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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12/12/1996 - 23/09/1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to methodology comparable to OECD TG 201 in compliance with GLP, without deviations that influence the quality of the results.
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Nominal: 0.5, 0.75, 1.1, 1.7 and 2.5 mg/l
- Sampling method: Duplicate 10-mL samples of each new WAF solution were collected at 0 hour and analyzed for OTNE by LSC. Equal volumes were removed from replicates A, B, and C of the control, vehicle blank, and each treatment level at 72 hours. The samples from the control, vehicle
blank, and each treatment level were composited and duplicate 10-mL sub-samples were analyzed for OTNE by LSC. In addition, duplicate 10-mL samples of the abiotic control solution were collected and analyzed at 72 hours.
- Sample storage conditions before analysis: No data
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The dosing solution was an isotopic dilution containing I4C-CPD 98663 and OTNE. The solution was prepared by quantitatively transferring a 1.0 mCi sample of 14C-CPD 98663 with OTNE to a volume of 10.0 mL. This standard solution was identified as PR-01554 and was analyzed by liquid scintillation counting (LSC) analysis to have an active ingredient concentration of 9.60 x 10^5 µg/mL. A 0.520-mL aliquot of this solution was brought to a 10.0 mL total volume with acetone to prepare the final dosing solution with a concentration of 4.99 x 10^4 µg/mL as analyzed by LSC. This solution was identified as R-01870 and was used to prepare all of the exposure solutions at each time period.

Test solutions were prepared individually for each treatment by adding an appropriate amount of the 4.99 x 10^4 µg/mL dosing solution to 2.0 L of dilution water in a 2-L volumetric flask. An additional volume of acetone was added to levels 1-4 to produce acetone concentrations equal to that in level 5 (50 µL/L). Each flask contained a Teflon-coated stir bar and the contents of the flasks were stirred for 20 ± 1 hours. Solutions were stirred so that a vortex of < 25 % of the volumetric depth was achieved. When stirring was terminated, the phases were allowed to separate for 1-1.5 hours. A portion of the water phase (water accommodated fraction - WAF) in each flask was removed via a glass siphon tube and used to fill the respective test flasks.
- Controls: yes, blanks and vehicle controls
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 50 ul/l
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No, phases were allowed to separate for 1-1.5 hours, after which only WAF's were used.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Scenedesmus subspicatus
- Strain: Utex no. 2594
- Source (laboratory, culture collection): Department of Botany, Culture Collection of Algae, University of Texas, Austin.
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: The parent culture was used to prepare individual cultures by transferring portions of the parent culture to autoclaved media in culture flasks. The prepared cultures were incubated at 24 ± 2°C in an environmental chamber under continuous fluorescent lighting. Periodically, new Scenedesmus subspicatus cultures were initiated using the parent stock or were cloned from an existing culture derived from the parent stock in 100 mL of algal nutrient medium. All cultures were maintained under the same conditions as those used for testing

ACCLIMATION
- Acclimation period: No data
- Culturing media and conditions (same as test or not): same as test
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
24.1 - 25.2 degrees Celsius
pH:
7.4 to 7.5 at test initiation and 10.2 - 10.3 at the conclusion of the exposure.
Nominal and measured concentrations:
Nominal: 0.5, 0.75, 1.1, 1.7 and 2.5 mg/l
Measured (0h): 0.554, 0.796, 1.23, 1.76 and 2.69 mg/l
Measured (72h): 0.496, 0.758, 1.10, 1.73 and 2.42 mg/l
Measured (mean): 0.53, 0.78, 1.2, 1.7 and 2.6 mg/l (106, 104, 109, 100 and 104% of nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass erlenmeyer with glass stopper, 125 ml filled with +/- 125 ml medium
- Aeration: No data
- Initial cells density: approximately 1.0 x 10^4 cells/ml
- Control end cells density: 21 x 10^4 cells/ml
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes, protocol meets the OECD Guideline 201. The test medium was a freshwater algal nutrient medium prepared with ABC reagent water.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The test medium was a freshwater algal nutrient medium prepared with ABC reagent water. After preparation, the medium was pH-adjusted to 7.5 ± 0.1 using 0.1 N NaOH and filtered through Millipore® 0.45-µm filters.
- Total organic carbon: < 1.00 mg/l
- Metals:
Al <0.044 mg/l
As <0.0050 mg/l
B <0.0058 mg/l
Cd <0.00042 mg/l
Cr <0.0013 mg/l
Co <0.0058 mg/l
Cu <0.0045 mg/l
Fe <0.015 mg/l
Pb <0.0034 mg/l
Hg <0.000023 mg/l
Ni <0.0078 mg/l
Se <0.0037 mg/l
Ag <0.00081 mg/l
Zn 0.0174 mg/l
- Pesticides: all <3.8 mg/l
- Culture medium different from test medium: No
- Intervals of water quality measurement: 72 hours

OTHER TEST CONDITIONS
- Sterile test conditions: No
- Adjustment of pH: Yes, pH adjusted to 7.5 using NaOH
- Photoperiod: Continuous light
- Light intensity and quality: 8600 +/- 10% lux, cool-white fluorescent light

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: light microscope and hemacytometer
- Chlorophyll measurement: No

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.5
- Range finding study
- Test concentrations: 0.1, 1.0 and 10.0 mg/l
- Results used to determine the conditions for the definitive study: based on cell counts, inhibition was <= 15% for all levels tested.
Reference substance (positive control):
no
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 2.6 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: radioactive compound
Basis for effect:
growth rate
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 2.6 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: radioactive compound
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 2.6 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: radioactive compound
Basis for effect:
growth rate
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 2.6 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: radioactive compound
Basis for effect:
biomass
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 2.6 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: radioactive compound
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 2.6 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: radioactive compound
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 2.6 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: radioactive compound
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Any stimulation of growth found in any treatment: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No, but measured concentrations at the highest concentration level are near the limit of solubility of the test substance.

Reported statistics and error estimates:
Statistical calculations of the 24-, 48-, and 72-hour EC50 and NOEC concentrations by SAS were based on area under the growth curve (EbC50) and growth rate (EC50) versus the mean measured OTNE concentrations. Prior to the EC50 and NOEC calculations, a Shapiro-Wilk's test and a Levene's test were conducted to test for normality and homogeneity of variance, respectively, over treatments at each time point. If the p values from the Shapiro-Wilk's and Levene's test were greater than 0.01, indicating normality and insignificant heterogeneity, then the analysis was performed on the non-transformed raw data. If the p values were less than 0.01 for some hour(s), then the raw data for each replicate were transformed using the square root of the raw data, the recommended transformation for count data. If both the raw data and transformed data have shown nonnormality or inequality of variance, then a nonparametric analysis of variance was performed using the ranks of the values .

The pH of the controls did deviate more than 1 pH unit which was a result of limited CO2 exchange die to the use of closed test vessels with minimal head space. The pH deviation of more than 1 pH unit did not affect the integrity of the test since acceptable growth (16x increase) was observed in the controls.

reported statistics(continued): Prior to the EC50 and NOEC calculations, the control groups were evaluated to determine whether or not they could be pooled by comparing the 72-hour means for the growth parameters of cell density, area under the growth curve, and growth rate. Tests for normality and homogeneity of variance were performed on the control data and then the planned comparison, or Least Significant Difference (LSD) test, was performed by inspecting the p value for the t-test between control means. The analysis showed no statistical significant difference (p > 0.05) between control groups, therefore, control groups were pooled for comparison to the treatment groups.

A one-way analysis of variance (ANOVA) (using PROC GLM in SAS) and a Dunnett's comparison to the pooled controls was conducted for each time point to determine the NOEC's. A one-tailed Dunnett's test was conducted at the 0.05 level of significance with the alternate hypothesis being that the area under the growth curve, and/or growth rate had been reduced in comparison to the pooled controls.

For the controls and each treatment group, the area under the growth curve and the growth rate were calculated by SAS.
Validity criteria fulfilled:
yes
Remarks:
Exponential increase in controls, variation within limits.
Conclusions:
The 72h-EC50 of OTNE towards Scenedesmus subspicatus is > 2.6 mg/l for both biomass and growth rate. The72h-NOEC based on growth rate is >= 2.6 mg/L.
Executive summary:

The toxicity op OTNE towards Scenedesmus subspicatus was investigated according to methodology comparable to international Guidelines (OECD Guideline 201) under GLP. Algae were exposed to water accommodated fractions (WAF) of 14C-labeled OTNE with nominal concentrations of 0.50, 0.75, 1.1, 1.7 and 2.5 mg/l in a static system for 72 hours. Validity criteria for the test were met. The 72h-EC50 for both growth rate and biomass was found to be > 2.6 mg/l. The 72h-NOEC was found to be >= 2.6 mg/l based on mean measured concentrations.

Description of key information

The toxicity op OTNE towards Scenedesmus subspicatus was investigated according to methodology comparable to international Guidelines (OECD Guideline 201) under GLP. Algae were exposed to water accommodated fractions (WAF) of 14C-labeled OTNE with nominal concentrations of 0.50, 0.75, 1.1, 1.7 and 2.5 mg/l in a static system for 72 hours. Validity criteria for the test were met. The 72h-EC50 for both growth rate and biomass was found to be > 2.6 mg/l. The 72h-NOEC was found to be 2.6 mg/l based on mean measured concentrations

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
2.6 mg/L

Additional information