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
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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
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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
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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
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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
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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
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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
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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.
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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.
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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.
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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);
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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).
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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;
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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.
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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
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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.
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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);
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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.
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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;
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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.
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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
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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.
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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);
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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).
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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).
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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.
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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).
------- |