N-(2-ethylhexyl)-8,9,10-trinorborn-5-ene-2,3-dicarboximide

Biodegradation in soil

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in soil, other

Remarks:

Aerobic

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

04 September 1991 to 24 March 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

Multiple aspects of study deviated from GLP, see complince statement on pg 3 & 4 of attached report for details.

Qualifier:

according to guideline

Guideline:

other: EPA Subdivision N Section 162-1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 307 (Aerobic and Anaerobic Transformation in Soil)

Deviations:

yes

Remarks:

Study completed pre-guideline: Rate of degradation should be done using 4 soils – only one was used here Generally should be conducted at 20°C, these were both done at 25°C Metabolites above 10% not identified due to MS limitations

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 835.4100 (Aerobic Soil Metabolism)

Deviations:

yes

Remarks:

Study completed pre-guideline: Rate of degradation should be done using 4 soils – only one was used here Generally should be conducted at 20°C, these were both done at 25°C Metabolites above 10% not identified due to MS limitations

GLP compliance:

yes

Remarks:

Exceptions made in compliance sttement (pg 3 and 4 of attached report)

Test type:

laboratory

Specific details on test material used for the study:

The 14C-hexyl-MGK 264 (Figure 2) was received from Amersham Corporation, Arlington Heights, Illinois, on October 2, 1990. The stated specific activity was 18.4 mCi/mmol (148,000 dpm/μg). Amersham's documentation stated that the radiochemical purity was 98 % . The batch number was JB3 l 63.

The 14C-norbornene-MGK 264 (Figure 2) was received from Amersham Corporation, Arlington Heights, Illinois, on August 6, 1991. The stated specific activity was 26 mCi/mmol (209,000 dpm/μg). Amersham's documentation stated that the radiochemical purity was 99 % . The batch number was VA3048.

Radiolabelling:

yes

Oxygen conditions:

aerobic

Soil classification:

not specified

Year:

1991

Soil no.:

#1

Soil type:

sandy loam

% Clay:

14

% Silt:

26

% Sand:

60

pH:

7.1

CEC:

9.4 meq/100 g soil d.w.

Bulk density (g/cm³):

1.49

Details on soil characteristics:

Note: %Organic Mattter = 0.6%, organic carbon not quoted in report.

Soil No.:

#1

Duration:

365 d

Soil No.:

#1

Initial conc.:

9.57 ppm

Based on:

test mat.

Remarks:

For Hexyl-14C-MGK264

Soil No.:

#1

Initial conc.:

8.7 ppm

Based on:

test mat.

Remarks:

For Hexyl-14C-MGK264

Parameter followed for biodegradation estimation:

test mat. analysis

Soil No.:

#1

Temp.:

25 dgrees C

Humidity:

The air was humidified by being passed through a gas washing bottle filled with water immediately before entering the test system.

Microbial biomass:

The microbial viability of the soils was confirmed at several of the sampling points and total plate counts were > 10^5 colonies/ g dry wright of soil at any given sample time

Details on experimental conditions:

The test system employed in this study is illustrated in Figure 1. A separate system was used for each of the two radiochemicals. Additionally, two control systems were set up, one for each of the two radiochemicals. The aeration vessel (metabolism vessel) was a standard tall form 3000-mL resin-pot containing a number of sample tubes. The sample tubes were pre-weighed 25 mm x 150 mm (50-mL) culture tubes containing 10.0 g (dry weight basis) of soil. All ground glass joints of the test system were treated with sealing wax to preserve the integrity of the closed system. The system was operated under positive pressure with a carrier gas flow rate between 10 and 100 mL/min. Air, free of C02 was used as a carrier gas. The air was humidified by being passed through a gas washing bottle filled with water immediately before entering the test system. A rubber expansion plug fitted into the large center hole of the resin-pot facilitated withdrawal of sample tubes. All aeration vessels were contained within an environmental control chamber maintained with dark conditions at a temperature of 25 ± 1 °C.

Soil No.:

#1

% Recovery:

107.8

St. dev.:

4.77

Remarks on result:

other: hexyl label

Soil No.:

#1

% Recovery:

101.66

St. dev.:

4.16

Remarks on result:

other: norbornene label

Key result

Soil No.:

#1

DT50:

264 d

Type:

(pseudo-)first order (= half-life)

Temp.:

25 °C

Remarks on result:

other: 14C-hexyl-MGK 264

Key result

Soil No.:

#1

DT50:

388 d

Type:

(pseudo-)first order (= half-life)

Temp.:

25 °C

Remarks on result:

other: 14C-norbornene-MGK 264

Key result

Soil No.:

#1

DT50:

341 d

Type:

(pseudo-)first order (= half-life)

Temp.:

25 °C

Remarks on result:

other: average for both labels

Transformation products:

yes

Remarks:

Transformation products were quantified however the lab was not able to attain identities of these degradates. These were less than 10% except for the 9 minute region which was not able to be identified by MS (GC or HPLC).

Details on transformation products:

Three regions of interest observed by HPLC were common to both the hexyl and norbomene test systems with retention intervals of 3-6 minutes, 7 minutes, and 9-9.5 minutes.

The 3-6 minute region for the 14C-hexyl-MGK 264 increased from 2.63 % of IMD at day 7 to 29. 71 % of IMD at day 365, while the 3-6 minute region for the 14C-norbornene-MGK 264 increased from 1. 68 % of IMD at day 30 to 20 .14 % of IMD at day 365. The 3-6 minute regions for both test systems were shown to contain at least six separate components by using an expanded HPLC gradient method. All components within the 3-6 minute region of interest were below 10% of IMD for both radiolabeled test substances.

The 7 minute region of interest for both test systems was observed on days 90, 120, and 180 in the acidified methanol extract of soil samples. The 7 minute region for the hexyl label occurred at 2.05% IMD on day 180, while the norbornene label occurred at 1.06% of IMD on day 180.

The 9 minute region of interest for the 14C-hexyl-MGK 264 increased from < 1 % of IMD atday 0 to 13.99% of IMD at day 365. The 9 minute region for the 14C-norbornene-MGK 264 increased from 1.21 % of IMD at day 0 to 12.72 % of IMD at day 365.

In summary degradate components observed for both 14C-hexyl and 14C-norbornene-MGK 264 were less than 10% of IMD except for the 9 minute region. Three attempts were made to identify the 9 minute region of interest by using gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry. However, despite best efforts the results obtained were inconclusive for identifying the material because of the small amount of the degradate material available.

Evaporation of parent compound:

not specified

Volatile metabolites:

yes

Remarks:

Ethylene Glycol Trap; 1 N H2S04 Trap and two 1 N KOH Trap

Residues:

yes

Remarks:

The efficiency of the oxidizer was determined to be > 95 % prior to sample combustion by combusting cellulose matrix with known levels of 14C-benzoic acid standard and determining the level of 14C-activity recovered versus the level applied.

Details on results:

The level of 14C-volatile degradation products was 0.00% of IMD in both the hexyl and norbornene systems at day 1 (the first measured interval). The cumulative 14C-volatile degradates increased to 12.85 % of the IMD (hexyl) and 7.24% ofIMD (norbornene) by the 365- day sample interval.
The majority of the 14C-volatile activity was found in the ethylene glycol traps for both the hexyl and norbornene systems.

In the hexyl system, cumulative 14C-volatiles trapped in the ethylene glycol accounted for 12.12% of IMD. The remainder of the 14C-volatile activity (0.73% of IMD) was in the first KOH trap.
In the norbornene system, 6. 78 % of IMD was trapped in ethylene glycol. The remainder of the 14C-volatile activity was in the H2S04 trap and in the first KOH trap at levels of 0.11 % and 0.34% of IMD, respectively.

Results with reference substance:

The nonextractable 14C-residues in the soil increased from 1.04% of IMD (hexyl) at day 1 and 1.84% of IMD (norbornene) at day 0 to 16.41 % of IMD (hexyl) and 13.45% of IMD (norbornene) at the day 120 sample interval. The 14C-nonextractable residues at the terminal sample interval for the hexyl and norbornene test systems accounted for 10.24% and 7.47% of IMD, respectively.

Conclusions:

MGK 264 would be expected to persist moderately under aerobic soil conditions in the environment. The calculated half-life for 14C-hexyl-MGK 264 was 294 days and for 14Cnorbornene-MGK 264 it was 388 days with an average half-life of 341 days.

MGK 264 metabolized in soil to yield at least 7 separate degradates of greater polarity than parent. Six of the components were less than 10 % of the IMD. Attempts were made to characterize the seventh component (retention time 9 minutes) as reported in the results and discussion section. The chromatographic profile of the degradation products of the molecule irrespective of label position were similar, suggesting that the hexyl and norbornene moieties of the molecule remain linked throughout the degradation process and that degradation products observed arose by transformations on the periphery of the molecule. It can be also inferred that the amide link probably remained intact in the major degradate.

Executive summary:

A 365-day aerobic soil metabolism study with14C-MGK 264 was conducted to address the general data requirements of the U.S. Environmental Protection Agency, Pesticide Assessment Guidelines, Subdivision N, Section 162-1 as required by 40 CFR 158.130.

Two separate lots of14C-MGK 264 that differed in the position of the radiolabel (hexyl and

norbornene) were tested separately with separate controls. The hexyl radiolabel was incorporated into the alkyl moiety of the test chemical, while the norbornene radiolabel was incorporated into the norbornene ring moiety of the test chemical. These two radiolabel positions were used in order to monitor independently the degradation of the individual moieties (the alkyl chain and the norbomene ring) of the test chemical and to evaluate if the amide bond remained intact. The aerobic soil metabolism study with14C-MGK 264 was conducted under dark conditions on sandy loam soil in an environmental chamber regulated at 25±1 °C. The study samples were treated with a sufficient amount of14C-MGK 264 stock solution to achieve a nominal concentration of 10 ppm. The actual concentration of MGK 264 in the test systems for14Chexyl-MGK 264 and14C-norbornene-MGK 264 after dosing was determined to be 9.57±0.18 ppm and 8.70±0.34 ppm, respectively. After dosing, the soils were incubated for 365 days under aerobic conditions by passing humidified, C02-free air through the test system.

 

MGK 264 would be expected to persist moderately under aerobic soil conditions in the environment. The calculated half-life for₁₄C-hexyl-MGK 264 was 294 days and for₁₄Cnorbornene-MGK 264 it was 388 days with an average half-life of 341 days.

MGK 264 metabolized in soil to yield at least 7 separate degradates of greater polarity than parent. Six of the components were less than 10 % of the IMD. The chromatographic profile of the degradation products of the molecule irrespective of label position were similar, suggesting that the hexyl and norbornene moieties of the molecule remain linked throughout the degradation process and that degradation products observed arose by transformations on the periphery of the molecule. It can be also inferred that the amide link probably remained intact in the major degradate.

Description of key information

Aerobic soil metabolism

A 365-day aerobic soil metabolism study with 14C-MGK 264 was conducted to assess the biotic degradation and mineralisation of the test item under at 25 ± 1 °C under dark conditions with a constant flow of humidified, C02-free air. Two separate lots of 14C-MGK 264 that differed in the position of the radiolabel (hexyl and norbornene) were used to conduct the study. Soil samples collected throughout the study were extracted with methanol and the extracts analyzed for 14C-MGK 264 and its degradates by HPLC, fraction collection, and LSC of fractions. Samples were collected at 0, 1, 3, 7, 14, 30, 60, 90, 120, 180, 270, 287 (norbornene only), 337 (hexyl only), and 365 days. As the study progressed and non-extractable residues increased, the extraction scheme was modified to include methanol/water, followed by methanol/0.1 N HCI.

 

The maximum concentration of extractable residues observed was 108.83 % (hexyl) of initial measured dose (IMD) at day 3 and 98.16% of IMD (norbornene) at day 0, and declined to a minimum of 84.12% of IMD (hexyl) and 77.01 % of IMD (norbornene) both at the day 120 sample interval.

 

Both nonextractable 14C-residues and 14C-volatile degradates increased throughout the 365-day sample interval, with the majority of the volatile activity being found in the ethylene glycol traps for both the hexyl and norbornene systems. Mass balance was maintained throughout the study with 14C-mass balance was 107.80 ± 4.77% and 101.66 ± 4.16% for the hexyl and norbornene test systems, respectively.

 

The parent compound for the hexyl test system decreased from 92.89% of IMD at day 0 to 39.92 % of IMD at day 365, and for the norbornene test system decreased from 92.41 % of IMD at day 0 to 47.82 % of IMD at day 365. One degradate was present at 12.72% IMD at day 365 in the hexy labelled material and could not be identified by MS, all other degradates observed were <10% IMD.

 

The half-life of MGK 264 was calculated using first-order kinetics. The half-lives for the 14Chexyl- and norbornene-MGK 264 was 294 days (r = 0.9607) and 388 days (r = 0.9556), respectively. The average half-life for both the labels is 341 days.

Anaerobic soil metabolism

A 90-day anaerobic soil metabolism study with 14C-MGK 264was conducted to assess the biotic degradation and mineralisation of the test item under at 25 ± 1 °C under dark conditions.The samples were incubated aerobically for 30 days by passing CO2 -free, humidified air through the test system until day 30 when the samples were flooded with water, and anaerobic incubation initiated by passing humidified nitrogen gas through the test system. Two separate lots of 14C-MGK 264 (hexyl and norbornene) were used to conduct the study. Soil samples collected throughout the study were extracted and analyzed for residues of 14C-MGK 264 and degradates by HPLC, fraction collection, and LSC of fractions. Samples were collected at 0, 1, 3, 7, 14, 30, 45, 60, 75, and 90 days. The aqueous phase (45-day sample onward) associated with samples after day 30 was separated from the soil residues by centrifugation and by decanting aqueous supernatant. The soil pellets were extracted with methanol.

 

The methanol-extractable soil residues were 98.35% (hexyl) and 98.17% (norbornene) of initial measured dose (IMD) at time 0 and declined to 91.34% (hexyl) and 94.04% (norbornene) at the day 30 sample interval. Between the day 45 sample interval (the first anaerobic sampling) and day 90, the methanol-extractable soil residues decreased and the aqueous phase recoveries increased slightly.Both nonextractable 14C-residues and 14C-volatile degradates increased throughout the 365-day sample interval, with the majority of the volatile activity being found in the ethylene glycol traps for both the hexyl and norbornene systems.

 

Neither 14C-hexyl-MGK 264 nor 14C-norbornene-MGK 264 degraded significantly in the test system and none of the degradate components exceeded 10 % of applied radioactivity in either the soil extracts or the aqueous phase.

 

The half-life determinations were calculated using first-order kinetics. The concentration of MGK 264 at day 90 was 67.39% (hexyl) and 79.15% (norbornene) of IMD. The half-life for the 14C-hexyl-MGK 264 was 174 days (r = 0.9423). The half-life for the 14C-norbornene-MGK 264 was 365 days (r = 0.8247). The average half-life for both the labels is 270 days.

Key value for chemical safety assessment

Half-life in soil:

341 d

at the temperature of:

25 °C