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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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
09.09.1991 - 07.07.1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP - Guideline study, tested with the source substance tetramethylthiuram disulfide (CAS No. 137-26-8). In accordance to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on an read-across substance
Qualifier:
according to guideline
Guideline:
other: BBA (Biologische Bundesanstalt /Federal Biological Research Centre/) Guidelines, Part IV, Section 5-1 (December 1990): Abbaubarkeit und Verbleib von Pflanzenschutzmitteln im Wasser/Sediment-System.
GLP compliance:
yes (incl. QA statement)
Remarks:
Swiss Federal Department of Interior
Radiolabelling:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
natural water / sediment
Details on source and properties of surface water:
River
- Details on collection: Water was sampled down to a depth of 10 to 30 cm with a plastic container. The sampling site was located about 1 to 2 m from firm land. After reception in the laboratory, the containers were kept at room temperature until sieving.
- Temperature (°C) at time of collection: 17.1°C
- pH at time of collection: 7.8-7.9
- Redox potential (mv) initial/final: 247, 225 mV
- Hardness (CaCO3): 10


Pond
- Details on collection : Water was sampled down to a depth of 10 to 30 cm with a plastic container. The sampling site was located about 1 to 2 m from firm land. After reception in the laboratory, the containers were kept at room temperature until sieving.
- Temperature (°C) at time of collection: 12.8°C
- pH at time of collection: 7.0-7.53
- Redox potential (mv) initial/final: 215, 59 mV
- Hardness (CaCO3): 21

Details on source and properties of sediment:
River sediment

- Details on collection: The sediment was collected with a plastic shovel from the top 5 to 10 cm of each system (river, pond)
- Textural classification (i.e. %sand/silt/clay):
Clay: 4.5%
Sand: 79.3%
Silt: 16.2%
- pH at laboratory: 7.39
- Redox potential (mv) initial/final: -114 (sampling site), -158 mV (end)
- CEC: 4.48 mVal N/100 g soil
- Biomass: 26.8 mg microbial C/100 mg dry soil

Pond sediment

- Details on collection: The sediment was collected with a plastic shovel from the top 5 to 10 cm of each system (river, pond)
- Textural classification (i.e. %sand/silt/clay):
Clay: 18.4%
Sand: 40.8%
Silt: 33.6%
- pH at laboratory: 7.08
- Redox potential (mv) initial/final: -417 (sampling site), -255 mV (end)
- CEC: 13.73 mVal N/100 g soil
- Biomass: 173.1 mg microbial C/100 mg dry soil
Duration of test (contact time):
101 d
Initial conc.:
1.195 other: mg 14C Thiram/L water
Parameter followed for biodegradation estimation:
radiochem. meas.
Details on study design:
TEST CONDITIONS
- Test solutions: The maximal recommended field rate for THIRAM application was 3.2 kg a.i./ha. The target application rate was then 1.1 mg 14C-THIRAM per litre water. Two application solutions were prepared, because the samplings at 0 and 6 hours were performed at the same day and the additional sampling was performed after 4 days. The labelled test item was dissolved in 5 mL acetone to prepare the first application solution and in 4 mL acetone to prepare the second application solution. The content of radioactivity was determined by LSC to be 1.295 x 109 and 1.062 x 109 dpm for the first and the second application, respectively. Based on the specific radioactivity of 71 µCi/mg, the amount of 14C-THIRAM was found to be 8.22 mg in 5 mL acetone (1st application) and 6.74 mg in 4 mL acetone (2nd application). 160 µL of application solution I and 180 µL of application solution II corresponding to 0.263 mg/flask (1.195 mg/L) and 0.3032 mg/flask (1.378 mg/L), respectively, were applied per each metabolism flask.
In the first application 1.195 mg of test item per litre water were added to both systems in duplicate for sampling days 0.25, 1, 2, 7, 14, 30 and 57. The second application was performed for sampling days 0, 4 and 101 with 1.378 mg 14C-THIRAM/L water. The results obtained for the samples of the second application were normalised to achieve the first application rate.
- Sample preparation: Water-sediment samples were prepared for the study by sieving (2 mm mesh, sediment), filtering (0.2 mm, water) and pre-incubation under humidified air at 20 ± 2.0°C in the dark to establish aerobic conditions. For each time interval duplicate test system flasks were prepared.- Test temperature: Aerobic incubation at 20 ± 2 °C, darkness

TEST SYSTEM
Before used, the water was filtered through a 0.2 mm sieve and the sediment was sieved through a 2.0 mm screen. The sieved sediments were stored overnight at 4°C, whereas the prepared water was kept at room temperature and aerated above the surface. Thereafter 220 mL of river water as well as of pond water, 140 g of wet river sediment and 158 g of wet pond sediment corresponding to 93.8 g and 91.6 g dry weight, respectively, were filled into 500 mL all-glass metabolism flasks (please see also Figure A7_1_2_2_2-3).
The study was run in duplicate. The flasks for each sampling interval were incubated in an air-conditioned room at 20 ± 2°C in the dark.
Each water-sediment test system was aerated on the water surface using a CO2-free and moistened air stream. During the acclimation period oxygen concentration, pH and redox potential of the water were measured at appropriate intervals. An equilibrium based on measured values was reached after about 18-20 days of incubation.
The target application rate was 1.1 mg 14C-THIRAM per litre water.
A pre-test was conducted to evaluate the trapping solvents, sampling methods as well as to assure an appropriate balance of radioactivity.


SAMPLING
- Sampling frequency: 0, 0.25, 1, 2, 4, 7, 14, 30, 57 and 101 days
Reference substance:
other: non labelled THIRAM: tetramethylthiuram disulphide
Reference substance:
other: tetramethylthiuram monosulphide (TMTM)
Reference substance:
other: Dimethyldithiocarbamic acid sodium salt dihydrate (DMDTC)
Reference substance:
other: Dimethyldithiocarbamic acid methyl ester (DMDTC-ME)
Reference substance:
other: Carbon disulfide (CS2 p.a.)
Compartment:
other: river
DT50:
1.9 d
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: r2=0.98812, DT90= 6.2 d
Compartment:
other: pond
DT50:
1.9 d
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: r2=0.99253, DT90=6.3 d
Transformation products:
no
Remarks:
Only minor transformation products were detected which were further mineralized to form CO2.
Details on transformation products:
The parent substance disappeared very rapidly from both river and pond systems. 14C-THIRAM represented less then 6% after only 7 days of incubation. The parent substance was not detected in any of the later sampling intervals, showing that 14C-THIRAM is rapidly degraded or adsorbed to the sediment.
14C-THIRAM was degraded mainly to 14CO2 and 14CS2, which reached 65.8% and 60.8% of the applied RA for river and pond systems, respectively, over the incubation period of 101 days.
Additional amounts of 14CO2 and 14CS2 were also detected in the water phase and in the sediment extracts. The maximal amount of 14CO2 detected in the water phase reached 18.1% (river, on day 7) and 19.2% (pond, on day 14) of the applied RA.
Additionally to the volatile compounds, the parent substance was degraded to a very high number of metabolites detected by HPLC. Radioactive fraction M1 represented the major metabolite and was mainly detected in the water phase. This very polar fraction showed the same retention time as the dissolved CS2 in water. The fraction M1 behaved also similarly to the formation of 14CS2 and reached in the river and pond systems a maximum concentration of 21.0% (20.6% in water and 0.4% in sediment) and 19.9% (19.2% in water and 0.7% in sediment) of the applied RA, respectively.
One radioactive fraction found mainly in water and two radioactive fractions found mainly in sediment extracts were identified as DMDTC-ME, TMTM and DMDTC, respectively, based on chromatographic comparison to the authentic reference substances. Each of these fractions did not exceed 5.5% of the applied RA.
In addition up to 12 unknown radioactive fractions were detected, but non of these fractions exceeded 10% of the applied RA. The main fractions M18 and M19 reached the maximum values of 7.5% and 9.7% of the applied RA in the river system and 6.3% and 7.2% in the pond system, respectively. These fractions disappeared very rapidly and represent less than 0.1% of the applied RA from day 14 to study termination for both systems.

14C-Thiram is very rapidly degraded, mainly to 14CS2, 14CO2 and to at least 20 minor metabolites, which were further degraded to CO2, other small fractions and disappeared by adsorption to sediment. The continuous formation of 14CO2 and the decrease of the amount of the bound residues in sediment, show that the non-extractable radioactivity is slowly released and further degraded and mineralised. The half-life of Thiram in the river as well as in the pond system was 1.9 days and the DT90 was 6.2 and 6.3 days for river and pond system, respectively.

The rapid conversion to 14CS2 and 14CO2 indicated that Thiram will not persist in aquatic environments.

Validity criteria fulfilled:
yes
Conclusions:
From the results of this study it can be concluded that 14C-THIRAM is very rapidly degraded, mainly to 14CS2, 14CO2 and to at least 20 minor metabolites, which were further degraded to CO2, other small fractions and disappeared by adsorption to sediment.
The continuous formation of 14CO2 and the decrease of the amount of the bound residues in sediment, show that the non-extractable radioactivity is slowly released and further degraded and mineralised.
The half-live of THIRAM in the river as well as in the pond system was 1.9 days and the DT90 was 6.2 and 6.3 days for river and pond system, respectively.
The rapid conversion to 14CS2 and 14CO2 indicated that THIRAM will not persist in aquatic environments.

Executive summary:

Materials and methods:The objective of the study was to provide information on the distribution and metabolism of14C-THIRAM in two different water/sediment systems under aerobic conditions according to BBA (Biologische Bundesanstalt /Federal Biological Research Centre/) Guidelines, Part IV, Section 5-1 (December 1990): Abbaubarkeit und Verbleib von Pflanzenschutzmitteln im Wasser/Sediment-System.

A water-sediment test systems was sampled from the river Rhine (Mumpf Zeltplatz, Aargau/Switzerland) and from the pond Judenweiher (Rheinfelden near Görbelhof, Aargau/Switzerland).
The study was performed in 0,5 L all-glass metabolism flasks. The water was filtered through a 0.2 mm sieve and the sediment was sieved through a 2.0 mm screen. The study was run in duplicate. The treated samples as well as the controls were prepared and incubated at 20 ± 2°C in the dark under aerobic conditions.

Each river and pond water-sediment system prepared for sampling on days 0.25, 1, 2, 7, 14, 30 and 57 was treated with14C-THIRAM in concentration of 1.195 mg/L water. The test systems prepared for sampling on days 0, 4 and 101 were treated with 1.378 mg14C-THIRAM/L water.
The results obtained for the samples of the second application were normalised to achieve the first application rate.

Water, sediment and volatile substances were worked up and analysed by High Performance Liquid Chromatography (HPLC).

Results and discussion: Radioactivity (RA) in the river and pond test system:

After treatment, the radioactivity (RA) in the river water as well as in the pond water decreased steadily from 107.5% of the applied RA on day 0 to 6.5% and 6.1% on day 101, respectively.

In the sediment the RA first increased up to a maximum of 29.6% (river) and 34.7% of the applied RA (pond) on day 14 and on day 30, respectively, then slowly decreased to 15.6% in the river system and to 23.9% of the applied RA in the pond system at the end of the incubation period.

The sediments were extracted with chloroform and with methanol. The extractable RA from sediments was low and increased continuously reaching maximum values of 3.9% for river and 7.5% for pond at days 14 and 30, respectively. Thereafter the extractable RA decreased to values of 1.8% at day 101 of incubation for both systems.

The degradation of14C-THIRAM to volatile compounds (14CO2and14CS2), which were trapped in the volatile traps, was very high and reached 65.8% and 60.8% of the applied RA for river and pond system, respectively, at study termination. In the river system the highest volatilisation rate was observed between days 7 and 30. The highest volatilisation rate for pond system was between days 7 and 14.

In the river system 56.1% of the RA applied was detected as14CO2and 9.6% as14CS2in the river at the study termination. Concurrently in the pond system 36.6% and 24.2% of the applied RA were detected as14CO2and14CS2, respectively.

The amount of14CO2in the NaOH trap was determined after precipitation with aqueous Ba(OH)2solution. The amount of14CO2increased with the incubation time and ranged from about 18% to 56% of the RA in the volatile traps of the river system, between days 4 and 14. At the study termination the value of 96% RA was reached. The corresponding values for the pond system ranged from 9% to 23%, respectively. On the last incubation day 97% of the RA was detected. The remaining volatile RA in the NaOH traps was considered to be  14CO2.

Also in the water phase as well as in sediment extracts from both systems volatiles compounds were observed. After precipitation with Ba(OH)2up to 57.5% (river) and 43.9% (pond) of the initial RA was detected in water as14CO2.
Total volatiles (in volatile traps and dissolved in water and sediment extracts) amounted to 70.6% and 67.3% of the RA applied at the study termination in the river and pond systems, respectively.

The total mean recoveries of radioactivity obtained during the whole incubation time were 95.6 ± 6.2% and 99.1% ± 6.1% of RA applied for the river and pond systems, respectively.

Endpoint:
biodegradation in water: sediment simulation testing
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP - Guideline study, tested with the source substance tetramethylthiuram disulfide (CAS No. 137-26-8). In accordance to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on an read-across substance
Qualifier:
according to guideline
Guideline:
other: BBA (Biologische Bundesanstalt /Federal Biological Research Centre/) Guidelines, Part IV, Section 5-1 (December 1990): Abbaubarkeit und Verbleib von Pflanzenschutzmitteln im Wasser/Sediment-System.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Swiss Department of Home Affairs
Radiolabelling:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
natural water / sediment
Details on source and properties of surface water:
- Details on collection: The water/sediment system was collected from the Rhine river (Sisseln, AG Switzerland). The water was sampled at a depth between 10 to 30 cm, and was transported in containers to the laboratory where it was stored at about 4°C until it was used.
- Temperature (°C) at time of collection: 15.8°C
- pH at time of collection: 8.03
- Redox potential (mv) initial/final: 215,202 mV
- Oxygen concentration initial/final: 10.3-10.1, 5.6-5.1 mg/L
- Hardness (CaCO3): 12
Details on source and properties of sediment:
- Details on collection: The water/sediment system was collected from the Rhine river (Sisseln, AG Switzerland). The sediment was sampled from the top 5 to 10 cm, and was transported in containers to the laboratory where it was stored at about 4°C until it was used.
- Textural classification (i.e. %sand/silt/clay):
Clay: 2.8%
Silt: 5.6%
Sand: 91.6%
- pH at time of collection: 7.35
- Organic carbon (%):
- Redox potential (mv) initial/final: -180, -199- - 275 mV
- CEC : 24.6 mVal N/100 g dry sediment
Duration of test (contact time):
10 d
Initial conc.:
16.26 µg/L
Based on:
act. ingr.
Parameter followed for biodegradation estimation:
radiochem. meas.
Details on study design:
TEST CONDITIONS
- Test solutions: The labelled 14C-THIRAM containing 75 MBq was dissolved in 1 mL acetone. To prepare the application solution, 30 µL were transferred into volumetric flask and diluted with acetone to 10 mL.
The content of radioactivity in the application solution was assayed in triplicate by LSC. For this purpose 3 x 50 µL were diluted to 10 mL with acetone and then 1.0 mL per flask was used to determine the radioactivity.
Based on this LSC-value (118583556 dpm/10 mL) an application volume of 460 µL was calculated, which correspond to 16.26 µg a.i./L water.
The two control flasks were applied with the same volume (460µL) of pure acetone.
- Sample preparation: Water-sediment samples were prepared for the study by sieving (2 mm mesh, sediment), filtering (0.2 mm, water) and pre-incubation under humidified air at 20 ± 2.0°C in the dark to establish aerobic conditions. For each time interval duplicate test systems (30 flasks in total) were prepared.
14 flasks were treated with radiolabeled THIRAM in acetone at a concentration of 16.26 µg a.i./L water, other 14 flasks were used as reserves and the last two as controls.
For sampling at time 0 hour each replicate test system was treated and immediately processed. For further sampling intervals, the test systems were incubated at 20 ± 2.0°C. The flasks were ventilated with CO2-free air directly after treatment. The outgoing stream was passed through three traps containing one empty trap followed by one with 50 ml 1M KOH dissolved in methanol and one ethylene glycol trap (50 ml).
- Test temperature: Aerobic incubation at 20 ± 2 °C, darkness

TEST SYSTEM
- Test concentration: The target value was calculated to be 16.0 µg 14C-THIRAM/L water or 8.48 µg active ingredient / 530 mL water (0.0895 MBq/sample, 5367840 dpm/sample, 10.55 MBq/mg specific radioactivity). The test item was applied in acetone (460µL per flask) at actual concentration of 16.26 µg a.i./L water or 8.62 µg a.i. per flask (5454844 dpm/flask)


SAMPLING
- Sampling frequency: 0.0, 0.25, 0.75, 2.0, 7.0 and 10.0 days
Reference substance:
other: non labelled THIRAM: tetramethylthiuram disulphide
Reference substance:
other: tetramethylthiuram monosulphide (TMTM)
Reference substance:
other: Dimethyldithiocarbamic acid sodium salt dihydrate (DMDTC)
Reference substance:
other: Dimethyldithiocarbamic acid methyl ester (DMDTC-ME)
Reference substance:
other: Carbon disulfide (CS2 p.a.)
DT50:
0.7 d
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: R2=0.9979, DT90= 2.5 d
Transformation products:
no
Remarks:
Only minor transformation products were detected which were further mineralized to form CO2.
Details on transformation products:
Partition of the water-phase indicated that with increasing incubation time more polar fractions appeared and remained in the water phase. Due to the low application rate, a special effort was given to characterize the polar fraction in the water phase labelled as MW. The radioactive fraction MW increased to 42.5% of the initial RA (6.9 µg/L) within 2 days and then decreased to 34.0% (5.5 µg/L) on day 10. This fraction was subsequently separated into at least 9 different unknown radioactive fractions, labelled as MW1 to MW9. None of these fractions could be identified by co-chromatography with a known reference compounds.
The main fraction was MW2, the only fraction which increased to 11.7% of RA applied on day 10. Other fractions reached their maximum value (≤ 8% of RA applied) before or at day 2 of incubation period and either decreased until day 10 or were not detectable.
DMDTC-ME was the only reference compound which was detected on day 7 and 10 with 1.2% and 0.8%, respectively. Another unknown, less polar fraction U was detected once on day 2 in the organic partition by HPLC and reached 1.2% of RA applied.

Thiram degraded fast from 96.7% or 15.7 ug/L on day 0 to 0.5% or 0.08 ug/L on day 10 which was just above the determination limit in this study. The degradation rate of Thiram was calculated using a first order kinetic model and the Optimum fit found. The degradation time (50% degradation of Thiram, DT-50 value) was 0.7 days and the DT-90 (90% degradation) value was 2.5 days. The calculted level of Thiram was 0.01% er 0.0016 ug/L on day 10.

Validity criteria fulfilled:
yes
Conclusions:
From the results of this study it can be concluded that under aerobic conditions THIRAM is very fast degraded to several polar minor metabolites and then mineralised to CO2.
The half-life of THIRAM was 0.7 days and the DT90 was 2.5 days.
Executive summary:

Materials and methods:The objective of the study was to provide information on the distribution and metabolism of14C-THIRAM in water/sediment systems according to BBA (Biologische Bundesanstalt /Federal Biological Research Centre/) Guidelines, Part IV, Section 5-1 (December 1990): Abbaubarkeit und Verbleib von Pflanzenschutzmitteln im Wasser/Sediment-System.

A water-sediment test system was sampled from the river Rhine (Sisseln, AG /Switzerland).
The study was performed in an open gas-flow-system in 1.0 L all-glass metabolism flasks. The water was filtered through a 0.2 mm sieve and the sediment was sieved through a 2.0 mm screen. The study was run in duplicate. 14 flasks with treated samples, 14 untreated flasks as reserves and 2 controls were prepared and incubated at 20 ± 2°C in the dark. Each water/sediment test system was treated with 460 µL of the radiolabelled14C-THIRAM in acetone. The actual radioactivity of the application solution was 5454844 dpm per flask. The test item was applicated at actual concentration of 16.26 µg a.i./L water or 8.62 µg a.i. per flask. Water, sediment and volatile substances were worked up and analysed by HPLC and TLC.

Results and discussion:14C-THIRAM was detected with 96.7% of the radioactivity applied (15.7 µg a.i./L) in the river water directly after treatment on day 0. This amount rapidly decreased to 51.6% (8.4 µg/L) on day 0.75 and was just slightly above the determination limit of 0.3% on day 10. Only 0.5% of applied RA (0.08 µg/L) was detected at the study termination.

The rate of degradation was calculated according to 1st-order kinetic model. 0.7 and 2.5 days were obtained for DT50and DT90, respectively. In water phase the level of radioactivity decreased very fast from 99.7% of applied radioactivity (16.2 µg parent equivalents per litre water) on day 0 to 25.2% (5.7 µg/L) on day 10. In sediment samples the radioactivity first increased from 0.6% (0.1 µg/L) on day 0 to 33.4% (5.4 µg/L) on day 7. Afterwards a slight decrease of RA up to 32.6% (5.3 µg/L) on day 10 was observed. The water phase was partitioned twice with dichloromethane. The RA in the dichloromethane phase decreased steadily to 5% at the end of the study. The radioactivity remaining in the water phase increased until day 2 to > 40% of the RA applied, but decreased thereafter until day 10 to about 30%. Volatile RA, including the14CO2dissolved in the water phase increased continuously during the incubation time. Within the first day the RA increased to 13.1% (2.1 µg parent equivalents per litre). Afterwards 23.6% of applied RA (correspond to 3.8 µg/L) were detected on day 2 and at the study termination 25.7% (4.2 µg/L).
No RA was found in the ethylene glycol traps. The radioactivity was retained in the 1M KOH in methanol and consisted mainly of14CO2. The amount of14CO2in the test system at the end of the study was 21.0% of the RA applied. At the same time other volatile substances reached only 4.7% (due to information of a former study probably14CS2). The mean recovery of radioactivity was 94.9 ± 2.9% of RA applied (15.4 ± 0.5 µg parent equivalents per L water). The individual recoveries were all between 92.0 and 101.5%.

Description of key information

Disulfiram is not expected to be persistent in aquatic environments (DT50 < 2 days).

Key value for chemical safety assessment

Additional information

No data on the biodegradation of disulfiram (CAS No. 97-77-8) in water and sediment were available. Therefore, a conclusion based on data from an analogue substance had to be drawn. Biodegradation in water/sediment systems of tetramethylthiuram disulfide (CAS No.137-26-8), substance structurally and composition related to tetraethylthiuram disulfide, was investigated in two studies.

These tests were conducted according to the German BBA Guidelines, Part IV, Section 5-1 (1990): “Degradability and metabolism of Plant Protection Products in Water/Sediment Systems”, under GLP conditions. A concentration of 1.195 mg14C-thiram/L water of the radiolabelled compound was incubated in the first study (1992) for 101 days.14C-thiram was very rapidly degraded, mainly to14CS2,14CO2and at least 20 minor metabolites, which were further degraded to CO2, other small fractions and disappeared by adsorption to sediment. The half life of the compound was 1.9 days. The non-extractable radioactivity was there further degraded and mineralised. In the second study (1995), 16.26 ug/L a.i. were incubated for 10 days under similar conditions, resulting in a rapid degradation (half life= 0.7 days) leading to several minor metabolites that further mineralised, being CO2the main degradation product.