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Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 - 22 Jan 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
insoluble test material was visible during the test.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom, date of inspection 21 Aug 2007
Test material information:
Composition 1
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test material was dispersed directly in water. 5, 16, 50, 160 and 500 mg of test material were each separately dispersed in approx. 250 mL of water and subjected to ultrasonication for approx. 15 min followed by mixing for 24 h. All test vessels were shielded from light during mixing. Synthetic sewage (16 mL), activated sewage sludge (200 mL) and water were added to a final volume of 500 mL to give the required concentrations of test item.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source of inoculum: Activated sludge was obtained on 22 Jan 2009 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, UK, which treats predominantly domestic sewage.
- Preparation of inoculum for exposure: The sludge sample was maintained on continuous aeration in the laboratory at 21 °C and was used on the day of collection.
- Suspended solids concentration: 3.6 g/L
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Hardness:
140 mg/L as CaCO3
Test temperature:
21 ± 1 °C
pH:
7.7 - 8.1
Nominal and measured concentrations:
Nominal: 0, 10, 32, 100, 320, 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: conical flasks
- Fill volume: 500 mL
- Aeration: 0.5 - 1 L clean, oil-free air per minute
- Amount of sludge per vessel: 200 mL
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Laboratory tap water was dechlorinated and partly softened before use.
- Total organic carbon: 1.041 mg/L
- Pesticides: < 0.001 to < 0.005 µg/L
- Chlorine: 0.297 mg/L
- Conductivity: 421 µS/cm
- Intervals of water quality measurement: The pH was measured at test start and prior to measurement of the oxygen consumption after 3 h contact time.

OTHER TEST CONDITIONS
- Photoperiod: normal laboratory lighting

EFFECT PARAMETERS MEASURED (with observation intervals if applicable)
- Observations were made on the test preparations throughout the test period.
- After 3 h contact time, an aliquot was removed from the conical flasks for the measurement of oxygen consumption.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
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
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
230 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Observations made at 0 hours (see Table 3) prior to the addition of activated sewage sludge and synthetic sewage showed that the test concentrations of 10, 32 and 100 mg/L contained slightly cloudy dispersions with small globules of test material dispersed throughout. The test concentrations of 320 and 1000 mg/L contained cloudy dispersions with an oily layer of test material on the surface and small globules dispersed throughout.
Observations made after 30 minutes and 3 hours contact time showed that the test concentrations of 10, 32 and 100 mg/L contained dark brown dispersions with small globules of test material dispersed throughout. The test concentrations of 320 and 1000 mg/L contained dark brown dispersions with an oily layer of test material on the surface and small globules dispersed throughout.
- In some instances, the initial and final dissolved oxygen concentrations were below those recommended in the test guidelines (6.5 and 2.5 mg O2/L, respectively).
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50 (3 h) 9.2 mg/L, 95% CL = 7.2 - 12 mg/L
Reported statistics and error estimates:
The percentage inhibition values were plotted against concentration, a line fitted using the Xlfit software (IDBS) and the EC15 and EC20 values for the test material and the EC15, EC20, EC50 and EC80 values for the reference material determined from the equation of the fitted line. The EC50 and EC80 values for the test material were determined by inspection of the inhibition of respiration rate data. 95% confidence limits were calculated for the reference material EC50 values only. The NOEC was taken as being the EC15 value.

 Definitive Test:

Oxygen consumption rates and percentage inhibition values for the control, test and reference materials are given in Table 1. The pH values of the test preparations at the start and end of the exposure period are given in Table 2, and observations made on the test preparations throughout the study are given in Table 3.

Percentage inhibition is plotted against concentration for the test and reference materials (Figures 1 and 2) (see attachment 1).

The following results were derived:

 

Mixture of PEMB and PE3MB

3,5-dichlorophenol

ECx(3 Hours)
(mg/L)

95% Confidence Limits (mg/L)

ECx(3 Hours)
(mg/L)

95% Confidence Limits (mg/L)

EC20

  690

-

    3.1

-

EC50

>1000

-

    9.2

7.2 - 12

EC80

>1000

-

27

-

NOEC

  230

-

-

-

It was not possible to obtain EC50or EC80values for the test material as no concentration tested resulted in greater than 50% inhibition.

It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L. Variation in respiration rates of controls 1 and 2 after 3 hours contact time was ± 4%.

The validation criteria for the control respiration rates and reference material EC50values were therefore satisfied.

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

Observations made at 0 hours (see Table 3) prior to the addition of activated sewage sludge and synthetic sewage showed that the test concentrations of 10, 32 and 100 mg/L contained slightly cloudy dispersion with small globules of test material dispersed throughout. The test concentrations of 320 and 1000 mg/L contained cloudy dispersions with an oily layer of test material on the surface and small globules dispersed throughout.

Observations made after 30 minutes and 3 hours contact time showed that the test concentrations of 10, 32 and 100 mg/L contained dark brown dispersions with small globules of test material dispersed throughout. The test concentrations of 320 and 1000 mg/L contained dark brown dispersions with an oily layer of test material on the surface and small globules dispersed throughout.

Table1              Oxygen Consumption Rates and Percentage Inhibition Values in the Definitive Test after 3 Hours Contact Time

Nominal

Concentration

(mg/l)

Initial O2

Reading

(mg O2/L)

Measurement Period

(minutes)

Final O2Reading

(mg O2/L)

O2Consumption Rates

(mg O2/L/min)

% Inhibition

Control

R1

6.3

7

2.8

0.50

-

 

R2

3.9

5

1.6

0.46

-

Test Material

10

6.4

7

2.9

0.50

[4]

 

32

4.7

5

2.4

0.46

4

 

100

3.8

5

1.7

0.42

13

 

320

4.1

5

2.0

0.42

13

 

1000

5.7

9

2.3

0.37

23

3,5-dichlorophenol

3.2

7.1

10

3.2

0.39

19

 

10

7.6

10

5.5

0.21

56

 

32

8.2

10

7.4

0.08

83


[Increase in respiration rate as compared to controls]

R1– R2= Replicates 1 to 2

Table2              pH Values of the Test Preparations at the Start and End of the Exposure Period

Nominal

Concentration

(mg/L)

pH

0 Hours

3 Hours

Control

R1

7.7

7.9

 

R2

7.7

7.8

Test Material

10

7.8

7.8

 

32

7.8

7.9

 

100

7.8

7.9

 

320

7.8

7.8

 

1000

7.7

7.8

3,5-dichlorophenol

3.2

7.7

7.9

 

10

7.8

8.0

 

32

7.8

8.1


R1– R2= Replicates 1 to 2

Table 3:              Observations on the Test Preparations Throughout the Test Period

Nominal

Concentration

(mg/L)

Observations on Test Preparations

0 Hours

30 Minutes

Contact Time

3 Hours

Contact Time

Control

R1

Dark brown dispersion

Dark brown dispersion

Dark brown dispersion

 

R2

Dark brown dispersion

Dark brown dispersion

Dark brown dispersion

Test Material

10

Slightly cloudy dispersion, small globules of test material dispersed throughout[1]

Dark brown dispersion, small globules of test material dispersed throughout

Dark brown dispersion, small globules of test material dispersed throughout

 

32

Slightly cloudy dispersion, small globules of test material dispersed throughout*

Dark brown dispersion, small globules of test material dispersed throughout

Dark brown dispersion, small globules of test material dispersed throughout

 

100

Slightly cloudy dispersion, small globules of test material dispersed throughout*

Dark brown dispersion, small globules of test material dispersed throughout

Dark brown dispersion, small globules of test material dispersed throughout

 

320

Cloudy dispersion, oily layer of test material on surface and small globules dispersed throughout*

Dark brown dispersion, oily layer of test material on surface and small globules dispersed throughout

Dark brown dispersion, oily layer of test material on surface and small globules dispersed throughout

 

1000

Cloudy dispersion, oily layer of test material on surface and small globules dispersed throughout*

Dark brown dispersion, oily layer of test material on surface and small globules dispersed throughout

Dark brown dispersion, oily layer of test material on surface and small globules dispersed throughout

3,5-dichlorophenol

3.2

Dark brown dispersion, no undissolved reference material visible

Dark brown dispersion, no undissolved reference material visible

Dark brown dispersion, no undissolved reference material visible

 

10

Dark brown dispersion, no undissolved reference material visible

Dark brown dispersion, no undissolved reference material visible

Dark brown dispersion, no undissolved reference material visible

 

32

Dark brown dispersion, no undissolved reference material visible

Dark brown dispersion, no undissolved reference material visible

Dark brown dispersion, no undissolved reference material visible


R1– R2= Replicates 1 to 2

[*]Observations prior to addition of activated sewage sludge and synthetic sewage.

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test material on the respiration of activated sewage sludge micro-organisms gave an EC50 (3 h) > 1000 mg/L. The NOEC (3 h) was 230 mg/L.
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", Method C.11 of Council Regulation (EC) No. 440/2008 and US EPA Draft Ecological Effects Test Guidelines OPPTS 850.6800.

Methods:

Activated sewage sludge was exposed to an aqueous dispersion of the test material at concentrations of 10, 32, 100, 320 and 1000 mg/L for a period of 3 hours at a temperature of approximately 21 °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 material, 3,5-dichlorophenol.

Results:

The effect of the test material on the respiration of activated sewage sludge gave a 3‑Hour EC50of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was230mg/L.

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

The reference material gave a 3-Hour EC50 value of 9.2 mg/L, 95% confidence limits 7.2 - 12 mg/L.

Conclusion:

The effect of the test material on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 230 mg/L.

Description of key information

EC50 (3 h) > 1000 mg/L and NOEC = 230 mg/L for activated sludge (OECD 209)

Key value for chemical safety assessment

EC50 or LC50 for microorganisms:
1 000 mg/L
EC10, LC10 or NOEC for microorganisms:
230 mg/L

Additional information

For the toxicity of the test item to microorganisms one key study according to OECD 209 and GLP is available (Showa Denko, 2009). Activated sludge from a sewage treatment plant treating predominantly domestic sewage was exposed to the test material for 3 hours. Based on the measurement of the respiration rate of the sludge samples, the EC50 (3 h) was established as > 1000 mg/L, the highest concentration tested. The NOEC was determined to be 230 mg/L.