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Diss Factsheets

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:
2017-05-15 to 2017-06-20
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
according to guideline
EU Method C.3 (Algal Inhibition test)
according to guideline
other: Guidance document on aquatic toxicity testing of difficult items and mixtures, OECD series on testing and assessment number 23, December 14, 2000
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
Cas Number:
Molecular formula:
Test material form:
other: wax
Details on test material:
Physical state: wax form
Appearance: clear, viscous, wax
Specific details on test material used for the study:
- Source and lot/batch No.of test material: I16DB1993
- Expiration date of the lot/batch: 2017-10-03 (retest date)
- Analytical purity: 88.4% (technical document DS-TEC-128833)

- Storage condition of test material: At room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: solubility in water: not indicated

Sampling and analysis

Analytical monitoring:
Details on sampling:
- Sampling method: Samples were taken at t=0 h, t= 24 h and t = 72 h from each test medium and from the control. Additionally, reserve samples of 3.2 mL were taken for possible analysis. At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
- Sample storage conditions before analysis: Stored in a freezer (< -15°C) until analysis.

Test solutions

Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Due to an expected low water solubility of the test item, a saturated solution (SS) was prepared at a loading rate of 100 mg/L. The preparation of test solutions started with melting a part of the test item at ~50°C. The required amount of melted test item was subsequently weighed onto a small watch glass and added to 1 litre of preheated (~50°C) test medium in a volumetric flask. The loading rate of 100 mg/L was subsequently magnetically stirred at room temperature for one day to ensure maximum dissolution of the test item in test medium. This resulted in a colourless solution that contained some undissolved material. The obtained mixture was allowed to settle for a short period (9 minutes for final test) where after the clear and colourless solution was collected by means of siphoning. The resulting SS was used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium.
After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL.

- Controls: yes
- Evidence of undissolved material (e.g. precipitate, surface film, etc): final test solutions were clear and colourless

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture
- Method of cultivation: Algal stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C. M1 medium was used.
- Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in M2 medium at a cell density of 1E+04 cells/mL. The pre-culture was maintained under the same conditions as used in the test. Cell density was measured before use.

- Pre-culture 3 days before start of the test

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
72 h

Test conditions

24 mg CaCO3/L
Test temperature:
22-23 °C
t=0h: 8.0-8.1
t=72h: 7.7-7.8
Dissolved oxygen:
not applicable
not relevant
not specifed
Nominal and measured concentrations:
nominal test concentrations: Control, 0.10, 1.0, 10.0 and 100% SS (1.0 and 100% SS not analysed)
measured test concentration t = 0 h: n.d.,0.0758, n.d., 7.63, n.d. mg/L
measured test concentration t = 24 h: n.d.,0.0707,n.d., 7.10, n.d mg/L
measured test concentration t = 72 h: n.d., 0.042,n.d., 5.78,

Final test:
nominal test concentrations: Control, 0.032, 0.10, 0.32, 1.0, 3.2 and 10% of the SS prepared at 100 mg/L
measured test concentration t = 0 h: n.d., 0.020, 0.0628, 0.204, 0.211, 0.636, 2.03, 6.33 mg/L
measured test concentration t = 24 h: n.d., 0.018, 0.0592, 0.193, 0.199, 0.597, 1.91, 6.01 mg/L
measured test concentration t = 72 h: n.d., 0.0070, 0.0250, 0.0855, 0.149, 0.302, 1.27, 4.83 mg/L
Details on test conditions:
- Test vessel: 100 mL all-glass flasks
- Type: capped vessels
- Material, size, headspace, fill volume: 100 mL all-glass flasks filled with 50 mL test solution
- Initial cells density: 10,000 cells/mL
- Control end cells density: 93.2 x 10,000 cells/mL (mean)
- No. of vessels per concentration apart from the highest concentration (replicates): 3
- No. of vessels per control and the highest concentration (replicates): 6
Final test:
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
1 extra replicate of each test group for sampling after 24 hours.
1 or 2 replicates of each test concentration without algae.

- Standard medium used: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis
- Detailed composition if non-standard medium was used:
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

- Source/preparation of dilution water: M2, according to the OECD 201 Guideline, formulated using Milli-RO water.
- Culture medium different from test medium: Yes (M1 versus M2). Three days before the start of the test the algal stock culture (culture in M1) was inoculated in the same culture medium (M2) used in the test. The culture was maintained under the same conditions as used in the test.
- Intervals of measurements: pH was measured at the beginning and at the end of the test. Temperature was continuously measured in a control vessel. At the end of the final test microscopic observations were performed on all test concentrations to observe for any abnormal appearance of the algae.

- Sterile test conditions: no information
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: using TLD-lamps (with a light intensity within the range of 98 to 105 µE.m-2.s-1)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]: At the beginning, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (pathlength = 20mm).
- Effect calculated parameters: specific growth rate and yield

- Spacing factor for test concentrations: 3.2
- Range finding study: yes
- Test concentrations: 0.1, 1.0, 10 and 100% of the SS (setup as combined range finder/limit test)
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Key result
72 h
Dose descriptor:
Effect conc.:
3.7 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% C.L. 3.6-3.8 mg/L
Key result
72 h
Dose descriptor:
Effect conc.:
1.5 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% C.L. 1.4-1.6 mg/L
Key result
72 h
Dose descriptor:
Effect conc.:
0.49 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- 72-h EC50 (yield) = 1.8 mg T001597/L (95% C.L. 1.6-2.0 mg/L)
- 72-h EC20 (growth rate) = 2.1 mg T001597/L (95% C.L. 2.0-2.2 mg/L)
- 72-h EC20 (yield) = 1.1 mg T001597/L (95% C.L. 0.70-1.3 mg/L)
- 72-h EC10 (yield) = 0.89 mg T001597/L (95% C.L.0.42-1.1 mg/L)
- 72-h NOEC (yield) = 0.49 mg T001597/L
Results with reference substance (positive control):
- Results with reference substance valid? yes
- 72-h EC50 (growth rate) = 0.99 mg/L (95% C.L. 0.97 to 1.00 mg/L)
- 72-h EC50 (yield) = 0.37 mg/L (95% C.L. 0.36 to 0.37 mg/L)
Reported statistics and error estimates:
Calculation of ECx values was based on probit analysis using linear maximum likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding average exposure concentrations of the test item.

The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Applicant's summary and conclusion

Validity criteria fulfilled:
A 72-h growth inhibition test with the unicellular green alga Pseudokirchneriella subcapitata was performed with the test substance T001597 according to the OECD guideline 201 (GLP conditions). It can be concluded that the test item had no significant inhibitory effect on the growth rate of Pseudokirchneriella subcapitata up to and including the test concentrations of 0.49 mg/L (NOEC) after the test period of 72 hours (measured as TWA). The 72-h ErC10 for growth rate inhibition was determined to be 1.5 mg/L with a 95% confidence interval ranging from 1.4-1.6 mg/L (measured as TWA). The 72-h ErC50 for growth rate inhibition was 3.7 mg/L, with a 95% confidence interval ranging from 3.6 to 3.8 mg/L (measured as TWA).
The results of the test can be considered reliable without restrictions.