Diallylamine

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

toxicity to microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 28 July 2015 and 26 August 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

.

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

yes

Remarks:

Deviation in stock solution preparation and achieving the 11.6 mg ATU/L. This deviation was considered to have no effect on the overall results of the study.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

- 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 test item was dissolved directly in water.
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 10, 32, 100, 320 and 1000 mg/L.

Vehicle:

no

Details on test solutions:

Deviation from Study Plan
A stock solution of 1.16g/L of ATU was prepared of which a 5 mL aliquot was added to each vessel to a final volume of 500 mL which gave a concentration of 11.6 mg ATU/L.
Although this was a deviation to the general study plan, this deviation was considered to have had no adverse effect on the study given that the correct final concentration of ATU in each vessel was obtained.

Test Item Preparation
The Sponsor indicated that the test item maybe volatile, therefore in order to determine if the test item was volatile once in solution and under aeration preliminary work was conducted (Please refer to Appendix 2 attached).
The test concentrations to be used in the definitive test were determined by a preliminary range-finding test.

Range-Finding Test
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 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.
* 250 and 25 mg/L stock solutions derived from pH adjusted 2500 mg/L stock solution (see Table 1 below)
The pH of the test item stock solutions were measured using a Hach HQ40d Flexi handheld meter (see Table 1 below) and adjusted if necessary to between pH 7.0 to pH 8.0.


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

A reference item, 3,5-dichlorophenol, was included in the range-finding test at concentrations of 3.2, 10 and 32 mg/L in order to confirm the suitability of the inoculum. A stock solution of 0.5 g/L was prepared by dissolving the reference item directly in water with the aid of ultrasonication for approximately 25 minutes. The pH of this stock solution was measured to be pH 5.4 and was adjusted to pH 7.3 using 1.0 M NaOH. The pH values were measured using a Hach HQ40d Flexi handheld meter. Aliquots (3.2, 10 and 32 mL) of the stock solution were removed and dispersed with activated sewage sludge (250 mL), synthetic sewage (16 mL) and water to a final volume of 500 mL to give the required concentrations of 3.2, 10 and 32 mg/L. The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution.

Definitive Test to Measure Total Respiration

Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 10, 32, 100, 320 and 1000 mg/L.

Test Item Preparation: The test item was dissolved directly in water. A nominal amount of test item (5000 mg) was dissolved in water and the volume adjusted to 2 liters to give a 2500 mg/L stock solution. A further nominal amount of test item (100 mg) was dissolved in water and the volume adjusted to 1 liter to give a 100 mg/L stock solution. Aliquots (50 and 160 mL) of the 100 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 concentrations of 10 and 32 mg/L. Similarly, aliquots (20, 64 and 250 mL) of the 2500 mg/L stock solution were used to prepare the test concentrations of 100, 320 and 1000 mg/L. All test concentrations were prepared in triplicate. The volumetric flasks containing the stock solutions were inverted several times to ensure homogeneity of the stock solutions.

The pH of the test item stock solutions was measured and adjusted to between pH 7.0 and 8.0 using Hach HQ40d Flexi handheld meter (see Table 6 below).
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.

Reference Item Preparations
For the purpose of the test a reference item, 3,5-dichlorophenol was used. A stock solution of 0.5 g/L was prepared by dissolving the reference item directly in water with the aid of ultrasonication for approximately 15 minutes. The pH of this stock solution was measured to be pH 5.6 and adjusted to pH 7.3 using 1.0 M NaOH. The pH values were measured using a Hach HQ40d Flexi handheld meter. Aliquots (3.2, 10 and 32 mL) of the stock solution were removed and dispersed with activated sewage sludge (250 mL), synthetic sewage (16 mL) and water to give the final concentrations of 3.2, 10 and 32 mg/L. The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution.

Preparation of Inoculum
The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC overnight prior to use in the test. On the day of collection the activated sewage sludge (10 liters) was fed synthetic sewage (500 mL). The pH of the sample on the day of the test was 7.3 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 prior to use.

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

Definitive Test to Measure Heterotrophic Respiration
Test Item Preparation: The test item was dissolved directly in water. A nominal amount of test item (5000 mg) was dissolved in water and the volume adjusted to 2 liters to give a 2500 mg/L stock solution. A further nominal amount of test item (100 mg) was dissolved in water and the volume adjusted to 1 liter to give a 100 mg/L stock solution. Aliquots (50 and 160 mL) of the 100 mg/L stock solution was dispersed with synthetic sewage (16 mL), allylthiourea (ATU) (5 mL), activated sewage sludge (250 mL) and water, to a final volume of 500 mL, to give the required concentrations of 10 and 32 mg/L. Similarly, aliquots (20, 64 and 250 mL) of the 2500 mg/L stock solution were used to prepare the test concentrations of 100, 320 and 1000 mg/L. All test concentrations were prepared in triplicate. The volumetric flasks containing the stock solutions were inverted several times to ensure homogeneity of the stock solutions.
The pH of the test item stock solutions was measured and adjusted to between pH 7.0 and 8.0 using Hach HQ40d Flexi handheld meter (see Table 11 below).

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.


3.5.3.3 Reference Item Preparation
For the purpose of the test a reference item, 3,5-dichlorophenol was used. A stock solution of 0.5 g/L was prepared by dissolving the reference item directly in water with the aid of ultrasonication for approximately 15 minutes. The pH of this stock solution was measured to be pH 5.7 and adjusted to pH 7.1 using 1.0 M NaOH. The pH values were measured using a Hach HQ40d Flexi handheld meter. Aliquots (3.2, 10 and 32 mL) of the stock solution were removed and dispersed with activated sewage sludge (250 mL), synthetic sewage (16 mL), allylthiourea (ATU) (5 mL) and water to give the final concentrations of 3.2, 10 and 32 mg/L. The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution.

For the purpose of the test a reference item, allylthiourea was also used. A stock solution of
1.16 g/L was prepared by dissolving the reference item directly in water with the aid of ultrasonication for approximately 5 minutes. The pH of this stock solution was measured to be pH 7.0 using a Hach HQ40d Flexi handheld meter. An aliquot (5 mL) of the stock solution was removed and dispersed in the test vessels, as described previously for the test item and 3,5-dichlorophenol, to give the final concentration of 11.6 mg/L. The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution.


3.5.3.4 Preparation of Inoculum
The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC overnight prior to use in the test. On the day of collection the activated sewage sludge (10 liters) was fed synthetic sewage (500 mL). The pH of the sample on the day of the test was 7.3 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 prior to use.
* rinsed three times with 20 mL deionized reverse osmosis water prior to drying in an oven

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 28 July 2015 for the range-finding test and on 24 August 2015 and 25 August 2015 for the definitive tests from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.

A synthetic sewage of the following composition, was added to each test vessel to act as a respiratory substrate:

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 liter of water with the aid of ultrasonication.

In the range-finding test, the pH of the synthetic sewage stock was pH 7.2. In the definitive tests, the pH of the synthetic sewage stock used to feed the activated sewage sludge to measure total respiration was pH 7.1 and the pH of the synthetic sewage stock used for the definitive test to measure total respiration was pH 6.8 and adjusted to pH 7.2 using 1.0 M NaOH. In the definitive test to measure heterotrophic respiration, the pH of the synthetic sewage stock used to feed the activated sewage sludge to measure heterotrophic respiration was pH 7.3 and the pH of the synthetic sewage stock used for the definitive test to measure total respiration was pH 7.1.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

The test water used for the range-finding and definitive tests was deionized reverse osmosis water containing less than 1 mg/L Dissolved Organic Carbon (DOC).

Test temperature:

between 20 and 22 ºC.

pH:

The pH of the test item stock solutions was measured and adjusted to between pH 7.0 and 8.0

Dissolved oxygen:

The oxygen concentrations in all vessels were measured after 30 minutes contact time. Please see Tables 7 and 12 below.

Salinity:

Not applicable

Nominal and measured concentrations:

Definitive test: 10, 32, 100, 320 and 1000 mg/L.

Details on test conditions:

Definitive Test to Measure Total Respiration
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 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. Two additional control vessels were then prepared prior to the test item vessels being prepared as described in the Test Item Preparation Section. Finally two further control vessels were prepared.

The test was conducted under normal laboratory lighting in a temperature controlled room at measured temperatures of between 20 and 22 ºC.


Definitive Test to measure Heterotrophic Respiration
Preparation of Test System: At time "0" 16 mL of synthetic sewage and 5 mL of allylthiourea (ATU) 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 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. Two additional control vessels were then prepared prior to the test item vessels being prepared as described in the Test Item Preparation Section. Finally two further control vessels were prepared.

The test was conducted under normal laboratory lighting in a temperature controlled room at measured temperatures of between 21 and 22 ºC.


Obserwations:
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.

pH Measurements
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.

Oxygen Concentration
The oxygen concentrations in all vessels were measured after 30 minutes contact time

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.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

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

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

45 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of nitrification rate

Remarks on result:

other: 95% CL: 38-53 mg/L

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 heterotrophic respiration

Details on results:

Definitive Test
The dissolved oxygen concentrations in all vessels after 30 minutes contact time are given in Table 7 for total respiration and Table 12 for heterotrophic respiration. Oxygen consumption rates and percentage inhibition values for the control, test and reference items after 3 hours contact time are given in Table 8 for total respiration and Table 13 for heterotrophic respiration. The pH values of the test preparations at the start and end of the exposure period are given in Table 9 for total respiration and Table 14 for heterotrophic respiration.

Inhibition of Respiration Rate for Total Respiration
The following results were derived:



Diallylamine 3,5-dichlorophenol

ECx (3 Hours) (mg/L) / 95% Confidence Limits (mg/L) ECx (3 Hours) (mg/L) / 95% Confidence Limits (mg/L)


EC10 23/ - 1.9/ -
EC20 45/ - 2.8/ -
EC50 >1000/ - 8 .6/ 6.7 - 11
EC80 >1000/ - 26/ -
NOEC -/ - -/ -



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

It was not possible to determine an EC50 value for the test item as no concentration tested resulted in greater than 50% inhibition.

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.

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.


 
Inhibition of Respiration Rate for Heterotrophic Respiration
The following results were derived:

Diallylamine 3,5-dichlorophenol

ECx (3 Hours) (mg/L) / 95% Confidence Limits (mg/L) ECx (3 Hours) (mg/L) /95% Confidence Limits (mg/L)


EC10 >1000/ - 5.0/ -
EC20 >1000/ - 6.7/ -
EC50 >1000/ - 16/ 13 - 20
EC80 >1000 - 40/ -
NOEC -/ - -/ -

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

It was not possible to determine an EC50 value for the test item as no concentration tested resulted in greater than 50% inhibition.

In some instances, the initial dissolved oxygen concentrations were outside that recommended in the test guidelines (7 mg O2/L). 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.

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.


Inhibition of Respiration Rate for Nitrification Respiration
The following results were derived:



Diallylamine 3,5-dichlorophenol

ECx (3 Hours) (mg/L) / 95% Confidence Limits (mg/L) ECx (3 Hours) (mg/L) /95% Confidence Limits (mg/L)


EC10 11/ - 0.21/ -
EC20 19/ - 0.37/ -
EC50 45/ 38 - 53 1.8/ 1.3 – 2.6
EC80 100/ - 9.2/ -
NOEC -/ - -/ -
The coefficient of variation of oxygen uptake in the control vessels was 13.1%. The validation criteria have therefore been satisfied.
The validation criterion for the reference item EC50 value was also satisfied.
 

Table 8     Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours Contact Time in the Definitive Test to Measure Total Respiration

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	4.7	4	2.2	37.50	-
	R2	4.6	5	1.9	32.40	-
	R3	4.4	4	2.0	36.00	-
	R4	4.2	3	2.4	36.00	-
	R5	4.6	4	2.2	36.00	-
	R6	5.1	5	2.2	34.80	-
Test Item	10 R1	4.6	5	1.8	33.60	5
	10 R2	4.0	3	2.3	34.00	4
	10 R3	4.7	5	1.9	33.60	5
	32 R1	4.9	5	2.3	31.20	12
	32 R2	5.2	6	2.3	29.00	18
	32 R3	5.0	6	1.9	31.00	13
	100 R1	4.6	5	2.6	24.00	32
	100 R2	4.9	6	2.6	23.00	35
	100 R3	6.0	9	2.5	23.33	34

 

Test Item	320 R1	6.1	10	2.7	20.40	42
	320 R2	6.3	10	2.8	21.00	41
	320 R3	6.2	10	2.7	21.00	41
	1000 R1	6.1	10	2.7	20.40	42
	1000 R2	6.1	10	2.8	19.80	44
	1000 R3	6.4	10	3.1	19.80	44
3,5-dichlorophenol	3.2	5.6	8	2.1	26.25	26
	10	5.8	10	2.8	18.00	49
	32	7.8	10	7.1	4.20	88

R₁– R₆= Replicates 1 to 6

 R₁– R₃= Replicates 1 to 3

Table 9     pH Values of the Test Preparations at the Start and End of the Exposure Period in the Definitive Test to Measure Total Respiration

Nominal Concentration (mg/L)		pH
		0 Hours	3 Hours
Control	R1	7.4	7.7
	R2	7.3	7.7
	R3	7.3	7.6
	R4	7.3	7.6
	R5	7.1	7.7
	R6	7.0	7.6
Test Item	10 R1	7.0	7.7
	10 R2	7.5	7.6
	10 R3	7.2	7.7
	32 R1	7.1	7.8
	32 R2	7.6	7.9
	32 R3	7.2	7.8
	100 R1	7.1	7.9
	100 R2	7.0	7.9
	100 R3	7.4	8.0

 

Test Item	320 R1	7.2	8.0
	320 R2	7.1	8.0
	320 R3	7.0	8.0
	1000 R1	7.1	7.9
	1000 R2	7.3	7.9
	1000 R3	7.2	7.9
3,5-dichlorophenol	3.2	7.2	8.0
	10	7.4	7.8
	32	7.1	7.7

R₁– R₆= Replicates 1 to 6

 R₁– R₃= Replicates 1 to 3

Table 13   Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours Contact Time in the Definitive Test to Measure Heterotrophic Respiration

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	6.1	10	2.6	21.00	-
	R2	6.3	10	2.8	21.00	-
	R3	6.7	10	3.4	19.80	-
	R4	6.8	10	3.6	19.20	-
	R5	6.5	10	3.0	21.00	-
	R6	6.6	10	3.3	19.80	-
Test Item	10 R1	6.4	10	3.0	20.40	0
	10 R2	5.5	10	2.3	19.20	5
	10 R3	6.4	10	3.1	19.80	2
	32 R1	6.5	10	3.2	19.80	2
	32 R2	5.9	10	2.5	20.40	0
	32 R3	3.8	4	2.3	22.50	[11]
	100 R1	5.7	8	3.2	18.75	8
	100 R2	6.8	10	3.2	21.60	[6]
	100 R3	6.8	10	3.5	19.80	2

  

Test Item	320 R1	5.9	8	3.0	21.75	[7]
	320 R2	6.0	9	2.8	21.33	[5]
	320 R3	6.8	10	3.3	21.00	[3]
	1000 R1	6.7	10	3.4	19.80	2
	1000 R2	6.2	10	2.8	20.40	0
	1000 R3	6.3	10	2.9	20.40	0
3,5-dichlorophenol	3.2	6.5	10	3.3	19.20	5
	10	6.1	10	3.2	17.40	14
	32	8.3	10	7.7	3.60	82

 

R₁– R₆= Replicates 1 to 6

[increase in respiration as compared to controls]

 R₁– R₃= Replicates 1 to 3

[increase in respiration as compared to controls]

 

Table 14   pH Values of the Test Preparations at the Start and End of the Exposure Period in the Definitive Test to Measure Heterotrophic Respiration

Nominal Concentration (mg/L)		pH
		0 Hours	3 Hours
Control	R1	7.2	8.0
	R2	7.4	8.0
	R3	7.4	8.1
	R4	7.4	8.1
	R5	7.2	8.1
	R6	7.2	8.0
Test Item	10 R1	7.3	8.1
	10 R2	7.2	8.0
	10 R3	7.4	8.1
	32 R1	7.4	8.1
	32 R2	7.4	8.1
	32 R3	7.5	8.1
	100 R1	7.6	8.1
	100 R2	7.3	8.1
	100 R3	7.2	8.1

  

Test Item	320 R1	7.4	8.0
	320 R2	7.5	8.0
	320 R3	7.4	8.1
	1000 R1	7.4	8.0
	1000 R2	7.4	7.9
	1000 R3	7.4	7.9
3,5-dichlorophenol	3.2	7.1	8.0
	10	7.1	7.6
	32	7.3	7.6

 

R₁– R₆= Replicates 1 to 6   R₁– R₃= Replicates 1 to 3

 

Validation Criteria

The coefficient of variation of oxygen uptake in the control vessels for total respiration was 4.9% and the specific respiration rate of the controls was 23.63 mg oxygen per gram dry weight of sludge per hour. The coefficient of variation of oxygen uptake in the control vessels for heterotrophic respiration was 3.9%. The validation criteria have therefore been satisfied.

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

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the total and heterotrophic respiration of activated sewage sludge gave a 3 Hour EC50 value of greater than 1000 mg/L.
The effect of the test item on the nitrification respiration of activated sewage sludge gave a 3 Hour EC50 value of 45 mg/L, 95% confidence limits 38 to 53 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…….

Following a preliminary range-finding test, activated sewage sludge was exposed to an aqueous solution of the test item at concentrations of 10, 32, 100, 320 and 1000 mg/L with and without the presence of Allythiourea (ATU) for a period of 3 hours at measured temperatures of between 20 to 22 °C with the addition of a synthetic sewage as a respiratory substrate.

 The rates of respiration (total respiration, heterotrophic respiration and nitrification respiration) were 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 total and heterotrophic respiration of activated sewage sludge gave a 3-Hour EC₅₀value of greater than 1000 mg/L.

 The effect of the test item on the nitrification respiration of activated sewage sludge gave a 3-Hour EC₅₀value of 45 mg/L, 95% confidence limits 38 to 53 mg/L. 

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

 

The test item gave the following 3-Hour EC₅₀values and 95% confidence limits. 

	Total Respiration		Heterotropic Respiration		Nitrification Respiration
	ECx (3 Hours) (mg/L)	95% Confidence Limits (mg/L)	ECx (3 Hours) (mg/L)	95% Confidence Limits (mg/L)	ECx (3 Hours) (mg/L)	95% Confidence Limits (mg/L)
EC10	23	-	>1000	-	11	-
EC20	45	-	>1000	-	19	38 – 53
EC50	>1000	-	>1000	-	45	-
EC80	>1000	-	>1000	-	100	-

The reference item gave the following 3-Hour EC₅₀values and 95% confidence limits. 

	Total Respiration		Heterotropic Respiration		Nitrification Respiration
	ECx (3 Hours) (mg/L)	95% Confidence Limits (mg/L)	ECx (3 Hours) (mg/L)	95% Confidence Limits (mg/L)	ECx (3 Hours) (mg/L)	95% Confidence Limits (mg/L)
EC10	1.9	-	5.0	-	0.21	-
EC20	2.8	-	6.7	-	0.37	-
EC50	8.6	6.7 - 11	16	13 - 20	1.8	1.3 – 2.6
EC80	26	-	40	-	9.2	-

 

The reference item gave a 3-Hour EC₅₀value of 8.6 mg/L, 95% confidence limits 6.7 to 11 mg/L for total respiration.

 The reference item gave a 3-Hour EC₅₀value of 16 mg/L, 95% confidence limits 13 to 20 mg/L for heterotrophic respiration.

 The reference item gave a 3-Hour EC₅₀value of 1.8 mg/L, 95% confidence limits 1.3 to 2.6 mg/L for nitrification respiration.

Description of key information

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…….

Following a preliminary range-finding test, activated sewage sludge was exposed to an aqueous solution of the test item at concentrations of 10, 32, 100, 320 and 1000 mg/L with and without the presence ofAllythiourea(ATU) for a period of 3 hours at measured temperatures of between 20 to 22 °C with the addition of a synthetic sewage as a respiratory substrate.

 

The rates of respiration (total respiration, heterotrophic respiration and nitrification respiration) were 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 total and heterotrophic respiration of activated sewage sludge gave a 3‑Hour EC₅₀ value of greater than 1000 mg/L.

 

The effect of the test item on the nitrification respiration of activated sewage sludge gave a 3‑Hour EC₅₀ value of 45 mg/L, 95% confidence limits 38 to 53 mg/L. 

 

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

 

The test item gave the following 3-Hour EC₅₀ values and 95% confidence limits.

	Total Respiration		Heterotropic Respiration		Nitrification Respiration
	ECx (3 Hours) (mg/L)	95% Confidence Limits (mg/L)	ECx (3 Hours) (mg/L)	95% Confidence Limits (mg/L)	ECx (3 Hours) (mg/L)	95% Confidence Limits (mg/L)
EC10	23	-	>1000	-	11	-
EC20	45	-	>1000	-	19	38 – 53
EC50	>1000	-	>1000	-	45	-
EC80	>1000	-	>1000	-	100	-

The reference item gave a 3-Hour EC₅₀ value of 8.6 mg/L, 95% confidence limits 6.7 to 11 mg/L for total respiration.

 

The reference item gave a 3-Hour EC₅₀ value of 16 mg/L, 95% confidence limits 13 to 20 mg/L for heterotrophic respiration.

 

The reference item gave a 3-Hour EC₅₀ value of 1.8 mg/L, 95% confidence limits 1.3 to 2.6 mg/L for nitrification respiration.

 

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

23 mg/L