Disodium 4-nitrophenyl phosphate

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2 August - 27 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Version / remarks:

2006

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Version / remarks:

Regulation (EC) No 440/2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 4-nitrophenyl phosphate

EC Number:

224-246-5

EC Name:

Disodium 4-nitrophenyl phosphate

Cas Number:

4264-83-9

Molecular formula:

C6H4NO6P.2Na

IUPAC Name:

disodium 4-nitrophenyl phosphate

impurity 1

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Oxidane

Test material form:

solid

Details on test material:

Batch Nr: 11674900
Storage: 2-8°C.

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source: Sludge from the aeration tank of the ARA Werdhölzli (CH-8048 Zürich), a municipal biological waste water treatment plant.
- Preparation of inoculum for exposure: The sludge was pre-conditioned for 5 days (aerated but not fed) to reduce the amount of O2 consumed by the blank controls. Therefore, the sludge was washed twice with tap water and once with test medium.
- Pretreatment: The activated sludge was used after sampling from the treatment plant without adaptation.
- Concentration of sludge: 30 mg/L dry matter in the final mixture.

Duration of test (contact time):

56 d

Initial test substance concentration

Initial conc.:

155 mg/L

Based on:

other: nominal test item concentration based on 94.5 mg ThOD/L

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of test medium: Aerobic mineral salts medium prepared with ultrapure water (conductivity: <1.5 µS/cm; DOC: <0.5 mg/L)
* mineral stock solution A (10mL/L): 8.5 g/L KH2PO4, 28.49 g/L K2HPO4.3H2O, 33.4 g/L Na2HPO4.2H2O, 0.5 g/L NH4Cl, pH 7.4
* mineral stock solution B (1 mL/L): 36.4 g/L CaCl2.2H2O
* mineral stock solution C (1 mL/L): 22.5 g/L MgSO4.7H2O
* mineral stock solution D (1 mL/L): 0.25 g/L FeCl3.6H2O
* Final test medium: 10 mL of solution A and 1 mL of solutions B, C and D per L of test medium

- Test temperature: 22±2°C, controlled at ± 1°C, in a thermostat cabinet
- pH: 7.4±0.2°C (measured prior to testing and if necessary adjusted with NaOH or HCl (except in flask C).
- Continuous darkness: yes, test bottles were in a thermostat cabinet.

TEST SYSTEM
- Test flasks: 510 mL glass bottles (tightly closed with manometric BOD measuring devices) containing a total volume of test solution of 200 mL. The bottles were equipped with stirring rods and butylrubber quivers which contain 2 pellets of sodium hydroxide each to absorb the produced CO2 from the head space.
- Test performed in duplicate (two test flasks)

After centrifugation, the sludge was suspended in test medium, at about 2 g/L dry matter. Before the test, this suspension was diluted down to 60 mg/L dry matter, i.e. twice the final concentration, since this suspension was diluted 1:1 (v:v) afterwards. The test item was dissolved in the test medium at a concentration twice of the final concentration to be achieved for the test. This stock solution was diluted with the sludge suspension 1:1 (v:v) to give a final test concentration of about 100 mg ThOD/l.

CONTROL AND BLANK SYSTEM
B: Inoculum blank (two replicates)
R: Procedure control (two replicates): 60.1 mg/L Sodium benzoate (100 mg ThOD/L)
C: Abiotic sterile control (one replicate): 156 mg/L test item (94.7 mg ThOD/L)
X: Toxicity control (one replicate): 156 mg/L test item and 60.1 mg/L reference item (total 195 mg oxygen demand/L)

SAMPLING
The test vessels were stirred by an inductive stirring system. During the test the O2 uptake was continuously measured with a manometric BOD measuring device. Temperature was recorded with a data logger.
At the end of the test, the pH was measured in all flasks except the abiotic sterile control (C).
Total elimination was determined in each test vessel (test suspension, blank and procedure control) based on the theoretical carbon content and the analysis of the dissolved organic carbon (DOC) concentrations at the end of the test.

STATISTICAL METHODS:
Values of % degradation were calculated for each test flask and day. The arithmetic mean of % degradation in each test flask on each day was calculated.

Reference substance

Reference substance:

other: Sodium benzoate

Remarks:

CAS 532-32-1

Results and discussion

% Degradation

% Degradation 1

Key result

Parameter:

% degradation (O2 consumption)

Value:

63

Sampling time:

28 d

Remarks on result:

other: test flask 1 after 28 d: 61.1% test flask 2 after 28 d: 65.5%

% Degradation 2

Parameter:

% degradation (O2 consumption)

Value:

ca. 80

Sampling time:

41 d

Remarks on result:

other: test flask 1 after 41 d: 82.9%, test flask 2 after 41 d: 74.1%

Details on results:

The biodegradability of 4-Nitrophenyl phosphate disodium salt based on O2 consumption was calculated to be 63% after 28 days, as compared to the theoretical oxygen demand (ThOD). An extension of the test showed that the biodegradability after 41 days was about 80%. The biodegradation reached about 36% at the end of the 10-d window (i.e. within 10 days after attainment of 10% degradation).
Biodegradation of the test item was observed without any lag-phase.

The respective concentrations of organic carbon at the beginning (theoretical value based on the test item’s carbon content and the test concentration, as mg TOC/L) and at the end of the test (56 days of incubation; mean measured value of the two replicates, as mg DOC/L) were, respectively:
- 31.0 and 2.18 for the test units (mean of two replicates)
- <0.5 and 1.75 for the blank control (mean of two replicates)
- 35.0 and 2.07 for the procedure control (mean of two replicates)
The total elimination calculated based on dissolved organic carbon (DOC) measurements reached 99% for both 4-Nitrophenyl phosphate disodium salt and sodium benzoate. This data is in line with the degradation calculated based on O2 consumption.

4-Nitrophenyl phosphate disodium salt (CAS no. 4264-83-9) did not reach the pass level of 60% for ready biodegradability in the Manometric Respirometry Test within the 10-d window and, therefore, cannot be termed as readily biodegradable. However, the pass level was reached within 28 days of incubation. The biodegradation further progressed constantly after 28 days until the end of the observation period, as demonstrated by the results of the test extension.

Any other information on results incl. tables

Procedure control:

The procedure control sodium benzoate reached 85% biodegradation after 14 days, thus confirming suitability of inoculum and test conditions.

Toxicity control:

At the applied initial test concentration of 156 mg/L the test item was not judged to have any inhibitory effect on the microbial population, since the biodegradation of the mixture (test item + reference item sodium benzoate) exceeded 25% within 14 days.

Abiotic steril control:

The oxygen consumption in the abiotic sterile control seems significant, as it is about half of the oxygen consumption of the blank control, which contains inoculum. However, since no flask can be compared to this sterile control, and since the temperature at the beginning of the test was not exactly 22°C but almost 23°C, it is difficult to assess whether 4 -Nitrophenyl phosphate disodium salt was actually abiotically degraded (by processes using O₂).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable, but failing 10-day window

Conclusions:

A ready biodegradability test (OECD 301F, Manometric Respirometry Test) using activated sludge of a municipal sewage treatment plant indicated that 4-Nitrophenyl phosphate disodium salt (CAS no. 4264-83-9) did not reach the pass level of 60% for ready biodegradability in the Manometric Respirometry Test within the 10-d window and, therefore, cannot be termed as readily biodegradable. However, the pass level was reached within 28 days of incubation. The biodegradation further progressed constantly after 28 days until the end of the observation period, as demonstrated by the results of the test extension. A biodegradation of about 80% was reached at day 41 based on O2 consumption.

Executive summary:

The biodegradability of 4-Nitrophenyl phosphate disodium salt (CAS no. 4264-83-9) exposed to microorganisms derived from activated sludge of a municipal sewage treatment plant was investigated under aerobic static exposure conditions, following the test guideline OECD 301 F. Both the mineralisation over the course of the test (based on O₂ consumption) and the total elimination at the end of the test (based on the determination of the dissolved organic carbon, DOC) were assessed. The biodegradability of 4-Nitrophenyl phosphate disodium salt based on O₂ consumption was calculated to be 63% after 28 days, as compared to the theoretical oxygen demand (ThOD). An extension of the test showed that the biodegradability after 41 days was about 80%. The biodegradation reached about 36% at the end of the 10 -d window (i.e. within 10 days after attainment of 10% degradation). Biodegradation of the test item was observed without any lag-phase.

The procedure control sodium benzoate reached 85% biodegradation after 14 days, thus confirming suitability of inoculum and test conditions. The total elimination calculated based on dissolved organic carbon (DOC) measurements reached 99% for both 4 -Nitrophenyl phosphate disodium salt and sodium benzoate. This data is in line with the degradation calculated based on O₂ consumption.

4-Nitrophenyl phosphate disodium salt (CAS no.4264-83-9) did not reach the pass level of 60% for ready biodegradability in the Manometric Respirometry Test within the 10-d window and, therefore, cannot be termed as readily biodegradable. However, the pass level was reached within 28 days of incubation. The biodegradation further progressed constantly after 28 days until the end of the observation period, as demonstrated by the results of the test extension. All validity criteria were fulfilled.