extended acclimation period, EPA recommends that the fish not be used. Type of food: [frozen brine shrimp, other] EPA recommends that fish be fed frozen brine shrimp twice per day to promote active reproduction and maintain body condition. Source of food: [............................] Frequency of feeding: [............................] times/day Details on feeding: [Describe any other relevant information about the feeding regime, including the amount given. Provide additional information if feeding conditions differed between the acclimation, pre-exposure, and definitive test periods.] Table 3: Fish Selection and Pre-Exposure Performance. Parameter Value(s) Details or Remarks Guideline Recommendations Age at test initiation: [...] months EPA recommends reproductively mature (sexually dimorphic) fish, 4.5 - 6 months old. Mean weight of males at test initiation (if determined): [...] g [Indicate whether the mean is based on a subsample of male fish or based on all male fish selected for the test.] EPA recommends that a subsample of fish be weighed before the test to estimate the mean weight for each sex. It is recommended
Parameter Minimum Maximum Mean Measurement Interval Guideline Recommendations Temperature (°C) EPA recommends temperature 25±1oC; inter-replicate and inter-treatment differentials should not exceed 1oC. pH EPA recommends pH 6.5 to 9.0. Dissolved oxygen (mg/L) EPA recommends dissolved oxygen (DO) >4.9 mg/L (>60% air saturation) Total alkalinity (mg/L as CaCO3) EPA recommends total alkalinity > 20 mg/L as CaCO3. Hardness [specify units] Total organic carbon (mg/L) EPA recommends that total organic carbon in dilution water be < 2 mg/L. Unionized ammonia (g/L) EPA recommends that unionized ammonia in the dilution water be < 1 g/L. Residual chlorine (g/L) EPA recommends that residual chlorine in dilution water be < 10 g/L. Other [specify] General recommendations for frequency of measurements: EPA recommends that temperature, pH, and dissolved oxygen be measured in all test tanks at least weekly and that hardness and alkalinity be measured in controls and in one tank at the highest test
4 Test concentration 2 0 1 2 3 4 Test concentration 3 0 1 2 3 4 Test concentration n 0 1 2 3 4 Positive control, if used 0 1 2 3 4 1 Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 2=Mild, 3=Moderate, 4=Severe. See Appendix E of the test guideline for reference.
Table 16: Additional Gonadal Histopathology Observations in Male [test organism]. Treatment (mg a.i./L) [measured] Additional Diagnostic Observations1 Severity Decreased Proportion of Spermatogonia Increased Vascular or Interstitial Proteinaceous Fluid Asynchronous Gonad Development Altered Proportions of Spermatocytes or Spermatids Granulomatous Inflammation n Incidence n Incidence n Incidence n Incidence n Incidence Control (dilution water only), if used 0 8 8 8 8 8 8 8 8 8 8 1 2 3 4 Solvent control, if used 0 1 2 3 4 Test concentration1 0 1 2 3 4 Test concentration 2 0 1
Table 17: Gonadal Histopathology in Female [test organism]. Treatment (mg a.i./L) [measured] Diagnostic Observations1 Severity Increased Oocyte Atresia Perifollicular Cell Hyperplasia/ Hypertrophy Decreased Yolk Formation n Incidence n Incidence n Incidence Control (dilution water only), if used 0 16 16 16 16 16 16 1 2 3 4 Solvent control, if used 0 1 2 3 4 Test concentration1 0 1 2 3 4 Test concentration 2 0 1 2 3 4 Test concentration 3 0 1 2 3 4 Test concentration n 0 1 2 3
4 Test concentration 2 0 1 2 3 4 Test concentration 3 0 1 2 3 4 Test concentration n 0 1 2 3 4 Positive control, if used 0 1 2 3 4 1 Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 2=Mild, 3=Moderate, 4=Severe. See Appendix E of the test guideline for reference. [Briefly summarize any effects on biochemical endpoints, including plasma vitellogenin (VTG), testosterone (T), and 17β-estradiol, if measured. Report all test concentrations at which effects were observed and the direction of the effect, i.e., apparent increase or decrease when compared to control.]
Table 19: Plasma Vitellogenin in [test organism]. Treatment (mg a.i./L) [measured] Plasma Vitellogenin (VTG) Males Females n Mean (ng/mL plasma) ±SD n Mean (ng/mL plasma) ±SD Control (dilution water only), if used 8 528 39 16 1.2x107 8.1x106 Solvent control, if used Test concentration 1 Test concentration 2 Test concentration 3 Test concentration n Positive control, if used Abbreviations: NA Not applicable. ND Not determined. SD Standard deviation.
Table 20: Plasma Sex Steroids in [test organism] (if measured). Treatment (mg a.i./L) [measured] Plasma Testosterone (T) Plasma 17β-estradiol (E2) Males Females Males Females n Mean (ng/mL plasma) ±SD n Mean (ng/mL plasma) ±SD n Mean (ng/mL plasma) ±SD n Mean (ng/mL plasma) ±SD Control (dilution water only), if used 8 9.8 0.9 16 3.0 0.2 8 0.4 0.1 16 6.1 1.2 Solvent control, if used Test concentration 1 Test concentration 2 Test concentration 3 Test concentration n Positive control, if used Abbreviations: NA Not applicable. ND Not determined. SD Standard deviation.
[Discuss qualitative observations of secondary sex characteristics and the incidence of behavioral effects or other clinical signs. Identify when during the study the effects were observed and whether the effects are considered treatment-related. Complete the following table, using a separate line for each type of observation. Add rows as necessary.] Table 21: Secondary Sex Characteristics and Clinical Signs in [test organism]. Treatment (mg a.i./L) [measured] Secondary Sex Characteristics and Clinical Signs1 Males Females Type n Incidence Type n Incidence Control (dilution water only), if used Light body color 8 1 Vertical bands 16 2 Solvent control, if used Test concentration1 Test concentration 2
Table 23: Reproductive and HPG Endpoints1,2 for Female [test organism] in the FSTRA with [test chemical]. Treatment (mg a.i../L) [measured] Fecundity Fert. Success Tubercle Score GSI Gonadal Staging and Histo. Plasma VTG Plasma T Plasma E2 % Diff. p % Diff. p Median p % Diff. p Effect? (Yes/No) % Diff. p % Diff. p % Diff. p Control (dilution water only), if used 0 NA 0 NA 0 NA 0 NA NA 0 NA 0 NA 0 NA Solvent control, if used Test conc. 1 Test conc. 2 Test conc. 3 Test conc. n Positive control, if used