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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 November 2011 - 23 March 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
22 July 2010
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark and under nitrogen.
- Stability under test conditions: Assumed stable.
- Solubility and stability of the test substance in the solvent/vehicle: N/A, applied as supplied.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: N/A

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: N/A
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material): Applied as supplied.

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable): N/A

OTHER SPECIFICS: No
Analytical monitoring:
no
Details on sampling:
- Concentrations: 10, 30, 100, 300 and 1000 mg/L
- Sampling method: In this test, a Co-ordinated Environmental Services (CES) Ltd automated respirometer and associated software was used to monitor the oxygen consumed by the mixtures following a 3- hour exposure phase.
Mixtures were incubated in 500 mL bottles fitted with an oxygen probe, a sinter to deliver air and a nitrogen gas nozzle. Aliquots of RO water, synthetic sewage and antifoam agent (silicone oil in water) were added to each bottle. Aliquots of the test substance were then added to the respective bottles to give final, nominal concentrations of 10, 30, 100, 300 and 1000 mg/L. Immediately prior to initiation of the instrument, the required volume of activated sludge was added to each bottle. The prepared mixtures were aerated and stirred for three hours in a thermostatically- controlled water bath, using an aerator connected to a laboratory supply of oil-free compressed air (one litre/minute). Following the exposure period, the aeration was stopped and the headspace of each bottle was flushed with nitrogen during the oxygen measurement phase. The instrument measured the amount of oxygen in the mixtures at one minute intervals for at least 15 minutes. The pH and temperature of the samples were measured at the start and end of the test.
- Sample storage conditions before analysis: N/A
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test substance were added to the required vessels to give nominal weights of 5, 15, 50, 150 and 500 mg/500 mL
- Controls: Activated sludge and synthetic sewage alone and reference/ positive control 3,5-DCP at nominal concentrations of 3, 10 and 32 mg/L.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): N/A
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): N/A
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: No
- Name and location of sewage treatment plant where inoculum was collected: A sample of activated sludge was obtained the day before the start of the test from Worlingworth Sewage Treatment Works (Suffolk, UK), which treats predominantly domestic waste.
- Method of cultivation: In the laboratory, the sample was maintained under aerobic conditions until required. The concentration of suspended solids in a homogenised sample was determined on the day of collection and immediately before the start of the test.
On the day of collection, an aliquot (10 mL) of the activated sludge was filtered through a dried and preweighed Whatman GF/C filter paper, which was then dried again at approximately 105°C for at least one hour, allowed to cool in a desiccator and reweighed. The mixed liquor suspended solids (MLSS) content of the activated sludge was then calculated. Synthetic sewage (50 mL/L) was added to the stock of activated sludge and this was aerated overnight.
On the day of the test, the MLSS content of the sludge was determined (in triplicate) and adjusted to 4 g/L by the addition of tap water. The pH and temperature of the sludge were also measured. Aliquots (200 mL) were then added to each mixture to give a final MLSS concentration of 1.6 g/L.
- Preparation of inoculum for exposure: See above
- Pretreatment: See above
- Initial biomass concentration: 1.6 g/L in the test vessels.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
The prepared mixtures were aerated and stirred for three hours in a thermostatically- controlled water bath, using an aerator connected to a laboratory supply of oil-free compressed air (one litre/minute). Following the exposure period, the aeration was stopped and the headspace of each bottle was flushed with nitrogen during the oxygen measurement phase. The instrument measured the amount of oxygen in the mixtures at one minute intervals for at least 15 minutes. The pH and temperature of the samples were measured at the start and end of the test.
Hardness:
Not determined
Test temperature:
20 ± 2 ºC
pH:
7.27 - 8.25
Dissolved oxygen:
Initial measurements = 6.25 - 7.74 mgO2/L
Salinity:
N/A
Conductivity:
N/A
Nominal and measured concentrations:
10, 30, 100, 300 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 mL bottles fitted with an oxygen probe, a sinter to deliver air and a nitrogen gas nozzle.
- Type (delete if not applicable): Closed
- Material, size, headspace, fill volume: 500 mL bottles filled to capacity.
- Aeration: 1 L/min (air)
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per positive control (replicates): 1
- Sludge concentration (weight of dry solids per volume): 1.6 g/L
- Weight of dry solids per volume of reaction mixture per unit of time: Not reported
- Nutrients provided for bacteria: 16 mL of synthetic sewage sludge added to each testing vessel.
- Biomass loading rate: Not reported
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: A sample of activated sludge was obtained the day before the start of the test from Worlingworth Sewage Treatment Works (Suffolk, UK), which treats predominantly domestic waste. In the laboratory, the sample was maintained under aerobic conditions until required. The concentration of suspended solids in a homogenised sample was determined on the day of collection and immediately before the start of the test.
On the day of collection, an aliquot (10 mL) of the activated sludge was filtered through a dried and preweighed Whatman GF/C filter paper, which was then dried again at approximately 105°C for at least one hour, allowed to cool in a desiccator and reweighed. The mixed liquor suspended solids (MLSS) content of the activated sludge was then calculated. Synthetic sewage (50 mL/L) was added to the stock of activated sludge and this was aerated overnight.
On the day of the test, the MLSS content of the sludge was determined (in triplicate) and adjusted to 4 g/L by the addition of tap water. The pH and temperature of the sludge were also measured. Aliquots (200 mL) were then added to each mixture to give a final MLSS concentration of 1.6 g/L.
- Particulate matter: Not reported
OTHER TEST CONDITIONS
- Adjustment of pH: Not required
- Photoperiod: Not reported
- Light intensity: Not reported
- Details on termination of incubation: Following the exposure period, the aeration was stopped and the headspace of each bottle was flushed with nitrogen during the oxygen measurement phase. The instrument measured the amount of oxygen in the mixtures at one minute intervals for at least 15 minutes. The pH and temperature of the samples were measured at the start and end of the test.
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The rate of respiration (specific respiration rate, Rs) of test, control and reference mixtures was calculated from continuous recordings of the measured oxygen level in test mixtures with time (up to 10 mins).
TEST CONCENTRATIONS
- Spacing factor for test concentrations: as per OECD 209 (paragraph 40)
- Justification for using fewer concentrations than requested by guideline: N/A
- Range finding study : No
- Test concentrations: N/A
- Results used to determine the conditions for the definitive study: N/A
Reference substance (positive control):
yes
Remarks:
3,5-DCP
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
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Not reported
- Effect concentrations exceeding solubility of substance in test medium: Not reported
- Adsorption (e.g. of test material to the walls of the test container): Not reported
- Blank controls oxygen uptake rate: The blank controls oxygen uptake rate was higher than 20 mg oxygen per one gram of activated sludge per hour in both tests
- Coefficient of variation of oxygen uptake rate in control replicates: The coefficient of variation (CV) of the blank respiration rates was less than 30 % in both tests
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: The EC50 of the reference item was within the acceptable range of 2 to 25 mg/L (actual = 11.34 mg/L).
- Other: N/A
Reported statistics and error estimates:
Where possible, the EC20, EC50 and EC80 (and 95 % confidence intervals) and no observed effect concentration (NOEC) of the test substance were calculated using SAFEstat programme SAS Proc NLIN (SAS Institute, 2002).

For the 3,5-dichlorophenol reference substance, only the EC50 was calculated.

The EC50 (and EC20 and EC80) was the concentration of the test or reference material at which the respiration rate is 50 % of mean control value (or 20 % and 80 % of the mean control value).

For substances that do not significantly inhibit the activity of the sludge the no observed effect concentration (NOEC) will be taken as the highest concentration tested.
Test item and 3,5-Dichlorophenol mixtures were analysed separately.

Table 1       DO concentrations, measurement times and specific respiration rates

 

Group

Nominal Conc.

(mg/L)

Rep.

Initial measured DO

(mg O2/L)

Final measured DO

(mg O2/L)

Measurement time

(mins)

Specific respiration rate

(Rs mg O2/gh)

Mean Rs

(mg O2/gh)

% inhibition or (% stimulation)

Mean % inhibition

Control

-

1

6.58

2.61

7

0.57

21.7

-

-

2

6.75

2.50

7

0.61

3

6.75

2.82

7

0.56

4

6.61

2.55

7

0.58

Test Item

10

1

6.51

2.28

6

0.71

23.4

(-22)

0

2

6.37

2.48

7

0.56

4

3

6.41

2.69

6

0.62

(-7)

4

6.25

2.59

6

0.61

(-5)

30

1

6.34

2.42

7

0.56

22.3

3

0

2

6.79

2.52

7

0.61

(-5)

3

6.73

2.70

7

0.58

1

4

6.67

2.89

6

0.63

(-9)

100

1

6.77

2.49

7

0.61

21.1

(-6)

3

2

6.54

2.53

7

0.57

1

3

6.43

2.68

7

0.54

7

4

6.59

2.36

8

0.53

9

300

1

6.65

2.46

7

0.60

23.5

(-3)

0

2

6.46

2.68

6

0.63

(-9)

3

6.31

2.26

6

0.68

(-17)

4

6.32

2.67

6

0.61

(-5)

1000

1

6.49

2.48

7

0.57

22.3

1

0

2

6.59

2.44

8

0.52

10

3

6.45

2.25

7

0.60

(-4)

4

6.68

2.53

6

0.69

(-19)

3,5-DCP

3

10

32

1

6.49

2.28

10

0.42

-

27

-

1

7.06

3.61

10

0.35

40

1

7.74

6.38

10

0.14

77

 

Validity criteria fulfilled:
yes
Conclusions:
At a concentration of 1000 mg/L, no inhibition of the total respiration rate was observed. The resulting EC50 and NOEC values were > 1000 and 1000 mg/L, respectively.
Executive summary:

OECD 209 (2012) - The inhibitory effects of Tetraesters of pentaerythritol with 2-methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid on the respiration of activated sludge was assessed in an OECD 209 test.

 

The activated sludge (with sewage feed at a final concentration of 1.6 g/L dry solids) was exposed to the test item under aerobic conditions for a period of 3 hours, after which time the respiration rate of the microorganisms was measured (via oxygen consumption). Three test groups were prepared;

 

a) Blank control vessels (4) were prepared containing activated sludge and synthetic sewage feed.

b) Test item flasks (4 per treatment group) were prepared containing activated sludge, synthetic sewage feed, anti-foaming agent (silicone oil) and test item. The test item was added directly to the sludge to achieve nominal concentrations of 10, 30, 100, 300 and 1000 mg/L.

c) Reference item flasks (3) were prepared containing activated sludge, synthetic sewage feed, anti-foaming agent and 3,5 -dichlorophenol, a known inhibitor of microbial respiration. The reference item was added directly to the sludge to achieve nominal concertation’s of 3.2, 10 and 32 mg/L (1 flask per concentration).

 

Upon preparation, the flasks were incubated aerobically for 3 h after which time the oxygen concentration in the solutions was measured for 6-8 minutes (10 minutes for low O2 consumption in reference item flasks). The respiration rate for each vessel was calculated and resulting inhibitory effects determined as a percentage versus the blank control vessels.

 

At a test concentration of 1000 mg/L, no inhibition of the total respiration rate was observed. The resulting EC50 and NOEC values were concluded to be > 1000 and 1000 mg/L, respectively.

 

The guideline validity criteria for the control and reference item vessels were satisfied. The study was therefore classified as acceptable and meets the requirement for Test Guideline OECD 209.

 

Description of key information

Inhibition to aquatic microorganisms: 3 h EC50 > 1000 mg/L and 3 h NOEC = 1000 mg/L; OECD 209; Dickinson, R. (2012)

Key value for chemical safety assessment

Additional information

OECD 209 (2012) - The inhibitory effects of Tetraesters of pentaerythritol with 2-methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid on the respiration of activated sludge was assessed in an OECD 209 test.

 

The activated sludge (with sewage feed at a final concentration of 1.6 g/L dry solids) was exposed to the test item under aerobic conditions for a period of 3 hours, after which time the respiration rate of the microorganisms was measured (via oxygen consumption). Three test groups were prepared;

 

a) Blank control vessels (4) were prepared containing activated sludge and synthetic sewage feed.

b) Test item flasks (4 per treatment group) were prepared containing activated sludge, synthetic sewage feed, anti-foaming agent (silicone oil) and test item. The test item was added directly to the sludge to achieve nominal concentrations of 10, 30, 100, 300 and 1000 mg/L.

c) Reference item flasks (3) were prepared containing activated sludge, synthetic sewage feed, anti-foaming agent and 3,5 -dichlorophenol, a known inhibitor of microbial respiration. The reference item was added directly to the sludge to achieve nominal concertation’s of 3.2, 10 and 32 mg/L (1 flask per concentration).

 

Upon preparation, the flasks were incubated aerobically for 3 h after which time the oxygen concentration in the solutions was measured for 6-8 minutes (10 minutes for low O2 consumption in reference item flasks). The respiration rate for each vessel was calculated and resulting inhibitory effects determined as a percentage versus the blank control vessels.

 

At a test concentration of 1000 mg/L, no inhibition of the total respiration rate was observed. The resulting EC50 and NOEC values were concluded to be > 1000 and 1000 mg/L, respectively.

 

The guideline validity criteria for the control and reference item vessels were satisfied. The study was therefore classified as acceptable and meets the requirement for Test Guideline OECD 209.