Aquatic Toxicology Lab 8 Analysis of Short-Term Chronic Test Data Ceriodaphnia dubia

1. Data Analysis a. Test Acceptability Criteria i. 80% survival in controls ii. average of 15 neonates per surviving female in controls b. Endpoints i. Mortality ii. Reproduction 2. Point Estimations - LC50 (a)Probit 3. Hypothesis testing - NOEC/LOEC a. null hypothesis - no difference i. when null hypothesis is shown to be false, the alternate hypothesis is assumed to be true b. alternate hypothesis - a significant difference exits i. the hypothesis that cannot be statistically tested or proven 4. Procedure a. refer to flowcharts on pages 153 and 159 of lab 7 handout b. 4 pieces of information needed to select appropriate analysis method i. concentrations where survival is significantly different from control (1)Fishers Exact Test ii. distribution of the data (1)shapiro-wilk's and chi-square iii. homogeneity of variance (1)Bartlett's, Hartley's, Cochran's, and Levene's tests iv. equality of replicates c. Survival i. see flowchart on p.153 ii. we will not include concentrations which are statistically significantly different from the controls iii. we will use Fisher's test to make this determination d. Reproduction i. see flow chart p.159 ii. NOEC/LOEC calculated using the mean number of neonates produced per adult including dead females (1)Concentrations with mortality significantly greater than controls (calculated using Fishers Exact Test) are not used iii. Depending on the distribution of the data, 4 tests will be used (1)Parametric i.e. Data are normal and variances are homogeneous (a)equal number of replicates (i) Dunnetts Procedure (b)unequal number of replicates (i) Bonferroni T-test

(2)Nonparametric i.e. data fail normality or homogeneity of variance tests or both (a)equal number of replicates (i) Steels Many-one Rank Test (b)unequal number of replicates (i) Wilcoxon Rank Sum iv. Minimum Significant Difference (MSD) (1)The minimum difference in mean reproductive output necessary for the null hypothesis (no difference) to be rejected.

(2)An important aspect of the data analysis (a)can vary due to changes in variability around control (3)Parametric test output has a MSD column (4)MSD cannot be calculated for nonparametric tests (5)Important because it normalizes a test result for variability in the controls and allows comparison of different tests . e. Download Toxstat from the Aquatic Tox Lab web page i. www.ias.unt.edu/~waller/aquatic_toxicology/laboratory ii. The file we want is toxstat.zip iii. Save toxstat.zip to either the H: drive or a floppy if you have one iv. Unzip the file as follows v. click the start button (1)click run (2)click browse (3)locate toxstat.zip file on either your h: drive or your floppy drive (a:) and click it (4)click open (5)click OK (6)this should begin the unzip program (7)save the files to the h: drive or you floppy drive f. Sample Data Set - Use Data Set 1 at the end of the handout . This is the same data set as in the lab 7 handout. i. Click on the start button ii. Click Run iii. Click on browse iv. Find Toxstat on the a: drive in the analysis\toxstat folders v. Click on OK and the following window appears

vi. Press any key to get to the next screen

vii.The first step in analysis is to determine if there are mortality differences between the exposed organisms and the controls. This is done using Fishers Exact Test. Press the F3 Key to get to the screen with the Fishers Exact Test option. Navigate through the options using the arrow keys and change the options using the space bar.

viii.Change the NO to YES by pressing the space bar to select the Fishers Exact Test and then press the F5 key to run the test. The data will be entered interactively. At the next screen (not shown) press E to enter data. The next screen will ask you to enter the data. Follow the prompts and you will have a screen that looks something like this:

ix. After all data are entered the results screens will appear. Note the highlighted statement regarding the statistical relationship between the control and concentration.

x. Press any key to continue until you get to the summary screen that looks like the following. This shows that mortality was significantly different between the 100% and control groups. This is indicated by the asterisk. What does this mean? We dont use the 100% group when calculating the NOEC/LOEC

xi. Now that we have tested for mortality differences we need to test for normality and homogeneity of variance. Toxstat will perform these analyses as well. Press F6 to return to the main menu. From the main menu press the F4 key to bring up the following screen.

xii.Select O to open a new file. Give the data a name, enter the number of groups including the control (do not include groups with significant mortality), a group ID (concentration), and finally enter the number of replicates per group. Use the arrow keys to move the highlighted box. To enter or change data, place the highlighter in the appropriate location and enter the data. The entry will appear at the top of the screen until you press enter. Once all group ID data are entered, press the F1 key to enter reproduction data.

xiii.The reproduction data entry page appears as follows. Enter the values for the total number of neonates produced by each mother in each group. When you have entered the data for one group, press the F6 key to move to the next group. If you want to check or change data in a previous group press the F5 key. Once all data are entered press the F8 key. Remember, dont enter 100%.

xiv.Press w on the next screen in order to write the data to a file. Name the file in order to write it to disk. Remember these rules! Some of you had trouble with this last time. (1)give it a full path (for example a:\myfolder\mydata.txt) (2)stick to the 8.3 naming convention (eight characters followed by a period and then three more characters) (3)Remember what you named it - write it down if you need to. xv.On the next screen (not shown in this handout) select R to return to the main menu. xvi.At the main menu press the F2 key in order to test for normality and heterogeneity of variance. Select all tests by highlighting NO and changing it to YES using the space bar.

xvii.Press the F5 key to run the tests. There will be a series of 5 screens, one for each test. Look at the last line for a statement regarding statistical significance. If any one of the tests fail for normality or heterogeneity, then you must follow the appropriate path on the flow chart and use the non-parametric method. This data set passes both the tests for normality and the tests for homogeneity of variance. Using this information and the flowchart on p. 159, determine the most appropriate analysis. Dunnett's test is most appropriate. xviii.Press F6 to return to the main menu. Press the F3 key to go to the summary statistics and tests of means menu. Turn off the Fisher's Exact test. Turn on the list data, summary statistics, and ANOVA - Dunnett's Test options and make sure that control<treatment is selected.

xix.Press the F5 key to run the analysis. There will be a couple of screens listing the data (not shown) followed by 2 screens of summary statistics on the raw data. Note the Coefficient of Variation on the second screen. This is a measure of reproductive variability adjusted for the total number of neonates produced.

xx.The next screen contains the output results of the analysis of variance. Note the statement about the F statistic at the bottom. Remember the null hypothesis is that there are no differences in mean reproduction between the exposed groups and the control. With this data set we are rejecting the null hypothesis and accepting the alternative that there are differences.

xxi.The next screen is a summary of the Dunnetts procedure to determine which groups are significantly different than the control. The groups with the asterisk next to them are

xxii. significantly different than the controls. Hence the NOEC is the highest concentrations not different than the controls and the LOEC is the lowest concentration significantly different than the controls.

xxiii.The final screen gives the MSD for this test.

g. Practice on Data Set 2, then analyze the data set from the test completed today. Be sure to use calculated concentration values instead of the percent values.

Test1 Replicate Concentration 1 Control 1.56% 3.12% 6.25% 12.50% 25.00% 27 32 39 27 10 2 30 35 30 34 13 3 29 32 33 36 7 4 31 26 33 34 7 5 16 18 36 31 7 6 15 29 33 27 10 7 18 27 33 33 10 8 17 16 27 31 16 0 9 14 35 38 33 12 10 27 13 44 31 2

D/0 D/0 D/0

0 D/0 D/0 D/0

0 D/0

Test 2 Replicate Concentration 1 Control 6.25% 12.5% 25% 50% 100% 35 35 32 34 24 2 39 32 34 34 3 4 5 6 7 8 9 10 32 34 33 22 18 30 35 30 37 35 31 19 22 35 34 36 32 31 32 23 24 40 35 31 32 32 34 33 33 32 26 21 21

D/23 28 D/24 22 22

24 22 D/20 21

D/3 D/10 14 D/11 12 D/5 14 14 15

Monday Lab Ceriodaphnia dubia Chronic Test Results Replicate Concentration 1 Control 6.25% 12.5% 25% 50% 100% 2 3 4 5 6 7 8 9 10 29 27 31 29 27 27 17 7 D D 31 33 37 32 26 37 32 27 28 18 25 27 25 34 36 30 31 24 32 28 23 25 30 31 14 D D D 5 D 15 4 D D 12 D D D D D

29 D/5 31 30 32 25 24 28 33 29

Tuesday Lab Ceriodaphnia dubia Chronic Test Results Replicate Concentration 1 Control 6.25% 12.5% 25% 50% 100% 25 24 D 16 9 D 2 17 22 20 11 D 3 28 32 27 D D 4 D 25 23 7 D 5 D 34 17 6 D 6 23 22 22 19 3 D 7 27 20 28 25 7 D 8 26 32 27 5 D 9 38 26 6 11 D 10 23 D/6 NA 13 1 D D/5 31 NA 25

NA 31