Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Ecotoxicological information

Toxicity to microorganisms

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 2 June 2011 and 3 June 2011.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols/ Current OECD Guidelines and to GLP standards, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Method C.11 of Commission Regulation (EC) No. 440/2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: US EPA Draft Ecological Effects Test Guidelines OPPTS 850.6800
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Details on sampling:
- Concentrations:
10, 32, 100, 320 and 1000 mg/l

- Sampling method:
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.

Vehicle:
no
Details on test solutions:
Test item preparation:
For the purpose of the test, the test item was dispersed directly in water.
Amounts of test item (5, 16, 50, 160 and 500 mg) were each separately dispersed in approximately 250 ml of water and subjected to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 hours in order to maximise the dissolved test item concentration. All test vessels were shielded from the 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 10, 32, 100, 320 and 1000 mg/l.

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

- Reference item preparation:
For the purpose of the test a reference item, 3,5-dichlorophenol 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 volutric flasks containing the reference item were inverted several tis to ensure homogeneity of the solutions.

- Evidence of undissolved material (e.g. precipitate, surface film, etc):
Observations made throughout the test period are given in Table 3 in any other information on results including tables section.
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
3 June 2011 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 7.5 and was 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 3.7 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.
Test temperature:
The test was conducted under normal laboratory lighting in a temperature controlled room at 21±1 Deg C.
pH:
Test item: 7.7 - 8.5
Dissolved oxygen:
Initial O2 reading: 4.8 - 8.7 mgO2/l
Final O2 reading: 2.1 - 8.4 mgO2/l
Salinity:
Not applicable.
Nominal and measured concentrations:
Nominal: 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 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±1DegC.

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 was 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.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference item, 3,5-dichlorophenol.


EVALUATION OF DATA:
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 – 2 RS / 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, 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 EC50 values 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.

Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
97 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: 79 - 120 mg/l
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
28 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
Oxygen consumption rates and percentage inhibition values for the control, test and reference items 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 (All tables are given in any other information on methdos and materials including tables section).

Percentage inhibition is plotted against concentration for the test and reference items (Figures 1 to 2) attached in backgriund material section.
The following results were derived:

1-(2-Hydroxyethyl)-2-Tall Oil-2-Imidazoline :
ECx (3 Hours) (mg/l) 95% Confidence Limits (mg/l)
EC20 35 -
EC50 97 79 - 120
EC80 300 -
NOEC 28 -


3,5-dichlorophenol:
ECx (3 Hours) (mg/l) 95% Confidence Limits (mg/l)
EC20 2.3 -
EC50 7.3 5.7 - 9.4
EC80 23 -
noec 1.9 -

Variation in respiration rates of controls 1 and 2 after 3 hours contact time was ± 7%.

The validation criteria for the control respiration rates and reference item EC50 values 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.



Results with reference substance (positive control):
- Results with reference substance valid?
Yes.

- Relevant effect levels:
The reference item gave a 3-Hour EC50 value of 7.3 mg/l, 95% confidence limits 5.7 – 9.4 mg/l.


Reported statistics and error estimates:
None.

Table 1              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

5.5

6

2.5

0.50

-

 

R2

4.0

3

2.3

0.57

-

Test Item

10

4.8

5

2.1

0.54

0

 

32

4.8

5

2.6

0.44

18

 

100

5.9

10

3.3

0.26

51

 

320

7.7

10

6.7

0.10

81

 

1000

8.7

10

8.4

0.03

94

3,5-dichlorophenol

3.2

5.7

8

2.6

0.39

27

 

10

7.0

10

4.9

0.21

61

 

32

8.3

10

7.6

0.07

87

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

7.4

8.1

 

R2

7.8

8.1

Test Item

10

7.7

7.9

 

32

7.8

8.0

 

100

7.8

8.1

 

320

7.9

8.4

 

1000

8.4

8.5

3,5-dichlorophenol

3.2

7.5

8.2

 

10

7.6

8.3

 

32

7.6

8.3

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

Very slightly cloudy dispersion with no undissolved test item visible*

Dark brown dispersion with no undissolved test item visible and a very small amount of foam on the surface

Dark brown dispersion with no undissolved test item visible and a very small amount of foam on the surface

 

32

Slightly cloudy dispersion with no undissolved test item visible*

Dark brown dispersion with no undissolved test item visible and a small amount of foam on the surface

Dark brown dispersion with no undissolved test item visible and a small amount of foam on the surface

 

100

Cloudy dispersion with no undissolved test item visible*

Dark brown dispersion with no undissolved test item visible and foam on the surface

Dark brown dispersion with no undissolved test item visible and foam on the surface

 

320

Cloudy dispersion, no undissolved test item visible*

Dark brown dispersion with no undissolved test item visible and foam on the surface

Dark brown dispersion with no undissolved test item visible and foam on the surface

 

1000

Very cloudy dispersion, no undissolved test item visible*

Dark brown dispersion with no undissolved test item visible and a large amount of foam on surface

Dark brown dispersion with no undissolved test item visible and a large amount of foam on surface

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 97 mg/l, 95% confidence limits 79 - 120 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 28 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 was designed to be compatible with 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 dispersion 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 time 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 EC50 of 97 mg/l, 95% confidence limits 79 - 120 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 28 mg/l.

The reference item gave a 3-Hour EC50value of 7.3 mg/l, 95% confidence limits 5.7 – 9.4 mg/l.

Description of key information

The effect of the test item on the respiration of activated sewage sludge gave a 3‑Hour EC50of 97 mg/l, 95% confidence limits 79 - 120 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 28 mg/l.

Key value for chemical safety assessment

EC50 for microorganisms:
97 mg/L
EC10 or NOEC for microorganisms:
28 mg/L

Additional information

Introduction.

A study was performed to assess the effect of the test item on the respiration of activated sewage sludge. The method followed was designed to be compatible with 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 dispersion 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 tiand 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 97 mg/l, 95% confidence limits 79 - 120 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 28 mg/l.

The reference item gave a 3-Hour EC50value of 7.3 mg/l, 95% confidence limits 5.7 – 9.4 mg/l.

Conclusion:

The effect of the test item on the respiration of activated sewage sludge gave a 3‑Hour EC50of 97 mg/l, 95% confidence limits 79 - 120 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was28mg/l.

The reference item gave a 3-Hour EC50value of 7.3 mg/l, 95% confidence limits 5.7 – 9.4 mg/l.