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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
9 - 19 Oct 2017
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
GLP compliance:

Test material

Constituent 1
Reference substance name:
Reaction mass of perfluoro(dimethyl - N - Butylamine ) and perfluoro (methyl - di - N - propylamine) and perfluoro (dimethyl - N - propylamine and 2,2,3,3,5,5,6,6, octafluoro-4-(trifluoromethyl)morpholine and perfluoro-N-pentane
EC Number:
Cas Number:
Reaction mass of perfluoro(dimethyl - N - Butylamine ) and perfluoro (methyl - di - N - propylamine) and perfluoro (dimethyl - N - propylamine and 2,2,3,3,5,5,6,6, octafluoro-4-(trifluoromethyl)morpholine and perfluoro-N-pentane
Specific details on test material used for the study:
- Source and lot/batch No.of test material: 002602456
- Physical state: colorless, clear liquid
- Expiration date of the lot/batch: 07 June 2020

- Storage condition of test material: at room temperature
- Stability under test conditions: test substance stable in water, but extremely volatile

Sampling and analysis

Analytical monitoring:
Limit test conducted at 100 mg/L (nominal) loading level, which is above the water solubility of the test material

Test solutions

Details on test solutions:
- Method: The test substance is poorly soluble and extremely volatile. Therefore, 42.5 mL air-tight vials were completely filled with medium and closed with a septum-sealed screw cap and parafilm. MTDID 22327 (2.5 μL) was then injected through the septum with gas-tight syringe. Vials were rotated slowly for seven days, and then centrifuged at 500g for 90 minutes to pull undissolved test substance into a single droplet at the bottom of the vial.
- Controls: Blank only
- Evidence of undissolved material: Test solutions were clear and colorless, with a visible small droplet of undissolved test substance at the bottom.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
- Common name: green algae
- Strain: NIVA CHL1
- Source: in-house laboratory culture.
- Age of inoculum (at test initiation): Three days
- Method of cultivation: Stock cultures were started by inoculating growth (M1) medium with algal cells from a pure culture on agar. The suspensions were continuously aerated in a climate room at a temperature of 21-24°C and light intensity of 60 to 120 μE/m²/s when measured in the photosynthetically effective wavelength range (400-700 nm). Growth (M1) medium (Nederlandse Praktijk Richtlijn no. 6505) was formulated using Milli-RO water (tapwater purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition.
NaNO3, 500 mg/l
K2HPO4∙3H2O, 52 mg/l
MgSO4∙7H2O, 75 mg/l
Na2CO3∙10H2O, 54 mg/l
C6H8O7∙H2O, 6 mg/l
NH4NO3, 330 mg/l
CaCl2∙2H2O, 35 mg/l
C6H5FeO7∙xH2O, 6 mg/l
H3BO3, 2.9 mg/l
MnCl2∙4H2O, 1.81 mg/l
ZnCl2, 0.11 mg/l
CuSO4∙5H2O, 0.08 mg/l
(NH4)6Mo7O24∙4H2O, 0.018 mg/l
- Acclimation period: Three days before the start of the test, fresh M2 medium was inoculated at a density of 1E4 cells/mL and was kept under the same conditions used in the test.

Study design

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

Test conditions

24 mg/L as CaCO3
Test temperature:
22 °C - 23 °C
7.3 - 7.7
Nominal and measured concentrations:
Nominal: 100 mg/L
Measured: not evaluated
Details on test conditions:
- Test vessel: Glass VOA vials closed with septa, completely filled. Fill volume ca. 42.5 mL. To initiate the test, a 0.85-mL aliquot of algal suspension was injected by syringe through the septum. A vent needle temporarily placed through the septum allowed an equal volume of medium to be displaced, after which syringe and vent needle were removed.
- Agitation: No
- Initial cells density: 1E+04 cells/mL
- Control end cells density: 126E+04 cells/mL
- No. of vessels per concentration (replicates): Six replicates per day, sacrificed for cell counts. Three vials with saturated solution but without algae.
- No. of vessels per control (replicates): Six replicates per day, sacrificed for cell counts.
- Standard medium used: No
- Detailed composition if non-standard medium was used: Standard OECD TG 201 test medium was adjusted for use in a sealed test vessel by increase of NaHCO3 to 300 mg/L and addition of 6 mM HEPES buffer, final pH 7.1± 0.3. Formulated using Milli-RO water (tapwater purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA).
- Source/preparation of dilution water: Growth medium
- Culture medium different from test medium: yes
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: Continuous
- Light intensity and quality: TLD lamps with a light intensity within the range of 75 to 82 μE/(m²∙s).
Test vessels were placed randomly in the incubator, and repositioned daily.
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cells were counted using a microscope and a counting chamber to determine inoculum density. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (pathlength = 10 mm) against an algal medium blank and the extra replicates for the treated solutions.
- Spacing factor for test concentrations: None
- Justification for using less concentrations than requested by guideline: Limit test
Reference substance (positive control):
Potassium dichromate (K2CrO7)

Results and discussion

Effect concentrationsopen allclose all
Key result
72 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
72 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: No effects on growth rate were observed at the only concentration tested.
Details on results:
Under the conditions of the study no inhibition of growth rate or inhibition of yield was observed at a loading rate of 100 mg/L.
- Exponential growth in the control: yes.
- Observation of abnormalities: Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.
- Other: The mean coefficient of variation for section-by-section specific growth rates in the control cultures exceeded 35% (i.e. 54%). These results were considered acceptable due to the unavoidable need to use sealed flasks with a volatile, low-soluble substance. The methodology was considered the best possible for MTDID 22327.
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: undissolved test substance present throughout study. This was necessary to maintain saturation of the volatile and low-soluble test material.
- Effect concentrations exceeding solubility of substance in test medium: yes
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 1.1 mg/L (growth rate). Historical range for the reference substance at the contract lab lies between 0.82 and 2.3 mg/L
- Other: The reference substance test was conducted within one month of testing of MTDID 22327.

Any other information on results incl. tables

Table 1. Growth rates (1/day) in the algal toxicity test
Loading rate (mg/L) Replicate Interval      
    0-24 h 24-48 h 48-72 h 0-72 h
0 (Control) 1 2.275 1.769 0.777 1.607
  2 2.809 1.289 0.641 1.58
  3 2.336 1.732 0.793 1.62
  4 2.41 1.717 0.718 1.615
  5 2.419 1.656 0.783 1.619
  6 2.368 1.896 0.6 1.621
Mean:   2.436 1.676 0.719 1.61
Std.Dev.:   0.19 0.2057 0.0813 0.0159
CV:   7.8 12.3 11.3 1
CV = 54%          
100 1 2.49 1.878 0.58 1.649
  2 2.548 1.832 0.413 1.597
  3 2.549 1.646 0.639 1.611
  4 2.483 1.669 0.76 1.637
  5 2.459 1.551 0.785 1.598
  6 2.591 1.446 0.738 1.592
Mean:   2.52 1.67 0.652 1.614
Std.Dev.:   0.0503 0.1637 0.1408 0.02386
CV:   2 9.8 21.6 1.5

Applicant's summary and conclusion

Validity criteria fulfilled:
Considered acceptable despite section by section coefficient of variation greater than 35% (i.e. 54%), because higher variation is expected with sealed-vessel methodology necessary for the test substance.
The 72-hour EC50 and EC10 of MTDID 22327 to green algae (Pseudokirchneriella subcapitata) exceeded a loading rate of 100 mg/L, a level which is greater than the maximum soluble concentration in the test medium, by OECD 201. No effects on growth rate were observed.
Executive summary:

The toxicity of MTDID 22327 to green algae (Pseudokirchneriella subcapitata) was assessed in a limit test according to OECD TG 201. The test substance is highly volatile. All tests were conducted in glass VOA vials, completely filled with medium and closed with caps. All transfers were made through a septum with a gas-tight syringe. A water soluble fraction was made at a loading rate of 100 mg/L, with residual undissolved test material allowed to remain in the test vials. Analytical measurement of test substance concentrations was not done, but the test substance was visibly present throughout the test. The 72-hour EC10 and EC50 were > 100 mg/L (nominal concentration),a level which was greater than the water solubility of MTID 22327 in the test medium. No effects on growth rate were observed.

The study was conducted according to internationally accepted test guidelines and in accord with GLP criteria. Variability in section-specific growth was above the limit given in OECD TG201, but is considered acceptable in a sealed-vessel protocol designed to contain a volatile and poorly-soluble test material. The study is considered reliable without restrictions and is suitable for Risk Assessment, Classification & Labeling, and PBT Analysis.