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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
4 Jun 2018 to 5 Jun 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The prediction of the skin sensitising potential of the two main constituents of DABCO TMR-31 (CAS 56968-08-2 and CAS 62568-43-8) was performed with BIOVIA Discovery Studio (TOPKAT) 4 .5, VEGA NIC 1.1.4 (CAESAR), OECD QSAR Toolbox 4.2, Toxtree 3.1.0 and DEREK Nexus 6.0.1. In addition, results from the Danish QSAR Database were also considered in this assessment. The TOPKAT model for skin sensitisation was extended by including data from the Envigo database
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Version / remarks:
2010
Deviations:
no
Principles of method if other than guideline:
NA (study was performed according to guideline).
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Batch: 99101243
Purity: 76.39%
Water content: 23.6%
Physical State/Appearance: Beige coloured paste
Expiry date: 23 November 2019
Storage: Room temperature in the dark under nitrogen
Analytical monitoring:
no
Details on sampling:
Measurement of the Respiration Rates:
At 3 hours contact time, an aliquot was removed from each vessel and poured into a measuring vessel (250 mL darkened glass Biological Oxygen Demand (BOD) bottle) and respiration rate measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe. The measuring vessel contents were stirred constantly by magnetic stirrer. Respiration rate for each vessel was measured over the linear portion of the oxygen consumption trace. 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.
Vehicle:
no
Details on test solutions:
Test Item Preparation:
Test concentrations were prepared by weighing (under non-actinic lighting) 5, 50 and 500 mg of test item and separately dispersing these amounts in deionized reverse osmosis water (~200 mL), before being subjected to ultrasonication (~15 minutes) followed by magnetic stirring (24 hours), at room temperature in order to maximize the dissolved test item concentration. Test vessels were shielded from the light during mixing. Synthetic sewage (16 mL), activated sewage sludge (250 mL) and water were added to a final volume of 500 mL to give the nominal test concentrations (corrected for purity) of 7.6, 76 and 760 mg/L (three replicates at 760 mg/L). The pH of test item dispersions was measured after stirring using a Hach HQ40d Flexi handheld meter and were adjusted to between pH 7.0 and 8.0 if necessary.


Reference Item Preparation:
A stock solution of 0.5 g/L of the reference item, 3,5 dichlorophenol, was prepared by dissolving 3,5 dichlorophenol directly in water with the aid of ultrasonication (~20 minutes). Stock solution pH was measured to be 5.6 (using a Hach HQ40d Flexi handheld meter) and was adjusted to pH 7.2 using 1.0 M NaOH. 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 stock solution was inverted several times to ensure homogeneity of the solution.
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 4 June 2018 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 continuously aerated in the laboratory at ~21°C overnight prior to use in the test. On the day of collection, the activated sewage sludge (7.5 litres) was fed synthetic sewage (375 mL) to act as a respiratory substrate. The pH of the sample on the day of the test was 7.4 (measured using a Hach HQ40d Flexi handheld meter). Determination of the suspended solids level of the activated sewage sludge was performed 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 three 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 ~105°C for at least 1-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.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Remarks on exposure duration:
NA
Post exposure observation period:
NA
Hardness:
Not specified
Test temperature:
20 to 21°C
pH:
0 hours: 7.3 to 8.0
3 hours: 7.1 to 7.8
Dissolved oxygen:
5.91 to 8.19 mg O2/L (>60% dissolved oxygen saturation in all vessels)
Salinity:
NA
Conductivity:
Not specified
Nominal and measured concentrations:
Nominal concentrations: 7.6, 76, 760 mg/L
Details on test conditions:
Preparation of test system:
At 0 hours, 16 mL of synthetic sewage was diluted with water to 250 mL and 250 mL of inoculum added in a 500 mL conical flask (first control) and the mixture was then continuously aerated with clean compressed air at a rate of 0.5 to 1.0 litre per minute for 3 hours. Thereafter, at 15 minute intervals the procedure was repeated for the second control, followed by the reference item vessels. The test item vessels and two further control vessels were then prepared. Testing was conducted under non actinic lighting in a temperature controlled room at measured temperatures of between 20 to 21°C.


Test preparation assessments:
Observations of test preparations were made throughout the test period, with observations of test item vessels at 0 hours made before addition of activated sewage sludge. The pH of test preparations was measured at test initiation and at the end of the 3 Hour incubation period, using a Hach HQ40d Flexi handheld meter. Oxygen concentrations in all vessels were measured after 30 minutes contact time.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 76 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
> 76 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.:
>= 76 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
Based on 0 hour observations made of the test vessel contents prior to addition of the activated sewage sludge inoculum, the test item appeared to be completely dissolved in the aqueous test medium at nominal concentrations up to and including 76 mg/L. At 760 mg/L, the test item formed a cloudy suspension, indicating it had only partially dissolved, thus introducing uncertainty regarding bioavailability.

No statistically significant toxic effects were shown at test concentrations of 7.6 and 76 mg/L, however statistically and biologically (>50%) significant toxic effects were shown at the nominal test concentration of 760 mg/L. Based on these results, a definitive test was not considered necessary.

Dissolved oxygen concentrations after 30 minutes contact time were >60% of the dissolved oxygen saturation level of 8.9 mg O2/L in all vessels.
Results with reference substance (positive control):
The 3-hour EC50 value for the reference item, 3,5-dichlorophenol, was 6.8 mg/L with 95% confidence limits 5.3 to 8.8 mg/L, thus confirming the acceptability of the inoculum used in the test system.
Reported statistics and error estimates:
Statistically significant toxic effects were not shown for 7.6 and 76 mg/L test concentrations. Although statistically and biologically (>50%) significant toxic effects were shown at the nominal test concentration of 760 mg/L, this concentration was beyond the limit of solubility, resulting in uncertainty regarding bioavailability of the test item.

Inhibition of respiration rate

 

Reaction mass of disodium N,N’-[(2-hydroxy-5-nonylphen-1,3-ylene)bis(methylene)[bis[N-methylaminoacetate] and sodium N-[(2-hydroxy-5-nonylphenyl)methyl]-N-methylaminoacetate

3,5-Dichlorophenol

ECx(3 Hours)
(mg/L)

95% Confidence Limits (mg/L)

ECx(3 Hours)
(mg/L)

95% Confidence Limits (mg/L)

 

EC10

>76

-

1.5

-

 

EC20

>76

-

2.2

-

 

EC50

>76

-

6.8

5.3 – 8.8

 

EC80

>76

-

21

-

 

NOEC

≥76

 

 

 

 

- = Not applicable 

pH values of the test item preparations after stirring and prior to the addition of inoculum in the range-finding test

 

Nominal Concentration
(mg/L)

pH

Prior to Adjustment

After Adjustment

Test Item

7.6

7.7

-

76

10.1

7.3

760 R1

11.0

7.8

760 R2

11.1

7.4

760 R3

11.1

7.7

 R = Replicate

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)

Dissolved Oxygen Saturation Level of 8.9 mg O2/L
(%)

Control

R1

6.33

71

R2

6.82

77

R3

6.75

76

R4

7.01

79

Test Item

10

6.12

69

100

5.91

66

1000 R1

7.09

80

1000 R2

8.06

91

1000 R3

7.57

85

3,5-Dichlorophenol

3.2

7.31

82

10

7.30

82

32

8.19

92

R = Replicate 

Oxygen consumption rates and percentage inhibition values after 3 hours contact time in the range-finding test

 

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

3.2

2

1.8

42.00

-

R2

3.3

2

2.1

36.00

-

R3

4.7

4

2.1

39.00

-

R4

3.9

3

2.0

38.00

-

Test Item

10

3.8

3

1.8

40.00

[3]

100

3.2

2

2.0

36.00

7

1000 R1

6.9

10

5.8

6.60

83

1000 R2

7.0

10

6.1

5.40

86

1000 R3

7.0

10

6.0

6.00

85

3,5-Dichlorophenol

3.2

5.5

8

1.9

27.00

30

10

6.5

10

3.9

15.60

60

32

7.3

10

6.7

3.60

91

 R = Replicate

- = Not applicable

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

7.8

7.4

R2

7.4

7.3

R3

8.0

7.5

R4

7.9

7.5

Test Item

7.6

7.4

7.3

76

7.3

7.1

760 R1

7.5

7.7

760 R2

7.4

7.6

760 R3

7.6

7.7

3,5-Dichlorophenol

3.2

7.7

7.8

10

7.7

7.8

32

7.4

7.4

 R = Replicate

Observations on the test preparations throughout the test period in 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

7.6

Pale yellow / brown dispersion, no undissolved test item visible

Dark brown dispersion, no undissolved test item visible

Dark brown dispersion, no undissolved test item visible

76

Pale yellow / brown dispersion, no undissolved test item visible

Dark brown dispersion, no undissolved test item visible

Dark brown dispersion, no undissolved test item visible

760 R1

Pale yellow / brown cloudy dispersion, with some foam at the surface

Dark brown dispersion, with a large layer of foam at the surface

Dark brown dispersion, with a large layer of foam at the surface

760 R2

Pale yellow / brown cloudy dispersion, with some foam at the surface

Dark brown dispersion, with a large layer of foam at the surface

Dark brown dispersion, with a large layer of foam at the surface

760 R3

Pale yellow / brown cloudy dispersion, with some foam at the surface

Dark brown dispersion, with a large layer of foam at the surface

Dark brown dispersion, with a large layer of foam at the surface

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

 

Validity criteria fulfilled:
yes
Conclusions:
No statistically or biologically (≥10%) significant inhibition of respiration of activated sewage sludge micro-organisms occurred at concentrations up to and including 76 mg/L, the highest tested concentration at which the test item appeared to be completely dissolved in the test medium. Inhibition >50% occurred at a nominal concentration of 760 mg/L that exceeded the apparent solubility limit of the test item under the conditions of the test. It is concluded that the 3 hour NOEC and EC10 are ≥76 and >76 mg/L, respectively. The EC50 could not reliably be determined, but it also exceeds 76 mg/L.
Executive summary:

The effect of the test item, Reaction mass of disodium N,N'-[(2-hydroxy-5-nonylphen-1,3-ylene)bis(methylene)]bis[N-methylaminoacetate] and sodium N-[(2-hydroxy-5-nonylphenyl)methyl]-N-methylaminoacetate, on the respiration of activated sewage sludge microorganisms was assessed at a GLP accredited laboratory according to the OECD Guideline 209, “Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)”.

Activated sewage sludge was exposed to aqueous dispersions of the test item at nominal concentrations of 7.6, 76 and 760 mg/L for a period of 3 hours at measured temperatures of 20 to 21°C with the addition of synthetic sewage as a respiratory substrate. Respiration rate was determined after 3 hours contact time and compared to data from an untreated control. Similar measurements were also made with the reference substance, 3,5-dichlorophenol, as a positive control.

The reference substance gave a 3‑hour EC50 value of 6.8 mg/L, with 95% confidence limits of 5.3 to 8.8 mg/L, thus confirming the acceptability of the inoculum used in the test system.

The test item showed no statistically or biologically (≥10%) significant inhibition of respiration of activated sewage sludge microorganisms at concentrations up to and including 76 mg/L, the highest tested concentration at which the test item appeared to be completely dissolved within the test medium. Inhibition >50% occurred at a nominal concentration of 760 mg/L that exceeded the apparent solubility limit of the test item under the conditions of the test. It is concluded that the 3‑hour NOEC and EC10 are ≥76 and >76 mg/L, respectively. The EC50 could not reliably be determined, but it also exceeds 76 mg/L. 

Description of key information

The effect of the test item, Reaction mass of disodium N,N'-[(2-hydroxy-5-nonylphen-1,3-ylene)bis(methylene)]bis[N-methylaminoacetate] and sodium N-[(2-hydroxy-5-nonylphenyl)methyl]-N-methylaminoacetate, on the respiration of activated sewage sludge microorganisms was assessed at a GLP accredited laboratory according to the OECD Guideline 209, “Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)” (Best, 2019).

Activated sewage sludge was exposed to aqueous dispersions of the test item at nominal concentrations (corrected for purity) of 7.6, 76 and 760 mg/L for a period of 3 hours at measured temperatures of 20 to 21°C with the addition of synthetic sewage as a respiratory substrate. Respiration rate was determined after 3 hours contact time and compared to data from an untreated control. Similar measurements were also made with the reference substance, 3,5-dichlorophenol, as a positive control.

The reference substance gave a 3‑hour EC50 value of 6.8 mg/L, with 95% confidence limits of 5.3 to 8.8 mg/L, thus confirming the acceptability of the inoculum used in the test system.

The test item showed no statistically or biologically (≥10%) significant inhibition of respiration of activated sewage sludge microorganisms at concentrations up to and including 76 mg/L, the highest tested concentration at which the test item appeared to be completely dissolved within the test medium. Inhibition >50% occurred at a nominal concentration of 760 mg/L that exceeded the apparent solubility limit of the test item under the conditions of the test. It is concluded that the 3‑hour NOEC and EC10 are ≥76 and >76 mg/L, respectively. The EC50 could not reliably be determined, but it also exceeds 76 mg/L. 

Key value for chemical safety assessment

EC50 for microorganisms:
76 mg/L
EC10 or NOEC for microorganisms:
76 mg/L

Additional information