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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:
13 - 16 Jul 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
Annex 5 corrected 28 July 2011
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
As amended by EC No. 761/2009
Qualifier:
according to
Guideline:
other: OECD series on testing and assessment number 23
Version / remarks:
Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Perfluorpropylvinylether (PPVE), MTDID 16437
- Substance type: pure active substance
- Physical state: liquid
- Analytical purity: 98.5%
- Storage condition of test material: At room temperature in the dark under nitrogen
- Other: Flush container with nitrogen after handling

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: Limit test, negative control also tested.
- Sampling method: Two extra test bottles each were made for the negative control and the water soluble fraction. Duplicate subsamples were transferred to new VOA vials and analyzed for each level at t=0 and t=72. 10 mL subsamples were taken from negative control. 0.3 mL subsamples were taken from the water soluble fraction and added to 9.7 mL blank medium in the new VOA vials.
- Sample storage conditions before analysis: Samples analyzed day of sampling

Test solutions

Vehicle:
no
Details on test solutions:
Therefore, 42.5mL air-tight vials were completely filled with medium and closed with a septum-sealed screw cap and parafilm. PPVE (2.8 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 dissolved test substance into a single drop at the bottom of the vial.
- Controls: Blank only
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): 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 lab culture.
- Age of inoculum (at test initiation): Three days
- Method of cultivation: - Method of cultivation: Stock cultures were maintained 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) 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 period: Three days before the start of the test, fresh medium was inoculated at a density of 1E4 cell/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:
21.7 and 22.8 °C
pH:
7.4 - 7.9
Nominal and measured concentrations:
Nominal: Control (0 mg/L), 100 mg/L
Measured: n.d., 458 μg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass VOA vials closed with PTFE-faced septa, completely filled. Fill volume ca. 42.5 m/L. 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: 119E+04 cells/mL
- No. of vessels per concentration (replicates): Six replicates per day, sacrificed for cell counts. Two additional flasks each were also assembled for analytical determination at 0 and 72 hours.
- No. of vessels per control (replicates): Six replicates per day, sacrificed for cell counts. Two additional flasks each were also assembled for analytical determination at 0 and 72 hours.

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.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Growth medium prepared in reverse-osmosis purified water (GEON Waterbehandeling, Berkel-Enschot, The Netherlands)
- 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 89 to 94 μE/(m²∙s). Test vessels were placed randomly in the incubator.

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 720 nm using a spectrophotometer with immersion probe (pathlength = 20 mm) against an algal medium blank.

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 concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.458 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 5.5% effect at only concentration tested
Remarks:
(EC50 exceeds medium solubility)
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 0.458 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: EC10 exceeds medium solubility
Details on results:
- Exponential growth in the control (for algal test): yes.
- Observation of abnormalities (for algal test): 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. 43.9%). This was considered unavoidable given the sealed flasks used to test a volatile, low-soluble substance. The results were considered as the ‘best possible’.
- 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: 1.3 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 14 days before the test substance assay.
Reported statistics and error estimates:
Growth rate data were normally distributed by Shapiro-Wilk's test, p(W) = 0.444 > 0.05. Variance of growth rate data was not homogeneous by Levene's test, p(F) = 0.038 < 0.05, but the normality requirement was fulfilled. The NOEC could not be determined by Welch t-test since a significant difference was shown (p(t) = 0.01). However, the inhibition of growth rate was 5.5%, and the effect was not biologically relevant (<10%).

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

9.725

52.702

123.345

2

1.0

9.410

49.819

115.444

3

1.0

10.036

52.948

108.070

4

1.0

10.051

47.624

120.316

5

1.0

9.419

43.202

111.940

6

1.0

9.984

46.470

134.870

Mean:

 

1.0

9.8

48.8

119.0

Std.Dev.:

 

0.0

0.3

3.8

9.5

CV:

 

0.0

3.1

7.7

8.0

 

 

 

 

 

458

1

1.0

8.620

31.124

76.835

2

1.0

8.278

24.303

86.891

3

1.0

8.292

37.835

117.368

4

1.0

8.572

23.434

112.074

5

1.0

8.672

27.693

76.068

6

1.0

8.726

30.148

86.959

Mean:

 

1.0

8.5

29.1

92.7

Std.Dev.:

 

0.0

0.2

5.3

17.8

CV:

 

0.0

2.3

18.1

19.2

 

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

2.275

1.690

0.850

1.605

2

2.242

1.667

0.840

1.583

3

2.306

1.663

0.713

1.561

4

2.308

1.556

0.927

1.597

5

2.243

1.523

0.952

1.573

6

2.301

1.544

1.060

1.635

Mean:

 

2.279

1.607

0.890

1.592

Std.Dev.:

 

0.031

0.074

0.118

0.026

CV:

 

1.400

4.600

13.200

1.600

CV = 43.9% for all section-specific control growth rates

 

 

 

 

1.3

1

2.154

1.284

0.904

1.447

2

2.114

1.077

1.274

1.488

3

2.115

1.518

1.132

1.588

4

2.149

1.006

1.565

1.573

5

2.160

1.161

1.010

1.444

6

2.166

1.240

1.059

1.488

Mean:

 

2.143

1.214

1.157

1.505

Std.Dev.:

 

0.023

0.181

0.235

0.062

CV:

 

1.100

14.900

20.300

4.100

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
test is considered best possible due to sealed-vessel method necessary for test substance
Conclusions:
The 72-hour EC50 and EC10 of PPVE to P. subcapitata are >458 µg/L by OECD TG 201. The NOEC was 458 µg/L. There were no effects at the limit of medium solubility.
Executive summary:

Toxicity of PPVE 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. Exposure concentrations were analytically determined in parallel test vials. Analytically determined concentrations increased somewhat during the test. Geometric mean concentration was used to express effect concentration. The 72-hour EC50 (growth rate) was >458 μg/L. Growth inhibition at this concentration was 5.5%, which is statistically significant but not biologically relevant (<10% effect). The 72-hour EC10 was >458 µg/L. The 72-hour NOEC was 458 µg/L. The effect concentrations exceed the water solubility of PPVE in the test medium.

The study was conducted according to internationally accepted test guidelines and in accord with GLP criteria, with test substance concentrations confirmed analytically. 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.