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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 - 20 June 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Stuy was conducted in accordance with International guidelines and in accordance with GLP. All guideline validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Version / remarks:
22 July 2010
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
not required
Remarks:
As it was not a requirement of the Test Guidelines, and due to the short-term nature of the study, no analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation
Details on sampling:
N/A
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item was dispersed directly in water
- Eluate: N/A
- Differential loading: N/A
- Controls: Test medium (maintained under identical conditions, minus test item)
- 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.):
Range-finder:
At 0 hours:
Nominal concentrations 10 - 100 mg/L: small particles of test item visible dispersed throughout and on the surface.
1000 mg/L: many large particles of test item visible on the surface and adhered to the sides of the vessel
At 30 minutes & 3 hours:
10 mg/L: few small particles of test item visible dispersed throughout
100 mg/L: small particles of test item visible dispersed throughout and a few particles adhered to the sides of the vessel
1000 mg/L: many particles of test item visible dispersed throughout, at the surface and adhered to the sides of the vessel

Definitive test:
0 hours (prior to addition of activated sewage sludge):
10 mg/L: small particles of test item visible dispersed throughout and at the surface
32 mg/L: particles of test item visible dispersed throughout and at surface
100 mg/L: large particles of test item visible dispersed throughout, at the surface and adhered to sides of vessel

30 minutes:
10 mg/L: few small particles of test item visible dispersed throughout
32 mg/L: particles of test item visible dispersed throughout
100 mg/L: large particles of test item visible dispersed throughout and some test item visible adhered to sides of vessel

3 hours:
10 mg/L: few small particles of test item visible dispersed throughout
32 mg/L: particles of test item visible dispersed throughout
100 mg/L: large particles of test item visible dispersed throughout and some test item visible adhered to sides of vessel
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: N/A
- Name and location of sewage treatment plant where inoculum was collected: Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.
- Method of cultivation: N/A
- Preparation of inoculum for exposure: 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 L) was fed synthetic sewage (500 mL).
- Pretreatment: Determination of the suspended solids level of the activated sewage sludgewas carried out by filtering a sample (100 mL) of the activated sewage sludge by suction through a pre-weighed GF/A filter paper (rinsed 3 times with 20 mL deionized reverse osmosis water prior to drying in an oven) 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 approximately 105 ºC for at least 1-Hour and allowed to cool before weighing. This process was repeated until a constant weight was attained.
- Initial biomass concentration: The suspended solids concentration was equal to 3.0 g/L prior to use.
Test type:
static
Water media type:
other: Deionized reverse osmosis water containing less than 1 mg/L Total Organic Carbon (TOC)
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
N/A
Hardness:
Not reported
Test temperature:
21 °C - 22 °C
pH:
7.2 - 7.8
Dissolved oxygen:
Above 60 % in all vessels at 30 mins contact time
Salinity:
Not reported
Conductivity:
Not reported
Nominal and measured concentrations:
Nominal: 10, 32 and 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: conical flask
- Type (delete if not applicable): open / closed
- Material, size, headspace, fill volume: 500 mL flask, 500 mL fill volume
- Aeration: yes, with clean, oil-free compressed air
- No. of vessels per concentration (replicates): five
- No. of vessels per control (replicates): four
- No. of vessels per vehicle control (replicates): N/A
- No. of vessels per abiotic control (replicates): N/A
- Sludge concentration (weight of dry solids per volume): 3.0 g/L (suspended solids)
- Weight of dry solids per volume of reaction mixture per unit of time: -
- Nutrients provided for bacteria: 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
- Biomass loading rate: N/A

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The test water used for the range-finding and definitive tests was deionized reverse osmosis water containing less than 1 mg/L Total Organic Carbon (TOC).
- Particulate matter: None

OTHER TEST CONDITIONS
- Adjustment of pH: yes (to between 7.0 and 8.0)
- Photoperiod: normal lighting
- Light intensity: normal laboratory lighting
- Details on termination of incubation: 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.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
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. Parameters: pH (measured at the test start following addition of activated sludge, and after 3 hours), O2 concentration (after 30 mins contact time), respiration rates (at 3 hours contact time).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Justification for using fewer concentrations than requested by guideline: Not relevant
- Range finding study: yes -
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 (three replicates for the test concentration of 1000 mg/L). The test item was dispersed directly in water.
Nominal amounts of test item (5, 50 and 500 mg (500 mg in triplicate)) were each separately dispersed in approximately 200 mL of deionized reverse osmosis water and subjected to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 hours, at room temperature, in order to maximize the dissolved test item concentration. All 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 required concentrations of 10, 100 and 1000 mg/L (three replicates at 1000 mg/L).
- Test concentrations: 10, 32 and 100 mg/L
- Results used to determine the conditions for the definitive study:
In the range-finding study, the dissolved oxygen concentrations after 30 minutes contact time in all vessels were above 60% of the dissolved oxygen saturation level of 8.9 mg O2/L.
No statistically significant toxic effects were shown at the test concentration of 10 mg/L, however statistically significant toxic effects were shown at the test concentrations of 100 and 1000 mg/L. Based on these results and after discussion with the Sponsor, it was considered necessary to perform a definitive test in order to obtain a NOEC for the test item. Test concentrations of 10, 32 and 100 mg/L were selected for the definitive test.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
32 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: N/A
- Effect concentrations exceeding solubility of substance in test medium: N/A
- Adsorption (e.g. of test material to the walls of the test container): Not reported
- Blank controls oxygen uptake rate: The specific respiration rate of the controls was 30.33 mg oxygen per gram dry weight of sludge per hour.
- Coefficient of variation of oxygen uptake rate in control replicates: The coefficient of variation of oxygen uptake in the control vessels was 6.5%
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: The EC50 value (3-Hour contact time) for the reference item was 7.2 mg/L.
Reported statistics and error estimates:
The following computerized system was used in the study: Shimadzu TOC; TOC measurement.
Range-finding results: No statistically significant toxic effects were shown at the test concentration of 10 mg/L, however statistically significant toxic effects were shown at the test concentrations of 100 and 1000 mg/L.
Definitive test: No statistically significant toxic effects were shown at the test concentrations of 10 and 32 mg/L, however statistically significant toxic effects (P < 0.05) were shown at the test concentration of 100 mg/L.

Table 1. Inhibition respiration rate with reference item

 

3,5-Dichlorophenol

ECx (3h)

(mg/L)

95% Confidence Limits (mg/L)

EC10

1.4

-

EC20

2.1

-

EC50

7.2

5.5 – 9.4

EC80

25

-

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.

Table 2. 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 (min)

Final O2 reading (mg O2/L)

O2 consumption rates (mg O2/L/hour)

Inhibition (%)

Control

R1

2.6

1

1.8

48.00

-

R2

4.0

2

2.5

45.00

-

R3

4.0

3

1.7

46.00

-

R4

3.8

2

2.2

48.00

-

Test item

10

3.1

1

2.3

48.00

[3]

 

100

4.3

4

1.7

39.00

17

1000 R1

5.0

5

2.0

36.00

23

1000 R2

4.2

3

2.3

38.00

19

1000 R3

5.0

5

2.1

34.80

26

3,5 Dichlorophenol

3.2

5.4

3

3.7

34.00

27

10

5.9

8

3.3

19.5

58

32

7.7

10

6.8

5.40

88

R = Replicate

- = N/A

[] = Increase in respiration rate compared to the controls

Table 3. Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours Contact Time in the Definitive Test

Nominal concentration (mg/L)

Initial O2 reading (mg O2/L)

Measurement period (min)

Final O2 reading (mg O2/L)

O2 consumption rates (mg O2/L/hour)

Inhibition (%)

Control

R1

3.1

2

1.5

48.00

-

R2

3.7

3

1.5

44.00

-

R3

3.7

2

2.2

45.00

-

R4

4.1

3

1.6

50.00

-

R5

4.8

4

2.0

42.00

-

R6

3.9

3

1.7

44.00

-

Test item

10 R1

3.5

2

2.0

45.00

1

10 R2

3.8

2

2.2

48.00

[5]

10 R3

3.0

2

1.5

45.00

1

10 R4

4.5

3

2.2

46.00

[1]

10 R5

3.8

2

2.2

48.00

[5]

32 R1

4.0

3

1.9

42.00

8

32 R2

4.9

4

2.1

42.00

8

32 R3

3.5

2

2.1

42.00

8

32 R4

4.5

3

2.2

46.00

[1]

32 R5

4.6

4

1.6

45.00

1

100 R1

4.7

4

2.1

39.00

14

100 R2

5.0

5

1.8

38.40

16

100 R3

6.0

7

1.9

35.14

23

 

100 R4

6.0

7

1.9

35.14

23

 

100 R5

5.1

5

1.9

38.40

16

3,5 Dichlorophenol

3.2

5.2

6

2.1

31.00

32

10

5.9

9

2.8

20.67

55

32

7.5

10

6.6

5.40

88

R = Replicate

- = N/A

[] = Increase in respiration rate compared to the controls

Validity criteria fulfilled:
yes
Conclusions:
No statistically significant toxic effects were shown at the test concentrations of 10 and 32 mg/L, however statistically significant toxic effects (P < 0.05) were shown at the test concentration of 100 mg/L. Based on these results the NOEC after 3 hours exposure was 32 mg/L.


Executive summary:

A study was performed on the test item, in accordance with OECD 209 (2010) in order to determine the effects on the test item on respiration of activated sewage sludge.

A preliminary range-finding test was conducted at nominal concentrations of 10, 100 and 1000 mg/L, alongside a control group. No statistically significant toxic effects were shown at the test concentration of 10 mg/L, however statistically significant toxic effects were shown at the test concentrations of 100 and 1000 mg/L. Based on the range-finding results, the definitive test concentrations used were 10, 32 and 100 mg/L, alongside a control group. The definitive test was deemed necessary in order to obtain a NOEC for the test item. The exposure duration of both tests was 3 hours, at a temperature between 21 and 22 ºC with the addition of a synthetic sewage respiratory substrate. A known inhibitor (3-5-dichlorophenol) was used as reference item.

No statistically significant toxic effects were shown at the test concentrations of 10 and 32 mg/L, however statistically significant toxic effects (P < 0.05) were shown at the test concentration of 100 mg/L. Based on these results the NOEC after 3 hours exposure was 32 mg/L. All validity criteria were satisfied for this study.

Description of key information

3 h NOEC: 32 mg/L; OECD 209; Best, N. (2018).

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
32 mg/L

Additional information

A study was performed on the test item, in accordance with OECD 209 (2010) in order to determine the effects on the test item on respiration of activated sewage sludge.

All validity criteria were satisifed in the study and thus can be used as a key study to support this endpoint. The test item was specifically assessed for its inhibitory effect at various concentrations, on micro-organisms from a plant primarily treating domestic sewage, through analysis of respiration rates.

A preliminary range-finding test was conducted at nominal concentrations of 10, 100 and 1000 mg/L, alongside a control group. No statistically significant toxic effects were shown at the test concentration of 10 mg/L, however statistically significant toxic effects were shown at the test concentrations of 100 and 1000 mg/L. Based on the range-finding results, the definitive test concentrations used were 10, 32 and 100 mg/L, alongside a control group.

In the definitive test, no statistically significant toxic effects were shown at the test concentrations of 10 and 32 mg/L, however statistically significant toxic effects (P < 0.05) were shown at the test concentration of 100 mg/L. Based on these results the NOEC after 3 hours exposure was 32 mg/L.