1,3-bis(trimethylsilyl)urea

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:

26 Jun - 18 Jul 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 (Carbon and Ammonium Oxidation))

Deviations:

yes

Remarks:

In some cases, the initial and final dissolved O2 concentrations were outside the recommended range. No adverse effects on the results are expected given that the O2 consumption rate was determined over the linear portion of the O2 consumption trace.

GLP compliance:

yes (incl. QA statement)

Remarks:

19 Nov 2018, The Department of Health of the Government of the United Kingdom

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: 50, 90, 160, 280 and 500 mg of test item (nominal, in triplicates) were each separately dispersed in approximately 200 mL of deionized reverse osmosis water, sealed with Nescofilm and ultrasonicated for approximately 15 min followed by magnetic stirring for 24 h at room temperature, shielded from light. Synthetic sewage (16 mL), activated sewage sludge (250 mL) wand water were added to a final volume of 500 mL to give the required final concentrations of 100, 180, 320, 560 and 1000 mg/L (three replicates each).
- Controls: Identical conditions but without test item.
- Evidence of undissolved material: Yes, after 0 h a few very small particles of test item were visible at the surface in the 100 mg/L and 180 mg/L samples.In all other samples, a slightly cloudy dispersion with particles of test item visible at the surface was noted at 0 h and some test item particles remained visible in the 560 and 1000 mg/L samples after 30 min and 3 h.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Inoculum collection: A mixed populaiton of activated sewage sludge was obtained on 26 Jun 2019.
- Name and location of sewage treatment plant where inoculum was collected: Severn Trent Water Plc sewage treatment plant (aeration stage), Leicestershire, UK, treating predominantly domestic sewage.
- Preparation of inoculum for exposure: The activated sewage sludge was maintained on continuous aeration in the laboratory at 21 °C overnight prior to use in the test.
- Initial biomass concentration: The suspended solids concentration of the obtained activated sewage sludge was 2.7 g/L and was adjusted to 3.0 g/L prior to the addition of synthetic sewage.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

approximately 20 °C

pH:

7.3 - 7.8 (at 0 h)
7.4 - 7.9 (after 3 h)

Dissolved oxygen:

5.69 - 6.15 mg O2/L (64 - 69% of saturation level 8.9 mg O2/L)

Nominal and measured concentrations:

100, 180, 320, 560 and 1000 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: Conical flask
- Fill volume: Synthetic sewage (16 mL), activated sewage sludge (250 mL) and water were added to a final volume of 500 mL to give the final concentrations.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Suspended solids concentration (weight of dry solids per volume): 2.7 g/L. The suspended solids concentration was adjusted to 3.0 g/L prior to the addition of synthetic sewage. The suspended solids level of the activated sewage sludge was checked prior to use to ensure the suspended solids level of the activated sewage sludge was 3.0 g/L.
- Nutrients provided for bacteria: Yes, a synthetic sewage (16 g peptone, 11 g meat extract, 3 g urea, 0.7 g NaCl, 0.4 g CaCl2.2H2O, 0.2 g MgSO4.7H2O, 2.8 g K2HPO4 dissolved in 1 L) was added as a respiratory substrate.
- Preparation of test system: 16 mL of synthetic sewage was diluted to 250 mL with water and 250 mL of inoculum (controls). Thereafter, at 15 min intervals, the procedure was repeated for the second control, reference item and test item vessels and four further control vessels. For the test item vessels, synthetic sewage (16 mL), activated sewage (250 mL) and water were added to the test item replicates (dispersed in approximately 200 mL deionized reverse osmosis water) to a final volume of 500 mL.
- Aeration: Yes, with clean, oil-free compressed air at a rate of 0.5 to 1.0 L per minute.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionized reverse osmosis water
- Particulate matter: < 1 mg/L Total Organic Carbon

OTHER TEST CONDITIONS
- Adjustment of pH: Yes, the pH of the test item dispersions was measured after stirring and adjusted to a pH between 7.0 and 8.0.
- Light intensity: Normal laboratory lighting

EFFECT PARAMETERS MEASURED:
- Measurement of respiration rates after 3 h exposure: 250 mL sample aliquots were transferred to darkened glass Biological Oxygen Demand bottles and the respiration rate was measured with a Yellow Springs dissolved oxygen meter fitted with a BOD probe. The rate of respiration was measured over the linear portion of the oxygen consumption trace or over an approximate 10 min period in cases of low oxygen consumption.
- Other: pH and dissolved oxygen was measured and observations on test item preparations were made throughout the test

TEST CONCENTRATIONS
- Spacing factor for test concentrations: approximately 1.75 - 1.8
- Range finding study : Yes
- Test concentrations of range-finder: 10, 100, 1000 mg/L
- Results used to determine the conditions for the definitive study: Yes. No statistically significant toxic effects were shown at the test concentrations of 10 and 100 mg/L. However, statistically significant toxic effects were shown at the test concentration of 1000 mg/L. It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

200 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

- Effect concentrations exceeding solubility of substance in test medium: The test item was not always fully disperesed and some particles remained visible in the test vessels.
- Blank controls oxygen uptake rate: 25.78 mg per gram dry weight of sludge per hour
- Coefficient of variation of oxygen uptake rate in control replicates: 5.88%

Results with reference substance (positive control):

- Results with reference substance valid? Yes, the reference item gave an EC50 (3 h) of 5 mg/L with acceptable confidence limits, confirming the acceptability of the inoculum used in the test system.

Reported statistics and error estimates:

The percentage inhibition values were plotted against the concentration for the reference item/test item, a line fitted using the X1fit software package (IDBS) and the EC10, EC20, EC50 and EC80 values determined from the equation for the fitted line. The EC50 and EC80 values for the test item were determined by inspection of the inhibition of respiration rate data. The 95% confidence limits were calculated for the reference item EC50 using the method of Litchfield and Wilcoxon (1949).
In order to determine the NOEC, a one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was carried out on the oxygen consumption data after 3 h to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

RESULTS

No statistically significant toxic effects were shown at the test concentration of 100 mg/L. However, statistically significant toxic effects (p < 0.05) were shown at the test concentrations of 180, 320, 560 and 1000 mg/L (nominal) but it was not possible to determine an EC50 value for the test item as no concentration tested resulted in greater than 50% inhibition. Based on these results, the NOEC (3 h) was 100 mg/L. It was considered unnecessary and unrealistic to test at concentrations above 1000 mg/L. The derived effect values are summarized in Table 1 and the oxygen consumption rates and percentage inhibition values after 3 h are summarized in Table 2.

 

Table 1. Derived effect values.

	[mg/L]	95% confidence limits [mg/L]
EC10 (3 h)	200	-
EC20 (3 h)	450	-
EC50 (3 h)	> 1000	-
EC80 (3 h)	> 1000	-
NOEC (3 h)	100	-

 

Table 2. Oxygen consumption rates and percentage inhibition values after 3 h contact time.

Nominal Concentration (mg/L)		Initial O2 Reading (mg O2/L)	Measurement Period (minutes)	Final O2 Reading (mg O2/L)	O2 Consumption Rates (mg O2/L/hour)	Inhibition (%)
Control	R1	5.8	6	2.0	38.00	-
	R2	4.5	4	2.1	36.00	-
	R3	5.1	5	2.0	37.20	-
	R4	4.3	3	2.4	38.00	-
	R5	6.0	5	2.5	42.00	-
	R6	5.6	5	2.2	40.80	-
100 mg/L	R1	4.7	4	2.1	39.00	[1]
	R2	5.0	5	1.9	37.20	4
	R3	4.8	4	2.3	37.50	3
180 mg/L	R1	5.9	7	1.9	34.29	11
	R2	5.3	5	2.3	36.00	7
	R3	5.4	6	1.9	35.00	9
320 mg/L	R1	5.3	6	1.9	34.00	12
	R2	5.5	6	2.3	32.00	17
	R3	6.1	8	2.1	30.00	22
560 mg/L	R1	5.6	7	2.1	30.00	22
	R2	5.6	7	2.0	30.86	20
	R3	5.5	5	3.0	30.00	22
1000 mg/L	R1	6.3	10	1.8	26.40	30
	R2	6.7	10	2.3	26.40	32
	R3	6.4	10	2.0	26.40	32

 

 VALIDITY CRITERIA

The study fulfilled the validity criteria defined by the guideline (Table 3).

 

Table 3: Validity criteria for OECD 209.

Criterion from the guideline	Outcome	Validity criterion fulfilled
The blank controls (without the test substance or reference substance) oxygen uptake rate should not be less than 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour.	The specific respiration rate of the controls was 25.78 mg oxygen per gram dry weight of sludge per hour.	Yes
The coefficient of variation of oxygen uptake rate in control replicates should not be more than 30% at the end of definitive test.	The coefficient of variation of oxygen uptake in the control vessels was 5.88%.	Yes

 

Validity criteria fulfilled:

yes

Description of key information

EC10 (3 h) = 200 mg/L (nominal, OECD 209)

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

200 mg/L

Additional information

The effects of 1,3-bis(trimethylsilyl)urea (CAS No. 18297-63-7) on the respiration of activated sewage sludge microorganisms was assessed with an experimental study according to OECD 209.

Domestic activated sewage sludge was exposed to an aqueous dispersion of the test item at nominal concentrations of 100, 180, 320, 560 and 1000 mg/L for 3 h in controlled conditions with the addition of a synthetic sewage as a respiratory substrate. A negative control and reference control were run in parallel.

After 3 h contact time, the respiration rate was determined by measurement of the Biological Oxygen Demand over the linear portion of the oxygen consumption trace and the obtained data was compared to data for the control and reference item. Observations were made on the test item preparations throughout the test and pH and dissolved oxygen were monitored.

After 0 h, a few very small particles of test item were visible at the surface in the 100 mg/L and 180 mg/L samples. In all other samples, a slightly cloudy dispersion with particles of test item visible at the surface was noted at 0 h and some test item particles remained visible in the 560 and 1000 mg/L samples after 30 min and 3 h.

No statistically significant toxic effects were shown at the test concentration of 100 mg/L. However, statistically significant toxic effects (p < 0.05) were shown at the test concentrations of 180, 320, 560 and 1000 mg/L but it was not possible to determine an EC50 value for the test item as no concentration tested resulted in greater than 50% inhibition. Based on these results, the EC10 (3 h) was 200 mg/L and the NOEC (3 h) was 100 mg/L.