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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 March 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date of GLP inspection: 19-21 July 2011, Date of Signature on GLP certificate: 31 August 2011
Analytical monitoring:
no
Details on sampling:
- Concentrations:
10, 32, 100, 320 and 1000 mg/l

- sampling method:
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. The pH of the control, reference item and test item 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.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
- Eluate:
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 liter 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 item vessels were prepared as described in Section 3.5.1. Finally 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.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC and was used on the day of collection. The pH of the sample was 8.0 and 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 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 one hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 3.0 g/L prior to use.


* rinsed three times with 20 mL deionized 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:
No data.
Test temperature:
The test was conducted in a temperature controlled room at 20±2 ºC.
pH:
8 (at start of test)
8.3-8.5 (at end of test)

See table.
Dissolved oxygen:
Not applicable
Salinity:
Not applicable.
Nominal and measured concentrations:
Range-Finding Test: 10, 100 and 1000 mg/L.
Details on test conditions:
- test water: The test water used for the test was deionized reverse osmosis water containing less than 1 mg/L Dissolved Organic Carbon (DOC).

Range-Finding Test
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. The test item was dissolved directly in water.

An amount of test item (2500 mg) was dissolved in water with the aid of ultrasonication for approximately 5 minutes and the volume adjusted to 1 liter to give a 2500 mg/L stock solution from which dilutions were made to give 250 and 25 mg/L stock solutions. An aliquot (200 mL) of the 25 mg/L stock solution was dispersed with synthetic sewage (16 mL), activated sewage sludge (250 mL) and water, to a final volume of 500 mL, to give the required concentration of 10 mg/L. Similarly, aliquots (200 mL) of the 250 mg/L and 2500 mg/L stock solutions were used to prepare the test concentrations of 100 and 1000 mg/L. The 1000 mg/L test concentration was prepared in triplicate. The volumetric flasks containing the stock solutions were inverted several times to ensure homogeneity of the stock solutions.
The pH of the test item stock solutions were measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter and adjusted to between pH 7.0 to pH 8.0.

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

A reference item, 3,5-dichlorophenol, was included in the range-finding test at concentrations of 3.2, 10 and 32 mg/L in order to confirm the suitability of the inoculum. A stock solution of 0.5 g/L was prepared by dissolving the reference item directly in water with the aid of ultrasonication for approximately 10 minutes. The pH of this stock solution was measured to be pH 6.2 and adjusted to pH 7.2 using 1.0 M NaOH. The pH values were measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter. Aliquots (3.2, 10 and 32 mL) of the stock solution were removed and dispersed with activated sewage sludge (250 mL), synthetic sewage (16 mL) and water to give the final concentrations of 3.2, 10 and 32 mg/L. The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution.

EFFECT PARAMETERS MEASURED
In order to calculate the inhibitory effect of the test and reference items the respiration rate was expressed as a percentage of the two control respiration rates.

The respiration rate, R, expressed in milligrams oxygen per liter 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 = is the oxygen concentration at the beginning of the selected section of the linear phase (mg/L);
Q2 = is the oxygen concentration at the end of the selected section of the linear phase (mg/L);
Δt = is 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/g/h) was deduced according to the following equation:
RS = R / SS
Where:
SS = is 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 = is 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.
The EC20, EC50 and EC80 values for the test item were determined by inspection of the inhibition of respiration rate data.
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).
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.:
> 100 - 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:
Range-Finding Test
Oxygen consumption rates and percentage inhibition values for the control, test and reference items are given in Table 2. The pH values of the test preparations at the start and end of the exposure period are given in Table 3 and observations made on the test preparations throughout the study are given in Table 4. The dissolved oxygen concentrations in all vessels after 30 minutes contact time are given in Table 5.
No statistically significant toxic effects were shown at the test concentrations of 10 and 100 mg/L (one test vessel each), however a statistically significant effect was observed on two out of three replicate vessels at 1000 mg/L. Based on these results and after discussion with the Sponsor, it was considered not necessary to perform a definitive test in order to assess whether the observed effect was due to toxicity or to other respiratory interactions with the tested substance or to estimate more precisely the NOEC.

Percentage inhibition is plotted against concentration for the reference item, 3,5-dichlorophenol (Figure 1).

The effect of the test item 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 between 100 mg/L and 1000 mg/L. It was, therefore, 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 0.02% and the specific respiration rate of the controls was 30.42 mg oxygen per gram dry weight of sludge per hour. The validation criteria have therefore been satisfied.
The validation criterion for the reference item EC50 value was also satisfied.
In some instances, the initial and final dissolved oxygen concentrations were below those recommended in the test guidelines (7.0 mg O2/L and 2.0 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.
The dissolved oxygen concentrations after 30 minutes contact time confirm that the dissolved oxygen concentrations 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 replicates R1 and R2 showing 57% and 55% saturation respectively and the test item 1000 mg/L replicate R2 vessel showing 51% 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.
It is not known what caused the lower inhibition value in the 1000 mg/L replicate R2 test vessel compared to the 1000 mg/L test vessels replicates R1 and R3, however the results from the 1000 mg/L test vessel replicates R1 and R3 showed statistically significant toxic effects compared to the control vessels.

Table 1. pH Values of the Test Item Stock Solutions prior to the Addition of Inoculum in the Range-Finding Test Nominal Concentration (mg/L)

 

Nominal concentration test item (mg/l)

pH*

Prior to adjustment

After adjustment

25

7.9

-

250

7.6

-

2500

8.5

7.3

 

* Measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter

- No adjustment necessary 

 

Table 2. Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours Contact Time in the Range-Finding Test

 

Nominal Concentration (mg/L)

Initial O2Reading

(mg O2/L)

Measurement Period (minutes)

Final O2Reading

(mg O2/L)

O2Consumption Rates (mg O2/L/hour)

% Inhibition

Control                     R1

R2

R3

R4

5.0

4

1.9

46.50

-

4.8

4

1.8

45.00

-

4.0

3

1.7

46.00

-

3.1

2

1.6

45.00

-

Test item                  10

100

1000 R1

1000 R2

1000 R3

4.3

3

2.1

44.00

4

4.5

4

1.7

42.00

8

3.9

3

2.0

38.00

17

4.2

3

2.0

44.00

4

4.5

4

1.8

40.50

11

Positive control        3.2

10

32

4.2

4

1.5

40.50

11

6.0

8

2.3

27.75

39

7.2

8

5.7

11.25

75

 

R1-R4 = Replicates 1-4

 

Table 3. pHValues of the Test Preparations at the Start and End of the Exposure Period in the Range-finding Test

 

Nominal Concentration (mg/L)

Initial O2Reading

(mg O2/L)

Measurement Period (minutes)

0 hours

3 hours

Control                     R1

R2

R3

R4

8.0

8.5

8.0

8.5

8.0

8.4

8.0

8.4

Test item                  10

100

1000 R1

1000 R2

1000 R3

8.0

8.5

8.0

8.4

8.0

8.4

8.0

8.3

8.0

8.4

Positive control        3.2

10

32

8.0

8.5

8.0

8.6

8.0

8.6

 

R1-R4 = Replicates 1-4

 

 

Table 4.Observations on the Test Preparations throughout the Test Period in the Range-Finding Test

 

 

Nominal Concentration (mg/L)

Observations on Test Preparations

0 hours*

30 minutes

Contact Time

3 hours

Contact Time

Control                     R1

 

R2

 

R3

 

R4

Pale yellow/brown dispersion

Dark brown dispersion

Dark brown dispersion

Pale yellow/brown dispersion

Dark brown dispersion

Dark brown dispersion

Pale yellow/brown dispersion

Dark brown dispersion

Dark brown dispersion

Pale yellow/brown dispersion

Dark brown dispersion

Dark brown dispersion

Test item                  10

 

 

 

 

100

 

 

 

1000 R1

 

 

 

1000 R2

 

 

 

1000 R3

Pale yellow/brown dispersion, no un-dissolved test item visible

Dark brown dispersion, no un-dissolved test item visible

Dark brown dispersion, no un-dissolved test item visible

Pale yellow/brown dispersion, no un-dissolved test item visible

Dark brown dispersion, no un-dissolved test item visible

Dark brown dispersion, no un-dissolved test item visible

Pale yellow/brown dispersion, no un-dissolved test item visible

Dark brown dispersion, no un-dissolved test item visible

Dark brown dispersion, no un-dissolved test item visible

Pale yellow/brown dispersion, no un-dissolved test item visible

Dark brown dispersion, no un-dissolved test item visible

Dark brown dispersion, no un-dissolved test item visible

Pale yellow/brown dispersion, no un-dissolved test item visible

Dark brown dispersion, no un-dissolved test item visible

Dark brown dispersion, no un-dissolved test item visible

Positive control        3.2

10

32

Pale yellow/brown dispersion, no un-dissolved reference item visible

Dark brown dispersion, no un-dissolved test item visible

Dark brown dispersion, no un-dissolved test item visible

Pale yellow/brown dispersion, no un-dissolved reference item visible

Dark brown dispersion, no un-dissolved test item visible

Dark brown dispersion, no un-dissolved test item visible

Pale yellow/brown dispersion, no un-dissolved reference item visible

Dark brown dispersion, no un-dissolved test item visible

Dark brown dispersion, no un-dissolved test item visible

 

*Observations made prior to the addition of activated sewage sludge

R1 – R4 = Replicates 1 to 4

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item 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 between 100 mg/L and 1000 mg/L. No statistically significant toxic effects were shown at the test concentrations of 10 and 100 mg/L (one test vessel each), however a statistically significant effect was observed on two out of three replicate vessels at 1000 mg/L. Based on these results and after discussion with the Sponsor, it was considered not necessary to perform a definitive test in order to assess whether the observed effect was due to toxicity or to other respiratory interactions with the tested substance or to estimate more precisely the NOEC.


This study is considered to be of sufficient adequacy and reliability to be used as a key study in accordance with Regulation (EC) No. 1907/2006 (REACH) and is considered to be sufficient for classification and labelling in accordance with Regulation (EC) No 1272/2008 (EU CLP). Therefore, no further testing is justified.

Description of key information

One key study exists. This study had been conducted in accordance with a recommended guideline (OECD 209) and under the conditions of GLP. As such, no further testing is considered necessary.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L

Additional information

The effect of the test item 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 between 100 mg/L and 1000 mg/L.