Prometryn

Bioaccumulation: aquatic / sediment

Link to relevant study record(s)

Reference

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8 Nov 1988

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)

GLP compliance:

yes

Radiolabelling:

yes

Details on sampling:

- Water: On each sampling day, approximately 500 mL of water were removed from each aquarium (control and treated) using 16 oz. polyethylene bottles. In order to provide sufficient sample for metabolite characterization, an additional 500 mL of water were collected on days 21 and 28 of the uptake phase and day 14 of the depuration phase.
- Fish: Fish were sampled during the uptake phase following the schedule in Table II. On these dates, three fish from each chamber were collected and pooled into control and treated samples. The fish were dissected into fillet/edible (muscle, skin, skeleton) and viscera/nonedible (fins, head, internal organs). Three additional fish from each test chamber were pooled into control and treated samples and reserved for whole fish analysis. Additional fish were collected for metabolite characterization on days 21 and 28 of the uptake phase and day 14 of the depuration phase. All samples were stored at -20°C until needed for analysis.

Vehicle:

yes

Remarks:

acetone

Details on preparation of test solutions, spiked fish food or sediment:

PRIMARY STOCK SOLUTION
- Solution Analysis: A 10 µL aliquot of the Primary Stock Solution was diluted to 50 mL with isooctane and designated the Primary Spiking Solution. A five replicate LSC analysis was performed, 50 µL per replicate, on the Primary Spiking Solution to determine the total activity in the Primary Stock Solution. Also, triplicate 1 µL injections of the Primary Spiking Solution were analyzed by GC to determine the concentration of the Primary Stock Solution. The five replicate LSC analysis of the Primary Spiking Solution averaged 4917 dpm/50 µL. Thus, the total radioactivity in the Primary Stock Solution was 7.38E+09 dpm or 3.22 mCi, which is 101% of the expected radioactivity. The triplicate GC analysis of 1 µL injections of the Primary Spiking Solution allowed calculation of the concentration of the Primary Stock Solution as 2.70 mg/mL.
- Radiopurity Analysis: The radiopurity of the Primary Stock Solution was determined by spotting 1 µL of the Primary Stock Solution in duplicate on a 20 x 20 cm silica gel TLC plate. On the same plate was streaked 10 µL of the non-radiolabelled Primary Standard (0.860 mg/mL). The plate was eluted in toluene: methanol (2:1 v/v). After elution, the plate was air dried and the cold spots were visualized using UV light. The plate was scanned on a Radiomatic Instruments Model RS Radio Thin-Layer Chromatograph (RTLC) Multi-Scanner to locate the radioactivity. After scanning, a custom computer program was used to calculate the radiopurity, which was determined to be 99% for 14C-substance.

SOLUTION ANALYSIS - BIOCONCENTRATION STOCK SOLUTION
The Bioconcentration Stock Solution was prepared by quantitatively transferring the entire contents of the 14C-substance Primary Stock Solution with acetone to a 100 mL volumetric flask containing 1,247 mg nonradiolabeled substance. The flask was then brought to volume with acetone. A 0.2 to 100 mL dilution of the Bioconcentration Stock Solution with iso-octane. This designated the 14C-substance Spiking Solution, and five 50 µL replicate aliquots were analyzed by LSC to determine the total activity present in the Bioconcentration Stock Solution. A 1 mL to 25 mL dilution of the 14C-substance Spiking Solution was made in iso-octane, and designated the the radiolabelled test substanceGC Test Solution. Triplicate 1 µL injections of this solution were analyzed by GC. The 14C-activity of the 14C-substance Spiking Solution was determined to be 7507 dpm/50 µL. The total 14C-activity in the Primary Stock Solution was 7.51E+09 dpm as calculated from the Spiking Solution LSC analysis. The average of the triplicate analysis of the GC test solution was 1.04 µg/mL. The concentration of the Primary Stock Solution was 13.0 mg/mL and the total number of µg present in the Primary Stock Solution was 1.30E+06 µg. From the total µg and the total 14C-activity present in the Bioconcentration Stock Solution, a working specific activity of 5,780 dpm/ µg was calculated.

SOLUTION PREPARATION - DILUTER STOCK SOLUTION.
Diluter stock solutions were prepared from the Bioconcentration stock (in acetone) as needed by transferring a 15.8 mL aliquot of the primary stock (13.0 mg/mL) to a 250 mL volumetric flask and diluting with acetone. The resultant diluter stock of 822 mg/L was then transferred to a brown glass bottle for use by the diluter toxicant injection system.

Test organisms (species):

Lepomis macrochirus

Details on test organisms:

The bluegill sunfish used in the bioconcentration study were obtained from Osage Catfisheries, Inc. in Osage Beach, Missouri. The fish were identified to species by the supplier. Confirmation of species identification was performed when necessary using the taxonomic keys developed by Eddy. All test fish were held in culture tanks on a 16-hour daylight photoperiod and observed for at least 14 days prior to testing. Fish culture techniques were basically those described by Brauhn et al. A daily record of fish observations during the holding period, along with any prophylactic and therapeutic disease treatments, is included in the raw data. During the holding, acclimation and test periods, the fish received Zeigler Brothers #1 Salmon Starter daily in an amount equivalent to approximately 3 percent of their body weight. A representative group of bluegill sunfish used for this experiment had an initial mean weight of 4.9 (±0.97) g and an initial mean standard length of 53 (±2.9) mm. Weight and length measurements were made just prior to initiation of the test.

Route of exposure:

aqueous

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

28 d

Total depuration duration:

14 d

Hardness:

206 - 275 ppm (Represents seasonal variation, difference from one month to the next, will not exceed 10%.)

Test temperature:

17 - 22°C (Represents seasonal variation, difference from one month to the next, will not exceed 10%.)

pH:

7.6 - 8.4 (Represents seasonal variation, difference from one month to the next, will not exceed 10%.)

Dissolved oxygen:

7.4 - 9.1 ppm (Represents seasonal variation, difference from one month to the next, will not exceed 10%.)

TOC:

0.9 - 2.8 ppm (Total monthly range of organic carbon.)

Conductivity:

500 - 650 pmhos/cm (Represents seasonal variation, difference from one month to the next, will not exceed 10%.)

Details on test conditions:

TEST SYSTEM
A modified proportional diluter system described by Mount and Brungs, with a Hamilton® Model 420 dual syringe dispenser, was used for the intermittent introduction of diluent water and 14C-substance into the 100-liter test aquaria containing around 70 liter of well water. Aerated well water (Table 1 in “Any other information on materials and methods incl tables”) was delivered to the glass aquaria at an average rate of approximately 380 mL/minute/ aquarium during the 28-day exposure period, an amount which was sufficient to replace the test volume approximately 7.7 times in a 24-hour period. The diluter system consisted of one 0.05 ppm nominal concentration treated aquarium and one control aquarium. The control aquarium also received a 0.10 mL aliquot of acetone which was equal to that delivered to the treated aquarium. The test aquaria were immersed in a water bath and held at 22°C (± 2°C) by electronically controlled submersible heating elements and circulating water. The diluter system was calibrated by volumetric measurements of mixing cell volumes. A 0.10 mL aliquot of 14C-substance diluter stock solution (822 mg/L) was delivered to 1640 mL of dilution water in the toxicant mixing cell to yield a nominal exposure concentration of 0.05 ppm.

TEST PROCEDURE - UPTAKE PHASE
The 14C-test solution was allowed to flow through the treated aquarium for an equilibration period of about 24 hours. The nominal test concentration was confirmed by radioanalysis before introducing the test fish and represented the day 0 sample. Well water was also allowed to flow through the control aquarium during this period. The uptake phase was initiated by transferring groups of 120 previously acclimated fish to each of the control and test chambers. This represented a biomass loading rate of 8.4 g/L initially. This rate was considered acceptable since the rate decreased steadily throughout the study and the test chambers were aerated for the duration of the study. These fish were observed initially and every 24 hours during the exposure period for any mortality and/or abnormal behavior. Excess food and fecal material was removed from the chambers as needed. Water and fish were sampled throughout the uptake period following the schedule outlined in Table 2 in “Any other information on materials and methods incl tables”. Fillet (edible) and viscera (nonedible) portions were sectioned. The water and tissue samples were radioassayed following the procedure outlined.

TEST PROCEDURE - DEPURATION PHASE
On day 28 of the exposure period, the addition of the 14test material was terminated. At the beginning of the depuration phase, the water in the test aquarium was siphoned and refilled twice using the following technique. The water in each test aquarium was removed by siphoning until
a depth of approximately 8 cm of water remained in each aquarium. The aquaria were filled to a volume of approximately 70 liters with uncontaminated well water (22°C), the chemical characteristics of which are described in Table 1 in “Any other information on materials and methods incl tables”. This water was then removed until a depth of approximately 8 cm of water remained and the aquaria were filled again with approximately 70 liters of uncontaminated well water. The fish were then exposed to flowing uncontaminated well water (22°C) for 14 days. During the depuration period, water and fish were sampled according to the schedule in Table 2 in “Any other information on materials and methods incl tables” and radioassayed.

CHEMICAL AND PHYSICAL TEST PARAMETERS
Water quality parameters of temperature, dissolved oxygen and pH were measured at 0-hour and throughout the study following the schedule provided in Table 2 in “Any other information on materials and methods incl tables”. Measurements of these selected parameters were made in the control and treated aquaria on each sampling date. The control and treated aquaria were aerated throughout the test to maintain dissolved oxygen levels at or above 40% saturation.

Nominal and measured concentrations:

- Nominal exposure concentration: 0.05 ppm
- Measured mean water concentration: 0.048 (±0.0057) ppm

Reference substance (positive control):

no

Remarks on result:

not measured/tested

Key result

Conc. / dose:

0.05 other: ppm

Temp.:

22 °C

Type:

BCF

Value:

77 other: ppm fish/ppm water

Basis:

whole body w.w.

Time of plateau:

8.2 d

Calculation basis:

steady state

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

2.5 d

Rate constant:

other: overall uptake rate constant

Remarks:

ppm in fish/ppm in water/day

Value:

22

Rate constant:

overall depuration rate constant (d-1)

Value:

0.28

Details on kinetic parameters:

See 'Details on results'.

Details on results:

PRELIMINARY DYNAMIC ACUTE TOXICITY STUDY
- In order to set the nominal test concentration for the bioconcentration study with the radiolabelled test substance, a 7-day, five level, dynamic toxicity study was conducted to determine the acute toxicity of the test substance to bluegill sunfish (Lepomis macrochlrus). The nominal concentrations were as follows: 0.3, 0.6, 1.1, 2.1 and 4.0 mg/L. Due to insufficient mortality to calculate an LC50, an EC50 was calculated based on total observed effects. The effects observed after 7 days of exposure were loss of equilibrium and quiescence. The EC50 for the test substance was 1.5 mg/L after 7 days. The 7-day no-observed effect concentration of the test substance was the nominal test concentration of 1.1 mg/L based on the lack of mortality and abnormal effects. The results of the preliminary dynamic acute toxicity study suggested that a 14C level of 0.05 mg/L, representing a number approximately 1/30 of the 7-day EC80 would be an appropriate test concentration for the bioconcentration study. This nominal concentration would be appropriate for evaluating the bioconcentration potential of the radiolabelled test substance. Also, the concentration was less than 1/10 of the EC 80 as suggested by EPA guidelines.

DEFINITIVE BIOCONCENTRATION STUDY
- The 14C-radioactivity calculated as ppm radiolabelled test substance in test water during the 28-day bioconcentration and 7-day depuration period is given in Table 1 in “Any other information on results incl tables”. Water concentrations ranged from 0.034 ppm to 0.051 ppm through 28 days of the bioconcentration (uptake) phase. The average water concentration (using the mean value for each sample day) during the uptake phase was 0.048 (±0.0057) ppm. The expected nominal concentration was 0.050 ppm for 14C-substance. The lowest water concentration (0.034 ppm) was measured at 4-hours and was considered due to the extent of the bioconcentration of the test substance in fish. The results of radioanalysis of the radiolabelled test substance in fillet (edible tissue) and viscera (nonedible tissue) during 28 days of constant exposure to the radiolabelled test substance and 14 days of depuration in clean water are given in Table 1 in “Any other information on results incl tables”. The tissue residues after 28 days of exposure were 2.6 ppm for fillet, 4.1 ppm for whole fish, and 5.2 ppm for viscera. These values corresponded to day 28 bioconcentration factors of 54, 85 and 110X, respectively. Daily bioconcentration factors for the uptake phase of the study ranged from 7.9 to 54X for fillet, 15 to 85X for whole fish, and 24 to 130X for viscera (Table 1 in “Any other information on results incl tables”). An analysis of depuration rates by day 14 of the elimination period (Table IV) showed 42, 73 and 83-percent depuration in fillet, whole fish and viscera, respectively. The depuration data indicated a gradual decrease in tissue concentration through day 7. Since no appreciable depuration occurred after day 7, it is likely that 14C-residues of the test substance had been incorporated into the fish tissue and little to no more depuration would be expected past 14 days. The uptake rate constant (K1), depuration rate constant (K2), time to one-half clearance, and steady-state bioconcentration factor (BCF) for whole fish were determined using the BIOFAC non-linear kinetic modeling computer program for the bioconcentration study with the radiolabelled test substance. The optimal parameter estimates were determined to be:
- Uptake Rate Constant (K2), ppm fish/ppm water/day: 22 (±1.6)
- Depuration Rate Constant (K2), day-1: 0.28 (±0.03)
- T (1/2) for Clearance, Days: 2.5 (±0.26)
- Bioconcentration Factor (BCF), ppm fish/ppm water: 77 (±9.9)
- Time to Reach 90% of Steady-State, Days: 8.2 (±0.86)
- The BIOFAC calculated BCF value for the radiolabelled test substance was 96-percent of the average bioconcentration factor of 80X for days 14, 21 and 28. No mortality or abnormal effects were observed during the conduct of the bioconcentration study. Minimum quantifiable limits (MQL) data for scintillation counting for water, fillet, whole fish and viscera for the radiolabelled test substance were 0.000492 ppm, 0.0217 ppm, 0.0211 ppm and 0.0221 ppm, respectively. Mean recovery data for the radiolabelled test substance in tissue sample oxidations were 97-98% for all tissue types.

ISOLATION AND CHARACTERIZATION OF 14C-RESIDUES IN WATER
Nominal water concentration of the radiolabelled test substance for the accumulation study was 0.05 ppm. Day 21 and 28 water samples were found to contain 0.051 and 0.049 ppm, respectively. The chemical nature of the 14C-residue in the water samples was investigated and provided information concerning the radioactive species to which the fish were exposed at the time of sampling. Preliminary solid phase extraction (SPE) indicated that the SPE technology was effective in removing the radiolabelled test substance related radioactivity from water. Using a 1 mL SPE column, a 50 mL sample of water could be extracted, leaving a radioactive residue in the post-extraction water which was below the quantifiable level. The extracted radioactivity could be liberated from the column by washing with methanol. It was found that if a total of 3 mL of methanol was used to elute the 14C-residue from the column, essentially 100% of the residue could be recovered in the methanol fraction. Table IX presents the overall distribution of 14C-residues in water. HPLC analysis of the SPE-extractable residues demonstrated the presence of only the radiolabelled test substance. The identity of the organic extractable water residue was further confirmed using GC/MS.

ISOLATION AND CHARACTERIZATION OF 14C-RESIDUES IN FISH FILLET
- Organic Extraction of Fillet Tissue: Day 21 and 28 fish fillet samples had 14C-residue levels of 2.3 and 2.6 ppm, respectively, representing bioconcentration factors of 49X and 54X. Preliminary extraction of fillet with acetone and ethyl acetate gave only about 39% of the available radioactivity in the organic-extractable fraction. Thus, extraction conditions were modified to use acetone followed by methanol for the extraction. Using this method, the treated fillet samples gave 49.5 and 40.0% of the initial residue in the organic-extractable fractions of day 21 and 28 fillet, respectively, while the radiolabelled test substance-fortified control sample gave 96.3% recovery under the same conditions. HPLC analysis of the organic-extractable residue demonstrated that parent was the major component of these mixtures. This was confirmed by GC/MS. However, in each case the HPLC raw data reveals a minor peak (Unknown B) at about 14 minutes (this peak is present below the limit of quantification for day 21. In the day 28 organic- extractable fraction. The test substance is found to represent 95.7% of the recovered radioactivity (76.1% recovery from HPLC), while 4.3% of the recovered radioactivity was present as Unknown B. Thus, the test substance and Unknown B represent 29.1 and 1.3% of the original fillet residue (0.757 and 0.034 ppm), respectively. The retention time observed for Unknown B coincides with that of metabolite M4. Recoveries obtained for HPLC injections of fillet extracts were in the range of about 70% of the injected amount. To determine whether or not a very nonpolar material was being retained on the column, analysis of these extracts by reverse phase TLC was performed. The results indicated that there was not a significant amount of radioactivity remaining on the origin, thus, the lack of accountability found for HPLC is not due to adherence of a very nonpolar metabolite to the column.
- Attempted Release of Fillet-Unextractable Residues by Hydrolysis: Attempts were made to release the tissue-bound residue using hydrolytic techniques. Acid hydrolysis (2N HCl, 60ºC, 4 hours) released very little radioactivity from the post-extraction tissues. The organic-extractable fractions contained <0.5% of the initial residue while the water-soluble fraction contained < 3%. Combustion analysis confirmed the presence of the remaining 45-58% of the initial tissue radioactivity in the post-hydrolysis tissue. In a single case, a stronger acid hydrolysis was attempted using 6N HCl at about 80°C for 19 hours. This successfully released some additional radioactivity, about 7.6% being extracted from acidic water, about 9.6% being extracted from neutral water and about 19.2% remaining in the water-soluble fraction. However, the low level of activity in these extracts hindered further analysis of the chemical nature of the extracted residues. Base hydrolysis of the post-acid-hydrolysis tissues gave varying results. For both day 21 and 28 tissues, the organic-extractable fraction contained <3% of the original radioactivity. However, in the case of day 21 tissue, 41.6% of the residue was found in the aqueous fraction, while the day 28 tissue gave only about 5% in that fraction. In each case, the residual tissue contained the remainder of the radioactivity. The difference noted for this single attempt at base hydrolysis is most likely the result of a difference in the actual methods employed rather than a fundamental difference in the tissue residues.

ATTEMPTED RELEASE OF FILLET-UNEXTRACTABLE RESIDUES BY PRONASE & DIGESTION
- One attempt was made to utilize the "Bligh-Dyer" method for extraction of day 28 fillet tissue. This
method removes fatty components using an initial hexane extraction. In this case, the bulk of the “organic soluble" test substance-related material is removed from the tissue by the hexane. Thus, 33.9% of the initial residue was extracted into the hexane fraction. HPLC analysis of this fraction produced results very similar to those found for the previous organic-extracts - including the low recovery from the column. Overall, this extraction procedure had no advantage over the previous procedure, and it was considerably more cumbersome. The residual tissue from this procedure was used in an attempt to liberate the unextractable residue using digestion with Pronase E, a general protease. This enzyme treatment succeeded in releasing significant radioactivity from the tissue. A negligible portion of the released material was found in the organic extractable fraction with essentially all of the released radioactivity found in the water-soluble fraction. These residues persisted in their water solubility after organic extraction under neutral, acidic and basic conditions. Further characterization of the residues solubilized by Pronase E was not undertaken.

ISOLATION AND CHARACTERIZATION OF 14C-RESIDUES IN FISH VISCERA
The 14C-residue levels of 5.3 and 5.2 ppm in day 21 and 28 viscera samples, respectively, represent a bioconcentration factor (BCF) of about 110X. These tissues were extracted and the nature of the organic-extractable residue was investigated. Viscera samples were extracted with acetone followed by 90/10 methanol/water. The resulting combined extract was reduced to aqueous and the residue was partitioned between ethyl acetate/hexane (5/1) and brine. The organic fraction was reduced in volume and analyzed by LSC and HPLC. An average of 63% of the viscera residue was found to be organic-extractable (67.2% for day 21 and 58.7% for day 28). Analysis of these extracts by HPLC indicates that the extracted material is almost completely parent (>97% test substance). In each case, a very small peak is observed at about 3 minutes. The identity of this minor peak was not pursued because it represents less than 2% of the total 14C-residue in viscera. GC/MS confirmed the identity of the major organic-extractable viscera residue as parent. The water-soluble fraction from viscera contained an average of 14.3% (approximately 0.75 ppm) relative to the original residue level. The tissue-bound residue represents about 23% or about 1.2 ppm relative to the original residue (by calculation). A single acid hydrolysis of the extracted solids was attempted using methanolic HCl at about 50°C for 4 hours. This procedure released only about 0.3% (0.016 ppm) relative to the original residue level. Due to the difficulty in releasing the unextractable residues, no further characterization of the tissue-unextractable residue was undertaken.

Table 1. Total 14C-Radioactivity Calculated as 14C-substance in Test Water and Fish Tissue During 28 Days Exposure and 14 Days Depuration with Bluegill Sunfish

Day		Total 14C concentration as 14C-substance (a)
		Water		Fillet		Whole fish		Viscera
		Actual ppm	Running mean	ppm	BCF (b)	ppm	BCF (b)	ppm	BCF (b)
Uptake	0 (c)	0.050	-	-	-	-	-	-	-
	0.17	0.034	0.042	0.33	7.9X	0.64	15 X	1.0	24 X
	1	0.047	0.044	0.78	18 X	2.1	48 X	3.3	75 X
	3	0.049	0.045	1.6	36 X	3.1	69 X	4.9	110 X
	7	0.051	0.046	1.6	35 X	3.8	83 X	4.5	98 X
	14	0.050	0047	1.8	38 X	3.5	74 X	5.9	130 X
	21	0.051	0.047	2.3	49 X	3.8	81 X	5.3	110 X
	28	0.049	0.048	2.6	54 X	4.1	85 X	5.2	110 X
Depuration	1	0.0011	-	1.9	-	2.3	-	2.5	-
	3	<MQL (d)	-	1.7	-	1.4	-	1.2	-
	7	<MQL (d)	-	1.5	-	1.1	-	0.97	-
	10	<MQL (d)	-	1.2	-	1.2	-	0.72	-
	14	<MQL (d)	-	1.5	-	1.1	-	0.87	-

(a)All values have been rounded to represent two significant figures following ABC S.O.P. #8.7.

(b)Daily bioconcentration factor (BCF) obtained by dividing the tissue concentration by the mean measured water concentration up to and including the respective sampling day (running mean).

Samples taken immediately prior to addition of fish.

(c)Below minimum quantifiable limits of 0.000492, 0.0217, 0.0211 and 0.0221 ppm for water, fillet, whole fish and viscera, respectively.

 

Table 2.Depuration of Total 14C Calculated as 14C-substance from Bluegill Sunfish During a 14-Day Clearance Period

Depuration Day	Fillet		Whole fish		Viscera
	Depuration concentration (b)	% Depuration	Depuration concentration (b)	% Depuration	Depuration concentration (b)	% Depuration
1	1.9	27	2.3	44	2.5	52
3	1.7	35	1.4	66	1.2	77
7	1.5	42	1.1	73	0.97	81
10	1.2	54	1.2	71	0.72	86
14	1.5	42	1.1	73	0.87	83

(a)All values have been rounded to two significant figures following ABC S.O.P. #8.7.

(b)Depuration rates are expressed as a percentage of the day 28 14C-substance concentrations of 2.6 ppm for fillet, 4.1 ppm for whole fish, and 5.2 ppm for viscera.

Note: The average minimum quantifiable limits (MQL) for fillet, whole fish and viscera were 0.0217,

0.0211 and 0.0221 ppm.

 

Table 3. Summary of Distribution of 14C-substance Equivalent Residues in Fish Fillet and Viscera

	Day 21		Day 28
	% Total Tissue residue	% of Recovered (a) Radioactiity	% Total Tissue residue	% of Recovered (a) Radioactiity
Fillet
Organic- extractable	49.5		40.0
Substance	34.4	100	29.1	95.7
Unknown B (a)	0	0	1.31	4.3
Tissue-Unextractable	45.5		57.5
Viscera
Organic- extractable	67.2		58.7
Substance	65.3	97.1	57.2	97.5
Unknown A (b)	1.95	2.9	1.48	2.5
Water-Soluble	15.8		12.9
Tissue-Unextractable	17.1		28.4

(a)Percent of the total radioactivity recovered during HPLC analysis of the organic-extractable fraction.

(b)Retention time 14 minutes, coincides with metabolite M4.

(c)Retention time 3 minutes.

Validity criteria fulfilled:

yes

Conclusions:

1. The test substance is accumulated in bluegill sunfish, under the conditions of a standard flow-through accumulation study, to the extent of about BCF=110X for viscera and BCF=54X for fillet.
2. Lack of significant decomposition of the radiolabelled test substance in water samples analyzed from days 21 and 28 of the uptake phase of the accumulation test indicated that the fish were exposed to intact parent.
3. The organic-extractable fractions of viscera and fillet samples contained similar profiles of parent and degradates (Table 3 in “Any other information on results incl tables”). The bulk of the organic-extractable residue (97.9 and 97.3% of the radioactivity recovered during HPLC analysis of fillet and viscera, respectively) is found in the form of parent. However, at Day 28, significant unextractable residue is found in both tissues, particularly in fillet (about 58% in fillet and 28% in viscera).
4. A variety of methods have been found to release part of the unextractable residue. Mild acid and base hydrolysis released relatively small amounts of the unextractable residue. Digestion with Pronase E released about 69% of the bound residue, suggesting that this residue is comprised of amino acid or protein conjugated materials.

Executive summary:

A dynamic 42-day study was conducted to evaluate the bioconcentration of 14C-substance in bluegill sunfish (Lepomis macrochirus). A flow-through proportional diluter system was used to maintain a mean water concentration of 0.048 (±0.0057) ppm 14C-substance for a 28-day exposure period. Radioanalysis of fillet, whole fish and visceral portions was performed throughout the exposure period. Tissue concentrations of 14C-substance ranged from 0.33 to 2.6 ppm for fillet, 0.64 to 4.1 ppm for whole fish, and 1.0 to 5.9 ppm for viscera during the uptake phase. Daily bioconcentration factors ranged from 7.9 to 54x, 15 to 85x, and 24 to 130x for fillet, whole fish and viscera, respectively. To measure the elimination of 14C-substance, the previously exposed test fish were placed in clean water for 14 days. Radioanalysis throughout the depuration period of 14 days indicated 42, 73 and 83 percent elimination from fillet, whole fish and viscera, respectively. The fillet concentration of 14C-substance dropped from 2.6 ppm after 28 days of exposure to 1.5 ppm by day 14 of the depuration period. Corresponding concentration decreases from 4.1 to 1.1 ppm in whole fish samples and from 5.2 to 0.87 ppm in viscera were observed. A non-linear two-compartment kinetic modelling computer program (BIOFAC) was used for analysis of the uptake/depuration data for whole fish. This method estimated an uptake rate constant (K1) of 22 (±1.6) ppm in fish/ppm in water/day, a depuration rate constant (K2) of 0.28 (±0.030) per day, the time for 50% clearance of 2.5 (±0.26) days, and a bioconcentration factor (BCF) of 77 (±9.9) ppm in fish/ppm in water. The time to reach 90% of steady-state was estimated to be 8.2 (±0.86) days. The calculated BCF value was 96% of the average observed whole fish bioconcentration factor (80X) for the radiolabelled test substance in fish at days 14, 21 and 28. No mortality or abnormal effects were observed in the test aquaria during the bioconcentration study. Water and fish tissue samples produced during the bioconcentration phase of this study were extracted and analysed by reverse phase HPLC and GC/MS. Water contained exclusively 14C-substance. The organic-extractable portion of the viscera residue consisted primarily of parent, at about 63% of the total viscera residue. Fillet tissue contained only about 45% organic-extractable residues. HPLC analysis of the organic-extractable residues showed an average of 98.6% of the recovered radioactivity was present as parent. Of the various methods attempted, Pronase E hydrolysis released the largest percentage (approximately 62%, released as water-soluble material) of the bound residue, suggesting that it was amino acid or protein conjugated specimen.

Description of key information

BCF = 77 in whole fish (bluegill sunfish) / ppm in water, EPA N165 -4, Forbis&Halls 1989

Key value for chemical safety assessment

BCF (aquatic species):

77 dimensionless

Additional information

A dynamic 42-day study was conducted to evaluate the bioconcentration of 14C-substance in bluegill sunfish (Lepomis macrochirus). A flow-through proportional diluter system was used to maintain a mean water concentration of 0.048 (±0.0057) ppm 14C-substance for a 28-day exposure period. Radioanalysis of fillet, whole fish and visceral portions was performed throughout the exposure period. Tissue concentrations of 14C-substance ranged from 0.33 to 2.6 ppm for fillet, 0.64 to 4.1 ppm for whole fish, and 1.0 to 5.9 ppm for viscera during the uptake phase. Daily bioconcentration factors ranged from 7.9 to 54x, 15 to 85x, and 24 to 130x for fillet, whole fish and viscera, respectively. To measure the elimination of 14C-substance, the previously exposed test fish were placed in clean water for 14 days. Radioanalysis throughout the depuration period of 14 days indicated 42, 73 and 83 percent elimination from fillet, whole fish and viscera, respectively. The fillet concentration of 14C-substance dropped from 2.6 ppm after 28 days of exposure to 1.5 ppm by day 14 of the depuration period. Corresponding concentration decreases from 4.1 to 1.1 ppm in whole fish samples and from 5.2 to 0.87 ppm in viscera were observed. A non-linear two-compartment kinetic modelling computer program (BIOFAC) was used for analysis of the uptake/depuration data for whole fish. This method estimated an uptake rate constant (K1) of 22 (±1.6) ppm in fish/ppm in water/day, a depuration rate constant (K2) of 0.28 (±0.030) per day, the time for 50% clearance of 2.5 (±0.26) days, and a bioconcentration factor (BCF) of 77 (±9.9) ppm in fish/ppm in water. The time to reach 90% of steady-state was estimated to be 8.2 (±0.86) days. The calculated BCF value was 96% of the average observed whole fish bioconcentration factor (80X) for the radiolabelled test substance in fish at days 14, 21 and 28. No mortality or abnormal effects were observed in the test aquaria during the bioconcentration study. Water and fish tissue samples produced during the bioconcentration phase of this study were extracted and analysed by reverse phase HPLC and GC/MS. Water contained exclusively 14C-substance. The organic-extractable portion of the viscera residue consisted primarily of parent, at about 63% of the total viscera residue. Fillet tissue contained only about 45% organic-extractable residues. HPLC analysis of the organic-extractable residues showed an average of 98.6% of the recovered radioactivity was present as parent. Of the various methods attempted, Pronase E hydrolysis released the largest percentage (approximately 62%, released as water-soluble material) of the bound residue, suggesting that it was amino acid or protein conjugated specimen.