This pathway stimulates the activity of maintenance functions such as digestion which involve smooth muscle and glands.  We were not able to detect these specific effects on parasympathetic function in the fry.  However, previous studies have documented human cases of diazinon exposure resulting in nausea and vomiting, abdominal cramps, slow pulse, diarrhea, pinpoint pupils, difficulty in breathing, and passing out (ATSDR, 1996).  In a few extreme cases high exposure has resulted in human death (ATSDR, 1996).

After the initial increase of mpm there was a steady decrease until one hour after the treatment.  Figure 1 only shows the decrease because the readings were taken at the ten minute observation period after the muscle fatigue set in.  After 30 minutes the fry were observed twitching with few short bursts and pivoting side to side, going in circles with no forward distance gained.  This is due to the depletion of ATP in the fatigued muscles in the fry.  After being exposed for 1 hour there was little to no movement due to almost total energy depletion.  This depletion ultimately resulted in death after 24 hours, with the exception of those treated with the smallest amount of diazinon.  The fry that survived 24 hours displayed weaker contractions and a decline in muscular performance. 

S. Beauvais, S. Jones, S. Brewer, and E. Little (2000) studied the neurotoxicity of diazinon and malathion to larval rainbow trout (Oncorhynchus mykiss) and their correlation with behavior and concluded that insecticides acting through cholinesterase inhibition adversely impacted learning, behavior, and schooling as well as caused both over-excitation and unresponsiveness.  This statement supports our explanations for the initial muscle spasms and the following lethargy.  It is possible that some fish have a compensatory mechanism that requires a threshold value to be reached before activated.   This mechanism protects the fish from biochemical injury for a short time period (S. Beauvais, S. Jones, S. Brewer, and E. Little, 2000).  This is a possible explanation for the slower average rate of decrease for 37.5 ppb diazinon treatment (represented in Figure 1).  This concentration may have been close to the threshold concentration needed to activate the protective mechanism in the fish.    

With more time and resources, it would be helpful to test for environmental levels of diazinon to compare with the concentrations used in this study.  Future research regarding diazinon effects on fry may concentrate more on the 10 minute period following exposure to diazinon.  In fact, it may be best to concentrate on measuring the first 3 minutes after exposure.   More observations should be made during the initial ten minutes of exposure because within this time period we qualitatively observed an increase in swimming movements.  It would have been beneficial to make qualitative observations at all treatment concentrations during the initial ten minutes of exposure.  Also, it may be beneficial to record the distance the fish swim more quantitatively. 

It would be interesting to study long term or permanent damage of diazinon on the developing stages of Pimephales promelas.  The fry exposed to 12.5 ppb diazinon recovered but did not behave normally after 24 hours.  Would these fry completely recover after a longer time period?  How would fry react to low chronic doses of diazinon throughout development to adult fish?  Diazinon typically degrades in the environment after 2 to 4 weeks (EXTOXNET, 2001), but repeat applications of the pesticide may cause chronic exposure for fish through agricultural runoff.  Chronic toxicity studies have been tested on rats and other mammals but are less common for fish and invertebrates. Field studies would include measuring diazinon concentrations at various runoff sites, residential or agricultural. 

Our experiment was significant because little is known about the response to diazinon in young or developing organisms.  Because of their smaller size and greater metabolic rates, we found these organisms had a much faster and profound response to the toxin.  In addition, the concentration of diazinon that caused negative effects was much lower than anticipated.  This shows that anywhere that insecticide runoff is present; it is possible that the organisms in that microenvironment will suffer negative effects from exposure to even low levels of diazinon.  The damage to these organisms, including the fathead minnow fry, is difficult to immediately detect and so often goes unnoticed.  The actual environmental cost does not emerge until effects are seen in organisms higher on the food chain.  Therefore the effects of diazinon are more detrimental than the commercial seller may indicate.  We believe further examination of the impacts of diazinon on developing aquatic organisms should be researched to better understand the effect of diazinon on these ecosystems.

We would like to thank Dr. Isabelle Girard and Dr. Ron Crunkilton for their help in supplying equipment and supporting our experiment.  We thank both Dr. Isabelle Girard and Dr. Chris Yahnke for reviewing our report and giving helpful advice. 

References

Agency for Toxic Substances and Disease Registry (ATSDR). 1996. Toxicological profile for diazinon. Atlanta, GA: U.S. Department of Health and Human Services, Public Health Service.

Allison, D., and R. Hermanutz. 1977. Toxicity of Diazinon to Brook Trout and Fathead Minnows. Ecological Research Series.

Beauvais, S., S. Jones, S. Brewer, and E. Little. 2000. Physiological measures of neurotoxicity of diazinon and malathion to larval rainbow trout (Oncorhynchus mykiss) and their correlation with behavior.  Archives of environmental contamination and toxicology. p 70-76.

Eisler, R. 1986.  Diazinon hazards to fish, wildlife, and invertebrates: a synoptic review. U. S. Fish Wildlife Biological Report No. 85 (1.9).  Laurel, MD: U.S. Department of the Interior, Fish, and Wildlife Services.

Environmental Protection Agency (EPA).  2000.  Diazinon summary.  Office of Pesticide Programs.  Retrieved May 18, 2004, from http://www.epa.gov/pesticides/op/diazinon/summary.htm. 

Extension Toxicology Network (EXTOXNET). 2001.  Pesticide Information Profile: Diazinon.  Retrieved 7 September, 2004, from http://pmep.cce.cornell.edu/profiles/extoxnet/carbaryl-dicrotophos/diazinon-ext.html. 

Friend, M. & Franson, J.C. 1998. Organophosphorus and Carbomate Pesticides. Field Manual of Wildlife Diseases: General Field Procedures and Diseases of Birds.  Washington D. C.: U.S. Department of the Interior and U.S. Geological Survey.  p 287-294.

Girard, I.  2004.  Personal interview.  Professor of Animal Physiology, University of Wisconsin-Stevens Point.