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Environmental fate & pathways

Biodegradation in water and sediment: simulation tests

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Reference
Endpoint:
biodegradation in water: simulation testing on ultimate degradation in surface water
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 309 (Aerobic Mineralisation in Surface Water - Simulation Biodegradation Test)
GLP compliance:
yes (incl. certificate)
Radiolabelling:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
natural water: freshwater
Details on source and properties of surface water:
The study was conducted using Biggesee natural lake water (North-Rhine-Westphalia, Germany) including 15 mg/L suspended matter.

- Site description: A freshwater lake fed by a stream from a weir on the river. Woodland around the lake.
- Date of collection: 30. October 2018, 8.15 a.m.
- Collection procedure: Immersion of container
- Sampling depth: 30 - 50 cm
- Storage conditions: Samples kept waterlogged at ca. 4 ºC in the dark
- Duration of storage (prior to use): 9 days at 4 ± 2°C
- Preparation prior to use: Filtered through 100 µm mesh

Physical and chemical properties of surface water
- Temperature at collection (°C): 6.6
- pH at collection: 7.48
- Oxygen concentration at collection (mg/L): 10.3
- Optical appearance: slightly turbid
- pH: 0 d (start of study) 7.99, 64 d (end of study) 8.34
- Oxygen concentration (mg/L): 0 d (start of study) 8.84, 64 d (end of study) 8.66
- Total carbon (TC, mg/L): 17.17
- Dissolved organic carbon (DOC, mg/L): 2.74
Details on source and properties of sediment:
The study was conducted using Biggesee natural lake water (North-Rhine-Westphalia, Germany) including suspended sediment.
The filtered water was amended by its own sediment. The sampled sediment was suspended by lake water and filtered using a 2 mm sieve to remove coarse products. Particles were sedimented after 30 minutes and the supernatant suspension was filtered through 100 µm mesh size. An aliquot of 300 g of the sedimented lake sediment was mixed with 600 mL filtrate. The suspension was shaken. After 1h the supernatant was removed and its dry contend was determined using an oven. A volume of 3 mL suspension contained a sediment dry weight of 5 mg. For a final sediment concentration of 15 mg/L in 250 mL sample volume a suspension volume of 2.25 mL was required. Therefore, 247.75 mL filtrate was mixed with 2.25 mL suspension solution to prepare a sediment amended lake water test sample.

Physical and chemical properties of sediment
- Sediment layer [cm]: 10 - 20
- Optical appearance: dark brown
- Organic carbon (Corg) [%]: 3.00
- Clay content [%]: 18.5
- Sand content [%]: 18.1
- Silt content [%]: 63.3
Details on inoculum:
The biological activity of the lake water test samples was characterised by the addition of the easily degradable compound 14C-sodium benzoate to control samples (blank controls). Concentration of 14C-sodium benzoate was 10 µg/L. Control samples were handled in analogy to the treated samples. The formation of 14CO2 was followed until the mineralisation level of approximately 75 % of the applied radioactivity [% AR] were reached. This was at 13 DAT (days after treatment). NaOH-containing traps were analyzed at 1, 2, 3, 6, 9 and 13 DAT. 14CO2 quantification was accomplished by liquid scintillation counting (LSC).
Duration of test (contact time):
64 d
Initial conc.:
9.5 µg/L
Based on:
test mat.
Initial conc.:
65 µg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
radiochem. meas.
Details on study design:
Test system
Incubation of subsamples was performed in a flow-through system which was placed in a dark, temperature controlled lab-room. A constant stream of water saturated surrounding air was passed over the subsamples in order to maintain aerobic conditions during the experiment. The outgoing gas was bubbled by means of a vacuum pump through three absorption traps in sequence containing ethylene glycol and two times 1 M NaOH in order to determine volatile products and the rate of mineralization (trapping of 14CO2).
For information on the abiotic transformation of the test substance additional sterilized samples (duplicates) were taken, processed and analysed at 7 days after treatment (DAT), and 63 DAT.

Treatment
Vessels (250 mL) for low concentration (9.5 µg/L) and high concentration (65 µg/L) were spiked with 27.90 µL (low) and 28.10 µL (high) of [phenyl-U-14C]ZMB2 application solution. The applications were made directly into the test samples by a pipette.
Intended nominal treatment rate at low concentration was 12.26 kBq (9.5 µg/L) and at high concentration 83.85 kBq (65 µg/L), respectively. Based on the mass balance of the 0d of incubation samples the actual concentration confirms generally to the indented concentration and a correction was not accomplished.

Sampling
Sampling was performed after the following incubation times: 0 d (immediately after application), 2 d, 6 d, 9 d, 15 d, 20 d, 28 d, 36 d, 57 d and 64 d after application (DAT). Additionally sterile samples were sampled after 7 d and 63 d.

Absorptions solutions
At each sampling time the absorption traps were sampled. Additionally at 30 days of incubation all absorption flasks were sampled and exchanged against new flasks for all samples. Immediately after sampling the volume of each trapping solution was measured and total radioactivity in each solution was determined by LSC.
Reference substance:
benzoic acid, sodium salt
Remarks:
14C-labelled, specific radioactivity: 130 mCi/mmol; 33 kBq/µg
Test performance:
Extraction of the test water
At sampling, the vessels were removed from the orbital shaker. The volume was determined and radioactivity measured by LSC. Afterwards, the samples were extracted 1 x using 50 mL and 4 x 30 mL diethyl ether to obtain the organic extract. The aqueous phase and organic extract were separated by means of a separation funnel. The organic extracts were combined, the volumes of combined extract and extracted water were determined and the radioactivity content in both phases was quantified by LSC.

Filtration of the sediment and combustion
The organic extract was filtered by a paper filter, the exact volume of the sediment free organic extract was measured and quantified by LSC. The same paper filter was used to filter the extracted water layer. The exact volume of the sediment free extracted water was measured and quantified by LSC. The filter was dried, combusted and subjected to radio-assaying.

Sample concentration for degradation product identification
The frozen (-20°C) sample 64 days of incubation, water layer, high test concentration, replicate A was thawed 33 days after sampling. An aliquot of 1 mL was separated and stored again at -20°C. The remaining 244 mL sample was concentrated by a stream of nitrogen to a final volume of 21.0 mL. The recovery of radioactivity after concentration was 90%. The sample was used for HR LC-MS/MS analytic.

Sample analysis
If a trigger value of 5% AR in the organic extracts or in the extracted water was exceeded both radio-TLC methods were accomplished. A sample aliquot was applied on TLC plate. To ensure sample stability on the TLC plate before the chromatographic run all samples were applied on one TLC plate within 1h.
Compartment:
biologically active treatment at end of test
% Recovery:
97.3
Remarks on result:
other: 9.5 µg/L ZMB2
Compartment:
biologically active treatment at end of test
% Recovery:
94.5
Remarks on result:
other: 65 µg/L ZMB2
Compartment:
abiotic control measured at end of test
% Recovery:
94.3
Remarks on result:
other: sterile sample, 9.5 µg/L
Compartment:
abiotic control measured at end of test
% Recovery:
91.4
Remarks on result:
other: sterile sample 65 µg/L
% Degr.:
89.6
Parameter:
radiochem. meas.
Sampling time:
64 d
Remarks on result:
other: 9.5 µg/L ZMB2
Remarks:
degradation is based on MB2 as ZMB2 decomposes rapidly to MB2 and zinc ions in water
% Degr.:
86.1
Parameter:
radiochem. meas.
Sampling time:
64 d
Remarks on result:
other: 65 µg/L ZMB2
Remarks:
degradation is based on MB2 as ZMB2 decomposes rapidly to MB2 and zinc ions in water
Compartment:
entire system
DT50:
12.86 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: for MB2 (9.5 µg/L ZMB2)
Remarks:
ZMB2 decomposes rapidly to MB2 and zinc ions in water
Compartment:
entire system
DT50:
24.6 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: for MB2 (65 µg/L ZMB2)
Remarks:
ZMB2 decomposes rapidly to MB2 and zinc ions in water (IUCLID 13.2)
Transformation products:
yes
No.:
#1
No.:
#2
No.:
#3
No.:
#4
No.:
#5
No.:
#6
Details on transformation products:
Short names of the metabolites: #1: MB2, #2: Desthio-MB2, #3: MB2 sulfonic acid, #4: MB2 acetic acid, #5: Hydroxy-MB2
Evaporation of parent compound:
no
Volatile metabolites:
yes
Remarks:
very low amounts 0.1 - 5.5% AR; The value of 5.5% AR is quite high and seems to be a contamination. A second contamination was found in the absorption trap of sample 20 DAT, high test concentration, replicate A (19.7% AR).
Residues:
yes
Remarks:
NER: At 0 DAT the radioactive residues on suspended matter are very low with only 0.2% AR (low conc.) and 0.1% AR (high conc.). At incubation end (64 DAT) the residues were 7.9 and 9.8% AR (low conc.) and 5.8 and 6.2% AR (high conc.).
Details on results:
In the 0 DAT samples, MB2 was found at mean levels of 89.4% AR (low test concentration) and 91.5% AR (high test concentration). MB2 degraded continuously over time. At 64 days of incubation the MB2 degraded to mean levels of 10.4% AR (low test concentration) and 13.9% AR (high test concentration).

Two major (>10% AR) degradation products were formed during incubation in low and high test concentration samples. They were reaching maximum main levels of 34,4% AR (Desthio-MB2, low test concentration) after 36 days of incubation, 50.1% AR (Desthio-MB2, high test concentration) after 64 days of incubation, 31,0% AR (MB2 sulfonic acid, low test concentration) after 28 days of incubation and 20.3% AR (MB2 sulfonic acid, high test concentration) after 57 days of incubation.
A further major degradation product MB2 acetic acid was only found for the low test concentration samples reaching a maximum main level of 10.0% AR after 64 days of incubation. In the high test concentration samples the maximum main level of MB2 acetic acid was 5.9% AR after 64 days of incubation.

Three further unknown degradation products were detected not exceeding a main level of 7.3% AR.
Results with reference substance:
Concentration of 14C-sodium benzoate was 10 µg/L. Control samples were handled in analogy to the treated samples. The formation of 14CO2 was followed until the mineralisation level of 75 % of the applied radioactivity [% AR] was reached. This was at 13 DAT (days after treatment).

Identification of radioactive residues in natural water at low concentration (9.5 µg/L) ZMB2 [%] (mean values)

 %AR / sampling days -->

 0d

 2d

 6d

 9d  15d  20d  28d  36d  57d  64d
  amount in extracs and extracted water

 
94.1

 92.0

 88.6

 83.5

 84.9

 82.9

 82.4

 76.9

 83.4

 83.7

   origin*

 -

1.6 

 2.8

 2.2

 3.4

 3.2

 3.1

 2.8

 3.4

 3.5

 MB2 (overlapping with Hydroxy-MB2)

89.4

82.4 

 65.9

 56.9

 42.0

 24.7

 14.6

 15.0

 13.6

 10.4

 Desthio-MB2

 
3.9

 5.7

 13.8

 15.5

 26.0

 33.7

 28.2

 34.4

 32.6

 27.5

 MB2 sulfonic acid

 0.8

 2.2

 5.1

 5.6

 11.4

 21.4

 31.0

 15.0

 21.1

 21.0

 MB2 acetic acid

 -

 -

 1.0

 3.3

 2.0

 -

 5.6

 2.4

 8.8

 10.0

 unknonwn degradation products (sum)

 -

 -

 -

 -

 -

 -

 -

 7.3

 3.9

 12.5

*radioactive residues remaining on origin and smear <Rf 0.1

Identification of radioactive residues in natural water at low concentration (65 µg/L) ZMB2 [%] (mean values)

 %AR / sampling days -->

 0d

 2d

 6d

 9d  15d  20d  28d  36d  57d  64d
  amount in extracs and extracted water

 94.9

 91.1

 88.9

 85.8

 88.4

 87.9

 87.0

 85.8

 87.2

 88.1

   origin*

 -

0.6 

 1.2

 1.1

 1.2

 0.8

 1.6

 2.5

 2.2

 1.8

 MB2 (overlapping with Hydroxy-MB2)

91.5

86.0

 79.2

 69.8

 67.4

 51.0

 47.6

 27.2

18.0

 13.9

 Desthio-MB2

-

 3.6

 5.8

 9.7

 13.6

 19.0

 23.8

 42.2

 41.0

 50.1

 MB2 sulfonic acid

 -

 1.0

 2.1

 3.7

 5.7

 13.5

 11.2

 12.4

 20.3

 10.8

 MB2 acetic acid

 -

 -

 0.6

 1.6

 0.5

 3.5

 2.8

 1.0

 5.7

 5.9

 unknown degradation products (sum)

 -

 -

 -

 -

 -

 -

 -

 0.6

 -

 5.7

*radioactive residues remaining on origin and smear <Rf 0.1

Validity criteria fulfilled:
yes
Remarks:
At 6 DAT, formation of 14CO2 exceeded 60 % AR showing the viability of the test system in both, the control samples and solvent control samples.
Conclusions:
ZMB2 decomposes to MB2 and zinc ion in water within seconds (see IUCLID section 13.2). Thus, the degradation of MB2 is described. The DegT50 value of MB2 (12° C) is 12.9 days (low concentration) and 24.6 days (high concentration). The geometric mean DT50 of the recommended optimisation was found to be 17.8 days for the parent compound.
Executive summary:

This study is concerned with the aerobic mineralisation of ZMB2 in natural surface water under controlled laboratory conditions. It was carried out according to the OECD-Guideline 309 "Aerobic Mineralisation in Surface Water – Simulation Biodegradation Test”. The incubation was performed using a 14C-labelled test item [phenyl-U-14C]zinc-4- and 5-methyl-2-mercaptobenzimidazole under aerobic conditions at 12 °C in the dark.

The study was conducted using Biggesee lake (North-Rhine-Westphalia) natural water amended with 15 mg/L suspended of its own sediment.

The biological activity of filtered lake water was characterised by the addition of the easily degradable compound 14C-sodium benzoate to control samples (blank controls). Concentration of 14C-sodium benzoate was 10 µg/L. Control samples were handled in analogy to the treated samples. The formation of 14CO2 was followed until the mineralisation level of 75 % of the applied radioactivity [% AR] was reached. This was at 13 DAT  (days after treatment). NaOH-containing traps were analysed at 1, 2, 3, 6, 9 and 13 DAT. 14CO2 quantification was performed by liquid scintillation counting (LSC). To investigate the influence of acetonitrile, which was used as solubilizer, further solvent control samples (lake water plus solvent plus 14C-sodium benzoate) were incubated under identical conditions.

Surface water was collected on 30 October 2018. Prior to filtering through 100 µm mesh it was stored for 9 days. During storage the samples were kept at ca. 4°C in the dark. An exchange of air with the environment was ensured.

Test system

Incubation of subsamples was performed in a flow-through system which was placed in a dark, temperature controlled lab-room. A constant stream of water saturated surrounding air was passed over the subsamples in order to maintain aerobic conditions during the experiment. The outgoing gas was bubbled by means of a vacuum pump through three absorption traps in sequence containing ethylene glycol and two times 1 M NaOH in order to determine volatile products and the rate of mineralization (trapping of 14CO2).

For information on the abiotic transformation of the test substance additional sterilized samples (duplicates) were taken, processed and analysed at 7 days after treatment (DAT), and 63 DAT.

Treatment

Vessels (250 mL) for low concentration (9.5 µg/L) and high concentration (65 µg/L) were spiked with 27.90 µL (low) and 28.10 µL (high) of [phenyl-U-14C]ZMB2 application solution. The applications were made directly into the test samples by a pipette.

Intended nominal treatment rate at low concentration was 12.26 kBq (9.5 µg/L) and at high concentration 83.85 kBq (65 µg/L), respectively. Based on the mass balance of the 0d of incubation samples the actual concentration confirms generally to the indented concentration and a correction was not accomplished.

Sampling and Processing

Sampling was performed after the following incubation times: 0 d (immediately after application), 2 d, 6 d, 9 d, 15 d, 20 d, 28 d, 36 d, 57 d and 64 d after application (DAT). Additionally sterile samples were sampled after 7 d and 63 d. After sampling, the water samples were extracted and worked-up. In addition, the corresponding absorption traps were removed and the trapping solutions were analysed.

Test system parameter

Test system parameters, pH and dissolved oxygen were measured throughout the study. The dissolved oxygen values were between 5.37 and 9.25 mg/L. The pH values were between pH 7.79 and pH 8.37

The biological activity of filtered lake water was characterised by the addition of the easily degradable compound 14C-sodium benzoate to control samples (blank controls, and blank control plus solvent methanol ) and following the formation of 14CO2. At 6 DAT, formation of 14CO2 exceeded 60 % AR showing the viability of the test system in both, the control samples and solvent control samples.

Results

Mass balance

The mass balances in all non-sterilised individual samples (both radiolabels) at low and high concentration ranged from 72.9 to 135.9 % AR, with an overall mean of 94.0 % AR. The mean mass balances in all sterilised samples at low and high concentration ranged from 91.0 to 94.3 % AR.

Non-extractable residues

At 0 DAT the radioactive residues on suspended matter are very low with only 0.2% AR (low test concentration) and 0.1% AR (high test concentration). The NER levels are raising over the incubation. At incubation end (64 DAT) the residues were 7.9 and 9.8% AR (low test concentration) and 5.8 and 6.2% AR (high test concentration).

Volatile degradation products

In the most of the ethylenglycol samples (24 samples) the radioactive residues were on trace level amount (<0.1% AR). At irregular intervals radioactivity between 0.1 and 9.6 % AR were found but not confirmed by the second replicate. Therefore these amounts of radioactivity should be contaminations during incubation or sample work up.

Very low amounts of applied radioactivity were found in the 1 M sodium hydroxide absorption solutions. At incubation end the amounts of radioactivity were 0.3% AR and 5.5% AR (low test concentration) and 0.1% AR (high test concentration). The value of 5.5% AR is quite high and seems to be a contamination. A second contamination was found in the absorption trap of sample 20 DAT, high test concentration, replicate A (19.7% AR).

Radioactive residues in surface water extracts (non-sterile samples)

The test substance ZMB2 itself was never found in any samples. Obviously in the presence of water it rapidly decomposes or dissociates forming MB2 and zinc species.

In the 0 DAT samples, the primary metabolite MB2 was found at mean levels of 89.4% AR (low test concentration) and 91.5% AR (high test concentration). MB2 degraded continuously over time. At 64 days of incubation the MB2 degraded to mean levels of 10.4% AR (low test concentration) and 13.9% AR (high test concentration). Two major (>10% AR) secondary degradation products were formed during incubation in low and high test concentration samples. They were reaching maximum main levels of 34,4% AR (Desthio-MB2, low test concentration) after 36 days of incubation, 50.1% AR (Desthio-MB2, high test concentration) after 64 days of incubation, 31,0% AR (MB2 sulfonic acid, low test concentration) after 28 days of incubation and 20.3% AR (MB2 sulfonic acid, high test concentration) after 57 days of incubation.

A further (third) major degradation product (MB2-acetic acid) was only found for the low test concentration samples reaching a maximum main level of 10.0% AR after 64 days of incubation. In the high test concentration samples the maximum main level of MB2 acetic acid was 5.9% AR after 64 days of incubation.

Three further unknown degradation products were detected (unknown 1, 2 and 3) not exceeding a main level of 7.3% AR.

Radioactive traces on the TLC base line (origin) were quite low not exceeding a main level of 3.5% AR.

Radioactive residues in surface water extracts (sterile samples)

For the sterilised samples, the level of MB2 was 83.4% AR (low test concentration) and 87.4% AR (high test concentration) in the 7 DAT samples. At 63 days of incubation the MB2 degraded to mean levels of 43.6% AR (low test concentration) and 65.4% AR (high test concentration).

At 63 days of incubation the degradation product Desthio-MB2 was formed to mean levels of 39.5% AR (low test concentration) and 17.6% AR (high test concentration).

All other degradation products formed only in small amounts after 63 days of incubation under sterile conditions not exceeding 6.0% AR.

Biotic degradation is generally an important factor for the degradation of MB2. However especially Desthio-MB2 is also formed in higher amounts under sterile conditions. Therefore abiotic degradation of MB2 must be considered as relevant.

Degradation products

MB2: ZMB2 decomposes to MB2 and zinc ion in water within seconds. Thus, the degradation of MB2 is described.

Desthio-MB2, MB2 sulfonic acid, MB2 acetic acid, Hydrox-MB2 and three unknown degradation products were detected.

Result

After 64 DAT 10.4 % of applied radioactivity were found assigned to MB2 in the low concentration sample and 13.9 % in the high concentration sample.The DegT50 value of MB2 (12° C) is 12.9 days (low concentration) and 24.6 days (high concentration). The geometric mean DT50 of the recommended optimisation was found to be 17.8 days for the parent compound.

Description of key information

Biodegradation in water and sediments (simulation test):

This study is concerned with the aerobic mineralisation of ZMB2 in natural surface water under controlled laboratory conditions. It was carried out according to the OECD-Guideline 309 "Aerobic Mineralisation in Surface Water – Simulation Biodegradation Test”. The incubation was performed using a 14C-labelled test item [phenyl-U-14C]zinc-4- and 5-methyl-2-mercaptobenzimidazole under aerobic conditions at 12 °C in the dark.

The study was conducted using Biggesee lake (North-Rhine-Westphalia) natural water amended with 15 mg/L suspended of its own sediment.

The biological activity of filtered lake water was characterised by the addition of the easily degradable compound 14C-sodium benzoate to control samples (blank controls). Concentration of 14C-sodium benzoate was 10 µg/L. Control samples were handled in analogy to the treated samples. The formation of 14CO2 was followed until the mineralisation level of 75 % of the applied radioactivity [% AR] was reached. This was at 13 DAT  (days after treatment). NaOH-containing traps were analysed at 1, 2, 3, 6, 9 and 13 DAT. 14CO2 quantification was performed by liquid scintillation counting (LSC). To investigate the influence of acetonitrile, which was used as solubilizer, further solvent control samples (lake water plus solvent plus 14C-sodium benzoate) were incubated under identical conditions.

Surface water was collected on 30 October 2018. Prior to filtering through 100 µm mesh it was stored for 9 days. During storage the samples were kept at ca. 4°C in the dark. An exchange of air with the environment was ensured.

Test system

Incubation of subsamples was performed in a flow-through system which was placed in a dark, temperature controlled lab-room. A constant stream of water saturated surrounding air was passed over the subsamples in order to maintain aerobic conditions during the experiment. The outgoing gas was bubbled by means of a vacuum pump through three absorption traps in sequence containing ethylene glycol and two times 1 M NaOH in order to determine volatile products and the rate of mineralization (trapping of 14CO2).

For information on the abiotic transformation of the test substance additional sterilized samples (duplicates) were taken, processed and analysed at 7 days after treatment (DAT), and 63 DAT.

Treatment

Vessels (250 mL) for low concentration (9.5 µg/L) and high concentration (65 µg/L) were spiked with 27.90 µL (low) and 28.10 µL (high) of [phenyl-U-14C]ZMB2 application solution. The applications were made directly into the test samples by a pipette.

Intended nominal treatment rate at low concentration was 12.26 kBq (9.5 µg/L) and at high concentration 83.85 kBq (65 µg/L), respectively. Based on the mass balance of the 0d of incubation samples the actual concentration confirms generally to the indented concentration and a correction was not accomplished.

Sampling and Processing

Sampling was performed after the following incubation times: 0 d (immediately after application), 2 d, 6 d, 9 d, 15 d, 20 d, 28 d, 36 d, 57 d and 64 d after application (DAT). Additionally sterile samples were sampled after 7 d and 63 d. After sampling, the water samples were extracted and worked-up. In addition, the corresponding absorption traps were removed and the trapping solutions were analysed.

Test system parameter

Test system parameters, pH and dissolved oxygen were measured throughout the study. The dissolved oxygen values were between 5.37 and 9.25 mg/L. The pH values were between pH 7.79 and pH 8.37

The biological activity of filtered lake water was characterised by the addition of the easily degradable compound 14C-sodium benzoate to control samples (blank controls, and blank control plus solvent methanol ) and following the formation of 14CO2. At 6 DAT, formation of 14CO2 exceeded 60 % AR showing the viability of the test system in both, the control samples and solvent control samples.

Results

Mass balance

The mass balances in all non-sterilised individual samples (both radiolabels) at low and high concentration ranged from 72.9 to 135.9 % AR, with an overall mean of 94.0 % AR. The mean mass balances in all sterilised samples at low and high concentration ranged from 91.0 to 94.3 % AR.

Non-extractable residues

At 0 DAT the radioactive residues on suspended matter are very low with only 0.2% AR (low test concentration) and 0.1% AR (high test concentration). The NER levels are raising over the incubation. At incubation end (64 DAT) the residues were 7.9 and 9.8% AR (low test concentration) and 5.8 and 6.2% AR (high test concentration).

Volatile degradation products

In the most of the ethylenglycol samples (24 samples) the radioactive residues were on trace level amount (<0.1% AR). At irregular intervals radioactivity between 0.1 and 9.6 % AR were found but not confirmed by the second replicate. Therefore these amounts of radioactivity should be contaminations during incubation or sample work up.

Very low amounts of applied radioactivity were found in the 1 M sodium hydroxide absorption solutions. At incubation end the amounts of radioactivity were 0.3% AR and 5.5% AR (low test concentration) and 0.1% AR (high test concentration). The value of 5.5% AR is quite high and seems to be a contamination. A second contamination was found in the absorption trap of sample 20 DAT, high test concentration, replicate A (19.7% AR).

Radioactive residues in surface water extracts (non-sterile samples)

The test substance ZMB2 itself was never found in any samples. Obviously in the presence of water it rapidly decomposes or dissociates forming MB2 and zinc species.

In the 0 DAT samples, the primary metabolite MB2 was found at mean levels of 89.4% AR (low test concentration) and 91.5% AR (high test concentration). MB2 degraded continuously over time. At 64 days of incubation the MB2 degraded to mean levels of 10.4% AR (low test concentration) and 13.9% AR (high test concentration). Two major (>10% AR) secondary degradation products were formed during incubation in low and high test concentration samples. They were reaching maximum main levels of 34,4% AR (Desthio-MB2, low test concentration) after 36 days of incubation, 50.1% AR (Desthio-MB2, high test concentration) after 64 days of incubation, 31,0% AR (MB2 sulfonic acid, low test concentration) after 28 days of incubation and 20.3% AR (MB2 sulfonic acid, high test concentration) after 57 days of incubation.

A further (third) major degradation product (MB2-acetic acid) was only found for the low test concentration samples reaching a maximum main level of 10.0% AR after 64 days of incubation. In the high test concentration samples the maximum main level of MB2 acetic acid was 5.9% AR after 64 days of incubation.

Three further unknown degradation products were detected (unknown 1, 2 and 3) not exceeding a main level of 7.3% AR.

Radioactive traces on the TLC base line (origin) were quite low not exceeding a main level of 3.5% AR.

Radioactive residues in surface water extracts (sterile samples)

For the sterilised samples, the level of MB2 was 83.4% AR (low test concentration) and 87.4% AR (high test concentration) in the 7 DAT samples. At 63 days of incubation the MB2 degraded to mean levels of 43.6% AR (low test concentration) and 65.4% AR (high test concentration).

At 63 days of incubation the degradation product Desthio-MB2 was formed to mean levels of 39.5% AR (low test concentration) and 17.6% AR (high test concentration).

All other degradation products formed only in small amounts after 63 days of incubation under sterile conditions not exceeding 6.0% AR.

Biotic degradation is generally an important factor for the degradation of MB2. However especially Desthio-MB2 is also formed in higher amounts under sterile conditions. Therefore abiotic degradation of MB2 must be considered as relevant.

Degradation products

MB2: ZMB2 decomposes to MB2 and zinc ion in water within seconds. Thus, the degradation of MB2 is described.

Desthio-MB2, MB2 sulfonic acid, MB2 acetic acid, Hydrox-MB2 and three unknown degradation products were detected.

Result

After 64 DAT 10.4 % of applied radioactivity were found assigned to MB2 in the low concentration sample and 13.9 % in the high concentration sample.The DegT50 value of MB2 (12° C) is 12.9 days (low concentration) and 24.6 days (high concentration). The geometric mean DT50 of the recommended optimisation was found to be 17.8 days for the parent compound.

Key value for chemical safety assessment

Half-life in freshwater:
17.8 d
at the temperature of:
12 °C

Additional information

Key value for chemical safety assessment given for MB2: ZMB2 decomposes to MB2 and zinc ion in water within seconds. Thus, the degradation of MB2 is described.