L-97-034-PB

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:

19th May - 17th June 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Pentaerythritol mixed esters with 2-methyl butyric acid, n-pentanoic acid, n-heptanoic acid, 3,5,5-trimethylhexanoic acid, n-octanoic acid, n-decanoic acid

Cas Number:

141686-49-9

IUPAC Name:

Pentaerythritol mixed esters with 2-methyl butyric acid, n-pentanoic acid, n-heptanoic acid, 3,5,5-trimethylhexanoic acid, n-octanoic acid, n-decanoic acid

Constituent 2

Reference substance name:

Trimethylol propane mixed esters with 2-methyl butyric acid, n-pentanoic acid, n-heptanoic acid, 3,5,5-trimethylhexanoic acid, n-octanoic acid, n-decanoic acid

Cas Number:

141686-50-2

IUPAC Name:

Trimethylol propane mixed esters with 2-methyl butyric acid, n-pentanoic acid, n-heptanoic acid, 3,5,5-trimethylhexanoic acid, n-octanoic acid, n-decanoic acid

Test material form:

liquid

Details on test material:

Identification: Hatcol ® 1772
Appearance/physical state: Clear colorless liquid
Purity: 100% (no solvent present)
Composition: 70% pentaerythritol esters described by CAS# 141686-49-9; 30% trimethylolpropane esters described by CAS# 141686-50-2

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

Inoculum:
A mixed population of activated sewage sludge micro-organisms was obtained on 18 May 1998 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Belper, Derbyshire, 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. A sub-sample of the washed sewage sludge was then removed and the suspended solids concentration determined.

Initial test substance concentration

Initial conc.:

ca. 10 mg/L

Based on:

COD

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Medium:
The mineral medium used in this study was that recommended in the OECD Guidelines.

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)/1. The study was carried out in a temperature controlled room at 21 ± 1°C, in darkness. Approximately 24 hours 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 litres by the addition of culture medium. The culture vessels were sealed and CO2-free air bubbled through the solution at a rate of approximately 40 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 de-gassed water.

Sampling and CO2 analysis:
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, 10, 14, 20, 22, 24, 27, 28 and 29 were analysed for CO2 immediately. The samples taken on days 6, 8 and 16 were stored deep frozen at -20°C prior to analysis. The samples taken on days 12 and 18 were also 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 degradation of the test material did not meet the 10 day window validation criterion given in the OECD Guidelines 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.

Toxicity determinations:
A toxicity control (test material and sodium benzoate) was included in the study to assess any toxic effect of the test material on the sewage sludge micro-organisms used in the study.

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

% Degradation

Key result

Parameter:

% degradation (CO2 evolution)

Value:

62

Sampling time:

28 d

Details on results:

The total CO2 evolution in the control vessels on day 28 was 45 mg/I (= 135 mg/3I). Although the CO2 evolution was in excess of 40 mg at the end of the test, this is considered not to affect the integrity of the study given that the upper level of 70 mg CO/I given in the OECD Guidelines was not exceeded, and that all other validation criteria were satisfied.

The test material attained 62% degradation after 28 days. However, despite attaining in excess of 60% the test material failed to satisfy the 10-day window validation criterion, whereby 60% degradation must be attained within 10 days of the degradation exceeding 10%, and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

The results of the inorganic carbon analysis of samples from both absorber vessels on day 29 confirmed that no significant amounts of CO2 were present in solution in the culture vessels as inorganic carbonate.

The toxicity control attained 81% degradation after 28 days thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the study.

BOD5 / COD results

Results with reference substance:

Sodium benzoate attained 91 % degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test material attained 62% biodegradation after 28 days. However, despite attaining in excess of 60% the test material failed to satisfy the 10-day window validation criterion 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)).

Results

The test material attained 62% biodegradation after 28 days. However, despite attaining in excess of 60% the test material failed to satisfy the 10-day window validation criterion and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Sodium benzoate attained 91 % degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.