5-octylbicyclo[2.2.1]hept-2-ene

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:

24 to 27 July 2018

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)

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Materia, Inc.
- Lot/batch No.of test material: RP367_ONB-D
- Expiration date of the lot/batch: 9 August 2018
- Purity test date: 9 August 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (15°C ± 10°C), in the dark
- Stability under test conditions: Assumed stable for the duration of the test
- Solubility and stability of the test substance in the solvent/vehicle: Soluble in water <0.001g/L, expected to be stable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:: None

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Both the preliminary range-finding test and the definitive test were prepared by the direct addition of measured amounts of test substance to dilution water and stirred.
In the main test, a nominal concentration (100 mg/L) was stirred for 23 hours and 5 minutes and allowed to settle for 4 hours and 30 minutes. After settling the first 100ml (approximately) of aqueous phase was removed (avoiding all settled and floating material) and discarded after filtering through Whatman 54 filter paper. The
remaining aqueous phase provided sufficient volumes for water quality measurements and testing.

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Added to the test system as an aquous phase due to low water solubility (<0.001g/L).

Analytical monitoring:

yes

Details on sampling:

- Concentration: Nominal (limit) concentration of 100 mg/L in the main test
- Sampling method: Duplicate samples were taken at the start and end of the 72 hour exposure period. Samples were taken from remaining test media after filling test vessels for 0 hours and pooled replicate flasks for 72 hours. Analysis of these samples for the verification of exposure concentrations was performed using Gas Chromatography Flame Ionisation Detection (GC/FID).
- Sample storage conditions before analysis: Ambient room temperature

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: In the main test (100 mg/L) the test concentration was stirred for 23 hours and 5 minutes and allowed to settled for 4 hours and 30 minutes. After settling, the first ca. 100ml of aqueous phase was removed (avoiding all settled and floating material) and discarded after filtering through Whatman 54 filter paper. The remaining aqueous phase provided sufficient volumes for water quality measurements and testing.
- Differential loading: All concentrations of the test substance are reported as nominal as received and are expressed as loading rates, following preparation of water accommodated fractions (WAF’s).
- Controls: Test concentrations: 0 (control) and 100 mg/L
Mean measured concentration: 0 (control) and 100 mg/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: strain CCAP 278/4 (received 15 May 2018).
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa, SAMS Ltd, Scottish Marine Institute, OBAN, Argyll, PA37 1QA, Scotland, United Kingdom
- Age of inoculum (at test initiation): Not specified, from a pre-culture growing in exponential phase.
- Method of cultivation: The stocks of algal culture were maintained, and the tests performed, in nutrient growth medium as per OECD 201 requirments.t

ACCLIMATION
- Acclimation period: Not specified
- Culturing media and conditions (same as test or not): yes
- Any deformed or abnormal cells observed: No

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Remarks on exposure duration:

72-hour (±2h) exposure in line with OECD 201 requirements

Post exposure observation period:

None

Hardness:

Not specified

Test temperature:

19.9 - 20.6°C

pH:

7.36 - 8.05

Nominal and measured concentrations:

Test concentrations: 0 (control), 100 mg/L
Mean measured concentration: 0 (control), 1.139 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250ml conical flask
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: Test volume: 100ml
- Initial cells density: Inoculum level adjusted to give an initial cell density of 1 x 10E04
cells/ml.
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of 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 7.15 (adjusted to: 8.08.
- Intervals of water quality measurement: 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 degreeC) 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
- Light intensity and quality: Illumination range within incubator throughout test: 5790 - 8050 Lux (Required: 6x10E03 - 8x10E03 Lux ± 15% of recorded mean)

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 sextuplicate at 24 hours for the control and triplicate thereafter, and in triplicate at 24, 48 and 72 hours (±2h) for the test concentrations. The cell counts were made using a haemocytometer and microscope.

TEST CONCENTRATIONS
- Range finding study: Yes
- Test concentrations: 0 (control), 0.1, 1.0, 10, 100mg/L
- Results used to determine the conditions for the definitive study: The duration of the preliminary study was 72 ± 2 hours. There was a single replicate at each concentration. Data from the preliminary test identified the 72-hour EC50 as being >100mg/L (by growth rate).

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1.139 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 1.139 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Observation of abnormalities (for algal test): The algal cells were examined microscopically during the determination of the cell density, all cells within the control and all test concentrations appeared normal, no morphological abnormalities were observed.
- Colour differences: When observed visually, the control conical flasks appeared colourless after 24 and 48 hours and pale green at 72 hours. The test conical flasks appeared colourless after 24 and 48 hours and pale green and colourless at 72 hours.

Results with reference substance (positive control):

A separate reference study (ENV 11396) was run from 16 to 19 January 2017 using potassium dichromate (K2Cr2O7) as a means of checking the test procedure and sensitivity of the test species. ISO 8692:2012 quotes the 72-hour ErC50 as 1.19mg/L (SD =0.27), the result obtained in the reference test should be in the range 0.65 and 1.73mg/L (mean value ± 2 x standard deviation). The ErC50 obtained was 1.06mg/L and all validity criteria were met.

Study Plan Deviations: Study plan section 9.4 Additional Study Details: Definitive test: states that the light should be at an “intensity in the range 6000 - 8000 lux at the level of the test flasks and will not vary by more than ± 15% from the mean value across all test vessels.” and that the temperature should be “21 to 24°C”.

A minimum lux value of 5790 and a maximum of 8050 lux was recorded, 210 lux below and 50 lux above the recommended range. The range was also outside the ± 15% recommended range. A minimum temperature of 20.5°C was also recorded, 0.5°C below the recommended limit.

Cell densities in the control cultures increased by the required amount (a factor of at least 16) within the 72-hour test period. Therefore, test sample concentration is considered to be the controlling factor in cell density changes in the test treatments. No morphological abnormalities were evident in any of the control vessels.

Validity criteria fulfilled:

yes

Conclusions:

EC50r >1.139 mg/l; NOEC >1.139 mg/l

Description of key information

EC50r >1.139 mg/l; NOEC >1.139 mg/l

Key value for chemical safety assessment

EC50 for freshwater algae:

1.139 mg/L

EC10 or NOEC for freshwater algae:

1.139 mg/L