p-nitrophenyl dihydrogen phosphate, compound with 2-amino-2-(hydroxymethyl)propane-1,3-diol (1:2)

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:

1 - 29 September 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

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

impurity 1

Reference substance name:

Methanol

EC Number:

200-659-6

EC Name:

Methanol

Cas Number:

67-56-1

Molecular formula:

CH4O

IUPAC Name:

methanol

impurity 2

Reference substance name:

Acetone

EC Number:

200-662-2

EC Name:

Acetone

Cas Number:

67-64-1

Molecular formula:

C3H6O

IUPAC Name:

acetone

impurity 3

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Oxidane

Constituent 1

Reference substance name:

p-nitrophenyl dihydrogen phosphate, compound with 2-amino-2-(hydroxymethyl)propane-1,3-diol (1:2)

EC Number:

269-198-6

EC Name:

p-nitrophenyl dihydrogen phosphate, compound with 2-amino-2-(hydroxymethyl)propane-1,3-diol (1:2)

Cas Number:

68189-42-4

Molecular formula:

C6H6NO6P.2C4H11NO3

IUPAC Name:

4-nitrophenyl dihydrogen phosphate - 2-amino-2-(hydroxymethyl)propane-1,3-diol (1:2)

Test material form:

solid

Details on test material:

Batch / Lot No. 46587100

Specific details on test material used for the study:

Batch no.: 46587100
Storage conditions: 2–8 °C, protected from light

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, domestic, non-adapted

Details on inoculum:

SOURCE:
From the aeration tank of the ARA Werdhölzli (CH-8048 Zürich), a municipal biological wastewater treatment plant.

PREPARATION OF INOCULUM FOR EXPOSURE:
The sludge was pre-conditioned to reduce the amount of O2 consumed in the blank controls. In the pre-conditioning procedure, the sludge was washed twice with tap water and once with test medium right after sampling from the wastewater treatment plant. After centrifugation, the sludge was suspended in test medium at 3–5 g/l dry matter and kept under constant aeration and stirring without feeding for five days, until test start. Before the start of the test the actual dry matter content of this suspension was determined. Based on the dry matter content of the sludge suspension it was diluted to 60 mg/l dry matter (i.e. twice as high as the final test concentration) and homogenised.

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):

28 d

Initial test substance concentration

Initial conc.:

72.1 mg/L

Based on:

other: nominal test item concentration based on 100 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: Analytical grade salts were dissolved in ultra-pure water:
* mineral stock solution A: 8.5 g/L KH2PO4, 28.49 g/L K2HPO4.3H2O, 33.4 g/L Na2HPO4.2H2O, 0.5
0 g/L NH4Cl
* mineral stock solution B: 36.4 g/L CaCl2.2H2O
* mineral stock solution C: 22.5 g/L MgSO4.7H2O
* mineral stock solution D: 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
The pH value of the test medium was adjusted to 7.4±0.2.
- Test temperature: 22±2°C, controlled at ±1 °C, in a thermostat cabinet in the dark
- pH: 7.4±0.2 (measured prior to testing and if necessary adjusted with NaOH or HCl (except in the abiotic sterile control)
- Continuous darkness: yes, test bottles were in a thermostat cabinet.

TEST SYSTEM:
- Test units: 510 ml glass flasks (tightly closed with manometric BOD measuring devices) containing a total volume of test solution of 200 ml. The flasks are equipped with stirring rods and butyl rubber quivers which contain two pellets of sodium hydroxide each to absorb the produced CO2 from the head space.
- Test performed in duplicate (two test flasks):The test item was applied by stock solution (test item in test medium; double concentrated to account for the addition of the sludge suspension).

CONTROL AND BLANK SYSTEM:
B: Blank control (two replicates): containing incolum and test medium
P: Procedure control (two replicates): containing inoculum, test medium and 60.2 mg/L reference
substance (100 mg ThOD/L)
X: Toxicity control (one replicate): containing inoculum, test medium, 72.5 mg/L test item and 60.2 mg/L reference substance (total 201 mg ThOD/L)
A: Abiotic sterile control (one replicate): containing inoculum, ultra-pure water, 0.04 mM HgCl2 (sterilizing agent to prevent microbial degradation) and 72.0 mg/L test item (99.9 mg ThOD/L)

SAMPLING:
The flasks were stirred by an inductive stirring system for the whole test period. During the test the O2 uptake (and by extension the biodegradation) was continuously measured with a manometric BOD measuring device. The temperature was recorded with a data logger. At the end of the test, the pH was measured, and samples for DOC analysis were taken, if applicable. At the end of the test, the pH was measured in all flasks except the abiotic sterile control (A). 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 total organic carbon concentration at the start of the test and the analysis of the dissolved organic carbon concentrations at the end of the test.

STATISTICAL METHODS:
No statistical analysis was performed.

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

% Degradation

Key result

Parameter:

% degradation (O2 consumption)

Value:

89

Sampling time:

28 d

Remarks on result:

other: test flask 1 after 28 d: 89% test flask 2 after 28 d: 89%

Details on results:

Based on O2 consumption the biodegradability of 4-NPP, di-TRIS was calculated to be 89% of the ThOD after 28 days and 75% at the end of the 10-day window (i.e. within 10 days after attainment of 10% degradation). Biodegradation of the test item was observed after a lag phase, as defined in the OECD guideline 301, of about two days.

Based on the determination of the organic carbon at the start and end of the test, the total elimination was calculated to be 96% for 4-NPP, di-TRIS and 99% for sodium benzoate. This data is consistent with the degradation determined based on O2 consumption.

BOD5 / COD results

Results with reference substance:

The procedure control with sodium benzoate (i.e. benzoic acid, sodium salt) reached a biodegradation of 80% after 14 days, thus confirming suitability of inoculum and test conditions.

Any other information on results incl. tables

TOXICITY CONTROL:

According to the OECD guideline 301 a substance is considered having inhibitory (i.e. toxic) effects on the inoculum if less than 25% degradation after 14 days are observed in the toxicity control. Since the biodegradation exceeded this pass level, it can be concluded that the test item does not have any significant toxic effects on the microbial population at the applied initial test concentration of 72.5 mg/l.

ABIOTIC STERIL CONTROL:

No degradation (0%) was observed in the abiotic sterile control. Therefore, it can be concluded that the test item is not degraded by oxygen consuming processes in the absence of microorganisms.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

4-NPP, di-TRIS (CAS no. 68189-42-4) reached the pass level of 60% biodegradation in the manometric respirometry test within the 10-day window and, therefore, can be termed as readily biodegradable.

Executive summary:

The biodegradability of 4-NPP, di-TRIS (CAS no. 68189-42-4) exposed to microorganisms derived from activated sludge of a municipal sewage treatment plant was investigated under aerobic static exposure conditions following the OECD guideline 301 F. The biodegradation was assessed based on O2 consumption compared to the theoretical oxygen demand ThOD.

Based on O2 consumption the biodegradation of 4-NPP, di-TRIS was calculated to be 89% of ThOD after 28 days and reached 75% at the end of the 10-day window (i.e. within 10 days after attainment of 10% degradation).

Biodegradation of the test item was observed after a lag phase, as defined in the OECD guideline 301, of about two days.

The procedure control with sodium benzoate reached a biodegradation of 80% after 14 days thus confirming suitability of inoculum and test conditions.

In addition, the total elimination was calculated for 4-NPP, di-TRIS and sodium benzoate based on the determination of the DOC at the end of the test. The obtained data is consistent with the degradation determined based on O2 consumption.

4-NPP, di-TRIS reached the pass level of 60% biodegradation in the manometric respirometry test within the 10-day window and therefore can be termed as readily biodegradable.

All validity criteria were fulfilled.