Registration Dossier

Diss Factsheets

Ecotoxicological information

Toxicity to microorganisms

Currently viewing:

Administrative data

Link to relevant study record(s)

Referenceopen allclose all

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted on 19 March 2010 exclusively
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

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 proposed source chemical (diammonium hydrogenorthophosphate) is highly soluble in water (> 10000 mg/L). In aqueous media soluble inorganic orthophosphates will dissociate to their ionic constituents; in this case ammonium and orthophosphate ions. Diammonium dihydrogenpyrophosphate will dissociate to ammonium cations and pyrophosphate anions. The pyrophosphate anions are unstable in aqueous solutions with the degree of instability varying according to pH. In distilled water they will hydrolyse slowly via abiotic mechanisms to orthophosphate. In natural waters a number of different processes can occur; abiotic hydrolysis, biotic degradation (as a result of the action of phosphatases which cleave pyrophosphates into orthophosphate subunits) and assimilation by organisms in the water. Thus the target substance (diammonium dihydrogenpyrophosphate) and the source substance (diammonium hydrogenorthophosphate) will be primarily absorbed as the same inorganic ions: ammonium and orthophosphate and are expected to behave in a similar manner under test conditions.
All (bio) transformation products of the source chemical are common to the target chemical and as such the data is considered to be adequate and reliable for use in the assessment of diammonium dihydrogenpyrophosphate for the ecotoxicity hazard assessment.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report attached.

3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report attached.

4. DATA MATRIX
See read-across justification report attached.

Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
not specified
Details on sampling:
Not applicable
Vehicle:
yes
Details on test solutions:
PREPARATION OF TEST SOLUTION

A stock solution of 0.5 g/l was prepared by adding 0.1252 g test substance to 250 ml of Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA). Thorough mixing (vortex) was used to accelerate dissolution and to ensure homogeneity. Volumes of the stock solution corresponding to the test concentration were then added to the test media. A concentration of 100 mg/l was tested in duplicate. Optimal contact between the test substance and test organisms was ensured applying continuous aeration and stirring.

Test organisms (species):
activated sludge
Details on inoculum:
Source: Municipal sewage treatment plant: ‘Waterschap de Maaskant', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.

Number of micro-organisms: Number of micro-organisms was determined as the amount of Mixed Liquor Suspended Solids (MLSS) per litre test medium.

Preparation of the sludge: The sludge was coarsely sieved, washed and diluted with ISO-medium. A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids (4.0 g/l of sludge, as used for the test). The pH was 7.9 on the day of testing. The batch of sludge was used one day after collection; therefore 50 ml of synthetic sewage feed was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.

Medium: Adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands) with the following composition:
CaCl2.2H2O 211.5 mg/l
MgSO4.7H2O 88.8 mg/l
NaHCO3 46.7 mg/l
KCl 4.2 mg/l
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Post exposure observation period:
10 minutes
Hardness:
no data
Test temperature:
Between 19.9 and 20.0 °C
pH:
no data
Dissolved oxygen:
no data
Salinity:
no data
Nominal and measured concentrations:
100 mg/L
Details on test conditions:
Contact time: 3 hours, during which aeration and stirring take place

Vessels: All glass, approximately 300 mL oxygen bottles and 1 litre test bottles

Milli-RO / Milli-Q water: Tap-water purified by reverse osmosis (Milli-RO) andsubsequently passed over activated carbon and ionexchange
cartridges (Milli-Q) (Millipore Corp., Bedford, Mass., USA).

Synthetic sewage feed:
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

Air supply: Clean, oil-free air.

Oxygen meter: WTW inolab Oxi 730 supplied with a WTW CellOx 325 oxygen electrode, electrolyte type ELY/G. Dissolved in Milli-Q water, made up to 1 litre and filtered. The pH was within 7.5 ± 0.5.

Recorder: Flatbed recorder SE 102 (Kipp and Zonen)

Performance of the test:
The synthetic sewage feed (16 ml), activated sludge (200 ml) and an adequate amount of the test
substance stock were mixed and made up to 500 ml with Milli-RO water in a 1 litre bottle. The mixture was aerated during the contact time, using a pipette as an aeration device.
After the 3-hour contact time, a well mixed sample of he contents was poured into a 300 ml oxygen bottle, and the flask was sealed with an oxygen electrode connected to a recorder, forcing the air out of the vessel. Oxygen consumption was measured and recorded for approximately 10 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer.
The pH and temperature were determined in the remaining part of the reaction mixture.
This procedure was repeated for the duplicate concentration. Two controls without test substance were tested in each test series, one at the start and
one at the end.

Table 1 in Section 'Any other information on materials and methods' below presents a detailed study design.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
Table 2 in Section 'Any other information on results incl. tables' below presents the results of the test.

No significant inhibition of respiration rate of the sludge was recorded at 100 mg Diammonium hydrogenorthophosphate per litre. The duplicate measurement confirmed the result of the first measurement. Therefore, no further testing was needed. Hence, the EC50 of Diammonium hydrogenorthophosphate exceeded 100 mg/l (based on nominal concentrations).
Results with reference substance (positive control):
Table 2 in Section 'Any other information on results incl. tables' below presents the results of the test.

The EC50 of 3,5-dichlorophenol was in the accepted range of 5-30 mg/l (6.0 mg/l, see also APPENDIX I).

Table 2: Controls (C) and Diammonium hydrogenorthophosphate (T): pH, oxygen concentration, oxygen consumption and percentage inhibition of the respiration rate

 

Flask

Concentration (mg/L)

Oxygen conc. At the start (approx. equal to mg O2/L)

Oxygen consumption (mg O2/l/h)

% Inhibition respiration rate

pH

Cstart T

0

6.5

71

-

7.8

Cend T

0

6.9

67

-

7.7

 

T 1

100

7.2

66

4

7.9

T 2

100

7.2

69

0

7.9

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this present test, Diammonium hydrogenorthophosphate was not toxic to waste water (activated sludge) bacteria at 100 mg/l. The 3-hour EC50 of Diammonium hydrogenorthophosphate exceeded 100 mg/l (based on nominal concentrations).
Executive summary:

The influence of Diammonium hydrogenorthophosphate on the respiration rate of activated sludge was investigated after a contact time of 3 hours.

The study procedures described in this report were based on the OECD guideline No. 209, 1984. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No. 440/2008 of 30 May 2008, Publication No. L142, Part C11 and ISO Standard 8192 (2007).

The batch of Diammonium hydrogenorthophosphate tested consisted of white crystals with a purity of 99.8%. A stock solution of 0.5 g/l was prepared in of Milli-RO water. Thorough mixing (vortex) was used to accelerate dissolution and to ensure homogeneity. Volumes of the stock solution corresponding to the test concentration were then added to the test media. A concentration of 100 mg/l was tested in duplicate. Optimal contact between the test substance and test medium was ensured applying continuous aeration and stirring during the 3-hour exposure period. Thereafter, oxygen consumption was measured and recorded for approximately 10 minutes.

No significant inhibition of respiration rate of the sludge was recorded at 100 mg Diammonium hydrogenorthophosphate per litre. The duplicate measurement confirmed the result of the first measurement. The EC50 of Diammonium hydrogenorthophosphate exceeded 100 mg/l (based on nominal concentrations). Since all criteria for acceptability of the test were met, this study was considered to be valid.

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

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 proposed source chemical (diammonium hydrogenorthophosphate) is highly soluble in water (> 10000 mg/L). In aqueous media soluble inorganic orthophosphates will dissociate to their ionic constituents; in this case ammonium and orthophosphate ions. Diammonium dihydrogenpyrophosphate will dissociate to ammonium cations and pyrophosphate anions. The pyrophosphate anions are unstable in aqueous solutions with the degree of instability varying according to pH. In distilled water they will hydrolyse slowly via abiotic mechanisms to orthophosphate. In natural waters a number of different processes can occur; abiotic hydrolysis, biotic degradation (as a result of the action of phosphatases which cleave pyrophosphates into orthophosphate subunits) and assimilation by organisms in the water. Thus the target substance (diammonium dihydrogenpyrophosphate) and the source substance (diammonium hydrogenorthophosphate) will be primarily absorbed as the same inorganic ions: ammonium and orthophosphate and are expected to behave in a similar manner under test conditions.
All (bio) transformation products of the source chemical are common to the target chemical and as such the data is considered to be adequate and reliable for use in the assessment of diammonium dihydrogenpyrophosphate for the ecotoxicity hazard assessment.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report attached.

3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report attached.

4. DATA MATRIX
See read-across justification report attached.

Reason / purpose for cross-reference:
read-across source
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
Table 2 in Section 'Any other information on results incl. tables' below presents the results of the test.

No significant inhibition of respiration rate of the sludge was recorded at 100 mg Diammonium hydrogenorthophosphate per litre. The duplicate measurement confirmed the result of the first measurement. Therefore, no further testing was needed. Hence, the EC50 of Diammonium hydrogenorthophosphate exceeded 100 mg/l (based on nominal concentrations).
Results with reference substance (positive control):
Table 2 in Section 'Any other information on results incl. tables' below presents the results of the test.

The EC50 of 3,5-dichlorophenol was in the accepted range of 5-30 mg/l (6.0 mg/l, see also APPENDIX I).

Table 2: Controls (C) and Diammonium hydrogenorthophosphate (T): pH, oxygen concentration, oxygen consumption and percentage inhibition of the respiration rate

 

Flask

Concentration (mg/L)

Oxygen conc. At the start (approx. equal to mg O2/L)

Oxygen consumption (mg O2/l/h)

% Inhibition respiration rate

pH

Cstart T

0

6.5

71

-

7.8

Cend T

0

6.9

67

-

7.7

 

T 1

100

7.2

66

4

7.9

T 2

100

7.2

69

0

7.9

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this present test, Diammonium hydrogenorthophosphate was not toxic to waste water (activated sludge) bacteria at 100 mg/l. The 3-hour EC50 of Diammonium hydrogenorthophosphate exceeded 100 mg/l (based on nominal concentrations).
Executive summary:

The influence of Diammonium hydrogenorthophosphate on the respiration rate of activated sludge was investigated after a contact time of 3 hours.

The study procedures described in this report were based on the OECD guideline No. 209, 1984. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No. 440/2008 of 30 May 2008, Publication No. L142, Part C11 and ISO Standard 8192 (2007).

The batch of Diammonium hydrogenorthophosphate tested consisted of white crystals with a purity of 99.8%. A stock solution of 0.5 g/l was prepared in of Milli-RO water. Thorough mixing (vortex) was used to accelerate dissolution and to ensure homogeneity. Volumes of the stock solution corresponding to the test concentration were then added to the test media. A concentration of 100 mg/l was tested in duplicate. Optimal contact between the test substance and test medium was ensured applying continuous aeration and stirring during the 3-hour exposure period. Thereafter, oxygen consumption was measured and recorded for approximately 10 minutes.

No significant inhibition of respiration rate of the sludge was recorded at 100 mg Diammonium hydrogenorthophosphate per litre. The duplicate measurement confirmed the result of the first measurement. The EC50 of Diammonium hydrogenorthophosphate exceeded 100 mg/l (based on nominal concentrations). Since all criteria for acceptability of the test were met, this study was considered to be valid.

Description of key information

One key study on an analogous substance exists. This study had been conducted in accordance with a recommended guideline (OECD 209) and under the conditions of GLP. As such, no further testing is considered necessary.

Key value for chemical safety assessment

EC50 for microorganisms:
100 mg/L

Additional information

Under the conditions of this present test, Diammonium hydrogenorthophosphate was not toxic to waste water (activated sludge) bacteria at 100 mg/l. The 3-hour EC50 of Diammonium hydrogenorthophosphate exceeded 100 mg/l (based on nominal concentrations).


Read-across in accordance with Annex XI, Section 1.5 of Regulation (EC) No. 1907/2006 (REACH) is justified on the following basis:


The proposed source chemical (diammonium hydrogenorthophosphate) is highly soluble in water (> 10000 mg/L). In aqueous media soluble inorganic orthophosphates will dissociate to their ionic constituents; in this case ammonium and orthophosphate ions. In aqueous media diammonium dihydrogenpyrophosphate will dissociate to ammonium cations and pyrophosphate anions. The pyrophosphate anions are unstable in aqueous solutions with the degree of instability varying according to pH. In distilled water they will hydrolyse slowly via abiotic mechanisms to orthophosphate. In natural waters a number of different processes can occur; abiotic hydrolysis, biotic degradation (as a result of the action of phosphatases which cleave pyrophosphates into orthophosphate subunits) and assimilation by organisms in the water. Thus the target substance (diammonium dihydrogenpyorophosphate) and the source substance (diammonium hydrogenorthophosphate) will be primarily absorbed as the same inorganic ions: ammonium and orthophosphate and are expected to behave in a similar manner under test conditions. 


 


 All (bio) transformation products of the source chemical are common to the target chemical and as such the data is considered to be adequate and reliable for use in the assessment of diammonium dihydrogenpyrophosphate for the ecotoxicity hazard assessment.