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Toxicity to microorganisms

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Reference
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:
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

The source chemical and sodium dihydrogenorthophosphate have the following similarities: 1. Both substances are inorganic salts of a monovalent cation from Group 1A of the periodic table, sodium or potassium, and phosphoric acid. Thus, they all share the Na+ or K+ cation and the 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 PO43- anion. 3. Sodium and potassium orthophosphates have been shown to have a similar toxicological profile and physicochemical nature and therefore this data is considered to be adequate and reliable for use in read-across. Any further testing would not be scientifically justified as all substances will dissociate to their anionic and cationic forms in natural waters and these ions (Na+, K+ and PO43-) are all ubiquitous and are not considered to pose a risk of ecotoxicity.

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 for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across: supporting information
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 monosodium phosphate on the respiration of activated sewage sludge micro-organisms was estimated to give 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:

The EC50 was estimated to be greater than 1000 mg/L for monosodium phosphate as found in the source study performed with dipotassium hydrogen phosphate. As explained in the justification for type of information, the differences in molecular structure between monosodium phosphate and dipotassium hydrogen phosphate are unlikely to lead to differences in the EC50 that are higher than the typical experimental error of the test method.

Description of key information

One key study to assess the toxicity to STP microorganisms of sodium dihydrogenorthophosphate exists, this study is conducted on an analogous substance. The study was performed under GLP conditions and to an appropriate guideline (OECD 209).
The additional supporting data provided were not considered to be adequate or reliable to fulfil neither the guideline nor the regulatory requirements. Further, there is no evidence within this data to suggest that essential validity criteria are met.
A number of literature papers relating to bioavailability and tolerance of inorganic phosphates in aquatic ecosystems are included in Section 6.6 to support the conclusions made in this endpoint.

Key value for chemical safety assessment

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

Additional information

The 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.

Read-across justification:

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

The source chemical and sodium dihydrogenorthophosphate have the following similarities:

1. Both substances are inorganic salts of a monovalent cation from Group 1A of the periodic table, sodium or potassium, and phosphoric acid. Thus, they all share the Na+ or K+ cation and the 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 PO43- anion.

3. Sodium and potassium orthophosphates have been shown to have a similar toxicological profile and physicochemical nature and therefore this data is considered to be adequate and reliable for use in read-across.

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