Diquat dibromide

Biodegradation in soil

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in soil: simulation testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 Nov 1987 to 5 Dec 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: EPA Guideline N-162-1

Deviations:

not specified

GLP compliance:

yes

Test type:

laboratory

Radiolabelling:

yes

Oxygen conditions:

aerobic

Soil classification:

USDA (US Department of Agriculture)

Year:

1987

Soil no.:

#1

Soil type:

sandy loam

% Clay:

16

% Silt:

22

% Sand:

62

% Org. C:

1.74

pH:

7.5

CEC:

12.4 meq/100 g soil d.w.

Bulk density (g/cm³):

1.41

Details on soil characteristics:

- Soil preparation: Stockton sandy loam soil with a moisture content of 3.01% was sieved through a 2 mm screen before applying to the test tube.

Soil No.:

#1

Duration:

274 d

Soil No.:

#1

Initial conc.:

2.677 mg/kg soil d.w.

Based on:

act. ingr.

Remarks:

cation

Parameter followed for biodegradation estimation:

CO2 evolution

radiochem. meas.

Soil No.:

#1

Temp.:

25.3 ± 0.4 ˚C

Humidity:

3.01%

Microbial biomass:

Day 0 - Baterial: 1.0E+6 (control); 3.2E+6 (treated) - Fungal: 6.0E+3 (control); 5.0E+5 (treated) After 274 days contact - Baterial: 5.8E+6 (control); 5.0E+6 (treated) - Fungal: 5.6E+4 (control); 4.5E+4 (treated)

Details on experimental conditions:

EXPERIMENTAL DESIGN
- Soil weight per test vessel: 10.000 ± 0.0001 g dry weight
- No. of replication controls: 19
- No. of replication treatments: 22
- No. of replicate for microbial activity analysis: 11
- Test vessel: Silanized 50 mL culture tubes
- Test system: All ground glass joints were treated with sealing wax to preserve the integrity of the closed system which was operated under positive pressure. Each sample was thoroughly mixed on a vortex type mixer for approximately one minute and then placed into a metabolism vessel in an environmental chamber.
- Details of traps for CO2 and organic volatile: Effluent air was passed through 250 mL solutions of ethylene glycol and 1N H2S04 to trap volatile organics, and duplicate 250 mL 1N KOH solutions to trap CO2.
- Control: An identical system containing undosed culture tubes was run concurrently as a control.
- Test material application: A 284 µg/L aliquot of 14C-labelled test substance stock solution containing 94.2 µg cation/mL was injected into each test tube.

SAMPLING DETAILS
- Sampling intervals: The study sediment and water were analyzed for microbial activity by conducting bacterial and fungal plate count analyses at the study initiation and at 6-months and 9-months after dosing. Duplicate soil sample tubes were removed from the metabolism vessel on each of the prescribed 11 sampling times; day 0, day 1, day 3, day 7, day 14, 1-month, 2-months, 3-months, 4-months, 6-months, and 9-months.

- Storage of samples: At -22 °C until the completion of the study period. One sample from each sampling period was then packed in dry ice and shipped to analysis facility where the samples were stored frozen at -15°C for a maximum of 2 months pending analysis.
At each sampling interval the ethylene glycol, and H2SO4 trapping solutions were transferred to 250 mL amber glass bottles. The KOH trapping solutions were transferred to 250 mL Nalgene bottles at each sampling interval also. Radioactivity in these trapping solutions was quantified by liquid scintillation counting.

Soil No.:

#1

% Total extractable:

91.4

% Non extractable:

8.6

% Recovery:

100

Parent/product:

parent

Key result

Soil No.:

#1

% Degr.:

< 5

Parameter:

radiochem. meas.

Remarks:

cation

Sampling time:

9 mo

Remarks on result:

other: No degradation of the test substance was observed.

Key result

Soil No.:

#1

DT50:

> 9 mo

Type:

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

Temp.:

25 °C

Remarks on result:

other: No degradation of the test substance was observed and thus no half life could be determined.

Transformation products:

no

Evaporation of parent compound:

not specified

Volatile metabolites:

no

Residues:

yes

Details on results:

An overview of the results is provided in Table 1 – Table 3 in ‘Any other information on results incl. tables’
Recovery and mass balance: The 14C-residues in the test system were characterized as volatile residues and extractable and non -extractable soil residues. The ten hour acid reflux with 18N H3SO4 was an effective means of separating the test substance from the soil as a maximum of 1.1% of the 14C-residues (0.03 µg test substance/g) was associated with the soil after refluxing. The remainder of the residues constituted the extractable soil residues for each sampling period. The mean fraction of soluble 14C-residues present as the test substance in the soil was 99.6% (std. dev. = 0.7%) for all sampling periods as determined by HPLC analysis of the appropriate extracts. The range of percent test substance in these extracts was 99.5 - 100.0%, with 100% of the 14C-residues characterized as the test substance at the termination (9 months) of the study. The range of MC-mass balance for this study is 99.2 - 107.1%.

Degradation: No quantity of 14C greater than background eluted from the HPLC column with retention times similar to those of the metabolites of the test substance. The test substance was the only 14C-labelled compounds detected in this study. As indicated by co-chromatography of the analytical test substance standard and visualization under ultraviolet light, the test substance residues were located on the TLC plates in the regions with Rf values of 0 - 0.38 on the cellulose plates and 0 - 0.23 on the silica plates. The remainder of the TLC plates were divided into 2 (cellulose ) or 3(silica) regions. The quantification of radioactivity in these regions confirms the HPLC analysis and indicate that the test substance is the only metabolite detected in this study.
The slope of the degradation curve for the test substance [time vs ln% (total test substance/initial dose)] is -0.000202. A confidence level of p = 0.3435 was calculated by SAS linear regression analysis program. This indicates that the slope of the degradation curve is not significantly different than zero; therefore, the data indicate that the test substance is not degrading in the test system. No degradation of the test substance was observed and thus no half-life could be determined. In addition, there was no 14C-labelled volatiles detected.

Table 1. HPLC analysis of soil and water samples

HPLC column recoveries of extracts
Sample day	Total DPM injected	Total DPM recovered	% Recovered	% as^athe test substance
0	249,433	250,293	100.4	99.5
1	367,350	346,450	94.3	97.5
3	491,475	526,124	107.1	100.0
7	543,750	545,668	100.3	100.0
14	383,800	380,838	99.2	99.9
1 month	443,100	450,469	101.6	99.8
2 month	720,400	725,028	100.7	99.8
3 month	501,625	528,284	105.3	100.0
4 month	408,450	419,067	102.5	99.7
6 month	632,475	673,099	106.5	99.7
9 month	547,125	575,195	105.1	99.5
14C Standard	512,230	512,230	100.0	100.0

* % as diquat by HPLC = [( DPM in test substance Peak )/(Total DPM Recovered )] x 100.

Table 2. Degradation rate of 14C-labelled test substance during test

	% of Dose as the test substance cation^a
Days after application	Extractable soil	Non-extractable soil	Total test substance	1n% total test substance
0	102.7	1.16	103.9	4.638
1	86.5	1.01	87.5	4.471
3	1053	0.33	105.7	4.661
7	96.8	0.26	97.2	4.529
14	101.9	0.26	102.6	4.627
1 month	98.1	0.28	98.4	4.588
2 month	97.8	0.78	98.6	4.591
3 month	104.3	0.62	104.9	4.653
4 month	101.2	0.17	101.4	4.618
6 month	94.3	0.40	94.7	4.551
9 month	92.1	0.36	92.5	4.523
			y = -0.000202x + 4.6058 r2 == 0.1000 p == 0.3435

As analyzed by HPLC, applied dose = 2.677 µg cation/g of soil.

Table 3. 14C-labelled volatiles in tapping solution in µg 14C-labelled cation

	Days after application
	1	3	7	14	31	62	91	123	183	275
Total µg test substance
Et	<0.024	<0.024	<0.024	<0.024	<0.024	<0.024	<0.024	<0.024	<0.024	<0.024
H	<0.024	<0.024	<0.024	<0.024	<0.024	<0.024	<0.024	<0.024	<0.024	<0.024
K1+K2	<0.024	<0.024	<0.024	<0.024	<0.024	0.025	<0.024	<0.024	0.028	<0.024
Total g soil in system	170	160	150	140	130	120	110	90	80	70
µg test substance/g of soil
Et	<0.024	<0.024	<0.024	<0.024	<0.024	<0.024	<0.024	<0.024	<0.024	<0.024
H	<0.024	<0.024	<0.024	<0.024	<0.024	<0.024	<0.024	<0.024	<0.024	<0.024
K1+K2	<0.024	<0.024	<0.024	<0.024	<0.024	0.025	<0.024	<0.024	0.028	<0.024
Accumulative µg test substance/g of soil
Et	<0.024	<0.024	<0.024	<0.024	<0.024	<0.024	<0.024	<0.024	<0.024	<0.024
H	<0.024	<0.024	<0.024	<0.024	<0.024	<0.024	<0.024	<0.024	<0.024	<0.024
K1+K2	<0.024	<0.024	<0.024	<0.024	<0.024	0.025	<0.024	<0.024	0.028	<0.024

Note: Et — Ethylene Glycol trapping solution

H = H2SO4 trapping solution

K1= First KOH trapping solution

K2 = Second KOH trapping solution

Note: Limit of detection = 2 x background = 0.024 µg cation (0.001 µg/g soil)

Conclusions:

The test substance has been shown to be stable under the conditions of an aerobic metabolism study conducted in sandy loam soil at a dose rate of 2.677 µg cation/g sediment. Both the TLC and HPLC analysis indicate that 14C-labelled residues were present solely as the test substance. As the concentration of the test substance remained constant during the study period, no valid half-life could be determined. Furthermore, no volatile products including 14CO2 were detected.

Executive summary:

The degradation of 14C radiolabelled test substance has been studied in Stockton sandy loam soil at a dose rate of 2.677 µg test substance (as cation)/g of soil. The study followed EPA Pesticide Assessment Guideline Section 162-1 and was incompliance with GLP criteria. Aerobic soils were incubated in darkness at 25.3 ± 0.4˚C for 9 months (274 days). Volatile and gaseous products from degradation were 'trapped' and radioactivity in soils was extracted and analszed. The degradation rate and the formation of degradation products of the test substance in soil was studied.

After 9 months, the range of 14C-mass balance for this study was 99.2 - 107.1%. Both the TLC and HPLC analysis indicated that 14C-residues were present solely as the parent compound (test substance) ie. no degradation occurred in soil over a period of 9 months. Furthermore, no volatile products including 14CO2 were detected. As a consequence, no degradation half-life could be determined. 

Description of key information

No measurable degradation after 9 months, 25 °C, EPA 162 -1 guideline, Johnston 1988

Additional information

The degradation of 14C radiolabelled test substance has been studied in Stockton sandy loam soil at a dose rate of 2.677 µg test substance (as cation)/g of soil. The study followed EPA Pesticide Assessment Guideline Section 162-1 and was incompliance with GLP criteria. Aerobic soils were incubated in darkness at 25.3 ± 0.4˚C for 9 months (274 days). Volatile and gaseous products from degradation were 'trapped' and radioactivity in soils was extracted and analszed. The degradation rate and the formation of degradation products of the test substance in soil was studied.

After 9 months, the range of 14C-mass balance for this study was 99.2 - 107.1%. Both the TLC and HPLC analysis indicated that 14C-residues were present solely as the parent compound (test substance) ie. no degradation occurred in soil over a period of 9 months. Furthermore, no volatile products including 14CO2 were detected. As a consequence, no degradation half-life could be determined.