cyproconazole (ISO); (2RS,3RS;2RS,3SR)-2-(4-chlorophenyl)-3-cyclopropyl-1-(1H-1,2,4-triazol-1-yl)butan-2-ol

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

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)

Version / remarks:

1982

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

yes

Details on sampling:

- Uptake phase: Water and fish were sampled throughout the uptake period following the schedule outlined in Table in ‘Any other information on materials and methods incl. tables’. The water and tissue samples were radio assayed.
- Depuration phase: Water and fish were sampled according to the schedule in Table in ‘Any other information on materials and methods incl. tables’

Vehicle:

yes

Remarks:

DMF

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

To prepare the 14 C-labelled test substance bioconcentration stock solution. 7.50 g of non-radiolabeled test substance were weighed into a 50 mL volumetric flask and dissolved in DMF. The combined 14C-labelled test substance stock solution was quantitatively transferred to the flask and brought to volume. A 250 µL aliquot of the bioconcentration stock solution was transferred to a 100 mL volumetric flask and then filled to volume with acetone. This was designated the spiking solution. A five replicate LSC analysis was performed on 50 µL aliquots of the spiking solution. The 14C-activity of the spiking solution was determined to be 8,510 DPM/50 µL. The total 14C-activity in the stock solution was 3.40E+09 DPM as calculated from the spiking solution LSC analysis. This measured DPM was used to determine the amount of 14C-labelled test substance in the bioconcentration stock solution.

A working specific activity of 453 DPM/µg was calculated by correcting the given specific activity for the amount of non-radiolabeled test substance added. The concentration of the 14C-labelled test substance bioconcentration stock solution was 150 mg/mL. Diluter stock solutions were prepared from the primary stock (in dimethylformamide) as needed by transferring an 8.0 mL aliquot of the primary stock to a 250 mL volumetric flask and diluting with dimethylformamide. The resultant diluter stock (5760 mg/L) was then transferred to a graduated brown glass bottle for use by the diluter toxicant injection system.

The diluter system consisted of one 0.30 mg/L nominal concentration aquarium and one control aquaria. The control aquarium also had an amount of dimethylformamide solvent (0.1 mL) delivered equal to that delivered in the exposure aquarium.

Test organisms (species):

Lepomis macrochirus

Details on test organisms:

TEST ORGANISM
- Common name: Bluegill sunfish
- Lot number: 5586
- Holding period: All test fish wen held in culture tanks on a 16-hour daylight photoperiod and observed for at least 14 days prior to testing. A daily record of fish observations during the holding period, along with any prophylactic and therapeutic disease treatments, is included in the raw data.
- Feeding before test: During the holding, acclimation and test periods, the fish received a standard commercial fish food daily in an amount equivalent to approximately 3 percent of their body weight.
- Length at study initiation: 64 ± 4.3 mm
- Weight at study initiation: 8.0 ± 1.7 g

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

Test temperature:

20 - 23 °C

pH:

8.0 - 8.3

Dissolved oxygen:

6.5 - 9.1 mg O2/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 L aquaria
- Type of flow-through: Proportional diluter
- Flow rate: Approximately 362 mL/minute/aquarium (replace approximately 70 L test volume approximately 7.4 times in a 24 hour period.)
- No. of fish per concentration: 120

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Aerated well water
- Hardness: 225 - 275 ppm (as CaCO3)
- pH: 7.8 - 8.3
- Temperature: 15 - 20°C
- Alkalinity: 325 - 375 ppm (as CaCO3)
- Dissolved oxygen: 9.2 - 10.1 ppm
- Conductivity: 700 μmhos/cm

OTHER TEST CONDITIONS
- Uptake phase: Before initiating the uptake portion of the study, the test solution was allowed to flow through the test aquaria for a 24-hour equilibration period.
- Depuration phase: On day 28 of the exposure period, the addition of the 14C-labelled test material was terminated. At the beginning of the depuration phase, the water in the test aquarium was siphoned and refilled twice. The water in each test aquarium was removed by siphoning until a depth of approximately three inches of water remained in each aquarium. The aquaria were filled to a volume of approximately 70 L with uncontaminated well water. This water was then removed until a depth of approximately three inches of water remained and the aquaria were filled again with approximately 70 L of uncontaminated well water. The fish were then exposed to flowing uncontaminated well water (22°C) for 14 days.


OBSERVATIONS
- Uptake phase: These fish were observed initially and every 24 hours during the exposure period for any mortality and/or adverse behaviour.

Preliminary Dynamic Acute Toxicity Study
- Methods: In order to set the test concentration for the bioconcentration study with 14C-labelled test substance, a 7-day dynamic toxicity study was conducted to determine the acute toxicity of the test substance to bluegill sunfish (Lepomis macrochirus). The 7-day no-observed effect concentration of the test substance was the nominal test concentration of 5.3 mg/L, based on the lack of mortality and abnormal effects at this concentration.
- Results: The results of the preliminary dynamic acute toxicity study suggested that a 14C level of 0.30 mg/L, would be an appropriate test concentration for the bioconcentration study. This nominal concentration would provide safe concentration for evaluating the bioconcentration potential of 14C-labelled test substance.

Nominal and measured concentrations:

- Nominal concentration: 0 (solvent control), 0.30 mg/L
- Measured concentration: 0 (solvent control), 0.27 mg/L

Reference substance (positive control):

no

Details on estimation of bioconcentration:

A steady-state approach was used for evaluating the bioconcentration study. The steady state bioconcentration factor (BCF) for the uptake period were determined by dividing the 14C-labelled test substance in tissue concentration by the mean 14C-labelled test substance concentration in water up to and including that day. The uptake rate constant (K1) and depuration rate constant (K2) were determined by the two-compartment non-linear kinetic model Dow BIOFAC computer program.

Remarks on result:

not measured/tested

Key result

Conc. / dose:

0.27 mg/L

Temp.:

20 °C

pH:

8.1

Type:

BCF

Value:

28 L/kg

Basis:

whole body w.w.

Time of plateau:

3 d

Calculation basis:

steady state

Remarks on result:

other: Whole fish

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

0.87 d

Rate constant:

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

Value:

22

Rate constant:

overall depuration rate constant (d-1)

Value:

0.79

Details on kinetic parameters:

The uptake rate constant (K1), depuration rate constant (K 2), time to one-half clearance, and steady-state bioconcentration factor (BCF) for whole fish were determined using the BIOFAC non-linear kinetic modelling computer program for the bioconcentration study. The optimal parameter estimates were determined to be:
- Uptake Rate Constant (K 1), ppm fish/ppm water/day: 22 ± 3.3
- Clearance Rate Constant (K 2), Day: 0.79 ± 0.12
- T (¼) for Clearance, Day: 0.87 ± 0.13
- Bioconcentration Factor (BCF), ppm fish/ppm water: 28 ± 6.0
- Time to Reach 90% of Steady-State, Days : 2.9 ± 0.45

Details on results:

An overview of the results is provided in Table 2 – Table 4 in ‘Any other information on results incl. tables’.

Water concentrations of the 14C-labelled test substance ranged from 0.24 mg/L to 0.30 mg/L 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 0.27 ± 0.021 mg/L. These concentrations compared favourably with the expected nominal concentration of 0.30 mg/L for 14C-labelled test substance.
The tissue residues after 28 days of exposure were 1.8 ppm for fillet, 5.9 ppm for whole fish, and 11 ppm for viscera. These values corresponded to day 28 bioconcentration factors of 6.7, 22 and 41, respectively. Daily bioconcentration factors for the uptake phase of the study ranged from 3.9 to 15 for fillet, 8.2 to 34 for whole fish, and 13 to 59 for viscera.
An analysis of depuration rates by day 14 of the elimination period showed > 83, 93 and 94 percent depuration in fillet, whole fish and viscera, respectively. The depuration data indicated an initial rapid decrease in tissue concentration. The fillet concentration of 14C-labelled test substance dropped from a day 28 uptake value of 1.8 ppm to less than minimum quantifiable limits (MQL) by day 14 of the depuration period. Whole fish levels decreased from 5.9 ppm on day 28 uptake to 0.41 ppm by the end of the study; whereas, viscera concentrations dropped from 11 ppm on day 28 uptake to 0.61 ppm by day 14 depuration.
Minimum quantifiable limits (MQL) data for scintillation counting for water fillet, whole fish and viscera for 14C-labelled test substance were 0.00716 ppm, 0.304 ppm, 0.299 ppm and 0.306 ppm, respectively. Mean recovery data for 14C-labelled test substance in tissue sample oxidations were 98% - 99% for all tissue types. 14C-Benzoic Acid instrument combustion recovery, in the absence of tissue, averaged 99% for the study.

A non-linear two-compartment kinetic modelling computer program (BIOFAC) was used for analysis of the uptake/depuration whole fish data.
This method estimated the uptake rate constant (K1) of 22 ± 3.3 ppm in fish/ppm in water/day, clearance rate constant (K2) of 0.79 ± 0.12 day , time for half clearance of 0.87 ± 0.13 days, bioconcentration factor (BCF) of 28 ± 6.0 ppm in fish/ppm in water and time to reach 90% of steady-state of 2.9 ± 0.45 days. The BIOFAC calculated BCF value was 128% of the average observed whole fish bioconcentration factor (26) for 14C-labelled test substance fish residues for days 3, 7, 14, 21 and 28.

Table 2. Total 14 C-test substance in Test Water and Fish Tissue During 28 Days Exposure and 14 Days Depuration with Bluegill Sunfish.

	Total 14C concentration as 14C labelled test substance a
	Water	Fillet		Whole fish		Viscera
Day	ppm	ppm	BCF b	ppm	BCF b	ppm	BCF b
Update
0 c	0.30
0.17	0.27	1.1	3.9	2.3	8.2	3.6	13
1	0.28	1. 6	5.7	5.3	19	7.7	28
3	0.29	2.8	10	5.9	21	13	46
7	0.25	4.2	15	6.7	24	9.5	34
14	0.25	2.2	8.1	9.1	34	16	59
21	0.24	2.1	7.8	7.3	27	11	41
28	0.26	1.8	6.7	5.9	22	11	41
Depuration
1	d	0.97		4.9		9.1
3	d	d		0.45		2.5
7	d	d		0.44		0.66
10	d	d		0.40		0.54
14	d	d		0.41		0.61

a. All values have been rounded to represent two significant figures.

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.

c. Samples taken immediately prior to addition of fish.

d. Below minimum quantifiable limit.

Table 3. Depuration of Total 14C Calculated as 14C-labelled test substance from Bluegill Sunfish During a 14-Day Clearance Period. 

	Fillet		Whole Fish a		Viscera a
Depuration day	Depuration concentration (ppm)	Percent depuration a	Depuration concentration (ppm) b	Percent depuration	Depuration concentration (ppm) b	Percent depuration
	0.97a	46	4.9	17	9. 1	17
3	<0.304	>83	0.45	92	2.5	77
7	<0.304	>83	0.44	93	0.66	94
10	<0.304	>83	0.40	93	0.54	95
14	<0.304	>83	0.41	93	0.61	94

a. These values have been rounded to two significant figures.

b Depuration rates are expressed as a percentage of the day 28 14C-labelled test substance concentrations of 1.8 ppm for fillet, 5.9 ppm for whole fish, and 11 ppm for viscera.

Note: The average minimum quantifiable limits (MQL) for fillet, whole fish and viscera were 0.304, 0.299 and 0.306 ppm, respectively.

Table 4. Recovery Data for Sample Oxidation of Tissue During the Bioconcentration study with Bluegill Sunfish Exposed to Total 14C Calculated as 14C-labelled test substance

	Percent Recovery of 14 C-labelled test substance
Day	Fillet c	Whole fish c	Viscera c	Percent recovery of 14C-labelled Benzoic acid c,d
Update
0.17, 1, 3	95	96	95	100
	94	96	98
7, 14, 21	99	99	96	98
	97	100	97
28 e	e	e	e	e
Depuration
1, 3	100	102	98	97
	101	101	100
7, 10, 14	100	98	101	101
	101	101	100
Mean	98	99	98	99
S.D.	2.7	2.3	2.1	1.8

a. Days that appear on the same line indicates that samples for these dates were processed together; therefore, only one set of recoveries were used for data sets shown.

b. Recovery samples were spiked with 8,510 DPM 14C-labelled test substance

c Values rounded to whole percentages.

d. Used to determine instrument recoveries.

e. Combusted with days 1 and 3 depuration samples.

Validity criteria fulfilled:

yes

Conclusions:

Based on the findings, the BCF was determined to be 28 L/kg in whole fish and the steady-state was reached on day 3. The overall uptake rate constant K1 was 22 ± 3.3 L/kg/day, and the overall depuration rate constant (K2) was 0.79 ± 0.12 /day. The DT50 of the substance was 0.87 ± 0.13 days.

Executive summary:

The bioaccumulation potential of the test substance was investigated in a flow-through study in accordance with EPA165-4 guideline and in compliance with GLP criteria. In this study, bluegill sunfish (Lepomis macrochirus) were exposed to radiolabelled test substance at concentrations of 0.27 mg/L for 28-day (uptake phase, 120 fish per concentration). 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).

The tissue residues after 28 days of exposure were 1.8 ppm for fillet, 5.9 ppm for whole fish, and 11 ppm for viscera. These values corresponded to day 28 bioconcentration factors of 6.7, 22 and 41, respectively. Daily bioconcentration factors for the uptake phase of the study ranged from 3.9 to 15 for fillet, 8.2 to 34 for whole fish, and 13 to 59 for viscera. An analysis of depuration rates by day 14 of the elimination period showed > 83, 93 and 94 percent depuration in fillet, whole fish and viscera, respectively. The fillet concentration of 14C-labelled test substance dropped from a day 28 uptake value of 1.8 ppm to less than minimum quantifiable limits (MQL) by day 14 of the depuration period. Whole fish levels decreased from 5.9 ppm on day 28 uptake to 0.41 ppm by the end of the study; whereas, viscera concentrations dropped from 11 ppm on day 28 uptake to 0.61 ppm by day 14 depuration. Minimum quantifiable limits (MQL) data for scintillation counting for water fillet, whole fish and viscera for 14C-labelled test substance were 0.00716 ppm, 0.304 ppm, 0.299 ppm and 0.306 ppm, respectively. Mean recovery data for 14C-labelled test substance in tissue sample oxidations were 98% - 99% for all tissue types. 14C-Benzoic Acid instrument combustion recovery, in the absence of tissue, averaged 99% for the study.

Based on the findings, the non-linear two-compartment kinetic modelling determined that BCF was 28 L/kg in whole fish and the steady-state was reached on day 3. The overall uptake rate constant K1 was 22 ± 3.3 L/kg/day, and the overall depuration rate constant (K2) was 0.79 ± 0.12 /day. The DT50 of the substance was 0.87 ± 0.13 days.

Description of key information

BCF = 28 L/kg, whole fish, Lepomis macrochirus, aqueous, freshwater, EPA OPP 165-4, Forbis 1986

Key value for chemical safety assessment

BCF (aquatic species):

28 L/kg ww

Additional information

One study is available for this endpoint. The flow-through study was conducted in accordance with EPA165-4 guideline and in compliance with GLP criteria (Reliability 1). Bluegill sunfish (Lepomis macrochirus) were exposed to radiolabelled test substance at concentrations of 0.27 mg/ L for 28-day (uptake phase, 120 fish per concentration). 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). The non-linear two-compartment kinetic modelling determined that BCF was 28 L/kg in whole fish and the steady-state was reached on day 3. The overall uptake rate constant K1 was 22 ± 3.3 L/kg/day, and the overall depuration rate constant (K2) was 0.79 ± 0.12 /day. The DT50 of the substance was determined to be 0.87 ± 0.13 days.