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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 March 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with REACH Annex XI, Section 1.5, of Regulation (EC) No. 1907/2006 (REACH) the standard testing regime may be adapted in cases where a grouping or read-across approach has been applied.

The similarities may be based on:
(1) a common functional group
(2) the common precursors and/or the likelihood of common breakdown products via physical or biological processes, which result in structurally similar chemicals; or
(3) a constant pattern in the changing of the potency of the properties across the category

(1) The source and target substances are both inorganic salts of a monovalent cation from Group 1A of the periodic table, sodium or potassium, and pyrophosphoric/orthophosphoric acid. Thus, they all share the Na+ or K+ cation and the P2O74-/PO43- anion as common functional groups.
(2) All members of the group will ultimately dissociate into the common breakdown products of the Na+ or K+ cations and the P2O74-/PO43- anion.
(3) Potassium and Sodium cations are essential micronutrients that are ubiquitous in the environment. As such, their uptake is tightly regulated and is therefore not considered to pose a risk for ecotoxicity. The pyrophosphate anion is unstable in aqueous solutions with the degree of instability varying according to pH. In distilled water pyrophosphates will hydrolyse slowly via abiotic mechanisms to inorganic phosphate. In natural waters a number of different processes can occur; abiotic hydrolysis, biotic degradation (as a result of the action of phosphatases which cleave pyrophosphate into orthophosphate subunits) and assimilation by organisms in the water all resulting in an ultimate breakdown product of orthophosphate. It is therefore deemed scientifically justified to avoid any further vertebrate testing and use the data from a study conducted on an orthophosphate (with either a potassium or sodium cation) for hazard assessment purposes.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report under Section 13 ‘Assessment Reports’.

3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report under Section 13 ‘Assessment Reports’.

4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’.
Reason / purpose:
read-across: supporting information
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)
Remarks:
Date of GLP inspection: 15 September 2009 Date of Signature on GLP certificate:26 November 2009
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES
- Melting point: >450°C
- Water solubility (under test conditions): very soluble (10,000 mg/L)
- Stability in water: dissociates to potassium cations and phosphate anions
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:

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 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 solutoions. 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 solution.
The control group was maintained under identical conditions but not exposed to the test item.
As 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.


- 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 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 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 throughout the test period (see Table 3 in any other information on results section) showed that at 0 hours, 30 minutes and 3 hours that the control vessels contained a dark brown dispersion and the reference item vessels contained a dark brown dispersion with no undissolved reference item visible.
Observations made of the test item vessels at 0 hours prior to the addition of activated sewage sludge and synthetic sewage showed that all test concentrations contained a clear colourless solution.
Observations made of the test item vessels after 30 minutes and 3 hours contact time showed that all test concentrations contained a dark brown dispersion with no undissolved test item visible.
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 7.1 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 4.0 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 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 (see in attached 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 (see in any other information on results 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". 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 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
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 (see in attached section).


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 (see in any other information on results 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 – 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 for the reference item 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 item were determined by inspection of the inhibition of respiration rate data.
95% confidence limits were calculated for the EC50 value for the reference item only 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.


TEST CONCENTRATIONS
10, 32, 100, 320 and 1000 mg/l

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
Remarks on result:
other: not specified
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 items in the definitive test are given in Table 1 (see in any other information on results section). The pH values of the test preparations at the start and end of the exposure period are given in Table 2 (see in any other information on results section) , and observations made on the test preparations throughout the study are given in Table 3 see in any other information on results section).
Percentage inhibition is plotted against concentration for the reference item, 3,5 dichlorophenol only (Figure 1 see in attached section).

The following results were derived:
Dipotassium hydrogenorthophosphate
ECx (3 Hours) (mg/l) 95% Confidence Limits (mg/l)
>1000 -
>1000 -
>1000 -
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 2.8 -
EC50 8.7 6.8 - 11
EC80 27 -
NOEC 2.3 -
Variation in respiration rates of controls 1 and 2 was ± 0% 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 8.3 mg/l, 95% confidence limits 6.8 - 11 mg/l.

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

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

5.7

6

2.2

0.58

-

 

R2

6.3

6

2.9

0.57

-

Test Item

10

6.0

6

2.5

0.58

[1]

 

32

5.7

5

2.8

0.58

[1]

 

100

5.4

5

2.5

0.58

[1]

 

320

6.0

6

2.5

0.58

[1]

 

1000

5.6

5

2.7

0.58

[1]

3,5-dichlorophenol

3.2

6.4

9

2.4

0.44

23

 

10

7.6

10

5.0

0.26

55

 

32

8.4

10

7.5

0.09

84


[Increase in respiration rate as compared to controls]

R1– R2= Replicates 1 to 2

Table2              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

7.3

7.8

 

R2

7.5

7.8

Test Item

10

7.5

7.8

 

32

7.5

7.8

 

100

7.5

7.8

 

320

7.7

7.8

 

1000

7.8

7.8

3,5-dichlorophenol

3.2

7.4

8.0

 

10

7.4

8.1

 

32

7.5

8.1


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

30 Minutes

Contact Ti

3 Hours

Contact Ti

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

Clear colourless solution, no undissolved test item visible*

Dark brown dispersion, no undissolved test item visible

Dark brown dispersion, no undissolved test item visible

 

100

Clear colourless solution, no undissolved test item visible*

Dark brown dispersion, no undissolved test item visible

Dark brown dispersion, no undissolved test item visible

 

320

Cloudy homogenous dispersion, no undissolved test item visible*

Dark brown dispersion, no undissolved test item visible

Dark brown dispersion, no undissolved test item visible

 

1000

Cloudy homogenous dispersion, 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

REFERENCES

Litchfield, J T and Wilcoxon, F (1949) A Simplified Method of Evaluating Dose-Effect Experints. J Pharmacol Exp Ther96, 99-113.

Xlfit, ID Business Solutions Ltd.

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.
This study is conducted according to the appropriate guidelines (EU ) and under the conditions of GLP and therefore the study is considered to be acceptable and to adequately satisfy both the guideline requirement and the regulatory requirement as a key study for this endpoint. Study is sufficient for classification and labelling purposes, in accordance with Regulation (EC) No. 1272/2008 (EU CLP).
Executive summary:

Introduction.

A study was perford to assess the effect of the test item on the respiration of activated sewage sludge. Thethod 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 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 greater than 1000 mg/l. The No

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 7.9 mg/l, 95% confidence limits 6.2 - 10 mg/l.

Description of key information

One key study to assess the toxicity of disodium dihydrogenpyrophosphate to STP microorganisms exists, this study is conducted on an analogous substance (see justification below). On this basis sodium and potassium pyrophosphates are not considered to be toxic to STP microorganisms. 
In addition, according to the Guidance on Information Requirements and Chemical Safety Assessment (chapter R.7b section R7.8.14), toxicity to STP microorganisms is best assessed using an “Activated Sludge Respiration Inhibition Test” according to OECD Guideline 209 as it is the most widely accepted indicator of the combined activity of sludge microorganism. According to the OECD 209 Guideline, the STP microorganism should be fed a synthetic sewage feed made by dissolving a number of inorganic compounds, including 2.8g K2HPO4 in IL of water which is fed to the microorganisms at a rate of 50 ml/day (containing 140 mg K2HPO4) if the sewage sludge is not to be used immediately. This indicates that phosphates are necessary for the survival of the sewage microorganism and therefore no further testing for this endpoint is required.

Key value for chemical safety assessment

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

Additional information

Rationale for read across:

In accordance with Annex XI, section 1.5 of Regulation (EC) No 1907/2006 (REACH) a read across approach may be used when substances have similarities based on the likelihood of common breakdown products via physical and biological processes, which result in structurally similar chemicals.

Sodium and potassium pyrophosphates are ionic in nature and therefore dissociate readily into cations and anions in water the toxicity of the both the cation and the anion must be addressed. Potassium and Sodium cations are essential micronutrients that are ubiquitous in the environment. As such, their uptake is tightly regulated and is therefore not considered to pose a risk for ecotoxicity. The pyrophosphate anion is unstable in aqueous solutions with the degree of instability varying according to pH. In distilled water pyrophosphates will hydrolyse slowly via abiotic mechanisms to inorganic phosphate. In natural waters a number of different processes can occur; abiotic hydrolysis, biotic degradation (as a result of the action of phosphatases which cleave pyrophosphate into orthophosphate subunits) and assimilation by organisms in the water all resulting in an ultimate breakdown product of orthophosphate.

It is therefore deemed scientifically justified to avoid any further vertebrate testing and use the data from a study conducted on an orthophosphate (with either a potassium or sodium cation) for risk assessment purposes (appropriate assessment factors to be considered).

The substance has the following properties:

Chemical name: dipotassium hydrogenorthophosphate

Chemical formula: H3O4P.2K

CAS number: 7758-11-4

Molecular weight: 175

Melting range: >450°C

Solubility in water considered to be very soluble (63.0 - 65.0% w/w at 20.0 ± 0.5°C) pH of solution: 10.1-10.7

It is therefore considered appropriate; due to the physicochemical nature of the substance tested and the read across arguments detailed, for this data to be used for read-across purposes for the following substances:

- disodium dihydrogenpyrophosphate

- trisodium hydrogen diphosphate

- tetrasodium pyrophosphate

- tetrapotassium pyrophosphate

Any further testing would not be scientifically justified as all substances would ultimately dissociate to their anionic and cationic forms in natural waters and these ions (Na+, K+ and PO43-(from P2O74-) are all ubiquitous and are not considered to pose a risk of ecotoxicity.