POLYPERINY

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:

1996-05-20 - 1996-07-24 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

under GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD Guidelines for Testing of Chemicals No. 301 B, Ready Biodegradability; CO2 Evolution Test, 1992.

Deviations:

yes

Remarks:

Analysis of the concentration, homogeneity and stability of the test material in the test solutions were not appropriate to the Test Guideline

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EU Commission Directive 92/69 EEC C.4-C (which constitutes Annex V of Council Directive 67/548/EEC)

Deviations:

yes

Remarks:

Analysis of the concentration, homogeneity and stability of the test material in the test solutions were not appropriate to the Test Guideline

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

- Storage condition of test material: room temperature

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic, adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): A mixed population of activated sewage sludge micro-organisms was obtained on 24 June 1996 from the aeration stage of the Severn Trent Water Pic sewage treatment plant at Belper, Derbyshire, UK, which treats predominantly domestic sewage.
- Preparation of inoculum for exposure: The sample of activated sewage sludge was maintained on continuous aeration upon receipt. A sample of the activated sewage sludge was washed three times by settlement and resuspension in culture medium to remove any excessive amounts of dissolved organic carbon (DOC) that may have been present. A sub-sample of the washed sewage sludge was then removed and the suspended solids concentration determined.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

10 mg/L

Based on:

IC (inorganic carbon)

Remarks:

An amount of test material (38.4 mg) was dispersed in inoculated culture medium and the volume adjusted to 3 litres to give a final concentration of 12.8 mg/l, equivalent to 10 mg carbon/I.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Preparation of inoculum
The sample of activated sewage sludge was maintained on continuous aeration upon receipt. A sample of the activated sewage sludge was washed three times by settlement and resuspension in culture medium to remove any excessive amounts of dissolved organic carbon (DOC) that may have been present. A sub-sample of the washed sewage sludge was then removed and the suspended solids concentration determined.
Culture medium:
Solution a KH2PO4 8.50 g/l
K2HPO4 21.75 g/l
Na2HPO4.2H2O 33.40 g/l
NH4CI 0.50 g/l, pH = 7.4
Solution b: CaCl2 27.50 g/l
Solution c: MgSO4.7H2O 22.50 g/l
Solution d: FeCI3.6H2O 0.25 g/l
To 1 litre (final volume) of purified water is 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.
Preparation of test system
The following test solutions were prepared and inoculated in 5 litre glass culture vessels each containing 3 litres of solution:
a) A control, in duplicate, consisting of inoculated culture medium.
b) The standard material (sodium benzoate), in duplicate, in inoculated culture medium to give a final test concentration of 10 mg carbon/I.
c) The test material, in duplicate, in inoculated culture medium to give a final test concentration of 10 mg carbon/I.
d) The test material plus the standard material in inoculated culture medium to give a final concentration of 20 mg carbon/I 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 study was carried out at a temperature of 21 'C in darkness.
Approximately 24 hours prior to the start of the study the vessels were filled with 2400 ml of culture medium and 23.7 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 litres by the addition of culture medium.
The culture vessels were sealed and CO2-free air bubbled through the solution at a rate of 70 ml/minute and stirred continuously by magnetic stirrer.
The CO2-free air was produced by sparging compressed air through the following series:
i) Three 500 ml Dreschel bottles filled with 350 ml 10N NaOH
ii) One 500 ml Dreschel bottle filled with 350 ml 0.025N Ba(OH)2
iii) One empty 500 ml Dreschel bottle to prevent liquid carry-over to the test vessels.
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 degassed water.
Samples (2 ml) were taken from the first CO2 absorber vessel on days 0, 1,2, 3, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 27, 28 and 29. The second absorber vessel was sampled on days 0 and 29.
The samples taken on days 0, 1,2, 3, 6, 8, 10, 14, 16, 20, 22, 24, 27, 28 and 29 were analysed for CO2 immediately. The samples taken on days 12 and 18 were stored deep frozen at -20' C, however, these samples were not analysed for CO2 as the results obtained from previous and subsequent analyses showed that the level of degradation of the test material did not increase during this time and therefore additional analyses were considered to be unnecessary.
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 analysed for CO2 using an Ionics 1555B TOC analyser and a Dohrmann DC-190 TOC analyser. Samples (40 or 50 //I) were injected into the IC (Inorganic Carbon) channel of the Total Carbon Analyser. Each analysis was carried out in triplicate.

Reference substance

Reference substance:

benzoic acid, sodium salt

Remarks:

An initial stock solution of 1000 mg/l was prepared by direct dispersion in culture medium and a 51.4 ml aliquot added to the test vessel to give a final test concentration of 17.7 mg/l, equivalent to 10 mg carbon/I.

Results and discussion

% Degradation

Key result

Parameter:

% degradation (CO2 evolution)

Value:

0

Sampling time:

29 d

Details on results:

See tables below.
Polyperin Y attained 0% degradation after 28 days and, therefore, cannot be considered as readily biodegradable under the strict terms and conditions of the OECD Guidelines. A test concentration of 10 mg C/l was employed in the study as an initial experiment performed at a test concentration of 20 mg C/l, showed the toxicity control to attain only 15% degradation after 14 days thereby indicating that the test material exhibited toxic effects on the activated sewage sludge microorganisms at this test concentration.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The study was conducted under GLP according to OECD 301B. The method is to be considered scientifically reasonable and suitable for the test item, the available information allows the conclusion that the test was properly conducted, all criteria for acceptability of the test were met, this study was considered to be valid. The test material attained 0% degradation 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:

Determination of 'ready' biodegradability: carbon dioxide (CO2) evolution test with the test item under GLP.

The study procedures described in this report were based on the OECD guideline No. 301 B, 1992. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No. 440/2008 of 30 May 2008, Publication No. L142, Part C.4-C.

The criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) was not met. In the toxicity control, the test item 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, the test item was designated as not readily biodegradable.