(R)-5-isopropyl-2-methylcyclohexa-1,3-diene

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:

2017-07-17 to 2017-08-18

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

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling method: 10 mL of the test item concentrations and the control were filled into a vial.
- Sample storage conditions before analysis: All samples were stored at 6 +- 2 °C until sample preparation and at room temperature until the start of the analysis, if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A saturated solution with a nominal loading of 4.37 mg/L test item was prepared with dilution water one day prior to the start of exposure. This dispersion was stirred for 24 hours with 1100 rpm at room temperature. After a separation phase of 1 hour, the saturated solution was removed by siphoning (from the approximate bottom of the flask) and used in the test.
- Controls: Negative control with test water but witout test item; positive control with the reference substance potassium dichromate
- Evidence of undissolved material: The solution was checked after stirring via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion). The Tyndall effect was negative.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: SAG 61.81
- Source: Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen
- Age of inoculum: 4 days old preculture, prepared in dilution water
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 2590 - 5180 lux for 24 hours per day

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol Ca+Mg/L (nominal, test medium)

Test temperature:

22.5 °C (mean)

pH:

8.06 to 9.42

Nominal and measured concentrations:

Nominal loading [mg/L]: 4.37; 0 (control), 2.56, 6.40, 16.0, 40.0, and 100% of the saturated solution
Geometric mean measured test item concentrations [µg/L]: 491, 125, 53.0, 21.7, 7.29, 0 (control)

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile headspace flasks
- Type: closed
- Volume, seals: Nominal volume = 59 mL; fill volume = 59 mL; with aluminium tops with PTFE seals
- Aeration: No, but agitation on a rotary shaker and oscillated at approx. 70 rpm
- Initial cells density:
Nominal: Approx. 5 x 10^3 - 10^4 cells/mL
Actual: 6839 cells/mL
- No. of vessels per concentration: 3
- No. of vessels per control: 6

GROWTH MEDIUM
- Standard medium used: Yes (OECD TG 201 medium), but additionally compounds were added to enable sufficient growth under conditions without headspace (NaHCO3, MES monohydrate)

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: No
- Photoperiod: 24 hours/day light
- Light intensity and quality: Approx. 4440 to 8880 lux, within +- 15% over incubation area

EFFECT PARAMETERS MEASURED:
- Chlorophyll measurement: Chlorophyll a -fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal.
- Other: Microscopic evaluation for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.5
- Range finding study: Yes
- Test concentrations: 3.10, 0.335 and 0.0309 mg/L (at the start of the range finding test)
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

465 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits: 133 - 491 µg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

264 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits: 51.9 - 491 µg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

147 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

biomass

Remarks on result:

other: 95% confidence limits: 80.2 - 334 µg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

13 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

biomass

Remarks on result:

other: 95% confidence limits: < 7.29 - 47.2 µg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

7.29 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

other: growth rate and biomass

Details on results:

- Exponential growth in the control: Yes
- Observation of abnormalities: No
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Any stimulation of growth found in any treatment: Yes, at the 7.29 µg/L level.
- Effect concentrations exceeding solubility of substance in test medium: Yes, in one replicate at the 7.29 µg/L level (geometric mean measured concentration).

Results with reference substance (positive control):

48h ErC50 (growth rate) = 0.498 mg/L;
48h EyC50 (yield) = 0.320 mg/L

Reported statistics and error estimates:

ECx-values with confidence intervals of grwoth rate and ield inhibition after 72 hours were calculated by sigmoidal dose-response. The NOEC and LOEC were determined by calculation of statistically significant differences of grwoth rates and yield. As a standard, One Way Analysis of Variance (ANOVA) and DUNNETT's test were used for NOEC/LOEC calculations.

Table 1: Measured concentrations and cercentage of the initial concentration of the test item.

Sampling date	2017-08-15 0 h	2017-08-18 72 h		Geometric mean
Start of analysis	15.08.2017	18.08.2017
Dilution level of the saturated solution [%]	TEST ITEM
	Meas. conc. [µg/L]	Meas. conc. [µg/L]	[%]	[µg/L]
100	571	421	74	491
40	159	97.6	61	125
16	66.2	42.4	64	53
6.4	26.8	17.6	66	21.7
2.56	10.6	6.70^1)	63	7.29
Control	< LOQ	< LOQ		< LOQ

Meas. conc. = Measured concentration of the test item

% = Percentage of the nominal concentration (10 µg test item/L)

LOQ = Measured concentration < 70% LOQ, therefore 1/2 LOQ (5 µg test item /L) taken into account for geometric mean

Table 2: Evaluation after 72 hours. Statistically significant differences of growth rate and yield compared to control values are marked (+), not significant diffrerences are marked (-).

Geometric mean measured test item concentration [µg/L]	Replicate No.	Growth rate [d-1]	Inhibition of growth rate [%]	Yiels [cells/mL]	Inhibition of yield [%]
491	1	0.659	58	42582	94
	2	0.662	58	43056	94
	3	0.703	55	49528	93
	Mean	(+) 0.675	57	(+) 45055	94
125	1	1.46	7	535382	27
	2	1.46	7	540707	27
	3	1.46	5	594107	19
	Mean	(+) 1.47	6	(+) 556732	25
53	1	1.44	8	503752	32
	2	1.47	6	549076	26
	3	1.46	7	534826	28
	Mean	(+) 1.45	7	(+) 529218	28
21.7	1	1.39	11	433528	41
	2	1.43	9	487893	34
	3	1.42	9	473468	36
	Mean	(+) 1.41	10	(+) 464963	37
7.29	1	1.52	2	655465	11
	2	1.56	1	718403	3
	3	1.57	0	745703	-1
	Mean	(-) 1.55	1	(-) 706524	4
Control	1	1.56		737042
	2	1.61		843256
	3	1.55		699443
	4	1.55		717467
	5	1.56		734204
	6	1.54		695200
	Mean	1.56		737769

negative values = growth stimulation

Validity criteria fulfilled:

yes

Conclusions:

The 72h ErC10 and the ErC50 (growth rate) of the test item was determined to be 0.264 and 0.465 mg/L, respectively. The 72h EyC10 and the EyC50 (yield) of the test item was determined to be 0.013 and 0.147 mg/L, respectively.

Executive summary:

To assess the ecotoxicological potential of the test item to aquatic algae (Pseudokircheriella subcapitata), an algal inhibition test according to OECD Guideline 201 and EU Method C.1 was carried out in compliance with GLP principles. The study was conducted under static conditions with an initial cell density of 6839 cells/mL. A saturated solution with a nominal loading of 4.37 mg test item /L was freshly prepared with dilution water one day prior to the start of exposure. This saturated solution was stirred for 24 hours with 1100 rpm at room temperature. The saturated solution and four dilution levels out of the stock solution were tested in geometrical with a dilution factor of 2.5. The geometric mean measured concentrations (analyzed by GC-MS) were 491, 125, 53.0, 21.7 and 7.29 µg/L. Three replicates were tested for each test item concentrations and six replicates for a negative control (with test media and inoculum, but without the test item). The suitability of the test system was confirmed by a positive control with the reference substance potassium dichromate. The effect values given are based on geometric mean measured concentration. In result, the 72h ErC10 and the ErC50 (growth rate) of the test item was determined to be 0.264 and 0.465 mg/L, respectively. The 72h EyC10 and the EyC50 (yield) of the test item was determined to be 0.013 and 0.147 mg/L, respectively. The 72h NOEC values for both growth rate and yield after 72 hours were 7.29 µg/L (0.00729 mg/L). For the reference substance, the 48h ErC50 for growth rate and yield was determined to be 0.498 mg/L and 0.320 mg/L, respectively. All validity criteria of the guidelines were fulfilled.

Description of key information

The 72h ErC10 and the ErC50 (growth rate) of the test item was determined to be 0.264 and 0.465 mg/L (geometric mean measured concentration), respectively. The 72h EyC10 and the EyC50 (yield) of the test item was determined to be 0.013 and 0.147 mg/L (geometric mean measured concentration), respectively.  

Key value for chemical safety assessment

EC50 for freshwater algae:

0.465 mg/L

EC10 or NOEC for freshwater algae:

0.264 mg/L

Additional information

To assess the ecotoxicological potential of the test item to aquatic algae (Pseudokirchneriella subcapitata), a valid Algal Inhibition Test according to OECD Guideline 201 and EU Method C.1 was carried out in compliance with GLP principles. The study was conducted under static conditions with an initial cell density of 6839 cells/mL. The geometric mean measured concentrations (analyzed by GC-MS) were 491, 125, 53.0, 21.7and 7.29 µg/L. Three replicates were tested for each test item concentration and six replicates for a negative control (with test media and inoculum, but without the test item). In result, the 72h ErC10 and the ErC50 (growth rate) of the test item was determined to be 0.264 and 0.465 mg/L, respectively. The 72h EyC10 and the EyC50 (yield) of the test item was determined to be 0.013 and 0.147 mg/L, respectively. The 72h NOEC values for both growth rate and yield after 72 hours were 7.29 µg/L (0.00729 mg/L).

The effect values are based on geometric mean measured concentrations.