Registration Dossier

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 April to 6 May 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in 2005 according to OECD Method 201 and EU Annex V test C.3 and in accordance with GLP. Study material is well characterized.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Test material is a extremely light tan colored powder which was received at testing laboratory on 15 February 2005 and stored at room temperature in the dark.

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Samples were taken from the control and each test group (replicates R1 -R3 pooled) at 0 and 72 hours for quantitative analysis. Duplicate samples were taken at 0 and 72 hours and stored at approximately -20°C for further analysis if necessary. Test samples were analyzed after filtration through glass wool to remove any agal cells.

Test solutions

Vehicle:
no
Details on test solutions:
For definitive study, aqueous solution of the test material at concentrations of 6.25, 12.5, 25, 50 and 100 mg/L (three replicate flasks per concentration) were used.

The test material was dissolved directly in culture medium with the aid of ultrasonication to develop a series of stock solutions which were separately mixed with algal suspension (250 mL) to give the required test concentrations of 6.25, 12.5,25, 50 and 100 mglL. The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity. The concentration and stability of the test material in the test preparations were verified by chemical analysis at 0 and 72 hours

The range-finding test was conducted by exposing Scenedesmus subspicatus cells to a series of nominal test concentrations of 1 .0, 10 and 100 mg/L for a period of 72 hours.

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Scenedesmus subspicatus (new name: Desmodesmus subspicatus)
- Strain: CCAP 276120
- Source (laboratory, culture collection): obtained from the Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland

- Cultures were maintained in the laboratory by the periodic replenishment of culture medium. The culture was maintained in the laboratory at a temperature of 21 +/- 1°C under continuous illumination (intensity approximately 7000 lux) and constant aeration.

- Culturing media and conditions (same as test or not): same as test

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h

Test conditions

Hardness:
No data
Test temperature:
Temperature was maintained at 24 +/-1°C throughout the test.
pH:
The pH values of test concentrations were as follows ( R1-R3 vessels):
6.25 mg/l pH 7.2 (0 hour) to 8.1 ( 72 hour)
12.5 mg/l pH 7.2 (0 hour) to 8.0 ( 72 hour)
25 mg/l pH 7.1 (0 hour) to 7.8 ( 72 hour)
50 mg/l pH 7.0 (0 hour) to 7.3 ( 72 hour)
100 mg/l pH 6.9 (0 hour) to 7.1 ( 72 hour)
The pH values of the control cultures were observed to increase from pH 7.4 at 0 hours to pH 8.2 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.
Dissolved oxygen:
no data
Salinity:
no data
Nominal and measured concentrations:
Analysis of the test preparations at 0 hours showed measured test concentrations to range from 99% to 105% of nominal. At 72 hours analysis of the test preparations showed a decline in measured test concentrations in the range of 32% to 66% of nominal. Overall the decline in measured test concentrations followed a concentration dependent pattern with the lowest test concentrations exhibiting the greatest decline in measured test concentration. Current regulatory advice is that in cases where a decline in measured concentrations is observed, geometric mean measured concentrations should be used for calculating EC5O values.
Details on test conditions:
For the range finding study the results showed no effect on growth at the test concentration of 1.0 mg/L but growth was observed to be reduced at 10 and 100 mglL. Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 6.25, 12.5,25, 50, and 100 mg/L.
The test was conducted in 250 mL glass conical flasks plugged with polyurethane foam bungs to reduce evaporation.
Three flasks each containing 100 ml of test preparation were used for the control and each treatment group.
At initiation of the test the culture contained a nominal cell density of l0^4 cells per mL.
The flasks were incubated at 24 +/- 1°C under continuous illumination (intensity approximately 7000 lux) and constantly shaken at approximately 150 rpm for 72 hours.
Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter Multisizer Particle Counter.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
60 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CL could not be defined
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CL
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
49 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CL could not be calculated
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 82 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
16 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CL
Details on results:
- Exponential growth in the control (for algal test): yes data show ed that the cell concentration of the control cultures increased by a factor of 61 after 72 hours.
- Observation of abnormalities (for algal test): All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures
- At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-hour test period all control, 6.25, 12.5 and 25mglL test cultures were observed to be green dispersions. The 50 and 100 mg/L test cultures were observed to be pale yellow/brown dispersions.
Results with reference substance (positive control):
The following data show that the cell concentration of the control cultures increased by a factor of 61 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.The pH values of the control cultures were observed to increase from pH 7.4 at 0 hours to pH 8.2 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.
Reported statistics and error estimates:
One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955) was carried out on the area under the growth curve data at 72 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999-2001). The geometric mean measured test concentrations of the samples were calculated using the measured test concentrations of replicates R1-R3 pooled. There were no statistically significant differences between the control, 6.25, 12.5 and 25 mg/L test concentrations (P>=0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) was 25 mg/L.

Any other information on results incl. tables

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 99% to 105% of nominal. At 72 hours analysis of the test preparations showed a decline in measured test concentrations in the range of 32% to 66% of nominal. This decline in measured test concentrations was in line with the preliminary stability analyses conducted that indicated that the test material was unstable in culture medium under test conditions. Overall the decline in measured test concentrations followed a concentration dependent pattern with the lowest test concentrations exhibiting the greatest decline in measured test concentration. Current regulatory advice is that in cases where a decline in measured concentrations is observed, geometric mean measured concentrations should be used for calculating EC5Ovalues. It was therefore considered justifiable to base the results on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data. The use of the geometric mean measured test concentrations in the calculation of the EC50 and NOEC values had no significant effect on the outcome of the study.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test material on the growth of Scenedesmus subspicatus has been investigated over a 72-Hour period and gave an EbC50 (72 h) value of 60 mg/L. At the maximum concentration tested (100 mg/L) exposure to the test material resulted in 30% inhibition of the 0 -72 hour growth rate and hence the ErC5O (0 -72 h) was greater than 100 mg/L. It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L. The No Observed Effect Concentration at 72 hours was 25 mg/L.
From the data both the growth (r) and the biomass (b) of Scenedesmus subspicatus (CCAP 276120) were affected by the presence of the test material over the 72-Hour exposure period.
Based on the geometric mean measured test concentrations the EbC50 (72 h) value was 49 mg/L and the ErC5O (0 -72 h) value was greater than 82 mg/L . The No Observed Effect Concentration at 72 hours was 16 mg/L.
Executive summary:

The key aquatic study was assigned reliability rating of 1 (reliable without restriction) and the results are considered valid. The effect of the test material on the growth of Scenedesmus subspicatus has been investigated over a 72 -hour period and gave an EbC50 (72h) value of 60 mg/L. At the maximum concentration tested (100 mg/L) exposure to the test material resulted in 30% inhibitoin of the 0 -72 hour growth rate and hence the ErC50 (0 -72h) was greater than 100 mg/L. It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L. The No Observed Effect Concentration at 72 hours was 25 mg/L. From the data both the growth (r) and the biomass (b) of Scenedesmus subspicatus (CCAP 276120) were affected by the presence of the test material over the 72 -hour period.

Based on the geometric mean measured test concentrations the EbC50 (72 h) value was 49 mg/L and the ErC5O (0 -72 h) value was greater than 82 mg/L . The No Observed Effect Concentration at 72 hours was 16 mg/L.