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:
1st November 2013 to 13th February 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Done under GLP and OECD methods

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Acid Off-white powder
Initially stored at room temperature in the dark for 4 days, followed by approximately 4 °C in the dark

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Sampling method: Samples of the algal populations were removed daily and cell concentrations determined for each
control and treatment group, using a Coulter® Multisizer Particle Counter.
- Sample storage conditions before analysis: Prior to the start of the test sufficient master culture was added to approximately 100 mL
volumes of culture media contained in conical flasks to give an initial cell density of approximately 103 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1 °C
until the algal cell density was approximately 10^4 - 10^5 cells/mL.

Test solutions

Vehicle:
no
Details on test solutions:
The range-finding test was conducted by exposing Pseudokirchneriella subcapitata
cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L for a period of
72 hours.

Definitive test based on the results of the range-finding test the following test concentrations were assigned to
the definitive test: 1.0, 3.2, 10, 32 and 100 mg/L.

The test item (nominal 100 mg) was dissolved in acetonitrile (100 mL) to prepare a stock solution with a nominal concentration of 1000 mg/L. This stock solution was further diluted with acetonitrile:water (50:50 v/v) to obtain a nominal 10 mg/L calibration standard.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium (Section 3.3). The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1 °C.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

To determine the potential effect of the test item on the appearance of algal cells, a sample was
removed from each test and control culture (replicates pooled) at the end of the test. The shape
and size of the algal cells was inspected microscopically and any abnormalities recorded.

All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 1.0 and 3.2 mg/L. However swollen cells were observed to be present in the test cultures at 10 mg/L and swollen cells and cell debris were observed to be present in the 32 and 100 mg/L test cultures.

Test conditions

Test temperature:
24 ± 1 °C
pH:
pH at 0 hrs: 7.3-7.5

pH at 73 hrs: 7.3-8.3
Nominal and measured concentrations:
Nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L

Analysis of these additional test preparations showed a decline in measured concentration in the range of 16% to 44% of nominal at 24 hours and in the range of less than 1% to 35% of nominal at 48 hours. A further decline in measured test concentration was observed at 72 hours in the range of 1% to 7% of nominal.

Chemical analysis of the 1.0, 10 and 100 mg/L test preparations at O hours (see Appendix 4) showed measured test concentrations to range from 82% to 99% of nominal. A decline in measured test concentration was observed after 72 hours in the range of less than the limit of quantification (LOQ), which was determined to be 0.0061 mg/L to 20% of nominal indicating that the test item was unstable under test conditions.

Given this decline in measured test concentrations it was considered justifiable to base the results on the time-weighted mean measured test concentrations in order to give a "worst case" analysis of the data.
Details on test conditions:
Prior to the start of the test sufficient master culture was added to approximately 100 mL
volumes of culture media contained in conical flasks to give an initial cell density of
approximately 103 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept
under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1 °C
until the algal cell density was approximately 10^4 - 10^5 cells/mL.

As in the range-finding test 250 mL glass conical flasks were used. Six flasks each containing
100 mL of test preparation were used for the control and three flasks each containing 100 mL
were used for each treatment group.
The control group was maintained under identical conditions but not exposed to the test item.
Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell
density of 9.34 x 105 cells per mL. Inoculation of 1 liter of test medium with 5.4 mL of this algal
suspension gave an initial nominal cell density of 5 x 103 cells per mL and had no significant
dilution effect on the final test concentration.
The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron
Version 2 incubator) at 24 ± 1 °C under continuous illumination (intensity approximately
7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at
approximately 150 rpm for 72 hours.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.98 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CL 0.86-1.1 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2.1 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.2 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CL 0.86-1.1 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.65 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
1.9 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.65 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CL 0.86-1.1 mg/L
Details on results:
All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 1.0 and 3.2 mg/L. However swollen cells were observed to be present in the test cultures at 10 mg/L and swollen cells and cell debris were observed to be present in the 32 and 100 mg/L test cultures.

At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control, 1.0 and 3.2 mg/L test cultures were observed to be green dispersions. The 10 mg/L test cultures were observed to be extremely pale orange/brown solutions, the 32 mg/L test cultures were observed to be slightly cloudy pale orange/brown solutions whist the 100 mg/L test cultures were observed to be cloudy orange/brown solutions
Results with reference substance (positive control):
The results from the positive control with potassium dichromate were within the normal ranges for this reference item.
Reported statistics and error estimates:
There were no statistically significant differences (P:2:0.05), between the control and 0.20 mg/L test concentration however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 0.20 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 0.65 mg/L.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and based on the time-weighted mean measured test concentrations.

The 0-72hr biomass EC50 of test substance was calculated to be 0.98 mg/L and NOEC 0.20 mg/L and LOEC 0.65 mg/L.

The 0-72hr growth rate EC50 of test substance was calculated to be 2.1 mg/L and NOEC 0.65 mg/L and LOEC 1.9 mg/L .