1,3-diethyl-2-thiourea

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:

Experimental dates:
¿ Range-finding test: from 16 to 18 August 2021
¿ Definitive test: from 18 to 21 October 2021

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))

Version / remarks:

the specific inhibition of oxygen uptake for ammonium oxidation (i.e. nitrification) was not determined. In the present study, the measured oxygen consumption includes both heterotrophic oxidation and nitrification.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

not required

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 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.

Vehicle:

no

Details on 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. Four control replicates and one replicate vessel per test concentration were prepared. The test item was dispersed directly in water.

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 equal to 42.7, 93.9, 206.6, 454.6 and 1000.0 mg/L.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Two different inoculum were used as both range-finding and definitive tests were carried out over two different time period: the inoculum was obtained on 16 August 2021 and on 18 October 2021 respectively from the activated sludge of the biological wastewater treatment plant from Mourenx (France, 64) which handles predominantly domestic sewage.
Both inoculum were then treated similarly: 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 chlorine free tap 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.
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 chlorine free tap 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:

-

Test temperature:

Range-finding test: range from 20.1 to 20.5°C (mean: 20.4°C)
Definitive test: range from 20.3 to 20.9°C (mean: 20.6°C)

pH:

pH measured were in the 7.5 – 7.7 range, no adjustment was performed.

Dissolved oxygen:

Range finding test: at T0, dissolved oxygen measurements were in the 5.53 - 7.84 mg O2/L range
Definitive test: at T0, dissolved oxygen measurements were in the 6.17 - 8.06 mg O2/L range

Salinity:

-

Conductivity:

-

Nominal and measured concentrations:

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.

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 equal to 42.7, 93.9, 206.6, 454.6 and 1000.0 mg/L.

Details on test conditions:

Synthetic sewage feed
The synthetic sewage feed was prepared to contain the following constituents per liter:
¿ 16 g peptone
¿ 11 g meat extract
¿ 3 g urea
¿ 0.7 g sodium chloride
¿ 0.4 g calcium chloride dihydrate CaCl2, 2H2O
¿ 0.2 g magnesium sulfate heptahydrate MgSO4, 7H2O
¿ 2.8 g di-potassium hydrogen phosphate K2HPO4
The final volume was filled up with ultra-pure water.

In order to obtain both a NOEC and EC10, four control replicates and five treatment concentrations in a geometric series with 2 replicates each were prepared fir the definitive test. 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 (100.0 mg) directly in previously heated water to aid dissolution, then the volume was 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. 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. The fifteen minutes interval was planned 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.

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.

The addition of the inoculum was considered as the start of the 3-hours 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 litre 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.

Reference substance (positive control):

yes

Remarks:

3, 5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

93.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

115 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

RESULTS
RANGE-FINDING TEST
Oxygen consumption over time for the reference and test items vessels is presented below. The total respiration rates and the percentage inhibition values compared to the control are presented below for both substances.
All validity criteria were fulfilled this range-finding test:
¿ 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 21.3%
¿ The 3h-EC50 for 3,5-dichlorophenol (22.0 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 activated sludge (dry weight of suspended solids) in an hour: the mean total respiration rate was 27.6 mg O2/g activated sludge (dry weight of suspended solids)/h
For total respiration, inhibitions in respiration were observed up to 20.6% at the highest tested concentration (i.e. 1000.0 mg/L).
Based on these results, the definitive test was conducted using the following range of concentrations: 42.7, 93.9, 206.6, 454.6 and 1000.0 mg/L.


DEFINITIVE TEST
Oxygen consumption over time for the reference and test items vessels is presented below. The total respiration rates and the percentage inhibition values compared to the control are presented below for both substances.
This test met all 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 14.3%
¿ The 3h-EC50 for 3,5-dichlorophenol (12.1 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 activated sludge (dry weight of suspended solids) in an hour: the mean total respiration rate was 25.3 mg O2/g activated sludge (dry weight of suspended solids)/h
Total respiration was inhibited at each tested concentrations as described below:

Test item nominal concentration
(mg/L) Inhibition of total respiration
(%)
42.7 -3.2
93.9 3.0
206.6 21.5
454.6 23.6
1000.0 27.5

Results with reference substance (positive control):

The 3h-EC50 for 3,5-dichlorophenol (12.1 mg/L) was between 2 and 25 mg/L for total respiration

Reported statistics and error estimates:

LOEC, NOEC and ECx for respiration inhibition were calculated using appropriate statistical methods with the software ToxRatPro Version 3.3.0

Table 1 Range-finding test: details of the test solutions preparation

Component	Control Fba & Fbb	Reference item concentrations (mg/L)			Test item concentrations (mg/L)			Control Fbc & Fbd
		Fc1 : 3.2	Fc2 : 10.0	Fc3 : 32.0	Ft1: 10.0	Ft2 : 100.0	Ft3: 1000.0
Sewage Feed (mL)	16	16	16	16	16	16	16	16
Reference item (mL)	0	1.6	5.0	16.0	0	0	0	0
Test item (mg)	0	0	0	0	5.3	51.2	502.3	0
Ultra-pure water (mL)	234	232.4	229.0	218.0	234	234	233.5	234
Activated sludge (mL)	250	250	250	250	250	250	250	250
Final volume (mL)	500	500	500	500	500	500	500	500
pH	7.6	7.6	7.7	7.7	7.6	7.6	7.6	7.6

 

Table 2 Definitive test: details of the test solutions preparation

Component	Control Fba & Fbb	Reference item concentrations (mg/L)			Test item concentrations (mg/L)					Control Fbc & Fbd
		Fc1 : 3.2	Fc2 : 10.0	Fc3 : 32.0	Ft1 a&b : 42.7	Ft2  a&b: 93.9	Ft3  a&b: 206.6	Ft4  a&b: 454.6	Ft5 a&b : 1000.0
Sewage Feed (mL)	16	16	16	16	16	16	16	16	16	16
Reference item (mL)	0	1.6	5.0	16.0	0	0	0	0	0	0
Test item (mg)	0	0	0	0	21.4	47.0	103.3	227.3	500	0
Ultra-pure water (mL)	234	232.4	229	218.0	234.0	234.0	234.0	234.0	234.0	234
Activated sludge (mL)	250	250	250	250	250	250	250	250	250	250
Final volume (mL)	500	500	500	500	500	500	500	500	500	500
pH	7.6	7.5	7.5	7.5	7.5	7.5	7.5	7.5	7.5	7.6

 

Table 3 Range-finding test: Oxygen measurements (mg O₂/L)

Time (min)	Control		Reference item			Test item			Control
	Fba	Fbb	Fc1: 3.2	Fc2: 10.0	Fc3: 32.0	Ft1: 10.0	Ft2: 100.0	Ft3: 1000.0	Fbc	Fbd
0	5.75	5.53	7.47	5.63	7.61	6.70	7.32	5.87	7.84	7.54
1	4.33	2.58	2.93	4.37	7.10	5.12	5.07	5.35	4.73	4.20
2	3.69	1.66	2.09	3.72	6.90	4.48	4.51	4.80	4.05	3.57
3	3.04	0.76	1.28	3.06	6.70	3.81	3.94	4.26	3.43	2.98
4	2.40	0.07	0.49	2.42	6.50	3.20	3.37	3.70	2.80	2.38
5	1.78	0.05	0.04	1.78	6.31	2.58	2.80	3.16	2.21	1.79
6	1.16			1.15	6.11	1.94	2.24	2.60	1.60	1.21
7	0.56			0.52	5.93	1.31	1.69	2.06	1.01	0.63
8	0.08			0.06	5.74	0.72	1.14	1.51	0.44	0.11
9	0.05				5.55		0.60	0.96
10	0.05				5.36

 

Table 4 Definitive test: Oxygen measurements (mg O₂/L)

Time (min)	Control		Reference item			Test item										Control
			Fc1: 3.2	Fc2: 10.0	Fc3: 32.0	Ft1: 42.7		Ft2: 93.9		Ft3: 206.6		Ft4: 454.6		Ft5: 1000.0
	Fba	Fbb				a	b	a	b	a	b	a	b	a	b	Fbc	Fbd
0	6.87	6.50	5.22	6.75	8.06	6.17	6.92	6.41	6.83	6.73	6.95	6.65	7.10	6.65	7.10	7.76	7.68
1	4.84	4.75	4.58	6.64	7.95	4.85	5.93	5.08	5.94	6.27	6.51	6.18	6.67	6.25	6.63	5.98	5.87
2	4.10	4.02	3.90	6.28	7.86	4.18	5.24	4.43	5.36	5.78	6.02	5.71	6.20	5.81	6.19	5.41	5.31
3	3.37	3.32	3.27	5.90	7.74	3.54	4.59	3.78	4.77	5.26	5.52	5.23	5.70	5.35	5.70	4.85	4.75
4	2.65	2.62	2.65	5.54	7.65	2.88	3.94	3.12	4.19	4.80	5.04	4.75	5.22	4.91	5.24	4.30	4.19
5	1.94	1.93	1.99	5.17	7.53	2.25	3.28	2.49	3.60	4.30	4.51	4.27	4.73	4.44	4.78	3.76	3.64
6	1.23	1.25	1.37	4.81	7.45	1.61	2.62	1.83	3.02	3.80	4.02	3.79	4.22	4.00	4.31	3.20	3.08
7	0.56	0.60	0.75	4.45	7.32	0.97	1.98	1.19	2.44	3.30	3.53	3.30	3.73	3.53	3.85	2.65	2.52
8				4.09	7.23	0.35	1.32	0.57	1.87	2.81	3.03	2.82	3.26	3.08	3.39	2.10	1.97
9				3.74	7.14		0.68		1.30	2.31	2.53	2.35	2.78	2.62	2.91	1.56	1.42
10				3.37	7.04				0.71	1.81	2.03	1.86	2.29	2.17	2.45	1.00	0.89

 

Table 5 Range-finding test: Respiration Rates & Percentage Inhibition

Treatment	Replicate	Oxygen measurements		Time window (min)*	Respiration Rate (mg O2/L/h)	Respiration Rate (mg O2/g/h)	% inhibition
		T0	T+"x" min
Control	Fba	4.33	1.16	5	38.04	25.36
	Fbb	2.58	0.76	2	54.60	36.40
	Fbc	4.73	1.01	6	37.20	24.80
	Fbd	4.20	1.21	5	35.88	23.92
Fc1 : 3.2		2.93	1.28	2	49.50	33.00	-19.5
Fc2 : 10.0		4.37	1.15	5	38.64	25.76	6.7
Fc3 : 32.0		7.10	5.36	9	11.60	7.73	72.0
Ft1 : 10.0		5.12	1.31	6	38.10	25.40	8.0
Ft2 : 100.00		5.07	1.14	7	33.69	22.46	18.7
Ft3 : 1000.00		5.35	1.51	7	32.91	21.94	20.6

*Time window used in respiration rate calculation

 

Table 6 Definitive test: Respiration Rates & Percentage Inhibition

Treatment	Replicate	Oxygen measurements		Time window (min)*	Respiration Rate (mg O2/L/h)	Respiration Rate (mg O2/g/h)	% inhibition per replicate	% inhibition average
		T0	T+"x" min
Control	Fba	4.84	1.23	5	43.32	28.88
	Fbb	4.75	1.25	5	42.00	28.00
	Fbc	5.98	1.00	9	33.20	22.13
	Fbd	5.87	1.42	8	33.38	22.25
Fc1 : 3.2		4.58	1.37	5	38.52	25.68	-1.4	-1.4
Fc2 : 10.0		6.64	3.37	9	21.80	14.53	42.6	42.6
Fc3 : 32.0		7.95	7.04	9	6.07	4.04	84.0	84.0
Ft1 : 42.7	Ft1 a	4.85	1.61	5	38.88	25.92	-2.4	-3.2
	Ft1 b	5.93	1.32	7	39.51	26.34	-4.1
Ft2 : 93.9	Ft2 a	5.08	1.19	6	38.90	25.93	-2.4	3.0
	Ft2 b	5.94	1.30	8	34.80	23.20	8.4
Ft3 : 206.6	Ft3 a	6.27	1.81	9	29.73	19.82	21.7	21.5
	Ft3 b	6.51	2.03	9	29.87	19.91	21.3
Ft4 : 454.6	Ft4 a	6.18	1.86	9	28.80	19.20	24.2	23.6
	Ft4 b	6.67	2.29	9	29.20	19.47	23.1
Ft5 : 1000	Ft5 a	6.25	2.17	9	27.20	18.13	28.4	27.5
	Ft5 b	6.63	2.45	9	27.87	18.58	26.6

*Time window used in respiration rate calculation

 

Validity criteria fulfilled:

yes

Remarks:

The coefficient of variation of oxygen uptake rate in the controls = 14.3% (<30%) The 3h-EC50 for 3,5dichlorophenol = 12.1 mg/L (between 2 and 25 mg/L) The blank controls oxygen uptake was 25.3 mg O2/g activated sludge (>20 mg oxygen/g activated sludge)

Conclusions:

ECx, 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
EC10 115.0 mg/L 17.6 751.5
EC50 ND ND ND
LOEC 206.6 mg/L NA NA
NOEC 93.9 mg/L NA NA

NA: Not Applicable
ND: Not Determined due to mathematical reasons or inappropriate data

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: 42.7, 93.9, 206.6, 454.6 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 and expressed 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).
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 14.3%
¿ The 3h-EC50 for 3,5-dichlorophenol (12.1 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 activated sludge (dry weight of suspended solids) in an hour: the mean total respiration rate was 25.3 mg O2/g activated sludge (dry weight of suspended solids)/h
ECx, 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
EC10	115.0 mg/L	17.6	751.5
EC50	ND	ND	ND
LOEC	206.6 mg/L	NA	NA
NOEC	93.9 mg/L	NA	NA

NA: Not Applicable

ND: Not Determined due to mathematical reasons or inappropriate data

Description of key information

The toxicity of 1,3-diethylthiourea was assessed in a reliable GLP Activated sludge respiration inhibition study performed according to OECD TG 209.
3h-NOEC = 93.9 mg/L
3h-EC10 = 115 mg/L

These results are in agreement with the results from the toxicity control of the biodegradation study performed according to OECD 301 guideline (NOEC = 2 mg/L).

Overall, the data obtained indicate that the substance is of low toxicity to micro-organisms 

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

93.9 mg/L