1-(3-chloropyridin-2-yl)-N-[4-cyano-2-methyl-6-(methylcarbamoyl)phenyl]-3-{[5-(trifluoromethyl)-2H-tetrazol-2-yl]methyl}-1H-pyrazole-5-carboxamide

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:

31 Oct 2014 - 09 Feb 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 305 (Bioaccumulation in Fish: Aqueous and Dietary Exposure) -I: Aqueous Exposure Bioconcentration Fish Test

Version / remarks:

adopted 2 October 2012

Deviations:

yes

Remarks:

The pH range was slightly out of the allowed range of ±0.5 units, but the pH was well within the overall allowed pH range.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1730 (Fish Bioconcentration Test)

Version / remarks:

Draft version (1996)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Arbeit, Integration und Soziales des Landes Nordrhein-Westfalen, Düsseldorf, Germany

Radiolabelling:

yes

Details on sampling:

- Sampling intervals/frequency for test organisms:
Fish samples – radioactivity measurement: On day 1, 3, 7, 10, 14, 21, 28, 29, 31, 35, 38 and 42 four fish per aquarium were killed and specific compartments were analysed
Fish samples – lipid content: On day 0, 28 and 42 four fish were sampled out of three aquaria; the lipid concentration was measured in the whole fish
Fish samples – biotransformation: For the biotransformation part 15 fish were sampled after 7 days and 14 days, respectively. The length and weight were measured and the fish were dissected into edible tissues and viscera/ non-edible parts. coarse pieces of the edibles or viscera of each day were combined and homogenised with a high speed blender. From these samples, a sub-sample was taken for extraction and analysis

- Sampling intervals/frequency for test medium samples: stock solutions and water samples from test chambers were sampled;
Water samples for the radioactivity measurements were taken at: a.) Day -1, 0, 1, 3, 7, 10, 14, 21, 28, 29, 31, 35, 38 and 42; b.) Day –1, 0, 1, 3, 7, 10 and 14; On the sampling days 3 aliquots of 10 mL sample and 10 mL Quickszint 401 were taken.
Water samples for the measurements of the biotransformation part were taken at Day 0, 1, 3, 7, 10, 14, 21, 28, 29, 31, 35, 38 and 42 → 2* 1.00 L; Samples from all sampling dates were analysed. b.) Day 7 and 14 (aquarium D) → 2 * 1.00 L
Stock solution samples for the radioactivity measurements were taken at: Day – 4; Day -1, freshly prepared and aged; Day 3, freshly prepared and aged; Day 10, freshly prepared and aged; Day 14, aged; Day 17, freshly prepared and aged; Day 24, freshly prepared and aged; Day 28, aged; Out of each stock solution 3 * 100 μL were taken and 9.9 mL test water and 10 mL Quickszint 401.
Stock solution samples for the measurements of the of the stability of the test item in the stock solutions were taken at: Day – 5, 14 and 28. Out of each stock solution 50 to 500 μL were taken

- Sample storage conditions before analysis: samples were stored deep-frozen until analysis

Vehicle:

yes

Remarks:

Dimethylformamide (DMF)

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

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The radioactive test compound was delivered in solid form and was solved in a volume of 50 mL acetonitrile containing 0.1% formic acid. From this solution the individual stock solutions for the study in DMF were prepared. After depletion of the stock solution in acetonitrile a further stock solution with a new aliquot of the radioactive test item of the same batch was prepared by dissolving the test compound in 10 mL acetonitrile containing 0.1% formic acid. The stock solutions introduced in the study were prepared by dissolving a certain amount of inactive test item and the in acetonitrile dissolved radioactive test item in DMF. A dosing system and flow-meters were used for the introduction of the substance and test water in 2000 mL mixing cells. The mixture was running continuously into the 100 L test aquaria to result in test concentrations of 0.03 and 0.3 mg/L, respectively.
- Controls: only solvent control: test medium without addition of the test item but with addition of the solvent
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): dimethylformamide (DMF)
- Concentration of vehicle in test medium (stock solution and final test solution(s) at different concentrations and in control(s)): 100 μL/L DMF, 1 μL/L acetonitrile and 0.001 μL formic acid

Test organisms (species):

Lepomis macrochirus

Details on test organisms:

TEST ORGANISM
- Common name: bluegill sunfish
- Source: Osage Catfisheries, Osage Beach, Missouri, USA (held in the laboratory since 2013-03-20)
- Length at study initiation (length definition, mean, range and SD): 7.7 cm (mean, bioconcentration part) and 10 cm (mean, biotransformation part fish)
- Weight at study initiation (mean and range, SD): and the mean body weight was 7.6 g (mean, bioconcentration part) and a mean body weight of 15.4 g (mean, biotransformation part fish).
- Health status: A prophylactic treatment with oxytetracyclin-hydrochloride (4g / 100L water) was performed immediately after the fish arrived in the laboratory. No mortality was noted 14 d prior to the test initiation.
- Feeding during test: yes
- Food type: Fish were fed with a commercial fish diet (e.g. Brutfutter Inicio (formerly Ecostart 17) BioMar, Denmark)
- Amount: a rate of 1 to 2% of fish body weight; amount of food was recalculated at regular intervals based on the mean body weight of sampled fish; feed was analysed for heavy metal and pesticide concentrations
- Frequency: daily

ACCLIMATION
- Acclimation period: After arrival until start of the test all fish were held in aquaria under flow-through conditions. All fish were held at a 16 to 8 hour light/dark period. The fish were fed daily with a commercial fish diet (e.g. Brutfutter Inicio (formerly Ecostart 17) BioMar, Denmark) at a rate of 1 to 2% of body weight. Based on the mean body weight of sampled fish the amount of food was recalculated at regular intervals. No mortality was noted 14 days prior to the test initiation. Only healthy fish (visual inspection for injuries, deformations, etc.) were used.

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:

2.7 ± 0.3 °dH

Test temperature:

22.8 - 23.3 °C (solvent control)
22.7 - 23.4 °C (0.03 mg/L)
22.6 - 23.4 °C (0.3 mg/L)

pH:

6.8 - 7.4 (solvent control)
6.8 - 7.4 (0.03 mg/L)
6.9 - 7.4 (0.3 mg/L)

Dissolved oxygen:

70 - 93% (solvent control)
67 - 91% (0.03 mg/L)
69 - 98% (0.3 mg/L)

TOC:

< 2.00 - 46.6 mg/L (solvent control)
< 2.00 - 45.7 mg/L (0.03 mg/L)
< 2.00 - 49.0 mg/L (0.3 mg/L)

Conductivity:

< 10.0 μS/cm

Details on test conditions:

TEST SYSTEM
Test vessel
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: Four glass aquaria with a size of 780 mm (L) x 360 mm (W) x 380 mm (H); filled up with water to 360 mm resulting in a water volume of 100 L
- Aeration: All aquaria were aerated until test termination in order to avoid <60% oxygen saturation. Aerated test water was transferred into the glass aquaria at an average rate of approximately 25 L / hour / aquarium during the exposure and the depuration period. This amount was sufficient to replace the approximately 100 liter test volume about 6 times in a 24 hour period.
- Type of flow-through (e.g. peristaltic or proportional diluter): A dosing system consisting of a ProMinentR mikro g/5a dispenser (for dosing of stock solution) and flow-meters (for water flow control) were used for the introduction of [pyrazole-carboxamide-14C]test item and test water in 2000 mL mixing cells. The mixture was running continuously into the 100 L test aquaria.
- Renewal rate of test solution (frequency/flow rate): The stock solutions with [pyrazole-carboxamide-14C] test item in dimethylformamide were transferred at a rate of 2.5 mL/h.
- No. of organisms per vessel: 70
- No. of vessels per concentration (replicates): one aquarium for 0.03 and 0.3 mg test item/L in the bioconcentration part of the test (Aquarium B and C, respectively) and 0.3 mg test item/L in the biotransformation part of the test (Aquarium D); Solvent control was performed in parallel (Aquarium A).
- Biomass loading rate: The biomass loading rate was between 0.77 - 0.84 g fish (wet weight) per litre of test medium per day.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water according to ISO
- Particulate matter: < 5 mg/L (total solids)
- chemical oxygen Demand: < 5mg/L
- Ionic concentrations: Ca2+ 0.384 mmol/L; Mg2+ 0.096 mmol/L; Na+ 0.148 mmol/L; K+ 0.015 mmol/L; Cl- 0.783 mmol/L; HCO3- 0.148 mmol/L; SO4- 0.096 mmol/L; Ratio of Na : K 10 : 1 (based on molarity);
- Total organic carbon: < 2.00 to 64.7 mg/L
- Inorganic contaminants: 1 μg/L Al, Zn; < 1 μg/L: As; Cr; Cu; Co; Fe; Pb; Ni; < 100 μg/L Bor; F- ; < 0.1 μg/L Cd; Hg; Ag; < 0.03 mg/L NH4+
- Pesticides: < 0.05 µg/L
- Chlorine (residual):< 0.01 mg/L
- Organochlorine Contaminants: < 0.01 µg/L
- Ca/mg ratio:4 : 1 (based on molarity)
- Intervals of water quality measurement: The temperatures, pH, the concentration of dissolved oxygen were measured in all treatment groups at test start (day 0) and once a week and continuously by a data logger in the control aquarium; The total organic carbon content was measured prior to test start, at test start, day 7, 14, 21, 28, 35 and 42. the total hardness was manually measured twice a week and the conductivity was hourly measured by a data logger

OTHER TEST CONDITIONS
- Photoperiod: 16 to 8 hour light/dark period
- Light intensity: warmwhite fluorescent light

RANGE-FINDING / PRELIMINARY STUDY
- The acute LC50 was above the limit of solubility in fish medium of 10 mg test item/L and fathead minnow. An early life stage test (OECD 210) with fathead minnow revealed only minor effects at a nominal concentration of 5 mg test item/L with an overall NOEC of 2.5 mg test item/L. Therefore, 0.03 and 0.3 mg test item/L (aquarium B and C) were chosen as test concentrations for the bioconcentration part of the study.

Nominal and measured concentrations:

0.03 and 0.3 mg/L (nominal)

Reference substance (positive control):

no

Lipid content:

7.31 %

Time point:

start of exposure

Remarks on result:

other: Mean

Lipid content:

6.45 %

Time point:

end of exposure

Remarks on result:

other: Mean

Lipid content:

5.79 %

Time point:

other: day 42 (after depuaration duration)

Remarks on result:

other: Mean

Lipid content:

6.88 %

Time point:

other: from day 0 to 28

Remarks on result:

other: mean lipid value used for lipid normalization

Conc. / dose:

0.03 mg/L

Temp.:

22.7 °C

pH:

7.3

Type:

BCF

Value:

0.176 dimensionless

Basis:

edible fraction

Calculation basis:

steady state

Remarks on result:

other: based on TRR

Conc. / dose:

0.03 mg/L

Temp.:

22.7 °C

pH:

7.3

Type:

BCF

Value:

1.31 dimensionless

Basis:

other: viscera tissue

Calculation basis:

steady state

Remarks on result:

other: based on TRR

Conc. / dose:

0.03 mg/L

Temp.:

22.7 °C

pH:

7.3

Type:

BCF

Value:

0.576 dimensionless

Basis:

whole body w.w.

Calculation basis:

steady state

Remarks on result:

other: based on TRR

Conc. / dose:

0.3 mg/L

Temp.:

22.6 °C

pH:

7.2

Type:

BCF

Value:

0.108 dimensionless

Basis:

edible fraction

Calculation basis:

steady state

Remarks on result:

other: based on TRR

Conc. / dose:

0.3 mg/L

Temp.:

22.6 °C

pH:

7.2

Type:

BCF

Value:

0.667 dimensionless

Basis:

other: viscera tissue

Calculation basis:

steady state

Remarks on result:

other: based on TRR

Conc. / dose:

0.3 mg/L

Temp.:

22.6 °C

pH:

7.2

Type:

BCF

Value:

0.136 dimensionless

Basis:

whole body w.w.

Calculation basis:

steady state

Remarks on result:

other: based on TRR

Conc. / dose:

0.03 mg/L

Temp.:

22.7 °C

pH:

7.3

Type:

BCF

Value:

0.217 dimensionless

Basis:

edible fraction

Calculation basis:

kinetic

Remarks on result:

other: based on TRR

Conc. / dose:

0.03 mg/L

Temp.:

22.7 °C

pH:

7.3

Type:

BCF

Value:

1.45 dimensionless

Basis:

other: viscera tissue

Calculation basis:

kinetic

Remarks on result:

other: based on TRR

Conc. / dose:

0.03 mg/L

Temp.:

22.7 °C

pH:

7.3

Type:

BCF

Value:

0.622 dimensionless

Basis:

whole body w.w.

Calculation basis:

kinetic

Remarks on result:

other: based on TRR

Conc. / dose:

0.3 mg/L

Temp.:

22.6 °C

pH:

7.2

Type:

BCF

Value:

0.181 dimensionless

Basis:

edible fraction

Calculation basis:

kinetic

Remarks on result:

other: based on TRR

Conc. / dose:

0.3 mg/L

Temp.:

22.6 °C

pH:

7.2

Type:

BCF

Value:

0.697 dimensionless

Basis:

other: viscera tissue

Calculation basis:

kinetic

Remarks on result:

other: based on TRR

Conc. / dose:

0.3 mg/L

Temp.:

22.6 °C

pH:

7.2

Type:

BCF

Value:

0.351 dimensionless

Basis:

whole body w.w.

Calculation basis:

kinetic

Remarks on result:

other: based on TRR

Rate constant:

growth-corrected half-life (d)

Remarks:

0.03 mg test item/L

Value:

2.12

Remarks on result:

other: edible tissue

Remarks:

Since no growth occurred the corrected values are similar to the respective non-corrected values

Rate constant:

growth-corrected half-life (d)

Remarks:

0.03 mg test item/L

Value:

1.15

Remarks on result:

other: viscera tissue

Remarks:

Since no growth occurred the corrected values are similar to the respective non-corrected values

Rate constant:

growth-corrected half-life (d)

Remarks:

0.03 mg test item/L

Value:

1.24

Remarks on result:

other: whole fish

Remarks:

Since no growth occurred the corrected values are similar to the respective non-corrected values

Rate constant:

growth-corrected half-life (d)

Remarks:

0.3 mg test item/L

Value:

6.74

Remarks on result:

other: edible tissue

Remarks:

Since no growth occurred the corrected values are similar to the respective non-corrected values

Rate constant:

growth-corrected half-life (d)

Remarks:

0.3 mg test item/L

Value:

1.11

Remarks on result:

other: viscera tissue

Remarks:

Since no growth occurred the corrected values are similar to the respective non-corrected values

Rate constant:

growth-corrected half-life (d)

Remarks:

0.3 mg test item/L

Value:

1.58

Remarks on result:

other: whole fish

Remarks:

Since no growth occurred the corrected values are similar to the respective non-corrected values

Rate constant:

growth rate constant (d-1)

Remarks on result:

not determinable

Remarks:

for all compartments

Rate constant:

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

Remarks:

0.03 mg test item/L

Value:

0.071

Remarks on result:

other: ±0.0421

Remarks:

edible tissue

Rate constant:

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

Remarks:

0.03 mg test item/L

Value:

0.875

Remarks on result:

other: ± 0.0501

Remarks:

viscera tissue

Rate constant:

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

Remarks:

0.03 mg test item/L

Value:

0.348

Remarks on result:

other: ± 0.0266

Remarks:

whole fish

Rate constant:

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

Remarks:

0.3 mg test item/L

Value:

0.019

Remarks on result:

other: ± 0.0511

Remarks:

edible tissue

Rate constant:

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

Remarks:

0.3 mg test item /L

Value:

0.437

Remarks on result:

other: ± 0.0378

Remarks:

viscera tissue

Rate constant:

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

Remarks:

0.3 mg test item /L

Value:

0.154

Remarks on result:

other: ± 0.0193

Remarks:

whole fish

Rate constant:

overall depuration rate constant (d-1)

Remarks:

0.03 mg test item/L

Value:

0.326

Remarks on result:

other: ± 0.0795

Remarks:

edible tissue

Rate constant:

overall depuration rate constant (d-1)

Remarks:

0.03 mg test item/L

Value:

0.602

Remarks on result:

other: ± 0.0608

Remarks:

viscera tissue

Rate constant:

overall depuration rate constant (d-1)

Remarks:

0.03 mg test item/L

Value:

0.6

Remarks on result:

other: ± 0.0719

Remarks:

whole fish

Rate constant:

overall depuration rate constant (d-1)

Remarks:

0.3 mg test item/L

Value:

0.103

Remarks on result:

other: ± 0.633

Remarks:

edible tissue

Rate constant:

overall depuration rate constant (d-1)

Remarks:

0.3 mg test item/L

Value:

0.627

Remarks on result:

other: ± 0.222

Remarks:

viscera tissue

Rate constant:

overall depuration rate constant (d-1)

Remarks:

0.3 mg test item/L

Value:

0.44

Remarks on result:

other: ± 0.189

Remarks:

whole fish

Rate constant:

growth-corrected depuration rate constant (d-1)

Remarks:

0.03 mg test item/L

Value:

0.326

Remarks on result:

other: ± 0.0795

Remarks:

edible tissue

Rate constant:

growth-corrected depuration rate constant (d-1)

Remarks:

0.03 mg test item/L

Value:

0.602

Remarks on result:

other: ± 0.0608

Remarks:

viscera tissue

Rate constant:

growth-corrected depuration rate constant (d-1)

Remarks:

0.03 mg test item/L

Value:

0.6

Remarks on result:

other: ± 0.0719

Remarks:

whole fish

Rate constant:

growth-corrected depuration rate constant (d-1)

Remarks:

0.3 mg test item/L

Value:

0.103

Remarks on result:

other: ± 0.633

Remarks:

edible tissue

Rate constant:

growth-corrected depuration rate constant (d-1)

Remarks:

0.3 mg test item/L

Value:

0.627

Remarks on result:

other: ± 0.222

Remarks:

viscera tissue

Rate constant:

growth-corrected depuration rate constant (d-1)

Remarks:

0.3 mg test item/L

Value:

0.44

Remarks on result:

other: ± 0.189

Remarks:

whole fish

Metabolites:

Characterisation and Identification of Metabolites in Fish and in Test Water:
In the biotransformation part of the study no metabolites were identified due to the low amount of radioactivity in fish and therefore no metabolic pathway was proposed.

Results with reference substance (positive control):

No reference substance was tested.

Details on results:

- Mortality of test organisms: mortality or other adverse effects/disease in both control and treated fish was less than 10 % at the end of the test.
- Behavioural abnormalities: no symptoms were recorded for the solvent control and both exposed aquaria
- Observations on body length and weight: mean body length 7.7 cm; mean body weight was 7.6 g
- Other biological observations: no mortalities or sublethal effects were recorded within the test duration
- Organ specific bioaccumulation: only low concentrations of the test item were found in the fish tissue which rapidly decreased in the depuration period.
- Mortality and/or behavioural abnormalities of control: no mortalities or sublethal effects were recorded within the test duration

The mortality or other adverse effects/disease in both control and treated fish is less than 10% at the end of the test. No mortalities or sublethal effects were recorded within the test duration.
The analysis of stock solutions of the test compound from all aquariums showed that the test item was stable in the stock solutions during the exposure phase. The HPLC chromatograms of the stock solutions before and after the exposure experiments showed a purity of greater or equal than 98 %.

In the uptake phase the measured concentrations at the lower treatment level of nominally 0.03 mg/L ranged between 26.1 and 35.3 μg/L with a mean of 32.1 μg/L. At nominally 0.3 mg/L the measured concentrations ranged from 260 to 387 μg/L with a mean value of 345 μg/L.
Details of biological and analytical results are summarized within the tables 1 -3 in section "Any other information on results incl. tables".

Reported statistics:

The uptake rate constant (Ku) and depuration rate constant (Kd) and the kinetic biocconcentration factor were determined by using the program Origin 8.6.0 G (64-bi) Sr3b99. This is a non-linear kinetic modelling programme which provides optional parameter estimates of rate constants k1 and k2 by utilising the actual (observed) bioconcentration study data. The kinetic bioconcentration factor (BCFK) was calculated using Origin 8.6.0G (64-bit) Sr3 b99 non-linear kinetic modelling computer program.

Analytical Results:

The analysis of stock solutions of the test compound from all aquariums showed that the test item was stable in the stock solutions during the exposure phase. On the basis of the results of this study it was concluded that the test item was metabolized in fish. Due to the low amount of radioactivity in fish, no metabolites were identified and therefore no metabolic pathway was proposed. In the uptake phase, the measured concentrations at the lower treatment level of nominal 0.03 mg/L ranged between 26.1 and 35.3 µg/L with a mean of 32.1 µg/L. At nominally 0.3 mg/L the measured concentrations ranged from 260 to 387 µg/L with a mean value of 345 µg/L.

Table 1: Distribution of radioactive residues in the different extracts

	edible parts of fish				viscera of fish
	day 7		day 14		day 7		day 14
TRR [mg/kg] =	0.048		0.059		0.117		0.124
Fraction	% TRR	mg/kg	% TRR	mg/kg	% TRR	mg/kg %	% TRR	mg/kg
Aqueous phase	100	0.048	100	0.059	67.5	0.079	72.1	0.089
Organic phase	n.q.	n.q	n.q	n.q	32.5	0.039	27.9	0.035
Total conventional extracted	100	0.048	100	0.059	100	0.117	100	0.124
Post extraction solids (PES)	n.q.	n.q.	n.q.	n.q.	n.q.	n.q.	n.q.	n.q.
Accountability	100	0.048	100	0.059	100	0.117	100	0.124

n.q: not quantified (< LOQ)

TRR: total radioactive residue

Table 2: Substance uptake and depuration constants and bioconcentration Factors

	0.03 mg/L			0.3 mg/L
Cw Chemical concentration in water [μg L-1]	32.1 ± 3.71			345 ± 37.8
	Edible tissue	viscera tissue	whole fish	Edible tissue	viscera tissue	whole fish
Cf Chemical concentration in fish at steady-state [mg kg-1]	0.00583 ± 0.00157	0.0432 ± 0.00593	0.0189 ± 0.00287	0.0376 ± 0.0233	0.230 ± 0.0192	0.108 ± 0.0246
BCFSS Steady-state BCF [L kg-1]	0.176 ± 0.0421	1.31 ± 0.146	0.576 ± 0.0663	0.108 ± 0.0716	0.667 ± 0.0681	0.316 ± 0.0751
BCFSSL Lipid normalized steady-state BCF [L kg-1]*	0.127	0.951	0.419	0.0785	0.167	0.230
BCFK Kinetic BCF [L kg-1]	0.217	1.45	0.622	0.181	0.697	0.351
k1 Overall uptake rate constant [L kg-1 day-1]	0.0709 ± 0.0112	0.875 ± 0.0501	0.348 ± 0.0266	0.0187 ± 0.0511	0.437 ± 0.0378	0.154 ± 0.0193
k2 Overall depuration rate constant [day-1]	0.326 ± 0.0795	0.602 ± 0.0608	0.600 ± 0.0719	0.103 ± 0.633	0.627 ± 0.222	0.440 ± 0.189
BCFKL Lipid-normalized kinetic BCF [L kg-1]	0.158	1.05	0.452	0.132	0.507	0.255
BCFKg Growth-corrected kinetic BCF [L kg-1]**	0.217	1.45	0.622	0.181	0.697	0.351
Kg Growth rate constant [day-1]***	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
k2g Growth-corrected depuration rate constant [day-1]**	0.326 ± 0.0795	0.602 ± 0.0608	0.600 ± 0.0719	0.103 ± 0.633	0.627 ± 0.222	0.440 ± 0.189
t1/2g Growth-corrected half-life [day]**	2.12	1.15	1.24	6.74	1.11	1.58
BCFKLG Lipid-normalized growth-corrected kinetic BCF [L kg-1]**	0.158	1.05	0.452	0.132	0.507	0.255

*Lipid normalization factor = 0.727 (based on mean lipid fraction (wet weight) of 6.88 %)

** Since no growth occurred the corrected values are similar to the respective non-corrected values

*** The growth rate constant (kg) could not be calculated due to the absence of fish growth within the

whole test duration

Table 3: Bioconcentration factors (BCF) based on TRR

0.03 mg/L (aquarium B)
Sampling day	BCF edible part		BCF viscera part		BCF whole fish
	mean*	SD	mean*	SD	mean*	SD
1	0.162	0.042	0.566	0.134	0.314	0.076
3	0.133	0.085	1.026	0.266	0.429	0.135
7	0.122	0.129	1.141	0.244	0.471	0.195
10	0.319	0.215	1.591	0.448	0.794	0.336
14	0.221	0.093	1.469	0.234	0.652	0.183
21	0.169	0.066	1.187	0.455	0.534	0.215
28	0.137	0.127	1.267	0.526	0.541	0.282
29	0.071	0.152	0.605	0.479	0.270	0.261
31	0.074	0.054	0.283	0.127	0.150	0.086
35	0.024	0.132	0.000	0.039	0.000	0.097
38	0.000	0.088	0.000	0.222	0.000	0.141
42	0.000	0.101	0.000	0.112	0.000	0.070
0.3 mg/L (aquarium C)
Sampling day	BCF edible part		BCF viscera part		BCF whole fish
	mean*	SD	mean*	SD	mean*	SD
1	0.148	0.053	0.468	0.156	0.262	0.058
3	0.065	0.114	0.449	0.128	0.194	0.123
7	0.117	0.093	0.519	0.269	0.258	0.101
10	0.244	0.155	0.943	0.560	0.500	0.257
14	0.060	0.113	0.597	0.225	0.250	0.118
21	0.190	0.056	0.733	0.148	0.398	0.084
28	0.073	0.036	0.673	0.240	0.302	0.098
29	0.002	0.081	0.287	0.337	0.118	0.190
31	0.119	0.096	0.160	0.104	0.133	0.093
35	0.137	0.051	0.000	0.085	0.031	0.032
38	0.000	0.031	0.000	0.097	0.000	0.037
42	0.000	0.278	0.156	0.444	0.000	0.351

*mean value based on 4 sampled fish

SD: standard deviation

Negative values were replaced by 0

The results from day 14 to 28 were used to calculate the steady-state BCF (BCFSS).

 

Validity criteria fulfilled:

yes

Description of key information

BCF < 2 (Lepomis macrochirus, OECD 305, BCF lipid normalized steady state as well as growth and lipid corrected kinetic BCF values from different fish tissues and the two different concentrations)

Key value for chemical safety assessment

BCF (aquatic species):

2 dimensionless

Additional information

One experimental study is available investigating uptake, depuration and metabolism of the test item by determining its uptake rate constant (Ku), depuration rate constant (Kd) and the bioconcentration factor (BCF), including the qualitative and quantitative characterization of the metabolites in fish and water (M-568718-01-1). Additionally, the biotransformation (metabolism) in fish was investigated by the qualitative and quantitative characterization of metabolites (≥ 10% of parent and/or ≥ 50 ppb). The study was performed according to OECD 305 and US EPA OCSPP 850.1730 under GLP conditions. The whole study was performed in a flow-through design with bluegill sunfish (Lepomis macrochirus) in glass aquaria (four aquaria in total). Fish were exposed to concentrations of 0.03 and 0.3 mg test item/L in the bioconcentration part of the test (Aquarium B and C, respectively) and 0.3 mg test item /L in the biotransformation part of the test (Aquarium D). Additionally, a solvent control was performed in parallel (Aquarium A). The bioconcentration part of the test consisted of two phases: the exposure (uptake) and post-exposure (depuration) phases. At start of the uptake phase (day 0) 70 randomly selected fish were transferred into aquaria B and C (exposure to 0.03 and 0.3 mg test item /L, respectively) and into aquarium A (solvent control). Afterwards they were transferred to pure test water without the test substance for the depuration phase. The concentration of the test item in the fish was followed through both phases of the test. The duration of the uptake phase was 28 d, followed by the depuration phase of 14 d. Water samples were collected during the exposure period of 28 d for aquarium C and 14 d for aquarium D. All water samples taken after day 28 were not further analyzed due to low radioactivity. The radioactive residues were extracted from water samples by solid phase extraction (SPE) with RP18. The residues were eluted with acetonitrile and methanol/tetrahydrofurane, concentrated and analyzed by HPLC with radiodetection. The radioactive concentrations in the water ranged from 250.5 to 391.7 µg/L. The parent compound was the only component in the water samples and was identified by comparison with the HPLC chromatogram of the stock solution. For the biotransformation part of the test, parent and the metabolites were analyzed for different tissues of the fish (edible and viscera part) after 7 and 14 d of exposure. No depuration phase was needed for this part of the study. The biotransformation part started on day 0 of the bioconcentration test (begin of uptake phase), with assignment of 30 fish to aquarium D. The test of biotransformation ended on day 14, when the last remaining fish were sampled. For this purpose, edible parts and viscera of fish were sampled on day 7 and 14, and were conventionally extracted with acetonitrile/water mixtures. No further radioactive residues were extracted by exhaustive extraction methods using microwave assistance and hydrochloric acid. The radioactivity in the remaining solids after exhaustive extraction was below LOD. The total radioactive residues (TRRs), were low and amounted to 0.048 mg/kg (day 7) and 0.059 mg/kg (day 14) in the edible parts and 0.117 mg/kg (day 7) and 0.124 mg/kg (day 14) in the viscera. Due to the low radioactivity, no metabolic profile was recorded for edible parts of fish and viscera. Instead, the radioactivity in the conventional extracts of the edible parts was characterised as polar by partition against n-hexane. The main radioactivity in viscera was found in the aqueous phase and was characterised as polar, while the radioactivity in the organic phases might be assigned to parent compound or non-polar metabolites. The lipid normalized steady-state (BCFSSL) was 0.419 in the lower and 0.230 in the higher concentration. The lipid-normalized growth-corrected kinetic BCF (BCFKLG) was 0.452 in the lower and 0.255 in the higher concentration. On the basis of the results of this study it was concluded that the test item was stable in water and metabolised in fish. Due to the low amount of radioactivity in fish, no metabolites were identified and therefore no metabolic pathway was proposed.