Inositol phosphates

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:

From April 7, 2009 to June 5, 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

A) The study was conducted according to OECD Guideline 301B, Method C.4-C of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110(Paragraph (m)). Compliance to GLP has also been claimed through the UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI2004/0994). B) Well-defined information were provided on: 1. Composition of the test substance (identity and purity) 2. Test method used 3. Test results 4. Definite conclusion

Justification for data waiving:

other:

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3100 (Aerobic Aquatic Biodegradation)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI2004/0994)

Test material

Test material information

Constituent 1

Reference substance name:

Inositol phosphates

EC Number:

700-206-7

Molecular formula:

As this substance is a UVCB substance and consists varying degree of components, a single molecular formula is not applicable

IUPAC Name:

Inositol phosphates

Constituent 2

Reference substance name:

Synthetic inositol phosphates

IUPAC Name:

Synthetic inositol phosphates

Details on test material:

- Name of test material (as cited in study report): Synthetic Inositol phosphates
- Physical state: Amber coloured liquid
- Lot/batch No.: 95816206JH-012
- Storage condition of test material: Room temperature in the dark

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
None

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Aeration tank of Severn Trent Water Plc Sewage Treatment Plant at Loughborough, Leicestershire, UK (treats predominantly domestic sewage)
- Storage conditions: Kept under aeration at a temperature of approx. 21 °C till the test
- Preparation of inoculum for exposure: Washed three times by settlement and resuspevsion in culture medium to remove any escessive amounts ofdissolved organic carbon
- Concentration of sludge: 3.1 g/L

Duration of test (contact time):

ca. 28 d

Initial test substance concentration

Initial conc.:

ca. 10 other: mg carbon/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation 1

Parameter followed for biodegradation estimation:

inorg. C analysis

Parameter followed for biodegradation estimation 2

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

TEST CONDITIONS
- Composition of 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

To 1 litre (final volume) of purified water was added the following volumes of solutions a-d:
10 mL of solution a
1 mL of solution b
1 mL of solution c
1 mL of solution d
- Test temperature: 21 °C
- Suspended solids concentration: 30 mg/L
- Continuous darkness: Yes


TEST SYSTEM
- Culturing apparatus: 5 L glass culturing vessels
- Method used to create aerobic conditions: The culture vessels were sealed and CO2-free air bubbled through the solution at a rate of approx. 40 mL/min. and stirred continuously by magnetic stirrer.
- Test performed in open system: No
- Details of trap for CO2 and volatile organics if used: 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 produced using purified de-gassed water.
- Other: Approx. 24 h prior to addition of the test and standard materials, the vessels were filled with 2400 mL of culture medium and 29 mL of inoculum and aerated overnight. On Day 0 the test and standard materials were added and the volume in all the vessels adjusted to 3 L by the addition of culture medium.


SAMPLING
- Sampling frequency:
CO2 analysis - Days 0, 2, 6, 8, 10, 14, 21, 28 and 29 from the control, standard and test material first CO2 absorber vessels. Days 0, 2, 6, 8, 10 and 14 from the toxicity control first CO2 absorber vessel. Days 0 and 29 from the control, standard and test material second CO2 absorber vessels. Day 0 from the toxicity control second CO2 absorber vessel.

DOC analysis - Day 0 from all culture vessels and Day 28 from the control, standard and test material.

- Sampling method:
CO2 analysis - Samples (300 or 50 µL) were injected into the IC (Inorganic Carbon) channel of the TOC analyser (Tekmar-Dohrmann Apollo 9000 TOC analyser and Shimadzu TOC-Vcsh TOC analyser). Inorganic carbon analysis occurs by means of the conversion of an aqueous sample to CO2 by orthophosphoric acid using zero grade air as the carrier gas. Calibration was done by standard solutions of sodium carbonate. Each analysis was carried out in triplicate.

DOC analysis - Samples (27, 13 or 50 µL) were injected into the Total Carbon (TC) and Inorganic Carbon (IC) channels of the TOC analyser (Shimadzu TOC-5050A TOC analyser and Shimadzu TOC-Vcsh TOC analyser). Total carbon analysis was carried out at 680 °C using a platinum-based catalyst and zero grade air as the carrier gas. Inorganic carbon analysis involves conversion by orthophosphoric acid at ambient temperature. Calibration was performed using standard solutions of potassium hydrogen phthalate and sodium carbonate in deionised water. Each analysis was carried out in triplicate.


CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes
- Toxicity control: 261.3 mL of the test material stock solution was dispersed in inoculated culture medium along with 51.4 mL of the sodium benzoate stock solution. The volume was adjusted to 3 L to give a final concentration of 174.2 mg test material/L plus 17.1 mg sodium benzoate/L, equivalent to a total of 20 mg carbon/L.

Reference substance

Reference substance:

benzoic acid, sodium salt

Remarks:

Sigma Aldrich Lot No. 096K1231

Results and discussion

Preliminary study:

The test material did not absorb to filter matrices or to activated sewage sludge.

Test performance:

No data

% Degradation

% Degradation 1

Parameter:

% degradation (inorg. C analysis)

Value:

ca. 90

Sampling time:

28 d

% Degradation 2

Parameter:

% degradation (DOC removal)

Value:

ca. 100

Sampling time:

28 d

Details on results:

The test material attained 90% degradation after 28 d and satisfied the 10 d window validation criterion, whereby 60% degradation must be attained within 10 d of the degradation exceeding 10%. The test material can therefore be considered to be readily biodegradable under the conditions of the experiment.

BOD5 / COD results

Results with reference substance:

The % biodegradation values of the reference material were as follows:
Day 14 - 73
Day 28 - 83
100% degradation was calculated from the results of DOC analyses.

Any other information on results incl. tables

Table 1: Percentage biodegradation values:

Day	% degradation Sodium Benzoate	% degradation test material	% degradation test material plus Sodium Benzoate toxicity control
0	0	0	0
2	36	16	37
6	67	63	68
8	67	64	73
10	65	75	71
14	73	75	66
21	84	83	-
28	87	97	-
29*	83	90	-

* Day 29 values corrected to include any carry-over of CO₂ detected in absorber 2.

Table 2: DOC values in the culture vessels on Days 0 and 28:

DOC* concentration
	Day 0		Day 28
Test vessel	mg C/L	% of nominal carbon content	mg C/L	% of initial carbon concentration	% degradation
Sodium Benzoate 10 mg C/L R1	10.3	103	<control	0	100
Sodium Benzoate 10 mg C/L R2	10.11	101	<control	0	100
Test Material 10 mg C/L R1	9.64	96	<control	0	100
Test Material 10 mg C/L R2	9.94	99	<control	0	100

R1-R2 = Replicates 1 and 2

* Corrected for control values

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

1. Total CO2 evolution on Day 28 was 31.75 mg/L 2. IC content of test material at the start of test was below 5% of the TC content 3. Difference between values for CO2 production at the end of test for replicate vessels was <20%

Interpretation of results:

readily biodegradable

Conclusions:

Based on the above results and under the conditions of the experiment, the test material is considered to be readily degradable as it attained 90% degradation within 28 d and the pass level of 60 % was reached within 10 d of exceeding the 10 % level.

Executive summary:

A study was performed to assess the ready biodegradability potential of synthetic inositol phosphates in an aerobic aqueous medium. The method followed OECD Guideline 301B, Method C.4-C of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110 (Paragraph (m)).

The test material, at a concentration of 10 mg carbon/L, was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in the dark at approximately 2^oC for 28 d.

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

The test material attained 90% degradation after 28 d and satisfied the 10 d window validation criterion, whereby 60% degradation was attained within 10 d of exceeding 10% degradation. The test material can therefore be considered to be readily biodegradable under the conditions of the experiment