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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Remarks:
Conducted according to guideline in effect at the time of study conduct
Qualifier:
according to
Guideline:
other: "Alga growth inhibition test, Daphnia sp. acute immobilisation test, and fish acute toxicity test" (Japanese notification, Yakushokuhatsu 0331 No.7, Heisei 23.03.29 Seikyoku No.5 , Kanpokihatsu No.110331009, March 31, 2011
Deviations:
no
Remarks:
Conducted according to guideline in effect at the time of study conduct
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: 99.7944%

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Preparation of standard solutions: About 45 mL dimethylsulfoxide was collected in a 50-mL volumetric bottle, and the bottle with dimethylsulfoxide was treated as a tare at weighing of the test substance. The test substance was collected into a 10-mL glass centrifuge tube from cylinder, then added into the 50-mL volumetric bottle by decantation and weighed subtracting the tare weight. After the weighing, this solution was filled up to 50 mL with dimethylsulfoxide. The concentration of the test substance was calculated based on the measured weight, and this solution was diluted sequentially with dimethylsulfoxide to prepare standard solutions of 4 concentrations within the range of approximately 10-1000 mg/L. Dimethylsulfoxide was used as the standard solution at 0 mg/L.

Analysis of Test Solutions: The concentrations of the test substance in all test solutions were measured at the beginning of exposure and at 24, 48 and 72 hours after the beginning of exposure by Gas Chromatography/Mass Spectromet1y (GC/MS).

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION:
1. Preparation of stock solution
For the preparation of the stock solution, 2.1 L of OECD medium (cooled in the refrigerator prior to use) was used as dilution water.
(1) 6.3 g of the test substance was collected into a 10-mL glass centrifuge tube from the cylinder. A 10-mL glass centrifuge tube, a stopper and tape for sealing the stopper were treated as a tare. The test substance was weighed subtracting the tare weight.
(2) The test substance was added into the dilution water by decantation while stirring with a magnetic stirrer, and then stirred for an additional 30 minutes.
(3) The resulting stock solution was allowed to rest at room temperature for 30 minutes and the supernatant was collected by decantation (about 1.7 L) prior to its use for the preparation of the test solutions.

2. Preparation of test solutions
For the preparation of the test solutions, dilution water was prepared by stirring 4 L of cooled OECD medium and allowing it to rest at room temperature in the same manner as the stock solution. For the preparation of the test solutions, the stock solution was mixed with dilution water in appropriate amounts for each concentration.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: ATCC22662
- Age of inoculum (at test initiation): 4 days

ACCLIMATION PERIOD: 4 days

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Test temperature:
22 ± 2°C
pH:
7.8 to 10.4
Nominal and measured concentrations:
Nominal concentrations: 0, 300, 540, 960, 1700 and 3000 mg/L
Time Weighted Mean Measured concentrations: 0, 1.50, 2.03 , 4.24, 7.02 and 14.4 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 mL Erlenmeyer flask with stopper, Iwaki, Asahi Glass Co., Ltd. (headspace volume is 490 mL, measured in the testing laboratory); 100 mL/vessel of test solution
- Initial cells density: 5 X 10e3 cells/mL
- Control end cells density: 4.5 X 10e5 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6


GROWTH MEDIUM
- Standard medium used: yes


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: 24 hour light
- Light intensity and quality: 60 to 65 µE/m2/s


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : measured at 24, 48 and 72
- Determination of cell concentrations: electronic particle counter
- Chlorophyll measurement: The colour of the test cultures was observed by comparison with the extra vessel of each test group.
- Other: At 72 hours after the beginning of exposure, the appearance of algae was observed through the microscope.

TEST CONCENTRATIONS:
- Range finding study data: : Range finding nominal test concentrations were: 0, 30, 300 and 3000 mg/L. Percent inhibition at 72 hours was 0, 2.9, 0.2 and 6%, respectively.

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
other: ErC50
Effect conc.:
> 14.4 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: The results were calculated from the time-weighted mean of the measured concentrations.
Duration:
72 h
Dose descriptor:
other: NOECr
Effect conc.:
4.24 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: The results were calculated from the time-weighted mean of the measured concentrations.
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): In all test groups, the colour of the test cultures (as observed with the naked eye) showed a tendency to get more greenish during the exposure period. By microscopic observation at the end of exposure, in all concentration groups neither unusual cell shape of algae (contraction, expansion, damaged cell etc.) nor agglutination was observed, and the algae looked normal compared with those in the control group.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: A rapid drop of the concentration was observed in the first 24 hour window after the beginning of exposure after which the concentration remained relatively constant. Given the high volatility of the test substance, the most likely reason for the decrease of its concentration was volatilization from the test solution and test culture. Test solutions before inoculation were colourless, and showed no suspended solids, no floating solids, and no precipitates in any of the test groups.
Reported statistics and error estimates:
EC50 was determined by the least squares linear regression analysis of points recognized as straight in the concentration-inhibition curves. 95% confidence limits were calculated.
Growth rates (0 - 72hr) of each concentration group were compared with the control group using Bartlett's test for homogeneity of variances. When the variance was homogeneous: an analysis of variance (1-way ANOVA), Dunnett's, Williams's or Scheffe's multiple comparison test (Parametric) was used.

When the variance is not homogeneous: Kruskal-Wallis rank sum test, Dunnett's, Williams's or Scheffe's multiple comparison test (Non parametric) were used.

Any other information on results incl. tables

Nominal

Concentration

mg/L

Mean Measured

Concentration

mg/L

Percent

Inhibition

Control

0

-

300

1.50

-0.3

540

2.03

0.1

960

4.24

1.3*

1700

7.02

3.2

3000

14.4

5.5*

* Indicates a significant difference from control

α = 0.01

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
This study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
72-hour ErC50 = > 14.4 mg/L
NOECr = 4.24 mg/L
Executive summary:

An acute, static 72-hour toxicity test was conducted with the algae Pseudokirchnerella subcapitata at nominal concentrations of 0, 300, 540, 960, 1700 and 3000 mg/L. The mean measured concentrations used in LC50 calculations were 0, 1.50, 2.03, 4.24, 7.02 and 14.4 mg/L. Because of the volatile nature of the test item, the test was performed in 500 mL Erlenmeyer flasks with stoppers. Control and test solutions were conducted with six and three replicates, respectively. In all the test groups, the temperature of the test solutions were 22 ± 2°C, the pH values were 7.8 to 10.4 and the biomass in the control cultures increased exponentially, fulfilling the test conditions.

Based on the mean measured concentrations, the 72-hour ErC50 was 14.4 mg/L and the NOECr was 4.24 mg/L.