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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 April 2010-16 April 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)
Deviations:
no
Qualifier:
according to guideline
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 (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Tris(methylphenyl) phosphite
EC Number:
247-119-6
EC Name:
Tris(methylphenyl) phosphite
Cas Number:
25586-42-9
Molecular formula:
C21H21O3P
IUPAC Name:
tris(methylphenyl) phosphite
Details on test material:
Name of test material (as cited in study report): TTP
- Substance type: Clear colourless liquid (determined at NOTOX)
- Physical state: Liquid
- Analytical purity: 92.1%
- Lot/batch No.: HR9C77938
- Expiration date of the lot/batch: 31 July 2010
- Stability under storage conditions: Stable
- Storage condition of test material: At room temperature in the dark under nitrogen
- Specific Gravity 1.18
-Stability in water No
-Solubility in water Not indicated

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Singular samples for analysis were taken from the control and the WSF prepared at 100 mg/l according to the schedule below.
Frequency at t=0 h, t=24 h and t=72 h
Volume 2 ml
Storage Not applicable, samples were analysed on the day of sampling.

Test solutions

Vehicle:
no
Details on test solutions:
The batch of TTP tested was a clear colourless liquid with a purity >99% and the substance was not completely soluble in test medium at the concentrations tested. Weighing was performed in a glove-box under nitrogen to avoid a reaction with moisture before initiation of the test. The time periods for stirring and settling were kept short based on the known potential for hydrolyses but long enough to ensure reaching maximum solubility.

Preparation of test solutions started with an aqueous mixture of 100 mg/l by adding a volume of 85 µl test substance to 1 litre of test medium. This mixture was magnetically stirred for a period of 4 hours. This resulted in a clear and colourless solution with a floating layer and precipitate. The dispersion was consequently left to settle for 1 hour after which the clear and colourless Water Soluble Fraction (WSF; middle fraction) was collected for testing. The lower test concentrations for the range-finding test were prepared by subsequent dilutions of the WSF in test medium. The final test solutions were all clear and colourless.

Test organisms

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 laboratory culture
- Age of inoculum (at test initiation): 4 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 in a climate room at a temperature of 21-24°C.

Study design

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

Test conditions

Hardness:
0.24 mmol/l (24 mg CaCO3/l)
Test temperature:
between 22.5 and 23.8°C.
pH:
Between 7.4 and 8.1
Nominal and measured concentrations:
Nominal concentration: A WSF prepared at a loading rate of 100 mg/l and dilutions containing 1 and 10% of the WSF (10 mg and 1 mg, respectively).
Measured concentration: At the start of the test, the actual test concentration in the WSF prepared at a loading rate of 100 mg/l corresponded to 0.70 mg/l. This level is greater than the water solubility of 5.85 ug/L because the WSF contains undissolved test substance. A similar value of 0.65 mg/L was reported in the water solubility preliminary test (NOTOX 491955). The measured concentrations decreased to 0.012 mg/l after 24 hours and further to 0.0054 mg/l after 72 hours of exposure. The latter measurement was in agreement with the limit of water solubility. Based on these results, the average exposure concentration was calculated to correspond to 0.036 mg/l.
Details on test conditions:
TEST SYSTEM
- Type: open
- Material, size, headspace, fill volume: 100 ml, normal headspace, 50 ml
- Aeration: no
- Initial cells density: 10000 cells/ml
- Control end cells density: 1202000 cells/ml
Replicates:
6 replicates of the control and the WSF 100 mg/l,
3 replicates of the WSF dilutions of 1 and 10%,
1 extra replicate of the control and the WSF 100 mg/l for sampling purposes,
1 or 2 replicates of each test concentration without algae.


GROWTH MEDIUM
- Standard medium used: yes
OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: TLD-lamps of the type ¿Cool-white¿ of 30 Watt, with a light intensity within the range of 63 to 72 µE.m-2.s-1.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
72 h NOErC, 72 h NOEbC, 72 h ErC50, 72 h EbC50

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Test concentrations: Control, a WSF prepared at a loading rate of 100 mg/l and dilutions containing 1 and 10% of the WSF
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Conc. based on:
test mat.
Basis for effect:
other: YIELD
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Conc. based on:
test mat.
Basis for effect:
other: YIELD
Details on results:
- Exponential growth in the control (for algal test): yes
- Effect concentrations exceeding solubility of substance in test medium: 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):
The EC50 for growth rate reduction (ERC50: 0-72h) was 1.2 mg/l with a 95% confidence interval ranging from 0.77 to 2.0 mg/l.
The EC50 for yield inhibition (EYC50: 0-72h) was 0.44 mg/l with a 95% confidence interval ranging from 0.37 to 0.54 mg/l.
Reported statistics and error estimates:
No significant reduction of growth rate was observed in the WSF prepared at 100 mg/l or in the two dilutions. No statistically significant reduction of growth rate was found at any of the concentrations tested (Bonferroni t test, a = 0.05).

A slight inhibition of yield (17%) was observed in the WSF prepared at 100 mg/l, while no inhibition was observed in the two dilutions. However, statistical analyses showed that the inhibition was not statistically significant (Bonferroni t test, a = 0.05).

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the present study with Pseudokirchneriella subcapitata, no significant reduction of growth rate was observed at a WSF prepared at a loading rate of 100 mg/l.

Both the EC50 for growth rate reduction (ERC50: 0-72h) and the EC50 for yield inhibition
(EYC50: 0-72h) exceeded a WSF prepared at a loading rate of 100 mg/l.