540/09-87-198 PESTICIDE ASSESSMENT GUIDELINES SUBDIVISION E HAZARD EVALUATION: WILDLIFE AND AQUATIC ORGANISMS Series 72-1 to 72-5 Aquatic Testing for Marine/Estuarine and Freshwater Fish and Invertebrates ADDENDUM 2 ON DATA REPORTING Prepared by: Curtis E. Laird Ecological Effects Branch Hazard Evaluation Division Edited by: Karen E. Warkentien Hazard Evaluation Division Project Manager: Elizabeth M. K. Leovey, PhD Hazard Evaluation Division Office of Pesticide Programs US Environmental Protection Agency December 1986 ------- -1- Subdivision E - Aquatic Testing for Marine/Estuarine and Freshwater Fish and Invertebrates Table of Contents of Addendum Discussion Page Introduction 1 Response to Public Conuients 1 Guidelines - Acute Toxicity Test for Freshwater Fish 4 Acute Toxicity Test for Freshwater Aquatic Invertebrates 10 Acute Toxicity Test for Shrimp 16 Acute Toxicity Test for Estuarine and Marine Fish 22 Oyster Embryo Test 28 Shell Deposition Study for Oyster 34 Fish Early Life-Stage 40 Aquatic Invertebrate Life-Cycle 49 Life-Cycle Tests for Fish 56 ------- -11- Table of Contents for Acute Toxicity Test for Freshwater Fish GUIDELINE Page Title/Cover Page ~ 4 Table of Contents 4 I. Summary/Introduction 4 II. Materials/Methods 5 III. Discussion and Results 7 IV. Conclusions 7 Tables 8 Table of Contents for Acute Toxicity Test for Freshwater Aquatic Invertebrates GUIDELINE Page Title/Cover Page 10 Table of Contents 10 I. Summary/Introduction 10 II. Materials/Methods 11 III. Discussion and Results 13 IV. Conclusions 13 Tables 14 ------- -111- Table of Contents for Acute Toxicity Test for Shrimp GUIDELINE Page Title/Cover Page 16 Table of Contents 16 I. Summary/Introduction 16 II. Materials/Methods 17 III. Discussion and Results 19 IV. Conclusions 19 Tables 20 Table of Contents for Acute Toxicity Test for Estuarine and Marine Fish GUIDELINE Page Title/Cover Page 22 Table of Contents 22 I. Summary/Introduction 22 II. Materials/Methods 23 III. Discussion and Results 25 IV. Conclusions 25 Tables 26 ------- -IV- Table of Contents for Oyster Embryo Test GUIDELINE Page Title/Cover Page 28 Table of Contents 28 I. Summary/Introduction 28 II. Materials/Methods 29 III. Discussion and Results 31 IV. Conclusions 31 Tables 32 Table of Contents for Shell Deposition Study for Oyster GUIDELINE Page Title/Cover Page 34 Table of Contents 34 I. Summary/Introduction 34 II. Materials/Methods 35 III. Discussion and Results 37 IV. Conclusions 37 Tables 38 ------- -V- Table of Contents for Fish Early Life-Stage GUIDELINE Page Title/Cover Page 40 Table of Contents 40 I. Summary/Introduction 40 II. Materials/Methods 41 III. Discussion and Results 43 IV. Conclusions 43 Tables 44 Table of Contents for Aquatic Invertebrate Life-Cycle GUIDELINE Title/Cover Page 49 Table of Contents 49 I. Summary/Introduction 49 II. Materials/Methods 50 III. Discussion and Results 52 IV. Conclusions 52 Tables 53 ------- -VI- Table of Contents for Life-Cycle Tests for Fish GUIDELINE Page Title/Cover Page 56 Table of Contents 56 I. Summary/Introduction 56 II. Materials/Methods 57 III. Discussion and Results 59 IV. Conclusions 59 Tables 60 ------- PESTICIDE ASSESSMENT GUIDELINES WILDLIFE AND AQUATIC ORGANISMS AQUATIC TESTING FOR MARINE/ESTUARINE AND FRESHWATER FISH AND INVERTEBRATES Subdivision E, Series 72-1 to 72-5 DATA REPORTING INTRODUCTION A. Purpose To provide a data reporting format for fish and invertebrate studies. B. Objective These Data Reporting Guidelines are designed to aid the petitioner/registrant in generating reports which are compatible with the Agency's review process. Data submitters are encouraged to submit complete reports following this guidance for efficient review by the Agency. This guidance pertains to the substance of the data report. PR Notice 86-5, effective on November 1, 1986 (available from the Registration Support and Emergency Response Branch, Office of Pesticide Programs, US EPA), pertains to the physical formating of reports (which are referred to as "studies") and submittal packages. Some of the re- quirements in PR Notice 86-5 are mandatory. RESPONSE TO PUBLIC COMMENTS The purpose of this seciton is to acknowledge and address the concerns ex- pressed in the letters of comments received by the Agency in response to the public notice in the FEDERAL REGISTER (51 FR 18660) of May 21, 1986. This addendum to the Pesticide Assessment Guidelines [Subdivision E] is to be considered an all-encompassing document. The Ecological Effects Branch (EEB) has reviewed the ten (10) comments submitted by the registrants and/or committees in regard to the Data Reporting Guidelines (DRGs). These Guidelines are not intended to create new data requirements, but to provide for consistent reporting of the necessary aquatic data required to perform a scientifically sound hazard assessment. This approach should eliminate most, if not all, of the recycling of submissions between EEB scientists and the registrant. The submitted comments pertain to either items common to all studies or a study specific item. Hence, this discussion has been arranged to consider the general comments first, followed by the specific comments. ------- -2- kGeneral Comments 1. Location of a reporting item - Considering the need to maintain a consistent format, revisions were made to the DRGs in response to these comments Whenever possible. 2. Composition of the test substance - is asking for the "Identity and Composition" of the test substance instead of the "Identity and Composition" of the impurities. Under the present guide- lines, these data are required under Subdivision E, Test Substance 70-4(2) (i). 3. Ingredient information from testing laboratories - It appears some sponsors do not make this information available to testing lab- oratories. In such a situation, the sponsor bears the responsibility since he is submitting the data for registration. 4. Telephone number of individual who can provide details of test - This item has been deleted from the DRG. 5. Time to implement new format - The registrant will be given sufficient time to implement the necessary changes in order to meet the recommended format standard. 6. Limited to DRG - Additional data can and should be reported, providing it is meaningful and use- ful in making a risk assessment. 7. Public comments - Ecological Effects Branch (EEB) is only addressing the comments relating to the nine aquatic studies. Study-Specific Comments 1. The following were changed as a result of comments. A. The shell deposition value has been changed from IC$Q to ECsg in ppm. B. The fish early life-cycle value has been changed from EC5Q to MATC in ppm. C. The aquatic invertebrate life-cycle value has been changed from EC5Q to MATC in ppm. D. Total and un-ionized ammonia has been deleted from fish early life-stage, aquatic invertebrate life-cycle, and fish life-cycle test. ------- -3- 2. The following were not changed as a result of comments. A. The water hardness, alkalinity, and conductivity are required under Subdivi- sion E, Sections 70-4(6)(ii)(E)(F) which calls for "[t]he source of dilution water, its chemical characteristics, and description of any pretreatment" and the "[m]ethods used for, and results of, all chemical analysis of water and all toxicant concentrations at beginning, during, and at end of tests, including validation studies and reagent blanks, if there is reason to sus- pect that the concentrations administered to the test water do not approxi- mate the actual concentrations." B. The no-observed-effect level (NOEL) for aquatic studies is required, except if the LC5o or £€50 has not been determined, as allowed by the specific testing requirement. ------- -4- PESTiaEE ASSESSMENT GUIDELINES WILDLIFE AND AQUATIC ORGANISMS Acute Toxicity Test for Freshwater Fish Subdivision E, Series 72-1 DATA REPORTING GUIDELINE The following describes the order and format for a study report item by item. TITLE/COVER PAGE Refer to PR Notice 86-5. TABLE OF CONTENTS The Table of Contents should indicate the overall organization of the study, including tables and figures. It must follow the title, data confidentiality, and GLP (if appropriate) pages as described in PR Notice 86-5. I. SUMMARY/INTRODUCTION A. Sponsor: (name of study owner); B. Name: (person(s) who can provide details of test procedures); C. Location of study; D. Location of raw data and final report; E. Material: (comnDn/trade name); F. Subject: (final or draft report, acute 96-hour IC^Qi and species tested); G. Test Doses: (control, concurrent vehicle control, #, #, #, #, # ppm); H. Test Dates: (initiation - date test started, termination and date of last day of observation); I. Length of Study: (hours); J. Results: (LC5Q value, 95% confidence limits, and no-observed-effect level); K. Tested Material: (chemical name, formulation, and percent active ingredient); L. Test Species/Strain: (both common and scientific name); ------- -5- M. Source of Organisms: (company and address); N. Weight/Length and Physical Condition at the Initiation of Study. II. MATERIAL/METHODS This section is a narrative, which would include the following items. A. Test Substance 1. Identification (this information may be provided by the laboratory or sponsor): a. Chemical name; b. Composition (qualitative and quantitative description); c. Percent active ingredient; d. Molecular structure; e. Source, lot number, or code; f. Identity and percent composition of impurities in the test substance. 2. Preparation of test solution (control/treatment): a. Any vehicle used to dissolve test material; b. Amount of vehicle added to control (if used); c. Description of method used to get test material into solution; d. Water source (reconstituted - if so, how prepared); e. Solubility of test material in ppm; f. Total amount of test material used; g. Temperature of water. B. Test Fish 1. Rationale for selection of species if the species used is different than that preferred in Subdivision E. a. Test species name (both scientific and common); b. History of test organisms (strain, diseases, and treat- ment). 2. Description of any pretest conditioning: a. Health: 1. Sickness; 2. Injuries; 3. Abnormalities; 4. Name of medication (if used); 5. Pretest diet. 3. Size/age/physical condition: a. Age (if known); b. Size (weight in grams and length in mm). ------- 4. Source/acclimation: a. Complete name and address of test fish supplier; b. Source of food and dilution water; c. Size of test container [in milliliters (mL)]; d. Temperature, pH, and dissolved oxygen, and name of equipment used to measure water quality; e. Feeding schedule, holding, and acclimation period; f. Test organisms from the same source (yes or no); g. Number of days held. C. Method 1. Test Vessels: a. Material type; b. Volume [in liters (L)]; c. Depth of test solution [in centimeters (cm)]; d. Size [in liters (L)]. 2. Test system: a. Source of dilution water; b. If flow-through, description of system and flow rate/day; c. Procedures used to prepare toxicant stock solution; d. Criteria used to determine effects. 3. Test design: a. Method used in assigning test organisms to test and control groups, and the number of replicates used; b. Number of fish per dose level and control group; c. Name of protocol followed during this test; d. Loading (weight per unit volume of water); e. Number of treatment levels used (nominal or measured); f. Length of exposure period; g. Type of control (positive, negative, or solvent control); h. Temperature; i. pH (when checked - see table 1); j. Lighting (time in hours and intensity in footcandles); k. Dissolved oxygen (see table 1); 1. Water hardness (expressed in mg/L as CaC03 - see table 1); m. Alkalinity (expressed in mg/L as CaCC>3 - see table 1); n. Conductivity (umhos/cm - see table 1); o. Range finding test results, concentrations, and mortality (if used); p. Aeration (yes or no); q. Water physical characteristic at the end of test: a. Water depth and volume; b. Temperature, pH, and dissolved oxygen; r. Concentration analysis (see table 1); s. Examination of fish physiology, locomotion, behavior and pathology; t. Mortalities during the test; ------- -7- D. Statistical Analysis 1. Cite references (author, title, journal, number, page, etc.). III. DISCUSSION AND RESULTS A. Include LCso value in ppm with 95% confidence limits (graphs, printouts, and other calculations should be attached to report). Provide raw mor- tality data (see table 2). B. Discuss the relationship, if any, between the physical factors, toxicant, and observation, and describe any precipitation and solubility problems. C. Provide statistical method used (cite references, author, etc.). D. Observation - Provide data on the following: v 1. Signs of intoxication - temporal onset, duration, and concentrations that showed effects; 2. The no-observed-effect level. IV. CONCLUSIONS This is a summation of the above section - Discussion/Results. ------- -8- TABLE 1 WATER QUALITY PARAMETERS Client : Test Material: Test Organism: Test Water : WATER QUALITY Conductivity Hardness Alkalinity Temperature pH umhos/cm mg/L as CaCC-3 mg/L as CaCO-} °C X X X X X Nominal Concentration Parameter Dissolved Oxygen (mg/L) pH Time Initial 48 hours 96 hours Initial 96 hours (mg/L) Control XXX Hig_h Medium XX X XX X XX X XX X XX X X X Low X X X X X ------- -9- TABLE 2 PERCENT MORTALITIES AND VALUES Client: Test Material: Test Organism: Test Water: PERCENT MORTALITY TEST MATERIAL NOMINAL CONCENTRATION (mg/L) Control 24 Hour #(#/#) #(#/#) #(#/#) #(#/#) #(#/#) #(f/#) 48 Hour #(#/#) #(#/#) #(#/#) #(#/#) #(#/#) #(#/#) 96 Hour !(#/#) #(#/#) #(#/#) #(#/#) #(#/#) #(#/#) 24 hour 48 hour 96 hour LC50 (mg/L) X 95% Low X Confidence Limits High X X X X X X X Observed Effects a floating b swimming at surface c lying on bottom d turned dark e loss of equilibrium ^ g\olping of air 9 etc. £/ How the percent of mortality table works. 1. #(#/#)a. 2. # = the total number of organisms tested per dose level. 3. f(#/#) = % mortality [No. dead/No, treated, e.g., 10% (1/10)] 4. a = the observed effects. ------- -10- PESTICIDE ASSESSMENT GUIDELINES WILDLIFE AND AQUATIC ORGANISMS Acute Toxicity Test for Freshwater Aquatic Invertebrates_ Subdivision E, Series 72-2 DATA REPORTING GUIDELINE The following describes the order and format for a study report item by item. TITLE/COVER PAGE Refer to PR Notice 86-5. v TABLE OF CONTENTS The Table of Contents should indicate the overall organization of the study, including tables and figures. It must follow the title, data confidentiality, and GLP (if appropriate) pages as described in PR Notice 86-5. I. SUMMARY/INTRODUCTION A. Sponsor: (name of study owner); B. Name: (person(s) who can provide details of test procedures); C. Location of study; D. Location of raw data and final report; E. Material: (common/trade name); F. Subject: (final or draft report, 48-hour LCso/ and species tested); G. Test Doses: (control, concurrent vehicle control, #, #, f, #, # ppm); H. Test Dates: (initiation, date test started, termination, and date of last day of observation); I. Length of Study: (hours); J. Results: (LC5Q value, 95% confidence limits, and no-observed-effect level); K. Tested Material: (chemical name, formulation, and percent active ingredient); L. Test Species/Strain: (both common and scientific name); M. Source of Organisms: (company and address); ------- -11- N. Age of Organisms at the Initiation of Study: (hours or life stage). MATERIAL/METHODS This section is a narrative, which would include the following items. A. Test Substance 1. Identification (this information may be provided by the laboratory or sponsor): a. Chemical name; b. Composition (qualitative and quantitative description); c. Percent active ingredient; d. Molecular structure; e. Source, lot number, or code; f. Identity and percent composition of impurities in the test substance. 2. Preparation of test solution (control/treatment): a. Any vehicle used to dissolve test material; b. Amount of vehicle added to control (if used); c. Description of method used to get test material into solution; d. Vfeter source (reconstituted - if so, how prepared); e. Solubility of test material in ppm; f. Total amount of test material used; g. Temperature of water. B. Test Organisms 1. Rationale for selection of species if the species used is different than that preferred in Subdivision E (if 1st instar were not used, then give stage of life in which organisms were tested): a. Test species name (both scientific and common); b. History of test organisms (strain, diseases, and treatment). 2. Description of any pretest conditioning: a. Health: 1. Sickness; 2. Injuries; 3. Abnormalities; 4. Name of medication (if used); 5. Pretest diet; 6. Number of days brood organisms were quarantined. 3. Size/age/physical condition: a. Age (in hours); b. Date of spawn. ------- -12- 4. Source/acclimation: a. Complete name and address of test species supplier; b. Source of food and dilution water; c. Volume of test container [in milliliters (mL)]; d. Photoperiod and lighting; e. Temperature, pH, dissolved oxygen, and name of equipment used to measure water quality; f. Chemical treatment (if used); g. Feeding schedule, holding and acclimation period; h. Test organisms from the same source (yes or no) ; i. Number of days adults were held. C. Method 1. Test Vessels: a. Material type; b. Volume of test solution [in milliliters (mL)]; c. Depth of test solution [in centimeters (cm)]; d. Size of test containers [in milliliters (mL)]. 2. Test system: a. Source of dilution water; b. If flow-through, description of system and flow rate/day; c. Procedures used to prepare toxicant stock solution; d. Criteria used to determine effects. 3. Test design: a. Method used in assigning test organisms to test and control groups, and the number of replicates used; b. Number of organisms per dose level and control group; c. Name of protocol followed during this test; d. Loading (number of organism per unit volume of water); e. Number of treatment levels used (nominal or measured); f. Length of exposure period; g. Type of control (positive, negative, or solvent control); h. Temperature; i. pH (when checked - see table 1); j. Lighting (time in hours and intensity in footcandles); k. Dissolved oxygen (see table 1); 1. Water hardness (expressed in mg/L as CaCC>3 - see table 1); m. Alkalinity (expressed in mg/L as CaCD3 - see table 1); n. Conductivity (umhos/cm - see table 1); o. Range finding test results, concentrations, and mortality (if used); p. Aeration (yes or no); q. Water physical characteristic at the end of test: 1. Water depth and volume; 2. Temperature, pH, and dissolved oxygen; ------- -13- r. Percent of death/effects at each dose level; s. Concentration analysis (see table 1); t. Examination of invertebrate physiology, locomotion, and behavior; • u. Mortalities during the test. D. Statistical Analysis 1. Cite references (author, title, journal, number, page, etc.). III. DISCUSSION AND RESULTS A. Provide LCso value in ppm with 95% confidence limits (graphs, printouts, and other calculations should be attached to report). Provide raw mortality data (see table 2). B. Discuss the relationship, if any, between the physical factors, toxicant, and observation, and describe any precipitation and solubility problems". C. Provide statistical method used (cite references, author, etc.). D. Observation - Provide the following data: 1. Signs of intoxication - temporal onset and duration, concentrations that showed effects; 2. The no-observed-effect level. IV. CONCLUSIONS This is a summation of the above section - Discussion/Results. ------- Client : Test Material: Test Organism: Test Water : -14- TABLE 1 WATER QUALITY PARAMETERS WATER QUALITY Conductivity Hardness Alkalinity Temperature umhos/cm mg/L as CaCO-^ mg/L as CaCO^ °C Parameter Time Control Nominal Concentration (mg/L) X X X X X Dissolved Oxygen (mg/L) pH Initial 48 hours Initial 48 hours X X X X High X X X X Medium X X X X Low X X X X ------- -15- TABLE 2 PERCENT MORTALITIES AND IC$Q VALUES Client : Test Material: Test Organism: Test Water : PERCENT MORTALITY TEST MATERIAL NOMINAL OR MEASURED CONCENTRATION (mg/L) Control X 24 Hour #(#/#) #(#/#) 48 Hour #(#/#) #(#/#) LC50 (mg/L) X #(#/#) #(#/#) 24 hour X X X #(#/#) #(#/#) #(#/#) #(#/f) 48 hour X X #(#/#) #(#/#) 95% Confidence Limits Low High X X X X Observed Effects a floating b lying on bottom c swimming near surface d etc. £/ How the percent of mortality table works. 1. #(#/#)a. 2. # = the total number of organisms tested per dose level. 3. #(#/#) = % mortality [No. dead/No, treated, e.g., 10% (1/10)]. 4. a = the observed effects. ------- -16- PESTICIDB ASSESSMENT GUIDELINES WILDLIFE AND AQUATIC ORGANISMS Acute Toxicity Test for Shrimp Subdivision E, Series 72-3 DATA REPORTING GUIDELINE The following describes the order and format for a study report item by item. TITLE/COVER PAGE Refer to PR Notice 86-5. v TABLE OF CONTENTS The Table of Contents should indicate the overall organization of the study, including tables and figures. It must follow the title, data confidentiality, and GLP (if appropriate) pages as described in PR Notice 86-5. I. SUMMARY/ INTRODUCTION A. Sponsor: (name of study owner); B. Name: (person(s) who can provide details of test procedures; C. Location of study; D. Location of raw data and final report; E. Material: (ccmmon/trade name); F. Subject: (final or draft report, 96-hour IC$Q, and species tested); G. Test Doses: (control, concurrent vehicle control, #, #, #, #, # ppm); H. Test Dates: (initiation, date test started, termination, and date of last day of observation); I. Length of Study: (days); J. Results: (LCso value, 95% confidence limits, and no-observed-effect level); K. Tested Material: (chemical name, formulation, and percent active ingredient); L. Test Species/Strain: (both common and scientific name); M. Source of Organisms: (company and address); ------- -17- N. Size/Age of Organisms at the Initiation of Study: (hours). II. MATERIAL/METHODS This section is a narrative, Which would include the following items. A. Test Substance 1. Identification (this information may be provided by the laboratory or sponsor): a. Chemical name; b. Composition (qualitative and quantitative description); c. Percent active ingredient; d. Molecular structure; e. Source, lot number, or code; f. Identity and percent composition of impurities in the test substance. 2. Preparation of test solution (control/treatment): a. Any vehicle used to dissolve test material; b. Amount of vehicle added to control (if used); c. Description of itethod used to get test material into solution; d. Water source (reconstituted - if so, how prepared); e. Solubility in ppm; f. Total amount of test material used; g. Salinity (o/oo). B. Test Shrimp 1. Rationale for selection of species if the species used is different than that preferred in Subdivision E: a. Test species name (both scientific and cannon); b. History of test organisms (strain, diseases, and treatment). 2. Description of any pretest conditioning: a. Health: 1. Sickness; 2. Injuries; 3. Abnormalities; 4. Pretest diet. 3. Size/age/physical condition: a. Age; b. Size; c. Date hatched (if known). ------- -18- 4. Source/acclimation; a. Complete name and address of test species supplier; .b. Source of food and dilution water; c. Volume of test container [in liters (L)]; d. Photoperiod and lighting; e. Temperature, pH, and dissolved oxygen, name of equipment used to measure water quality; f. Chemical treatment (if used); g. Percent mortality; h. Feeding schedule; i. Test organisms from the same source (yes or no); j. Acclimation period (in days). C. Method 1. Test Vessels: a. Material type; b. Volume [in liters (L)]; c. Depth of test solution [in centimeters (cm)]; d. Size [in centimeters (cm)]. 2. Test system: a. Source of dilution water; b. If flow-through, description of system and flow rate/day; c. Procedures used to prepare toxicant stock solution; d. Criteria used to determine effects. 3. Test design: a. Method used in assigning test organisms to test and control groups; b. Number of organisms per dose level and control group; c. Number of days food was withheld prior to test; d. Loading (weight of organism per unit volume of water); e. Number of treatment levels used (nominal or measured); f. Length of treatment period; g. Type of control (positive, negative, or solvent control); h. Temperature; i. pH (when checked - see table 1); j. Lighting (time in hours and intensity in footcandles) ; k. Dissolved oxygen (see table 1); 1. Range finding test results, concentrations, and mortality (if used); m. Aeration (yes or no); n. Water physical characteristic at the end of test: a. Water depth and volume; b. Temperature, pH, and dissolved oxygen; o. Percent of death/effects at each dose level; p. Name of protocol followed during the test; q. Salinity (o/oo). ------- -19- D. Statistical Analysis 1. Cite references (author, title, journal, number, page, etc.). III. DISCUSSION AND RESULTS A. Provide IC$Q value in ppm with 95% confidence limits (graphs, printouts, and other calculations should be attached to report). Provide raw mortality data (see table 2). B. Discuss the relationship if any, between the physical factors, toxicant, and observation, and describe any precipitation and solubility problems. C. Provide statistical method used (cite references, author, etc.). D. Observation - Provide the following data: v 1. Signs of intoxication - temporal onset, duration, and concentrations that showed effects; 2. The no-observed-effect level. IV. CONCLUSIONS This is a summation of the above section - Discussion/Results. ------- -20- TABLE 1 WATER QUALITY PARAMETERS Client : Test Material: Test Organism: Test Water : WATER QUALITY Temperature 6 hours, 12 hours, etc. Nominal Concentration Parameter Dissolved Oxygen (mg/L) pH Salinity o/oo Time Initial 48 hours 96 hours Initial 96 hours Initial 96 hours Control X X X X X X X X High X X X X X X X (mg/L) X Medium X X X X X X Low X X X X X ------- -21- TABLE 2 PERCENT MORTALITIES AND LC50 VALUES Client : Test Material: Test Organism: Test Water : PERCENT MORTALITY TEST MATERIAL NOMINAL CONCENTRATION (mg/L) Control X 24 Hour #(#/#) #(#/#) #(#/#) #(#/#) #(#/#) #(#/#) 48 Hour #(#/#) #(#/#) #(#/#) #(#/#) #(#/#) #(#/#) 96 Hour #(#/#) #(#/#) #(#/#) #(#/#) #(#/#) #(#/#) 24 hour 48 hour 96 hour LC50 (mg/L) 95% Confidence Limits Low High X X X X X X Observed Effects a floating b swimming at surface c lying on bottom ^ turned dark e etc. £ How the percent of mortality table works. 1. #(#/#)a. 2. I = the total number of organisms tested per dose level. 3. #(#/#) = % mortality [No. dead/No, treated, e.g., 10% (1/10)] 4. a = the observed effects. ------- -22- PESTICTDE ASSESSMENT GUI KLINES WILDLIFE AND AQUATIC ORGANISMS Acute Toxicity Test for Estuarine and Marine Fish Subdivision E, Series 72-3 DATA REPORTING GUIDELINE The following describes the order and format for a study report item by item. TITLE/COVER PAGE Refer to PR Notice 86-5. TABLE OF CONTENTS The Table of Contents should indicate the overall organization of the study, including tables and figures. It must follow the title, data confidentiality, and GLP (if applicable) pages as described in PR Notice 86-5. I. SUMMARY/ INTRODUCTION A. Sponsor: (name of study owner); B. Name: (person(s) who can provide details of test procedures); C. Location of study; D. Location of raw data and final report; E. Material: (common/trade name); F. Subject: (final or draft report, acute 96-hour LCso* and, species tested); G. Test Doses: (control, concurrent vehicle control, #, f, #, f, #, and # ppm); H. Test Dates: (initiation - date test started, termination - date of last day of observation); I. Length of Study: (hours); J. Results: (LC5Q value, 95% confidence limits, and no-observed-effects level); K. Tested Material: (chemical name, formulation, and percent active ingredient); L. Test Species/Strain: (both common and scientific name); ------- -23- M. Source of Organisms: (company and address); N. Weight/Length and Physical Condition at the Initiation of Study. II. MATERIAL/METHODS This section is a narrative, which would include the following items. A. Test Substance 1. Identification (this information may be provided by the laboratory or sponsor): a. Chemical name; b. Composition (qualitative and quantitative description); c. Percent active ingredient; d. Molecular structure; e. Source, lot number, or code; f. Identity and percent composition of impurities in the test substance. 2. Preparation of test solution (control/treatment): a. Any vehicle used to dissolve test material; b. Amount of vehicle added to control (if used); c. Description of method used to get test material into solution; d. Water source (reconstituted - if so, how prepared); e. Solubility of test material in ppm; f. Total amount of test material used; g." Temperature of water; h. Salinity (o/oo). B. Test Fish 1. Rationale for selection of species if the species used is different than that preferred in Subdivision E: a. Test species name (both scientific and common); b. History of test organisms (strain, diseases, and treatment). 2. Description of any pretest conditioning: a. Health: 1. Sickness; 2. Injuries; 3. Abnormalities; 4. Name of medication (if used); 5. Pretest diet; 6. Number of days brood fish were quarantined; ------- -24- 3. Size/age/physical condition: a. Age (if known); b. Size (weight in grams and length in mm). 4. Source/acclimation: a. Complete name and address of test fish supplier; b. Source of food and dilution water; c. Size of test container [in milliliters (mL)]; d. Temperature, pH, dissolved oxygen, and name of equipment used to measure water quality; e. Feeding schedule, holding, and acclimation period; f. Test organisms from the same source (yes or no); g. Number of days held; h. Salinity (o/oo). C. Method 1. Test Vessels: a. Material type; b. Volume [in liters (L)]; c. Depth of test solution [in centimeters (cm)]; d. Size [in liters (L)]. 2. Test system: a. Source of dilution water; b". If flow-through, description of system and flow rate/day; c. Procedures used to prepare toxicant stock solution; d. Criteria used to determine effects. 3. Test design: a. Method used in assigning test organisms to test and control groups, and the number of replicates used; b. Number of fish per dose level and control group; c. Name of protocol followed during this test; d. Loading (weight per unit volume of water); e. Number of treatment levels used (nominal or measured); f. Length of exposure period; g. Type of control (positive, negative, or solvent control); h. Temperature; i. pH (when checked - see table 1); j. Lighting (time in hours and intensity in footcandles); k. Dissolved oxygen (see table 1); 1. Range finding test results, concentrations, and mortality (if used); m. Aeration (yes or no); n. Water physical characteristic at the end of test: 1. Water depth and volume; 2. Temperature, pH, and dissolved oxygen; ------- -25- o. Concentration analysis; p. Examination of fish physiology, locomotion, behavior and pathology; q. Mortalities during the test. D. Statistical Analysis 1. Cite references (author, title, journal, number, page, etc.). III. DISCUSSION AND RESULTS A. Provide LCso value in ppm with 95% confidence limits (graphs, printouts, and other calculations should be attached to report). Provide raw mortality data (see table 2). B. Discuss the relationship, if any, between the physical factors, toxicant, and observation, and describe any precipitation and solubility problems. v C. Provide statistical method used (cite references, author, etc.). D. Observation - Provide data on the following: 1. Signs of intoxication - temporal onset, duration, and concentrations that showed effects; 2. The no-observed-effect level. |IV. CONCLUSIONS This is a summation of the above section - Discussion/Results. ------- -26- TAELE 1 WATER QUALITY PARAMETERS Client: Test Material: Test Organism: Test Water: WATER QUALITY Dissolved Oxygen (mg/L) PH Temperature Initial 48 hours 96 hours Initial 96 hours CO 6 Control X X X X X hrs, 12 High X X X X X hrs, etc Medium X X X X X Low X X X X X ------- -27- TABLE 2 PERCENT MORTALITIES AND LCs0 VALUES Client : Test Material: Test Organism: Test Water : PERCENT MORTALITY TEST MATERIAL NOMINAL CONCENTRATION (mg/L) Control X 24 Hour #(#/#) #(#/#) #(#/#) #(#/#) #(#/#) #(#/#) 48 Hour #(#/#) #(#/#) #(#/#) #(#/#) #(#/#) #(#/#) 96 Hour #(#/#) #(#/#) #(#/#) #(#/#) #(#/#) #(#/#) 24 hour 48 hour 96 hour LC5o (mg/L) 95% Low Confidence Limits High X XX X XX X XX Observed Effects a floating b swimming at surface c lying on bottom ^ turned dark e loss of equilibrium f gulping of air 9 etc. £/ How the percent of mortality table works. 1. #(#/#)a. 2. # = the total number of organisms tested per dose level. 3. #(#/#) = % mortality [No. dead/No, treated, e.g., 10% (1/10)] 4. a = the observed effects. ------- -28- PESTICICE ASSESSMENT GUIDELINES WILDLIFE AND AQUATIC ORGANISMS Oyster Embryo Test Subdivision E, Series 72-3 DATA REPORTING GUIDELINE The following describes the order and format for a study item by item. TITLE/COVER PAGE Refer to PR Notice 86-5. TABLE OF CONTENTS The Table of Contents should indicate the overall organization of the study, including tables and figures. It must follow the title, data confidentiality, and GLP (if appropriate) pages as described in PR Notice 86-5. I. SUMMARY/INTRODUCTION A. Sponsor: (name of study owner); B. Name: (person(s) who can provide details on test procedures); C. Location of study; D. Location of raw data and final report; E. Material: (common/ trade name); F. Subject: (final or draft report, 48-hour ECso and species tested); G. Test Doses: (control, concurrent vehicle control, #, f, #, #, and # ppm); H. Test Dates: (initiation, date test started, termination and date of last day of observation); I. Length of Study: (days); J. Results: (ECso value, 95% confidence limits, and no-observed-effect level); K. Tested Material: (chemical name, formulation, and percent active ingredient); L. Test Species/Strain: (both common and scientific name); M. Source of Organisms: (company and address); ------- -29- N. Stage of Organisms at the Initiation of Study: (hours). II. MATERIAL/METHODS This section is a narrative, which would include the following items. A. Test Substance 1. Identification (this information may be provided by the laboratory or sponsor): a. Chemical name; b. Composition (qualitative and quantitative description); c. Percent active ingredient; d. Molecular structure; e. Source, lot number, or code; f. Identity and percent composition of impurities in the test substance. 2. Preparation of test solution (control/treatment): a. Any vehicle used to dissolve test material; b. Amount of vehicle added to control (if used); c. Description of method used to get test material into solution; d. Water source (reconstituted - if so, how prepared); e. Solubility in ppm; f. Total amount of test material used; g. Salinity (o/oo). B. Test Oyster 1. Rationale for selection of species if the species used is different than that preferred in Subdivision E: a. Test species name (both scientific and common); b. History of test organisms (strain, diseases, and treatment). 2. Description of fertilization process and method used to collect embryos. 3. Size/age: a. Stage of life at initiation of test; b. Date of spawn; c. List of abnormalities (if any occurred). 4. Source/acclimation: a. Complete name and address of test species supplier; b. Source of food and water; c. Volume of test container [in liters (L)]; d. Photopericd and lighting; e. Temperature, pH, dissolved oxygen, and name of equipment used to measure water quality; ------- -30- f. Chemical treatment (if used); g. Percent mortality; h. Feeding schedule; i. Test organisms from the same source (yes or no); j. Salinity (o/oo). C. Method 1. Test vessels: / a. Material type; b. Volume [in liters (L)]; c. Volume of test solution [in centimeters (cm)]; d. Size [in centimeters (cm)]. 2. Test system: a. Source of dilution water; b. If flow-through, description of system and flow rate/day; c. Procedures used to prepare toxicant stock solution; d. Criteria used to determine effects; e. Duplication of treatment levels (yes or no). 3. Test design: a. Method used in assigning test organisms to test and control groups; b. Number of fertilized eggs per liter of test solution; c. Loading (weight of organism per unit volume of water); d. Number of treatment levels used (nominal or measured); e. Length of treatment period; f. Type of control (positive, negative, or solvent control); g. Temperature (prior to and during spawning); h. pH (when checked - see table 1); i. Lighting (time in hours, intensity in footcandles, and transition period between light and dark); j. Dissolved oxygen (see table 1); k. Range finding test results (if used); 1. Aeration (yes or no); m. Water physical characteristics at the end of test: 1. Water depth and volume; 2. Temperature, pH, dissolved oxygen, and salinity; n. Name of protocol used during the test; o. How embryos collected and preserved; p. Number of normal developed larvae at the end of observation; q. Number of deformed larvae at the end of observation period. D. Statistical Analysis A. Cite references (author, title, journal, number, page, etc.). ------- I1- -31- DISCUSSION AND RESULTS A. Provide £€50 value in ppm with 95% confidence limits (graphs, printouts, and other calculations should be attached to report). Provide raw mortality data (see table 2). B. Discuss the relationship, if any, between the physical factors, toxicant, and observation, and describe any precipitation and solubility problems. C. Provide statistical method used (cite references, author, etc.). D. Observation - Provide the following data: 1. Signs of intoxication - temporal onset and duration, and concentrations that showed effects? 2. The no-observed-effect level. IV. CONCLUSIONS This is a summation of the above section - Discussion/Results. ------- Client : Test Material: Test Organism: Test Water : -32- TABLE 1 WATER QUALITY PARAMETERS WATER QUALITY Temperature Dissolved Oxygen (mg/L) PH (°C) Initial 48 hours Initial 48 hours 6 hrs, 12 hrs, etc. High X X X X X X X X Medium X X X X Low X X X X ------- -33- TABLE 2 PERCENT REDUCTION OF NORMAL LARVAE AND VALUES Client : Test Material: Test Organism: No. Per Cone.: Test Water : PERCENT REDUCTION OF NORMAL LARVAE TEST MATERIAL NOMINAL OR MEASURED CONCENTRATION (mg/L) Control 24 Hour #(#/#) 48 Hour #(#/#) X X X X #(#/#) #(#/#) #(#/#) #(#/#) #(#/#) #(#/#) #(#/#) #(*/*) X *(*/#) #(#/#) 24 hour 48 hour EC50 (mg/L) 95% Low Confidence Limits High X X X X X X Observed Effects a floating b dead c lying on bottom d etc. £/ How the percent of mortality table works. 1. #(f/#)a. 2. # = the total number of organisms tested per dose level. 3. #(#/#) = % mortality [No. dead/No, treated, e.g., 10% (1/10)]. 4. a = the observed effects. ------- -34- PESTICIDE ASSESSMENT GUIDELINES WILDLIFE AND AQUATIC ORGANISMS Shell Deposition Study for Oyster Subdivision E, Series 72-3 DATA REPORTING GUIDELINE The following describes the order and format for a study report item by item. TITLE/COVER PAGE Refer to PR Notice 86-5. TABLE OF CONTENTS The Table of Contents should indicate the overall organization of the study, including tables and figures. It must follow the title, data confidentiality, and GLP (if appropriate) pages as described in PR Notice 86-5. I. SUMMARY/INTRODUCTION A. Sponsor: (name of study owner); B. Name(s) of principal investigator(s); C. Location of study; D. Location of raw data and final report; E. Material: (common/trade name); F. Subject: (final or draft report, 96-hour £€50, and species tested); G. Test Doses: (control, concurrent vehicle control, #, #, #, #, # ppm); H. Test Dates: (initiation, date test started, termination, and date of last day of observation); I. Length of Study: (days); J. Results: (EC$Q value, 95% confidence limits, and no-observed-effect level); K. Tested Material: (chemical name, formulation, and percent active ingredient); L. Test Species/Strain: (both common and scientific name); M. Source of Organisms: (company and address); ------- -35- N. Size of Organisms at the Initiation of Study: (mm). II. MATERIAL/METHODS This section is a narrative, which would include the following items. A. Test Substance 1. Identification (this information may be provided by the laboratory or sponsor): a. Chemical name; b. Composition (qualitative and quantitative description)? c. Percent active ingredient; d. Molecular structure; e. Source, lot number, or code; f. Identity and percent composition of impurities in the test substance. 2. Preparation of test solution (control/treatment): a. Any vehicle used to dissolve test material; b. Amount of vehicle added to control (if used); c. Description of method used to get test material into solution; d. Water source (reconstituted - if so, how prepared); e. Solubility of test material in ppm; f. Total amount of test material used; g. Salinity (o/oo). B. Test Oyster 1. Rationale for selection of species if the species used is different than that preferred in Subdivision E: a. Test species name (both scientific and common); b. History of test organisms (strain, diseases, and treatment). 2. Description of any pretest conditioning: a. Health: 1. Sickness; 2. Injuries; 3. Abnormalities; 4. Pretest diet; 3. Size/age/physical condition: a. Height (in mm); b. Abnormalities (if any occurred). ------- -36- 4. Source/acclimation: a. Complete name and address of test species supplier; b. Source of food and water; c. Size of test container [in liters (L)]; d. Photoperiod and lighting; e. Temperature, pH, dissolved oxygen, and name of equipment used to measure water quality; f. Chemical treatment (if used); g. Percent mortality; h. Feeding schedule; i. Test organisms from the same source (yes or no); j. Salinity and acclimation period in days. C. Method 1. Test Vessels: a. Material type; b. Volume [in liters (L)]; c. Depth of test solution [in centimeters (on)]; d. Size [in liters (L)]. 2. Test system: a. Source of dilution water; b. If flow-through, description of system and flow rate/day; c. Procedures used to prepare toxicant stock solution; ' d. Criteria used to determine effects. 3. Test design: a. Method used in assigning test organisms to test and control groups; b. Number of organisms per dose level; c. Loading (weight of organism per unit volume of water); d. Number of treatment levels used (nominal or measured); e. Length of treatment period; f. Type of control (positive, negative, or solvent control); g. Temperature (how often measured); h. pH (when checked - see table 1); i. Lighting (time in hours, intensity in footcandles, and transition period between light and dark); j. Dissolved oxygen (see table 1); k. Range finding test results (if used - include sample size, test levels, and mortality data); 1. Aeration (yes or no); m. Water physical characteristic at the end of test: 1. Water depth and volume; 2. Temperature, pH, and dissolved oxygen; n. Percent of death/effects at each dose level; o. Name of protocol followed during the test; p. How peripheral valve edge removed; q. Method used to measure oyster size. ------- -37- D. Statistical Analysis 1. Cite references (author, title, journal, number, page, etc.)* III. DISCUSSION AND RESULTS A. Provide ECso value in ppm with 95% confidence limits (graphs, printouts, and other calculations should be attached to report). Provide raw mortality data (see table 2). B. Discuss the relationship, if any, between the physical factors, toxicant, and observation, and describe any precipitation and solubility problems. C. Provide statistical method used (cite references, author, etc.). D. Observation - Provide the following data: 1. Signs of intoxication - temporal onset and duration, and concentrations that showed effects; 2. The no-observed-effect level. IV. CONCLUSIONS This is a summation of the above section - Discussion/Results. ------- Client : Test Material: Test Organism: Test Water : -38- TABLE 1 WATER QUALITY PARAMETERS WATER QUALITY Temperature Parameter Dissolved Oxygen (mg/L) pH Salinity 0/00 6 hours, Time Initial 48 hours 96 hours Initial 96 hours Initial 96 hours 12 hours, etc. Control X X X X X X X Nominal X X High X X X X X X X Concent rat ion (mg/L) X X Medium X X X X X X Low X X X X X ------- -39- TABLE 2 PERCENT CHANGE IN SHELL GROWTH AND EC$Q VALUES Client : Test Material: Test Organism: Test Water : PERCENT CHANGE IN SHELL GROWTH TEST MATERIAL NOMINAL OR MEASURED CONCENTRATION (mg/L) Control 24 Hour #(#/#) #(#/#) #(#/#) #(#/f) #(#/#) #(#/#) 48 Hour #(#/#) #(#/#) #(#/#) #(#/#) #(#/#) #(#/#) 96 Hour #(#/#) #(#/#) #(#/#) #(#/#) #(#/#) #(#/#) EC50 (mg/L) 95% Low Confidence Limits High 24 hour X X X 48 hour 96 hour X X X X X X Observed Effects a floating b dead c no growth d etc. V How the percent of change in shell growth table works. 1. #(#/#)a- 2. # = the total number of organisms tested per dose level. 3. #(#/#) = % mortality [No. dead/No, treated, e.g., 10% (1/10)] 4. a = the observed effects. ------- -40- PESTICICE ASSESSMENT GUIDELINES WILDLIFE AND AQUATIC ORGANISMS Fish Early Life-Stage Subdivision E, Series 72-4 DATA REPORTING GUIDELINE The following describes the order and format for a study report item by item. TITLE/COVER PAGE Refer to PR Notice 86-5. TABLE OF CONTENTS The Table of Contents should indicate the overall organization of the study, including tables and figures. It must follow the title, data confidentiality, and GLP (if appropriate) pages as described in PR Notice 86-5. I. SUMMARY/INTRODUCTION A. Sponsor: (name of study owner); B. Name(s) of principal investigator(s) C. Location of study; D. Location of raw data and final report; E. Material: (common/trade name); F. Subject: (final or draft report, fish early life-stage, and species tested); G. Test Doses: (control, concurrent vehicle control, #, #, #, #, # ppm); H. Test Dates: (initiation, date test started, termination and date of last day of observation); I. Length of Study: (days); J. Results: (MATC value ppm and no-observed-effects level); K. Tested Material: (chemical name, formulation, and percent active ingredient); L. Test Species/Strain: (both common and scientific name); M. Source of Eggs: (company and address); ------- N. Stage of Eggs development at the Initiation of Study. MATERIAL/METHODS This section is a narrative, which would include the following items. A. Test Substance 1. Identification (this information may be provided by the laboratory or sponsor): a. Chemical name; b. Composition (qualitative and quantitative description); c. Percent active ingredient; d. Molecular structure; e. Source, lot number, or code; f. Identity and percent composition of impurities in the test substance. 2. Preparation of test solution (control/treatment): a. Any vehicle used to dissolve test material; b. Amount of vehicle added to control (if used); c. Description of method used to get test material into solution; d. Water source (reconstituted - if so, how prepared); e. Solubility in ppm; f. Total amount of test material used. B. Test Fish 1. Rationale for selection of species if the species used is different than that preferred in Subdivision E (if eggs were not used, give stage of life cycle in which organisms were tested): a. Test species name (both scientific and common); b. History of test organisms (strain, diseases, and treatment). 2. Description of any pretest conditioning: a. Health: 1. Sickness; 2. Injuries; 3. Abnormalities; 4. Name of medication (if used); 5. Pretest diet; 6. Number of days brood organisms were quarantined. 3. Size/age/physical condition: a. Age of unfertilized eggs and milt at the beginning of test; b. Description of procedures used to strip eggs and milt; c. Description of the procedures used in storage and handling of eggs; d. Description of the fertilization process. ------- -42- 4. Source/acclimation: a. Complete name and address of test species supplier; b. Source of food and dilution water; c. Size of test container [in liters (L)]; d. Number of females used per batch; e. Temperature, pH, and dissolved oxygen; f. Description of methods used initially to determine different stages of development; g. Percent mortality (brood fish); h. Feeding schedule, holding and acclimation period; i. Test organisms from the same source (yes or no); j. Number of males used per batch of eggs. C. Method 1. Test Vessels: a. Material type; b. Volume [in milliliters (mL)]; c. Depth of test solution [in centimeters (cm)]; d. Size of vessels [in centimeters (cm)]. 2, Test system: a. Source of dilution water; b. If flow-through, description of system construction material, system, and flow rate/day; c. Procedures used to prepare toxicant stock solution; d. Criteria used to determine effects. 3. Test design: a. Method used in assigning test organisms to test and control groups; b. Number of eggs per dose level; c. Name of protocol followed during this test; d. Loading (number of organism per unit volume of water); e. Number of treatment levels used (nominal or measured); f. Length of exposure period; g. Type of control (positive, negative, or solvent control); h. Temperature; i. pH (when checked - see table 2); j. Lighting (time in hours and intensity in footcandles); k. Dissolved oxygen (see table 2); 1. Water hardness (expressed in mg/L as CaCC>3 - see table 2); m. Alkalinity (expressed in mg/L as CaCC>3 - see table 2); n. Conductivity (umhos/cm - see table 2); o. Range finding test results, concentrations, and mortality (if used); p. Aeration (yes or no); ------- q. Water physical characteristic at the end of test: 1. Water depth and volume; 2. Temperature, pH, dissolved oxygen, and name of equipment used to measure the water quality; r. Period food was withheld prior to termination; s. Number of normal fry at the end of test (see table 3); t. Concentration analysis (see table 1); u. Examination of fish physiology, locomotion, and behavior; v. Length and weight (see table 5); w. Mortalities during the test; x. Fry survival and growth; y. The larvae fish released from the retaining cup when batch was ( ) % complete or ( ) hours has elapsed; z. Description of method used to keep eggs clean. D. Statistical Analysis 1. Cite references (author, title, journal, number, page, etc.). III. DISCUSSION AND RESULTS A. Provide MATC value in ppm (graphs, printouts, and other calculations should be attached to report). Provide raw mortality data (see table 3). B. Discuss the relationship, if any, between the physical factors, toxicant, and observation. C. Provide statistical method used (cite references, author, etc.). D. Observation - Provide the following data: 1. Signs of intoxication - temporal onset and duration; 2. The no-observed-effect level. IV. CONCLUSIONS This is a summation of the above section - Discussion/Results. ------- TABIE 1 MEASURED CONCENTRATION OF TEST COMPOUND IN WATER TO WHICH (Test fish name) (Scientific Name) WERE EXPOSED FOR # DAYS Nominal Concent rat ion (mg/L) Mean + SD Number of Range Samples Water Control 11 -- " (Detected) " Solvent Control Detected or not ------- BIS- WATER QUALITY CHARACTERISTICS MEASURED DURING EXPOSURE OF (TEST FISH) EMBRYOS AND lARVAE TO (TEST COMPOUND) Water Quality Number of Characteristic Mean + SD Range Measurements Temperature (°C) " + " Dissolved Oxygen " + " (mg/L) pH "~ -' Total Hardness " + " (mg/L as Total Alkalinity (mg/L as CaC03) Specific Conductance (umhos/cm) ------- -46- TABLE 3 SURVIVAL OF (TEST FISH NAME) AT HATCH (DAY #), AT # DAYS POST-HATCH, AFTER EXPOSURE TO SEVERAL (XINCENTRATIONS OF (TEST COMPOUND) Mean Measured Concentration (mg/L) Tank # # # # # Water Control Solvent Control # # # I # # # # # # # # # # Percent Larvae Survival At Hatch on Day 1 (Mean) * I # * # # * I # * # # * # # * # # * # # Percent Survival At # Days Post-Hatch (Mean) * # # * # # * # # * # # * I # * # # * # # Percent Survival At # Days Post-Hatch (Mean) * # # * 1 # * I # * # # * # - 1 * # 1 * # # ------- -47- TABLE 4 LENGTH AND WEIGHT CF [TEST FISH NAME] AT # DAYS POST-HATCH AFTER EXPOSURE TO SEVERAL CONCENTRATIONS OF (TEST COMPOUND) Mean Weight (g) Measured Total Length (mm) Replicate (N) Concentration Mean + SD (N) Average (mg/L) Tank (Treatment Mean + SD) (Treatment Average) # # # # # ter Control •Ivent Control # # # # # # # # # # f # * # # + * + (# T t + # + (# + f + # + (# + # + 1 + (t ± # + f + (#± f + # + (f 7 # + # + (# + # # *) # f f) # # #) * # f) # # f) # f #) # # f) # + # + (# + # + # + <# ± # + # + (#i f + # + (# + # + # + <*± # + * + (f i t + # + (t + # # #) # # f) # # i) # # t) # f #) # # f) f f t) a The mean length of these fish was/was not significantly less than the mean length of fish in solvent control group according to a (statistic name) test (P < 0.05). ------- -48- TABLE 5 LENGTH AND WEIGHT CF [TEST FISH NAME] AT # DAYS POST-HATCH AFTER EXPOSURE TO SEVERAL CONCENTRATIONS OF (TEST COMPOUND) Mean Weight (g) Measured Total Length (mm) Replicate (N) Concentration Mean + SD (N) Average (mg/L) Tank (Treatment Mean + SD) (Treatment Average) # # ft # ft # # # ft # # # \ # # # Water Control # f Solvent Control ft ft ft ft (# # # (ft # # (ft # # (# # # (# ft # (ft # ft (ft + # + ft T ft) + ft + ft T ft) + ft + ft 7 ft) + ft + ft ± *) + ft + ft ±*> + ft + ft ± *) + ft + ft + ft) (#) (ft) (ft) (ft) (ft) (ft) (ft) (ft) (ft) (ft) (ft) (ft) (ft) (ft) f + ft + (ft 7 ft + ft + (ft + ft + ft + (ft 7 ft + ft + (ftT ft + ft + (ft + ft + ft T (ft + ft + ft + (# + # ft ft) ft ft ft) ft ft ft) ft ft ft) ft ft ft) ft ft ft) ft ft ft) (#) (ft) (ft) (ft) (ft) (ft) (ft) (ft) (ft) (ft) (ft) (ft) (ft) (ft) a Mean lengths and mean weights of the groups of fish exposed to (test compound) were/were not significantly different than the mean length of fish in water and solvent control groups accord- ing to a (statistic name) test ( ). ------- PESTICIDE ASSESSMENT GUIDELINES WILDLIFE AND AQUATIC ORGANISMS Aquatic Invertebrate Life-Cycle Subdivision E, Series 72-4 DATA REPORTING GUIDELINE The following describes the order and format for a study report item by item. TITLE/COVER PAGE Refer to PR Notice 86-5. TABLE OF CONTENTS The Table of Contents should indicate the overall organization of the study, including tables and figures. It must follow the title, data confidentiality, and GLP (if appropriate) pages as described in PR Notice 86-5. I. SUMMARY/INTRODUCTION A. Sponsor: (name of study owner); B. Name: (person(s) who can provide details of test procedures); C. Location of study; D. Location of raw data and final report; E. Material: (common/trade name); F. Subject: (final or draft report, invertebrate life-cycle, and species tested); G. Test Doses: (control, concurrent vehicle control, #, #, #, #, # ppm); H. Test Dates: (initiation, date test started, termination, and date of last day of observation); I. Length of Study: (days); J. Results: (MATC value in ppm and no-observed-effects level); K. Tested Material: (chemical name, formulation, and percent active ingredient); L. Test Species/Strain: (both common and scientific name); ------- -50- M. Source of Organisms: (company and address); N. Age of Organisms at the Initiation of Study: (hours). II. MATERIAL/METHODS This section is a narrative which would include the following items. A. Test Substance 1. Identification (this information may be provided by the laboratory or sponsor): a. Chemical name; b. Composition (qualitative and quantitative description); c. Percent active ingredient; d. Molecular structure; e. Source, lot number, or code; f. Identity and percent composition of impurities in the test substance. 2. Preparation of test solution (control/treatment): a. Any vehicle used to dissolve test material; b. Amount of vehicle added to control (if used); c. Description of method used to get test material into solution; d. Water source (reconstituted - if so, how prepared); e. Solubility in ppm; .f. Total amount of test material used; B. Test Invertebrate 1. Rationale for selection of species if the species used is different than that preferred in Subdivision E: a. Test species name (both scientific and common); b. History of test organisms (strain, diseases, and treatment). 2. Description of any pretest conditioning: a. Health: 1. Sickness; 2. Injuries; 3. Abnormalities; 4. Mortalities (%); 5. Pretest diet. 3. Size/age/physical condition: a. Age at initiation of test (in hours); b. Date spawned; c. List of any abnormalities. ------- -51- 4. Source/acclimation: a. Complete name and address of test species supplier; b. Source of food and dilution water; c. Size of test container [in milliliters (mL)]; d. Temperature, pH, and dissolved oxygen; e. Percent mortality; f. Feeding schedule; g. Test organisms from the same source (yes or no); h. Number of days adults were held in laboratory prior to use of offspring. C. Method Materials Belong To- " ""* O.PPT Lr-n-y ' ' 1. Test Vessels: 4C"!. V H-t:-.r.-.-.i.' P-T (\*'z.rf?i\ a. Material type; b. Volume [in milliliters (mL)]; c. Volume of test solution [in centimeters (cm)]; d. Size of vessel [in milliliters (mL)]. 2. Test system: a. Source of dilution water; b. If flow-through, description of system and flow rate/day; c. Procedures used to prepare toxicant stock solution; d. Criteria used to determine effects. 3. Test design: a. Method used in assigning test organisms to test and control groups; b. Number of organisms per dose level; c. Name of protocol followed during this test; d. Loading (number of organisms per unit volume of water); e. Number of treatment levels used (nominal or measured); f. Length of exposure period; g. Type of control (positive, negative, or solvent control); h. Temperature; i. pH (When checked - see table 1); j. Lighting (time in hours and intensity in footcandles); k. Dissolved oxygen (see table 1); 1. Water hardness (expressed in mg/L as CaCC>3 - see table 1); m. Alkalinity (expressed in mg/L as CaCC>3 - see table 1); n. Conductivity (umhos/cm - see table 1); o. Range finding test results, concentrations, and mortality (if used); p. Aeration (yes or no); q. Water physical characteristic at the end of test: 1. Water depth and volume; 2. Temperature, pH, and dissolved oxygen; r. Number of normal organisms at the end of test (see table 3); s. Concentration analysis (see table 1); ------- -52- t. Examination of invertebrate physiology, locomotion, behavior, and pathology; u. Organisms count/measurement (see table 3); v. Mortalities during the test; x. If static system was used, how often test solution renewed. D. Statistical Analysis 1. Cite references (author, title, journal, number, page, etc.). III. DISCUSSION AND RESULTS A. Provide MATC value in ppm (graphs, printouts, and other calculations should be attached to report). Provide raw mortality data (see table 3). B. Discuss the relationship, if any, between the physical factors, toxicant, and observations. C. Provide statistical method used (cite references, author, etc.). D. Observation - Provide the following data: 1. Detailed record of spawning, fertility, and fecundity; 2. Signs of intoxication - temporal onset and duration; 3. The no-observed-effect level and reproductive effects. IV. CONCLUSIONS This is a summation of the above section - Discussion/Results. ------- -53- TABLE 1 WATER QUALITY CHARACTERISTICS MEASURED DURING EXPOSURE OF (TEST ORGANISMS) TO (TEST COMPOUND) Water Quality Characteristic Mean + SD Range Number of Measurements Temperature (°C) Dissolved Oxygen (mg/L) PH Total Hardness (mg/L as CaC03) Total Alkalinity (mg/L as CaCO3) Specific Conductance (umhos/cm) ------- -54- TABLE 2 PERCENT MORTALITIES AND MATC VALUES Client : Test Material: Test Organism: Test Water : PERCENT MORTALITY TEST MATERIAL NOMINAL CONCENTRATION (mg/L) Control Day # #(#/#) #(#/#) #(#/#) #(#/#) #(#/#) #(#/#) Day # #(#/#) #(#/#) #(#/#) #(#/#) #(#/#) #(#/#) Day # Day # Chronic Results MATC (mg/L) No effect level 95% Confidence Limits Low High Observed Effects a floating k swimming near surface c lying on bottom ^ turned dark e etc. £ How the percent of mortality table works. 1. f(#/#)a. 2. # = the total number of organisms tested per dose level. 3. #(#/#) = % mortality [No. dead/No, treated, e.g., 10% (1/10)] 4. a = the observed effects. ------- -55- TABLE 3 INVERTEBRATE MEASUREMENT/COUNT I OF CONCENTRATIONS t OF ADULTS TOUNG/ALIVE/DEAD LENGTH # # # # # # # # # f # # # * # # f # CONTROLS # # # # # # # # * # # f ------- -56- PESTICIDE ASSESSMENT GUIDELINES WILDLIFE AND AQUATIC ORGANISMS Life-Cycle Tests for Fish Subdivision E, Series 72-5 DATA REPORTING GUIDELINE The following describes the order and format for a study report item by item. TITLE/COVER PAGE Refer to PR Notice 86-5. TABLE OF CONTENTS The Table of Contents should indicate the overall organization of the study, including tables and figures. It must follow the title, data confidentiality» and GLP (if appropriate) pages as described in PR Notice 86-5. I. SUMMARY/INTRODUCTION A. Sponsor: (name of study owner); B. Name: (person(s) who can provide details of test procedures); C. Location of study; D. Location of raw data and final report; E. Material: (common/trade name); F. Subject: (final or draft report, fish life-cycle, and species tested); G. Test Doses: (control, concurrent vehicle control, #, #, #, #, # ppm); H. Test Dates: (initiation, date test started, termination, and date of last day of observation); I. Length of Study: (days); J. Results: (MATC value and no-observed-effects level); K. Tested Material: (chemical name, formulation, and percent active ingredient); L. Test Species/Strain: (both common and scientific name); M. Source of Organisms: (company and address); ------- -57- N. Stage of Organisms at the Initiation of Study: (hours). 11. MATERIAL/METHODS This section is a narrative, which would include the following Items. A. Test Substance 1. Identification (this information may be provided by the laboratory or sponsor): a. Chemical name? b. Composition (qualitative and quantitative description); c. Percent active ingredient; d. Molecular structure; e. Source, lot number, or code; f. Identity and percent composition of impurities in the test substance. 2. Preparation of test solution (control/treatment): a. Any vehicle used to dissolve test material; b. Amount of vehicle added to control (if used); c. Description of method used to get test material into solution; d. Water source (reconstituted - if so, how prepared); e. Solubility of test material in ppm; f. Total amount of test material used. B. Test Fish 1. Rationale for selection of species if the species used is different than that preferred in Subdivision E (if eggs were not used, give stage of life in which organisms were tested): a. Test species name (both scientific and cannon); b. History of test organisms (strain, diseases, and treatment). 2. Description of any pretest conditioning: a. Health: 1. Sickness; 2. Injuries; 3. Abnormalities; 4. Pretest diet; 5. Number of days brood fish were quarantined. 3. Size/age/physical condition: a. Age of unfertilized eggs and milt (in hours); b. Description of the procedures used to strip the eggs and milt; c. Description of the procedures used in handling and storage of eggs; d. Description of the fertilization procedures. ------- -58- 4. Source/acclimation: . a. Complete name and address of test species supplier; b. Source of food and dilution water; c. Size of test container [in liters (L)]; d. Number of females used per batch; e. Temperature, pH, dissolved oxygen, and name of equipment used to measure water quality; f. Description of methods used initially to determine different stages of development; g. Percent mortality (brood fish); h. Feeding schedule, holding, and acclimation period; i. Test organisms from the same source (yes or no); j. Number of males used per batch of eggs. C. Method 1. Test vessels: a. Material type; b. Volume [in milliliters (mL)]; c. Volume of test solution [in milliliters (mL)]; d. Size [in centimeters (cm)]. 2. Test system: a. Source of dilution water; b. If flow-through, description of system construction material, system, and flow rate/day; c. Procedures used to prepare toxicant stock solution; d. Criteria used to determine effects. 3. Test design: a. Method used in assigning test organisms to test and control groups and number of replicates used; b. Number of eggs per dose level; c. Name of protocol followed during this test; d. Loading (number of eggs per unit volume of water - if known); e. Number of treatment levels used (nominal or measured); f. Length of exposure period; g. Type of control (positive, negative, or solvent control); h. Temperature; i. pH (when checked - see table 2); j. Lighting (time in hours and intensity in footcandles); k. Dissolved oxygen (see table 2); 1. Water hardness (expressed in mg/L as CaC03 - see table 2); m. Alkalinity (expressed in mg/L as CaCO3); n. Conductivity (umhos/cm - see table 2); o. Range finding test results, concentrations, and mortality (if used); p. Aeration (yes or no); ------- -59- q. Water physical characteristic at the end of test: 1. Water depth and volume; 2. Temperature, pH, and dissolved oxygen; r. Period food was withheld prior to termination; s. Number of normal fry at the end of test (see tables" 3a and 3b); t. Concentration analysis (see table 1); u. Examination of fish physiology, locomotion, and behavior; v. Organisms length and weight (see table 5); w. Mortalities and effects during the test; x. If static system was used, how often test solution renewed; y. Fry survival, growth, and deformities. D. Statistical Analysis 1. Cite references (author, title, journal, number, page, etc.). III. DISCUSSION AND RESULTS A. Provide maximum acceptable toxic concentration (MATC) expressed as the range between the lowest observed effect concentration and the highest observed no effect concentration in ppm. B. Discuss the relationship, if any, between the physical factors, toxicant, and observation. C. Provide statistical method used (cite references, author, etc.) IV. CONCLUSIONS This is a summation of the above section - Discussion/Results. ------- -60- TABIE 1 MEASURED CONCENTRATION OF TEST COMPOUND IN WATER TO WHICH (Test fish name) (Scientific Name) WERE EXPOSED FOR # DAYS Nominal Concentration (mg/L) Mean + SD Range Number of Samples + T Water Control " " (Detected) " " Solvent Control Detected or not ------- -61- TABLE 2 WATER QUALITY" CHARACTERISTICS MEASURED DURING EXPOSURE OF (TEST FISH) EMBRYOS AND LARVAE TO (TEST COMPOUND) Water Quality Number of Characteristic Mean + SD Range Measurements Temperature (eC) " + Dissolved Oxygen " + (mg/L) pH Total Hardness " + (mg/L as CaC03) Total Alkalinity " + (mg/L as CaCO3) Specific Conductance " + (umhos/cm) ------- -62- TABLE 3a SURVIVAL OF (TEST FISH NAME) AT HATCH (DAY #), AT # DAYS POST-HATCH, AT # DAYS POST-HATCH AND SWIM-UP AFTER - EXPOSURE TO SEVERAL CONCENTRATIONS OF (TEST COMPOUND) Mean Measured Concentration (mg/L) Tank ft * ft # * # # * # # * # # * # Water Control ft t Solvent ft Control # Percent Percent Percent Larvae Survival Survival Survival At # Days At # Days At Hatch on Post-Hatch Post-Hatch Day # (Mean) (Mean) (Mean) # # # # # # ft # I ft # # ft # # * # # * ft # * ft t * # t # ft # * # t * ft # # # # # # # ft # # # ft # * # # # # # ft ft * ft ft ft # ft # Percent At Swim-up At # Days Post-Survival (Mean) # ft # # # ft # # # # # # # ft ft # ft # # ft # ------- -63- TABLE 3b (F2) SURVIVAL OF (TEST FISH NAME) AT HATCH (DAY #), AT # DAYS POST-HATCH, AT + DAYS POST-HATCH AND SWIM-UP AFTER EXPOSURE TO SEVERAL CONCENTRATIONS OF (TEST COMPOUND) Mean Measured Concent rat ion (mg/L) Tank # # # # # Water Control Solvent Control # # # # # # # # # # # # # # Percent Percent Larvae Survival Survival At # Days At Hatch on Post-Hatch Day # (Mean) (Mean) # # # # # * # # # # # # # # # # # # * # # # # # # ft 1 # t # # # f # f # # # # # 1 # Percent Percent Survival At Swim-up At # Days At # Days Post-Hatch Post-Survival (Mean) (Mean) * # # * # # * # f # # f * # # * # # * # f # # # # f # # # # # # # # # # # # # # # # ------- -64- TABLE 4 LENGTH AND WEIGHT CF [TEST FISH NAME] AT # DAYS POST-HATCH AFTER EXPOSURE TO SEVERAL CONCENTRATIONS CF (TEST COMPOUND) Mean Weight (g) Measured Total Length (mm) Replicate (N) Concentration Mean + SD (N) Average (mg/L) Tank (Treatment Mean + SD) (Treatment Average) # # ft # # Water Control Solvent Control # # # ft ft # ft # # ft ft ft ft ft # + # + (# T f + ft + (ft + ft + # + (# + ft + ft + (ft + ft + ft + (ft + ft + ft + (ft + ft + ft + (ft + # ft ft) ft ft ft) ft ft ft) ft ft ft) ft ft ft) ft ft ft) ft ft ft) # + # + ft + ft + ft + (ft + ft + ft T (ft + ft + ft + (ft + ft + ft + (ft ? ft + ft + (ft + ft + ft + (ft + # ft ft ft ft ft) ft ft ft) ft ft ft) ft ft ft) ft ft ft) ft ft ft) a The mean length of these fish was/was not significantly less than the mean length of fish in solvent control group according to a (statistic name) test (P < 0.05). ------- -65- TABLE 5 LENGTH AND WEIGHT CF [TEST FISH NAME] AT f DAYS POST-HATCH AFTER EXPOSURE TO SEVERAL CONCENTRATIONS OF (TEST COMPOUND) Mean Measured Concentration (mg/L) Tank f # # # # ter Control ilvent Control # # # # # # f # # f f # # # Total Length (mm) Mean + SD (N) (Treatment Mean + SD) # + # f + # (# ± #) # + # # + # (# ± #) # + t f + # (# ±#) # + # # + # (# + #) t + # t + f (# + f) # + # # + # (# + #) # + # # + # (# + t) (#) (#) (#) (#) (#) (#) (#) (#) (#) (#) (#) (#) (#) (#) Weight (g) Replicate (N) Average (Treatment Average) # + # t + # (# ± #) # + # # + t (# + # # + # # + f (# ±f) f + # # + (# + # ) # + # # + # (t ±# ) t + 1 # + # (#±D # + # # + 1 (# + #) (#) (#) (#) (#) (#) (#) (t) (#) (#) (#) (t) (t) (t) (#) a The mean length of these fish was/was not significantly less than the mean length of fish in solvent control group according to a (statistic name) test (P < 0.05). ------- |