rel-1-[(1R,6S)-2,2,6-trimethylcyclohexyl]hexan-3-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:

16 Jan 2017 - 27 Jan 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:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

03 Jan 2017

Specific details on test material used for the study:

Hysandol called Timberol in the test report may be a multi of the cis and trans-isomer, while Hysandol is a mono-trans isomer. The cis isomer is expected to have the same results for this endpoint because it is stereo isomer.

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control, 1.00, 3.16, 10.0, 31.6, 100% of saturated solution prepared at nominal loading rate of 2.00 mg test item/L.
- Sampling method: All concentration levels and the control were analytically verified via GC-MS after 0 (start of exposure) and 72 hours (end of the exposure). Separate replicates for each test item concentration and control for the test item analysis at the beginning of the exposure were prepared with algae. The samples for the test item analysis after 72 hours were prepared with algae and incubated under test conditions.
- Sample storage conditions before analysis: All original samples were stored at 6 ± 2 °C until sample preparation. Prepared samples were stored at room temperature in an autosampler until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A saturated solution with a nominal loading of 2.00 mg/L test item was prepared once with dilution water. The test item was placed on to the surface of the demineralized water with a pipette. The solution was stirred for 30 minutes at 30 °C (1100 rpm) and then for 30 minutes (1100 rpm) at room temperature. According to the results obtained from the non-GLP preliminary test, the preparation of the saturated solution is considered to be appropriate. The saturated solution was taken from the bottom and was used for testing. The saturated solution was checked after stirring via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion), which was negative.
- The saturated solution and further four dilution levels out of the saturated solution were tested in a geometrical series with a dilution factor of square root 10 (approximately 3.16): 1.00 - 3.16 -10.0 - 31.6 - 100% of the saturated solution.
- Controls: Six replicates (without test item) were incubated under the same conditions as the test concentrations.
- Dilution water: according to OECD TG 201 medium, with addition of NaHCO3 (250 mg/L) and MES monohydrate (2665.6 mg/L) to enable sufficient growth under conditions without headspace.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: HINDAK, SAG 61.81
- Source: Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen Nikolausberger Weg 18, D-37073 Göttingen
- Age of inoculum (at test initiation): a 3 d old preculture, prepared in dilution water, was used as inoculum.
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 2590 - 5180 lux corresponding to 35 - 70 uE m-2 S-1 for 24 hours per day.
- Culture medium: Nutrient medium Z according to Lüttge et al 1994.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Dilution water: 0.24 mmol Ca+Mg/L

Test temperature:

21.5-23 (mean 22.3) °C

pH:

Start: 8.14-8.27
End (72 h): 9.41-9.62

Nominal and measured concentrations:

Nominal: 1.00, 3.16, 10.0, 31.6, 100% of saturated solution with a nominal loading of 2.00 mg/L
Geometric mean measured test item concentrations: 1.79, 11.6, 63.5, 200, 1000 ug/L (See Table 1 in 'Any other information on results incl. tables' for measured concentrations).

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile headspace flasks, volume: 59 mL, with aluminium tops with PTFE seals.
- Aeration: No
- Initial cells density: Nominal: approximately 5 x 10^3 - 10^4 cells/mL; Actual: 6305 cells/mL
- Control end cells density: 566 x 10^3 cells/mL (mean, N=6)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, OECD TG 201 medium

TEST MEDIUM / WATER PARAMETERS
- Test medium: medium according to OECD TG 201
- Culture medium different from test medium: yes, Nutrient medium Z
- Intervals of water quality measurement: The pH-values at the start of exposure were measured in one additional replicate of each test item concentration and the control. At the end of exposure, the pH-values were measured from pooled samples of each test item concentration and the control. The room temperature was measured continuously. Light intensity was measured prior to the start of exposure.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous light
- Light intensity and quality: Approximately 4440 to 8880 lux, corresponding to 60 to 120 uE*m-2*s-1

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. No self-fluorescence was found in the saturated solution (100%) in the preliminary range finding test.
- Other: Microscopic evaluation of the cells at the start and the end of the incubation period was carried out. The cells were checked 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: Square root 10 (approx 3.16)
- Range finding study test concentrations: 1.00, 10.0, 100 % of saturated solution prepared at nominal loading 2 mg/L
- Results used to determine the conditions for the definitive study: yes.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (performed October 2016)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 1 000 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

200 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control: yes
- Microscopic evaluation of the cells at the start and end of exposure revealed no morphological abnormalities.
- See Table 2 in 'Any other information on results incl. tables' for details on growth rate and inhibition per concentration.
- See attached illustration for Cell densities for each concentration at 0-72h.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50 for growth rate inhibition, with headspace: 0.435 mg/L (95% confidence interval 0.414-0.458)
- EC50 for growth rate inhibition, without headspace: 0.811 mg/L (95% confidence interval 0.763-0.883)
- Validity range for EC50: 0.782 ± 0.534

Reported statistics and error estimates:

The NOEC and LOEC were determined by calculation of statistically significant differences of growth rates. As a standard, One Way Analysis of Variance (ANOVA) and Dunnett’s test were used for NOEC/LOEC calculations. When running a One Way Analysis of Variance, a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance tests are 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05.

Table 1: Measured Concentrations, Percentage of the Initial Concentrations and Geometric Mean of TIMBEROL® in Fresh Medium (0 hours) and Old Medium (72 hours) with Algae

Sampling date	2017-01-23 0 hours fresh medium	2017-01-26 72 hours old medium		Geometric mean measured test item concentration [ug/L]
Start of analysis	2017-01-23	2017-01-26
Dilution level of the saturated solution [%]	TIMBEROL®
	Meas. conc. [ug/L]	Meas. conc. [ug/L]	%
100	1074	931	87	1000
31.6	264	151	57	200
10.0	134(2)	30.1	22	63.5
3.16	17.9	7.47	42	11.6
1.00	3.20(2)	<LOQ		1.79(1)
Control	<LOQ	<LOQ

Meas. Conc: measured concentration of the test item, enrichment and dilution factors taken into account

%: percentage of the initial measured concentration of the test item

LOQ: limit of quantification (2 ug test item/L)

(1): values <LOQ were taken into account with ½ LOQ

(2): reanalyzed on 2017-01-26 with new dilution factor

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

Geometric mean measured test item concentration   [ug/L]	Replicate No.	Growth rate [d^-1]	Inhibition of growth rate [%]
1000	1	1.42	5
	2	1.37	8
	3	1.38	8
	Mean	1.39 (+)	7
200	1	1.45	3
	2	1.48	2
	3	1.47	2
	Mean	1.47 (-)	2
63.5	1	1.58	-5
	2	1.58	-6
	3	1.51	-1
	Mean	1.56 (+)*	-4
11.6	1	1.53	-2
	2	1.59	-6
	3	1.52	-1
	Mean	1.55	-3
1.79	1	1.55	-4
	2	1.52	-1
	3	1.58	-6
	Mean	1.55	-4
Control	1	1.50
	2	1.51
	3	1.45
	4	1.52
	5	1.51
	6	1.52
	Mean	1.50

*: statistically significant due to growth stimulation

Validity criteria fulfilled:

yes

Remarks:

See 'Overall remarks' for details on validity criteria.

Conclusions:

Regarding the growth rate, the test item did not cause inhibitory effects higher than 7 % at the saturation level. The EC50 and EC10 for inhibition of growth rate after 72 hours were both > 1000 ug/L. The NOEC-value for inhibition of growth rate after 72 hours was 200 ug/L. All effect levels were based on geometric mean measured test item concentrations.

Executive summary:

In a 72 -h toxicity study conducted according to OECD guideline 201 and GLP principles, freshwater algae (Pseudokirchneriella subcapitata) were exposed to the test item at 1.00, 3.16, 10.0, 31.6 and 100% of a saturated solution prepared at a nominal loading of 2 mg/L. At t=0 the concentrations were: 1074, 264, 134, 17.0 and 3.2 ug/l, respectively. After 72 hours these concentrations decreased to 931, 151, 30.1, 7.47 and < LoQ ug/l, respectively. In view of the decrease with ca 50% (leaving out the highest and the lowest) geometric mean measured concentrations of 1.79, 11.6, 63.5, 200 and 1000µg/L, were used to derive the effect values. The test item did not cause inhibitory effects on the growth rate higher than 7 % at the highest test concentration, but this was statistically significantly different than the control. The NOEC is 200µg/L but not considered biologically relevant, the EC10 and EC50 are both >1000µg/L. The study is considered to be reliable without restrictions.

Description of key information

In a 72 -h toxicity study conducted according to OECD guideline 201 and GLP principles, freshwater algae (Pseudokirchneriella subcapitata) were exposed to the test item at 1.00, 3.16, 10.0, 31.6 and 100% of a saturated solution prepared at a nominal loading of 2 mg/L. At t=0 the concentrations were: 1074, 264, 134, 17.0 and 3.2 ug/l, respectively. After 72 hours these concentrations decreased to 931, 151, 30.1, 7.47 and < LoQ ug/l, respectively. In view of the decrease with ca 50% (leaving out the highest and the lowest) geometric mean measured concentrations of 1.79, 11.6, 63.5, 200 and 1000µg/L, were used to derive the effect values. The test item did not cause inhibitory effects on the growth rate higher than 7 % at the highest test concentration, but this was statistically significantly different than the control. The NOEC is 200µg/L but not considered biologically relevant, the EC10 and EC50 are both >1000µg/L. The study is considered to be reliable without restrictions.