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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 February 2008 to 27 February 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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)
Analytical monitoring:
no
Details on sampling:
- Concentrations:
Range finding: nominal test concentrations of 100 and 1000 mg/L
Main study: a single test concentration of 1000 mg/L (three replicate vessels)

- Sampling method:
Observations were made on the test preparations throughout the test period, and the pH of the control, reference material and test material preparations at 0 hours and prior to measurement of the oxygen consumption rate after 3 hours contact time.

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

- Method:
Range-finding Test:
In the range-finding test activated sewage sludge micro-organisms were exposed to nominal test concentrations of 100 and 1000 mg/l. The test material was dispersed directly in water.

Amounts of test material (50 and 500 mg) were each separately dispersed in approximately 250 mL of water and subjected to ultrasonication for approximately 15 minutes. 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 100 and 1000 mg/L.

Definitive Test:
Based on the results of the range-finding test a "limit test" was conducted at a concentration of 1000 mg/l (three replicate vessels) to confirm that at this concentration no effect on respiration of the activated sewage sludge was observed.
For the purpose of the definitive test, the test material was dispersed directly in water.
An amount of test material (500 mg) was dispersed in approximately 250 mL of water and subjected to ultrasonication for approximately 15 minutes. Synthetic sewage (16 mL), activated sewage sludge (200 mL) and water were added to a final volume of 500 mL to give the required concentration of 1000 mg/L.

This method of preparation was performed for the three separate replicate test vessels.

- 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 material being added. The test material vessels were preparedas described in Section "Definitive Test". Finally a second control was prepared.

As each vessel reached 30 minutes 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. After measurement the contents of the BOD bottle were returned to the test vessel.

This procedure was repeated after 3 hours contact time. The test was conducted under normal laboratory lighting in a temperature controlled room at 21±1 °C.

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


- Evidence of undissolved material (e.g. precipitate, surface film, etc):
Observations made throughout the test period showed that all test vessels contained a light brown dispersion with white particles of test material dispersed homogenously throughout and on the surface at 0 hours, 30 minutes and 3 hours contact time.
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 22 February 2008 for the range-finding test and on 27 February 2008 for the definitive test 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 7.8 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.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Post exposure observation period:
Not applicable.
Hardness:
The test water used for both the range-finding and definitive tests 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±1ºC.
pH:
The pH values of the test were 7.5 to 8.0.
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:
1000 mg/L.
Details on test conditions:
TEST SYSTEM

- Test vessel: 500 mL conical flask
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 300 mL water and 200 mL innoculum
- Aeration: yes, 0.5 - 1 litre per minute
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

TEST MEDIUM / WATER PARAMETERS
Test medium:
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.

Water parameters:
- Source/preparation of dilution water: Laboratory tap water dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex water softener).
- Total organic carbon: min: 1.090 mg/L, average: 1.429 mg/L, max: 1.900 mg/L
- Metals: Al: < 10.596 µg/L; Cu: <0.021 mg/L; Fe: <28.038 µg/L; Pb: <1.113 µg/L; Mn: <1.723 µg/L: Ni: <2.325 µg/L and Na: 24.875 mg/L
- Pesticides: min: 0.000 mg/L, average: 0.019 mg/L, max: 0.092 mg/L
- Chlorine: (Total) min: 0.020, average: 0.274, max: 0.510
- Conductivity: min: 289.0000 µS/cm, average: 403.577 µS/cm, max: 504.000 µS/cm (at 20°C)
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH:
No adjustment made.

- 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 materials 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 material only, a line fitted using the Xlfit software package (IDBS) and the EC20, EC50 and EC80 values determined from the equation for the fitted line.

The EC20, 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 EC50 value for the reference material after 3 hours contact time using the method of Litchfield and Wilcoxon (Litchfield and Wilcoxon 1949).

The No Observed Effect Concentration (NOEC) was determined by inspection of the inhibition of respiration rate data.

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.


- Range finding study

- Test concentrations:
In the range-finding test activated sewage sludge micro-organisms were exposed to nominal test concentrations of 100 and 1000 mg/L. The test material was dispersed directly in water.
Using this approach the effect of an excess amount of undissolved test material can be assessed in order to determine whether the test material has any effect on the activated sewage sludge micro-organisms exoenzymes, or whether the uptake of undissolved test material by processes such as phagocytosis has any adverse effect on the activated sewage sludge.

- Results used to determine the conditions for the definitive study:

Based on the results of the range-finding test a "limit test" was conducted at a concentration of 1000 mg/l (three replicate vessels) to confirm that at this concentration no effect on respiration of the activated sewage sludge was observed.
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.:
1 000 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: no
- Effect concentrations exceeding solubility of substance in test medium: no

Range-finding Test
Data supplied indicated the water solubility of the test material to be 1.5 x 10-3 g/L. Although the test material was not in solution in the range-finding test, no significant effect on respiration was observed at either test concentration employed and given the extremely low water solubility of the test material it was considered that no inhibition would occur at this concentration. It was therefore considered justifiable to conduct the definitive test at a single nominal test concentration of 1000 mg/L which was in excess of the water solubility of the test material.

Definitive Test
The following results were derived:

4MDBS:
ECx (30 Minutes) (mg/L) 95% Confidence Limits (mg/L) ECx (3 Hours)(mg/L) 95% Confidence Limits (mg/L)
EC20 >1000 - >1000 -
EC50 >1000 - >1000 -
EC80 >1000 - >1000 -
NOEC 1000 - 1000 -

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

3,5-dichlorophenol:
ECx (30 Minutes) (mg/L) 95% Confidence Limits (mg/L) ECx (3 Hours) (mg/L) 95% Confidence Limits (mg/L)
EC20 3.9 - 3.7 -
EC50 11 - 10 8.0 - 13
EC80 32 - 27 -

It was not possible to obtain 95 % confidence limits for the reference material EC50 value after 30 minutes contact time as the data generated did not fit the models available for calculation.
Variation in respiration rates of controls 1 and 2 after 30 minutes was ± 3%, and ± 2% after 3 hours 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 10 mg/L, 95% confidence limits 8.0 - 13 mg/L.

- Other:
None.
Reported statistics and error estimates:
None.
Validity criteria fulfilled:
yes
Conclusions:
The effect of the test material 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 1000 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 C11 of EEC Commission Directive 88/302/EEC and US EPA Draft Ecological Effects Test Guidelines OPPTS 850.6800.

Methods. Following a preliminary range-finding test, activated sewage sludge was exposed to an aqueous dispersion of the test material at a concentration of 1000 mg/L (three replicate flasks) 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 30 minutes and 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 EC50 of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/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 10 mg/L, 95 % confidence limits 8.0 - 13 mg/L.

Endpoint:
activated sludge respiration inhibition testing
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
It is proposed that the structural similarity and properties of the target substance and the structural analogue (sources substance) are sufficiently close for there to be a reasonable expectation of similar effects.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source chemical:
1-(2,6-bis(4-tolyl)-1,3-dioxano(5,4-d)-1,3-dioxan-4-yl)ethane-1,2-diol (EC 402-950-5, CAS 87826-41-3)

Target chemical:
2,6-bis(4-ethylphenyl)perhydro-1,3,5,7-tetraoxanaphth-4-ylethane-1,2-diol (EC 406-176-9, CAS 79072-96-1)

3. ANALOGUE APPROACH JUSTIFICATION
Based on the structural similarity of the source substances and target substance, similarity of physic-chemical properties and similarity in experimental (eco)toxicological test data it is concluded that target substance and the structural analogue (source substance) are sufficiently close for there to be a reasonable expectation of similar effects, for the endpoints where results have been read-across.

4. DATA MATRIX
Please see 'Read-across justification to support the REACH registration of EC 406-176-9' document attached in section 13.
Reason / purpose:
read-across source
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.:
1 000 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: no
- Effect concentrations exceeding solubility of substance in test medium: no

Range-finding Test
Data supplied indicated the water solubility of the test material to be 1.5 x 10-3 g/L. Although the test material was not in solution in the range-finding test, no significant effect on respiration was observed at either test concentration employed and given the extremely low water solubility of the test material it was considered that no inhibition would occur at this concentration. It was therefore considered justifiable to conduct the definitive test at a single nominal test concentration of 1000 mg/L which was in excess of the water solubility of the test material.

Definitive Test
The following results were derived:
EC20 (30 min): >1000 mg/L
EC50 (30 min): >1000 mg/L
EC80 (30 min): >1000 mg/L
NOEC (30 min): 1000 mg/L

EC20 (3 hr): >1000 mg/L
EC50 (3 hr): >1000 mg/L
EC80 (3 hr): >1000 mg/L
NOEC 3 hr): 1000 mg/L

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

3,5-dichlorophenol:
ECx (30 Minutes) (mg/L) 95% Confidence Limits (mg/L) ECx (3 Hours) (mg/L) 95% Confidence Limits (mg/L)
EC20 3.9 - 3.7 -
EC50 11 - 10 8.0 - 13
EC80 32 - 27 -

It was not possible to obtain 95 % confidence limits for the reference material EC50 value after 30 minutes contact time as the data generated did not fit the models available for calculation.
Variation in respiration rates of controls 1 and 2 after 30 minutes was ± 3%, and ± 2% after 3 hours contact time.
The validation criteria for the control respiration rates and reference material EC50 values were therefore satisfied.
Results with reference substance (positive control):
Yes
Validity criteria fulfilled:
yes
Conclusions:
The effect of the test material 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 1000 mg/L.
Executive summary:

Introduction.

A study (on structural analogue substance EC 402 -950 -5) 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 C11 of EEC Commission Directive 88/302/EEC and US EPA Draft Ecological Effects Test Guidelines OPPTS 850.6800.

Methods.

Following a preliminary range-finding test, activated sewage sludge was exposed to an aqueous dispersion of the test material at a concentration of 1000 mg/L (three replicate flasks) 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 30 minutes and 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 was1000mg/L.

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

The reference material gave a 3-Hour EC50value of 10 mg/L, 95 % confidence limits 8.0 - 13 mg/L.

Based on these results, it is proposed that the target substance (EC 406-176-9) would not show toxicity to microorganims up to 1000 mg/L in an activated sludge respiration inhibition test.

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Range-finding study: 03 October 1997. Definitive study: 16 October 1997.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Details on sampling:
As each vessel reached 30 min contact time, an aliquot was removed from the conical flask and poured into the measuring vessel and the rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe.
Vehicle:
no
Details on test solutions:
For the purpose of the definitive study the test material was prepared by a direct dispersion in water. An amount of test material was dispersed in approximately 250 mL of water and subjected to ultrasonication for approximately 30 min. Synthetic sewage, activated sewage sludge and water were added to a final volume of 500 mL to give the test concentration of 1000 mg/L.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of 21 °C and was used on the day of collection. The pH of the sample was 7.7 and the suspended solids equal to 3.8 g/L prior to use.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Post exposure observation period:
The rate of respiration was determined after 30 min and 3 h.
Hardness:
100 mg/L as CaCO3
Test temperature:
21 °C
Nominal and measured concentrations:
Range-finding study: series of nominal test concentrations of 100 and 1000 mg/L
Definitive study: 1000 mg/L
Details on test conditions:
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 500 mL conical flask (first control). Thereafter, at 15 min intervals the procedure was repeated with appropriate amounts of the reference material being added. Finally a second control was prepared. As each vessel reached 30 minutes contact time an aliquot was removed from the conical flask and poured into the measuring vessel 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 an approximate 10 min period. This procedure was repeated after 3 h contact time. The study was conducted under normal laboratory lighting in a temperature controlled room at 21 °C.
- Definitive study
- No. of vessels per concentration (replicates): 3
- Aeration: yes; the mixture was aerated with compressed air via narrow bore glass tubes at a rate of approximately 0.5 - 1 L/ min
- The control group was maintained under identical conditions but not exposed to the test material.
- The exposure conditions for each flask were as described in “PREPARATION OF TEST SYSTEM”.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: laboratory tap water dechlorinated by passage through an activated carbon filter.

OTHER TEST CONDITIONS
- Light intensity: The study was conducted under normal laboratory lighting

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: series of nominal test concentrations of 100 and 1000 mg/L
- To prepare the test series amounts of test material were each separately dispersed in water with the aid of ultrasonication for a period of 30 min. Synthetic sewage, activated sewage sludge and water were added to each flask giving the test concentrations of 100 and 1000 mg/L. The control group was maintained under identical conditions but not exposed to the test material. The exposure conditions for each flask were as described in “PREPARATION OF TEST SYSTEM”, however oxygen consumption rates were measured after 3 hours contact time only.
- The control group was maintained under identical conditions but not exposed to the test material.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
30 min
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:
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
Details on results:
Range-finding study
- No significant effect on respiration was observed at either concentration employed. Based on this information, a single test concentration, in triplicate, of 1000 mg/L was selected for the definitive study.

Definitive study
- Variation in respiration rates of controls 1 and 2 after 30 min: ± 4 %.
- Variation in respiration rates of controls 1 and 2 after 3 hours: ± 2 %.
- The validation criteria for the control respiration rates and reference material EC50 values have been satisfied.
- It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.
Results with reference substance (positive control):
- Results with reference substance valid? Yes.
3,5-dichlorophenol EC50 (30 minutes): 10 mg/l
3,5-dichlorophenol EC50 (3 hours): 8 mg/l

Oxygen consumption rates and percentage inhibition values in the definitive study after 30 min contact time

Concentration (mg/L)

O2Consumption rates (mg O2/L/min)

% inhibition

Control

R1

0.47

-

R2

0.51

-

Test Material

1000 R1

0.48

2

1000 R2

0.51

[4]

1000 R3

0.54

[10]

Reference Material

3.2

0.45

8

10

0.22

55

32

0.09

82

[increase in respiration as compared to controls]; R1- R3= Replicates

Oxygen consumption rates and percentage inhibition values in the definitive study after 3 h contact time

Concentration (mg/L)

O2Consumption rates (mg O2/L/min)

% inhibition

Control

R1

0.49

-

R2

0.51

-

Test Material

1000 R1

0.48

4

1000 R2

0.49

2

1000 R3

0.51

[2]

Reference Material

3.2

0.44

12

10

0.17

66

32

0.07

86

[increase in respiration as compared to controls]; R1- R3= Replicates

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test material on the respiration of activated sewage sludge microorganisms gave a 3-Hour ECS0 of greater than 1000 mg/l.
Executive summary:

Methods

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 No. 209 "Activated Sludge Respiration Inhibition Test".

Procedure

The test material was aerated for a period of 3 h at 21°C in the presence of activated sewage sludge with the addition of a synthetic sewage as a respiratory substrate. The rate of respiration was determined after 30 min and 3 h 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 EC50 of greater than 1000 mg/L.

Endpoint:
activated sludge respiration inhibition testing
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
It is proposed that the structural similarity and properties of the target substance and the structural analogue (sources substance) are sufficiently close for there to be a reasonable expectation of similar effects.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source chemical:
1,3:2,4-bis-O-(3,4-dimethylbenzylidene)-D-glucitol (EC 413-110-2, CAS 135861-56-2)

Target chemical:
2,6-bis(4-ethylphenyl)perhydro-1,3,5,7-tetraoxanaphth-4-ylethane-1,2-diol (EC 406-176-9, CAS 79072-96-1)

3. ANALOGUE APPROACH JUSTIFICATION
Based on the structural similarity of the source substances and target substance, similarity of physic-chemical properties and similarity in experimental (eco)toxicological test data it is concluded that target substance and the structural analogue (source substance) are sufficiently close for there to be a reasonable expectation of similar effects, for the endpoints where results have been read-across.

4. DATA MATRIX
Please see 'Read-across justification to support the REACH registration of EC 406-176-9' document attached in section 13.
Reason / purpose:
read-across source
Duration:
30 min
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:
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
Details on results:
Range-finding study
- No significant effect on respiration was observed at either concentration employed. Based on this information, a single test concentration, in triplicate, of 1000 mg/L was selected for the definitive study.

Definitive study
- Variation in respiration rates of controls 1 and 2 after 30 min: ± 4 %.
- Variation in respiration rates of controls 1 and 2 after 3 hours: ± 2 %.
- The validation criteria for the control respiration rates and reference material EC50 values have been satisfied.
- It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.
Results with reference substance (positive control):
- Results with reference substance valid? Yes.
3,5-dichlorophenol EC50 (30 minutes): 10 mg/l
3,5-dichlorophenol EC50 (3 hours): 8 mg/l
Validity criteria fulfilled:
yes
Conclusions:
The effect of the test material on the respiration of activated sewage sludge microorganisms gave a 3-Hour ECS0 of greater than 1000 mg/l.
Executive summary:

Methods

A study was performed to assess the effect of the test material (structural analogue source substance EC 413-110-2)

on the respiration of activated sewage sludge. The method followed that described in the OECD Guidelines for Testing of Chemicals No. 209 "Activated Sludge Respiration Inhibition Test".

Procedure

The test material was aerated for a period of 3 h at 21°C in the presence of activated sewage sludge with the addition of a synthetic sewage as a respiratory substrate. The rate of respiration was determined after 30 min and 3 h 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 EC50 of greater than 1000 mg/L.

Based on these results, it is proposed that the target substance (EC 406-176-9) would not show toxicity to microorganims up to 1000 mg/L in an activated sludge respiration inhibition test.

Description of key information

The toxicity to microorganisms of the substance (EC 406-176-9) has been assessed by reading across the results of studies conducted on two structurally similar analogue substances.

It is proposed that these results can be used in the assessment of the target substance (EC 406-176-9).

Source Substance;1,3:2,4-bis-O-(3,4-dimethylbenzylidene)-D-glucitol (EC-413 -110 -2):

Methods

A study was performed to assess the effect of the test material (EC 413-110-2) on the respiration of activated sewage sludge.

Procedure

The test material was aerated for a period of 3 h at 21°C in the presence of activated sewage sludge with the addition of a synthetic sewage as a respiratory substrate. The rate of respiration was determined after 30 min and 3 h 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.

Source Substance;1-(2,6-bis(4-tolyl)-1,3-dioxano(5,4-d)-1,3-dioxan-4-yl)ethane-1,2-diol (EC 402-950-5):

A study (on EC 402-950-5) was performed to assess the effect of the test material on the respiration of activated sewage sludge. 

 

Methods.

Following a preliminary range-finding test, activated sewage sludge was exposed to an aqueous dispersion of the test material at a concentration of 1000 mg/L (three replicate flasks) 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 30 minutes and 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 3Hour EC50 of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L.

 

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

 

Key value for chemical safety assessment

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

Additional information

Based on the two read-across substance studies, it is proposed that the target substance (EC 406-176-9) would not show toxicity to microorganims up to 1000 mg/L in an activated sludge respiration inhibition test.