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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:
2009-02-09 - 2009-04-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
OECD Guideline for Testing of Chemicals No. 201 'Alga, Growth Inhibition Test' (2006)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
EU Method C.3 'Algal inhibition test' (1992)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Ministry for environment, agriculture and consumerism of North Rhine-Westphalia
Analytical monitoring:
yes
Details on sampling:
- Concentrations: nominal 1.0 mg/L and control
- Sampling method: Unicate samples of the control and duplicate samples of the test concentration were taken for GC analysis after 0, 24, 48 and 72 hours of test duration.
- Sample storage conditions before analysis: Routinely, the samples were analysed immediately. Only in exceptional cases, they were stored overnight deep frozen and protected from light.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 1.3 mg of the test item were added to 1 L of dilution water, then treated for 1 h in an ultrasonic bath and afterwards stirred for 24 h on a magnetic stirrer. Finally undissolved particles of the test item were removed by filtration using a folded filter of pore size 7-12 µm. The pH was measured to be pH 6.0.
- Controls: Yes, dilution water only.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Desmodesmus subspicatus (formerly Scenedesmus subspicatus)
- Strain: 86.81 SAG
- Source (laboratory, culture collection): Strain of the test species obtained form "The Collection of Algal Cultures" of the Institute of Plant Physiology at the University of Göttingen (Germany)
- Maintenance and acclimatisation: Exponentially-growing stock cultures were maintained in the test facility under constant temperature conditions (23 +/- 2 °C) at a light intensity in the range 60 - 120 µE x m-2 x s-1 (measured in the range 400 to 700 nm using a spherical quantum flux meter). The nutrient medium (according to BRINGMANN & KÜHN, 1977) was renewed once a week. Cell density measurements were made using a microcellcounter, Sysmex F300, Digitana.

PREPARATION OF PRE-CULTURES
Pre-cultures were set up three days before the start of the test. They were grown under identical exposure conditions as the stock cultures, except from the use of a different nutrient medium.

TEST CULTURES
The algal inocula for a test were taken from an exponentially-growing preculture and were mixed with the nutrient medium to make up to a final cell density of about 5000 cells per millilitre in the test medium.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
No post exposure observation period is described.
Hardness:
1.3 °dH (22.5 mg/L CaCO3)
Test temperature:
21 - 25 °C
pH:
test start: 7.9 - 8.1
test end: 8.8 - 8.4
Nominal and measured concentrations:
nominal: 1 mg/L (nominal)
geometric mean measured: 0.0085 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type: closed with cotton stoppers
- Material, size, headspace, fill volume: 300 mL
- Initial cells density: approximately 5000 cells per millilitre
- Control end cells density: 578330 cells per millilitre
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- Cell density measurements:

GROWTH MEDIUM
- Medium used: Nutritient medium
- Detailed composition: please see "Any other information on materials and methods"

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Culturing apparatus: Light chamber in which a temperature in the range 21 °C to 25 °C was maintained at +/- 2 °C, and continuous uniform illumination was provided in the spectral range 400 to 700 nm. Temperature was measured and recorded daily.
- Light intensity and quality: At the average of the test solutions, a light intensity in the range 60 - 120 µE. xm-2 x s -1, or an equivalent range of 4000 to 8000 lux, was used. Light intensity was checked before start of the study.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Cell densities were recorded at 24 hour intervals.
- Determination of cell concentrations: Cell densities were measured in a microcell counter (Sysmex F300, Digitana) by taking small aliquots from each test flask for measurements, which were not replaced.
Reference substance (positive control):
not required
Remarks:
according to OECD 201
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.009 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.009 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 0.009 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 0.009 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Reported statistics and error estimates:
All calculations were carried out using the statistics program ToxRatPro Version 2.10 (released 19.02.2009). For the calculation all algae counts were divided by a factor of 10000.
Validity criteria fulfilled:
yes
Conclusions:
The study was conducted under GLP according to OECD Guideline 201 and EU Method C.3 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or deviations from the guidelines, the validity criteria were met. Hence, the results can be considered as reliable to assess the toxicity of the test substance towards algae.
Exponentially growing cells of the freshwater green algae Desmodesmus subspicatus were exposed to the test item added to water at a limit concentration of nominally 1 mg/L, representing the limit of water solubility, for a period of 72 hours under defined conditions. At this concentration no inhibition of algal yield and growth rate was observed at the end of the 72 hour study period, relative to control cultures grown under identical conditions. The 72 h ErC50 and the 72 h EyC50 were determined to be greater than the limit concentration of nominal 1.0 mg/L, corresponding to a geometric mean measured concentration of 0.0085 mg/L. Accordingly, no toxic effects against algae were observed at the limit of water solubility under test conditions.
Executive summary:

The acute toxicity of bis-(2,6 -diisopropylphenyl)carbodiimide to aquatic algae was tested according to OECD guideline 201 and EU Method C.3 in a static freshwater test with Desmodesmus subspicatus (former name: Scenedesmus subspicatus) as test organism. The study was conducted under certificated GLP compliance.

Because of the low solubility of the test item, a limit test was performed at the limit of water solubility under test conditions in order to demonstrate that the EC50 values (based on yield as well as on growth rate) are greater than this concentration. Two range-finding tests were conducted, which provided the concentration to be used in the main test. The final test concentration was chosen as nominal 1 mg/L, whereby direct weighing was done for preparation. For the test item concentration as well as for the control 6 replicates were prepared. The algal inocula for the experiment were taken from an exponentially growing pre-culture and were mixed with the nutrient medium to make up to a final cell density of about 5000 cells per milliliter in the test medium. The algae were exposed for a period of 72 hours and the cell densities were measured at 24 hour intervals. Inhibition of the algal population was measured as reduction in growth rate and yield, relative to control cultures grown under identical conditions. All calculations were carried out using a statistical software programme (ToxRatPro Version 2.10). The maintenance of test item concentrations was proven by analytical measurements. For the test item concentration as well as for the control 6 replicates were prepared. The algal inocula for the experiment were taken from an exponentially growing pre-culture and were mixed with the nutrient medium to make up to a final cell density of about 5000 cells per milliliter in the test medium. The algae were exposed for a period of 72 hours and the cell densities were measured at 24 hour intervals. Inhibition of the algal population was measured as reduction in growth rate and yield, relative to control cultures grown under identical conditions. All calculations were carried out using a statistical software programme (ToxRatPro Version 2.10). The maintenance of test item concentrations was proven by analytical measurements.

Analysis of the yield and growth rate of the algal population within 72 h exposure period gave the following results: ErC50(72h) > 0.0085 mg/L and EyC50(72 h) > 0.0085 mg/L, as well as NOEC(r) 0.0085 mg/L and NOEC(y) 0.0085 mg/L. The results are expressed in terms of geometric mean measured concentrations. Accordingly, no toxic effects against algae were observed at the limit of water solubility under these exposure conditions.

Description of key information

Toxicity to aquatic algae and cyanobacteria: ErC50 (72h) > 0.0085 mg/L (geometric mean measured) for Desmodesmus subspicatus (static, OECD 201, GLP)

Key value for chemical safety assessment

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

Additional information

The acute toxicity of bis-(2,6 -diisopropylphenyl)carbodiimide to aquatic algae was tested according to OECD guideline 201 and EU Method C.3 in a static freshwater test with Desmodesmus subspicatus (former name: Scenedesmus subspicatus) as test organism. The study was conducted under certificated GLP compliance.

Because of the low solubility of the test item, a limit test was performed at the limit of water solubility under test conditions in order to demonstrate that the EC50values (based on yield as well as on growth rate) are greater than this concentration. Two range-finding tests were conducted, which provided the concentration to be used in the main test. The final test concentration was chosen as nominal 1 mg/L. For the test item concentration as well as for the control 6 replicates were prepared. The algal inocula for the experiment were taken from an exponentially growing pre-culture and were mixed with the nutrient medium to make up to a final cell density of about 5000 cells per milliliter in the test medium. The algae were exposed for a period of 72 hours and the cell densities were measured at 24 hour intervals. Inhibition of the algal population was measured as reduction in growth rate and yield, relative to control cultures grown under identical conditions. All calculations were carried out using a statistical software programme (ToxRatPro Version 2.10). The maintenance of test item concentrations was proven by analytical measurements.

Analysis of the yield and growth rate of the algal population within 72 h exposure period gave the following results: ErC50(72h) > 0.0085 mg/L and EyC50(72 h) > 0.0085 mg/L, as well as NOEC(r) 0.0085 mg/L and NOEC(y) 0.0085 mg/L. The results are expressed in terms of geometric mean measured concentrations. Accordingly, no toxic effects against algae were observed at the limit of water solubility under these exposure conditions.

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