3a,4,4a,5,8,8a,9,9a-octahydro-4,9:5,8-dimethano-1H-benz[f]indene

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:

8 January 2019 (Experimental start) 12 April 2019 (Experimental Completion)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

yes

Remarks:

Illumination, max of "8250 lux" was recorded. Min & max of 5900 & 8210 Lux, outside ±15%. Test vol of 200ml was used. Temperature was 20.8 to 21.6°C. Column used: Eclipse XDB Phenyl (250 x 4.6;5µm). These deviations had no impact on study integrity.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

3a,4,4a,5,8,8a,9,9a-octahydro-4,9:5,8-dimethano-1H-benz[f]indene

EC Number:

230-512-1

EC Name:

3a,4,4a,5,8,8a,9,9a-octahydro-4,9:5,8-dimethano-1H-benz[f]indene

Cas Number:

7158-25-0

Molecular formula:

C15H18

IUPAC Name:

3a,4,4a,5,8,8a,9,9a-octahydro-4,9:5,8-dimethano-1H-benz[f]indene

Test material form:

solid

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: RP229-0714
- Expiration date of the lot/batch: 26 November 2019
- Purity test date: 26 November 2018 (CoA)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (15ºC ± 10ºC), no protection from light
- Stability under test conditions: Assumed stable for the duration of the study
- Solubility and stability of the test substance in the solvent/vehicle: Stable in water (solubility in water 0.00538 g/litre at 20°C).
- 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: Each test concentration was stirred for 23 hours and 8 minutes then allowed to settle for 4 hours and 15 minutes. After settling, the first Ca. 100ml of aqueous phase was removed (avoiding all settled and floating material) and discarded. The remaining aqueous phase provided sufficient volumes for water quality measurements and testing. The only practicable method to prepare the 0.1 and 1mg/L test concentrations was by dilution (by addition of 2.5 and 25ml of the
10mg/L preparation to 250ml of dilution water respectively).

FORM AS APPLIED IN THE TEST (if different from that of starting material) Test substance administered as a solution in water. 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).

OTHER SPECIFICS:
- measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical is added: Initial pH was tested at 0 Hours in the control (8.1±0.1). The pH range in control and test concentrations throughout testing were 8.05 - 8.80. Due to the spiking method used, it was only practical to take the water qualities on single
replicates at each concentrations at 0 hours and on pooled replicates at the end of the 72-hour test period.

No reference to osmolality or any precipitate within the report.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0 (control), 0 (solvent control), 50mg/L as a WAF. After consultation with the Sponsor, a 50mg/L limit test was conducted as a 100mg/L solvent stock
was unachievable due to the physical characteristics of the test material and solvent volume requirements as defined in OECD 201 as (must “not exceed 100μl/L”).

- Sampling method: 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).
- Analysis of test substance: Samples were taken at the start and end of the 72 hour exposure period. Samples were taken from a separate vessel prepared in the same way as the test vessels, but without algal inoculation at 0 hours. A “dummy” replicate was placed in the incubator at test conditions for the 72 hour exposure period. Analysis of these samples for the verification of exposure concentrations was performed using HPLC.

- Sample storage conditions before analysis: Samples tested as soon as possible after sampling.

Test solutions

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 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: 0 (control, OECD test media), 0 (solvent control, OECD Test media /DCM)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant):
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 50mg/L limit test as a 100mg/L solvent stock was
unachievable due to the physical characteristics of the test material and solvent volume requirements as defined in OECD 201 as (must “not exceed 100μl/L”)

- Evidence of undissolved material (e.g. precipitate, surface film, etc.): When observed visually, the control, solvent control and 50mg/L conical flasks appeared clear and
colourless after 24 hours and green after 48 and 72 hours. Also, at 72 hours the 50mg/L conical flasks had visible white precipitate on the bottom of the vessels.

- Other relevant information: None

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4 (received 15 May 2018).
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa, SAMS Research Services Ltd, Scottish Marine Institute, OBAN, Argyll, PA37 1QA, Scotland, United Kingdom
- Age of inoculum (at test initiation): Not specified
- Method of cultivation: Algal test inoculum: From a pre-culture growing in exponential phase. Inoculum level adjusted to give an initial cell density of 1 x 10-4 cells/ml. Culture conditions: Temperature: 21.2 – 21.6ºC. Illumination: 6.04 – 8.25 kLux continuous white light, Orbiting: set to 200rpm.


ACCLIMATION
- Acclimation period: Not specified. From a pre-culture growing in exponential phase. Inoculum level adjusted to give an initial cell density of 1 x 10-4 cells/ml. Culture conditions: Temperature: 21.2 – 21.6ºC. Illumination: 6.04 – 8.25 kLux continuous white light, Orbiting: set to 200rpm.
- Culturing media and conditions (same as test or not): yes
- Any deformed or abnormal cells observed: No

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Remarks on exposure duration:

The study objective was to determine the 72-hour effect concentration (EC), defined as the concentration that inhibits algae growth by given percent after a 72-hour exposure according to OECD test guideline 201.

Post exposure observation period:

No post exposure observation period

Test conditions

Test temperature:

Temperature range within incubator throughout test: 20.8 - 21.6°C (Required: 21 - 24±2°C)

pH:

Initial pH at 0 Hours: 8.05 (Required in control at the start of the test: 8.1 ±0.1)
pH range in control and test concentrations throughout test: 8.05 - 8.80

Nominal and measured concentrations:

Nominal concentration 50 mg/L as a limit test. The results for the analytical confirmation of exposure concentrations indicate that the measured concentrations remained within 80-120% of nominal. Therefore all concentrations of the test substance are reported as nominal as received after analytical confirmation.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250ml conical flask
- Type: open system
- Material, size, headspace, fill volume: Test volume: 200ml (headspace 50 mL)
- Aeration: No
- Initial cells density: Inoculum level adjusted to give an initial cell density of 1 x 10-4 cells/ml based upon inoculation volume.
- Control end cells density: Mean of 96.7 x 10-4 cells/ml (Control). Mean of 90.3 x 10 -4 cells/ml (Solvent control).
- No. of vessels per concentration (replicates): Six replicates at the limit test concentration of 50 mg/L.
- No. of vessels per control (replicates): Six control flasks.
- No. of vessels per solvent control (replicates): Six solvent control flasks.

GROWTH MEDIUM
- Standard medium used: yes, sterilised deionised water with added nutrients according to the OECD 201

TEST MEDIUM / WATER PARAMETERS

Culture medium: According to OECD 201 guideline.
Dilution water: Deionised; sterilised by autoclaving at 120°C for 15 minutes.
Method of preparation: Sterile nutrient stock solutions were prepared and added to the dilution water to obtain the culture medium. The test was carried out without adjustment of the pH after addition of the test substance to the media. The pH was 8.05 at test initiation.

Composition of culture medium: Nutrient Final concentration in culture medium (mg/L)

Stock Solution 1 (10ml/L) NH4Cl 15
MgCl2.6H2O 12
CaCl2.2H2O 18
MgSO4.7H2O 15
KH2PO4 1.6

Stock Solution 2 (1ml/L) FeCl3 0.038
Na2EDTA.2H2O 0.1

Stock Solution 3 (1ml/L) H3BO3 0.185
MnCl2 4H2O 0.415
ZnCl2 0.003
CoCl2.6H2O 0.0015
CuCl22H2O 10-5
Na2MoO4.2H2O 0.007
Stock Solution 4
(1ml/L) NaHCO3 50

- 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 according to OECD 201 to deionised water (sterilised by autoclaving at 120°C for 15 minutes) at a pH of
7.52.
- Intervals of water quality measurement: Due to the spiking method used, it was only practical to take the water qualities on single replicates at each concentrations at 0 hours and on pooled replicates at the end of the 72-hour test period

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Photoperiod: 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.
- Light intensity and quality: Illumination: 6.04 – 8.25 kLux continuous white light. In the definitive test: states that the light intensity will be in the range of “6000 - 8000 lux” and “will not vary by more than ± 15%”. A minimum light value was recorded as 5900 Lux and a maximum value of 8210 Lux, 100 lux below and 210 lux
above the recommended range. The light values were also outside the ±15% variation range. This was considered to hvae had no impact on the study integrity or validity

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cell counts were made using a haemocytometer and light microscope. Cell densities were measured microscopically by direct
cell counts on each test and control replicate, in sextuplicate at 24 hours for the control and solvent control and triplicate thereafter, and in triplicate at 24, 48 and 72
hours (±2h) for the test concentration.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Not applicable, only a single limit concentration of 50 mg/L used in the main test.
- Justification for using less concentrations than requested by guideline: A single 50mg/L limit test concentration was conducted as a 100mg/L solvent stock was
unachievable due to the physical characteristics of the test material and solvent volume requirements as defined in OECD 201.

- Range finding study: yes
- Test concentrations: A range finding test was conducted at concentrations of 0 (control), 0.1, 1.0, 10, 100mg/L. The duration of the preliminary study was 72 ± 2
hours. There was a single replicate at each concentration.
- Results used to determine the conditions for the definitive study: Data from the preliminary test identified the 72-hour EC as being between 1-10mg/L (by growth rate) and 1-10mg/L (by yield). In the definitive test concentrations between 0.625 to 50 mg/L did not give an EC50, therfore the main test was conducted as a limit test at 50 mg/L. 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). The higher concentration of 100mg/L solvent stock was unachievable due to the physical characteristics of the test material and solvent volume
requirements as defined in OECD 201 as (must “not exceed 100μl/L”).

Reference substance (positive control):

not required

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks:

Determined by direct observation

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

>= 50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks:

Determined by direct observation

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): None
- Unusual cell shape: None
- Colour differences: When observed visually, the control, solvent control and 50mg/L conical flasks appeared clear and colourless after 24 hours and green after 48 and 72 hours.
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Any stimulation of growth found in any treatment: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: At 72 hours the 50mg/L conical flasks had visible white precipitate on the bottom of the vessels.
- Effect concentrations exceeding solubility of substance in test medium: No

Reported statistics and error estimates:

Statistical data
EC(r)10 value = >50mg/L*
EC(r)20 value = >50mg/L*
EC(r)50 value = >50mg/L*
Determined by Linear Interpolation, using ToxCalc v5.0. * Not possible to calculate confidence limits.


NOEC(r) = 50mg/L
LOEC(r) = >50mg/L
Determined by direct observation. Following tests for normality of distribution and equal variances, using ToxCalc v5.0.

Any other information on results incl. tables

Results: All study validity criteria were met. The results are summarised in the table below.

 

Exposure Period (hours)	ErCxvalue (mg/L) (95% confidence limits)			EyCxvalue (mg/L) (95% confidence limits)
	ErC10	ErC20	ErC	EyC10	EyC20	EyC
0 to 72	>50^*	>50^*	>50^*	18.7^(1)^*	37.4^(1)^*	>50^*
NOEC (0-72h)	50mg/L (Determined by direct observation)^$			<50mg/L (Determined by direct observation^$
Statistical methods used in ToxCalc v5.0: Linear Interpolation ^(1)Extrapolated value – value below lowest concentration tested which was 50mg/L. *Confidence limits not possible to determine. ^$Following Shapiro-Wilk’s Test for normality of distribution and F-Test which indicated equal variances. All concentrations of the test substance are reported as nominal as received after analytical confirmation. All results in this study are calculated from the measured cell densities of the solvent control.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The 72 hour EC(r) and EC(y) of Tricylopentadiene (Distilled) to Pseudokirchneriella subcapitata were >50mg/L and >50mg/L (determined by Linear Interpolation) respectively.
The 0 to 72-hour NOEC(r) was 50mg/L (determined by direct observation) and the 0-72 hour LOEC was >50mg/L (determined by direct observation).

The results indicate that the measured concentrations remained within 80-120% of nominal (actual values 93.667 – 98.365%). Therefore effect concentrations are reported as nominal concentrations of Tricylopentadiene (Distilled) as received.