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Ecotoxicological information

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:
31 Aug - 10 Dec 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline study under GLP, analytical confirmation of test substance concentration, test substance concentrations declined over time.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): MTDID 948, (E)-1,1,1,2,3,4,5,5,5-nonafluoro-4-(trifluoromethyl)pent-2-ene
- Substance type: pure active substance
- Physical state: liquid
- Analytical purity: 99%
- Expiration date of the lot/batch: 09 Aug 2016
- Stability under test conditions: volatile, vapour pressure: 35464Pa at 293K
- Storage condition of test material: at room temperature

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: All test concentrations and controls, at t = 0hr and 72 hrs
- Sampling method: Because the substance is extremely volatile, test chambers were not sampled directly. Instead, duplicate, abiotic vials were set up and held under test conditions for each time point. An appropriate volume (0.25 mL to 10 mL) was taken from the test solution and transferred via gas-tight syringe through the septum to a VOA vial closed with a bonded septum cap. The vial had contained sufficient blank medium for a total volume of 10 mL after addition of the sample.
- Sample storage conditions before analysis: None, samples analyzed immediately

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Test substance at 100 mg/L in test medium was shaken for seven days at 100 RPM in completely filled, septum-closed VOA vials. Vials were then centrifuged for 90 min at ca. 500xg to consolidate the test substance into a single droplet at the bottom of the vial above the Water Soluble Fraction (WSF). Lower dilutions were made by using a gas-tight syringe to inject precise volumes of the WSF into completely filled, septum-closed VOA vials, with vent needles to allow drainage of blank medium.
- Controls:Blank test medium
- Evidence of undissolved material: Test solutions were clear and colorless except the 100% WSF, which contained a droplet of undissolved test substance.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- 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∙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
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:
yes
Total exposure duration:
72 h

Test conditions

Hardness:
24 mg/L as CaCO3
Test temperature:
21.8 - 22.5 °C
pH:
7.3 - 7.5
Nominal and measured concentrations:
Nominal: Control (0 mg/L), 4.6%, 10%, 22%, 46% and 100% of the saturated solution (i.e., 100 mg/L)
Measured:
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass VOA vials, completely filled with PTFE-faced septa. Fill volume ca. 42.5 m/L
- Agitation: Yes, during incubation the algal cells were kept in suspension by continuous shaking
- Initial cells density: 1E+04 cells/mL
- Control end cells density: 59.6E+04 cells/mL
- No. of vessels per concentration (replicates): Three replicates per day, sacrificed for cell counts. Two algae-free flasks were also assembled for analytical determination at 72 hours.
- No. of vessels per control (replicates): Six replicates per day, with two additional algae-free flasks or analytical determination at 72 hours.

GROWTH MEDIUM
- Standard medium used: yes, cells grown in M1. Preacclimation in adjusted M2 before preliminary tests only.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Standard medium (adjusted M2) prepared in reverse osmosis purified water. M2 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 102 to 105 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 test concentration closest to the EC50 to observe for any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Range finding study: yes, done after initial limit test
- Test concentrations: Control, 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 concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
35 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Remarks:
trans isomer
Basis for effect:
growth rate
Remarks on result:
other: 95% CI, 32 - 38 µg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
17.2 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Remarks:
trans isomer
Basis for effect:
growth rate
Remarks on result:
other: 95% CI, 13 - 20 µg/L
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
17 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Remarks:
trans isomer
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control: yes (Table 2, 3, 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 highest concentration 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 τ=48h and τ = 72 h (Fig. 1) 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: 1.5 mg/L. 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 7 days after the test substance assay.
Reported statistics and error estimates:
Growth rate data were normally distributed by Shapiro-Wilk's test, p(W) = 0.692 > 0.01. Variance of growth rate data were homogeneous by Levene's test, p(F) = 0.023 > 0.01. The NOEC was determined by Williams multiple sequential t-test to be 3.3 µg/L. Because the inhibition of growth rate was 9.7%, or <10%, the effect was not biologically significant and the NOEC was 17 µg/L.

Any other information on results incl. tables

 

Table 2, Individual cell densities in the Algal toxicity test

Time-weighted average concentration (µg/L)

Replicate

Time

0 h

24 h

48 h

72 h

Control

1

1.0

3.408

13.183

63.237

2

1.0

4.118

15.396

56.763

3

1.0

3.985

21.905

61.493

4

1.0

3.992

16.267

55.376

5

1.0

4.113

13.332

61.895

6

1.0

4.026

13.122

59.035

Mean:

 

1.0

3.9

15.5

59.6

Std.Dev.:

 

0.0

0.3

3.4

3.1

CV:

 

0.0

6.8

21.8

5.2

 

 

 

 

 

1.3

1

1.0

3.541

15.788

62.758

2

1.0

3.920

12.457

68.815

3

1.0

4.139

12.671

68.541

Mean:

 

1.0

3.9

13.6

66.7

Std.Dev.:

 

0.0

0.3

1.9

3.4

CV:

 

0.0

7.8

13.7

5.1

 

 

 

 

 

3.1

1

1.0

4.384

12.391

49.908

2

1.0

3.789

11.338

69.907

3

1.0

2.973

13.101

57.684

Mean:

 

1.0

3.7

12.3

59.2

Std.Dev.:

 

0.0

0.7

0.9

10.1

CV:

 

0.0

19.1

7.2

17.0

 

 

 

 

 

3.3

1

1.0

3.450

11.129

44.187

2

1.0

4.039

12.692

69.601

3

1.0

3.695

11.552

51.176

Mean:

 

1.0

3.7

11.8

55.0

Std.Dev.:

 

0.0

0.3

0.8

13.1

CV:

 

0.0

7.9

6.9

23.9

 

 

 

 

 

 

17

1

1.0

3.033

10.925

33.470

2

1.0

2.675

7.526

35.540

3

1.0

3.137

3.673

53.946

Mean:

 

1.0

2.9

7.4

41.0

Std.Dev.:

 

0.0

0.2

3.6

11.3

CV:

 

0.0

8.2

49.2

27.5

 

 

 

 

 

 

48

1

1.0

3.745

7.199

2.241

2

1.0

3.553

5.873

4.214

3

1.0

3.637

5.132

3.435

Mean:

 

1.0

3.6

6.1

3.3

Std.Dev.:

 

0.0

0.1

1.0

1.0

CV:

 

0.0

2.6

17.3

30.1

Table 3, Growth rates (1/day) in the algal toxicity test

Time-weighted average concentration (µg/L)

Replicate

Interval

0-24 h

24-48 h

48-72 h

0-72 h

Control

1

1.226

1.353

1.568

1.382

2

1.415

1.319

1.305

1.346

3

1.383

1.704

1.032

1.373

4

1.384

1.405

1.225

1.338

5

1.414

1.176

1.535

1.375

6

1.393

1.182

1.504

1.359

Mean:

 

1.369

1.356

1.362

1.362

Std.Dev.:

 

0.0715

0.1938

0.2114

0.0175

CV:

 

5.2

14.3

15.5

1.3

 

CV = 12% for all section-specific control growth rates

 

 

1.3

1

1.264

1.495

1.38

1.38

2

1.366

1.156

1.709

1.41

3

1.42

1.119

1.688

1.409

Mean:

 

1.35

1.257

1.592

1.4

Std.Dev.:

 

0.0792

0.2071

0.1842

0.0174

CV:

 

5.9

16.5

11.6

1.2

 

 

 

 

 

3.1

1

1.478

1.039

1.393

1.303

2

1.332

1.096

1.819

1.416

3

1.09

1.483

1.482

1.352

Mean:

 

1.3

1.206

1.565

1.357

Std.Dev.:

 

0.1962

0.2416

0.2246

0.0563

CV:

 

15.1

20

14.4

4.2

 

 

 

 

 

3.3

1

1.238

1.171

1.379

1.263

2

1.396

1.145

1.702

1.414

3

1.307

1.14

1.488

1.312

Mean:

 

1.314

1.152

1.523

1.33

Std.Dev.:

 

0.079

0.0168

0.1642

0.0773

CV:

 

6

1.5

10.8

5.8

 

 

 

 

 

 

17

1

1.11

1.282

1.12

1.17

2

0.984

1.034

1.552

1.19

3

1.143

0.158

2.687

1.329

Mean:

 

1.079

0.825

1.786

1.23

Std.Dev.:

 

0.084

0.5905

0.8095

0.0867

CV:

 

7.8

71.6

45.3

7

 

 

 

 

 

 

48

1

1.32

0.654

-1.167

0.269

2

1.268

0.503

-0.332

0.479

3

1.291

0.344

-0.401

0.411

Mean:

 

1.293

0.5

-0.633

0.387

Std.Dev.:

 

0.0264

0.1546

0.4634

0.1074

CV:

 

2

30.9

73.1

27.8

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The 72-hour EC50 (growth rate) of HFP kinetic dimer to Pseudokirchneriella subcapitata was 35 (95% CI, 32 - 38 µg/L) µg/L based on geometric mean concentration of the trans isomer. The corresponding 72-h NOEC (growth rate) was 17 µg/L, and the EC10 was also 17 µg/L (95% CI, 13 - 20 µg/L). Test conducted per OECD TG201.
Executive summary:

Toxicity of HFP kinetic dimer 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 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. Dilutions of the 100 mg/L stock had no residual undissolved material. Exposure concentrations with respect to the trans isomer were analytically determined in parallel test vials. Analytically determined concentrations declined throughout the test except in the undiluted test solution containing undissolved material. Geometric mean concentrations were used to calculate effect concentration. The 72-hour EC50 (growth rate) was 35 µg/L. The 72-hour EC10 and the 72-hour NOEC were 17 mg/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, declining as much as 80% in some test vessels. 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 without restrictions. It is suitable for Risk Assessment, Classification & Labeling, and PBT Analysis.