4-(2-butenylidene)-3,5,5-trimethylcyclohex-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 Mar 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, Germany (03.01.2017)

Analytical monitoring:

yes

Remarks:

GC-MS

Details on sampling:

- Concentrations: All loading levels and the control at the beginning (0 h) and end (48 h) of exposure.
- Sampling method: Separate replicates for each test item loading and control were prepared for the test item analysis at the beginning of the exposure with algae.
- 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. Prepared extracts were stored at 6 ± 2 °C until measurement, if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Preparation of aqueous water accommodated fractions (WAFs) by directly pipetting an appropriate amount of the test item into a glass bottle with an appropriate amount of dilution water for each loading. This dispersion was shaken for 24 h with 20 rpm at room temperature. After a separation phase of 1 h, the WAF was removed by siphoning from the centre of the flask.
- Controls: Six replicates (without test item) were exposed under the same conditions as the test loadings.
- Evidence of undissolved material: The test media were clear throughout the exposure phase.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Unicellular green algae
- Age of inoculum (at test initiation): 4 d (preculture in dilution water)
- Method of cultivation: Fresh stocks are prepared monthly on Z-Agar and kept at a light intensity of 2567 - 5130 lux (35 - 70 µE*m^-2*s^-1) for 24 h per day. The algae are cultured in 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

Test temperature:

22.5 °C (mean value)

pH:

Control: 8.15 (0 h), 9.33 (72 h)
Treatments: 8.00 - 8.12 (0 h), 8.01 - 9.35 (72 h)

Nominal and measured concentrations:

Control, 1.00, 3.16, 10.0, 31.6, and 100 mg/L (nominal)
< LOQ, 0.382, 1.02, 4.23, 12.7, and 38.3 mg/L (measured, 0 h)
< LOQ, 0.592, 0.869, 3.83, 11.8, and 30.2 mg/L (measured, 72 h)

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile headspace flasks (59 mL) with aluminium tops and PTFE seals, filled with 59 mL test volume.
- Type: Closed
- Initial cells density: 6839 cells/mL
- Control end cell density: 539731 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Yes (OECD medium), according to OECD testing guideline 201

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: According to the guidelines
- Culture medium different from test medium: Culture medium same as test medium. The preculture was prepared in dilution water (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 loading and the control. At the end of exposure, the pH-values were measured from the pooled samples of each test item loading and the control. Room temperature was measured continuously and light intensity was measured prior to the start of exposure.

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Photoperiod: Continuous light (24 h/d)
- Light intensity and quality: 5857 lux (mean value)

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Cell density was measured daily (after 0, 24, 48, and 72 h) by determination of chlorophyll-a fluorsecence using a fluorometer (Microplate Reader Chameleon V), with excitation at 436 nm and emission at 685 nm. Dilution water was used as a background signal. No auto-fluorescence was found in the WAF with a nominal loading rate of 100 mg/L (preliminary range-finding test).
- Microscopic evaluation: At the start (0 h) and end (72 h) of the incubation period.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.16
- Range finding study: Yes, non-GLP
- Test concentrations: 1.00, 10.0, and 100.0 mg/L (nominal)
- Results used to determine the conditions for the definitive study: Yes, 100% growth rate inhibition was observed after 72 h at a nominal loading level of 100 mg/L and 10% growth rate inhibition at 10 mg/L nominal loading rate.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

8.18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval 7.03 - 9.52 mg/L

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

20.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval 18.8 - 21.6 mg/L

Details on results:

- Exponential growth in the control: Yes
- Observation of abnormalities: No morphological abnormalities were observed
- Any observations (e.g. precipitation) 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 toxicity of the reference item fell within the valid range of the test facility SOPs.
- ErC50 (72 h): 0.435 mg/L (with headspace), 0.811 mg/L (without headspace)
- Other: The growth inhibition test with the reference item is carried out in regular intervals. The most recent tests dated from 11- 14 Oct 2016 (with headspace) and 10 - 13 Oct 2016 (without headspace).

Reported statistics and error estimates:

EL10/20/50 values with confidence intervals of growth rate and yield inhibition after 72 h were calculated by sigmoidal dose-response regression and third order polynomial regression. The NOEL and LOEL were determined by calculation of statistically sifnificant differences of growth rate and yield. The following statistical tests were conducted: Shapiro-Wilk´s test on normal distribution (significance level 0.01), Levene´s test on variance homogeneity (significance level 0.01), Trend Analysis (contrasts) on monotonicity (significance level 0.05), Step-down Jonckheere-Terpstra test (significance level 0.05).

VALIDITY CRITERIA

The test fulfilled the validity criteria (Table 1).

 

Table 1: Validity criteria.

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.	Cell growth in the control cultures increased 79-fold (specific growth rate 1.46/d).	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%	The mean coefficient of variation for section-by-section specific growth rates was 27.1% in the control cultures.	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.	The coefficient of variation of the average specific growth rates during the whole test in replicate control cultures was ≤ 7%	Yes

 

ANALYTICAL RESULTS

The samples at the start of the exposure (0 h) were prepared on the sampling day (liquid-liquid extraction) but measured 48 h later (extracts were stored at 6 ± 2 °C). The stability of sample extracts was demonstrated in a separate stability test conducted from 19 – 26 Apr 2017.

The measured concentrations of the four main components of the test item at the end of the exposure were in the range of 79 – 155% of the measured initial concentrations (Table 2). All effect concentrations were based on the nominal test item loading levels.

 

Table 2. Measured concentrations and percent of the nominal concentrations.

Nominal test item loading [mg/L]	Nominal sum of four major peaks of the test item [mg/L]	Meas. conc. [mg/L]	Meas. conc. [mg/L]	%
100	51.3	38.3	30.2	79
31.6	16.2	12.7	11.8	93
10.0	5.13	4.23	3.83	91
3.16	1.62	1.02	0.869	85
1.00	0.513	0.382	0.592	155
Control		< LOQ	< LOQ

Meas. conc.= measured concentration of the four major peaks

%  = percent of the initial measured concentration of the four major peaks

LOQ = Limit of Quantification (0.05 mg/L test item)

 

BIOLOGICAL RESULTS

The effects of the test item on cell growth are summarized in Table 3.

Table 3. Evaluation after 72 h. Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-).

Nominal test item loading [mg/L]	Replicate n°	Growth rate [d^-1]	Inhibition of growth rate [%]	Yield [cells/mL]	Inhibition of yield [%]
100	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
31.6	1	0.327	78	11414	98
	2	0.272	81	8629	98
	3	0.391	73	15239	97
	Mean	(+) 0.330	77	(+) 11761	98
10.0	1	1.28	12	307517	42
	2	1.26	14	289756	46
	3	1.20	18	241682	55
	Mean	(+) 1.24	15	(+) 279652	48
3.16	1	1.42	2	478541	10
	2	1.43	2	487641	8
	3	1.35	7	386899	27
	Mean	(+) 1.40	4	(+) 451027	15
1.00	1	1.46	0	538817	-1
	2	1.38	5	416861	22
	3	1.43	2	487758	8
	Mean	(-) 1.42	2	(-) 481145	10
Control	1	1.49		594118
	2	1.45		526586
	3	1.47		556022
	4	1.47		549555
	5	1.44		503442
	6	1.41		467627
	Mean	1.46		532892

Negative inhibition = growth stimulation

n.a. = data not determinable

Validity criteria fulfilled:

yes

Remarks:

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

Conclusions:

Based on the outcome of the Growth Inhibition Test, the ErL10 (72 h) is 8.18 mg/L and the ErL50 (72 h) is 20.2 mg/L (nominal loading rates, OECD 202, P. subcapitata).

Description of key information

ErL10 (72 h) = 8.18 mg/L (nominal loading rate, OECD 201, P. subcapitata)

ErL50 (72 h) = 20.2 mg/L (nominal loading rate, OECD 201, P. subcapitata)

Key value for chemical safety assessment

EC50 for freshwater algae:

20.2 mg/L

EC10 or NOEC for freshwater algae:

8.18 mg/L

Additional information

There is one study available, in which the toxicity of the substance to aquatic algae was investigated according to the OECD guideline 201 and GLP.

The substance is a UVCB and five water accommodated fractions (WAFs) with nominal test item loading levels ranging from 1.0 to 100.0 mg/L in a geometric series with a separation factor of 3.16 were prepared by shaking each loading level for 24 h (20 rpm) and removing the aqueous phase (WAF) by siphoning from the center of the flask after a 1 h separation phase. Pseudokirchneriella subcapitata at an initial cell density of 6839 cells/mL was exposed to these 5 loading rates for 72 h under static conditions. The concentration of the test item in the test vessels were verified by GC-MS analysis of the four main constituents of the substance at the start (0 h) and end (72 h) of the test.

The measured initial concentrations ranged from 0.513 to 51.3 mg/L. The measured concentrations of the test item at the end of the exposure ranged from 79 to 155% of the initial measured concentrations. All effect values were based on the nominal loading levels.

After 72 h, growth inhibition was observed and an ErL10 (72 h) of 8.18 mg/L as well as an ErL50 (72 h) of 20.2 mg/L were determined.