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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Link to relevant study record(s)

Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-12-16 to 2019-12-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
2011
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Batch number: K51496539
Analytical monitoring:
yes
Details on sampling:
- Preparation of the samples: The sample and the control were diluted with acetonitrile containing 0.2% ortho phosphoric acid (factor 2) directly after sampling.
- Sample storage: All samples were stored at room temperature until start of analysis, if necessary. Prepared samples were stored at room temperature until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A saturated solution with a nominal loading rate of 100 mg test item/L was prepared once at room temperature 24 ± 1 hour prior to the start of the exposure. An appropriate amount of the test item was weighed out with an appropriate amount of dilution water. The saturated solution was stirred for 24 ± 1 hours (1100 rpm, room temperature) with a magnetic stirrer. Undissolved particles were removed by membrane filtration (membrane filter 0.45 µm). The filter was saturated in order to avoid adsorption during the filtration. The first 25 mL of the filtrate were discarded. The filtration was interrupted for 15 minutes to allow for adsorption and saturation of the filter material with dissolved test item. Thereafter, the filtration was continued. The next 25 mL were discarded. The following filtrate, i.e. the saturated solution, was used in the test. During filtration, the filter was always kept covered. The saturated solution was checked via laser beam (Tyndall effect) for undissolved test item. The Tyndall effect was negative.
- Differential loading: One limit loading rate was tested.
- Controls: Six replicates (without test item) were exposed under the same conditions as the test concentrations; Potassium dichromate was tested as a reference item
- Evidence of undissolved material: No.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: CCAP 278/4 (axenic)
- Source: Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Dunstaffnage Marine Laboratory Dunbeg, OBAN; Argyll PA37 1QA; Scotland, UK
- Age of inoculum: A four days old preculture, prepared in dilution water, was used as inoculum.
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounts 2567 - 5130 lux for 24 hours per day.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
none
Hardness:
32 mg CaCO3/L
Test temperature:
21.8 °C
pH:
7.88 -9.42
Dissolved oxygen:
not specified
Salinity:
not applicable
Conductivity:
137 µS/cm
Nominal and measured concentrations:
nominal: 100 mg/L
measured: < LOQ
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type: closed, sealed with cotton wool plugs
- Material, size, fill volume: glass, 250 mL, 100 mL
- Aeration: passive; test containers were placed on a rotary shaker and oscillated at approximately 70 rpm
- Initial cells density: approximately 5 x 10^3 - 10^4 cells/mL in nominal, measured to be 5416 cells/mL
- No. of vessels per concentration: 6
- No. of vessels per control: 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Ultrapure water was used to prepare the dilution water.
- Conductivity: max. 0.1 µS/cm

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: 24 hours/day light
- Light intensity and quality: Approximately 4440 to 8880 lux, corresponding to 60 to 120 µE*m-2*s-1; light homogeneity within ± 15% over incubation area

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: counting chamber; fluorimeter
- Chlorophyll measurement: The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. No self-fluorescence was found in the preliminary range finding test at the saturated solution (100 mg/L).
- Microscopic evaluation: Microscopic evaluation of the cells at the start and the end of the incubation period was carried out. The cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not applicable (limit test)
- Range finding study: A non-GLP preliminary range finding test under static conditions over a period of 72 hours with headspace was conducted at the test facility with a saturated solution with a nominal loading rate of 100 mg/L. Out of a saturated solution two dilution levels were prepared with the concentrations 1.00 and 10.0% of the saturated solution. In the range finding test, two replicates per concentration and four for the control were tested. All concentration levels were visually clear throughout the exposure period.
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
< 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
< 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
LOELR
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EL20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
- Exponential growth in the control: yes, increase by 244-fold (specific growth rate 1.83 day^- 1)
- Observation of abnormalities: no
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Any stimulation of growth found in any treatment: no
- Any observations 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):
ErC50 = 0.940 mg/L (nominal), 95% confidence interval: 0.732 - 1.33
EyC50 = 0.660 mg/L (nominal), 95% confidence interval: 0.393 - 0.700
Reported statistics and error estimates:
EL-values of the growth rate and yield inhibition were estimated empirically based on the results of the single treatment level (limit test design). NOEL/LOEL were determined by calculation of statistical significance of growth rate and yield. As a standard, a t-test was used for NOEL/LOEL calculations. When running a t-test a Normality test and an Equal Variance test were done first. The Shapiro-Wilk-Test was used to test for normal distribution of data. The Brown-Forsythe test was done for analysis of variance homogeneity. P-values for both Normality and Equal Variance tests are 0.05. The α-value (acceptable probability of incorrectly concluding that there is a difference) is α=0.05.

Table 1: Cell densities.

Nominal test item loading [mg/L] Replicate No. Cell denisty [cells/mL]
0 hours 24 hours 48 hours 72 hours
100 mg/L 1 5416 16320 129219 969007
2 5416 17676 134321 1057848
3 5416 19046 139257 1089636
4 5416 16113 152284 1072842
5 5416 17513 152936 1173950
6 5416 17878 149168 1172928
Mean 5416 17424 142864 1089369
Control 1 5416 23131 1740.34 1324712
2 5416 21210 162915 1263473
3 5416 22301 171619 1314733
4 5416 22841 180849 1374853
5 5416 20499 175884 1284502
6 5416 23525 173956 1363023
Mean 5416 22251 173210 1320883

Table 2: Evaluation after 72 hours. Statistically significant differences

of growth rates and yield compared to control values are marked (+),

not significant differences are marked (-).

Nominal test item loading [mg/L] Replicate No. Growth rate [d-1] Growth rate inhibition [%] Yield [cells/mL] Inhibition of yield [%]
100 1 1.73 6 963591 27
2 1.76 4 1052432 20
3 1.77 3 1084220 18
4 1.76 4 1067426 19
5 1.79 2 1168534 11
6 1.79 2 1167512 11
Mean (+)* 1.77 4 (+) 1083953 18
Control 1 1.83   1319296  
2 1.82   1258057  
3 1.83   1309317  
4 1.85    1369437   
5 1.82   1279086   
6  1.84   1357607  
Mean 1.83   1315467  

* = statistically significant but biologically not relevant (inhibition < 5%)

Table 3: Section-by-Section and Average Specific Growth Rates of the Control Group (0 -72 hours).

  Replicate No. Specific growth rate [d1] section-by-section Mean (0 - 72 hours) SD± CV [%] Mean CV [%]
0 - 24 hours 24 - 48 hours 48 - 72 hours
Control 1 1.45 2.02 2.03 1.83 0.33 18 20
2 1.37 2.04 2.05 1.82 0.392 21.6
3 1.42  2.04  2.04  1.83  0.360  19.7 
4 1.44 2.07 2.03 1.85 0.353 19.1
5 1.33 2.15 1.99 1.82 0.434 23.8
6 1.47 2 2.06 1.84 0.325 17.6
  Mean 1.83  
SD ± 0.01
CV [%] 0.6

SD = Standard deviation

CV = Coefficient of variation

Table 4: Analytical results.

Sampling date Fresh Media 0 hours Old Media 72 hours
Nominal loading of the test item in the saturated solution [%]
  Meas. Conc. [µg/L] Meas. Conc. [µg/L]
100 < LOQ < LOQ
QC 35.8 34.0
Control < LOQ < LOQ

Meas. conc. = measured concentration of the test item, dilution factors taken into account

LOQ = limit of quantification of the analytical method (30 µg test item/L)

QC = quality control (30 µg test item/L)

Table 5: Validity criteria

Validity Criterion Required This study
Increase of the cell growth in the control cultures Exponentially, > 16-fold corresponding to a specific growth rate of 0.92 day-1 244-fold (specific growth rate 1.83 day-1)

Mean coefficients of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3) in the

control cultures

< 35% 20.00%

Coefficient of variation of average specific growth rates during the whole

test period in replicate control cultures

< 7% 0.60%
Validity criteria fulfilled:
yes
Conclusions:
In a limit test according to OECD Guideline 201 and EU Method C.3 with the unicellular freshwater green alga Pseudokirchneriella subcapitata, the 72-hour NOEC (growth rate) of the test item was determined to be 100 mg/L in nominal. The 72-hour ErL10 and ErL50 for the growth rate were >100 mg/L (nominal). The 72-hour EyL50 based on the yield was determined to be >100 mg/L (nominal). The 72-hour NOEC (yield) and EyL10 (yield) were found to be below 100 mg/L (nominal).
Executive summary:

The toxicity of the test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined in a limit-test according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016/Method C.3. The study was conducted over a period of 72 hours under static conditions with an initial cell density of 5416 cells/mL. A saturated solution with a nominal loading rate of 100 mg test item/L was prepared once at room temperature 24 ± 1 hour prior to the start of the exposure. The saturated solution was stirred for 24 ± 1 hours (1100 rpm, room temperature) with a magnetic stirrer. Undissolved particles were removed by membrane filtration. The saturated solution was checked via laser beam (Tyndall effect) for undissolved test item. The Tyndall effect was negative and no undissolved test material was hence detected. Six replicates were tested for the limit loading and six for the control. The test media were clear throughout the test period. The environmental conditions were within the acceptable limits and all validity criteria of the test guidelines were fulfilled. The limit loading rate and the control were analytically verified by HPLC-DAD at the start and the end of the exposure. The measured concentrations of the saturated solution of the test item in the fresh and in the old media were below the Limit of Quantity (LOQ). All effect values given are thus based on the nominal loading of the test item. In result, the 72-hour NOEL for the growth rate was determined to be 100 mg/L. The differences of growth rate were calculated to be statistically significant, but considered biologically not relevant since the inhibition was below 5%. Consequently, the 72-hour ErL10 and ErL50 were determined to be >100 mg/L. The 72-hour NOEL (yield) and EyL10 (yield) were found to be <100 mg/L. The 72-hour EyL50 was determined to be >100 mg/L.

Description of key information

In a GLP-compliant limit test according to OECD Guideline 201 and EU Method C.3 with the unicellular freshwater green alga Pseudokirchneriella subcapitata, the 72-hour NOEL (growth rate) of the test item was determined to be 100 mg/L in nominal. The 72-hour ErL10 and ErL50 were >100 mg/L in nominal (reference 6.1.5-1).

Key value for chemical safety assessment

Additional information

Key information

The toxicity of the test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined in a limit-test according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016/Method C.3. The study was conducted over a period of 72 hours under static conditions with an initial cell density of 5416 cells/mL. A saturated solution with a nominal loading rate of 100 mg test item/L was prepared once at room temperature 24 ± 1 hour prior to the start of the exposure. The saturated solution was stirred for 24 ± 1 hours (1100 rpm, room temperature) with a magnetic stirrer. Undissolved particles were removed by membrane filtration. The saturated solution was checked via laser beam (Tyndall effect) for undissolved test item. The Tyndall effect was negative and no undissolved test material was hence detected. Six replicates were tested for the limit loading and six for the control. The test media were clear throughout the test period. The environmental conditions were within the acceptable limits and all validity criteria of the test guidelines were fulfilled. The limit loading rate and the control were analytically verified by HPLC-DAD at the start and the end of the exposure. The measured concentrations of the saturated solution of the test item in the fresh and in the old media were below the Limit of Quantity (LOQ). All effect values given are thus based on the nominal loading of the test item. In result, the 72-hour NOEL for the growth rate was determined to be 100 mg/L. The 72-hour ErL10 and ErL50 were >100 mg/L. The 72-hour NOEL (yield) and EyL10 (yield) were found to be below 100 mg/L. The 72-hour EyL50 (yield) was determined to be >100 mg/L (reference 6.1.5 -1).