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Hydrolysis

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Reference
Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1995-11-07 - 1996-07-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EPA Guideline Subdivision N 161-1 (Hydrolysis)
Deviations:
no
GLP compliance:
yes
Remarks:
U.S. EPA Good Laboratory Practice Standards (40 CFR Part 160)
Specific details on test material used for the study:
[14C]Ferbam (specific activity of 63.6 mCi/mmole) and [13C]ferbam were synthesized by PTRL East, Inc. Purity analyses were performed by proton nuclear magnetic resonance spectroscopy (NMR). The actual [14C]ferbam test substance used for purity assay by NMR was used for dosing following transfer to acetonitrile solvent. Non-radiolabelled reference substances for tetramethylthiuram disulfide (TMTD), tetramethylthiourea (TMTU) and carbon disulfide (CS2) were obtained from Aldrich Chemical Company (Milwaukee, WI). Tetramethylthiuram monosulfide (TMTM) was obtained from Lancaster Synthesis, Inc. (Windham, NH). 14CS2 and 14CO2 (sodium bicarbonate salt) were obtained from Sigma Chemical Company (St. Louis, MO). Non-radiolabelled standards of TMTD, TMTM, TMTU and CS2 were analyzed by HPLC (qualitatively) prior to use in the study and following the last sampling to confirm stability.
Solvents used were HPLC-grade or higher quality and were obtained from Fisher Scientific (Pittsburgh, PA). Aqueous buffered solutions were prepared with HPLC-grade water with specifications of 0.001 AU ultraviolet absorbance and 1-ppm residue after evaporation. Deionized ultrafiltered (DIUF) water was also obtained from Fisher Scientific. Trypticase soy agar and potato dextrose agar were obtained from Difco Laboratories (Detroit, MI). Safety-Solve™ scintillation cocktail (Research Products International, Mount Prospect, IL) was used for all liquid scintillation counting (LSC) and 14C-radiocarbon detection by high performance liquid chromatography (HPLC). All other chemicals used were reagent-grade or higher quality.
Radiolabelling:
yes
Analytical monitoring:
not specified
Details on sampling:
Sampling and Sample Storage
No samples were stored from the portion of the study conducted to determine the degradation rate of ferbam in aqueous buffered solutions. For the study to generate degradation products, flasks were removed from the incubator following 1, 2,4, 7, 14, 21 and 30 days of incubation. At each of these intervals, including Day 0, flasks were connected to a series of volatile traps via the Teflon® outlet valve. The head space of each flask was flushed for approximately one hour with air being pulled through trap solutions. Traps consisted of two gas dispersion tubes containing 50 ml of methanolic KOH followed by one containing 50 ml of EG, in series. Following flushing at Day 0, 1,2,4,7,14 and 30, aliquots of the solutions were sampled for quantitation of degradation products. At these intervals, flasks were sonicated for approximately 15 minutes prior to flushing. After flushing, three 50-ul aliquots were removed from each flask via the mini-inert valve with a syringe for direct radioassay of the test solution. Five ml of solution was then removed with a syringe and a 2-ml aliquot placed in a scintillation vial for pH determination using a Corning Model 240 pH meter. The remaining 3-ml aliquot was placed in a 7-ml, silylated, foil-covered vial containing 3 ml of chloroform. For sampling at Day 4 through Day 30, the sample was placed in an empty vial and 3 ml of chloroform added with the syringe used for sampling the aqueous hydrolysis solutions. The solution was partitioned immediately by vortexing. The aqueous phase was then transferred to a separate 7-ml, silylated, foil-covered vial. Aliquots (3 x 50 ul) of the aqueous and organic phases were taken for radioassay. The remaining organic and aqueous phases were analyzed by HPLC. A 7-ml aliquot (5 ml for sample analysis and 2 ml for pH determination) was removed from the pH 9 aqueous buffer solution, Replicate B only, at Day 14 to provide a larger sample size for exploring alternative methodology for quantitation of products. This aliquot was partitioned as above with chloroform (1:1, v:v).
Following 30 days of incubation, the remainder of the solutions in the flasks were sampled. In addition to sampling as above, four 1-ml aliquots of each buffered test solution were removed for confirmation of sterility. A 1-ml aliquot of each buffered solution was used for culturing duplicate plates of trypticase soy agar and potato dextrose agar. The trypticase soy agar plates were incubated at approximately 35°C for 1 day and the potato dextrose agar plates at approximately 25°C for 2 days. A 15-ml aliquot of the remaining solution for each buffer solution was transferred to a 15-ml, silylated, foil-covered storage vial fitted with a Teflon®-lined septum cap. In addition, at Day 30, vials containing the organic and aqueous partition phases were fitted with Teflon®-lined septum caps.

Trap Solution Precipitation with Barium Chloride
Trapped radiocarbon was found primarily in the methanolic KOH solutions with the initial trap containing most of the radiocarbon. In addition to radiocarbon trapped in solution, radiocarbon was also found in the sintered glass frit of the methanolic KOH trap dispersion tube as precipitated salt. After removal of the methanolic KOH solution, the dispersion tube frit was rinsed with approximately 15 ml of DIUF water by drawing water into the frit and evacuating several times. The water rinses were added to the methanolic KOH trap solutions. Precipitation of the methanolic KOH trap solutions and water rinses with barium chloride was performed following adjustment of water:methanol content of the solutions to 1:1 (v:v) with DIUF water.
Buffers:
Aqueous buffered solutions (500-ml volumes) were prepared at nominal pHs of 5, 7 and 9 as follows:
pH 5 (Acetic Acid - Sodium Acetate):
- 73 ml of 0.1M acetic acid were added to 50 ml of 0.1M NaOH
- HPLC-grade water was added for a final volume of 500 ml
- adjusted to pH 5 with acetic acid
pH 7 (Potassium Dihydrogen Phosphate):
- 11.2 ml of 0.1 M potassium dihydrogen phosphate were added to 12.9 ml of 0.1M sodium hydrogen phosphate
- HPLC-grade water was added for a final volume of 500 ml
- adjusted to pH 7 with HC1, KOH or NaOH
pH 9 (Sodium Borate - Hydrochloric Acid):
- 23 ml of 0.04M HC1 were added to 250 ml of 0.01M sodium borate decahydrate
- HPLC-grade water was added for a final volume of 500 ml
- adjusted to pH 9 with 0.5N NaOH
After preparation, the pH of each buffered solution was measured with a Corning Model 240 or 340 meter (Corning, New York). Buffer solutions were then sterilized by passing the solution through a 0.2-micron filter (Corning, Inc., Corning, New York). Buffer solutions were stored at approximately 25°C prior to use.
Details on test conditions:
Preparation of Test Solutions for Determination of Degradation Rates
Solutions of [13C]ferbam and TMTD were prepared in acetonitrile at a concentration of 1 mg/ml. For determination of the absorption spectra of ferbam and TMTD, aliquots of the 1 mg/ml solutions were diluted with acetonitrile to prepare a solution of 10 ppm. For determination of the linearity of the ferbam absorption response with concentration (lambda = 400 nm), aliquots of the 1 mg/ml solution in acetonitrile were diluted with acetonitrile to prepare solutions of 10, 7.5, 5.0, 2.5 and 1.0 ppm. Aqueous buffered solutions and DIUF water were fortified directly in a quartz cuvette with 10 jxl of the 1 mg/ml ferbam solution for determination of the degradation rate (1% acetonitrile co-solvent). Immediately following fortification, the cuvette was capped, inverted, shaken and placed in the UV spectrophotometer. The above experiment was repeated at a ferbam concentration of 50 ppm (50 (ul, 5% acetonitrile co-solvent) in pH 7 and 9 buffered solutions.

Preparation of Test Solution for Generation of Degradation Products
A solution of [14C]ferbam (specific activity of 59.96 mCi/mmole, 144.14 (uCi/mg) was prepared in chloroform-d? and assayed for identity and purity by NMR. A treatment solution was prepared by nitrogen evaporation of this chloroform solution to dryness and solubilization of the solid [14C]ferbam in acetonitrile (ACN). Flasks were prepared by adding 64 ml of pH 5,7 and 9 buffer to separate 250-ml, foil-covered Erlenmeyer flasks previously sterilized by autoclaving (121 °C, 15 psi, approximately 30 minutes). Duplicate flasks were prepared for each buffer solution. Flasks were equipped with inlet and outlet Teflon® valves such that the head space could be flushed to remove volatile products. In addition, each flask was equipped with a Teflon® mini-inert valve with septum which allowed dosing and sampling with a syringe while the flask remained sealed. Following introduction of the buffer, four 1-ml aliquots were removed from each flask for microbial assays to confirm sterility of the buffer solutions.
To the remaining 60 ml of buffer in each sealed flask, an aliquot (450 ul) of treatment solution was injected via the septum port. Based on radioassay of the treatment solution (45,377 dpm/jxl), the dose rate was 1.1 ppm. An additional 150 uI of acetonitrile was then added to increase solubilization of ferbam in the buffer solutions (total co-solvent concentration of 1% by volume). Each flask was then sonicated for approximately 15 minutes. Each flask was then purged with air under negative pressure with the air being pulled through a series of volatile collection traps. Traps consisted of two gas dispersion tubes containing 50 ml of IN potassium hydroxide in methanol (methanolic KOH) followed by one containing 50 ml of ethylene glycol (EG) in series. Following flushing, flasks were incubated (Incubator Model 307, Fisher Scientific, Arcada, CO) in the dark at 25.0 ± 0.2°C throughout the study.

Test System
The test system for the kinetic portion of the study consisted of solutions of [13C]ferbam contained in quartz cuvettes. The test system for the portion of the study to generate degradation products consisted of aqueous buffered solutions treated with [14C]ferbam contained in flasks. All test systems were identified by PTRL East, Inc. project number, sample identification, pH of the test solution, date and personnel
Duration:
30 d
pH:
5
Temp.:
25 °C
Remarks:
refer to different fields in materials & methods
Duration:
30 d
pH:
7
Temp.:
25 °C
Remarks:
refer to different fields in materials & methods
Duration:
30 d
pH:
9
Temp.:
25 °C
Remarks:
refer to different fields in materials & methods
Number of replicates:
not specified
Positive controls:
not specified
Negative controls:
not specified
Statistical methods:
Means, standard deviations and/or standard errors were determined for temperature measurements, LSC and HPLC data. Degradation rates were determined by linear regression analysis.
Preliminary study:
not specified
Test performance:
not specified
Transformation products:
yes
No.:
#1
No.:
#2
No.:
#3
No.:
#4
Details on hydrolysis and appearance of transformation product(s):
not specified
Key result
pH:
7
Temp.:
25 °C
DT50:
7.8 min
St. dev.:
0.94
Type:
(pseudo-)first order (= half-life)
Key result
pH:
5
Temp.:
25 °C
DT50:
12.1 min
St. dev.:
0.991
Type:
(pseudo-)first order (= half-life)
Key result
pH:
9
Temp.:
25 °C
Remarks on result:
other: not determinable since ferbam degraded instantly
Other kinetic parameters:
not specified
Details on results:
Purity of 14C-Ferbam
As a result of the instability of ferbam in either reverse or normal phase HPLC or TLC chromatographic systems, purities of [13C]ferbam, used for determination of degradation rates, and [14C]ferbam, used for generation of degradation products, were established by NMR spectroscopy. The actual contents of NMR tube containing [14C]ferbam were subsequently used for preparation of the dosing solution for the portion of the study to generate degradation products.
Non-radiolabelled reference substances were analyzed qualitatively by HPLC prior to initiation of the study and following the last sampling interval for determination of stability at the test site.

Degradation Rates of 13C-Ferbam in Buffered Solutions
UV scans of ferbam and TMTD solutions at 10-ppm concentration in acetonitrile (200 - 500 nm) were corrected for the acetonitrile solvent background. A standard curve was generated from UV absorbance measurements at 400 nm of acetonitrile solutions containing 10, 7.5, 5.0, 2.5 and 1.0 ppm of ferbam. Assuming degradation products are not formed that absorb at 400 nm, the linear correlation of absorbance and concentration of ferbam permits measurement of the amount remaining in solution. Since it is feasible that degradation products could be formed that absorb at 400 nm, rates determined by this procedure can only be interpreted as upper limits of the degradation rate. A further limitation of the method is the assumption that the UV absorbance at 400 nm is not suppressed by solvent interactions.
Kinetic profiles for ferbam at 10-ppm concentration in pH 5 buffered aqueous solution and in DKJF water (1% acetonitrile co-solvent) were meassured (lambda = 400 nm). In pH 5 buffer, ferbam degraded with a half-life of 12.1 minutes (r2 = 0.9912) whereas in DIUF water, the half-life was 31.0 minutes (r2 = 0.9686). Although highly correlated with first-order degradation, the kinetic profile for DIUF water appeared more linear than expected for first-order degradation. This potentially indicates a more complex dissipation mechanism than can be ascribed to first-order hydrolytic degradation, e.g., hydrolysis accompanied by dissociation of ferbam, a metal complex, into the metal cation (Fe+3, Fe(DTC)+2, Fe(DTC)2+) and dimethyldithiocarbamate anions (DTC-).
Absorbance of ferbam at 10-ppm in pH 7 and 9 buffer solutions was too low for reliable quantitation. The kinetic profile for ferbam at 50-ppm concentration in pH 7 buffered aqueous solutions (5% acetonitrile co-solvent) is was determined (lambda = 400 nm). In pH 7 buffer, ferbam degraded with a half-life of 7.8 minutes (r2 = 0.940). In pH 9 buffer, ferbam appeared to degrade (or dissociate) immediately; no absorbance could be measured for ferbam.

Degrádate Generation Phase - Schedule of Sampling Events and Temperature Measurements
For the phase of the study to generate degradation products, sampling was performed following 0, 1, 2,4,7, 14 and 30 days of incubation. Incubator temperatures during the 30-day period were 25.0 ± 0.2°C (mean ± standard deviation).

Degrádate Generation Phase - pH and Sterility of Test Solutions
The pH of each pH 5, 7 and 9 hydrolysis test solution for the incubation to generate degradation products was measured at each sampling interval. The pH values were stable over the study period. The mean pH values (average of replicate solutions) at initiation (Day 0) in the pH 5, 7 and 9 solutions were 5.11, 7.04 and 9.10, respectively. The mean pH values following 30 days of incubation were 5.08, 7.00 and 8.92 for these solutions, respectively. Sterility of the pH 5,7 and 9 test solutions was determined at Day 0 and Day 30 of the study. No microbial growth was observed following culturing of Day 0 and Day 30 aliquots of these solutions on the potato dextrose agar and trypticase soy agar. It was concluded that the hydrolysis solutions were sterile throughout the 30-day incubation phase.

Degrádate Generation Phase - Radiocarbon Material Balance
For the 30-day incubation, total radiocarbon recoveries for buffer solutions of pH 5, 7 and 9 were 94.1 ± 1.8, 91.7 ± 1.7 and 96.0 ± 1.2%, respectively (mean +- standard deviation for all sample intervals). Material balance was determined by comparison of radiocarbon recovered at sampling (in solution and in the volatile traps) plus that removed at all prior intervals compared to the initial applied radiocarbon.

Degradation of [14C]Ferbam
[14C]Ferbam hydrolyzed rapidly in buffer solutions of pH 5, 7 and 9. However, there were notable differences in subsequent degradation of hydrolysis products. In pH 5, 7 and 9 aqueous buffers, the primary products of ferbam degradation were TMTD, DTC-, carbon disulfide (CS2) and carbon dioxide (CO2). Concentrations following 30 days of incubation in pH 5 buffer were 53.2, 11.2, 29.7 and 4.1% of the test system radiocarbon, respectively. TMTD concentrations was greatest immediately following dosing (70.2%) and decreased with incubation time. DTC" slowly increased with incubation time reaching a maximum of 11.2% following 30 days. CS2, found both in the volatile traps and to a lesser extent in solution, increased with incubation time. CS2 in solution decreased over the 30-day incubation period. In addition to TMTD, DTC- and CS2, results of BaCl2 precipitation of the methanolic KOH traps indicated the presence of C02 although in considerably lesser amounts than CS2.
At pH 7, concentrations of TMTD, DTC-, CS2 and CO2 following 30 days of incubation were 6.7, 24.3, 59.8 and 8.3%, respectively. As in pH 5 buffer, the concentration of TMTD was greatest immediately following dosing (56.8%) and decreased with incubation time (6.7% following 30 days). DTC- increased in concentration with incubation time reaching a maximum of 24.3% of the test system following 30 days. CS2 was found both in solution and in the volatile traps. Immediately following dosing, CS2 in solution constituted 23.4% the test system radiocarbon. The amount in solution decreased with incubation time (0% by Day 30) whereas the amount in the volatile traps increased (59.8% at 30 days). In addition to TMTD, DTC- and CS2, results of BaCl2 precipitation of the methanolic KOH traps indicated the presence of CO2 although in considerably lesser amounts than CS2.
At pH 9, concentrations of TMTD, DTC-, CS2 and CO2 following 30 days of incubation were 2.3, 79.9, 9.8 and 7.3, respectively. As in pH 5 and 7 buffer, the concentration of TMTD was greatest immediately following dosing (46.2%) and decreased with incubation time (2.3% following 30 days). DTC" increased in concentration with incubation time reaching a maximum of 79.9% of the test system following 30 days. CS2 was found both in solution and in the volatile traps. Immediately following dosing, CS2 in solution constituted 39.6% of the test system radiocarbon. The amount in solution decreased with incubation time (0% by Day 30) whereas the amount in the volatile traps increased (9.8% at Day 30). These results indicate that the CS2 formed in solution was capable of reaction with other components present to contribute to the formation of DTC-. In addition to TMTD, DTC" and CS2, results of BaCl2 precipitation of the methanolic KOH traps indicated the presence of CO2 although in considerably lesser amounts than CS2.
In summary, ferbam degraded rapidly in aqueous solutions of pH 5,7 and 9 with initial formation of TMTD. TMTD showed a pH-dependent rate of degradation to other products. TMTD was most stable under acidic (pH 5) conditions and was the major product following 30 days of incubation. Under neutral (pH 7) and basic (pH 9) conditions, TMTD degraded more rapidly to DTC". At pH 7, DTC- subsequently degraded to CS2 and lesser amounts of CO2. CS2 was the major terminal product of degradation under neutral conditions. DTC" was relatively stable in pH 9 buffer and was the major terminal product following 30 days of incubation.

Confirmation of Degrádate Identity TMTD
TLC analysis was used to confirm HPLC characterization of TMTD in pH 5,7 and 9 Day 14, Replicates A and B, Organic Partition Phase. Although the presence of TMTD was confirmed in all samples, instability of components of the organic partition phase on silica gel was noted. Instability increased with increasing pH. This also correlated with the amount of plate-applied radiocarbon since the organic phase contained decreasing amounts of radiocarbon with increasing pH at Day 14 (approximately 60, 14 and 11% of the test system respectively for pH 5,7 and 9, mean of replicates).
For the organic partition phase of the pH 5 buffer, only TMTD was observed by HPLC (59.9-62.1 % of the test system at Day 14). By TLC, TMTD constituted from 87.9 to 94.5% of plate-recovered radiocarbon for Replicates A and B in the two one-dimensional TLC systems. Total recovery from the plates was adequate to permit quantitative interpretation, i.e., from 91.2 to 94.8% of plate-applied radiocarbon. For the organic partition phase of the pH 7 buffer, HPLC showed TMTD as the primary component (13.3-15.7% of the test system at Day 14) with significantly lower amounts of CS2. By TLC, TMTD constituted 75.2 to 81.9% of the plate-recovered radiocarbon. However, total plate recovery was not adequate to permit quantitative interpretation of these plates, i.e., plate recoveries were 82.0 to 88.6% of the plate-applied radiocarbon. For the organic partition phase of the pH 9 buffer, HPLC showed TMTD as the primary component (8.5-13.9% of the test system, approximately 68 to 78% of injected radiocarbon) with lesser amounts of DTC-. By TLC, TMTD constituted 16.5 to 32.9% of the plate-recovered radiocarbon. Total radiocarbon recovery from the plates was however not adequate to permit quantitative interpretation of these plates, i.e., 73.4 to 79.5%. The remaining radiocarbon, probably including DTC", was primarily origin-bound material.
In summary, TLC was adequate to confirm the presence of TMTD in the organic partition phase of pH 5,7 and 9 buffer solutions. Quantitative interpretation of TLC was only possible for the pH 5 buffer solution as a result of instability of components on silica gel.

Dithiocarbamate Anion
Under basic conditions, the polar product formed from hydrolysis of ferbam was the dominant product following 30 days of incubation. Under neutral conditions, this product was relatively unstable and further degraded to CS2. The hydrolysis of dithiocarbamate metal complexes to the dithiocarbamate anion (DTC") under basic conditions (0.25M ethylenediamine tetraacetic acid, 0.45M NaOH) is reported in the literature (Gustafsson, et.al) and is the basis of methodology to quantitatively analyze dithiocarbamate metal complexes. Cochromatography of the polar product, remaining in the aqueous phase following partitioning of pH 7 and 9 hydrolysis samples with chloroform, with dithiocarbamate anion was achieved using a reverse phase semi-preparative column with an acetonitrile: water mobile phase. Retention of the dithiocarbamate anion was marginal as a result of its high water solubility. HPLC radiochromatograms of the post-partitioned aqueous phases of Day 30, Replicate A and B, pH 7 and Day 14, Replicate A and B, pH 9 solutions are presented in Figures 30-33. The polar product was also shown to degrade to CS2 upon acidification, behavior consistent with the presence of DTC-
To provide additional confirmation of the polar product as the dithiocarbamate anion, solutions of a mixture of [13C] and [14C]ferbam were prepared at a nominal concentration of 1 mg/ml in aqueous 10 mM NaOH and pH 9 borate buffer. The solutions were allowed to react at 35°C for approximately 3 days after which they were partitioned (1:1, v:v) with chloroform to remove any residual un-reacted ferbam, TMTD or other organo soluble degradation products. Negative ion liquid chromatography/mass spectrometry (LC/MS) was performed on these samples by direct infusion to confirm the presence of DTC-. In negative ion mode, the major ion observed for both solutions was m/e 123 corresponding to the [13C]DTC- anion (M). In addition, a [13C]methylated adduct at m/e 139 was observed (M+13CH3> as well as a fragment ion at m/e 107 corresponding to loss of a 13C-methyl group (M-13CH3). Comparable spectra were obtained for the reaction product in both NaOH and borate buffer (pH 9). LC/MS spectra of the NaOH and borate buffer solutions are presented in Figures 37 and 38, respectively. LC/MS by direct infusion was attempted for the pH 9, Day 30, aqueous partition phase but was unsuccessful as a result of the low concentration (approximately 0.80 ppm) and instability of the dithiocarbamate anion.

Sample Stability
Analyses were performed on all samples immediately following sample collection. Therefore, no sample storage stability data are required to support the results presented.
Results with reference substance:
not specified
Validity criteria fulfilled:
yes
Conclusions:
Aqueous buffered solutions of [13C]ferbam at pH 5, 7 and 9 were prepared at a nominal concentrations of 10 ppm or 50 ppm. UV spectrophotometry at 400 nm was utilized to monitor ferbam degradation. Ferbam degraded most rapidly in pH 7 and 9 solutions. The degradation rate in pH 9 buffer could not be determined since ferbam degraded instantly (within 10 seconds of solution preparation). In pH 5 and 7 buffered solutions, degradation half-lives were 12.1 (r2 = 0.9912) and 7.8 minutes (r2 = 0.940), assuming first-order degradation kinetics. Degradation was slower in DIUF water with a half-life of 31.0 minutes (r2 = 0.9686).
Ferbam degraded rapidly in aqueous solutions of pH 5, 7 and 9 with initital formation of TMTD. TMTD showed a pH-dependent rate of degradation to other products. TMTD was most stable under acidic (pH 5) conditions and was the major product following 30 days of incubation. Under neutral (pH 7) and basic (pH 9) conditions, TMTD degraded mor rapidly to DTC-. At pH7 DTC- subsequently degraded to CS2 and lesser amounts of CO2. CS2 was the major terminal product of degradation und neutral condititons. DTC- was relatively stable in pH 9 buffer and was the major terminal product following 30 days of incubation.
Executive summary:

This study was designed and conducted according to the U.S. EPA Pesticide Assessment Guidelines Subdivision N, Series 161-1and under GLP to establish the significance of hydrolysis as a route of degradation for ferbam and to quantify significant degradation products formed by hydrolysis of [14C]ferbam.

Ferbam was sufficiently unstable in either reverse or normal phase high performance liquid chromatography (HPLC) or thin layer chromatography (TLC) to preclude these methods for determination of purity of the test substance. As a result of this instability, the purity of [14C]ferbam and [13C]ferbam were determined in a chloroform-d3 solution by proton nuclear magnetic resonance spectroscopy (NMR).

Ferbam degraded sufficiently rapid in aqueous media to preclude conventional methods for determination of its degradation rate. In addition, ferbam was sufficiently unstable that it did not appear as a single discrete moiety in high performance liquid chromatography (HPLC) or thin layer chromatography (TLC) using commercially available separation media. However, ferbam did absorb light in the UV region at wavelengths considerably beyond that for typical organic compounds (>320 nm). The absorption spectra of tetramethylthiuram disulfide (TMTD), a probable degradation product, showed a maximum absorption at 278 nm but minimal absorption at wavelengths >320 nm. Absorption at 400 nm was utilized to monitor ferbam degradation in aqueous buffered solutions of pH 5, 7 and 9, and in deionized ultrafiltered (DIUF) water. Solutions of [13C]ferbam and TMTD were prepared in acetonitrile at a concentration of 1 mg/ml. For determination of the absorption spectra of ferbam and TMTD, aliquots of the 1 mg/ml solutions were diluted with acetonitrile to prepare a solution of 10 ppm. For determination of the linearity of the ferbam absorption response with concentration (k = 400 nm), aliquots of the 1 mg/ml solution in acetonitrile were diluted with acetonitrile to prepare solutions of 10, 7.5, 5.0, 2.5 and 1.0 ppm. Aqueous buffered solutions and DIUF water were fortified directly in a quartz cuvette with 10 |il of the 1 mg/ml ferbam solution for determination of the degradation rate (1% acetonitrile co-solvent). Immediately following fortification, the cuvette was capped, inverted, shaken and placed in the UV spectrophotometer. This experiment was repeated at a ferbam concentration of 50 ppm (50 jxl, 5% acetonitrile co-solvent) in pH 7 and 9 buffered solutions. Degradation was most rapid in pH 7 and 9 buffered solutions. The degradation rate in pH 9 buffer could not be determined since ferbam degraded instantly (within 10 seconds of solution preparation). In pH 5 and 7 buffered solutions, degradation half-lives were 12.1 (r2 = 0.9912) and 7.8 minutes (r2 = 0.9405), assuming first-order degradation kinetics. Degradation was slower in DIUF water with a half-life of 31.0 minutes (r2 = 0.9686).

To assess degradation products of ferbam, aqueous buffered solutions at pH 5, 7 and 9 were prepared in flasks, capable of being flushed to remove volatile products, by addition of 60 ml of sterile buffer followed by addition of 450 \i\ of [14C]ferbam in acetonitrile. The acetonitrile co-solvent concentration was 1.0%. Duplicate flasks were prepared for each buffered solution at a concentration of 1.1 ppm of [14C]ferbam. Flasks were flushed with air for one hour following 21 days of incubation at 25 ± 1°C and just prior to sampling following 0, 1,2,4, 7, 14 and 30 days. Head space gases in the flasks were flushed through traps consisting of two methanolic KOH traps (IN KOH) and one ethylene glycol trap in series. Following flushing at each sample interval, aliquots of the buffered solutions were removed for characterization of radiolabeled components by high performance liquid chromatography (HPLC). During the study period, samples were maintained at a temperature of 25.0 ± 0.2°C (mean + standard deviation).

In pH 5, 7 and 9 aqueous buffers, the primary products of ferbam degradation were TMTD, dithiocarbamate anion (DTC-), carbon disulfide (CS2) and carbon dioxide (CO2). Concentrations following 30 days of incubation in pH 5 buffer were 53.2, 11.2,29.7 and 4.1% of the applied radiocarbon, respectively. At pH 7, concentrations following 30 days were 6.7, 24.3, 59.8 and 8.3%, respectively, whereas at pH 9 concentrations were 2.3, 79.9, 9.8 and 7.3% of the applied radiocarbon. From the degradation profiles at pH 5,7 and 9, ferbam appeared to degrade initially to TMTD. TMTD was most stable under acidic (pH 5) conditions and was the major product following 30 days of incubation. Under neutral (pH 7) and basic (pH 9) conditions, TMTD degraded more rapidly to DTC-. At pH 7, DTC- subsequently degraded to CS2 with lesser amounts of CO2. CS2 was the major terminal product of degradation under neutral conditions. DTC- was relatively stable in pH 9 buffer and was the major terminal product following 30 days of incubation.

Description of key information

This study was designed and conducted according to the U.S. EPA Pesticide Assessment Guidelines Subdivision N, Series 161-1and under GLP to establish the significance of hydrolysis as a route of degradation for ferbam and to quantify significant degradation products formed by hydrolysis of [14C]ferbam.

Ferbam degraded most rapidly in pH 7 and 9 solutions. The degradation rate in pH 9 buffer could not be determined since ferbam degraded instantly (within 10 seconds of solution preparation). In pH 5 and 7 buffered solutions, degradation half-lives were 12.1 (r2 = 0.9912) and 7.8 minutes (r2 = 0.940), assuming first-order degradation kinetics. Degradation was slower in DIUF water with a half-life of 31.0 minutes (r2 = 0.9686).

Ferbam degraded rapidly in aqueous solutions of pH 5, 7 and 9 with initial formation of TMTD. TMTD showed a pH-dependent rate of degradation to other products. TMTD was most stable under acidic (pH 5) conditions and was the major product following 30 days of incubation. Under neutral (pH 7) and basic (pH 9) conditions, TMTD degraded more rapidly to DTC-. At pH7 DTC- subsequently degraded to CS2 and lesser amounts of CO2. CS2 was the major terminal product of degradation und neutral conditions. DTC- was relatively stable in pH 9 buffer and was the major terminal product following 30 days of incubation.

Key value for chemical safety assessment

Half-life for hydrolysis:
7.8 min
at the temperature of:
25 °C

Additional information

Key: Nixon, 1996

This study was designed and conducted according to the U.S. EPA Pesticide Assessment Guidelines Subdivision N, Series 161-1and under GLP to establish the significance of hydrolysis as a route of degradation for ferbam and to quantify significant degradation products formed by hydrolysis of [14C]ferbam.

Ferbam was sufficiently unstable in either reverse or normal phase high performance liquid chromatography (HPLC) or thin layer chromatography (TLC) to preclude these methods for determination of purity of the test substance. As a result of this instability, the purity of [14C]ferbam and [13C]ferbam were determined in a chloroform-d3 solution by proton nuclear magnetic resonance spectroscopy (NMR).

Ferbam degraded sufficiently rapid in aqueous media to preclude conventional methods for determination of its degradation rate. In addition, ferbam was sufficiently unstable that it did not appear as a single discrete moiety in high performance liquid chromatography (HPLC) or thin layer chromatography (TLC) using commercially available separation media. However, ferbam did absorb light in the UV region at wavelengths considerably beyond that for typical organic compounds (>320 nm). The absorption spectra of tetramethylthiuram disulfide (TMTD), a probable degradation product, showed a maximum absorption at 278 nm but minimal absorption at wavelengths >320 nm. Absorption at 400 nm was utilized to monitor ferbam degradation in aqueous buffered solutions of pH 5, 7 and 9, and in deionized ultrafiltered (DIUF) water. Solutions of [13C]ferbam and TMTD were prepared in acetonitrile at a concentration of 1 mg/ml. For determination of the absorption spectra of ferbam and TMTD, aliquots of the 1 mg/ml solutions were diluted with acetonitrile to prepare a solution of 10 ppm. For determination of the linearity of the ferbam absorption response with concentration (k = 400 nm), aliquots of the 1 mg/ml solution in acetonitrile were diluted with acetonitrile to prepare solutions of 10, 7.5, 5.0, 2.5 and 1.0 ppm. Aqueous buffered solutions and DIUF water were fortified directly in a quartz cuvette with 10 |il of the 1 mg/ml ferbam solution for determination of the degradation rate (1% acetonitrile co-solvent). Immediately following fortification, the cuvette was capped, inverted, shaken and placed in the UV spectrophotometer. This experiment was repeated at a ferbam concentration of 50 ppm (50 jxl, 5% acetonitrile co-solvent) in pH 7 and 9 buffered solutions. Degradation was most rapid in pH 7 and 9 buffered solutions. The degradation rate in pH 9 buffer could not be determined since ferbam degraded instantly (within 10 seconds of solution preparation). In pH 5 and 7 buffered solutions, degradation half-lives were 12.1 (r2 = 0.9912) and 7.8 minutes (r2 = 0.9405), assuming first-order degradation kinetics. Degradation was slower in DIUF water with a half-life of 31.0 minutes (r2 = 0.9686).

To assess degradation products of ferbam, aqueous buffered solutions at pH 5, 7 and 9 were prepared in flasks, capable of being flushed to remove volatile products, by addition of 60 ml of sterile buffer followed by addition of 450 \i\ of [14C]ferbam in acetonitrile. The acetonitrile co-solvent concentration was 1.0%. Duplicate flasks were prepared for each buffered solution at a concentration of 1.1 ppm of [14C]ferbam. Flasks were flushed with air for one hour following 21 days of incubation at 25 ± 1°C and just prior to sampling following 0, 1,2,4, 7, 14 and 30 days. Head space gases in the flasks were flushed through traps consisting of two methanolic KOH traps (IN KOH) and one ethylene glycol trap in series. Following flushing at each sample interval, aliquots of the buffered solutions were removed for characterization of radiolabeled components by high performance liquid chromatography (HPLC). During the study period, samples were maintained at a temperature of 25.0 ± 0.2°C (mean + standard deviation).

In pH 5, 7 and 9 aqueous buffers, the primary products of ferbam degradation were TMTD, dithiocarbamate anion (DTC-), carbon disulfide (CS2) and carbon dioxide (CO2). Concentrations following 30 days of incubation in pH 5 buffer were 53.2, 11.2,29.7 and 4.1% of the applied radiocarbon, respectively. At pH 7, concentrations following 30 days were 6.7, 24.3, 59.8 and 8.3%, respectively, whereas at pH 9 concentrations were 2.3, 79.9, 9.8 and 7.3% of the applied radiocarbon. From the degradation profiles at pH 5,7 and 9, ferbam appeared to degrade initially to TMTD. TMTD was most stable under acidic (pH 5) conditions and was the major product following 30 days of incubation. Under neutral (pH 7) and basic (pH 9) conditions, TMTD degraded more rapidly to DTC-. At pH 7, DTC- subsequently degraded to CS2 with lesser amounts of CO2. CS2 was the major terminal product of degradation under neutral conditions. DTC- was relatively stable in pH 9 buffer and was the major terminal product following 30 days of incubation.

Supporting Information: Warren, 1987

A hydrolysis study was conducted according to EPA Guideline Subdivision N 161-1 (Hydrolysis) and under GLP conditions with 14C-Ferbam in four aqueous buffered solutions: pH 5, pH 7, pH 9A and pH 9B at a nominal test concentration of 10 ppm. Quantification of total 14C-Ferbam residue in the test system was measured by liquid scintillation counting analysis (LSC). Characterization and quantification of Ferbam was by spectrophotometric analysis.

The pH 5 buffered test system (acetic acid/sodium acetate) was conducted for 24 hours. The estimated half-life of 14C-Ferbam in the pH 5 test system was 3 hours.

The pH 7 test system was conducted for 26 hours. No parent Ferbam was observed at the 2 hour sampling interval, indicating rapid hydrolysis of the test material. It was not possible to estimate a half-life based on the first two time points in which parent Ferbam was observed. It can only be stated that the half-life of C-Ferbam in the pH 7 test system was between 1 and 2 hours.

In the pH 9A test system (boric acid/sodium borate) no parent Ferbam was defected in the 0 hour sampling. This indicates that the half-life of C-Ferbam in the pH 9A test system was so rapid (on the order of 5 to 10 minutes) that the compound had completely hydrolyzed before analysis could be made.

The pH 9B test system (glycine/NaOH) was conducted for 27 hours. No parent Ferbam was observed at the last sampling interval (27 hours). The parent Ferbam appeared to degrade rapidly, although some variability in the data was observed, possibly due to a buffer interaction with the parent test material. Even though the half-life of the 14C-Ferbam in the pH 9B test system could not be calculated, it was estimated to be less than 27 hours.