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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 08 November to 14 November 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was performed according to internationals guidelines with GLP statement. All validity criteria were fulfilled. This study was assessed as reliable without restriction.
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EEC Commission Diractive 87/302/EEC
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
2nd December 2002
Specific details on test material used for the study:
- Storage condition of test material: Approximately 4 °C under nitrogen in the dark
Analytical monitoring:
not required
Details on sampling:
Not Applicable
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: An amount of test material (500 mg) was dispersed in approximately 250 ml of water and subjected to ultrasonication for approximately 30 minutes. Synthetic sewage (16 ml), activated sewage sludge (200 ml) and water were added to a final volume of 500 ml to give the required concentration of 1000 mg/l. This method of preparation was performed for three separate replicate test vessels.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Observations made throughout the test period showed that at the test concentration of 1000 mg/l, large oily globules of test material were visible on the surface of each vessel at 0 hours, 30 minutes and 3 hours contact time.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- 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 and was used on the day of collection. The pH of the sample was 7.6 measured using a WTW pHiOxi 3401 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 deionised reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105°C for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 4.0 g/l prior to use.
- Initial biomass concentration: 4.0 g/l
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Remarks on exposure duration:
none
Post exposure observation period:
Not Applicable
Hardness:
Test water ca. 100 mg/l as CaCO3
Test temperature:
21 ± 1 °C (performed in a temperature controlled room). Test water and activated sewage sludge were also maintained at this temperature.
pH:
To see results in a Table, please refer to the Table 6.1.7/1 in "Remarks on results incl. tables and figures"
pH measured at the end of exposure period (after 3 hours):
- Controls: 8.05 ± 0.07
- Test Material: 7.8 ± 0.1
- 3,5-dichlorophenol: 8.2 ± 0.1
Dissolved oxygen:
See Table 6.1.7/1 in "Remarks on results incl. tables and figures"
Salinity:
Not Applicable
Nominal and measured concentrations:
Nominal concentrations: 1000 mg/l.
Maesured concentration not available/Not required by the OECD guideline 209
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 ml darkened glass Biological Oxygen Demand (BOD) bottles
- Material, size, headspace, fill volume: Yellow Springs dissolved oxygen meter
- Aeration: yes
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 2


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water (dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened
(Elga Nimbus 1248D Duplex water softener) giving water with a total hardness of approximately 100 mg/l as CaC03).
- Total organic carbon: Not Applicable
- Particulate matter: Not Applicable
- Metals: below the specified limits
- Pesticides: below the specified limits
- Chlorine: dechlorinated water
- Alkalinity: Not required
- Ca/mg ratio: Not Applicable
- Conductivity: Not Applicable/Not required in OECD Guideline 209
- Culture medium different from test medium: Not Applicable
- Intervals of water quality measurement: Not required

OTHER TEST CONDITIONS
- Adjustment of pH: not required

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Oxygen consumption

TEST CONCENTRATIONS
- Spacing factor for test concentrations: a unique concentration of 1000 mg/l was tested
- Justification for using less concentrations than requested by guideline: See below
- Range finding study
- Test concentrations: 100 and 1000 mg/l
- Results used to determine the conditions for the definitive study: No significant effect on respiration was observed at any of the test concentrations employed. A study to determine the General Physico-chemical Properties of the test material (Safepharm Laboratories Project Number 161/439) indicated the water solubility value of the test material to be 0.093 mg/l. Given the extremely low water solubility it was considered that no inhibition
would occur at this level as no significant inhibition was observed in the range-finding test. It was therefore considered justifiable to conduct the definitive test at a single nominal test concentration of 1000 mg/l in excess of the water solubility level of the test material. A single test concentration, in triplicate, of 1000 mg/l was selected for the definitive test.
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
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% CL:Not Applicable
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
Remarks:
respiration rate
Remarks on result:
other: 95% CL:Not Applicable
Details on results:
1000 mg/l of test material did not significantly decrease oxygen consumption rates.
See Table 6.1.7/2 in "Remarks on results including tables and figures"
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Observations made throughout the test period showed that at the test concentration of 1000 mg/l, large oily globules of test material were visible on the surface of each vessel at 0 hours, 30 minutes and 3 hours contact time.
Results with reference substance (positive control):
- Results with reference substance valid? YES
- Relevant effect levels: 3 hours- EC50: 10.0 mg/l
- Other: none
Reported statistics and error estimates:
In order to calculate the inhibitory effect of the test and reference materials the respiration rate was expressed as a percentage of the two control respiration rates.
% inhibition=[1- 2RS/(RC1+RC2)]100 (with RS = O2 consumption rate for test or reference sample and RC1+RC2 = O2 consumption rates for controls 1 and 2).
The percentage inhibition values were plotted against concentration for the reference material only, a line fitted using the Xlfit software package (IDBS 2002) and the EC50 values determined from the equation for the fitted line.
The EC50 values for the test material were determined by inspection of the inhibition of respiration rate data.

Table 6.1.7/1 : pH Values of the Test Preparations at the End of the Exposure

 

Nominal Concentration (mg/l)

pH at the end exposure

Control

R1

R2

 

8.1

8.0

Test Material

1000 R1

1000 R2

1000 R3

 

8.1

8.0

8.0

3,5-dichlorophenol

3.2

10

32

 

8.3

8.3

8.4

 

Table 6.1.7/2: Oxygen Consumption Rates and Percentage Inhibition Values in the Definitive Test after 3 Hours Contact

Nominal Concentration (mg/l)

Initial O2reading

(mg O2/l)

Measurement Period (minutes)

Final O2Reading (mg O2/l)

O2Consumption Rates (mg O2/l/min)

% Inhibition

Control

R1

R2

 

6.4

6.5

 

10

7

 

1.9

2.5

 

0.45

0.43

 

-

-

Test Material

1000 R1

1000 R2

1000 R3

 

3.5

3.3

4.1

 

4

3

4

 

1.8

2.0

2.5

 

0.43

0.43

0.40

 

2

2

9

3,5-dichlorophenol

3.2

10

32

 

 

6.7

7.2

7.7

 

 

10

10

10

 

 

2.9

5.0

6.8

 

 

0.38

0.22

0.09

 

 

14

50

80

(R1-R3 = Replicates)

 

In some instances, the initial and final dissolved oxygen concentrations were below those recommended in the test guidelines (between approx. 6.5 mg O2/1and 2.5 mg02/1). 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.

 

Relatively low initial oxygen readings were observed in the test vessels, see Table 2.

This could possibly be due to insoluble material coating the membrane of the oxygen probe. This was considered not to have had a significant effect on the results of the test as no significant inhibition of respiration was observed in the test vessels throughout the test and the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace. It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/l.

Validity criteria fulfilled:
yes
Remarks:
the two control respiration rates are within 15% of each other and EC50 of 3,5 dichlorophenol is in the accepted range of 5-30 mg/l
Conclusions:
The effect of the test material on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 of greater than 1000 mg/I. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/I.
Executive summary:

Introduction. A study was performed to assess the effect of the test material on the respiration of activated sewage sludge. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 209 "Activated Sludge, Respiration Inhibition Test", EEC Commission Directive87/302/EECand US EPA Draft Ecological Effects Test Guidelines OPPTS 850.6800.

 

Methods. Following a preliminary range-finding test, activated sewage sludge was exposed to an aqueous dispersion of the test material at a concentration of 1000 mg/l (three replicate flasks) for a period of 3 hours at a temperature of 21°C with the addition of a synthetic sewage as a respiratory substrate.

The rate of respiration was determined after 30 minutes and 3 hours contact time and compared to data for the control and a reference material, 3,5-dichlorophenol.

 

Results.The effect of the test material on the respiration of activated sewage sludge gave a 3-Hour EC50 of greater than 1000 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/l. The reference material gave a 3-Hour EC50 value of 10 mg/l.

Description of key information

OECD Guideline 209, EEC Commission Directive 87/302/EEC, US EPA Draft Ecological Effects Test Guidelines OPPTS 850.6800, GLP, key study, validity 1:

3-Hour EC50 > 1000 mg/L; 3-Hour NOEC = 1000 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L
EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

One key study, performed according to OECD Guideline No 209, EEC Commission Directive 87/302/EEC and US EPA Draft Ecological Effects Test Guidelines OPPTS 850.6800 with GLP statement, is available to assess the effect of the test material on the respiration of activated sewage sludge.

Following a preliminary range-finding test, activated sewage sludge was exposed to an aqueous dispersion of the test material at a concentration of 1000 mg/L (three replicate flasks) for a period of 3 hours at a temperature of 21°C with the addition of a synthetic sewage as a respiratory substrate. The rate of respiration was determined after 30 minutes and 3 hours contact time and compared to data for the control and a reference material, 3,5-dichlorophenol.

 The effect of the test material on the respiration of activated sewage sludge gave a 3-Hour EC50 of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L.

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