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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 May 2012 to 24 May 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP 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.
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
There was an exception to GLP compliance: No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. This exception is considered not to affect the purpose or integrity of the study.
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: 5, 50 and 500 (in triplicate) mg of test material were each separately dispersed in approximately 200 mL of deionised reverse osmosis water and subjected to ultrasonication for approximately 15 minutes and stirred with a magnetic stirrer for 24 hours. All test vessels were shielded from the light during mixing. 16 mL of synthetic sewage, 250 mL of activated sewage sludge and water were added to a final volume of 500 mL to give the required nominal concentrations of 10, 100 and 1000 mg/L.
- Controls: The positive control 3,5-dichlorophenol was prepared as a 0.5 g/L stock solution by dissolving directly into water with the aid of ultrasonication (20 minutes). The pH of this stock solution was measured to be 5.4 and was adjusted to pH 7.0 using 1.0 M NaOH. Aliquots (3.2, 10 and 32 mL) of the stock solution were removed and dispersed with activated sludge (250 mL), synthetic sewage (16 mL) and water to give the final concentrations of 3.2, 10 and 32 mg/L.
A negative control was also included.
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 23 May 2012 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 for exposure: The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 °C overnight prior to use in the test. On the day of collection the activated sewage sludge (10 litres) was fed synthetic sewage sludge (450 mL).
- Suspended solids: 3 g/L
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20 ± 2 °C
pH:
7.0 - 7.3 (after 0 hours exposure); 7.8 - 7.9 (after 3 hours exposure)
Dissolved oxygen:
4.9 - 6.0 mg O₂/L in the test material vessels after 30 minutes of contact time.
Nominal and measured concentrations:
Nominal test concentrations of 10, 100 and 1000 mg/L.
Details on test conditions:
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.

The pH of the synthetic sewage stock used to feed the activated sewage sludge and used for the study was 6.8 and adjusted to 7.0 using 1.0 M NaOH. The pH value was measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter.

Preparation of Test System
At time "0" 16 mL of synthetic sewage was diluted to 250 mL with water and 250 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 for the second control followed by the reference item vessels with appropriate amounts of the reference item being added. The test material vessels were prepared and two further control vessels were 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 7.0 mg O2/L and 2.0 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.

OTHER TEST CONDITIONS
The test was conducted under normal laboratory lighting. The test material was dispersed directly in water. The control group was maintained under identical conditions but not exposed to the test material.


EFFECT PARAMETERS MEASURED
Observations were made on the test preparations throughout the test period. Observations of the test material vessels at 0 hours were made prior to addition of activated sewage sludge. The pH of the control, reference item and test material preparations was measured at 0 hours and prior to measurement of the oxygen consumption rate after 3 hours contact time. The oxygen concentrations in all vessels were measured after 30 minutes contact time.

- Validity of the test:
The results of the study are considered valid if
(i) the EC50 (3-hour contact time) for 3,5-dichlorophenol lies within the range 2 to 25 mg/L
(ii) the specific respiration rate of the blank controls should not be less than 20 mg oxygen per gram dry weight of sludge per hour
(iii) the coefficient of variation of oxygen uptake rate in control replicates should not be more than 30 % at the end of the test.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
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
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
Remarks:
respiration rate
Details on results:
Oxygen consumption rates and percentage inhibition values for the control, test and reference items are given in Table 1.
The dissolved oxygen concentrations after 30 minutes contact time in all vessels were above 60 to 70 % of the dissolved oxygen saturation level of 8.9 mg O2/L with the exception of control replicate R1 showing 47 % saturation and the 10 mg/L test material vessel showing 55 % saturation. This deviation was considered to have had no adverse effect on the study given that all oxygen consumption values were measured/calculated over the linear portion of the traces.

No statistically significant toxic effects were shown at any of the test concentrations employed. It was therefore considered justifiable not to perform a definitive test.

It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.

The coefficient of variation of oxygen uptake in the control vessels was 1.8 % and the specific respiration rate of the controls was 26.60 mg oxygen per gram dry weight of sludge per hour. The validation criteria were therefore satisfied.
Results with reference substance (positive control):
The following results were derived:

3 h EC20 4.0 mg/L
3 h EC50 11 mg/L (95 % Confidence Limits 8.7 - 14 mg/L)
3 h EC80 31 mg/L

The validation criterion for the reference item EC50 value was satisfied.
Reported statistics and error estimates:
Evaluation of Data
The respiration rate, R, expressed in milligrams oxygen per litre per hour (mg O2/L/h), was calculated from the linear part of the recorded oxygen decrease graph according to the following equation:
R = [(Q1 - Q2) / Δt] x 60

Where:
Q1 = the oxygen concentration at the beginning of the selected section of the linear phase (mg/L)
Q2 = the oxygen concentration at the end of the selected section of the linear phase (mg/L)
Δt = the time interval between the beginning and end of the selected section of the linear phase (min)

The specific respiration rate, RS, expressed as the amount of oxygen consumed per gram dry weight of sludge per hour (mg O2/g/h) was deduced according to the following equation:
RS = R / SS

Where:
SS = the concentration of suspended solids in the test mixture (g/L)

The percentage inhibition was calculated according to the following equation:
% inhibition = [1 – (R / Rbc)] x 100

Where:
Rbc = the mean respiration rate of the blank controls.

The percentage inhibition values were plotted against concentration for the reference item only, a line fitted using the Xlfit software package (IDBS) and the EC20, EC50 and EC80 values determined from the equation for the fitted line.
95 % confidence limits were calculated for the reference item EC50 value using the method of Litchfield and Wilcoxon (Litchfield and Wilcoxon, 1949).

One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was carried out on the oxygen consumption data after 3 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Table 1: 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 O2 Reading

(mg O2/L)

O2 Consumption Rates

(mg O2/L/hour)

% Inhibition

Control

R1

4.7

4

2.0

40.50

-

R2

4.8

4

2.2

39.00

-

R3

4.8

5

1.5

39.60

-

R4

4.7

4

2.0

40.50

-

Test Material

10

4.4

3

2.3

42.00

[5]

100

5.2

5

1.7

42.00

[5]

1000R1

4.8

3

2.8

40.00

0

1000R2

5.0

4

2.2

42.00

[5]

1000R3

5.6

5

2.2

40.80

2

Reference Item

3.2

6.0

7

1.9

35.14

12

10

7.3

10

4.0

19.80

50

32

7.9

10

6.5

8.40

79

[Increase in respiration rate as compared to controls]

R1 - R4 = replicates 1 to 4

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the study, the effect of the test material on the respiration of activated sewage sludge micro-organisms gave a 3 hour EC50 value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L.
Executive summary:

The effect of the test material on the respiration of activated sewage sludge was investigated in accordance with the standardised guideline OECD 209.

During the study activated sewage sludge was exposed to an aqueous dispersion of the test material at concentrations of 10, 100 and 1000 mg/L (3 replicates of the 1000 mg/L concentration) for a period of 3 hours at a temperature of 20 ± 2 °C with the addition of a synthetic sewage as a respiratory substrate. 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.

Under the conditions of the test the 3 hour EC50 was determined to be in excess of 1000 mg/L and the NOEC was determined to be 1000 mg/L. It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L. The reference item gave a 3 hour EC50 value of 11 mg/L (95 % confidence limits: 8.7 - 14 mg/L).

Description of key information

EC50 > 1000 mg/L, NOEC = 1000 mg/L, OECD 209, Roulstone 2012

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

The effect of the test material on the respiration of activated sewage sludge was investigated in accordance with the standardised guideline OECD 209.

During the study activated sewage sludge was exposed to an aqueous dispersion of the test material at concentrations of 10, 100 and 1000 mg/L (3 replicates of the 1000 mg/L concentration) for a period of 3 hours at a temperature of 20 ± 2 °C with the addition of a synthetic sewage as a respiratory substrate. 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.

Under the conditions of the test the 3 hour EC50 was determined to be in excess of 1000 mg/L and the NOEC was determined to be 1000 mg/L. It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L. The reference item gave a 3 hour EC50 value of 11 mg/L (95 % confidence limits: 8.7 - 14 mg/L).

The study was performed in accordance with a standardised guideline and in line with GLP. The study was reported to a high standard, sufficient to assess the accuracy of the reported conclusions. The study was therefore assigned a reliability score of 1 in accordance with the criteria as defined by Klimisch (1997) and considered suitable for assessment as an accurate reflection of the test material.