(4E)-4-(3,3,4-trimethylcyclopentylidene)butanal

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:

From 10 October 2019 to 22 October 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Temperatures recorded continuously were situated between 17.7 and 20.7°C throughout the test (average value: 19.7°C), which was slightly lower than the requirements value: 20 ± 2 °C. This minor deviation was considered not to affect the results of the test as no impact was observed on the blank controls (all validity criteria of the test have been fulfilled).

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Deviations:

yes

Remarks:

Temperatures recorded continuously were situated between 17.7 and 20.7°C throughout the test (average value: 19.7°C), which was slightly lower than the requirements value: 20 ± 2 °C.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Temperatures recorded continuously were situated between 17.7 and 20.7°C throughout the test (average value: 19.7°C), which was slightly lower than the requirements value: 20 ± 2 °C.

GLP compliance:

yes (incl. QA statement)

Remarks:

Inspection dates: 29-30-31 May 2018 / Signature date: 15 November 2018

Specific details on test material used for the study:

- Appearance: From very light yellow to yellow liquid
- Storage condition of test material: Room temperature. Keep only in the original container in an appropriate place.
- Water solubility: 50.3 mg/L at 20°C (LPL 2020, OECD 105/EU A.6, slow-stirring method)
- Vapour pressure: 5.0 Pa at 20°C and 8.7 Pa at 25°C (LPL 2020, OECD 104/EU A.4, effusion method, Knudsen cell)
- Log Kow: 4.3 at 25°C (LPL 2020, OECD 123/EU A.23, slow-stirring method)

Analytical monitoring:

not required

Vehicle:

no

Details on test solutions:

- Administration of the test and reference substances: The test vessels were 1 L cylindrical glass bottles closed with PTFE screw caps. A magnetic stirring bar was placed in each test vessel. Blank controls were prepared with 16 mL synthetic sewage feed and 234 mL test water. Test mixtures were prepared by addition of the required amounts of test item in pre-filled test vessels (with synthetic sewage feed and test water). 3,5-dichlorophenol (3,5-DCP) was used as a reference (positive control) in order to check the procedure. The 3,5-DCP stock solution was diluted with test water as necessary to obtain the required test concentrations into reference mixtures after filling with synthetic sewage and water. Then, 250 mL of sludge suspension were sequentially added in all test vessels. After addition of the inoculum, the test vessels were immediately closed and the mixtures were stirred under aeration for 3 hours.
- Test mixtures: Based on the results of the range-finding test, test solutions were prepared to obtain the following nominal test concentrations: 10, 100 and 1000 mg/L in order to determine
a concentration with no significant effect and/or to demonstrate that EC50 was higher than 1000 mg/L.
- Controls: Test water without test item but treated in the same way as the test mixtures. The test item was not known to have strong reducing properties, therefore it was judged not necessary to perform abiotic controls.
- Reference mixtures: The 3,5-DCP stock solution was diluted to obtain the following nominal concentrations (spaced by a factor of approximately 1.68): 5.0, 8.4, 14.1, 23.7 and 40.0 mg/L

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Origin: Activated sludge was collected (14/10/2019) from a wastewater treatment plant (Aureilhan, France) receiving predominantly domestic sewage.
- Pretreatment: Upon arrival in the test facility, the sludge was sieved (using a 1 mm2 mesh sieve), washed 4 times with chlorine-free tap water and resuspended in tap water. The activated sludge was aerated and fed with the synthetic sewage feed (35 mL per L of sludge suspension) until the next day (final test). The dry weight of the sludge suspension was determined, then the sludge was diluted in chlorine-free tap water to obtain the required sludge solids concentration of approximately 3.0 g/L.
- Initial biomass concentration: The activated sludge concentration in all test, reference and blank mixtures was approx. 1.5 g/L of suspended solids

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

Temperatures recorded continuously were situated between 17.7 and 20.7°C throughout the test (average value: 19.7°C), which was slightly lower than the requirements laid down in the guidelines (20 ± 2 °C) during 40 minutes. This minor deviation was considered not to affect the results of the test as no impact was observed on the blank controls (all validity criteria of the test have been fulfilled).

pH:

The pH of the reaction mixtures at the end of the test period ranged from 7.728 to 8.097.

Salinity:

Not applicable

Nominal and measured concentrations:

- Nominal concentrations: 10, 100, 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: All-glass bottles of approximately 1 L capacity closed with PTFE screw caps, containing 500 mL of reaction mixture.
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 1 L capacity; 500 mL of reaction mixture
- Aeration: All suspensions were aerated and stirred during the exposure period.
- No. of vessels per concentration (replicates): 5 replicates bottles per test concentration containing synthetic sewage feed, test water, test item and inoculum (test mixtures)
- No. of vessels per control (replicates): 6 replicates bottles containing synthetic sewage feed, test water and inoculum (blank controls)
- No. of vessels per positive control (replicates): 1 replicate per reference substance concentration containing synthetic sewage feed, test water, reference substance and inoculum (reference
mixtures)
- Sludge concentration (weight of dry solids per volume): The activated sludge concentration in all test, reference and blank mixtures was approx. 1.5 g/L of suspended solids.
See details on content of test vessels in the Table 6.1.7/1 in "Any other information on materials and methods incl. tables'.

TEST MEDIUM / WATER PARAMETERS
- Test water: Deionised water, containing less than 1 mg/L DOC (Dissolved Organic Carbon).
- Test medium The medium was prepared by adding each of the following constituents at the stated amounts to each litre of ultrapure water:
Peptone = 16g; Meat extract = 11g; Urea = 3g; NaCl = 0.7g; CaCl2.2H2O = 0.4g; MgSO4.7H2O = 0.2g; K2HPO4 = 2.8g.
The pH of this solution was 7.14, in accordance with the requirements 7.5 +/- 0.5. This nutrient solution was refrigerated and stored in the dark before use.

OTHER TEST CONDITIONS
- Adjustment of pH: no

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Dissolved O2 (DO): After the exposure, samples from each mixture were poured into BOD bottles to continuously measure the oxygen concentration using an oxygen electrode and determine the rate of decrease of dissolved O2. The measurement of total oxygen uptake inhibition was determined only (carbon and ammonium oxidation not separated).

TEST CONCENTRATIONS
- Range finding study : A range-finding test was conducted between September 9 and September 10, 2019 to determine the range of concentrations for the definitive test. Microorganisms from activated sludge were exposed to the nominal test concentrations 10 (duplicates), 32, 100 (duplicates), 320 and 1000 mg/L (duplicates) and to a control. Test procedures and conditions were similar to those applied in the final test. All validity criteria were met (CV of oxygen uptake rate in control replicates: 9.0%; mean oxygen uptake rate in the blank controls: 30.8 mg O2/g dry weight activated sludge/h; EC50 of 3,5-DCP for total respiration: 15.1 mg/L).

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

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

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

After 3 hours of exposure, a slight effect on the respiration rate of activated sludge was observed at 100 and 1000 mg/L (average inhibition of 13.0 and 9.6%, respectively). Based on these results, the highest nominal test concentration without observed effects (= NOEC) was 10 mg/L; the 3-hour EC50 was estimated to be ≥ 1000 mg/L
.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: The EC50 of 3,5-DCP was 11.5 mg/L for total respiration and thus remained within the prescribed range of 2 to 25 mg/L.

Reported statistics and error estimates:

Statistical analyses were performed by ToxRat.

Table 6.1.7/2: Range-finding test results (respiration rate and inhibition at the end of the test)

		Total respiration (RT)
Content	Concentration (mg.L^-1)	Oxygen uptake rate (mg O2.g^-1.h^-1)	Inhibition (%)
Control (1)	0	29.650	-
Control (2)	0	30.849	-
Ref (1)	5.00	27.821	9.770
Ref (2)	8.40	28.299	8.219
Ref (3)	14.20	14.357	53.436
Ref (4)	23.80	7.021	77.228
Ref (5)	40.00	4.677	84.833
Test item	10.72	33.351	-8.167
Test item	11.22	28.027	9.100
Test item	33.72	28.956	6.089
Test item	100.08	26.584	13.781
Test item	101.02	25.140	18.463
Test item	320.66	28.485	7.617
Test item	999.2	26.589	13.764
Test item	1001.92	24.106	21.817
Control (3)	0	34.645	-
Control (4)	0	28.190	-

Table 6.1.7/3: Definitive test results (oxygen uptake rate, inhibition, and pH at the end of the test)

Content	Concentration (mg.L^-1)	Oxygen uptake rate (mg O2.g^-1.h^-1)	Inhibition (%)	pH
Control (1)	0	26.247	-	7.950
Control (2)	0	26.755	-	7.944
Ref (1)	5.00	23.763	17.389	7.887
Ref (2)	8.40	19.932	30.708	7.844
Ref (3)	14.20	10.413	63.799	8.018
Ref (4)	23.80	5.105	82.252	8.048
Ref (5)	40.00	3.691	87.169	8.097
Control (3)	0	27.985	-	7.966
Control (4)	0	28.394	-	7.917
Test item	10.28	26.471	7.975	7.945
Test item	11.96	28.832	-0.233	7.876
Test item	11.02	30.270	-5.233	7.782
Test item	10.54	29.438	-2.338	7.889
Test item	11.34	29.357	-2.057	7.946
Test item	100.16	26.080	9.333	7.976
Test item	100.50	24.509	14.795	8.066
Test item	101.44	24.868	13.546	8.055
Test item	101.16	24.641	14.337	8.080
Test item	101.44	n.d.*	n.d.	7.756
Test item	1000.12	32.743	-13.831	7.728
Test item	1001.86	23.468	18.414	7.781
Test item	1000.04	25.602	10.996	7.737
Test item	999.94	23.852	17.078	7.760
Test item	1000.18	24.368	15.286	7.754
Control (5)	0	29.693	-	7.984
Control (6)	0	33.519	-	7.808

Validity criteria fulfilled:

yes

Remarks:

The validity of the test was demonstrated: Oxygen uptake rate in the blank controls >20 mg O2/g/h; Coefficient of variation of oxygen uptake rate in control replicates <30%; EC50 = 11.5 mg/L for 3,5-DCP (within the range 2-25 mg/L for total respiration).

Conclusions:

After 3 hours of exposure, a slight effect on the respiration rate of activated sludge was observed at 100 and 1000 mg/L (average inhibition of 13.0 and 9.6%, respectively). No effect on the respiration rate was observed at 10 mg/L.
Therefore, under the experimental conditions and based on nominal concentrations, the 3h-EC50 value was estimated to be higher than 1000 mg/L. The NOEC value was 10 mg/L and the LOEC 100 mg/L.

Executive summary:

This study was performed according to OECD Guideline 209 and EU Method C.11 with GLP compliance to assess the effects of the test substance on microorganisms from activated sludge.

Following a preliminary range-finding test, microorganisms from activated sludge were exposed to the test substance at nominal concentrations of 10, 100 and 1000 mg/L (five replicates for each concentration) and to six blank controls in order to determine a concentration with no significant effect and to demonstrate that EC50 was higher than 1000 mg/L. Furthermore, an exposure of the test organism to chemical 3,5 -dichlorophenol (3,5 -DCP) was performed in order to check the sensitivity of the batch of activated sludge used on the day of exposure. All the mixtures contained an activated sludge concentration of about 1.5 g/L of suspended solids. After 3 hours of exposure under aeration and stirring, samples of each mixture were poured into BOD bottles to continuously measure the oxygen concentration using an oxygen electrode and determine the rate of decrease of dissolved O2. The measurement of total oxygen uptake inhibition was determined only (carbon and ammonium oxidation not separated).

After 3 hours of exposure, a slight effect on the respiration rate of activated sludge was observed at 100 and 1000 mg/L (average inhibition of 13.0 and 9.6%, respectively). No effect on the respiration rate was observed at 10 mg/L.

Therefore, under the experimental conditions and based on nominal concentrations, the 3h-EC50 value was estimated to be higher than 1000 mg/L. The NOEC value was 10 mg/L and the LOEC 100 mg/L.

Description of key information

OECD Guideline 209, EU Method C.11, GLP, Key study, validity 1:

3h-EC50 (activated sludge) > 1000 mg/L based on nominal concentrations.

3h-NOEC (activated sludge) = 10 mg/L based on nominal concentrations.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

10 mg/L

Additional information

One key study is available to assess the toxicity of the registered substance to microorganisms.

This study (LPL, 2020) was performed according to OECD Guideline 209 and EU Method C.11 with GLP compliance. Following a preliminary range-finding test, microorganisms from activated sludge were exposed to the registered substance at nominal concentrations of 10, 100 and 1000 mg/L (five replicates for each concentration) and to six blank controls in order to determine a concentration with no significant effect and to demonstrate that EC50 was higher than 1000 mg/L. After 3 hours of exposure, a slight effect on the respiration rate of activated sludge was observed at 100 and 1000 mg/L (average inhibition of 13.0 and 9.6%, respectively). No effect on the respiration rate was observed at 10 mg/L. Therefore, under the experimental conditions and based on nominal concentrations, the 3h-EC50 value was estimated to be higher than 1000 mg/L and the NOEC value was determined at 10 mg/L.