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Short-term toxicity to aquatic invertebrates

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short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1997-03-13 to 1997-03-15
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
other: FDA Environmental Assessment Technical Assistance Handbook '4.08, Springborn Laboratories Protocol #:070794/FDA 4.08 DM-SA
Version / remarks:
Protocol Amendments #1 and #2 dated 13 March 1997 and 26 August 2004, respectively.
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
5-[2-ethoxy-5-(4-methylpiperazin-1-yl)sulfonylphenyl]-1-methyl-3-propyl-4H-pyrazolo[4,3-d]pyrimidin-7-one;2-hydroxypropane-1,2,3-tricarboxylic acid
Cas Number:
Molecular formula:
5-[2-ethoxy-5-(4-methylpiperazin-1-yl)sulfonylphenyl]-1-methyl-3-propyl-4H-pyrazolo[4,3-d]pyrimidin-7-one;2-hydroxypropane-1,2,3-tricarboxylic acid
Test material form:
Specific details on test material used for the study:
- Source and lot No.of test material: Sponsor and DJM01001
- Purity test date: 1996-10-28

- Solubility and stability of the test substance in the solvent/vehicle: Slightly soluble in water

- Treatment of test material prior to testing: Dilution to a super saturated solution of 50 mg active ingredient / L to make a stock solution.
- Final dilution of a dissolved solid, stock liquid or gel: Dilutions to 6.3, 13, 25, 50 and 100% of a 50 mg/L (nominal) filtered, supersaturated stock solution

FORM AS APPLIED IN THE TEST (if different from that of starting material)
6.3, 13, 25, 50 and 100% of a 50 mg/L solution

Sampling and analysis

Analytical monitoring:
Details on sampling:
- Concentrations: 6.3, 13, 25, 50 and 100% of a 50 mg/L solution
- Sampling method: During the definitive exposure period, water samples were removed from two replicate solutions of each treatment level and the controls at 0, 24 and 48 hours for the analysis of UK-92,480 concentration. Samples analyzed at the 0- and 24-hour sampling intervals were removed from replicates E and F, while samples analyzed at test termination were removed from replicates A and B. Each exposure solution sample was collected from the approximate midpoint of the test vessel with a volumetric pipet. Quality control (QC) samples, formulated in the test dilution water at concentrations of UK-92,480 similar to the nominal treatment range, were prepared at each sampling interval and remained with the exposure solution samples throughout the analytical process. Results of the analyses of the QC samples were used to judge the precision and quality control maintained during the analysis of exposure solution samples.

Total hardness concentrations were measured by the EDTA titrimetric method and total alkalinity concentrations were determined by potentiometric titration to an endpoint of pH 4.5 (APHA et al., 1989). Specific conductivity was measured with a Yellow Springs Instrument Company (YSI) Model #33 salinity-conductivity-temperature meter and probe. The pH was measured with a Hanna Model HI9024 pH meter and combination electrode. Dissolved oxygen concentration was measured with a YSI Model #57 dissolved oxygen meter and probe. Daily temperature was measured with a Fisher Scientific alcohol thermometer. Temperature was continuously monitored throughout this study using a Fisher Scientific Min-Max thermometer.

- Sample storage conditions before analysis: Samaples were analyzed immediately.

Test solutions

Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Direct weight addition
- Controls: negative water control was used
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): water
- A film was noted in the 15 mg/L concentration throughout the 48 hour exposure period

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
- Common name: Daphnids
- Strain/clone: Daphnia magna
- Source: Labratory cultures maintained at Springborn
- Feeding during test: 2 mL per day
- Food type: unicellular green algae, Ankistrodesmus falcatus
- Amount: 2 mL/vessel
- Frequency: Once per day (4 x 10^7 cells/min)

- Acclimation period: The daphnid culture has existed in this water for multiple generations
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: unicellular green algae, Ankistrodesmus falcatus, 2 mL/vessel
- Feeding frequency: Once per day (4 x 10^7 cells/min)
- Health during acclimation (any mortality observed): None

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
48 h
Post exposure observation period:

Test conditions

180 mg/L as CaCO3 for the duration of the test
Test temperature:
19-20 oC throughout the exposure period
7.3 to 8.1 throughout the exposure period
Dissolved oxygen:
7.4 to 9.5 % saturation
120-130 mg/L as CaCO3 for the duration of the test
400-500 (µmhos/cm)
Nominal and measured concentrations:
Nominal Concentrations: 0 (solvent control), 6.3, 13, 25, 50 and 100% of a 50 mg/L solution
Measured Concentrations:
Measured Concentration (mg/L)
Nominal Concentration (%) 0 hour 24 hour 48 hour Mean (SD)

Control <0.25 <0.26 <0.26 <0.29 <0.29 Not Applicable
Solvent Control: <0.25 <0.25 <0.26 <0.26 <0.29 <0.29 Not Applicable
6.3 0.95 0.97 0.98 1.0 1.0 1.0 0.99 (0.03)
13 2.4 2.4 2.4 2.5 2.4 2.5 2.4 (0.03)
25 4.9 4.8 4.9 4.9 4.8 4.9 4.9 (0.03)
50 9.6 9.7 9.7 9.8 9.7 9.7 9.7 (0.05)
100 16 16 15 16 15 14 15 (0.90)
QC #1 (1.00) 0.978 0.971 0.992
(% recovery) (97.8) (97.1) (99.2)
QC #2 (5.00) 5.00 5.07 5.01
(% recovery) (100) (101) (100)
QC #3 (16.0) 16.2 16.1 16.0
(% recovery) (101) (101) (100)
Details on test conditions:
- Test vessel: 1000 mL glass beakers
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 500 mL of test solution with a depth of 12.7 cm with a surface area of 79 cm^2.
- Aeration: No
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 6

- Source/preparation of dilution water: Well water supplemented by Town of Wareham water
- Total organic carbon: 0.69 mg/L
- Particulate matter:
- Metals:
- Pesticides: <0.53 ug/L
- Chlorine: 25 mg/L
- Alkalinity: 120 mg/L of CaCO3
- Ca/mg ratio: 10.3 mg/L
- Conductivity: 500 µmhos/cm
- Salinity:
- Culture medium different from test medium: They are the same.
- Intervals of water quality measurement: Monthly

- Adjustment of pH: No
- Photoperiod: 16 h light / 8 h dark
- Light intensity: The test area was illuminated with Duro Test Vita-Lite and Sylvania Coolwhite7 fluorescent bulbs at an intensity range of 60 to 70 footcandles at the solutions surface. Light intensity was measured with a General Electric Type 217 light meter.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :


- Test concentrations:
First preliminary Study: Nominal treatment level solutions of 500, 100, 50 and 10 mg/L
Second preliminary Study: Nominal concentrations of 5, 10, 50 and 100 mg/L.
- Results used to determine the conditions for the definitive study:
First preliminary Study: Several hours following preparation of the treatment level solutions, undissolved test article was noted on the bottom of the test vessels and at the surface of the 500, 100 and 50 mg/L treatment level solutions. A film was observed on the surface of the 1000 mg/L treatment level solution following 24 hours of exposure. The 10 mg/L treatment level solution was clear and colorless throughout the exposure period. Immobilization of 5, 75, 65, 100 and 100%, was observed among daphnids to the 10, 50, 100, 500 and 1000 mg/L treatment level solutions, respectively. In an effort to eliminate the presence of undissolved test article in the test vessels, a second preliminary study was conducted.
Second preliminary Study: A film was observed at the surface of the 50 and 100 mg/L treatment level solutions following 48 hours of exposure. The 5 and 10 mg/L treatment level solutions were clear and colorless throughout the exposure period. At test termination, immobilization of 100 and 70% was observed among daphnids exposed to the 50 and 100 mg/L solutions, respectively. No immobilization or sublethal effects were observed among daphnids exposed to the 5 and 10 mg/L treatment levels.
Combined Results: Based on the results of the two preliminary tests, the functional water solubility of the test article in dilution water was determined to be ≥10 mg/L and ≤50 mg/L, nominal. Utilizing these data and the dose response data observed during these preliminary studies, nominal concentrations of 6.3, 13, 25, 50 and 100% of a 50 mg/L (nominal) filtered, supersaturated stock solution were selected for the definitive study.
Reference substance (positive control):

Results and discussion

Effect concentrationsopen allclose all
Key result
24 h
Dose descriptor:
Effect conc.:
ca. 15 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
Key result
48 h
Dose descriptor:
Effect conc.:
ca. 14 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
Key result
48 h
Dose descriptor:
Effect conc.:
ca. 9.7 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
Details on results:
Throughout the 48-hour study, the 15 mg/L treatment level was observed to be clear and colorless with a film of undissolved test article on the surface of the test solutions. Treatment levels ≤ 9.7 mg/L were clear and colorless throughout the exposure period. At test termination (48 hours), immobilization of 75% was observed among organisms exposed to the highest treatment level tested (15 mg/L). In addition, adverse effects (e.g., lethargy) were observed among all of the mobile daphnids exposed to this treatment level. No immobilization or adverse effects (e.g., lethargy) were observed among daphnids exposed to the remaining treatment levels tested (0.99 to 9.7 mg/L) or the controls.

Daphnids exposed to the 15 mg/L exposure solutions did not exhibit effects typical of complications associated with physical toxicity (e.g., organisms caught on particulate matter orfloating on the surface of the test solution). Additionally, analytical recoveries were consistent and established the expected concentration gradient. Therefore, the effects observed during the 48 hour test are representative of the exposure of daphnids to UK-92,480 at the limit of functional solubility
of the test material in dilution water.

Based on the results of this study, the 48-hour EC50 value for daphnids exposed to UK-92,480 was estimated by nonlinear interpolation to be 14 mg/L (corresponding 95% confidence interval of 9.7 to 15 mg/L). The No-Observed-Effect Concentration (NOEC) established for this study was determined to be 9.7 mg/L

- Mortality of control: None
- Other adverse effects control: none
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: For the 15 mg/L mean measured concentration, A film of undissolved test article was observed on the surface of the test solutions throughout the 48-hour exposure period.
Reported statistics and error estimates:
A computer program (Stephan, 1982, personal communication) was used to calculate the EC50 values and 95% confidence intervals.

Three statistical methods were available in the computer program: moving average angle analysis, probit analysis and nonlinear interpolation with 95% confidence limits calculated by binomial probability. Moving average angle and probit analyses yield statistically sound results only if at least two concentrations produce immobilization of between 0 and 100% of the test organism population. The selection of reported EC50 values and 95% confidence intervals was based upon an examination of the data base and the results of the computer analysis. Selection criteria included:
1) The establishment of a concentration-effect (immobilization) relationship
2) The number of concentrations causing partial responses
3) The span of responses bracketing the EC50 value.
If two or more statistical methods produced acceptable results, then the method which yielded the smallest 95% confidence interval was selected. The No-Observed-Effect Concentration (NOEC) during the 48-hour exposure period was also determined. The NOEC is defined as the highest concentration tested at and below which there were no toxicant related immobilization or physical and behavioral abnormalities (e.g., lethargy) with respect to the control organisms.

Statistical Results 95% Confidence Interval
Time EC50 (mg/L) Lower (mg/L) Upper (mg/L)
24 hour 15 9.7 Upper confidence level could not be calculated.
48 hour 14 9.7 15

Applicant's summary and conclusion

Validity criteria fulfilled:
The test substance aquatic invertebrate tosicity levels were adequately determined by this study which followed the appropiate critera.