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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 June 2011.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Qualifier:
according to
Guideline:
other: Method C.11 of Commission Regulation (EC) No. 440/2008
Deviations:
no
Qualifier:
according to
Guideline:
other: US EPA Draft Ecological Effects Test Guidelines OPPTS 850.6800
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Details on sampling:
- Concentrations:
10, 32, 100, 320 and 1000 mg/L

- Sampling method:
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.
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 and synthetic sewage. The pH of the control, reference item and test item preparations were measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter at 0 hours and prior to measurement of the oxygen consumption rate after 3 hours contact time.
Vehicle:
no
Details on test solutions:
Test item preparation:
For the purpose of the test, the test item was dissolved directly in water. At the request of the Sponsor all test concentrations were corrected for a water content of 4.3%
An amount of test item (2090 mg) was dissolved in water and the volume adjusted to 1 litre to give a 2000 mg ai/l stock solution from which dilutions were made to give 200 and 20 mg ai/l stock solutions. An aliquot (250 ml) of the 20 mg ai/l stock solution was dispersed with synthetic sewage (16 ml), activated sewage sludge (200 ml) and water, to a final volume of 500 ml, to give the required concentration of 10 mg ai/l. Similarly, aliquots (80 and 250 ml) of the 200 and 2000 mg ai/l stock solutions were used to prepare the test concentrations of 32, 100, 320 and 1000 mg ai/l. The volumetric flasks containing the stock solutions were inverted several times to ensure homogeneity of the stock solution.

Reference item preparation:
For the purpose of the test a reference item, 3,5-dichlorophenol was used. Two stock solutions of 50 and 160 mg/l were prepared by dissolving the reference item directly in water with the aid of ultrasonication for approximately 20 minutes. Aliquots (10 and 100 ml) of the 160 mg/l stock solution were removed and dispersed with activated sewage sludge, synthetic sewage and water to give the final concentrations of 3.2 and 32 mg/l. Similarly, a 100 ml aliquot of the 50 mg/l stock solution was used to prepare the 10 mg/l concentration. The volumetric flasks containing the reference item were inverted several times to ensure homogeneity of the solutions.


- Differential loading:
Not applicable.

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

- Chemical name of vehicle (organic solvent, emulsifier or dispersant):
Not applicable.

- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)):
Not applicable.

- Evidence of undissolved material (e.g. precipitate, surface film, etc):
Observations made throughout the test period are given in Table 3 in any other information on results including tables section)
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Test Species
A mixed population of activated sewage sludge micro-organisms was obtained on 13 June 2011 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 21ºC and was used on the day of collection. The pH of the sample was 7.4 measured using a WTW pH/Oxi 340I pH and dissolved oxygen 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 deionised reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105ºC 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 3.7 g/l prior to use.

* rinsed three times with 20 ml deionised reverse osmosis water prior to drying in an oven

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
Not applicable.
Hardness:
The test water used for the test was laboratory tap water dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex water softener) giving water with a total hardness of approximately 140 mg/l as CaCO3. After dechlorination and softening the water was then passed through a series of computer controlled plate heat exchangers to achieve the required temperature.
Test temperature:
The test was conducted under normal laboratory lighting in a temperature controlled room at 21±1 Deg C.
pH:
The pH values of the test preparations at the start and end of the exposure period are given in Table 2 in any other information on results including tables section.
Dissolved oxygen:
In some instances, the initial and final dissolved oxygen concentrations were below those recommended in the test guidelines (6.5 mg O2/l and 2.5 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.
Salinity:
Not applicable.
Nominal and measured concentrations:
Nominal concentrations of 10, 32, 100, 320 and 1000 mg/L
Details on test conditions:
Test system:
At time "0" 16 ml of synthetic sewage was diluted to 300 ml with water and 200 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 approximately 0.5 – 1 litre per minute. Thereafter, at 15 minute intervals the procedure was repeated with appropriate amounts of the reference item being added. The test item vessels were prepared as described above. Finally a second control was prepared.
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 approximately 6.5 mg O2/l and 2.5 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.
The test was conducted under normal laboratory lighting in a temperature controlled room at 21±1°C.


Synthetic 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 litre of water with the aid of ultrasonication.
OTHER TEST CONDITIONS
- Adjustment of pH:
The pH values of the test preparations at the start and end of the exposure period are given in Table 2 in any other information on results including tables section

- Photoperiod:
3 hours.

- Light intensity:
Normal laboratory lighting.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
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.

Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
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
Remarks on result:
other: not specified
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
750 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: not specified
Details on results:
Oxygen consumption rates and percentage inhibition values for the control, test and reference items are given in Table 1. The pH values of the test preparations at the start and end of the exposure period are given in Table 2, and observations made on the test preparations throughout the study are given in Table 3. All tables are given in any other information on results including tables section) Percentage inhibition is plotted against concentration for the test and reference items (Figures 1 to 2 attached in background material section).
The following results were derived:

The following results were derived:
Poly[oxy(methyl-1,2-ethanediyl)], alpha-[2-[bis(phosphonomethyl)amino]methylethyl]-omega-[2-[bis(phosphonomethyl)amino]methylethoxy]-sodium salt:
ECx (3 Hours) (mg ai/l) 95% Confidence Limits (mg ai/l)
EC20 930 -
EC50 >1000 -
EC80 >1000 -
NOEC 7500 -



3,5-dichlorophenol:
ECx (30 Minutes) (mg/l) 95% Confidence Limits (mg/l)
EC20 2.1 -
EC50 6.1 5.1 - 8.5
EC80 21 -
NOEC 1.7
Variation in respiration rates of controls 1 and 2 after 3 hours contact time was ± 2%.
The validation criteria for the control respiration rates and reference item EC50 values were therefore satisfied.
It was not possible to determine EC50 or EC80 values for the test item as no concentration tested resulted in greater than 50% inhibition. It was also not possible to obtain 95% confidence limits for the test item EC50 value as the data generated did not fit the models available for calculation.

Values obtained for the pH of the test preparations at the end of the exposure period (see Table 2 in any other information on results including tables section), showed a significantly lower pH value at the test concentration of 1000 mg ai/l compared to the other test vessels. This may be considered to be a contributing factor to the toxic nature of the test item but is considered not to affect the integrity of the study, as the purpose was to determine the effect that the test item would have on a waste water treatment facility where pH adjustment of the incoming effluent would not occur.


Results with reference substance (positive control):
- Results with reference substance valid?
Yes.

- Relevant effect levels:
The reference item gave a 3-Hour EC50 value of 6.6 mg/l, 95% confidence limits 5.1 – 8.5 mg/l.


- Other:
None.

Table1              Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours Contact Time

Nominal

Concentration (mg/L)

Initial O2

Reading

(mg O2/l)

Measurement Period

(minutes)

Final O2Reading

(mg O2/l)

O2Consumption Rates

(mg O2/l/min)

% Inhibition

Control

R1

4.6

6

1.7

0.48

-

 

R2

4.5

6

1.5

0.50

-

Test Item

   10

4.3

5

1.9

0.48

2

 

   32

4.4

6

1.6

0.47

4

 

 100

4.4

6

1.6

0.47

4

 

 320

5.3

8

1.6

0.46

6

 

1000

5.6

8

2.6

0.38

22

3,5-dichlorophenol

3.2

6.3

10

2.8

0.35

29

 

10

7.1

10

5.4

0.17

65

 

32 mg

7.9

10

7.3

0.06

88


R1– R2= Replicates 1 to 2

Table2              pH Values of the Test Preparations at the Start and End of the Exposure Period

Nominal

Concentration (mg/L)

 

pH

0 Hours

3 Hours

Control

R1

7.6

8.0

 

R2

7.6

8.0

Test Item

10

7.5

7.9

 

32

7.4

8.0

 

100

7.3

7.9

 

320

6.9

7.8

 

1000

6.3

7.1

3,5-dichlorophenol

3.2

7.6

8.1

 

10

7.5

8.2

 

32

7.5

8.2


R1– R2= Replicates 1 to 2

Table3              Observations on the Test Preparations Throughout the Test Period

Nominal

Concentration (mg/L)

 

Observations on Test Preparations

0 Hours

30 Minutes

Contact Time

3 Hours

Contact Time

Control

R1

Dark brown dispersion

Dark brown dispersion

Dark brown dispersion

 

R2

Dark brown dispersion

Dark brown dispersion

Dark brown dispersion

Test Item

10

Clear colourless solution, no undissolved test material visible*

Dark brown dispersion, no undissolved test material visible

Dark brown dispersion, no undissolved test material visible

 

32

Clear colourless solution, no undissolved test material visible*

Dark brown dispersion, no undissolved test material visible

Dark brown dispersion, no undissolved test material visible

 

100

Clear colourless solution, no undissolved test material visible*

Dark brown dispersion, no undissolved test material visible

Dark brown dispersion, no undissolved test material visible

 

320

Clear colourless solution, no undissolved test material visible*

Dark brown dispersion, no undissolved test material visible

Dark brown dispersion, no undissolved test material visible

 

1000

Clear colourless solution, no undissolved test material visible*

Dark brown dispersion, no undissolved test material visible

Dark brown dispersion, no undissolved test material visible

3,5-dichlorophenol

3.2

Dark brown dispersion, no undissolved reference material visible

Dark brown dispersion, no undissolved reference material visible

Dark brown dispersion, no undissolved reference material visible

 

10

Dark brown dispersion, no undissolved reference material visible

Dark brown dispersion, no undissolved reference material visible

Dark brown dispersion, no undissolved reference material visible

 

32

Dark brown dispersion, no undissolved reference material visible

Dark brown dispersion, no undissolved reference material visible

Dark brown dispersion, no undissolved reference material visible

R1– R2= Replicates 1 to 2

*Observations made prior to the addition of synthetic sewage and activated sewage sludge

Validity criteria fulfilled:
yes
Conclusions:
The 3‑hour EC50 for respiration inhibition was > 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 750 mg/L.
Executive summary:

A study was perford 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 (1984) No 209 "Activated Sludge, Respiration Inhibition Test", Method C.11 of Commission Regulation (EC) No. 440/2008 and US EPA Draft Ecological Effects Test Guidelines OPPTS 850.6800.

Activated sewage sludge was exposed to an aqueous solution of the test item at concentrations of 10, 32,100, 320 and 1000 mg/L for a period of 3 hours.  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.

The 3‑hour EC50 for respiration inhibition was > 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 750 mg/L.  The reference item 3-Hour EC50 was 6.6 mg/L.

Description of key information

A single key sgtudy is submitted in support of this endpoint, conducted in accordance with OECD 209. The 3‑hour EC50 for respiration inhibition was > 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 750 mg/L.  The reference item 3-Hour EC50 was 6.6 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L
EC10 or NOEC for microorganisms:
750 mg/L

Additional information

A study was perford 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 (1984) No 209 "Activated Sludge, Respiration Inhibition Test", Method C.11 of Commission Regulation (EC) No. 440/2008 and US EPA Draft Ecological Effects Test Guidelines OPPTS 850.6800.

Activated sewage sludge was exposed to an aqueous solution of the test item at concentrations of 10, 32,100, 320 and 1000 mg/L for a period of 3 hours.  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.

The 3‑hour EC50 for respiration inhibition was > 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 750 mg/L.  The reference item 3-Hour EC50 was 6.6 mg/L.