Reaction products of 1-(substitutedphenyl)urea coupled with diazotated potassium sodium substituted-5-{[2-(substituted)ethyl]sulfonyl}benzenesulfonate, further condensed with 2,4,6-trichloro-1,3,5-triazine, further converted with disubstituted benzene-1,4-disulfonic acid in aq. sodium hydroxide

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:

The study was conducted on 13 October 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Analytical monitoring:

no

Details on sampling:

Not applicable

Vehicle:

yes

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. The test item was dissolved directly in water.

A nominal amount of test item (2500 mg) was dissolved in water with the aid of ultrasonication for approximately 10 minutes and the volume adjusted to 1 liter to give a 2500 mg/L stock solution from which dilutions were made to give 250 and 25 mg/L stock solutions. An aliquot (200 mL) of the 25 mg/L stock solution was dispersed with synthetic sewage (16 mL), activated sewage sludge (250 mL) and water, to a final volume of 500 mL, to give the required concentration of 10 mg/L. Similarly, aliquots (200 mL) of the 250 mg/L and 2500 mg/L stock solutions were used to prepare the test concentrations of 100 and 1000 mg/L. The 1000 mg/L test concentration was prepared in triplicate. The volumetric flasks containing the stock solutions were inverted several times to ensure homogeneity.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

A mixed population of activated sewage sludge micro-organisms was obtained on 13 October 2015 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.

Preparation of Inoculum
The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC and was used on the day of collection. The pH of the sample was 7.7 measured using a Hach HQ40d Flexi handheld meter. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the activated sewage sludge by suction through a pre-weighed GF/A filter paper* using a Buchner funnel which was then rinsed 3 times with 10 mL of deionized reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105 ºC for at least one hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 3.0 g/L.

* rinsed three times with 20 mL deionized reverse osmosis water prior to drying in an oven

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

No data

Test temperature:

Approximately 19 °C

pH:

The pH of test preparations was measured at the test start (i.e. after the addition of activated sludge) and at the end of the 3-Hour incubation period using a Hach HQ40d Flexi handheld meter (range: pH 7.6 - 8.2)

Dissolved oxygen:

The oxygen concentrations in all vessels were measured after 30 minutes contact time (range: 6.1 - 8.6 mg O2/mL after 30 minutes contact)

Salinity:

No data

Nominal and measured concentrations:

Nominal concentrations of 10, 100 and 1000 mg/L

Details on test conditions:

Test Water
The test water used for the test was deionized reverse osmosis water containing less than 1 mg/L Dissolved Organic Carbon (DOC).

Procedure
Range-Finding Test
Test Item Preparation
As it was not a requirement of the Test Guidelines, no analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

The control group was maintained under identical conditions but not exposed to the test item.

Preparation of Test System
At time "0" 16 mL of synthetic sewage was diluted to 250 mL with water and 250 mL of inoculum added in a 500 mL conical flask (first control). The mixture was aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of 0.5 to 1.0 liter per minute. Thereafter, at 15 minute intervals the procedure was repeated for the second control followed by the reference item vessels with appropriate amounts of the reference item being added. Finally two further control vessels were prepared.


Evaluations
Observations
Observations were made on the test preparations throughout the test period. Observations of the test item vessels at 0 hours were made prior to addition of activated sewage sludge.


Measurement of the Respiration Rates
As each vessel reached 3 hours contact time an aliquot was removed from the conical flask and poured into the measuring vessel (250 mL darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe. The contents of the measuring vessel were stirred constantly by magnetic stirrer. The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace (where possible between 7 mg O2/L and 2 mg O2/L). In the case of a rapid oxygen consumption, measurements may have been outside this range but the oxygen consumption was always within the linear portion of the respiration curve. In the case of low oxygen consumption, the rate was determined over an approximate 10 minute period.


Data Evaluation
Calculation of the Oxygen Uptake Rates
The respiration rate, R, expressed in milligrams oxygen per liter per hour (mg O2/L/h), was calculated from the linear part of the recorded oxygen decrease graph according to the following equation:

R = [(Q1 - Q2) / Δt] x 60

Where:
Q1 = the oxygen concentration at the beginning of the selected section of the linear phase (mg/L);
Q2 = the oxygen concentration at the end of the selected section of the linear phase (mg/L);
Δt = the time interval between the beginning and end of the selected section of the linear phase (min).

The specific respiration rate, RS, expressed as the amount of oxygen consumed per gram dry weight of sludge per hour (mg O2/g/h) was deduced according to the following equation:

RS = R / SS

Where:
SS = the concentration of suspended solids in the test mixture (g dry weight/L).

Calculation of Percentage of Inhibition
The percentage inhibition was calculated according to the following equation:

% inhibition = [1 – (R/Rbc)] x 100

Where:
Rbc = the mean respiration rate of the blank controls.

ECX and NOEC
The percentage inhibition values were plotted against concentration for the reference item only, a line fitted using the Xlfit software package (IDBS) and the EC10, EC20, EC50 and EC80 values determined from the equation for the fitted line.

The EC10, EC20, EC50 and EC80 values for the test item were determined by inspection of the inhibition of respiration rate data.

95% confidence limits were calculated for the reference item EC50 value using the method of Litchfield and Wilcoxon (Litchfield and Wilcoxon, 1949).

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 for the range-finding test after 3 hours for the control and all test concentrations 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).

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

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

Remarks:

respiration rate

Details on results:

Range-Finding Test
The dissolved oxygen concentrations after 30 minutes contact time in all vessels were above 60 to 70% of the dissolved oxygen saturation level of 8.9 mg O2/L.

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.
Percentage inhibition is plotted against concentration for the reference item, 3,5 dichlorophenol.


Inhibition of Respiration Rate
It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.

No statistically significantly toxic effects were shown at the test concentrations of 10 and 100 mg/L, however statistically significantly toxic effects (P < 0.05) were shown at the test concentration of 1000 mg/L.

In some instances, the initial and final dissolved oxygen concentrations were outside those recommended in the test guidelines (7 mg O2/L and 2 mg O2/L respectively). This was considered to have had no adverse effect on the results of the study given that in all cases the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace.

Results with reference substance (positive control):

The 3 h EC10 was 2.0 mg/L.
The 3 h EC20 was 2.9 mg/L.
The 3 h EC50 was 8.3 mg/L, with 95 % confidence limits of 6.5 - 11 mg/L.
The 3 h EC80 was 24 mg/L.

Reported statistics and error estimates:

No data

Validation Criteria

The coefficient of variation of oxygen uptake in the control vessels was 0.29% and the specific respiration rate of the controls was 23.30 mg oxygen per gram dry weight of sludge per hour. The validation criteria have therefore been satisfied.

The validation criterion for the reference item EC₅₀value was also satisfied. 

Inhibition of Respiration Rate

The following results were derived: 

	FAT40871/A TE		3,5-dichlorophenol
	ECx (3 Hours) (mg/L)	95% Confidence Limits (mg/L)	ECx (3 Hours) (mg/L)	95% Confidence Limits (mg/L)
EC10	>100	-	2.0	-
EC20	>100	-	2.9	-
EC50	>1000	-	8.3	6.5 - 11
EC80	>1000	-	24	-

pH Values of the Test Item Stock Solutions prior to the Addition of Inoculum in the Range-Finding Test

Nominal Concentration (mg/L)		pH
		Prior to Adjustment	After Adjustment
Test Item	25	7.2	-
	250	7.1	-
	2500	6.6	7.1

 -     no adjustment required

Dissolved Oxygen Concentrations of the Test Preparations after 30 Minutes Contact Time in the Range-Finding Test

Nominal Concentration (mg/L)		Dissolved Oxygen Concentration (mg O2/L)	Expressed as a Percentage of a Dissolved Oxygen Saturation Level of 8.9 mg O2/L
Control	R1	6.7	75
	R2	7.1	80
	R3	6.1	69
	R4	6.3	71
Test Item	10	6.3	71
	100	6.7	75
	1000 R1	6.5	73
	1000 R2	8.0	90
	1000 R3	6.0	67
3,5-dichlorophenol	3.2	6.9	78
	10	7.7	87
	32	8.6	97

R₁– R₄= Replicates 1 to 4

Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours Contact Timein the Range-Finding Test

Nominal Concentration (mg/L)		Initial O2 Reading (mg O2/L)	Measurement Period (minutes)	Final O2Reading (mg O2/L)	O2Consumption Rates (mg O2/L/hour)	% Inhibition
Control	R1	5.7	6	2.2	35.00	-
	R2	5.7	6	2.2	35.00	-
	R3	5.5	6	2.0	35.00	-
	R4	5.1	5	2.2	34.80	-
Test Item	10	5.2	5	2.2	36.00	[3]
	100	5.6	6	2.2	34.00	3
	1000 R1	5.3	6	2.2	31.00	11
	1000 R2	6.7	10	2.1	27.60	21
	1000 R3	5.6	7	1.9	31.71	9
3,5-dichlorophenol	3.2	5.8	8	2.1	27.75	21
	10	7.3	10	4.9	14.40	59
	32	8.2	10	7.4	4.80	86

[Increase in respiration rate as compared to controls]

R₁– R₄= Replicates 1 to 4

pH Values of the Test Preparations at the Start and End of the Exposure Period in the Range-Finding Test

Nominal Concentration (mg/L)		pH
		0 Hours	3 Hours
Control	R1	7.7	7.8
	R2	7.7	7.9
	R3	7.8	7.8
	R4	7.8	7.8
Test Item	10	7.8	7.8
	100	7.8	7.8
	1000 R1	7.6	7.8
	1000 R2	7.7	7.9
	1000 R3	7.6	7.8
3,5-dichlorophenol	3.2	7.7	8.0
	10	7.7	8.2
	32	7.7	8.2

R₁– R₄= Replicates 1 to 4

Observations on the Test Preparations throughout the Test Periodin the Range-Finding Test

Nominal Concentration (mg/L)		Observations on Test Preparations
		0 Hours*	30 Minutes Contact Time	3 Hours Contact Time
Control	R1	Pale yellow/brown dispersion	Dark brown dispersion	Dark brown dispersion
	R2	Pale yellow/brown dispersion	Dark brown dispersion	Dark brown dispersion
	R3	Pale yellow/brown dispersion	Dark brown dispersion	Dark brown dispersion
	R4	Pale yellow/brown dispersion	Dark brown dispersion	Dark brown dispersion
Test Item	10	Dark yellow/orange dispersion, no undissolved test item visible	Dark brown dispersion, no undissolved test item visible	Dark brown dispersion, no undissolved test item visible
	100	Dark orange dispersion, no undissolved test item visible	Dark orange/brown dispersion, no undissolved test item visible	Dark orange/brown dispersion, no undissolved test item visible
	1000 R1	Dark red dispersion, no undissolved test item visible	Dark red/brown dispersion, no undissolved test item visible	Dark red/brown dispersion, no undissolved test item visible
	1000 R2	Dark red dispersion, no undissolved test item visible	Dark red/brown dispersion, no undissolved test item visible	Dark red/brown dispersion, no undissolved test item visible
	1000 R3	Dark red dispersion, no undissolved test item visible	Dark red/brown dispersion, no undissolved test item visible	Dark red/brown dispersion, no undissolved test item visible
3,5-dichlorophenol	3.2	Pale yellow/brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible
	10	Pale yellow/brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible
	32	Pale yellow/brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible

*Observations made prior to the addition of activated sewage sludge

R₁– R₄= Replicates 1 to 4

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of greater than 1000 mg/L. The 3 Hour EC10 value was greater than 100 mg/L.

Executive summary:

Introduction

A study was performed to assess the effect of the test item on the respiration of activated sewage sludge. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2010) No. 209 "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)".

 

Methods…….

Activated sewage sludge was exposed to an aqueous solution of the test item at concentrations of 10, 100 and 1000 mg/L (3 replicates of the 1000 mg/L test concentration) for a period of 3 hours at a measured temperature of approximately 19°C with the addition of a synthetic sewage as a respiratory substrate under static conditions.

 

The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference item, 3,5-dichlorophenol.

 

Results…….

The effect of the test item on the respiration of activated sewage sludge gave a 3‑Hour EC₅₀value of greater than 1000 mg/L. The 3‑Hour EC₁₀value was greater than 100mg/L.

It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.

The reference item gave a 3-Hour EC₅₀value of 8.3 mg/L, 95% confidence limits 6.5 to 11 mg/L.

Description of key information

Activated sewage sludge was exposed to an aqueous solution of the test item at concentrations of 10, 100 and 1000 mg/L for a period of 3 hours at 19°C with the addition of a synthetic sewage as a respiratory substrate under static conditions.

 

The effect of the test item on the respiration of activated sewage sludge gave:

- 3‑Hour EC₅₀value of greater than 1000 mg/L. 

- The 3‑Hour EC₁₀value was greater than100mg/L.

It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.

It can be conclude that the test item is not toxic and have no effect on the respiration of activated sewage sludge.

Key value for chemical safety assessment

EC50 for microorganisms:

100 mg/L