1-[[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl]methyl]-1H-1,2,4-triazole

Biodegradation in water and sediment: simulation tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: simulation testing on ultimate degradation in surface water

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 Mar 2013 to 02 Aug 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 309 (Aerobic Mineralisation in Surface Water - Simulation Biodegradation Test)

Version / remarks:

April 2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

yes

Remarks:

chlorophenyl ring labelling

Oxygen conditions:

aerobic

Inoculum or test system:

natural water: freshwater

Details on source and properties of surface water:

The study was conducted using Tuckahoe Lake natural water. The natural water was sampled on May 08, 2013 from Tuckahoe Lake in Caroline County, Maryland. The water was scooped from the lake and placed into a 20L container. Full details of the sampling dates and the locations are given in Table 1 in "Any other information on materials and metods incl tables". After receipt, the water was stored at 4 ± 2°C except for one storage day where the recorded high temperature was 7.5°C. Prior to characterization and dispensing, the water was passed through a 100 μm sieve. Prior to dosing, the sterilized test vessels including water were autoclaved twice (60 minute cycles at approximately 128ºC), allowing for a light/dark cycle in between. The key characteristics of the water-sediment systems are summarized in Table 2 in "Any other information on materials and metods incl tables". Test system parameters, pH, dissolved oxygen and turbidity were measured throughout the study. The data is shown in Table 3 in "Any other information on materials and metods incl tables".

Duration of test (contact time):

62 d

Initial conc.:

10 µg/L

Based on:

act. ingr.

Remarks:

low test concentration

Initial conc.:

95 µg/L

Based on:

act. ingr.

Remarks:

high test concentration

Parameter followed for biodegradation estimation:

CO2 evolution

radiochem. meas.

other: high performance liquid chromatography (HPLC)

Details on study design:

The experimental design is summarized in Table 3 in 'Any other information on materials and methods incl tables'.

Reference substance:

benzoic acid, sodium salt

Compartment:

natural water: freshwater

% Recovery:

116

St. dev.:

6.9

Remarks on result:

other: 10 µg/L - Tuckahoe Lake Water Subjected to Diffuse Light (12 hr light/dark cycle) for the substance

Compartment:

natural water: freshwater

% Recovery:

107.2

St. dev.:

3

Remarks on result:

other: 95 µg/L - Tuckahoe Lake Water Subjected to Diffuse Light (12 hr light/dark cycle) for the substance

Compartment:

natural water: freshwater

% Recovery:

122.2

Remarks on result:

other: 95 µg/L sterilized sample- Tuckahoe Lake Water Subjected to Diffuse Light (12 hr light/dark cycle) for the substance

Parent/product:

parent

Compartment:

total system

% Degr.:

40.4

Parameter:

radiochem. meas.

Sampling time:

62 d

Remarks on result:

other: test substance, low concentration (10 μg/L)

Parent/product:

parent

Compartment:

total system

% Degr.:

2.4

Parameter:

radiochem. meas.

Sampling time:

62 d

Remarks on result:

other: test substance, high concentration (95 μg/L)

Key result

Compartment:

natural water: freshwater

DT50:

78.29 d

Type:

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

Temp.:

20 °C

Remarks on result:

other: 10 µg/L [14C]-test substance

Remarks:

Natural water/ 12 hour light/dark cycle

Transformation products:

yes

Details on transformation products:

- Transformation Product 1 (TP1): TP1 was the target of this analysis, together with confirmation of parent, based on the percentage of the dose that TP1 represented. TP1 yielded a protonated molecular ion at m/z 258/60. This showed the characteristic isotopic pattern for a molecule containing two chlorine atoms. The accurate mass confirmed the elemental formula to within 0.7 ppm. The base peak ion at m/z 370 together with other non-chlorine containing ions originated from co-eluting endogenous material that it was not possible to remove completely by background subtraction. The protonated molecular ion lost the elements of imidazole to yield the ion at m/z 189/91. It is clear that the product ion mass spectra are almost identical for TP1 and M1. Thus, TP1 is confirmed as M1.
- Transformation Product 2 (TP2): TP2 was detected and structurally characterized but was not present above the trigger level requiring structural identification. The data is presented here for completeness. TP2 yielded a protonated molecular ion at m/z 256/58 and showed the characteristic isotopic pattern for a dichlorinated molecule. The accurate mass measurement confirmed the elemental formula to within 0.2 ppm. TP2 yielded a protonated molecular ion at m/z 256/58. The spectrum showed low ion abundance that reflected its low level in the sample. The product ion spectrum is much clearer and the structural assignments for the major ions are shown on the figure. The molecule represents the ketone (M2) related to TP1 (M1). It is not possible from the spectra to determine if TP1 is the precursor of TP2 or vice versa.
- Proposed degradation pathway: The major degradate observed in all incubation groups was M1.

Evaporation of parent compound:

no

Volatile metabolites:

yes

Remarks:

See in 'Any other information on results incl tables'

Residues:

yes

Remarks:

See in 'Any other information on results incl tables'.

Details on results:

- DegT50 of substance in natural water systems: Estimation of the degradation rate (DT50) was only possible for the low dose samples subjected to diffuse light. The DegT50 value for the low dose (10 μg/L) samples subjected to diffuse light was 78.3 days. The results of the kinetic analysis are shown in Table 1 in 'Any other information on results incl tables'.
- DegT50 of substance in sterilized test samples: The degradation of the substance in sterilized test samples indicated that degradation was not entirely biotic.

MASS BALANCE
- The mass balances are presented in Table 2 and Table 3 in 'Any other information on results incl tables'. A summary of the mean mass balance of applied radioactivity (AR) values is presented in Table 4 in 'Any other information on results incl tables'. The exaggerated 14CO2 levels are believed to have artificially contributed to the total % AR recovered causing mass balances to be higher than normal in some cases.

Results with reference substance:

- Degradation of sodium 14C benzoate: The degradation of 14C-Benzoic acid to CO2 (74.5% AR at 14 DAT for both groups) indicated a viable microbial population.

RADIOCHEMICAL PURITY

- The radiochemical purity of the primary stock solution of ¹⁴C-substance was determined by TLC (and found to be approximately 97%). The radiochemical purity of the treatment solution was determined by HPLC before and after treatment. The RCP of the stock solution both pre- and post-dosing was 100%.

 

TREATMENT RATE

- The definitive application rates for water were 1 and 9.5 µg per unit for the low and high dose rates, respectively. With water volumes of 100 mL, this is equivalent to 10 and 95 µg/L respectively.

 

TEST SYSTEM PARAMETERS

- Test system parameters, dissolved oxygen, turbidity and pH, were measured throughout the study. The data is shown in Table 5.

 

VOLATILE DEGRADATION PRODUCTS

- The amounts of carbon dioxide produced are presented in Table 2 and Table 3. A summary of the mean amounts of carbon dioxide produced is presented in Table 6. The elevated amounts of ¹⁴CO₂ reported for the low dose samples treated at 10 µg/L are believed to have been derived from natural background (chemiluminescence) originating from the KOH trapping solution. Therefore, contrary to the values reported, mineralization likely occurred at much lower levels or not at all.

 

RADIOACTIVE RESIDUES

- The water was subjected to liquid-liquid extraction and the organic eluent was concentrated under nitrogen and analyzed by HPLC. Over the duration of the study, the parent compound decreased to a mean level of 60% AR by 62 DAT for samples treated at 10 µg/L and subjected to diffuse light. M1 was the only metabolite found at >5% AR, reaching maximum levels of 35.5 and 41.8% AR at 55 and 62 DAT, respectively in the 10 µg/L samples. One minor unknown characterized as M2 was also detected at <5%. Little degradation occurred over the course of the study in sterile or non-sterile samples treated at 95 µg/L and exposed to diffuse light. Over the duration of the study, the parent compound decreased to a mean level of about 89% AR by 62 DAT for samples treated at 10 µg/L and subjected to continuous darkness. M1 was the only metabolite found at > 5% AR, reaching a mean maximum level of 8.8% AR by 62 DAT in the 10 µg/L samples. Little degradation occurred over the course of the study in sterile or non-sterile samples treated at 95 µg/L when subjected to continuous darkness. For sterilized samples, in the presence and absence of light, parent was present at a mean level of 98.3% AR at 62 DAT. Microbes and algae present in the non-sterile samples treated at the high dose may have been stunned by the elevated concentration of the test material thus rendered unable to fully participate in the degradation process. Full results are presented in Table 7 and Table 8.

 

STRUCTURAL ASSIGNMENTS

- Confirmation of Sample 528E-106-G1/A/R13 EX-CONC: The ESI(+) LC/MS analysis of this sample showed the presence of one major and one minor transformation product together with confirmation of parent.

- Parent: The substance yielded a base peak protonated molecular ion at m/z 342/44. The characteristic chlorine isotopic pattern for a dichlorinated molecule was observed. The accurate mass confirmed the elemental formula to within 1.3 ppm. It is clear that the product ion mass spectra and retention times of parent in the two samples are almost identical which confirms parent (substance) in G1-A-R13.

 

Table 1. Summary of DegT50 Values for Low Dose Samples Subjected to Diffuse Light

System	Test concentration (µg/L)	SFO
		DegT50(days)	k	Chi^2	r^2	Prob > t
^14C-chlorophenyl substance
Natural water/ 12 hour light/dark cycle	10	78.29	0.008854	3.345	0.7694	2.002E-05

 

Table 2. Distribution and Recovery of Radioactivity: Tackahoe Lake Water Subjected to Diffuse Light (12 hr light/dark cycle) for ¹⁴C-chlorophenyl- substance

a) Incubation Group 1/Subgroup A – test samples, low concentration (10µg/L)

Fraction	Rep.	Incubation time (DAT)
		0	14	28	37	44	55	62
Water	A	106.3	108.5	112.0	111.2	109.0	109.2	111.7
	B	100.4	107.1	110.7	107.4	113.9	110.4	111.0
	Mean	103.4	107.8	111.3	109.3	111.4	109.8	111.3
^14CO2	A	NA	3.0	5.7	8.7	9.0	11.3	12.4
	B	NA	1.8	5.9	7.3	10.1	9.9	10.1
	Mean	NA	2.4	5.8	8.0	9.5	10.6	11.2
TOTAL	A	106.3	111.5	117.7	119.8	118.0	120.5	124.1
	B	100.4	108.9	116.3	114.7	123.9	120.3	121.1
	Mean	103.4	110.2	117.1	117.3	121.0	120.4	122.6
Mean ± SD		116.0± 6.9% AR

NA – Not Applicable

 

b) Incubation Group 1/Subgroup B – test samples, high concentration (95 µg/L)

Fraction	Rep.	Incubation time (DAT)
		0	14	28	37	44	55	62
Water	A	103.3	99.0	108.4	107.1	110.7	106.1	108.1
	B	105.7	105.0	104.9	105.9	109.8	109.3	107.5
	Mean	104.5	102.0	106.6	106.5	110.3	107.7	107.8
^14CO2	A	NA	0.3	0.8	0.8	1.0	1.0	1.3
	B	NA	0.3	0.5	0.8	1.2	1.1	1.3
	Mean	NA	0.3	0.6	0.8	1.1	1.1	1.3
TOTAL	A	103.3	99.3	109.2	107.9	111.7	107.1	109.4
	B	105.7	105.3	105.4	106.7	111.0	110.4	108.7
	Mean	104.5	102.3	107.3	107.3	111.4	108.8	109.0
Mean ± SD		107.2 ± 3.0% AR

NA – Not Applicable

 

c) Incubation Group 1/Subgroup C – sterilized samples, high concentration (95µg/L)

Fraction	Rep.	Incubation time (DAT)
		62
Water	A	119.5
	B	122.8
	Mean	121.2
^14CO2	A	1.0
	B	1.1
	Mean	1.1
TOTAL	A	120.6
	B	123.9
	Mean	122.2

 

d) Incubation Group 1/Subgroup D – reference samples, ¹⁴C-Benzoic acid (10 µg/L)

Fraction	Rep.	Incubation time (DAT)
		3	6	8	10	14
^14CO2 (Cumulative)	A	30.5	31.0	67.8	70.6	76.5
	B	44.1	60.7	65.5	68.8	72.5
	Mean	37.3	45.8	66.7	69.7	74.5

*Cumulative 14CO2 evolved confirms degradation of the reference substance.

 

Table 3. Distribution and Recovery of Radioactivity: Tuckahoe Lake Water Subjected to Continuous Darkness for ¹⁴C-chlorophenyl-substance.

a) Incubation Group 2/Subgroup A – test samples, low concentration (10 µg/L)

Fraction	Rep.	Incubation time (DAT)
		0	62
Water	A	106.0	105.0
	B	103.2	117.9
	Mean	104.6	111.4
^14CO2	A	NA	11.9
	B	NA	10.6
	Mean	NA	11.2
TOTAL	A	106.0	116.9
	B	103.2	128.5
	Mean	104.6	122.7
Mean ± SD		113.7    ± 12.8% AR

 

b) Incubation Group 2/Subgroup B– test samples, high concentration (95 µg/L)

Fraction	Rep.	Incubation time (DAT)
		0	62
Water	A	103.7	107.6
	B	104.3	112.5
	Mean	104.0	110.0
^14CO2	A	NA	1.2
	B	NA	1.6
	Mean	NA	1.4
TOTAL	A	103.7	108.8
	B	104.3	114.1
	Mean	104.0	111.5
Mean ± SD		107.7    ± 5.3% AR

 

c) Incubation Group 2/Subgroup C – sterilized samples, high concentration (95 µg/L)

Fraction	Rep.	Incubation time (DAT)
		62
Water	A	115.6
	B	116.6
	Mean	116.1
^14CO2	A	1.0
	B	1.2
	Mean	1.1
TOTAL	A	116.6
	B	117.8
	Mean	117.2

 

d) Incubation Group 2/Subgroup D – reference samples,¹⁴C-Benzoic acid (10 µg/L)

Fraction	Rep.	Incubation time (DAT)
		3	6	8	10	14
^14CO2 (Cumulative)	A	43.8	57.7	58.3	60.2	70.5
	B	54.5	65.7	71.7	75.4	78.3
	Mean	49.1	61.7	65.0	67.8	74.5

*Cumulative ¹⁴CO₂ evolved confirms degradation of the reference substance.

Table 4.Summary of the mean mass balance of applied radioactivity (AR) values.

Mean Total radioactivity (for duplicate samples per time point)	Group 1: Diffuse light (12h light/dark cycle)Tuckahoe Lake Water (10 µg/L): 103.4 to 122.6% AR Tuckahoe Lake Water (95 µg/L): 102.3 to 111.4% AR Sterilized Tuckahoe Lake Water (95 µg/L): 122.2% AR   Group 2: Continuous darkness Tuckahoe Lake Water (10 µg/L): 104.6 to 122.7% AR Tuckahoe Lake Water (95 µg/L): 104.0 to 111.5% AR Sterilized Tuckahoe Lake Water (95 µg/L): 117.2% AR
Mean Recovery at 0 DAT	Group 1: Diffuse light (12h light/dark cycle)Tuckahoe Lake Water (10 µg/L): 103.4% AR Tuckahoe Lake Water (95 µg/L): 104.5% AR   Group 2: Continuous darkness Tuckahoe Lake Water (10 µg/L): 104.6 % AR Tuckahoe Lake Water (95 µg/L): 104.0% AR
Overall Mean Recovery by group (all samples for all time points)	Group 1: Diffuse light (12h light/dark cycle)Tuckahoe Lake Water (10 µg/L): 116.0% AR Tuckahoe Lake Water (95 µg/L): 107.2% AR Sterilized Tuckahoe Lake Water (95 µg/L): 122.2% AR   Group 2: Continuous darkness Tuckahoe Lake Water (10 µg/L): 113.7% AR Tuckahoe Lake Water (95 µg/L): 107.7% AR Sterilized Tuckahoe Lake Water (95 µg/L): 117.2% AR

 

Table 5. Physico-chemicalParameters

Group 1: Diffuse Light (12 hr light/dark cycle)

Time point	Replicate	Parameters
		Tuckahoe Lake Water
		Dissolved Oxygen (mg/L)	pH	Turbidity (NTU)
Day 0	1	10.4	6.8	14.7
	2	10.4	6.7	14.6
Day 14	1	9.0	8.0	17.3
	2	8.9	7.9	17.4
Day 28	1	9.1	7.7	15.6
	2	9.3	8.0	15.6
Day 37	1	9.0	7.1	15.6
	2	8.9	7.3	13.5
Day 44	1	9.0	7.9	10.3
	2	9.0	7.9	7.5
Day 55	1	9.1	8.1	10.6
	2	9.0	7.9	8.2
Day 62	1	8.7	8.0	7.2
	2	8.7	7.9	5.3

 

Group 2: Continuous Darkness

Time point	Replicate	Parameters
		Tuckahoe Lake Water
		Dissolved Oxygen (mg/L)	pH	Turbidity (NTU)
Day 0	1	10.3	6.8	14.7
	2	10.5	6.8	14.5
Day 14	1	8.9	7.7	18.1
	2	9.0	7.6	17.2
Day 28	1	8.8	7.8	13.6
	2	8.7	7.6	13.4
Day 37	1	8.8	7.4	18.9
	2	8.7	7.4	20.0
Day 44	1	8.8	7.8	14.8
	2	8.7	7.8	15.2
Day 55	1	8.7	7.7	14.1
	2	8.8	7.7	14.0
Day 62	1	8.6	7.8	13.4
	2	8.6	7.8	11.2

 

 

Table 6.Summary of the mean mass balance of applied radioactivity (AR) values.

Small amounts of radioactivity were evolved as volatile products throughout the course of the study.
^14CO2 evolved at end of study	Group 1: Diffuse Light (12h light/dark cycle)Tuckahoe Lake Water (10 µg/L): 11.2% AR Tuckahoe Lake Water (95 µg/L): 1.3% AR Sterilized Tuckahoe Lake Water (95 µg/L): 1.1% AR   Group 2: Continuous darkness Tuckahoe Lake Water (10 µg/L): 11.2% AR Tuckahoe Lake Water (95 µg/L): 1.4% AR Sterilized Tuckahoe Lake Water (95 µg/L): 1.1% AR

 

Table 7. Summary of Characterization / Identification of Radioactive Residues in Tuckahoe Lake Water Subjected to Diffuse Light (12 hr light/dark cycle) for ¹⁴C-chlorophenyl substance

a) Incubation Group 1/Subgroup A – test samples, low concentration (10 µg/L)

	Rep	Incubation time (DAT)
		0	14	28	37	44	55	62
Substance	A	113.0	97.7	90.5	76.9	85.5	52.6	45.0
	B	106.9	95.4	87.3	87.3	78.3	78.1	74.2
	Mean	109.9	96.5	88.9	82.1	81.9	65.3	59.6
M2	A	ND	ND	2.3	2.5	0.8	2.6	4.0
	B	ND	ND	1.9	2.0	0.8	1.7	ND
	Mean	ND	ND	2.1	2.3	0.8	2.1	4.0
M1	A	ND	2.0	8.1	14.6	11.2	35.5	41.8
	B	ND	2.5	9.8	10.4	17.9	16.6	25.9
	Mean	ND	2.2	8.9	12.5	14.5	26.1	33.8
Total	A	113.0	99.7	100.8	94.1	97.4	90.7	90.8
	B	106.9	97.9	99.0	99.7	96.9	96.4	100.1
	Mean	109.9	98.8	99.9	96.9	97.2	93.5	95.4

ND – Not Detected, *Expressed as % AR

 

b) Incubation Group 1/Subgroup B – test samples, high concentration (95 µg/L)

	Rep	Incubation time (DAT)
		0	14	28	37	44	55	62
Substance	A	108.3	98.9	93.1	99.5	100.3	95.1	97.4
	B	109.1	98.4	98.3	100.1	99.5	94.6	97.8
	Mean	108.7	98.6	95.7	99.8	99.9	94.9	97.6
@ 20.1 min	A	ND	ND	ND	0.6	ND	ND	ND
	B	ND	ND	ND	ND	ND	ND	ND
	Mean	ND	ND	ND	0.6	ND	ND	ND
M1	A	ND	ND	0.4	0.9	2.1	1.6	1.6
	B	ND	ND	1.2	0.6	0.4	1.2	1.7
	Mean	ND	ND	0.8	0.7	1.3	1.4	1.6
Total	A	108.3	98.9	93.5	100.9	102.4	96.7	99.0
	B	109.1	98.4	99.4	100.7	100.0	95.8	99.4
	Mean	108.7	98.6	96.5	100.8	101.2	96.3	99.2

ND – Not Detected, *Expressed as % AR, **In cases of one replicate resulting in an ND value, the single value was carried as mean value

 

c) Incubation Group 1/Subgroup C – sterilized samples, high concentration (95 µg/L)

	Rep	Incubation time (DAT)
		62
Substance	A	97.2
	B	102.6
	Mean	99.9
M2	A	1.2
	B	ND
	Mean	1.2
M1	A	1.4
	B	ND
	Mean	1.4
Total	A	99.9
	B	102.6
	Mean	101.2

ND – Not Detected, *Expressed as % AR, **In cases of one replicate resulting in an ND value, the single value was carried as mean value.

 

Table 8. Summary of Characterization / Identification of Radioactive Residues in Tuckahoe Lake Water Subjected to Continuous Darkness for ¹⁴C-chlorophenyl substance

 

a) Incubation Group 2/Subgroup A – test samples, low concentration (10 µg/L)

	Rep	Incubation time (DAT)
		0	62
Substance	A	112.5	88.2
	B	112.0	88.8
	Mean	112.2	88.5
M1	A	ND	8.9
	B	ND	8.7
	Mean	ND	8.8
Total	A	112.5	97.2
	B	112.0	97.5
	Mean	112.2	97.3

ND – Not Detected, *Expressed as % AR

 

b) Incubation Group 2/Subgroup B – test samples, high concentration (95 µg/L)

	Rep	Incubation time (DAT)
		0	62
Substance	A	111.1	100.9
	B	108.2	98.1
	Mean	109.7	99.5
M1	A	ND	1.0
	B	ND	2.5
	Mean	ND	1.7
Total	A	111.1	101.8
	B	108.2	100.6
	Mean	109.7	101.2

ND – Not Detected, *Expressed as % AR

 

c) Incubation Group 2/Subgroup C – sterilized samples, high concentration (95 µg/L)

	Rep	Incubation time (DAT)
		62
Substance	A	94.3
	B	99.1
	Mean	96.7
M2	A	2.3
	B	ND
	Mean	2.3
M1	A	4.3
	B	2.4
	Mean	3.3
Total	A	100.9
	B	101.5
	Mean	101.2

ND – Not Detected, *Expressed as % AR, **In cases of one replicate resulting in an ND value, the single value was carried as mean value.

Validity criteria fulfilled:

yes

Conclusions:

The DegT50 values of the substance at a test concentration of 10 µg/L in natural freshwater water system was determined to be 78 days. Degradation of the substance was seen only at low levels or not at all in high dose test vessels (95 μg/L) under both incubation scenarios.

Executive summary:

The mineralization rate and the route and rate of degradation of [¹⁴C-chlorophenyl]- substance were investigated in Tuckahoe Lake natural water in an aerobic laboratory test according to the OECD TG 309 and in compliance with GLP. Significantly more degradation of the substance was observed in natural water dosed at a low concentration (10 μg/L) and subjected to diffuse light than degradation in low dose test vessels (10 μg/L) subjected to continuous darkness. Degradation of the substance was seen only at low levels or not at all in high dose test vessels (95 μg/L) under both incubation scenarios. M1 was the only major metabolite found, reaching a maximum level of 41.8% of applied radioactivity in low dose test vessels (10 μg/L) subjected diffuse light. Mineralization was a very minor route of degradation with CO₂ accounting for < 2% AR in vessels dosed at 95 μg/L. Contrary to the values reported, mineralization likely occurred at much lower levels or not at all in the vessels dosed at 10 μg/L. The DegT50 values of the substance at a test concentration of 10 µg/L in natural freshwater water system was determined to be 78 days. Degradation of the substance was seen only at low levels or not at all in high dose test vessels (95 μg/L) under both incubation scenarios.

Reference 2

Endpoint:

biodegradation in water: sediment simulation testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 Dec 1998 to 29 Jul 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Richtlinie fur die Prufung von Pflanzenschutzmitteln im Zulassungsverfahren, Teil IV, 5-1, Abbaubarkeit und Verbleib von Pflanzenschutzmitteln im Wasser / Sediment-System.

Version / remarks:

Dezember 1990, Biologische Bundesanstalt fur Land- und Forstwirtschaft, Bundesrepublik Deutschland,

GLP compliance:

yes

Radiolabelling:

yes

Remarks:

14C-triazole labelled

Oxygen conditions:

aerobic

Inoculum or test system:

natural water / sediment: freshwater

Details on source and properties of surface water:

Pond (Froschweiher; Rheinfelden, AG / Switzerland) Rhine river (Mohlin, AG / Switzerland). Water was sampled on September 11, 1998. Water was sampled down to a depth of 10 - 30 cm with a plastic container. The sampling sites were located about 1 - 3 m from shore. The system parameters measured at the sampling sites are given in Table 1 in “Any other information on materials ad methods incl tables”. After transportation the Rhine and pond water were aerated and the sediments were covered with the corresponding water. Water and sediment were kept at room temperature until filling into the flasks. The water was specified for Ntotal, Ptotal, pH, redox potential, oxygen content, hardness and TOC. The corresponding data are given in Table 1 in “Any other information on materials ad methods incl tables”.

Details on source and properties of sediment:

Pond (Froschweiher; Rheinfelden, AG / Switzerland) Rhine river (Mohlin, AG / Switzerland). Sediments were sampled on September 11, 1998. The sediment was sampled with a shovel from the top 5 - 10 cm. The sampling sites were located about 1 - 3 m from shore. The sediments were directly sieved into plastic containers. The system parameters measured at the sampling sites are given in Table 1 in “Any other information on materials ad methods incl tables”. After transportation the Rhine and pond water were aerated and the sediments were covered with the corresponding water. Water and sediment were kept at room temperature until filling into the flasks. The sediment was specified for its water content, organic carbon, Ntotal, Ptotal, pH, redox potential, cation exchange capacity, particle size and microbial biomass. The corresponding data are given in Table 1 in “Any other information on materials ad methods incl tables”.

Duration of test (contact time):

175 d

Initial conc.:

0.127 other: kg/ha

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

radiochem. meas.

Details on study design:

EXPERIMENTAL DESIGN
- Apparatus and Preparation: An aquatic system consists of sediment and the corresponding water as a static system. Preparation of the aquatic test system: Sediment was filled into a 1-litre glass metabolism flask to a height of about 2 - 2.5 cm (102.7 ± 0.3 g and 153.7 ± 0.3 g based on sediment dry weight for pond and Rhine, respectively). Afterwards 502.9 ± 3.6 mL pond and 503.5±4.1 mL Rhine water were layered upon the corresponding sediment, reaching a height of about 6 cm. After preparation, the aquatic systems were equilibrated in climatic chambers under aeration with air. During equilibration and throughout the whole study pH and oxygen content of the water and redox potential of water and sediment were followed. The effluent air was passed through a trapping system consisting of one absorption bottle containing 50 mL ethylene glycol and one absorption bottle containing 50 mL 2 N NaOH.
- Number of Samples: Treatment rate 0.127 kg / ha (Rhine aquatic system: 24 (reserve samples included), Pond aquatic system: 18 (reserve samples included)), Treatment rate 0.625 kg / ha (3 per aquatic system), Untreated: Biomass: 3 per aquatic system and Reference flasks: 3 per aquatic system).
- Water volume: The water volume was checked in about weekly intervals during the first month and thereafter in about two-week intervals. Pure water (bidistilled or adequate quality) was
added when necessary.
- Incubation Temperature: The aquatic systems were incubated in the dark at 20 ± 2 °C. The temperature was continuously monitored during incubation.
- Aeration: The systems were continuously aerated with moistened air at a flow rate of about 30 mL/min.

TREATMENT AND INCUBATION
- Prior to the preparation of the treatment solutions, an amount of about 10.0 mg 14C-substance was dissolved in 5 mL of acetone (stock solution). Thereafter, three dilutions in acetone were prepared and analysed for amount of radioactivity, radiochemical purity and amount of substance. The accurate amount of the test substance determined by liquid scintillation counting (LSC) was calculated to be 10.26 mg. For application, a stock solution volume of 2.5 mL was made up to 5 mL with acetone (treatment solution). Individual aliquots of the treatment solution (about 95 µL for each
sample) were placed into vials and kept deep-frozen until application. For application of high dose samples, a volume of 239.2 µL of the stock solution was used and also kept deep-frozen until application. The individual aliquots were evenly applied onto the water surface of the aquatic systems by means of a Hamilton syringe. Finally, all application devices were rinsed, the radioactivity determined by LSC, and the actual amount applied calculated. The total amount of the substance applied to each flask was 0.0994 ± 0.0006 mg for Pond aquatic system and 0.0996 ± 0.0006 mg for Rhine aquatic system. These amounts corresponded to a field rate of about 0.127 kg / ha (EEC), taking into account a surface area of 78.5 cm2 in the incubation flasks. The concentration of acetone was < 0.1 % of the amount of water. After completion of the treatment, the incubation flasks were connected to the air-flow systems and incubated in climatic chambers. During incubation, the systems were aerated and gently stirred from the top without disturbing the sediment.

Compartment:

natural water / sediment: freshwater

% Recovery:

98.1

Remarks on result:

other: Values are given in % of the applied radioactivity at the end of the experiment

Remarks:

For all samples of pond aquatic system the total recovery ranged from 98.1 % to 107.4 % of the applied radioactivity

Compartment:

natural water / sediment: freshwater

% Recovery:

100

Remarks on result:

other: Values are given in % of the applied radioactivity at the end of the experiment

Remarks:

For all samples of Rhine aquatic system the total recovery ranged from 98.9 % to 105.9 % of the applied radioactivity

Parent/product:

parent

Compartment:

total system

% Degr.:

22.4

Parameter:

radiochem. meas.

Sampling time:

175 d

Remarks on result:

other: Pond Aquatic System

Parent/product:

parent

Compartment:

total system

% Degr.:

16.3

Parameter:

radiochem. meas.

Sampling time:

175 d

Remarks on result:

other: Rhine Aquatic System

Key result

Compartment:

natural water / sediment: freshwater

DT50:

6.4 d

Type:

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

Temp.:

20 °C

Remarks on result:

other: Pond water phase under aerobic conditions

Key result

Compartment:

natural water / sediment: freshwater

DT50:

484.7 d

Type:

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

Temp.:

20 °C

Remarks on result:

other: Pond total system under aerobic conditions

Key result

Compartment:

natural water / sediment: freshwater

DT50:

5.5 d

Type:

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

Temp.:

20 °C

Remarks on result:

other: Rhine water phase under aerobic conditions

Key result

Compartment:

natural water / sediment: freshwater

DT50:

635.9 d

Type:

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

Temp.:

20 °C

Remarks on result:

other: Rhine total system under aerobic conditions

Transformation products:

yes

Details on transformation products:

- Characterisation of Parent Compound and Metabolites:
- Parent Compound: The nature of the parent compound was confirmed by HPLC/MS and HPLC co-chromatography.
- Metabolite M3: M3 was tentatively characterised by HPLC and 2D-TLC co-chromatography as M3.
- Metabolite M1: M1 was tentatively characterised by HPLC and 2D-TLC co-chromatography as M1.
- Metabolite M4: M4 was tentatively characterised by HPLC and 2D-TLC co-chromatography as M4.
- Pattern of Metabolites in Pond and Rhine River Aquatic Systems: In both aquatic systems, besides the parent compound, up to eight minor metabolites were found. On average, metabolites M3, M1, Unknown 1, and M4 reached their highest concentrations with 2.8 - 2.9 %, 3.1 - 5.0 %, 3.1 - 4.4 %, and 2.1 - 2.3 %, respectively, after 90 to 175 days. All other metabolites (Unknown 2 - Unknown 5) ranged between < LD and 1.3 %.
- Concentration of the substance and Metabolites in Water: For Pond Water The amount of the substance decreased from 98.1 % (day 0) to 0.9 % (day 175) of the applied radioactivity. The detected metabolites reached at highest 3.3 % (M3, day 119) of the applied radioactivity. For Rhine River Water: The amount of the substance decreased from 96.5 % (day 0) to 2.0 % (day 175) of the applied radioactivity. The detected metabolites reached at highest 2.3 % (M3, day 175) of the applied radioactivity.
- Concentration of the substance and Metabolites in the Sediment: For Pond Sediment: In the pond sediment the substance increased from 2.0 % of the applied radioactivity (day 0) to 91.0 % (day 33) and dropped to 76.8 % at the end of the study. The detected metabolites reached at highest 3.0 % (M1, day 175) of the applied radioactivity. For Rhine River Sediment: in the river sediment the substance increased from 2.0 % of the applied radioactivity (day 0) to 85.5 % (day 33) and was 81.7 % at the end of the study. The detected metabolites reached at highest 4.0 % (M1, day 119) of the applied radioactivity.
- Concentration of the substance and Metabolites in the Total Aquatic Systems: For Pond Aquatic System: In the pond aquatic system the substance decreased from 100.2 % of the applied radioactivity (day 0) to 77.6 % at the end of the study. The detected metabolites reached at highest 4.4 % (Unknown 1, day 90) of the applied radioactivity. For Rhine River Aquatic System: In the river aquatic system the substance decreased from 98.5 % of the applied radioactivity (day 0) to 83.7 % at the end of the study. The detected metabolites reached at highest 5.0 % (M1, day 119) of the applied radioactivity.

Evaporation of parent compound:

no

Volatile metabolites:

yes

Remarks:

See 'Any other information on results incl tables'.

Residues:

not specified

Details on results:

DEGRADATION / DISSIPATION HALF-LIVES OF THE SUBSTANCE IN POND AND RHINE RIVER AQUATIC SYSTEMS
- The dissipation half-life of the substance from Rhine river and pond water was in the range of 5.5 - 6.4 days (Table 1 in 'Any other information on results incl tables'). The degradation half-life in the total systems (which practically represented the degradation in the sediment) was 485 days for pond and 636 days for Rhine river. A reliable half-life estimation of the metabolites was not possible due to their low concentrations.

PURITY AND STABILITY OF THE TEST SUBSTANCE
- Purity: The purity of the radio-labelled test compound was found to be 98.5 % (HPLC).
- Stability: HPLC-analysis of radio-labelled test substance in the application solution about six months after storage in the deep freezer (-20 °C) showed a purity of 100 %. Thus, the test substance proved to be stable in the vehicle.

MICROBIAL BIOMASS
At study start the microbial biomass of Rhine and Pond sediment was 46.01 and 56.89 mg microbial carbon / 100 g sediment, respectively, demonstrating viability of the test systems.

REDOX POTENTIAL, OXYGEN CONTENT, PH AND TEMPERATURE OF THE AQUATIC SYSTEM
During the equilibration and incubation period sediment and water of the aquatic systems were monitored for redox potential and temperature . Additionally, the water was monitored for oxygen content and pH. The values obtained for redox potential, oxygen content, and pH in the reference samples during equilibration showed stability before application (day -5) for both aquatic systems. The mean values obtained for redox potential (142 - 189 mV) and oxygen content
(6.6 - 6.7 mg/L) of the water of both aquatic systems during incubation indicated aerobic conditions in the water, whereas the redox potential in sediment samples confirmed anaerobic conditions (-330 - -295 mV). For both aquatic systems the pH ranged from 7.75 - 7.80 at treatment to 8.30 - 8.48 at the end of the study. The recorded water temperatures were in agreement with the target value of 20 °C.

RECOVERY AND DISTRIBUTION PATTERN OF RADIOACTIVITY
The data discussed hereafter refer to mean values of Series A and B. In addition, the individual data are listed in the corresponding tables.
- Recovery: For all samples of pond and Rhine aquatic system the total recovery ranged from
98.1% to 107.4% of the applied radioactivity (Tables 2 - 3 in 'Any other information on results incl tables').
- Radioactivity in the Water: For Pond Water: After treatment, the amount of radioactivity in the pond water decreased from 98.1% at day 0 to 10.7% at day 33 (Table 2 in 'Any other information on results incl tables'). From day 63 on the radioactivity slowly decreased from 8.4 % to 7.1 % at day 175. For Rhine River Water: After treatment, the amount of radioactivity in the Rhine water decreased from 96.5 % at day 0 to 10.0% at day 33 (Table 3 in 'Any other information on results incl tables'). From day 63 on the radioactivity remained at a level between 6.7 % and 8.6 % and amounted to 8.4% at day 175.
- Radioactivity in the Sediment: For Pond Sediment: The results are shown in Table 2 in 'Any other information on results incl tables'. Immediately after application of the test substance, the extractable amount of radioactivity was 2.0% of the applied dose. It reached a maximum of 91 % (day 33), and slowly decreased to 83.0% the end of the study. The amount of non-extractable radioactivity increased to 7.9% of the applied dose at day 90 and was 70% at the end of the study. For Rhine River Sediment: The results are shown in Table 3 in 'Any other information on results incl tables'. Immediately after application of the test substance, the extractable amount of radioactivity was 2.0% of the applied dose and increased to a plateau in the range of 86.2% to 88.7% from day 33 onwards until the end of the study. The amount of non-extractable radioactivity increased to 10.1% of the applied dose at day 90 and was 9.1% at the end of the study.

VOLATILES
In pond and river aquatic systems volatile radioactivity was formed in very low amounts. The volatiles increased to a maximum of 0.4% of the applied radioactivity at day 175. Due to this low concentration, they were not further analysed.

Table 1. Disappearance / Degradation Rates of the substance in Pond and Rhine River Aquatic Systems Incubated under Aerobic Conditions.

Parameter*	Pond		Rhine
	Water Phase	Total System	Water Phase	Total System
A (% applied)	97.9	100.7	96.4	96.4
k (1/days)	0.1084	0.0014	0.1270	0.0011
DT50 (days)	6.4	484.7	5.5	635.9
DT90 (days)	21.2	1610.2	18.1	2112.5
Chi2	8.2487	8.1665	11.9717	8.4615

MicroCal Origin Vers. 5.0 was used for this evaluation. The dissipation of the substance in the sediments corresponded to that in the total systems, due to the low concentrations of the substance in the water from day 63 onwards. Therefore, the dissipation half-life of the substance in the sediment was not calculated.

Table 2. Balance of Radioactivity Applied to Pond Aquatic System Treated with ¹⁴C-substance.

Series A

Incubation Time (days)	Volatiles*	Water	Extractables	Non-extractables	Recovery
0	n.p.	97.2	2.9	0.6	100.7
14	<0.1	21.7	80.1	3.2	105.1
33	0.2	10.7	91.0	3.2	105.1
63	0.2	7.3	88.5	4.8	100.8
90	0.3	9.9	89.3	7.9	107.4
119	0.3	6.3	90.0	7.8	104.3
175	0.4	6.9	83.3	7.5	98.1

 

Series B

Incubation Time (days)	Volatiles*	Water	Extractables	Non-extractables	Recovery
0	n.p.	99.0	1.1	0.2	100.4
14	<0.1	23.2	80.3	2.9	106.5
63	0.2	9.5	89.0	4.0	102.7
119	0.4	10.3	90.5	6.9	108.1
175	0.5	7.4	82.7	7.6	98.2

 

Mean of Series A and B

Incubation Time (days)	Volatiles*	Water	Extractables	Non-extractables	Recovery
0	n.p.	98.1	2.0	0.4	100.6
14	<0.1	22.5	80.2	3.1	105.8
33	0.2	10.7	91.0	3.2	105.1
63	0.2	8.4	88.7	4.4	101.7
90	0.3	9.9	89.3	7.9	107.4
119	0.3	8.3	90.3	7.3	106.2
175	0.4	7.1	83.0	7.6	98.1

(Values are given in % of the applied radioactivity; * Volatiles trapped in NaOH and ethylene glycol; n.p.: not performed)

 

 

Table 2. Balance of Radioactivity Applied to Rhine Aquatic System Treated with ¹⁴C-substance.

Series A

Incubation Time (days)	Volatiles*	Water	Extractables	Non-extractables	Recovery
0	n.p.	96.5	2.2	0.4	99.0
14	0.4	21.5	76.7	2.0	100.6
33	0.4	11.5	88.8	4.1	104.7
63	0.3	5.5	89.9	4.6	99.6
90	0.5	8.7	84.3	10.3	103.8
119	0.3	7.0	87.3	9.7	104.3
175	0.4	7.4	92.1	8.3	108.1

 

Series B

Incubation Time (days)	Volatiles*	Water	Extractables	Non-extractables	Recovery
0	n.p.	96.6	1.8	0.4	98.8
14	0.1	11.1	86.6	3.1	100.9
33	0.2	8.5	88.6	4.7	101.9
63	0.3	7.8	86.5	5.1	99.7
90	0.3	6.4	88.1	9.8	104.6
119	0.3	10.2	88.1	8.9	107.5
175	0.4	9.3	82.9	9.9	102.6

 

Mean of Series A and B

Incubation Time (days)	Volatiles*	Water	Extractables	Non-extractables	Recovery
0	n.p.	96.5	2.0	0.4	98.9
14	0.2	16.3	81.6	2.6	100.7
33	0.3	10.0	88.7	4.4	103.3
63	0.3	6.7	87.9	4.8	99.7
90	0.4	7.6	86.2	10.1	104.2
119	0.3	8.6	87.7	9.3	105.9
175	0.4	8.4	87.5	9.1	105.3

(Values are given in % of the applied radioactivity; * Volatiles trapped in NaOH and ethylene glycol; n.p.: not performed)

Validity criteria fulfilled:

yes

Conclusions:

The substance dissipated from the water phase of aquatic systems with half-lives between 5.5 - 6.4 days. For the total aquatic system the DT50-values were 485 days (pond) and 636 days (river). Up to eight minor metabolites were detected with maximum concentrations not exceeding 5% of the applied radioactivity for any of them. Volatile products evolved were < 0.4%. The amount of bound residues formed was 7.9 - 10.1% of the applied radioactivity.

Executive summary:

The objectives of the study were to provide information on the rate of degradation and metabolism of the substance, and on the rate of formation and decline of its major degradation products in two aerobic aquatic model systems: river and pond, according to the BBA Guideline Part IV, 5-1 and in compliance with GLP. For this purpose, natural sediment/water samples (Rhine river and pond water) were treated with ¹⁴C-triazole labelled substance at a concentration corresponding to a field rate of 0.127 kg/ha and incubated at 20 °C. Prior to application the aquatic systems were equilibrated for about eight weeks until parameters (redox potential, pH, oxygen content) reached stability. After application the radioactivity in the pond water decreased from 98.1% (day 0) to 7.1% (day 175). For Rhine river water the corresponding values were 96.5% (day 0) and 8.4% (day 175).The extractable radioactivity from sediments first increased to 91.0 % (pond) and 88.7 % (Rhine river) at day 33 and then slowly decreased during incubation to 83.0 % (pond) and 87.5 % (river) of the applied radioactivity at day 175. The non-extractable radioactivity of the pond sediment increased to 7.9 % (day 90) and was 7.6% at the end of incubation. For the Rhine river sediment, the corresponding data were 10.1% (day 90) and 9.1% (day 175). Volatiles were formed in very low amounts. During the whole study the volatile radioactivity was < 0.4% of the applied radioactivity. Therefore, no further analysis was performed. The total recovery ranged for all samples from 98.1 to 107.4% of the applied radioactivity. The concentration of the substance in the total aquatic system decreased from 100.2% (day 0, pond) and 98.5 % (day 0, Rhine river) to 77.6% (pond) and 83.7% (Rhine) at the end of the study. In both aquatic systems, besides the parent compound, up to eight minor metabolites were found. On average, metabolites M1, M3, and M4 reached their highest concentrations with 2.8 - 2.9%, 3.1 - 5.0%, 3.1 - 4.4%, and 2.1 - 2.3%, respectively, after 90 to 175 days. All other metabolites (M4, Unknown 2 - Unknown 4) ranged between < LD and 1.3%. The dissipation half-life of the substance from Rhine river and pond water was in the range of 5.5 - 6.4 days. The degradation half-life in the total systems was 485 days for pond and 636 days for Rhine river. In conclusion, the substance dissipated from the water phase of aquatic systems with half-lives between 5.5 - 6.4 days. For the total aquatic system, the DT50-values were 485 days (pond) and 636 days (river). Up to eight minor metabolites were detected with maximum concentrations not exceeding 5% of the applied radioactivity for any of them. Volatile products evolved were < 0.4%. The amount of bound residues formed was 7.9- 10.1% of the applied radioactivity.

Description of key information

All available data was assessed. The studies that follow standard test conditions are included here as the key studies. The other studies are included as supporting information.

DT50 = 78.29 days,  freshwater; aerobic, 20˚C, OECD TG 309, Matthews & Schäffer 2013  

DT50 = 555 days,  freshwater/sediment; aerobic, 20˚C, BBVA Part IV, Reischmann 1999a

Key value for chemical safety assessment

Half-life in freshwater:

78.29 d

at the temperature of:

20 °C

Half-life in freshwater sediment:

555 d

at the temperature of:

20 °C

Additional information

Two studies follow standard test conditions are included as the key studies. One of the studies is a simulation testing on ultimate degradation in surface water (Matthews & Schäffer 2013, Reliability 1). Another study is a simulation testing on biodegration in water and sediment system (Reischmann 1999a, Reliability 1). The effect values from these studies were used in the CSA. The test condition, results and effect values are summarised in the Table below.

Test system	Test conditions	Mineralisation / Non- extractable residues / Major metabolites (% applied)	Compartment or Concentration	DT50 [d] – Kinetic model	Author / Year
Tuckahoe Lake natural water	Aerobic (ca 100 mL per test vessel; continuous stirring; rates: 10 µg/L and 95 µg/L, 20˚C )	Miner: < 2 % (both treatments) Maj, met: 41.8% AR for M1 (62 DAT, 10 µg/L, with diffuse light) 8.8% AR for M1 (62 DAT, 10 µg/L, in darkness)	Water	78.29 (10 µg/L treatment)	Matthews & Schäffer 2013
		DT50 for CSA	Surface Water	78.29
River water and sediment	Aerobic (2-2.5cm sediment overlaid by 0.5 L water; surface area 80 cm2; rate: 0.127 kg ai/ha,20°C)	Miner.: <0.4% Non-extr.: 10.1% (90 DAT) Maj. met. (>10% AR): Noneb	Water	5.4 – SFO	Reischmann, 1999a
River water and sediment	Aerobic (2-2.5cm sediment overlaid by 0.5 L water; surface area 80 cm2; rate: 0.127 kg ai/ha, 20°C)	Miner.: <0.4% Non-extr.: 10.1% (90 DAT) Maj. met. (>10% AR): Noneb	Total system	636 – SFO (extrapolated)	Reischmann, 1999a
Pond water and sediment	Aerobic (2-2.5cm sediment overlaid by 0.5 L water; surface area 80 cm2; rate: 0.127 kg ai/ha, 20°C)	Miner.: <0.4% Non-extr.: 7.9% (90 DAT) Maj. met. (>10% AR): Noneb	Water	6.4 – SFO	Reischmann, 1999a
Pond water and sediment	Aerobic (2-2.5cm sediment overlaid by 0.5 L water; surface area 80 cm2; rate: 0.127 kg ai/ha,20°C)	Miner.: <0.4% Non-extr.: 7.9% (90 DAT) Maj. met. (>10% AR): Noneb	Total system	485 – SFO (extrapolated)	Reischmann, 1999a
		Geomean DT50 for CSA	Water/sediment (Total system)	555

Six biodegradation studies using water and sediment system without following standard test conditions are used as supporting information. The water-sediment system test conditions, results and effect values were included in the Table below.

Test system	Test conditions	Mineralisation / Non- extractable residues / Major metabolites (% applied)	Compartment or Concentration	DT50 [d] – Kinetic model	Author / Year
River water and sediment	Anaerobic (2-2.5cm sediment overlaid by 0.5 L water; surface area 80 cm2; rate: 0.01 & 0.095 mg/L, 20°C)	Miner.: <0.1% Non-extr.: 6.2% (179 DAT) Maj. met. (>10% AR): None	Water	7.9 – 2 comp. model	Reischmann, 1999b
River water and sediment	Anaerobic (2-2.5cm sediment overlaid by 0.5 L water; surface area 80 cm2; rate: 0.01 & 0.095 mg/L, 20°C)	Miner.: <0.1% Non-extr.: 6.2% (179 DAT) Maj. met. (>10% AR): None	Total system	No significant degradation	Reischmann, 1999b
Flooded rice fields water and sediment	Aerobic (set up: not specified sediment amount and 500 mL water; rate: 10 ppm, 21 – 27 ˚C) Anaerobic (set up: 175.9 g sediment and 750 mL water; rate: 8.8 ppm, 24.5 ± 7.5 ˚C)	Miner.: <0.1% Maj. met. (>10% AR): None	water	Not determined	Spare 1984
Flooded rice fields water and sediment	Aerobic (set up: not specified sediment amount and 500 mL water; rate: 10 ppm, 21 – 27 ˚C) Anaerobic (set up: 175.9 g sediment and 750 mL water; rate: 8.8 ppm, 24.5 ± 7.5 ˚C)	Miner.: <0.1% Maj. met. (>10% AR): None	Total system	Not determined	Spare 1984
Rice paddy sediment and standing water	Anaerobic (25 g sediment, 71 mL water, rate: 10 mg/L, nitrogen atmosphere, 25°C, glucose amendment before treatment)	Miner.:<0.7% Non-extr.: 29.6% (368 DAT) Maj. met. (>10% AR): None	Total system	37 d - SFO	Das, 1987
Loam / Mississippi / from rice paddy	Aerobic (25 ˚C; 25 g sediment, 71.4 mL water; 36.9 ppm in soil phase, equivalent to 12.6 ppm in water phase, 6 mo.)	Miner.: 1.9 % Extr.: 91% (180 DAT) Maj. met. (>10% AR): None	Total system	34	Das 1988
Clay / Sharkey clay / Missippi	Aerobic (25˚C; 20 g soil, 20 mL water; 10.6 ppm; 30 days)	Miner.: < 2.0% Non-extr.: 5.5% (30 DAT) Maj. met. (>10% AR): None	Total system	426.8	Das 1992a
Clay / Sharkey clay / Missippi	Anaerobic (25˚C; 20 g soil, 20 mL water; 10.6 ppm; 360 days)	Miner.: < 4.0% Non-extr.: 6.6% (360 DAT) Maj. met. (>10% AR): None	Total system	363	Das 1992b