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

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:

2018-07-22 to 2019-08-10

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The test solutions were subjected for active ingredient analysis using the validated analytical method. For main study, six replicates for control and three replicate for treatment groups were prepared. 100 mL of test samples from each replicates were drawn and mixed together for each group. Collected samples were centrifuged at 2000 rpm for 10 minutes in order to remove algal cell. The representative samples were divided into two equal portions.
One portion (150 mL) was sent for test concentration analysis at 0 and 72 h and the second portion (150 mL) was stored at -20 ± 5 ºC and stored until report finalisations.

Vehicle:

no

Details on test solutions:

A quantity of approximately 18 mg sclareolide was dissolved in OECD medium 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 17.8, 35.6, 71.1, 152.9, and 320.0 mL from the stock A were taken and diluted to 320 mL with OECD medium in respective sterile glass beakers of 600 mL capacity to obtain the nominal concentrations levels of 0.5, 1.0, 2.0, 4.3, and 9.0 mg sclareolide/L, respectively. After sampling for analytical monitoring the remaining volume was transferred in 250 mL capacity conical flask.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: ATCC 22662
- Source (laboratory, culture collection): American Type Culture Collection, 10801, University of Boulevard, Manassas, Virginia, 20110-2209, USA
- Method of cultivation: Static condition

PRE-CULTURE
The pre-culture was prepared 2 days prior to the commencement of the study by transferring 18.0 mL from the latest sub culture into a new culture vessel. The pre-culture was incubated under the same conditions as those required for the test and were used when growing exponentially (5 x 10^3 - 10^4 cells/mL). The temperature for pre-culture ranged between 22 - 23 °C.

Mean initial cell concentration was derived from 3 samples taken from the pre-culture. The cell concentration of the algal stock culture was determined as 1755000 cells/mL by manual counting using a haemocytometer and a microscope. Each replicate was inoculated with 1.0 mL of algal culture to obtain the required cell concentration (approximately 5 x 10^3- 10^4 cells/mL).

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

N/A

Hardness:

not reported

Test temperature:

21 - 23 °C

pH:

7.55 – 7.83

Dissolved oxygen:

not reported

Salinity:

N/A

Conductivity:

not reported

Nominal and measured concentrations:

A preliminary range finding study was conducted with the test concentration levels of 0.0 (control), 0.01, 0.10, 1.00, 5.00, 9.00 mg of sclareolide/L. Two replicates were run for each concentration levels of the test item and three replicates for the control.
Based on the results of preliminary range finding study, the test concentrations selected for the main study were 0.0 (control), 0.5, 1.0, 2.0, 4.3, and 9.0 mg sclareolide/L (geometric factor 2.1). Three replicates were run for each concentration of the test item and six replicates for the control group.

Main study:
- Nominal test concentrations: 0 (control), 0.5, 1.0, 2.0, 4.3, and 9.0 mg sclareolide/L
- Measured test concentrations: 0 (control), 0.51, 1.01, 2.07, 4.38 and 9.79 mg sclareolide/L

Details on test conditions:

TEST SYSTEM
- Initial mean number of cells: 5484 cells/mL
- Culturing Apparatus: 250 mL sterile conical flask
- Mean Illumination (lux): 6383 – 6523 (± 15% of the mean value)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (OECD medium)

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: The cell concentration of each replicate was determined at a regular interval of 24 h, by manual counting using a haemocytometer and a microscope.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.52 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

1.41 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.24 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.91 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

4.47 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

>= 9 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.91 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

1.41 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.25 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: biomass, growth rate and yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: biomass, growth rate and yield

Details on results:

Results of the tested concentrations:
The percent inhibition of biomass was 1.45, 24.73, 35.24, 41.98, and 88.90 for 0.5, 1.0, 2.0, 4.3, and 9.0 mg sclareolide/L, respectively.
The percent inhibition of growth rate was 0.29, 5.48, 8.21, 9.94, and 45.24 for 0.5, 1.0, 2.0, 4.3, and 9.0 mg sclareolide/L, respectively.
The percent inhibition of yield was 1.65, 24.08, 33.90, 39.38, and 90.14 for 0.5, 1.0, 2.0, 4.3, and 9.0 mg sclareolide/L, respectively.
Based on results of statistical analysis, the NOEC and LOEC values for biomass (cell growth), growth rate, and yield was 0.5 and 1.0 sclareolide/L, respectively.

Validity criteria was fulfilled as:
- The cell concentration in control cultures was increased by a factor of 148.0 times within the three-day test period.
- The coefficient of variation of average specific growth rate during the whole test period (0-3) in replicate control culture was 0.60%.
- The mean coefficient of variation for day 0-1, 1-2, and 2-3 in control culture was 2.70%.

Results with reference substance (positive control):

The percent inhibition of biomass was 0.82, 29.27, 82.92, 98.36, and 100.23 for test concentration levels 0.1 , 0.3, 0.8, 2.0, and 5.0 mg/L, respectively.
The percent inhibition of growth rate was -0.28, 6.64, 39.00, 82.71, and 93.64 for test concentration levels 0.1, 0.3, 0.8, 2.0, and 5.0 mg/L, respectively.
The percent inhibition of yield was 0.44, 30.95, 87.46, 99.19, and 99.75 for test concentration levels 0.1, 0.3, 0.8, 2.0, and 5.0 mg/L, respectively.

EbC10 for biomass inhibition (0 - 72 h): 0.20 mg/L
ErC10 for growth rate inhibition (0 - 72 h): 0.33 mg/L
EyC10 for yield inhibition (0 - 72 h): 0.20 mg/L
EbC20 for biomass inhibition (0 - 72 h): 0.26
ErC20 for growth rate inhibition (0 - 72 h): 0.50 mg/L
EyC20 for yield inhibition (0 - 72 h): 0.27 mg/L
EbC50 for biomass inhibition (0 - 72 h): 0.46 mg/L
ErC50 for growth rate inhibition (0 - 72 h): 1.07 mg/L
EyC50 for yield inhibition (0 - 72 h): 0.48 mg/L
NOEC for biomass, growth rate and yield: 0.1 mg/L
LOEC for biomass, growth rate and yield: 0.3 mg/L

Validity criteria fulfilled:

yes

Conclusions:

The growth rate inhibition effect concentrations for the inhibition of growth rate were ErC10 = 2.52 mg/L, ErC20 = 4.47 mg/L and ErC50 = > 9 mg/L.
The biomass inhibition effect concentrations for the biomass inhibition were EbC10 = 0.91 mg/L, EbC20 = 1.41 mg/L and EbC50 = 3.24 mg/L.
The yield inhibition effect concentrationsfor the yield were EyC10 = 0.91 mg/L, EyC20 = 1.41 mg/L and EyC50 = 3.25 mg/L.
Based on results of statistical analysis, the NOEC and LOEC value for biomass (cell growth), growth rate, and yield was 0.5 and 1.0 mg sclareolide/L, respectively.

Executive summary:

A GLP compliant algae growth inhibition test was conducted on sclareolide according to OECD 201 guideline. Test item was checked for solubility and precipitation in OECD media prior to the preliminary study. Based on the results, 9 mg/L was selected as the highest test concentration for preliminary range finding study and acetone as the solvent. Exponentially growing cultures of P. subcapitata were used. A preliminary range finding study was conducted with the test concentrations of 0.0 (control), 0.01, 0.10, 1.00, 5.00, 9.00 mg/L. Two replicates were run for each concentration levels of the test item and three replicates for the control. Based on the results of preliminary range finding study, the test concentrations selected for the main study were 0.0, 0.5, 1.0, 2.0, 4.3, and 9.0 mg/L (geometric factor 2.1). Analytical concentrations of active ingredient at the end of the study were 0.0, 0.51, 1.01, 2.07, 4.38 and 9.79 mg sclareolide/L. Six replicates for control and three replicate for treatment groups were prepared for main study and algal cultures were assessed for their growth by visual cell count at 24, 48, and 72 h. Potassium dichromate was used as a positive control. The EC10,EC20,and EC50were calculated using the Probit analysis. NOEC and LOEC were determined by a suitable statistical procedure for multi sample comparison. Data of biomass, specific growth rate and yield were subjected to Bartlett’s test to meet the homogeneity of variance before conducting Analysis of Variance (ANOVA) and Dunnett’s Test through in-house developed, validated computer software. Stability of the test item in the test media was monitored through the study. The study met all the validity criteria. A concentration-effect relationship was observed and results were statistically analyzed to obtain effect concentrations. The growth rate inhibition effect concentrations for the inhibition of growth rate were ErC10 = 2.52 mg/L, ErC20 = 4.47 mg/L and ErC50 = > 9 mg/L. The biomass inhibition effect concentrations were EbC10= 0.91 mg/L, EbC20= 1.41 mg/L and EbC50= 3.24 mg/L. The yield inhibition effect concentrations were EyC10= 0.91 mg/L, EyC20= 1.41 mg/L and EyC50= 3.25 mg/L. Based on results of statistical analysis, the NOEC and LOEC value for biomass (cell growth), growth rate, and yield was 0.5 and 1.0 mg/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

9 mg/L

EC10 or NOEC for freshwater algae:

2.52 mg/L