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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-10-09 to #
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
28 July 2011
Deviations:
yes
Remarks:
1)autoclaving of stock solution for 20 min (required 15 minutes); uncritical, as goal of sterility is achieved. 2)exp III: temp. not within desired range; uncritical as normal growth observed. 3)exp II: measured start cell conc.
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
07 December 2015
Deviations:
yes
Remarks:
1)autoclaving of stock solution for 20 min (required 15 minutes); uncritical, as goal of sterility is achieved. 2)exp III: temp. not within desired range; uncritical as normal growth observed. 3)exp II: measured start cell conc.
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Nominal Concentration Test Item: Blank control, 0.1, 0.32, 1.0, 3.2, 10 g/L
- Sampling method: Each sample was measured in duplicate or triplicate, respectively (depending on the variation between the measured values).
- Sample storage conditions before analysis: not reported
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The water-accommodated fractions (WAF) were prepared for the test. This was done by mixing the nominal loads of 10 / 3.2 / 1 / 0.32 / 0.1 g/L resp. 10.64 / 3.40 / 1.06 / 0.34 / 0.11 mL/L test item (based on a density of 0.94 g/mL stated in the MSDS) with the corresponding amount of algal medium (demineralised water enriched with minerals but without algae) and stirring moderate for 24 hours. The lower phase was used unfiltered as test solutions.
- Controls: The positive control was treated in the same way as the test item solution.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): no vehicle used
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): in Experiment 1, no further specification
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: /; Scientific name: Desmodesmus subspicatus
- Strain: 86.81 SAG
- Source (laboratory, culture collection): January 2016 by MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen)
- Pre-culture and inoculum: The algae are kept as stock culture on solid agar at 2 - 8 °C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was prepared. Four days before the start of each test, an aliquot of the permanent culture was brought into nutrient medium and incubated under continuous lighting for 96 hours. The resulting culture grew exponentially. Before usage, the pre-culture was checked for the absence of cell aggregates and the cell number of culture was determined.

ACCLIMATION
- Acclimation period: 4 d
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
no
Test temperature:
19.8 - 22.6 °C
pH:
Control: 7.5
Test item: 7.5 - 7.6
Nominal and measured concentrations:
0.1 / 0.32 / 1.0 / 3.2 / 10 g/L nominal concentrations
31.4, 19.5, n.c., 29.1, 36.3 mg/L geometric mean (Experiment I)
5.1, 14.7, 17.6,11.1, 15.0 mg/L geometric mean (Experiment III)
Due to the test items properties, a poorly soluble UVCB, the scattering measured DOC values are not unusual. The solubility of such a substance in test medium is not always comparable to the solubility in demineralised water in spite of the same handling and the same equipment as in a study for water solubility.
Details on test conditions:
TEST SYSTEM
- Test vessel: glass flasks, total volume 65 mL (each filled with 45±1mL)
- Type (delete if not applicable): open (covered with perforated plastic foil acting as a stopper)
- Exposure conditions: The test vessels are kept in the light (5000 lux at temperatures: 19.8 – 22.6 °C (third experiment), 21.7 – 23.6 °C (first experiment). The pH was measured in every prepared solution. After the test, the pH value in treatments and blank control was measured again.
- Initial cells concentration: 2.4 *10^3 cells/mL (respectively 3.7 *103 cells/mL for experiment I)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6


TEST MEDIUM / WATER PARAMETERS
- OECD TG 201 medium according to OECD 201 (2006)

OTHER TEST CONDITIONS
- Sterile test conditions: All solutions were sterilised before use. Stock solutions I and III and the deionised water were sterilized using an autoclave. Stock solutions II and IV were sterilized using filtration.
- Adjustment of pH: no

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Cell concentration: measured at the beginning of the test with an electronic particle counter in the blank control solution. This measured value was used as start cell concentration for all replicates. Before the start of incubation and every 24 hours, the cell number was determined with an electronic particle counter.

PERFORMANCE OF THE STUDY
- For each treatment, 200 mL (250mL in experiment I) of the respective test item solution was mixed with the necessary amount of algal pre-culture (2.06 mL, in experiment I 1.223 mL) to achieve a cell concentration of 2.4 *103 cells/mL (respectively 3.7 *103 cells/mL for experiment I). For the blank control, 350 mL nutrient medium was used instead of test item solution and mixed with the necessary amount of algal pre-culture (3.60 mL; in exper-iment I 1.713 mL).
The real actual cell concentration at the beginning of the test was measured with an elec-tronic particle counter in the blank control solution. This measured value was used as start cell concentration for all replicates.
In these mixtures, the pH-value was measured. The pH was measured in every prepared solution. Samples for the analytical determination were taken from the test solutions and control solutions.
The test vessels were filled with 45 ± 1 mL of the respective test solution and incubated open (covered with perforated plastic foil acting as a stopper) for 72 hours, shaken on an orbital shaker to keep the algae in suspension. Before the start of incubation and every 24 hours, the cell number was determined with an electronic particle counter. After the test, the pH value in treatments and blank control was measured again.
At the end of the test, the treatments were examined microscopically in order to assess the appearance of the algae and detect abnormalities (e.g. caused by the exposure to the test item).
The content of DOC in the test vessels was measured at the start and at the end of the tests.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9)
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
5.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
14.7 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
12 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % CL = 7.7 - 19 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
5.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other:
Remarks:
yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
14.7 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other:
Remarks:
yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
4.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95 % CL = 1.7 - 13 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
20 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95 % CL = 4.7 - 77 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 15 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Exact value not determined due to mathematical reasons or inappropriate data
Details on results:
- Exponential growth in the control (for algal test): yes
- Microscopical Observations: Normal and Healthy Appearance of the Algae (Experiment III)
- Unusual cell shape: not reported
- Colour differences: not reported
- Flocculation: not reported
- Adherence to test vessels: not reported
- Any stimulation of growth found in any treatment: not reported
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Because of the poor solubility of the test item, the correlation between nominal concentra-tion and measured concentration was weak. Therefore, the determination of the results was based on the geometric mean of the measured concentrations.

Results with reference substance (positive control):
The 72h-EC50s of potassium dichromate (K2Cr2O7, CAS No. 7778-50-9) were determined in a separate reference test. The values lay within the range of the laboratory (growth rate 0.73 - 1.10 mg/L, yield 0.21 – 0.66 mg/L).
Reported statistics and error estimates:
Calculation of results was performed with the help of validated software (Microsoft Ex-cel®). The estimation of the biological data was accomplished using the software Tox-Rat® Professional, version 3.2.1.

Table 1: Inhibition values (experiment III)

Nominal concentration in g/L

% Inhibition

Growth Rate (0-72h)

Yield (0-72h)

Blank control

0

0

0.1

77.96

97.65

0.32

5.05

19.56

1.0

5.63

21.53

3.2

11.73

39.47

10

21.73

60.36

Validity criteria fulfilled:
yes
Remarks:
All validity criteria were met. No observations were made which might cause doubts concerning the validity of the study outcome. The result of the test can be considered valid.
Conclusions:
The 72-h ErC10 of the test item in Pseudokirchneriella subcapitata is 12 mg/L (95 % C.I. 7.7 – 19 mg/L; measured values). The 72 h ErC50 was not determined, but must be above 15 mg/L as the highest measured concentration equaled 15 mg/L in the test showing 17 % Inhibition of growth rate)).
Executive summary:

In a 72-hour acute toxicity study, cultures of Desmodesmus subspicatus were exposed to VOELOFA Monomer at nominal concentrations of 0.1 / 0.32 / 1.0 / 3.2 / 10 g/L under static conditions in accordance with the OECD test guideline 201. Three experiments were performed. In the 1stexperiment all validity criteria were met. However, mistakenly at the lowest concentration 100 mg/L no algae pre-culture was added and at the concentration 1 g/L, only the IC was measured at the beginning instead of IC and TC because of a wrong measuring program. So, no value for the DOC concentration in treatment 1 g/L at the beginning of the first experiment was available.

Because of incomplete data the experiment was repeated. Nevertheless, all data of the first experiment are stated in this report.

In the 2ndexperiment one of the validity criteria was not met. Therefore, a 3rdexperiment was performed. The results of the invalid experiment are not stated in this report, but will be kept together with the other raw data in the GLP-archive of the test facility.

Because all validity criteria were met the biological result were based only on the result of the 3rdexperiment. Significant inhibition of algal growth was observed in all concentrations except for the lowest concentrated treatment.

Because of the poor solubility of the test item, the correlation between nominal concentration and measured concentration was weak. Therefore, the determination of the results was based on the geometric mean of the measured concentrations.

The NOEC, EC10 and EC50 values based on growth rate were 5.1, 12 and > 15 mg/L (meas.), respectively. An exact value for the 72 h ErC50 was not determined, but must be above 15 mg/L as the highest measured concentration equaled 15 mg/L in the test showing 17 % Inhibition of growth rate)).

Based on the results of this study, VOELOFA Monomer would be classified as Aquic Chronic 3 in accordance with the EU CLP Regulation. 

This study is classified as acceptable and satisfies the guideline requirements for a growth inhibition study with aquatic algae.

 

Result Synopsis

Test Organism: Desmodesmus subspicatus

Test Type (Flowthrough, Static, Static Renewal): Static

 

72 h ErC50: >15 mg/L (n.d., highest measured conc: 15 mg/L showing 17 % Inhibition of growth rate));

72 hr ErC10: 12 mg/L (95 % C.I. 7.7 - 19 mg/L)

72 hr LOErC: 14.7 mg/L

72 hr NOErC: 5.1 mg/L

Endpoint(s) Effected: Growth rate

 

72 hr EyC50: 20 mg/L (95 % C.I. 4.7 - 77 mg/L)

72 hr EyC10:  4.6 mg/L (95 % C.I. 1.7 - 13 mg/L)

72 hr LOEyC: 14.7 mg/L

72 hr NOEyC: 5.1 mg/L

Endpoint(s) Effected: Yield

Description of key information

In a 72-hour algae growth inhibition test cultures of Pseudokirchneriella subcapitata were exposed to the test item at nominal concentrations of 0.0 (control), 0.1, 0.32, 1.0, 3.2 and 10 g test item/L under static conditions in accordance with the OECD 201 (2006) and GLP.

 

The following effect levels - based on the inhibition of growth rate and geometric mean of measured concentrations - are calculated:

- 72 h ErC50: >15 mg/L (n.d., highest measured conc: 15 mg/L showing 17 % Inhibition of growth rate));

- 72-h ErC10: 12 mg/L (95 % C.I.7.7 – 19 mg/L);

- 72 h NOErC: 5.1 mg/L and

- 72 h LOErC: 14.7 mg/L.  

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
12 mg/L

Additional information