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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Ethylene glycol monopropyl ether
- Substance type: production sample
- Analytical purity: 99.82%
- Lot/batch No.: TXEG
- Storage condition of test material: room temperature in the dark

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours. Cell count samples were taken at 0, 25, 48 and 72 hours and the cell densities determined using a Coulter Multisizer Particle Counter. The pH of the control and 100 mg/l test concentration was determined at initiation of the test and after 72 hours exposure. The pH was measured using a WTW pH 320 pH meter. The temperature within the incubator was recorded daily.

Test solutions

Vehicle:
no
Details on test solutions:
The range-finding test was conducted using a series of nominal test concentrations of 1.0, 10 and 100 mg/I. An amount of test item (50 mg) was dissolved in culture medium and the volume adjusted to 500 ml to give a 100 mg/l stock solution from which a series of dilutions was made to give further stock solutions of 10 and 1.0 mg/l. Based on the result of the range-finding test a "limit test" was conducted at a concentration of 100 mg/l. An amount of test item (100 mg) was dissolved in culture medium and the volume adjusted to 1 litre to give a 100 mg/l stock solution. The stock solution and the prepared concentration were inverted several times to ensure adequate mixing and homogeneity. The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours.

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), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1°C. 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 104 - 105 cells/ml.

Study design

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

Test conditions

Test temperature:
24 ± 1°C
pH:
Control: 7.5 at test intitation, 8.0 at 72 hours
100 mg/L: 7.5 at test intitation, 7.9 at 72 hours
Nominal and measured concentrations:
Limit test at nominal concentration of 100 mg/L. Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 89% to 105% of nominal and so it was considered justifiable to estimate the EC50 values in terms of the nominal test concentration only.
Details on test conditions:
In the definitive test 250 ml glass conical flasks were used. Six flasks each containing 100 ml of test preparation were used for the control and 100 mg/l 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 characterised by a cell density of 3.72 x 10e5 cells per ml. Inoculation of 1 litre of test medium with 11 ml of
this algal suspension gave an initial nominal cell density of 4 x 10e3 cells per ml and had no significant dilution effect on the final test concentration.
The flasks were plugged with polyurethane foam bungs and incubated 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.:
> 100 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata to the test item during the range-finding test showed no effect on growth at the test concentrations of 1.0, 10 and 100 mg/l. Chemical analysis of the 100 mg/l test preparation at a and 72 hours showed measured test concentrations of 104% and 99% of nominal respectively were obtained indicating that the test item was stable under test conditions. The data from the definitive test also demonstrated that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata were not affected by the presence of the test item at a nominal test concentration of 100 mg/l over the 72-Hour exposure period.
Results with reference substance (positive control):
A positive control used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/l. Exposure conditions and data evaluation for the positive control were similar to those in the definitive test. Exposure of Pseudokirchneriella subcapitata to the reference item gave the following results:
ErC50 (0 - 72 h): 0.82 mg/I, 95% confidence limits 0.73 - 0.93 mg/I
EyC50 (0 - 72 h): 0.47 mg/l, 95% confidence limits 0.43 - 0.51 mg/I

No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/l
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/l
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/l
Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/l

Reported statistics and error estimates:
A Student's t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) was carried out on the growth rate and yield data after 72 hours for the control and the 100 mg/l test concentration to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package.

Any other information on results incl. tables

Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Concentration (mg/l)  Replicate Growth Rate (cells/ml/hour)  Yield (cells/ml) 
0-72 h  % Inhibition  0-72 h  % Inhibition* 
Control  R1  0.070    6.13E+05   
R2  0.063  3.74E+05 
R3  0.068  5.16E+05 
R4  0.072  7.04E+05 
R5 0.069  5.67E+05 
R6  0.069  5.74E+05 
Mean  0.069  5.58E+05 
SD 0.003  1.10E+05 
100 R1  0.072 [4] 7.11E+05  
R2  0.062 10 3.53E+05
R3  0.066 4 4.50E+05
R4  0.071 [3] 6.62E+05
R5 0.067 3 5.08E+05
R6  0.067 3 4.89E+05
Mean  0.068 2 5.29E+05 5
SD 0.004   1.34E+05  

* In accordance with the OECD test guideline only the mean value for yield is calculated

R1 - R6 =Replicates 1 to 6

SD =Standard Deviation

[Increase in growth as compared to controls]

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The data show that the cell concentration of the control cultures increased by a factor of 130 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 mean coefficient of variation for section by section specific growth rate for the control cultures was 12% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%. The coefficient of variation for average specific growth rate for the control cultures over the test period (0 - 72 h) was 4% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.
Executive summary:

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 440/2008. Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at a concentration of 100 mg/L (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter Multisizer Particle Counter. Exposure of Pseudokirchneriella subcapitata to the test item gave EC50 values of greater than 100 mg/L and correspondingly the No Observed Effect Concentration was 100 mg/L. It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L. Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 89% to 105% of nominal and so the results are based on nominal test concentrations only.