(octahydro-4,7-methano-1H-indenyl)methyl 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:

2018-04-19 to 2018-

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

Details on test solutions:

RANGE-FINDING TEST: PREPARATION OF TEST SOLUTIONS
In the range-finding test, activated sewage sludge micro-organisms were exposed to a series of nominal test concentrations of 10, 100 and 1000 mg/L. Two replicate vessels were prepared per treatment. The test item was dispersed directly in water.

The reference item, 3,5-dichlorophenol, was included at concentrations of 3.2, 10 and 32 mg/L in order to confirm the suitability of the inoculum. A stock solution at 1 g/L was prepared by dissolving the reference item (101 mg) directly in previously heated water to aid dissolution, the volume was then made up to 100 mL with ultra-pure water. Aliquots of the stock solution were added to activated sewage sludge, synthetic sewage and water to achieve final concentrations of 3.2, 10 and 32 mg/L, each with a single replicate.

At the start of the test, 16 mL of synthetic sewage feed were made up to 250 mL with ultra-pure water and 250 mL activated sludge inoculum were added to the first blank control vessels Fba (final suspended solid of nominally 1.5 g/L/dw). Thereafter, at time intervals of about fifteen minutes, the procedure was repeated with test vessels in which 16 mL of synthetic sewage were made up to 250 mL with the appropriate amount of reference or test item diluted in ultra-pure water. As for the control, 250 mL of activated sludge inoculum were then added. Fifteen minutes after the preparation of the last treatment, the last blank control vessel Fbb was prepared at last. The interval lasted fifteen minutes so that the 10 min incubation period with O2 depletion monitoring could take place exactly 3 hours after start of incubation of each test vessel.

Details including weights of test item, volumes of constituent and final volumes are shown in Table 1 (see "any other information on materials and methods incl. tables").

Upon addition of the microbial inoculum, the pH was measured in order to identify if there was any variation in pH caused by the addition of the test item. The pH was not adjusted.

DEFINITIVE TEST: PREPARATION OF TEST SOLUTIONS
The definitive test was conducted using a range of concentrations deduced from the preliminary range-finding test: activated sewage sludge micro-organisms were exposed to a series of nominal test concentrations of 95.3 171.5, 308.6, 555.6 and 1000.0 mg/L. In order to obtain both a NOEC and an EC50, four control replicates and five treatment concentrations in a geometric series with 2 replicates each were prepared. The test item was dispersed directly in ultra-pure water. The same inoculum as for the range-finding test was used.

The reference item, 3,5-dichlorophenol, was included at concentrations of 3.2, 10 and 32 mg/L in order to confirm again the suitability of the inoculum (test solutions were prepared with a single replicate from the same stock solution as the range-finding test).

The test protocol was identical to the one described for the range-finding test, except that more test item concentrations were tested.

Details including weights of test item, volumes of constituent and final volumes are shown in Table 2 (see "any other information on materials and methods incl. tables").

Upon addition of the microbial inoculum the pH was measured in order to identify if there was any variation in pH caused by the addition of the test item. The pH was not adjusted.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

INOCULUM
RANGE-FINDING TEST
The inoculum was obtained on 01 October 2018 from the activated sludge of the biological wastewater treatment plant from Mourenx (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 2 days.

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 deionised water until the recommended guideline value of 3 g/L ± 10% was achieved.


DEFINITIVE TEST
The inoculum was obtained on 28 May 2018 from the activated sludge of the biological wastewater treatment plant from Mourenx (France, 64) which handles predominantly domestic sewage.

On arrival at the laboratory, the activated sludge (around 20L) was centrifuged (1000g for 10 minutes) and the solid sewage inoculum pellet was re-suspended in 6L deionised water. 300 mL synthetic sewage feed was then added. Sewage sludge was maintained under aerated conditions for a maximum of 2 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 6-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:

¿ Range-finding test: mean measured temperature: 21.1°C, min.: 20.9°C, max.: 21.4°C
¿ Definitive test: mean measured temperature: 20.5°C, min.: 20.1°C, max.: 21.2°C.

pH:

See "any other information on results incl. tables".

Nominal and measured concentrations:

Nominal concentrations
RANGE-FINDING TEST
Test item 10.0, 100.0, 1000.0 mg/L
Reference item 3.2, 10.0, 32.0 mg/L

DEFINITIVE TEST
Test item 95.3, 171.5, 308.6, 555.6, 1000.0 mg/L
Reference item 3.2, 10.0, 32.0 mg/L

Details on test conditions:

See "any other information on materials and methods incl. tables".

Key result

Duration:

3 h

Dose descriptor:

NOEC

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:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

See "any other information on results incl. tables".

Results with reference substance (positive control):

See "any other information on results incl. tables".

Results and discussion

Table3Respiration Rates and Percentage Inhibition: Reference item (Range-Finding test)

Oxygen measurements (mg O2/L)
Time (min)	Control Fba	Nominal concentration (mg/L)			Control Fbb
		Fc1: 3.2	Fc2: 10.0	Fc3: 32.0
0	8.04	7.92	8.64	8.12	8.51
1	2.82	5.02	6.77	7.43	3.73
2	2.30	4.40	6.39	7.23	3.13
3	1.78	3.91	6.02	7.06	2.62
4	1.26	3.44	5.63	6.89	2.04
5	0.75	2.98	5.26	6.76	1.46
6	0.25	2.49	4.88	6.58	0.88
7	0.05	2.02	4.51	6.40	0.32
8		1.55	4.12	6.23	0.05
9		1.07	3.74	6.08
10		0.59	3.37	5.90
pH at T0	7.5	7.5	7.5	7.5	7.5

  

Percentage Inhibition
Treatment	Oxygen measurements		Time window used in respiration rate calculation (min)	Respiration Rate (mg O2/L/h)	Respiration Rate (mg O2/g/h)	% Inhibition
	T0	T+"x" min
Control Fba	2.82	1.26	3.0	31.20	20.80
Control Fbb	3.73	1.46	4.0	34.05	22.70
Fc1: 3.2	5.02	1.07	8.0	29.63	19.75	9.2
Fc2: 10.0	6.77	3.37	9.0	22.67	15.11	30.5
Fc3: 32.0	7.43	5.90	9.0	10.20	6.80	68.7

 

Table4Respiration Rates and Percentage Inhibition: Test item (Range-finding test)

Oxygen measurements (mg O2/L)
Time (min)	Control Fba	Nominal concentration (mg/L)						Control Fbb
		Ft1: 10.0		Ft2: 100.0		Ft3: 1000.0
		a	b	a	b	a	b
0	8.04	8.77	8.15	8.70	8.11	8.68	8.02	8.51
1	2.82	6.59	5.53	2.70	2.38	4.81	3.16	3.73
2	2.30	6.07	4.87	2.28	1.71	4.44	2.37	3.13
3	1.78	5.66	4.37	1.86	1.28	4.10	1.99	2.62
4	1.26	5.18	3.87	1.44	0.85	3.74	1.61	2.04
5	0.75	4.72	3.37	1.03	0.43	3.38	1.23	1.46
6	0.25	4.25	2.87	0.61	0.06	3.02	0.86	0.88
7	0.05	3.77	2.37	0.20		2.65	0.48	0.32
8		3.31	1.87	0.05		2.29	0.14	0.05
9		2.83	1.33			1.93	0.02
10		2.37	0.83			1.55
pH at T0	7.5	7.5	7.5	7.5	7.5	7.5	7.5	7.5

 

 

Treatment	Replicate	Oxygen measurements		Time window used in respiration rate calculation (min)	Respiration Rate (mg O2/L/h)	Respiration Rate (mg O2/g/h)	% inhibition per replicate	% inhibition mean
		T0	T+"x" min
Control	Fba	2.82	1.26	3.0	31.20	20.80
	Fbb	3.73	1.46	4.0	34.05	22.70
Ft1: 10.00	a	6.59	2.37	9.0	28.13	18.76	13.8	8.6
	b	5.53	1.33	8.0	31.50	21.00	3.4
Ft2: 100.00	a	2.70	1.03	4.0	25.05	16.70	23.2	22.4
	b	1.71	0.43	3.0	25.60	17.07	21.5
Ft3: 1000.00	a	4.81	1.55	9.0	21.73	14.49	33.4	32.0
	b	2.37	0.86	4.0	22.65	15.10	30.6

Table5Respiration Rates and Percentage Inhibition: Reference item (Definitive test)

Oxygen measurements (mg O2/L)
Time (min)	Control		Nominal concentration (mg/L)			Control
			Fc1: 3.2	Fc2: 10.0	Fc3: 32.0
	Fba	Fbb				Fbc	Fbd
0	6.94	6.86	6.42	7.07	8.16	5.19	4.88
1	6.60	4.56	4.65	5.95	7.90	3.42	3.32
2	2.49	3.48	3.65	5.35	7.63	2.31	1.96
3	1.42	2.41	2.69	4.72	7.45	1.21	1.00
4	0.29	1.30	1.72	4.11	7.27	0.15	0.09
5	0.04	0.28	0.74	3.46	7.07	0.05
6		0.06	0.05	2.84	6.88
7				2.22	6.70
8				1.59	6.52
9				0.85	6.34
10				0.33	6.25
pH at T0	7.6	7.6	7.7	7.6	7.6	7.7	7.7

 

Treatment	Replicate	Oxygen measurements		Time window used in respiration rate calculation (min)	Respiration Rate (mg O2/L/h)	Respiration Rate (mg O2/g/h)	% inhibition per replicate
		T0	T+"x" min
Control	Fba	2.49	0.29	2.0	66.00	44.00
	Fbb	4.56	1.30	3.0	65.20	43.47
	Fbc	3.42	1.21	2.0	66.30	44.20
	Fbd	3.32	1.00	2.0	69.60	46.40
Fc1 : 3.2		4.65	1.72	3.0	58.60	39.07	12.2
Fc2 : 10.0		5.95	2.22	6.0	37.30	24.87	44.1
Fc3 : 32.0		7.63	6.34	7.0	11.06	7.37	83.4

 

Table 6. Respiration Rates and Percentage Inhibition: Test item (Definitive test)

Oxygen measurements (mg O2/L)
Time (min)	Control		Nominal concentration (mg/L)										Control
			Ft1: 95.3		Ft2: 171.5		Ft3: 308.6		Ft4: 555.6		Ft5: 1000.0
	Fba	Fbb	a	b	a	b	a	b	a	b	a	b	Fbc	Fbd
0	6.94	6.86	5.97	6.03	5.92	6.01	5.72	5.87	5.56	5.67	5.31	5.91	5.19	4.88
1	6.60	4.56	0.07	0.08	0.09	0.47	0.09	0.11	0.08	0.40	0.73	0.07	3.42	3.32
2	2.49	3.48				0.04		0.03		0.04	0.07		2.31	1.96
3	1.42	2.41											1.21	1.00
4	0.29	1.30											0.15	0.09
5	0.04	0.28											0.05
6		0.06
7
8
9
10
pH at T0	7.6	7.6	7.7	7.7	7.7	7.7	7.7	7.7	7.7	7.7	7.7	7.7	7.7	7.7

 

Treatment	Replicate	Oxygen measurements		Time window used in respiration rate calculation (min)	Respiration Rate (mg O2/L/h)	Respiration Rate (mg O2/g/h)	% inhibition per replicate	% inhibition mean
		T0	T+"x" min
Control	Fba	2.49	0.29	2.0	66.00	44.00
	Fbb	4.56	1.30	3.0	65.20	43.47
	Fbc	3.42	1.21	2.0	66.30	44.20
	Fbd	3.32	1.00	2.0	69.60	46.40
Ft1 : 95.3	a	NA	NA	NA	NA	NA	NA	NA
	b	NA	NA	NA	NA	NA	NA
Ft2 : 171.5	a	NA	NA	NA	NA	NA	NA	NA
	b	NA	NA	NA	NA	NA	NA
Ft3 : 308.6	a	NA	NA	NA	NA	NA	NA	NA
	b	NA	NA	NA	NA	NA	NA
Ft4 : 555.6	a	NA	NA	NA	NA	NA	NA	NA
	b	NA	NA	NA	NA	NA	NA
Ft5 : 1000.0	a	NA	NA	NA	NA	NA	NA	NA
	b	NA	NA	NA	NA	NA	NA

NA: Not Applicable

Range-finding test

Oxygen measurements for each time point used to calculate the total respiration rate and the percentage inhibition values compared to the control are presented inTable3for the reference item and inTable4for the test item.

 

For total respiration, no significant inhibitions in respiration were observed at the two lowest tested concentrations. At the highest tested concentration (i.e. 1000 mg/L), inhibition reached 32.0% compared to the control and was statistically significant. It was thus decided to conduct the definitive test at the following test item concentration: 95.3 171.5, 308.6, 555.6 and 1000.0 mg/L.

 

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 test. It was determined to be 6.2%

§ The 3h-EC50 for 3,5-dichlorophenol (18.0 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. The mean total respiration rate was 21.8 mg O₂/g of activated sludge (dry weight of suspended solids)/h

Definitive test

Oxygen measurements for each time point used to calculate the total respiration rate and the percentage inhibition values compared to the control are presented inTable5for the reference item and inTable6for the test item.

 

As shown inTable6, total respiration was not inhibited at any tested concentration. On the contrary, endogenous respiration seems enhanced in test item groups compared to the control.

  

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 test. It was determined to be 2.9%

§ The 3h-EC50 for 3,5-dichlorophenol (11.5 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. The mean total respiration rate was 44.5 mg O₂/g activated sludge (dry weight of suspended solids)/h

Environmental conditions

Measured pH were in the 7.5-7.7 range, no adjustment was performed.

 

The ambient temperature during the testwas measured as follows: 

 

§ Range-finding test:mean measured temperature: 21.1°C, min.: 20.9°C, max.: 21.4°C

§ Definitive test:mean measured temperature: 20.5°C, min.: 20.1°C, max.: 21.2°C.

Validity criteria fulfilled:

yes

Conclusions:

During the definitive test, no inhibition of the total reparation was observed up to the highest tested concentration of 1000 mg/L. LOEC and NOEC values under the conditions of this test were thus estimated to be as follows:

3h-NOEC =1000.0 mg/L
3h-LOEC >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. Only total respiration inhibition was assessed in this study.

 

The definitive test was conducted at the following nominal concentrations:95.3, 171.5, 308.6, 555.6 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 andexpressed as mg O₂/L/h for the linear part of the graphs (then converted in mg O₂/g activated sludge dry weight of suspended solids/h).

 

 

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 test. It was determined to be 2.9%

§ The 3h-EC50 for 3,5-dichlorophenol (11.5 mg/L) was between 2 and 25 mg/L for total respiration

§ The blank controls oxygen uptake was no less than 20 mg oxygen/g of activated sludge (dry weight of suspended solids) in an hour. The mean total respiration rate was 44.5 mg O₂/g activated sludge (dry weight of suspended solids)/h

 

 

As no inhibition was recorded during the definitive test, no ECx could be statistically determined. LOEC and NOEC values under the conditions of this test were estimated to be as follows:

 

3h-NOEC =1000.0 mg/L

3h-LOEC >1000.0 mg/L

Description of key information

A study assessing the activated sludge respiration inhibition toxicity of the test item was conducted in accordance with the OECD 209 Test Guideline and GLP requirements.

The 3h-NOEC was determined to be equal or greater than the 1000 mg/L (highest tested concentration).

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L