phosphonic acid, [2-(4-aminophenyl)-1-hydroxyethylidene]bis-, monosodium salt

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:

The study was conducted between 17 September 2018 and 05 December 2018.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

This deviation was considered to have had no effect on the study given that the validation criteria for the procedure control vessel were satisfied.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Deviations:

yes

Remarks:

This deviation was considered to have had no effect on the study given that the validation criteria for the procedure control vessel were satisfied.

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3110 (Paragraph (m))

Deviations:

yes

Remarks:

This deviation was considered to have had no effect on the study given that the validation criteria for the procedure control vessel were satisfied.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

phosphonic acid, [2-(4-aminophenyl)-1-hydroxyethylidene]bis-, monosodium salt

EC Number:

828-215-2

Cas Number:

172796-84-8

Molecular formula:

C8H12NO7P2Na

IUPAC Name:

phosphonic acid, [2-(4-aminophenyl)-1-hydroxyethylidene]bis-, monosodium salt

impurity 1

Reference substance name:

4-aminophenylacetic acid

EC Number:

214-828-7

EC Name:

4-aminophenylacetic acid

Cas Number:

1197-55-3

Molecular formula:

C8H9NO2

IUPAC Name:

4-aminophenylacetic acid

impurity 2

Reference substance name:

EBP dimer

Molecular formula:

C16H22N2O12P4

IUPAC Name:

EBP dimer

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Identification: Phosphonic acid, [2-(4-aminophenyl)-1-hydroxyethylidene]bis-,monosodium salt (“EBP”)
CAS Number 172796-84-8
Batch: CHPC071917EBP
Purity: >98% pure
Physical State/Appearance: Beige colored powder
Expiry Date: 01 July 2019
Storage Conditions: Room temperature in the dark

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

Inoculum
A mixed population of activated sewage sludge micro-organisms was obtained on 05 November 2018 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

Preparation of Inoculum
The activated sewage sludge sample was washed twice by settlement and re-suspension in mineral medium to remove any excessive amounts of Dissolved Organic Carbon (DOC) that may have been present. The washed sample was then maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC and used on the day of collection. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the washed activated sewage sludge by suction through pre-weighed GF/A filter paper* using a Buchner funnel. Filtration was then continued for a further 3 minutes after rinsing the filter three successive times with 10 mL of deionized reverse osmosis water. The filter paper was then dried in an oven at approximately 105 ºC for at least 1-Hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 2.4 g/L prior to use.
* Rinsed 3 times with 20 mL deionized reverse osmosis water prior to drying in an oven

Medium
The mineral medium used in this study was that recommended in the OECD Guidelines.
The deionized reverse osmosis water used for the preparation of the mineral medium and the mineral medium used for the test contained less than 1 mg/L Total Organic Carbon (TOC).
Mineral Medium
Solution a KH2PO4 8.50 g/L
K2HPO4 21.75 g/L
Na2HPO4.2H2O 33.40 g/L
NH4Cl 0.50 g/L
pH = 7.4
Solution b CaCl2 27.50 g/L
Solution c MgSO4.7H2O 22.50 g/L
Solution d FeCl3.6H2O 0.25 g/L
(In order to avoid having to prepare solution (d) immediately before use, one drop of concentrated HCl per liter was added as a preservative).
To 1 liter (final volume) of purified water* was added the following volumes of solutions a to d
10 mL of Solution a
1 mL of Solution b
1 mL of Solution c
1 mL of Solution d

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

33.2 mg/L

Based on:

test mat.

Remarks:

10 mg carbon/L

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Preliminary Work
In order to investigate whether the test item adsorbed to filter matrices and/or the activated sewage sludge, the following work was conducted and samples analyzed for Dissolved Organic Carbon (DOC)/Total Organic Carbon (TOC) using a Shimadzu TOC-VCPH TOC analyzer. During sample preparation, samples are either filtered or centrifuged to remove sewage sludge solids.
A nominal amount of test item (200 mg) was dissolved in mineral medium (1 liter) to give a 200 mg/L stock solution. Two samples were taken for analysis; one untreated (TOC) and one filtered (DOC) through a 0.45 μm Gelman AcroCap filter (discarding the initial 5 mL used to pre-condition the filter). A further nominal amount of test item (200 mg) was dissolved in mineral medium and inoculated at a concentration of 30 mg suspended solids (ss)/L prior to adjusting to a final volume of 1 liter. Two samples were taken for DOC analysis; one after filtration through a 0.45 μm Gelman AcroCap filter (discarding the initial 5 mL used to pre-condition the filter) and the other after centrifugation at 4000 g for 15 minutes. Control samples were prepared by inoculating mineral medium (1000 mL) at a suspended solids level of 30 mg ss/L and then filtering or centrifuging as per the test item samples.
In order to confirm the results shown from this work, samples from a further 200 mg/L stock solution were analyzed one untreated (TOC) and one filtered (DOC) through a 0.45 μm Gelman AcroCap filter (discarding the initial 5 mL to pre-condition the filter).

Test Item Preparation
The results from the preliminary work gave results of 75 and 76% of nominal respectively for the initial 200 mg/L samples analyzed neat and filtered and 83 and 84% of nominal from the further samples prepared for TOC analysis. Therefore in order to ensure that the test item vessels were dosed correctly the required amount of test item was dispersed directly in mineral medium with the aid of ultrasonication.
An initial test was terminated after 14 days due to failure of the procedure control to attain greater than 60% biodegradation by Day 14 of the test and additionally there was greater than 20% variation between the inorganic carbon values for the procedure control vessels Replicates 1 and 2.
An amount of test item (99.6 mg) was dispersed in approximately 400 mL of mineral medium with the aid of ultrasonication (15 minutes) prior to dispersal in inoculated mineral medium. The volume was adjusted to 3 liters to give a final concentration of 33.2 mg/L, equivalent to 10 mg carbon/L.
A test concentration of 10 mg carbon/L was employed in the test following the recommendations of the Test Guidelines.

Reference Item Preparation
A reference item, sodium benzoate (C6H5COONa), was used to prepare the procedure control vessels. An initial stock solution of 1000 mg/L was prepared by dissolving the reference item directly in mineral medium. An aliquot (51.4 mL) of this stock solution was added to the test vessel containing inoculated mineral medium prior to the volume being adjusted to 3 liters to give a final test concentration of 17.1 mg/L, equivalent to 10 mg carbon/L. The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution.

Toxicity Control
A toxicity control, containing the test item and sodium benzoate, was prepared in order to assess any toxic effect of the test item on the sewage sludge micro-organisms used in the test.
An amount of test item (99.6 mg) was dispersed in approximately 400 mL of mineral medium with the aid of ultrasonication (15 minutes) prior to dispersal in inoculated mineral medium. An aliquot (51.4 mL) of the sodium benzoate stock solution was also added to the test vessel and the volume adjusted to 3 liters to give a final concentration of 33.2 mg test item/L plus 17.1 mg sodium benzoate/L, equivalent to a total of 20 mg carbon/L.

Preparation of Test System
The following test preparations were prepared and inoculated in 5 liter test culture vessels each containing 3 liters of solution:
a) An inoculated control, in duplicate, consisting of inoculated mineral medium.
b) The procedure control containing the reference item (sodium benzoate), in duplicate, in inoculated mineral medium give a final concentration of 10 mg carbon/L.
c) The test item, in duplicate, in inoculated mineral medium to give a final concentration of 10 mg carbon/L.
d) The test item plus the reference item in inoculated mineral medium to give a final concentration of 20 mg carbon/L to act as a toxicity control (one vessel only).
Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/L. The test was carried out in a temperature controlled room at temperatures of between 21 °C and 24 °C for 28 days, in darkness.
Approximately 24 hours prior to addition of the test and reference items the vessels were filled with 2400 mL of mineral medium and 37.5 mL of inoculum and aerated overnight. On Day 0 the test and reference items were added and the pH of all vessels measured using a Hach HQ40d Flexi handheld meter and the pH was adjusted to pH 7.4 ± 0.2 using diluted hydrochloric acid or sodium hydroxide solution prior to the volume in all of the vessels being adjusted to 3 liters by the addition of mineral medium, which had been purged overnight with CO2-free air. The inoculum control vessels were prepared in a similar manner without the addition of test item or reference item.
The test vessels were sealed and CO2-free air bubbled through the solution at a rate of 20 to 100 mL/minute per vessel and stirred continuously by magnetic stirrer.
The CO2-free air was produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb®) granules.
The CO2 produced by degradation was collected in two 500 mL Dreschel bottles containing 350 mL of 0.05 M NaOH. The CO2 absorbing solutions were prepared using purified water.

Assessments
Observations
The appearance of the test preparations was recorded on Days 0, 7, 14, 21 and 28.

pH Measurements
The pH of the test preparations was determined on Day 0 and on Day 28 prior to acidification with hydrochloric acid, using a Hach HQ40d Flexi handheld meter.

Inorganic Carbon Analysis
Samples (2 mL) were taken from the first CO2 absorber vessels on Days 0, 2, 6, 8, 10, 14, 21, 28 and 29. The second absorber vessels were sampled on Days 0 and 29.
All samples were analyzed for Inorganic Carbon (IC) immediately with the exception of the Day 0 Absorber 2 samples.
The remainder of all samples with the exception of the Day 0 Absorber 1 and 2 and Day 29 Absorber 2 samples were frozen for further analysis if required.
On Day 28, 1 mL of concentrated hydrochloric acid was added to each vessel to drive off any inorganic carbonates formed. The vessels were resealed, aerated overnight and the final samples taken from both absorber vessels on Day 29.
The samples were analyzed for IC using either a Shimadzu TOC-VCSH TOC analyzer, a Shimadzu TOC-VCPH TOC analyzer or a Shimadzu TOC-LCSH TOC analyzer. Samples (50 or 135 μL) were injected into the IC channel of the TOC analyzer. IC analysis occurs by means of the conversion of an aqueous sample to CO2 by orthophosphoric acid or 2M HCl using zero grade air as the carrier gas. Calibration was by reference solutions of sodium carbonate (Na2CO3). Each analysis was carried out in at least triplicate with three replicates being used in the calculation.

Inorganic Carbon/Total Carbon Ratio
Samples (30 mL) were removed from the test item vessels on Day 0 prior to the addition of the test item in order to calculate the IC content in the test media. The samples were filtered through 0.45 µm Gelman AcroCap filters (first approximate 5 mL used to pre condition the filter was discarded) prior to DOC analysis. Samples (30 mL) were also removed from the inoculum control vessels on Day 0 and filtered through 0.45 µm Gelman AcroCap filters (first approximate 5 mL used to pre condition the filter was used) prior to DOC analysis.
Inorganic Carbon/Total Carbon analysis of the test item dispersions after dosing was not possible due to the low near nominal results from the preliminary work performed to measure the TOC of neat and filtered samples. Therefore in order to ensure that the test item vessels were dosed correctly the required amount of test item was dispersed directly in mineral medium with the aid of ultrasonication.
The samples were analyzed for IC and Total Carbon (TC) using a Shimadzu TOC VCPH TOC Analyzer. Samples (50 µL) were injected into the TC and IC channels of the TOC analyzer. TC analysis is carried out at 680 °C using a platinum based catalyst and zero grade air as the carrier gas. IC analysis involves conversion by orthophosphoric acid at ambient temperature. Calibration was performed using reference solutions of potassium hydrogen phthalate (C8H5KO4) and sodium carbonate (Na2CO3) in deionized water. Each analysis was carried out in at least triplicate with three replicates being used in the calculation.

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

Preliminary study:

The results from the preliminary work gave results of 75 and 76% of nominal respectively for the initial 200 mg/L samples analyzed neat and filtered and 83 and 84% of nominal from the further samples prepared for TOC analysis. Therefore in order to ensure that the test item vessels were dosed correctly the required amount of test item was dispersed directly in mineral medium with the aid of ultrasonication.

Test performance:

The total CO2 evolution in the inoculum control vessels on Day 28 was 28.04 mg/L and therefore satisfied the validation criterion given in the OECD Test Guidelines.
The IC content of the test item suspension in the mineral medium at the start of the test was below 5% of the TC content and hence satisfied the validation criterion given in the OECD Test Guidelines.
The difference between the values for CO2 production at the end of the test for the replicate vessels was <20% and hence satisfied the validation criterion given in the OECD Test Guidelines.
Acidification of the test vessels on Day 28 followed by the final analyses on Day 29 was conducted according to the methods specified in the Test Guidelines. This acidification effectively kills the micro organisms present and drives off any dissolved CO2 present in the test vessels. Therefore any additional CO2 detected in the Day 29 samples originated from dissolved CO2 that was present in the test vessels on Day 28 and hence the biodegradation value calculated from the Day 29 analyses is taken as being the final biodegradation value for the test item.
The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed an increase in all replicate vessels with the exception of inoculum control Replicate 1 and test item Replicate 2.
The IC analysis of the samples from the second absorber vessels on Day 29 confirmed that no significant carry-over of CO2 into the second absorber vessels occurred.
The toxicity control attained 34% biodegradation after 14 days and 35% biodegradation after 28 days thereby confirming that the test item did not exhibit an inhibitory effect on the sewage treatment micro organisms used in the test.

% Degradation

Key result

Parameter:

% degradation (CO2 evolution)

Value:

10

Sampling time:

28 d

Details on results:

See "Any Other Information on Results"

BOD5 / COD results

Results with reference substance:

Sodium benzoate attained 67% biodegradation after 14 days with greater than 60% degradation being attained in a 10-day window. After 28 days 80% biodegradation was attained. These results confirmed the suitability of the inoculum and test conditions and satisfied the validation criterion given in the OECD Test Guidelines.

Any other information on results incl. tables

Inorganic Carbon Values on Each Analysis Occasion

Day	Inorganic Carbon (mg IC)
	Inoculum Control				Procedure Control				Test Item				Toxicity Control
	R1		R2		R1		R2		R1		R2		R1
	Abs1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2
0	0.70	0.35	0.58	0.58	0.35	1.17	0.35	1.40	0.58	1.40	0.35	1.40	0.35	1.40
2	6.96	-	7.19	-	18.21	-	26.10	-	8.93	-	10.44	-	25.99	-
6	11.88	-	12.11	-	27.57	-	32.52	-	12.11	-	13.95	-	33.79	-
8	12.96	-	15.48	-	29.81	-	36.92	-	14.33	-	14.56	-	36.23	-
10	15.39	-	16.07	-	33.75	-	37.51	-	17.33	-	16.65	-	38.07	-
14	17.34	-	20.51	-	38.08	-	39.78	-	19.95	-	21.87	-	39.55	-
21	21.86	-	23.43	-	44.28	-	42.93	-	25.12	-	23.89	-	45.18	-
28	22.62	-	23.29	-	46.14	-	43.23	-	26.99	-	24.42	-	42.56	-
29	22.49	1.51	23.60	1.97	46.87	2.09	46.54	2.09	27.95	2.09	23.27	2.09	43.57	2.09

R  = Replicate

Abs = CO₂absorber vessel

Percentage Biodegradation Values

Day	Biodegradation (%)
	Procedure Control	Test Item	Toxicity Control
0	0	0	0
2	50	9	32
6	60	3	36
8	64	1	37
10	66	4	37
14	67	7	34
21	70	6	38
28	72	9	33
29*	80	10	35

*Day 29 values corrected to include any carry-over of CO₂detected in Absorber 2

Total and Inorganic Carbon Values in the Culture Vessels on Day 0

Test vessel	Total Carbon* (mg/L)	Inorganic Carbon^*(mg/L)	IC Content (% of TC)
Test Item 10 mg C/L R1	9.92**	0.08	1
Test Item 10 mg C/L R2	10.00^**	0.09	1

R = Replicate

* Corrected for control values

** TC value given is the sum of the TC value obtained from analysis and the nominal TC contribution of the test item

pH Values of the Test Preparations on Days 0 and 28

Test Vessel	pH
	Day 0 Pre-Adjustment	Day 0 Post-Adjustment	Day 28
Inoculum Control R1	7.8	7.4	7.4
Inoculum Control R2	7.7	7.4	7.4
Procedure Control R1	7.7	7.4	7.4
Procedure Control R2	7.7	7.4	7.4
Test Item R1	7.7	7.4	7.4
Test Item R2	7.7	7.4	7.4
Toxicity Control	7.7	7.4	7.3

R = Replicate

Observations on the Test Preparations Throughout the Test Period

Test Vessel		Observations on Test Preparations
		Day 0	Day 7	Day 14	Day 21	Day 28
Inoculum Control	R1	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion
	R2	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion
Procedure Control	R1	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible
	R2	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible
Test Item	R1	Light brown dispersion, no undissolved test item visible	Light brown dispersion, no undissolved test item visible	Light brown dispersion, no undissolved test item visible	Light brown dispersion, no undissolved test item visible	Light brown dispersion, no undissolved test item visible
	R2	Light brown dispersion, no undissolved test item visible	Light brown dispersion, no undissolved test item visible	Light brown dispersion, no undissolved test item visible	Light brown dispersion, no undissolved test item visible	Light brown dispersion, no undissolved test item visible
Toxicity Control		Light brown dispersion, no undissolved test or reference item visible	Light brown dispersion, no undissolved test or reference item visible	Light brown dispersion, no undissolved test or reference item visible	Light brown dispersion, no undissolved test or reference item visible	Light brown dispersion, no undissolved test or reference item visible

R = Replicate

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item attained 10% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Executive summary:

Introduction

A study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No. 301B, "Ready Biodegradability; CO₂ Evolution Test" referenced as Method C.4-C of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3110 (Paragraph (m)).

Methods

The test item, at a concentration of 10 mg carbon/L, was exposed to activated sewage sludge micro-organisms with mineral medium in sealed culture vessels in the dark at temperatures of between 21 °C and 24 °C for 28 days.

The biodegradation of the test item was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference item, sodium benzoate, together with a toxicity control were used for validation purposes.

Results

The test item attained 10% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Sodium benzoate attained 67% biodegradation after 14 days with greater than 60% degradation being attained in a 10-day window. These results confirmed the suitability of the inoculum and test conditions and satisfied the validation criterion given in the OECD Test Guidelines.