Di-tert-pentyl peroxide

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:

June 2019

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

Version / remarks:

Some differences to the recommended method were conducted in agreement with the sponsor's representative:
1. the specific inhibition of oxygen uptake for ammonium oxidation (i.e. nitrification) was not determined
2. the test vessels replicates were in a number below the requisite number.
These differences are not considered to affect the outcome or the achievement of the study objectives and are fully reported in the present Study Report.

Deviations:

yes

Remarks:

No preliminary range-finding test were conducted. This deviation was not considered to have affected the outcome or the achievement of the study objectives.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

Not applicable

Vehicle:

no

Details on test solutions:

Activated sewage sludge micro-organisms were exposed to a series of nominal test concentrations of 51.4, 108.0, 226.8, 476.2 and 1000 mg/L. In order to obtain both a NOEC and EC50, four controls and five treatment concentrations in a geometric series with 2 replicates were prepared. The test item was dispersed directly in ultra-pure 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 (100 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 Fbd was prepared at last. The fifteen minutes interval was done 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 the 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.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

The inoculum was obtained on 13 May 2019 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 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

Hardness:

no data

Test temperature:

mean measured temperature : 21.5°C (min 21.4°C - max 21.7°C)

pH:

in the range 7.5 – 7.6 (see "any other information on results incl. tables")

Dissolved oxygen:

Parameter of interest in this test

Salinity:

not relevant

Nominal and measured concentrations:

No range finding test performed.
Nominal test concentrations : 51.4, 108.0, 226.8, 476.2 and 1000 mg/L

Details on test conditions:

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

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

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

Details on results:

Inhibition rates decrease with increasing test item concentration, suggesting the test item has no adverse effects on the activated sludge respiration. The statistical analysis of the data did not show significant effects: NOEC >= 1000.0 mg/L.
See "any other information on results incl. tables"

Results with reference substance (positive control):

The 3h-EC50 for 3,5-dichlorophenol (22.2 mg/L) was between 2 and 25 mg/L for total respiration.
See "any other information on results incl. tables"

Table 1 : Respiration Rates and Percentage Inhibition : test item

Oxygen measurements (mg O2/L)
Time (min)	Control		Nominal concentration (mg/L)										Control
			Ft1: 51.4		Ft2: 108.0		Ft3: 226.8		Ft4: 476.2		Ft5: 1000.0
	Fba	Fbb	a	b	a	b	a	b	a	b	a	b	Fbc	Fbd
0	8.82	9.21	9.24	8.60	9.16	8.50	9.11	8.44	9.01	8.32	8.86	8.16	8.91	8.04
1	6.14	6.29	6.35	5.87	7.40	5.55	6.24	5.31	5.69	5.37	4.74	5.25	5.73	5.51
2	5.56	5.71	5.77	5.25	6.98	4.95	5.60	4.66	4.95	4.71	4.08	4.57	5.10	4.87
3	5.05	5.21	5.24	4.68	6.40	4.37	5.00	4.06	4.32	4.09	3.47	3.96	4.47	4.29
4	4.53	4.70	4.70	4.16	5.78	3.81	4.43	3.46	4.00	3.48	2.83	3.33	3.85	3.70
5	4.02	4.18	4.17	3.62	5.24	3.23	3.84	2.87	3.09	2.86	2.20	2.71	3.23	3.11
6	3.51	3.69	3.61	3.07	4.73	2.66	3.22	2.26	2.44	2.24	1.57	2.09	2.60	2.54
7	3.01	3.16	3.07	2.53	4.15	2.08	2.65	1.66	1.80	1.63	0.95	1.45	1.96	1.97
8	2.50	2.66	2.52	1.98	2.24	1.51	2.06	1.05	1.19	1.02	0.33	0.83	1.37	1.39
9	1.99	2.14	2.00	1.44	1.55	0.92	1.47	0.46	0.57	0.40	0.06	0.23	0.69	0.84
10	1.48	1.61	1.42	0.91	0.97	0.37	0.86	0.04	0.09	0.04			0.12	0.28
pH at T0	7.6	7.6	7.6	7.6	7.6	7.6	7.6	7.6	7.6	7.6	7.6	7.6	7.6	7.6

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 Fb	a	6.14	1.48	9.0	31.07	20.71
	b	6.29	1.61	9.0	31.20	20.80
	c	5.73	1.37	7.0	37.37	24.91
	d	5.51	1.39	7.0	35.31	23.54
Ft1 : 51.4	a	6.35	1.42	9.0	32.87	21.91	2.6	2.1
	b	5.87	1.44	8.0	33.23	22.15	1.5
Ft2 : 108.0	a	7.40	4.15	6.0	32.50	21.67	3.7	0.5
	b	5.55	1.51	7.0	34.63	23.09	-2.6
Ft3 : 226.8	a	6.24	1.47	8.0	35.78	23.85	-6.0	-7.1
	b	5.31	1.05	7.0	36.51	24.34	-8.2
Ft4 : 476.2	a	5.69	1.19	7.0	38.57	25.71	-14.3	-12.4
	b	5.37	1.02	7.0	37.29	24.86	-10.5
Ft5 : 1000.0	a	4.74	1.57	5.0	38.04	25.36	-12.8	-12.7
	b	5.25	1.45	6.0	38.00	25.33	-12.6

Table 2 : Respiration rates and percentage inhibition : reference item

 

Oxygen measurements (mg O2/L)
Time (min)	Control		Nominal concentration (mg/L)			Control
	Fba	Fbb	Fc1: 3.2	Fc2: 10.0	Fc3: 32.0	Fbc	Fbd
0	8.82	9.21	8.64	9.22	8.61	8.91	8.04
1	6.14	6.29	5.54	6.70	7.60	5.73	5.51
2	5.56	5.71	4.92	6.20	7.40	5.10	4.87
3	5.05	5.21	4.35	5.75	7.20	4.47	4.29
4	4.53	4.70	3.79	5.32	6.99	3.85	3.70
5	4.02	4.18	3.25	4.89	6.79	3.23	3.11
6	3.51	3.69	2.68	4.45	6.59	2.60	2.54
7	3.01	3.16	2.12	4.00	6.40	1.96	1.97
8	2.50	2.66	1.57	3.55	6.21	1.37	1.39
9	1.99	2.14	1.03	3.11	6.01	0.69	0.84
10	1.48	1.61	0.51	2.65	5.82	0.12	0.28
pH at T0	7.6	7.6	7.6	7.6	7.5	7.6	7.6

   

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 Fb	a	6.14	1.48	9.0	31.07	20.71
	b	6.29	1.61	9.0	31.20	20.80
	c	5.73	1.37	7.0	37.37	24.91
	d	5.51	1.39	7.0	35.31	23.54
Fc1 : 3.2		5.54	1.03	8.0	33.83	22.55	-0.3
Fc2 : 10.0		6.70	2.65	9.0	27.00	18.00	20.0
Fc3 : 32.0		7.60	5.82	9.0	11.87	7.91	64.8

 

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 Table 2 for the reference item and in Table 1 for the test item.

Total respiration was inhibited at each tested concentrations as described below:

Test item nominal concentration (mg/L)	Inhibition of total respiration (%)
51.4	2.1
108.0	0.5
226.8	-7.1
476.2	-12.4
1000.0	-12.7

Inhibition rates decrease with increasing test item concentration, suggesting the test item has no adverse effects on the activated sludge 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 test: it was determined to be 9.3%

- The 3h-EC50 for 3,5-dichlorophenol (22.2 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 22.5 mg O2/g of activated sludge (dry weight of suspended solids)/h

Validity criteria fulfilled:

yes

Conclusions:

Inhibition rates decrease with increasing test item concentration, suggesting the test item has no adverse effects on the activated sludge respiration. The statistical analysis of the data did not show significant effects: NOEC >= 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 definitive test was conducted at the following nominal concentrations: of 51.4, 108.0, 226.8, 476.2 and 1000 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 test: it was determined to be 9.3%

- The 3h-EC50 for 3,5-dichlorophenol (22.2 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 22.5 mg O2/g of activated sludge (dry weight of suspended solids)/h

A decreasing of the inhibition rates with increasing test item concentration was observed, suggesting the test item has no adverse effects on the activated sludge respiration. The statistical analysis of the data did not show significant effects: NOEC >= 1000.0 mg/L

Description of key information

The toxicity to microorganisms was tested on the di-tert-amyl peroxyde to activated sludge in a reliable GLP study following OECD 209 and guidance on the testing of difficult substances.

No effect up to the highest concentration tested of 1000 mg/L, which is above the water solubility, was observed. So NOEC >= 1000 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L