Allyl (cyclohexyloxy)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:

30 May - 02 Jun 2016

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

GLP compliance:

yes (incl. QA statement)

Remarks:

Staatliches Gewerbeaufsichtsamt Hildesheim, Germany

Analytical monitoring:

yes

Details on sampling:

- Concentrations: control, 10, 20, 40, 80, and 160 mg/L (nominal)
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until the start of the analysis, if necessary. Prepared samples were stored in the autosampler at room temperature until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution of 160 mg/L was freshly prepared with dilution water 24 h prior to the start of the exposure. Before preparation, the pH of the dilution water was adjusted to pH 4.02. For preparation, the density of the test item was taken into account. For the preparation of the stock solution the test item was pipetted under the surface of the dilution water. The stock solution was stirred (1100 rpm) for 24 h at room temperature with a minimized headspace. Thereafter, the pH was adjusted to pH 8.05.
- Application: Addition of appropriate volumes of the test solutions to the replicate test vessels.
- Controls: Yes, dilution wate without test substance.
- Evidence of undissolved material: No, the test solutions were clear throughout the test period.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: unicellular freshwater green alga
- Strain: SAG 61.81
- Source: Sammlung der Algenkulturen (SAG), Göttingen, Germany
- Age of inoculum (at test initiation): 3 d

CULTIVATION
- Method: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 2567 - 5130 lux, corresponding to 35 - 70 µE * m-2 * s-1 for 24 h per day.
- Culture medium: Nutrient 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 (dilution water)

Test temperature:

22.0 °C (mean), 21.5 °C (min), 22.5 °C (max)

pH:

0 h: 8.05 (control), 8.01 - 8.06 (treatments)
72 h: 9.17 (control), 7.93 - 9.22 (treatments)

Nominal and measured concentrations:

nominal: control, 10.0, 20.0, 40.0, 80.0, and 160 mg/L
measured: control, 5.02, 10.7, 23.9, 54.1, and 118 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: sterile headspace glass flasks
- Type (delete if not applicable): closed with aluminum tops with PFTE seals
- size, headspace, fill volume: Volume = 59 mL; Test volume = 59 mL; Headspace = 0 mL
- Initial cell density: 5E03- 5E04 cells/mL (nominal), 6305 cells/mL (actual)
- Control end cells density: 532044 cells/mL (72 h)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Other: Seperate replicates for each measuring were prepared.

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

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guidelines (OECD TG 201 medium)
- pH = 8.1 ± 0.2
- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH-value was measured at the start of exposure (in one additional replicate of each test concentration level and the control) and at the end of exposure (from pooled samples of each test concentration level and the control). Room temperature was measured continuously. Light intensity was measured prior to the start of exposure.

OTHER TEST CONDITIONS
- Adjustment of pH: yes, the pH was adjusted to pH 8.05
- Photoperiod: 24 h light/d
- Light intensity and quality: 4807 - 5684 lux, mean: 5338 lux (n = 24), within ± 15% over incubation area

- Other: The test containers were placed on a rotary shaker (SM 30 A, Bühler) and oscillated at approximately 70 rpm. The test containers were positioned randomly and repositioned daily.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Fluorometer, Microplate Reader Chameleon V, Hidex with Software Micro Win 4.41 (excitation: 436 nm, emission: 685 nm)
- Chlorophyll a-fluorescence: 0, 24, 48, and 72 h
- Microscopic evaluation of the cells: at 0 and 72 h

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range-finding study: yes, non-GLP, Erlenmeyer flasks, static test with headspace in OECD TG 201 medium
- Test concentrations range-finding test: 0.1, 1, 10, and 100 mg/L
- Results used to determine the conditions for the definitive study: yes, 2% growth rate inhibition at 0.1 mg/L (nominal) and 58% growth rate inhibition at 100 mg/L (nominal) after 72 h

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

69.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 65.7 - 73.9 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

30.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 26.2 - 34.2 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

23.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

36.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CI: 33.5 - 39.9 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

11.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CI: < 5.02 - 15.8 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

5.02 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, specific growth rate: 1.48 day-1
- Observation of abnormalities: no morpholocial abnormalities (microscopic evaluation, at 0 and 72 h)
- Any stimulation of growth found in any treatment: no
- Any observations that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no, water solubility = 1655 mg/L (20 °C, pH 5.2)

Results with reference substance (positive control):

- Results with reference substance valid? yes
- ErC50 (72 h): 0.460 mg/L (nominal)

Reported statistics and error estimates:

The NOEC/LOEC was determined by calculation of statistical significance of growth rate and yield. The following statistical tests were conducted:
Hypothesis tests: Monotonicicty was tested by trend analysis by contrasts for growth rate and yield (significance level 0.05).
Shapiro-Wilk´s test on normal distribution was done with a significance level of 0.01 for growth rate and yield.
Levene´s test on variance homogeneity was done with a significance level of 0.01 for growth rate and yield.
William´s Multiple Sequential t-test was done with significance level of 0.05 for growth rate and yield.

Calculations were carried out using software Excel (Microsoft Corportaion), SigmaPlot (SPSS Inc.), GraphPad Prism (GraphPad Software, Inc.), ToxRat (ToxRat Solutions GmbH).

No auto-fluorescence of the test substance was found in the preliminary range-finding test at the nominal concentration 100 mg/L.

Table 1: Evaluation after 72 h. Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-).

Geometric mean measured test item concentrations [mg/L]	Replicate n°	Growth rate [d^-1]	Inhibition of growth rate [%]	Yield [cells/mL]	Inhibition of yield [%]
118	1	n.a.	100	n.a.	100
	2	n.a.	100	n.a.	100
	3	n.a.	100	n.a.	100
	Mean	(+)    n.a.	100	(+)    n.a.	100
54.1	1	1.05	29	141080	73
	2	0.958	35	105335	80
	3	0.999	32	120118	77
	Mean	(+)  1.00	32	(+) 122178	77
23.9	1	1.38	7	388853	26
	2	1.37	8	374420	29
	3	1.42	4	433308	18
	Mean	(+)   1.39	6	(+) 398860	24
10.7	1	1.42	4	435958	17
	2	1.43	3	453126	14
	3	1.43	3	458426	13
	Mean	(+)   1.43	4	(+) 449170	15
5.02	1	1.47	1	507392	3
	2	1.46	1	500062	5
	3	1.46	1	503473	4
	Mean	(-)^1   1.46	1	(+)^1503642	4
Control	1	1.47		514355
	2	1.49		547788
	3	1.48		533298
	4	1.48		521712
	5	1.48		534482
	6	1.46		502797
	mean	1.48		525739

n.a. = data not determinable.

1) not biologically significant

Table 2: TOC Content of the Test Media after 0 and 72 h.

Nominal test item concentration [mg/L]	TOC [mg C/L]
	After 0 h (without algae)	After 72 h (without algae)
40.0	26.2	27.1
Control	< 2.00	< 2.00

LOQ = Limit of Quantification (2 mg/L)

 

The TOC results showed that the carbon content was stable during the exposure period.

Table 3: Measured Concentrations and Percentage of the Measured Concentrations of the test substance in fresh medium (0 h) and old medium (72 h) with and without algae.

Sampling date	2016-05-30 0 h (with algae)			2016-06-002 72 h (with algae)			2016-06-02 72 h (without algae)		Geometric mean measured concentration (with algae) [mg/L]
Start of analysis	2016-05-30			2016-06-02			2016-06-02
Nominal test item [mg/L]	Test substance
	meas. conc. [mg/L]	[%]	meas. conc. [mg/L]		[%]	meas. conc. [mg/L]		[%]
160	140	87	100		62	107		67	118
80	71.1	89	41.2		51	48.0		60	54.1
40	35.1	88	16.3		41	23.7		59	23.9
20	17.3	86	6.63		33	11.8		59	10.7
10	8.83	88	2.85		29	5.75		57	5.02
Control	< LOQ		< LOQ			< LOQ			< LOQ

Meas.conc. = measured concentration of the test item (dilution factor taken into account)

[%] = percentage of the nominal concentration of the test item

LOQ = Limit of Quantification (0.100 mg test item/L)

The measured concentrations of the test substance at the start were in the range of 86 - 89%. At the end of the test, the test substance concentrations with algae were in the range of 29 - 62% and without algae in the range of 57 - 67%. Therefore, all effect values given are based on the geometric mean measured test item concentrations of the test substance with algae.

VALIDITY CRITERIA:

Table 4: 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.	84-fold (specific growth rate 1.48 per day)	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%	20.7 %	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%.	0.68 %	yes

Validity criteria fulfilled:

yes

Remarks:

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

Conclusions:

The study, showed that the substance inhibits the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 h of exposure. The ErC50 (72 h) and ErC10 (72 h) values were 69.2 and 30.2 mg/L (geom. mean meas.), respectively.

Executive summary:

The toxicity of the substance to aquatic algae was conducted according to OECD guideline 201 and GLP. Pseudokirchneriella subcapitata was used as the test organism. The green algae was exposed to nominal concentrations of 10.0, 20.0, 40.0, 80.0, and 160 mg/L and a control corresponding to 5.02, 10.7, 23.9, 54.1, and 118 mg/L mean measured concentrations. The test duration was 72 h. An analytical monitoring via TOC analysis was performed in order to verify the concentrations in all test item concentrations and in the control. The measured concentrations of the test substance at the start were in the range of 86 - 89%. At the end of the test, the test substance concentrations with algae were in the range of 29 - 62% and without algae in the range of 57 - 67%. Therefore, all effect values given are based on the geometric mean measured test item concentrations of the test substance with algae at the end of the test. Observed effects resulted in an ErC50 (72 h) of 69.2 mg/L and an ErC10 (72 h) of 30.2 mg/L (both geom. mean meas.).

Description of key information

ErC50 (72 h) = 69.2 mg/L and a ErC10 (72 h) = 30.2 mg/L (both geom. mean meas. P. subcapitata, OECD 201)

Key value for chemical safety assessment

EC50 for freshwater algae:

69.2 mg/L

EC10 or NOEC for freshwater algae:

30.2 mg/L

Additional information

One study is available, in which the toxicity of the substance to aquatic algae was tested. The study was conducted according to OECD guideline 201 and GLP. Pseudokirchneriella subcapitata was used as the test organism. The green algae was exposed to nominal concentrations of 10.0, 20.0, 40.0, 80.0, and 160 mg/L and a control corresponding to 5.02, 10.7, 23.9, 54.1, and 118 mg/L mean measured concentrations. The test duration was 72 h. An analytical monitoring via TOC analysis was performed in order to verify the concentrations in all test item concentrations and in the control. The measured concentrations of the test substance at the start were in the range of 86 - 89%. At the end of the test, the test substance concentrations with algae were in the range of 29 - 62% and without algae in the range of 57 - 67%. Therefore, all effect values given are based on the geometric mean measured test item concentrations of the test substance with algae at the end of the test. Observed effects resulted in an ErC50 (72 h) of 69.2 mg/L and an ErC10 (72 h) of 30.2 mg/L (both geom. mean meas.).