Reaction mass of 1,1,2,2,3,3,3-heptafluoro-N-(heptafluoropropyl)-N-(pentafluoroethyl)propan-1-amine and 1,1,2,2,3,3,3-heptafluoro-N-(heptafluoropropyl)-N-(trifluoromethyl)propan-1-amine and 1,1,2,2,3,3,3-heptafluoro-N,N-bis(heptafluoropropyl)propan-1-amine

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:

09-Oct-2017 to 19-Oct-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 1,1,2,2,3,3,3-heptafluoro-N-(heptafluoropropyl)-N-(pentafluoroethyl)propan-1-amine and 1,1,2,2,3,3,3-heptafluoro-N-(heptafluoropropyl)-N-(trifluoromethyl)propan-1-amine and 1,1,2,2,3,3,3-heptafluoro-N,N-bis(heptafluoropropyl)propan-1-amine

EC Number:

920-778-3

Cas Number:

2176446-38-9

Molecular formula:

constituents: CnF(2n+3)N with n= 7-9

IUPAC Name:

Reaction mass of 1,1,2,2,3,3,3-heptafluoro-N-(heptafluoropropyl)-N-(pentafluoroethyl)propan-1-amine and 1,1,2,2,3,3,3-heptafluoro-N-(heptafluoropropyl)-N-(trifluoromethyl)propan-1-amine and 1,1,2,2,3,3,3-heptafluoro-N,N-bis(heptafluoropropyl)propan-1-amine

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): MTDID 50908
- Physical state: Colourless liquid
- Storage condition of test material: At room temperature

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0002712253
- Physical state: colorless liquid
- Expiration date of the lot/batch: 07 June 2020
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- 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:

no

Remarks:

The study was conducted as a limit test at 100 mg/L (nominal) loading level, which is above the water solubility of the test material

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- 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 50908 (2.4 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 drop 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:

TEST ORGANISM
- 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:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Hardness:

24 mg/L as CaCO3

Test temperature:

22°C - 23°C

pH:

7.4 - 7.7

Nominal and measured concentrations:

Nominal: Control (0 mg/L), 100 mg/L
Measured: not evaluated

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass VOA vials closed with PTFE-faced 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: 131E+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.
GROWTH MEDIUM
- 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).
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Growth medium
- Culture medium different from test medium: yes
OTHER TEST CONDITIONS
- 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.
TEST CONCENTRATIONS
- Spacing factor for test concentrations: None
- Justification for using less concentrations than requested by guideline: Limit test

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

No effects on growth rate were observed at the single concentration tested.

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Details on results:

Under the conditions of the study with Pseudokirchneriella subcapitata exposed to MTDID 50908 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. 48%). 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 50908.
- 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. Undissolved test substance was observed throughout the test.

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: Reference substance toxicity assay conducted within one month of the test substance assay.

Any other information on results incl. tables

 Table 1, Individual cell densities in the algal toxicity test (x 10^4 cells/mL)

Loading rate (mg/L)	Replicate	Time
		0 h	24 h	48 h	72 h
0 (Control)	1	1	10.563	52.988	124.804
	2	1	10.840	52.679	133.489
	3	1	11.776	53.828	130.787
	4	1	10.262	55.176	133.769
	5	1	10.773	57.636	133.208
	6	1	10.712	70.132	126.882
Mean:		1	10.8	57.1	130.5
Std.Dev.:		0	0.5	6.6	3.8
CV:		0	4.7	11.6	2.9
100	1	1	11.049	54.988	129.765
	2	1	10.166	62.392	131.595
	3	1	12.900	50.080	143.701
	4	1	11.702	51.700	118.790
	5	1	10.666	57.367	123.868
	6	1	9.170	59.101	141.467
Mean:		1	10.9	55.9	131.5
Std.Dev.:		0	1.3	4.6	9.7
CV:		0	11.7	8.3	7.4

Table 2, 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	2.357	1.613	0.857	1.609
	2	2.383	1.581	0.93	1.631
	3	2.466	1.52	0.888	1.625
	4	2.328	1.682	0.886	1.632
	5	2.377	1.677	0.838	1.631
	6	2.371	1.879	0.593	1.614
Mean:		2.381	1.659	0.832	1.624
Std.Dev.:		0.0462	0.124	0.1211	0.0098
CV:		1.9	7.5	14.6	0.6
CV = 48% for all section-specific control growth rates
100	1	2.402	1.605	0.859	1.622
	2	2.319	1.814	0.746	1.627
	3	2.557	1.356	1.054	1.656
	4	2.46	1.486	0.832	1.592
	5	2.367	1.682	0.77	1.606
	6	2.216	1.863	0.873	1.651
Mean:		2.387	1.634	0.856	1.626
Std.Dev.:		0.1173	0.1936	0.1092	0.0246
CV:		4.9	11.8	12.8	1.5

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

Considered acceptable despite section by section coefficient of variation greater than 35% (i.e. 48%), because higher variation is expected with sealed-vessel methodology necessary for the test substance.

Conclusions:

The 72-hour EC50 and EC10 of MTDID 50908 to P. 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 TG 201. No effects on growth were observed.

Executive summary:

Toxicity of MTDID 50908 to the freshwater alga Pseudokirchneriella subcapitata was assessed in a limit test according to OECD201. The test substance is highly volatile. All tests were conducted in glass VOA vials, completely filled with medium and closed with a PTFE-faced septum cap. All transfers were made through the septum with 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 loading rate was well above the solubility limit of the test material in water, and visible undissolved test substance was observed 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 50908 in the test medium. No effects on growth 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 test is considered reliable without restrictions. It is suitable for Risk Assessment, Classification & Labeling, and PBT Analysis.