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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 12 September 2013 and 11 October 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Identification: GL500
Description: Clear, colourless liquid
Batch: 20130510
Purity: Component A: C16H22O4: 6%
Component B: C20H30O4: 38%
Component C: C24, H38, O4: 56%
Expiry date: 10 May 2018
Storage Conditions: Room temperature in the dark
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
A mixed population of activated sewafe sludge micro-organisms was obtained on 11 September 2-13 from the aeration stage of the Severn Trent Water Plc sewage treatment plat at Loughborough, Leicestershire, UKm which treats predominantly domestic sewage.

The activated sewage sludge sample was washed twice by settlement and resuspension 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 (10 mL) of the washed activated sludge by suction through pre-weighed GF/A filter paper, rinsed three times with 20 mL deionized reverse osmosis water prior to drying in an oven, 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):
28 d
Initial conc.:
13.7 mg/L
Based on:
test mat.
Remarks:
Equivalent to 10 mg carbon/L
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
The test item was dispersed directly in mineral medium. The mineral medium used in this study was that recommended in the OECD guidelines.

An amount of test item (41.2 mg) was dispersed in approximately 4000 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 15 minutes) prior to dispersal in inoculated mineral medium. The volume was adjusted to 3 liters to give a final concentration of 13.7 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.

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 with the aid of ultrasonication for approximately 15 minutes. 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 litres 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 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 amount of test item (41.2 mg) was dispersed in approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 15 minutes) prior to dispersal in innoculated mineral medium. An aliquot (51.4 mL) of the sodium bezoate stock soltion was also added to the test vessel and the volume adjusted to 3 liters to give a final concentration of 13.7 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 3 test preparations were prepared and inoculated in 5 liter test cuture vessels, each containing 3 liters of solution:
(a) An inoculated control, in duplicate, consisting of inoculated mineral medium
(b) The procedure control containing the reference item (sodium benzoate), in duplicate, in inoculated mineral medium to give a final concentration of 10 mg carbon/L.
(c) The test item, in duplicate, in inoculated mineral medium to give a final concentration of 10 mg carbon/L.
(d) The test item 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).

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 oom at approximately 21 ºC, in darkness.

Approximately 24 hours prior to addition of thetest item and reference items the vessels were filled with 2400 mL of mineral medium and 31.0 mL of inoculum and aerated overnight. On Day 0 the test and referene items were added and the pH of all vessels measured. 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 per vessel and stirred continuously by magnetic stirrer.

The CO2 produced by degradation was collected in two 500 mL Dreschel bottles containing 350 mL of 0.05 M NaOH.

Observations:
The appearance of the test preparations was recorded on Days 0, 4, 14, 18 and 25.
The pH of the test measurements was determined on Day 0 and day 28 prior to acidification with hydrochloric acid.

IC Analysis:
Samples (2 mL) were taken from the first CO2 absorber vessels on Days 0, 1, 4, 6, 8, 11, 14, 21, 28 and 29. The second absorber vessels were all sampled on Days 0 and 29. The samples taken on Days 0, 4, 8, 11, 14, 21, 28 and 29 were analyzed for IC immediately. The samples taken on Days 1 and 6 were stored frozen prior to analysis. On day 28, 1 ml of 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.

Validation Criteria:
The results of the degradation test are considered valid if in the same test the reference item yields ≥ 60% degradation by Day 14.
the test item may be considered to be readily biodegradable if ≥ 60% degradation is attained within 28 days. This level of degradation must be reached within 10 days of biodegradation exceeding 10%.
The toxicity control (test item and sodium benzoate) should attain ≥ 25% degradation by Day 14 for the test item to be considered as non-inhibitory.
The test is considered valid if the difference of the extremes of replicate values of production of CO2 at the time the plateau is reached, at the end of the test or at the end of the 10-day window, as appropriate, is less than 20%.
The total CO2 evolution in the inoculum control vessels t the end of the test should not normally exceed 40 mg/L medium, however values up to 70 mg/L are acceptable.

Reference substance:
other: Sodium benzoate
Test performance:
The total CO2 evolution in the inoculum control vessels on Day 28 was 27.43 mg/L and therefore satisfied the validation criterion.
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.
The difference between the values for CO2 production at the end of the test for the replicate vessels was <20% and ence satisfied the validation criterion.
Key result
Parameter:
% degradation (CO2 evolution)
Value:
41
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 speecified 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 decreaser in all replicate vessels. This decrease was considered to be due to sampling/analytical variation in the day 28 samples. The inoculum control replicate values dropped causing a large drop in the reference, test and toxicity control vessels observed in the day 29 results. 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 41% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

The toxicity control attained 60% biodegradation after 14 days and 70% 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.

Sodium benzoate attained 68% biodegradation after 14 days and 61% after 28 days thereby confirming the suitability of the innoculum and test conditions. The slight decrease in biodegradation between days 14 and 18 was considered to be due to sampling/analytical variation.
Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test item attained 41% 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 aerobi 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-organisims with mineral medium in sealed culture vessels in the dark at approximately 21 ºC for 28 days.

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

Results

The test item attained 41% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 15 August 2016 and 12 September 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 302 C (Inherent Biodegradability: Modified MITI Test (II))
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Test substance: GL500
Chemical name: 1,4-Benzenedicarboxylic acid, mixed Bu and 2-ethylhexyl diester (CAS name) Reaction product of terephthalic acid, 2-ethylhexanol and 1-butanol (IUPAC name)
CAS: 1571954-81-8 (A: 1962-75-0, B:1429441-82-6, C: 6422-86-2)
Chemical formula: A: C16H22O4 B: C20H30O4 C: C24H38O4
Molecular weight: A: 278 B: 334 C: 390
Purity: 99.8 %
Lot number: GLFG160607
Re-certification date: Jul. 01, 2017
Appearance: Colorless liquid
Storage conditions: Room temperature (25°C),no contact with air and light
Water solubility 2.19 mg/L (209r
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
Activated sludge, surface soil and surface water were sampled from ten sites distributed in four districts throughout Nanjing city, such as Chengdong, Chengbei, and Jiangning. 1 L of the sludge, soil and water were collected and mixed thoroughly together. After removing floating matter, the mixture was allowed to stand and then the supernatant was filtrated through filter paper. After that the filtrate was adjusted to pH 7.0 with sodium hydroxide or phosphoric acid. Finally an appropriate volume of the filtrate was transferred to a fill-and-draw activated sludge vessel and aerated for about 23.5 h (Batch No.: IN201607071).

Thirty minutes after stopping the aeration, about one third of the whole volume of supernatant was discarded. Then an equal volume of synthetic sludge (a solution at pH 7.0 containing 0.1% each of glucose, peptone and potassium orthophosphate) was added into the settled material which was then aerated again. This procedure was repeated once per day during one month.

Before use the mixture was allowed to stand, and the supernatant was removed. A small quantity of sludge was taken to be centrifuged (2500 r/min×10 min) and then weighed. Then the sludge was dried in the oven and weighed again in order to calculate the content of dry sludge was 10.9%. At last 18.35 g of centrifuged sludge was diluted 0.5 L with basal culture medium (BSM) to get an activated sludge suspension with a concentration of 4000 mg/L (dry basis).
Duration of test (contact time):
28 d
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Preparation of the Test Medium
The Based Salt Medium (BSM) was prepared by adding 3 mL of each of the following parent solutions prepared in pre-aerated deionized water to 1 litre of deionized water.10 L BSM was prepared (Batch No.: BSM201608151).
Parent solution A g/L
KH2PO4 (potassium dihydrogen phosphate) 8.50
K2HPO4 (dipotassium hydrogen phosphate) 21.75
Na2HPO4.12H2O (disodium phosphate dodecahydrate) 44.60
NH4Cl (ammonium chloride) 1.70
The pH of this solution was 7.30
Parent solution B
CaCl2 (calcium chloride) 27.50
Parent solution C
MgSO4.7H2O (magnesium sulphate heptahydrate) 22.50
Parent solution D
FeCl3.6H2O (iron (III) chloride hexahydrate) 0.25

Preparation of Solutions of the Test Substance
Test Substance was added to the test bottles directly.

A parent solution of the reference substance (sodium benzoate) at 1002 mg/L: 1.0022 g of reference substance was dissolved in 1 L volumetric flask with BSM.

Test Conditions
(1) Average concentration of test chemicals: about 30 mg/L.
(2) Concentration of reference chemicals: 100 mg/L (W/V)
(3) Concentration of activated sludge in “test” and “blank inoculum control”: 100 mg/L (W/V)
(4) Concentration of activated sludge in “reference”: 30 mg/L (W/V)
(5) Test temperature: (25±2) ºC
(6) Period: 28 days
(7) pH: 7.31-7.62
(8) Stir vigorously with mechanical stirrer.

Test Procedure
The following test substance will be added to the BOD bottles 1”abiotic control”, bottles 2-4 “test”:

Bottle 1 2 3 4
Test substance (g) 0.0104 0.0106 0.0097 0.0092
Concentration of test substance (mg/L) 34.7 35.3 32.3 30.7

Bottle 1 designated as “abiotic control” was then filled to a final volume of 300 mL with deionised water. Concentration of test chemical was about 30 mg/L.
For bottles 2-4 designated as “test”, 7.5 mL of activated sludge suspension were added to each of these after which they were filled to a final volume of 300 mL with BSM. The concentration of activated sludge was 100 mg/L and concentration of test chemical was 50 mg/L.
For bottle 5 designated as “procedure control”, 30 mL parent solution of reference substance and 2.25 mL of activated sludge suspension were added. Bottle was then filled to a final volume of 300 mL with BSM. The concentration of activated sludge was 30 mg/L and concentration of the reference chemical was 100 mg/L.
For bottle 6 designated as “blank inoculum control”, 7.5 mL of activated sludge suspension was added and bottle was then filled to a final volume of 300 mL with BSM. The concentration of activated sludge was 100 mg/L.
The equipment was then assembled, checked for air-tightness, stirrers were started and measurement of oxygen uptake under conditions of darkness began.
The temperature, the operation of the stirrer and recorder was checked daily. Any changes in colour of the contents of the vessels were recorded. The BOD for the six bottles were determined and recorded.
After the 28 days of testing, concentration of GL500 in the testing bottles was analysed.

Analysis of COD
The COD was determined to substitute the ThOD which cannot be calculated as the test substance is mixture.

Parent solution of test substance (1168 mg/L): 0.0584 g test substance was weighted and made up to 50 mL with acetone.

19.9 mg/L and 40.3 mg/L samples were prepared by adding 34 μL and 69 μL of Parent solution of the test substance (1168 mg/L) into test tubes, with solvent was dried, Then 2 mL COD reagents were added, and 3 parallel for each concentration. Blank control was prepared by above method expect for Parent solution of the test substance substituted by solvent.

Heat all samples at 150ºC for 120 minutes, and then determine the COD of samples after cool them to room temperature.
Reference substance:
other: Sodium benzoate
Test performance:
The test was valid because the level of biodegradation of the reference substance, sodium benzoate, was 76.3 % after 7 days (> 40%), and 81.8 % after 14 days (> 65%), and recovery rate of residual amount of the test compound in the “abiotic control” test was found to be more than 10% after 28 days.
Key result
Parameter:
% degradation (O2 consumption)
Value:
81.7
Sampling time:
28 d
Details on results:
Test solutions were prepared in an inorganic salts medium, inoculated with a number of micro-organisms collected from 10 places in Nanjing city. During the test, the temperature was kept at (25±2) ºC.

The BOD results showed that inherent biodegradation of the test substance (GL500) was 62.1 % after 28 days based on ThODNH3.
Based on the residue analysis, inherent biodegradation of the test substance (GL500) was 81.7% during the testing period.
Results with reference substance:
The level of biodegradation of the reference substance, sodium benzoate, was 76.3 % after 7 days (> 40%), and 81.8 % after 14 days (> 65%)
Validity criteria fulfilled:
yes
Interpretation of results:
inherently biodegradable, fulfilling specific criteria
Conclusions:
The BOD results showed that inherent biodegradation of the test substance (GL500) was 62.1 % after 28 days based on ThOD.
Based on the residue analysis, inherent biodegradation of the test substance (GL500) was 81.7% during the testing period.
Executive summary:

The inherent biodegradation test on the test substance (GL500) was performed according to “The guidelines for the testing of chemicals” SEPA(HJ/T 153 -2004), “The guidelines for the testing of chemicals, Degradation and Accumulation” (the 2nd edition) (Beijing: China Environment Press. 2013), and Procedure of the ‘Guidelines for Testing of Chemicals’ of the OECD: “Inherent Biodegradability: Modified MITI Test (II)” (1981).

Test solutions were prepared in an inorganic salts medium, inoculated with a number of micro-organisms collected from10 places in the city. During the test, the temperature was kept at (25±2) ºC. The test was valid because the level of biodegradation of the reference substance sodium benzoate was 76.3% after 7 days (> 40%), and 81.8 % after 14 days (> 65%), and the recovery rate of residual amount of the test compound in the “abiotic control” is found to be more than 10% after 28 days.

The BOD results showed that inherent biodegradation of the test substance (GL500) was 62.1 % after 28 days.

Based on the residue analysis, inherent biodegradation of the test substance (GL500) was 81.7 % during the testing period.

Description of key information

The test item attained 41% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Based on the residue analysis, inherent biodegradation of the test substance (GL500)was 81.7 % during the testing period.

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable

Additional information