Pirimiphos-methyl

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:

09 Apr 2007 to 10 Jul 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 305 (Bioconcentration: Flow-through Fish Test)

Version / remarks:

1996

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: Testing Guidelines for Toxicology Studies and in compliance with TCCA Good Laboratory Practice Standards and Test Guidelines: Bioaccumulation Test Method

Version / remarks:

Issued by National Institute of Environmental Research, Korea, December 19, 2006

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: JMAFF Test Guidelines

Version / remarks:

Issued by Japan Ministry of Agriculture, Forestry and Fisheries, Japan, April 2, 2007.

Deviations:

not specified

GLP compliance:

yes

Radiolabelling:

yes

Details on sampling:

- Determination of radiochemical activity and stability: Because each 100 mL of stock solution was used for approximately 3 days, 1 mL of high concentration stock solution was taken at 0, 1, 2, 4, 8, 12, 24, 48, and 72 hours with duplication to confirm the stability of stock solution.

- LSC validation and recovery of test substance: The LSC calibration for its validation was performed by using unquenched standard. It was performed periodically during the study and was successful for calibration. A count time of 5 min was used for all water and solubilized fish samples. Background values of water and fish samples were used with those of solvent control samples at each sampling date to subtract from low and high concentration exposed samples.

- Determination of 14C-labelled test substance concentration in water and fish: Although the sampling dates of water were the same with those of fish in the protocol, water sampling and analysis were performed at least 5 times per week. Three aliquots of 5 mL were taken from the middle of each test tank and placed into 20 mL scintillation vial. Then 12 mL of cocktail was added to each vial, shaken and then measured by the means of LSC for total radioactivity. Four fish from each test tank were taken at 0 (1 hr), 1, 2, 4, 7, 10, 14, 17, 21, 24 and 28 days during the uptake phase and 0 (6 hr), 1, 2, 3, 7 and 14 days during the depuration phase. Individual weight and length were recorded first. To determine the concentration of bound residue in fish, each 10 fish from solvent control and high concentration tank were taken at 25 days after expose.

- Determination of lipid content in fish: Lipid content was determined in each 5 fish from the control tank on days 0 and 42 and from the high concentration tank on days 28 and 42.

Vehicle:

yes

Remarks:

acetonitrile

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

- Preparation of test solution and dosing system: Stock solutions were prepared by mixing labeled and non-labeled compounds (1:1). For high concentration stock solution, 3 mL (5 mg, 12.4 MBq) of 10 times diluted radio-chemical standard solution dissolved in acetonitrile and 5 mL of non-labeled standard solution (1,000 mg/L) were added into 100 mL vol-flask. After evaporation of acetonitrile with N2 gas, 10 drops (approximately 0.17 g) of surfactant (HCO-40) was added and then the 100 mL vol-flask was filled up with deionised water. Low concentration stock solution was prepared by adding 0.3 mL (0.5 mg, 1.24 MBq) of 10 times diluted radio-chemical standard solution dissolved in acetonitrile and 0.5 mL of non-labeled standard solution (1,000 mg/L). A solvent control stock solution was prepared by adding 10 drops of HCO-40 only. Above stock solutions were renewed periodically during the uptake phase. Each stock solution was injected with flow rate of 20 μL/min and diluted with 10,000-fold with dilution water having flow rate of 200 mL/min to provide the test concentration. Dosing of all stock solutions were stopped after the uptake phase (28 days). Fish in all test tanks were transferred to the clean water tanks with flowing dilution water only after the uptake phase was completed.

Test organisms (species):

Oryzias latipes

Details on test organisms:

TEST ORGANISM
- Common name: Killifish
- Source: Obtained from the test facility
- Length at study initiation: 3.13 ± 0.05 cm (low concentration); 3.17 ± 0.08 cm (high concentration)
- Weight at study initiation: 0.2291 ±0.0291 g (low concentration); 0.2359 ± 0.0345 g (high concentration)

Feeding during test
- Food type: Commercially prepared food diet
- Amount and frequency: 1 to 2% of the total biomass daily throughout the test. Uneaten food and feces were removed by siphon.

ACCLIMATION
- Acclimation period: 7 days
- Acclimation conditions (same as test or not): Same as 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:

46.3 - 46.7 mg/L as CaCO3

Test temperature:

23.6 ˚C

pH:

7.0 - 7.1

Dissolved oxygen:

6.8 - 6.9 mg O2/L

TOC:

< 0.5 mg/L (detection limit)

Details on test conditions:

TEST SYSTEM
- Test vessel: 25 L tank
- Flow rate: The flow rates of dilution water and stock solutions were 200 mL/min and 20 μL/min, respectively, which exchanged approximately 11.5 times of the test tank volume per day. The flow rates of stock solutions and dilution water were checked both 48 and 24 hours before the start of the test and then at least 5 times per week during the test. The flow rates combined dilution water and stock solution were in the range of 197 - 204, 198 - 202, and 197 - 202 mL/min for solvent control, low and high concentrations, respectively.
- No. of vessels per concentration: 1
- No. of vessels per solvent control:1


TEST MEDIUM / WATER PARAMETERS
- Temperature, dissolved oxygen (DO) and pH: Water temperature, DO and pH were recorded at least 5 times per week throughout the test.
- Total hardness: Total hardness was measured at the first day and the last day of experimental period in the control and one test tank at the higher concentration.
- Total organic carbon (TOC): The TOC throughout the test was measured at both 48 and 24 hours before the start of the test and at least once a week.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours darkness (controlled by an automatic timer)

Nominal and measured concentrations:

Nominal concentrations: 0 (negative control), 1 µg/L, and 10 µg/L

Reference substance (positive control):

no

Lipid content:

7.1 %

Time point:

other: 28 days of exposure

Remarks on result:

other: exposure level: 10 µg/L

Lipid content:

6.3 %

Time point:

end of exposure

Remarks on result:

other: exposure level: 10 µg/L

Lipid content:

7 %

Time point:

end of exposure

Remarks on result:

other: Solvent control

Lipid content:

7.2 %

Time point:

start of exposure

Remarks on result:

other: Solvent control

Key result

Temp.:

23 °C

pH:

7

Type:

BCF

Value:

1 264 dimensionless

Basis:

whole body w.w.

Time of plateau:

4 d

Calculation basis:

steady state

Remarks:

from day 4 to day 28

Remarks on result:

other: Mean calculated value

Conc. / dose:

1 µg/L

Temp.:

>= 23.2 - <= 24.2 °C

pH:

7

Type:

BCF

Value:

1 251.2 dimensionless

Basis:

whole body w.w.

Time of plateau:

4 d

Calculation basis:

steady state

Remarks:

from day 4 to day 28

Conc. / dose:

10 µg/L

Temp.:

>= 22.3 - <= 24.2 °C

pH:

7

Type:

BCF

Value:

1 276.9 dimensionless

Basis:

whole body w.w.

Time of plateau:

4 d

Calculation basis:

steady state

Remarks:

from day 4 to day 28

Conc. / dose:

1 µg/L

Temp.:

>= 23.2 - <= 24.2 °C

pH:

7

Type:

BCF

Value:

1 200.1 dimensionless

Basis:

whole body w.w.

Time of plateau:

42 d

Calculation basis:

kinetic

Conc. / dose:

10 µg/L

Temp.:

>= 22.3 - <= 24.2 °C

pH:

7

Type:

BCF

Value:

1 200.5 dimensionless

Basis:

whole body w.w.

Time of plateau:

42 d

Calculation basis:

kinetic

Remarks on result:

not measured/tested

Rate constant:

overall uptake rate constant (L kg-1 d-1)

Remarks:

Ku

Value:

684.041

Remarks on result:

other: low concentration

Rate constant:

overall uptake rate constant (L kg-1 d-1)

Remarks:

Ku

Value:

1 008.41

Remarks on result:

other: high concentration

Rate constant:

overall depuration rate constant (d-1)

Remarks:

Kd

Value:

0.57

Remarks on result:

other: low concentration

Rate constant:

overall depuration rate constant (d-1)

Remarks:

Kd

Value:

0.84

Remarks on result:

other: high concentration

Details on kinetic parameters:

See Table 5 in 'Any other information on results incl. tables'

Details on results:

An overview of the results is provided in Table 1 - Table 6 in 'Any other information on results incl. tables'.

- Stability of stock solutions: The recovered rate was average 108.1 ± 5.6% (99.3 - 114.8%). This result means that the 14C-labelled test substance was stable in water based medium at least for 3 days.

- Recovery test of 14C-labelled test substance: The recoveries of 14C-labelled test substance from fish were 111.6 ± 12.4% and 118.2 ± 7.3% at two different concentration levels. The concentrations recovered from water during the equilibration phase were 88.9 ± 1.3% and 99.1 ± 8.5% for low concentration and 98.2 ± 2.5% and 90.5 ± 0.7% for high concentration.

- 14C-labelled test substance concentration in water and fish: The concentration of 14C-labelled test substance from test tanks was determined by LSC during the uptake phase. The ranges were 0.45 - 0.55 and 4.44 - 5.47 µg/L for low and high concentration, respectively. Those values could be converted to 0.90 - 1.10 and 8.89 - 10.71 µg/L based on the ratio of labeled and non-labeled to 1:1. The average concentrations during steady-state, from day 4 to day 28, were 0.50 ± 0.03 µg/L and 5.22 ± 0.14 µg/L for low and high concentration, respectively. The results showed just the concentration of labeled compound detected without the concentration of non-labeled compound.
The 14C-labelled test substance was rapidly accumulated in killifish. The steady-state reached after 4 days exposure in both low and high concentration levels. The average concentration at steady-state of high concentration level was 6665.8 ± 728.2 μg/kg, which was calculated by excluding the value of 21 days sample to sustain the coefficient variation within 20%. For low concentration level, there was high variation at steady-state from 4 days to 28 days. Although the concentration of 21 days sample in low concentration level was excluded, the coefficient variation was 29.2%. However, these results were considered to be acceptable as there were not a statistically significant difference resulting from the One Way ANOVA analysis. Also, there was a statistically significant difference (P ≤ 0.001) between low and high concentration groups. The concentrations of the bound and extractable residues in fish at steady-state (25 days) were 99.4 and 4919.3 µg/kg, respectively. These values were 2 and 98% for the concentration of total radioactive residue and 1.5 and 73.8% for the average concentration at steady-state in high concentration, respectively. From the analysis of solvent extractable solution, no significant peak to be considered as metabolite was found in RI-HPLC chromatogram.

- Determination of BCF: Bioconcentration factor at steady-state (BCFss) was calculated from the ratio of Cfish/Cwater. The BCFss were 1251.2 and 1276.9 for low and high concentration level, respectively. Uptake and depuration rate constants (ku and Kd) were calculated. Their values showed 684.041 and 0.570 for low concentration and 1008.410 and 0.840 for high concentration, respectively. Therefore, the ratios of ku/kd (BCFk) were 1200.1 and 1200.5 for low and high concentration, respectively.

- Determination of lipid contents: The lipid contents were determined from control fish at day 0 and 42 and from exposed fish to high concentration at day 28 and 42 with each 5 replications. The average lipid contents were 7.2 and 7.0% for control fish and 7.1 and 6.3% for exposed fish.

Table 1. Stability of 14C-labelled test substance in the high concentration stock solution

	Radioactivity (dpm/10 µL)
Hours	1	2	Average	% Recovery
0	1616	1656	1636	111.3
1	1312	1608	1460	99.3
2	1576	1648	1612	109.7
4	1672	1704	1688	114.8
8	1608	1744	1676	114.0
12	1544	1424	1484	101.0
24	1368	1776	1572	106.9
48	1664	1624	1644	111.8
72	1496	1568	1532	104.2
Mean ± SD				108.1 ± 5.6
Coefficient variation (%)				5.2

Nominal activity: 1470 dpm/10 µL

Table 2. Recovery of 14C-labelled test substance in fish

Level	Nominal (dpm)	Detected (dpm)^1	% recovery	Mean SD
Low	125	118.86	95.1	111.6 ± 12.4
		147.40	117.9
		154.59	123.7
		136.91	109.5
High	250	287.49	115.0	118.2 ± 7.3
		274.12	109.6
		305.05	122.0
		315.35	126.1

1. Values were adjusted by those of control

Table 3. Concentration of 14C-labelled test substance in water

Phase	Time (days)	Low (mean ± SD)		High (mean ± SD)
		dpm/mL^1	µg/L^3	dpm/mL^1	µg/L^3
Equilibration	-2	65.30 ± 0.98	0.44 ± 0.01	722.79 ± 18.56	4.91 ± 0.13
	-1	72.95 ± 6.28	0.50 ± 0.04	665.86 ± 5.41	4.52 ± 0.04
Uptake	0	66.68 ± 2.93	0.45 ± 0.02	653.92 ± 5.27	4.44 ± 0.04
	1	68.81 ± 6.79	0.47 ± 0.05	731.09 ± 6.34	4.97 ± 0.04
	2	75.26 ± 4.52	0.51 ± 0.03	776.89 ± 6.32	5.28 ± 0.04
	3	70.29 ± 1.13	0.48 ± 0.01	774.80 ± 3.47	5.26 ± 0.02
	4^2	71.14 ± 1.25	0.48 ± 0.01	728.04 ± 8.59	4.95 ± 0.06
	5	67.36 ± 2.04	0.46 ±0.0l	805.10 ± 13.44	5.47 ± 0.09
	7	72.48 ± 2.68	0.49 ± 0.02	778.00 ± 6.72	5.29 ± 0.05
	8	75.76 ± 3.40	0.51 ± 0.02	796.05 ± 10.23	5.41 ± 0.07
	9	73.55 ± 1.81	0.50 ± 0.01	767.42 ± 9.48	5.21 ±0.06
	10	72.76 ± 0.26	0.49 ± 0.01	768.45 ± 8.84	5.22 ± 0.06
	11	71.65 ± 5.18	0.49 ± 0.04	763.13 ± 4.11	5.19 ± 0.03
	14	70.69 ± 1.45	0.48±0.Ql	785.41 ± 3.10	5.34 ± 0.02
	15	71.42 ± 3.85	0.49 ± 0.03	772.92 ± 3.07	5.25 ± 0.02
	17	72.24 ± 4.26	0.49 ± 0.03	723.28 ± 4.74	4.91 ± 0.03
	18	76.64 ± 3.37	0.52 ± 0.02	755.56 ± 5.57	5.13 ± 0.04
	21	78.60 ± 5.50	0.53 ± 0.04	774;68 ± 7.18	5.26 ± 0.05
	22	72.30 ± 5.81	0.49 ± 0.04	758.43 ± 0.28	5.15 ± 0.01
	23	69.23 ± 4.09	0.47 ± 0.03	765.69 ± 8.68	5.20 ± 0.06
	24	66.56 ± 0.94	0.45 ± 0.01	764.19 ± 12.46	5.19 ± 0.09
	25	77.63 ± 5.26	0.53 ± 0.04	774.73 ± 5.45	5.26 ±0.04
	28	81.04 ± 8.08	0.55 ± 0.06	788.51 ± 14.52	5.36 ± 0.10
Average uptake concentration (ng/ml)		0.49 ± 0.03 (5.39%)^4		5.18 ± 0.22 (4.23%)
Average steady-state concentration (ng/ml)		0.50 ± 0.03 (5.31%)		5.22 ± 0.14 (2.72%)

1. Values were adjusted by those of control samples

2. Bold-face means the times of steady-state

3. Values were not included the concentration of non-labeled compound

4. Numbers in parentheses are the Coefficient Variation (CV).

Table 4. Concentration of 14C-labelled test substance in fish

Phase	Time (days)	Low (mean ± SD)		High (mean ± SD)
		dpm/mL^1	µg/L^3	dpm/mL^1	µg/L^3
Uptake	0	12.9 ± 0.7	87.8 ± 4.8	142.4± 7.4	967.5 ± 50.4
	1	35.6 ± 7.2	241.6 ± 49.2	375.8 ± 80.2	2553.1 ± 545.1
	2	61.8 ± 8.0	420.1 ± 54.0	810.2 ± 484.6	5505.0 ± 3292.8
	4^2	79.5 ± 29.4	540.2 ± 199.6	1046.4 ± 222.7	7109.7 ± 1513.1
	7	78.3 ± 38.0	531.7 ± 258.4	1147.2 ± 542.5	7794.4 ± 3685.8
	10	73.1 ± 26.4	496.7 ± 179.7	939.6± 371.0	6383.9 ± 2521.0
	14	113.6 ± 77.3	772.2 ± 525.1	909.0 ± 334.7	6176.0 ± 2273.8
	17	66.4 ± 10.6	451.4±71.7	864.3 ± 350.5	5872.2 ± 2381.6
	21	56.7 ± 9.1	385.6 ± 62.0	477.0 ± 279.9	3240.7 ± 1901.6
	24	142.0 ± 73.6	965.1 ± 499.8	1071.3 ± 276.9	7279.1 ± 1881.4
	28	91.5 ± 27.7	621.8 ± 188.0	889.7 ± 290.2	6045.0 ± 1971.8
Depuration	28.25	65.3 ± 31.l	443.5 ± 211.4	649.5 ± 244.8	4412.9 ± 1663.l
	29	30.9 ± 9.4	210.3 ± 63.7	372.8± 145.1	2533.1 ± 985.5
	30	6.5 ± 2.3	44.3 ± 15.5	70.0 ± 27.9	475.4± 189.5
	31	4.4 ± 2.2	30.2 ± 14.9	52.6 ± 20.4	357.4 ± 138.9
	35	4.1 ± 2.4	27.8 ± 16.6	14.7 ± 8.2	99.9 ± 55.9
	42	0.9 ± 0.4	6.4 ± 2.7	3.4 ± 0.5	23.3 ± 3.6
Average steady-state concentration (pg/mg)		625.6 ± 182.5 (29.2%)^4		6665.8 ± 728.2 (10.9%)

1. Values were adjusted by those of control samples

2. Bold-face means the times of steady-state

3. Values were not included the concentration of non-labeled compound

4. Numbers in parentheses are the coefficient variation (CV).

Table 5. Bioconcentration factors (BCF)

Level	Ku	Kd	BCFk	BCFss
Low concentration	684.041(193.481)^1	0.570 (0.173)	1200.1	1251.2
High concentration	1008.410(468.577)	0.840 (0.412)	1200.5	1276.9

1. Numbers in parentheses are the approximate standard errors of the parameters

 

Table 6. Lipid contents

		Lipid contents (%)
Test tank	Exposure time (day)	1	2	3	4	5	Mean ± SD
Solvent Cont.	0	8.7	6.7	9.0	5.2	6.3	7.2 ± 1.6
	42	8.1	9.4	4.1	3.6	9.8	7.0 ± 2.9
High Cone.	28	7.3	8.7	7.0	6.1	6.6	7.1 ± 1.0
	42	7.9	3.9	5.5	8.6	5.7	6.3 ± 1.9

 

 

Validity criteria fulfilled:

yes

Remarks:

See Validity of the study in 'Any other information on materials and methods incl. tables'

Conclusions:

During the uptake phase, the concentration of 14C-labelled test substance was rapidly increased in fish, reaching a steady-state within 4 days, the BCFss for total radioactive residues in whole fish were 1251.2 and 1276.9 for low and high concentrations, respectively. Thus, the mean BCFss of these two concentrations was 1264 for the whole fish. The BCFk based on the uptake and depuration rate constants were 1200.1 and 1200.5 for low and high concentrations, respectively. During the depuration phase, 14C-labelled test substance was rapidly depurated from fish. More than 50% of the average concentration at steady-state was depurated within 1 day in both low and high concentration. Also, more than 95% of the average concentration at steady-state was depurated after 2 days.
In conclusion, although the test substance showed to have limited bioconcentration potential in killifish (Oryzias latipes), the bioconcentrated residues were rapidly eliminated in clean water. Therefore, bioconcentration potential of the test substance is considered to be low unless organism is exposed for a prolonged period.

Executive summary:

The bioaccumulation potential of the test substance was investigated in a study in accordance with OECD TG 305 and in compliance with GLP criteria. In this study, killifish (Oryzias latipes) were exposed to radiolabelled and non-radiolabelled (ratio of 1:1) test substance at concentrations of 1 and 10 µg/L in a flow-through during a 28-day uptake phase. Afterwards, fish were transferred to aquaria without the test substance in a 14-day depuration phase. Specific activities were confirmed by liquid scintillation counting (LSC) then radio-HPLC. The recoveries of 14C-labelled test substance from fish were 111.6 ± 12.4% and 118.2 ± 7.3% at the two different concentration levels. The concentrations recovered from water during the equilibration phase were 88.9 ± 1.3% and 99.1 ± 8.5% for low concentration and 98.2 ± 2.5% and 90.5 ± 0.7% for high concentration. The concentration of 14C-labelled test substance in water ranges were 0.45 - 0.55 and 4.44 - 5.47 µg/L for low and high concentration, respectively. Those values could be converted to 0.90 - 1.10 and 8.89 - 10.71 µg/L based on the ratio of labeled and non-labeled to 1:1.

The 14C-labelled test substance was rapidly accumulated in killifish. The steady-state reached after 4 days exposure in both low and high concentration levels. The average concentration at steady-state of high concentration level was 6665.8 ± 728.2 μg/kg, which was calculated by excluding the value of 21 days sample to sustain the coefficient variation within 20%. For low concentration level, there was high variation at steady-state from 4 days to 28 days. Although the concentration of 21 days sample in low concentration level was excluded, the coefficient variation was 29.2%. However, these results were considered to be acceptable as there were not a statistically significant difference resulting from the One Way ANOVA analysis. Also, there was a statistically significant difference (P ≤ 0.001) between low and high concentration groups. The concentrations of the bound and extractable residues in fish at steady-state (25 days) were 99.4 and 4919.3 µg/kg, respectively. These values were 2 and 98% for the concentration of total radioactive residue and 1.5 and 73.8% for the average concentration at steady-state in high concentration, respectively. From the analysis of solvent extractable solution, no significant peak to be considered as metabolite was found in RI-HPLC chromatogram. The lipid contents were determined from control fish at day 0 and 42 and from exposed fish to high concentration at day 28 and 42 with each 5 replications. The average lipid contents were 7.2 and 7.0% for control fish and 7.1 and 6.3% for exposed fish.

The bioconcentration factor at steady-state (BCFss) was calculated from the ratio of Cfish/Cwater. The BCFss values were 1251.2 and 1276.9 for the low and high concentration levels, respectively. Uptake and depuration rate constants (ku and Kd) were also calculated. These values showed 684.041 and 0.570 for low concentration and 1008.410 and 0.840 for high concentration, respectively. Therefore, the ratios of ku/kd (BCFk) were 1200.1 and 1200.5 for low and high concentration, respectively. After the depuration phase, 0.3-1.0% of residues of the test material remained in the test organisms. Based on the findings, it was determined that the mean BCFss was determined to be 1264.

Description of key information

BCF = 1264, whole fish, Oryzias latipes, aqueous, freshwater, OECD TG 305, Seo 2007

Key value for chemical safety assessment

BCF (aquatic species):

1 264 dimensionless

Additional information

The bioaccumulation potential of the test substance was investigated in a study in accordance with OECD TG 305 and in compliance with GLP criteria. In this study, killifish (Oryzias latipes) were exposed to radiolabelled and non-radiolabelled (ratio of 1:1) test substance at concentrations of 1 and 10 µg/L in a flow-through during a 28-day uptake phase. Afterwards, fish were transferred to aquaria without the test substance in a 14-day depuration phase. Specific activities were confirmed by liquid scintillation counting (LSC) then radio-HPLC. The recoveries of 14C-labelled test substance from fish were 111.6 ± 12.4% and 118.2 ± 7.3% at the two different concentration levels. The concentrations recovered from water during the equilibration phase were 88.9 ± 1.3% and 99.1 ± 8.5% for low concentration and 98.2 ± 2.5% and 90.5 ± 0.7% for high concentration. The concentration of 14C-labelled test substance in water ranges were 0.45 - 0.55 and 4.44 - 5.47 µg/L for low and high concentration, respectively. Those values could be converted to 0.90 - 1.10 and 8.89 - 10.71 µg/L based on the ratio of labelled and non-labelled to 1:1.

The 14C-labelled test substance was rapidly accumulated in killifish. The steady-state reached after 4 days exposure in both low and high concentration levels. The average concentration at steady-state of high concentration level was 6665.8 ± 728.2 μg/kg, which was calculated by excluding the value of 21 days sample to sustain the coefficient variation within 20%. For low concentration level, there was high variation at steady-state from 4 days to 28 days. Although the concentration of 21 days sample in low concentration level was excluded, the coefficient variation was 29.2%. However, these results were considered to be acceptable as there were not a statistically significant difference resulting from the One Way ANOVA analysis. Also, there was a statistically significant difference (P ≤ 0.001) between low and high concentration groups. The concentrations of the bound and extractable residues in fish at steady-state (25 days) were 99.4 and 4919.3 µg/kg, respectively. These values were 2 and 98% for the concentration of total radioactive residue and 1.5 and 73.8% for the average concentration at steady-state in high concentration, respectively. From the analysis of solvent extractable solution, no significant peak to be considered as metabolite was found in RI-HPLC chromatogram. The lipid contents were determined from control fish at day 0 and 42 and from exposed fish to high concentration at day 28 and 42 with each 5 replications. The average lipid contents were 7.2 and 7.0% for control fish and 7.1 and 6.3% for exposed fish.

The bioconcentration factor at steady-state (BCFss) was calculated from the ratio of Cfish/Cwater. The BCFss values were 1251.2 and 1276.9 for the low and high concentration levels, respectively. Uptake and depuration rate constants (ku and Kd) were also calculated. These values showed 684.041 and 0.570 for low concentration and 1008.410 and 0.840 for high concentration, respectively. Therefore, the ratios of ku/kd (BCFk) were 1200.1 and 1200.5 for low and high concentration, respectively. After the depuration phase, 0.3-1.0% of residues of the test material remained in the test organisms. Based on the findings, it was determined that the mean BCFss was determined to be 1264.