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Environmental fate & pathways

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:
Experimental start date 11 April 2017. Experimental completion date 10 May 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
yes
Remarks:
Please see any other information on materials and methods section.
Qualifier:
according to guideline
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
yes
Remarks:
Please see any other information on materials and methods section.
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
yes
Remarks:
Please see any other information on materials and methods section.
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
1-(methylamino)-4-[(3-methylphenyl)amino]anthraquinone
EC Number:
229-059-2
EC Name:
1-(methylamino)-4-[(3-methylphenyl)amino]anthraquinone
Cas Number:
6408-50-0
Molecular formula:
C22H18N2O2
IUPAC Name:
1-(methylamino)-4-[(3-methylphenyl)amino]anthraquinone
Test material form:
solid: particulate/powder
Details on test material:
Identification: 1-(methylamino)-4-[(3-methylphenyl)amino] anthraquinone
Physical state/Appearance: dark red powder
Batch: 702W01
Purity: 99.4%
Expiry Date: 08 February 2020
Storage Conditions: room temperature in the dark
Intended use/Application: industrial chemical
Specific details on test material used for the study:
Identification: 1-(methylamino)-4-[(3-methylphenyl)amino] anthraquinone
Physical state/Appearance: dark red powder
Batch: 702W01
Purity: 99.4%
Expiry Date: 08 February 2020
Storage Conditions: room temperature in the dark
Intended use/Application: industrial chemical

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
Inoculum
A mixed population of activated sewage sludge micro-organisms was obtained on 10 April 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 3.8 g/L prior to use.
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
13 mg/L
Based on:
test mat.
Details on study design:
Preliminary Solubility Work
Information provided by the Sponsor indicated that the test item was insoluble in water. The following preliminary solubility/dispersibility work was performed in order to determine the most suitable method of preparation:
i) Ultrasonication and High Shear Mixing: A nominal amount of test item (100 mg) was dispersed in 1 liter of deionized reverse osmosis purified water with the aid of shaking by hand for approximately 1 minute, this formed an extremely pale grey dispersion with particles of test item visible dispersed throughout, settled on the bottom and floating on the surface. After ultrasonication for 30 minutes a pale grey dispersion was formed with particles of test item visible dispersed throughout, settled on the bottom and floating on the surface. This was then subjected to high shear mixing (approximately 7500 rpm, 15 minutes) and formed a pale blue/ grey dispersion with particles of test item visible floating at the surface. Test item was visible adhered to the side of the vessel.
This work confirmed that the test item was insoluble in water. Therefore the following additional solubility work was conducted to ascertain the best method to employ in the biodegradation test.
i) Ultrasonication: A nominal amount of test item (50 mg) was dispersed in approximately 400 mL of mineral media with the aid of ultrasonication for 15 minutes. The volume was then adjusted to a final volume of 3 liters with mineral media. This formed an extremely pale grey dispersion with particles of test item visible dispersed throughout, settled on the bottom and floating at the surface.
ii) High Shear Mixing: A nominal amount of test item (50 mg) was dispersed in approximately 400 mL of mineral media with the aid of high shear mixing (approximately 7500 rpm, 15 minutes). The volume was then adjusted to a final volume of 3 liters with mineral media. This formed pale blue/grey dispersion with particles of test item visible floating at the surface. Test item was visible adhered to the side of the vessel used for mixing.
iii) Preliminary Solution in a Volatile Solvent: The addition of a test item solvent stock to glass fibre filter paper was attempted.
A nominal amount of test item (1000 mg) was dissolved in chloroform (10 mL) with the aid of shaking by hand for 10 seconds, and formed an extremely dark blue solution. An aliquot (450 μL) of this solvent stock solution was dispensed to filter paper. The solvent was allowed to evaporate to dryness for approximately 15 minutes. The filter paper was then added to approximately 400 mL of mineral medium and subjected to high shear mixing (approximately 7500 rpm, 5 minutes). The volume was then adjusted to 3 liters with mineral medium. This formed a dark blue dispersion containing small broken up pieces of filter paper visible dispersed throughout with a layer of broken up filter paper/ test item visible at the surface.
From the preliminary solubility work and following the recommendations of the International Standards Organisation (ISO 10634, 1995) it was concluded that the best testable dispersion was found to be obtained when using the preliminary solution in a volatile solvent 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 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 (1000 mg) was dissolved in 10 mL of chloroform to give a 1000 mg/10 mL solvent stock solution. An aliquot (390 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 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 5 minutes) prior to addition to inoculated mineral medium. The volume was then adjusted to 3 liters to give a final concentration of 13.0 mg/L, equivalent to 10 mg carbon/L. The volumetric flask containing the solvent stock solution was inverted several times to ensure homogeneity of the solution.
A filter paper**was added to each control vessel in order to maintain consistency between the test and procedure control vessels. Chloroform (390 μL) was dispensed onto each filter paper and evaporated to dryness for approximately 15 minutes. The filter paper was dispersed in approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 5 minutes) prior to addition to each vessel.
A test concentration of 10 mg carbon/L was employed in the test following the recommendations of the Test Guidelines.
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, 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 and the volume 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. Chloroform (390 μL) was dispensed onto each filter paper and evaporated to dryness for approximately 15 minutes. The filter paper was dispersed in approximately 400 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 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.
An aliquot (390 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 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 5 minutes) 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 13.0 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 paper* to 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 with chloroform evaporated to dryness 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 temperatures of between 22 and 26 °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 23.7 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/min 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.
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

% Degradation
Parameter:
% degradation (CO2 evolution)
Value:
0
Sampling time:
28 d
Details on results:
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 a decrease in all replicate vessels with the exception of toxicity control. This decrease was considered to be due to sampling/analytical variation. 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.
Statistical analysis of the Day 29 IC values for the control and test item vessels showed there were no statistically significant differences (P≥ 0.05) between the control and the test item. 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 32% biodegradation after 14 days and 41% 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.

BOD5 / COD results

Results with reference substance:
Sodium benzoate attained 71% biodegradation after 14 days and 72% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Any other information on results incl. tables

Percentage Biodegradation Values

Day % Biodegradation
Procedure Control Test Item Toxicity Control
0 0 0 0
2 44 0 22
6 62 2 29
8 67 1 29
10 60 6 34
14 71 0 32
21 74 3 37
28 92 5 37
29* 72 0 41

* Day 29 values corrected to include any carry-over of CO2 detected in Absorber 2

Validation Criteria

The total CO2 evolution in the inoculum control vessels on Day 28 was 29.83 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 (see Table 3) 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.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
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.
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)).

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 and 26 °C for 28 days.

Following the recommendations of the International Standards Organisation (ISO 10634, (1995)), the test item was dissolved in an auxiliary solvent prior to being adsorbed onto a filter paper and subsequent dispersal in test media. 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.

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.

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