1-[2-(2-chloroethoxy)phenylsulfonyl]-3-(4-methoxy-6-methyl-1,3,5-triazin-2-yl)urea;triasulfuron (ISO)

Bioaccumulation: aquatic / sediment

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

10 Sep 1990 to 22 Oct 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 165-4 (Laboratory Studies of Pesticide Accumulation in Fish)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

yes

Details on sampling:

- Sampling intervals/frequency for test organisms: 5 fish were collected from treatment aquarium on days 1, 3, 7, 10, 14, 21, 25 and 28 of exposure, and on days 1, 3, 7, 10 and 14 of depuration. Tissue from the solvent control aquarium was sampled on day 28 of exposure and day 14 of depuration.
- Sampling intervals/frequency for test medium samples: 7 separate days during the week prior to test initiation. During the in-life phase, on days 0, 1, 3, 7, 10, 14, 21, 25 and 28 of exposure, and on days 1, 3, 7, 10 and 14 of depuration. Water from the solvent control on days 0 and 28 of exposure and on day 14 of depuration.
- Sample storage conditions before analysis: Not reported.
- Details on sampling and analysis of test organisms and test media samples (e.g. sample preparation, analytical methods):Concentrations of the substance in the water samples were determined by high performance liquid chromatography. Each vial for the treated fish sample was then placed in a liquid scintillation spectrophotometer and the disintegrations per minute determined.

Vehicle:

no

Test organisms (species):

Lepomis macrochirus

Route of exposure:

aqueous

Evaluation of the Bioconcentration Factor
Analyses of the test solution samples removed on each of the seven days before the exposure was initiated resulted in measured concentrations which averaged 111% of the nominal concentration. Based on these results, the exposure was initiated. The bioconcentration study exposing bluegill to a nominal concentration of 1.4 mg/Land a solvent control solution was terminated after 28 days of exposure and 14 days of depuration. Throughout the study, undissolved test material was observed in the dilution system and the test aquaria. As undissolved material was observed, it was removed. During both the exposure and depuration periods, no mortality occurred among the test organisms. In general, the fish appeared healthy and exhibited normal behavior throughout the study. During the 42 day study, the measured water quality parameters varied minimally between test aquaria and remained within acceptable ranges for the maintenance of bluegill. Results of the water quality measurements made in the test aquaria during this test are summarized in Table 1. Continuous monitoring of the temperature in the treatment aquaria throughout the definitive test resulted in a temperature range of 16 - 18 ºC. Characterizations of the dilution water on day 1 of exposure established a hardness of 32 mg/L as CaCO3. The concentrations of 14C-residues, calculated as test substance (phenyl ring label), measured in the exposure solution during the 28 day exposure period and the 14 day depuration period are presented in Table 2. The concentration in the exposure solution remained relatively constant throughout the 28 day exposure period with a mean (standard deviation) of 1.5 (± 0.1) mg test substance (phenyl ring label)/L. No detectable levels of 14C-residues (< 0.049 mg/L) were present in the depuration aquarium during the 14 day depuration period. Analyses of the solvent control solution during the exposure period also indicated no detectable levels of 14C-residues. Analysis by HPLC on days 10 and 25 of exposure indicated that the 14C measurements were a reliable indication of the quantity of test substance present in the exposure aquarium. HPLC analyses resulted in measured concentrations of 1.1 mg/L on day 10 and 1.3 mg/L on day 25. These may be compared with 1.5 mg/L on day 10 and 1.6 mg/L on day 25 by total 14 C assay. The concentrations of 14C-residues in tissue measured in the edible, non-edible and whole body portions of the bluegill are also presented in Table 2. The concentrations of 14 C-residues measured in the edible, non-edible and whole body tissue portion reached steady state (equilibrium) by day 1 of the exposure period. Steady state was determined by comparing all sample interval measurements by an ANOVA program. Due to the extremely low uptake of test substance (phenyl ring label) by bluegill in this study, many of the tissue samples collected and analyzed contained total 14C-residues below the detection limit. For calculations, the detection limit was used as the tissue concentration for those samples below detection. The mean steady state concentration of test substance (phenyl ring label) in edible and non-edible tissue was 0.70 mg/kg and 4.0 mg/kg, respectively. The analytical results for the edible and non­edible tissue portions of the fish were input into a computer program to calculate the tissue residue concentration on a whole body basis. The mean steady state whole body tissue concentration was 2.1 mg/kg. Analyses of solvent control fish tissue for 14 C-residues at pretreatment and day 28 of the exposure period established that no detectable levels were present (Table 3). Based on a mean measured concentration of 1.5 mg/l (± 0.1) in the exposure solution during the 28-day exposure period and the mean steady state tissue concentrations, bioconcentration factors were calculated. The bioconcentration factors (BCF) of test substance (phenyl ring label) in bluegill tissue were 0.46 X in edible tissue (muscle); 2.6 X in non-edible tissue (viscera and carcass); and 1.3 X in whole body tissue. Predicted bioconcentration factors for edible, non-edible and whole body fish tissue were also calculated. With a computer program the uptake (Ku) and the depuration constant (Kd) for each tissue type was determined. Ku/Kd established the predicted BCF. Results of the polar (acetonitrile) solvent and the nonpolar (hexane) solvent extractions of edible tissue samples revealed that 1.2 µg of the 14 C-residues accumulated in the exposure were extractable with acetonitrile. None were extractable with hexane and none could be quantified as non-extractable. Due to the extremely low bioconcentration factors, insufficient 14 C­ residues were available in tissue removed from day 28 of exposure.

Depuration Phase
Half-life (50% elimination) of the 14C-residues present in the whole body tissue of bluegill on the last day of exposure could not be determined due to the extremely low bioconcentration factors observed. Due to the low tissue concentrations measured on day 28 of exposure, depuration data was calculated based upon the detection limit concentrations of tissue found at the end of exposure which indicated complete elimination of 14C-residues from all tissue types by the first day of the 14-day depuration period. Half-life, or the time when 50% of the accumulated 14 C-residues were eliminated, was reached by day 1.

Metabolite Identification
Procedures used and results of the metabolite identification phase of the study, performed at the sponsor. Since the bioconcentration factors were extremely low, full identification by mass spectrometry was impossible. However, based upon two­ dimensional TLC and HPLC with radiometric detection, metabolism of test substance was determined to be minimal. The residue concentrations in the edible tissue were not sufficient for extensive investigation and efforts were concentrated on characterization of the residues in the nonedible tissue. Parent test substance was shown to be the major component of 14 C-residues found in non-edible tissue (Day 14 = 98.1%; Day 28 = 56.7%). Cleavage of the sulfonyl urea bridge was minimal, and suggested that this pathway is a minor one. Additionally, some other polar metabolites were observed in non-edible tissue extracts.

Table 1. Water Quality Determinations Made During 28 Days of Exposure  and  14  Days  of  Depuration  With  Bluegill  (Lepomis macrochirus) and test substance (Phenyl Ring Label).

		Mean (Standard Deviation)
	Dissolved oxygen (mg/L)	Temperature (ºC)	pH (range)
Exposure aquarium	8.5 a (0.2) N=29	17 (±1) N=29	7.2-7.8
Depuration Aquarium	9.3 b (0.2) N=15	17 (±1) N=15	7.4-7.6
Solvent Control aquarium d	8.8 c (0.3) N=44	17 (±1) N=44	7.2-7.6

a 88%  of  saturation at 17 ºC

b 96% of  saturation  at 17 ºC

c 95% of saturation at 17 ºC

d Solvent control values are cumulative and include both exposure and depuration phase.

 

Table 2. 14C-Residue Concentrations, Calculated as test substance (Phenyl Ring Label), Measured in Fish Tissue Portions and Exposure Solution Water During 28 Days of Exposure of Bluegill (Lepomis macrochirus) to test substance (Phenyl Ring Label) and During 14 Days of Depuration.

Test day	Water concentration (mg/L)	Tissue concentration (mg/kg) b
		Edible (Muscle)	Non-Edible (Carcass/ Viscera)	Whole body a
Exposure
1	1.4	<0.09	0.17	0.12
	1.5	<0.24	<0.34	0.28
	1.4	<0.15	<0.23	0.19
		<0.14	<0.22	0.17
		<0.16	<0.24	0.19
Mean	1.4	0.16	0.24	0.19
SD	0.1	0.05	0.06	0.06
3	1.5	3.6	6.8	4.8
	1.4	<0.63	0.92	0.77
	1.1	0.35	5.4	2.4
		0.27	0.34	0.29
		<0.25	<0.41	0.31
Mean	1.4	1.0	2.8	1.7
SD	0.1	1.4	3.1	1.9
7	1.5	<0.16	1.3	0.59
	1.7	0.49	67 c	29
	1.6	0.14	0.26	0.18
		<0.23	0.54	1.37
		0.32	0.60	0.44
Mean	1.6	0.27	14	6.1
SD	0.1	0.14	30	13
10	1.4	0.31	0.54	0.42
	1.5	0.17	10	3.9
	1.5	<0.16	0.5	0.29
		8.6	12	9.9
		3.5	3.7	3.6
Mean	1.5	2.5	5.4	3.6
SD	<0.1	3.7	5.4	3.9
14	1.5	<0.25	SL d	NA a
	1.4	<0.23	3.5	1.6
	1.5	0.24	0.65	0.41
		<0.20	0.59	0.36
		<0.36	0.92	0.60
Mean	1.5	0.26	1.4	0.74
SD	<0.1	0.06	1.4	0.59
21	1.5	<0.21	1.7	0.78
	1.5	<0.083	0.66	0.30
	1.5	<0.28	0.33	0.30
		<0.13	0.66	0.36
		<0.16	0.89	0.43
Mean	1.5	0.17	0.84	0.43
SD	<0.1	0.08	0.51	0.20
25	1.7	<0.11	0.43	0.23
	1.6	<0.32	6.3	3.0
	1.6	<0.25	1.0	0.60
		<0.16	0.84	0.44
		<0.23	0.41	0.31
Mean	1.6	0.21	1.8	0.91
SD	<0.1	0.08	2.5	1.2
28	1.7	<0.15	0.89	0.45
	1.7	<0.33	<0.46	0.39
	1.6	3.4	3.3	3.4
		<0.53	7.5	3.8
		0.54	8.4	4.3
Mean	1.7	0.99	4.1	2.4
SD	<0.1	1.36	3.7	1.9
Depuration
1	<0.049	<0.082	0.31	NA
	<0.049	0.13	0.82	0.38
	<0.049	<0.077	0.41	NA
		<0.21	<0.35	NA
		<0.080	0.43	NA
3	<0.049	<0.28	<0.42	NA
	<0.049	<0.44	<0.55	NA
	<0.049	0.36	0.69
		0.53	<0.69	NA
		<0.44	1.1	NA
7	<0.049	<0.24	<0.32	NA
	<0.049	<0.25	<0.34	NA
	<0.049	<0.25	<0.34	NA
		<0.22	<0.35	NA
		<0.22	<0.37	NA
10	<0.049	<0.27	<0.43	NA
	<0.049	0.17	<0.21	NA
	<0.049	<0.24	<0.35	NA
		0.21	<0.28	NA
		0.11	0.17
14	<0.049	<0.20	<0.32	NA
	<0.049	<0.33	<0.39	NA
	<0.049	<0.18	<0.30	NA
		<0.25	<0.36	NA
		<0.22	<0.31	NA

a Based on calculations using sample weights  and tissue concentrations of 14 C-residues measured in  the edible and non-edible tissue portions.

b The mean steady state concentration of test substance (phenyl ring label) in edible and non-edible tissue was 0.70 mg/kg and 4.0 mg/kg, respectively.

c Sample may have been contaminated during sample collection and analysis.

d Sample lost.

e Not applicable since whole body calculation was not performed.

NOTE - Values presented for whole body 14C-residue concentration have been calculated from the edible and non-edible tissue concentrations using the detection limit in instances where one or more tissue portion was determined to be below the detection limit. Mean and standard deviation calculations were performed similarly.

 

Table 3. 14C-Residue Concentrations,Calculated as test substance (Phenyl Ring Label), Measured in Fish Tissue Portions and Solvent Control Water Solution During 42 Days of Maintenance During Bioconcentration Study.

Exposure Test Day	Water- Concentration(mg/L)	Tissue Concentration (mg/kg) b
		Edible (Muscle)	Non-edible (Carcass/Viscera)	Whole Body
0	< 0.049	< 0.20	< 0.29	NA
or	< 0.049	< 0.36	< 0.42	NA
pretreatment		< 0.27	< 0.36	NA
		< 0.46	< 0.53	NA
		<  0.38	< 0.42	NA
28	< 0.049	0.48	< 0.25	NA
	< 0.049	< 0.27	< 0.37	NA
		<  0.19	< 0.29	NA
		< 0.27	< 0.34	NA
		<  0.17	< 0.28	NA
Depuratlon Test Day
14	< 0.049	< 0.28	< 0.37	NA
	< 0.049	< 0.20	< 0.33	NA
		< 0.20	< 0.31	NA
		<  0.19	< 0.23	NA
		< 0.26	< 0.35	NA

a Based on radiometric analysis of duplicate samples.

b Based on radiometric analysis of the tissue portions of five (5) fish.

 

Table 4. 14C-Residue Concentrations, Calculated as test substance (Phenyl Ring Label), Measured in Extractions of Edible (Muscle) Tissue of Bluegill (Lepomis macrochirus) After 28 Days of Continuous Aqueous Exposure to test substance (Phenyl Ring Label).

	Replicate 1	Replicate 2	Replicate 3
Grams edible tissue extracted	10.4372	10.5708	10.0952
µg hexane extractable	0	0	0
µg acetonitrile extractable	1.6	0.91	1.2

 

Table 5. BCF, non lipid content normalized

Tissue type	Ku/Kd	Predicted BCF	Measured BCF
Edible	0.35/0.70	0.50	0.46
Non-edible	1.6/0.56	2.8	2.6
Whole Body	0.84/0.59	1.4	1.3

Validity criteria fulfilled:

not specified

Conclusions:

In a bioaccumulation study following EPA 165-4 bluegill sunfish (Lepomis macrochirus) reached steady state bioconcentration factors of 0.46 for edible tissue and 2.6 for non-edible tissue. This corresponds to whole body bioconcentration factor of 1.3. Depuration of accumulated residues occurred rapidly, with the half-life of the residues less than 1 day. No normalisation was performed for lipid content to the BCF factor.

Executive summary:

The bioaccumulation potential of the 14C-phenyl-ring-labelled test substance was investigated in a flow-through system in accordance with EPA 165-4 and in compliance with GLP criteria. Bluegill sunfish (Lepomis macrochirus) were continuously exposed to a nominal concentration of 1.4 mg/L test substance (phenyl ring label) (1/100 of the estimated functional solubility based on previous static exposure at SLI) for 28 days, after which 35 fish were transferred to flowing, uncontaminated water for a 14-day depuration period. During both the exposure and depuration periods, no mortality occurred among test organisms. The concentration of 14C-residues in the exposure solution was determined at 9 intervals during the exposure period and at 5 intervals during the depuration period by radiometric methods. The concentration of 14C-residues in fish tissue portions was determined at the same intervals. Radiometric analyses of the exposure water and fish tissue revealed the following. The concentration of 14C-residues in the exposure solution remained relatively constant throughout the 28-day exposure period at a mean (± S.D.) measured concentration of 1.5 (± 0.1) mg/L test substance (phenyl ring label). Concentrations of 14C-residues present in the water of the depuration aquarium remained < 0.049 mg/L (the limit of radiometric detection) throughout the 14-day depuration period. The concentration of 14C-residues in the edible tissue (filets and bone) of bluegill reached steady state by day 1. The mean steady-state bioconcentration factor for test substance (phenyl ring label) in the edible tissue of bluegill was determined to be 0.46X. Model calculations based on the mean measured water and individual edible tissue concentrations (exposure phase only) predicted a bioconcentration factor of 0.50X. The concentration of 14C-residues in the non-edible tissue of the exposed bluegill reached steady state by day 1. The mean steady-state bioconcentration factor for test substance (phenyl ring label) in the non-edible tissue of bluegill was determined to be 2.6X. Model calculations based on the mean measured water and individual non-edible tissue concentrations (exposure phase only) predicted a bioconcentration factor for non-edible tissue of 2.8 X. The concentration of 14C-residues calculated for the whole body tissue of bluegill exposed to test substance (phenyl ring label) reached steady state by day 1. The mean steady-state bioconcentration factor for test substance (phenyl ring label) in the whole body of bluegill was determined to be 1.3 X. Model calculations based on the mean measured water and individual whole body tissue concentrations (exposure phase only) predicted a bioconcentration factor for the whole body of 1.4 X. Half-life (50% elimination) of the 14C-residues present in the whole body tissue of bluegill on the last day of exposure could not be determined due to the extremely low bioconcentration factors observed. Many of the tissue concentrations were determined to be less than the detection limit. Of the accumulated 14C-residues in edible tissue of bluegill exposed 28 days to test substance (Phenyl ring label), none was extractable with a nonpolar solvent (hexane); 1.2 µg was extractable with a polar solvent (acetonitrile) from approximately 10 grams of tissue; and the non-extractable quantity could not be determined, since the initial pre-extraction tissue collected at day 28 contained no measurable 14C-residues. Since the bioconcentration factors were extremely low, full identification of 14C­ residues extracted from tissue was impossible. However, two-dimensional TLC and HPLC with radiometric detection were used to identify 14C-residues as primarily test substance (Day 1 4 = 98.1%; Day 28 = 56.7%). Overall results showed that metabolism of test substance in bluegill sunfish was not extensive.

Reference 2

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

7 Nov 1990 to 22 Jan 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 165-4 (Laboratory Studies of Pesticide Accumulation in Fish)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

yes

Test organisms (species):

Lepomis macrochirus

Test type:

flow-through

Metabolites:

Yes, M13

Tissue Residues

The radioactive levels in the edible and nonedible tissues are given in Table 1. Regardless of the label, ppm equivalents to 14C-test substance were very low. For the phenyl labeled edible tissues, the ppm values were 0.23 ppm and 0.18 ppm at days 14 and 28, respectively. Similarly, the ppm levels in the triazine labeled edible tissues were 0.33 at day 14 and were less than the limit of detection at day 28. The phenyl labeled nonedible tissues contained 6.46 ppm equivalents at day 14.After 28 days, the ppm values decreased to 2.55 ppm. Nonedible tissues with the triazine ring label had similar ppm levels of 2.79 and 3.00 on days 14 and 28, respectively. Acetonitrile/water extractions resulted in all the 14C being extractable, regardless of the label or time. For the phenyl labeled tissues, the day 14 sample contained 38.7% organic solubles and 61.3% aqueous solubles. By day 28, 52.3% of the radio­ activity was organic soluble, while 47.7% remained in the aqueous fraction. Partitioning of the extracts with the triazine ring label resulted in most of the radioactivity partitioning into the organic fraction, 66.9% and 79.9% on days 14 and 28, respectively. The aqueous fraction contain 33.1% and 20.1% for the 14 and 28 day tissues, respectively. After 14 days, most of the phenyl labeled 14C was found in the organic fraction, 78.2%, while only 21.8% remained in the aqueous fraction. After 28 days, the amount of 14C in the organic fraction was reduced to 40%, while 60% was found in the aqueous fraction. For the 14 day extract from the triazine label, 62.5% of the radioactivity partitioned into the organic fraction while 37.5% remained in the aqueous fraction. The 28 day sample was also partitioned. Radioactive levels above the limit of detection were observed in the organic fraction, but due to the extremely low levels of 14C, quantitative analysis was not possible.
Silica SepPak Purification: The organic fractions of edible tissues from both labels were partially puri­ fied for 2D-TLC analysis using Silica SepPaks. The levels of 14C were extremely low, therefore it was not possible to quantitate recoveries from the SepPaks. Analyses showed that the Methanol #1 and #2 Eluates contained test substance. These fractions were combined and characterized by 2D-TLC to confirm the presence of parent in the edible tissues. These analyses characterize some, but not all the 14C present in the organic fraction of the edible tissues.

C18 Bond Elut Purification

Aqueous fractions from the nonedible tissues were partially purified on a C18 Bond Elut cartridge. Recoveries were good for all samples ranging from 84.9%-100.0%. For the phenyl labeled samples, the Methanol #1 Eluates were further analyzed by 2D-TLC and HPLC. The Methanol #1 Eluates from the triazine labeled samples were characterized by 2D-TLC, but there was not sufficient 14 C-present for HPLC analysis. The Water Eluate from the phenyl labeled day 28 nonedible aqueous fraction was characterized by 2D-TLC and HPLC. There was not sufficient 14e-material for analysis of the Water Eluate from the triazine labeled day 14 nonedible aqueous fraction. The aqueous fraction of the day 28 phenyl labeled edible tissue was also purified on a C18 Bond Elut. Due to the extremely low levels of radio­ activity found in the tissue, quantitative recoveries could not be determined. The Methanol #1 eluate and the Water Eluate were subjected to 2D-TLC analysis. There was not sufficient 14C present in the other aqueous fractions of the edible tissues for further analysis.

 

Thin Layer Chromatography
The organic fractions from the nonedible tissues were chromatographed directly by 2D-TLC. There was one 14C region present, zone 1, which cochromatographed with parent, test substance. Two-dimensional TLC was also used to analyze the partially purified organic fractions of the edible tissues (MetLanol Eluates from Silica SepPaks). In the 2D-TLC autoradiograms obtained from 14 day and 28 day samples from the phenyl label, zone 1 was the only component detected. In the 2D-TLC profile of the triazine labeled organic fraction of the 14 day edible tissue, zone 1 or parent was the predominant component. A minor, unknown metabolite, zone 5, was found only in the triazine label. A trace of zone 2 was detected. Zone 2 cochromatographed with M2. Analysis of the organic fraction of the 28 day edible tissue sample was not successful due to extremely low levels of 14 C. The Methanol #1 Eluates from the Cl8 Bond Elut purification of the nonedible tissue aqueous fractions were characterized by 2D-TLC. Parent (zone 1) was the only component in the 14 day sample. The profile for the 28 day sample was slightly more complex. Zone 1, zone 3, and polar material at the origin were present. Zone 3 cochromatographed with the M13 standard. These 2D-TLC profiles were similar to those obtained from the phenyl label. The sample taken after 14 days of exposure contained parent (zone 1), M13 (zone 3) and polar material at the origin. In the 28 day samples, zone 1 was the only component. The Water Eluate (Cl8 Bond Elut) from the phenyl­ labeled 28 day nonedible tissue was also characterized by 2D-TLC. A trace of radioactivity was seen in zone 1 and at the origin. Attempts were made to characterize 14C in the edible tissue aqueous fraction. The Methanol #1 Eluate and the Water Eluate from the Cl8 Bond Elut clean-up of the phenyl labeled day 28 aqueous fraction were analyzed by 2D-TLC. Due to the extremely low He levels and the large amount of natural products, the results were inconclusive. There was not sufficient 14 C in other aqueous fractions from the edible tissues for further characterization. These 2D-TLC data indicate parent was the predominant component in the nonedible tissues regardless of the time. A minor metabolite which cochromatographed with M13 was detected, indicating that possible hydroxylation of the phenyl ring has occurred. The polar material found at the origin may be the result of conjugation of the phenyl ring. Parent was also detected in the edible tissues. A trace of M2 was observed in the triazine labeled edible tissue. The corresponding phenyl moiety, M3, was not detected in the phenyl labeled tissues. Instead, hydroxylation of the phenyl moiety might have occurred producing M13. This explains the radioactive zone at the origin for he phenyl labeled tissue, and the slight difference in the radioactive distribution between organic and aqueous fraction. These data indicate that cleavage of the sulfonyl urea bridge was a minor metabolic pathway.

High Pressure Liquid Chromatography
The organic fractions of the nonedible tissue were further characterized by HPLC. The 14C histogram obtained from phenyl labeled samples exposed for 14 and 28 days, respectively. There was one predominant component, Peak 1. This peak had the same retention time as test substance. A small amount of polar material eluted in the solvent front for the 14 day tissue. The HPLC profile of the triazine labeled organic fractions was similar to the phenyl labeled samples. Peak 1 was the major component; low levels of the polar peaks 2 and 3 were found. To confirm that Peak 1 was parent, cochromatography of triazine-14C-test substance with the 14 day samples from each label was performed. Peak 1 cochromatographed with triazine-14 C-test substance for both samples, thus confirming the TLC results. There was not sufficient radioactivity in the organic fractions of the edible tissue for HPLC characterization. The aqueous fractions after C18 Bond Elut purification were also characterized by HPLC. Only the phenyl labeled samples were present in sufficient quantities for analysis. In the HPLC profile of the Methanol #1 Eluate from the 14 day sample the major component was Peak 1 or parent. The HPLC profiles of the Methanol #1 Eluate and the Water Eluate from the aque­ ous fraction of the 28 day phenyl labeled sample. Some parent was present, Peak 1. Other polar components were also found in Peaks 2 and 3. Very low levels of these metabolites made further analysis impossible. These HPLC data confirm the 2D-TLC data indicating that parent was the major component in the nonedible tissues, regardless of the time. Peaks 2 and 3 con­ tain polar metabolites, possibly conjugates of the phenyl ring.

 

Quantitation of Metabolites
The organic and aqueous fractions of the nonedible tissues were quantitated by 2D-TLC. Regardless of the label, parent was the major component comprising 54.5-100.0% of the total 14C (1.39-6.46 ppm). Quantifiable levels of a minor metabolite, M13, were detected only in the triazine labeled day 14 sample. It accounted for 6.5% of the dose (0.18 ppm). M13 was also observed in the day 28 phenyl labeled sample, but it was below the limit of detection. Polar material was found on the origin, 10.8% and 14.8% of the dose (0.30 ppm and 0.38 ppm) and in Quadrant III, 2.9-11.9% of the dose (0.08-0.30 ppm). HPLC quantitation was performed on the organic and aqueous fractions of the phenyl labeled nonedible tissues and the organic fractions of the triazine labeled nonedible tissues. These results agree very closely with the 2D-TLC quantitation results. Parent was the major component, accounting for 58.9% and 96.1% of the dose (1.50 ppm and 6.21 ppm) at days 28 and 14, respectively. Polar components were also present accounting for 0.3-34.0% of the dose (0.01-0.87 ppm). Parent accounted for 62.3% and 76.1% of the dose at days 14 and 28, respectively. Peak 2 and peak 3 contained 3.8% and 4.6% of the dose (0.11 and 0.13 ppm), respectively. A summary of the quantitation of the phenyl labeled nonedible tissues is shown in Table 2. Parent accounted for 98.1% of the dose (6.34 ppm) at day 14 and decreased to 56.7% (1.45 ppm) at day 28. A summary of the quantitation results from the triazine labeled tissues is given in Table 3. Parent accounted for 74.4% of the dose (2.08 ppm) at day 14 and 92.5% of the dose (2.78 ppm) at day 28. M13 comprised 6.5% of the total 14C (0.18 ppm) at Day 14 and was not detected at day 28. There were not sufficient radioactive levels in the edible tissues for quantitative analyses.

 

Table 1. Radioactivity found in tissues of bluegill sunfish expressed as ppm equivalents to 14C-test substance.

Label	Tissue	Exposure time	ppm
Phenyl	Edible Tissue	Day 14	0.23
	Edible Tissue	Day 18	0.18
	Nonedible Tissue	Day 14	6.46
	Nonedible Tissue	Day 18	2.55
Triazine	Edible Tissue	Day 14	0.33
	Edible Tissue	Day 18	<*
	Nonedible Tissue	Day 14	2.79
	Nonedible Tissue	Day 18	3.00

<*Less than the limit of detection, twice background dpms.

 

Table 2. Summary of quantitation of metabolites in nonedible tissue from bluegill sunfish dosed with phenyl- 14C-test substance.

Metabolite	Day 14		Day 28
	% Dose	ppm	% Dose	ppm
Test substance	98.1	6.34	56.7	1.45
M13	<*	<*	<*	<*

<*Less than the limit of detection, twice background dpms.

Quantitation results are an average of TLC and HPLC quantitation.

 

Table 3. Summary of quantitation of metabolites in nonedible tissues from bluegill sunfish exposed to triazine-14C-test substance.

Metabolite	Day 14		Day 28
	% Dose	ppm	% Dose	ppm
Test substance	74.4	2.08	92.5	2.78
M13	6.4	0.18	<*	<*

<*Less than the limit of detection, 0.08 ppm..

Quantitation results were obtained by averaging the 2D-TLC and HPLC results from the organic fractions and adding these values to the 2D-TLC results from the aqueous fraction

Validity criteria fulfilled:

not specified

Conclusions:

In a bioaccumulation study in bluegill sunfish (Lepomis macrochirus) following EPA 165-4, the bioconcentration factor (BCF) were calculated to be 1.2 in whole fish (bluegill sunfish; steady-state by day 1) at exposure conc. of 1.4 - 1.5 mg/L. No normalisation was performed for lipid content to the BCF factor.

Executive summary:

The bioaccumulation potential of the 14C-triazine-ring-labelled and 14C-phenyl-ring-labelled test substance was investigated in a flow-through system in accordance with EPA 165-4 and in compliance with GLP criteria. One set of bluegill sunfish was exposed to phenyl and a second set was exposed to triazine labeled 14C-test substance at 1.4-1.5 mg/L for 28 days. The distribution of radioactivity in the edible and nonedible tissues was similar for both labels. The ppm equivalents in the edible tissues were low for the 14 and 28 day samples, ranging from below the limit of detection to 0.33 ppm. In the nonedible tissue, ppm values were slightly higher ranging from 2.55 ppm to 6.46 ppm for days 14 and 28. These very low levels of radioactivity made mass spectral identification of metabolites impossible. However, attempts were made to characterize these metabolites by chromatographic methods. Acetonitrile/water extractions resulted in 100% extractables for all samples. Methylene chloride partitioning of the phenyl labeled extracts showed that the organic soluble C in the nonedible tissues accounted for 38.7-52.3% of the total radioactivity. The aqueous fractions contained 47.7%-61.3% of the radioactivity. The triazine labeled nonedible tissues contained higher levels in the organic fractions, 66.9-79.9% of the 14C, with only 20.1%-33.1% remaining in the aqueous fraction. The edible tissues from the phenyl ring label contained 40.0%-78.2% organic solubles and 21.8%-60.0% aqueous solubles. The levels in the organic fractions of the triazine labeled edible tissues were 62.5%-100% of the radioactivity. The aqueous fractions contained <*%-37.5% of the total 14c. The ppm levels in the edible tissue were not sufficient for extensive investigation and efforts were concentrated on characterization of the residues in the nonedible tissue. Two dimensional-TLC characterization of the organic fractions of the nonedible tissues showed that unchanged test substance was the pre dominant component. Partially purified organic fractions of the edible tissues also contained parent. A trace of M2 was detected in the edible tissue indicating minimal cleavage of the sulfonyl urea bridge had occurred. The aqueous fractions of the nonedible tissues were partially purified in a C18 Bond Elut, then subject to 2D-TLC analysis. Results indicated that unchanged parent was the predominant component in most of the aqueous samples. M13 was detected in samples from both labels. Unknown polar metabolites were also found at the origin. HPLC confirmed the 2D-TLC results indicating that test substance was the major component in the nonedible tissues. Other polar components such as peak 2 and peak 3 were detected in the aqueous fractions. Their identity was not determined due to very low 14C levels. Quantitation of the nonedible tissues by 2D-TLC and HPLC indicate that parent accounted for 56.7-98.1% of the total 14C. M13 comprised 6.5% of the dose in the day 14 triazine labeled nonedible tissue. It was observed in the phenyl labeled tissues but was below the limit of quantitation. Based on the 2D-TLC and HPLC data, metabolism of test substance in bluegill sunfish was not extensive. Parent was shown to be the major component in the nonedible tissues, regardless of time. Cleavage of the sulfonyl urea bridge was minimal, indicating that this was a minor metabolic pathway. Hydroxylation of the phenyl ring to form M13 and other polar metabolites was a possible route of metabolism. Because of the extremely low levels of radioactivity present, mass spectral analysis was not feasible for confirmation of these metabolites.

Description of key information

1.3 in whole fish (bluegill sunfish; steady-state by day 1) at exposure conc. of 1.5 mg/L, clearance time ≤ 1 day (US EPA 165-4, Fackler 1992)

1.2 in whole fish (bluegill sunfish; steady-state by day 1) at exposure conc. of 1.2 mg/L, clearance time ≤ 1 day (US EPA 165-4, Fackler 1991)

Key value for chemical safety assessment

BCF (aquatic species):

1.3

Additional information

Low log Pow values <3 over a range of pH values were observed in a valid GLP study according to OECD TG 107 (Burkhard 1994), indicating a low bioaccumulation potential of the substance. 

Two bioaccumulation studies in fish are available (Fackler 1991 & Fackler 1992) and presented. Both available studies followed standard test guidelines and complied with GLP (Reliability 1). In the first study (Fackler 1991), bluegill sunfish (Lepomis macrochirus) were exposed to the 14C-phenyl-ring-labelled test substance at nominal concentrations of 1.4 – 1.5 mg/L for 28 days uptake phase and 14 days depuration. The bioconcentration factor (BCF) were calculated to be 1.2 in whole fish (bluegill sunfish; steady-state by day 1) at exposure conc. of 1.4 - 1.5 mg/L. No normalization for the lipid content was performed. In the second study (Fackler 1992), bluegill sunfish (Lepomis macrochirus) were exposed to the 14C-triazine-ring- and 14C-phenyl-labelled test substance at nominal concentrations of 1.4 – 1.5 mg/L for 28 days uptake phase and 14 days depuration (35 fish). The test conditions were as following: pH 6.8 – 7.3, 16.9 – 17.1 °C, 16 hours light -8 hours darkness cycle. The water conditions were 30 - 35 mg/L total hardness (as CaCO3) and 0.3 – 1.9 mg/L TOC. The steady state bioconcentration factors of 0.46 for edible tissue and 2.6 for non-edible tissue were determined. This corresponds to whole body bioconcentration factor (BCF) of 1.3. No normalization for the lipid content was performed.