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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-07-31 to 2015-08-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: A14JB3442
- Expiration date of the lot/batch: 2016-09-30 (retest date)
- Purity test date: 2014-10-01

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- 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 = 0.21 g/L

Analytical monitoring:
yes
Details on sampling:
- Sampling method: singular samples (3.0 mL) were taken before the start of the test and after 72 hours from each test medium and from the control.
- Sample storage conditions before analysis: the samples were stored in a freezer until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A supersaturated stock solution (SS) with a loading rate of nominal 100 mg/L was prepared. The test item was mixed into the test water using magnetic stirring for 2 days at room temperature in the dark. This resulted in a clear and colourless dispersion that contained undissolved floating material and precipitate.The supersaturated stock solution was filtered through a 0.45 µm membrane filter (Whatman; RC55). The filter was preconditioned with a small volume of test solution that was discared. The resulting SS was used as the highest test concentration. The lower test concentrations were prepared by subsequent dilutions in test medium. The final test solutions were all clear and colourless. Note that the control solution was also filtered to minimise treatment related effects.After preparation, volume of 50 mL were added to each replicate of the respective test concentration.
- Controls: yes
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): The final test solutions were all clear and colourless
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- 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
- Acclimation period: 3 days (pre-culture) cells from the algal stock culture were inoculated in M2 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.
- Culturing media and conditions (same as test or not): same as the test
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
24 mg CaCO3/L
Test temperature:
21-22°C
pH:
At the start of the test: 7.9 - 8.3
At the end of the test: 8.0 - 8.1
Dissolved oxygen:
not reported
Salinity:
not applicable
Nominal and measured concentrations:
* combined limit/range-finding test:
nominal test concentrations: 1.0 and 100% of the SS prepared at a concentration of 100 mg/l
mean measured concentrations: 0.916-0.926 mg/L and 91.6-92.8 mg/L

* final test:
nominal test concentrations: 0, 1.0, 3.2, 10, 32 and 100% of the SS prepared at 100 mg/l
mean measured concentrations (mg/L): n.d.-0.0057, 0.956-0.965, 3.04-3.06, 9.31-9.37, 29.4-29.7, 93.3

The mean measured concentrations (calculated as the average over all measurements per test concentration) ranged from 99 to 101% of nominal.
Details on test conditions:
TEST SYSTEM
- Test vessel: flasks
- Type (delete if not applicable): 100 mL capped vessels, all-glass
- Material, size, headspace, fill volume: 100 mL flasks filled with 50 mL test suspension
- Initial cells density: 10,000 cells/mL
- Control end cells density: 144.5 x 10,000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (OECD 201)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO water (tapwater purified with reverse osmosis)
- Culture medium different from test medium: no
- Intervals of water quality measurement:
* temperature: continuously
* pH: at the beginning and the end of the test

OTHER TEST CONDITIONS
- Sterile test conditions: no information
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: range: 81-85 µE.m-².s-1 by TLD-lamp

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Shimadzu Spectrophotometer UV-1800 including UVProbe 2.33 software
- effect calculated parameters: specific growth rate

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations: 1.0 and 100% of the SS prepared at a concentration of 100 mg/l
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
86 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 82-90
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
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: clear test medium
- 72-h EC50 (biomass) = 37 mg T001036/L.
- 72-h EC10 (biomass) = 15 mg T001036/L.
- 72-h NOEC (biomass) = 10 mg T001036/L.
- 72-h EC10 (growth) = 31 mg T001036/L (95% C.L.: 28-35)
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50 (biomass) = 0.46 mg/L
- EC50 (growth rate) = 1.3 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 nominal concentrations of the test item. The calculations were performed with ToxRat Professional v. 3.0.0 (ToxRat Solutions® GmbH, Germany).
Validity criteria fulfilled:
yes
Conclusions:
A 72-h growth inhibition test with the unicellular green alga Pseudokirchneriella subcapitata was performed with the test substance T001036 according to the OECD 201 guideline (GLP conditions). The results were based on nominal concentrations. The 72-h EC50 (growth rate) was determined to be 86 mg T001036/L (95% CL= 82-90 mg/L). The 72-h NOEC and the 72-h EC10 (growth rate) (ErC10) were determined to be 10 mg T001036/L and 31 mg T001036/L respectively. The results of the test can be considered reliable without restrictions.

Description of key information

The study of Migchielsen (2016), investigating the acute toxicity of T001036 to algae according to OECD guideline 201, was considered as the key study for endpoint coverage. The 72-h ErC10 and 72-h ErC50 for growth rate were determined to be 31 mg/L and 86 mg/L respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:
86 mg/L
EC10 or NOEC for freshwater algae:
10 mg/L

Additional information

A well-designed growth inhibition study was performed by Migchielsen (2016) in which Pseudokirchneriella subcapitata was exposed to 1.0, 3.2, 10, 32 and 100% of a SS prepared at nominally 100 mg/l of test substance (five concentrations tested + 1 control) during 72 hours. Analyses showed that measured test item concentrations were in agreement with the nominal concentrations (93-97%, despite the filtration) and that they remained stable during the test period. Hence, it was justified to base all further results and the toxicity parameters on nominal concentrations.

The measured 72-hour ErC50 was determined to be 31 mg/L, the 72- hour NOErC and ErC10 were 10 mg/L and 31 mg/L respectively.

Based on the results it can be considered that T001036 is harmful to algae.