reaction mass of [(1R*,5S*)-3,3,5-trimethylcyclohexyl] acetate and [(1S*,5S*)-3,3,5-trimethylcyclohexyl] acetate

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:

02 - 06 Mar 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Remarks:

Staatliches Gewerbeaufsichtsamt Hildesheim, Hildesheim, Germany

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 3.125, 6.25, 12.5, 12.5, 25.0, 50.0, 100% of a saturated stock solution (analysed at 0 and 72 h)
- Sample storage conditions before analysis: Samples were analysed directly.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution of 40 mg/L was freshly prepared before start of exposure. 43.5 µL test item/L was directly introduced into a measuring flask using a pipette and complete filled with dilution water. The stock solution was stirred at approx. 1100 rpm for 2 h at room temperature.
- Eluate: no
- Differential loading: no
- Controls: yes, test medium control
- Evidence of undissolved material: Presence of undissolved test item during preparation of test concentrations and during the test was not observed. The stock solution was checked with a laser beam (Tyndall effect) for undissolved test item.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: SAG 61.81
- Source (laboratory, culture collection): Sammlung von Algenkulturen (SAG) Pflanzenphysiologisches Institut der Universität Göttingen, Göttingen, Germany
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar in the laboratory (light intensity 35-70 µE*m-2*s-1 for 24 h/day.
- Age of inoculum (at test initiation): 3 d (pre-culture prepared in dilution water)

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 - 23 °C

pH:

8.11 - 8.19 (0 h)
8.25 - 9.36 (72 h)

Nominal and measured concentrations:

nominal: control, 3.125, 6.25, 12.5, 12.5, 25.0, 50.0, 100%
measured:

Details on test conditions:

TEST SYSTEM
- Test vessel: headspace flasks with aluminium top and PTFE seals
- Type: sealed
- Material, size, headspace, fill volume: glass, 119 mL, headspace: 0 mL, fill volume: 119 mL
- Aeration: constantly shaken on a rotary shaker (70 rpm)
- Initial cells density: 5087 cells/mL
- Control end cells density: 585148 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, according to guideline

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous light
- Light intensity and quality: 87.3 (mean) µE*m-2*s-1, CV [%]: 6.51;Fluorescent tubes, OSRAM L 36 W/11-865, cool daylight

EFFECT PARAMETERS MEASURED
- Chlorophyll measurement: Chlorophyll-a measurement was used for determination of cell density. Daily measurement at an excitation of 436 nm, emission at 685 nm. Dilution water was used as a background signal. No self-fluorescence was found in the definitive test at the test item concentration of 32.2 mg/L.
- Other: Microscopic observations of cells at the beginning and end of the study (unusual cell shape, color differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells were observed).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
- Test concentrations: 0.21, 2.10, 21.0 mg/L (nominal)
- Results used to determine the conditions for the definitive study: 29% inhibition of growth at 21 mg/L (nominal), 2% inhibition of growth at 2.10 mg/L (nominal), 5% inhibition of growth at 0.210 mg/L (nominal). The measured concentrations after 72 h were 10.3 mg/L, 1.10 mg/L and 0.018 mg/L.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

5.06 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 4.34 - 5.88 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

15.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 14.7 - 16.8 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.32 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CI: 1.47 - 3.12 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6.23 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CI: 5.32 - 7.64 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): In the highest test concentration the algae cells appeared rounder and paler than in the control.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: ErC50 (72 h) 0.749 mg/L (with headspace), 0.774 mg/L (without headspace); EyC50 (72 h) 0.282 mg/L (with headspace), 0.502 mg/L (without headspace)

Reported statistics and error estimates:

The ECx values with confidence intervals were calculated by sigmoidal dose-response regression. The NOEC was determined by calculation of statistically significant differences of growth rates and yield. As a standard, one way analysis of variance (ANOVA) and Dunnett’s test were used for NOEC calculation. When running an ANOVA, a normality test and an equal variances test were done first. P-values for the normality and equal variances test are 0.05. The alpha value is 0.05.

Table 1: Inhibition of growth an yield after 72 h

Geometric mean of measured test concentrations [mg/L]	Replicate	Growth rate [d-1]	Inhibition of growth rate [%]	Yield [cells/mL]	Inhibition of yield [%]
32.2	1	< LOQ	< LOQ	< LOQ	< LOQ
	2	< LOQ	< LOQ	< LOQ	< LOQ
	3	< LOQ	< LOQ	< LOQ	< LOQ
	Mean	n.a.	n.a.	n.a.	n.a.
14.0	1	0.84	47	58151	90
	2	0.89	44	68857	88
	3	0.93	41	77675	87
	Mean	0.89	44	66228	88
5.53	1	1.44	9	382379	34
	2	1.40	11	335370	42
	3	1.46	8	398874	31
	Mean	1.44	9	372208	36
3.94	1	1.39	12	326244	44
	2	1.43	10	364033	37
	3	1.45	8	390532	33
	Mean	1.42	10	360270	38
1.80	1	1.57	0	567214	2
	2	1.57	1	558904	4
	3	1.63	-3	660228	-14
	Mean	1.59	-1	595449	-3
1.00	1	1.54	2	517728	11
	2	1.58	0	578002	0
	3	1.59	0	592302	-2
	Mean	1.57	1	562677	3
Control	1	1.58		583772
	2	1.55		520425
	3	1.57		553792
	4	1.59		591456
	5	1.60		607888
	6	1.61		623035
	Mean	1.58		580061

Table 2: Measured concentrations after 0 and 72 h.

Nominal	Measured after 0 h [mg/L]	Measured after 0 h [%]	Measured after 72 h [mg/L]	Measured after 72 h [%]
40	31.5	79	32.9	82
20	14.3	72	13.6	68
10	6.59	66	4.64	46
5	4.15	83	3.74	75
2.5	2.02	81	1.60	64
1.25	1.12	90	0.90	72
Control	< LOQ		< LOQ

Validity criteria fulfilled:

yes

Conclusions:

This study resulted in an ErC50 (72 h) of 15.7 mg/L and an ErC10 (72 h) of 5.06 mg/L (72 h) (geom. mean measured concentration) based on inhibition of growth rate of P. subcapitata (OECD 201, GLP).

Description of key information

ErC10 (72 h): 5.06 mg/L based on inhibition of growth of P. subcapitata (OECD 201)
ErC50 (72 h): 15.7 mg/L based on inhibition of growth of P. subcapitata (OECD 201)

Key value for chemical safety assessment

EC50 for freshwater algae:

15.7 mg/L

EC10 or NOEC for freshwater algae:

5.06 mg/L

Additional information

One experimental study is available investigating the effects of the substance to freshwater algae. The study was performed according to OECD 201 (GLP) under static conditions using Pseudokirchneriella subcapitata as test organism. Nominal test concentrations of 6.25, 12.5, 25.0, 50.0 and 100% of a saturated stock solution as well as a control were tested. The actual exposure concentrations were verified by GC/FID analysis at the beginning and after 72 h. Measured test item concentrations did not remain within ± 20% of the nominal concentrations. Thus, the effect concentrations were based on the geometric mean of the measured concentrations. At the end of the study an ErC10 (72 h) of 5.06 mg/L (95% CI: 4.34 - 5.88 mg/L) and an ErC50 (72 h) of 15.7 mg/L (95% CI: 14.7 - 16.8 mg/L) were calculated.