(2Z)-2-phenylhex-2-enenitrile

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15-09-2008 to 15-10-2008

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 209 (Activated Sludge, Respiration Inhibition Test

Version / remarks:

(4 April 1984)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: Novermber 2007; signature: June 2008

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 5.01, 16.06, 50.44, 160.10 and 500.16 mg of test item were each separately dispersed in approximately 284 mL tap water. The test item was mixed into the tap water by ultrasonic treatment for fifteen minutes and intense stirring for 24 hours at room temperature in the dark to dissolve a maximum amount of the test item and/or disperse it as homogeneously as possible. No emulsifiers or solvents were used. Synthetic sewage waste water (16 mL), activated sludge (200 mL) were added to give the required concentrations of 10, 32, 100, 320 and 1000 mg/L.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None reported

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Source: A mixed population of activated sewage sludge microorganisms was obtained from the aeration stage of the sewage treatment plant at (ARA Ergolz II, Füllinsdorf, Switzerland), which treats predominantly domestic sewage wastewater.
- Preparation of inoculum for exposure: The sludge was washed twice with tap water by centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated. The pH of the sample on the day of the test was adjusted from 8.2 to 7.0.
- Pretreatment: During the holding period of two days prior to use, the sludge was fed daily with 50 mL synthetic wastewater (details within the full study report) per litre and was kept at room temperature under continuous aeration until use. Before use, the dry weight of the activated sludge was measured again in the inoculum used for the test.
- Initial biomass concentration: The suspended solids concentration was determined to be 3.0 g (dry weight) / L prior to use.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

As each vessel reached 3 hours contact time, an a well-mixed sample of each test medium was poured into a flask after three hours incubation time, and was not further aerated. Then the dissolved oxygen concentration was measured with an oxygen electrode and an oxygen meter and continuously recovered. The contents of the measuring vessel were stirred constantly by magnetic stirrer. The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace (where possible between approximately 2.5 to 6.5 mg O2/L and 2.0 mg O2/L). In the case of a rapid oxygen consumption, measurements may have been outside this range but the oxygen consumption was always within the linear portion of the respiration curve. In the case of the low oxygen consumption, the rate was determined over a period of at least 10 minutes.

Test temperature:

20 ± 1 °C

pH:

0 hours: control: 7.1 - 7.3 and test groups: 7.2 - 7.4 and reference item: 7.1 - 7.2
3 hours: control: 7.9 - 8.0 and test groups: 7.9 - 8.0 and reference item: 8.0

Dissolved oxygen:

0 minutes: control: 8.2 - 8.5 mgO2/L and test groups: 7.8 - 8.3 mgO2/L and reference item: 8.1 - 8.3 mgO2/L
3 hours:control: 8.1 - 8.3 mgO2/L and test groups: 8.1 - 8.5 mgO2/L and reference item: 8.2 - 8.4 mgO2/L

Nominal and measured concentrations:

Definitive test: nominal: 0 (control) 10, 32, 100, 320 and 1000 mg/L
Reference item was completed: nominal: 5, 16, 50 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 2000 mL glass flasks
- Type (delete if not applicable): Open, vessel continuously aerated with seal.
- Aeration: Yes, with intense stirring on magnetic stirrers but without foaming.
- Type of flow-through (e.g. peristaltic or proportional diluter): Not reported.
- Renewal rate of test solution (frequency/flow rate): None.
- No. of vessels per concentration (replicates): Single.
- No. of vessels per control (replicates): Single (negative) and single (reference item)
- Nitrification inhibitor used (delete if not applicable): Not applicable.
- Biomass loading rate: See table.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: See table.
- Culture medium different from test medium: Not applicable.
- Intervals of water quality measurement: Temperature, pH and dissolved oxygen values were determined in all test media and controls; prior to and at the end of the 3 hour incubation period.

OTHER TEST CONDITIONS
- Adjustment of pH: The pH of the inoculated sludge was measured and adjusted at the start of the test only to pH 7.0, if necessary. The test item dispersions were not adjusted for pH.
- Light intensity: The test was conducted under normal laboratory lighting

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Biological Oxygen Demand. Monitor the oxygen consumed by the test and control mixtures following a 3-hour exposure phase.

TEST CONCENTRATIONS
- Test concentrations: Definitive study: 0, (control) 10, 32, 100, 320 and 1000 mg/L (nominal)
- Range finding study: Yes.
- Results used to determine the conditions for the definitive study: Yes. The range finding study was not reported but the final test item concentrations were selected based on the preliminary results. The spacing factor for the test item concentrations was in accordance with the guideline (spacing factor: 3.2)

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other:

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

156 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95% Confidence Limits 17 - 319 mg/L

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

98 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95% Confidence Limits 5 - 219 mg/L

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None.
- Effect concentrations exceeding solubility of substance in test medium: No.
- Adsorption (e.g. of test material to the walls of the test container): Not reported.
- Blank controls oxygen uptake rate: 0.883 to 0.905 mgO2/L/min; mean: 0.894 mgO2/L/min (deviation: 2.6)
- Coefficient of variation of oxygen uptake rate in control replicates: The oxygen consumption rates of the two controls (run at the start and at the end of the test) differed only by 3% (guideline, recommended maximum variation: 15%).

Results with reference substance (positive control):

- Results with reference substance valid?: Yes.
- Relevant effect levels: the EC50 for 3,5-dichlorophenol was: total respiration: actual 11 (C.I. – ) mg/L. This was within the expected range: 5 to 30 mg/L. Full information is provided in the full study report.

Reported statistics and error estimates:

Effect levels were estimated using Probit analysis (Finney, D.J., 1971) and/or using statistical programmes (consistent with Davies, R.G, 1971).

Table 1.0 – composition of the test media

Vessel	Final Nominal concentration (mg/L)	Synthetic waste water (mL)	Amount of test item (mg)*	Aliquot of stock solution (mL)**	Activated sludge (mL)	Tap water (mL)
Inoculum Control
A	0	16	-	-	200	284
Reference Item
1	5	16	-	5	200	279
2	16	16	-	16	200	268
3	50	16	-	50	200	234
Test item
4	10	16	5	-	200	284
5	32	16	16	-	200	284
6	100	16	50	-	200	284
7	320	16	160	-	200	284
8	1000	16	500	-	200	284
Inoculum Control
B	0	16	-	-	200	284

* : amounts directly weighed into the designated test vessels (rounded values)

** : stock solution of reference item

 

Table 2.0 – Definitive test results

Vessel	Nominal concentration (mg/L)	Oxygen consumption rate (mg O2 / L / min)	Inhibition (%)	pH		Oxygen concentration (mg O2 / L)
				start*	end*	start*	end*
Inoculum Control
A	0	0.883		7.1	7.9	8.5	8.1
B	0	0.905		7.3	8.0	8.2	8.3
Reference Item
1	5	0.748	16.4	7.1	8.0	8.1	8.4
2	16	0.289	67.7	7.2	8.0	8.3	8.4
3	50	0.093	89.6	7.2	8.0	8.2	8.2
Test item
4	10	0.871	2.6	7.2	7.9	7.8	8.1
5	32	0.940	-5.1	7.3	8.0	8.3	8.5
6	100	0.752	15.9	7.3	7.9	7.8	8.5
7	320	0.574	35.8	7.4	7.9	7.9	8.4
8	1000	0.498	44.3	7.3	7.9	7.9	8.5

* : start and end of the 3-hour incubation period

- % inhibition: increased oxygen consumption rate related to the control

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, the 3 hour EC50 for total inhibition was > 1000 mg/L. The EC20 was 156 (C.I. 17 - 319) mg/L. All effect levels were based on nominal test item concentrations.

Executive summary:

The effect on respiration rate of activate sludge was examined using a method according to OECD TG 209 and EU Method C.11 in accordance with GLP. A definitive test was conducted examining total respiration only was carried out under static conditions. The test item concentrations were 0 (control) 10, 32, 100, 320 and 1000 mg/L for a period of three hours at a temperature of 20 ± 1 °C with the addition of synthetic sewage as respiratory substrate. The respiration rates of the control, reference item (3,5-dichlorophenol) and test item replicates were measured after a contact time of three hours, and the inhibitory effects of the test and reference item were determined in comparison to the control respiration rates. The variation of oxygen uptake in the inoculum control vessels was 3%. The three-hour 50% effect concentration for 3,5-DCP was estimated to be EC50 11 mg/L. This confirmed the suitability of the activated sludge and method used. All validity criteria were considered to have been satisfied. Saturation concentration (equilibrium) under the test conditions was reached and resulted in less than 50% inhibition after three hours contact time. Thus, the 3-hour EC50 and EC80 are clearly higher than the water solubility limit of the test item under the test conditions, or higher than 1000 mg/L. Under the conditions of this study, the test item total respiration EC20 was 156 (C.I. 17 – 319) mg/L, EC50 was > 1000 mg/L. The NOEC was 98 (C.I. 5 – 219) mg/L. All effect levels were based on nominal test item concentrations.

Description of key information

ASRIT: EC50 = > 1000 mg/L (nominal), 20 ± 2°C, OECD TG 209, 2008

ASRIT: EC80 = > 1000 mg/L (nominal, 20 ± 2°C, OECD TG 209, 2008

ASRIT: EC20 = 156 mg/L (C.I. 17 - 319 mg/L) (nominal), 20 ± 2°C, OECD TG 209, 2008

ASRIT: NOEC = 98 mg/L (C.I. 5 - 219 mg/L) (nominal), 20 ± 2°C, OECD TG 209, 2008

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

98 mg/L

Additional information

Key data : OECD TG 209, 2008 : The effect on respiration rate of activate sludge was examined using a method according to OECD TG 209 and EU Method C.11 in accordance with GLP. A definitive test was conducted examining total respiration only was carried out under static conditions. The test item concentrations were 0 (control) 10, 32, 100, 320 and 1000 mg/L for a period of three hours at a temperature of 20 ± 1 °C with the addition of synthetic sewage as respiratory substrate. The respiration rates of the control, reference item (3,5-dichlorophenol) and test item replicates were measured after a contact time of three hours, and the inhibitory effects of the test and reference item were determined in comparison to the control respiration rates. The variation of oxygen uptake in the inoculum control vessels was 3%. The three-hour 50% effect concentration for 3,5-DCP was estimated to be EC50 11 mg/L. This confirmed the suitability of the activated sludge and method used. All validity criteria were considered to have been satisfied. Saturation concentration (equilibrium) under the test conditions was reached and resulted in less than 50% inhibition after three hours contact time. Thus, the 3-hour EC50 and EC80 are clearly higher than the water solubility limit of the test item under the test conditions, or higher than 1000 mg/L. Under the conditions of this study, the test item total respiration EC20 was 156 (C.I. 17 – 319) mg/L, EC50 was > 1000 mg/L. The NOEC was 98 (C.I. 5 – 219) mg/L. All effect levels were based on nominal test item concentrations.