Methyl 4(or 1)-isopropyl-1(or 4)-methylbicyclo[2.2.2]oct-5-ene-2-carboxylate

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:

2017

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)

Version / remarks:

2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.1, 0.316, 1.00, 3.16, 10.0, 31.6 and 100 % dilution level of the saturated solution and control were analytically verified
- Sampling method: Samples taken at 0 hours (without algae) and 72 hours (with algae)
- Sample storage conditions before analysis: Samples were stored at 6 ± 2 ºC

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Two stability tests were conducted prior to the main study. In the definitive test, a saturated solution with a nominal loading of 14 mg/L test item was prepared by placing the test item on to the surface of the dilution water with a pipette. The saturated solution was stirred at 1100 rpm with a magnetic stirrer at room temperature for 24 ± 1 hour. After completion of the stirring the saturated solution was removed from the homogenous phase, from the bottom of the glass and was used for testing. Stock solution diluted with test water to create test series.
- Eluate: Dilution water
- Controls: OECD TG 201 medium only
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): None
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): Not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): The saturated solution was checked after stirring via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion), which was negative

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: Hindak SAG 16.81
- Source (laboratory, culture collection): Commercial supplier
- Age of inoculum (at test initiation): 4 days old
- Method of cultivation: Fresh stocks prepared monthly at test facility using nutrient medium

ACCLIMATION
- Acclimation period: Not applicable
- Culturing media and conditions (same as test or not): Fresh stocks are prepared every month on Z-Agar at a light intensity of 2590 - 5180 lux for 24 hours per day.
- Any deformed or abnormal cells observed: None reported

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Nominal: 0.24 mmol Ca+Mg/L.

Test temperature:

22 - 23 ºC

pH:

Test start: 8.01 - 8.14
Test termination: 8.08 - 9.60

Dissolved oxygen:

Not reported

Nominal and measured concentrations:

Nominal: 0.100, 0.316, 1.00, 3.16, 10.0, 31.6 and 100 % saturated solution
Measured (0 hours): 0.0131, 0.0401, 0.124, 0.414, 1.3, 4.21, 15.1 mg/L
Measured (72 hours): 0.0136, 0.0443, 0.134, 0.483, 1.44, 4.18, 13.8 mg/L

Details on test conditions:

TEST SYSTEM

- Test vessel: Sterile headspace flasks with aluminium tops
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 59 mL
- Aeration: None reported
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable
- Renewal rate of test solution (frequency/flow rate): Not applicable
- Initial cells density: 6305 cells/mL
- Control end cells density: 628200 (mean of 6 replicates)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): Not applicable

GROWTH MEDIUM

- Standard medium used: yes
- Detailed composition if non-standard medium was used: Not applicable

TEST MEDIUM / WATER PARAMETERS

- Source/preparation of dilution water: OECD 201 medium
- Total organic carbon: Not reported
- Particulate matter: Not reported
- Metals: As per OECD TG 201 medium method
- Pesticides: None reported
- Chlorine: As per OECD TG 201 medium method
- Alkalinity: Not reported
- Ca/mg ratio: Not reported
- Conductivity: Not reported
- Culture medium different from test medium: Yes
- Intervals of water quality measurement: pH measured at start and end of the test

OTHER TEST CONDITIONS

- Sterile test conditions: yes
- Adjustment of pH: None reported
- Photoperiod: Continuous illumination
- Light intensity and quality: 4440 – 8880 lux
- Salinity (for marine algae): Not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Cell density daily, microscopic evaluation of cells at start and end of test
- Chlorophyll measurement: Measured daily via Chlorophyll a-fluorescence

TEST CONCENTRATIONS

- Spacing factor for test concentrations: Dilution factor of 3.16
- Justification for using less concentrations than requested by guideline: Not applicable
- Range finding study: Yes
- Test concentrations: 0.1, 1, 10 and 100 % saturated solution
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

13.8 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % confidence interval: 8.93 - > 15.1 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.78 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % confidence interval: 2.01 - 3.35 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.37 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95% confidence interval: 2.52 - 4.45 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.71 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95 % confidence interval: 1.15 - 2.40 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.3 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: Inhibition of growth rate and yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): None
- Any stimulation of growth found in any treatment: Yes
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: ErC50 with headspace: 0.435 mg/L (95% CI: 0.414 - 0.458 mg/L); ErC50 without headspace: 0.811 mg/L (95% CI: 0.763 - 0.883 mg/L); EyC50 with headspace: 0.230 mg/L (95% CI: 0.179 - 0.322 mg/L); EyC50 without headspace: 0.387 mg/L (95% CI: 0.347 - 0.428 mg/L)

Reported statistics and error estimates:

EC10-, EC20- and EC,50-values with confidence intervals of growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression.

The NOEC and LOEC were determined by calculation of statistically significant differences of growth rates and yield by One Way Analysis of Variance (ANOVA) (α =0.05) and Dunnett’s test (α =0.05). A normality test (p = 0.05) and an equal variance test (p = 0.05) were performed prior to the ANOVA analysis.

Summary of analytical measurements:

Dilution level of the saturated solution (%)	Initial measured Concentration (mg/L)	72 hour measured concentration	72 hour recovery rate (%)
100	15.1	13.9	92
31.6	4.21	4.18	99
10.0	1.30	1.44	111
3.16	0.414	0.483	117
1.00	0.124	0.134	108
0.316	0.0401	0.0443	111
0.100	0.0131	0.0136	104
Control	< LoQ	<LoQ

Evaluation of growth rate and yield at 72 hours

Initial measured concentration (mg/L)	Replicate No.	Growth rate (d^-1)	Inhibition of growth rate (%)	Yield (cells/mL)	Inhibition of yield (%)
15.1	1	0.853	44	75245	88
	2	0.725	53	49256	92
	3	0.675	56	41487	93
	Mean	0.751*	51	55329*	91
4.21	1	1.25	18	264253	58
	2	1.19	22	217570	65
	3	1.18	23	212073	66
	Mean	1.21*	21	231299	63
1.30	1	1.55	-1	661563	-6
	2	1.58	-3	711741	-14
	3	1.47	4	510718	18
	Mean	1.53	0	628007	-1
0.414	1	1.58	-3	707090	-14
	2	1.59	-4	736746	-18
	3	1.56	-2	671373	-8
	Mean	1.58	-3	705070	-13
0.124	1	1.52	1	593963	4
	2	1.56	-2	675940	-9
	3	1.48	3	532819	14
	Mean	1.52	1	600907	3
0.0401	1	1.60	-5	770658	-24
	2	1.62	-6	800399	-29
	3	1.61	-5	793577	-28
	Mean	1.61	-5	788211	-27
0.0131	1	1.62	-6	807305	-30
	2	1.62	-6	813282	-31
	3	1.57	-2	694912	-12
	Mean	1.60	-5	771833	-24
Control	1	1.54	N.A.	640984	N.A.
	2	1.58	N.A.	711713	N.A.
	3	1.54	N.A.	640392	N.A.
	4	1.54	N.A.	625959	N.A.
	5	1.51	N.A.	581870	N.A.
	6	1.48	N.A.	530451	N.A.
	Mean	1.53	N.A.	621895	N.A.

N.A. = not applicable; * statistically significantly different

Validity criteria fulfilled:

yes

Remarks:

Increase of cell growth in control >16 fold (100 fold); mean coefficient of variation section by section specific growth rates <35% (27.9%); coefficient of variation average specific growth rate in controls <7% (2.14%)

Conclusions:

The 72-hour EyC50 and EyC10 were calculated to be 3.37 mg test item/L and 1.71 mg test item/L, respectively. The 72-hour ErC50 and ErC10 values were calculated to be 13.8 mg test item/L and 2.78 mg test item/L, respectively

Executive summary:

The toxicity of the test item to the green algae Pseudokirchneriella subcapitata was determined in a study following OECD guideline 201. Algae were exposed to 0.100, 0.316, 1.00, 3.16, 10, 31.6 and 100 % dilutions of a saturated solution, equivalent to initial measured concentrations of 0.0131, 0.0401, 0.124, 0.414, 1.30, 4.21 and 15.1 mg test item/L alongside a control. The 72 -hour ErC50 and ErC10 values were calculated to be 13.8 mg test item/L and 2.78 mg test item/L, respectively. The 72-hour EyC50 and EyC10 were calculated to be 3.37 mg test item/L and 1.71 mg test item/L, respectively.

The study is a GLP compliant, guideline experimental study available as an unpublished report. There are no restrictions and it is fully adequate for assessment (Klimsch 1).

Description of key information

The 72 -hour ErC50 and ErC10 values were calculated to be 13.8 mg test item/L and 2.78 mg test item/L, respectively. The 72-hour EyC50 and EyC10 were calculated to be 3.37 mg test item/L and 1.71 mg test item/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

3.37 mg/L

EC10 or NOEC for freshwater algae:

1.71 mg/L

Additional information

The toxicity of the test item to the green algae Pseudokirchneriella subcapitata was determined in a study following OECD guideline 201 (2017). Algae were exposed to 0.100, 0.316, 1.00, 3.16, 10, 31.6 and 100 % dilutions of a saturated solution, equivalent to initial measured concentrations of 0.0131, 0.0401, 0.124, 0.414, 1.30, 4.21 and 15.1 mg test item/L alongside a control. The 72 -hour ErC50 and ErC10 values were calculated to be 13.8 mg test item/L and 2.78 mg test item/L, respectively. The 72-hour EyC50 and EyC10 were calculated to be 3.37 mg test item/L and 1.71 mg test item/L, respectively.

The study is a GLP compliant, guideline experimental study available as an unpublished report. There are no restrictions and it is fully adequate for assessment (Klimsch 1).