(2Z)-2-phenylhex-2-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:

11-05-2018 to 03-09-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met..

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23 (2000)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: July 2016; signature: January 2017

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Definitive test: nominal concentrations: 0 (control), 0.25, 0.55, 1.21, 2.66, 5.86 and 12.88 mg/L.
Spacing factor (2.2) determined from range-finding test and physico-chemical properties of the substance.
Controls: test water without test item but treated in the same way as the test item solutions.
Singular samples for possible analysis were taken from all test concentrations and the control; Frequency at t=0 h, t=24 h and t=72 h
- Sampling method: Single samples for analysis were taken from the control and all test concentrations at the start and at the end of the test (t=48h)
- Sample storage conditions before analysis: Samples were used on day of sampling and analysed as soon as possible after their preparation.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock preparation: The mixing vessel was a cylindrical glass bottle sealed with screw cap and fitted with a drain port near the bottom for drawing off the saturated stock solution. The volume of the mixing vessel was approximately 1 L. A magnetic stirring bar was placed in the vessel and approx. 1 L test water was added. Then an excess of the test item (approx. 100 mg) was carefully added directly to the surface of the test water. The mixing vessel was thereafter closed immediately. The mixing was initiated with the vortex in the centre extending maximally around 10% vessel depth from the top to the bottom of the vessel. After 23 hours of gentle stirring in the dark at room temperature, the contents of the vessel was allowed to stand undisturbed for at least 1 hour before use. The first 100 mL were discarded via the drain port. This was subject to analysis. The stock solution was diluted with test water as necessary to obtain the required test concentrations based on measured concentration of the stock solution (ca. 18 mg/L).
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable.
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): stock-solution: ca. 18 mg/L ; final test solutions (range finder): nominal concentrations: 0 (control), 0.32, 1.0, 3.2, 10.0 and 21.0 mg/L ; final test solutions (definitive): nominal concentrations: 0 (control), 0.25, 0.55, 1.21, 2.66, 5.86 and 12.88 mg/L.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): None. Turbidity testing indicated absence of undissolved test item.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Museum National d’Histoire Naturelle - 12, rue Buffon, Case N°19, 75005 Paris, France ; In-house laboratory culture.
- Age of inoculum (at test initiation): 2 to 4 days (in pre-culture under the same conditions as the test).
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 23 ± 2°C.

ACCLIMATION
- Acclimation period: 2 to 4 days pre-culture.
- Culturing media and conditions (same as test or not): Yes. OECD medium from OECD TG 201 : Annex 3
The stock solutions 1 and 3 were sterilised by autoclaving (120 °C, 15 min), then stored in the dark at 4°C. The stock solutions 2 and 4 were sterilised by membrane filtration (mean pore diameter 0.2 μm). The growth medium was prepared by adding an appropriate volume of the stock solutions 1-4 to water. To 500 mL of sterilised water were added: - 10 mL of stock solution 1, 1 mL of stock solution 2, 1 mL of stock solution 3, 1 mL of stock solution 4. Then, the mixture was made up to 1000 mL with sterilised water. The pH of this solution was approximately in the range of 8.1 ± 0.2.
- Any deformed or abnormal cells observed: None reported.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

Not reported ; Ca+Mg: 0.24 mmol/l (24 mg CaCO3/L) in this type of test system

Test temperature:

During the exposure period the temperature measured in the incubator was maintained between 23.2 and 23.4 °C. Temperature remained within the limits prescribed by the protocol (21-24°C, constant within 2°C).

pH:

0 hours: pH 8.3 ± 0.1 ; 72 hours: pH 8.30 - 9.34 (definitive test concentrations) and pH 9.75 (controls). pH did not vary more than 1.5 units.

Nominal and measured concentrations:

Range-finder test: Nominal: 0 (control), 0.32, 1.0, 3.2, 10.0 and 21.0 mg/L
Final test: Nominal: 0 (control), 0.25, 0.55, 1.21, 2.66, 5.86 and 12.88 mg/L
Equivalent geometric mean measured concentrations: 0 (control), 0.27, 0.80, 1.71, 2.93, 6.11 and 13.02 mg/L.
See table 3 for nominal and measured concentrations at initial exposure and during the course of the test.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass.
- Type (delete if not applicable): Closed - Static
- Material, size, headspace, fill volume: Glass, 100 mL, containing ca. 100 mL test solution minimum headspace; closed airtight (capped)
- Aeration: Vessel shaken continuously.
- Initial cells density: ca. 5 x 10^3 cells/mL
- Control end cells density: Mean (of replicates after 72 hours) 63.5 x10^4 cells/mL [127-fold increase within 72 hours. The specific growth rate was determined at 1.61 day-1, i.e. greater than 0.92 day-1]
- No. of vessels per concentration (replicates): 3 replicates of each test concentration
- No. of vessels per control (replicates): 6 replicates of the control
- No. of vessels per vehicle control (replicates): Not applicable.

GROWTH MEDIUM
- Standard medium used: Yes. OECD medium from OECD TG 201 : Annex 3
- Detailed composition if non-standard medium was used: See above. Since the test was performed in sealed conditions, additional sodium bicarbonate was added to test water to insure a satisfactory CO2 supply for the algal growth (for all treatments and inoculum suspension): 7 mL of NaHCO3 was added to the sterilised water during test water reconstitution (instead of 1 mL) to obtain a final concentration of 350 mg/L.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Not reported. Presumed to be either RO-Water
- Culture medium different from test medium: No. OECD medium from OECD TG 201 : Annex 3 was utilised modified as above.
- Intervals of water quality measurement: Not reported.

OTHER TEST CONDITIONS
- Sterile test conditions: No.
- Adjustment of pH: No. (Did not vary by more than 1.5 log units).
- Photoperiod: 24 hours ; continuous
- Light intensity and quality: 4440 to 8880 Lux
- Salinity (for marine algae): Not applicable.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cell numbers were counted daily by microscope using a counting chamber.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2. In definitive test justified from the results of the range finding study.
- Justification for using less concentrations than requested by guideline: Not applicable.
- Range finding study: Yes.
- Test concentrations: Three replicates per concentration were exposed to dilutions representing Nominal: 0 (control), 0.32, 1.0, 3.2, 10.0 and 21.0 mg/L in the range-finding test. In the definitive test 5 concentrations were used at Nominal: 0 (control), 0.25, 0.55, 1.21, 2.66, 5.86 and 12.88 mg/L
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.166 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 0.976 - 1.386 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.49 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CL: 0.394 - 0.593 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.657 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 0.417- 0.817 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.142 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CL: 0.079 - 0.201 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.27 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): Yes. [127-fold increase within 72 hours. The specific growth rate was determined at 1.61 day-1, i.e. greater than 0.92 day-1]
- Observation of abnormalities (for algal test): Microscopic observations at the end of the test did not report any unusual observations.
- Unusual cell shape: No.
- Colour differences: None.
- Flocculation: Not reported.
- Adherence to test vessels: Not reported.
- Aggregation of algal cells: No.
- Other:
- Any stimulation of growth found in any treatment: No.
- Effect concentrations exceeding solubility of substance in test medium: No.

Results with reference substance (positive control):

- Results with reference substance valid?: Yes.
- EC50: The EC50 for growth rate inhibition (72h-ERC50) was 0.98 mg/L
- Other: The sensitivity of the test system was in agreement with the guidelines (expected 72h-EC50: 0.92 mg/L to 1.46 mg/L).

Reported statistics and error estimates:

The evaluation of the effects was based on measured concentrations. The software utilised was ToxRat® Professional.
Full details of the probit analysis are reported in the full study report.

Table 1. Mean cell densities (x10^4 cells/mL) during the definitive test

	Replicate	Nominal concentration (mg/L)
		Control	0.25	0.55	1.21	2.66	5.86	12.88
t=24h	1	4.0	2.0	1.2	1.6	0.8	0.8	0.4
	2	2.8	0.8	2.0	0.8	0.4	0.4	0.4
	3	3.2	1.6	2.4	0.8	0.4	1.2	0.4
	4	3.6
	5	2.8
	6	3.6
	Mean	3.3	1.5	1.9	1.1	0.5	0.8	0.4
	Std. Dev.	0.48	0.61	0.61	0.46	0.23	0.40	0.00
t=48h	1	13.6	6.0	8.0	0.8	0.4	0.0	0.4
	2	10.8	15.6	6.8	2.0	0.0	1.2	0.4
	3	16.4	14.4	8.4	3.6	0.4	0.4	0.0
	4	12.0
	5	19.6
	6	15.6
	Mean	14.7	12.0	7.7	2.1	0.3	0.5	0.3
	Std. Dev.	3.21	5.23	0.83	1.40	0.23	0.61	0.23
t=72h	1	48.8	36.0	24.8	1.2	0.0	0.0	1.2
	2	48.0	40.4	22.4	1.6	0.8	0.0	0.4
	3	68.8	56.0	28.0	0.8	1.2	2.4	0.8
	4	72.0
	5	76.0
	6	67.2
	Mean	63.5	44.1	25.1	1.2	0.7	0.8	0.8
	Std. Dev.	12.05	10.51	2.81	0.40	0.61	1.39	0.40

 

Table 2. Mean specific growth rate during the definitive test

	Replicate	Nominal concentration (mg/L)
		Control	0.25	0.55	1.21	2.66	5.86	12.88
t=0h - t=24h	1	2.079	1.386	0.875	1.163	0.470	0.470	-0.223
	2	1.723	0.470	1.386	0.470	-0.223	-0.223	-0.223
	3	1.856	1.163	1.569	0.470	-0.223	0.875	-0.223
	4	1.974
	5	1.723
	6	1.974
	Mean	1.888	1.006	1.277	0.701	0.008	0.374	-0.223
	Std. Dev.	0.1463	0.4778	0.3593	0.4002	0.4002	0.5555	0.0000
	% Inh.	-	46.7	32.4	62.9	99.6	80.2	111.8
t=0h - t=48h	1	1.652	1.242	1.386	0.235	-0.112	-0.805	-0.112
	2	1.536	1.720	1.305	0.693	-0.805	0.438	-0.112
	3	1.745	1.680	1.411	0.987	-0.112	-0.112	-0.805
	4	1.589
	5	1.834
	6	1.720
	Mean	1.679	1.548	1.367	0.638	-0.343	-0.160	-0.343
	Std. Dev.	0.1090	0.2650	0.0553	0.3790	0.4002	0.6226	0.4002
	% Inh.	-	7.9	18.6	62.0	120.4	109.5	120.4
t=0h - t=72h	1	1.527	1.426	1.301	0.292	-0.536	-0.536	0.292
	2	1.521	1.464	1.267	0.388	0.157	-0.536	-0.074
	3	1.641	1.573	1.342	0.157	0.292	0.523	0.157
	4	1.657
	5	1.675
	6	1.634
	Mean	1.609	1.487	1.304	0.279	-0.029	-0.183	0.125
	Std. Dev.	0.0673	0.0764	0.0372	0.1161	0.4444	0.6116	0.1852
	% Inh.	-	7.6	19.0	82.7	101.8	111.4	92.3

 

Table 3. Measured concentrations versus nominal concentrations: final test

Nominal concentration (mg/L)	Expected nominal concentration in test item (mg test item/L )	Start (t=0h)		End (t=72h)		Relative loss to initial value (t=0h - t=72h) (%)	Geometric mean measured concentrations
		Meas. Conc. (mg/L)	% nominal	Meas. Conc. (mg/L)	% nominal		(mg/L)	% nominal
Control	0	Abs.	N.A.	Abs.	N.A.	N.A.	N.A.	N.A.
0.25	0.25	0.26	104	0.29	116	-11	0.27	108
0.55	0.54	0.96	178	0.67	124	30	0.80	148
1.21	1.19	1.91	161	1.53	129	20	1.71	144
2.66	2.62	3.06	117	2.81	107	8	2.93	112
5.86	5.78	6.54	113	5.71	99	13	6.11	106
12.88	12.69	13.59	107	12.47	98	8	13.02	103

N.A.: not applicable

% = Percent of expected nominal concentration in test item

Abs.= Absence: concentrations below the LOQ (0.11 mg/L) and the LOD (0.03 mg/L)

Validity criteria fulfilled:

yes

Conclusions:

The test item 72h-ErC50 for growth rate reduction was 1.166 (C.I. 0.976 – 1.386) mg/L based on GMM concentrations. The corresponding ErC10 was 0.657 (C.I. 0.417 – 0.817) mg/L and the NOEC was 0.270 mg/L.

Executive summary:

The algal growth inhibition to Pseudokirchneriella subcapitata, was carried out according to OECD TG 201 Freshwater Alga and Cyanobacteria, Growth Inhibition Test and EU Method C.3 guidelines under GLP. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours. Following a preliminary range finding study at 0.32, 1.0, 3.2, 10.0 and 21.0 mg/L nominal concentrations, a definitive study was conducted under closed static conditions and minimum headspace with an initial cell density of 5 x10^3 cells/mL. Five test item solutions were employed with a concentrations of 0 (control), 0.25, 0.55, 1.21, 2.66, 5.86 and 12.88 mg/L. Three replicates were tested for each test item concentration and six replicates for the control under constant illumination and shaking at a temperature of 23 ± 2 °C. Specific analyses of samples taken from all test concentrations at the start and the end of the test revealed that test item levels were stable, with losses of test item < 20%, except for the nominal concentration of 0.55 mg/L where losses of approximately 30% were observed. Test item concentrations were satisfactorily maintained within ± 20% of the nominal concentrations throughout the test, except for geometric means of the measured concentrations of 0.55 and 1.21 mg/L that were higher compared to their corresponding nominal concentrations (148 and 144%, respectively). On this basis the effect concentrations were based on equivalent geometric mean measured concentrations of: 0 (control), 0.27, 0.80, 1.71, 2.93, 6.11 and 13.02 mg/L. Environmental conditions were within the acceptable limits. In the control, cell density increased by an average factor of >16 within 72 hours. The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35%. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7%. The study met the acceptability criteria prescribed by the guidelines.The test item 72h-ErC50 for growth rate reduction was 1.166 (C.I. 0.976 – 1.386) mg/L based on GMM concentrations. The corresponding ErC10 was 0.657 (C.I. 0.417 – 0.817) mg/L and the NOEC was 0.270 mg/L.

Description of key information

EC50 (aquatic algae; growth rate) = 1.166 (C.I. 0.976 – 1.386) mg/L based on GMM concentrations, 72 hour, freshwater, OECD TG 201, 2018

EC10 (aquatic algae; growth rate) = 0.657 (C.I. 0.417 – 0.817) mg/L based on GMM concentrations, 72 hour, freshwater, OECD TG 201, 2018

NOEC (aquatic algae; growth rate) = 0.270 mg/L based on GMM concentrations, 72 hour, freshwater, OECD TG 201, OECD TG 201, 2018

Key value for chemical safety assessment

EC50 for freshwater algae:

1.166 mg/L

EC10 or NOEC for freshwater algae:

0.27 mg/L

Additional information

Key data : OECD TG 201, 2018 : The algal growth inhibition to Pseudokirchneriella subcapitata, was carried out according to OECD TG 201 Freshwater Alga and Cyanobacteria, Growth Inhibition Test and EU Method C.3 guidelines under GLP. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours. Following a preliminary range finding study at 0.32, 1.0, 3.2, 10.0 and 21.0 mg/L nominal concentrations, a definitive study was conducted under closed static conditions and minimum headspace with an initial cell density of 5 x10^3 cells/mL. Five test item solutions were employed with a concentrations of 0 (control), 0.25, 0.55, 1.21, 2.66, 5.86 and 12.88 mg/L. Three replicates were tested for each test item concentration and six replicates for the control under constant illumination and shaking at a temperature of 23 ± 2 °C. Specific analyses of samples taken from all test concentrations at the start and the end of the test revealed that test item levels were stable, with losses of test item < 20%, except for the nominal concentration of 0.55 mg/L where losses of approximately 30% were observed. Test item concentrations were satisfactorily maintained within ± 20% of the nominal concentrations throughout the test, except for geometric means of the measured concentrations of 0.55 and 1.21 mg/L that were higher compared to their corresponding nominal concentrations (148 and 144%, respectively). On this basis the effect concentrations were based on equivalent geometric mean measured concentrations of: 0 (control), 0.27, 0.80, 1.71, 2.93, 6.11 and 13.02 mg/L. Environmental conditions were within the acceptable limits. In the control, cell density increased by an average factor of >16 within 72 hours. The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35%. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7%. The study met the acceptability criteria prescribed by the guidelines.The test item 72h-ErC50 for growth rate reduction was 1.166 (C.I. 0.976 – 1.386) mg/L based on GMM concentrations. The corresponding ErC10 was 0.657 (C.I. 0.417 – 0.817) mg/L and the NOEC was 0.270 mg/L.