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Biodegradation in water and sediment: simulation tests

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Reference
Endpoint:
biodegradation in water: sediment simulation testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 308 (Aerobic and Anaerobic Transformation in Aquatic Sediment Systems)
Version / remarks:
In accordance with ECHA's decision, the anaerobic test was not performed.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Radiolabelling:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
natural sediment: freshwater
Details on source and properties of surface water:
Not applicable.
Details on source and properties of sediment:
Test Systems
Source of Test Systems
Sediment and water systems were collected from two sites in the United States. The first test system was collected from Goose River, North Dakota, and the second test system was collected from Golden Lake, North Dakota. Both test systems were collected on 28 September 2017 by Agvise Laboratories (Northwood, North Dakota). The test systems were received at EAG Laboratories - Hercules on 3 October 2017. Sediments were collected from the top 5 cm of the sediment layer with free access to air. Water samples were collected from the same sites at the same time as the sediment collections.
On arrival at EAG Laboratories - Hercules, the systems were stored refrigerated in the dark until use in the study. The water layers were sieved through a 0.2 mm sieve and sediment layers were sieved through a 2 mm sieve prior to use in the study.
Locations of the sites are presented below:
Sampling Site GPS Coordinates
Goose River (GR) N47°43.779, W97°37.312
Golden Lake (GL) N47°33.744, W97°38.452

Characterization of the Test Systems
The moisture content of the sediments was determined by using a Metter Toledo HB43-S moisture analyzer with five determinations for each sediment. Physicochemical characterization of the water and sediment was performed by Agvise Laboratories, Northwood, ND. A summary of the water and sediment characteristics is presented in Table 1.

Microbiological Viability of Test System
The sediments used in this study were analyzed for microbial viability (microbial biomass) prior to use (shortly following receipt), at the beginning of the incubation (experimental start) and following the final sampling of the study. Microbial biomass results are presented in Table 1.
Details on inoculum:
Not applicable.
Duration of test (contact time):
103 d
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
1. Protocol
The study was performed according to the appended study protocol entitled "Aerobic Aquatic Metabolism of [14C]copper bis (dibutyldithiocarbamate) (CDBC) in Two Test Systems". It was designed and conducted following OECD TG 308 (April 2002) Aerobic and Anaerobic Transformation in Aquatic Sediment Systems.

2. Test Substance
The test substance, [14C]CDBC, was supplied by EAG Laboratories (Columbia, Missouri) and received at EAG Laboratories – Hercules on 25 May 2017. A second shipment of the same batch of test substance was received at EAG Laboratories – Hercules on 14 December 2018. The test substance was received at EAG Laboratories – Hercules as a solid and was dissolved in acetonitrile prior to use in the study. HPLC purity determination was performed prior to application, which indicated that the test substance had a purity of 98.0%. The test substance was stored frozen (-20°C) when not in use.
Common Name: CDBC
Chemical Formula: C18H36CuN2S4

3. Analytical Reference Standard
An analytical reference standard was shipped from EAG Laboratories – Columbia and stored frozen upon receipt at EAG Laboratories - Hercules. The reference standard solution was prepared by adding acetonitrile and THF (approximate ratio of 75/25, v/v) to a weighed amount of the standard in an amber glass vial, to obtain a final concentration of approximately 10 mg/mL. The reference standard was stored frozen when not in use.

4. Test Systems
4.1 Source of Test Systems
Sediment and water systems were collected from two sites in the United States. The first test system was collected from Goose River, North Dakota, and the second test system was collected from Golden Lake, North Dakota. Both test systems were collected on 28 September 2017 by Agvise Laboratories (Northwood, North Dakota). The test systems were received at EAG Laboratories - Hercules on 3 October 2017. Sediments were collected from the top 5 cm of the sediment layer with free access to air. Water samples were collected from the same sites at the same time as the sediment collections. On arrival at EAG Laboratories - Hercules, the systems were stored refrigerated in the dark until use in the study. The water layers were sieved through a 0.2 mm sieve and sediment layers were sieved through a 2 mm sieve prior to use in the study.
Locations of the sites are presented in the table below:

Sampling Site GPS Coordinates
Goose River (GR) N47°43.779, W97°37.312
Golden Lake (GL) N47°33.744, W97°38.452

4.2 Characterization of the Test Systems
The moisture content of the sediments was determined by using a Metter Toledo HB43-S moisture analyzer with five determinations for each sediment. Physicochemical characterization of the water and sediment was performed by Agvise Laboratories, Northwood, ND. A summary of the water and sediment characteristics is presented in Table 1. Results of the pH, dissolved oxygen (DO), and oxidation/reduction potential (ORP) measurements (taken during sampling) are presented in Table 2.

4.3 Microbiological Viability of Test System
The sediments used in this study were analyzed for microbial viability (microbial biomass) prior to use (shortly following receipt), at the beginning of the incubation (experimental start) and following the final sampling of the study. Microbial biomass results are presented in Table 1. Sediment viability was evaluated using the substrate induced respiration rate (SIR) method (production rate of CO2 gas after glucose addition).

unique sample designation (e.g. “Sample #1”).

5 Preliminary Testing
A number of preliminary experiments were conducted to determine the optimal extraction procedure of CDBC, as well as to investigate the stability properties of CDBC in various organic solvents or in the presence of various metal ions.

5.1 Preliminary Sample Extractions
A number of samples were setup in the same manner as for the definitive study (using different batches of sediment/water from the same sites) for sampling immediately following dosing. Samples were dosed with CDBC and water layers decanted. Sediment layers were extracted with a number of extraction solvents, including acetonitrile, acetonitrile/water (4/1, v/v), ethyl acetate, tetrahydrofuran (THF), acetone, dichloromethane (DCM), hexane, and methanol. Water layers and extracts were radioassayed by LSC to determine recoveries. Selected extracts were analyzed by HPLC to determine the extent of degradation of CDBC. Selected post-extracted sediments were combusted on a biological oxidizer to determine recoveries.

5.2 Stability of CDBC in Organic Solvents
To examine the stability of CDBC in organic solvents, dilutions of CDBC were prepared in 4-mL amber vials. These dilutions were prepared using acetonitrile, THF, acetone, methanol, DCM, ethyl acetate, and hexane. The dilutions were allowed to stand at room temperature for 1 hour (acetonitrile and THF) or for more than 2 hours (all other solvents), then analyzed by HPLC. The dilutions in DCM, hexane, and ethyl acetate were evaporated to dryness and reconstituted with acetonitrile prior to HPLC analysis.

5.3 Stability of CDBC in the Presence of Metal Ions
To determine the effect (if any) of the presence of metal ions on the degradation of CDBC, three samples were prepared in FLPE centrifuge bottles. Two of these bottles contained CuCl, Pb(CH3COO)•3H2O, and CrCl3•6H2O in water such that each contained 1 mg of the relevant metal (copper, lead, and chromium); the third bottle contained only water. All three bottles were dosed with CDBC, a stir bar added to each, and the samples placed on stir plates for constant stirring. An aliquot of acetone (100 mL) was added to one of the samples containing the metal ions. Samples were stirred for 2 hours and analyzed by HPLC to determine the extent of degradation of CDBC in the presence or absence of metal ions, and whether acetone affects the rate of degradation of CDBC in the presence of metal ions. Samples were also analyzed after 7 days of refrigerated storage to evaluate the potential effects of storage on degradation of CDBC.

5.4 Preparation of Test Systems
Approximately 50 g (dry weight equivalent) of the sediments were weighed into amber bottles fitted with caps containing Teflon-lined septa. The corresponding water was added to each sample to form a water layer. For the GL test system, the ratio of water to sediment was approximately 3:1 by height. For the GR test system, an amount of water was added to achieve a final ratio (water to sediment) greater than 3:1 by weight, since this sediment type was less dense (higher wet/dry ratio) and therefore achieving the 3:1 ratio of water to sediment could not be done by height. Instead, 50 g dry weight of sediment (approx. 104.6 g sediment, of which approx. 54.6 g was entrained water) and 120 mL water were used, for a total of 174.6 mL water (approx. water:sediment ratio of 3.5:1).
The resulting sediment layers had heights of 3.5 cm (GR) and 2 cm (GL). The resulting water layers had heights of 4.5 cm (GR) and 7 cm (GL).
The samples were connected to one EG trap and two 10% aqueous NaOH traps via Teflon tubing. The 10% aqueous NaOH and ethylene glycol traps were filled with 20 mL of solution and fitted with caps containing Teflon-lined septa. The samples were connected to humidifiers and peristaltic pumps that constantly pulled air through the sample water layers. Empty vials were placed between the sample and EG trap, between the EG and NaOH #1 trap, and after the NaOH #2 trap to prevent potential contamination. Samples were kept in a controlled temperature chamber and pre incubated at 20 ± 2°C in the dark for at least 8 days before application.

The study samples were setup as follows:

Test System Number of Treated Samples Number of Untreated Samples
Goose River (GR) 18 2 surrogates + 4 biomass
Golden Lake (GR) 18 2 surrogates + 4 biomass

Surrogate samples were prepared for representative aerobicity measurements throughout the study.

5.5 Dose Rate and Preparation of Dose Solutions
The target dose rate was 10 µCi per vessel (approximately 22 million dpm per sample). The dosing solution was prepared by diluting an aliquot (702 µL) of the stock [14C]CDBC test substance (in acetonitrile) in a total of 5800 µL acetonitrile.

5.6 Study Dosing
Aliquots of the dose solution (100 µL) were applied via glass syringe directly to the sample water layer surface. The dosed samples were returned to the controlled temperature chamber and maintained at 20 ± 2 °C in the dark. The time 0 samples were processed immediately after dosing. Aliquots (100 µL x 2) of the application solution were taken before, during, and after the dosing process; each aliquot was divided among 10 LSC vials and radioassayed by LSC to determine the application rate.

5.7 Sampling Procedure
The schedule of events is shown in Table 3. At each sampling time, duplicate samples for each test system were removed from the constant temperature chamber. Samples were taken at time 0 and after 1, 7, 14, 34, 62, and 103 days of incubation. After removing the samples from the controlled temperature chamber, the pH and oxidation/reduction potential (ORP) were taken in the water and sediment layers. The dissolved oxygen (DO) of the water layers was measured in the middle of the layer. Aerobicity measurements were taken from the duplicate surrogate samples which were prepared for each test system. Water layers were decanted into graduated cylinders containing 150 mL of methanol. The volumes were measured and aliquots (3 x 1 mL) were taken for LSC radioassay.
For the time 0 samples, sediment layers were transferred to pre-weighed fluorinated centrifuge bottles (250 mL capacity). Aliquots of methanol (15 mL) and hexane (100 mL) were used to aid in the transfer of sediment to the centrifuge bottle. Samples were shaken on a Wrist-Action shaker for 30 minutes, followed by centrifugation at 4,000 rpm for 5 minutes. The supernatant was decanted into a graduated cylinder and the sediment extracted twice more with 100 mL hexane as above. The supernatants were combined in the graduated cylinder, the volumes of the organic and aqueous layers were measured and recorded, and aliquots were taken of the organic (1 mL x 3) and aqueous (20 µL x 3) layers for radioassay by LSC. The sediment was then further extracted three times with 100 mL ethyl acetate with shaking and centrifugation as above. The supernatants were decanted into separate graduated cylinders, volumes measured and aliquots (1 mL x 3) taken for radioassay by LSC.
For the 1 day samples, sediment layers were transferred to pre-weighed fluorinated centrifuge bottles (250 mL capacity). Aliquots of methanol (15 mL) and hexane (100 mL) were used to aid in the transfer of sediment to the centrifuge bottle. Samples were shaken on a Wrist-Action shaker for 30 minutes, followed by centrifugation at 4,000 rpm for 5 minutes. The supernatant was decanted into a graduated cylinder, the volumes of the organic and aqueous layers were measured and recorded, and aliquots were taken of the organic (1 mL x 3) and aqueous (0.2 mL x 3) layers for radioassay by LSC. The sediment was then further extracted twice with 75 mL ethyl ether with shaking and centrifugation as above. The supernatants were combined in a graduated cylinder, the volumes of the organic and aqueous layers were measured and recorded, and aliquots were taken of the organic (1 mL x 3) and aqueous (0.2 mL x 3) layers for radioassay by LSC. The sediment was finally extracted twice with 100 mL ethyl acetate with shaking and centrifugation as above. The supernatants were decanted into separate graduated cylinders, volumes measured and aliquots (1 mL x 3) taken for radioassay by LSC.
For all remaining samplings (7-103 day), sediment layers were transferred to pre-weighed fluorinated centrifuge bottles (250 mL capacity). Aliquots of methanol (15 mL) and hexane (100 mL) were used to aid in the transfer of sediment to the centrifuge bottle. Samples were shaken on a Wrist-Action shaker for 30 minutes, followed by centrifugation at 4,000 rpm for 5 minutes. The supernatant was decanted into a graduated cylinder, the volumes of the organic and aqueous layers were measured and recorded, and aliquots were taken of the organic (1 mL x 3) and aqueous (0.2 – 1 mL x 3) layers for radioassay by LSC. The sediment was further extracted three times using 75 mL of ethyl acetate each time, with shaking and centrifugation as above. The supernatants from the three extractions were combined in a graduated cylinder, volumes of the organic and aqueous layers were measured and recorded, and aliquots were taken of the organic (1 mL x 3) and aqueous (0.02 – 0.2 mL x 3) layers for radioassay by LSC.
To further characterize the 103 day post-extracted sediment, one further extraction was undertaken using 100 mL methanol. Bottles were shaken on a Wrist-Action shaker for 30 minutes, followed by centrifugation at 4,000 rpm for 5 minutes. Supernatants were decanted, volumes measured, and aliquots (1 mL x 3) taken for radioassay by LSC. Following decant of final extract, the centrifuge bottles (with caps) were weighed and recorded on the sampling worksheet to determine post-extracted sediment (PES) weight.
The volumes of the EG and NaOH trap solutions were measured and aliquots (3 x 1 mL for EG trap solutions and 3 x 0.5 mL for NaOH trap solutions) were taken for LSC radioassay. The NaOH trap aliquots were quenched with 3 mL methanol prior to LSC radioassay. In preparation for HPLC analysis, water layers and sediment extracts were concentrated under nitrogen or via Speed-Vac prior to injection when necessary.

5.8 Combustion and Radioassay
The extracted sediment residues were weighed and aliquots (0.25 – 0.5 g x 4) were combusted to determine the levels of unextracted radiocarbon. Combustions were carried out using a Harvey Biological Oxidizer (model OX-500 or OX-600) and the 14CO2 generated was trapped with cocktail (R. J. Harvey Instrument Corporation). The 14C content was determined by LSC.
All LSC assays of extracts and solutions utilized 5 mL or 15 mL of Safety Solve scintillation cocktail (Research Products International Corp.) in 7 mL or 20 mL standard polyurethane counting vials, respectively, and Beckman LS 5000 CE or LS 6000 IC liquid scintillation spectrometers. Computer-constructed quenched curves, derived from a series of ten sealed quenched standards, automatically converted cpm to dpm. Typical parameters were as follows: counting efficiency, 96%; background, 35 DPM; counting time, 1 to 5 minutes.

5.9 Sample Storage and Storage Stability
All samples were extracted on the same day of collection. Initial HPLC analysis of the samples was performed within 3 days of collection. All samples and standard solutions were stored frozen (< 0°C) when not in use.

6 DT50 and DT90 Using Non-linear Regression Calculations (KinGUI software):
The percent CDBC at each time point was based on the HPLC analysis of the water layers and combined sediment extracts for each test system. DT50 and DT90 for the parent compound were calculated using CAKE software, in consideration of the FOCUS Work Group on Degradation Kinetics (References 3 and 4). To this end, a modeling program (CAKE Model 3.3) was used to evaluate the study data (parent and three largest metabolites for each set) using single first-order (SFO), first-order multi-compartment (FOMC), double first-order in parallel (DFOP), and hockey stick (HS) kinetic models. The models were used to generate DT50 and DT90 values for CDBC. Acceptability was determined by inspection of residuals and the graphical representation of the degradation curve, in addition to the chi-squared error percent (¿2) and coefficient of determination (r2).

7 Statistical Methods
Means, standard deviations, relative standard deviations and single first-order kinetics were the only statistical analyses performed on the data. Many tables were constructed with Microsoft® Excel software.
Key result
Compartment:
natural water / sediment: freshwater
DT50:
6.4 d
Type:
other: hockey stick
Temp.:
20 °C
Remarks on result:
other: Goose River, total system
Key result
Compartment:
natural water / sediment: freshwater
DT50:
10.4 d
Type:
other: hockey stick
Temp.:
20 °C
Remarks on result:
other: Golden Lake, total system
Key result
Compartment:
natural water / sediment: freshwater
DT50:
13.6 d
Type:
other: Hockey stick
Temp.:
12 °C
Remarks on result:
other: Goose River, total system
Remarks:
Using equation from ECHA's guidance R7.b (v4.0, p. 222)
Key result
Compartment:
natural water / sediment: freshwater
DT50:
22.1 d
Type:
other: Hockey Stick
Temp.:
12 °C
Remarks on result:
other: Golden Lake, total system
Remarks:
Using equation from ECHA's guidance R7.b (v4.0, p. 222)
Transformation products:
yes
No.:
#1
No.:
#2
No.:
#3
No.:
#4
No.:
#5
Details on transformation products:
Goose river
CDBC degraded to a large number of metabolites in the presence of GR water and sediment. These metabolites were separated into 14 regions and/or peaks in order to track frequently observed metabolites. The largest of these components was a peak eluting at 34 minutes (henceforth "RT34", transformation product #4), which represented an average of 3.3% AR at time 0, increased to a maximum average of 8.2% AR at 7 days, and represented 4.3% AR at the end of the study (103 days). The next largest of these components was a peak eluting at 27 minutes (henceforth "RT27"), which represented an average of 1.6% AR at time 0, increased to a maximum average of 7.6% AR at 7 days, and represented 3.6% AR at the end of the study. The 3rd largest component was a peak eluting at 31 minutes (henceforth "RT31"), which represented an average of 0.5% AR at time 0, increased to a maximum average of 6.2% AR at 34 days, and represented 2.5% AR at the end of the study. None of the other 11 peaks/regions exceeded 10% AR in any single sample or 5% AR in consecutive sampling intervals.

Decay Times (DT50) of parent compound and degradation products in the total system:

CDBC: 6.4 days at 20°C, i.e. 13.6 days at 12°C
RT27: 3.12 days, i.e. 6.6 days at 12°C (this is not RT21)
RT31: 31.5 days, i.e. 66.9 days at 12°C
RT34: 39.7 days, i.e. 84.3 days at 12°C


Goose lake
The metabolite profile for the GL test system closely resembled the observed profile for the GR test system. RT34 (transformation product #4) was the largest metabolite observed in the GL test system, increasing from an average of 3.3% AR at time 0 to a maximum average of 10.2% AR at 14 days, then decreasing to 3.5% AR at the end of the study. The next largest component was a peak eluting at 21 minutes (henceforth "RT21") which represented 1.2% AR at time 0, increased to a maximum average of 9.8% AR at 14 days, and represented 1.5% AR at the end of the study. The 3rd largest component was RT31, which represented 4.3% AR at time 0, increased to a maximum average of 8.5% AR at 14 days, and decreased to 1.7% AR at the end of the study. The 4th largest component was a peak eluting at 18 minutes (henceforth "RT18"), which represented 0.5% AR at time 0, increased to a maximum average of 6.9% AR at 7 days, and decreased to 2.7% AR at the end of the study. The 5th largest component was the region of polar components eluting within the first 10 minutes of the run. This region represented 2.7% AR at time 0, increased to a maximum average of 6.7% AR at 14 days, and decreased to 4.1% AR at the end of the study. None of the other 9 peaks/regions exceeded 10% AR in any single sample or 5% AR in consecutive sampling intervals.

Decay Times (DT50) of parent compound and degradation products in the total system:

CDBC: 10.4 days at 20°C, i.e. 22.1 days at 12°C
RT21: 10.7 days, i.e 22.7 days at 12°C. (this is not RT27)
RT31: 31.6 days, i.e. 67.1 days at 12°C
RT34: 26 days, i.e., 55.2 days at 12°C


All DT50 were well below 120 days. No degradation product identified during the study is likely to be persistent or very persistent. The equation for temperature correction was the one recommended in ECHA's guidance R.7b (v4.0) : DT50(X°C) = DT50(T°C)* exp[(Ea/R)x(1/X - 1/T)] where T is the test temperature (20°C or 285K) and X is the target temperature (12°C or 293K).
Evaporation of parent compound:
no
Volatile metabolites:
no
Residues:
yes
Remarks:
Unextracted residues were around 35% of applied radioactivity after 103 days incubation.
Details on results:
1 Radiochemical Purity and Stability of [14C]CDBC
The radiopurity of the purified test substance was determined by HPLC to be 98.0% prior to use in the study. The stability of the test substance under conditions of administration was confirmed by HPLC analysis of the post-dosing water layer, which indicated radiopurity of 100%.

2 Application Rate
The target dose rate was 22 million dpm (10 µCi) per vessel. Aliquots (n = 6) of the application solution were taken to confirm the treatment rate of 22262666 dpm (10.03 µCi) per vessel (0.3% relative standard deviation) for both test systems. The 1 day samples were dosed separately, and duplicate aliquots of the application solution were taken to confirm the treatment rate of 23381875 dpm (10.53 µCi) per vessel for both test systems.

3 Characteristics of the Test Systems
The microbial biomass of the sediments were determined upon arrival, at the experimental start and after the final sampling of the study. The results are shown in Table 3. The total organic content (TOC) of the water and sediment was also measured upon arrival, at the start and after the final sampling of the study. The results indicated that the microbial biomass as percent of organic carbon exceeded 1% throughout the study, indicating that the test systems remained viable throughout the study (Table 1).
Throughout the study, the pH in the water layers averaged 8.21 for the GR test system and 8.29 for the GL test system. The pH in the sediment layers averaged 6.79 for the GR test system and 7.52 for GL test system throughout the study. The dissolved oxygen content in water layers averaged 8.41 ppm for the GR test system (individually ranging from 4.79 to 12.67 ppm) and 7.91 ppm for the GL test system (ranging from 3.48 to 14.12 ppm), indicating aerobic conditions. The water layer ORP values standardized to Eh averaged 311 mV in the GR test system (ranging from 271 to 346 mV) and 312 mV in the GL test system (ranging from 221 to 526 mV). ORP in the sediment layers averaged -92 mV for the GR test system (ranging from -167 to -2.5 mV) and -126 mV for the GL test system (ranging from -189 to -56 mV) (Table 2).

4 Radiocarbon Distribution and Mass Balance
The mass balance was based on the sum of radiocarbon in the water layers, sediment extracts, volatile traps and residual sediment radiocarbon (post-extracted sediment), and is expressed as percent of applied radiocarbon (AR). Mass balance for the study is presented in Table 4 and Table 5 for the two test systems.

4.1 Goose River (GR), North Dakota Test System
The mass balance for [14C]CDBC in the GR test system averaged 93.1 ± 3.7% AR. Individual sample recoveries ranged from 86.5 to 98.1% AR. The time 0 sample exhibiting mass balance less than 90% AR is attributed to a fluctuation in dose or to potential instability in the water layer, as the samples were immediately decanted upon dosing. The 103 day samples exhibiting mass balance less than 90% AR are attributed to loss of entrained 14CO2 from the water and sediment layer upon sampling, as the formation of 14CO2 was significant by the end of the study, as evidenced by the recoveries in NaOH volatile traps (discussed below). At time 0, the average radiocarbon recovered in the water layers and the sediment extracts was 64.3 and 18.8% AR, respectively. Recovery in water layers decreased to an average of 2.5% AR by the end of the incubation period (103 days). Extractable radiocarbon (from sediment) increased to a maximum average of 53.9% AR at 34 days, then decreased to 27.2% AR at the end of the incubation (103 days) (Table 4). Post-extracted sediment residues represented 9.6% AR at time 0, increased to a maximum average of 38.1% AR at 62 days, and represented 35.8% AR at the end of the study (Table 4). A significant amount of mineralization was detected in the NaOH traps (as 14CO2), increasing to a maximum average of 22.0% AR at 103 days. Recoveries of organic volatiles trapped by EG were less than 1% AR throughout the study (Table 4).

4.2 Golden Lake (GL), North Dakota Test System
The mass balance for [14C]CDBC in the GL test system averaged 92.9 ± 3.8% AR. Individual sample recoveries ranged from 87.7 to 98.7% AR. The time 0 sample exhibiting mass balance less than 90% AR is attributed to a fluctuation in dose or to potential instability in the water layer, as the samples were immediately decanted upon dosing. The 103 day samples exhibiting mass balance less than 90% AR are attributed to loss of entrained 14CO2 from the water and sediment layer upon sampling, as the formation of 14CO2 was significant by the end of the study, as evidenced by the recoveries in NaOH volatile traps (discussed below). At time 0, average radiocarbon recovered in the water layers and the sediment extracts was 56.4 and 24.6% AR, respectively. Recovery in water layers decreased to an average of 4.5% AR by the end of the incubation period (103 days). Extractable radiocarbon (from sediment) increased to a maximum average of 64.8% AR at 7 days, then decreased to 27.3% AR at the end of the study (103 days) (Table 5). Post-extracted sediment residues represented an average of 9.2% AR at time 0 and increased to a maximum average of 34.3% AR at the end of the study (103 days). A significant amount of mineralization was detected in the NaOH traps (as 14CO2), increasing to a maximum average of 21.6% AR at the end of the study (103 days). Recoveries of organic volatiles trapped by EG were at most 1.7% AR throughout the study (Table 5).

5 Degradation of [14C]CDBC in Water/Sediment Systems Under Aerobic Conditions
The percent of CDBC in water layers and sediment extracts was determined by HPLC. Results are summarized in Table 6 and Table 7 for the GR and GL test systems, respectively.

5.1 Goose River (GR), North Dakota Test System
[14C]CDBC degraded immediately upon addition to the GR test system, representing an average of 66.1% AR in the time 0 samples. This behavior was observed during preliminary testing, and the final sampling procedure was chosen to minimize the degradation of CDBC during the sample dosing and sampling procedure. Degradation of CDBC continued following application, decreasing from an average of 66.1% AR at time 0 to an average of 3.0% AR at the end of the aerobic incubation (103 days). Amounts of CDBC in the water layers declined rapidly from an average of 64.3% AR at time 0 to an average of 8.1% AR after 7 days of incubation, and represented 0.1% AR at the end of the study (103 days). Recovery of CDBC in the sediment layers increased from an average of 1.8% AR at time 0 to a maximum average of 29.7% AR after 34 days of incubation, then declined to average 2.9% AR at the end of the study.
CDBC degraded to a large number of metabolites in the presence of GR water and sediment. These metabolites were separated into 14 regions and/or peaks in order to track frequently observed metabolites. The largest of these components was a peak eluting at 34 minutes (henceforth "RT34"), which represented an average of 3.3% AR at time 0, increased to a maximum average of 8.2% AR at 7 days, and represented 4.3% AR at the end of the study (103 days). The next largest of these components was a peak eluting at 27 minutes (henceforth "RT27"), which represented an average of 1.6% AR at time 0, increased to a maximum average of 7.6% AR at 7 days, and represented 3.6% AR at the end of the study. The 3rd largest component was a peak eluting at 31 minutes (henceforth "RT31"), which represented an average of 0.5% AR at time 0, increased to a maximum average of 6.2% AR at 34 days, and represented 2.5% AR at the end of the study. None of the other 11 peaks/regions exceeded 10% AR in any single sample or 5% AR in consecutive sampling intervals.

5.2 Golden Lake (GL), North Dakota Test System
[14C]CDBC degraded immediately upon addition to the GL total test system, representing an average of 58.3% AR in the time 0 samples. Degradation of CDBC continued following application, decreasing from an average of 58.3% AR at time 0 to an average of 7.3% AR at the end of the aerobic incubation (103 days). Amounts of CDBC in the water layers declined rapidly from an average of 56.4% AR at time 0 to an average of 13.4% AR after 7 days of incubation, and represented 0.1% AR at the end of the study. Recovery of CDBC in the sediment layers increased from an average of 2.0% AR at time 0 to a maximum average of 28.1% AR after 7 days of incubation, then declined to average 7.2% AR at the end of the study.
The metabolite profile for the GL test system closely resembled the observed profile for the GR test system. RT34 was the largest metabolite observed in the GL test system, increasing from an average of 3.3% AR at time 0 to a maximum average of 10.2% AR at 14 days, then decreasing to 3.5% AR at the end of the study. The next largest component was a peak eluting at 21 minutes (henceforth "RT21") which represented 1.2% AR at time 0, increased to a maximum average of 9.8% AR at 14 days, and represented 1.5% AR at the end of the study. The 3rd largest component was RT31, which represented 4.3% AR at time 0, increased to a maximum average of 8.5% AR at 14 days, and decreased to 1.7% AR at the end of the study. The 4th largest component was a peak eluting at 18 minutes (henceforth "RT18"), which represented 0.5% AR at time 0, increased to a maximum average of 6.9% AR at 7 days, and decreased to 2.7% AR at the end of the study. The 5th largest component was the region of polar components eluting within the first 10 minutes of the run. This region represented 2.7% AR at time 0, increased to a maximum average of 6.7% AR at 14 days, and decreased to 4.1% AR at the end of the study. None of the other 9 peaks/regions exceeded 10% AR in any single sample or 5% AR in consecutive sampling intervals.

6 Confirmation of [14C]CDBC and Identification of Unknown Metabolites by LC/MS
The confirmation of CDBC were performed by high-resolution accurate-mass LC/MS (HR-AM-LCMS) analysis of a representative sample from each system. Additionally, identification of three metabolites (RT18, RT27, and RT34) was performed using definitive samples as well as samples dosed at an exaggerated rate (to provide more mass for analysis).

7 Confirmation of 14CO2 in NaOH Traps
The supernatants of the NaOH trap solutions that were precipitated with saturated BaCl2 (aq.) contained negligible radiocarbon, suggesting that the radiocarbon in the NaOH trap solutions was 14CO2 and had precipitated to Ba14CO3.

8 Characterization of Post-Extracted Residues
The 103 day PES were further characterized by extracting with methanol, such that the sediment was extracted with solvents of differing polarity and dielectric constants. Methanol extracts contained 2.3 – 3.1% AR for the GR test system, and 3.1 – 3.5% AR for the GL test system. The post-methanol extracted sediment was further partitioned into components soluble in acid, fulvic acid (FA) fraction, base, humic acid (HA) fraction, and the insoluble humin fraction. Generally, the major part of the unextracted residues were in the insoluble humin fraction (averages of 20.5% AR for the GR test system and 19.0% AR for the GL test system). Fulvic acid recoveries averaged 8.4% AR for the GR test system, and 8.3% AR for the GL test system. Fulvic acids represent organic acids in sediment that are the product of microbial degradation in sediment and are themselves resistant to further biodegradation. Recovery in the humic acid fraction averaged 4.2% AR for the GR test system, and 3.8% AR for the GL test system. Humic acids are similar to fulvic acids but typically have a larger molecular weight and lower oxygen content.
The data generated during the study demonstrates that the bound material is not generally remobilizable. Only a small fraction (approximately 10%) of the bound material at the end of the study was mobilized upon addition of the methanol extraction, and having performed extractions with several solvents of differing polarity and dielectric constants, any further extractions would be expected to mobilize an even smaller fraction of the bound material. Additionally, the extractability increased to a maximum at 7-34 days, then decreased at the end of the study. This, in combination with increasing formation of 14CO2 during the study, would indicate that the bound material is generated primarily by binding of metabolites rather than by binding of the test substance.

9 Degradation Rate of [14C]CDBC in Aerobic Water/Sediment Systems
The DT50, and DT90 results in the total test system and water layers are summarized in table 8. Kinetic models included CDBC as well as the three largest metabolites observed for each total test system (RT27, RT31, and RT34 for the GR test system, and RT21, RT31, and RT34 for the GL test system). Kinetic models for water layer alone included only CDBC. The best-fit kinetic models were determined to be hockey stick (HS) for the total test system and single first-order (SFO) for the water layer alone based on visual fit of the data, r2 and chi-2 values. The double first-order in parallel (DFOP) and first-order multi-compartment (FOMC) models were also used.


Table 2a: Dissolved Oxygen, pH and ORP Measurements of the Aerobic Test Systems. GR Test System

GR Water Layer Aerobicity Measurements

Time Point

DO

ORP

pH

EhA

pE 7B

pH + pE

(days)

(ppm)

(mV)

 

(mV)

 

 

T0

6.11

52.9

8.53

297

5.0

13.5

7.72

80.9

8.42

325

5.5

13.9

1 day

7.28

56.5

7.64

301

5.1

12.7

7.75

81.1

7.75

325

5.5

13.3

7 day

5.30

46.0

8.49

290

4.9

13.4

4.79

45.0

8.55

289

4.9

13.5

14 day

12.67

45.8

8.55

290

4.9

13.5

11.87

55.4

8.24

300

5.1

13.3

34 day

9.18

79.9

8.16

324

5.5

13.7

8.29

66.3

7.92

311

5.3

13.2

62 day

10.84

77.2

7.90

322

5.4

13.3

9.16

92.5

8.05

337

5.7

13.8

103 day

9.83

101.3

7.88

346

5.8

13.7

9.67

95.6

8.10

340

5.7

13.8

 

GR Sediment Aerobicity Measurements

Time Point

ORP

pH

EhA

pE 7B

pH + pE

(days)

(mV)

 

(mV)

 

 

T0

-129.7

6.67

115

1.9

8.6

-151.3

6.72

93

1.6

8.3

1 day

-70.4

6.40

174

2.9

9.3

-116.7

6.94

128

2.2

9.1

7 day

-79.3

6.56

165

2.8

9.4

-99.1

6.56

145

2.4

9.0

14 day

-134.1

7.24

110

1.9

9.1

-122.1

6.62

122

2.1

8.7

34 day

-103.9

6.43

140

2.4

8.8

-38.8

6.78

206

3.5

10.3

62 day

-33.7

6.82

211

3.6

10.4

-83.6

7.24

161

2.7

9.9

103 day

-2.5

6.96

242

4.1

11.1

-10.8

7.01

234

4.0

11.0

AORP + 244.3 mV

BEh ÷ 59.2 mV

Table 2b: Dissolved Oxygen, pH and ORP Measurements of the Aerobic Test Systems. GL Test System

GL Water Layer Aerobicity Measurements

Time Point

DO

ORP

pH

EhA

pE 7B

pH + pE

(days)

(ppm)

(mV)

 

(mV)

 

 

T0

6.92

50.1

8.37

294

5.0

13.4

4.07

-23.1

8.15

221

3.7

11.9

1 day

7.96

60.4

8.28

305

5.2

13.5

7.30

55.2

8.53

300

5.1

13.6

7 day

6.47

64.8

8.45

309

5.2

13.7

5.40

99.8

8.40

344

5.8

14.2

14 day

14.12

52.8

7.79

297

5.0

12.8

10.10

29.9

8.02

274

4.6

12.6

34 day

9.79

74.3

8.48

319

5.4

13.9

9.32

67.5

8.61

312

5.3

13.9

62 day

9.04

70.4

8.36

315

5.3

13.7

9.33

81.5

8.46

326

5.5

14.0

103 day

10.21

72.8

8.22

317

5.4

13.6

10.05

88.3

8.28

333

5.6

13.9

 

GL Sediment Aerobicity Measurements

Time Point

ORP

pH

EhA

pE 7B

pH + pE

(days)

(mV)

 

(mV)

 

 

T0

-185.2

7.70

59

1.0

8.7

-175.0

7.65

69

1.2

8.9

1 day

-118.5

7.66

126

2.1

9.8

-143.0

7.59

101

1.7

9.3

7 day

-132.6

7.59

112

1.9

9.5

-185.6

7.62

59

1.0

8.6

14 day

-125.3

7.58

119

2.0

9.6

-189.3

7.62

55

0.9

8.5

34 day

-82.8

6.95

162

2.7

9.7

-71.3

6.67

173

2.9

9.6

62 day

-70.3

7.78

174

2.9

10.7

-63.3

7.92

181

3.1

11.0

103 day

-56.2

7.44

188

3.2

10.6

-61.6

7.32

183

3.1

10.4

AORP + 244.3 mV

BEh ÷ 59.2 mV

Table 4: Material Balance for the Aerobic Degradation of [14C]CDBC in the Goose River (GR) North Dakota Test System.

GR Mass Balance as Percent of Applied Radiocarbon

Time Point

Rep

Water Layer

Sediment Extracts

EG Trap

NaOH Traps

Post-Extracted Sediment

Total

T0

A

54.7

28.5

NA

NA

14.9

98.1

B

73.9

9.0

4.2

87.1

Average

64.3

18.8

NA

NA

9.6

92.6

1 day

A

79.0

12.9

0.0

0.0

5.4

97.3

B

77.3

10.1

0.0

0.0

6.9

94.3

Average

78.2

11.5

0.0

0.0

6.2

95.8

7 day

A

9.7

57.7

0.1

0.2

25.2

92.9
B

16.7

49.9

0.1

0.4

28.4

95.5
Average

13.2

53.8

0.1

0.3

26.8

94.2

14 day

A

12.7

48.2

0.5

1.6

29.5

92.5

B

12.4

50.8

0.3

2.1

30.5

96.1

Average

12.6

49.5

0.4

1.9

30.0

94.3

34 day

A

5.0

54.2

0.2

6.3

30.1

95.8

B

6.9

53.5

0.9

6.9

28.0

96.2

Average

6.0

53.9

0.6

6.6

29.1

96.0

62 day

A

2.8

28.3

0.3

19.1

41.1

91.6

B

3.6

30.6

0.7

21.2

35.0

91.1

Average

3.2

29.5

0.5

20.2

38.1

91.4

103 day

A

2.4

33.3

0.5

17.8

35.0

89.0

B

2.5

21.0

0.3

26.1

36.6

86.5

 

Average

2.5

27.2

0.4

22.0

35.8

87.8

NA: Not Applicable

Average

93.1

Std. Dev.

3.7

 

 

Table 5: Material Balance for the Aerobic Degradation of [14C]CDBC in Golden Lake (GL) North Dakota Test System.

GL Mass Balance as Percent of Applied Radiocarbon

Time Point

Rep

Water Layer

Sediment Extract

EG Trap

NaOH Traps

Unextracted Sediment

Total

T0

A

54.4

25.7

NA

NA

11.9

92.0

B

58.3

23.5

6.4

88.2

Average

56.4

24.6

NA

NA

9.2

90.1

1 day

A

88.8

4.6

0.0

0.0

1.7

95.1

B

85.6

5.7

0.0

0.0

2.0

93.3

Average

87.2

5.2

0.0

0.0

1.9

94.2

7 day

A

15.4

70.4

0.1

0.1

12.6

98.6

B

17.7

59.2

0.1

0.1

18.9

96.0

Average

16.6

64.8

0.1

0.1

15.8

97.3

14 day

A

17.8

58.2

0.3

0.4

20.5

97.2

B

20.5

58.2

0.3

1.0

18.7

98.7

Average

19.2

58.2

0.3

0.7

19.6

98.0

34 day

A

6.1

44.1

0.6

13.3

25.9

90.0

B

6.1

41.3

0.5

11.7

30.5

90.1

Average

6.1

42.7

0.6

12.5

42.7

90.1

62 day

A

9.3

34.4

1.7

19.0

30.1

94.5

B

5.3

29.9

0.6

18.5

35.8

90.1

Average

7.3

32.2

1.2

18.8

33.0

92.3

103 day

A

4.8

28.5

1.3

21.2

33.7

89.5

B

4.1

26.1

0.7

21.9

34.9

87.7

 

Average

4.5

27.3

1.0

21.6

34.3

88.6

NA: Not Applicable

Average

92.9

Std. Dev.

3.8

 

Table 6: Product Balance of [14C]CDBCand Metabolites for the Aerobic Degradation in Goose River (GR) North Dakota Test System.

Product Balance in Total System (Water and Sediment Combined)

GR Total System Product Balance Expressed as Percent of Applied Radiocarbon

Incubation Time (days):

0

1

7

14

34

62

103

CDBC

Rep A

57.6

78.7

37.5

27.8

36.8

7.8

4.1

Rep B

74.6

76.7

29.0

26.6

25.3

4.6

1.8

Average

66.1

77.7

33.3

27.2

31.1

6.2

3.0

Others1

Rep A

22.8

12.7

27.4

31.3

17.7

22.2

30.6

Rep B

7.2

10.2

35.6

34.5

27.8

28.6

20.8

Average

15.0

11.5

31.5

32.9

22.8

25.4

25.7

Un-Analyzed Hexane Extract Water Layer

Rep A

2.8

0.3

1.3

1.3

1.0

0.9

0.7

Rep B

1.1

0.4

1.1

1.4

1.6

0.8

0.6

Average

2.0

0.4

1.2

1.4

1.3

0.9

0.7

Un-Analyzed Ethyl Acetate Extract Water

Rep A

NA

NA

1.2

0.5

3.7

0.2

0.3

Rep B

NA

NA

0.9

0.7

5.7

0.2

0.3

Average

NA

NA

1.1

0.6

4.7

0.2

0.3

Unanalyzed Ethyl Ether Extract Water

Rep A

NA

0.2

NA

NA

NA

NA

NA

Rep B

NA

0.1

NA

NA

NA

NA

NA

Average

NA

0.2

NA

NA

NA

NA

NA

Volatile Organics (Ethylene Glycol Traps)

Rep A

NA

0.0

0.1

0.5

0.2

0.3

0.5

Rep B

NA

0.0

0.1

0.3

0.9

0.7

0.3

Average

NA

0.0

0.1

0.4

0.6

0.5

0.4

CO2

Rep A

NA

0.0

0.2

1.6

6.3

19.1

17.8

Rep B

NA

0.0

0.4

2.1

6.9

21.2

26.1

Average

NA

0.0

0.3

1.9

6.6

20.2

22.0

Bound Residues

Rep A

14.9

5.4

25.2

29.5

30.1

41.1

35.0

Rep B

4.2

6.9

28.4

30.5

28.0

35.0

36.6

Average

9.6

6.2

26.8

30.0

29.1

38.1

35.8

1Other peaks represented at most 12.3% AR

Table 6 (cont.): Product Balance of [14C]CDBC and Metabolites for the Aerobic Degradation of Goose River (GR) North Dakota Test System.

Product Balance in Water Layer

GR Water Layer Product Balance Expressed as Percent of Applied Radiocarbon

Incubation Time (days):

0

1

7

14

34

62

103

CDBC

Rep A

54.7

76.1

7.6

0.9

0.6

0.0

0.1

Rep B

73.9

74.5

8.5

2.3

2.2

0.2

0.0

Average

64.3

75.3

8.1

1.6

1.4

0.1

0.1

Others1

Rep A

0.0

2.9

2.1

11.8

4.4

2.8

2.3

Rep B

0.0

2.8

8.2

10.1

4.7

3.4

2.5

Average

0.0

2.9

5.2

11.0

4.6

3.1

2.4

1Other peaks represented at most 5.3% AR


Table 6 (cont.): Product Balance of [14C]CDBC and Metabolites for the Aerobic Degradation of Goose River (GR) North Dakota Test System.

Product Balance in Sediment Layer

GR Sediment Layer Product Balance Expressed as Percent of Applied Radiocarbon

Incubation Time (days):

0

3

10

30

43

62

103

CDBC

Rep A

2.9

2.6

29.9

26.9

36.2

7.8

4.0

Rep B

0.7

2.2

20.5

24.3

23.1

4.4

1.8

Average

1.8

2.4

25.2

25.6

29.7

6.1

2.9

Others1

Rep A

22.8

9.8

25.3

19.5

13.3

19.4

28.3

Rep B

7.2

7.4

27.4

24.4

23.1

25.2

18.3

Average

15.0

8.6

26.4

22.0

18.2

22.3

23.3

1Other peaks represented at most 11.5% AR

 

Table 7: Product Balance of [14C]CDBCand Metabolites for the Aerobic Degradation in Golden Lake (GL) North Dakota Test System.

Product Balance in Total System (Water and Sediment Combined)

GL Total System Product Balance Expressed as Percent of Applied Radiocarbon

Incubation Time (days):

0

3

10

30

43

62

103

CDBC

Rep A

57.3

75.0

48.9

20.5

26.3

10.7

9.1

Rep B

59.3

63.7

34.0

25.1

19.1

3.8

5.5

Average

58.3

69.4

41.5

22.8

22.7

7.3

7.3

Others1

Rep A

20.3

18.2

34.8

54.2

20.1

31.9

23.5

Rep B

20.3

27.2

40.7

52.1

25.4

30.4

23.7

Average

20.3

22.7

37.8

53.2

22.8

31.2

23.6

Un-Analyzed Hexane Extract Water Layer

Rep A

2.5

0.2

1.6

1.0

1.0

1.0

0.6

Rep B

2.2

0.3

1.8

1.3

0.9

0.8

0.6

Average

2.4

0.3

1.7

1.2

1.0

0.9

0.6

Un-Analyzed Ethyl Acetate Extract Water

Rep A

NA

NA

0.5

0.3

2.8

0.1

0.1

Rep B

NA

NA

0.4

0.2

2.0

0.2

0.4

Average

NA

NA

0.5

0.3

2.4

0.2

0.3

Un-Analyzed Ethyl Ether Extract Water

Rep A

NA

0.0

NA

NA

NA

NA

NA

Rep B

NA

0.1

NA

NA

NA

NA

NA

Average

NA

0.1

NA

NA

NA

NA

NA

Volatile Organics (Ethylene Glycol Traps)

Rep A

NA

0.0

0.1

0.3

0.6

1.7

1.3

Rep B

NA

0.0

0.1

0.3

0.5

0.6

0.7

Average

NA

0.0

0.1

0.3

0.6

1.2

1.0

CO2

Rep A

NA

0.0

0.1

0.4

13.3

19.0

21.2

Rep B

NA

0.0

0.1

1.0

11.7

18.5

21.9

Average

NA

0.0

0.1

0.7

12.5

18.8

21.6

Bound Residues

Rep A

11.9

1.7

12.6

20.5

25.9

30.1

33.7

Rep B

6.4

2.0

18.9

18.7

30.5

35.8

34.9

Average

9.2

1.9

15.8

19.6

28.2

33.0

34.3

1Other peaks represented at most 11.8% AR

Table 7 (cont.): Product Balance of [14C]CDBCand Metabolites for the Aerobic Degradation in Golden Lake (GL) North Dakota Test System.

Product Balance in Water Layer

GL Water Layer Product Balance Expressed as Percent of Applied Radiocarbon

Incubation Time (days):

0

1

7

14

34

62

103

CDBC

Rep A

54.4

73.4

15.4

3.6

0.0

0.4

0.2

Rep B

58.3

63.0

11.3

8.3

0.0

0.0

0.0

Average

56.4

68.2

13.4

6.0

0.0

0.2

0.1

Others1

Rep A

0.0

15.4

0.0

14.2

6.1

8.9

4.6

Rep B

0.0

22.6

6.4

12.2

6.1

5.3

4.1

Average

0.0

19.0

3.2

13.2

6.1

7.1

4.4

1Other peaks represented at most 8.3% AR

Table 7 (cont.):Product Balance of [14C]CDBCand Metabolites for the Aerobic Degradation in Golden Lake (GL) North Dakota Test System.

Product Balance in Sediment Layer

GL Sediment Layer Product Balance Expressed as Percent of Applied Radiocarbon

Incubation Time (days):

0

3

10

30

43

62

103

CDBC

Rep A

2.9

1.6

33.5

16.9

26.3

10.3

8.9

Rep B

1.0

0.7

22.7

16.8

19.1

3.8

5.5

Average

2.0

1.2

28.1

16.9

22.7

7.1

7.2

Others1

Rep A

20.3

2.8

34.8

40.0

14.0

23.0

18.9

Rep B

20.3

4.6

34.3

39.9

19.3

25.1

19.6

Average

20.3

3.7

34.6

40.0

16.7

24.1

19.3

1Other peaks represented at most 11.8% AR

Table 8: kinetics of 14CDBC degradation

 

Test System

Compartment

Kinetic Model*

DT50
(days)

DT90
(days)

¿2 Error %

r2

Goose River (GR)
North Dakota

Total System

SFO

9.8

32.6

25.8

0.8364

DFOP

6.8

97.2

21.7

0.8825

HS

6.4

95.8

19.3

0.9022

FOMC

7.5

140.0

21.6

0.8627

Water Layer

SFO

3.1

10.3

28.3

0.9250

DFOP

3.1

10.3

33.7

0.9250

HS

3.1

10.3

33.6

0.9256

FOMC

2.0

6.5

30.6

0.9249

Golden Lake (GL)
North Dakota

Total System

SFO

12.7

42.0

22.0

0.8929

DFOP

10.0

96.9

16.1

0.9084

HS

10.4

95.4

13.9

0.9235

FOMC

10.4

120.0

15.1

0.9042

Water Layer

SFO

3.9

13.0

25.0

0.9348

DFOP

3.9

13.0

29.7

0.9348

HS

3.9

13.4

29.7

0.9349

FOMC

2.6

8.5

27.0

0.9348

Validity criteria fulfilled:
yes
Conclusions:
Mass balance for the study, defined as the sum of radiocarbon in the water layers, sediment extracts, post-extracted sediment, sodium hydroxide traps, and ethylene glycol traps for organic volatiles averaged 93.1 ± 3.7% AR (GR) and 92.9 ± 3.8 (GL) over the course of the study.
CDBC immediately degraded upon addition to the aerobic test systems. CDBC represented averages of 66.1 and 58.3% AR at time 0 in the GR and GL test systems, respectively. CDBC declined to averages of 27.2 and 22.8% AR after 14 days of incubation in the GR and GL test systems, respectively. CDBC represented 3.0 and 7.3% AR at the end of the study (103 days) in the GR and GL test systems, respectively.
Numerous components were detected in HPLC analysis of water layers and sediment extracts. Several of these components were identified by LC/MS during the study.
Disappearance times (DTs) were determined for CDBC in both the total system compartment (water and sediment layers combined) and the water layer compartment using SFO, DFOP, HS, and FOMC kinetics, following the FOCUS approach and using CAKE software. For the total test system, the HS model was used as it provided a better fit to the experimental data (including parent and three largest metabolites). The DT50 of CDBC in the total system was 6.4 days for the GR test system and 10.4 days for the GL test system. For the water layer alone, the SFO model was used as it provided a better fit to the experimental data (parent alone). The DT50 of CDBC in the water layer was 3.1 days for the GR test system and 3.9 days for the GL test system.
Recovery in post-extracted sediment increased during the incubation for both test systems, increasing to maxima of 38.1 and 34.3% AR after 62 and 103 days of incubation in the GR and GL test systems, respectively. Recovery in post-extracted sediment represented 35.8% AR at the end of the study in the GR test system. Unextracted sediment residues were fractionated into fulvic and humic acid fractions and the insoluble humin, and the majority of the available radiocarbon was recovered in the insoluble humin fraction.
Generation of 14CO2(g) was significant in both test systems, reaching maximum averages of 22.0 and 21.6% AR at the end of the study for the GR and GL test systems, respectively. The NaOH traps from the final time point were further characterized by barium carbonate precipitation to confirm the presence of 14CO2. Negligible amounts of organic volatiles were detected in the EG traps, representing at most 1.7% AR for all samples.
[14C]CDBC degraded rapidly in sediment and water under aerobic conditions, with DT50 values of 6.4 and 10.4 days in the GR and GL test systems, respectively. CDBC dissipated primarily by formation of numerous metabolites, 14CO2, and unextracted sediment residues.
Executive summary:

The overall objective of this study was to evaluate the rate and route of degradation of [14C]copper bis(dibutyldithiocarbamate) (henceforth CDBC ; CAS number 13927-71-4) in two water/sediment systems under aerobic conditions in accordance with the following guideline: OECD 308 (Aerobic and Aerobic Transformation in Aquatic Sediment Systems).

Water/sediment systems were freshly collected from two sites in North Dakota, USA (Goose River and Golden Lake). The water/sediment samples were treatedwith [14C]CDBC and incubated in the dark at 20°C for periods of up to 103 days. The samples were prepared in amber bottles and connected to two 10% aqueous NaOH traps to capture CO2and one ethylene glycol (EG) trap to capture other volatile organic compounds. The samples were connected to their respective traps via Teflon tubing and were continuously aerated under a closed system. Radioassay was performed on samples at designated intervals by liquid scintillation counting (LSC).

[14C]CDBC and metabolites were identified and quantified by high performance liquid chromatography (HPLC) of water layers and sediment extracts with co-injection of a CDBC analytical reference standard. The HPLC assignment of [14C]CDBC was confirmed/identified by high resolution accurate mass liquid chromatography/mass spectrometry (HR-AM-LC/MS).

Mass balance for the study was defined as the sum of the radiocarbon in the water layers, sediment extracts, post-extracted sediment combustions, and volatile traps. For the Goose River test system (designated as GR), the average mass balance of radiocarbon was 93.1 ± 3.7% AR (applied radiocarbon), with individual sample recoveries ranging from 86.5 to 98.1% AR. Three samples during the study exhibited a material balance of between 87.1 and 89.0% AR, one of which was a time 0 sample, and the other two of which were the 103 day samples. The 103 day samples likely exhibited mass balance < 90% AR due to loss of dissolved and/or entrained14CO2during sample workup.

For the Golden Lake test system (designated as GL), the study average for mass balance radiocarbon recoveries was 92.9 ± 3.8% AR (sample range 87.7 – 98.7% AR). As with the GR test system, three GL samples were observed with recoveries between 87.7 and 89.5% AR, one of which was a time 0 sample and the other two of which were 103 day samples, for the reasons stated above. Radiocarbon in the water layers decreased in both test systems from averages of 64.3 and 56.4% AR at time 0 for the GR and GL test systems, respectively, to averages of 2.5 and 4.5% AR at the end of the study (103 days) for the GR and GL test systems, respectively. Radiocarbon in the sediment extracts increased from 18.8 and 24.6% AR at time 0 to 53.8 and 62.7% AR at 7 days, and represented 27.2 and 27.3% AR at 103 days in the GR and GL test systems, respectively.

A significant amount of14CO2was detected in the sodium hydroxide traps, increasing to averages of 22.0% AR (GR) and 21.6% AR (GL) at 103 days. Negligible radiocarbon (<0.1% AR) was detected in the EG traps for organic volatiles during the study for both test systems.

Post-extraction residues (PER) in sediments averaged 9.6 and 9.2% AR at time 0 in the GR and GL test systems, respectively. PER formation was significant in both test systems, increasing to averages of 35.8 and 34.3% AR at 103 days in the GR and GL test systems, respectively.

Preliminary testing indicated that degradation of CDBC was rapid and significant immediately upon addition to the aerobic test systems and this behavior was also observed in the definitive study. [14C]CDBC was detected at 66.1 and 58.3% AR (for the total water/sediment system) at time 0 for the GR and GL test systems, respectively. The parent compound rapidly degraded, representing 27.2 and 22.8% AR at 14 days in the GR and GL test systems, respectively, and represented 3.0 and 7.3% AR at the end of the study (103 days) in the GR and GL test systems, respectively.

A table summarizing the balance and distribution of the study is shown below.

 

Goose River Test System

Golden Lake Test System

Overall mean material balance (%AR)

93.1 ± 3.7% AR

92.9 ± 3.8% AR

Material balance, individual sample value range

86.5 – 98.1% AR

87.7 – 98.7% AR

Recovery in water layer at Day 0

64.3% AR

56.4% AR

Recovery in water layer at Day 103

2.5% AR

4.5% AR

Recovery in sediment extract at Day 0

18.8% AR

24.6% AR

Recovery in sediment extract at Day 103

27.2% AR

27.3% AR

Recovery in sodium hydroxide traps (for14CO2) at Day 103

22.0% AR

21.6% AR

Recovery in PER at Day 0

9.6% AR

9.2% AR

Recovery in PER
at Day 103

35.8% AR

34.3% AR

[14C] CDBC in the total test system at Day 0

66.1% AR

58.3% AR

[14C] CDBC in the total test system at 14 Day

27.2% AR

22.8% AR

[14C] CDBC in the total test system at 103 Day

3.0% AR

7.3% AR

Disappearance times (DT50, and DT90) for the degradation of [14C]CDBC in the two test systems were calculated using the CAKE program (version 3.3) following the FOCUS approach. The calculated disappearance times of [14C]CDBC in the total system and water layer, are presented in the following table. The best-fit kinetic model was determined based on visual fit of the data, r2and ¿2 values.


Dissipation of [14C]CDBC

Test System

Compartment

Best-fit Kinetic Model

DT50(days)

DT90(days)

Goose River (GR)

Total System

HS

6.4

95.8

Water Layer

SFO

3.1

10.3

Golden Lake
(GL)

Total System

HS

10.4

95.4

Water Layer

SFO

3.9

13.0

SFO = Single First-Order; HS = Hockey Stick

[14C]CDBC degraded primarily by formation of a number of degradation products, formation of non-extractable sediment residue, and mineralization to14CO2.Several of these unknown metabolites were identified by high-resolution accurate-mass liquid chromatography with mass spectrometry (HR-AM-LC/MS) in the course of the study.

Description of key information

The overall objective of this study was to evaluate the rate and route of degradation of copper bis(dibutyldithiocarbamate) (henceforth CDBC ; CAS number 13927-71-4) in two water/sediment systems under aerobic conditions in accordance with the following guideline: OECD 308 (Aerobic and Anaerobic Transformation in Aquatic Sediment Systems). Tests were performed in two water/sediment systems (Goose River GR and Golden Lake GL). CDBC and degradation products were identified and quantified by high performance liquid chromatography (HPLC).

Preliminary testing indicated that degradation of CDBC was rapid and significant immediately upon addition to the aerobic test systems and this behavior was also observed in the definitive study. CDBC was detected at 66.1 and 58.3% AR (for the total water/sediment system) at time 0 for the GR and GL test systems, respectively. The parent compound rapidly degraded, representing 27.2 and 22.8% AR at 14 days in the GR and GL test systems, respectively, and represented 3.0 and 7.3% AR at the end of the study (103 days) in the GR and GL test systems, respectively.

None of the degradation product exceeded 10% AR in any single sample or 5% AR in consecutive sampling intervals.

Disappearance times (DT50 and DT90) for the degradation of CDBC in the two test systems were calculated using the CAKE program (version 3.3) following the FOCUS approach.. The best-fit kinetic model was determined based on visual fit of the data, r2and chi-2 values.


Dissipation of [14C]CDBC at 20°C

Test System

Compartment

Best-fit Kinetic Model

DT50(days)

DT90(days)

Goose River (GR)

Total System

HS

6.4

95.8

Water Layer

SFO

3.1

10.3

Golden Lake
(GL)

Total System

HS

10.4

95.4

Water Layer

SFO

3.9

13.0

SFO = Single First-Order; HS = Hockey Stick

CDBC degraded primarily by formation of a number of degradation products, formation of non-extractable sediment residue, and mineralization to CO2. Several of these unknown metabolites were identified by high-resolution accurate-mass liquid chromatography with mass spectrometry (HR-AM-LC/MS) in the course of the study.

Disappearance times (DT50 and DT90) for the three largest metabolites in the total system were calculated using the CAKE program (version 3.3) following the FOCUS approach. The calculated disappearance times in the total system are presented in the table below.

Decay Times (DT50 and DT90) of the three largest degradation products in the total system:

20°C

12°C

Test System Metabolite name Kinetic Model Compartment DT50 (days) DT90 (days) DT50 (days) DT90 (days)
GR RT21 HS Total System 3,12 10,4 6,6 22,1
GR RT31 HS Total System 31,5 105 66,9 223,1
GR RT34 HS Total System 39,7 132 84,3 280,5
GL RT21 HS Total System 10,7 35,5 22,7 75,4
GL RT31 HS Total System 31,6 105 67.1 223.1
GL RT34 HS Total System 26 86,2 55.2 183.1

Key value for chemical safety assessment

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

Additional information