Phenylphosphonic acid

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:

16 Aug 2017 to 05 Nov 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:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Phenylphosphonic acid

EC Number:

216-388-1

EC Name:

Phenylphosphonic acid

Cas Number:

1571-33-1

Molecular formula:

C6H7O3P

IUPAC Name:

phenylphosphonic acid

Test material form:

solid: particulate/powder

Details on test material:

Lot number LKF6065

Specific details on test material used for the study:

Appearance: White crystalline powder
Batch: LKF6065
Test item storage: At room temperature protected from light
Stable under storage conditions until: 09 December 2017 (expiry date)

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, industrial (adaptation not specified)

Details on inoculum:

Source: The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.

Treatment: The freshly obtained sludge was used immediately. The concentration of suspended solids was determined to be 4.2 g/L in the concentrated sludge. Before use, the sludge was allowed to settle (33 minutes) and the supernatant liquid was used as inoculum at the amount of 10 mL/L of mineral medium.

Reason for selection: The test has been accepted internationally for determining the 'ready' biodegradability of test items under aerobic conditions.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

26.5 mg/L

Based on:

test mat.

Remarks:

corresponding to 12 mg TOC/L

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS

Test Procedure and Conditions
Test duration 28 days for the inoculum blank and test item (last CO2 measurement on day 29). 14 days for the positive and toxicity control (last CO2 measurement on day 15). During the test period, the test media were aerated and stirred continuously.
Test vessels: 2 litre brown coloured glass bottles.
Milli-RO water: Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon.

Stock solutions of mineral components:
A)8.50 g KH2PO4
21.75 g K2HPO4
67.20 g Na2HPO4.12H20
0.50 g NH4Cl
dissolved in Milli-RO water and made up to 1 litre, pH 7.4 +/- 0.2
B)22.50 g MgSO4.7H2O dissolved in Milli- RO water and made up to 1 litre.
C)36.40 g CaCl2.2H2O dissolved in Milli-RO water and made up to 1 litre.
D)0.25 g FeCl3.6H2O dissolved in Milli-RO water and made up to 1 litre.

Mineral medium: 1 litre mineral medium contains: 10 mL of solution (A), 1 mL of solutions (B) to (D) and Milli- RO water.
Barium hydroxide: 0.0125 M Ba(OH)2 (Boom, Meppel, The Netherlands), stored in a sealed vessel to prevent absorption of CO2 from the air.
Synthetic air (CO2 < 1 ppm)1: A mixture of oxygen (ca. 20%) and nitrogen (ca. 80%) was
passed through a bottle, containing 0.5 - 1 litre 0.0125 M Ba(OH)2 solution to trap CO2 which might be present in small amounts. The synthetic air was passed through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min).
Illumination: The test media were excluded from light.

Preparation of Bottles
Pre-incubation medium: The day before the start of the test (day -1) mineral components, Milli-RO water (ca. 80% of final volume) and inoculum (1% of final volume) were added to each bottle. This mixture was aerated with synthetic air overnight to purge the system of CO2.

Type and number of bottles: Test suspension: containing test item and inoculum (2 bottles).
Inoculum blank: containing only inoculum (2 bottles)
Positive control: containing reference item and inoculum (1 bottle).
Toxicity control: containing test item, reference item and inoculum (1 bottle).
Preparation: At the start of the test (day 0), test and reference item were added to the bottles containing the microbial organisms and mineral components. The volumes of suspensions were made up to 2 litres with Milli-RO water, resulting in the mineral medium described before. Three CO2-absorbers (bottles filled with 100 mL 0.0125 M Ba(OH)2) were connected in series to the exit air line of each test bottle.

Determination of CO2
Experimental CO2 production: The CO2 produced in each test bottle reacted with the barium hydroxide in the gas scrubbing bottle and precipitated out as barium carbonate. The amount of CO2 produced was determined by titrating the remaining Ba(OH)2 with 0.05 M standardized HCl (1:20 dilution from 1 M HCl (Titrisol® ampoule), Merck, Darmstadt, Germany).
Measurements: Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until day 28, for the inoculum blank and test item. Titrations for the positive and toxicity control were made over a period of at least 14 days. Each time the CO2-absorber nearest to the test bottle was removed for titration; each of the remaining two absorbers were moved one position in the direction of the test bottle. A new CO2-absorber was placed at the far end of the series. Phenolphthalein (1% solution in ethanol, Merck) was used as pH-indicator.

On the penultimate day, the pH of respective test suspensions was measured and 1 mL of concentrated HCl (37%, Merck) was added to the bottles of the inoculum blank and test suspension. The bottles were aerated overnight to drive off CO2 present in the test suspension. The final titration was made on day 15 (positive and toxicity control) and on day 29 (remaining vessels).

Theoretical CO2 production: The theoretical CO2 production was calculated from the molecular formula.

Measurements and Recordings
pH: At the start of the test (day 0) and on the penultimate day (day 14 for the positive and toxicity control and day 28 for the inoculum blanks and test item), before addition of concentrated HCl.
Temperature of medium: Continuously in a vessel with Milli-RO water in the same room.

CONTROL AND BLANK SYSTEM
Test suspension: containing test item and inoculum (2 bottles).
Inoculum blank: containing only inoculum (2 bottles)
Positive control: containing reference item and inoculum (1 bottle).
Toxicity control: containing test item, reference item and inoculum (1 bottle).


STATISTICAL METHODS:

Reference substance

Reference substance:

other: sodium acetate

Results and discussion

% Degradation

Parameter:

% degradation (CO2 evolution)

Value:

>= 1 - <= 3

Sampling time:

28 d

Details on results:

Details of results can be found in the attached background material section below. The
Theoretical CO2 production (ThCO2) of Phenyl Phosphonic Acid was calculated to be 1.67 mg CO2/mg.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

In conclusion, Phenyl Phosphonic Acid was not readily biodegradable under the conditions of the modified Sturm test presently performed.

Executive summary:

The objective of the study was to evaluate the non-volatile test item Phenyl Phosphonic Acid for its ready biodegradability in an aerobic aqueous medium with microbial activity introduced by inoculation with the supernatant of activated sludge; Carbon dioxide (CO2)evolution test (modified Sturm test).

The study procedures described in this report were in compliance with the OECD guideline No. 301 B, 1992.

Phenyl Phosphonic Acid was a white crystalline powder with a purity of 100.0% (assay). The test item was tested in duplicate at a concentration of 26.5 mg/L, corresponding to 12 mg TOC/L. The organic carbon content was based on the molecular formula. The Theoretical CO2 production (ThCO2) of Phenyl Phosphonic Acid was calculated to be 1.67 mg CO2/mg.

The study consisted of six bottles:  

- 2 inoculum blanks (no test item),

- 2 test bottles (Phenyl Phosphonic Acid),

- 1 positive control (sodium acetate) and

- 1 toxicity control (Phenyl Phosphonic Acid plus sodium acetate).

Since Phenyl Phosphonic Acid was easily soluble in water the test media were prepared using a stock solution of 1 g/L in Milli- RO water. Aliquots of 55 mL of the clear and colourless stock solution were added to the test item bottles A and B and to the toxicity control. These test bottles contained medium with microbial organisms. The volumes of suspensions were made up to 2 litres with Milli-RO water. The test solutions were continuously stirred during the test, to ensure optimal contact between the test item and the test organisms. Test duration was 28 days for the inoculum blank and test item (last CO2 measurement on day 29) and 14 days for the positive and toxicity control (last CO2 measurement on day 15).

The relative biodegradation values calculated from the measurements performed during the test period revealed no biological relevant biodegradation of Phenyl Phosphonic Acid (3% and 1%, based on ThCO2).

In the toxicity control, Phenyl Phosphonic Acid was found not to inhibit microbial activity.

Since all criteria for acceptability of the test were met, this study was considered to be valid.

In conclusion, Phenyl Phosphonic Acid was designated as not readily biodegradable.