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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:
31-Oct-2017 to 11-November-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
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Specific details on test material used for the study:
- Source and lot/batch No.of test material: 116B2018
- Physical state: clear and colorless liquid
- Analytical purity: 96.3%
- Expiration date of the lot/batch: 31 August 2018
- Purity test date: 15 Nov 2016
- Storage condition of test material: at room temperature container flushed with nitrogen

Sampling and analysis

Analytical monitoring:
Details on sampling:
- Concentrations: negative control and saturated solution (limit test)
- Volume: 42.5 mL, a complete vial (see sampling method)
- Sampling method: Duplicate extra sample vials were made to be sacrificed for analytic monitoring.
- Sample storage conditions before analysis: Samples were analyzed on the day of sampling.

Test solutions

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. PPVE-2 (2.5 μL) was then injected through the septum with gas-tight syringe. Thereafter the septum was sealed using parafilm. 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: Test solutions contained a visible small droplet of undissolved test substance at the bottom of the vials.

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): Four 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: Four days before the start of the test, fresh adjusted OECD 201 medium was inoculated at a density of 1E+04 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:
21°C - 22°C
7.3 - 7.8
Nominal and measured concentrations:
Nominal: Control (0 mg/L), 100 mg/L
Measured: 1.8 µg/L (time weighted average)
Details on test conditions:
- Test vessel: Glass VOA vials closed with PTFE-faced septa, completely filled. Fill volume ca. 42.5mL. To initiate the test, a 0.85-mL aliquot of algal suspension was added to each replicate. Algal cells were introduced as quickly as possible, and the volume of media introduced with the algae was as small as possible yet sufficient to fill the exposure vials completely before the caps were replaced.
- Agitation: No
- Initial cells density: 1E+04 cells/mL
- Control end cells density: 132E+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 76 to 82 μE/(m²∙s). Test vessels were placed randomly in the incubator, and repositioned daily.
- 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) against an algal medium blank and the extra replicates for the treated solutions. Test vials were sacrificed for each density measurement.
- Spacing factor for test concentrations: None
- Justification for using less concentrations than requested by guideline: Limit test
Reference substance (positive control):
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Key result
72 h
Dose descriptor:
Effect conc.:
> 1.8 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: 1.8 µg/L was considered the maximum soluble concentration in test medium. Test conducted at a loading rate of 100 mg/L.
Key result
72 h
Dose descriptor:
Effect conc.:
> 1.8 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: 1.8 µg/L was considered the maximum soluble concentration in test medium. Test conducted at a loading rate of 100 mg/L.
72 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth rate
72 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
Under the conditions of the study with Pseudokirchneriella subcapitata exposed to PPVE-2, a 2.3% inhibition of growth rate was observed at 1.8 µg/L, which was considered the maximal solubility of PPVE-2 in the test media in a test conducted at a loading rate of 100 mg/L. This result is statistically, but not biologically relevant. The study report did not include an EC10 dose descriptor, but since the inhibition of growth rate did not exceed 10%, the EC10 effect concentration is by definition > 1.8 µg/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.
- 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: 0.86 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 63 days after the initiation of the test substance assay.
Reported statistics and error estimates:
Growth rate data were normally distributed by Shapiro-Wilk's test, Shapiro-Wilk´s W = 0.968, p(W) = 0.890 (>0.01). Variance of growth rate data were homogeneous by Levene's test, p(F) = 0.084 (> 0.01). NOEC was determined using the Two-sample t-test porecedure (α=0.05, one-sided, smaller). Statistical calculations were done in ToxRat Professional v.3.2.1. (ToxRat Solutions ® GmbH, Germany).

Any other information on results incl. tables

Table 2, Individual cell densities in the algal toxicity test (x10E+04 cells/mL)

Loading rate (mg/L)  Replicate Time      
    0 h 24 h 48 h 72 h
 0 (Control) 1 1 5.436 41.619 112.353
  2 1 5.054 38.819 135.263
  3 1 5.048 27.249 119.437
  4 1 5.122 29.942 159.965
  5 1 5.532 27.722 121.613
  6 1 5.656 29.172 142.801
Mean:   1 5.3 32.4 131.9
Std.Dev.:   0 0.3 6.2 17.7
CV:   0 5.0 19.1 13.4
100 1 1 5.217 38.1 114.529
  2 1 5.858 35.693 116.789
  3 1 5.819 28.677 126.515
  4 1 5.341 29.127 125.790
  5 1 5.296 29.071 106.698
  6 1 6.083 32.629 111.684
Mean:   1 5.6 32.2 117.0
Std.Dev.:   0 0.4 4.0 7.9
CV:   0 6.5 12.3 6.7

Table 3, 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 1.693 2.036 0.993 1.574
  2 1.62 2.039 1.248 1.636
  3 1.619 1.686 1.478 1.594
  4 1.634 1.766 1.676 1.692
  5 1.711 1.612 1.479 1.6
  6 1.733 1.64 1.588 1.654
Mean:   1.668 1.796 1.41 1.625
Std.Dev.:   0.05 0.1936 0.2496 0.0437
CV:   3 10.8 17.7 2.7
CV = 13% for all section-specific control growth rates          
100 1 1.652 1.988 1.101 1.58
  2 1.768 1.807 1.185 1.587
  3 1.761 1.595 1.484 1.613
  4 1.675 1.696 1.463 1.612
  5 1.667 1.703 1.3 1.557
  6 1.805 1.68 1.23 1.572
Mean:   1.721 1.745 1.294 1.587
Std.Dev.:   0.0644 0.1371 0.1536 0.0223
CV:   3.7 7.9 11.9 1.4

Applicant's summary and conclusion

Validity criteria fulfilled:
Control cell density increased by a factor of > 16 (i.e. 132); The mean CV for section-by-section specific growth rate in the controls was < 35% (i.e. 13%); the CV of average specific growth rates in the whole test period for controls was < 7% (i.e. 3%).
The 72-hour EC50 & EC10 (growth rate) of PPVE-2 to Pseudokirchneriella subcapitata was beyond the range tested (nominal concentration of 100 mg/L, measured 1.8 µg/L, dissolved TWA). The test was conducted per OECD TG 201.
Executive summary:

The toxicity of PPVE-2 to the freshwater alga Pseudokirchneriella subcapitata was assessed in a limit test according to OECD201. The test substance is volatile, therefore all tests were conducted in glass VOA vials, completely filled with medium and closed with a PTFE-faced septum cap. 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, and visible undissolved test substance was observed throughout the test. Analytical measurement of test substance concentrations was conducted, to confirm the loading rate was well above the solubility limit of the test material in water. The time weighted average (TWA) concentration of the PPVE-2 in the test vials was 1.8 µg/L. The 72-hour EC10 and EC50 were > 100 mg/L (nominal concentration), and therefore greater than the water solubility of PPVE-2 in the test medium. A statistically significant growth rate inhibition of 2.3% was observed in the 100 mg/L (nominal) test, however this level of inhibition is not biologically relevant. The study was conducted according to internationally accepted test guidelines and in accord with GLP criteria. The test is considered reliable without restrictions, and the results are suitable for Risk Assessment, Classification & Labeling, and PBT Analysis.