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

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 Jun 2017 - 29 Jun 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

03 January 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, 6.25%, 12.5%, 25.0%, 50.0% and 100% of Saturated Solution of a nominal concentration of 2 mg/L.
- Sampling method: At the start of the exposure and at renewal (0 and 24 hours), samples of the fresh media were taken after preparation of all test item concentrations and analyzed. At renewal and at the end of the exposure (24 and 48 hours), samples of the 24-hours old media were taken directly from the test vessels and analyzed.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until the start of the analysis, if necessary. Prepared samples were stored in the autosampler at room temperature until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A saturated solution with a nominal concentration of 2 mg/L of the test item, which corresponds to 2.22 uL/L of the test item (relative density (D20/4): 0.9001 was taken into account), was prepared one day prior to the start of the exposure (at -24 hours) and one day prior to the renewal of the test solutions (at 0 hours) in a brown glass flask. The glass flask was filled with 40 g glass beads (diameter: 4 mm, supplier: VWR) and up to the top with the dilution water having no headspace (total volume: 2275 mL). The test item was pipetted into the dilution water and the brown glass flask was closed immediately with a screw cap with a septum. This dispersion was shaken for 24 hours with 20 rpm at room temperature. After completion of shaking, the dispersion was allowed to stand for approximately 1 hour for separation of undissolved test item. Thereafter, the saturated solution was removed from the approximate center of the water body. The saturated solution was checked via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion). No Tyndall effect was observed in any approach. The saturated solution was used as the highest concentration level and as a stock solution for the preparation of further dilution levels by dilution with dilution water.
- Dilution water: same as Culture medium.
- Controls: Dilution water without test item incubated under the same conditions as the test groups. The control solution was prepared without test item following the same method as specified for the saturated solution.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain/clone: Daphnia magna STRAUS (Clone 5)
- Source: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany.
- Feeding during test: No
- Age at start of test: Less than 24 hours old daphnids from a healthy stock were used for the study. Juvenile daphnids were removed from the culture vessels at the latest 24 hours before the start of the exposure and discarded. The juveniles born within the following period of max. 24 hours preceding the exposure were used for the test. No first brood progeny was used for the test.
- No acclimatization was necessary, because the composition of the dilution water was equivalent to the culture medium.

CULTURING
- Culture: In glass vessels (2 - 3 L capacity) at Noack Laboratorien GmbH with approximately 1.8 L culture medium, at 20 ± 2 °C, in an incubator, 16 hours illumination, light intensity of max. 1500 lx
- Culture medium: Elendt M4, according to OECD 202, Annex 3 (2004), modified to a total hardness of 160 to 180 mg CaC03/L.
- Feeding: The culture daphnids are fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus, with an algae cell density of > 106cells/mL. The algae are cultured at the test facility.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

165-174 mg CaCO3/L

Test temperature:

19-20 °C

pH:

Fresh media at start: 7.68-7.73
Fresh media at renewal (24 h): 7.46-7.96
Old media at renewal (24 h): 7.62-8.18
Old media at end (48 h): 7.79-8.12

Dissolved oxygen:

Fresh media at start: 8.14-9.22 mg/L
Fresh media at renewal (24 h): 8.67-9.17 mg/L
Old media at renewal (24 h): 4.74-8.23 mg/L
Old media at end (48 h): 7.22-8.43 mg/L

Conductivity:

423-435 uS/cm

Nominal and measured concentrations:

Nominal: 6.25 - 12.5 - 25.0 - 50.0 - 100% of the saturated solution
Geometric mean measured test item concentrations: 0.0760-0.165-0.321 - 0.613 - 1.16 mg/L. See Table 1 in 'Any other information on results incl. tables' for Measured concentrations of the test item during the test.

Details on test conditions:

TEST SYSTEM
- Test vessel: Sealed glass flasks (4.5 (ID) x 9.5 (H) cm) with screw were used and filled up to the top with the test solutions (no headspace). A test volume of approximately 130 mL was provided in each test vessel.
- Aeration: No
- Renewal of test solution: after 24 h. A second set of test vessels were filled up to the top with the freshly prepared test solutions and the daphnids were transferred by pipette.
- No. of organisms per vessel: 5 daphnids
- No. of vessels per concentration (replicates): 4 vessels
- No. of vessels per control (replicates): 4 vessels

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Culture medium, M4.
- Culture medium different from test medium: No
- Intervals of water quality measurement: Prior to the start of the exposure (0 hours) and the renewal of the test solutions (24 hours), the water quality parameters (i.e. pH-value, dissolved oxygen concentration, temperature, conductivity and total hardness) of the dilution water were measured. At the start of the exposure and at renewal (0 and 24 hours), the water quality parameters of the fresh media (i.e. pH-value, dissolved oxygen concentration) were measured in one additional replicate (without daphnids) per concentration level and control. At renewal and at the end of the exposure (24 and 48 hours), the water quality parameters of the old media were measured in all replicates (containing daphnids) per concentration level and control. The incubator temperature was recorded throughout the period of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16/8 hours light/dark cycle
- Light intensity: Diffuse light, light intensity of max. 1500 lx

EFFECT PARAMETERS MEASURED:
- Immobilization was determined in all groups after 24 and 48 hours. An animal was considered immobile, if it was not able to swim in the water phase within 15 seconds after gentle agitation of the test vessel. Other adverse effects did not appear.

RANGE-FINDING STUDY
- Test concentrations: Control, 10% and 100% of saturated solution prepared at a nominal loading rate of 2 mg/L
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate, performed June 2017

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.696 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: None observed
- All tested concentration levels were visually clear throughout the exposure period. No Tyndall effect was observed in the saturated solution (observations directly after preparation at 0 and 24 hours). No immobility or any adverse effects were observed at the two lowest concentration levels (0.0760 and 0.165 mg/L) and in the control.
- See Table 2 in 'Any other information on results incl. tables' for the details on immobility.
- See attached illustration for the Concentration-Effect Relationship curve if the test item after 48 h.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: EC50: 2.05 mg/L (95% confidence limits: 1.80-2.33 mg/L), which falls within the validity range for this clone (0.6-2.4 mg/L according to AQS P 9/2 (02/2000)).

Reported statistics and error estimates:

The EC10- and the EC50-values (after 24 and 48 hours of exposure) were calculated by sigmoidal dose-response regression with the software GraphPad Prism. The respective 95% confidence limits were calculated from the standard error and the t-distribution. All calculations were carried out from the best-fit values with the software GraphPad Prism. The concentration-effect relationships are shown graphically.
The EC50-value for the reference item and its 95% confidence limits were calculated accordingly.

Table 1: Measured Concentrations of the Test Item TIMBEROL® during the Definitive Test

Sampling date	2017-06-27 0 hours Start of the exposure interval	2017-06-28 24 hours End of the exposure interval		2017-06-28 24 hours Start of the exposure interval	2017-06-29 48 hours End of the exposure interval		Geometric mean measured test item concentration [mg/L]
Start of analysis	2017-06-27	2017-06-28		2017-06-28	2017-06-29
Dilution level of the saturated solution [%]	TIMBEROL®
	Meas. conc. [mg/L]	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	Meas. conc. [mg/L]	%
100*	1.50	1.08	72	1.27	0.881(2)	69	1.16
50.0	0.725	0.554	76	0.657	0.535	81	0.613
25.0	0.380	0.298	78	0.359	0.261	73	0.321
12.5	0.186	0.146	79	0.176	0.155	88	0.165
6.25	0.0803(1)	0.0681	85	0.0914	0.0667	73	0.0760
Control	<LOQ	<LOQ		<LOQ	<LOQ

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

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

LOQ: limit of quantification of the analytical method (0.002 mg test item/L)

(1): reanalyzed on 2017-06-28

(2): mean value of two samples, which were analyzed with different dilution factors

*: saturated solution

Table 2: Immobilization Rates after 24 and 48 hours of Exposure in the Definitive Test

(n = 20, divided into 4 replicates with 5 daphnids each)

Geometric mean measured concentration of the test item [mg/L]	IMMOBILIZATION [%]
	24 hours					48 hours
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
1.16	80	80	60	60	70	80	100	100	100	95
0.613	40	40	0	20	25	60	40	20	40	40
0.321	0	0	0	0	0	0	20	20	0	10
0.165	0	0	0	0	0	0	0	0	0	0
0.0760	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

MV: mean value

Validity criteria fulfilled:

yes

Remarks:

See 'Overall Remarks' for details on validity criteria.

Conclusions:

Based on the geometric mean measured concentrations of the test item, the 48 hours- EC50 for Daphnia magna was 0.696 mg/L (95% confidence limits: 0.611 - 0.839 mg/L).

Executive summary:

In a 48-h semi-static acute toxicity test conducted according to OECD guideline 202, Daphnia magna were exposed to the test item at nominal concentrations of 6.25, 12.5, 25.0, 50.0, and 100% of a Saturated Solution prepared at 2 mg/L, corresponding to geometric mean measured concentrations of 0.0760, 0.165, 0.321, 0.613, and 1.16 mg/L. During the 24 hours exposure the substance decreased to ca 70 -80% of the fresh solutions. No immobility was observed in the control and the two lowest concentrations. The 48h-EC50 for Daphnia magna exposed to the test item was 0.696 mg/L based on geometric mean measured concentrations (95% conficence limits: 0.611 -0.839 mg/L). The study is considered to be reliable without restrictions.

Description of key information

In a 48-h semi-static acute toxicity test conducted according to OECD guideline 202, Daphnia magna were exposed to the test item at nominal concentrations of 6.25, 12.5, 25.0, 50.0, and 100% of a Saturated Solution prepared at 2 mg/L, corresponding to geometric mean measured concentrations of 0.0760, 0.165, 0.321, 0.613, and 1.16 mg/L. During the 24 hours exposure the substance decreased to ca 70 -80% of the fresh solutions. No immobility was observed in the control and the two lowest concentrations. The 48h-EC50 for Daphnia magna exposed to the test item was 0.696 mg/L based on geometric mean measured concentrations (95% conficence limits: 0.611 -0.839 mg/L). The study is considered to be reliable without restrictions.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.696 mg/L