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EC number: 220-239-6 | CAS number: 2682-20-4
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
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- Boiling point
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- Vapour pressure
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- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
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- Additional toxicological data
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Biodegradation in water and sediment: simulation tests
Some information in this page has been claimed confidential.
Administrative data
- Endpoint:
- biodegradation in water: sediment simulation testing
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- Not stated in report
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP/Guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 002
- Report date:
- 2002
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 308 (Aerobic and Anaerobic Transformation in Aquatic Sediment Systems)
- Deviations:
- not specified
- Qualifier:
- according to guideline
- Guideline:
- EPA Subdivision N Pesticide Guideline 162-4 (Aerobic Aquatic Metabolism)
- Deviations:
- not specified
- GLP compliance:
- yes
Test material
- Test material form:
- other: Not stated in report
- Details on test material:
- RH-24573 (also known as RH-573) labeled at the 4- and 5-carbon positions of the molecule, was used in the study. The test material Lot No. was 724.03 and had a radiopurity of 99.88% with a specific activity of 39.05 mCi/g, equivalent to 86691 disintegrations per minute per microgram (dpm/ug). The radiopurity of the test substance was reassessed after preparation of the dosing solution using high performance liquid chromatography (HPLC) analysis. Fractions from the entire run of the HPLC injection of the dosing solution were collected in polyethylene counting vials and were submitted for radioassay. Quantitation was done by liquid scintillation counting (LSC). The radiopurity was determined to be 99.4%.
Constituent 1
- Specific details on test material used for the study:
- Details on properties of test surrogate or analogue material (migrated information):
Not applicable - Radiolabelling:
- yes
Study design
- Oxygen conditions:
- aerobic
- Inoculum or test system:
- natural water / sediment
- Details on source and properties of surface water:
- Two sediments and their associated water were used for this study. Their sources are described as follows: (1) Almshouse was obtained from Neshaminy Creek, intersection of Almshouse and Militia Hill Roads, Doylestown Township, Bucks County, Pennsylvania, USA (2) Cedar Hill was obtained from Park Creek, underneath the Cedar Hill Road bridge, Horsham Township, Montgomery County, Pennsylvania, USA.
- Details on source and properties of sediment:
- Two sediments and their associated water were used for this study. Their sources are described as follows: (1) Almshouse was obtained from Neshaminy Creek, intersection of Almshouse and Militia Hill Roads, Doylestown Township, Bucks County, Pennsylvania, USA (2) Cedar Hill was obtained from Park Creek, underneath the Cedar Hill Road bridge, Horsham Township, Montgomery County, Pennsylvania, USA.
- Details on inoculum:
- Not applicable
- Duration of test (contact time):
- 30 d
Initial test substance concentration
- Initial conc.:
- 1 mg/L
- Based on:
- act. ingr.
- Parameter followed for biodegradation estimation:
- test mat. analysis
- Details on study design:
- Preliminary Study
Sediment sample from each collection site was taken from the upper layer of the sediment (0-10 cm thickness). Associated water was collected from the same site and at the same time as the sediment. The sediment was wet-sieved through a #10, USA Standard Testing Sieve, with a 2-mm mesh grating. The moisture content of each sediment prior to dosing was determined by weighing duplicate 10g samples of the sieved sediment into aluminum pans and then drying the sediment samples in a 100C oven. Samples were weighed every ~2 hours or overnight until a stable dry weight was obtained.
Sediment equivalent to ~30 g (Almshouse) and ~50 g (Cedar Hill) on a dry weight basis [dwb] were weighed into 8oz glass jars. Sediment water corresponding to a 3:1 volume:weight; v:w [dwb] ratio) water;sediment was added to each jar. The jars were fitted with a two-hole rubber stopper then connected in series to allow air to enter and exit each jar. Zero-grade moistened air was purged through the system from the first jar to the last jar, blowing air over the water surface while maintaining minimal disturbance of the sediment layer. The jars were incubated in the dark at 20 +/- 1C in a constant temperature room for acclimation. After the acclimation period, ten samples for each sediment were treated at a nominal concentration of ~1 ppm. Two of the samples prepared per sediment will serve as negative control flasks.
A volume of 50 ul (Almshouse samples) and 84 ul (Cedar Hill samples) of dosing solution (equivalent to a treatment rate of 1 parts per million (ppm) of RH-573) were applied to the water surface using an electronic pipette. The water layer was gently stirred using a Bio-Probe, avoiding the disturbance of the sediment during the mixing. The jars were stoppered and were connected in series to an air supply as previously described. The last jar in the series was connected to a flask with a 2oz jar contining 50 ml of 1 N potassium hydroxide (KOH) to trap any 14CO2 and acidic volatiles generated by the entire series of sample jars. The samples were incubated in the dark at 20 +/- 1C in a constant temperature room.
The sampling intervals in the preliminary study were at Time 0, Day 0.17, Day 1, and Day 3 post treatment. At each sampling, duplicate flasks for each sediment/water were harvested for analysis and processed.
Definitive Study
A total of twenty-two 8oz glass jars per sediment/water were prepared for the definitive phase of the study. Samples were prepared by weighing 87.89 g (+/-0.2g) of the Almshouse sediment and 85.59 g (+/-0.6g) of the Cedar Hill sediment, which are equivalent to 40 g and 60 g of sediment, respectively, on a dry weight basis. These weights gave a sediment layer of 2.2 cm for the Almshouse sediment and 2.4 cm for the Cedar Hill sediment. A volume of 72 ml of the Almshouse water and 155 ml of the Cedar Hill water (corresponding to a 3:1 (v;w {dw b} ratio) water:sediment: were added to the respective sediment set. The samples were set-up for an acclimation period of seven days (Cedar Hill) and ten days (Almshouse).
Six additional jars for each sediment/water were prepared for the measurement of key parameters such as pH, total organic carbon (TOC), dissolved oxygen, redox potential, and microbial biomass (sediment only ) at different stages of the study. These characterization samples were maintained in the same manner as the definitive samples; however, the water-sediment systems were not dosed with the test substance and the effluent gases were not trapped.
After the acclimation period, twenty jars per set were dosed with 67 ul (Almshouse) and 100 ul (Cedar Hill) of the dosing solution, which was equivalent to a treatment rate of 1.04 ppm for the Almshouse samples and 1.02 ppm for the Cedar Hill samples. The remaining two jars from each set were not dosed and served as negative controls. The radiolabeled test substance was applied to the water layer of each sample using an electronic pipette. The water layer was gently mixed using a Bio-Probe, making sure that disturbance of the sediment layer is as little as possible. The jars were stoppered and then connected in series with zero-grade moistened air blowing over the water surface. A flask with a jar of 50 ml 1 N KOH was added at the end of each series of sample jars to collect any 14CO2 and/or acidic volatiles evolved. The samples were incubated in the dark at 20 +/-1C in a constant temperature room.
Sampling for the Almshouse sediment/water was performed on 0, 0.04, 0.13, 0.21, 1, 1.3, 2. 7, and 30 days post-treatment. Sampling from the Cedar Hill sediment/water was performed on 0, 0.17, 0.25, 1, 1.3, 2, 3, 7, and 30 days post treatment.
Two additional jars containing the Almshouse sediment and water were prepared for the high dose treatment (2 ppm) of 14C-RH-573. The high-dosed samples were used for the isolation of larger quantities of the metabolites generated by the incubated samples for metabolite identification. These samples were prepared, dosed, and incubated the same way as the 1 ppm-dosed definition samples. The 2 ppm-dosed samples were harvested 2 days post treatment.
When quantitative recoveries of the applied dose were not obtained from the Cedar Hill Day 30 harvest, the Day 30 interval for this sediment/water set was repeated. Two jars of the Cedar Hill sediment and water were prepared and acclimated as previously described for the initial set of Cedar Hill samples. The duplicate samples were dosed with 85 ul of the 14C-RH-573 dosing solution, equivalent to a treatment rate of 1.01 ppm. After dosing, the two jars were stoppered and then connected in series with zero-grade moistened air blowing over the water surface. A flask with a jar of 50 ml of 1 N KOH was added at the end of the two sample jars. The samples were incubated in the dark at 20 +/- 1C in a constant temperature room. The KOH traps were harvested and recharged with fresh KOH at regular intervals similar to the initial set of the Cedar Hill samples. The duplicate samples were harvested at 30 days after dosing.
Trapping of 14C Volatiles
Off gases from the jars were trapped in 1 N KOH. The KOH in the traps were assayed by LSC at each harvest interval and in-between harvest intervals during the study and were replaced with vials containing fresh KOH. Duplicate aliquots were taken from each sample and 15 mlof Hionic-Fluor was added for radioassay. The presence of 14CO2 in the KOH samples was confirmed by a barium hydroxide [Ba(OH)2] precipitation test.
Characterization of Water-Sediment Samples
Key parameters for both water and sediment such as temperature, pH, total organic carbon (TOC), O2 concentration, redox potential and microbial biomass (on sediment only) were measured at different stages of the study using the characterization samples.
Analyses for total organic carbon (TOC) were done by Galbraith Laboratories, Inc. in Knoxville, Tennessee (Almshouse samples) and by Agvise Laboratories, Inc. (Cedar Hill) samples. Dissolved oxygen, redox potential, pH and microbial plate counts were done in-house at Rohm and Haas.
Determination of Microbial Plate Counts
One gram of the sediment was asceptically removed from the sample bottle and placed in a sterile scintillation vial. Sterile pH 7 phosphate buffer (9 ml) was added to the vial. This is the 1/10 dilution of the sediment sample-referred to as the first dilution. The vial was shaken to mix the contents. Consecutive 1:10 serial dilutions in pH 7 phosphate buffer solution were performed on the first dilution sample, giving 1/100, 1/1000, 1/10000, 1/100000 dilutions of the original sediment sample.
An aliquot of each dilution (0.1 ml) was streaked on a Trypticase Soy Broth Agar for bacterial quantitation. The plates were incubated at 30C for 2 days. Another aliquot of each dilution (0.1 ml) was streaked on a Potato Dextrose Agar for fungal quantitation. The plates were incubated at 25C for 6 days.
Validation of 14CO2 in KOH Traps by Barium Salt Precipitation
To confirm that the radioactivity trapped by the 1 N KOH solution was 14CO2, duplicate aliquots (0.5 ml) from various intervals were transferred into microcentrifuge tubes (1.5 ml volume). Approximately 0.5 ml of saturated BaCl2 was added to the KOH solution and shaken using a vortex to mix the sample thoroughly. A small amount (0.1 ml) of 2 M potassium carbonate (K2CO3) was then added, and the samples were vortexed again. The microcentrifuge tubes were centrifuged for 5 minutes at 3000 rpm using an Eppendorf Centrifuge. The supernatant was pipetted into a scintillation vial and 15 ml of Hionic-Fluor cocktail was added. Water (0.5 ml) was added to the precipitate, and the contents of the microcentrifuge tube were transferred to a scintillation vial using a Pasteur pipet. A second 0.5 ml portion of water was again added to the microcentrifuge tube to ensure complete transfer to the precipitate. Both water rinses were combined in the scintillation vial and 15 ml of Hionic-Fluor cocktail was added to the vial for LSC.
Reference substance
- Reference substance:
- not specified
Results and discussion
Half-life of parent compound / 50% disappearance time (DT50)open allclose all
- Compartment:
- entire system
- DT50:
- 11 h
- Type:
- (pseudo-)first order (= half-life)
- Temp.:
- 20 °C
- Remarks on result:
- other: Almshouse sediment
- Compartment:
- entire system
- DT50:
- 34 h
- Type:
- (pseudo-)first order (= half-life)
- Temp.:
- 20 °C
- Remarks on result:
- other: Cedar Hill sediment
- Mineralization rate (in CO2):
- 18 other: percent in Cedar Hill sediment/water and 28% in Almshouse sediment/water
- Transformation products:
- yes
Identity of transformation products
- No.:
- #5
- Details on transformation products:
- Analyses of Extracted Fractions
The initial EtOAc/MeOH fractions resulting from the Almshouse supernatant (water phase) fractionation (Day 0-Day 0.21) were analyzed using reversed-phase (RP)-HPLC. Analyses of the rest of the EtOAc/MeOH, CH2Cl2/MeOH/HCl, and MeOH/KOH extracts from both sets of samples were conducted using normal-phase TLC (NP-TLC). This also provided quantitation of parent compound and metabolites.
EtOAc/MeOH fractions (from the water phase)
Analyses of the EtOAc/MeOH extracts by TLC showed that on Day 0 through Day 1.3 of the Almshouse samples and Day 0 through Day 3 of the Cedar Hill samples, mainly parent compound was detected. Parent compound decreased rapidly throughout the study for the Almshouse, down to 21.3% by Day 1 then 0.2% by Day 30. The decline of parent compound in the Cedar Hill samples went from an average of 52.4% of the applied radioactivity on Day 1 to 1.3% by Day 7 then 0.5% by Day 30. In addition to parent compound, six metabolites were observed in the Almshouse and nine metabolites were observed in the Cedar Hill. These additional metabolites were detected by TLC but not one exceeded 3% of the total applied radioactivity.
In the EtOAc/MeOH fractions where greater than 10% of the dosed 14C was attributed to RH-573, confirmation of parent compound by HPLC and LC/MS was done. The EtOAc/MeOH fractions from Day 0 through Day 1.3 of the Almshouse and Day 0 through Day 3 of the Cedar Hill were each analyzed by LC/MS using atmoshperic pressure chemical ionization (APCI) in the positive ion mode to confirm the presence of parent compound. The LC/MS chromatograms of these samples showed one major radioactive (RAM) peak with a retention time of ~7.5 minutes. The mass spectra obtained for this peak showed the molecular ion at m/z 116, indicating a molecular weight of 115 atomic mass unit (amu) corresponding to the molecular weight (MW) of RH-573. The LC/MS chromatograms and mass spectra of the major component observed from all of the EtOAc/MeOH sample extracts analyzed were consistent with the LC/MS chromatogram and mass spectrum of the RH-573 standard.
Therefore, it was concluded that the peak at ~7.5 minutes were parent (RH-573).
CH2Cl2/MeOH/HCl fractions (from the water phase)
Parent compound and six other minor metabolites (Met-1, Met-2, Met-4, Met-6a, Met-6b, and Met-7) found in the Almshouse CH2Cl2/MeOH/HCl fractions accounted for less than 5% of the applied radioactivity. Parent compound was observed at 22.4% of the applied dose on Day 0.25 and less than 6% in all of the other sampling intervals of the Cedar Hill CH2Cl2/MeOH/HCl fractions. LC/MS analysis of the Day 0.25 replicate samples showed that the major radioactive peak in these samples has a molecular ion at m/z 116 (APCI, positive ion mode; Ion Trap [IT-LC/MS]), confirming its identity as RH-573. Four other minor metabolites (Met-1, Met-3, Met-5, and Met-6) were observed in the Cedar Hill CH2Cl2/MeOH/HCl fractions, most not exceeding 4% of the applied dose.
One major metabolite was found in both Almshouse and Cedar Hill CH2Cl2/MeOH/HCl fractions. The major metabolite, labeled Met-5 in the Almshouse samples, matched the Rf value of Met-4 in the Cedar Hill samples. Metabolite 5 reached a maximum average of 19.2% of the applied dose at Day 1.3 for the Almshouse and 19.6% after 2 days in the Cedar Hill CH2Cl2/MeOH/HCl fractions. The total amount of Metabolite 5 found in the extractable fractions reached a high of 23.5% in the Almshouse and 20.5% in the Cedar Hill at Day 2.
HPLC injections of the CH2CL2/MeOH/HCl fractions from Almshouse Day 0.13 through Day 7 samples and from Cedar Hill Day 1 thorugh Day 7 samples isolated two major components, one at 15.2 minutes and the other at 20.1 minutes. The 15.2 minute fraction (Fraction 1) matched the retention time of Rh-573 on the HPLC and was analyzed by IT-LC/MS (APCI, positive ion mode). The mass spectra showed a molecular ion at m/z 116 for the radioactive peak at 7.13 minutes, corresponding to the molecular weight of RH-573 thereby confirming its identity. The 20.1 minute fraction (Fraction 2) from both the Almshouse and Cedar Hill CH2Cl2/MeOH/HCl fractions, possibly matching Metabolite 5 seen on the TLC chromatograms, was submitted for LC/MS analysis for confirmation of its identity.
TLC analysis of the CH2Cl2/MeOH/HCl fraction from the high dose Almshouse sediment/water (labeled 573Alm-D2AB-Sup-SPE-CH2Cl2/MeOH) showed the same major metabolite, Metabolite 5, observed in the CH2Cl2/MeOH/HCl and MeOH/KOH fractions from the 1 ppm dosed samples. LC/MS analysis of the CH2Cl2/MeOH/HCl fractions is described in the following seciton.
Identification of Metabolite 5
The intial identificaiton of Metabolite 5 was done by electrospray ionization (ESI)-LC/MS analysis (Micromass Quattro-SQ) using the CH2Cl2/MeOH/HCl fraction from the high dose Almshouse sediment/water (573Alm-D2AB-Sup-SPE-CH2Cl2/MeOH). Injection of this fraction showed only one radioactive (RAM) peak with a retention time of 5.6 minutes. The LC/ESI-MS negative ion spectrum of this 5.6 minute RAM peak shows a molecular ion at m/z 164. Further analysis by a positive ion ESI-LC/MS spectrum showed a protonated molecular ion at m/z 166. Both the negative and positive ion data indicate a MW of 165 for this metabolite. The negative ion spectrum for the m/z 164 peak shows the most abundant ion to be m/z 164 with a sulfur isotope ion at m/z 166, indicative of probably one sulfur in the molecule. Exact mass measurement was made using electrospray ionization-Fourier-transform Ion Cyclotron Resonance-mass spectrometry (ESI-FTICR-MS; Bruker) with flow injection. The exact mass of the 164 ion was measured to be 164.0023 indicating an elemental formula of C4H6NO4S- for the m/z 164 ion and a molecular formula of C4H7NO2S (and a molecular mass of 165.0096) for the compound. The proposed structure for this metabolite, identified as 2(methyl carbamoyl)-1-oxoethane sulfinic acid.
The 20.1 minute HPLC fraction (Fraction 2) isolated from the Almshouse and Cedar Hill CH2Cl2/MeOH/HCl fractions were also analyzed by ESI-LC/MS (Micromass LCT) for comparison with the metabolite identified in the CH2Cl2/MeOH/HCl fraction from the high dose Almshouse sediment/water. Two radioactive peaks at 6.0 minutes and 6.7 minutes were observed for Fractoin 2 from Almshouse (labeled ALM-Metabolite 5). The mass spectrum (ESI, negative mode) showed two m/z 164 peaks corresponding to the retention time of these two RAM peaks, with presence of a m/z 125 component co-eluting with the 6.0 minute peak and a m/z 164 peak. Eact mass measurement of each of the ions was made using internal calibraion by infusing the calibration solution post HPLC column with the HPLC eluent at 7 ul/min and using the 168.9888 ion as the lock mass. The exact mass of the 164 ion in the 6.0 minute peak was measured to be 164.0081, indicating an elemental formula of C4H6NO4S- for the m/z 164 and a molecular fomula of C4H7NO4S ( and a molecular mass of 165.0096) for the compound. These data matched the data obtained from the FTICR-LC/MS analysis of Metabolite 5 above and is the same MW 165 compound and the same structure. The exact mass of the m/z 164 ion eluting at 6.7 minutes was measured to be 164.0060, also indicating an elemental formula of C4H6NO4S- and a molecular formula of C4H7NO4S (and a molecular mass of 165.0096). This MS 165 metabolite at 6.7 minutes is proposed to be an isomer of the m/z 164 ion eluting at 6.0 minutes.
The exact mass of the 125 ion was measured to be 124.9984. Close examination of the exact mass spectrum shows a sulfur isotope ion suggesting the presence of one sulfur in the compound and the molecular formula of C2H6O4S.
Fragmentation data at a cone voltage of 55 volts shows a m/z 97 fragment ion from the m/z 125 ion. No fragmentation was observed for the m/z 164 ion from the 6.0 minute peak and the 6.7 minute peak.
Fragment 2 from Cedar Hill labelled CED-Metabolite 5 showed only one radioactive peak at 5.6 minutes, which correlated with a m/z 164 peak and a m/z 125 peak seen in the negative ESI analysis. The negative ion data indicates two components co-eluting at the 5.6 minute RAM peak. The 5.6 minute appears to match the 6.0 minute peak from ALM-Metabolite 5 sample. Eact mass measurement was also done on both ions, as previously described for th ALM-Metabolite 5 sample. The exact mass of the m/z 164 was measured to be 164.0040, indicating an elemental formula of C4H6NO4S- for the m/z 164 and a molecular formula of C4H7NO4S (and a molecular mass of 165.0096) for the compound. These data fit the same proposed structure for Metabolite 5 from 573Alm-D2AB-Sup-SPE-CH2Cl2/MeOH (high dose) already identified above.
The exact mass of the m/z 125 ion was measured to be 124.9984, indicating a molecular formula of C2H6O4S (close examination of the exact mass spectrum also shows a sulfur isotope ion indicating the presence of one sulfur in the compound). Identical data was obtained for this ion to that obtained from the m/z ion seen co-eluting with the m/z 164 ion in the 6.0 minute peak of ALM-Metabolite 5.
In order to further establish the identity of Metabolite 5, a portion of the isolated Metabolite 5 was derivatized using diazomethane. A mass spectrum of the derivatized material using Finnegan LCQ Deca Ion Trap (IT-LC/MS, APCI in positive ion mode) showed one radioactive peak with a protonated molecular ion at m/z 180. This corresponds to a molecular weight of 179 amu suggesting the structure of the methylated 2(methyl carbamoyl)-1-oxoethane sulfinic acid.
MeOH/KOH fractions (from the sediment)
TLC analysis of the extracts showed several metabolites. Met-3 in the Almshouse and Met-2 in the Cedar Hill matched the Rf value of RH-573. Parent compound was detected at a maximum level of 1.1% (Day 0.21) in the Almshouse and 0.6% (Day 2) in the Cedar Hill. Other than parent, nine metabolites were observed from the Almshouse and ten metabolites were observed from the Cedar Hill samples. The major metabolite found in the Almshouse MeOH/KOH fractions, Met-5 (Met-4 in the Cedar Hill), have the same Rf value as Metabolite 5. Met-5 reached a maximum level of 3.2% of the applied radioactivity in the Almshouse at Day 2 and 1.0% in the Cedar Hill at Day 30. The other metabolites were detected in low levels for both sediment-water samples, with none exceeding 1.0% of the dosed 14C. With only less than 7% of the total applied radioactivity extracted into the MeOH/KOH fractions and the low amount of activity present in these extracts, no further identification was possible.
Analyses of Post Extracted Solids (PES)
The CaCl2 extraction released only up to 4.3% (Day 7A) of the applied 14C-residues in the Almshouse PES from the initial 60.2% activity left in the PES. The radioactivity extracted by 0.25 N HCl into the Hydrolysate-1 fraction only reached a high of 4.8% for the Almshouse PES (Day 7A and Day 30A). The Hydrolysate-1 fraction was subsequently partitioned with EtOAc but the majority of the radioactivity stayed with the Aqueous-1 fraction (up to 4.1%). The radioactivity in the Hydrolysate-2 (NaOH extractable) for the Almshouse was characterized to be associated between fulvic acid (maximum of 6.6%) and humic acid (high of 18.1%). The humiins fraction contained a maximum value of 28.7% of the applied radioactivity (equivalent to approximately 48% of the total PES).
The Cedar Hill PES showed a similar distribution pattern of extraction of the PES as the Almshouse samples. With as high as 62.6% of radioactivity in the PES fraction (Day 30A-repeat), only 5.5% was extracted by CaCl2. Low levels (2.4% - 4.7%) were extracted by the acid reflux, which partitioned mostly into the aqueous fraction (2.0% - 4.0%). The fulvic acid fraction contained up to 6.6% of the total applied radioactivity and a maximum amount of 15.7% was associated with the humic acid fraction. The majority of the radioactivity in the Cedar Hill PES appears to be incorporated into the humins fraction (up to 30.2%). - Evaporation of parent compound:
- not specified
- Volatile metabolites:
- yes
- Residues:
- yes
- Details on results:
- Definitive Study
The initial extraction (Day 0) showed that all of the applied readioactivity remained in the supernatant fraction for both sediment-water systems (RH-573 is very water-soluble). However, the percentage of 14C in the Almshouse supernatant fraction (water phase) decreased very quickly with time, from an average of 104.9% on Day 0 to 53.4% by Day 1, and subsequently to less than 18% by Day 7. The amount of the applied radioactivity remaining in the Cedar Hill water fraction also decreased rapidly, on an average, from 105.2% on Day 0 to 83.1% by Day 1 then down to 41.1% by Day 7. By Day 30, the Almshouse water fraction had an average of 5.8% of the applied radioactivity and the Cedar hill had 9.3% of the applied dose remaining in the water fraction. As seen in the preliminary study, after SPE clean-up of the water fraction, the amount of activity seen in the EtOAC/MeOH eluates decreased with time while vise-versa, the activity seen in the CH2Cl2/MeOH/HCl eluates increased with time up to Day 2 then decreased again by Day 30, In the Almshouse supernatant fraction, all of the applied radioactivity was initially eluted by EtOAC/MeOH, beginning at an average of 105.9% at Day 0 then rapidly declining to 1.9% by Day 30. Concurrently, the amount of applied dose observed in the Almshouse CH2Cl2/MeOH/HCl eluates started at an average of 4.1%, increasing to 31.9% by Day 2 and then declining to 2.6% by Day 30. The amount of applied dose eluted by EtOAC/MeOH from the Cedar Hill supernatant fraction started at an average of 104.6% at time 0 and decreased to 2.6% by Day 30. The Cedar Hill CH2Cl2/MeOH/HCl eluted activity started at an average of 3.0% of the applied radioactivity and increasing to a maximum average of 27.6% at Day 2 and to a final level of 5.3% at Day 30. All throughout the 30-day study, less than 1.5% of the total applied radioactivity in the supernatant fraction eluted through the aqueous fraction of the SPE procedure.
The amount of radioactivity extracted from the Almshouse sediment into the MeOH/KOH fraction gradually increased to an averaged maximum of 6.7% of the applied radioactivity after 2 days and then slowly declined to 3.2% by Day 30. The amount of the applied dose extracted from the Cedar Hill sediment by MeOH/KOH reached an averaged maximum of 4.4% of the applied activity by Day 7 and then went back down to 3.3 % of the applied dose by Day 30.
The average level of 14C residues remaining in the PES fraction reached a maximum of 59.4% (Day 7) for Almshouse and 61.5% (Day 30) of the applied dose for Cedar Hill.
The evolved volatiles from the study, collected as 14CO2, increased with time, reaching a maximum of 27.9% of the total applied radioactivity for the Almshouse and 18.4% for the Cedar Hill by Day 30. The presence of 14CO2 in the KOH solution was confirmed by Ba(OH)2 precipitation test. Essentially all of the radioactivity in the KOH solution (>94%) was in the precipitate fraction, which proves the presence of 14CO2.
Total 14C Recovery
Quantitative recoveries were obtained for both the Almshouse and Cedar Hill samples throughout the testing period. The overall average recovery of the applied radioactivity was 101.8% for the Almshouse samples and 99.2% for the Cedar Hill samples.
Half-life Calculation of the Parent Compound
The half-life (T1/2) was determined by regression analysis using pseudo-first order kinetics (i.e., the natural log of the percentage of RH-573 remaining in the sample versus incubation time. The rate constant (k) needed to determine half-life was the slope of the line.
The half-life for RH-573 in the Almshouse sediment/water, calculated based on the first two days of data, was 0.46 day (11.0 hours) and 4.7 days (112 hours) when calculated over the entire 30 days.
The half-life of RH-573 in the Cedar Hill was 1.4 days (34 hours) over the first 7 days of data and 4.5 days (109 hours) over the entire 30-day duration of the study.
The half-lives for two different time spans for both sediment/water were calculated for a comparison. However, the half-life calculated over 2 days provided the best fit for the Almshouse sediment/water with an excellent coefficient of correlation (0.99380), indicating a good first-order kinetics for the test compound. The best fit for the Cedar Hill was the half-life calculated over 7 days with a coefficient of correlation of 0.9810. At these sampling points (day 2 for the Almshouse and Day 7 for the Cedar Hill), more than 90% of the test substance had already dissipated by transformation and volatilization. Quantitation of parent compound beyond these sampling points may not be as accurate due to the low levels found (0.7% for the Almshouse and 0.9% for the Cedar Hill).
Proposed Metabolic Pathway
As seen in previous studies (References 4,5 and 6), metabolism of RH-573 involves cleavage of the isothiazolone ring bond and the subsequent formation of polar molecules such as N-methyl malonamic acid, 2(methyl carbamoyl)-1-oxoethane sulfinic acid, malonamic acid,and malonic acid. Results of this study showed increasing amount of 14CO2 produced with time and the dramatic increase of 14C-bound activity associated in the sediments. These indicate that parent molecule is being rapidly metabolized into a more polar molecule and is getting incorporated into the soil matrix (PES).
References
4 Reynolds, J.L., Aerobic Aquatic Metabolism of [14C]-RH-573, Study No. XBL91006, Rohm and Haas Technical Report No. 34 -94 -122.
5 Krzeminski, S.F., Brackett, C.K, Fisher, J.D., and Spinnler, J.F., Fate of Microbicidal 3 -Isothiazolone Compounds in the Environment: Products of Degradation. Agric. Food. Chem 23:1068 (1975)
6 Williams, T. and Jacobson, A. Environmental Fate of Isothiazolone Biocides. CORROSION/99, Paper Number 99303 (NACE International, 1999) - Results with reference substance:
- No data.
Any other information on results incl. tables
Preliminary Work
A preliminary study was performed on both the Almshouse and Cedar Hill sediment and water. This study was conducted to obtain information on the half-life of the test substance on each sediment/water in order to establish an appropriate sampling interval regime and the duration of the test. Data from a previous aerobic aquatic metabolism study of RH-573 (Reference 4) had already demonstrated that at a higher dose concentration (5 ppm), the rate of decline of RH-573 was slower due to its inhibition of microbial activity in the water-sediment system. The results of that study established that 1 ppm is an appropriate rate to use for the study.
Distribution of Radioactivity Throughout the Study
Preliminary Study
The amount of 14C activity remaining in the supernatant fraction (water phase) decreased from an average of 112.0% at Day 0 to 34.8% at Day 3 for the Almshouse sediment-water and from an average of 105.5% at Day 0 to 42.9% at Day 3 for the Cedar Hill sample. Solid phase extraction (SPE) of the supernatant fractions showed a pattern of elution relative to the incubation time. The amount of radioactivity from the Almshouse supernatant eluted by EtOAC/MeOH decreased from an average 101.5% at Day 0 down to an average of 4.6% by Day 3 while vise-versa, the activity eluted by the CH2Cl2/MeOH/HCl increased from an average of 5.8% at Day 0 to 24.6% by Day 3. The Cedar Hill sample showed a similar pattern, the EtOAC/MeOH activity decrasing from an average of 102.0% at Day 0 down to 9.9% by Day 3 while the CH2Cl2/MeOH/HCl eluted activity increased from an average of 3.9% at Day 0 to 24.6% by Day 3.
The level of radioactivity extracted from the residual solids with MeOH/KOH reached an average maximum of 10.0% of the applied dose for the Almshouse and 3.6% for the Cedar Hill by Day 3. Volatiles, including 14CO2, increased to an average maximum of 7.9% for the Almshouse and 4.3% for the Cedar Hill by Day 3. Production of 14CO2 indicates cleavage of hte isothiazolone ring and subsequent oxidation of the resulting alkyl metabolites. Results of the preliminary study suggested a short half-life (9 hours for Almshouse and 18 hours for Cedar Hill). The data points and half-life plots identified sampling intervals of 0, 0.04, 0.13, 0.21, 1, 1.3, 2, 7, and 30 days for the Almshous sediment water samples and sampling intervals of 0, 0.17, 0.25, 1, 1.3, 2, 3, 7, and 30 days for the Cedar Hill sediment water samples.
References
4 Reynolds, J.L., Aerobic Aquatic Metabolism of [14C]-RH-573, Study No. XBL91006, Rohm and Haas Technical Report No. 34 -94 -122.
Applicant's summary and conclusion
Validity criteria
- Validity criteria fulfilled:
- yes
- Conclusions:
- Under aerobic aquatic conditions, RH-573 degrades rapidly when applied to sediment/water maintained at 20C. Ultimately, the sediment residues are mineralized, converted to 14CO2 and a sizeable amount of the radioactivity becomes incorporated into the bound residues. After the exhaustive extraction procedures conducted on the post extracted solids (PES), the remaining bound residues are characterized as covalently bound into the soil matrix and therefore unavailable for bioaccumulation.
Other than parent and 14CO2, the major metabolite found in both Almshouse and Cedar Hill sediment/water is the 2(methyl carbamoyl)-1 -oxoethane sulfinic acid which accounted for up to 23.5% of the applied dose. The production of 14CO2 indicates cleavage of the isothiazolone ring bond as demonstrated in previous studies. For both sediment/water, no other detected metabolite exceeded 9% of the applied radioactivity.
The half-life of RH-573 in the Almshouse sediment/water is 0.46 day and 1.4 days for the Cedar Hill sediment/water. - Executive summary:
The rate of degradation of RH-24573 (also known as RH-573) was examined in two sediment-water systems at a treatment concentration of 1 ppm. The study was conducted under aerobic conditions at 20C in the dark. The half-life of parent compound in the Almahouse sediment/water, calculated over the first 2 days, was 0.46 day and over the entire 30 days was 4.7 days. The half-life of parent compound in the Cedar Hill sediment/water, calculated over 7 days, was 1.4 days and over the 30 day duration of the study was 4.5 days.
Duplicate samples of the 1ppm-dosed sediment/water were harvested and extracted at days 0, 0.04, 0.13, 0.21, 1, 1.3, 2, 7 and 30 days post-treatment for the Cedar Hills samples. Harvest extracts were analyzed using thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC) in conjunction with liquid scintillation counting (LSC) and liquid chromatography/mass spectrometry (LC/MS). The post extracted soils (PES) were analyzed for total radioactivity by LSC following combustion. Quantitative recoveries of the total applied radioactivity were obtained throughout the testing period. The overall averaged recovery for 14C-RH-573 from the Almshouse sediment/water was 101.8% and from the Cedar Hill sediment/water was 99.2%.
The total amount of applied dose remaining in the supernatant (water phase) decreased quickly with time, from an average of 105% at Day 0 to less than 6% for the Almshouse and less than 10% for the Cedar Hill after 30 days. Parent compound and its metabolites were extracted from the water phase using solid phase extraction (SPE) and from the sediment using MeOH/0.15 N KOH. The MeOH/0.15 N KOH mixture extracted a maximum average of 6.7% (by Day 2) of applied dose from the Almshouse solids and a maximum amount of 4.4% (by Day 7) from the Cedar Hill solids. The total amount of radioactivity remaining as parent in the extracted fractions (supernatant and sediment extracts) decreased from 106% at zero time to 5% after 2 days to less than 1% after 30 days for the Almshouse sediment/water. The amount of parent compound in the Cedar Hill extracted fractions (supernatant and sediment extracts) also decreased from an average of 101% at Day 0 down to 3% by Day 7 to a final value of less than 1% by Day 30. As the amount of radioactivity in the water layer decreased with time, the level of radioactivity in the PES increased to a maximum average of 59% at Day 7 for the Almshouse and 62% at Day 30 for the Cedar Hill. Evolved volatiles trapped in the KOH solution (confirmed as CO2 by barium salt precipitation) increased to an averaged maximum of 28% in the Almshouse sediment/water and 18% for the Cedar Hill sediment/water by Day 30.
Other than parent and 14CO2, the major metabolite found in the extractable residues from both sediment/water (labeled as Met-5 in the Almshouse and Met-4 in the Cedar Hill) was identified as 2(methylcarbamoyl)-1 -oxoethane sulfinic acid by LC/MS and confirmed by derivatization using diazomethane. The overall concentration of sulfinic acid reached a maximum level of 23.5% of the applied radioactivity in the Almshouse sediment/water and 20.5% of the dose in the Cedar Hill by Day 2. Other minor metabolites were detected in both sediment/water but their levels never exceeded 9% of the applied dose at any sampling interval. There were no metabolites observed whose concentration was continuously increasing through the end of the study.
Selected samples of the PES fraction from each sediment/water were subjected to various extraction procedures. Refluxing overnight with 0.02 N CaCl2 released only 3% - 6% of the total applied radioactivity from the PES, which ranged from 27% - 63%. Acid hydrolysis (0.25 N HCl) released another 2% - 5% of the remaining radioactivity which when partitioned with EtOAc remained mostly in the Aqueous fraction (2% - 4%). Shaking the residual solids overnight with base (0.5 N NaOH) extracted 9% - 25% of the applied dose. Further characterization of the basic fraction showed that the amount of radioactivity associated with fulvic acid ranged from 4% - 7% and 5% - 18% of the total applied radioactivity is associated with Humic acid. Unextractable bound resideues associated with soil humins totaled up to 30% of the applied dose.
The decreased 14C-activity remaining in the water phase and the increased activity in the bound residues as the incubation time increased, along with the production of 14CO2 suggest that the metabolic pathway for RH-24573 involves cleavage of the isothiazolone ring bond and the formation of polar metabolites. The presence of 14CO2 can only arise by ring cleavage and subsequent oxidation. Thus, the metabolic pathway reported in previous studies involving ring cleavage and subsequent oxidation of the rsulting alkyl compounds is also demonstrated in this study.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
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