Reaction mass of 1,2,3,4,4a,5,6,7-octahydro-2,5,5-trimethyl-2-naphthol and 4-(2,6,6-trimethyl-2-cyclohexen-1-yl)butan-2-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 Sep 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 including the control at 0 h (test start), 24 h, 48 h, and 72 h (test end).
- Sampling method: For test item analysis at the beginning of the exposure, separate replicates for each test item concentration and control were prepared with algae. The samples for the test item analysis after 24, 48, and 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: Five dilution levels were prepared by dilution of the stock solution with a nominal concentration of 100 mg/L test item (dilution factor 10^(1/2)). The stock solution was prepared 1 d prior to the start of exposure by stirring the dispersion for 24 h at 1100 rpm at room temperature.
- 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 green alga
- Age of inoculum (at test initiation): A 3 d old preculture prepared in dilution water was used as inoculum for the test.
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar at 2590 - 5180 lux for 24 h/d.
- 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:

22.5 °C (mean room temperature)

pH:

0 h: 8.24 (control), 7.93 - 8.01 (treatments)
72 h: 9.36 (control), 8.08 - 9.29 (treatments)

Nominal and measured concentrations:

Control, 0.800, 2.53, 8.00, 24.3, and 80.0 mg/L (nominal)
Control, 0.813, 2.35, 6.53, 22.2, and 64.4 mg/L (time-weighted average)

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile 59 mL flasks without headspace (59 mL test volume), sealed with aluminium tops and PTFE
- Initial cell density: 6839 cells/mL
- Control end cell density: 612085 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

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

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD medium 201
- Culture medium different from test medium: Culture medium: Nutrient medium Z; Test medium: OECD medium 201 (dilution water)
- 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.

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Photoperiod: 24 h light/d
- Light intensity and quality: 4440 - 8880 lux, 60 - 120 µE*m^-2*s^-1
- Other: The test vessels were randomly placed on a rotary shaker oscillating at approximately 70 rpm and were repositioned daily.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Chlorophyll a-fluorescence
- Chlorophyll measurement: At 0, 24, 48, and 72 h using a Fluorometer (Microplate Reader Chameleon V, Hidex). Excitation: 436 nm; Emission 685 nm
- Microscopic evaluation: At test start (0 h) and end (72 h)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.16
- Range finding study: Yes, one non-GLP preliminary range-finding test was conducted.
- Test concentrations: Control, 1.00, 10.0, and 100 mg/L (nominal)
- Results used to determine the conditions for the definitive study: Yes, after 72 h 100% growth rate inhibition was recorded at 100 mg/L.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

8.72 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 7.45 - 13.0

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

6.1 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 4.96 - 6.41 mg/L

Details on results:

- Exponential growth in the control: Yes, cell growth in the control cultues increased 89-fold (specific growth rate 1.50/d)
- Observation of abnormalities: No morphological abnormalities
- Any stimulation of growth found in any treatment: Growth stimulation was observed at 0.813 and 2.35 mg/L (time weighted average test item concentration)
- Any observations that might cause a difference between measured and nominal values: The test media were clear throughout the exposure.
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? The results of the reference item test were in the valid range of 0.773 ± 0.540 mg/L.
- ErC50 (72 h): 0.559 mg/L (with headspace), 0.811 mg/L (without headspace).
- Other: The reference test was conducted with potassium dichromate from 18 - 21 Apr 2017 (with headspace) and 10 - 13 Oct 2016 (without headspace).

Reported statistics and error estimates:

The EC values were calculated by sigmoidal dose-response regression. The NOEC/LOEC was determined by calculation of statistical significance of growth rate and yield using the following tests:
- Shapiro-Wilk's test on normal distribution (significance level = 0.01)
- Levene's Test on variance homogeneity (significance level = 0.01)
- Trend analysis by contrasts on monotonicity of Concentration/Response (significance level = 0.05)
- Williams Multiple t-test (significance level = 0.05)

ECx CALCULATION FOR GROWTH RATE USING TIME WEIGHTED AVERAGE TEST ITEM CONCENTRATION
- Equation: Sigmoidal dose-response (variable slope). Y = Bottom + (Top - Bottom)/(1+10^((log EC50-X)*HillSlope))
- Transform: x = log(x)
- Fitting results: Bottom = -1.005, Top = 100.2, log EC50 = 0.9392, HillSlope = 5.941, EC50 = 8.717, EC10 = 6.101
- Goodness of fit: Degrees of freedom: 11; R square: 0.9985; Absolute Sum of Squares: 49.66, Sy.x: 2.125

VALIDITY CRITERIA

The study met the validity criteria laid down by 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.	Increase of cell growth in control: 89-fold, specific growth rate: 1.50/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 in the control: 25.5%	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.	Coefficient of variation of average specific growth rates in control culture replicate s: 1.45%	Yes

 

ANALYTICAL RESULTS

The measured test item concentrations were 77 – 111% of the nominal values at the start of exposure at 0 h, 79 – 108% after 24 h, 79 – 100% after 48 h and 74 – 90% after 72 h (Table 2). All effect values are expressed as time weighted average test item concentrations.

Table 2. Measured concentrations and percentage of the nominal concentrations of test item.

nominal test item concentration [mg/L]	0 h		24 h		48 h		72 h
	meas. conc. [mg/L]	[%]	meas. conc. [mg/L]	[%]	meas. conc. [mg/L]	[%]	meas. conc. [mg/L]	[%]
80.0	70.7	88	63.5	79	63.2	79	62.7	78
25.3	19.8	78	20.8	82	25.4	100	21.8	86
8.00	6.17	77	6.54	82	7.05	88	5.90	74
2.53	2.42	95	2.39	95	2.43	96	2.04	80
0.800	0.889	111	0.864	108	0.779	97	0.717	90
control	< LOQ		< LOQ		< LOQ		< LOQ

Meas. conc. = measured concentration of the test item

% = percentage of the nominal concentration of the test item

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

 

BIOLOGICAL RESULTS

After 72 h growth inhibition by the test item was observed (Table 3). The NOEC (72 h) and LOECD (72 h) for inhibition of growth rate and yield was 2.35 mg/L and 6.53 mg/L, respectively.

 

Table 3. Evaluation after 72 hours.

Time weighted average test item concentration	Replicate no.	Growth rate	Inhibition of growth rate	Yield	Inhibition of yield
[mg/L]		[d^-1]	[%]	[cells/mL]	[%]
64.4	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
22.2	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
6.53	1	1.24	17	275120	55
	2	1.31	12	342008	43
	3	1.29	14	316494	48
	Mean	(+) 1.28	15	(+) 311207	49
2.35	1	1.44	4	512360	15
	2	1.49	0	595283	2
	3	1.51	-1	621032	-3
	Mean	(-) 1.48	1	(-) 576225	5
0.813	1	1.54	-3	678879	-12
	2	1.53	-2	671066	-11
	3	1.56	-4	734912	-21
	Mean	(-) 1.54	-3	(-) 694952	-15
Control	1	1.54		681600
	2	1.50		613073
	3	1.48		572841
	4	1.49		587236
	5	1.50		606841
	6	1.48		569885
	Mean	1.50		605246

Negative values = growth stimulation

N.a. = not applicable

Validity criteria fulfilled:

yes

Remarks:

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

Conclusions:

The guideline study resulted in an ErC50 (72 h) of 8.72 mg/L and an ErC10 (72 h) of 6.10 mg/L for aquatic algae (OECD 201, P. subspicatus).

Executive summary:

The short-term toxicity of the test item to aquatic invertebrates was assessed according to OECD guideline 202 and GLP. In a static test using closed test vessels without headspace, Daphnia magna was exposed to five nominal test item concentrations ranging from 1.25 to 20.0 mg/L in a geometric series with a dilution factor of 2. Test item concentrations in all treatment levels were analytically verified in fresh media at the beginning of the test (0 h) and in aged media at the end of the test (48 h) by GC-MS. The measured test item concentrations in fresh media at 0 h ranged from 1.08 to 17.1 mg/L. The measured test item concentrations in aged media at 48 h ranged from 91 – 100% of the measured initial concentrations in the fresh media, indicating that test item concentrations were stable throughout the duration of the test. Therefore, all effect concentrations were based on the measured initial concentrations of the test item. After 48 h immobilization was observed and an EC50 (48 h) of 8.30 mg/L was obtained.

Description of key information

ErC10 = 6.10 mg/L (measured, time weighted average, OECD 201, S. subspicatus)

ErC50 = 8.72 mg/L (measured, time weighted average, OECD 201, S. subspicatus)

Key value for chemical safety assessment

EC50 for freshwater algae:

8.72 mg/L

EC10 or NOEC for freshwater algae:

6.1 mg/L

Additional information

There is one study available, in which the toxicity of the test item to aquatic algae was assessed according to OECD 201 and GLP.

In a static test, P. subcapitata at an initial cell density of 6839 cells/mL was exposed to five concentration levels in a geometric series with a separation factor of 10^(1/2) ranging from 0.80 – 80.0 mg/L (nominal) in closed test vessels without headspace for 72 h. Test item concentrations in all treatment levels and the control were analytically verified by GC-MS at the start of exposure (0 h) and after 24, 48 and 72 h.

The measured concentrations at the start of exposure (0 h) ranged from 77 to 111% of the nominal values and were 79 – 108% of nominal after 24 h, 79 – 100% after 48 h and 74 – 90% after 72 h, indicating that the test item was stable for the duration of the test. All effect values were expressed as time weighted average of measured test item concentrations.

 After 72 h, the test item was found to inhibit cell growth and an ErC10 (72 h) of 6.10 mg/L as well as an ErC50 (72 h) of 8.72 mg/L was obtained.