(R)-6-(isopropyl)-3-methylcyclohex-2-en-1-one

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:

21 - 24 Feb 2017

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

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

Analytical monitoring:

yes

Details on sampling:

- Concentrations: control, 0.316, 1.0, 3.16, 10.0, 31.6, and 100 mg/L (at 0 and 72 h)
- Sampling method: Separate replicates for each test item concentration and control for the test item analysis at the beginning of the exposure were prepared with algae. The samples for the test item analysis after 72 h 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 and at room temperature until the start of the analysis (on an autosampler), if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Six test solutions in a geometric series with a factor of √10 (3.16) were freshly prepared with dilution water by adding an appropriate amount of the test item. For preparation, the density of the test item was taken into account. The dilution level 0.316 mg/L was prepared out of the test solution with the concentration level 100 mg/L. The test solutions were shaken manually at room temperature until the test item was completely dissolved.
- Controls: Six replicates without test item.
- Evidence of undissolved material: The test media were clear throughtou the test period.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: unicellular green microalga
- Strain: SAG 61.81
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity is adjusted to 2590 - 5180 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)
- Age of inoculum (at test initiation): A 4 d old preculture, prepared in dilution water, was used as inoculum.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol Ca+Mg/L (OECD medium = dilution water)

Test temperature:

mean room temperature: 23 °C (min.: 22.0, max 23.0 °C)

pH:

0 h: 8.05 - 8.16
72 h: 8.21 - 9.42

Nominal and measured concentrations:

control, 0.316, 1.00, 3.16, 10.0, 31.6, and 100 mg/L (nominal)
< LOQ, 0.817, 0.79, 2.48, 8.87, 28.2, and 90.8 mg/L (geometric mean measured)

Details on test conditions:

TEST SYSTEM
- Test vessel: sterile headspace flasks
- Type: closed with aluminum tops with PTFE seals
- Material, size, headspace, fill volume: Volume: 59 mL; Test volume: 59 mL
- Initial cells density: 7143 cells/mL
- Control end cells density: 511835 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Yes, OECD medium.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD medium, according to the guidelines
- Culture medium different from test medium: Yes, culture medium (nutrient medium Z) different from test medium (OECD medium).
- Intervals of water quality measurement: The pH-values at the start of exposure were measured in one additional replicate of each test item concentration and the control. At the end of exposure, the pH-values were measured from pooled samples of each test item concentration and the control. The room temperature was measured continuously. Light intensity was measured prior to the start of exposure.

OTHER TEST CONDITIONS
- Photoperiod: 24 h light/day
- Light intensity and quality: 4440 - 8880 lux, corresponding to 60 - 120 µE*m^(-2)*s^(-1), light homogeneity within ± 15% over incubation area
- Other: The flasks 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, Mikrotex Laborsysteme GmbH)
- Chlorophyll measurement: Chlorophyll a-fluorescence was measured daily, at wavelenghts 436 nm (excitation) and 685 nm (emission). Dilution water was used as background signal.
- Microscopic evaluation: Cells were microscopically analysed at the start and the end of the incubation period.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.16
- Range finding study: Yes, a non-GLP preliminary range-finding test was conducted in a closed system without headspace under static condition for 72 h.
- Test concentrations: 1.0, 10.0, and 100 mg/L
- Results used to determine the conditions for the definitive study: Yes, the dilution levels are based on the results of the preliminary range-finding test.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

26.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

95% confidence interval 14.7 - 79.7 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

62.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

95% confidence interval: 41.1 - 85.5 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.48 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: growthrate and yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

39 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.63 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: Yes, a 72-fold increase of the cell growth (1.42 d^-1) was observed in the control cultures (required: ≥ 16-fold).
- Observation of abnormalities: No morphological abnormalities were observed at the start and the end of the exposure.
- Any observations that might cause a difference between measured and nominal values: The test media were clear throughout the test period.
- 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 toxicity is in the valid range following test facility SOPs. The toxicity of the reference item potassium dichromate to the unicellular freshwater green alga P. subcapitata was tested from 11 - 14 Oct 2016 (with headspace) and on 10 - 13 Oct 2016 (without headspace).
- ErC50 (72 h) = 0.435 mg/L, 95% confidence interval 0.414 - 0.458 mg/L (with headspace), 0.811 mg/L, 95% confidence interval 0.763 - 0.883 mg/L (without headspace)

Reported statistics and error estimates:

ECx-values with confidence intervals of growth rate inhibition and for yield inhibition after 72 h were calculated by sigmoidal dose-response regression.
The NOEC and LOEC were 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/LOEC calculations. When running ANOVA, a Normality test and Equal Variance test were done first with p-values of 0.05 for both tests. The alpha value is 0.05.
Calculations were carried out with the software Excel (Microsoft Corporation), SigmaPlot (SPSS Inc.), and GraphPad Prism (GraphPad Software Inc.)

The study meets the validity criteria of the guideline.

CHLOROPHYLL A FLUORESCENCE

No self-fluorescence of the samples was found in the range-finding test at the nominal concentration of 100 mg/L test item. Dilution water was used as a background signal.

ANALYTICAL RESULTS

The measured concentrations of the test item in the fresh media (0 h) were between 76 and 87% of the nominal values. The measured concentrations in the old media (72 h) were between 73 and 95% of the nominal values (Table 1). Therefore, the geometric mean measured test item concentrations were calculated.

The results of the lowest test concentration (nominal 0.316 mg/L) were comparable to that of the second lowest concentration (nominal 1.00 mg/L). Erroneously, this test group was applied the same way as the second lowest test concentration.

Table 1. Measured concentrations and percentage of the nominal concentration and geometric mean in fresh medium (0 h) and old medium (72 h) with algae.

Sampling date	2017-02-21 0 h fresh medium		2017-02-24 72 h old medium
Start of extraction	2017-02-21		2017-02-24
Date of GC-analysis^1)	2017-03-03		2017-03-03
Nominal test item concentration [mg/L]	Measured concentration [mg/L]	%	Measured concentration [mg/L]	%	Geometric mean measured concentration [mg/L]
100	86.5	86	95.3	95	90.8
31.6	27.3	86	29.1	92	28.2
10.0	8.72	87	9.03	90	8.87
3.16	2.66	84	2.31	73	2.48
1.00	0.759	76	0.823	82	0.79
0.316	0.820^2)	-^2)	0.815^2)	-^2)	0.817^2)
Control	< LOQ		< LOQ

Measured concentration            = measured concentration of the test item (dilution factor taken into account)

%                                              = percentage of the nominal test item concentration

LOQ                                         = limit of quantification (50 µg test item/L)

1)                                              = reanalyzed due to technical problems

2)                                              = due to an error during application the test item amount for the nominal test item concentration 1.00 mg/L was applied.

BIOLOGICAL RESULTS

All effect values are given based on the geometric mean measured test item concentrations.

 

Table 2. Evaluation of biological data 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 concentration [mg/L]	Mean growth rate [d^-1]	Mean inhibition of growth rate [%]
90.8	(+) -0.120	100
28.2	(+) 1.27	11
8.87	(+) 1.36	4
2.48	(-) 1.39	2
0.79	(+) 1.35	5
0.817	(-) 1.43	0
control	1.42

 

 

 

Description of key information

ErC10 (72 h) = 26.8 mg/L (geom. mean measured, OECD 201, P. subcapitata)

ErC50 (72 h) = 62.2 mg/L(geom. mean measured, OECD 201, P. subcapitata)

Key value for chemical safety assessment

EC50 for freshwater algae:

62.2 mg/L

EC10 or NOEC for freshwater algae:

26.8 mg/L

Additional information

There is one GLP study available investigating the toxicity of the substance to freshwater algae according to the OECD guideline 201.

In a static test, P. subcapitata at an initial cell density of 7143 cells/mL was exposed to 0.316, 1.0, 3.16, 10.0, 31.6, and 100 mg/L (nominal) test item concentrations for 72 h. The test item concentrations were analytically verified by GC-MS at the beginning (0 h) and at the end of the test (72 h).

The test media were clear throughout the test period. The measured concentrations of the test item were between 76 and 87% of the nominal values at 0 h and between 73 and 95% of the nominal values at 72 h. Therefore, the geometric mean measured concentration was used for the evaluation.

After 72 h, the test item was found to inhibit the growth of the freshwater green algae. The obtained effect values are an ErC10 (72 h) of 26.8 mg/L and an ErC50 (72 h) of 62.2 mg/L (geometric mean measured concentration).