Di-tert-dodecyl disulphide

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:

27-03-2017 to 07-06-2017

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

At the end of the incubation period, a well-mixed sample of each solution (following their order of preparation) were poured into a BOD bottle.

Vehicle:

no

Details on test solutions:

Activated sewage sludge micro-organisms were exposed to a series of nominal test concentrations of 62.5, 125.0, 250.0, 500.0 and 1000.0 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 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 the suitability of the inoculum: a stock solution at 1 g/L was prepared by dissolving the reference item (100.5 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 feed 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 control vessel (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 couple in which 16 mL of synthetic sewage feed 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 second control vessel 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.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

The inoculum was obtained on 27 March 2017 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 10L) 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 3 days and fed daily with synthetic sewage feed (at 50 mL/L).

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 did remain constant at 20°C within ± 2°C during the test: mean measured temperature: 21.6°C, min.: 20.9°C, max.: 22.0°C.

pH:

All measured pH reported were in the range 7 – 8.

Nominal and measured concentrations:

nominal concentrations of test item: 62.5, 125.0, 250.0, 500.0 and 1000.0 mg/ L

Details on test conditions:

The addition of the inoculum was considered as the start of the 3-hour incubation time period.

All solutions were incubated at 20 +/- 2°C in darkness for 3 hours in a temperature controlled room. Solutions were mixed by stirring and aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of 0.5 to 1.0 liter per minute during 3 hours. At the end of the incubation period, a well-mixed sample of each solution (following their order of preparation) were poured into a BOD bottle in which dissolved oxygen concentrations were measured at 1 min intervals for up to a maximum period of 10 min or until the oxygen concentration felt below 1 mg/L.

The temperature was monitored throughout the test period.

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.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on 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 Table 1 and table 2.

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

Test item nominal concentration Inhibition of total respiration
(mg/L) (%)

62,5 1.4
125.0 6.1
250.0 4.2
500.0 2.1
1000.0 0.3

The statistical analysis showed no significant inhibition.

Results with reference substance (positive control):

The 3h-EC50 for 3,5-dichlorophenol to activated sludge was estimated under the conditions of the test as follows: 22.1 mg/L for total respiration.

Reported statistics and error estimates:

The statistical analysis showed no significant inhibition.

Table 1: Oxygen measurements of test item

Oxygen measurements (mg O2/L)
Time (min)	Control		Nominal concentration (mg/L)										Control
			62.5		125.0		250.0		500.0		1000.0
	a	b	a	b	a	b	a	b	a	b	a	b	c	d
0	7.72	7.69	7.54	7.40	7.59	7.20	7.44	7.20	7.45	7.86	7.80	7.78	8.81	7.63
1	6.53	6.34	5.75	5.74	5.54	5.90	5.45	5.05	5.48	5.09	5.17	5.05	6.61	6.44
2	5.93	5.74	5.18	5.21	4.99	5.31	4.92	4.40	4.94	4.49	4.61	4.43	6.02	5.91
3	5.33	5.10	4.61	4.71	4.46	4.78	4.39	3.90	4.39	3.91	4.05	3.83	5.46	5.37
4	4.73	4.49	4.02	4.19	3.91	4.22	3.84	3.31	3.83	3.32	3.46	3.23	4.88	4.83
5	4.14	3.86	3.44	3.67	3.38	3.65	3.29	2.73	3.25	2.73	2.90	2.62	4.30	4.28
6	3.53	3.24	2.84	3.16	2.84	3.08	2.74	2.13	2.67	2.14	2.31	1.92	3.71	3.73
7	2.93	2.60	2.26	2.64	2.30	2.49	2.17	1.54	2.08	1.54	1.72	1.41	3.12	3.17
8	2.32	1.97	1.67	2.11	1.75	1.90	1.60	0.95	1.49	0.94	1.14	0.80	2.52	2.60
9	1.71	1.34	1.07	1.56	1.22	1.31	1.04	0.36	0.90	0.35	0.54	0.18	1.93	2.03
10	1.09	0.71	0.48	1.06	0.67	0.71	0.47	ND	0.32	ND	0.03	ND	1.32	1.45
pH at T0	7.50	7.49	7.49	7.50	7.51	7.38	7.38	7.38	7.39	7.37	7.39	7.39	7.50	7.49

 Table 2: Percentage inhibition of test item

Treatment	Replicate	Oxygen measurements		Minutes retained	Respiration Rate (mg O2/L/h)	Respiration Rate (mg O2/g/h)	% inhibition per replicate	% inhibition mean
		T0	T+"x" min
Control	a	6.53	1.08	9.0	36.33	24.22
	b	6.34	1.34	8.0	37.50	25.00
	c	6.61	1.32	9.0	35.27	23.51
	d	6.44	1.45	9.0	33.27	22.18
62.5	a	5.75	1.07	8.0	35.10	23.40	1.4	1.4
	b	5.74	1.06	8.0	35.10	23.40	1.4
125.0	a	5.54	1.22	8.0	32.40	21.60	9.0	6.1
	b	5.90	1.31	8.0	34.43	22.95	3.3
250.0	a	5.45	1.04	8.0	33.08	22.05	7.1	4.2
	b	5.05	1.54	6.0	35.10	23.40	1.4
500.0	a	5.48	1.49	7.0	34.20	22.80	3.9	2.1
	b	5.09	1.54	6.0	35.50	23.67	0.3
1000.0	a	5.17	1.14	7.0	34.54	23.03	2.9	0.3
	b	5.05	1.41	6.0	36.40	24.27	-2.3

 

Validity criteria fulfilled:

yes

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.
As no significant inhibition was recorded, no ECx could be statistically determined. LOEC and NOEC values under the conditions of this test were calculated to be as follows for the test item:

95% confidence level
Lower limit Upper limit

NOEC =1000.0 mg/L NA NA
LOEC >1000.0 mg/L NA NA

NA: Not Applicable

Executive summary:

This study was designed to determine the effects of the test item on sewage micro-organisms by measuring the respiration rate in a 3-hour 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: 62.5, 125.0, 250.0, 500.0 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 andexpressed 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 2.0%

 - The 3h-EC50 for the reference item (18.5 mg/L) passed the validity criterion of the guideline set 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: 34.1 mg O₂/g of activated sludge (dry weight of suspended solids)/h

  

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

                                         95% confidence level

                                         Lower limit       Upper limit

NOEC      =1000.0 mg/L       NA                     NA

LOEC       >1000.0 mg/L       NA                     NA

NA: Not Applicable

 

NA: Not Applicable

Description of key information

The toxicity to microorganisms has been measured according to OECD 209. No significant inhibition was recorded: no ECx could be statically determined. Under the conditions of the test, LOEC and NOEC values are respectively > 1000 and = 1000 mg/L.