Reaction products of triethanolamine esters of polyphosphoric acids with alkyl derivatives of pyridine

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:

20th January - 17th April 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

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

Principles of method if other than guideline:

In view of the difficulties associated with the evaluation of the biodegradability of organic compounds with low water solubility, a modification to the standard method of preparation of the test concentration was performed. An approach endorsed by the International Standards Organisation (IS0,1995) and the published literature (Handley et al, 2002) is to adsorb the test item onto an inert support prior to dispersion in the test vessels. Using this method the test item is evenly distributed throughout the test medium and the surface area of test item exposed to the test organisms is increased thereby increasing the potential for biodegradation.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of triethanolamine esters of polyphosphoric acids with alkyl derivatives of pyridine

EC Number:

939-981-3

IUPAC Name:

Reaction products of triethanolamine esters of polyphosphoric acids with alkyl derivatives of pyridine

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

A mixed population of activated sewage sludge micro-organisms was obtained on 24 February 2014 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage. The activated sewage sludge sample was washed twice by settlement and resuspension in mineral medium to remove any excessive amounts of dissolved organic carbon (DOC) that mayhave been present. The washed sample was then maintained on continuous aeration in the laboratory at a temperature of approximately 21°C 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 preweighed 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 °C 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 2.8 g/L prior to use. Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/L.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

10 other: mg carbon/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

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

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 liter (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
- pH: 7.4
- pH adjusted: yes
- Aeration of dilution water: Continuous
- Suspended solids concentration: 2.8 g/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 5 litre culture vessels
- Number of culture flasks/concentration: 1 per condition (5 in total) 30 mg/L
- Method used to create aerobic conditions: The test vessels were sealed and CO2-free air bubbled through the solution at a rate of 30 to 100 mL/min per vessel and stirred continuously by magnetic stirrer

TEST SYSTEM
- Culturing apparatus: 5 litre culture vessels
- Number of culture flasks/concentration: 2 per condition, except toxicity control (9 in total); 30 mg/L
- Method used to create aerobic conditions: The test vessels were sealed and CO2-free air bubbled through the solution at a rate of 30 to 100 mL/min per vessel and stirred continuously by magnetic stirrer
- Measuring equipment: Tekmar-Dohrmann Apollo 9000 TOC analyzer or a Shimadzu TOC-VcsH TOC analyzer

SAMPLING
- Sampling frequency: Days 0, 2, 6, 8, 10, 14, 21, 28 and 29
- Sampling method: Inorganic carbon/total organic carbon
- Sample storage before analysis: room temperature in the dark

CONTROL AND BLANK SYSTEM
- Inoculum blank: An inoculated control, in duplicate, consisting of inoculated mineral medium plus 500 mg silica gel.
- Toxicity control: The test item plus the reference item in inoculated mineral medium plus 500 mg silica gel to give a final concentration of 20 mg carbon/L to act as a toxicity control (one vessel only).
- Other: Propylene glycol, in duplicate, in inoculated mineral medium plus 500 mg silica gel to give a final concentration of 3 .16 mg/L.

STATISTICAL METHODS:
Statistical analysis of the Day 29 inorganic carbon values for the propylene glycol inoculum control and test item vessels was carried out using a Student's t-test to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001 ).

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

% Degradation

Key result

Parameter:

% degradation (CO2 evolution)

Value:

0

Sampling time:

28 d

BOD5 / COD results

Results with reference substance:

Sodium benzoate attained 79% biodegradation after 14 days and 93% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Any other information on results incl. tables

Day							% Biodegradation
							Procedure Control								Test Item								Toxicity Control
0							0								0								0
2							30								6								31
6							51								6								39
8							63								2								42
10							69								0								40
14							79								0								36
21							81								0								32
28							87								0								40

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

This substance demonstrated zero biodegradation over 28 days.

Executive summary:

The test item attained 0% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B. Statistical analysis of the Day 29 inhibitory concentration values for the propylene glycol inoculum control and test item vessels showed there were no statistically significant differences (P>0.05) between the control and the test item vessels. The test item was therefore considered not to have a toxic effect on the sewage sludge micro-organisms used in the study and this was confirmed by the toxicity control results. The toxicity control attained 36% biodegradation after 14 days and 38% 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.