diethyl 1-(2,4-dichlorophenyl)-5-methyl-4,5-dihydro-1H-pyrazole-3,5-dicarboxylate

Biodegradation in water and sediment: simulation tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: sediment simulation testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 Nov 1992 - 04 Mar 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: BBA Part IV, 5-1 (1990)

Qualifier:

according to guideline

Guideline:

EPA Subdivision N Pesticide Guideline 162-4 (Aerobic Aquatic Metabolism)

GLP compliance:

yes

Radiolabelling:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

natural water / sediment

Details on source and properties of surface water:

Sampling of inoculum (sediment plus supernatant water). see below

Details on source and properties of sediment:

Sampling of inoculum (sediment plus supernatant water):

- Details on collection: The sediment and the corresponding surface water were taken with a pail from two sites near Frankfurt which were not in the immediate vicinity of agricultural areas. Locations with flat water was chosen.
- Source I:
River Nidda (old branch)
D-60529 Frankfurt-Nied
south-west of the highway crossing Frankfurt- West
- Source II:
Gravel pit near
D-65933 Frankurt-Schwanheim at the border of the wild-life reserve
Schwanheimer Dunen

- Storage length: Acclimatication period between sampling of the inoculum and the start of incubation: ca. 3 weeks at about 22 - 26°C.

Details on inoculum:

- Preparation of inoculum for exposure:
The sediment and the corresponding water were again combined and filled into cylindrical, brown dark-glass bottles with a volume of 500 ml up to a height of approx. 2 - 2.5 cm sediment and approx. 6 cm water. These heights are recommended in the German BBA guideline IV, 5-1. Based on the internal diameter of the bottles of 8.4 cm and the measured bulk density of the separated dried sediments, the composition of the inoculum
in one bottle was calculated as:

* System River Nidda: ca. 95 g sediment (dry weight) and 341 ml water
* System Gravel Pit: ca.145 g sediment (dry weight) and 341 ml water

Due to the natural moisture content of the sediments the following portions were actually
combined to yield the required composition:

* System River Nidda: ca. 156 g sediment (moist weight) and 280 ml water
* System Gravel Pit: ca. 181 g sediment (moist weight) and 305 ml water

Duration of test (contact time):

101 d

Initial conc.:

29 µg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

radiochem. meas.

test mat. analysis

Details on study design:

TEST CONDITIONS
After application of the 14C-test substance the incubation was conducted at approx. 20°C in the dark in static systems with appropriate traps to collect volatiles and 14C-carbon dioxide.

SAMPLING
- Sampling frequency: Duplicate samples were taken for analysis by days 0, 0.25, 1, 2, 7, 14, 30, 36, 59 and 101 after application.
- Sampling method: For each sample, the supernatant and the sediment were separated by centrifugation followed by the extraction of the sediment with aqueous acetonitrile. The non-extractable residues were quantified by combustion of the air-dried sediments after extraction. All extracts were analysed by radio-HPLC using reversed phase gradient elution. The metabolites detected were identified by co-elution with authentic reference standard material.

Compartment:

entire system

DT50:

1.1 d

Type:

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

Remarks on result:

other: Gravel-pit; DT90: 7d; r²= 0.9138

Compartment:

water

DT50:

0.9 d

Type:

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

Remarks on result:

other: Gravel-pit; DT90:8d; r²=0.9105

Compartment:

entire system

DT50:

1.1 d

Type:

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

Remarks on result:

other: Nidda; DT90: 7d; r²=0.5850

Compartment:

water

DT50:

0.6 d

Type:

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

Remarks on result:

other: Nidda; DT90: 11d; r²=0.8848

Transformation products:

yes

No.:

#1

No.:

#2

No.:

#3

No.:

#4

Details on transformation products:

see remarks on results

Findings:

 

Material balances and distribution of radioactivity between water and sediment:

The overall mean recovery of radioactivity was more than 97% of total applied radioactivity for all samples in the course of the study. The absorption to the sediment was rapid for the sediment with a higher clay and silt content,i.e.Nidda, resulting in 53.8% of the total dose in the water and a portion of 47.9% being extractable from the sediment of day 0 samples. For the gravel-pit systems, 88.2% were found in the water directly after application besides 12.8% radioactivity extractable from the sediment.

By day 101, the total amount of extractable radioactivity in the total systems was still high with values of 83.5% and 89.4% (Nidda / Gravel-pit, mean values of two replicates, respectively). Consequently, the values for non-extractable radioactivity were low at this time point with 17.2% for the Nidda and 6.0% of total applied dose for the Gravel-pit system. 1.6% (Nidda) and 2.1% (Gravel-pit) of the radiolabel applied were detected as¹⁴C-carbon dioxide by the end of the incubation period, day 101. Other volatiles were formed to a negligible extent (< 0.1%). By day 101, 41.7% / 79.4% (Nidda / Gravel-pit) of the applied dose were detected in the water phase. The total radioactivity in the sediments had increased to 59.0% / 16.0% at this sampling interval.

 

Metabolic profiles:

The predominant portion of parent compound AE F107892 was rapidly hydrolysed to values below 2% of applied dose in both water/sediment systems after seven days. Hydrolysis at one ester group resulted in the formation of the two isomeric monocarboxylic acids AE F113225 and AE F114952 as predominant metabolites in the early phase of metabolism. Their peak values in the water were 49.9% (Nidda, day 2) and 74.9% (Gravel-pit, day 7) for AE F113225 and 12.4% / 17.3% (Nidda / Gravel-pit) for AE F114952 by days 2 and 7, respectively.

The dicarboxylic acid AE F109453 and the pyrazole carboxylic acid AE F094270 were formed successively later in the course of the test. The dicarboxylic acid AE F109453 resulted from stepwise hydrolysis of the parent compoundviathe isomeric monocarboxylic acid precursors. AE F109453 was further converted by a combined decarboxylation/elimination step (formal loss of carbonic acid) into AE F094270. The latter compound was not observed in abiotic hydrolysis, indicating that this component is the result of a microbially induced transformation.

A maximum value of 44.5% for AE F109453 was reached in total system Nidda by day 36 with a decline to 11.1% by the end of the study. In contrast, AE F109453 showed no decline in system Gravel-pit within the course of the study. As a consequence, the maximum values of 46.5% of total applied dose for this compound were observed at the last sampling interval. The same applies for AE F094270 in either test system with maximum values of 62.4% / 27.1% (Nidda / Gravel-pit) by the end of the study (day 101).
Consequently, no (apparent) disappearance times could be calculated for the latter components in every system. Other minor components accounted for less than 5.1% of applied radiolabel for a single component in the course of the study.

Disappearance Times of the parent compound mefenpyr-diethyl and main metabolites formed in aerobic aquatic metabolism:

The calculations were performed with an EXCEL spreadsheet by application of a simple first order kinetics. For metabolites, this reflects a worst case estimation due to the “apparent” character as the calculated curve represents only the degradation part. It does not take into account the portion of metabolite resulting from the simultaneous formation.

Time course of the degradation of AE F107892 in total system Nidda, formation of metabolites [% of applied radioactivity]¹

Day	AE F107892	AE F113225	AE F114952	AE F109453	AE F094270	Non-extractable	^14CO2	Total
0	87.8	10.3	3.0	-	-	2.3	n.d.	104.0
0.25	77.8	16.9	5.1	-	-	1.5	n.d.	101.5
1	61.3	32.9	6.9	-	-	2.5	n.d.	99.8
2	20.4	62.0	14.1	1.9	-	4.1	n.d.	102.0
7	0.3	61.9	11.9	14.1	0.2	8.3	< 0.1	106.4
14	2.4	57.3	12.7	8.7	4.0	8.1	0.3	98.5
30	-	51.9	5.5	15.7	7.0	18.5	0.2	102.2
36	-	24.9	-	44.5	18.1	15.1	0.2	105.5
59	-	14.3	-	33.8	28.6	24.4	0.5	104.1
101	-	4.3	-	11.1	62.4	17.2	1.6	102.3

¹: Mean value of two replicates

n.d. = not determined

Time course of the degradation of AE F107892 in total system Gravel-pit, formation of metabolites (% of applied radioactivity)¹

Day	AE F107892	AE F113225	AE F114952	AE F109453	AE F094270	Non-extractable	^14CO2	Total
0	98.4	3.5	0.2	-	-	1.2	n.d.	102.3
0.25	86.0	10.5	2.4	-	-	0.4	n.d.	102.7
1	34.8	50.4	12.0	0.4	-	0.9	n.d.	99.4
2	43.9	48.2	11.1	-	-	0.8	n.d.	101.7
7	3.1	82.8	18.5	2.2	0.4	1.1	< 0.1	106.6
14	1.4	77.8	12.5	3.0	4.4	0.9	< 0.1	99.9
30	-	60.9	8.9	23.8	6.6	1.2	0.1	101.3
36	-	72.8	7.4	8.5	8.2	2.3	0.2	96.5
59	-	59.2	3.1	15.6	18.3	3.2	0.4	102.1
101	-	9.6	3.8	46.5	27.1	6.0	2.1	97.6

¹: Mean values of two replicates

n.d. = not determin

Disappearance times of AE F107892 and its major metabolites in two water/sediment systems

Water/sediment system / Component and Phase	Gravel-pit			Nidda
Total system	DT50[days]	DT90[days]	r^2	DT50[days]	DT90[days]	r^2
AE F107892	1.1	3.6	0.9709	1.1	3.5	0.9813
AE F113225	61.2	203.3	0.9718	36.8	122.1	0.9681
AE F114952 (P1)^1	21.5	71.3	0.9791	18.2	60.6	0.9450
AE F109453	-	-	-	39.3	130.7	0.9919
AE F094270	-	-	-	-	-	-
Water phase	DT50[days]	DT90[days]	r^2	DT50[days]	DT90[days]	r^2
AE F107892	0.9	2.9	0.9499	0.6	1.8	0.5640
AE F113225	61.3	203.6	0.9677	30.9	102.6	0.9626
AE F114952 (P1)^1	17.1	57.0	0.9877	20.1	66.6	0.9334
AE F109453	-	-	-	37.8	125.7	0.9949
AE F094270	-	-	-	-	-	-
Sediment	DT50[days]	DT90[days]	r^2	DT50[days]	DT90[days]	r^2
AE F107892	1.3	4.4	0.9974	1.1	3.5	0.9982
AE F113225	41.2	136.9	0.9882	24.3	80.6	0.9974
AE F114952 (P1)^1	-	-	-	6.6	21.8	0.9915
AE F109453	-	-	-	52.0	172.7	0.9588
AE F094270	-	-	-	-	-	-

Note:   Values for metabolites were calculated using the approach described in the footnote. The DT values for metabolites should be therefore regarded as “apparent” disappearance times.

- : Calculation not possible as no decline was observed in the course of the study.

¹: By an addendum to the report (see list of study description above), the structure of an unknown component reported as Peak “P1” in the original report was assigned to the structure of the mono-carboxylic acid AE F114952.

Apparent half-lives were derived by the input of the maximum value for start plus the values of successive sampling intervals observed in the course of the experiments. From the values a non-cumulative degradation curve can be derived, similar in shape to the degradation curve of the parent substance. However, the actual measured and observed concentration of a given metabolite in a sample is the result of two dynamic processes, a degradation and a formation part in parallel. The calculated curve represents only the degradation part and does not take into account the portion of metabolite resulting from the simultaneous formation. It is a consequence of this simplified approach that apparent half-lives are usually longer than those derived from a full kinetic evaluation. The values should therefore be regarded as a worst case.

Description of key information

Half-life < 2 days (water-sediment system)

Half-life < 1 day (water phase)

Key value for chemical safety assessment

Half-life in freshwater:

1 d

at the temperature of:

20 °C

Half-life in freshwater sediment:

2 d

at the temperature of:

20 °C

Additional information

The dissipation from the water phase and the kinetics of metabolic breakdown of U-14C radiolabelled test substance was studied in two different water/sediment systems (System River Nidda: clay silt; System Gravel Pit: sand). The parent compound was rapidly degraded by microbially induced processes. The metabolic pathway was similar in both water/sediment systems. The determined half-lives for the entire system were < 2 days. The half-lives for the water phase were < 1 day. 

Hydrolysis of the lipophilic parent compound results in the formation of mono-and dicarboxylic acids and their salts. With respect to their hydrophilic nature under the conditions of pH in the environment, a risk for an accumulation of such polar material in aquatic organisms can be excluded.