1,1,2,3,3-pentafluoro-3-(heptafluoropropoxy)prop-1-ene

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:

12 Oct - 15 Dec 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

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:

1,1,2,3,3-pentafluoro-3-(heptafluoropropoxy)prop-1-ene

EC Number:

817-766-4

Cas Number:

91891-42-8

Molecular formula:

C6F12O

IUPAC Name:

1,1,2,3,3-pentafluoro-3-(heptafluoropropoxy)prop-1-ene

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: MA-3-HO-ANL 1600356
- Expiration date of the lot/batch: 31 Aug 2020
- Purity test date: 05 Feb 2018
- Purity: 96.9%
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature container flushed with nitrogen
- Stability under test conditions: Yes

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All concentrations sampled.
- Volume: 42.5 mL, one complete vial (see sampling method).
- Sampling method: Three extra sample vials for each concentration were made, without inoculation to be sacrificed for analytic monitoring (one each for t=0h, t=24h, and t=72 h) and incubated under similar conditions to the inoculated vials.
- Sample storage conditions before analysis: Samples were analyzed on the day of sampling.

Test solutions

Vehicle:

no

Details on test solutions:

- Method: The test substance is volatile. Therefore, 42.5mL air-tight vials were completely filled with medium and closed with a septum-sealed screw cap. MA-3 (2.5 µL) was then injected through the septum with gas-tight syringe to achieve a loading rate of 100 mg/L. Thereafter the septum was sealed using parafilm. Vials were rotated slowly for four days, and then centrifuged at 500g for 90 minutes to pull dissolved test substance into a single drop at the bottom of the vial. A dilution series was also made of additional 100% saturated samples. Dilutions were made by injecting exact volumes of the saturated solution through the septa of vials that were complete filled with medium. Excess medium was allowed to drain through a vent needle during injection.
- Controls: Blank only
- Evidence of undissolved material: Test solutions were clear and colorless. Undiluted (100% saturated) samples contained a visible small droplet of undissolved test substance at the bottom of the vials.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture
.
- Method of cultivation: Stock cultures were maintained in M1 medium (Nederlandse Praktijk Richtlijn no. 6505).
NaNO3, 500 mg/l
K2HPO4, 39.5 mg/l
MgSO4∙7H2O, 75 mg/l
Na2CO3, 20 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
Cells for the final test were taken directly from stock culture. Algae were acclimated only for preliminary testing. Three days before the start of the test, cells from the algal stock culture were inoculated in culture medium (M2 according to OECD 201) at a cell density of 1e+04 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

24 mg/L as CaCO3

Test temperature:

21 - 23 °C

pH:

7.5 - 7.8

Dissolved oxygen:

n/a

Salinity:

n/a

Conductivity:

n/a

Nominal and measured concentrations:

see Table 1

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass VOA vials, completely filled and closed with septa. Fill volume ca. 42.5 m/L
- Agitation: No
- Initial cells density: 1E+04 cells/mL
- Control end cells density: 114.3E+04 cells/mL
- No. of vessels per concentration (replicates): Three replicates per day, sacrificed for cell counts.
- No. of vessels per control (replicates): Six replicates per day, sacrificed for cell counts.
GROWTH MEDIUM
- Standard medium used: yes, cells grown in M1. Preacclimation in adjusted M2 before test.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Standard medium (adjusted M2) prepared in reverse osmosis purified water. M2 (per OECD 201) was adjusted by addition of 300 mg/L NaHCO3 and 6 mM HEPES buffer, final pH 7.1 ± 0.3
- Ca/mg ratio: 1
- Culture medium different from test medium: yes
OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: Continuous
- Light intensity and quality: Fluorescent (TL-D) lamps with a light intensity within the range of 84 to
85 μE/(m²∙s). Test vessels were placed randomly and randomly repositioned every day.
EFFECT PARAMETERS MEASURED:
- 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 cuvettes (pathlength = 10 mm).
- Appearance: at the end of the test, microscopic examination was done on the 10% dilution to observe for any abnormal appearance of the algae.
TEST CONCENTRATIONS
- Range finding study: yes, done after initial limit test
- Test concentrations: Control, undiluted WSF, and 1.0% and 10% dilutions of WSF
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

270 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI, 154 - 620 µg/L

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

6.9 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI, 2.1 - 14 µg/L

Details on results:

- Exponential growth in the control: yes (Table 3, 4, Fig. 1)
- Observation of abnormalities: None observed. Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to 10% saturated solution (the lowest concentration showing a significant effect on cell density) when compared to the control.
- Any stimulation of growth found in any treatment: no
- Effect concentrations exceeding solubility of substance in test medium: no
- Other: Test vials were sacrificed at each time point. Decline in average cell number between time points does not reflect a reduction in cell count within the same test chambers.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: 1.05 mg/L. Historical range for the reference substance at the contract lab lies between 0.82 and 2.6 mg/L
- Other: Reference substance toxicity assay conducted between the rangefinder and the definitive study, appr 36 days before the definitive test.

Reported statistics and error estimates:

Growth rate data were normally distributed by Shapiro-Wilk's test, p(W) = 0.047 > 0.01. Variance of growth rate data were not homogeneous by Levene's test, p(F) = 0.007 < 0.01. The NOEC could not determined due to excessive variability between exposure levels relative to biological effect. The EC10 was determined instead. EC10 and EC50 were determined based on probit analysis using linear maximum likelihood regression with the percentages of growth rate inhibition v. logarithms of the corresponding time-weighted average concentrations. Analysis was done using ToxRat Professional v3.2.1 (ToxRat Solutions GmbH, Germany)

Any other information on results incl. tables

Table 3, cell densities

Time (hr)	Replicate	Control	0.20 µg/L	5.4 µg/L	6.8 µg/L	21 µg/L	301 µg/L
0	1	1.000	1.000	1.000	1.000	1.000	1.000
	2	1.000	1.000	1.000	1.000	1.000	1.000
	3	1.000	1.000	1.000	1.000	1.000	1.000
	4	1.000
	5	1.000
	6	1.000
	Mean	1.0	1.0	1.0	1.0	1.0	1.0
24	1	6.709	5.221	5.221	4.649	3.262	2.267
	2	4.032	4.490	8.490	4.439	4.350	2.569
	3	4.865	3.987	3.631	4.725	6.309	1.348
	4	4.146
	5	4.356
	6	2.359
	Mean	4.4	4.6	5.8	4.6	4.6	2.2
48	1	18.188	32.523	34.348	20.109	23.537	4.821
	2	18.322	34.634	20.459	23.581	11.282	5.081
	3	24.510	28.497	29.935	21.852	11.384	6.983
	4	11.759
	5	28.656
	6	23.581
	Mean	20.8	31.9	28.2	21.8	15.4	5.6
72	1	111.464	131.173	119.681	103.738	18.029	18.532
	2	111.388	130.511	102.955	57.306	26.278	11.428
	3	98.904	134.423	100.539	87.298	65.669	6.054
	4	116.552
	5	128.635
	6	118.816
	Mean	114.3	132.0	107.7	82.8¹	36.7¹	12.0¹

1, statistically significant effect on yield

Table 4, section and average growth rates

Section	Replicate	Control	0.20 µg/L	5.4 µg/L	6.8 µg/L	21 µg/L	301 µg/L
0-24 hr	1	1.903	1.653	1.653	1.537	1.182	0.966
	2	1.394	1.502	2.139	1.49	1.47	0.944
	3	1.582	1.383	1.29	1.553	1.842	0.299
	4	1.422
	5	1.472
	6	0.858
	Mean	1.439	1.513	1.694	1.527	1.498	0.736
24-48 hr	1	0.997	1.829	1.884	1.465	1.976	0.607
	2	1.514	2.043	0.88	1.67	0.953	0.682
	3	1.617	1.967	2.11	1.531	0.59	1.645
	4	1.042
	5	1.884
	6	2.302
	Mean	1.559	1.946	1.624	1.555	1.173	0.978
48-72 h	1	1.813	1.395	1.248	1.641	-0.267	1.347
	2	1.805	1.327	1.616	0.888	0.846	0.811
	3	1.395	1.551	1.212	1.385	1.752	-0.143
	4	2.294
	5	1.502
	6	1.617
	Mean	1.738¹	1.424	1.359	1.305	0.777	0.671
0-72 hr	1	1.571	1.626	1.595	1.547	0.964	0.973
	2	1.571	1.624	1.545	1.349	1.09	0.812
	3	1.531	1.634	1.537	1.49	1.395	0.6
	4	1.586
	5	1.619
	6	1.593
	Mean	1.579²	1.628	1.559	1.462	1.149	0.795
Percent	Inhibition	─	-(3.1)	1.2	7.4	27	50³

1, CV of control section rates, 26%

2, CV of control average growth rates, 1.8%

3, effect statistically significant

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

exponential increase >16/test period (114), <7% CV for average growth rate (1.8%), <35% CV for section specific growth rate (26%)

Conclusions:

MA-3 72-h EC50, 270 µg/L; MA-3 72-h EC10, 6.9 µgL (OECD 201)

Executive summary:

Toxicity of MA-3 to the freshwater alga Pseudokirchneriella subcapitata was assessed 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 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. Dilutions of the 100 mg/L stock had no residual undissolved material. Exposure concentrations were analytically determined in parallel test vials. Analytically determined concentrations declined throughout the test except in the undiluted test solution containing undissolved material. Time-weighted average concentrations were used to calculate effect concentration. The 72-hour EC50 (growth rate) was 270 µg/L. The 72-hour EC10 was 6.9 µg/L.

The study was conducted according to internationally accepted test guidelines and in accord with GLP criteria, with test substance concentrations were confirmed analytically. Test substance concentrations were not stable during the test. The highest test concentration, at which the bulk of effects were observed, was a different treatment than the dilutions (it is possible that the EC50 would have been higher or not attained if all samples were treated identically). The study is deemed reliable with restrictions. It is suitable for Risk Assessment, Classification & Labeling, and PBT Analysis.