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Environmental fate & pathways

Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 E (Ready biodegradability: Modified OECD Screening Test)
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
A mixed population of sewage treatment micro-organisms was obtained on 22 August 2016 from the final effluent stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage. The sample of effluent was filtered through coarse filter paper (first approximate 200 mL discarded) and maintained on aeration in a temperature controlled room at 21 ± 1 ºC prior to use.
Duration of test (contact time):
28 d
Initial conc.:
20 mg/L
Based on:
other: carbon
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Test Item Preparation
Following preliminary solubility work and the recommendations of the International Standards Organisation (ISO 10634: 1995) and in the published literature (Handley et al, 2002) the test item was dissolved in an auxiliary solvent prior to adsorption onto filter paper*. High shear mixing was also applied to break up the filter paper containing the test item. 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.
A nominal amount of test item (500 mg) was dissolved in 10 mL of acetone to give a 500 mg/10 mL solvent stock solution. An aliquot (200 μL) of this solvent stock solution was dispensed onto a filter paper* and the solvent allowed to evaporate to dryness for approximately 15 minutes. The filter paper was dispersed in approximately 350 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 5 minutes) prior to the addition of the inoculum (5 mL) and adjusting to a final volume of 500 mL to give a final concentration of 20 mg/L.
A test concentration of 20 mg carbon/L was employed in the test following the recommendations of the Test Guidelines and from the information from the ASRI study (EC50 value 66 mg/L, Envigo study number HK47LS).
A filter paper* was added to each control vessel in order to maintain consistency between the test and control vessels. Acetone (200 μL) was dispensed onto each filter paper and evaporated to dryness for approximately 15 minutes. The filter paper was dispersed in approximately 350 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 5 minutes) prior to addition to each vessel.
As it was not a requirement of the Test Guidelines, no analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Reference Item Preparation
A reference item, aniline (C6H5NH2), 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 with the aid of ultrasonication for approximately 10 minutes. An aliquot (50 mL) of this stock solution was diluted with mineral medium (445 mL) and the inoculum (5 mL), to give the test concentration of 100 mg/L. The volumetric flask containing the stock solution was inverted several times to ensure homogeneity.
A filter paper* was added to each control vessel in order to maintain consistency between the test and procedure control vessels. Acetone (200 μL) was dispensed onto each filter paper and evaporated to dryness for approximately 15 minutes. The filter paper was dispersed in approximately 350 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 5 minutes) prior to addition to each vessel.

Toxicity Control
A toxicity control, containing the test item and aniline, was prepared in order to assess any toxic effect of the test item on the sewage treatment micro-organisms used in the test.
An aliquot (200 μL) of the test item solvent stock solution was dispensed onto a filter paper* and the solvent allowed to evaporate for approximately 15 minutes. The filter paper was dispersed in approximately 350 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 5 minutes) ) prior to the addition of the inoculum (5 mL) and an aliquot (50 mL) of the 1000 mg/L aniline stock solution (see Section 3.5.3) adjusting to a final volume of 500 mL to give a final concentration 20 mg test item/L and 100 mg aniline/L.

Preparation of Test System
The following test preparations were prepared and inoculated in 500 mL bottles:
a) Three replicate bottles containing inoculated mineral medium plus a filter paper* to act as the inoculum control.
b) Two replicate bottles containing inoculated mineral medium and the reference item, aniline plus a filter paper**, at a concentration of 100 mg/L.
c) Three replicate bottles containing inoculated mineral medium and the test item on a filter paper*at a concentration of 20 mg/L.
d) Two replicate bottles containing inoculated mineral medium the reference item, aniline, at a concentration of 20 mg/L and the test item on a filter paper* at a concentration of 100 mg/L to act as toxicity control vessels.

All vessels were inoculated with the prepared inoculum at a rate of 1% v/v.
On Day 0 the test and reference items were added and the pH of all vessels measured using a Hach HQ40d Flexi handheld meter.
In order to confirm that the aniline and test item stock solutions were prepared correctly, a diluted, 100 mg/L stock solution (in reverse osmosis water) was also sampled for Dissolved Organic Carbon (DOC) analysis.
All remaining inoculum control, test item, procedure control and toxicity control vessels were placed in a CES Multi-Channel Aerobic Respirometer.
The system consists of a sample flask sealed by a sensor head/CO2 trap immersed in a temperature controlled water bath. The samples were stirred for the duration of the test with a magnetically coupled stirrer.
As biodegradation progresses, the micro-organisms convert oxygen to carbon dioxide which is absorbed into the ethanolamine solution (50% v/v) causing a net reduction in gas pressure within the sample flask (see Figure1). The pressure reduction triggers the electrolytic process, generating oxygen and restoring the pressure in the sample flask. The magnitude of the electrolyzing current and the duration of the current is proportional to the amount of oxygen supplied to the micro-organisms. The data generated from the respirometer’s own battery backed memory was collected on the hard disk drive of a non-dedicated computer.
The test was conducted in diffuse light at a temperature of between 21 and 22 ºC.
On Day 28, two inoculum control, one procedure control, two test item and one toxicity control vessel were sampled for pH.
The remaining vessels which were not sampled were discarded and are not reported. Additional replicate vessels were prepared and incubated in order that in the event of a leak in the test system a replicate vessel could be discarded without jeopardizing the integrity of the test.

Oxygen Consumption Measurements
The daily Biological Oxygen Demand (BOD) values for the inoculum control, test item, procedure control and the toxicity control were monitored.
Physico-chemical Measurements
The temperature of the water bath was recorded daily.
pH Measurements
In order to confirm whether the pH of the test preparations changed, the pH was measured using a Hach HQ40d Flexi handheld meter on Days 0 and 28.
Reference substance:
aniline
Parameter:
% degradation (CO2 evolution)
Value:
0
Sampling time:
28 d
Details on results:
Validation Criteria
The mean BOD of the inoculated mineral medium (control) was 27.39 mg O2/L after 28 days and therefore satisfied the validation criterion given in the OECD Test Guidelines.
The pH of the inoculated test item vessels on Day 28 were 7.7 and 7.6 and hence satisfied the validation criterion given in the OECD Test Guidelines.
The difference between extremes of replicate BOD values at the end of the test was less than 20% and therefore satisfied the validation criterion given in the OECD Test Guidelines.

Biodegradation
The test item attained 0% biodegradation after 28 days, calculated from the oxygen consumption values, and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.
The toxicity control attained 35% biodegradation after 14 days and 40% biodegradation after 28 days. However there were statistically significant differences (P≥0.05) between the control and test item oxygen consumption values on Day 28. The test item was therefore considered to have had an inhibitory effect on the sewage treatment micro-organisms used in the test.
Care should therefore be taken with the interpretation of the biodegradation results as the test item has exhibited inhibitory effects at the test concentration of 20 mg/L.
Aniline (procedure control) attained 75% biodegradation after 14 days and 82% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.
Results with reference substance:
Aniline (procedure control) attained 75% biodegradation after 14 days and 82% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.
Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The test item 1,2-Ethanediamine, N-(2-aminoethyl)-, reaction products with glycidyl tolyl ether attained 0% biodegradation after 28 days and therefore cannot be considered as readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.
Executive summary:

The test item 1,2-Ethanediamine, N-(2-aminoethyl)-, reaction products with glycidyl tolyl ether attained 0% biodegradation after 28 days and therefore cannot be considered as readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.

Description of key information

The test item 1,2-Ethanediamine, N-(2-aminoethyl)-, reaction products with glycidyl tolyl ether attained 0% biodegradation after 28 days and therefore cannot be considered as readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information