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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:
2012-01-20 to 2012-04-27
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
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Samples were taken from the control and the 100 mg/L test group (replicates R1-R6 pooled) at 0 and 72 h for quantitative analysis of test substance concentration.
- Duplicate samples were taken.
- All samples were stored at approximately -20°C prior to analysis.
Vehicle:
no
Details on test solutions:
- 200 mg test substance was dissolved in culture medium and the volume adjusted to 1 L to prepare the single 100 mg/L test solution to be used in the definitive test.
- The test solution was inverted several times to ensure adequate mixing and homogeneity.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland
- Master cultures were maintained in the lab by periodic replenishment of culture medium and were kept under constant aeration and illumination at 21°C.
- Age of inoculum (at test initiation): ~3 days (preculture, in log growth phase at the time of inoculation of the test)
- Initial cell density in test: 5E+03 cells/mL (nominal)

ACCLIMATION
- Acclimation period: none
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: none noted
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
None
Hardness:
ca. 15 mg CaCO3/L (calculated)
Test temperature:
24 +/- 1 °C
pH:
7.4-8.3
Dissolved oxygen:
N/A
Salinity:
N/A
Nominal and measured concentrations:
Nominal: Control and 100 mg/L
Measured: 99-102% of nominal
Details on test conditions:
TEST SYSTEM
- Test vessel: conical flask
- Type (delete if not applicable): plugged with polyurethane foam bungs
- Material, size, headspace, fill volume: glass, 250 mL, 100 mL fill volume
- Aeration: by shaking only
- Flasks were incubated using an INFORS Multitron Version 2 incubator and were constantly shaken at ca. 150 rpm
- Initial cells density: 5.84E+03 cells/mL (mean measured)
- Control end cells density: 1.33E+06 cells/mL (mean measured)
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse osmosis purified deionised water
- A standard test medium was used
- Culture medium different from test medium: no
- Intervals of water quality measurement: batch
- pH was measured at the start and end of testing. Temperature was recorded daily.

OTHER TEST CONDITIONS
- Adjustment of pH: not after addition of test item
- Photoperiod: continuous illumination
- Light intensity and quality: warm white light - 7000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): cell density, at 0, 24, 48 and 72 h
- Determination of cell concentrations: Coulter Multisizer Particle Counter

TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations in range finding study: 0.1, 1.0, 10, 100 mg/L
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Same results when based on yield
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): none
Results with reference substance (positive control):
72-h ErC50 = 1.4 mg/L (95% CI = 1.2-1.7 mg/L)
This result was within the normal range for this reference item.
Reported statistics and error estimates:
Student's t-test incorporating Bartlett's test for homogeneity of variance was used to identify statistically significant differences (p < 0.05) between test and control groups.
Validity criteria fulfilled:
yes
Conclusions:
In this study a growth rate based 72-h EC50 of > 100 mg/L was obtained for the freshwater green alga Pseudokirchneriella subcapitata. No significant differences in growth rate nor yield were identified between the single test concentration and the control group. Therefore the 72-NOEC was >= 100 mg/L. Measured test substance concentrations were in agreement with nominal concentrations. The results of the test can therefore be considered reliable.
Executive summary:

In a 72-h toxicity study, the freshwater alga Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum) was exposed to N-methylmorpholine oxide in a limit test at 100 mg/l under static conditions in accordance with OECD guideline 201. Nominal concentrations were confirmed by analytical measurements. No effects on growth rate were observed. Therefore, the 72-h NOEC value was determined at >= 100 mg/L and the EC50value at >100 mg/L, both based on growth rate. No abnormalities were noted.

This toxicity study is classified as acceptable and satisfies the guideline requirement for OECD 201 toxicity study.

Results synopsis

Test organism: Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Test type: Static

72 h EC50(growth rate): > 100 mg/L

72 h NOEC (growth rate): >= 100 mg/L

Description of key information

In a 72-h toxicity study, the freshwater alga Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum) was exposed to N-methylmorpholine oxide in a limit test at 100 mg/l under static conditions in accordance with OECD guideline 201. Nominal concentrations were confirmed by analytical measurements. No effects of the test substance on growth rate were observed. Therefore, the 72-h NOEC value was determined at >= 100 mg/L and the EC50value at >100 mg/L, both based on growth rate. 

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L

Additional information

Short-term toxicity of NMMO to algae was investigated by exposing Desmodesmus subspicatus during 72 hours in a static test, according to the OECD guideline 201. This resulted in an EC50(72h) value of > 100 mg/L and an NOEC(72h) >= 100 mgL, both based on growth rate.