Reaction mass of (2E)-Tridec-2-enenitrile and (2Z)-Tridec-2-enenitrile and (3E)-Tridec-3-enenitrile and (3Z)-Tridec-3-enenitrile

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:

from 2017-03-31 to 2017-08-09

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

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Sampling method: The concentrations of the test substance in the test item solutions and the control were analytically verified after 0 (start of exposure), 24 hours, 48 hours and 72 hours (end of the exposure). Separate replicates for each test item concentration and the control were prepared without algae for the test item analysis at the beginning of the exposure. The samples for the test item analysis after 24, 48 and 72 hours were prepared like the test replicates and incubated under test conditions. Additionally, the adsorption on glass of the highest test item concentration was measured after 72 hours. The additional replicates are needed for the test item analysis because the replicates used for the determination of the alga cell density had to be homogenized before the measurement, which may lead to a decrease of test item concentration when determined from these replicates.
- Preparation of the measured samples: The test item concentrations were diluted with acetonitrile and / or demineralized water prior to analysis. The last dilution step took place in a SPME vial with at least approx. 300 g sodium chloride/L, either added to the vial or already included in the dilution medium. 100 µL acetonitrile were added to the control and all test item concentration vials before sealing.
- Preparation of the adsorption samples: One test vessel (volume: appr. 60 mL) of the 100 % saturated solution was emptied, rinsed with dilution water and extracted with 2 mL acetonitrile (ultrasonic for appr. 30 s), corresponding to an enrichment factor appr. 30. 100 µL of the extract were added to 10 mL demineralized water (dilution factor 100) in a SPME vial containing approx. 1 g sodium chloride and analyzed.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until the start of the analysis. Prepared samples were stored in the autosampler at room temperature protected from light, in the tightly closed original container until analysis.
- Concentrations: All concentration levels and the control were analytically verified.
- Preparation of 1 x LOQ, five samples of the dilution water (9.9 mL each) fortified samples in a headspace vial containing approx. 3 g sodium chloride were spiked with 100 µL of the test item solution (5 µg/L in acetonitrile) to reach a concentration of nominal 0.05 µg/L and were analyzed directly.
- Preparation of 10000 x LOQ, five samples of dilution water (4.95 mL each) were spiked with 50 µL of the test item solution (50 mg/L in acetonitrile) to reach a concentration of nominal 500 µg/L. For measuring, the samples were further diluted with acetonitrile and demineralized water in two steps with dilution factor 200. The last dilution step took place in a 20 mL headspace vial containing approx. 3 g sodium chloride.
Two blank replicates of the dilution water were prepared with approx. 3 g sodium chloride, added with 100 µL acetonitrile and analyzed directly.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A saturated solution with a nominal loading of 1 mL test item/L was prepared once with demineralized water 96 ± 1 hours prior to the start of the exposure. The test item was placed onto the surface of the demineralized water with a pipette. A slow stirring procedure was applied. Gentle stirring (to avoid formation of an emulsion) was carried out with a magnetic stirrer at 30 ± 2 °C (current: 29.1 to 30.5 °C) for 96 ± 1 hours. After a separation phase of 1 hour at room temperature the saturated solution was taken from the homogeneous phase. The saturated solution was checked after stirring via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion). Then the components of the dilution water were added to the saturated solution. The saturated solution was clear and colorless.
- Test concentrations: The saturated solution and seven dilution levels out of the saturated solution were tested in a geometrical series with a dilution factor of approximately 3.16: The dilution levels are based on the results of a preliminary range finding test (non-GLP, flasks without headspace).
- Controls: Blank control performed with test solution without test item exposed under the same conditions as the test concentrations. The positive control was performed with the reference substance potassium dichromate.
- Evidence of undissolved material: No

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green alga
- Source: Sammlung von Algenkulturen (SAG). Pflanzenphysiologisches Institut der Universität Göttingen (Göttingen, Germany)
- Age of inoculum (at test initiation): A four days old preculture, prepared in dilution water, was used as inoculum.
- Culture medium: Nutrient medium Z according to Lüttge et al. (1994)
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 2590 - 5180 lux corresponding to 35 - 70 µE x m-2 x s-1 for 24 hours per day.

ACCLIMATION
- Culturing media and conditions: Same as the test
- Any deformed or abnormal cells observed: No

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

pH:

8.18 - 9.53

Nominal and measured concentrations:

Nominal concentration: 0.0316, 0.100, 0.316, 1.00, 3.16, 10.0, 31.6 and 100 % of the saturated solution (1 mL test item/L)
Initial measured test item concentrations (0 hours): < LOQ, 0.202 - 0.583 - 2.14 - 6.09 - 20.0 - 57.3 - 173 - 442 µg/L. At test end, the test item concentrations were in the range of < LOQ - 57% of the initial values of all concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile headspace flasks with aluminum tops with PFTE seals
- Type: closed
- Volume: 59 mL
- Aeration: No
- Initial cells density: Nominal: approximately 5 x 1000 - 10 000 cells/mL (Actual: 6839 cells/mL)
- No. of vessels per concentration (replicates): 3 (separate replicates for each measuring were prepared)
- 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: Demineralized water
- Culture medium different from test medium: No
- Intervals of water quality measurement: The pH-value at the start of exposure was measured in one additional replicate of each test item concentration and the control. At the end of exposure, the pH-value was measured from pooled samples of each test item concentration and the control after measurement of the cell density. The room temperature was measured continuously. Light intensity was measured prior to the start of exposure.

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: No
- Photoperiod: 24 hours/day light
- Light intensity and quality: Approximately 4440 to 8880 lux, corresponding to 60 to 120 µE*m-2*s-1;
- Light homogeneity: Within ± 15 % over incubation area
- Incubation: Test containers were positioned randomly and repositioned daily.
- Agitation: Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.

EFFECT PARAMETERS MEASURED: Inhibition of growth rate and yield of the algae after 0, 24, 48 and 72 hours.
- Determination of cell concentrations: Microscopic evaluation of the cells at the start and the end of exposure (72 hours) was carried out. The cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.
- Chlorophyll measurement: The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. No self-fluorescence was found in the preliminary range finding test in the saturated solution.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.16
- Range finding study
Test concentrations: A non-GLP preliminary range finding test in a closed bottle system under static conditions over a period of 72 hours was conducted at the test facility with a saturated solution of the test item with a nominal loading level of 1 mL/L (nominal 836.6 mg/L). Out of the saturated solution four dilution levels were tested prepared by diluting the saturated solution by factor 100, 1000 and 10000 with dilution water. The dilution levels of 1, 0.1, 0.01 and 0.001 % of the saturated solution were visually clear throughout the exposure period.
- Results used to determine the conditions for the definitive study: After 72 hours the EC10 and the EC50 were determined to be greater than 1 % saturated solution (nominal 8.366 mg/L, corresponding to a measured concentration of 2.38 µg/L)

Reference substance (positive control):

yes

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

20 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

59.4 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % C.L. (54.6 - 65.3 mg/L)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

106 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % C.L. (100 - 112 mg/L)

Details on results:

- Exponential growth in the control: Yes. 89-fold (specific growth rate 1.50 day-1)
- Observation of abnormalities: Not observed
- Unusual cell shape: Not observed
- Colour differences: Not observed
- Flocculation: Not observed
- Adherence to test vessels: Not observed
- Aggregation of algal cells: Not observed
- Other:
The mean coefficients of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3) in the control cultures was determined to be 16.9 %
Coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was determined to be 1.77 %
- Any stimulation of growth found in any treatment: Not observed
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Not observed
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid?: Yes
- EC50: After 72 hours the ErC50 was determined to be 0.435 mg/L with Headspace and 0.811 mg/L without Headspace (Valid Range (average ± 3 x SD): 0.782 ± 0.534 mg/L).
After 72 hours the EyC50 was determined to be 0.230 mg/L with Headspace and 0.387 mg/L without Headspace (Valid Range (average ± 3 x SD): 0.410 ± 0.288 mg/L)

Reported statistics and error estimates:

EC10-, EC20- and EC50-value with confidence intervals of growth rate and yield inhibition after 72 hours was calculated by sigmoidal dose-response regression. 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 a = 0.05.

Table 1: Measured Concentrations and Percentage of the Initially Measured Concentrations of the test substance

Sampling Date	0 hours	24 hours		48 hours		72 hours
Saturated solution of the test item [%]	Meas. Conc. [µg/L]	Meas. Conc. [µg/L]	% initial	Meas. Conc. [µg/L]	% initial	Meas. Conc. [µg/L]	% initial
100	442	318	72	359	81	251	57
31.6	173	78.6	45	29.0	17	< LOQ	-
10	57.3	22.6	39	< LOQ	-	< LOQ	-
3.16	20.0	3.26	16	< LOQ	-	< LOQ	-
1.0	6.09	0.0774	1.3	< LOQ	-	< LOQ	-
0.316	2.14	< LOQ	-	< LOQ	-	< LOQ	-
0.100	0.583	< LOQ	-	< LOQ	-	< LOQ	-
0.0316	0.202	< LOQ	-	< LOQ	-	< LOQ	-
Control	< LOQ		< LOQ		< LOQ		< LOQ

 

Meas. conc = Measured concentration of the test item (dilution factor taken into account)

% = Percentage of the initially measured concentration of the test item (0 hours)

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

 

 

 

Table 2: Cell densities 

Measured initial test item concentration	Replicate	Cell density [cells/mL]
[mg/L]	No.	0 hours	24 hours	48 hours	72 hours
442	1	6839	< LOQ	< LOQ	< LOQ
	2	6839	< LOQ	< LOQ	< LOQ
	3	6839	< LOQ	< LOQ	< LOQ
	Mean	6839	n.a	n.a	n.a
173	1	6839	8118	7717	13221
	2	6839	8349	8428	12009
	3	6839	6637	6064	10725
	Mean	6839	7701	7403	11985
57.3	1	6839	23906	69151	445856
	2	6839	23637	80264	418878
	3	6839	19055	68455	359568
	Mean	6839	22199	72623	408101
20.0	1	6839	29565	147281	572054
	2	6839	29867	139814	657347
	3	6839	25647	128602	492379
	Mean	6839	28360	138566	573927
6.09	1	6839	33612	187098	542911
	2	6839	33986	168413	579604
	3	6839	27485	155451	508853
	Mean	6839	31694	170321	543789
2.14	1	6839	31818	162414	574483
	2	6839	29653	171415	584256
	3	6839	27839	143916	476286
	Mean	6839	29770	159248	545008
0.583	1	6839	26812	173381	590196
	2	6839	35520	189181	585777
	3	6839	27663	164902	566905
	Mean	6839	29998	175821	580959
0.202	1	6839	34607	191031	653339
	2	6839	30525	176793	621884
	3	6839	26669	150254	509409
	Mean	6839	30600	172693	594877
Control	1	6839	32649	179262	610678
	2	6839	35461	188754	637246
	3	6839	35827	177565	681136
	4	6839	33258	184968	596135
	5	6839	31839	161531	598652
	6	6839	23909	161589	536509
	Mean	6839	32117	175612	610059

 

< LOQ = below the limit of quantification (2736 cells/mL)

n.a. = data not determinable

 

 

 

 

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 (-). 

Measured initial test item concentration	Replicate	Cell density [cells/mL]
[mg/L]	No.	0 hours	24 hours	48 hours	72 hours
442	1	< LOQ	100	< LOQ	100
	2	< LOQ	100	< LOQ	100
	3	< LOQ	100	< LOQ	100
	Mean	(+) n.a.	100	(+) n.a.	100
173	1	0.220	85	6382	99
	2	0.188	87	5170	99
	3	0.150	90	3886	99
	Mean	(+) 0.186	88	(+) 5146	99
57.3	1	1.39	7	439017	27
	2	1.37	8	412039	32
	3	1.32	12	352729	42
	Mean	(+) 1.36	9	(+) 401262	33
20.0	1	1.48	1	565215	6
	2	1.52	-2	650508	-8
	3	1.43	5	485540	20
	Mean	(+) 1.47	1	(-) 567088	6
6.09	1	1.46	3	536072	11
	2	1.48	1	572765	5
	3	1.44	4	502014	17
	Mean	(+) 1.46	3	(+) 536950	11
2.14	1	1.48	1	567644	6
	2	1.48	1	577417	4
	3	1.44	5	469447	22
	Mean	(+) 1.46	3	(+) 538169	11
0.583	1	1.49	1	583357	3
	2	1.48	1	578938	4
	3	1.47	2	560066	7
	Mean	(+) 1.48	1	(-) 538169	5
0.202	1	1.52	-2	646500	-7
	2	1.50	0	615045	-2
	3	1.44	4	560066	17
	Mean	(+) 1.49	1	(-) 588038	3
Control	1	1.50		603839
	2	1.51		630407
	3	1.53		674297
	4	1.49		589296
	5	1.49		591813
	6	1.45		529665
	Mean	1.50		603220

 

 

 

Validity criteria fulfilled:

yes

Conclusions:

In this study, the test substance was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on measured initial test item concentrations): The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 106 (100 - 112) µg/L and 65.5 (58.4- 127) µg/L, respectively. The EC10-values with 95 % confidence intervals for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 59.4 (54.6 - 65.3) µg/L and 37.2 (< 0.202- 55.3) µg/L, respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 20.0 µg/L, respectively.

Executive summary:

The toxicity of the test substance to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016 C.3. The aim of the study was the determination of NOEC, LOEC, EC10- , EC20- and EC50-values of growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 6839 cells/mL. With regard to the volatility of the test item, glass flasks without headspace were used to reduce losses of the test item. Eight concentrations were tested in a geometrical series with a dilution factor of approximately 3.16: 0.0316 - 0.100 - 0.316 -1.00 - 3.16 -10.0 - 31.6 -100 % of the saturated solution, corresponding to the measured initial test item concentrations 0.202 - 0.583 - 2.14 - 6.09 - 20.0 - 57.3 - 173-442 µg/L. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits.

The test solutions were clear throughout the test period. The concentrations of the test substance in the test item solutions and the control were analytically verified via GC-MS at the test start, after 24, 48 and 72 hours. The measured concentrations of the test substance at the start were in the range of 0.202 - 442 µg/L. At test end, the test item concentrations were in the range of < LOQ - 57 % of the initial values of all concentrations. Adsorption to the glass wall was investigated at the end of the exposure for the 100 % saturated solution and was found to be negligible. The daily analysis of all test groups showed a rapid decrease of test item concentrations. Therefore, the evaluation based on measured initial concentrations was considered the most fitting evaluation.

Description of key information

The 72h-ErC50 was determined to be 106 µg/L, the 72h-NOEC was determined to be 20 µg/L and the 72h-ErC10 was determined to be 59.4 µg/L in a study conducted on the fresh algae Pseudokirchneriella subcapitata according to OECD 201 (2011).

Key value for chemical safety assessment

EC50 for freshwater algae:

106 µg/L

EC10 or NOEC for freshwater algae:

59.4 µg/L

Additional information

In a key static growth inhibition test according to OECD 201 (2011), the fresh algae Pseudokirchneriella subcapitata was exposed over 72 hours to the test substance. The ErC50 was determined to be 106 µg/L, the 72h-ErC10 was determined to be 59.4 µg/L and the 72h-NOEC was determined to be 20 µg/L. All effect values given are based on the measured initial test item concentrations and the validity criteria were fulfilled.