Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
9th December 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted 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. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
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)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Sampling and analysis

Analytical monitoring:
no
Details on sampling:
- Concentrations:
test concentrations of 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 material vessels at 0 hours were made prior to addition of activated sewage sludge and synthetic sewage. The pH of the control, reference material and test material preparations were measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter at 0 hours and prior to measurement of the oxygen consumption rate after 30 minutes contact time.

- Sample storage conditions before analysis:
not specified in report

Test solutions

Vehicle:
no
Details on test solutions:

For the purpose of the test, the test material was dispersed directly in water.
Amounts of test material (5, 16, 50, 160 and 500 mg) were each separately added to synthetic sewage (16 ml), activated sewage sludge (200 ml) and water to a final volume of 500 ml to give the required concentrations of 10, 32, 100, 320 and 1000 mg/l.
The control group was maintained under identical conditions but not exposed to the test material.


- Eluate:
At time "0" 16 ml of synthetic sewage was diluted to 300 ml with water and 200 ml of inoculum added in a 1 litre beaker (first control). The mixture was stirred via magnetic stirring. Thereafter, at 15 minute intervals the procedure was repeated with appropriate amounts of the reference material being added. The test material vessels were prepared as described above. Finally a second control was prepared.

As each vessel reached 30 minutes contact time an aliquot was removed from the beaker 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 vessel was measured over the linear portion of the oxygen consumption trace (where possible between approximately 6.5 mg O2/l and 2.5 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.

The test was conducted under normal laboratory lighting in a temperature controlled room at 21±1 ºC
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 and synthetic sewage. The pH of the control, reference material and test material preparations were measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter at 0 hours and prior to measurement of the oxygen consumption rate after 30 minutes contact time.

- Differential loading:
Not applicable.

- Controls:
The control group was maintained under identical conditions but not exposed to the test material.

- Chemical name of vehicle (organic solvent, emulsifier or dispersant):
Not applicable.

- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)):
Not applicable.

- Evidence of undissolved material (e.g. precipitate, surface film, etc):
Observations made at 0 hours (see Table 3 in overall remarks and attachements) prior to the addition of activated sewage sludge and synthetic sewage showed that the test concentrations of 10 and 32 mg/l contained a clear colourless water column with a few small globules of test material visible on the surface. For the test concentration of 100 mg/l an oily layer of test material was visible on the surface. The test concentration of 320 mg/l had a slight oily layer with a few globules of test material visible. An oily layer and globules of test material were visible on the surface of the 1000 mg/l test vessel.
Observations of the dosed activated sludge mixtures made after 30 minutes contact time showed the test concentrations of 10 and 32 mg/l to contain a dark brown dispersion with no undissolved test material. The test concentrations of 100, 320 and 1000 mg/l contained dark brown dispersions with a few small globules of test material visible on the surface. These observations confirm that there was not a substantial loss of test material (via volatilization) following 30 minutes of stirring/suspension in the activated sludge test mixtures.

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Test Species
A mixed population of activated sewage sludge micro-organisms was obtained on
9 December 2009 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
The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21ºC and was used on the day of collection. The pH of the sample was 7.4 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 deionised reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105ºC 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 4.2 g/l prior to use.


Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
30 min
Post exposure observation period:
Not applicable.

Test conditions

Hardness:
Test Water
The test water used for the definitive test was laboratory 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 140 mg/l as CaCO3. After dechlorination and softening the water was then passed through a series of computer controlled plate heat exchangers to achieve the required temperature. Typical water quality characteristics for the tap water as supplied, prior to dechlorination and softening, are given in Appendix 1 (attached ).
Test temperature:
The test was conducted under normal laboratory lighting in a temperature controlled room at 21±1 Deg C.
pH:
The pH values of the test preparations at the start and end of the exposure period are given in Table 2 in overall remarks and attachments.
Dissolved oxygen:
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.

The relatively low initial oxygen reading observed at the highest concentration (1000 mg/l) was considered to be due to the insoluble test material coating the membrane of the oxygen probe. The EC50 value for the test material was therefore taken as being greater than 320 mg/l due to the extremely low start and finish oxygen readings taken over just one minute for the 1000 mg/l test vessel.
The relatively large increase in the respiration rate observed in the 320 mg/l test vessel, see Table 1 in any other information on results, was considered to be due to the insoluble material coating the membrane of the oxygen probe.
Salinity:
Freshwater used
Nominal and measured concentrations:
10, 32, 100, 320 and 1000 mg/l
Details on test conditions:

TEST SYSTEM
Preparation of test system
At time "0" 16 ml of synthetic sewage was diluted to 300 ml with water and 200 ml of inoculum added in a 1 litre beaker (first control). The mixture was stirred via magnetic stirring. Thereafter, at 15 minute intervals the procedure was repeated with appropriate amounts of the reference material being added. The test material vessels were prepared as described in the section on details on test solutions. Finally a second control was prepared.
As each vessel reached 30 minutes contact time an aliquot was removed from the beaker 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 vessel was measured over the linear portion of the oxygen consumption trace (where possible between approximately 6.5 mg O2/l and 2.5 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.
The test was conducted under normal laboratory lighting in a temperature controlled room at 21 ± 1ºC.
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 and synthetic sewage. The pH of the control, reference material and test material preparations were measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter at 0 hours and prior to measurement of the oxygen consumption rate after 30 minutes contact time.

TEST MEDIUM / WATER PARAMETERS
The test water used for the definitive test was laboratory 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 140 mg/l as CaCO3. After dechlorination and softening the water was then passed through a series of computer controlled plate heat exchangers to achieve the required temperature. Typical water quality characteristics for the tap water as supplied, prior to dechlorination and softening, are given in Appendix 1 attached.


OTHER TEST CONDITIONS

- Photoperiod:
30 minutes

- Light intensity:
Normal laboratory lighting.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
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 – 2 RS ] x 100
RC1 + RC2
where
RS = oxygen consumption rate for test or reference sample
RC1 + RC2 = oxygen consumption rates for controls 1 and 2

The percentage inhibition values were plotted against concentration, a line fitted using the Xlfit software package (IDBS) and the EC15, EC20, EC50 and EC80 values (for the reference material only) determined from the equation for the fitted line.
The EC80 value for the test material was determined by inspection of the inhibition of respiration rate data.
95% confidence limits were calculated for the reference material EC50 values using the method of Litchfield and Wilcoxon (Litchfield and Wilcoxon 1949).
Whilst the Test Guidelines do not specify procedures for determination of a No Observed Effect Concentration (NOEC), a NOEC was determined which is equivalent to the EC15 value determined from an inhibition response curve and percentage inhibition values for the test material.
The results of the study are considered valid if (i) the two control respiration rates are within 15% of each other and (ii) the EC50 (30-minute contact time) for 3,5-dichlorophenol lies within the range 5 to 30 mg/l.



TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2

- Justification for using less concentrations than requested by guideline:
Na

Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol

Results and discussion

Effect concentrationsopen allclose all
Duration:
30 min
Dose descriptor:
EC50
Effect conc.:
> 320 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: not specified
Duration:
30 min
Dose descriptor:
NOEC
Effect conc.:
320 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: not specified
Details on results:

Definitive Test
Oxygen consumption rates and percentage inhibition values for the control, test and reference items are given in Table 1 in any other information on results section. Percentage inhibition is plotted against concentration for the reference material (See Figure 1 in overall remarks and attachments section).

The following results were derived from the described experimental procedure. Whereas some loss of the volatile test material components may have occurred during preparation and incubation of the test mixtures, a similar degree of loss of these materials could be expected upon their discharge to and treatment within a typical activated sludge wastewater treatment plant. Therefore, the results are expected to give a reasonable approximation of the potential effects of the test material on operation of such wastewater treatment processes.


The following results were derived:
Reaction Products of C3 alcohols and C3 alkenes

ECx (30 Minutes) 95% Confidence Limits (mg/l)
(mg/l)
EC20 >320 -
EC50 >320 -
EC80 >320 -
NOEC 320 -



3,5-dichlorophenol:
ECx (30 Minutes) (mg/l) 95% Confidence Limits (mg/l)
EC20 3.2 -
EC50 9.8 8.0 - 12
EC80 31 -

Variation in respiration rates of controls 1 and 2 was ± 5% after 30 minutes a contact time.
The validation criteria for the control respiration rates and reference material EC50 values were therefore satisfied.




Results with reference substance (positive control):

- Results with reference substance valid?
Yes.

- Relevant effect levels:
The reference material gave a 3-Hour EC50 value of 11 mg/l, 95% confidence limits 9.0 - 13 mg/l.

- Other:
None.
Reported statistics and error estimates:
None.

Any other information on results incl. tables

Table1              Oxygen Consumption Rates and Percentage Inhibition Values after 30 Minutes Contact Time

 

Nominal

Concentration

(mg/l)

Initial O2

Reading

(mg O2/l)

Measurent Period

(minutes)

Final O2Reading

(mg O2/l)

O2Consumption Rates

(mg O2/l/min)

% Inhibition

Control

R1

6.2

7

1.8

0.63

-

 

R2

5.0

4

2.2

0.70

-

Test Material

10

5.5

5

2.1

0.68

[2]

 

32

4.1

3

2.0

0.70

[5]

 

100

5.7

6

1.8

0.65

2

 

320

6.1

5

1.8

0.87

[31]

 

1000

1.7

1

0.9

*

*

3,5-dichlorophenol

3.2

4.7

5

2.0

0.54

19

 

10

7.7

10

4.6

0.31

53

 

32

7.9

10

6.6

0.13

80


[Increase in respiration rate as compared to controls]

R1– R2= Replicates 1 to 2

*Values not calculated due to very low start and finish oxygen concentrations

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

Nominal

Concentration

(mg/l)

pH

0 Hours

30 Minutes

Control

R1

7.6

7.7

 

R2

7.8

7.8

Test Material

10

7.7

7.9

 

32

7.7

7.7

 

100

7.8

7.8

 

320

7.8

7.8

 

1000

7.8

7.7

3,5-dichlorophenol

3.2

7.5

7.7

 

10

7.6

7.9

 

32

7.7

7.9


R1– R2= Replicates 1 to 2


Table3              Observations on the Test Preparations Throughout the Test Period

Nominal

Concentration

(mg/l)

Observations on Test Preparations

0 Hours Contact Time

30 Minutes

Contact Time

Control

R1

Dark brown dispersion

Dark brown dispersion

 

R2

Dark brown dispersion

Dark brown dispersion

Test Material

10

Few small globules of test material visible on surface of a clear colourless water column*

Dark brown dispersion, no undissolved test material visible

 

32

Few small globules of test material visible on surface of a clear colourless water column *

Dark brown dispersion, no undissolved test material visible

 

100

Slight oily layer of test material visible on surface of a clear colourless water column *

Few small globules of test material on the surface of a dark brown dispersion

 

320

Slight oily layer with a few globules of test material visible on surface of a clear colourless water column *

Few small globules of test material on the surface of a dark brown dispersion

 

1000

Oily layer and globules of test material visible on surface of a clear colourless water column *

Few small globules of test material on the surface of a dark brown dispersion

3,5-dichlorophenol

3.2

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

 

32

Dark brown dispersion, no undissolved reference material visible

Dark brown dispersion, no undissolved reference material visible


R1– R2= Replicates 1 to 2

*Observations made prior to addition of activated sewage sludge and synthetic sewage

Concentration-Response Curve: 3,5-dichlorophenol – 30 Minutes Contact Time see attached figure 1

See atached Appendix1 For Typical Water Quality Characteristics


Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test material on the respiration of activated sewage sludge micro-organisms gave a 30-minute EC50 of greater than 320 mg/l. The No Observed Effect Concentration (NOEC) after 30 minutes exposure was 320 mg/l.
Executive summary:

Introduction.

A study was perford 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 Commission Regulation (EC) No. 440/2008 and US EPA Draft Ecological Effects Test Guidelines OPPTS 850.6800.

Methods. 

Due to the volatile nature of the test material, the test duration was reduced from 3 hours, as specified in the Test Guidelines, to 30 minutes as it was considered likely that a longer test duration would result in significant losses of test material from the test system. In order to minimise any losses of test material from the test system, rather than vigorous aeration of the test vessels, the test preparations were kept in suspension by stirring via magnetic stirrers. 

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 30 minutes 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 30 minutes contact tiand 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 30-Minute EC50 of greater than 320 mg/l. 

The EC50 value for the test material was taken as being greater than 320 mg/l and not 1000 mg/l. This was due to the extremely low start and finish oxygen reading taken over just one minute for the 1000 mg/l test vessel which was considered to be due to the insoluble test material coating the membrane of the oxygen probe. 

Whilst the Test Guidelines do not specify procedures for determination of a No Observed Effect Concentration (NOEC), a NOEC was determined which is equivalent to the EC15 value determined from an inhibition response curve and percentage inhibition values for the test material. The No Observed Effect Concentration (NOEC) after 30 minutes exposure was greater than 320 mg/l.

The reference material gave a 30-minute EC50value of 9.8 mg/l, 95% confidence limits 7.6 - 13 mg/l.

CONCLUSION:

The effect of the test material on the respiration of activated sewage sludge gave a 30-Minute EC50 of greater than 320 mg/l. The No Observed Effect Concentration (NOEC) after 30 minutes exposure was 320 mg/l.