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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:
November - December 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
28 July 2011
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: hydrogenious 005
- Expiration date of the lot/batch: >3 years, unlimited shelf life
- Purity: > 95%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at ambient temperature (15 ± 10°C), no protection from light
- Stability under test conditions: stable
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0, 100 mg/l
- Sampling method: Duplicate samples of the loading rates were taken at the start and end of the 72 hour exposure period. The samples were taken from remaining test media after filling test vessels for 0 hours and pooled replicate flasks for 72 hours, after filtering using 0.45um syringe filter to remove algal cells
- Sample storage conditions before analysis: no storage
Vehicle:
no
Details on test solutions:
The test concentrations (loading rates) for both the range-finding and definitive tests were prepared separately following the direct addition method as described in the OECD guidance document on aquatic toxicity testing of difficult substances and mixtures no. 23. Calculated weights of test sample were weighed into aspirators containing media to provide enough solution for testing and subsequent water quality measurements. The loading rates were stirred on magnetic stirrers for 22 hours in the range-finding test and 70 hours and 15 minutes in the definitive test and left to settle for 4 hours and 20 minutes in the range-finding test and 26 hours and 45 minutes in the definitive test. After settling, the aqueous phase was drawn off for testing and filtered through Whatman 54 filters, discarding the first 100-120ml of solution and thereby saturating the filter before removal of test dilutions.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Raphidocelis subcapitata
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa, SAMS Research Services Ltd, Scottish Marine institute, UK, received 22 April 2016
- Age of inoculum (at test initiation): pre-culture growing in exponential phase, no further details mentioned
- Method of cultivation: Temperature: 22.1-22.4°C, illumination: 6020-7350 lux continuous white light, orbiting: set to 200 rpm, culture media: de-ionised water with added nutrients according to guideline

ACCLIMATION
- Acclimation period: not applicable
- Culturing media and conditions (same as test or not): same as in test
- Any deformed or abnormal cells observed: nothing mentioned
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
21.7 - 22.4°C
pH:
7.40 - 8.15
Nominal and measured concentrations:
0, 100 mg/l nominal
All concentrations of the test substance are reported as nominal as received, expressed as loading rates from direct addition since the test substance was insoluble in water. The substance was not detected by HPLC analysis in the exposure dilutions at 72 hours and was below LOD at 0 hours.
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 ml conical flask
- Material, size, headspace, fill volume: 100 ml test volume in test vessel
- Aeration: orbital shaking, 200 rpm
- Initial cells density: 1x10E4 cells/ml
- Control end cells density: 80x10E4 cells/ml
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): n.a.

GROWTH MEDIUM
- Standard medium used: yes, according to guideline

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: de-ionised water, sterilised by autoclaving at 120°C for 15 minutes
- Culture medium different from test medium: no
- Intervals of water quality measurement: not mentioned

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: yes, medium adjusted to pH 8.15 at start of test
- Photoperiod: continuously
- Light intensity and quality: 6040 – 6980 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cell densities were measured microscopically by direct cell counts on each test and control replicate in triplicate at 24, 48 and 72 hours (±2h). The cell counts were made using a haemocytometer and microscope.
- Test condition qualtiy measurements: The temperature (to 0.1°C) and light intensity (Lux) within the incubator was recorded at the beginning of the study, after 24, 48 hours and at the end of the 72-hour test period. The pH (to 0.01) and temperature (to 0.1°C) were recorded for each test and control solution at the beginning of the test and on the pooled replicates at the end of the 72-hour test period.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: n.a., limit test
- Justification for using less concentrations than requested by guideline: limit test
- Range finding study
- Test concentrations: 0 (control), 0.1, 1.0, 10 and 100mg/l
- Results used to determine the conditions for the definitive study: no adverse effect on algal growth up to the highest loading rate tested (100mg/l)
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: determined by homoscedastic t-test
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no abnormalities observed (cells microscopically examined, all cells appeared normal)
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: n.a.
- Effect concentrations exceeding solubility of substance in test medium: n.a.
Reported statistics and error estimates:
The results of the homoscedastic t-test (1-tailed, P=0.05), following tests to determine normality of distribution and equality of variance, indicated no significant effects on growth expressed as growth rate and increase in biomass (cell yield) at the loading rate of 100mg/l

see picture below

Validity criteria fulfilled:
yes
Remarks:
The measured control cell density increase was recorded as 80.0, corresponding to a specific growth rate of 1.455 per day. The variation coefficient for the 72h period was 4.47% and for each time period 16.52%.
Conclusions:
The 72-hour ErL50 and EyL50 of dibenzylbenzene, ar-methyl derivative, hydrogenated to Raphidocelis subcapitata were both >100mg/l (determined by Linear Interpolation). The 72-hour NOEL(r) was determined to be 100mg/l and the 0-72 hour LOEL was >100 mg/l (by homoscedastic t-test, 1-tail, P=0.05).
Executive summary:

The test was performed according to the OECD 201 (2011) guideline and in compliance with GLP. The test substance was insoluble in water therefore preparation of loading rates was performed by direct addition and an extended mixing period according to OECD Guidance Document 23. The growth curves demonstrated that the algae in the control and at 100mg/l dibenzylbenzene, ar-methyl derivative, hydrogenated were in logarithmic growth for the duration of the study. The algal cells were examined microscopically during the determination of cell concentration. All cells within the control and test loading rate of 100mg/l appeared normal, no abnormalities were observed. All validity criteria for the definitive test were met. The test substance, dibenzylbenzene, ar-methyl derivative, hydrogenated, was not detected by HPLC-UV analysis in the freshly prepared exposure solutions, or in samples taken after 72-hours exposure (no peaks were seen at the 100mg/l loading rate). All concentrations of the test substance therefore are reported as nominal as received, expressed as loading rates from direct addition.

The 72-hour ErL50 and EyL50 of dibenzylbenzene, ar-methyl derivative, hydrogenated to Raphidocelis subcapitata were both >100mg/l (determined by Linear Interpolation). The 72-hour NOEL(r) was determined to be 100mg/l and the 0-72 hour LOEL was >100 mg/l (by homoscedastic t-test, 1-tail, P=0.05).

Description of key information

The test was performed according to the OECD 201 (2011) guideline and in compliance with GLP. The test substance was insoluble in water therefore preparation of loading rates was performed by direct addition and an extended mixing period according to OECD Guidance Document 23. The growth curves demonstrated that the algae in the control and at 100mg/l dibenzylbenzene, ar-methyl derivative, hydrogenated were in logarithmic growth for the duration of the study. The algal cells were examined microscopically during the determination of cell concentration. All cells within the control and test loading rate of 100 mg/l appeared normal, no abnormalities were observed. All validity criteria for the definitive test were met. The test substance, dibenzylbenzene, ar-methyl derivative, hydrogenated, was not detected by HPLC-UV analysis in the freshly prepared exposure solutions, or in samples taken after 72-hours exposure (no peaks were seen at the 100 mg/l loading rate). All concentrations of the test substance therefore are reported as nominal as received, expressed as loading rates from direct addition.

The 72-hour ErL50 and EyL50 of dibenzylbenzene, ar-methyl derivative, hydrogenated to Raphidocelis subcapitata were both >100mg/l (determined by Linear Interpolation). The 72-hour NOEL(r) was determined to be 100 mg/l and the 0-72 hour LOEL was >100 mg/l (by homoscedastic t-test, 1-tail, P=0.05).

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
100 mg/L

Additional information