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:
2017-03-06 to 2017-06-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2011
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Sampling and analysis

Analytical monitoring:
yes
Remarks:
LC-MS/MS
Details on sampling:
- Concentrations:
All concentration levels and the control were analysed at the start (0 hours) and at the end of the exposure (72 hours) with algae. Without algae the test item concentrations 6.40, 16.0 and 40.0 mg/L were analysed after incubation under test conditions at the end of the exposure.

- Sample storage conditions before analysis: All original samples were stored at room temperature before preparation. Prepared samples were stored in an autosampler at room temperature until analysis.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 100 mg test item/L, freshly prepared with dilution water. The stock solution was agitated until the test item was completely dissolved.
- Controls: Stock solution without test item
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green alga
- Strain: Pseudokirchneriella subcapitata HINDAK, SAG 61.81
- Source (laboratory, culture collection): Sammlung von Algenkulturen (SAG) Pflanzenphysiologisches Institut der Universitat Gottingen Nikolausbergerweg 18, D-37073 Gottingen
- Method of cultivation:Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 2567 - 5130 lux for 24 hours per day.
- Age of inoculum (at test initiation): A three days old preculture, prepared in dilution water, was used as inoculum.
ACCLIMATION
- Culturing media and conditions (same as test or not): same as test; Nutrient medium Z according to LÜTTGE et al. (1994)

Study design

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

Test conditions

Test temperature:
21 - 24 °C, controlled at ± 2 “C
pH:
8.1 ± 0.2
Nominal and measured concentrations:
nominal concentrations: 100, 40.0, 16.0, 6.40, 2.56 mg/L
geometric mean measured concentrations: 102, 16.7, 1.99, 1.20, 1.32 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Sterile Erlenmeyer flasks (vol. 250 mL) with cotton wool plugs
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: size: 250 mL, fill volume: 100 mL
- Initial cells density: 6839 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD TG 201 medium
- Culture medium different from test medium: yes, cultivation was performed in Nutrient medium Z according to LÜTTGE and the test was performed in dilution water

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no, except the highest concentration 100 mg/L which was adjusted to pH 8.1
- Photoperiod: 24 hours/day light
- Light intensity and quality: Approximately 4440 to 8880 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: fluorimeter
- Chlorophyll measurement: yes, Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm

TEST CONCENTRATIONS
- Spacing factor for test concentrations: A geometric series with a dilution factor of 2.5
- Range finding study: yes
- Test concentrations: 100, 10, 1 mg/L
- Results used to determine the conditions for the definitive study: Growth rate inhibition and yield inhibition significantly occurred at 100 mg/L
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
59.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 102 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: pH corrected
Key result
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
35 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
23.8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
16.7 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
102 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: not reported
- Colour differences: not reported
- Adherence to test vessels: not reported
- Aggregation of algal cells: not reported
- 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
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50: 0.435 (0.414 - 0.458) validity range: 0.763 ± 0.514
Reported statistics and error estimates:
The NOEC and LOEC were determined by calculation of analyses statistically significant differences of growth rates and yield.
The following statistical tests were conducted: Shapiro-Wilk’s test on normal distribution was done with a significance level of 0.01.
Levene’s test on variance homogeneity was done with a significance level of 0.01.
Student-t test on significant differences was done with a significance level of 0.05 for yield (pH control).
Dunnett’s multiple t-test procedure was done with a significance level of 0.05 for yield.
Multiple sequentially-rejective Welsh-t-test after Bonferroni-Holm was calculated with a significance level 0.05 for growth rate.

Any other information on results incl. tables

Table 2: pH-values

Geometric mean measured concentration [mg/L]

pH value start

0 hours

pH value end

72 hours

pH control (100 mg/L nominal)

8.09

8.33 

102

3.93

4.78 

16.7

7.32

8.60 

1.99

7.73

8.84 

1.20

7.86

8.92 

1.32

7.94

9.15 

control

8.01

9.31 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In the present study according to OECD Guideline 201 (2011), GLP, Pseudokirchneriella subcapitata were incubated with the following nominal concentrations: 100, 40.0, 16.0, 6.40, 2.56 mg/L corresponding to the geometric mean measured concentrations: 102, 16.7, 1.99, 1.20, 1.32 mg/L, respectively, for 72 h. The growth rate of Pseudokirchneriella subcapitata after incubation with 102 mg/L of the test item resulted in 100% inhibition. Therefore, the solution with the highest concentration was adjusted to pH 8.1. Under these conditions no growth inhibition occurred. The very low pH in the unadjusted test solution was considered to be responsible for the growth inhibition. The measured concentrations of the test item were between 18% and 118% of the nominal value. Bacterial contaminations were found in the concentrations 6.40 and 16.0 mg/L. In an additional stability test with sterilized test solutions the test item concentrations remained in the range of e 20% of the nominal values. Thus, the decrease of test item in the definitive test is most probably due to microbial degradation by bacteria and algae.
Under the present conditions the following effect values based on growth rate were obtained:
NOEC 16.7 mg/L
EC50 neutralized > 102 mg/L
EC50 un-neutralized 59.3mg/L
EC20 35.0 mg/L
EC10 23.8 mg/L