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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:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: OECD method 301C. No data on GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
Deviations:
not specified
GLP compliance:
not specified
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
- Concentration of activated sludge: 30 mg/L
Duration of test (contact time):
4 wk
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Parameter:
% degradation (O2 consumption)
Value:
4
Sampling time:
4 wk
Remarks on result:
other: Direct analysis: HPLC
Parameter:
% degradation (O2 consumption)
Value:
2
Sampling time:
4 wk
Remarks on result:
other: Direct analysis: HPLC

Indirect analysis: BOD:

-1%, 0%, 0%

Validity criteria fulfilled:
not specified
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The substance is considered as not readily biodegradable.
Executive summary:

A ready biodegradability study was conducted according to OECD 301 C (MITI-I). The test period was 4 weeks. The BOD after the 4 weeks was 0%. The direct analysis showed the following results:

HPLC: 4%, 2%, 2%

The substance is considered as not readily biodegradable.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: EU method C.4-C. GLP study.
Qualifier:
according to guideline
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
yes
Remarks:
(no data available to assess the total CO2 evolution in the inoculum blank at the end of the test)
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Coarse-filtered supernatant of homogenised activated sludge obtained from Canterbury Sewage Treatment Works.
Duration of test (contact time):
28 d
Initial conc.:
20 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Test temperature: 20 ± 1 ºC
- Aeration of dilution water: 30 ml/min of CO2-free air.
- Inoculum: 10 ml/L


TEST SYSTEM
- Number of culture flasks/concentration: 61 and 62 mg of the test substance were added to duplicate vessels to give a nominal test concentration of 20 mg/L.
- Measuring equipment: CO2 evolution was determined by titrating the contents of the Ba(OH)2.8H2O absorber bottles proximal to the units against standard HCl. In order to measure CO2 trapped as inorganic carbonates in the medium, the contents of each Sturm vessel were acidified with 1 ml concentrated H2SO4 on Day 27 of the test. All three absorber bottles were titrated on Day 28.


SAMPLING
- Sampling frequency: The extent of biodegradation in each unit was determined every few days.


CONTROL AND BLANK SYSTEM
- Inoculum blank: Controls containing inoculated mineral medium only were included in the test run.

Reference substance:
benzoic acid, sodium salt
Remarks:
(20 mg/L)
Parameter:
% degradation (CO2 evolution)
Value:
0 - 2
Sampling time:
28 d
Results with reference substance:
The reference compound sodium benzoate was extensively degraded, with net carbon dioxide evolution of 83-86% ThCO2 at the end of the test.
Validity criteria fulfilled:
not specified
Remarks:
(no data available to assess the total CO2 evolution in the inoculum blank at the end of the test)
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
A negligible proportion (0 -2% ThCO2) of the test substance was mineralised over the 28 day test period. On the basis of these data, the test substance is not "readily biodegradable".
Executive summary:

The aims of this study were to determine any inhibitory effects of the test substance on activated sludge respiration and to assess its "ready biodegradability" using the EEC modified Sturm test. A negligible proportion (0 -2% ThCO2) of the test substance was mineralised over the 28 day test period. On the basis of these data, the test substance is not "readily biodegradable".

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:
other: Guideline Study
Qualifier:
according to guideline
Guideline:
other: OECD 314B
Deviations:
yes
Remarks:
Outlined in Methods
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
other: aerobic and anaerobic
Inoculum or test system:
other: activated sludge, adapted and non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Samples of activated sludge and raw sewage taken from an oxidation ditch at the municipality of Driebergen, the Netherlands. The activated sludge was transported in plastic bottles and aerated until use.
- Preparation of inoculum for exposure: The dry weight of the sludge was adjusted to 2.5-3.0 g/L by the addition of tap water if the concetration was too high or allowing the sludge to settle and decanting if the concentration was too low.
- Pretreatment: For sterile control, the sludge was sterilized using heat and mercury chloride. The confirmation of sterility was done by placing a few drops of the supernatant on agar plates and incubating at 20 degrees C for 1-3 weeks, after which the formed colonies were counted.
- Concentration of sludge: adjusted to 2.5-3.0 g/L
Duration of test (contact time):
28 d
Initial conc.:
0.01 mg/L
Based on:
other: Liquid Scintillation Counting
Initial conc.:
0 mg/L
Based on:
test mat.
Initial conc.:
0.001 mg/L
Based on:
test mat.
Initial conc.:
0.01 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Parameter followed for biodegradation estimation:
radiochem. meas.
Parameter followed for biodegradation estimation:
test mat. analysis
Details on study design:
TEST CONDITIONS
- Test temperature: 19.0-20.6 degrees C (mean of 19.7 degrees C)
- Continuous darkness: yes


TEST SYSTEM
- Culturing apparatus: Closed test vessels with a volume of 2 L with 1 L of total sludge per vessel.
- Number of culture flasks/concentration: 1
- Method used to create aerobic conditions: The biotic systems were fed continuously with CO2 free moisturized air.
- Measuring equipment: HPLC
- Test performed in open system: No
- Details of trap for CO2 and volatile organics if used: The biotic vessels were connected to gas trapping systems which consisted of (1) One empty bottle, (2) One bottle containing 200 mL of ethylene glycol and (3) Two bottles, each containing 200 mL of 1.5M KOH solution for trapping CO2.
- Other:


SAMPLING
- Sampling frequency: Biotic activated sludge at 1h, 4h, 1d, 2d, 3d, 7d, 14d, 21d, and 28d. For abiotic sludge at 1h, 4h, 1d, 7d, and 28d.
- Sampling method:
Gas Traps: Two separate 1 mL samples were taken from each gas trap, placed in 20 mL scintillation vials with 10mL Ultima Gold and the radioactivity was counted by LSC.
Sampling for measurement of dissolved 14CO2: Two 5 mL samples of mixed liquor were taken from the test vessels and placed in biometer flasks with 2 mL of 1.5 mL KOH solution dispensed in the headspace. One mL of 18% HCl was injected and the biometer flask closed and incubtaed in the dark overnight. After, 10 mL of Ultima Gold was added to the KOH solution in the scintillation vials and the radioactivity measured.
Samples for determination of total activity in the mixed liquor: At each time point, two 1 mL samples were taken from each test vessel. The radioactivity in these samples was counted by LSC.
Samples for chemical analysis: Four 5 mL mixed liquor samples (three reserves) were taken from each test vessel. The samples were placed in centrifuge tubes and flash forzen by immersion into a liquid nitrogen bath. The samples were stored frozen until farther analysis.


CONTROL AND BLANK SYSTEM
- Abiotic sterile control: Sludge sterlized with heat and mercury chloride. The sterility of the sludge was confirmed prior to start of the test.
Preliminary study:
In the preliminary test the recovery remained between 90 and 110% TAR. Feasibility work revealed that methanol with 5% formic acid was the most suitable solvent. With this extraction method, 80-90% of the applied radioactivity is extractable from the solid residue, whereas the fraction that remains in the aqueous phase as well as the bound residue are both below 10% TAR. The recovery from the volatile traps is negligible. It was necessary to concentrate pooled extracts of the solid residue for detection on HPLC-RAD. When DMSO was added, a good recovery during the evaporation step was established. By re-dissolving the remaining DMSO fraction in 100 uL of Eluent A and 50 uL of Eluent B a chromatography and detection on HPLC-RAD was established. HPLC-RAD analysis of the extracts from the solid residue revealed that after 7 days of incubation, the estimated concentration of AN-2 was only 20% TAR. There was one metabolite eluting just before AN-2 that reached an estimated concentration of 30% TAR and one metabolite eluting just after AN-2 that reached an estimated concentration of 40% TAR. The concentration of these metabolites was increasing at the end of the test, which indicates that they are rather persistent. Based on this result, the DT50 of AN-2 was estimated to be approximately 3 days.
Test performance:
During the acclimatization and pre-adaptation phase, the room temperature varied between 18.5 and 21.5 degrees C (average 19.8 degrees C). During the incubation, the room temperature varied between 19.0 and 20.6 degrees C (average value 19.7 degrees C). On the agar plates on which drops of supernatant of sterilized sludge of the abiotic test system were placed before the start of the test and subsequently incubated for three weeks no colonies were observed. This confirmed that the abiotic system was sterile.
Parameter:
% degradation (test mat. analysis)
Value:
> 99
Sampling time:
0.04 h
Remarks on result:
other: already after 0.4 h no original test substance could be detected anymore
Conclusions:
It can be concluded that the primary degradation of the test material is rapid.
Executive summary:

An activated sludge die away test was carried out with radiolabelled AN-2 (4,4’-methylenebis(2,6-di-tert-butylphenol). The test, which was based on the outcome of a preliminary experiment, was carried out in glass round bottom test vessels. Two biotic test systems were prepared: One system contained 11 non-adapted activated sludge and one system contained 11 pre-adapted activated sludge. These biotic systems were connected to a air supply and a gas trap series consisting of one empty bottle, one bottle with ethylene glycol and two bottles containing 1.5 M KOH solution. One abiotic test system was prepared that contained 11 abiotic, sterilized sludge. This system was closed and not connected to an air flow.

The test substance has a water solubility of less than 0.01 mg.L-1. As a first estimate this was taken as the target test substance concentration. At the same time this is the lowest test substance concentration that allows proper HPLC analysis of solids extracts. The test substance was dosed from a stock solution in ethanol. The stock solution was dosed slowly, while the activated sludge was gently shaken, yielding a final test substance concentration of 0.01 mg.L-1(0.072 MBq.L-1).

The test systems were incubated in the dark at 20±2°C and shaken continuously. Air was forced through the test systems by a pump. At various time points during the incubation, samples were taken from the mixed liquor (ML) and the gas traps. The total radioactivity in the samples was determined, and the ML samples were further processed to determine the distribution between the phases and to determine primary degradation. The aqueous phase of the ML and the solid phase mixed liquor suspended solids (MLSS) were separated by centrifugation. The radioactivity in the mixed liquor was determined and samples were analyzed by HPLC to determine if primary degradation occurred. The solids were extracted by using methanol/formic acid (95:5 v/v %). The amount of radioactivity in the extracts and remaining on the solids was determined and the extracts were concentrated and analyzed by HPLC.

The primary degradation of AN-2 was very rapid with estimated 50% disappearance times (DT50 values) of <1 hour in the biotic system with non-adapted sludge and the abiotic system. In the biotic system with pre-adapted sludge, the estimated DT50 value was <4days. An overview of estimated degradation rates of AN-2 is given in the Table below.

Table:Overview of estimated degradation rates of AN-2 in various test systems.

Test System

PreliminaryTest

Biotic System, with non-adapted sludge

Biotic System, with pre-adapted sludge

Abiotic System

DT50

app. 3 d

<1 h

<4 d

<1 h

DT90

n.d.*

<1 h

App. 7 d

<1 h

K

0.2 d-1

<0.7 h-1

< 0.2 d-1

<0.7 h-1

*not determined

From the rapid degradation rate in the biotic test system with non-adapted sludge and in the abiotic test system, it can be concluded that the degradation of AN-2 is abiotic. However, sludge characteristics still seem to play a very important role in the degradation rate. In this study three batches of sludge were studied: one batch for the preliminary test, one batch that was used for the biotic system with pre-adapted sludge in the definitive test and one batch that was used for the biotic system with non-adapted sludge and the abiotic system in the definitive test. The degradation may be catalysed by a certain chamical substance that was only present in the latter batch of sludge, which would be an explanation for the difference in degradation rate between the different systems. For a structurally related compound *2,6-di-tert-butyl-p-cresol(BHT), CAS 128-37-0), it is known that the stability in the environment and the formation of degradation products depend on several parameters like presence of atmospheric or dissolved oxygen, irradiation, pH-value, temperature and the presence of traces of heavy metals (metal oxides). This could also be the case for AN-2. In spite of the different degradation rates in the different batches of sludge, it can be concluded that the primary degradation of AN-2 is rapid.

Although identification of degradation products was not within the scope of the present study, the results give some indications with respect to the route of degradation: the fact that the degradation products have a retention time that does not differ much from the parent indicates that there were only slight modifications of the molecule (possibly de-methylation). Formation of phenolic moieties and hydrolysis are considered unlikely. Further elucidation of the molecular structure will be necessary to obtain more details about the exact route of the degradation.

An overview of the degradation products per system is given in the table below. In the biotic system with non-adapted sludge, two major transient degradation products (M1 and M2) and two major persistent degradation products (M3 and M4) were formed. In the biotic system with pre-adapted sludge, more degradation products were formed and M4 was not a major persistent degradation product, but a minor transient degradation product. It can be concluded that M4 was degraded in the pre-adapted sludge, whereas it was not degraded in non-adapted sludge. M7 is a major degradation product in the abiotic system, whereas it was not found in the biotic system with non-adapted sludge and it was a major transient degradation product in the biotic system with pre-adapted sludge. This indicates that M7 can only be degraded via a biotic route. M8 is a degradation product that is typical for pre-adapted sludge: it was not found in the other two test systems. M9 was only detected in the abiotic system, which indicates that also M9 can only be degraded via a biotic route.

Table:Overview of degradation products in various test systems

Test System

Approximate RetentionTime (min.)

Biotic System, with non-adapted sludge

Biotic System, with pre-adapted sludge

Abiotic System

M1

20.9

Major, transient

n.d.

n.d.

M2

24.6

Major, transient

Major, transient

Major, transient

M3

26.4

Major, persistent

Major, persistent

Major, but seems to degrade slowly

M4

25.4

Major, persistent

Minor, transient

Major, persistent*

M5

23.8

n.d.

Minor, transient

n.d.

M6

29.3

n.d.

Minor, transient

n.d.

M7

30.6

n.d.

Minor, transient

Major, persistent

M8

25.7

n.d.

Major, persistent

n.d.

M9

27.2

n.d.

Major, persistent

n.d. = not detected

*Indicative, the concentrations at the end of the test were above 10%, but the peaks in the chromatograms had areas close to the background.

In all test systems, total recovery was higher than the target recovery of the guideline of 85-110 % TAR. It is difficult to explain the high recovery. It may be that, compared to the test system, the samples contained a slightly higher concentration of solids (to which the test substance was adsorbed). In the test systems with non-adapted and pre-adapted sludge, there were occasions at the beginning of the test, where the recovery was very high. The test substance is very poorly water soluble. In this test system, major part adsorbed to solids. It is likely that some equilibration time was needed, in which the test substance was not homogeneously distributed in the test system.

From the very first sampling point (t = 1 h), the major part of the radioactivity was recovered from the solids. Most of the radioactivity that was adsorbed to the solids was extractable. The amount of14CO2that was formed was negligible.
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Internationally accepted method, EPI-Suite, EPA (USA)
Justification for type of information:
QSAR prediction: migrated from IUCLID 5.6
Principles of method if other than guideline:
QSAR calculation by EPI-Suite, EPA (USA) v4.10 / BIOWIN v4.10 (attached QSAR Prediction Reporting Format, QPRF)
GLP compliance:
no
Parameter:
probability of ready biodegradability (QSAR/QSPR)
Remarks on result:
not readily biodegradable based on QSAR/QSPR prediction

Probability of Rapid Biodegradation (BIOWIN v4.10):


Biowin1 (Linear Model) : 0.0959


Biowin2 (Non-Linear Model) : 0.0005


Expert Survey Biodegradation Results:


Biowin3 (Ultimate Survey Model): 1.4501 (recalcitrant)


Biowin4 (Primary Survey Model) : 2.6321 (weeks-months)


MITI Biodegradation Probability:


Biowin5 (MITI Linear Model) : -0.1702


Biowin6 (MITI Non-Linear Model): 0.0014


Anaerobic Biodegradation Probability:


Biowin7 (Anaerobic Linear Model): -1.7978


Ready Biodegradability Prediction: NO

Validity criteria fulfilled:
not applicable
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
Ready Biodegradability Prediction: NO
Executive summary:

The substance is estimated to be not readily biodegradable based on the calculation conducted with EPI-Suite, EPA (USA) v4.10 / BIOWIN v4.10.

Description of key information

The key study is an activated sludge die-away biodegradation test  using non-adapted and adapted activated sludge. This study resulted in a rapid degradation of the test substance in particular under abiotic and non-adapted conditions, indicating that the substance itself is rapidly degraded. However as almost no mineralisation was observed and the substance was not readily biodegradable in standard tests on ready biodegradations, further investigations on the degradation products are proposed,

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable

Additional information

The key study is an activated sludge die-away biodegradation test using non-adapted and adapted activated sludge. This study resulted in a rapid degradation of the test substance in particular under abiotic and non-adapted conditions, indicating that the substance itself is rapidly degraded. However as almost no mineralisation was observed and the substance was not readily biodegradable in standard tests on ready biodegradations, further investigations on the degradation products are proposed. In a first approach the substance can be regarded as inherently biodegradable.

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