Reaction mass of 1-[(1R*,6S*)-2,2,6-trimethylcyclohexyl]hexan-3-ol and 1-[(1S*,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:

23 - 26 Jan 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

One study is available, in which the toxicity of test item to aquatic algae was assessed according to OECD 201 and GLP.
In a static test, the unicellular freshwater green alga P. subcapitata at an initial cell density of 6305 cells/mL was exposed to five concentration levels in a geometric series with a dilution factor of approx. 3.16 for 72 h. The test media were prepared by dilution of a saturated solution with a nominal loading level of 2.00 mg/L test item to obtain treatment levels of 1.00, 3.16, 10.0, 31.6 and 100% of the saturated solution. Test item concentrations were analytically verified by GC-MS at the beginning (0 h) and end (72 h) of exposure.
At 0 h, the measured test item concentrations ranged from 3.20 – 1074 µg/L. After 72 h, the recovery rates ranged from < LOQ to 87% of the initial, measured values. Therefore, the geometric mean measured concentrations were calculated and used to express effect concentrations. The corresponding geometric mean measured test item concentrations calculated for the treatment levels were 1.79, 11.6, 63.5, 200, and 1000 µg/L. After 72 h of exposure, neither morphological abnormalities nor significant growth inhibition was recorded. The derived ErC10 (72 h) and ErC50 (72 h) are both > 1000 µg/L (geom. mean measured), corresponding to > 100% of the saturated solution.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Staatliches Gewerbeaufsichtsamt Hildesheim, Hildesheim, Germany (03 Jan 2017)

Analytical monitoring:

yes

Remarks:

GC-MS

Details on sampling:

- Concentrations: Control and all concentration levels were analytically verified at the start and end of exposure (0 and 72 h): 1.00, 3.16, 10.0, 31.6, and 100% of saturated solution (nominal loading 2.00 mg/L)
- Sampling method: At the beginning of exposure, separate replicates for each test item concentration and control were prepared with algae. The samples for 72 hours were prepared with algae and incubated under test conditions.
- Sample storage conditions before analysis: All 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 test item/L was prepared with dilution water by stirring for 30 min at 30 °C (1100 rpm) and then for 30 min (1100 rpm) at room temperature. The saturated solution was taken from the bottom and was used for testing.
- Differential loading: No, the saturated solution was used as stock solution for the preparation of four further dilution levels in a geometric series with a dilution factor of ca. 3.16 to prepare test solutions at 1.00, 3.16, 10.0, 31.6, and 100% of the saturated solution.
- Controls: Dilution water without test item.
- Evidence of undissolved material: No undissolved material was detected in the saturated solution (negative Tyndall effect).

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Unicellular freshwater green alga
- Age of inoculum (at test initiation): 3 d old preculture, prepared in dilution water.
- Method of cultivation: Fresh stocks are prepared monthly on Z-agar and kept at 2590 - 5180 lux (35 - 70 µE*m^-2*s^-1) for 24 h/d. Algae are cultured in nutrien medium Z, according to Lüttge et al. (1994).

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol Ca+Mg/L

Test temperature:

22.3 °C (mean)

pH:

8.14 - 8.27 (0 h)
9.41 - 9.62 (72 h)

Nominal and measured concentrations:

Control, 1.0, 3.16, 10.0, 31.6, and 100% of saturated solution (nominal)
Control, 1.79, 11.6, 63.5, 200, and 1000 µg/L (geom. mean measured)

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile, 59 mL headspace flasks with aluminium tops and PTFE seals
- Initial cell density: 6305 cells/mL
- Control end cells density: 566630 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Yes, nutrient medium Z according to Lüttge et al. 1994

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD TG 201 medium, according to the guidelines
- Culture medium different from test medium: Algae were cultured in nutrient medium Z and the test was conducted with dilution water (OECD 201 medium).
- Intervals of water quality measurement: pH values were measured at the start (in one additional replicate) and end of exposure (from pooled samples). Room temperature was measured continuously. Light intensity was measured before test start.

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Photoperiod: 24 h light/d
- Light intensity and quality: 4440 - 8880 lux (60 - 120 µE*m^-2*s^-1)
- Other: The test flasks were randomly placed on a rotary shaker, oscillated at approx. 70 rpm and repositioned daily.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations (chlorophyll a- fluorescence): After 0, 24, 48, and 72 h using a fluorometer (Microplate Reader Chameleon V, Hidex, with an excitation of 436 nm and emission of 685 nm)
- Microscopic evaluation: At 0 and 72 h
- Other: No auto-fluorescence of the saturated solution (100%) was detected in the preliminary range-finding test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Approx. 3.16
- Range finding study: A non-GLP preliminary range finding test was conducted in a closed bottle system without headspace under static conditions.
- Test concentrations: 1.00, 10.0, and 100% of saturated solution
- Results used to determine the conditions for the definitive study: Yes. After 72 h, the inhibition of growth rate was 8% at the 100% of saturated solution concentration level and 1% in the 1.00% of saturated solution concentration level.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

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

Remarks on result:

other:

Remarks:

concentration corresponding to 100% of sat. solution

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

Remarks on result:

other:

Remarks:

concentration corresponding to 100% of sat. solution

Details on results:

- Exponential growth in the control: Yes, cell growth increased 90-fold (specific growth rate: 1.50 per day)
- Observation of abnormalities: No morphological abnormalities were recorded
- Any stimulation of growth found in any treatment: Yes, at the treatments levels with geom. mean measured concentrations of 1.79, 11.6, and 63.5 µg/L
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No undissolved test item was detected in the saturated solution.
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes, the reference item test with potassium dichromate produced results in the valid range of 0.782 ± 0.534 mg/L (ErC50).
- ErC50 (72 h): 0.435 mg/L (with headspace), 0.811 mg/L (without headspace)

Reported statistics and error estimates:

EC10 and EC20 values of yield inhibition were calculated by straight line. EC10, EC20, and EC50 values for growth rate and yield were determined directly from results.
FITTING RESULTS GROWTH INHIBITION AFTER 72 HOURS
1) One Way Analysis of Variance:
Normality Test (Shapiro-Wilk): passed (p = 0.676); Equal Variance Test: passed (p = 0.829)
The differences in the mean values among the treatment groups are greater than would be expected by chance ( p < 0.001).
2) Multiple Comparisons versus Control Group (Dunnett's Method)
Significant difference for the geometric mean meausred test item concentrations of 63.5 and 1000 µg/L compared to the control. The statistical significance of the concentration 63.5 µg/L was caused by growth stimulation.

VALIDITY CRITERIA

The study met the validity criteria of the guideline (Table 1).

Table 1: Validity criteria for OECD 201.

Criterion from the guideline	Outcome	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	90-fold (specific growth rate 1.50 day^-1)	Yes
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%	11.9%	Yes
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.	1.76%	Yes

 

 ANALYTICAL RESULTS

The measured concentrations of the test item were 3.20 – 1074 mg/L at the start of the exposure (0 h) and < LOQ to 87% of initial values at the end of exposure (72 h) (Table 2). Therefore, exposure concentrations were expressed as the calculated geometric mean measured concentrations.

Table 2. Measured concentrations, percentage of the initial concentrations and geometric mean of the test item in fresh medium (0 h) and old medium (72 h) with algae.

dilution level of the saturated solution [%]	meas. conc. [µg/L]	meas. conc. [µg/L]	%	Geometric mean measured concentration [µg/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	<LOQ

Meas. Conc.       = measured concentration of the test item

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

LOQ                   = limit of quantification (2 µg/L)

1)                        = values < LOQ were taken into account with ½ LOQ

2)                        = reanalyzed with new dilution factor

 

BIOLOGICAL RESULTS

Growth rate and yield inhibition is summarized in Table 3.

Table 3. Evaluation after 72 hours. Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-).

geometric mean measured test item concentration [µg/L]	replicate n°	growth rate [d^-1]	inhibition of growth rate [%]	yield [cells/mL]	inhibition of yield [%]
1000	1 2 3	1.42	5	441946	21
		1.37	8	381929	32
		1.38	8	388103	31
	mean	(+) 1.39	7	(+) 403993	28
200	1 2 3	1.45	3	482258	14
		1.48	2	519863	7
		1.47	2	514535	8
	mean	(-)1.47	2	(-) 505552	10
63.5	1	1.58	-5	709469	-27
	2	1.58	-6	717362	-28
	3	1.51	-1	582980	-4
	mean	(+)* 1.56	-4	(+)* 669937	20
11.6	1	1.53	-2	613538	-10
	2	1.59	-6	732557	-31
	3	1.52	-1	595694	-6
	mean	(-) 1.55	-3	(-) 647263	-16
1.79	1	1.55	_4	661997	-18
	2	1.52	-1	589070	-5
	3	1.58	-6	723987	-29
	mean	(-) 1.55	-4	(-) 658351	-18
Control	1	1.50		557412
	2	1.51		572578
	3	1.45		477240
	4	1.52		589746
	5	1.51		575933
	6	1.52		589041
	mean	1.50		560325

*  = statistically significant due to growth stimulation

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Conclusions:

The test resulted in both an ErC10 (72 h) and ErC50 (72 h) of > 1000 µg/L (> 100% of the saturated solution, OECD 201, P. subcapitata), indicating that the substance does not cause inhibitory effects on cell growth of unicellular green algae up to the saturation level.

Description of key information

No effects up to saturation level (ErC10 (72 h) and ErC50 (72 h) > 1000 µg/L (geom. mean measured, 100% saturated solution, OECD 201, P. subcapitata)

Additional information

One study is available, in which the toxicity of test item to aquatic algae was assessed according to OECD 201 and GLP.

In a static test, the unicellular freshwater green alga P. subcapitata at an initial cell density of 6305 cells/mL was exposed to five concentration levels in a geometric series with a dilution factor of approx. 3.16 for 72 h. The test media were prepared by dilution of a saturated solution with a nominal loading level of 2.00 mg/L test item to obtain treatment levels of 1.00, 3.16, 10.0, 31.6 and 100% of the saturated solution. Test item concentrations were analytically verified by GC-MS at the beginning (0 h) and end (72 h) of exposure.

At 0 h, the measured test item concentrations ranged from 3.20 – 1074 µg/L. After 72 h, the recovery rates ranged from < LOQ to 87% of the initial, measured values. Therefore, the geometric mean measured concentrations were calculated and used to express effect concentrations. The corresponding geometric mean measured test item concentrations calculated for the treatment levels were 1.79, 11.6, 63.5, 200, and 1000 µg/L. After 72 h of exposure, neither morphological abnormalities nor significant growth inhibition was recorded. The derived ErC10 (72 h) and ErC50 (72 h) are both > 1000 µg/L (geom. mean measured), corresponding to > 100% of the saturated solution.