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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
Study period:
This study was conducted between 01 August 2017 and 30 August 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Version / remarks:
EC No. 440/2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Information as provided by the Sponsor.
Identification: 4,4'-(1,3-Phenylenediisopropylidene) diphenylcyanate
Batch: FAR366085A
Purity: not supplied
Physical state/Appearance: yellow viscous liquid
Expiry Date: 29 September 2019
Storage Conditions: approximately 4 ºC in the dark
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
A mixed population of activated sewage sludge micro organisms was obtained on 31 July 2017 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, 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. The washed sample was then maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC 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 pre weighed 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 ºC 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 2.9 g/L prior to use.
Duration of test (contact time):
ca. 28 d
Initial conc.:
ca. 12.5 mg/L
Based on:
test mat.
Initial conc.:
ca. 10 mg/L
Based on:
IC (inorganic carbon)
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Preliminary Solubility Work
Information provided by the Sponsor indicated that the test item was practically insoluble in water. Therefore preliminary solubility/dispersibility work was performed in order to determine the most suitable method of preparation
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 weighed onto filter paper** prior to dispersal in mineral medium. 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 (37.5 mg) was weighed onto a filter paper prior to addition to inoculated mineral medium. The volume was then adjusted to 3 liters to give a final concentration of 12.5 mg/L, equivalent to 10 mg carbon/L.
A test concentration of 10 mg carbon/L was employed in the test following the recommendations of the Test Guidelines.

Reference Item Preparation
A reference item, sodium benzoate (C6H5COONa), 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. An aliquot (51.4 mL) of this stock solution was added to the test vessel containing inoculated mineral medium prior to the volume being adjusted to 3 liters to give a final test concentration of 17.1 mg/L, equivalent to 10 mg carbon/L. The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution.
A filter paper was added to each vessel in order to maintain consistency between the test and procedure control vessels.

Toxicity Control
A toxicity control, containing the test item and sodium benzoate, was prepared in order to assess any toxic effect of the test item on the sewage sludge micro-organisms used in the test.
A nominal amount of test item (37.5 mg) was weighed onto a filter paper* prior to addition to the test vessel containing inoculated mineral medium. An aliquot (51.4 mL) of the sodium benzoate stock solution was also added to the test vessel and the volume was adjusted to 3 liters to give a final concentration of 12.5 mg test item/L plus 17.1 mg sodium benzoate/L equivalent to a total of 20 mg carbon/L.

Preparation of Test System
The following test preparations were prepared and inoculated in 5 liter test culture vessels each containing 3 liters of solution:
a) An inoculated control, in duplicate, consisting of inoculated mineral medium plus a filter paper.
b) The procedure control containing the reference item (sodium benzoate), in duplicate, in inoculated mineral medium plus a filter papert o give a final concentration of 10 mg carbon/L.
c) The test item on a filter paper, in duplicate, in inoculated mineral medium to give a final concentration of 10 mg carbon/L.
d) The test item on a filter paper plus the reference item in inoculated mineral medium to give a final concentration of 20 mg carbon/L to act as a toxicity control (one vessel only).
A filter paper was added to the inoculum control and procedure control vessels in order to maintain consistency between these vessels and the test item vessels.
Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/L. The test was carried out in a temperature controlled room at approximately 22 °C to 24”C, in darkness.
Approximately 24 hours prior to addition of the test and reference items the vessels were filled with 2400 mL of mineral medium and 31 mL of inoculum and aerated overnight. On Day 0 the test and reference items were added and the pH of all vessels measured using a Hach HQ40d Flexi handheld meter. If necessary, the pH was adjusted to pH 7.4 ±0.2 using diluted hydrochloric acid or sodium hydroxide solution prior to the volume in all the vessels being adjusted to 3 liters by the addition of mineral medium which had been purged overnight with CO2 free air.
The test vessels were sealed and CO2 free air bubbled through the solution at a rate of 30 to 100 mL/minute per vessel and stirred continuously by magnetic stirrer.
The CO2 free air was produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb®) granules.
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 water.

Assessments
Observations
The appearance of the test preparations was recorded on Days 0, 6, 13, 20 and 27.

pH Measurements
The pH of the test preparations was determined on Day 0 and on Day 28 prior to acidification with hydrochloric acid, using a Hach HQ40d Flexi handheld meter.
Reference substance:
benzoic acid, sodium salt
Test performance:
The total CO2 evolution in the inoculum control vessels on Day 28 was 26.27 mg/L and therefore satisfied the validation criterion given in the OECD Test Guidelines.
The IC content of the test item suspension in the mineral medium at the start of the test was below 5% of the TC content and hence satisfied the validation criterion given in the OECD Test Guidelines.
The difference between the values for CO2 production at the end of the test for the replicate vessels was <20% and hence satisfied the validation criterion given in the OECD Test Guidelines with the exception of the inoculum control vessels where the difference was approximately 24%.

Key result
Parameter:
% degradation (CO2 evolution)
Value:
0
Sampling time:
28 d
Remarks on result:
other: not readily biodegradable
Details on results:

Acidification of the test vessels on Day 28 followed by the final analyses on Day 29 was conducted according to the methods specified in the Test Guidelines. This acidification effectively kills the micro organisms present and drives off any dissolved CO2 present in the test vessels. Therefore any additional CO2 detected in the Day 29 samples originated from dissolved CO2 that was present in the test vessels on Day 28 and hence the biodegradation value calculated from the Day 29 analyses is taken as being the final biodegradation value for the test item.
The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed an increase in all replicate vessels with the exception of test item Replicate 1 and 2 and the toxicity control vessel.
Inorganic carbon analysis of the samples from the second absorber vessels on Day 29 confirmed that no significant carry-over of CO2 into the second absorber vessels occurred.
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.
However care should be taken in the interpretation of these results as the toxicity control vessel did not pass the validation criterion whereby it should attain equal to or greater than 25% biodegradation by Day 14 of the study for the test item to be considered non-inhibitory.
The toxicity control attained 12% biodegradation after 14 days and 2% biodegradation after 28 days thereby confirming that the test item did exhibit an inhibitory effect on the sewage treatment micro-organisms used in the test at a concentration of 10 mg carbon/L. The decrease in biodegradation between Days 14 and 28 was considered to be due to the inhibitory nature of the test item resulting in the CO2 evolution in the toxicity control vessel being less than that in the inoculum control vessels.
Sodium benzoate attained 68% biodegradation after 14 days with greater than 60% degradation being attained in a 10-Day window. After 28 days 76% biodegradation was attained. These results confirmed the suitability of the inoculum and test conditions and satisfied the validation criterion given in the OECD Test Guidelines.

Percentage Biodegradation Values:

Day

Biodegradation
(%)

Procedure Control

Test Item

Toxicity Control

0

0

0

0

2

22

0

19

6

66

0

21

8

75

0

25

10

64

6

0

14

68

0

12

21

75

0

7

28

78

0

6

29*

76

0

2


*Day 29 values corrected to include any carry-over of CO2detected in Absorber 2

Validity criteria fulfilled:
yes
Remarks:
Sodium benzoate attained 68% biodegradation after 14 days with greater than 60% degradation being attained in a 10-Day window. After 28 days 76% biodegradation was attained. These results satisfied the validation criterion
Interpretation of results:
not readily biodegradable
Conclusions:
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.
Care should be taken in the interpretation of these results as the toxicity control vessel did not pass the validation criterion whereby it should attain equal to or greater than 25% biodegradation by Day 14 of the study for the test item to be considered non-inhibitory.
Executive summary:

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; CO2Evolution 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)).

Methods

The test item, at a concentration of 10 mg carbon/L, was exposed to activated sewage sludge micro-organisms with mineral medium in sealed culture vessels in the dark at temperatures of between 22 °C to 24 °C for 28 days.

Following the recommendations of the International Standards Organisation (ISO 10634, (1995)) and the published literature (Handleyet al, 2002), the test item was weighed onto a filter paper*prior to dispersal in test media. Using this method the test item is 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.

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

Results

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.

Care should be taken in the interpretation of these results as the toxicity control vessel did not pass the validation criterion whereby it should attain equal to or greater than 25% biodegradation by Day 14 of the study for the test item to be considered non-inhibitory.

 


*GF/A (70 mm diameter)

Description of key information

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information