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

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Definitive (main) test: 30 July 2019 (Experimental start) to 1 August 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

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

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: Room temperature (ambient, 15ºC ± 10ºC), in the dark
- Stability under test conditions: Assumed stable for the duration of the study
- Solubility and stability of the test substance in the solvent/vehicle: The test item was insoluble in the test media and therefore dosed with a carrier solvent, Dichloromethane (DCM). Assumed stable for the duration of the study.
- 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: Prepared by the direct addition of test item in solvent stock solutions

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Prepared by the direct addition of test item in solvent stock solutions

OTHER SPECIFICS:
- measurement of pH and precipitate in the culture medium to which the test chemical is added:
Definitive test: Initial pH at 0 Hours: 7.36 (adjusted to 7.45) (Required: 6-9). pH range in control and test concentrations throughout test: 7.46 – 7.76.
No precipitate present as test item solutions were prepared by the direct addition of test item in solvent stock solutions.

- other information:
Initial Dissolved Oxygen (DO) at 0 Hours: 8.10mg/l, DO range in control and test concentrations throughout the test: 8.05 – 9.16mg/l (Required: ≥3mg/l)
Temperature range within incubator throughout test: 18.9 –19.8°C (Required: 20±2°C maintained to ±1°C).
Illumination: 16 hours - Light, 8 hours - dark cycle
Water quality measurements: The pH (to 0.01), dissolved oxygen mg/l (to 0.01) and temperature (to 0.1°C) measurements were taken at 0 hours on remaining test solution, and at 48-hour endpoint on pooled replicates for each test and control solution. Temperature of incubation was recorded at 0, 24 and 48 hours.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The test was carried out at concentrations of 0.02, 0.08, 0.32, 1.28 and 5.12mg/l. Additional sacrificial test vessels were prepared without Daphnia. These solutions were extracted and analysed to verify the initial concentrations of Tricyclopentadiene (Distilled) in the test at 0hrs. Once extracted, the samples were concentrated by a factor of 10 to final concentrations of 0.2, 0.8, 3.2, 12.8 and 51.2 mg/l prior to analysis.

- Sampling method: A single method of analysis using High Performance Liquid Chromatography (HPLC) was used to determine the measured concentration of Tricyclopentadiene (Distilled) in the toxicity test exposure solutions.

- Sample storage conditions before analysis: Analytical samples unable to be processed within an acceptable time-frame were frozen and stored immediately after collection until use.

Test solutions

Vehicle:

yes

Remarks:

TCPD was insoluble in the test media and therefore dosed with a carrier solvent, Dichloromethane (DCM).

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Information provided by the Sponsor indicated that the sample was insoluble in water. Therefore, both the prelim and the definitive test were prepared by the direct addition of test item in solvent stock solutions.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): None.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Originally sourced from a clone of D. magna genotype 5
- Justification for species other than prescribed by test guideline: None, species used as per OECD 202 guideline
- Age at study initiation (mean and range, SD): ≤ 24 hours.
- Stage and instar at study initiation: Not specified
- Source: Actual source prior to arrival at Chemex (in 2014) not specified.
- Feeding during test : Daphnids were not fed during the test period.
- Food type: Each working day, Daphnia were fed a suspension of Unicellular green algae (Chlorella vulgaris)
- Amount: Not specified
- Frequency: Each working day

ACCLIMATION
- Acclimation period: Not specified
- Acclimation conditions (same as test or not): Same
- Type and amount of food: Daphnia were fed a suspension of Unicellular green algae (Chlorella vulgaris), quantity not specified
- Feeding frequency: Each working day
- Health during acclimation (any mortality observed): All Daphnia were healthy, no mortality observed.


METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS: Not specified

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

The objective was determine the 48-hour EC50 according to OECD test guideline 202.

Test conditions

Test temperature:

Temperature: 20 ± 2°C. Temperature range within incubator throughout test: 18.9 – 19.8°C

pH:

Stabilised between pH 6 and 9 before commencement of the test. pH range in control and test concentrations throughout test: 7.46 – 7.76

Dissolved oxygen:

Dissolved oxygen: >60% (or 3mg/l) Absolute Saturation Volume. Initial Dissolved Oxygen at 0 Hours: 8.10mg/l.
DO range in control and test concentrations throughout test: 8.05 – 9.16mg/l (Required: ≥3mg/l)

Nominal and measured concentrations:

All concentrations of the test substance are reported as nominal as received. As Tricyclopentadiene (Distilled) was insoluble in test media it was therefore dosed with a carrier solvent, Dichloromethane (DCM).

Details on test conditions:

TEST SYSTEM
- Test vessel: 40ml glass dishes.
- Type (delete if not applicable): Open, however to limit aerial contamination and reduce evaporative losses, the vessels were covered with a perspex sheet.
- Material (Glass dishes), size (40 mL)l, fill volume (40 mL)
- Volume of solution: 30 mL (Dose Range Finding Test), 40 mL (Definitive test)
- Aeration: Not specified, but oxygen saturation above accepted guideline parameters
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per solvent control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: De-ionised water
- Culture medium different from test medium: No, Daphnia were cultured, and the tests performed, in Elendt M4 reconstituted Daphnia water.
- Intervals of water quality measurement: Not specified

OTHER TEST CONDITIONS
- Adjustment of pH: Initial pH at 0 Hours: 7.36 (adjusted to 7.45) (Required: 6-9). pH range in control and test concentrations during test: 7.46 – 7.76
- Photoperiod: : 16 hours - light, 8 hours - dark cycle
- Light intensity: Not specified

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): The number of immobilised Daphnia were recorded after 24 and 48-hour exposure periods.

SOLVENT CONTROL PERFORMED: Yes , all test substance concentrations are reported as nominal as received. TCPD was insoluble in the test media and therefore dosed with a carrier solvent Dichloromethane (DCM).

RANGE-FINDING STUDY
- Test concentrations: (0) Control, Solvent control, 0.01, 0.10, 1.0 and 10 mg/L TCPD
- Results used to determine the conditions for the definitive study: Data from the preliminary test identified the 48-hour EC50 as being between 0.1-1.0mg/l. The concentrations selected for the definitive study were: (0) Control, Solvent control, 0.02, 0.08, 0.32, 1.28 and 5.12 mg/l.

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

1.321 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mortality

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.08 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mortality

Effect concentrations 3

Key result

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

0.32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mortality

Details on results:

- Behavioural abnormalities: Daphnia were examined at 24 and 48-hours for abnormal behaviour during the determination of immobility. Abnormalities were observed during the test period.
- Other biological observations: Daphnia trapped in meniscus (mobile), Daphnia trapped in meniscus (immobile)
- Mortality of control: None
- Other adverse effects control: None
- Immobilisation of control: One Daphnia imobilised at 24 and 48 hours
- Immobilisation of solvent control: One Daphnia immobilised at 24 hours, and two at 48 hours.
- Abnormal responses: None
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No, all test solutions prepared by direct addition of test item in solvent stock solutions.
- Effect concentrations exceeding solubility of substance in test medium: None

Results with reference substance (positive control):

- Results with reference substance valid? Yes, a separate GLP study was conducted (June 2019) to monitor the sensitivity of the test animals and test procedures. The reference study gave a 24 hour EC50 of 1.07mg/l of potassium dichromate which was within the acceptable range 0.6 mg/l to 2.1 mg/l as specified in Technical Corrigendum to ISO6341.

Reported statistics and error estimates:

Where possible, the EC10 and EC50 values with 95% confidence limits and NOEC were calculated according to the statistical methods of ToxCalc™ Version 5.0 “Comprehensive Toxicity Data Analysis and Database Software”, copyright 1994-1996.

The 24-hour EC50 and 48-hour EC50 of Tricyclopentadiene (Distilled) to Daphnia magna were 1.679mg/l (determined by Maximum Likelihood Probit (P = 0.11), using ToxCalc v5.0) and 1.321mg/l (Determined by Determined by Maximum Likelihood Probit (P = 0.38), using ToxCalc v5.0), respectively.

The 0 to 24-hour NOEC and LOEC were 0.08mg/l and 0.32mg/l respectively (determined by Bonferroni t-test with Arcsin square root transformation, 1-tail, P=0.05, following tests for normality of distribution and equal variances, using ToxCalc v5.0)

Any other information on results incl. tables

Cumulative immobilisation

Nominal Conc (mg/l)	No. immobilised		No. Immobilised
	24 hours	48 hours	24 hours	48 hours
0 (Control)	1	1	5	5
Solvent control	1	1	5	10
0.02	2	2	10	10
0.08	1	1	5	5
0.32	10	9	50	45
1.28	10	11	50	55
5.12	12	12	60	70

Observations

Nominal Conc (mg/l)	Recorded observations
	24 hours	48 hours
0 (Control)	19NS 19NS	19NS 19NS
Solvent control	18NS, 1TM	18NS
0.02	18NS	17NS, 1TM
0.08	14NS, 5TM	15NS, 4TI
0.32	6NS, 4TM	9NS, 2TI
1.28	6NS, 3TM, 1TI	6NS, 3TM
5.12	4NS, 4TM	4NS, 1TM, 1TI

Key:

S	Sedimentation	Ms	Movement Slower
D	Debris attached to Daphnia	TM	Trapped in Meniscus (mobile)
LI	Lighter Colouration	TI	Trapped in Meniscus (immobile)
NS	No Signs of Abnormality	SM	DaphniaSmaller
-	All Daphnia immobilised

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

The definitive test (conducted 30th July to 1st August 2019) was performed according to OECD guideline 202 (2004) and met all validity criteria.

Conclusions:

The definitive test conducted from 30th July to 1st August 2019 was performed according to OECD guideline 202 (2004) and met all validity criteria.

The 48-hour EC50 of Tricyclopentadiene (Distilled) to Daphnia magna was 1.321mg/l (determined by Maximum likelihood-probit).
The 0 to 48-hour NOEC and LOEC were 0.08mg/l and 0.32mg/l respectively (determined by Dunnett’s Test with Arcsin square root transformation).
Analytical confirmation of nominal test concentrations by HPLC was performed.