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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.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:
18 April 2010 to 19 April 2010
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
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
not required
Details on sampling:
At it was not a requirement of the Test Guidelines, no analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
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.
The control group was maintained under identical conditions but not exposed to the test item.

- 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): For the purpose of the test, the test item was dispersed directly in water.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Yes, undissolved particle in all concentrations.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture:
A mixed population of activated sewage sludge micro-organisms was obtained on 19 April 2010 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic 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.

- Preparation of inoculum for exposure:
The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC and was used on the day of collection. The pH of the sample was 7.5 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 105 ºC 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.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
not reported
Hardness:
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.
Test temperature:
The test was conducted under normal laboratory lighting in a temperature controlled room at 21±1°C.
pH:
At 0 hours: 7.0-7.1
At 3 hours: 7.5-8.0
Dissolved oxygen:
Initial reading: 3.9-7.6 mg O2/l
Final reading: 1.7-6.6 mg O2/l

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:
n/a
Nominal and measured concentrations:
Nominal: 10, 32, 100, 320, 1000 mg/l
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 ml conical flask
- Material, size, headspace, fill volume: At time "0" 16 ml of synthetic sewage was diluted to 300 ml with water and 200 ml of inoculum
- Aeration: 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.


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution 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.

OTHER TEST CONDITIONS
- Photoperiod: In a darkened vessel under normal laboratory lighting for the duration of the test.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
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
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
44 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
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: EC50 = 8.6 mg/l (CL: 6.7-11); NOEC = 2.6 mg/l
Reported statistics and error estimates:
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.
The EC15, EC20, EC50 and EC80 values for the test item were determined by inspection of the inhibition of respiration rate data.
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.

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.

Percentage inhibition is plotted against concentration for the test and reference items (Figures 1 to 2 attached)

The following results were derived:

 

CASNo 818-08-6

3,5-dichlorophenol

ECx(3 Hours)
(mg/l)

95% Confidence Limits (mg/l)

ECx(3 Hours)
(mg/l)

95% Confidence Limits (mg/l)

EC20

    60

-

  2.9

-

EC50

>1000

-

  8.6

6.7 - 11

EC80

>1000

-

26

-

NOEC

    44

-

  2.4

-

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

It was not possible to determine EC50 or EC80 values for the test item as no concentration tested resulted in greater than 50% inhibition. It was also not possible to obtain 95% confidence limits for the test item EC50 value as the data generated did not fit the models available for calculation.

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.

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 O2 Reading

(mg O2/l)

O2 Consumption Rates

(mg O2/l/min)

% Inhibition

Control

R1

5.4

7

1.7

0.53

-

 

R2

4.7

5

2.1

0.52

-

Test Item

10

3.9

4

2.0

0.48

9

 

32

3.9

4

1.9

0.50

5

 

100

4.8

8

1.9

0.36

31

 

320

5.2

8

1.9

0.41

22

 

1000

5.5

9

2.1

0.38

28

3,5-dichlorophenol

3.2

5.8

9

1.9

0.43

18

 

10

7.0

10

5.1

0.19

64

 

32

7.6

10

6.6

0.10

81

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

7.0

7.5

 

R2

7.1

7.6

Test Item

10

7.1

7.6

 

32

7.1

7.6

 

100

7.1

7.5

 

320

7.1

7.6

 

1000

7.1

7.6

3,5-dichlorophenol

3.2

7.0

7.8

 

10

7.0

8.0

 

32

7.0

8.0

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 water column with undissolved test item visible on surface*

Dark brown dispersion with small amounts of undissolved test item visible on surface

Dark brown dispersion with small amounts of undissolved test item visible on surface

 

32

Slightly cloudy dispersion with some undissolved test item visible on surface and dispersed throughout *

Dark brown dispersion with some undissolved test item visible on surface and dispersed throughout

Dark brown dispersion with some undissolved test item visible on surface and dispersed throughout

 

100

Slightly cloudy dispersion with test item visible on surface and on bottom of vessel*

Dark brown dispersion with undissolved test item visible dispersed throughout

Dark brown dispersion with undissolved test item visible dispersed throughout

 

320

Slightly cloudy dispersion with a large amount of undissolved test item on surface and on bottom of vessel*

Dark brown dispersion with undissolved test item visible on surface and bottom of vessel and dispersed throughout and adhered to sides of the vessel

Dark brown dispersion with undissolved test item visible on surface and bottom of vessel and dispersed throughout and adhered to sides of the vessel

 

1000

Slightly cloudy dispersion with a large amount of undissolved test item visible on surface and on bottom of vessel and adhered to sides of vessel*

Dark brown dispersion with a large amount of undissolved test item visible on surface and bottom of vessel and dispersed throughout and adhered to sides of the vessel

Dark brown dispersion with a large amount of undissolved test item visible on surface and bottom of vessel and dispersed throughout and adhered to sides of the vessel

3,5-dichlorophenol

3.2

Dark brown dispersion, with no undissolved reference item visible

Dark brown dispersion, with no undissolved reference item visible

Dark brown dispersion, with no undissolved reference item visible

 

10

Dark brown dispersion, with no undissolved reference item visible

Dark brown dispersion, with no undissolved reference item visible

Dark brown dispersion, with no undissolved reference item visible

 

32

Dark brown dispersion, with no undissolved reference item visible

Dark brown dispersion, with no undissolved reference item visible

Dark brown dispersion, with 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 44 mg/l.
Executive summary:

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.

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.

The effect of the test item 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 44 mg/l.

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

The reference item gave a 3-hour EC50 value of 8.6 mg/l, 95% confidence limits 6.7 - 11 mg/l.

Description of key information

The following study was submitted to address the endpoint Toxicity to Microorganisms:

Clarke, N (2010) ASSESSMENT OF THE INHIBITORY EFFECT ON THE RESPIRATION OF ACTIVATED SEWAGE SLUDGE. Testing Laboratory: Harlan Laboratories Ltd, Shardlow Business Park, London Road, Shardlow, Derbyshire, DE72 2GD, UK. Owner company: Organo Tin REACH Consortium, c/o ReachCentrum, Avenue E. Van Nieuwenhuyse 6, B-1160, Brussels, BELGIUM. Report No. 3109/0024. Report date: 2010-05-21

The study was performed on the test material dibutyltin oxide. The study was assigned a reliability score of 1.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L

Additional information

The study Clarke N (2010) was performed in accordance with the OECD Guideline 209 and in compliance with GLP. The study was therefore assigned a reliability score of 1 and considered adequate for assessment. 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.


The effect of the test item 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 44 mg/l.


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


The reference item gave a 3-hour EC50 value of 8.6 mg/l, 95% confidence limits 6.7 - 11 mg/l.