(E)-1-(2,4,4-trimethyl-2-cyclohexen-1-yl)-2-buten-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:

04 - 07 Jul 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 concentration levels and the control at the beginning and end of exposure (0 and 72 h)
- Sampling method: Separate replicates for each test item concentration and the 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 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 10 mg/L (nominal) was prepared with dilution water 1 d before test start by stirring for 24 h at 1100 rpm and room temperature. The stock solution and four dilution levels out of the stock solution were tested in a geometrical series with a dilution factor of 3.16.
- Controls: Dilution water without test item incubated under the same conditions as the test concentrations.
- Evidence of undissolved material: The test media were clear throughout the exposure phase.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: unicellular freshwater green algae
- Strain: SAG 61.81
- Age of inoculum (at test initiation): A 4 d old preculture, prepared in dilution water (OECD medium 201) was used as inoculum
- Method of cultivation: Fresh stocks are prepared every month on Z-agar and held in 24 h light/d at 2567 - 5130 lux. 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 (OECD medium 201/dilution water)

Test temperature:

22.5 °C (mean room temperature)

pH:

0 h: 8.02 (control), 7.89 - 8.06 (treatments)
72 h: 9.37 (control), 7.94 - 9.49 (treatments)

Nominal and measured concentrations:

Control, 0.1000, 0.316, 1.000, 3.16, and 10.0 (nominal)
< LOQ, 0.0858, 0.248, 0.891, 2.68, and 9.21 mg/L (geometric mean meausred)

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile 59 mL glass flasks with aluminium tops and PTFE seals without headspace
- Initial cell density: 6839 cells/mL
- Control end cell density: 883225 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

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

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD medium 201, according to guideline
- pH: 8.1 ± 0.2
- Culture medium different from test medium: Culture medium: Nutrient medium Z ; Dilutuion water for test medium: OECD 201 medium
- Intervals of water quality measurement: The pH was measured at the start of exposure in one additional replicate of each test item concentration and the control and at the end of exposure in pooled samples of each test item concentration and the control. Room temperature was measured continuously. Light intensity was measured prior to the start of exposure.

OTHER TEST CONDITIONS
- Photoperiod: 24 h light/d
- Light intensity and quality: 5608 lux (mean), CV = 5.36%
- Others: The test vessels were randomly positioned on a rotary shaker (70 rpm) and repositioned daily.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Chlorophyll a fluorescence was measured by Fluorometer (Microplate Reader Chameleon V, Hidex with software) with an excitation at 436 nm and an emission at 685 nm after 0, 24, 48, and 72 h
- Microscopic evaluation: The cells were visually screened by microscope at the start (0 h) and end (72 h) of exposure.
- Other: No autofluorescence was detected in the dilution water.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.16
- Range finding study: Yes
- Test concentrations: Control, 0.5, 5.0, and 50.0 mg/L
- Results used to determine the conditions for the definitive study: Yes, after 72 h 100% growth rate inhibition was observed in the 50.0 mg/L treatment and 76% in the 5.0 mg/L treatment level.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.14 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 1.04 - 1.24 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.45 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 2.38 - 2.51 mg/L

Details on results:

- Exponential growth in the control: Yes, an increase of 129-fold was recorded, corresponding to a specific growth rate of 1.62 /d
- Observation of abnormalities: No
- Any observations that might cause a difference between measured and nominal values: The test media were clear throghout the exposure phase.
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes, the result of the reference test was in the valid range of 0.773 ± 0.540 defined by the guideline.
- ErC50 (72 h) with headspace: 0.559 mg/L potassium dichromate, 95% confidence interval: 0.538 - 0.581 mg/L
- ErC50 (72 h) without headspace: 0.811 mg/L potassium dichromate, 95% confidence interval: 0.763 - 0.883 mg/L
- Other: The most recent reference tests were performed from 18 - 21 Apr 2017 (with headspace) and 10 - 13 Oct 2017 (without headspace).

Reported statistics and error estimates:

Calculations were performed with ToxRat Professional (ToxRat Solutions GmbH) software. EC values with confidence intervals were calculated by sigmoidal dose-response regression using the geometric mean measured test item concentration. The NOEC and LOEC were determined by calculation of statistically significant differences of growth rates and yield. The following tests were performed: Shapiro-Wilk's test on normal distribution (significance level: 0.01), Levene's test on variance homogeneity (significance level: 0.01), Trend analysis of Monotonicity of Concentration/Response (significance level: 0.05), Williams Multiple t-test (significance level: 0.05).

ECx CALCULATION OF GROWTH RATE

- EQUATION: Sigmoidal dose-response (variable slope), Y = Bottom + (Top - Bottom)/(1 + 10^(logEC50 - X) * HillSlope))
- TRANSFORM: x = log(x)
- BEST-FIT VALUES: Bottom = -1.627, Top = 103.0, logEC50 = 0.3927, HillSlope = 2.681; EC50 = 2.447, EC20 = 1.496, EC10 = 1.138
- GOODNESS OF FIT: Degrees of Freedom: 11, R square: 0.9991, Absolute Sum of Square: 22.09, Sy.x = 1.417

VALIDITY CRITERIA

The study met the validity criteria of the guideline (Table 1).

 

Table 1: 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.	129-fold increase of cell growth in the control cultures (specific growth rate = 1.62 /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 was 30.7%.	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.	The coefficient of variation of average specific growth rates during the test was 0.67%.	Yes

 

 

ANALYTICAL RESULTS

The measured concentrations of the test item ranged from 84 – 102% of the nominal concentration at the start of the test (0 h) and from 74 to 83% of nominal concentrations at the end of the test (72 h). Therefore, the geometric mean measured test item concentrations were calculated (Table 2).

 

Table 2. Measured concentrations and percentage of the nominal concentration of the substance

nominal test item concentration [mg/L]	meas. conc. [mg/L]	%	meas. conc. [mg/L]	%	geometric mean [mg/L]
10.0	10.2	102	8.34	83	9.21
3.16	2.82	89	2.55	81	2.68
1.0	0.993	99	0.80	80	0.891
0.316	0.264	84	0.233	74	0.248
0.100	0.0947	95	0.0777	78	0.0858
Control	< LOQ		< LOQ		< LOQ

Meas. conc. = measured concentration of the test item

% = percentage of the nominal concentration

LOQ = limit of quantification (10 µg/L)

 

BIOLOGICAL RESULTS

After 72 h of exposure, growth inhibition was observed (Table 3). Microscopic evaluation revealed no morphological abnormalities. All effect values (with 95% confidence intervals) are based on the geometric mean measured test item concentrations and were as follows:

-       NOEC (72 h) = 0.248 (growth inhibition and yield)

-       LOEC (72 h) = 0.891 (growth inhibition and yield)

-       ErC10 (72 h) = 1.14 mg/L (1.04 – 1.24 mg/L)

-       EyC10 (72 h) = 0.758 mg/L (0.636 – 0.848 mg/L)

-       ErC50 (72 h) = 2.45 mg/L (2.38 – 2.51 mg/L)

-       EyC50 (72 h) = 1.21 mg/L (1.07 – 1.54 mg/L)

Table 3. Evaluation after 72 hours. 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]	replicate n°	Growth rate [d^-1]	Inhibition of growth rate [%]	Yield [cells/mL]	Inhibition of yield [%]
9.21	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
2.68	1	0.741	54	56320	94
	2	0.703	57	49508	94
	3	0.677	58	45359	95
	Mean	(+) 0.707	56	50396	94
0.891	1	1.54	5	677954	23
	2	1.55	4	707331	19
	3	1.54	5	685503	22
	Mean	(+) 1.54	5	(+) 690263	21
0.248	1	1.66	-2	980444	-12
	2	1.67	-3	1008768	-15
	3	1.68	-4	1042651	-19
	Mean	(-) 1.67	-3	1010621	-15
0.0858	1	1.62	0	876688	0
	2	1.63	-1	904895	-3
	3	1.62	0	873704	0
	Mean	(-) 1.62	0	885096	-1
Control	1	1.62		874581
	2	1.62		879614
	3	1.62		886929
	4	1.60		825893
	5	1.63		913322
	6	1.62		877976
	Mean	1.62		876386

 

Validity criteria fulfilled:

yes

Remarks:

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

Conclusions:

The guideline test for the toxicity of the substance to aquatic algae resulted in an ErC10 (72 h) of 1.14 mg/L and an ErC50 (72 h) of 2.45 mg/L (geometric mean measured, OECD 201, P. subcapitata).

Executive summary:

The toxicity ofthe substance to aquatic algae was assessed according to OECD guideline 201 and GLP. Pseudokirchneriella subcapitata at an initial cell density of 6839 cells/mL was exposed to five concentration levels of the test item in a geometric series with a separation factor of 3.16 and a control for 72 h in closed bottles without headspace. Test item concentrations were analytically verified by GC-MS at the beginning (0 h) and at the end of the test (72 h). The measured test item concentrations at the start of exposure were 84 – 102% of the nominal values and 74 – 83% at the end of exposure. Therefore, all effect values are based on geometric mean measured concentrations. After 72 h growth inhibition was observed with the following effect values: ErC10 (72 h) = 1.14 mg/L and ErC50 (72 h) = 2.45 mg/L.

Description of key information

ErC10 (72 h) = 1.14 mg/L (geom. mean measured, OECD 201, P. subcapitata)

ErC50 (72 h) = 2.45 mg/L (geom. mean measured, OECD 201, P. subcapitata)

Key value for chemical safety assessment

EC50 for freshwater algae:

2.45 mg/L

EC10 or NOEC for freshwater algae:

1.14 mg/L

Additional information

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

 

Pseudokirchneriella subcapitata at an initial cell density of 6839 cells/mL was exposed to five concentration levels of the test item in a geometric series with a separation factor of 10^(1/2) and a control for 72 h in closed bottles without headspace. Test item concentrations were analytically verified by GC-MS at the beginning (0 h) and at the end of the test (72 h).

 

The measured test item concentrations at the start of exposure were 84 – 102% of the nominal values and 74 – 83% at the end of exposure. Therefore, all effect values are based on geometric mean measured concentrations. After 72 h growth inhibition was observed with the following effect values: ErC10 (72 h) = 1.14 mg/L and ErC50 (72 h) = 2.45 mg/L.