Reaction mass of (1RS)-1-[(1RS)-3,3-dimethylcyclohexyl]ethyl [(2RS,3RS)-3-ethyloxiran-2-yl]acetate and (1RS)-1-[(1RS)-3,3-dimethylcyclohexyl]ethyl [(2SR,3SR)-3-ethyloxiran-2-yl]acetate and (1RS)-1-[(1SR)-3,3-dimethylcyclohexyl]ethyl [(2RS,3RS)-3-ethyloxiran-2-yl]acetate and (1RS)-1-[(1SR)-3,3-dimethylcyclohexyl]ethyl [(2SR,3SR)-3-ethyloxiran-2-yl]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:

22-01-2015 to 28-02-2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, 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)

Remarks:

inspected June 2013; signature: November 2013

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All relevant concentration levels and the control were analytically verified via GC-MS at the start (0 hours), 24 hours and at the end of the exposure (72 hours). Definitive Test: Nominal concentrations: 0 (control), 0.5, 1.0, 2.0, 4.0 and 8.0 mg/L ; equivalent geometric mean measured concentrations: 0 (control), 0.319, 0.632, 1.35, 2.60 and 5.92 mg/L
- Sampling method: Analytical evaluation of the concentrations of the test item were carried out via GC-MS from freshly prepared media after 0 hours (with algae), 24 hours (with algae) and old test media after 72 hours (with algae) of exposure. Three replicates per concentration and six for the control (without test item) were prepared. Separate replicates for each measuring time were prepared. All samples for the test item analysis were taken from additionally prepared replicates with algae. The method was validated prior to this study according to SANCO 3029/99 rev.4 (2000).
- Sample storage conditions before analysis: All original samples were stored at 6 ± 2 °C if necessary. Prepared samples were stored in an autosampler at room temperature until analysis.

Vehicle:

no

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: HINDÁK, SAG 61.81
- Source (laboratory, culture collection): Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen, Germany
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 35-70 µE·m-2·s-1 for 24 hours per day.

ACCLIMATION
- Acclimation period: No. However, a three days old preculture, prepared in dilution water, was used as inoculum.
- Culturing media and conditions (same as test or not): No. Culture Medium: Nutrient medium Z according to LÜTTGE et al. (1994) Botanica Acta, Journal of the German Botanical Society, No. 3 Volume 107 page 111-186 (June 1994), THIEME-VERLAG.
Dilution water: (mg/L) - NH4Cl 15 ; MgCl2.6 H2O: 12 ; CaCl2.2 H2O: 18 ; MgSO4.7H2O: 15 ; KH2PO4: 1.6 ; FeCl3.6H2O: 0.064 ; Na2EDTA.2H2O: 0.1 ; H3BO3: 0.185 ; MnCl2.4H2O: 0.415 ; ZnCl2: 3x10-3 ; Na2MoO4.2H2O: 7x10-3 ; CoCl2.6H2O: 1.5x10-3 ; CuCl2.2H2O: 1x10-5 ; NaHCO3: 50 ; NaHCO3* : 250 ; MES monohydrate*: 2665.6 ; pH 8.1 +/- 0.2. This medium has a nominal hardness of 0.24 mmol Ca+Mg/L.
* additional compounds were added to enable sufficient growth under conditions without headspace.
- Any deformed or abnormal cells observed: None reported.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

Ca+Mg: 0.24 mmol/L

Test temperature:

Nominal range: 21 - 24 °C, controlled at ± 2°C - measured room temperature values were min: 22 ; max 23.0 and mean 22.5°C

pH:

0 hours: pH 8.00 ± 0.1 (and 8.24 in control); 72 hours: pH 9.22-9.35 (definitive test concentrations) and pH 9.51 (controls). pH did not vary more than 1.5 units.

Nominal and measured concentrations:

Preliminary test: 0 (control), 0.08, 0.8, 8.0 mg/L as a nominal test item concentration (non-GLP range finding test)
Definitive Test: Nominal concentrations: 0 (control), 0.5, 1.0, 2.0, 4.0 and 8.0 mg/L ;
equivalent geometric mean measured concentrations: 0 (control), 0.319, 0.632, 1.35, 2.60 and 5.92 mg/L
See table 3 for nominal and measured concentrations at initial exposure and during the course of the test.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass.
- Type: Closed - Static. Sterile headspace flasks
- Material, size, headspace, fill volume: volume: 119 mL, with aluminium tops with PFTE seals. Minimum headspace.
- Aeration: Vessel shaken continuously. Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.
- Initial cells density: nominal: 5 x 10^3 - 10^4 and current: 6153 cells/ml
- Control end cells density: Mean (of replicates after 72 hours) 646676 cells/ml (or ca. x105 increase in cell density)
- No. of vessels per concentration (replicates): 3 replicates of each test concentration; 1 extra replicate of each test group for sampling purposes
- No. of vessels per control (replicates): 6 replicates of the control
- No. of vessels per vehicle control (replicates): Not applicable.

GROWTH MEDIUM
- Standard medium used: Yes. adjusted-Medium M2.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Prepared according to guidelines (see 'Details on test organisms' field for more details on composition).
- Culture medium different from test medium: Yes. (see 'Details on test organisms' field)
- Intervals of water quality measurement: Start and end of the test period.

OTHER TEST CONDITIONS
- Sterile test conditions: No.
- Adjustment of pH: No.
- Photoperiod: 24 hours ; continuous
- Light intensity and quality: 60 - 120 µE/m2/s ; within ± 15 % over incubation area
- Salinity (for marine algae): Not applicable.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- 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 background signal. No self-fluorescence was found at the concentration level of 8.00 mg/L in the range finding test.
- Other: Initial cell density: Microscopic evaluation of the cells was carried out at the start and end of the exposure. The cells were checked for any unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation and adherence of algae to test containers or aggregation of algae cells.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.0. In definitive test justified from the results of the range finding study.
- Justification for using less concentrations than requested by guideline: Not applicable.
- Range finding study: Yes.
- Test concentrations: Two or more replicates per concentration were exposed to dilutions representing 0 (control), 0.08, 0.8, 8.0 mg/L based on nominal test item concentration in a preliminary test. One extra test vessel per concentration without algae was used as background for the determination of the algal cell density at each time interval.
- Results used to determine the conditions for the definitive study: Yes.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 5.92 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% C.I.: - mg/L ; geometric mean measured concentrations

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 5.92 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% C.I.: - mg/L ; based on geometric mean measured concentrations

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 5.92 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% C.I.: - mg/L ; based on geometric mean measured concentrations

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.74 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% C.I.: 0.671 - 3.87 mg/L ; based on geometric mean measured concentrations

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.35 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: based on geometric mean measured concentrations

Details on results:

- Exponential growth in the control (for algal test): Yes.
- Observation of abnormalities (for algal test): Microscopic evaluation of the cells at the start of the incubation period and at test end revealed no morphological abnormalities in the test item concentration or control.
- Unusual cell shape: No.
- Colour differences: None.
- Flocculation: Not reported.
- Adherence to test vessels:
- Aggregation of algal cells: No.
- Other:
- Any stimulation of growth found in any treatment: Yes. At low concentrations 1.00 mg/L nominal test item concentration a growth rate and yield inhibition was negative at 72h. This was low dose stimulation and was limited and was taken into account in the data analysis and effect level calculations.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Not applicable. The measured concentrations of the test item at the start of exposure were in the range of 81-98% and after 72 hours in the range of 47-59% of the nominal values. Therefore, the geometric mean measured concentrations were used for calculations.
- Effect concentrations exceeding solubility of substance in test medium: No.

Results with reference substance (positive control):

- Results with reference substance valid?: Yes.
- EC50: The EC50 for growth rate reduction (ERC50: 0-72h) was 0.774 mg/L with a 95% confidence interval ranging from 0.711 to 1.08 mg/L without headspace. The EC50 for yield inhibition (EYC50: 0-72h) was 0.502 mg/L with a 95% confidence interval ranging from 0.400 to 0.583 mg/L without headspace.
The results with headspace and without headspace were within the test facility SOPs (historic values).
- Other: The sensitivity of the test system was in agreement with the historical data.

Reported statistics and error estimates:

EC10-, EC20- and EC50-values with confidence intervals of growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression. The NOEC/LOEC were determined by calculation of statistically significant differences of growth rate and yield inhibition. 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 alpha-value (acceptable probability of incorrectly concluding that there is a difference) is alpha = 0.05.

Table 1. Results of the Preliminary Range Finding Test (non GLP, 0 - 72 hours)

Nominal test item concentration [mg/L]	Growth Rate Inhibition [%]	Yield Inhibition [%]
8.00	11	42
0.80	0	-2
0.08	3	14

Negative values = growth stimulation

 

Table 2. Percentage reduction in growth rate and inhibition of yield in the definitive test

Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-)

Nominal test item concentration	Replicate		Growth Rate	Inhibition of Growth Rate		Yield	Inhibition of Yield
[mg/L]			[d-1]	[%]		[Cells/mL]	[%]
5.92	1		1.40	10		405359	37
	2		1.41	9		416712	35
	3		1.45	6		475104	26
	Mean	(+)	1.42	8	(+)	432392	32
2.60	1		1.49	4		527036	18
	2		1.49	4		523587	18
	3		1.51	2		570689	11
	Mean	(+)	1.50	4	(+)	540437	16
1.35	1		1.49	4		525594	18
	2		1.50	3		555636	13
	3		1.52	2		583547	9
	Mean	(-)	1.50	3	(-)	554926	13
0.632	1		1.58	-2		701021	-9
	2		1.59	-3		721186	-13
	3		1.63	-5		817398	-28
	Mean	(-)	1.60	-3	(+)*	746535	-17
0.319	1		1.50	3		549302	14
	2		1.54	1		618482	3
	3		1.52	2		580787	9
	Mean	(-)	1.52	2	(-)	582857	9
Control	1		1.54			618482
	2		1.50			546950
	3		1.60			736803
	4		1.53			606628
	5		1.55			644385
	6		1.58			689889
	Mean		1.55			640523

 * statistical significance caused by stimulation of growth

Table 3. Measured Concentrations and Percentage of the nominal Concentration of the Test Item

Sampling	0 hours Start of the exposure interval		48 hours End of the exposure interval		Geometric Mean Measured
Nominal test item concentration [mg/L]	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%
8.00	7.47 #1	93	4.69	59	5.92	74
4.00	3.54 #1	89	1.91	48	2.60	65
2.00	1.96	98	0.931	47	1.35	68
1.00	0.832	83	0.480	48	0.632	63
0.50	0.407	81	0.250	50	0.319	64
0.00 (Control)	< LOQ		< LOQ		< LOQ

Limit of quantification LOQ = 0.1 mg/L test item

#1 : mean of two replicates

Validity criteria fulfilled:

yes

Conclusions:

The test item 72h-EC50 (growth rate reduction) was greater than 5.92 (C.I. – ) mg/L. The corresponding EC10 was greater than 5.92 (C.I. – ) mg/L and the NOEC was 1.35 mg/L.

Executive summary:

The algal growth inhibition to Pseudokirchneriella subcapitata, was carried out according to OECD TG 201 Freshwater Alga and Cyanobacteria, Growth Inhibition Test and EU Method C.3 guidelines under GLP. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours. The study was conducted under static conditions with an initial cell density of 6153 cells/mL. With regard to the volatility of the test item, glass flasks without headspace were used to reduce losses of the test item. Five test item solutions with a dilution factor of 2 (nominal) and concentrations of 0 (control), 0.5, 1.0, 2.0, 4.0 and 8.0 mg/L were freshly prepared with dilution water. The stock solution was stirred for 2 hours with 1100 rpm at room temperature from stock solution. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits. The concentrations of the test item were analytically verified via GC-MS at test start (0 hours), after 24 hours and at the end (72 hours) of the exposure. The measured of the test item at the start were in the range of 81-98 %. After 72 hours, the measured concentrations recovery ranges were 47-59 %. All effect values given are based on geometric mean measured concentrations of test item. The validity criteria of the test guideline were fulfilled. The EC50 for growth rate reduction (72h-ErC50) was > 5.92 (C.I. – ) mg/L. The corresponding EC10 was > 5.92 (C.I. – mg/L and the NOEC was 1.35 mg/L.

Description of key information

ErC50 (algae; growth rate) = > 5.92 (C.I. - ) mg/L ; based on geometric mean measured concentrations, 72hour-freshwater, OECD TG 201, 2015

ErC10 (algae; growth rate) = > 5.92 (C.I. - ) mg/L ; based on geometric mean measured concentrations, 72hour-freshwater, OECD TG 201, 2015

NOEC (algae; growth rate) = 1.35 mg/L ; based on geometric mean measured concentration, 72hour-freshwater, OECD TG 201, 2015

Key value for chemical safety assessment

EC50 for freshwater algae:

5.92 mg/L

EC10 or NOEC for freshwater algae:

1.35 mg/L

Additional information

Key study : OECD TG 201, 2015 : The algal growth inhibition to Pseudokirchneriella subcapitata, was carried out according to OECD TG 201 Freshwater Alga and Cyanobacteria, Growth Inhibition Test and EU Method C.3 guidelines under GLP. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours. The study was conducted under static conditions with an initial cell density of 6153 cells/mL. With regard to the volatility of the test item, glass flasks without headspace were used to reduce losses of the test item. Five test item solutions with a dilution factor of 2 (nominal) and concentrations of 0 (control), 0.5, 1.0, 2.0, 4.0 and 8.0 mg/L were freshly prepared with dilution water. The stock solution was stirred for 2 hours with 1100 rpm at room temperature from stock solution. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits. The concentrations of the test item were analytically verified via GC-MS at test start (0 hours), after 24 hours and at the end (72 hours) of the exposure. The measured of the test item at the start were in the range of 81-98 %. After 72 hours, the measured concentrations recovery ranges were 47-59 %. All effect values given are based on geometric mean measured concentrations of test item. The validity criteria of the test guideline were fulfilled. The EC50 for growth rate reduction (72h-ErC50) was > 5.92 (C.I. – ) mg/L. The corresponding EC10 was > 5.92 (C.I. – mg/L and the NOEC was 1.35 mg/L.