Prometryn

Biodegradation in water and sediment: simulation tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: sediment simulation testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Feb 10 1998 to Mar 24 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Qualifier:

equivalent or similar to guideline

Guideline:

other: Richtlinie für die Prüfung von Pflanzenschutzmitteln, Teil IV, 5-1, Abbaubarkeit und Verbleib von Pflanzenschutzmitteln im Wasser/Sediment-System. Biologische Bundesanstalt für Land- und Forstwirtschaft Bundesrepublik Deutschland

Version / remarks:

Dezember 1990

GLP compliance:

yes

Radiolabelling:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

natural water / sediment: freshwater

Details on source and properties of surface water:

- Sampling of Water: The pond system was sampled from Froscheweiher at Rheinfelden (AG,Switzerland), the river system was sampled from Rhine River at Möhlin (AG,Switzerland). Water was sampled down to a depth of 5 to 10 cm with a plastic container. The sampling site was located about 5 m from the shore. Water was transported to the laboratory in sealed plastic containers. Before use, the water was passed through a 0.2 mm.
- Analysis of Water: The temperature of water, oxygen concentration, pH and redox potential were measured at the surface and about 5 cm above the sediment before sampling. At the testing facilities water was characterised for total N, total P, TOC and hardness. All data are summarised in Tables 1 and 2.
- Preparation and Equilibration of the Test System: About two months before application the sieved sediment was filled into 1-liter incubation flasks to a height of about 2 cm, corresponding to mean wet weights of 249 - 251g for Rhine and pond sediment. Finally, exactly 500 mL of the corresponding pond and Rhine water phases were added to reach a height of the water layer of about 6 cm. After preparation, the aquatic systems were equilibrated in climatic chambers at 19.5 ± 0.5°C under aerobic conditions in the dark. Gentle agitation of the water phase without disturbing the sediment was achieved by means of a suspended special magnetic stirrer. The systems were continuously ventilated with moistened air (about 10-50 mL/min). The effluent gas stream was passed through a trapping system consisting of two bottles with 2 N NaOH (50 mL each).

Details on source and properties of sediment:

- Sampling of Sediment: The pond system was sampled from Froscheweiher at Rheinfelden (AG,Switzerland), the river system was sampled from Rhine River at Möhlin (AG, Switzerland). Sediment was sampled with a shovel from the top 5 to 10 cm of each system. The sampling site was located about 5 m from the shore. Sediment were transported to the laboratory in sealed plastic containers. Before use, sediment were passed through a 2 mm sieve. Thereafter, colloidal particles were allowed to settle in the dark at room temperature for about two days.
- Analysis of Sediment: Sediments were characterised for cation exchange capacity, organic carbon content, particle size distribution, total N, total P5, dry weight and microbial biomass. All data are summarised in Tables 1 and 2.
- Microbial Biomass Determination in the Sediments: The microbial biomass in the sediment was determined using the respiratory method of Anderson and Domsch (Data are given in Tables 1 and 2).
- Preparation and Equilibration of the Test System: About two months before application the sieved sediment was filled into 1-liter incubation flasks to a height of about 2 cm, corresponding to mean wet weights of 249 - 251g for Rhine and pond sediment. Finally, exactly 500 mL of the corresponding pond and Rhine water phases were added to reach a height of the water layer of about 6 cm. After preparation, the aquatic systems were equilibrated in climatic chambers at 19.5 ± 0.5°C under aerobic conditions in the dark. Gentle agitation of the water phase without disturbing the sediment was achieved by means of a suspended special magnetic stirrer. The systems were continuously ventilated with moistened air (about 10-50 mL/min). The effluent gas stream was passed through a trapping system consisting of two bottles with 2 N NaOH (50 mL each).

Duration of test (contact time):

268 d

Initial conc.:

0.794 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

radiochem. meas.

Details on study design:

TREATMENT AND SAMPLING
- Preparation of the application solution: The 14C-labelled test substance was dissolved in 10 mL acetone and its amount was determined by liquid scintillation counting (LSC) to be 12.57 mg/10 mL (stock solution). About 7 mL of the stock-solution were mixed with 20.53 mg unlabeled substance. The radioactivity was determined by LSC and the specific activity calculated to be 0.57 MBq/mg. For application, appropriate volumes (about 100 µL) of the stock solution corresponding to 405 µg 14C-substance were placed into tarred vials (application solution) which were weighted afterwards.
- Treatment and Incubation of the Test Systems: A volume of about 100 µL of application-solution was added to the water layer with a 0.1 mL Hamilton syringe. The vials containing the application-solution were rinsed, the radioactivity of the rinsing solution determined and the applied amount of the test solution calculated. Thus the total amount of the substance applied to each aquatic system was about 397 µg corresponding to a concentration of 0.794 ppm (mg/L) with respect to the water phase. This concentration corresponded to a field rate of 2.5 kg a.i./ha assuming a homogeneous distribution of the compound within the water layer down to a depth of 30 cm. The concentration of acetone was lower than 0.1 % with respect to the amount of water present. After treatment the incubation flasks were connected to the metabolism apparatus and incubated at 20°C.
- Sampling: Water and sediment samples were taken for analysis directly after treatment and up
to 268 days after treatment. The absorption solutions were exchanged weekly during the first month, thereafter in two weeks intervals.
- Monitoring of Various Parameters: During equilibration and incubation pH, redox potential and oxygen concentration were measured in the water phase. Additionally the redox potential was determined in the sediment (Table 1 in “Any other information on results incl tables”). The ambient incubation temperature was continuously recorded. The water volume of each incubated flask was checked in about one to two weeks intervals. The microbial biomass of the sediments was determined at the start of the study (Tables 1 and 2 in “Any other information on materials and methods incl tables”).

EXTRACTION PROCEDURE AND SAMPLE PREPARATION
- At each sampling date, the water phase was decanted and passed through a filter to separate the remaining suspended matter from water.
- Water Samples: The radioactivity in the water phase was measured by liquid scintillation counting
(1000 µL aliquots). Amounts of 50-80 mL of the appropriate water phase were concentrated under reduced pressure to about 5 mL by use of a rotary evaporator, the radioactivity determined and analysed by HPLC and in addition for selected samples TLC was performed.
- Sediment Samples: After decanting the water, the sediments were first extracted under shaking (300 rpm for 20 min) with about 250 mL acetone and then two times with 250 mL acetone/water 80/20 v/v at room temperature for 20 min. After each extraction step the samples were centrifuged at 1800 rpm and at 20°C for 10 min. Finally all extracts were combined and the radioactivity determined by LSC. Thereafter, 80-400 mL of each extract were concentrated to about 2 ml under reduced pressure and submitted to LSC and HPLC analysis. Finally selected sediments were submitted to Soxhlet extraction or directly quantified by combustion followed by LSC. For Soxhlet extraction, sediments were refluxed with 350 mL acetonitrile for 8 hours in a Soxhlet apparatus. Since only low amounts of radioactivity were extracted under such conditions no subsequent analysis was performed. Radioactivity of the extracts was measured by LSC. Residual radioactivity remaining in the homogenised extracted sediments was determined by combustion of aliquots followed by LSC. For this purpose about up to 1.0 g of dried sediment was combusted.
- Reflux Extraction: Selected sediment samples, after Soxhlet extraction were submitted to reflux extraction procedures. Two subsequent extraction steps were performed with the individual soil samples. The first extraction step was performed by refluxing about 20 g (dry weight) of the corresponding sediments with 100 mL acetonitrile/water (4/1 v/v) for two hours (at 80°C). Thereafter, the solution was centrifuged at 1800 rpm for ten minutes, the supernatant decanted and its radioactivity measured in a liquid scintillation counter. The second step was done by refluxing the same soil with acetonitrile/1N HCI (9/1 v/v) at a temperature of 80°C for two hours. After a further centrifugation step (same conditions as above) the supernatant was decanted and aliquots measured in the liquid scintillation counter.
- Organic Matter Fractionation: The fractionation of soil organic matter was performed. For selected samples, portions of about 20 g of soil after harsh (acid) extraction of the corresponding soils were mixed with 0.5 N aqueous NaOH solution (ratio about 1:3 w/v) and extracted by shaking at room temperature for about 24 hours at 200 rpm followed by centrifugation at 2500 rpm for 10 min. The supernatant was decanted and the precipitate was one time extracted with water by shaking for 2 hours. Water and sediment were again separated by centrifugation. The supernatant was decanted and combined with the latter supernatant. Concentrated HCI was added until a pH < 1 was reached. The resulting suspension was centrifuged as described above, the supernatant decanted and the volume determined (fulvic acid fraction). The remaining solid (humic acid fraction) was dissolved in 25 mL of 0.1 N NaOH and radioassayed by LSC. The humin fraction was determined by combustion of the residual radioactivity remaining in the soil after alkali treatment (humin fraction) and by substraction of the radioactivity determined for fulvic and humic acid from the initial radioactivity present in the sample before reflux extraction.

Compartment:

natural water / sediment: freshwater

% Recovery:

91.1

Remarks on result:

other: Mean Balance of Radioactivity During Degradation of 14C-substance in the Pond Aquatic System under Aerobic Conditions at 20 °C after 268 days

Remarks:

For the detailed table see Tables 2 and 3 in "Any opther information on results incl tables".

Compartment:

natural water / sediment: freshwater

% Recovery:

100

Remarks on result:

other: Mean Balance of Radioactivity During Degradation of 14C-substance in the Rhine-river Aquatic System under Aerobic Conditions at 20 °C after 268 days

Remarks:

For the detailed table see Tables 2 and 3 in "Any opther information on results incl tables".

Key result

Compartment:

natural water / sediment: freshwater

DT50:

110.4 d

Type:

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

Temp.:

20 °C

Remarks on result:

other: in total pond system under aerobic conditions

Key result

Compartment:

natural water / sediment: freshwater

DT50:

235.8 d

Type:

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

Temp.:

20 °C

Remarks on result:

other: in the total rhine system in total pond system under aerobic conditions

Compartment:

natural water: freshwater

DT50:

5.3 d

Type:

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

Temp.:

20 °C

Remarks on result:

other: in Pond under aerobic conditions

Compartment:

natural sediment: freshwater

DT50:

176.5 d

Type:

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

Temp.:

20 °C

Remarks on result:

other: in Pond under aerobic conditions

Compartment:

natural water: freshwater

DT50:

10.9 d

Type:

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

Temp.:

20 °C

Remarks on result:

other: in Rhine water under aerobic conditions

Compartment:

natural sediment: freshwater

DT50:

381.1 d

Type:

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

Temp.:

20 °C

Remarks on result:

other: in Rhine under aerobic conditions

Mineralization rate (in CO2):

0.4 other: %

Transformation products:

not measured

Details on transformation products:

PATTERN AND IDENTITY OF METABOLITES
- Water Phase: The parent compound decreased within 268 days from 91.4 and 95.9% to 3.2 and 10.2% in the water phase of pond and Rhine-river samples, respectively. Besides the parent compound 5 metabolites were formed in both systems (M1, M3, M4, M2, M5). In Rhine-river water only M2 was formed as major metabolite, reaching 11.1% at day 268. M3 accounted for maximally 5.9% at day 58, all other metabolites were at maximum in the range of 0.3 - 3.4%. In pond water M2 was the major metabolite reaching 7.6% at day 268. All other metabolites were only formed in maximum amounts of 0.3 to 3.6%.
- Sediment: On day 14 and 30 after treatment substance reached its maximum value in the sediment extracts of pond (48.7%) and Rhine-river (49.3%) samples, respectively. After 268 days of incubation it decreased to 25.6 and 37.6% in the same sediment extracts, respectively. Besides the parent compound, 5 metabolites were formed in the sediments of both systems (M1, M3, M4, M2, M5). In pond sediment only M2 was formed as major metabolite, reaching 10.5% at day 268. All other metabolites were at maximum in the range of 0.3 - 2.3%. In Rhine-river sediment M2 was the major metabolite reaching 6.8% at day 268. All other metabolites were only formed in minor amounts (at maximum 0.5 - 1.4%).
- Total System: After 268 days the substance amounted to 28.8% and 47.9% in the pond and Rhine-river aquatic systems, respectively. M2 was formed as major metabolite in both systems and reached at maximum 18.1% and 18.0% at the end of the study in pond and Rhine-river samples, respectively. All other metabolites (M1, M3, M4, M5) were maximally formed in amounts of 0.1 and 7.3%.
- Identification of Metabolites: Parent and metabolites M2, M3, and M4 were identified by co-chromatography on 2D-TLC and HPLC. Metabolite M5 was only characterised by HPLC, therefore the structure of M5 can not be definitively assigned to the reference compound M5.
- Metabolic Pathway of the substance in Aquatic Systems: In aquatic systems incubated under aerobic conditons at 20°C the substance was degraded according to the metabolic pathways shown in Figure 28 yielding mainly M2. The substance was also degraded by oxidation to the sulfoxide M3 or by dealkylation to M4.

Evaporation of parent compound:

no

Remarks:

See in "Details on results"

Volatile metabolites:

no

Remarks:

See in "Details on results"

Residues:

yes

Remarks:

See in "Details on results"

Details on results:

RECOVERY AND DISTRIBUTION OF RADIOACTIVITY
- Recovery of Radioactivity: The results reported in % of the radioactivity applied are given in Tables 2 and 3 in “Any other information on results incl tables”. Total mean recovery for the whole study was 95.9% ± 6.6%. The average recovery of the radioactivity after sample work-up was 97.0% ± 1.5% indicating that practically no radioactivity was lost during solvent evaporation.
- Radioactivity in the water: After application, the amount of radioactivity in the pond and Rhine water decreased significantly within the incubation time. After 268 days 16.6 and 29.8% of the totally applied radioactivity were determined in pond and Rhine water, respectively (Table 2 and 3 in “Any other information on results incl tables”).
- Radioactivity in the Sediment: Extractable radioactivity was at the beginning of the study 8.3 and 4.3% in the pond and Rhine-river sediment, respectively. It steadily increased to 51.0 and 51.4% after 14 and 58 days, respectively. At the end of the incubation time the extractable radioactivity amounted to 39.2 and 47.2% in the pond and Rhine-river sediments, respectively. Amounts extractable by Soxhlet extraction were below 3.0% after 182 days (Tables 2 and 3 in “Any other information on results incl tables”). The non-extractable radioactivity in pond and Rhine-river sediments increased to 34.9 and 26.0% at the end of the study (268 days), respectively (Table 2 and 3 in “Any other information on results incl tables”). When non extractables from day 182 were submitted to reflux extractions, 5.7% and 3.3 % were found to be extractable by neutral and acidic procedures for pond and Rhine-river sediments, respectively. The radioactivity still remaining in the sediments fractionated into 5.5 and 3.4% fulvic acid, 3.3 and 1.0% humic acid and 12.8 and 7.2% humin for pond and Rhine-river sediments, respectively.
- Volatile Radioactivity (14CO2): The mineralisation rate of the substance in the sediment water systems was very low accounting for 0.4% and 0.5% of the applied radioactivity at the end of the study in pond and Rhine-river samples, respectively. For details see Tables 2 and 9 in “Any other information on results incl tables”.

RATES OF DISSIPATION AND DEGRADATION OF THE SUBSTANCE
- The results are summarised in Table .
- Disappearance Rate of the substance from Water: The substance disappeared from the water layer of the pond and Rhine aquatic system with a DT-50 of 5.3 and 10.9 days, respectively. The corresponding DT-90 values were 160.7 and 294.4 days.
- Degradation Rate of the substance in Sediment: The degradation of the substance in the sediments of both aquatic systems was found to be slow. The DT-50 observed was 176.5 and 381.1 days for pond and Rhine sediments, respectively. Observed DT-90 values were in the same sediments 586 and 1266 days.
- Degradation Rate of the substance in Total System: The substance was degraded slowly with half-life times of 110.4 and 235.8 days in the pond and Rhine-river total aquatic systems, respectively. The corresponding DT-90 values were 366.7 and 783.2 days.

RADIOCHEMICAL PURITY AND STABILITY OF THE TEST SUBSTANCE

According to HPLC chromatograms of the application solution before treatment the test substance showed a radiochemical purity of 97.6%. The stability of the test compound was proven by re-analysis of an aliquot of the application solution after treatment. Its purity was still 97.3% thus proving the stability of the test substance in the vehicle during treatment.

Table 1. Physicochemical Characteristics at Sampling and Before Treatment.

Mean values
Date	Redox potential (Eobs)**		Temp	pH	O2 Water
	Water	Sediment
	mV		[^oC]		[mg/L]
Pond	100	-388	20.1	8.4	8.1
Rhine	97	-370	20.2	8.3	8.3

**: Electrode used: Ag/AgCI/1M KNOg/0.01M KCI; Eref = 342 mV. The redox potential referred to the hydrogen electrode is calculated as follows: Eh - Eobs + Eref; Eh: Oxidation-reduction potential referred to the hydrogen scale (mV). Eobs: Observed oxidation-reduction potential of the Ag/AgCI reference electrode (mV). Eref: Redox potential of the reference electrode as related to the hydrogen electrode = 342 mV.

 

Physicochemical Characteristics of Water and Sediments at Sampling

Sediment		Date	Redox potential [Eobs]**		Temp	pH	O2 water
			Water	Sediment
			[mV]		[°C]		[mg/L]
Rhine	Sampling Laboratory	15.10.97	81		9.3	8.1	10.4
		22.10.97	165	-406	21.5	8.0	8.6
Pond	Sampling Laboratory	15.10.97	84		10.5	7.4	5.8
		22.10.97	53	-337	21.4	8.2	8.3

Physicochemical Characteristics of the Aquatic Systems before Treatment

	Date	Redox potential [Eobs]**		Temp	pH	O2 water
		Water	Sediment
		[mV]		[°C]		[mg/L]
Rhine	23.12.97	100	-470	21.0	8.1	8.1
	20.01.98	101	-470	21.0	8.2	8.2
	17.02.98	94	-540	20.4	8.2	8.1
Pond	23.12.97	99	-465	21.2	8.2	7.8
	20.01.98	98	-460	20.5	8.1	7.9
	17.02.98	97	-460	21.2	8.2	7.9

 

**: Electrode used: Ag/AgCI/1M KN03/0.01M KCI; Eref= 342 mV.

The redox potential referred to the hydrogen electrode is calculated as follows:

Eh = Eobs + Eref

Eh: Oxidation-reduction potential referred to the hydrogen scale (mV).

Eobs: Observed oxidation-reduction potential of the Ag/AgCI reference electrode (mV)

Eref: Redox potential of the reference electrode as related to the hydrogen electrode = 342 mV.

 

Table 2. Mean Balance of Radioactivity During Degradation of 14C-substance in the Pond Aquatic System under Aerobic Conditions at 20 °C.

Time	CO2	Water Layer	Extractables			Non-extractable	Recovery
			Cold	Soxhlet	Total
[day]			[% radioactivity applied]
0	n.d.	91.4	8.3	n.d.	8.3	1.2	100.9
1	n.d.	79.7	16.2	n.d.	16.2	1.6	97.6
3	n.d.	57.4	33.8	n.d.	33.8	2.0	93.2
7	<0.05	50.6	45.8	n.d.	45.8	4.4	100.7
14	<0.05	34.4	51.0	n.d.	51.0	9.6	95.0
35	<0.05	49.5	40.0	n.d.	40.0	5.1	94.7
58	0.2	29.8	50.5	n.d.	50.5	15.3	95.7
91	0.2	21.7	50.2	n.d.	50.2	26.2	98.3
120	0.3	16.9	42.9	1.2	44.2	23.4	84.8
149	0.2	19.9	40.1	n.d.	40.1	19.9	73.2
182	0.5	19.5	44.2	2.5	46.6	27.3	93.8
268	0.4	16.6	39.2	n.d.	39.2	34.9	91.1

n.d.: not detected

 

Table 3. Mean Balance of Radioactivity During Degradation of 14C-substance in the Rhine-river Aquatic System under Aerobic Conditions at 20 °C.

Time	CO2	Water Layer	Extractables			Non-extractable	Recovery
			Cold	Soxhlet	Total
[day]			[% radioactivity applied]
0	n.d.	95.9	4.3	n.d.	4.3	0.5	100.7
1	n.d.	85.0	16.1	n.d.	16.1	1.1	103.9
3	n.d.	77.3	24.7	n.d.	24.7	1.3	103.4
7	<0.05	59.7	38.9	n.d.	38.9	2.3	103.3
14	<0.05	49.7	41.8	n.d.	41.8	3.9	95.3
30	<0.05	41.4	50.6	n.d.	50.6	5.0	98.3
58	0.1	34.4	51.4	n.d.	51.4	7.4	93.3
91	0.2	32.8	51.0	n.d.	51.0	11.8	99.2
120	0.3	30.3	49.7	1.5	51.1	13.1	94.8
149	0.4	32.2	45.2	n.d.	45.2	16.4	94.1
182	0.5	29.1	46.7	2.2	48.9	15.2	93.8
268	0.5	29.8	47.2	n.d.	47.2	26.0	103.4

n.d.: not detected

 

Table. Rates of Dissipation of the substance from Water Layer and Sediment of the Aquatic Systems and Disappearance Times of the substance from the Total Systems  

System	Compartment	Rate Constant (1/days )		Disappearance time (days)
		k01 or k1	k02	DT-50	DT-90
Pond aerobic	Water	0.323	0.0091	5.3	160.7
	Sediment	0.00393		176.5	586.2
	Total System	0.00628		110.4	366.7
Rhine aerobic	Water	0.1626	0.0049795.3	10.9	294.4
	Sediment	0.001819	176.5	381.1	1266
	Total System	0.00294	110.4	235.8	783.2

Validity criteria fulfilled:

yes

Conclusions:

Under aerobic conditions at 20°C the substance rapidly dissipates from the water layer. Degradation was relatively slow. M2 was the only major metabolite formed, reaching about 18% of the applied radioactivity in both systems at the end of the study. Finally degradation was leading to bound residues (26.0- 34.9%). Mineralisation to 14CO2 was very low (0.4-0.5%).

Executive summary:

The degradation of the test substance in aquatic freshwater/sediment systems was determined in the following study conducted in accordance with BBA part IV, 5-1 guideline and in compliance with GLP criteria. The objectives of the study were to provide information on the rate of degradation of the substance, the rate of formation and decline of degradation products and the metabolism in two differentaquatic model systems. For this purpose two natural sediment/water samples were treated with 14C-labelled substance corresponding to a field rate of 2.5 kg a.i./ha and incubated under aerobic conditions at 20°C in the laboratory. The amount of radioactivity in the aqueous phase declined from 91.4 and 95.9% at day 0 to 16.6 and 29.8% after 268 days in pond and Rhine-river systems, respectively. The extractable radioactivity from the sediment increased up to 51.0%and 51.4% after 14 and 58 days in pond and Rhine-river systems, respectively. At the end of the study the extractable amount decreased to 39.2 and 47.2% in the same systems, respectively. Non-extractables amounted to 34.9 and 26.0% after 268 days in pond and Rhine-river systems, respectively. When non-extractables of pond samples from day 182 were submitted to reflux extraction procedures 2.8% were found to be extractable by neutral and a further 0.5% by acidic procedures. The radioactivity still remaining with the soil fractionated into 5.5, 3.3, and 12.8% fulvic acid, humic acid and humin, respectively. Reflux extraction of the non-extractable radioactivity of Rhine-river sediments released 4.6% by neutral and a further 1.1% by acidic procedures. The radioactivity still remaining with the soil fractionated into 3.4, 1.0 and 7.2% fulvic acid, humic acid and humin, respectively. Volatiles in form of carbon dioxide were formed in verysmall amounts, reaching only 0.4 and 0.5% of the applied radioactivity after 268days. The total recovered radioactivity ranged from 73.2 to 103.9%. In both aquatic systems besides the parent molecule only M2 was formed in significant amounts. At the end of the study M2 reached 18.1 and 18.0% in pond and Rhine-river samples,respectively. All other metabolites, M1, M3,M4 and M5, were maximally formed in amounts of 0.1 to 7.3%. The substance disappeared from the water layer of the pond and Rhine aquatic system with a DT-50 of 5.3 and 10.9 days, respectively. The corresponding DT-90 values were 160.7 and 294.4 days. The degradation of the substance in the sediments of both aquatic systems was found to be slow. The DT-50 observed was 176.5 and 81.1 days for pond and Rhine sediments, respectively. Observed DT-90 values were in the same sediments 586 and 1266 days. Consequently, the substance was degraded slowly with half-life times of 110.4 and 235.8 days in the pond and Rhine-river total aquatic systems, respectively. The corresponding DT-90 values were 366.7 and 783.2 days.

Reference 2

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 Apr 1985 to 22 Jul 1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

The systems used consisted of natural waters (Rhine river (I), pond (II)) and 1% of a sediment. The substance was added to the systems resulting in an initial concentration of 10 mg/L. The study was carried out in an all-glass metabolism apparatus with open gas-flow system.

GLP compliance:

yes

Radiolabelling:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

natural water / sediment: freshwater

Details on source and properties of surface water:

- System I: Rhine-river water + 1% sandy loam. See more information in Table 1 in “Any other information on materials and methods incl tables”.
- System II: Pond water + 1% hydrosoil (pond soil). See more information in Table 1 in “Any other information on materials and methods incl tables”.
- Preparation: Before use, the water and respective soil were passed through a 0.2 mm and 2.0 mm sieve, respectively. Thereafter, the components of the systems were characterized.
- Water: pH: 8.6 (Rhine-river) and 8.4 (Pond)

Details on source and properties of sediment:

- System I: Rhine-river water + 1% sandy loam. See more information in Table 1 in “Any other information on materials and methods incl tables”.
- System II: Pond water + 1% hydrosoil (pond soil). See more information in Table 1 in “Any other information on materials and methods incl tables”.
- Preparation: Before use, the water and respective soil were passed through a 0.2 mm and 2.0 mm sieve, respectively. Thereafter, the components of the systems were characterized.
- Oxygen content (mg/L): 9.4 (Rhine-river) and 8.9 (Pond)

Duration of test (contact time):

77 d

Initial conc.:

10 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

radiochem. meas.

Details on study design:

TREATMENT
- About one day before treatment with 14C-substance, the aquatic systems were established by mixing 500 mL of the respective water with 5 g (dry weight basis) of the corresponding soil in the 1 liter glass bottles. After continuous stirring (200 rpm) with a magnetic bar molten in glass and aeration for one day, the 14C-labelled test article was added from a stock solution prepared as follows: The 14C-labelled test article was dissolved in 10.0 mL acetone and its accurate amount determined by radioactivity measurements to be 32.40 mg. Thereafter, the following amounts of labelled and unlabelled material were mixed to obtain the desired quantity of diluted radioactive material, including an excess: Total of 42.15 mg substance (from which 32.4 mg were of 32.2 mCi/g and 9.75 mg were of analytical grade). The unlabelled material was dissolved in acetone, the 10.0 mL acetone containing the 14C- labelled material quantitatively added and the whole mixture made up with acetone to 25.0 mL. The specific radioactivity of 14C-substance was determined to be 24.75 mCi/g. Aliquots of each 0.300 mL of the dissolved radioactive material were then applied dropmise by means of a Hamilton syringe to the respective flasks. During application, the system was continuously stirred. The concentration of 14C-substance amounted to 0.51 mg per 500 mL (1 ppm). An equal volume of acetone was added to the control flasks, Incubation; The flasks were incubated in an air-conditioned room at 25 degrees centigrade in the dark. During incubation system was continuously stirred (about 200 RPM) and ventilated with air at a flow rate of 60 mL/min.

SAMPLING
- Absorption solutions (NaOH, 2-methoxy-ethanol): The absorption solutions were monitored for 14CO2 and volatiles at day 8 14 21, 28 35, 49, 63 and 77.
- pH and oxygen concentration in the test medium: pH and oxygen concentration was measured in the water one day before treatment, directly after the treatment and at day 8, 14, 21, 28, 35, 49, 63 and 77.
- Water and soil: Samples of 5.0 mL water each were taken directly after the treatment and 3, 14, 21, 28, 35, 49, 63 and 77 days after application. Soil samples were analysed after termination of the study at day 77.
- Microbial biomass: Samples of water (2.5 mL) were taken for determination of microbial biomass directly after the treatment and after 35, 49 and 77 days of incubation.

ISOLATION OF RADIOACTIVITY FROM WATER AND SOIL
- Water samples: Aqueous samples were taken 10 minutes after the magnetic stirrer had been stopped and the radioactivity in 500 microlitre aliquots was determined by direct liquid scintillation
counting. Additionally, 10 mL aliquots of the aqueous samples were concentrated about 10-fold by lyophilization and subjected to TLC.
- Soil samples: At the end of the study flask were separated by centrifugation for 10 minutes at
3000 RPM. The radioactivity in the water was determined and analysed as described before.
water and soil in the metabolism. The soil was then extracted five times by shaking with acetone-water (8+2, v/v). The radioactivity in the extracts was determined by liquid scintillation counting. After partitioning with CH2C12, only the organic phase was further analysed for number and amounts of metabolites by TLC. Additionally extracted soil samples were dried at room temperature, homogenized in a mortar and then the residual radioactivity determined by combustion of aliquots.

DETERMINATION OF MICROBIAL BIOMASS
- Microbial biomass was determined for fungi/yeasts and bacteria after removal of soil sediments by the dilution agar plate counts method. For this purpose, one mL of the aqueous samples was diluted with 9 mL of a 1 % peptone solution (sterile) followed by further dilution steps performed as described below.
- To 0.5 mL of the diluted material in petri-dishes 9.0 mL of an adequate agar solution (43 degrees centigrade) was added and agar and aqueous sample thoroughly mixed. After solidification of the agar, the plates were exposed under adequate condition The agar used and the conditions of exposure were as follows.
- Total plate counts (bacteria): Standard Plate Counts Agar was dissolved in bidistilled water (23 5 g/L) its pH adjusted to 7.0 and sterilized for 20 minutes at 120 degrees centigrade. Thereafter, the temperature of the agar was lowered to 45 degrees centigrade overnight in a water bath. Exposure of the plates was performed at 37 degrees centigrades. The number of colonies developed on the plates was controlled after 24 and 48 hours of exposure.
- Plate counts of yeast and fungi: The agar used was the Rose-Bengal-Chloramphenicol Agar Base.It was dissolved in bidistilled water (32.0 g per litre), the pH adjusted to 5.5 and sterilized as described. After cooling, plating was performed. The plates were exposed at 25 degrees centigrade and the number of colonies formed enumerated after 72 hours of exposure.

% Degr.:

94

Parameter:

radiochem. meas.

Sampling time:

77 d

Remarks on result:

other: Rhine-riv . water + 1 % sandy loam

% Degr.:

97.5

Parameter:

radiochem. meas.

Sampling time:

77 d

Remarks on result:

other: Pond water + 1 % sandy loam

Key result

Compartment:

natural water / sediment: freshwater

DT50:

90 d

Type:

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

Temp.:

20 °C

Remarks on result:

other: Rhine river total system under aerobic conditions

Key result

Compartment:

natural water / sediment: freshwater

DT50:

35 d

Type:

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

Temp.:

20 °C

Remarks on result:

other: Pond total system under aerobic conditions

Transformation products:

not specified

Details on transformation products:

CHARACTERIZATION OF RADIOACTIVITY
- Elimination of substance from Water: In the Rhine-river water, up to 40% elimination of the parent molecule was observed within 77 days of incubation. By extrapolation, a half-life of 90 days could be estimated. In system II (Pond), the elimination half-life was found to be about 35 days, i.e. the amount of substance found at the same time was 45% of the amount initially applied. Finally after 77 days, the concentration of substance in the latter system had decreased to 0.26 ppm corresponding to 25.5% of its initial amount.
- Pattern of Metabolites in the Water: The metabolite pattern in the water samples is presented in Table 4 in “Any other information on results incl tables”. Besides the parent molecule, mainly two metabolites M4 and M2 were found in both systems. M2 and M4 progressively increased from day 21 on and amounted in system I to 16.3% and 17 4% after 77 days, respectively. In system II, M4 increased from 4.8%. after 8 days up to 29.7% after 49 days, and then declined to 21.7% at day 77. In the same system, M2 was detected from day 21 on (5.2%) and amounted up to 29.7%. at day 77. Additionally, small amounts (up to 6.1%) of M5 and M11 were detected after 77 days in system I and II, respectively.
- Pattern of Metabolites in the Soil: At the end of the degradation study the soil was separated from the water and analysed for extractables and number of metabolites in the extractables. The results are summarized in Tables 2 and 5 in “Any other information on results incl tables”.
- Analysis of the extractable radioactivity in the sandy loam (System I): The amount of extractable radioactivity in the sandy loam accounted, on average, for 3.7% Analysis by TLC showed besides the parent molecule (1.1%) the presence of two metabolites (M4 and M2), both being less than 1.0%.
- Analysis of the extractable radioactivity in the pond soil (System II): In the extractables of the pond soil, mainly the parent molecule was found (4.5%). In addition, two further metabolites (M4 and M2) were found in similar amounts of 1.9% and 2.1%, respectively. In conclusion it can be stated that the substance is eliminated from the Rhine-river aquatic system with a halflife of about 90 days whereas its elimination from the pond system was more rapid with a half-life of about 35 days. The occurrence of the major metabolites indicates two main metabolic pathways, i.e. N-dealkylation and ring-hydroxylation. However, M4 from day 49 to day 77 in system II suggests a more complex degradation pattern M4 and M2 the decrease of metabolite.

Evaporation of parent compound:

no

Volatile metabolites:

no

Residues:

not specified

Details on results:

RECOVERY OF RADIOACTIVITY
- Radioactivity in the Water Phase: Elimination of radioactivity from the water of the aquatic systems is presented in Table 1 in “Any other information on results incl tables”.
- Rhine-river water: The table shows that the radioactivity in the Rhine-river water remained almost unchanged during the 77 days incubation period. On average, 96.3* and 100.1*% of the radioactivity applied were found at day 0 and 77, respectively. The slight increase in the concentration of radioactivity in the water was due to evaporation losses of the water during incubation.
- Pond water: In pond water, a small change in the level of radioactivity was observed as compared to the level of 88 5 % at day 0, i.e. 21 days after the treatment 83 1 7. of the radioactivity applied were found in the water and after 77 days on average 81.7% of the dose applied were found in the water.
- Balance of Radioactivity in the Aquatic Systems: The balance of radioactivity in the various aquatic systems at the end of the study is presented in Tables 2 and 3 in “Any other information on results incl tables”. On average, 73.8 % and 60.4 % of the radioactivity applied were recovered from Rhine-river and pond water, respectively. From the respective hydrosoils, 3.7% and 12.8% of the radioactivity applied could be extracted. The corresponding figures for non-extractable radioactivity were found to be 3.1% and 12.8%. The amount of 14CO2 (Table 4) accounted, on average, in system I (Rhine-river water + 1% sandy loam) and system II (Pond water + 1% pond soil) for 0.1% and 0.2%, respectively. Volatile radioactivity (2- methoxy-ethanol trap) ranged from 0.% to 0.3%. Total recoveries amounted, on average to 94.0% and 97.5% for system I and II, respectively.
* The discrepancy between X-values of the radioactivity in the respective water was due to the fact that subsamples were removed during the study accounting, on average, for 13.0 and 11.2% for system I (Rhine-river) and II (Pond), respectively. Furthermore, water evaporation losses were not taken into account when calculating total-radioactivity.

INFLUENCE OF THE TEST SUBSTANCE ON AQUATIC MICROORGANISMS
- pH and oxygen concentration: No significant difference in pH and oxygen concentration in the water of treated and untreated test systems was observed. The pH and 02-concentration ranged on average from 8.1±0.2 to 8.6±0.2 (pH) and from 7.3±0.8 to 8.6±0.6 mg 02/L, respectively.
- Microbial plate counts: No significant influence of the substance on the number of bacteria and fungi/yeasts was observed between the treated and untreated aquatic systems (Rhine-river water/sandy loam). Total plate counts (bacteria) of the treated samples accounted for 3.400.000 and 110.000 microorganisms per mL at the beginning and at the end of the study, respectively. In the untreated system, the number of microorganisms amounted to 3.100.000 and 150.000 at the beginning and after 77 days, respectively. The number of fungi and yeasts per mL in treated system I accounted for 94 and 0 at the beginning and at the end of the study, respectively. The corresponding figures of the untreated systems were 69 and 12. In treated system II (pond water/pond soil) total plate counts amounted to 5.300 and 780.000 microorganisms per mL at the beginning and end of the study, respectively In the untreated samples, plate counts were found to be 4.700 and 1.190.000 at the beginning and end of the study, respectively. The number of fungi/yeasts determined at the start and after 77 days for the treated samples amounted to 26 and 20 microorganisms per mL of water, respectively. The corresponding figures of the untreated systems amounted to 36 and 16 microorganisms per mL. In conclusion no negative influence of the substance on aquatic microorganisms was observed.

Table 1. Concentration of 14C-substance in the aqueous phase of the aquatic systems (%-values are based on the radioactivity applied, ppm-values are based on the amount of parent molecule found and on the volume of water unit: mg/L).

Time (days)	System I (Rhine-riv water+1% sandy loam)				System II (Pond water +1% pond soil)
	Replicate		Mean		Replicate		Mean
	A	B
	%	%	%	ppm	%	%	%	ppm
0	96.5	96.1	96.3	0.96	87.4	89.5	88.5	0.89
8	107.7	93.8	100.8	1.00	81.1	70.5	75.8	0.76
14	96.5	96.6	96.6	0.97	65.5	62.5	63.5	0.64
21	90.5	102.8	96.7	0.97	57.9	53.6	55.8	0.56
28	96.9	94.9	95.9	0.96	49.5	49.2	49.4	0.49
35	69.1	73.4	71.3	0.71	44.7	46.6	45.7	0.46
49	67.1	70.1	68.6	0.69	31.8	45.2	38.5	0.39
63	63.0	66.8	64.9	0.65	23.1	32.0	27.6	0.28
77	53.8	66.7	60.3	0.60	24.5	26.4	25.5	0.26

 

Table 2. Balance of radioactivity in aquatic systems 77 days after treatment with 14C-substance (values in % of the radioactivity applied).

	System I (Rhine-riv water+1% sandy loam)			System II (Pond water +1% pond soil)
	Replicate		Mean	Replicate		Mean
	A	B		A	B
Aqueous phase	73.9	73.7	73.8	60.6	60.2	60.4
Hydrosoil
Extractable	3.7	3.6	3.7	11.5	14.0	12.8
Non-Extractable	3.2	3.0	3.1	13.0	12.6	12.8
Volatiles
NaOH trapped (14CO2)	0.1	<0.1	0.1	0.2	0.1	0.2
2-methoxy- thanol trapped	0.4	0.2	0.3	0.2	<0.1	0.1
Subsamples removed	13.2	12.8	13.0	11.4	11.0	11.2
Total	94.5	93.3	94.0	96.9	97.9	97.5

 

Table 4.Pattern of metabolites in water samples of the aquatic system I and II treated with 14C-sybstance (%-values represent mean of two replicates).

 

Rf in SS V	Identity	0	8	14	21	28	35	49	63	77
		System I Rhine-riv water+1% sandy loam
0.81	Substance	96.3	100.8	96.6	78.8	76.0	71.3	68.6	64.9	60.3
0.72	M4	n.d.	n.d.	n.d.	11.9	13.4	13.9	13.5	16.2	16.3
0.50	M2*	n.d.	n.d.	n.d.	6.0	6.5	11.0	14.8	14.1	17.4
0.42	M11	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
0.25	M5**	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	1.5	3.2	6.1
TOTAL		96.3	100.8	96.6	96.7	95.9	96.2	98.4	98.4	100.1
		System II Pond water +1% pond soil
0.81	Substance	88.5	75.8	63.5	55.8	49.4	45.7	35.8	27.6	25.5
0.72	M4	n.d.	4.8	19.6	22.1	23.5	26.0	29.7	22.0	21.7
0.50	M2*	n.d.	n.d.	n.d.	5.2	10.4	9.5	13.3	25.8	29.3
0.42	M11	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	4.3	5.2
0.25	M5**	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
TOTAL		88.5	80.6	83.1	83.1	83.3	81.2	81.5	79.7	81.7

* In this solvent system, the reference compound M2 cannot be separated optimally from M13

** Identified on grounds of similar Rf- values (0.29- 0.36) without co -chromatography .

n.d. Not detected

 

Table 5. Pattern of metabolites in the extractables of the sandy loam (System I) and pond soil (System II) (values given are expressed in % of the radioactivity applied).

Rf in SS V	Identity	System I			System II
		Replicate		Mean	Replicate		Mean
		A	B		A	B
0.81	Susbtance	1.1	1.1	1.1	3.4	5.5	4.5
0.72	M4	0.9	0.8	0.8	1.5	2.3	1.9
0.50	M2	0.2	0.3	0.3	2.2	2.0	2.1
-	n.a.*	1.5	1.4	1.5	4.4	4.2	4.3
TOTAL		3.7	3.6	3.7	11.5	14.0	12.8

* n.a.= not analysed: radioactivity remaining in the aqueous phase after partition with CH2Cl2

Validity criteria fulfilled:

not specified

Conclusions:

It can be stated that the substance is eliminated from the Rhine-river aquatic system with a halflife of about 90 days whereas its elimination from the pond system was more rapid with a half-life of about 35 days. The occurrence of the major metabolites indicates two main metabolic pathways, i.e. N-dealkylation and ring-hydroxylation. However, M4 from day 49 to day 77 in system II suggests a more complex degradation pattern M4 and M2 the decrease of metabolite.

Executive summary:

In the present study, the elimination of the 14C-labelled substance and the number and nature of metabolites formed in two aquatic model systems under laboratory conditions was investigated for 77 days of incubation. The systems used consisted of natural waters (Rhine river (I), pond (II)) and 1% of a sediment. The substance was added to the systems resulting in an initial concentration of 10 mg/L. The substance was eliminated from the Rhine-river water by 31.4% after 49 days and 39.7% after 77 days, i.e. with an approximate half-life of 90 days In the pond water. The substance was eliminated faster with a half-life of 35 days, i.e. 61.5% and 74.5% of the of the parent molecule were eliminated from the system after 49 days and 77 days, respectively. Besides the parent molecule, two major metabolites, i.e. M4 and M2 were found in both systems. In system I, (Rhine-water), M4 ranged from 13.9%. at day 35 to 16.3% at day 77. In system II (Pond water), this compound ranged from 4.8% to 21.7% at day 8 and day 77, respectively, with a maximum of 29.7% after 49 days. The compound M2 ranged from 11.0% to 17.4% in system I at day 35 and day 77 and from 5.2% to 29.3% in system II at day 21 and the end of the study, respectively. In addition, the metabolites M5 in system I and M1 in system II amounted to 6.1% and 5.2% at the end of the study, respectively. Extraction of the soil sediments after 77 days of incubation showed that 3.7% and 12.8% of the radioactivity applied could be extracted from the soil of system I and II, respectively. Analysis of the extractable radioactivity showed the presence of 1.1% and 4.5% of the radioactivity applied in form of the parent molecule for system I and II, respectively. Two metabolites (M4 and M2) were found in both systems, ranging from 0.8 to 1.9% and from 0.3 to 2.1%. respectively. Non-extractables accounted, on average, in the respective systems for 3.1% and 12.8% In addition small amounts of 14CO2 and volatile radioactivity trapped by 2- methoxy-ethanol ranged on average from 0.1% to 0.3% in both systems indicating some mineralization of the substance. The recovery of radioactivity observed in all experiments ranged on average from 94.0% to 97.5%. Finally, the influence of the substance on aquatic microorganisms was investigated by measuring the number of microorganisms. No significant influence of the substance on the number of microorganisms was observed.

Description of key information

- Freshwater: The DT50 in freshwater ranged from 35 to 90 days (geometric mean of 56 days), no guideline followed (similar to OECD TG 309), Ellgehausen 1986

- Freshwater sediment: The DT50 in total system freshwater ranged from 110.4 to 235.8 days (geometric mean of 161 days), BBA Part IV, 5 -1, Schulze-Aurich 2000

Key value for chemical safety assessment

Half-life in freshwater:

56 d

at the temperature of:

25 °C

Half-life in freshwater sediment:

161 d

at the temperature of:

20 °C

Additional information

The study by Ellgehausen (1985) investigated the degradation of the 14C-labelled test substance in two aquatic systems (Rhine water and Pond water) in the laboratory incubated for 77 days at a constant temperature of 25 °C in the dark. The test systems consisted of 0.5 L of natural water with 1% (5 g) of natural soil added as a sediment. The test substance was added at a concentration of 1.0 mg/L. Elimination of the test substance in the Rhine water was measured to be 31.4% after 49 days and 39.7% after 77 days (half-life: 90 days). In the second system (Pond water), the elimination of test substance ranged from 4.8% to 21.7% at day 8 and day 21 (half-life of 35 days), respectively. Extraction from the sediment layer after 77 days of incubation showed residues of 3.7% and 12.8% of the test substance for the Rhine and Pond test systems, respectively. The overall recovery in the experiment ranged from 94% to 97.5%.

The study by Schulze-Aurich (2000) investigated the degradation of the 14C-labelled test substance in two aquatic water-sediment systems (Rhine water and Pond water) in the laboratory incubated for 268 days at a constant temperature of 20 °C in the dark. The test systems consisted of 0.5 L of natural water underlain by 250 g of the natural sediment from these water bodies (ca. 2cm of sediment layer in the test vessels). The amount of radioactivity in the aqueous phase declined from 91.4% and 95.9% at day 0 to 16.6% and 29.8% after 268 days in the Pond and Rhine systems, respectively. The extractable radioactivity from the sediment increased up to 51.0% and 51.4% after 14 and 58 days in Pond and Rhine systems, respectively. Non-extractables amounted to 34.9% and 26.0% after 268 days in Pond and Rhine systems, respectively. The total recovered radioactivity ranged from 73.2% to 103.9%.