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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:
2012-05-21 - 2012-05-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
2006
Deviations:
no
Qualifier:
according to
Guideline:
ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)
Version / remarks:
2004
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2009
Deviations:
no
Qualifier:
according to
Guideline:
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000.
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
CYANOX® 1790 Antioxidant
IUPAC nomenclature - Tris(4-tert-butyl-3-hydroxy-2,6-dimethylbenzyl) isocyanurate
Synonym - [tris(4-t-butyl-3-hydroxy-2,6-dimethyl-benzyl)-s-triazine-2,4,6-(1H,3H,5H) trione]
Lot WP1031901
Appearance - White powder, odorless
CAS No. 40601-76-1
Molecular Formula - C42H57N3O6
Molecular Weight - 699.92 g/mole
Purity > 99%
Analytical monitoring:
yes
Details on sampling:
Samples for possible analysis were taken from all test concentrations and the control according to the schedule below.

Frequency: at t=0 h, t=24 h and t=72 h
Volume: 2 ml
Storage: Samples were stored in a freezer until analysis.

At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.

Compliance with the Quality criteria regarding maintenance of actual concentrations was demonstrated by running a test vessel at the highest substance concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.

Additionally, singular reserve samples of 2 ml were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
The batch of CYANOX® 1790 Antioxidant tested was an off white powder with a purity >96% and the substance was not completely soluble in test medium at the loading rate initially prepared.

Preparation of test solutions started with a loading rate of 100 mg/l a 2-day period of magnetic stirring. The obtained mixture was filtered through a 0.45 µm membrane filter (RC55, Whatman) and used as the highest concentration. The lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. The final test solutions were all 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
10E4 cells/ml.


Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): in-house culture
- Age of inoculum (at test initiation): 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 10E4 cells/ml.
- 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 (60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm) in a climate room at a temperature of 21-24°C.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
no
Hardness:
0.24 mmol(24 mg CaCO3/L)
Test temperature:
21.9 and 24.5°C
pH:
8.0-8.2
Dissolved oxygen:
not measured
Salinity:
not measured
Nominal and measured concentrations:
Solutions containing 0.1, 1.0, 10 and 100% of a 0.45 µm filterate prepared at a loading rate of 100 mg/l.

Samples taken from all groups were analysed. The actual concentration in the undiluted filtrate was 0.012 mg/l at the start of the test. Note that this concentration is below the limit of quantification of the analytical method, i.e. 0.1 mg/l, and therefore, is only indicative. However, it was close to the provided solubility of the test substance in water, i.e. <0.02 mg/l. The measured concentration in this group dropped below the limit of detection of the analytical method, i.e. below 0.005 mg/l, after 24 hours of exposure. In the lower groups the actual concentrations were below the limit of detection from the start of the test.
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 ml, all-glass, containing 50 ml of test solution
- Type (delete if not applicable): open
- Initial cells density: 10e4 cell/ml
- Control end cells density: 3015000 cells/ml
- No. of vessels per concentration (replicates): 6 for limit concentration, 3 for lower concentrations
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): not applicable
- Incubation: Capped vessels were distributed at random in the incubator and as such were daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.


GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- M2; according to the OECD 201 Guideline, formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA)

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continous
- Light intensity and quality: 72 to 83 uE.m-2.s-1.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [ spectrophotometer], t=0, 24, 48 and 72 h
- 72 h NOErC, 72 h NOEyC, 72 h ErC50, 72 h EyC50


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Range finding study: the study was performed as a combined limit/range-finding test
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.012 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.012 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.012 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.012 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
- Exponential growth in the control (for algal test): yes
- Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 0.98 mg/l with a 95% confidence interval ranging from 0.74 to 1.3 mg/l (growth rate reduction)
Reported statistics and error estimates:
- For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used.
- Statistical analysis of the data was not needed as the effects recorded were not significant (<10%).
- No EC50 and EC10-values could be calculated because the test substance proved to be non-toxic (EC10 > maximum soluble concentration).
Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the present study, no reduction of growth rate or inhibition of yield was recorded at any of the concentrations.
Executive summary:

Following OECD TG 201 and under GLP conditions, no reduction of growth rate or inhibition of yied was recorded with Pseudokirchneriella subcapitatata at any of the concentrations of Cyanox 1790 Antioxidant tested.

The EC50 for growth rate reduction (ERC50: 0-72h) and the EC50 for yield inhibition (EYC50: 0-72h) were beyond the range tested, i.e. exceeded the initially measured concentration of 0.012 mg/l.

The NOEC for both growth rate reduction and yield inhibition was 0.012 mg/l, i.e. solubility limit in medium.

Due to the very low solubility of CYANOX® 1790 Antioxidant in water, concentration levels that might be toxic for algae could not be reached.

Description of key information

In a 72-h aquatic toxicity study in algae, the test material produced no significant reduction in the growth rate up to the highest concentration tested.

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L
EC10 or NOEC for freshwater algae:
100 mg/L

Additional information

In a 72 -h study conducted according to OECD Guideline 201, the test material did not produce significant toxicity in the freshwater algae species, Pseudkirchnerella subcapitata, up to a highest nominal concentration of 100 mg/L. The measured actual concentration of the test material at the start of testing was 0.012 mg/L. The EC50 was reported as > 0.012 mg/L. The NOEC was reported as 0.012 mg/L. Key values for purposes of the chemical safety assessment will be reported as the nominal concentration (100 mg/L).