3,3,5-trimethylcyclohexyl acrylate

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:

2016-11-10 to 2017-

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

activated sludge, domestic

Details on inoculum:

The inoculum was obtained on 21 November 2016 from the activated sludge of the biological wastewater treatment plant from Abidos (France, 64) which handles predominantly domestic sewage.

On arrival at the laboratory, the activated sludge (around 15L) was centrifuged (1000g for 10 minutes) and the solid sewage inoculum pellet was re-suspended in 10L deionised water. 500 mL synthetic sewage feed was then added. Sewage sludge was maintained under aerated conditions for a maximum of 4 days and fed daily with synthetic sewage feed.

In order to obtain the required sludge suspended solids concentration of 3 g/L in the stock solution to be used in the experiments, the solid content of the 10-L sludge solution was determined by removing a sub-sample of homogenised sludge and drying in an oven at approximately 105°C. The suspended solids concentration was subsequently adjusted with the source water until the recommended guideline value of 3 g/L ± 10% is achieved.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

The ambient temperature remained constant at 20°C within ± 2°C during the test:
• Room 1: mean measured temperature: 21.3°C, min.: 20.9°C, max.: 21.8°C.
• Room 2: mean measured temperature: 21.6°C, min.: 21.4°C, max.: 21.6°C.

pH:

Measured pH were in the range 7.5 – 7.6, no adjustment was performed.

Nominal and measured concentrations:

Nominal concentrations: 10, 100, 1000 mg/L

Reference substance (positive control):

yes

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

inhibition of total respiration

Range-finding test

 Reference item results

Oxygen measurements for each time point used to calculate the total respiration rate and the percentage inhibition values compared to the control are presented in the tables below:

Respiration Rates and Percentage Inhibition: Reference item (Range-Finding test)

Oxygen measurements (mg O2/L)
Time (min)	Control a	Nominal concentration (mg/L)			Control b
		3.2	10.0	32.0
0	8.15	7.52	7.66	8.20	7.56
1	6.53	5.66	6.70	8.05	6.54
2	5.30	4.80	6.18	7.87	5.45
3	4.36	3.91	5.65	7.66	4.60
4	3.31	3.04	5.12	7.48	3.73
5	2.24	2.15	4.61	7.29	3.01
6	1.17	1.28	4.08	7.08	2.36
7	0.17	0.44	3.58	6.91	1.74
8			3.07	6.72	1.13
9			2.55	6.52	0.55
10			2.05	6.33
pH at T0	7.6	7.6	7.5	7.5	7.5

Percentage Inhibition
Treatment	Oxygen measurements		Length of time used in respiration rate calculation (minutes)	Respiration Rate (mg O2/L/h)	Respiration Rate (mg O2/g/h)	% Inhibition
	T0	T+"x" min
Control a	5.30	2.24	3.0	61.20	40.80
Control b	6.54	1.74	6.0	48.00	32.00
3.2	5.66	2.15	4.0	52.65	35.10	3.6
10.0	6.70	2.05	9.0	31.00	20.67	43.2
32.0	8.05	6.33	9.0	11.47	7.64	79.0

 

Test item results

Oxygen measurements for each time point and treatment used to calculate the total respiration rate and the percentage inhibition values compared to the control are presented in the tables below:

Respiration Rates and Percentage Inhibition: Test item (Range-finding test)

Oxygen measurements (mg O2/L)
Time (min)	Control c	Nominal concentration (mg/L)						Control d
		10.0		100.0		1000.0
		a	b	a	b	a	b
0	8.07	7.56	7.58	7.38	7.36	7.31	7.25	7.49
0.5				3.35	3.30		4.39
1	6.57	4.93	5.31	2.80	2.23	4.56	3.61	6.94
1.5				2.34	1.65		3.15
2	5.78	3.97	4.29	1.90	1.12	3.96	2.78	6.42
2.5				1.46	0.61		2.42
3	4.98	2.99	3.29	1.02	0.17	3.33	2.06	5.89
3.5				0.59			1.68
4	4.18	2.04	2.31	0.18		2.71	1.27	5.37
4.5							0.93
5	3.40	1.09	1.35			2.08	0.58	4.85
5.5							0.23
6	2.59	0.17	0.40			1.48		4.31
6.5
7	1.79					0.86		3.80
7.5
8	1.02					0.26		3.28
8.5
9	0.22							2.76
9.5
10								2.23
pH at T0	7.5	7.5	7.6	7.5	7.6	7.6	7.6	7.6

Treatment	Replicate	Oxygen measurements		Length of time used in respiration rate calculation (minutes)	Respiration Rate (mg O2/L/h)	Respiration Rate (mg O2/g/h)	% inhibition per replicate	% inhibition mean
		T0	T+"x" min
Control	c	6.57	1.79	7.0	40.97	27.31
	d	6.94	2.23	9.0	31.40	20.93
10.00	a	4.93	2.04	3.0	57.80	38.53	-59.7	-62.8
	b	5.31	2.31	3.0	60.00	40.00	-65.8
100.00	a	3.35	1.90	1.5	58.00	38.67	-60.3	-116.9
	b	3.30	1.65	1.0	99.00	66.00	-173.6
1000.00	a	4.56	2.08	4.0	37.20	24.80	-2.8	-28.7
	b	4.39	2.06	2.5	55.92	37.28	-54.5

 

No inhibition in respiration was observed at any tested concentrations as confirmed with the statistical analysis. On the contrary, the respiration rate determined in all exposed test flasks is higher than in control group. The test item seems to be favourable to the activated sludge respiration.

 

 Definitive test

From the results obtained in the preliminary range-finding test and in accordance with the Sponsor’s representative, no definitive test was conducted.

Validity criteria / quality criteria

The oxygen uptake and the coefficient of variation of oxygen uptake rate in control replicates were as follows:

 

Test substance	Oxygen uptake (Rs) (mg O2/g of activated sludge (dry weight of suspended solids)/h)			Coefficient of variation of oxygen uptake rate
	Control a	Control b	Mean
Reference item	40.8	32.0	36.4	17.1%
Test item	27.3	20.9	24.1	18.7%

 

The 3h-EC₅₀for 3,5-dichlorophenol to activated sludge was estimated under the conditions of the test as follows: 25.0 mg/L for total respiration.

This test met the validity criteria of the guideline:

 § The coefficient of variation of oxygen uptake rate in the controls should be no more than 30% at the end of the definitive test: it was determined to be 17.1% for the reference item test and 18.7% for the test item assay

 § The 3h-EC50 for 3,5-dichlorophenol (25.0 mg/L) was between 2 and 25 mg/L for total respiration

 § The blank controls oxygen uptake was not less than 20 mg oxygen/g of activated sludge (dry weight of suspended solids) in an hour: 36.4 and 24.1 mg O₂/g of activated sludge (dry weight of suspended solids)/h

Validity criteria fulfilled:

no

Conclusions:

This study was designed to determine the inhibition of respiration rates of an activated sludge by the test item following the procedure described in the corresponding OECD guideline 209.

No inhibition in respiration was observed at any tested concentrations as confirmed with the statistical analysis. On the contrary, the respiration rate determined in all exposed test flasks is higher than in control group. The test item seems to be favourable to the activated sludge respiration: in accordance with the Sponsor’s representative, no definitive test was conducted.

LOEC and NOEC values under the conditions of this test were thus calculated to be respectively >1000.0 mg/L and =1000.0 mg/L.

Executive summary:

This study was designed to determine the effects of the test item on sewage micro-organisms by measuring the respiration rate in 3 hours test according to the OECD Guideline 209 (July 2010).

 

At the request of the sponsor, the inhibition of oxygen uptake by ammonium oxidation (i.e. nitrification) was not determined, so that only total respiration inhibition was assessed in this study.

 

The test was conducted at the following nominal concentrations:10.0, 100.0 and 1000.0 mg/L.A reference item (3, 5-dichlorophenol) was tested as a reference inhibitory substance at the following nominal concentrations: 3.2, 10.0 and 32.0 mg/L.

 

The prepared vessels were aerated for 3h, after which dissolved oxygen concentrations were measured at 1 min intervals for up to a maximum period of 10 min. The respiration rate for each vessel was calculated from the measured values of recorded oxygen concentrations versus time and expressed as mg O₂/L/h for the linear part of the graphs (then converted in mg O₂/g of activated sludge dry weight of suspended solids/h).

 

The definitive test met the validity criteria of the guideline detailed as follows:

 

§ The coefficient of variation of oxygen uptake rate in the controls should be no more than 30% at the end of the definitive test: it was determined to be 17.1% for the reference item test and 18.7% for the test item assay

 

§ The 3h-EC50 for 3,5-dichlorophenol (25.0 mg/L) was between 2 and 25 mg/L for total respiration

 

§ The blank controls oxygen uptake was not less than 20 mg oxygen/g of activated sludge (dry weight of suspended solids) in an hour: 36.4 and 24.1 mg O₂/g activated sludge (dry weight of suspended solids)/h

 

No inhibition in respiration was observed at any tested concentrations as confirmed with the statistical analysis. On the contrary, the respiration rate determined in all exposed test flasks was higher than in the control group. This suggests that the test item could be favourable to the activated sludge respiration. In accordance with the Sponsor’s representative, no definitive test was conducted.

 

LOEC and NOEC values under the conditions of this test were thus calculated to be respectively>1000.0 mg/L and =1000.0 mg/L.

Description of key information

A study assessing the respiration inhibition effects of 3,3,5-TRIMETHYLCYCLOHEXYL ACRYLATE (CAS 86178-38-3) to an activated sludge was conducted in accordance with the OECD 209 Test Guideline and GLP requirements.

The 3h NOEC was determined to be equal to the highest concentration tested: 1000 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L