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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-02-15 to 2016-03-16
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 201 (Alga, Growth Inhibition Test)
according to guideline
EU Method C.3 (Algal Inhibition test)
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Batch M15BB1451
Test item storage conditions At room temperature
Stable under storage conditions until 30 January 2021 (expiry date)
The characterization and stability of the test item have been performed according
Analytical monitoring:
Details on sampling:
- Sampling method: samples were taken before the start of the test and after, 24h and 72 hours from all test concentrations and from the control. The filter used for preparation of the SS was kept for possible analysis of the residue. A volume of 1.9 mL was taken. At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling. Reserve samples of 1.9 mL were taken from all test solutions for possible analysis.
- Sample storage conditions before analysis: stored in a freezer (< -15°C) until analysis.
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preparation of test solutions started with the highest concentration of 100 mg/L applying a 10-minutes period of ultrasonic waves, followed by an approximately 3-hour period of magnetic stirring to accelerate the dissolution of the test item in test medium (in the combined limit/range-finding test, an additional 8-minute period with ultrasonic waves was applied after stirring period). The pH of the obtained solution was adjusted from 9.1 to 8.5 in the combined limit/range-finding test and from 8.9 to 8.6 in the final test with 1M HCL (Merck, Darmstadt, Germany). Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All final test solutions were clear and colourless. 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
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis 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: Algae 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.

- Acclimation period: 3 days
- Culturing media and conditions (same as test or not): same as the test. 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
Test type:
Water media type:
Limit test:
Total exposure duration:
72 h
24 mg CaCO3/L
Test temperature:
8.0 - 8.2
Dissolved oxygen:
not reported
not applicable
Nominal and measured concentrations:
nominal test concentrations final test: 0.46, 1.0, 2.2, 4.6 and 10 mg/L
measured test concentration final test t= 0 h: 0.464, 1.03, 1.04, 2.27, 4.43, 10.0 mg/L
measured test concentration final test t= 24h: 0.464, 1.03, 1.04, 2.26, 4.46, 10.0 mg/L
measured test concentration final test t= 72h: 0.425, 0.969, 0.963, 2.30, 4.49, 10.2 mg/L
Details on test conditions:
- Test vessel: glass flasks
- Type (delete if not applicable): capped vessels
- Material, size, headspace, fill volume: 100 mL all-glass flasks filled with 50 mL test solution
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): no flow-through system applied
- Renewal rate of test solution (frequency/flow rate): no renewal
- Initial cells density: 10,000 cells/mL
- Control end cells density: 137.1.5 x 10,000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

- Standard medium used: yes

- Source/preparation of dilution water: M2 according to the OECD 201 guideline, formulated using Milli-RO water
- Culture medium different from test medium: no
- Intervals of water quality measurement: temperature measured continuously, pH at the beginning and the end of the test.

- Sterile test conditions: no information
- Adjustment of pH: none
- Photoperiod: continuous illumination
- Light intensity and quality: 60 to 120 μE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm, using TLD-lamps.

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.
- effect calculated parameters: specific growth rate and yield

- Spacing factor for test concentrations: x 2.2

- Range finding study
- Test concentrations: 0.1, 1.00, 10 mg/L
- Results used to determine the conditions for the definitive study: yes.
Reference substance (positive control):
potassium dichromate
72 h
Dose descriptor:
Effect conc.:
1.82 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: CL 95%: 1.81-1.83 mg/L
72 h
Dose descriptor:
Effect conc.:
1 mg/L
Nominal / measured:
meas. (arithm. mean)
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:
- EC50 (yield) = 1.37 mg T000750 /L (CL 95% 1.36-1.39 mg/L)
- EC10 (growth rate) = 1.12 mg T000750 / L (95% C.L. 1.10-1.13 mg/L).
- EC10 (yield) = 0.41 mg T000750 / L (95% C.L. 0.40-0.41 mg/L).
- NOEC (yield) = n.d.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 72-h EC50 (growth rate) = 1.0 mg/L
Reported statistics and error estimates:
For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller) or inhibition of yield (Multiple Sequentially-rejective Welsh-t-test After Bonferroni-Holm, α=0.05, onesided, smaller). Additionally, the EC10 and EC20 were determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993. 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 nominal concentrations of the test item.The calculations were performed with ToxRat Professional v. 3.2.1 (ToxRat Solutions® GmbH, Germany).
Validity criteria fulfilled:
A 72-h growth inhibition test with the unicellular green alga Pseudokirchneriella subcapitata was performed wit the test substance T000750 according to the OECD guideline 201 (GLP conditions).
Under the test conditions with Pseudokirchneriella subcapitata, T000750 inhibited growth rate of this fresh water algae species significantly at a concentration of 2.2 mg/L. The 72-h ECr50 was determined to be 1.82 mg/L
The results of the test can be considered reliable without restrictions.

Description of key information

The study of Tabor-kaplon (2016), investigating the acute toxicity of T000750 in zebra fish according to OECD guideline 201, was considered as the key study for endpoint coverage.  A LC50 of 1.82 mg/L (based on nominal test concentrations) was found after an exposure of 72 hours. Based on the results it can be considered that T000750 is toxic to algae.

Key value for chemical safety assessment

EC50 for freshwater algae:
1.82 mg/L

Additional information