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Environmental fate & pathways

Biodegradation in water and sediment: simulation tests

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Endpoint:
biodegradation in water: simulation testing on ultimate degradation in surface water
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 2019 - March 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report Date:
2020

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 309 (Aerobic Mineralisation in Surface Water - Simulation Biodegradation Test)
Principles of method if other than guideline:
The robust study summary (RSS) provided gives an overview of test results available to date. The identification of metabolites is still on-going. An update of this RSS including the metabolite identification is foreseen May 2020.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Selcia Ltd,
- Lot/batch number of test material: 6957KEP010-12
- Expiration date of the lot/batch: End of February 2020 (Set arbitrary to 1 year upon receipt by Fraunhofer IME); recertification by Fraunhofer IME by HPLC until February 27, 2021
- Purity test date: 15 February 2019

RADIOLABELLING INFORMATION
- Radiochemical purity: 99.1 %
- Specific activity: 6.25 MBq/mg
- Locations of the label: 2 outer benzene rings
- Expiration date of radiochemical substance: End of February 2020 (Set arbitrary to 1 year upon receipt by Fraunhofer IME); recertification by Fraunhofer IME by HPLC until February 27, 2021

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Store <- 15 °C, keep away from food and drink; store in a dry, well-ventilated area.
- Stability under storage conditions: Verified and confirmed
- Stability under test conditions: Degradation during test itself has been observed .However, stability in stock solutions has been demonstrated
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: not applicable

Radiolabelling:
yes

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
natural water: freshwater
Details on source and properties of surface water:
- Details on collection (e.g. location, sampling depth, contamination history, procedure): Biggesee (Bigge Lake) in Olpe, Germany. It is a drinking water reservoir without industrial or domestic inputs (waste water treatment plants). The drainage area is mostly grassland so that agricultural input is low.
After the sampling, the water was tranported to Fraunhofer IME. Upon arrival at Fraunhofer IME the surface water was passed through a 100 µm filter to remove coarse particles.
- date of collection: 11 October 2019
- sampling depth: 15-25 cm
- Storage conditions: aerobic conditions, in the dark at 4°C (max 7d)
- Storage length: maximally 1 month

- Temperature (°C) at time of collection: 11.3°C
- pH at time of collection: 7.34
- Electrical conductivity: not determined
- Redox potential (mv) initial/final: 257.5/236.8 (12°C sample set)
259.6/235.3 (20°C sample set; final value measured at day 42)
- Oxygen concentration (mg/l) initial/final: 9.29/9.00 (12°C sample set)
8.45/8.24 (20°C sample set; final value measured at day 42)
- Hardness (CaCO3): not determined
- Dissolved organic carbon: 4.05 mg/L
- Biomass: 5.5 BOD mg O2/L

- Water filtered: yes
- Type and size of filter used, if any: 100 µm filter
Details on source and properties of sediment:
Not applicable
Details on inoculum:
Not applicable
Duration of test (contact time):
60 d
Initial test substance concentrationopen allclose all
Initial conc.:
8 µg/L
Based on:
test mat.
Remarks:
nominal test concentration
Initial conc.:
40 µg/L
Based on:
test mat.
Remarks:
nominal test concentration
Parameter followed for biodegradation estimation:
CO2 evolution
radiochem. meas.
test mat. analysis
Details on study design:
TEST CONDITIONS
- Volume of test solution/treatment: 250 mL in erlenmeyers of 0.5 L
- Composition of medium: fresh water as described above
- Additional substrate: no
- Solubilising agent (type and concentration if used): organic co-solvent acetonitrile (MeCN)
- Test temperature:
- 12°C: sample set of low concentration (8 µg/L) and high concentration (40 µg/L) for determination of degradation kinetics
- 20°C: sample set of high concentration (40 µg/L) for determination of metabolites
For justification of test temperature selection, please see document attached as background material.
- pH initial/final: 7.9/7.8
- pH adjusted: no
- Aeration of dilution water: by a gentle flow-through humidified air
- Suspended solids concentration:
- Continuous darkness: yes/no
- Any indication of the test material adsorbing to the walls of the test apparatus: no visual observations. This has also been confirmed by analysisof a rinsing solution


TEST SYSTEM
- Culturing apparatus: flow-through syste. The vessel will be closed with ground joint caps equipped with a gas inlet and outlet. The inlet will be connected gas-tight to a gas supply, which continuously supplies moistened air in order to ensure aerobic conditions during incubation. The lids outlet will be connected gas-tight to two adsorption traps with 2 N NaOH in series. The outgoing gas will pass through these bottles in order to trap 14CO2. Vessels will be placed on orbital shaker and gently shaken in order to facilitate oxygen transfer into the liquid phase.
- Number of culture flasks/concentration: 2 replicates per concentration and per sampling point.
- Method used to create aerobic conditions: Vessels will be placed on orbital shaker and gently shaken in order to facilitate oxygen transfer into the liquid phase. Moreover, samples are aerated by a gentle flow-through of humidified air.
- Method used to create anaerobic conditions: not applicable
- Method used to control oxygen conditions: not applicable

- Test performed in closed vessels due to significant volatility of test substance: closed vessels with ground joint caps equipped with a gas inlet and outlet.
- Test performed in open system: No
- Details of trap for CO2 and volatile organics if used: t wo adsorption traps with 2 N NaOH in series


SAMPLING
- Sampling frequency: 0, 1, (3), 7, 14, 21, 28, 41, 55, 60 days
- Sampling method used per analysis type: sacrifical sampling

DESCRIPTION OF CONTROL AND/OR BLANK TREATMENT PREPARATION
CONTROL AND BLANK SYSTEM
- Sterility check if applicable: yes. Sterilised subsamples will be prepared in duplicate per incubation temperature in order to be able to distinguish between microbial and chemical transformation of the test item. The sterilised samples (heat sterilization by means of an autoclave) will be applied using a Laminar flow cabinet. Only sterilised glasses and pipettes will be used for the application. After application, the sterile samples will be sealed gas-tight and placed on an orbital shaker to insure a gently agitation of surface water.
- Sample storage before analysis: not applicable, direct analysis
- Inoculum blank: Non-treated samples, called blank controls, have been incubated under identical conditions as the samples with the test substance. These samples are used to monitor the pH and the DO values during the incubation period. The values have been determined at every sampling time point.
- Reference substance: 14C-sodium benzoate. Samples applied with 14C-sodium benzoate as reference substance have been prepared in duplicate per incubated temperature and incubated under identical conditions as the samples with the test substance. Sodium benzoate is easily degraded under aerobic conditions. These reference samples were prepared to ensure or demonstrate sufficient microbial activity of the test water.

STATISTICAL METHODS: see "other info"
Reference substance
Reference substance:
benzoic acid, sodium salt
Remarks:
14C labelled

Results and discussion

Mean total recoveryopen allclose all
Compartment:
entire system
% Recovery:
95.1
St. dev.:
2
Remarks on result:
other: 12°C, high concentration sample set
Remarks:
on day 60
Compartment:
entire system
% Recovery:
83.1
St. dev.:
9.9
Remarks on result:
other: 12°C; low concentration sample set
Remarks:
on day 55 as measurement on day 60 failed
% Degradationopen allclose all
Key result
% Degr.:
71.03
St. dev.:
8.56
Parameter:
test mat. analysis
Remarks:
radio-HPLC
Sampling time:
60 d
Remarks on result:
other: 12°C, high concentration sample set
Key result
% Degr.:
92.2
St. dev.:
2.26
Parameter:
test mat. analysis
Remarks:
radio-HPLC
Sampling time:
55 d
Remarks on result:
other: 12°C, low concentration sample set
Half-life of parent compound / 50% disappearance time (DT50)open allclose all
Key result
Compartment:
entire system
DT50:
11.8 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: low concentration sample set
Key result
Compartment:
entire system
DT50:
28.5 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: low concentration sample set
Transformation products:
not specified
Remarks:
the identification of the transformation products is on-going and will be updated when full report is available (May 2020)
Details on transformation products:
details will be provided when full report is available.
Evaporation of parent compound:
no
Details on results:
TEST CONDITIONS
- Aerobicity, moisture, temperature and other experimental conditions maintained throughout the study: Yes
- Anomalies or problems encountered (if yes): yes, to be completed

TRANSFORMATION PRODUCTS
information on transformation products will be completed when final report is available.


TOTAL UNIDENTIFIED RADIOACTIVITY (RANGE) OF APPLIED AMOUNT
information on transformation products will be completed when final report is available.

EXTRACTABLE RESIDUES
- % of applied amount at day 0: 99.4 % (12°C; low conc) and 104.3 % (12°C; high)
- % of applied amount at end of study period: 38% (low after 55d, sum of EA and DCM extracts; additional 2.1% in rinsing solution and filter extracts); 78.7% (high at day 60, sum of EA and DCM extracts; additional 3.5% in rinsing solution and filter extracts)

NON-EXTRACTABLE RESIDUES
- % of applied amount at day 0: 0.7 % (12°C; low conc), 0.3 % (12°C; high conc)
- % of applied amount at end of study period: 27.1% (12°C low after 55d, sum of extracted water and filter combustion); 12.3% (high at day 60; sum of extracted water and filter combustion)

MINERALISATION
- % of applied radioactivity present as CO2 at end of study: 2.7 to 0.5 % for the low and high concentration sample set respectively

VOLATILIZATION
- % of the applied radioactivity present as volatile organics at end of study: None

STERILE TREATMENTS
- Transformation of the parent compound: Yes. 35.8% parent recovered after 62d
- Formation of transformation products: Yes. Comparable to non sterile samples
- Formation of extractable and non-extractable residues: 92.1 % exctractable, 5.9% non extractable (found in extracted water)
- Volatilization: No. Closed sample

RESULTS OF SUPPLEMENTARY EXPERIMENT (if any):
It was observed that the mass balance deviates more from the desired 90-100 % as the study proceeds. Therefore additional efforts were undertaken to trace back the missing radio-activity and to improve the mass balance.
The additional efforts were:
- Extraction of filtered biosolids and afterwards combustion of filter.
- Rinsing of glassware
Results with reference substance:
Reference substance: sodium benzoate (14C labelled)
12°C standard reference sample: 45% mineralization after 14d
12°C solvent control (1% MeCN) reference sample: 61% mineralization after 14d
20°C solvent control (1% MeCN) reference sample: 63% mineralization after 14d

Any other information on results incl. tables

For better interpretation of the results we refer to the extraction scheme attached in the background material section.

LOW CONCENTRATION SAMPLE SET, 12°C

Mass balance

low concentration sample set, at 12°C   % of applied radio-activity by incubation time (days)
Rep 0 1 7 14 21 28 35 41 55 59
Organic extract (EA extraction) 1 99.8 100.3 96.8 74.9 60.5 57.5 40.3 31,4 19.2 na
2 98.9 98.4 101.5 73.4 63.4 61.8 32.9 32.5 34.6 na
mean 99.4 99.4 99.1 74.2 61.9 59.7 40.3 32.0 26.9 na
Aq. sample after EA extraction 1 0.8 2.1 10.8 21.2 28.0 28.6 37.2 44.3 43.1 na
2 0.6 1.9 8.9 21.1 28.4 26.1 39.4 38.4 48.8 na
mean 0.7 2.0 9.9 21.2 28.2 27.3 38.3 41.3 45.9 na
Alkaline traps 1 0.0 0.2 0.3 0.3 0.8 0.8 1.4 2.3 5.0 4.5
2 0.0 0.2 0.6 0.3 0.6 0.8 0.0 0.5 0.5 2.0
mean 0.0 0.2 0.5 0.3 0.7 0.8 0.7 1.4 2.7 3.3
Filter extract 1 na na na na na na na 1.3 1.4 1.4
2 na na na na na na na 1.7 2.6 0.9
mean na na na na na na na 1.5 2.0 1.1
Filter combustion 1 na na na na na na na 4.5 4.4 7.7
2 na na na na na na na 4.4 6.4 5.8
mean na na na na na na na 4.4 5.4 6.8
Rinsing solution 1 0.0 0.0 0.0 0.0 0.2 0.0 1.7 0.6 0.1 0.2
2 0.0 0.0 0.0 0.0 4.6 0.0 0.6 0.4 0.1 2.4
mean 0.0 0.0 0.0 0.0 2.4 0.0 1.1 0.5 0.1 1.3
Total 1 100.7 102.6 107.9 96.4 89.9 86.9 80.6 84.4 73.2 13.8
2 99.5 100.4 111.0 94.9 97.0 88.7 72.8 77.8 93.0 11.2
mean 100.1 101.5 109.5 95.7 93.2 87.8 76.7 81.1 83.1 12.5

The extraction with DCM is not included in the mass balance, but is important for the determination of parent and metabolites. The results are given below:

  % of applied radio-activity by incubation time (days)
Rep 0 1 7 14 21 28 35 41 55 59
DCM extract 1 na na na na na 11.8 12.0 16.0 16.7 na
2 na na na na na 10.7 10.9 24.1 5.4 na
mean na na na na na 11.2 12.0 20.0 11.1 na
Aq. sample after EA and DCM extraction 1 na na na na na 9.9 13.0 18.0 15.2 17.4
2 na na na na na 9.6 15.3 14.0 28.2 23.6
mean na na na na na 9.8 14.1 16.0 21.7 20.5

Results radio-HPLC evaluation on EA + DCM extract samples

  % of applied radio-activity by incubation time (days)
Rep 0 1 7 14 21 28 35 41 55 59
Parent peak in EA + DCM extract 1 99.8 93.5 83.1 32.3 30.1 12.2 5 5.8 4 na
2 98.9 91 95.4 26.5 39.9 9.1 5 7.2 7.3 na
mean 99.4 92.2 89.2 29.4 35 10.6 5 6.5 5.6 na

Results on the metabolites will be provided when full report is available.

HIGH CONCENTRATION SAMPLE SET, 12°C

Mass balance

high concentration sample set, at 12°C   % of applied radio-activity by incubation time (days)
Rep 0 1 3 7 14 21 28 35 41 56 60
Organic extract (EA extraction) 1 103.7 99.0 99.5 96.3 82.6 76.8 70.5 71.5 80.6 76.8 71.9
2 104.9 102.0 97.4 95.9 91.9 77.8 74.4 64.3 86.9 73.4 69.7
mean 104.3 100.5 98.5 96.1 87.2 77.3 72.5 67.9 83.7 75.1 70.8
Aq. sample after EA extraction 1 0.2 0.9 2.6 7.3 12.8 17.7 17.1 18.0 20.3 18.6 13.2
2 0.3 1.0 2.6 5.1 9.4 20.4 16.8 22.6 17.6 19.4 22.8
mean 0.3 1.0 2.6 6.2 11.1 19.1 17.0 20.3 18.9 19.0 18.0
Alkaline traps 1 0.0 0.0 0.1 0.0 0.1 0.6 0.8 0.5 0.3 0.8 0.3
2 0.0 0.0 0.0 0.0 0.0 0.4 1.0 0.0 0.0 0.0 0.6
mean 0.0 0.0 0.1 0.0 0.1 0.5 0.9 0.2 0.1 0.4 0.5
Filter extract 1 na na na na na na na na 2.2 1.9 2.0
2 na na na na na na na na 2.5 1.7 0.8
mean na na na na na na na na 2.3 1.8 1.4
Filter combustion 1 na na na na na na na na 2.7 3.4 2.8
2 na na na na na na na na 1.6 3.3 2.0
mean na na na na na na na na 2.2 3.3 2.4
Rinsing solution 1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.9 1.0 0.5 3.0
2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.8 1.2 0.3 1.2
mean 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.8 1.1 0.4 2.1
Total 1 103.9 100.0 102.3 103.6 95.5 95.1 88.4 90.9 107.1 102.0 93.1
2 105.2 103.1 100.0 100.9 101.4 98.6 92.2 87.7 109.9 98.1 97.1
mean 104.6 101.5 101.1 102.3 98.4 96.8 90.3 89.3 108.5 100.0 95.1

The extraction with DCM is not included in the mass balance, but is important for the determination of parent and metabolites. The results are given below:

  % of applied radio-activity by incubation time (days)
Rep 0 1 3 7 14 21 28 35 41 56 60
DCM extract 1 na na na na na na na 7.7 9.1 6.7 6.1
2 na na na na na na na 8.5 5.9 7.3 9,7
mean na na na na na na na 8.1 7.5 7.0 7.9
Aq. sample after EA and DCM extraction 1 na na na na na na 5.2 5.3 5.6 5.2 4.3
2 na na na na na na 5.0 6.2 4.1 5.2 15.6
mean na na na na na na 5.1 5.8 4.8 5.2 9.9

Results radio-HPLC evaluation on EA + DCM extract samples

  % of applied radio-activity by incubation time (days)
Rep 0 1 3 7 14 21 28 35 41 56 60
Parent peak in EA + DCM extract 1 97.2 92.3 97.2 90.3 68.6 62.7 31.6 33.1 23.4 34.7 32.5
2 93.2 94.4 95.1 85.7 67.2 58.7 40.2 37.1 35.9 34.6 18.4
mean 95.2 93.3 96.1 88.0 67.9 60.7 35.9 35.1 29.7 34.6 25.5

Information on the metabolites will be provided when full report is available.

Sterile samples:

Sterile samples were run with high concentration test substance. As for the biotic samples, degradation of R898 has been observed. This leads to the conclusion that primary degradation may occur not only via biotic, but also via abiotic pathways. Further degradation of the primary metabolites does involve microbial processes.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
R898 is not persistent in water. The DT50 values observed for the parent were 11.8 and 28.5 days for the low and high concentration samples set, incubated at 12°C. The metabolite identification is pending.
Executive summary:

The degradation potential of R898 in freshwater has been assessed according to OECD 309, surface water simulation testing. For this study radio-labelleled R898 was used, with the label present on the two outer rings of the molecule. The study has been performed using freshwater sampled at Biggesee, Germany.

For the purpose of determination of DT50 of the parent, the main incubation temperature was set at 12°C. At this temperature two sample sets have been assessed: a low concentration (8 µg/L) and high concentration (40 µg/L) sample set. Samples were incubated for a total incubation period of 60 days and sampling was performed on 0, 3, 7, 14, 21, 28, 41, 55 and 60 days.

Additionally, for the purpose of metabolite identification, a sample set of the high test concentration (40 µg/L) has been incubated at 20°C for 60 days. As this sample set is related to metabolite identification, the results will be added when full report is available.

12°C:

At start of the experiment all parent material could be found in the organic (ethyl acetate) extract. No degradation was observed in the first 7 days of the experiment in both the low and the high concentration samples. As of day 14 significant degradation was observed. With increasing degradation, more and more radio-activity was observed in the aqueous sample. This shows that the metabolites are more polar as compared to the parent compound. Around day 41 an additional filtration step has been introduced to filter of the biosolids formed. About 2 -3 % of parent was found in the filter extracts.

For the low concentration sample set the mass balance at the end of the experiment was 83.1 % (after 55 days), while for the higher sample set the mass balance yielded 95 %. This coincides with the fact that more degradation was observed in the low concentration sample set.

Fitting the pseudo first-order kinetics to the parent concentrations (using CAKE model) results in following DT50 values: 11.8 days for the low concentration sample set and 28.5 days for the high concentration sample set. For both the conclusion is hence that the substance is not persistent in freshwater.

Information on the metabolites will be added in May 2020

Sterile samples

Sterile samples were run with high concentration test substance. As for the biotic samples, degradation of R898 has been observed. This leads to the conclusion that primary degradation may occur not only via biotic, but also via abiotic pathways. Further degradation of the primary metabolites does involve microbial processes.

In conclusion, based on the available results, DAPD is considered to be not persistent in freshwater with both DT50 values well below the cut-off of 40days.