1-cyclohexyl-1H-pyrrole-2,5-dione

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 July to 22 December 2017

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)

Deviations:

yes

Remarks:

The temperature in the algae incubator dropped 0.4°C below the allowed range but had no effect on the outcome or intergrity of the result.

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Nippon Shokubai Co. Ltd., Kogin Building, 4-1-1 Koraibashi, Chuo-ku, Osaka 541-0043, Japan. Lot/batch number: 5K21
- Expiration date of the lot/batch: 26 May 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (15 +/- 10°C). Protected from light, 40°C or lower (SDS)
- Stability in container after opening: Stable
- Solubility and stability of the test substance in the solvent/vehicle: Stability in water: - Undergoes Hydrolysis


OTHER SPECIFICS:
- Appearance (specified): Solid White Flake
- Appearance (observed): White Solid Flakes
- Solubility (specified): 0.3g/L (0.03g/100ml)
- 0.03g/100g water at 25°C (SDS)
- Homogeneity (specified): Homogeneous
- Homogeneity (observed): Homogeneous flakes of various sizes

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Dilution water: The stocks of algal culture were maintained, and the tests performed, in nutrient growth medium (OECD 201) which was prepared by adding appropriate amounts of nutrient stocks to deionised water (sterilised by autoclaving at 120°C for 15 minutes) at a pH of 8.30 (adjusted to 8.20) (See 2.5 Test methods & conditions).
- Preparation of test solutions: Information provided by the Sponsor indicated that the sample was soluble at 0.3g/L (0.03g/100ml), 0.03g/100g water at 25°C (SDS). Stock solutions were prepared for both the range-finding and definitive tests by addition of the test sample directly to nutrient growth medium in a volumetric flask of adequate volume. Calculated volumes of the stock solution were added to nutrient growth medium and made to volume to prepare the test concentrations and provide enough solution for testing and subsequent water quality measurements.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa, SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland, PA37 1QA

- Pre-culture conditions (prior to the test start):
- Temperature: 21.1 – 21.5ºC
- Illumination: 6440 - 7930 lux continuous white light
- Orbiting: set to 200rpm
- Culture media: Deionised water with added nutrients according to OECD 201.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

± 2 hours

Test temperature:

Temperature range within incubator throughout test: 20.6 - 21.5°C

pH:

Initial pH at 0 Hours: 8.20
pH range in control and test concentrations throughout test: 7.45 - 8.20

Nominal and measured concentrations:

Nominal; 0 (control), 0.1, 0.32, 1.0, 3.2, 10 and 32mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: conical flask
- Material, size, headspace, fill volume: glass, 250 mL, 100 mL test volume
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): None
- Initial cells density: 1xE04
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: sterilised deionised water with added nutrients according to OECD 201.
- Intervals of water quality measurement: The temperature (to 0.1ºC) 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.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: 72h
- Light intensity and quality: 6000-7910 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. Six counts were taken of each control replicate at 24 hours and 3 counts were taken of each control replicate at 48 and 72 hours (±2h). 3 counts were taken for each test concentration replicate at 24, 48 and 72 hours (±2h). The cell counts were made using a haemocytometer and microscope.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 0.3
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations:
- Results used to determine the conditions for the definitive study:

Reference substance (positive control):

yes

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.646 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% Confidence Limits

Remarks:

(0.471 - 0.884mg/l)

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.1 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

determined by Bonferroni t Test

Details on results:

Test observations:
- All cells within the control and all test concentrations appeared normal, no morphological abnormalities were observed.
- When observed visually, the control and all test conical flasks appeared colourless after 24 hours.
- At 48 hours the control and 0.1mg/l appeared slightly green with all other concentrations clear and colourless.
- At 72 hours, the control and 0.1mg/l appeared pale green, 0.32 and 1.0mg/l appeared slightly green and 3.2, 10 and 32mg/l were clear and colourless. The lack of green colouration in the 3.2, 10 and 32mg/l test concentrations indicates a lack of algal growth throughout the 72 hour test period.

Validity criteria fulfilled:

yes

Conclusions:

The ErC50 was found to be 0.646 mg/L. This is also regarded as a NOEC and gives a classifciation of Aquatic chronic 3.

Description of key information

The ErC50 was found to be 0.646 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.646 mg/L

EC10 or NOEC for freshwater algae:

0.646 mg/L