USER'S GUIDE PROBIT PROGRAM VERSION 1.5 ECOLOGICAL MONITORING RESEARCH DIVISION ENVIRONMENTAL MONITORING SYSTEMS LABORATORY U. S.

ENVIRONMENTAL PROTECTION AGENCY CINCINNATI, OHIO 45268

I. INTRODUCTION This computer program was prepared under contract with The U. S. Environmental Protection Agency. The program was designed for the analysis of mortality data from acute and short-term chronic toxicity tests with fish and other aquatic life, performed with effluents, receiving waters, and reference toxicants by regulatory agencies and permittees under the National Pollutant Discharge Elimination System, abbreviated NPDES. The toxicity test methods and examples of the statistical analysis of test data are described in detail in the current editions of the following three major EMSL methods manuals: a. Methods for measuring the acute toxicity of effluents and receiving waters to freshwater and marine organisms. Environmental Monitoring Systems Laboratory, U.S. Environmental Protection Agency, Cincinnati, Ohio 45268. b. Short-term methods for estimating the chronic toxicity of effluents and receiving waters to freshwater organisms. Environmental Monitoring Systems Laboratory, U. S. Environmental Protection Agency, Cincinnati, Ohio 45268. c. Short-term methods for estimating the chronic toxicity of effluents and receiving waters to marine and estuarine organisms. Environmental Monitoring Systems Laboratory, U. S. Environmental Protection Agency, Cincinnati, Ohio 45268.

Users who are unfamiliar or lack experience with the basic premises of the calculations should carefully read the discussion of Probit Analysis in the toxicity test manuals listed above and, if necessary, seek the assistance of a statistician before attempting to use this software or interpret the results. It is the user's responsibility to ensure that the data meet the assumptions of the statistical test. Although this user's guide is designed to help the users understand the limitations of the program and how the data must be entered, undoubtedly there will be instances where the results may not appear to be correct. evaluated before use. If you should have any difficulty or questions concerning this software, write or call the Ecological Monitoring Research Division, Environmental Monitoring Systems Laboratory, U.S. Environmental Protection Agency, Cincinnati, Ohio 45268 (Phone: 513-569-7401; Fax: 513-569-7609). II. FILES ON THE DISTRIBUTION DISKETTE: There are three files on the diskette under subdirectory PROBIT: A. PROBIT.EXE - The program used to calculate the LC/EC endpoints. This is the only file needed to execute the Probit program on an IBM - compatible PC. B. README-P.1ST - This user's guide in ASCII text format. C. README-P.WP5 - This user's guide in WordPerfect 5.1 format. III. INSTALLATION A. Make a backup copy of the PROBIT program files on the distribution diskette, and store the original diskette in a safe place. B. The PROBIT program can be executed directly from a diskette, if necessary, or from the hard disk. To execute from the hard disk, create an appropriate (sub) directory on the hard disk and use the DOS "COPY" command to copy the files from the diskette to the new (sub) directory. Therefore, the results should be carefully

IV. PROGRAM OVERVIEW Toxicity test data must be entered from the keyboard, and can be edited, if necessary, by the user. The data are not saved by the program except as a part of the analysis output. The program provides for "abbreviated" or "full" input/output, and output to the printer or to a disk file. The abbreviated input/output is designed specifically for the limited input/output needs of the effluent toxicity monitoring program. The full input/output option provides output containing several endpoints and statistical parameters as well as a plot of the Probit values. The program performs the following functions: A. Provides input data editing capability. B. Adjusts the response proportions for control mortality. C. Calculates the Chi-square statistic for heterogeneity, and tests it against the tabular (critical) value as one indicator of how well the data fit the model. D. Calculates LC/EC endpoints and associated 95% confidence limits. E. Provides data analysis output to the printer or to a disk file. V. PROGRAM RESTRICTIONS The following restrictions apply to the PROBIT program: A. The number of exposure concentrations, exclusive of the control, must be no less than 3 and no greater than 20. B. There must be at least two exposure concentrations for which the percent responding is greater than 0% and less than 100%. C. Data entry must begin with the lowest exposure concentration. VI. PROGRAM EXECUTION

You have been supplied with a compiled version of the program. C:\PROBIT) type: PROBIT and press the "enter" key. A. Program Input and Analysis Options

To execute the

program, from the appropriate (sub) directory DOS prompt (i.e. A:\PROBIT or

When the program is entered, you are presented with the following options: 1. Do you wish abbreviated (A) or full (F) input/output? 2. Output to printer or disk file (P/D)? If output to a disk file is chosen, you are prompted to enter a complete DOS filename, including extension. 3. Data entry/editing options Selection of option "A", abbreviated input/output, determines the format of both the input and output. Use of this option is recommended for NPDES permitcompliance test data analysis because the data input routine is simple and direct, and the output is limited to the two Probit endpoints most commonly calculated for the effluent monitoring program: LC/EC1 and LC/EC50. Under the "A" option, a plot of the Probit values is omitted. Following completion of data entry, the program prints the data to asks if you wish to modify the values. the screen and If you wish to change any of the

concentration mortality data, the program will prompt you to enter the observation number to be edited. This observation number appears in the column to the left of the concentration levels. 4. Test information requested includes the toxicant exposure concentrations, and the number of organisms exposed and responding (mortalities) in the control and at each toxicant exposure concentration. The test data can be edited immediately after all test values have been input, as illustrated in the examples below. 5. Note that you may halt execution of the program at any time by pressing the "CTRL" key and the "BREAK" key at the same time. B. Data Analysis Computations performed by the program are based on Finney (1971), Probit Analysis, Cambridge University Press.

C. Program output Output is provided immediately via the printer, (P), or stored in an ASCII format disk file (D), depending on the option selected. No data analysis output is printed on the screen for either option. The abbreviated version (A) of the program provides the following output: 1. A table of the test concentrations, observed responses, and responses adjusted for control mortality (if any), using Abbott's Formula. 2. Calculated Chi-square statistic for heterogeneity of variance, and the tabular value at the 0.05 level. 3. Estimated LC/EC1 and LC/EC50, and associated 95% confidence limits. 4. Warning messages, where appropriate, such as: a. "No convergence in 25 iterations. This usually means that the data do not fit the Probit model." b. "Significant heterogeneity exists. This means that the endpoints calculated for the data may not be valid." c. "Slope not significantly different from zero. LC fiducial (confidence) limits cannot be computed." d. "Not converging. This usually means that only one concentration is on the linear portion of the concentration - response curve." The full version (F) of the program provides the following output in addition to that provided by the abbreviated (A) version: 1. Estimates of the normal population parameters, mu and sigma. 2. Estimates of the regression model slope and intercept as well as the spontaneous response rate. The associated 95% confidence limits are also provided. 3. A table of several LC/EC endpoints, ranging from LC/EC1 to LC/EC99 along with the associated 95% confidence limits. 4. A plot of the adjusted probits and predicted regression line.

VII. EXAMPLES OF DATA ANALYSIS Data on the survival of fathead minnows data from a 48-h acute effluent toxicity test are used to illustrate the abbreviated data input and analysis output for the Probit Analysis program. The data are listed below in Table 1. Examples of program input and output are provided in Figures 1 and 2, respectively. Figure 3 illustrates the program's data editing facility. Figure 4 illustrates the full output option. TABLE 1. FATHEAD MINNOW MORTALITY DATA FROM A 48-HOUR ACUTE EFFLUENT TOXICITY TEST FOR PROBIT ANALYSIS ____________________________________________________ Conc. (% Effl.) Control 6.25 12.5 25.0 50.0 100.0 Number Exposed 20 20 20 20 20 20 0 3 9 20 20 Number Responding 1

____________________________________________________

___________________________________________________

FIGURE 1. EXAMPLE OF PROBIT PROGRAM ABBREVIATED DATA INPUT USING THE DATA IN TABLE 1 _________________________________________________________________________ _____

EPA PROBIT ANALYSIS PROGRAM USED FOR CALCULATING LC/EC VALUES Version 1.5

Do you wish abbreviated (A) or full (F) input/output? A Output to printer (P) or disk file (D)? P Title ? Fathead Minnow Data This program is provided to calculate the following endpoints: 1. LC50 from acute toxicity tests, 2. LC1 from fish embryo/larval tests, 3. EC50 from sea urchin fertilization tests Responders (number responding) are defined as: 1. Number of dead organisms in acute toxicity tests, 2. Number of dead plus deformed organisms in fish (short-term chronic) embryo/larval tests, 3. Number of eggs not fertilized in sea urchin (short-term chronic) fertilization tests. Number responding in the control group = ? 1 Number of animals exposed in the concurrent control group = ? 20 Number of exposure concentrations, exclusive of controls ? 5

_________________________________________________________________________ FIGURE 1. EXAMPLE OF PROBIT PROGRAM ABBREVIATED DATA INPUT USING THE DATA IN TABLE 1 (CONTINUED) _________________________________________________________________________ Input data starting with the lowest exposure concentration Concentration = ? 6.25 Number responding = ? 0 Number exposed = ? 20 Concentration = ? 12.5 Number responding = ? 3 Number exposed = ? 20 Concentration = ? 25.0 Number responding = ? 9 Number exposed = ? 20 Concentration = ? 50.0 Number responding = ? 20 Number exposed = ? 20 Concentration = ? 100 Number responding = ? 20 Number exposed = ? 20

Number Number ______ 1 2 3 4 5 Conc. 6.2500 12.5000 25.0000 50.0000 100.0000 0 3 9 20 20

Number Exposed

Resp. 20 20 20

______ ______ _______

20 20

Do you wish to modify your data ? N The number of control animals which responded = 1 The number of control animals exposed = 20 Do you wish to modify these values ? N _________________________________________________________________________ FIGURE 2. EXAMPLE OF PROBIT ANALYSIS OUTPUT FROM THE DATA INPUT IN FIGURE 1. OUTPUT IS DIRECTED TO THE PRINTER. _________________________________________________________________________ Fathead Minnow Data Proportion Observed Number Conc. Exposed Control 6.2500 12.5000 25.0000 50.0000 100.0000 20 20 20 20 20 20 Resp. 1 0 3 9 20 20 Responding Controls Number Proportion Adjusted for Responding

0.0500 0.0000 0.1500 0.4500 1.0000 1.0000

0.0000 -.0363 0.1191 0.4300 1.0000 1.0000

Chi - Square for Heterogeneity (calculated) Chi - Square for Heterogeneity (tabular value at 0.05 level) Fathead Minnow Data Estimated LC/EC Values and Confidence Limits Exposure Point LC/EC 1.00 LC/EC 50.00 Conc. 9.338 24.045 95% Confidence Limits Lower 3.530 18.597 Upper 13.554 29.333

= =

3.340 7.815

_________________________________________________________________________ NOTE: The data analysis output table is directed to the printer. This table is not displayed on the screen. FIGURE 3. EXAMPLE OF PROBIT PROGRAM ABBREVIATED DATA INPUT WITH DATA EDITING USING THE DATA IN TABLE 1. OUTPUT IS DIRECTED TO A DISK FILE. _________________________________________________________________________

 EPA PROBIT ANALYSIS PROGRAM USED FOR CALCULATING LC/EC VALUES Version 1.5

Do you wish abbreviated (A) or full (F) input/output? A Output to printer (P) or disk file (D)? D File name for output? TEST-P.OUT Title ? Fathead Minnow Data This program is provided to calculate the following endpoints: 1. LC50 from acute toxicity tests, 2. LC1 from fish embryo/larval tests, 3. EC50 from sea urchin fertilization tests Responders (number responding) are defined as: 1. Number of dead organisms in acute toxicity tests, 2. Number of dead plus deformed organisms in fish (short-term chronic) embryo/larval tests, 3. Number of eggs not fertilized in sea urchin (short-term chronic) fertilization tests. Number responding in the control group = ? 1 Number of animals exposed in the concurrent control group = ? 5 Input data starting with the lowest exposure concentration Concentration = ? 6.25 Number responding = ? 0 Number exposed = ? 20

_________________________________________________________________________ FIGURE 3. EXAMPLE OF PROBIT PROGRAM ABBREVIATED DATA INPUT WITH DATA EDITING USING THE DATA IN TABLE 1. OUTPUT IS DIRECTED TO A DISK FILE (CONTINUED) _________________________________________________________________________ Concentration = ? 12.5 Number responding = ? 3 Number exposed = ? 20 Concentration = ? 25.0 Number responding = ? 9 Number exposed = ? 20 Concentration = ? 50 Number responding = ? 15 Number exposed = ? 20 Concentration = ? 100 Number responding = ? 20 Number exposed = ? 20 Number Number ______ 1 2 3 4 5 Conc. 6.2500 12.5000 25.0000 50.0000 100.0000 0 3 9 15 20 Number Exposed Resp. 20 20 20 20 20

______ ______ _______

Do you wish to modify your data ? Y Observation Number to be modified ? 4 Concentration = ? 50 Number responding = ? 20 Number exposed = ? 20

Number Number ______ 1 2 3 4 5 Conc. 6.2500 12.5000 25.0000 50.0000 100.0000 0 3 9 20 20

Number Exposed

Resp. 20 20 20

______ ______ _______

20 20

Do you wish to modify your data ? N _________________________________________________________________________ FIGURE 3. EXAMPLE OF PROBIT PROGRAM ABBREVIATED DATA INPUT WITH DATA EDITING USING THE DATA IN TABLE 1. OUTPUT IS DIRECTED TO A DISK FILE (CONTINUED) _________________________________________________________________________ The number of control animals which responded = 1 The number of control animals exposed = 5 Do you wish to modify these values ? Y Number of responders in the control group = ? 1 Number of animals exposed in the concurrent control group = ? 20 The number of control animals which responded = 1 The number of control animals exposed = 20 Do you wish to modify these values ? N Output stored in TEST-P.OUT _________________________________________________________________________ NOTE: As in the previous example, the data analysis output table is not displayed on the screen. The output is sent to the disk output file, TEST-P.OUT. A hard copy of the disk file can be obtained using the DOS "PRINT" command. To display the file contents on the screen, issue the "TYPE" command from the DOS prompt.

FIGURE 4. EXAMPLE OF PROBIT PROGRAM FULL DATA INPUT OPTION USING THE DATA IN TABLE 1 _________________________________________________________________________ EPA PROBIT ANALYSIS PROGRAM USED FOR CALCULATING LC/EC VALUES Version 1.5

Do you wish abbreviated (A) or full (F) input/output? F Output to printer (P) or disk file (D)? P Title ? Fathead Minnow Data This program is provided to calculate the following endpoints: 1. LC50 from acute toxicity tests, 2. LC1 from fish embryo/larval tests, 3. EC50 from sea urchin fertilization tests. Responders (number responding) are defined as: 1. Number of dead organisms in acute toxicity tests, 2. Number of dead plus deformed organisms in fish (short-term chronic) embryo/larval tests, 3. Number of eggs not fertilized in sea urchin (short-term chronic) fertilization tests. Number responding in the control group = ? 1 Number of animals exposed in the concurrent control group = ? 20 Number of exposure concentrations, exclusive of controls ? 5 _________________________________________________________________________ FIGURE 4. EXAMPLE OF PROBIT PROGRAM FULL DATA INPUT OPTION USING THE DATA IN TABLE 1 (CONTINUED) _________________________________________________________________________ Input data starting with the lowest exposure concentration

Concentration = ? 6.25 Number responding = ? 0 Number exposed = ? 20 Concentration = ? 12.5 Number responding = ? 3 Number exposed = ? 20 Concentration = ? 25.0 Number responding = ? 9 Number exposed = ? 20 Concentration = ? 50.0 Number responding = ? 20 Number exposed = ? 20 Concentration = ? 100 Number responding = ? 20 Number exposed = ? 20 Number Number ______ 1 2 3 4 5 Conc. 6.2500 12.5000 25.0000 50.0000 100.0000 0 3 9 20 20 Number Exposed Resp. 20 20 20 20 20

______ ______ _______

Do you wish to modify your data ? N The number of control animals which responded = 1 The number of control animals exposed = 20 Do you wish to modify these values ? N _________________________________________________________________________ FIGURE 5. EXAMPLE OF PROBIT PROGRAM FULL ANALYSIS OUTPUT OPTION USING THE DATA INPUT IN FIGURE 4 Fathead Minnow Data

Conc.

Number Exposed 20 20 20 20 20 20

Number Resp. 1 0 3 9 20 20

Observed Proportion Responding 0.0500 0.0000 0.1500 0.4500 1.0000 1.0000 = 3.340

Control 6.2500 12.5000 25.0000 50.0000 100.0000

Proportion Responding Adjusted for Controls 0.0000 -.0363 0.1191 0.4300 1.0000 1.0000

Predicted Proportion Responding 0.0350 0.0005 0.0538 0.5381 0.9641 0.9998

Chi - Square for Heterogeneity (calculated) Mu Sigma = = 1.381032 0.176582 Estimate Std. Err.

Chi - Square for Heterogeneity (tabular value at 0.05 level)

= 7.815

Parameter Intercept Slope

95% Confidence Limits ( -6.621536, ( 2.990552, 0.979748) 8.335608) 0.091402)

---------------------------------------------------------------------2.820894 1.939103 5.663080 1.363535 0.035031

Spontaneous Response Rate

0.028761 ( -0.021339,

Fathead Minnow Data Estimated LC/EC Values and Confidence Limits Exposure Point LC/EC 1.00 LC/EC 5.00 LC/EC 10.00 LC/EC 15.00 LC/EC 50.00 LC/EC 85.00 LC/EC 90.00 LC/EC 95.00 LC/EC 99.00 Conc. 9.338 12.319 14.280 15.777 24.045 36.647 40.489 46.935 61.918 95% Confidence Limits Lower 3.530 5.893 7.717 9.233 18.597 29.964 32.625 36.706 45.156 Upper 13.554 16.560 18.495 19.978 29.333 53.838 63.907 83.067 137.735

FIGURE 5. EXAMPLE OF PROBIT PROGRAM FULL ANALYSIS OUTPUT OPTION USING THE DATA INPUT IN FIGURE 4 (CONTINUED) _________________________________________________________________________ Fathead Minnow Data PLOT OF ADJUSTED PROBITS AND PREDICTED REGRESSION LINE Probit 10+ 9+ 8+ 7+ 6+ 5+ 4+ 3+ - . o o

. . .. .. ... ... .. .... ... ... ... ... ... o ... .... o ... .. . . ... .... ....

-. 2+ 1+ 0+o -+--------------+--------+---------+---------+--------+--------------+EC01 EC10 EC25 EC50 EC75 EC90 EC99 ________________________________________________________________________