Reaction mass of 1-(3,3-dimethylcyclohexyl)ethyl acetate and 2,6,6-trimethylcycloheptanol 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:

March 25 - June 13, 2019

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)

Version / remarks:

2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Series on Testing and Assessment No. 23 (Guidance Document on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals

Version / remarks:

2nd edition; 2019

GLP compliance:

yes (incl. QA statement)

Remarks:

Swiss Federal Office of Public Health, Consumer protection directorate, Notification authority for chemicals, Bern, Switzerland

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The control and loading rates of 0.32, 1.0, 3.2, 10, 32 and 100 mg/L were analysed analytically. The analytical samples of the lowest concentration with the loading rate 0.10 mg/L were not analyzed as this loading rate was below the NOEC determined in this test and thus not relevant for the interpretation of the biological results.
- Sampling method: Duplicate samples without algae were taken from each loading rate and the control at the start of the test. At the end of the test (after 72 h), samples (containing algae) were taken in duplicate from all loading rates and from the control. For sampling from the aged test media, the contents of the respective replicates were combined prior to sampling.
- Sample storage conditions before analysis: All samples were stored frozen (at -20 ± 5 °C) immediately after sampling.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Water Accommodated Fractions (WAFs) with individual dispersions of the test item with the loading rates of 1.0, 3.2, 10, 32 and 100 mg/L were prepared. Thereafter the dispersions were stirred for 3 hours to dissolve the different components of the test item as far as possible within a reasonable time period. After stirring, the dispersions were let to settle for 30 minutes and thereafter the lower phases were filtered through a 0.45 µm membrane filter. The filter was saturated by at least 200 mL test medium and the negative pressure of the filtration unit was reduced to a minimum to avoid losses of the volatile test item. These undiluted filtrates were tested as WAFs. Due to technical reasons (operational constraints on the precision of direct pipetting of test item), the WAFs with the lower loading rates of 0.32 mg/L and 0.10 mg/L were prepared as dilutions of the WAF with the loading rate of 1.0 mg/L.
- Eluate: no
- Differential loading: yes
- Controls: yes, test water without test item

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: 61.81 SAG
- Source (laboratory, culture collection): supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, Germany)
- Age of inoculum (at test initiation): four days
- Method of cultivation: An inoculum culture was set up four days before the start of the exposure. The algae were cultivated under the test conditions. The inoculum culture was diluted threefold one day before the start of the test to ensure that the algae were in the exponential growth phase when used to inoculate the test solutions.

ACCLIMATION
- Acclimation period:
- Culturing media and conditions (same as test or not): same as test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

test water: 0.15 mmol/L (= 15 mg/L as CaCO3)

Test temperature:

23 °C

pH:

control: 7.5 - 7.7
test media: 7.5 - 7.6

Nominal and measured concentrations:

nominal loading rates: control, 0.1, 0.32, 1.0, 3.2, 10, 32, 100 mg/L
measured (0 h): < LOQ, not measured, 0.24, 0.71, 1.9, 5.1, 11, 19 mg/L
measured (72 h): < LOQ, not measured, 0.13, 0.46, 1.5, 3.8, 10, 16 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type: closed
- Material, size, headspace, fill volume: glass, without headspace, 60 mL
- Aeration: constantly shaken
- Initial cells density: 5000 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: according to guideline

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: LED light, approx. 75 μE s-1 m-2

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter (Cell Counter CASY TT, OLS, Bremen, Germany) for cell number in pre-culture. The algal biomass in the samples was determined by fluorescence measurement (SpectraMax I3x, Molecular Devices Ltd, Wokingham Berkshire/UK). The measurements were performed at least in duplicate at an excitation of 440 nm and emission of 680 nm.
- Cell shape: The shape and size of the algal cells were visually inspected.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations: control, 0.1, 1.0, 10, 100 mg/L loading rate
- Results used to determine the conditions for the definitive study: 0, 0.8, 2.3, 17, 97 % inhibition of average growth after 72 h

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

11 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CI: 10-12 mg/L

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

52 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CI: 50-53 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:

EL10

Effect conc.:

1.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95 % CI: 1.4-1.8

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

9.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95 % CI: 9.2-10

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.32 mg/L

Nominal / measured:

nominal

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): The microscopic examination of the algal cells at the highest test concentration of 100 mg/L at the end of the test showed a disfiguration of most of the algal cells as compared to the control. However, no difference was determined between the algae growing at the loading rates of 32 and 10 mg/L and the algal cells in the control.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: 1.1 mg/L (tested May 2019)
- Other: sensitivity of the test system was within the range recommended by the guideline (72-hour EC50 for the growth rate 0.92 1.5 mg/L)

Reported statistics and error estimates:

The 72-hour EL10, EL20 and EL50 values for the inhibition of average growth rate and yield and their 95% confidence intervals were calculated by Probit Analysis using linear maximum likelihood regression. For the determination of the LOEL and NOEL, the average growth rate and yield at the test concentrations were compared to the control values by Williams t-test and Welch t test after Bonferroni-Holm-adjustment, where appropriate. Statistical analysis was performed using ToxRat Professional®.

Table 1: Inhibition of growth (µ)

Treatment Loading Rate	Average Growth Rate µ [day^-1] and Inhibition Ir[%]
	0-24 h		0-48 h		0-72 h
[mg/L]	µ [day^-1]	Ir[%]	µ [day^-1]	Ir[%]	µ [day^-1]	Ir[%]
Control	1.787	0.0	1.828	0.0	1.790	0.0
0.10	1.829	-2.3	1.803	1.4	1.771	1.1
0.32	1.802	-0.8	1.811	0.9	1.773	1.0
1.0	1.764	1.3	1.809	1.0	1.747*	2.4
3.2	1.558*	12.8	1.712^#	6.4	1.722*	3.8
10	1.251*	30.0	1.506^#	17.6	1.566*	12.5
32	1.214*	32.1	1.200^#	34.4	1.242*	30.6
100	0.585*	67.3	0.465^#	74.6	0.486*	72.9

*: Mean value statistically significantly lower than in the control (according to Williams t-test one-sided smaller,a= 0.05).

#: Mean value statistically significantly lower than in the control (according to Welch t-test, one-sided smaller,a= 0.05).

Table 2: Inhibition of Yield (Y)

Treatment Loading Rate	Yield Y (x 10^4) and Inhibition Iy[%]
	0-24 h		0-48 h		0-72 h
[mg/L]	Y	Iy[%]	Y	Iy[%]	Y	Iy[%]
Control	3.71	0.0	28.01	0.0	158.83	0.0
0.10	3.91	-5.5	26.56	5.2	149.81	5.7
0.32	3.75	-1.2	27.02	3.5	150.67	5.1
1.0	3.60	3.1	26.88	4.0	139.29^#	12.3
3.2	2.79*	24.7	22.02^#	21.4	129.32^#	18.6
10	1.89*	49.0	14.35^#	48.8	80.61^#	49.2
32	1.79*	51.8	7.47^#	73.3	30.07^#	81.1
100	0.59*	84.1	1.17^#	95.8	2.46^#	98.5

*: Mean value statistically significantly lower than in the control (according to Williams t-test one-sided smaller,a= 0.05)

#: Mean value statistically significantly lower than in the control (according to Welch t-test, one-sided smaller,a= 0.05).

Table 3: Measured concentrations of WAF loading rates

Sampling Day/ Sample Age [d/h]	Water Accomodated 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	Control	n.d.	0.1	<LOQ	-
(fresh)	0.32	2.39	0.1	0.239	-
	1.0	7.12	0.1	0.712	-
	3.2	19.3	0.1	1.93	-
	10	50.9	0.1	5.09	-
	32	107	0.1	10.7	-
	100	186	0.1	18.6	-
3/72	Control	n.d.	0.1	<LOQ	n.a.
(aged)	0.32	1.34	0.1	0.134	56
	1.0	4.55	0.1	0.455	64
	3.2	14.6	0.1	1.46	76
	10	38.3	0.1	3.83	75
	32	103	0.1	10.3	96
	100	161	0.1	16.1	86

LOQ: 0.0400 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 72-hour EL10 and EL50 values for the inhibition of growth rate were found to be 52 and 11 mg/L, respectively. Since WAFs were tested, all effect values were based on the nominal loading rates of the test item.

Description of key information

ErL10 (72 h) = 11 mg/L (nominal, OECD 201)
ErL50 (72 h) = 52 mg/L (nominal, OECD 201)

Key value for chemical safety assessment

EC50 for freshwater algae:

52 mg/L

EC10 or NOEC for freshwater algae:

11 mg/L

Additional information

One experimental study is available investigating the toxicity of the test item to aquatic algae. The impact of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72-hour test according to OECD 201 (2011) under GLP conditions. Since the test item is a complex mixture consisting of several constituents with differently low solubility, Water Accommodated Fractions (WAFs) with the loading rates of 0.10, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L were tested to assess the toxicity of the test item to algae. A control group (test water without test item) was tested in parallel. For preparation of the WAFs, individual dispersions of the test item with the loading rates of 1.0, 3.2, 10, 32 and 100 mg/L were prepared. Thereafter the dispersions were stirred for 3 hours to dissolve the different components of the test item as far as possible within a reasonable time period. After stirring, the dispersions were let to settle for 30 minutes and thereafter the lower phases were filtered through a 0.45 µm membrane filter. These undiluted filtrates were tested as WAFs. Due to technical reasons (operational constraints on the precision of direct pipetting of test item), the WAFs with the lower loading rates of 0.32 mg/L and 0.10 mg/L were prepared as dilutions of the WAF with the loading rate of 1.0 mg/L. The test item gave a chromatographic profile consisting of several peaks. The analytical measurements of the soluble components were based on the sum of these peaks. At the start of the test, the measured concentrations of the analyzed components of the test item in the test media with loading rates of 0.32, 1.0, 3.2, 10, 32 and 100 mg/L were 0.24, 0.71, 1.9, 5.1, 11 and 19 mg/L, respectively. The measured concentrations at the end of the test were in the range of 56 and 96 % of the initially measured values. The 72-hour EL10 and EL50 values for the inhibition of growth rate were found to be 52 and 11 mg/L, respectively. Since WAFs were tested, all effect values were based on the nominal loading rates of the test item. The preparation of the WAFs and procedure of evaluation of data of WAFs is based on recommendations of the OECD Guidance Document No. 23 on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals, 2019.