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Reference
Endpoint:
activated sludge respiration inhibition testing
Remarks:
(Includes evaluation of heterotrophic and nitrification respiration inhibition.)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 October 2017 to ****
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Deviations:
yes
Remarks:
see below
Principles of method if other than guideline:
Guideline deviation:
The total and heterotrophic respiration inhibition was evaluated using one set of vessels in which oxygen consumption was measured before and after amendment with a nitrification inhibitor (ATU), rather than with two discrete sets of vessels (i.e. with and without ATU) incubated for 3 hours as described in the OECD Guideline 209. This deviation is not considered to have adversely impacted the outcome of the study because it reduces the inherent variability within the test system and the validity criteria for the controls and reference substance were met for all respiration endpoints.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
-Purity: >93%
-Description: Dark Amber Liquid
-Storage: Room temperature (15 to 30°C) under nitrogen
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Each treatment vessel contained a synthetic sewage concentrate, dechlorinated tap water, microbial inoculum, test or reference substance (blank controls had neither) and a nitrification inhibitor (for heterotrophic respiration assessment) to achieve total test volumes of 250 mL.

The test substance was weighed into individual foil boats and added directly to test vessels to achieve nominal concentrations of 1, 10 and 100 mg/L (duplicate) and 1000 mg/L (triplicate). Immediately after preparation, the pH of each vessel was determined and adjusted to within 7.5 ± 0.5 g/L with 40% NaOH, as necessary.

The reference substance, 3,5-dichlorophenol (3,5-DCP), was added the test vessels as a prepared aqueous stock solution (0.50 g/L) to achieve nominal concentrations of 0.1, 2.0 and 40 mg/L (single vessel for each).
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: sludge return line at Burley Menston sewage treatment works (West Yorkshire, UK); collected on 10 October 2017.
- Method of cultivation: stored for 2 days before use; maintained at 20 ± 2°C and fed daily with synthetic sewage concentrate at a rate of 50 mL/L.
- Initial biomass concentration: 6.07 g/L (suspended solids concentration determined gravimetrically following homogenisation); adjusted at the start of the test with dechlorinated tap water to a nominal range of 3 ± 0.3 g/L.
- Preparation of inoculum for exposure: The pH was determined to be 6.45, and was adjusted to 7.44 with sodium hydroxide (NaOH).
- Pretreatment: none.
- Test initiation: Test vessels were inoculated in three batches of five at 30 minute intervals. Each prepared batch consisted of one control vessel and either four test substance vessels or one test substance and three reference substance vessels, as appropriate.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Post exposure observation period:
Following oxygen consumption measurements to determine total respiration rates, a nitrification inhibitor (N-allylthiourea, ATU), was added as an aqueous stock solution (2.32 g/L) to the prepared blank control, test substance and reference substance vessels to give a final concentration of ca. 11.6 mg ATU/L. Oxygen consumption in the absence of nitrification was measured at least 10 minutes after amendment with ATU to assess heterotrophic respiration. Nitrification respiration was calculated as the difference between total respiration and heterotrophic respiration.
Hardness:
Not specified
Test temperature:
20 +/- 2°C
pH:
7.05 - 7.43
Dissolved oxygen:
Aerated; see tabulated oxygen consumption data
Salinity:
Not applicable
Conductivity:
Not specified
Nominal and measured concentrations:
Nominal test concentrations:
- test substance: 1, 10, 100 and 1000 mg/L
- reference substance: 0.1, 2.0 and 40 mg/L
Details on test conditions:
The study was conducted as a combined range finding / limit test. A definitive test was not conducted in this study because as no significant inhibition was observed for total, nitrification and heterotrophic respiration in the presence of test substance during the combined range finding / limit test.

TEST SYSTEM
- Test vessel: glass flasks (containing a total volume of 250 mL)
- No. of vessels per concentration (replicates):
- test substance: 1, 10 and 100 mg/L (duplicate) and 1000 mg/L (triplicate)
- reference substance: 0.1, 2.0 and 40 mg/L (single vessel for each)
- No. of vessels per control (replicates): three; duplicate measurements were taken for each replicate control vessel, one at the start and another at the end of each run, to bracket each run
- No. of vessels per vehicle control (replicates): not applicable
- No. of vessels per abiotic control (replicates): not applicable
- Aeration: yes
- Nutrients provided for bacteria: pre-made synthetic sewage concentrate (SI093) purchased from Strathkelvin Instruments prepared in water (250 mL)
- Nitrification inhibitor used: N-allylthiourea (ATU)
- Biomass loading rate: Following inoculation, each vessel contained a nominal suspended solids concentration of ca. 1.5 g/L.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: mains tap water
- Particulate matter: not specified

OTHER TEST CONDITIONS
- Adjustment of pH: see above.
- Photoperiod: not specified
- Light intensity: not specified
- Details on termination of incubation: At the end of the 3 hour incubation period, a portion (20 mL) of each test preparation was transferred to an appropriate sample tube containing a PTFE stirrer. The dissolved oxygen (DO) electrode was sealed in the neck of the flask, ensuring air was completely excluded. The flask contents were stirred at a constant rate during DO measurements. Oxygen consumption was measured over a period of up to 10 minutes.
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
Basis for effect:
other: inhibition of total, heterotrophic and nitrification respiration
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Basis for effect:
other: inhibition of total, heterotrophic and nitrification respiration
Details on results:
Dissolved oxygen measurements were plotted over time. Respiration rates (mg/L/h) were determined from the gradient of the linear portion of the graph between 2.0 mg O2/L and 7.0 mg O2/L, where possible. The graphical data and calculated respiration rates were generated using Strathkelvin Strathtox software.

Respiration rates and percent inhibition for the test substance are summarized in Table 1.

The blank control respiration rate was ≥ 20 mg O2/g/h. The coefficient of variation of the blank control respiration rates were ≤ 30%.

The EC50 values for total, heterotrophic and nitrification respiration were concluded to be greater than 1000 mg/L. The no observed effect concentrations (NOEC) for total, heterotrophic and nitrification respiration were therefore determined to be 1000 mg/L.
Results with reference substance (positive control):
Respiration rates and percent inhibition for the reference substance are summarized in Table 2. The 3-hour EC50 values for the reference item, 3,5-dichlorophenol (3,5-DCP), were determined to be 3.5 mg/L (total respiration), 12.1 mg/L (nitrification respiration) and 1.1 mg/L (heterotrophic respiration).
Reported statistics and error estimates:
Statistical analysis was performed using the CETIS program v 1.8.6.8.

As less than 50% inhibition was observed in the presence of test substance, the EC50 for total, heterotrophic and nitrification respiration could not be determined and is therefore concluded to be greater than the highest concentration tested. The EC50 for the reference substance was based on a statistical analysis (linear interpolation) of concentration versus effect in total respiration and nitrification respiration.

Linear interpolation analysis was performed to estimate total, heterotrophic and nitrification respiration EC10, EC20 and EC50 values for the reference substance and test substance, where possible. A parametric-control versus treatment test (Dunnett multiple comparison test) was performed to estimate total, heterotrophic and nitrification respiration no observed effect concentrations (NOEC) for the test substance.

Table1: Respiration Rates and Percent Inhibition for the Test Substance

Test Substance

Nominal

Concentration

(mg/L)

Rep

Total

Respiration

Rate
(mg/L/h)

Heterotrophic

Respiration Rate*
(mg/L/h)

Nitrification

Respiration Rate** 
(mg/L/h)

Total Respiration

Inhibition
(%)

Heterotrophic

Respiration

Inhibition
(%)

Nitrification

Respiration

Inhibition
(%)

Control

A

60.6

30.4

30.2

 

 

 

 

A

66.7

31.3

35.4

 

 

 

 

B

55.5

27.8

27.7

 

 

 

 

B

61.9

29.8

32.1

 

 

 

 

C

60.8

29.7

31.1

 

 

 

 

C

67.4

30.5

36.9

 

 

 

 

Mean

62

30

32

1

A

60.3

28.3

32.0

 

 

 

 

B

56.4

27.7

28.7

 

 

 

 

Mean

58

28

30

6.1

6.4

5.8

10

A

61.7

30.6

31.1

 

 

 

 

B

57.9

27.1

30.8

 

 

 

 

Mean

60

29

31

3.8

3.6

4.0

100

A

61.3

30.3

31.0

 

 

 

 

B

58.4

29.3

29.1

 

 

 

 

Mean

60

30

30

3.7

0.4

6.8

1000

A

61.2

30.9

30.3

 

 

 

 

B

58.2

29.3

28.9

 

 

 

 

C

59.8

29.3

30.5

 

 

 

 

Mean

60

30

30

3.9

0.3

7.2

Rep: Replicate vessel

- Not Applicable

* Respiration in the presence of a nitrification inhibitor, N-allylthiourea (11.6 mg ATU/L).

** Calculated as the difference of total respiration rate and heterotrophic respiration rate.

 

  

Table 2: Respiration Rates and Percent Inhibition for the Reference Substance

Reference Substance

Nominal

Concentration

(mg/L)

Total

Respiration

Rate
(mg/L/h)

Heterotrophic

Respiration

Rate*
(mg/L/h)

Nitrification

Respiration

Rate**
(mg/L/h)

Total Respiration

Inhibition
(%)

Heterotrophic

Respiration

Inhibition
(%)

Nitrification

Respiration

Inhibition
(%)

0.1

62.4

30.1

32.3

-0.4

-0.6

-0.2

2.0

35.7

28.6

7.1

42.6

4.4

78.0

40

6.4

4.5

1.9

89.7

85.0

94.1

- Not Applicable

* Respiration in the presence of a nitrification inhibitor, N-allylthiourea (11.6 mg ATU/L).

** Calculated as the difference of total respiration rate and heterotrophic respiration rate.
Validity criteria fulfilled:
yes
Conclusions:
No statistically significant effects were observed on respiration of activated sludge at the maximum limit concentration of the test substance following a 3-hour exposure. The EC50 values for total, heterotrophic and nitrification respiration were concluded to be greater than 1000 mg/L. The no observed effect concentrations (NOEC) for total, heterotrophic and nitrification respiration were determined to be 1000 mg/L.
Executive summary:

The study was conducted to determine the no observable effect concentration (NOEC) and, where possible, the effect concentration for an x% effect (e.g. EC10, EC20, EC50) for activated sludge micro-organisms exposed to the test substance in accordance with OECD Test Guideline 209.

The activated sludge inoculum was collected from the sludge return line at Burley Menston Sewage Treatment Works (West Yorkshire, U.K.), which has a predominantly domestic catchment. The point of collection was selected to ensure that the activated sludge sample was relatively free of exogenous material.

A combined range finding / limit test, employing nominal test concentrations of 1, 10, 100 and 1000 mg/L, was undertaken to determine the appropriate concentration levels for a definitive test. A definitive test was not conducted in this study because no statistically significant inhibition was observed for total, nitrification and heterotrophic respiration during the combined range finding / limit test following a 3-hour exposure to the test substance.

The validity criteria were met in this range finding / limit test and the data are therefore considered valid.

The EC50 values for total, heterotrophic and nitrification respiration were concluded to be greater than 1000 mg/L. The no observed effect concentrations (NOEC) for total, heterotrophic and nitrification respiration were therefore determined to be 1000 mg/L.

The 3-hour EC50 values for the reference item, 3, 5-dichlorophenol, were determined to be 3.5 mg/L (total respiration), 12.1 mg/L (nitrification respiration) and 1.1 mg/L (heterotrophic respiration).

Description of key information

In an OECD Guideline No. 209 study, conducted according to GLP, no significant inhibition oftotal, heterotrophic or nitrification respirationwas observed following a 3-hour exposure of activated sewage sludge to the test substance, resulting in an EC50 value of greater than 1000 mg/L.  The No Observed Effect Concentration (NOEC) following the 3-hour exposure was 1000 mg/L (Smithers Viscient (ESG) Ltd, ****).

Key value for chemical safety assessment

Additional information