Reaction mass of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9RS)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide and of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9SR)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide; isopyrazam

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:

5 Sep 2006 to 19 Dec 2006

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:

June 1996

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1730 (Fish Bioconcentration Test)

Version / remarks:

April 1996

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

yes

Details on sampling:

- Determination of residue concentration in fish tissues by LSC: Samples of four fish were taken at each sampling time, terminated and individual weights were recorded. The fish were dissected into edible and non-edible portions. The portions of each tissue type from the four fish were then pooled and weighed into 50 or 100 mL glass bottles. Soluene was added to each sample (43 g equivalent to 50 mL) and the sealed vials were rotated on a roller for at least 48 hours until all tissues were solubilised. Three samples weighing between 0.402 - 1.199 g of solubilised fish tissues were then weighed into LSC vials. Then 0.5 mL acetic acid and 10 mL scintillator were added. Fish were taken from the test concentration on days 0 (1 h), 0.25 (6 h), 1, 2, 3, 7, 10 14, 21, and 28 to demonstrate when steady state concentration was reached. Fish were also removed from the blank control on days 0, 28 and 42. The solubilised fish samples were analysed for 14-C residues using LSC.
- Characterisation and quantzfication of parent residue in fish tissues by TLC: Once steady state (day 25) had been reached 24 fish were removed from the test tank for analysis of test item residues in whole fish and the determination of the lipid content. The sample was split into two sub-samples of 12 fish; one sub-sample was analysed, the other sub-sample was stored frozen as a back-up.
- Lipid content by gravimetric analysis: Lipid content was determined in eight fish from the control tank on days 0 and 42. Lipid content was also determined in test fish on day 25 (steady state); a sub-sample was taken from fish taken for residue analysis.


- Determination of test concentration in water by LSC: Three aliquots of 10 mL were taken from the centre of the relevant test vessel and placed into a 20 mL scintillation vial. Then 10 mL of OptiPhase HiSafe was added to each vial, shaken and then measured by the means of Liquid Scintillation Counting (LSC) for total radioactivity. Samples of the test concentration were taken prior to the addition of fish (equilibrium phase) on days -2 and -1. During the uptake phase samples were taken on a daily basis from the test concentration. Following the start of depuration samples, were taken after 1, 3 and 6 hours on day 28, then on days 29, 30, 3 1 32 and 42. Samples of the blank control on days -2, 0, 28 and 42 were taken to confirm the absence of radioactivity.
- Quantification of parent in water by TLC: Duplicate samples of 100 mL were taken on days - 1, 0, 7, 14, 2 1, 25, in water by TLC: 28 and 35 for the quantification of test item.
- Sample receipt and preparation: On the day of receipt in the Metabolism section, the fish were divided into subsamples ranging between 5 to 10 g.
- Sample storage conditions before analysis: Samples placed in a freezer (≤ -10 ˚C) and stored frozen prior to analysis.

Vehicle:

yes

Remarks:

DMF

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

The application solution was prepared by adding 1.5 mg (3.2 MBq) of 14C-test item to a 5 L mixture of dimethylformamide (DMF) and deionised water (1:9 v/v) to give a 300 µg/L solution.
- Controls: A solvent control application solution was prepared containing 5 L of DMFIdeionised water (1:9 v/v) only. Both application solutions were renewed periodically during the study.
- Dosing system: Both tanks were dosed during the uptake phase (days 0 to 28).

Test organisms (species):

Lepomis macrochirus

Details on test organisms:

TEST ORGANISM
- Common name: Bluegill sunfish
- Batch: BG/2006/02
- Supplier: Aquatic Research Organisms, 1 Lafayette Road, PO Box 1271, Hampton, NH 0843-1271, US.
- Length at study initiation: 5.1 ± 0.6 cm
- Weight at study initiation: 1.9 ± 0.7 g
- Food type (during test): Gamma Brine shrimp
- Amount: Between 1 - 2% body weight
- Batch mortality during 7 days preceding test: 0.3%

ACCLIMATION
- Acclimation period: 12 days
- Acclimation conditions: Same as test
- Environmental parameters: During acclimation, the temperature ranged from 21.5 - 21.9 ˚C, pH from 7.02 - 7.24 and dissolved oxygen from 84 - 92% air saturation.

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:

- Control: 200.0 - 2 7.4 mg CaCO3/L
- Test substance treated groups: 204.4 - 216.0 mg CaCO3/L

Test temperature:

- Control: 21.9 - 22.4
- Test substance treated groups: 21.9 - 22.6°C

pH:

- Control: 6.97 - 8.32
- Test substance treated groups: 6.89 - 8.24

Dissolved oxygen:

- Control: 78.8 - 111.6%
- Test substance treated groups: 90.1 - 110.2%

TOC:

- Control: 1.7- 7.3
- Test substance treated groups: 1.3 - 7.4 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 150 L glass tanks
- Type: Open soft
- Fill volume: Approximately 128 L of test solution
- Flow rate: The application solutions (flow rate 0.72 mL/min) were then diluted 1000-fold with dilution water (flow rate 43 L/h, giving eight water exchanges a day) to provide the test concentration and the solvent control; both containing 100 µL of DMF per litre of dilution water.
- No. of organisms per vessel: 100
- No. of vessels per concentration: 1
- No. of vessels per control: 1

TEST MEDIUM / WATER PARAMETERS
- Preparation of dilution water: The dilution water was prepared by mixing mains water with de-ionised water to give a total hardness of 100 - 250 mg CaCO3/L. The mixed water then passes through an activated carbon filter and a UV sterilizer to produce the dilution water. Analysis of representative samples of dilution water was conducted on at least a biannual basis for potential toxicants, pesticides, particulate matter, total organic carbon and metals.
- Intervals of water parameter measurement: Water temperature, dilution water flow and flow of application solution (only during uptake phase) were recorded on a daily basis in both test tanks. The temperature was additionally monitored continuously by means of a data logger in both tanks throughout the study phase. The dissolved oxygen, pH and TOC were measured at least once a week. Water hardness was measured at the start of the test and at the end of depuration.

OTHER TEST CONDITIONS
- Photoperiod: A 16-hour light : 8-hour dark photoperiod was maintained with a 30 minute dawn/dusk transition period.

Nominal and measured concentrations:

- Nominal concentration: 0.3 µg/L
- Measured concentration: 0.26 - 0.31 µg/L

Reference substance (positive control):

no

Lipid content:

6 %

Time point:

other: day 0

Remarks on result:

other:

Remarks:

control

Lipid content:

5.9 %

Time point:

other: day 25

Remarks on result:

other:

Remarks:

test substance treated group

Lipid content:

5 %

Time point:

other: day 42

Remarks on result:

other:

Remarks:

control

Key result

Conc. / dose:

0.3 µg/L

Temp.:

22 °C

pH:

7.8

Type:

BCF

Value:

360 L/kg

Basis:

whole body w.w.

Time of plateau:

42 d

Calculation basis:

kinetic

Remarks on result:

other: 5% lipid normalised; calculated outside of the study

Conc. / dose:

0.3 µg/L

Temp.:

22 °C

pH:

7.8

Type:

BCF

Value:

406 L/kg

Basis:

whole body w.w.

Time of plateau:

28 d

Calculation basis:

kinetic

Remarks on result:

other: reported in the original study

Conc. / dose:

0.3 µg/L

Temp.:

22 °C

pH:

7.8

Type:

BCF

Value:

391 L/kg

Basis:

whole body w.w.

Time of plateau:

28 d

Calculation basis:

steady state

Remarks on result:

other: 5% lipid normalised; calculated outside of the original study

Conc. / dose:

0.3 µg/L

Temp.:

22 °C

pH:

7.8

Type:

BCF

Value:

441 L/kg

Basis:

whole body w.w.

Time of plateau:

28 d

Calculation basis:

steady state

Remarks on result:

other: reported in the original study

Rate constant:

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

Value:

1 215.2

Rate constant:

overall depuration rate constant (d-1)

Value:

2.99

Details on results:

An overview of the results is provided in Table 1 - Table 10 in 'Any other information on results incl. tables'.
- Radiochemical purity and stability of test Item: Aliquots of the stock solution of the test item were analysed by TLC using both normal and
reversed-phase solvent systems. The radiochemical purity was determined at 97.6% three days before the start of the uptake phase and 95.4% 101 days after the start of the uptake phase. This confirmed the stability of test item throughout the study.
- Test Item Concentration in Water: The concentration of the test substance equivalents was determined by LSC during the accumulation phase and ranged from 0.26 - 0.31 µg/L, with an average concentration of 0.28 µg/L. The actual range was 93% - 111% of the average accumulation concentration. The average concentration during steady state was 0.29 µg/L. The levels of radioactivity decreased to a level of 0.02 µg/L the test susbstance equivalents within six hours after start of depuration. After 24 hours and throughout the rest of the depuration phase the measured concentration was not detectable. The radioactivity was characterised by TLC on days -1, 0, 7, 14, 21, 25 and 28 and confirmed the presence of > 95% test substance. During depuration (day 35), no test substance could be measured.
- Total radioactive residues (TRR) in fish tissues: 14C-residues were rapidly concentrated in both edible and non-edible portions of the fish. The concentrations reached a constant plateau after 10 days exposure. At steady state (days 10 - 28), the measured concentrations of test item equivalents in the edible, non-edible and in the whole fish tissues were 17, 283 and 128 µg/kg, respectively. At steady state, the concentration of the test item in the whole fish was within ± 25% of the mean steady state concentration for at least 4 successive samples taken at intervals of at least 3 days. The required ± 20 range of the mean could not be achieved (samples on days 10 and 14 gave 122 and 77%, respectively, of the mean steady state concentration). However, these values were considered to be acceptable as they were only slightly outside the criterion.
- Residue identification in fish tissues: Total fish tissue samples from day 25 of the accumulation phase were analyzed to determine the amount of parent test item. The extractability of radioactivity with organic and aqueous solvents at ambient temperature of the total fish tissues was >98% (unextractables 1.8%). Analysis of the extracts showed a low test item mean concentration (11.9%). Various unidentified residues were found in the organic and in the aqueous extraction phase. Unassigned radioactivity was found at a level of 28.2%. Non extractable radioactivity was determined at a level of 1.8%.
- Lipid Determination: The lipid content was determined in fish tissues from control fish at day 0 and day 42 and from exposed fish at day 25 and mean lipid levels were 6.0, 5.0 and 5.9% wlw respectively.
- Estimation of the bioconcentration factors: The measured bioconcentration factor (BCFss) was determined at steady state by comparing the concentrations of total 14C-residues as test item equivalents in whole fish tissues to the average concentration in water at steady state. The BCFss based on total 14C-residues in whole fish tissues at steady state was 441. The calculated bioconcentration factor (BCFk) was estimated from the ratio of the uptake rate constant to the depuration rate constant. The BCFk was estimated to be 406 in whole fish. The BCFparent (test item) based on the residue pattern in fish tissues at steady state (day 25) was 55.
- Uptake and depuration constants (ku and kd): After termination of the uptake phase, 14C-residues were rapidly eliminated from the edible tissues, non-edible and whole fish tissues giving DT-90 values of 1.76, 1.10 and 1.15 days. Uptake constants were 944.6, 1849.4 and 1215.2 for edible, non-edible and whole fish tissues respectively. Depuration constants were 15.8, 2.13 and 2.99 for edible, non-edible and whole fish tissues respectively.

Table 1. Test item equivalents in the test concentration

Phase	Incubation [days]	Radioactivity [µg equiv/L]	% of mean accumulation concentration	% of mean steady state concentration
Uptake	-2	0.26	93	90
	-1	0.26	93	90
	0	0.26	93	90
	1	0.26	93	90
	2	0.27	96	93
	3	0.28	100	97
	4	0.28	100	97
	5	0.29	104	100
	6	0.29	104	100
	7	0.26	93	90
	8	0.29	104	100
	9	0.28	100	97
	10	0.30	107	103
	11	0.27	96	93
	12	0.28	100	97
	13	0.30	107	103
	14	0.30	107	103
	15	0.27	96	93
	16	0.27	96	93
	17	0.26	93	90
	18	0.30	107	103
	19	0.30	107	103
	20	0.30	107	103
	21	0.29	104	100
	22	0.29	104	100
	23	0.28	100	97
	24	0.28	100	97
	25	0.29	104	100
	26	0.29	104	100
	27	0.30	107	103
	28	0.31	111	107
Depuration
	28.04	0.05	18	17
	28.13	0.03	11	10
	28.25	0.02	7	7
	29	n/d	n/a	n/a
	30	n/d	n/a	n/a
	31	n/d	n/a	n/a
	32	n/d	n/a	n/a
	42	n/d	n/a	n/a
Average accumulation		0.28	Not detectable	n/d
Average steady state		0.29	Not applicable	n/a
SD accumulation		0.015	RSD accumulation[%]	5.19
SD steady state		0.014	RSD steady state [%]	4.69
Average accumulation +20%		0.34	Average accumulation -20%	0.22
Average steady state +20%		0.35	Average steady state -20%	0.23

 

Table 2. Quantity of parent in the test concentration

Day	Concentration based on total radioactivity	Parent content
	(µg/L)	(%)
-1	0.26	96.1
0	0.26	98.6
7	0.26	96.4
14	0.30	95.8
21	0.29	96.7
25	0.29	96.6
28	0.31	96.6
35	n/d	n/a

n/d Not detectable

n/a Not applicable

 

Table 3: Uptake/depuration constants ku and kd

			Phase
Portion	Accumulation			Depuration
	Ku	Kd	C01	C02	Kd01	Kd02
Edible	944.6 (606.3)	15.7784 (10.3221)	26.3224 (6.2827)	0.2676 (3.6223)	1.3102 (0.7018)	0.02 (-)
Non-edible	1849.4 (1291.6)	2.1252 (1.5491)	196.3 (12.1929)	3.8749 (5.2007)	2.0910 (0.3359)	1E-08 (-)
Total	1215.2 (685.1)	2.9905 (1.7385)	108.8 (7.4420)	1.7629 (3.2471)	1.9984 (0.3520)	1E-08 (-)

() Numbers in parentheses are the approximate standard errors of the parameters

-       

 

Table 4: BCF estimates, DT50 and DT90

Portion	BCF	BCF parent	BCFk	DT1-50 (d)	DT1-90 (d)	DT2-50 (d)	DT2-90 (d)
Edible	59	-	59.9	0.53	1.76	34.66	115.13
Non-edible	976	-	870.3	0.33	1.10	n/d	n/d
Total	441	55	406.4	0.35	1.15	n/d	n/d

n/d Not displayed (see main appendix text for reasons why

-       

 

Table 5. test item equivalents in fish from the test concentration

	Incubation			Fish portions
Phase		Time	Edibles	Non- edibles	Total
		[days]	[µg/kg]	[µg/kg]	[µg/kg]
Accumulation		0	8.4	35	21
		0	14	99	55
			15	226	115
		2	24	193	104
		3	15	172	88
		7	14	156	74
	Steady State	10	19	327	156
	Steady State	14	15	252	98
	Steady State	21	16	242	113
	Steady State	25	18	329	152	Depuration as % Steady State
	Steady State	28	17	263	120	Edibles [%]	Non-edibles [%]	Total [%]	Day [d]
Depuration		28.04	20	178	98	-18	37	23	0.04
		28.25	28	133	76	-65	53	41	0.25
		29	2.6	14	8.1	85	95	94	1
		30	1.3	9.2	4.7	92	97	96	2
		31	1.3	7.1	3.7	92	97	97	3
		35	1.3	6.3	3.3	92	98	97	7
		42	1.0	4.0	2.0	94	99	98	14

 

Average at steady state:	17	283	128
Upper limit +20%	20	340	154
Lower limit -20%	14	226	102

 

Table 6. Parent and residue identification in tissues from fish from the test concentration

		Subsample 1		Subsample 2		Mean
TRR by summation mg/kg		0.139 1		0.123 1		0.131 1
TRR by LSC quantification mg/kg		0.128 2		0.128 2		0.128 2
Origin of Component	Component	% TRR 10	Residue 10 (mg/kg)	% TRR 10	Residue 10 (mg/kg)	% TRR 10	Residue 10 (mg/kg)
Chromatographed	Test item	13.4	0.018	10.3	0.013	11.9	0.016
	Organo-soluble Unknowns 4	7.3	0.010	6.8	0.008	7.1	0.009
	Aqueous soluble Unknowns 5	55.7	0.077	56.8	0.070	56.3	0.074
	Baseline/ solvent front 6	0.0	0.000	0.0	0.000	0.0	0.000
	Unassigned (remainder) 7	28.2	0.039	28.1	0.034	28.2	0.037
Not Chromatographed	Unextracted 8	2.0	0.003	1.5	0.002	1.8	0.002
	Gains on Fractionation 9	6.6	0.008	3.5	0.004	5.3	0.007
	Total	100.0	0.139	100.0	0.123	100.0	0.131

1 TRR determined by summation of radioactivity present in the extracts and debris following solvent extraction.

2 TRR determined by direct quantification employing LSC.

4 At least 2 discrete components, neither? > 5.3 & 4.9 % TRR (0.007 & 0.006 mg/kg) respectively.

5 At least 3 discrete components, none > 30.8 & 31.4 % TRR (0.043 & 0.038 mg/kg) respectively.

6 Baseline or solvent front material on TLC.

7 Percentages of all discrete components subtracted from the total percentage of TRR chromatographed. i.e. areas of streaked radioactivity and areas in between discrete bands.

8 Radioactivity remaining in the debris after the initial extraction.

9 The net cumulative incremental gains/losses during analysis. Calculated as (sum of all components) - 100.0%.

10 The slight discrepancy between the %TRR and residue figures within the data is due to rounding.

 

Table 7. Lipid content of fish

Sample		Lipid Content(% w/w)
		1st sub-sample	2nd sub-sample	Mean
Steady state	Treated	5.6	6.2	5.9
Day0	Control	5.9	6.1	6.0
Final Day	Control	5.0	5.0	5.0

 

5% lipid normalisation

The BCFk is 406 L/kg.

According to Table 7, the mean lipid content = (5.9% +6.0% +5.0%)/3 = 5.63%

Thus, the 5% lipid normalised BCFss = 406 L/kg * (5%/5.63%) = 360 L/kg.

The calculation is accroding to OECD TG 305 with regards on lipid correction.

Validity of the study

- The temperature variation during the uptake and depuration phases was less than h 2°C.

- The concentration of dissolved oxygen did not fall below 60% saturation during the whole study period.

- The concentrations of the test item (based on total radioactivity) in the water of the test aquaria were maintained within h 20% of the mean of the measured values during the accumulation phase.

- Fish showed no sub-lethal symptoms or mortality during the accumulation and depuration phases.

Validity criteria fulfilled:

yes

Remarks:

See validity of the study in ‘Any other information on results incl. tables’

Conclusions:

In a bioaccumulation study in bluegill sunfish (Lepomis macrochirus) following OECD TG 305 and OPPTS 850.1730, the kinetic bioconcentration factor (BCFk) were calculated to be 406 L/kg. After 5% lipic content normalization, the BCFk value was 360 L/kg.

Executive summary:

The bioaccumulation potential of the 14C-phenyl-ring-labelled test substance was investigated in a flow-through system in accordance with OECD TG 305 and OPPTS 850.1730 and in compliance with GLP criteria. In this study, bluegill sunfish (Lepomis macrochirus; 100 fish per concentration) were exposed to the 14C-labelled test substance at a nominal test concentration of 0.3 µg/L (measured concentration during the accumulation phase was 0.26 - 0.31 µg/L) for 28 days uptake phase and 14 days depuration. In addition, a negative control and a solvent control (DMF) groups were included as well and tested under the same condition. The test conditions were as following: pH 6.89 – 8.32, 21.9 – 22.6 °C, 16 hours light -8 hours darkness cycle with 30-minute dawn and dusk transition periods. The water conditions were 200.0 - 217.4 mg/L total hardness (as CaCO3), 1.3 – 7.4 mg/L TOC and 78.8 – 111.6% of saturation level. Fish tissue were sampled on day 0, 0.25, 1, 2, 3, 7, 10, 14, 21 and 28 days in the uptake phase, and on days 29, 30, 31, 35 and 42 during the depuration phase for total radioactivity residues analysis. Control fish were sampled on days 0, 28 and 42. The analysis was performed using LSC. Parent residues in whole fish were determined during steady state (on day 25) by TLC. The lipid content of the control fish was determined on days 0 and 42 and treated fish at steady state (on day 25). Water samples were taken on day -2, -1 days, then daily during the uptake phase. Following the start of the depuration phase, water samples were taken after 1, 3 and 6 hours, then daily from days 29 to 32. A final water sample was taken on day 42. Water samples were taken from the solvent control on days -2, 0, 28 and 42. These samples were analysed by TLC and LSC.

The results show that the14C-residues were rapidly concentrated in both edible and non-edible portions of the fish. The concentrations reached a constant plateau after 10 days exposure. At steady state (days 10 - 28), the measured concentrations of the test substance equivalents in the edible, non-edible and in the whole fish tissues were 17, 283 and 128 µg/kg, respectively. During the 14-day depuration phase, the 14C-residues were rapidly eliminated from the fish tissues with > 95% reduction after 2 days depuration and 98% after 14 days in dilution water only. The mean lipid content of fish on days 0, 42 and during steady state was 6.0, 5.0 and 5.9% w/w, respectively. The overall mean lipid content was 5.6% w/w. The calculated depuration time for 90% of the bioconcentrated residues (DT90) for the whole fish was 1.15 days. Based on these findings, the update rate constants were calculated to be 1215.2 L/kg/day for the whole fish tissues. The depuration rate constants were calculated to be 2.99/day for the whole fish tissues. The kinetic bioconcentration factor (BCFk) and steady state bioconcentration factor (BCFss) were determined to be 406 and 441 L/kg, respectively, for the whole fish wet weight. After 5% lipid content normalisation, the BCFk and BCFss values were 360 and 391 L/kg, respectively.

Description of key information

In a bioaccumulation study in bluegill sunfish (Lepomis macrochirus) the kinetic bioconcentration factor (BCFk) were calculated to be 406 L/kg, After 5% lipid content normalisation, the BCFk value was 360 L/kg , OECD TG 305 and OPPTS 850.1730, Behsen & Albuquerque 2007

Key value for chemical safety assessment

BCF (aquatic species):

360 L/kg ww

Additional information

One study is available for this endpoint which followed standard test guideline and complied with GLP (Reliability 1). In this study (Behsen & Albuquerque 2007), bluegill sunfish (Lepomis macrochirus) were exposed to the 14C-phenyl-ring-labelled test substance at nominal concentrations of 0.3 µg/L (measured concentration during the accumulation phase was 0.26 - 0.31 µg/L) for 28 days uptake phase and 14 days depuration (100 fish per concentration). In addition, a negative control and a solvent control (DMF) groups were included as well and tested under the same condition. The test conditions were as following: pH 6.89 – 8.32, 21.9 – 22.6 °C, 16 hours light -8 hours darkness cycle with 30-minute dawn and dusk transition periods. The water conditions were 200.0 - 217.4 mg/L total hardness (as CaCO3), 1.3 – 7.4 mg/L TOC and 78.8 – 111.6% of saturation level. The kinetic bioconcentration factor (BCFk) and steady state bioconcentration factor (BCFss) were determined to be 406 and 441 L/kg, respectively, for the whole fish wet weight. After 5% lipid content normalisation, the BCFk and BCFss values were 360 and 391 L/kg, respectively.