[3aR-(3aα,5aβ,9aα,9bβ)]decahydro-3a,6,6,9a-tetramethylnaphth[2,1-b]furan-2(1H)-one

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

Study period:

2018-07-22 to 2019-08-02

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)

Version / remarks:

adopted on April 13, 2004.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

SAMPLING AND ANALYSIS
80 mL of test samples from each replicates were drawn and mixed together for each group at 0 and 48 h. The samples were divided into two equal portions. One portion (160 mL) was sent for test concentration analysis and the second portion (160 mL) was stored at -20 ± 5 ºC temperature till the study completion.

Vehicle:

no

Details on test solutions:

A quantity of 18 mg sclareolide was dissolved in reconstituted water and made up to 2000 mL with reconstituted water to obtain the test concentration of 0.009 mg/mL (stock A). Stock solution was kept for sonication for an hour. After sonication, test solution was kept for 24 h stirring. Volumes of 14.4, 26.0, 49.1, 92.4 and 173.3 mL from the stock A were taken and diluted to 260 mL with reconstituted water in respective glass beakers of 600 mL capacity to obtain at 0.5, 0.9, 1.7, 3.2 and 6.0 mg sclareolide/L, respectively.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
Healthy Daphnia magna (showing no signs of stress such as presence of males and ephippia, delay in the production of the first brood, discolouration, etc. and having normal behavior) less than 24 h old (second instar) Cultured and bred in the Laboratory of Ecotoxicology, Jai Research Foundation (originally procured from the MicroBio Test Inc, Belgium).
The sensitivity of the test system and reliability of the experimental technique has been previously validated with potassium dichromate as a positive control.

ACCLIMATION
- Gravid females were acclimatised to the test conditions for a minimum period of 48 h. During this period, Daphnia were fed with live algal cells (Pseudokirchneriella subcapitata). Freshly hatched daphnids belonging to the same stock (less than 24 h-old) were collected with the help of a micropipette and was used for the study. Feed was not provided during the exposure period.
- Acclimatization conditions: In a glass bottle containing 1000 mL of reconstituted water at the mean temperature of 20.0 ± 0.1 °C, mean dissolved oxygen level of 8.71 ± 0.04 mg/L as air saturation value, mean pH of 7.45 ± 0.03 measured daily and kept under a 16 h light and 8 h dark photoperiod during acclimatisation. The total hardness measured was 184.8 mg/L as CaCO3 and light intensity measured was 1340 Lux, on the day of acclimatisation.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

N/A

Hardness:

173.6 mg/L as CaCO3 (at 0 h)

Test temperature:

19.6 - 20.8 °C (at 0 and 48 h)

pH:

7.20 - 7.73 (at 0 and 48 h)

Dissolved oxygen:

7.28 - 8.23 mg/L (at 0 and 48 h)

Salinity:

N/A

Conductivity:

not reported

Nominal and measured concentrations:

A preliminary range finding study was conducted with a total of 60 daphnids, divided into six groups of 10 daphnids per group. The concentrations selected for the preliminary range finding study were 0.0 (control), 0.01, 0.1, 1.0, 5.0, and 9.0 mg sclareolide/L. The percent immobility observed were 0, 0, 0, 10, 100, and 100% at the test concentrations of 0.0 (control), 0.01, 0.1, 1.0, 5.0, and 9.0 mg sclareolide/L, respectively.

Based on the results of the preliminary range finding study, five nominal concentrations 0.5, 0.9, 1.7, 3.2 and 6.0 mg sclareolide/L (Geometric factor 1.9) along with control were selected for the main study.

Nominal: 0 (control), 0.5, 0.9, 1.7, 3.2, and 6.0 mg/L
Measured (at 48 h): 0 (control), 0.4, 0.9, 1.8, 3.5 and 6.5 mg/L

Details on test conditions:

TEST SYSTEM
- The whole study was conducted in a specially designed low temperature water bath preset to a water temperature of 20.0 °C (constant within ± 1°C). Uniform temperature in the water chamber of the water bath was obtained by maintaining a slow flow of water by a motor. The beakers containing the test solutions with daphnids were kept in the water bath. The beakers were held in position by special clamps provided within the chamber of the water bath.

OTHER TEST CONDITIONS
- Adjustment of pH: The pH did not vary by more than 1.5 units in any of the tests.
- Photoperiod: A photoperiod of 16 h light and 8 h darkness cycle was maintained with an automatic timer attached to the water bath. Lighting was provided via fluorescent tubes attached on the lid of the low temperature water bath.
- Light intensity: 1310 and 1350 Lux

PREPARATION OF TEST CONCENTRATIONS
- A quantity of 18 mg sclareolide was dissolved in reconstituted water and made up to 2000 mL with reconstituted water to obtain the test concentration of 0.009 mg/mL (stock A). Stock solution was kept for sonication for an hour. After sonication, test solution was kept for 24 h stirring. Volumes of 14.4, 26.0, 49.1, 92.4 and 173.3 mL from the stock A were taken and diluted to 260 mL with reconstituted water in respective glass beakers of 600 mL capacity to obtain at 0.5, 0.9, 1.7, 3.2 and 6.0 mg sclareolide/L, respectively.

DAPHNID SELECTION (for main study)
Second instar daphnids (less than 24 h old) were separated from the adults in a culture of daphnia. This culture had previously been acclimatised to the study conditions and was immediately transferred to labelled glass beakers (five for each replicate). Transfer was performed using a micropipette having a capacity of 100 - 1000 μL. The mobility of the test organisms was verified immediately after their introduction by gentle swirling of the test container with visual inspection.

IMMOBILITY AND BEHAVIOURAL SYMPTOMS
- All test daphnids were observed for immobility and abnormal behaviour or appearance, at 0, 24, and 48 h of exposure. Mobility of the daphnids was assessed by gently swirling the test container for 15 seconds and observing their swimming behaviour. Daphnids unable to swim during the agitation of the test container and remaining settled on the water surface or settled at the bottom of the test vessel were recorded as immobile.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

1.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

3.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

behaviour

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

0.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

behaviour

Details on results:

IMMOBILITY AND BEHAVIOURAL OBSERVATION
- Percent immobilisation observed at 24 h was 0, 0, 10, 40, and 95 at 0.5, 0.9, 1.7, 3.2 and 6.0 mg/L, respectively. No immobility was observed in any replicate of the control group.
- Percent immobilisation observed at 48 h was 0, 5, 50, 85, and 100 at 0.5, 0.9, 1.7, 3.2 and 6.0 mg/L, respectively. No immobility was observed in any replicate of the control group.
- At 24 h, lethargy was observed at 1.7, 3.2 and 6.0 mg/L. No immobility or behavioural symptom was observed in other dose groups and control.
- At 48 h, lethargy was observed at 0.9, 1.7 and 3.2 mg/L. All daphnids were found immobile at 6.0 mg/L. No immobility or behavioural symptom was observed in other dose groups and control.
- The highest tested concentration level of sclareolide causing no immobilisation was 0.5 mg/L and the lowest tested concentration level causing 100% immobilisation within the 48 h test period was 6.0 mg/L.
- No sign of disease or stress (discolouration or unusual behaviour, such as trapping at the surface of water) was observed in any replicate of the control group.

Results with reference substance (positive control):

Results with reference substance valid?: Yes
Relevant effect levels:
24 h EC50: 0.5 mg/L
48 h EC50: 0.4 mg/L

Reported statistics and error estimates:

The 24 and 48 h EC50 and the associated 95% confidence limits were calculated following the Probit analysis method.

Validity criteria fulfilled:

yes

Conclusions:

The highest tested concentration level of the sclareolide causing no immobilisation and the No Observed Effect Concentration (NOEC) was 0.5 mg/L and the lowest tested concentration level causing 100% immobilisation within the 48 h test period was 6.0 mg/L. The Lowest Observed Effect Concentration (LOEC) was 0.9 mg/L over the 48 h exposure period of the sclareolide. The 24 and 48 h EC50 of the sclareolide was determined 3.1 and 1.9 mg/L, respectively.

Executive summary:

A GLP compliant acute immobilization study was conducted on sclareolide using Daphnia magna according to OECD 202 guideline. Based on results of the preliminary range finding study, concentration levels selected for the main study were 0 (control), 0.5, 0.9, 1.7, 3.2, and 6.0 mgsclareolide/L. In the main study, 48 h static toxicity test procedure was followed, comprising six groups along with control. Each group consisted of four replicates with 5 daphnids per replicate.

The stability of sclareolide in test media was performed during method validation. Test media was analysed for sclareolide active ingredient concentration and stability to monitor the concentration level of test solution at 0 and 48 h, during the main study. The active ingredient concentration of sclareolidein test media was within acceptable limit (>80% of nominal concentration). The study conditions met all validity criteria.

The highest tested concentration level of the sclareolide causing no immobilisation and the No Observed Effect Concentration (NOEC) was 0.5 mg/L and the lowest tested concentration level causing 100% immobilisation within the 48 h test period was 6.0 mg/L.The Lowest Observed Effect Concentration (LOEC) was 0.9 mg/L over the 48 h exposure period of the sclareolide.The 24 and 48 h EC₅₀ of the sclareolide was determined 3.1 and 1.9 mg/L, respectively.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Remarks:

48-hr

Effect concentration:

1.9 mg/L