Reaction mass of 5,9-Undecadienal-2,6,10-trimethyl-, (5E)- and 5,9-Undecadienal-2,6,10-trimethyl-, (5Z)-

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 Jan - 22 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)

Version / remarks:

2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Staatliches Gewerbeaufsichtsamt Hildesheim, Germany

Analytical monitoring:

yes

Details on sampling:

- Concentrations: control and all test item concentrations
- Sampling method: samples were taken at the start of the exposure (0 hours), after 24, 48 h and at the end of the exposure (72 h); separate replicates were prepared without algae for the test item analysis at the beginning of the test;
- Sample storage conditions before analysis: samples were stored at room temperature

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: stock solution with test item was prepared and stirred for 30 min (1100 rpm, room temperature) with a magnetic stirrer; test item concentrations were performed with the appropriate dilution of the stock solution.
- Controls: yes, test medium control

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: SAG 61.81
- Age of inoculum (at test initiation): four days old preculture, prepared in dilution water, was used as inoculum
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar.

ACCLIMATION
- Culturing media and conditions: not the same as the test (stock culutres on Z-Agar; Nutrient medium Z according to Lüttge et al. (1994); Light intensity amounted to 2567 - 5130 lux for 24 hours per day.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol Ca+ Mg/L

Test temperature:

22.0 - 23.0 °C (mean value 22.5 °C)

pH:

8.22 - 9.15 (control)
8.11 - 9.29 (test item concentrations)

Nominal and measured concentrations:

Control, 0.240, 0.427, 0.759, 1.35 and 2.40 mg/L
Control, 0.0130, 0.0494, 0.0970, 0.196 and 0.499 mg/L (based on time weigted average)

Details on test conditions:

TEST SYSTEM
Test vessel:
- Type: closed (volatile substance)
- Material, size, headspace, fill volume: Sterile headspace flasks, volume: 59 mL, with aluminium tops with PTFE seals
- Agitation: Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm
- Initial cells density: approx 5 x 10 E+03 - 10 E+04 cells/mL (nominal) and 6839 cells/mL (current)
- Control end cells density: 383255 cells/mL (mean)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD TG 201 medium additional compounds were added to enable sufficient growth under conditions without headspace
- Culture medium different from test medium: yes
- Intervals of water quality measurement: pH-values at the start of exposure were measured in one additional replicate; at the end of exposure, the pH-values were measured from pooled samples; room temperature was measured continuously; light intensity was measured prior to the start of exposure

OTHER TEST CONDITIONS
- Photoperiod: 24 hours/day light
- Light intensity and quality: Approximately 4440 to 8880 lux, corresponding to 60 to 120 µE*m-2*s-1, within ± 15% over incubation area

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Fluorometer: Microplate Reader Chameleon V (Hidex) with Software Micro Win 4.41 (Mikrotec Laborsysteme GmbH) fluorescence values were related to cell density values according to a calibration curve
- Chlorophyll measurement: daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 4th root out of 10
Range finding study
- Test concentrations: control, 0.240 and 2.40 mg/L
- Results used to determine the conditions for the definitive study: after 72 the inhibition of growth rate was -5% at 0.240 mg/L and 17% at 2.40 mg/L. The dilution levels of the main test are based on these results.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.449 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 0.449 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.354 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% Confidence Intervals

Remarks:

0.218 - 0.499 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.196 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: grotwh rate and yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.499 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.499 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.193 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95 % Confidence Intervals

Remarks:

0.130 - 0.325 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.137 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95 % Confidence Intervals

Remarks:

0.0885 - 0.225 mg/L

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities: no
- Others: Biological and analytical results are summarized within the tables 1 to 5.

Results with reference substance (positive control):

- EC50 (72 h): 0.436 mg/L (with headspace)
- EC50 (72 h): 0.811 mg/L (without headspace)

Reported statistics and error estimates:

ECx-values with confidence intervals of growth rate inhibition and for yield inhibition after 72 hours were calculated by sigmoidal dose-response.
NOEC/LOEC was determined by calculation of statistical significance of growth rates and yield. As standard One Way Analysis of Variance (ANOVA) and Dunnett's test was used for NOEC/LOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance tests are 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) is alpha=0.05.

Table 1: Results of the non-GLP range finding test

Nominal test item concentration [mg/L]	Growth Rate Inhibition [%]	Yield Inhibition [%]
2.40	17	55
0.240	-5	-25

 

Results of the main test

Table 2: Cell Densities

Time weighted average test item concentration	Replicate	Cell density [cells/mL]
[mg/L]	No.	0 hours	24 hours	48 hours	72 hours
	1	6839	10450	32845	227184
0.499	2	6839	13393	29679	249832
	3	6839	9747	40061	226511
	Mean	6839	11197	34195	234509
	1	6839	14414	65418	293078
0.196	2	6839	15324	61040	339777
	3	6839	13914	49992	303553
	Mean	6839	14551	58817	312136
	1	6839	11992	75132	419715
0.0970	2	6839	15301	78345	394698
	3	6839	12077	53117	295887
	Mean	6839	13123	68865	370100
	1	6839	17592	87111	450497
0.0494	2	6839	18303	102139	484731
	3	6839	15795	91354	384866
	Mean	6839	17230	93535	440031
	1	6839	17987	117401	493275
0.0130	2	6839	17507	90037	465507
	3	6839	16082	112274	399409
	Mean	6839	17192	106571	452730
	1	6839	21686	118957	414390
	2	6839	19175	115031	417374
	3	6839	20035	111145	346931
Control	4	6839	17601	112737	394113
	5	6839	16571	87251	367837
	6	6839	18362	97896	358884
	Mean	6839	18905	107170	383255

  

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 (-).

Time weighted average test item concentration	Replicate	Growth rate	Inhibition of growth rate	Yield	Inhibition of yield
[mg/L]	No.	[d-1]	[%]	[cells/mL]	[%]
	1	1.17	13	220345	41
	2	1.20	11	242993	35
0.499	3	1.17	13	219672	42
	Mean	(+) 1.18	12	(+) 227670	40
	1	1.25	7	286239	24
	2	1.30	3	332938	12
0.196	3	1.26	6	296714	21
	Mean	(-) 1.27	5	(-) 305297	19
	1	1.37	-2	412876	-10
	2	1.35	-1	387859	-3
0.0970	3	1.26	6	289048	23
	Mean	(-) 1.33	1	(-) 363261	3
	1	1.40	-4	443658	-18
	2	1.42	-6	477892	-27
0.0494	3	1.34	0	378027	0
	Mean	(-) 1.39	-3	(-) 433192	-15
	1	1.43	-6	486436	-29
	2	1.41	-5	458668	-22
0.0130	3	1.36	-1	392570	-4
	Mean	(-) 1.40	-4	(-) 445891	18
	1	1.37		407551
	2	1.37		410535
	3	1.31		340092
Control	4	1.35		387274
	5	1.33		360998
	6	1.32		352045
	Mean	1.34		376416

negative inhibition = increase of growth

Table 4: Measured Concentrations and Percentage of the Nominal Concentration of the test item in Fresh Medium (0 hours) to Old Medium (72 hours)

Sampling date	2017-02-14 0h		2017-02-15 24 h		2017-02-16 48 h		2017-02-17 72 h		2017-02-17 72 h
Start of analysis	2017-02-14 (Start of the exposure) (with algae)		2017-02-15 (with algae)		2017-02-16 (with algae)		2017-02-17 (End of the exposure) (with algae)		2017-02-17 (without algae)
Nominal test item concentration [mg/L]	Test Item
	Meas. conc. [mg/L]	[%]	Meas. conc. [mg/L]	[%]	Meas. conc. [mg/L]	[%]	Meas. conc. [mg/L]	[%]	Meas. conc. [mg/L]	[%]
2.40	1.44	60	1.20	50	0.371	15	0.0538	2	0.328 2)	14
1.35	0.863	64	0.659	49	0.145	11	0.00728	1	0.167	12
0.759	0.460	61	0.465	61	0.0748	10		< LOQ	0.0933	12
0.427	0.246	58	0.224	52	0.0281	7		< LOQ	0.0750 2)	18
0.240	0.123	51	0.107	44		< LOQ 1)		< LOQ	0.0335	14
Control	< LOQ		< LOQ		< LOQ		< LOQ		< LOQ

Meas. conc. = Measured concentration of the test item (dilution factor taken into account), mean value of two injections

% = Percent of the nominal concentration of the test item

LOQ = Limit of quantification (3 µg test item /L)

1) = Reanalysed on 2017-02-17

2) = Reanalysed on 2017-02-22

Table 5: Time Weighted Average Mean Measured Test Item Concentrations of the test item

Nominal test item concentration [mg/L]	Time Weighted Average Mean Measured Test Item Concentration [mg/L]
2.40	0.499
1.35	0.196
0.759	0.0970
0.427	0.0494
0.240	0.0130
Control	< LOQ

LOQ = Limit of quantification (3 µg test item /L)

Validity criteria fulfilled:

yes

Conclusions:

The substance was found to inhibit the growth rate of the freshwater green alga Pseudokirchneriella subcapitata after 72 h of exposure. The test was conducted according to the OECD guideline 201 (GLP). ErC10- and ErC50-value were found to be 0.354 mg/L and >0.499 mg/L (based on the time weighted average), respectively.

Description of key information

ErC50 (72 h) > 0.449 mg/L (Pseudokirchneriella subcapitata, OECD 201, based on time weighted average)

ErC10 (72 h) = 0.354 mg/L (Pseudokirchneriella subcapitata; OECD 201, based on time weighted average)

Key value for chemical safety assessment

EC50 for freshwater algae:

0.449 mg/L

EC10 or NOEC for freshwater algae:

0.354 mg/L

Additional information

One experimental study is available investigating the toxicity of the substance to aquatic freshwater algae. The study was conducted according to OECD 201 (GLP) using the freshwater algae Pseudokirchneriella subcapitata. In a static test system, the test organism was exposed to five nominal concentrations of 0.240, 0.427, 0.759, 1.35 and 2.40 mg/L and a control corresponding to time weighted average of 0.0130, 0.0494, 0.0970, 0.196 and 0.499 mg/L. The concentrations of the test item in all test vessels and the control were analytically verified. The measured concentrations in the fresh media (0 h) were in the range of 51 to 64% of the nominal values at all tested concentration levels. The measured concentrations in the old media (72 h) were in the range of 12 to 18% of the nominal values of all concentrations (samples without algae). Therefore, all effect values given are based on the time weighted avergae test item concentrations. The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were > 0.449 mg/L (TWA). The EC10 values were 0.354 mg/L (TWA) and 0.137 mg/L (TWA) for growth rate and yield, respectively.