PESTICIDE TOXICOLOGY. Introduction. Materials and Methods. Warning about handling pesticides. Experimental design

Transcription

1 PESTICIDE TOXICOLOGY Introduction Around the world, insects do tremendous damage to agricultural crops and destroy economically important resources such as forest. Insects are also important vectors of diseases that can be either incapacitating or lethal to humans and other animals. Put this all together and it s not surprising that we have attempted to eliminate what is really only a small group of insects that are a nuisance. As recently as 100 years ago, mechanical and cultural methods were the only tools available to control insects. The end of World War II brought new chemicals that replaced these measures and by the early 1960s use of organophosphates such as DDT had become wide spread. The dangers of relying only on pesticides to control insect pests is becoming clear, and only beginning to have an impact on our agricultural practices. Even with the use of integrated pest management and alternative control strategies the development of new chemicals, whether synthetic, or of natural origins will still be tools for control. This requires an understanding of the chemical's toxicity and a way to measure that, so that differences between them can be identified. During this laboratory you will be introduced to some common criteria, and methods, for assessing insecticide toxicity. Stringent standards and criteria for experimental designs for toxicological testing must be met and are governed by international bodies like the World Health Organization (WHO) and in Canada, Agriculture Canada. The criteria you will be following today are adaptations of procedures that might be used by researchers. Restraints of time and materials have us using non-standard exposure times and conditions. Insects haven t reacted passively to our chemical attack. They have become resistant to a variety of pesticides by using an elaborate detoxification system that has been part of their genetic make up since they and plants started their co-evolutionary dance many millions of years ago. Being ably to accurately quantify some measure of toxicity and follow any changes in the susceptibility of an insect to the poison provides a means of determining if resistance has occurred in a pest population. This is particularly important because failure to recognize resistance will result in the application of ever increasing doses of the pesticide to achieve control. Increased dosages to control insects only increase resistance! Your lab write up will follow the form and instructions for authors who wish to publish in the scientific journal: Pesticide Biochemistry and Physiology. Your write up should be in the form of a scientific paper submitted to that journal. You can find copies in the library Materials and Methods Warning about handling pesticides The acute dermal LD50 in rats for permethrin is >,000 mg/kg and >4,000 mg/kg for Malathions. These values are high and as such the chemicals are not present, during these experiments, at dangerous levels. This doesn't mean you can be careless. Avoid contacting and exposure to the impregnated filter paper disks as you would in all laboratory techniques involving chemicals. The concentrations on the filter paper are as % (weight/volume) and the filter paper diameter is known. What is the dose per unit surface area? Experimental design The test animal for our experiment is the Rice weevil, Sitophilus oryzae and we will be testing a pesticide in the presence and absence of a second chemical. Depending on availability from our supplier the pesticide will be either malathione or permethrin, the second chemical is an unknown. The compounds have been dissolved in acetone and applied to Whatman #1 filter papers with either pesticide on its own, or in combination with a fivefold larger amount of the unknown. The Acetone was allowed to evaporate and the filter papers place in petri dish test arenas. Pesticide Toxicology BIO 3333 Entomology Houseman

2 Experimental set-up The insect colony is reared in whole wheat seeds and adults will be available chilled, on ice. It s important to place only live insects in the test arenas. To do this, allow some of the weevils to warm up and as the crawl up to the rim of the container gently remove them to a second container. From this second container that contains only live insects place 5 (or approximately 5) adults into the exposure arena and close it. Weevils are quite active and their characteristic snout is an ideal tool to reach under the edge of the lid and pry it open to escape. To prevent this keep a weight on the tip of the weevil filled dishes at all times. We need a precise count of how many weevils are in each chamber. When you have filled 4 or 5 chambers (about half the concentration range being tested). Count the number of weevils in the chamber that are alive and dead. Use a felt marker to place the information on the dish 3L D (3 Living and Dead as an example). We need to be accurate at this point so that you don't attribute their death to the treatment when you look for mortality at the end of the exposure period. Why are these particular dilutions and concentrations of the chemicals used? Once you have completed this initial count and marked on the plates put an elastic band around the dishes to insure that the insect do not crawl out from underneath. We will be using two different exposure times. One is at four hours and the other at 4 hours later. For the four hour exposure we ll count the insects in the afternoon lab. For the 4 hour exposure we ll need volunteers to come to the lab at 10:00 the following morning. Please make an effort to show up. The more people we have the faster the task will get done. The criteria for mortality As mentioned mortality will be assessed four and twenty four hours after exposure. It is essential that we have a uniform method for scoring mortality. The criterion of mortality we ll use for this study is knockdown and is defined as a loss of leg movement and the inability to stand or walk. Tap the side of the petri dish on the bench, to dislodge the insects, give it a gently shake to spread the weevils evenly over the filter paper surface. Anyone moving and walking around is alive. Some insects may be on their backs and kicking frantically to try and roll over, that s alive as well, but we ll score that as moribund. You may find it helpful to place a small mark with a highlighter over what look like dead insects and then waiting to see if they are still underneath the mark a few minutes later. REPEAT this with the sample to insure that you successfully count all living, moribund, or dead animals. Once mortality has been assessed count the total number of insects, subtract the number for those that died from manipulation and determine the % mortality for each treatment. Be sure the number of animals in the treatment and the number dead from treatment on the table of class results. Did you notice anything about the insects that were dead, or those that were close to death and unable to cling to the paper? Pesticide Toxicology - - BIO 333 Entomology Houseman

3 Results: Probit Analysis The data curves in this experiment, and the one on feeding behavior lab, are S-shaped curves. Just as there were extremely low levels of the sugars that were undetected by the flies there are low concentrations of the pesticide that have no affect on most of the weevils. At the highest possible concentrations the pesticide kills all the weevils and even with increasing concentration the results remains as 100% mortality. Increasing the dose of the pesticide won t kill any more insects and the curve levels off. Between the two extremes is a gradual rise to a linear region of the curve followed by another gradual leveling off. When Biologists use statistics to compare dependent and independent variables, the data is usually in the form of a straight lines. In the fly lab you plotted the linear region and estimated a 50% response threshold from a series of lines for different sugars. The more data points in the linear region the more robust the statistics. Because biological data often plots onto curves, data transformations are used to produce straighter lines on which the statistical analysis can be done. One transformation typical of toxicity testing is the probit analysis that we will be doing in this lab. The simplest explanation is the probit transform increases the linear region of the S shaped curve and you ll use the linear region to calculate an LD 50. This time we are going to be much more precise about how we ll be doing it. We ll uses the statistical regression of the probit analysis to determine the error around the estimate of the LD 50, and then see of the two values, are significantly different from each other. Standard error of the determination, comparison of slopes and elevation is adapted from Biostatistical Analysis second edition, Jerrold H. Zar. Prentice-Hall (1984). Two common data curves are the S-shaped cumulative distribution curve and the normal curve, and both are related to each other Cumlative distribution Normal distribution Probits Fig. 1. Relationship between the normal distribution curve and the Cumulative distribution curve Probits are simply deviations from the mean of a normal distribution with a standard deviation of 1 and a mean of 5. You can convert between probabilities and probits by reading values from the cumulative distribution curve in Figure 1. So when half of the insects die in your sample (a value of 0.5 on the y-axis), that corresponds to a probit value of 5. Likewise, if 10% of your insects die, that equals a probit value of about 3.7. Probits are used to turn complicated curves Pesticide Toxicology BIO 3333 Entomology Houseman

4 (like the S-shaped curve in Figure 1) into straighter lines are easier to work with. Be warned this transformation increases the region of linearity, it still tails off at both ends like the original curve! Calculating % Mortality using Abbott s formula Some of the insects died in the controls containing acetone carrier by itself and the acetone carrier in the presence of the unknown compound. We need to account for death that hasn t occurred because of the pesticide using Abbott s formula. x - y % mortality = 100 x where : x = % survivorship in the control group (concentration of pesticide = 0) y = % survivorship in the experimental group Determining the probit values Once you have % mortality you can convert your values to probits using the probit calculator supplied on the web site. Enter the % mortality and read off the value. Again be warned the probit transformation only works for the near linear and linear parts of the curve. The calculation of probit for values like 100% mortality and 0% have no meaning. Determining LD 50 and the error of the estimate Plot probit values against the log concentration of the treatment and locate the linear part of the plot to determine the LD 50 for each of the two treatments. To do this you will need to calculate the equation of the line and also whether there is in a linear relationship that is significant using some very basic statistics. Microsoft Excel contains the tools you need, but they may not have been installed. The Analysis tools are part of an add-in and you ll find the Add-in menu under Tools. For your convenience we ve provided an Excel spreadsheet with an analysis similar to the one that you will be doing. Be warned, yes another warning, the samples are linear X,Y plots yours involve logarithmic transforms. Every straight line is defined by the usual equation of a line. y = bx+ a Where the slope of the line is given by the value b and the Y intercept or the elevation of the line is the value a when X=0. Simple manipulation of the equation and you can solve X when y=50. It get s trickier when we want to know the error in the estimate, or the confidence interval. To do that you ll need K using the t-value for the confidence interval, number of degrees of freedom for your regression and the standard error of the X. K b t = S b Pesticide Toxicology BIO 333 Entomology Houseman

5 You can use either T-tables to obtain the t-value or use the handy function INVT available in Excel using 0.05 for the 95% confidence interval, and the appropriate degrees of freedom, in this case N. Once K is determined then calculate the confidence interval X by ( i Y) t ( Y S i Y) + ± K K K 1 1 Y X n x In this equation X = X and Y n = Y n x can be determined using the DEVSQ function in Excel. Remember that as you make these final calculations you have used the log10 of the numbers and any results from these equations will have to have the antilog applied to them. Comparing two lines In addition to knowing the error in any measurement along the line biologists often have to know if two lines are different. They do that by first testing if their slopes are different, if they are then the lines are different. If the slopes aren t different they may be parallel lines. One may above the other or to the left and in these cases their elevations have to be tested for a significant difference. In this case a second test is required. Comparing the slope You have two regression lines, one for each of the two different pesticide treatments that you've investigated. Now you want to determine whether there is a difference between these two treatments, a difference between the lines. To do this you will use a T-Test and the t value it generates to see if the slopes b 1 and b of the lines are the same. In this analysis you are testing whether b1=b. Or, does the slope of curve 1 equal the slope of curve? b t 1 b = S b1 b The denominator in the equation is the standard error of the difference between the two regression coefficients. In our experiment the sum of the squares, again is calculated using DEVSQ function and is the same for both of the curves being tested S b1 S YX, b = x ( ) p ( S YX, ) p ( ) ( 1 x ) and the numerator you see here is the pooled residual square and is calculated as: S (Residual SS) ( YX, ) 1 Residual SS p = ( ) (Residual DF) 1 + ( Residual DF) SS is sum of squares DF is degrees of freedom that is the sum of the two residual degrees of freedom. From all of this you get a t value and use either T-tables to check for significance or use the handy function TDIST to determine what is the threshold T-value. If your computed T value is greater then the slopes differ. If the slopes differ then the lines are different. Comparing the elevation Pesticide Toxicology BIO 3333 Entomology Houseman

6 If the slopes are the same it doesn t mean that the lines are, it only tells us that the two lines are parallel to each other. One could be above the other or too the left and each of these example results in different Y-intercepts, or elevations for the curves. We have a number of calculations to do: Sum of the squares of X for common regression Sum of the cross products for the common regression Sum of the scares of Y for the common regression Residual SS for the common regression Residual degrees of Freedom Residual MS for the common regression A c = ( x ) + ( x ) 1 B c = ( xy) + ( xy 1 ) C c = ( y ) + ( y ) 1 B SS c = C c c A c DF c = n 1 + n 3 S Y X SS ( ) c c = DF c t = B c ( Y 1 Y ) ( X1 X A c ) ( ) 1 c ( X 1 X ) S Y X n 1 n A c Once again look up the value of t to determine if the elevations are the same. Pesticide Toxicology BIO 333 Entomology Houseman

7 Discussion Discuss the results you have obtained. What is the toxicity of the pesticide? Did the unknown chemical have an effect on the toxicity of the pesticide that we used? What would this type of chemical be called? The chemical we used has a molecular weight of and is a liquid with a density of It is insoluble in water and miscible in organic solvents. Can you guess what it is from what you may have read in the literature? What are sources of error in the experiment? Are their improvements that could be carried out to decrease these errors? Incorporate the answers to other questions contained in the hand out in this section. Pesticide Toxicology BIO 3333 Entomology Houseman