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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-10-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: 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
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Sponsor's identification: Amine C8
Description: dark brown liquid
Batch number: T7-271109
Purity: 99.65%
Date received: 21 September 2010
Expiry date: 31 December 2011
Storage conditions: room temperature in the dark

Certificate of Analysis given in Appendix 1 (see overall remarks and attachments section)
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 and synthetic sewage. The pH of the control, reference item and test item 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 3 hours contact time.

- Sample storage conditions before analysis:
not specified in report
Vehicle:
no
Details on test solutions:
Method:

Definitive Test:
For the purpose of the test, the test item was dissolved directly in water.
An amount of test item (2000 mg) was dissolved in water with the aid of ultrasonication for approximately 15 minutes and the volume adjusted to 1 litre to give a 2000 mg/l stock solution from which dilutions were made to give 200 and 20 mg/l stock solutions. An aliquot (250 ml) of the 20 mg/l stock solution was dispersed with synthetic sewage (16 ml), activated sewage sludge (200 ml) and water, to a final volume of 500 ml, to give the required concentration of 10 mg/l. Similarly, aliquots (80 and 250 ml) of the 200 and 2000 mg/l stock solutions were used to prepare the test concentrations of 32, 100, 320 and 1000 mg/l. The volumetric flasks containing the stock solutions were inverted several times to ensure homogeneity of the stock solutions.


Eluate:
At time "0" 16 ml of synthetic sewage was diluted to 300 ml with water and 200 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 with appropriate amounts of the reference item being added. The test item vessels were prepared as described above. Finally a second control was 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 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± Deg C.
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 and synthetic sewage. The pH of the control, reference item and test item 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 3 hours contact time.


Differential loading:
Not applicable.

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

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 on the test preparations throughout the study are given in Table 3 (attached in any other information on materials and methods incl. tables section)
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Laboratory culture:
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 7.7 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 3.9 g/l prior to use.

* rinsed three times with 20 ml deionised 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:
The test water used for the 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 2 (attached in overall remarks and attachments section)
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 (attached in any other information on materials and methods incl. tables section)
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.
Salinity:
Not applicable.
Nominal and measured concentrations:
10, 32, 100, 320 and 1000 mg/l
Details on test conditions:
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 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 with appropriate amounts of the reference item being added. The test item vessels were prepared as described in Details of test solutions section. Finally a second control was 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 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 Deg C.
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 and synthetic sewage. The pH of the control, reference item and test item 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 3 hours contact time.


TEST MEDIUM / WATER PARAMETERS
Dechlorinated tap water


OTHER TEST CONDITIONS
Adjustment of pH:
The pH values of the test preparations at the start and end of the exposure period are given in Table 2 (attached in any other information on materials and methods incl. tables section)

Photoperiod:
3 hours.

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 items the respiration rate was expressed as a percentage of the two control respiration rates.
% inhibition = [ 1- 2RS / RC1 + RC2] x 100
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 for the reference item only, a line fitted using the Xlfit software package (IDBS) and the EC15, 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).
The No Observed Effect Concentration (NOEC) was taken as being the EC15 value.
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 (3-Hour contact time) for 3,5-dichlorophenol lies within the range 5 to 30 mg/l.


TEST CONCENTRATIONS
Spacing factor for test concentrations: 3.2


Range finding study
Not Applicable

Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
other: EC20
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: not specified
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: not specified
Duration:
3 h
Dose descriptor:
other: EC80
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: not specified
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: 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 (attached in any other information on materials and methods incl. tables section). The pH values of the test preparations at the start and end of the exposure period are given in Table 2 (attached in any other information on materials and methods incl. tables section) and observations made on the test preparations throughout the study are given in Table 3 (attached in any other information on materials and methods incl. tables section).
Percentage inhibition is plotted against concentration for the reference item (Figure 1, attached in overall remarks and attachments section).
The following results were derived:

Amine C8:
ECx (3 Hours) (mg/l) 95% Confidence Limits (mg/l)
EC20 >1000 -
EC50 >1000 -
EC80 >1000 -
NOEC 1000 -

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

3,5-dichlorophenol:
ECx (3 Hours) (mg/l) 95% Confidence Limits (mg/l)
EC20 2.3 -
EC50 7.0 5.4 - 9.0
EC80 22 -
NOEC 1.9

Variation in respiration rates of controls 1 and 2 after 3 hours contact time was ± 1%.
The validation criteria for the control respiration rates and reference item EC50 values were therefore satisfied.




Results with reference substance (positive control):
Reference item preparation
For the purpose of the test a reference item, 3,5-dichlorophenol (Sigma-Aldrich Batch No. 04621CJ) was used. Two stock solutions of 50 and 160 mg/l were prepared by dissolving the reference item directly in water with the aid of ultrasonication for approximately 20 minutes. Aliquots (10 and 100 ml) of the 160 mg/l stock solution were removed and dispersed with activated sewage sludge, synthetic sewage and water to give the final concentrations of 3.2 and 32 mg/l. Similarly, a 100 ml aliquot of the 50 mg/l stock solution was used to prepare the 10 mg/l concentration. The volumetric flasks containing the reference item were inverted several times to ensure homogeneity of the solutions.


- Results with reference substance valid?
Yes.

- Relevant effect levels:
The reference item gave a 3-Hour EC50 value of 7.0 mg/l, 95% confidence limits 5.4 - 9.0 mg/l.

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

 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.

Table1              Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours 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

6.7

10

2.3

0.44

-

 

R2

6.8

10

2.3

0.45

-

Test Item

10

6.6

10

2.4

0.42

6

 

32

5.8

8

1.9

0.49

[10]

 

100

6.0

9

1.8

0.47

[6]

 

320

5.9

9

2.1

0.42

6

 

1000

5.6

9

1.6

0.44

1

3,5-dichlorophenol

3.2

6.9

10

3.7

0.32

28

 

10

7.8

10

6.1

0.17

62

 

32

8.4

10

7.9

0.05

89


[Increase in respiration rate as compared to controls]

R1¿ R2= Replicates 1 to 2

Table 2              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

7.6

8.1

 

R2

7.7

8.1

Test Item

10

7.7

8.1

 

32

7.8

8.0

 

100

8.0

8.0

 

320

8.6

8.1

 

1000

9.3

8.5

3,5-dichlorophenol

3.2

7.6

8.2

 

10

7.6

8.3

 

32

7.6

8.4

 


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 Item

10

Clear colourless solution, no undissolved test item visible*

Dark brown dispersion, no undissolved test item visible

Dark brown dispersion, no undissolved test item visible

 

32

Extremely pale brown solution, no undissolved test item visible *

Dark brown dispersion, no undissolved test item visible

Dark brown dispersion, no undissolved test item visible

 

100

Extremely pale brown solution, no undissolved test item visible *

Dark brown dispersion, no undissolved test item visible

Dark brown dispersion, no undissolved test item visible

 

320

Pale brown solution, no undissolved test item visible *

Dark brown dispersion, no undissolved test item visible

Dark brown dispersion, no undissolved test item visible

 

1000

Pale brown solution, no undissolved test item visible *

Dark brown dispersion, no undissolved test item visible

Dark brown dispersion, no undissolved test item visible

3,5-dichlorophenol

3.2

Dark brown dispersion, no undissolved reference item visible

Dark brown dispersion, no undissolved reference item visible

Dark brown dispersion, no undissolved reference item visible

 

10

Dark brown dispersion, no undissolved reference item visible

Dark brown dispersion, no undissolved reference item visible

Dark brown dispersion, no undissolved reference item visible

 

32

Dark brown dispersion, no undissolved reference item visible

Dark brown dispersion, no undissolved reference item visible

Dark brown dispersion, no undissolved reference item visible


R1¿ R2= Replicates 1 to 2

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

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 of greater than 1000 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/l.
Executive summary:

Introduction.


A study was performed to assess the effect of the test item 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. 


Activated sewage sludge was exposed to an aqueous solution of the test item at concentrations of 10, 32, 100, 320 and 1000 mg/l for a period of 3 hours at a temperature of 21 ± 1°C with the addition of a synthetic sewage as a respiratory substrate.


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


Results.


The effect of the test item 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 was1000mg/l.


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


The reference item gave a 3-Hour EC50value of 7.0 mg/l, 95% confidence limits
5.4 - 9.0 mg/l.


 


Conclusion


The effect of the test item 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 1000 mg/l.

Description of key information

An activated sludge inhibition test (Clarke, 2010 -  report 41004022) carried out according to the OECD guideline 209 with activated sludge resulted in a 3h-EC50 of >1000 mg/L and a 3h-NOEC of 1000 mg/L. The study is given a Klimisch score of 1, was conducted under GLP and is considered as the key study for endpoint coverage.

Key value for chemical safety assessment

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

Additional information

The study of Clarke (2010 -  report 41004022) was performed to assess the effect of the test item on the respiration of activated sewage sludge. The method followed that described in the OECD guideline 209.


Activated sewage sludge was exposed to an aqueous solution of the test item at concentrations of 10, 32, 100, 320 and 1000 mg/l for a period of 3 hours at a temperature of 21 ± 1°C with the addition of a synthetic sewage as a respiratory substrate.


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


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