5-ethylidene-8,9,10-trinorborn-2-ene, manufacture of, by-products from

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Principles of method if other than guideline:

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed ( OECD 2019). A series of solvent stock solutions was prepared in dimethylformamide in a series of nominal concentrations. An aliquot of each solvent stock solution was separately dispersed in test water with the aid of magnetic stirring fto give the desired nominal testconcentrations.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Duplicate samples were taken from the solvent control and each test group from the bulk test preparation at 0 and 24 hours and from the pooled replicates (R1 to R4) at 24 and 48 hours for quantitative analysis.
- Sample storage conditions before analysis: All samples were placed in universal vials containing 5 mL of THF supplied by Chemistry and stored refrigerated prior to analysis. Duplicate samples were taken at 0, 24 and 48 hours and stored refrigerated for further analysis if necessary.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Range finder tests were carried out with and without solvent. The method preferred for th e definitive test used Dimethyl formamide as the vehicle as the substance alone could not be suitably solubilised using a water accomodated fraction approach. Aliquots of stock solution were diluted in test medium to give required final test concentrations.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Dimethyl formamide (DMF)
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 10g/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no. The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24, 48 hours.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Age at study initiation (mean and range, SD): adults
- Stage and instar at study initiation: first instar
- Source: in house breeding. Culture conditions ensured that reproduction was by parthenogenesis.
- Age of parental stock (mean and range, SD):
- Feeding during test : no


METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS: Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing.
- Food type: mixture of algal suspension (Pseudokirchneriella subcapitata) and GEMMA Micro 300 fish food suspension
- Frequency: daily

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

20-21 °C

pH:

8.0 to 8.1 across all replicates throughout study

Dissolved oxygen:

8.8 - 9.1mg O2/L across all replicates throughout study

Salinity:

not applicable

Conductivity:

not measured

Nominal and measured concentrations:

Nominal: 0.04, 0.09, 0.2, 0.5, 1.2 mg/L
Measured (geometric mean): 0.018, 0.075, 0.15, 0.32, 0.99 mg/L

Details on test conditions:

TEST SYSTEM
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 150mL, vessel completely filled.
- Volume of solution: 150mL
- Aeration: No
- Renewal rate of test solution (frequency/flow rate): renewed at 24hrs
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4 (100 μL/L of DMF)
- Biomass loading rate:

TEST MEDIUM / WATER PARAMETERS
- diluent water considered not to contain any contaminant that would affect the integrity or outcome of the study.
- Test medium: Reconstituted Water – Elendt M4 Medium, containing salts, trace elements and vitamins.
- Water Hardness: 2.5 mmol/L (= 250 mg/L as CaCO3)
- Alkalinity: 0.9 mmol/L

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16hrs light, 8hr dark with 20min dawn/dusk transitions
- Light intensity: between 200 and 1200 lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilized if they were unable to swim within 15 seconds after gentle agitation.

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Study 1 using water accomodated fraction with nominal loading of 1.0, 10, 100mg/L. Significant immobilisation at all loadings
- Study 2 using water accomodated fraction with nominal loading of 0.001, 0.01, 0.1, 1.0mg/L. Stability of test substance could not be confirmed.
- Study 2 using water accomodated fraction with stock solution with nominal loading of 1.0mg/L which was diluted to produce test concentrations of 0.1 and 0.01mg/L. Inconsistent results obtained.
- Study 4 using stock solution of substrate in dimethyl formamide (10g/L) diluted to produce test solution strengths as per test 2. This approach was deemed satisfactory (only ~50% substrate loss) and was sufficient to determine test concentrations for definitive study. Substance loss was a reason why a semi-static approach was used.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

0.88 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (total fraction)

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.11 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (total fraction)

Basis for effect:

mobility

Details on results:

- Other biological observations: reduced mobility seen at concentrations below lethality.
- Mortality of control: <10%
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no. All solutions observed to be colourless throughout study with no sign of precipitation.
- Effect concentrations exceeding solubility of substance in test medium:no

Results with reference substance (positive control):

- Results with reference substance valid? Yes. Last conducted on tes system 7 months previously.
- Relevant effect levels:
- Limit test: no
- Dose-response test: yes (0.32, 0.56, 1.0, 1.8, 3.23 mg/L)
- EC50 (48hr): 0.71 mg/L

Reported statistics and error estimates:

95% confidence interval of 48hr EC50: 0.055 - 0.19 mg/L

Validation criteria:

• No more than 10% of the control daphnids showed immobilization or other signs of disease or stress (e.g. discoloration or unusual behavior such as trapping at the surface water).

• The dissolved oxygen concentration at the end of the test was greater than 3 mg/L in the control and test vessels.

Validity criteria fulfilled:

yes

Executive summary:

In a guideline GLP study, the 48hr EC50 in daphnia magna of the substance THI-TM was determined to be 0.11mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.11 mg/L