Reaction mass of 7,7-dimethyl-2-methylidenebicyclo[2.2.1]heptane and (1R)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane and (1S)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane and (1S)-2,6,6-trimethylbicyclo[3.1.1]hept-2-ene

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:

06 January 2018 - 01 February 2018

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:

- Concentrations: 0.0 (control), 0.0 (vehicle control), 10.0, 14.5, 21.0, 30.5 and 44.2 mg/L
- Sampling method: A known volume of sample (10 mL) was taken in a separating funnel and to this 30 mL of dichloromethane was added.
- Sample storage conditions before analysis: not applicable

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A quantity of 100.0, 145.0, 210.0, 305.0 and 442.0 mg was dissolved in acetone and volume was made up to 1 mL with acetone to achieve test concentrations of 100.0 (Stock A), 145.0 (Stock B), 210.0 (Stock C), 305.0 (Stock D) and 442.0 (Stock E) mg/mL. Volume of 12 µL from the stock solutions A, B, C, D and E and 1000 µL algal culture were taken and diluted to 120 mL with sterile algal culture medium in respective flasks to obtain the nominal test concentrations of 10.0, 14.5, 21.0, 30.5 and 44.2 mg test item/L.
- Controls: For control group, only 1000 µL alga culture was transferred using a micropipette and diluted to 120 mL with sterile algal culture medium and for vehicle control group, 12 µL acetone and 1000 µL algal culture were taken and diluted to 120 mL with sterile algal culture medium.
- Chemical name of vehicle: acetone
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 0.1mL/L

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- 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

ACCLIMATION
- Acclimation period: 48 h
- Culturing media and conditions (same as test or not): OECD medium was used. The pre-culture was incubated under the same conditions as those required for the test and were used when growing exponentially (5 x 10E+03 - 10E+04 cells/mL). The temperature for pre-culture ranged between 21-23ºC.
- Any deformed or abnormal cells observed: not reported.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

None

Test temperature:

21-23ºC

pH:

7.50-7.68

Nominal and measured concentrations:

Nominal concentrations: 0.0 (control), 0.0 (vehicle control), 10.0, 14.5, 21.0, 30.5 and 44.2 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: sterile conical flask
- Material, size, headspace, fill volume: glass, 250 mL capacity, 120 mL fill volumen
- Aeration: The cells in the cultured flasks were maintained in suspension by agitating the test flasks continuously at 100 ± 2 rpm, using an orbital shaker during the test period.
- Initial cells density: 5 x 10E+03 - 10E+04 cells/mL
- Control end cells density: 532083 cells/mL (control) and 533333 cells/mL (vehicle control)
- No. of organisms per vessel: 6896 (at 0h)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 6

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

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: the OECD medium was prepared as per the OECD guideline 201.
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Photoperiod: Constant illumination
- Light intensity and quality: 6550 - 6583 lux (± 15% of mean value) obtained with an universal UV-free white fluorescent lamp.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The cell concentration of each replicate was determined once, at a regular interval of 24 h, by manual counting using a haemocytometer (Hausser Scientific, U.S.A.) and a binocular microscope (Nikon)
- Other:
Temperature: measured at 24 h intervals.
Light intensity: measured at 24 h intervals.
pH: measured at the beginning and at the end of the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: geometric factor of 1.45
- Justification for using less concentrations than requested by guideline: Based on immobility results at the highest concentration, i.e. 50 mg test item/L, obtained in a preliminary range finding study.
- Range finding study
- Test concentrations: 0.0 (control), 0.0 (vehicle control), 0.01, 0.1, 1.0, 10.0 and 50.0 mg/L
- Results used to determine the conditions for the definitive study: The percent inhibition of biomass was 1.39, 2.54, 2.82, 3.69 and 93.31, and the percent inhibition of growth rate was 0.00, 0.15, 0.31, 0.15 and 109.91 at the test concentrations of 0.01, 0.1, 1.0, 10.0 and 50.0 mg/L, respectively.

Reference substance (positive control):

yes

Remarks:

(potassium dichromate)

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

16.75 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (95% CL: 14.94 – 18.77)

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

20.53 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (95% CL: 18.83– 22.39)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

30.31 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (95% CL: 28.29 – 32.48)

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

14.12 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

(Yield)

Remarks on result:

other: (95% CL: 13.20 – 15.12)

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

16.19 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

(Yield)

Remarks on result:

other: (95% CL: 15.29 – 17.14)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

21.02 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

(Yield)

Remarks on result:

other: (95% CL: 19.95 – 22.15)

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

14.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: (biomass (cell growth), growth rate and yield)

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: (biomass (cell growth), growth rate and yield)

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): not reported.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none

Results with reference substance (positive control):

- Results with reference substance valid? yes
- 72h-ErC50: 1.29 mg/L (95% CL: 1.12-1.49)
- 72h-EyC50: 0.47 mg/L (95% CL: 0.41-0.54)

Reported statistics and error estimates:

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.
The EC10, EC20, and EC50 were calculated using the Probit analysis method (Finney, 1971).

Table 1: Mean Values of Algal Biomass, Yield, Specific Growth Rate

Group	Test Concentration (mg/L)	Mean Number of Cells Count/mL at					Specific Growth Rate (0 - 3)
		0 h	24 h	48 h	72 h	Yield
G1	0.0 (Control)	6896	28750	119167	532083	525187	1.45
G2	0.0 (vehicle control)		30000	118750	533333	526437	1.45
G3	10.0		29167	115833	532500	525604	1.45
G4	14.5		18333	82500	425833*	418937*	1.37*
G5	21.0		12500	71667	195000*	188104*	1.11*
G6	30.5		6667	34167	59167*	52271*	0.71*
G7	44.2		2500	8333	17500*	10604*	0.31*

*= Significantly lower than control at 1% level (p≤0.01)

Table 2: Percentage (%) Inhibition of Biomass, Growth Rate, and Yield

Group	Test Concentration (mg/L)	Percentage (%) Inhibition of
		Biomass (EbC) 0 - 72 h	Growth Rate (ErC) 0 - 72 h	Yield (EyC)0 - 72 h
G1	0.0 (Control)	-	-	-
G2	0.0 (vehicle control)	-	-	-
G3	10.0	1.05	0.00	0.16
G4	14.5	25.53	5.13	20.42
G5	21.0	58.70	23.18	64.27
G6	30.5	86.64	50.66	90.27
G7	44.2	99.41	78.64	97.99

Note: Growth rate is a logarithmic term. Therefore, EbC and ErC values are not numerically comparable.  All values mentioned above are based on the nominal concentrations selected for the study

Table 3: EC10, EC20 and EC50 Values

Exposure Time (72 h)	EbCxValue for Biomass Inhibition	Concentration (mg/L)	95% Confidence Limits (mg/L)		Regression Equation (y = a + bx)
			Lower Limit	Upper Limit
	EbC10	13.12	12.14	14.18	y = -4.22 + 7.10
	EbC20	15.13	14.18	16.14
	EbC50	19.88	18.85	20.97
Exposure Time (72 h)	ErCxValue for Growth Rate Inhibition	Concentration (mg/L)	95% Confidence Limits (mg/L)		Regression Equation (y = a + bx)
			Lower Limit	Upper Limit
	ErC10	16.75	14.94	18.77	y = -2.37 + 4.98
	ErC20	20.53	18.83	22.39
	ErC50	30.31	28.29	32.48
Exposure Time (72 h)	EyCxValue for YieldInhibition	Concentration (mg/L)	95% Confidence Limits (mg/L)		Regression Equation (y = a + bx)
			Lower Limit	Upper Limit
	EyC10	14.12	13.20	15.12	y = -4.82 + 7.42
	EyC20	16.19	15.29	17.14
	EyC50	21.02	19.95	22.15

Validity criteria fulfilled:

yes

Remarks:

(increase of biomass in control during 72h > 16 fold; coefficient of variation of the mean specific growth rate among replicates in control (t0-t72) < 7%; the mean of the replicate coefficients of variation in section-by-section growth rate < 35%)

Conclusions:

In the Pseudokirchneriella subpicata green algae toxicity test the 72h-ErC50 of the test substance was determined to be 30.31 mg/L.

Executive summary:

An Algae Growth Inhibition Test was performed with Pseudokirchneriella subpicata green algae on the test substance over a period of 72 h in static conditions according to OECD Guideline 201, following GLP. OECD medium was used for algal culturing. In the previous solubility study the test item was found to be insoluble in OECD medium and acetone was selected as the vehicle. A preliminary range finding study, with 2 replicates for test item and 3 replicates for controls, was conducted with the test concentrations of 0.0 (control), 0.0 (vehicle control), 0.01, 0.1, 1.0, 10.0 and 50.0 mg/L, resulting in percent inhibitions of growth rate of 0.00, 0.15, 0.31, 0.15 and 109.91, respectively. Based on these results, the test concentrations selected for the main study were 0.0 (control), 0.0 (vehicle control), 10.0, 14.5, 21.0, 30.5 and 44.2 mg/L (geometric factor 1.45). Three replicates were taken for each concentration of the test item and six replicates for control group and vehicle control group. 1 mL of algal culture was inoculated into the test vessel for each replicate to obtain an initial mean cell concentration of 6986 cells/mL. A validated analytical method based on gas chromatography was used to monitor the concentration and stability of the main active ingredients in the test solution at 0 and 72 h. The active ingredient concentration in the test media was within acceptable limit (> 80% of nominal concentration). All validity criteria were fulfilled. The growth rate inhibition effect concentrations with their respective 95% lower and upper confidence limits for the inhibition of growth rate were ErC10 = 16.75 (14.94 – 18.77) mg/L,  ErC20 = 20.53 (18.83 – 22.39) mg/L and ErC50 = 30.31 (28.29 – 32.48) mg/L. The yield inhibition effect concentrations with their respective 95% lower and upper confidence limits for the yield were EyC10 = 14.12 (13.20 – 15.12) mg/L, EyC20 = 16.19 (15.29 – 17.14) mg/L and EyC50 = 21.02 (19.95 – 22.15) mg/L. The LOEC (72h) and NOEC (72h) for growth rate and yield were 14.5 and 10 mg/L respectively.

Description of key information

Key study. Test method according to OECD 201, GLP study. In the Pseudokirchneriella subpicata green algae toxicity test the 72h-ErC50 of the test substance was determined to be 30.31 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

30.31 mg/L

EC10 or NOEC for freshwater algae:

10 mg/L

Additional information

Key study. An Algae Growth Inhibition Test was performed withPseudokirchneriella subpicatagreen algae on the test substance over a period of 72 h in static conditions according to OECD Guideline 201, following GLP. OECD medium was used for algal culturing. In the previous solubility study the test item was found to be insoluble in OECD medium and acetone was selected as the vehicle. A preliminary range finding study, with 2 replicates for test item and 3 replicates for controls, was conducted with the test concentrations of 0.0 (control), 0.0 (vehicle control), 0.01, 0.1, 1.0, 10.0 and 50.0 mg/L, resulting in percent inhibitions of growth rate of 0.00, 0.15, 0.31, 0.15 and 109.91, respectively. Based on these results, the test concentrations selected for the main study were 0.0 (control), 0.0 (vehicle control), 10.0, 14.5, 21.0, 30.5 and 44.2 mg/L (geometric factor 1.45). Three replicates were taken for each concentration of the test item and six replicates for control group and vehicle control group. 1 mL of algal culture was inoculated into the test vessel for each replicate to obtain an initial mean cell concentration of 6986 cells/mL. A validated analytical method based on gas chromatography was used to monitor the concentration and stability of the main active ingredients in the test solution at 0 and 72 h. The active ingredient concentration in the test media was within acceptable limit (> 80% of nominal concentration). All validity criteria were fulfilled. The growth rate inhibition effect concentrations with their respective 95% lower and upper confidence limits for the inhibition of growth rate were ErC10 = 16.75 (14.94 – 18.77) mg/L,  ErC20 = 20.53 (18.83 – 22.39) mg/L and ErC50 = 30.31 (28.29 – 32.48) mg/L. The yield inhibition effect concentrations with their respective 95% lower and upper confidence limits for the yield were EyC10 = 14.12 (13.20 – 15.12) mg/L, EyC20 = 16.19 (15.29 – 17.14) mg/L and EyC50 = 21.02 (19.95 – 22.15) mg/L. The LOEC (72h) and NOEC (72h) for growth rate and yield were 14.5 and 10 mg/L respectively.