Reaction mass of 1-[rac-(1R,6S)-2,2,6-trimethylcyclohexyl]pentan-3-ol isomer 1 and 1-[rac-(1S,6S)-2,2,6-trimethylcyclohexyl]pentan-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:

from 2018-07-05 to 2019-01-08

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:

Duplicate samples were taken from each treatment at test start (samples without algae) and at the end of the test (samples with algae). For sampling at the end of test, the replicates were pooled. Immediately after sampling, 1 mL of internal standard was added to each sample, which was then stored frozen (at -20 ± 5 °C).

Vehicle:

no

Details on test solutions:

A Water Accommodated Fraction (WAF) was prepared by direct addition of the test item to the test water to obtaine the required concentration. Using slow-stirring method the test solutions were prepared in fully filled vessels closed with glass stoppers and stirred for 96 hours before the start of the main test. They were slowly stirred in the dark at room temperature.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: No. 61.81 SAG
- Source: Collection of Algal Cultures, SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen, Germany
- Age of inoculum (at test initiation): 3 days

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.15 mmol/L (= 15 mg/L as CaCO3)

Test temperature:

23 °C

pH:

7.6 - 7.8

Nominal and measured concentrations:

Water Accommodated Fractions (WAFs) with the loading rates of 0.10, 0.32, 1.0, 3.2 and 10 mg/L
Measured concentration: at the beginning 0.094, 0.27, 0.80, 3.6 and 4.0 mg/L, at the end 60 to 121 % of the initially measured values

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type: tightly sealed with glass stoppers
- Material, size, headspace, fill volume: glass, 60 mL, no headspace, completely filled
- Aeration: no
- Initial cells density: 5000 cells/mL
- Control end cells density: 110.76E04 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: AAP medium prepared according to the guidelines

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: yes, to 7.5 with 1 M sodium hydroxide solution
- Photoperiod: continuous illumination
- Light intensity and quality: 69 µE/sm², LED lights

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: electronic particle counter
- Determination of biomass: fluorescence measurement at an excitation of 440 nm and emission of 680 nm

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: 3 replicates per concentration and control.
- Test concentrations: 0.1, 1.0, 10 and 100 mg/L

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

0.61 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CI: 0.53 – 0.71 mg/L

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 32 % inhibition was observed at the loading rate of 10 mg/L

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

0.17 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

yield

Remarks on result:

other: 95 % CI: 0.14 – 0.20 mg/L

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

1.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

yield

Remarks on result:

other: 95 % CI: 1.1 – 1.4 mg/L

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

< 0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

<= 0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

Since the test item consists of several constituents with different solubility, the absolute amount of test item could not be analytically determined and the measured test item concentrations should be regarded as approximated values. Therefore, all reported biological results were based on the loading rates of the test item.

Results with reference substance (positive control):

The 72-hour EC50 for growth rate in the reference test was 1.3 mg/L (April 2018).

Growth rate

At the loading rates of 0.10 and 0.32 mg/L the mean inhibition compared to the control was 2.1 and 2.0 %, respectively. This statistical significance (results of Williams’ t-test, one-sided smaller, α = 0.05, Table 2) was caused by the very low variability between replicates within these loading rates and the control (coefficient of variation of 1.6, 0.9 and 1.3 %, respectively). However, this statistically significant finding was not considered as a biologically relevant toxic effect, since the mean inhibition compared to the control was below 10 %. Moreover, the EL10 for growth rate was calculated to be 0.61 mg/L. At the higher loading rate of 1.0 to 10 mg/L, the mean inhibition compared to the control was in the range of 14 to 32 % and was statistically significantly different from the control. Therefore, the 72-hours NOELR for growth rate was determined to be at the loading rate of 0.32 mg/L.

Table 1: Average Growth Rates (µ)

Treatment/ Loading Rate [mg/L]	Average Growth Rate μ[day-1] and Inhibition Ir [%]
	0-24 h		0-48 h		0-72 h
	μ [1/day]	Ir [%]	μ [1/day]	Ir [%]	μ [1/day]	Ir [%]
Control	1.327	0.0	1.640	0.0	1.683	0.0
0.10	1.289	2.9	1.615	1.5	1.648(*)	2.1
0.32	1.170	11.9	1.591	3.0	1.649(*)	2.0
1.0	1.131	14.8	1.387*	15.4	1.454*	13.6
3.2	0.943*	29.0	1.186*	27.7	1.190*	29.3
10	0.892*	32.8	1.083*	34.0	1.142*	32.1

*: Mean value statistically significantly lower than in the control (according to Williams’ t-test one-sided smaller, α = 0.05)

(*): Mean value statistically significantly different from the control due to very low variability of results, however not estimated as a biologically relevant toxic effect, see Section 7 (according to Williams t-test, one-sided smaller, α = 0.05).

Yield

At the loading rates of 0.10 to 10 mg/L, the mean inhibition compared to the control was in the range of 9.8 to 81 % and was statistically significantly different from the control (results of Williams’ t-test, one-sided smaller, α = 0.05, Table 3). The EL10 for this endpoint was calculated to be 0.17 mg/L. Therefore, the72-hours NOELR for yield was determined to be lower than the lowest tested loading rate of 0.10 mg/L.

 Table 2. Yield (Y)

Treatment/ Loading Rate [mg/L]	Yield Y (x 104) and Inhibition Iy [%]
	0-24 h		0-48 h		0-72 h
	Y	Iy [%]	Y	Iy [%]	Y	Iy [%]
Control	2.02	0.0	18.26	0.0	110.52	0.0
0.10	1.92	5.0	17.40	4.7	88.62*	9.9
0.32	1.59	21.2	16.49*	9.7	99.72*	9.8
1.0	1.50	25.9	10.74*	41.2	55.23*	50.0
3.2	1.12*	44.7	6.94*	62.0	24.60*	77.7
10	1.03*	49.2	5.55*	69.9	21.27*	80.8

*: Mean value statistically significantly lower than in the control (according to Williams’ t-test one-sided smaller, α = 0.05)

The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the highest loading rate of 10 mg/L and the algal cells in the control. The shape and size of the algal cells were obviously not affected by the test item up to at least this loading rate. All test media were clear solutions throughout the test period.

Table 3. Analytical results for Test Samples

Sampling Day/ Sample Age [d/h]	Water Accommodated Fraction Loading Rate mg Test Item/L	Measured Concentration of Test Item x	Sample Preparation Factor F	Determined Concentration of Test Item c [mg/L]	% of Initially Measured [%]
0/0 (fresh)	Control 0.10 0.32 1.0 3.2 10.0	n.d. 0.940 2.65 7.97 36.0 40.3	0.1 0.1 0.1 0.1 0.1 0.1	<LOQ 0.0940 0.265 0.797 3.60 4.03
3/72 (aged)	Control 0.10 0.32 1.0 3.2 10.0	n.d. 5.69 7.95 6.02 28.8 38.7	0.02 0.02 0.02 0.1 0.1 0.1	<LOQ 0.114 0.159 0.602 2.88 3.87	121 60 76 80 96

LOQ: 0.0432 mg test item / L

n.d. = not detected

n.a. = not applicable

The tabulated values of the samples represent rounded results obtained by calculation using the exact raw data.

Validity criteria fulfilled:

yes

Conclusions:

The EL50 of the test item after 72-h exposure was determined to be >10 mg/L and respectively the EL10 was 0.61 mg/L based on the growth rate.

Executive summary:

The impact of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72-hour static test according to the OECD 201 and the Commission Regulation (EC) No 761/2009, C.3. In order to assess the toxicity of the test item as a complex mixture containing different sparingly soluble compounds to algae, Water Accommodated Fractions (WAFs) with the loading rates of 0.10, 0.32, 1.0, 3.2 and 10 mg/L were tested. Additionally, a control group (test water without test item) was tested in parallel. As the test item is a volatile substance, the test was performed using glass stoppered Erlenmeyer flasks (closed system) completely filled with test medium, minimizing the air space in the flasks and avoiding potential losses of test item by evaporation. Slow-stirring method for the preparation of the test item solutions was used. The solutions were stirred slowly at room temperature for 96 hours. At the start of the test, the measured concentrations in the test media with the loading rates of 0.10, 0.32, 1.0, 3.2 and 10 mg/L were 0.094, 0.27, 0.80, 3.6 and 4.0 mg/L, respectively. At the end of the test, 60 to 121 % of the initially measured values were determined.

Since the test item consists of several constituents with different solubility, the absolute amount of test item could not be analytically determined and the measured test item concentrations should be regarded as approximated values. Therefore, all reported biological results were based on the loading rates of the test item. The EL50 of the test item after 72-h exposure was determined to be >10 mg/L and respectively the EL10 was 0.61 mg/L based on the growth rate. Based on yield the EL50 was determined to be 1.2 mg/L and respectfully the EL10 was determined to be 0.17 mg/L.

Description of key information

The EL50 of the test item after 72-h exposure was determined to be >10 mg/L and respectively the EL10 was 0.61 mg/L based on the growth rate.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

0.61 mg/L

Additional information

The impact of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72-hour static test according to the OECD 201 and the Commission Regulation (EC) No 761/2009, C.3. In order to assess the toxicity of the test item as a complex mixture containing different sparingly soluble compounds to algae, Water Accommodated Fractions (WAFs) with the loading rates of 0.10, 0.32, 1.0, 3.2 and 10 mg/L were tested. Additionally, a control group (test water without test item) was tested in parallel. As the test item is a volatile substance, the test was performed using glass stoppered Erlenmeyer flasks (closed system) completely filled with test medium, minimizing the air space in the flasks and avoiding potential losses of test item by evaporation. Slow-stirring method for the preparation of the test item solutions was used. The solutions were stirred slowly at room temperature for 96 hours. At the start of the test, the measured concentrations in the test media with the loading rates of 0.10, 0.32, 1.0, 3.2 and 10 mg/L were 0.094, 0.27, 0.80, 3.6 and 4.0 mg/L, respectively. At the end of the test, 60 to 121 % of the initially measured values were determined.

Since the test item consists of several constituents with different solubility, the absolute amount of test item could not be analytically determined and the measured test item concentrations should be regarded as approximated values. Therefore, all reported biological results were based on the loading rates of the test item. The EL50 of the test item after 72-h exposure was determined to be >10 mg/L and respectively the EL10 was 0.61 mg/L based on the growth rate. Based on yield the EL50 was determined to be 1.2 mg/L and respectfully the EL10 was determined to be 0.17 mg/L.