(1'R,2R)-2-Methyl-4-(2',2',3'-tri-nal and methylcyclopent-3'-en-1'-yl)-4-pent-enal

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:

05-03-2001 to 28-03-2001

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:

other: EEC Commission Directive 87/302/EEC

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: February 2000; signature: April 2000

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: For the purpose of the definitive test, the test item was: (i) dispersed directly in water.
Within (i): amounts of the test item (50, 90, 160, 280 and 500 mg test item) was dispersed in approximately 250 mL of water and subjected to ultrasonication (approximately 30 minutes). Synthetic sewage (16 mL), activated sewage sludge (200 mL) and water were added to a final volume of 500 mL to give the required concentration of 100, 180, 320, 560 and 1000 mg/L.
- Eluate: Not applicable.
- Differential loading: Not applicable.
- Controls: For positive control - reference item: Two stock solutions of 50 and 160 mg/L were prepared by dissolving the reference material directly in water with the aid of ultrasonic disruption. Aliquots (10 and 100 mL) of the 160 mg/L stock solution were removed and dispersed with activated sewage sludge, synthetic sewage and water to give the final concentrations of 3.2 and 32 mg/L. A 100 mL aliquot of the 50 mg/L stock solution was used to prepare the 10 mg/L concentration. The flasks were inverted several times to ensure homogeneity of the solutions.
- 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)): (i) direct dispersion: to give the required concentration of 100, 180, 320, 560 and 1000 mg/L.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Within (i) observations indicated the test item was visible as undissolved test item and as globules or an oily slick on the surface. With globules of test item also visible on the surface. This was considered to be due to the insoluble nature of the test item in the test medium.

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 Loughborough, UK (full details available in the full study report), which treats predominantly domestic sewage wastewater.
- Preparation of inoculum for exposure: The activated sewage sludge sample was maintained on continuous aeration in the laboratory at temperature of 21°C and was used on the day of collection. The pH of the sample was 7.4.
- Pretreatment: Not applicable.
- Initial biomass concentration: The suspended solids concentration in the inoculum was determined to be 4.0 g (dry weight) / L prior to use. In the preparation of the test system 200 mL inoculum was dissolved in 16 mL synthetic sewage and 300 mL water. Applicant assessment indicates: this should yield a final suspended solids concentration of ca. 1.5 to 1.6 g/L.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Remarks on exposure duration:

Respiration was measured at 30 minutes and 3 hours using a dissolved oxygen meter BOD probe

Post exposure observation period:

As each vessel reached 30 minutes contact time, an aliquot was removed from the conical flask and poured into the measuring vessel and the rate of respiration measured. The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace for an approximate 10 minute period (between approximately 7.9 mg O2/L and 1.7 mg O2/L). After measurement the contents of the BOD bottle were returned to the test vessel. This was repeated after 3 hours contact time.

Test temperature:

21 °C

pH:

3 hours: control: 7.8 and test groups: 7.7 and reference item: 7.9 - 8.1

Dissolved oxygen:

30 minutes: control: 5.9 – 6.9 mgO2/L and test groups: direct dispersion: 5.5 – 6.2 mgO2/L and reference item: 6.2 -7.5 mgO2/L
3 hours: control: 6.0 – 6.6 mgO2/L and test groups: direct dispersion: 6.5 – 6.8 mgO2/L and reference item: 6.7 – 7.9 mgO2/L

Nominal and measured concentrations:

Range finder: nominal: 0 (control), 1.1, 10.5, 100 and 1000 mg/L (conducted in two separate range finders) and Reference item was completed: nominal: 3.2 and 32 mg/L
Definitive test (direct dispersion): nominal: 0 (control), 100, 180, 320, 560 and 1000 mg/L
Reference item was completed: nominal: 3.2, 10, 32 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 mL glass flasks
- Type (delete if not applicable): Open, vessel continuously aerated with seal.
- Aeration: Yes, with 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): Definitive test (direct dispersion): single
- No. of vessels per control (replicates): control: duplicate ; triplicate (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: Test water used for both the range-finding and definitive tests was laboratory tap water dechlorinated by passage through an activated carbon filter and partly softened through a Duplex Water Softener giving total hardness 100 mg/L CaCO3. Water quality characteristics is reported in the full study report.
- 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: No. pH at end of test was in range 7.7 to 7.87 in controls and treatment groups ; 7.9 to 8.1 in the reference item groups
- 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 30-minute and 3-hour exposure phase.

TEST CONCENTRATIONS
- Test concentrations: Definitive test (direct dispersion): nominal: 0 (control), 100, 180, 320, 560 and 1000 mg/L
Reference item was completed: nominal: 3.2, 10, 32 mg/L
Further post study: Definitive test (direct dispersion): nominal: 0, 100 and 180 mg/L
Reference item was completed: nominal: 3.2 and 32 mg/L
- Range finding study: Yes.
- Results used to determine the conditions for the definitive study: Yes.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

180 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: direct dispersion

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: direct dispersion

Duration:

30 min

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:

direct dispersion

Remarks on result:

other: EC50 was found to be greater than the highest concentration employed in the test

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Not applicable. Nominal values only were utilised in the study. Observations indicated the test item was visible as undissolved test item and as globules or an oily slick on the surface. With globules of test item also visible on the surface. This was considered to be due to the insoluble nature of the test item in the test medium.
- Effect concentrations exceeding solubility of substance in test medium: Yes.
- Adsorption (e.g. of test material to the walls of the test container): Not reported.
- Blank controls oxygen uptake rate: 0.34 to 0.38 mgO2/L/min
- Coefficient of variation of oxygen uptake rate in control replicates: The oxygen consumption rates of the two controls differed by 6% (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 6.3 (C.I. – ) mg/L. This was within the expected range: 5 to 30 mg/L. Full information is provided in the full study report. In the post-study evaluations at 3.2 and 32 mg/L reference item: this again was in the expected range of 5 to 30 mg/L.

Table 1.0 – Definitive test results plus post study follow on work

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	-		7.8	8.5	8.1
B	0	0.905	-		7.8	8.2	8.3
Reference Item
1	3.2	0.748	25		7.9	8.1	8.4
2	10	0.289	69		8.0	8.3	8.4
3	32	0.093	89		8.1	8.2	8.2
Test item
4	100	0.871	[3]		7.7	7.8	8.1
5	180	0.940	50		7.7	8.3	8.5
6	320	0.752	50		7.7	7.8	8.5
7	560	0.574	53		7.7	7.9	8.4
8	1000	0.498	50		7.7	7.9	8.5
Inoculum Control
A	0	0.50	-			6.3	1.3
B	0	0.42	-			6.5	2.7
Reference Item
1	3.2	0.35	24			6.4	2.3
2	10	0.01	98			6.8	4.2
Test item
1	100	0.42	24			6.4	2.9
2	180	0.26	98			7.6	7.9

* : 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 180 mg/L based on nominal test item concentrations prepared by direct dispersion. The NOEC was 100 mg/L.

Executive summary:

The effect on respiration rate of activate sludge was examined using a method according to OECD TG 209 in accordance with GLP. Following a preliminary range finding tests (conducted over a range of test concentrations of 1.1, 10.5, 100 and 1000 mg/L), activated sewage sludge was exposed in a definitive limit test to the test item at concentrations of 100, 180, 320, 560 and 1000 mg/L prepared by direct dispersion for a period of 3 hours at 21°C with the addition of synthetic sewage as a respiratory substrate examining total respiration under static conditions. 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 6%. Due to observations of a flat dose response in the definitive test at 180, 320 and 560 mg/L further post study work using 0, 100 and 180 mg/L plus reference item at 3.2 and 32 mg/L were conducted dispersed directly in water. The three-hour 50% effect concentration for 3,5-DCP was estimated to be EC50 6.3 mg/L. This confirmed the suitability of the activated sludge and method used. In some instances, the initial and final dissolved oxygen concentrations were below those recommended in the test guidelines (6.5 mg O2/L and 2.5 mg O2/L respectively). This was considered to have had no adverse effect on the results of the study given that in all cases the oxygen consumption rate was determined over the linear portion of the trace. All validity criteria were considered to have been satisfied. Observations made throughout the study period, showed that at all test concentrations employed undissolved test item was visible as globules or an oily slick of test item on the surface of the test preparations. This was due to the insoluble nature of the test material in the test medium. After 3 hours contact time the 180, 320, 560 and 1000 mg/L test concentrations all exhibited approximately 50% inhibition of respiration rate. Post study work conducted at concentrations of 100 and 180 mg/L, confirmed that the flat response observed in the definitive study was a true effect. It was considered that this effect was possibly due to the presence of dispersed test material exhibiting an effect on the activated sewage sludge micro-organisms exoenzymes or phagocytosis processes. Under the conditions of this study, the test item total respiration EC50 was 180 mg/L. The NOEC was 100 mg/L based on nominal test item concentrations.

Description of key information

ASRIT: EC50 = 180 mg/L (nominal), 21°C, OECD TG 209, 2001

ASRIT: NOEC = 100 mg/L (nominal), 21°C, OECD TG 209, 2001

Key value for chemical safety assessment

EC50 for microorganisms:

180 mg/L

EC10 or NOEC for microorganisms:

100 mg/L

Additional information

Key study : OECD TG 209, 2001 : The effect on respiration rate of activate sludge was examined using a method according to OECD TG 209 in accordance with GLP. Following a preliminary range finding tests (conducted over a range of test concentrations of 1.1, 10.5, 100 and 1000 mg/L), activated sewage sludge was exposed in a definitive limit test to the test item at concentrations of 100, 180, 320, 560 and 1000 mg/L prepared by direct dispersion for a period of 3 hours at 21°C with the addition of synthetic sewage as a respiratory substrate examining total respiration under static conditions. 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 6%. Due to observations of a flat dose response in the definitive test at 180, 320 and 560 mg/L further post study work using 0, 100 and 180 mg/L plus reference item at 3.2 and 32 mg/L were conducted dispersed directly in water. The three-hour 50% effect concentration for 3,5-DCP was estimated to be EC50 6.3 mg/L. This confirmed the suitability of the activated sludge and method used. In some instances, the initial and final dissolved oxygen concentrations were below those recommended in the test guidelines (6.5 mg O2/L and 2.5 mg O2/L respectively). This was considered to have had no adverse effect on the results of the study given that in all cases the oxygen consumption rate was determined over the linear portion of the trace. All validity criteria were considered to have been satisfied. Observations made throughout the study period, showed that at all test concentrations employed undissolved test item was visible as globules or an oily slick of test item on the surface of the test preparations. This was due to the insoluble nature of the test material in the test medium. After 3 hours contact time the 180, 320, 560 and 1000 mg/L test concentrations all exhibited approximately 50% inhibition of respiration rate. Post study work conducted at concentrations of 100 and 180 mg/L, confirmed that the flat response observed in the definitive study was a true effect. It was considered that this effect was possibly due to the presence of dispersed test material exhibiting an effect on the activated sewage sludge micro-organisms exoenzymes or phagocytosis processes. Under the conditions of this study, the test item total respiration EC50 was 180 mg/L. The NOEC was 100 mg/L based on nominal test item concentrations.