3-(5,5,6-trimethylbicyclo[2.2.1]hept-2-yl)cyclohexan-1-ol

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:

Study Initiation Date: October 25, 2021 to Study Completion Date: January 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 2.7 mg/L, 6.48 mg/L, 15.55 mg/L, 37.32 mg/Land 89.58 mg/L
- Sampling method: The test concentrations were prepared in OECD TG 201 medium by dilution of stock and transferred 100 ml of each test solution into 250 ml Erlenmeyer flasks.

Vehicle:

yes

Remarks:

Acetone

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 25.4 mg of test item was dissolve in 25 µL acetone and made up to 250 mL in a volumetric flask using OECD medium (stock).
- Controls: OECD medium
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Acetone
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 0.1 ml/l (10 µL)

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: ATCC
- Source (laboratory, culture collection): Initially procured from “American Type Culture Collection”, Chromachemie Laboratory Private Limited, later test system was sub-cultured in the test facility.
- Age of inoculum (at test initiation): 3 days before start of the test.
- Method of cultivation:The required volume of OECD growth medium was prepared as per OECD 201, test system was sub-cultured using OECD medium.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21.3 – 23 °C

pH:

7.5 - 7.8

Nominal and measured concentrations:

Nominal

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL conical flasks
- Type (delete if not applicable): open / closed Sterilized, flasks were covered with cap
- Material, size, headspace, fill volume: Conical flasks were made of glass. Volume capacity 250 mL.
- Aeration: no aeration
- Initial cells density: 10000 cells/ ml
- Control end cells density: 10000 cells/ml
- No. of organisms per vessel: 10000 cells/mL
- No. of vessels per concentration (replicates): Range finding study: 3 replicates/test item concentration
Main Study: 3 replicates/test item concentration
- No. of vessels per control (replicates): 6 replicates for control group during range finding and main study.
- No. of vessels per vehicle control (replicates):6 replicates for control group during range finding and main study.

GROWTH MEDIUM
- Standard medium used: yes Yes (OECD Medium as per OECD 201)

- Culture medium different from test medium: NO


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH:no
- Photoperiod:Continuous light for test system
- Light intensity and quality:Average: 6716-6734 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The cells were counted using Haemocytometer under illumination of the microscope at 24, 48, and 72 h after inoculation.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study Yes
- Test concentrations: 0 (control), 2.70, 6.48, 15.55, 37.32, 89.58 mg/L
- Results used to determine the conditions for the definitive study: Based on the results of range finding test following concentrations of 0 (control), 2.70, 6.48, 15.55, 37.32, 89.58 mg/L were selected for the main study as significant changes (inhibition algal growth rate) were observed in treatment groups during 72h test period.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

39.76 mg/L

95% CI:

> 32.88 - < 46.64

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

13.32 mg/L

95% CI:

> 11.3 - < 15.34

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other:

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

15.55 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Other details not known

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

6.48 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Other details not known

Details on results:

- Exponential growth in the control (for algal test): yes Yes (52.3 times of the initial cell count in control and vehicle control, respectively)

Results with reference substance (positive control):

- Results with reference substance valid Yes
- EC50:
ErC50 (72h) = 0.65 mg/L (growth rate) (95% C. I. = 0.55 to 0.75 mg/l)
EyC50 (72h) = 0.26 mg/L (yield) (95% C. I. = 0.22 to 0.30 mg/l)

Reported statistics and error estimates:

72h EC50 value with 95% confidence limits (upper limit, lower limit) were calculated by probit analysis using NCSS Software 2019, version 19.0.3.
The NOEC and LOEC value were calculated using the One-way Analysis of Variance - Kruskal - Wallis - Comparison Z-Value test.

Validity criteria fulfilled:

yes

Remarks:

The algal cells in the solvent control increased by 52.3 times which was more than 16 times of the initial cell count.The mean coefficient for growth rate for control culture was 27.98%. The coefficient of variation of avg specific growth rate was 0.5%.

Conclusions:

The effect of a test chemical on freshwater algae was studied as per OECD 201 test guidelines, the 72-h EC50 of a test chemical to alga on growth rate and yield is 39.76 mg/L (95%. C. I. = 32.88 to 46.64), 13.32 mg/L (95% C. I. 11.3 to 15.34 mg/l), respectively. 72-h LOEC and NOEC of the test chemical to alga on growth rate was 15.55 mg/L and 6.48mg/l, respectively.

Executive summary:

The study was conducted to assess the effects of the test chemical on the growth of green Algae, Pseudokirchneriella subcapitata. This study was performed in compliance with OECD Guideline for Testing of chemicals OECD NO. 201, Adopted by the Council on 23 March 2006 and Council Regulation (EC) No. 440/2008, Annex Part C.3 'Freshwater Algae and Cyanobacteria, Growth Inhibition Test. Solubility of the test item was performed by weighing 103.0 mg of the test chemical in a glass vial and dissolving it in 10 µL of acetone and transferred it into the 100 ml volumetric flask and made up to the mark using natural water for solubility check. The stability of the test chemical in natural water determined by analyzing the test concentrations of 1 and 100 mg/L at O hour, 24 hours, 48 hours and 72 hours showed that the test chemical concentration remained 80% to 120% (98.27 % to 100.75 % for 1 mg/Land 98.87 % to 99.51 % for 100 mg/L) with respect to initial measured concentration and hence the dose verification for definitive test was performed at the beginning and at the termination of the test. The test concentrations were prepared in OECD TG 201 medium by dilution of stock and transferred 100 ml of each test solution into 250 ml Erlenmeyer flasks. Three replicates per test item concentration and six replicates for the controls were used for both range finding test and definitive test. The controls and treatment flasks were inoculated with Algae, Pseudokirchneriella subcapitata (pre-cultured for 3 days) and tested at an initial cell density of 10000 cells per ml, incubated under test condition for 72 hours The study was performed by a static method. The cells were counted using an Improved Neubaur's Haemocytometer under illumination of the microscope at 24, 48 and 72 hour after inoculation. A range finding test was conducted at test chemical concentrations of 5, 10, 25, 50 and 100 mg/L along with control groups without test chemicals. No significant changes in the appearance of algae cells were observed in controls and in the test concentrations during 72 hours test period. At the end of 72-hour observation, 1.95 %, 3.36 %, 5.40 %, 27.50 %, and 85.34 % in terms of the percent inhibition of growth rate and 7.27 %, 12.18 %, 18.86 %, 66.01 % and 98.23 % % in terms of the percent inhibition of yield was observed in the test concentrations of 5, 10, 25, 50 and 100 mg/L respectively. Based on the results of the range finding test, the definitive test was conducted at test chemical concentrations of 2.70, 6.48, 15.55, 37.32 and 89.58 mg/L in a geometric series with a factor of 2.4 along with control groups without test chemical. No significant changes in the appearance of algae cells were observed in controls and in the test concentrations during 72 hours test period. At the end of 72-hour observation, 1.69 %, 9.32 %, 18.74 %, 35.20 % and 84.73 % in terms of the percent inhibition of growth rate and 6.58 %, 31.44 %, 53.37 %, 76.60 % and 98.38 % % in terms of the percent inhibition of yield was observed in the test concentrations of 2.70, 6.48, 15.55, 37.32 and 89.58 mg/L respectively. During the Definitive test period, all the flasks were incubated in the room under test condition. The pH of the solvent control at the test start was 8.0 and at the termination of the test was 8.1 and therefore did not vary more than 1.5 units during the study. The pH of all the tested concentrations was 7.7 to 7.8 at the beginning of the test and 7.5 to 7.6 at the test termination. The room temperature (Average) was between 21.3°C and 22.8°C. The temperature of the control and test concentration at the beginning was between 22.3°C and 22.8C and at test termination was between 22.5°C and 23.0°C. The average intensity of light ranged from 6716 to 6734 Lux and the % CV ranged from 0.29 % to 1.50 %, which proved that the light intensity is maintained within ±15% from the average light intensity.

The test chemical available in the test medium (Acetone + natural water) was determined by a validated GC method. The test chemical concentration of test chemical in the test medium at the initiation (0 hour) was between 98.26 % and 99.04 % and the end of test (96 hours) was between 98.55 % and 99.92 % for 2.7 and 89.58 mg/L of the nominal test concentrations. The concentration of the test chemical has been satisfactorily maintained within 80 to 120% of the nominal concentration during the exposure period. Based on the results of this study, the ErCso (percent inhibition of growth rate) value for 72 hours of the test chemical was found to be 39.76 mg/L with 95% confidence limits between 32.88 mg/L and 46.64 mg/L. The EyCs (percent inhibition of yield) value for 72 hours of the test chemical was found to be 13.32 mg/L with 95% confidence limits between 11.30 mg/L and 15.34 mg/L. The LOEC and NOEC (inhibition of growth rate and yield) value for 72 hours was 15.55 mg/L and 6.48 mg/L. The results observed in this study meets all the validity criteria as per OECD 201 test guideline. Based on the ErC50 value, it can be concluded that the substance is likely to be hazardous to aquatic algae and can be considered to be classified as aquatic chronic category 3 as per the CLP classification criteria.

Description of key information

The study was conducted to assess the effects of the test chemical on the growth of green Algae, Pseudokirchneriella subcapitata. This study was performed in compliance with OECD Guideline for Testing of chemicals OECD NO. 201, Adopted by the Council on 23 March 2006 and Council Regulation (EC) No. 440/2008, Annex Part C.3 'Freshwater Algae and Cyanobacteria, Growth Inhibition Test. Solubility of the test item was performed by weighing 103.0 mg of the test chemical in a glass vial and dissolving it in 10 µL of acetone and transferred it into the 100 ml volumetric flask and made up to the mark using natural water for solubility check. The stability of the test chemical in natural water determined by analyzing the test concentrations of 1 and 100 mg/L at O hour, 24 hours, 48 hours and 72 hours showed that the test chemical concentration remained 80% to 120% (98.27 % to 100.75 % for 1 mg/Land 98.87 % to 99.51 % for 100 mg/L) with respect to initial measured concentration and hence the dose verification for definitive test was performed at the beginning and at the termination of the test. The test concentrations were prepared in OECD TG 201 medium by dilution of stock and transferred 100 ml of each test solution into 250 ml Erlenmeyer flasks. Three replicates per test item concentration and six replicates for the controls were used for both range finding test and definitive test. The controls and treatment flasks were inoculated with Algae, Pseudokirchneriella subcapitata (pre-cultured for 3 days) and tested at an initial cell density of 10000 cells per ml, incubated under test condition for 72 hours The study was performed by a static method. The cells were counted using an Improved Neubaur's Haemocytometer under illumination of the microscope at 24, 48 and 72 hour after inoculation. A range finding test was conducted at test chemical concentrations of 5, 10, 25, 50 and 100 mg/L along with control groups without test chemicals. No significant changes in the appearance of algae cells were observed in controls and in the test concentrations during 72 hours test period. At the end of 72-hour observation, 1.95 %, 3.36 %, 5.40 %, 27.50 %, and 85.34 % in terms of the percent inhibition of growth rate and 7.27 %, 12.18 %, 18.86 %, 66.01 % and 98.23 % % in terms of the percent inhibition of yield was observed in the test concentrations of 5, 10, 25, 50 and 100 mg/L respectively. Based on the results of the range finding test, the definitive test was conducted at test chemical concentrations of 2.70, 6.48, 15.55, 37.32 and 89.58 mg/L in a geometric series with a factor of 2.4 along with control groups without test chemical. No significant changes in the appearance of algae cells were observed in controls and in the test concentrations during 72 hours test period. At the end of 72-hour observation, 1.69 %, 9.32 %, 18.74 %, 35.20 % and 84.73 % in terms of the percent inhibition of growth rate and 6.58 %, 31.44 %, 53.37 %, 76.60 % and 98.38 % % in terms of the percent inhibition of yield was observed in the test concentrations of 2.70, 6.48, 15.55, 37.32 and 89.58 mg/L respectively. During the Definitive test period, all the flasks were incubated in the room under test condition. The pH of the solvent control at the test start was 8.0 and at the termination of the test was 8.1 and therefore did not vary more than 1.5 units during the study. The pH of all the tested concentrations was 7.7 to 7.8 at the beginning of the test and 7.5 to 7.6 at test termination. The room temperature (Average) was between 21.3°C and 22.8°C. The temperature of the control and test concentration at the beginning was between 22.3°C and 22.8C and at test termination was between 22.5°C and 23.0°C. The average intensity of light ranged from 6716 to 6734 Lux and the % CV ranged from 0.29 % to 1.50 %, which proved that the light intensity is maintained within ±15% from the average light intensity. The test chemical available in the test medium (Acetone + natural water) was determined by a validated GC method. The test chemical concentration of test chemical in the test medium at the initiation (0 hour) was between 98.26 % and 99.04 % and the end of test (96 hours) was between 98.55 % and 99.92 % for 2.7 and 89.58 mg/L of the nominal test concentrations. The concentration of the test chemical has been satisfactorily maintained within 80 to 120% of the nominal concentration during the exposure period. Based on the results of this study, the ErCso (percent inhibition of growth rate) value for 72 hours of the test chemical was found to be 39.76 mg/L with 95% confidence limits between 32.88 mg/L and 46.64 mg/L. The EyCs (percent inhibition of yield) value for 72 hours of the test chemical was found to be 13.32 mg/L with 95% confidence limits between 11.30 mg/L and 15.34 mg/L. The LOEC and NOEC (inhibition of growth rate and yield) value for 72 hours was 15.55 mg/L and 6.48 mg/L. The results observed in this study meets all the validity criteria as per OECD 201 test guideline. Based on the ErC50 value, it can be concluded that the substance is likely to be hazardous to aquatic algae and can be considered to be classified as aquatic chronic category 3 as per the CLP classification criteria.

Key value for chemical safety assessment

EC50 for freshwater algae:

39.76 mg/L

EC10 or NOEC for freshwater algae:

6.48 mg/L

Additional information

The study was conducted to assess the effects of the test chemical on the growth of green Algae, Pseudokirchneriella subcapitata. This study was performed in compliance with OECD Guideline for Testing of chemicals OECD NO. 201, Adopted by the Council on 23 March 2006 and Council Regulation (EC) No. 440/2008, Annex Part C.3 'Freshwater Algae and Cyanobacteria, Growth Inhibition Test. Solubility of the test item was performed by weighing 103.0 mg of the test chemical in a glass vial and dissolving it in 10 µL of acetone and transferred it into the 100 ml volumetric flask and made up to the mark using natural water for solubility check. The stability of the test chemical in natural water determined by analyzing the test concentrations of 1 and 100 mg/L at O hour, 24 hours, 48 hours and 72 hours showed that the test chemical concentration remained 80% to 120% (98.27 % to 100.75 % for 1 mg/Land 98.87 % to 99.51 % for 100 mg/L) with respect to initial measured concentration and hence the dose verification for definitive test was performed at the beginning and at the termination of the test. The test concentrations were prepared in OECD TG 201 medium by dilution of stock and transferred 100 ml of each test solution into 250 ml Erlenmeyer flasks. Three replicates per test item concentration and six replicates for the controls were used for both range finding test and definitive test. The controls and treatment flasks were inoculated with Algae, Pseudokirchneriella subcapitata (pre-cultured for 3 days) and tested at an initial cell density of 10000 cells per ml, incubated under test condition for 72 hours The study was performed by a static method. The cells were counted using an Improved Neubaur's Haemocytometer under illumination of the microscope at 24, 48 and 72 hour after inoculation. A range finding test was conducted at test chemical concentrations of 5, 10, 25, 50 and 100 mg/L along with control groups without test chemicals. No significant changes in the appearance of algae cells were observed in controls and in the test concentrations during 72 hours test period. At the end of 72-hour observation, 1.95 %, 3.36 %, 5.40 %, 27.50 %, and 85.34 % in terms of the percent inhibition of growth rate and 7.27 %, 12.18 %, 18.86 %, 66.01 % and 98.23 % % in terms of the percent inhibition of yield was observed in the test concentrations of 5, 10, 25, 50 and 100 mg/L respectively. Based on the results of the range finding test, the definitive test was conducted at test chemical concentrations of 2.70, 6.48, 15.55, 37.32 and 89.58 mg/L in a geometric series with a factor of 2.4 along with control groups without test chemical. No significant changes in the appearance of algae cells were observed in controls and in the test concentrations during 72 hours test period. At the end of 72-hour observation, 1.69 %, 9.32 %, 18.74 %, 35.20 % and 84.73 % in terms of the percent inhibition of growth rate and 6.58 %, 31.44 %, 53.37 %, 76.60 % and 98.38 % % in terms of the percent inhibition of yield was observed in the test concentrations of 2.70, 6.48, 15.55, 37.32 and 89.58 mg/L respectively. During the Definitive test period, all the flasks were incubated in the room under test condition. The pH of the solvent control at the test start was 8.0 and at the termination of the test was 8.1 and therefore did not vary more than 1.5 units during the study. The pH of all the tested concentrations was 7.7 to 7.8 at the beginning of the test and 7.5 to 7.6 at test termination. The room temperature (Average) was between 21.3°C and 22.8°C. The temperature of the control and test concentration at the beginning was between 22.3°C and 22.8C and at test termination was between 22.5°C and 23.0°C. The average intensity of light ranged from 6716 to 6734 Lux and the % CV ranged from 0.29 % to 1.50 %, which proved that the light intensity is maintained within ±15% from the average light intensity. The test chemical available in the test medium (Acetone + natural water) was determined by a validated GC method. The test chemical concentration of test chemical in the test medium at the initiation (0 hour) was between 98.26 % and 99.04 % and the end of test (96 hours) was between 98.55 % and 99.92 % for 2.7 and 89.58 mg/L of the nominal test concentrations. The concentration of the test chemical has been satisfactorily maintained within 80 to 120% of the nominal concentration during the exposure period. Based on the results of this study, the ErCso (percent inhibition of growth rate) value for 72 hours of the test chemical was found to be 39.76 mg/L with 95% confidence limits between 32.88 mg/L and 46.64 mg/L. The EyCs (percent inhibition of yield) value for 72 hours of the test chemical was found to be 13.32 mg/L with 95% confidence limits between 11.30 mg/L and 15.34 mg/L. The LOEC and NOEC (inhibition of growth rate and yield) value for 72 hours was 15.55 mg/L and 6.48 mg/L. The results observed in this study meets all the validity criteria as per OECD 201 test guideline. Based on the ErC50 value, it can be concluded that the substance is likely to be hazardous to aquatic algae and can be considered to be classified as aquatic chronic category 3 as per the CLP classification criteria.