4-(2-chlorophenyl)-N-cyclohexyl-N-ethyl-5-oxo-4,5-dihydro-1H-tetrazole-1-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:

13 Apr - 19 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:

adopted version of 1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1730 (Fish Bioconcentration Test)

Version / remarks:

1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 72-6 (Aquatic Organism Accumulation Tests)

Version / remarks:

1982

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 165-4 (Laboratory Studies of Pesticide Accumulation in Fish)

Version / remarks:

1982

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

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

Radiolabelling:

yes

Details on sampling:

- Sampling intervals/frequency for test organisms:
Part 1 of study: Fish were sampled throughout the uptake- and depuration period. At each sampling date, four fish from each chamber were collected and processed individually. The fish were dissected into edible (fillet = body, muscle, skin, skeleton) and viscera/nonedible (viscera = head, fins, internal organs) parts. Sampling was done on days 1, 3, 7, 10, 15, 21, and 28 during exposure; on days 29, 31, 35, 38, and 42 during depuration.
Part 2 of study: On each sampling day (day 7 and day 15) fifteen fish at a time were sampled in order to study the biotransformation of the test item.
- Sampling intervals/frequency for test medium samples: On each sampling day, three samples of 10 mL water were removed from each aquarium. The concentrations of the test item in test medium were calculated based on liquid scintillation counting of triplicate 10-mL samples pipetted directly from control and each test tank to vials. To each sample 10 mL of scintillation cocktail (Quickszint 401, Fa. Zinsser Analytik GmbH, Germany) were added. Radioactivity was measured (expressed as disintegrations per minute, dpm) and concentrations of equivalents calculated. For the determination of metabolites in water samples of 500 mL were taken from the high concentration level of part 1 (aquarium C) and from part 2 (aquarium D).
- Sample storage conditions before analysis: The samples were deep-frozen until analysis.
- Details on sampling and analysis of test organisms and test media samples (e.g. sample preparation, analytical methods):
Part 1 of study: Samples were transferred into pre-weighed vials suitable for further handling. After determination of the weight 20 mL of BTS-450 (Beckman Tissue-Solubilizer-450) were added to each fish sample. Solutions were incubated in a drying oven for at least 5 days at 50 °C and were slightly agitated once a day during this period. After incubation two aliquots each with 1 mL of the homogenized solution were removed from each vessel and were mixed with 200 μL HCl (5 N). The mixture was shaken
and 7 mL Quickszint 401 were added. Afterwards the radioactivity (expressed as disintegrations per minute, dpm) was measured in the radio analysis laboratory in order to determine the TRR (total radioactivity residues) in fish. On day 0, day 28 and day 42 four additional fish were taken from each aquarium to determine the lipid content after a modified method based on Bligh & Dyer (1959).
Part 2 of study: Fish were dissected into edible and viscera parts and than transferred into vials. After determination of the wet weight the samples were investigated in the test site for Metabolism/Environmental Fate.

Vehicle:

yes

Remarks:

Dimethylformamide

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

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 100 μL/L dilution water (= 0.01 vol.-%) were used in this study as solvent carrier. Thus the nominal a.s. concentrations in the stock solutions B, C and D were:
Part 1 of study (Bioconcentration and Depuration):
a) Flask A: solvent (Dimethylformamide)
b) Flask B: 99.47 mg in a 2.00 L volume, whole radioactivity of 40 MBq, is resulting in 50 mg/L (nominal) stock solution and 0.005 mg/L (nominal) in the aquarium after 1:10,000 dilution.
c) Flask C: 999.45 mg in a 2.00 L volume, whole radioactivity of 40 MBq, is resulting in 500 mg/L (nominal) stock solution and 0.05 mg/L (nominal) in the aquarium after 1:10,000 dilution.
Part 2 of study (Biotransformation):
d) Flask D: 598.98 mg in a 1.20 L volume, whole radioactivity of 80 MBq, is resulting in 500 mg/L (nominal) stock solution and 0.05 mg/L (nominal) in the aquarium after 1:10,000 dilution.

- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Dimethylformamide
- Concentration of vehicle in test medium (stock solution and final test solution(s) at different concentrations and in control(s)): 100 μL/L dilution water (= 0.01 vol.-%). In such a concentration dimethylformamide is not acutely toxic to fish and well accepted
by the test guidelines.

Test organisms (species):

Lepomis macrochirus

Details on test organisms:

TEST ORGANISM
- Common name: Bluegill sunfish
- Strain: lot F 2/07 (used for the bioconcentration part), lot F 6/06 (used for the biotransformation part)
- Source: Osage Catfisheries, Inc., Osage Beach, Missouri, USA; Lot F 2/07 was delivered on 2007-01-31 by airfreight, Lot F 6/06 was delivered on 2006-06-28 by airfreight.
- Length at study initiation (length definition, mean, range and SD): 6.0 ± 0.6 cm (mean) (Part 1 of study); 10.8 ± 0.7 cm (mean) (Part 2 of study)
- Weight at study initiation (mean and range, SD): 3.8 ± 1.1 g (mean) (Part 1 of study); 20.4 ± 3.5 g (mean) (Part 2 of study)
- Lipid content at test initiation (mean and range, SD): 6.81%
- Description of housing/holding area: All test fish were held in culture tanks on a 16-hour daylight photoperiod. Fish culture techniques were primarily those described by Brauhn & Schoettger (1975): Aquisition and Culture of Research Fish: Rainbow Trout, Fathead Minnows, Channel Catfish and Bluegills. Environmental Protection Agency, Ecological Research Series EPA-660/3-75-011.
- Feeding during test
- Food type: Standard fish-feed (Pro Aqua Brutfutter, Skretting, Germany)
- Amount: 2 percent of mean body-weight (During Part 1 test period (bioconcentration and depuration)), 1 percent of mean body-weight (During Part 2 of the study (biotransformation)); The amount of feed was re-calculated once per week

ACCLIMATION
- Acclimation period: at least 14 days
- Type and amount of food: Standard fish-feed (Pro Aqua Brutfutter, Skretting, Germany); 2 percent of mean body-weight; The amount of feed was re-calculated once per week
- Health during acclimation (any mortality observed): No mortality was noted 14 days prior to the test initiation, and all unsuitable fish (i.e., injured, deformed, etc.) were eliminated from the test prior to the assignment of test groups.

Route of exposure:

aqueous

Justification for method:

aqueous exposure method used for following reason: Aqueous route of exposure is used based on the good water solubility of the test substance, its stability in water and the low potential for adsorption

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

28 d

Total depuration duration:

14 d

Hardness:

48 mg CaCO3/L (40 - 60 mg CaCO3/L)

Test temperature:

23.6 °C - 25.3 °C (mean: 24.4 °C - 25.1 °C)
23.8 °C - 25.4 °C (mean: 24.6 °C) (temperature inside the control aquarium)

pH:

6.7 - 7.0 (mean: 6.8 - 6.9)

Dissolved oxygen:

72 - 99% (mean: 87 - 93%)

TOC:

31.4 - 44.5 mg/L
(throughout the whole biological part of the study, all measured TOC values in the test vessels did not exceed the concentration of organic carbon originating from the test item and from the solubilising agent (nominal sum TOC is about 46 mg/L for all test levels including control) by more than 10 mg/L as expected by OECD guideline)

Details on test conditions:

TEST SYSTEM
- Test vessel:
Material, size, headspace, fill volume: 100 L test glass aquaria
- Aeration: yes; All test chambers have been aerated throughout the test.
- Type of flow-through (e.g. peristaltic or proportional diluter): see test system under "Any other information on materials and methods incl. tables" below
- Renewal rate of test solution (frequency/flow rate): 25 L/hour/aquarium
- No. of aquaria: 4 (one control (aquarium A), two different treatment levels (aquaria B+C), one aquarium for biotansformation (D))
- No. of organisms per vessel: 60 (Part 1 of study); 30 (Part 2 of study)
- No. of vessels per concentration (replicates): 1
- No. of vessels per control / vehicle control (replicates): 1
- Biomass loading rate: 2.3 g fish/L and 0.38 g fish/L/day (Part 1 of study) 1.0 g fish/L/day (Part 2 of study)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted Water (Composition according to ISO)
- Particulate matter: < 5 mg/L
- Metals: < 1 µg/L
- Pesticides: < 0.05 µg/L
- Chlorine: < 10 µg/L
- Ca/Mg ratio: 4:1
- Intervals of water quality measurement: Water quality parameters of dissolved oxygen, temperature and pH were measured initially and throughout the study in each aquarium once a week. In addition, the daily temperature-fluctuation was measured continously in the control tank and recorded as hourly mean values. TOC was measured at the beginning of the test and then once a week. For details see Tables 3, 4, and 5 under "Any other information on results incl. tables" below.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): The fish were observed initially and every 24 hours on working days for mortality and/or adverse behaviour.

RANGE-FINDING / PRELIMINARY STUDY
- Results used to determine the conditions for the definitive study: Exposure levels were determined based on the results of previously conducted fish toxicity tests

Nominal and measured concentrations:

0.005 mg/L and 0.05 mg/L (part 1 of the study) (nominal)
0.05 mg/L (part 2 of the study) (nominal)

Reference substance (positive control):

no

Details on estimation of bioconcentration:

BASIS FOR CALCULATION OF BCF
In evaluating the data obtained from the bioconcentration study the apparent steady state bioconcentration factor was calculated as the ratio of concentration in fish and in water.
- Estimation software: The results were calculated using a spreadsheet (TM Microsoft Excel 97).

Lipid content:

7.94 %

Time point:

other: overall mean (day 0-28)

Lipid content:

6.81 %

Time point:

start of exposure

Lipid content:

9.07 %

Time point:

other: Day 28

Lipid content:

11.15 %

Time point:

end of exposure

Key result

Type:

BCF

Value:

71 dimensionless

Basis:

whole body w.w.

Calculation basis:

steady state

Key result

Type:

BCF

Value:

54 dimensionless

Basis:

normalised lipid fraction

Remarks:

whole fish, normalised to 6% lipid content

Calculation basis:

steady state

Conc. / dose:

0.005 mg/L

Type:

BCF

Value:

153.6 dimensionless

Basis:

edible fraction

Calculation basis:

kinetic

Conc. / dose:

0.005 mg/L

Type:

BCF

Value:

530.1 dimensionless

Basis:

non-edible fraction

Remarks:

viscera

Calculation basis:

kinetic

Conc. / dose:

0.005 mg/L

Type:

BCF

Value:

289.8 dimensionless

Basis:

whole body w.w.

Calculation basis:

kinetic

Conc. / dose:

0.05 mg/L

Type:

BCF

Value:

116.9 dimensionless

Basis:

edible fraction

Calculation basis:

kinetic

Conc. / dose:

0.05 mg/L

Type:

BCF

Value:

520.1 dimensionless

Basis:

non-edible fraction

Remarks:

viscera

Calculation basis:

kinetic

Conc. / dose:

0.05 mg/L

Type:

BCF

Value:

275.5 dimensionless

Basis:

whole body w.w.

Calculation basis:

kinetic

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

0.42 d

Remarks on result:

other: half-life for 0.05 mg/L test level

Remarks:

After 14 days in uncontaminated water 100 % of the mean plateau radioactivities were depurated from whole fish in both test levels.

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

0.55 d

Remarks on result:

other: half-life for 0.005 mg/L test level

Remarks:

After 14 days in uncontaminated water 100 % of the mean plateau radioactivities were depurated from whole fish in both test levels.

Rate constant:

other: uptake rate constant (Ku) (1/day)

Remarks:

for edible parts

Value:

146.2

Rate constant:

other: uptake rate constant (Ku) (1/day)

Remarks:

for viscera

Value:

860.3

Rate constant:

other: uptake rate constant (Ku) (1/day)

Remarks:

whole fish

Value:

363

Rate constant:

other: uptake rate constant (Ku) (1/day)

Remarks:

for edible parts

Value:

86.8

Rate constant:

other: uptake rate constant (Ku) (1/day)

Remarks:

for viscera

Value:

1 027.5

Rate constant:

other: uptake rate constant (Ku) (1/day)

Remarks:

whole fish

Value:

452.3

Rate constant:

other: depuration rate constant (Kd) (1/day)

Remarks:

for edible parts

Value:

0.952

Rate constant:

other: depuration rate constant (Kd) (1/day)

Remarks:

for viscera

Value:

1.62

Rate constant:

other: depuration rate constant (Kd) (1/day)

Remarks:

whole fish

Value:

1.25

Rate constant:

other: depuration rate constant (Kd) (1/day)

Remarks:

for edible parts

Value:

0.742

Rate constant:

other: depuration rate constant (Kd) (1/day)

Remarks:

for viscera

Value:

1.98

Rate constant:

other: depuration rate constant (Kd) (1/day)

Remarks:

whole fish

Value:

1.64

Details on kinetic parameters:

- Uptake rate constant k(u) (1/day): 146.2 - 860.3 (nominal test level 0.005 mg/L), 86.8 - 1027.5 (nominal test level 0.05 mg/L) (for details see Table 1 under "any other information on results incl. tables" below)
- Depuration rate constant k(d) (1/day): 0.952 - 1.62 (nominal test level 0.005 mg/L), 0.741 - 1.98 (nominal test level 0.05 mg/L) (for details see Table 1 under "any other information on results incl. tables" below)
- Computation / data analysis: OriginTM 6.0 computer programme (OriginTM 6.0 by Microcal Software, Inc. MicrocalTM)

Metabolites:

The objective of Part 2 of the study was to investigate the nature of residues in edible and non-edible tissues (viscera) of fish under flow-through conditions and to quantify the metabolites to the extent possible. Investigations on metabolites were carried out with fish from a separate test group (aquarium D) of the bioconcentration study. These fish were exposed to the test item for 7 and 15 days.
The parent compound (i.e the test item) accounted for 77 - 96% of the radioactivity in the profile of all water samples after extraction and concentration. In the samples collected during the exposure phase of fish, two metabolites were detected with ca. 1 - 12% of the TRR. The parent compound and metabolite [trade name]-sulfate were identified by HPLC co-chromatography. Metabolite CPT was assigned by its retention time and chromatographic comparison. These metabolites are probably mainly formed by metabolism of fish since they were not present at the beginning (day 0) of the exposure phase.
Between 92% and 97% of the total radioactive residue (TRR) in edibles and viscera could be identified by LC-MS/MS and 1H-NMR spectroscopy. 2 - 7% of the TRR were characterised by their behaviour during extraction and RP-HPLC analysis. The metabolites characterised accounted for 1 - 13 peaks in the HPLC profiles. No single peak not identified was >3% of the TRR.
The major compound detected in edible fish samples was the metabolite CPT followed by the active ingredient test item. CTP accounted for ca. 56 - 61% of
the TRR in edibles. Ca. 30 - 34% were represented by the parent compound. In viscera, the three metabolites CPT, [trade name]-sulfate and [trade name]-glucuronide were all in the range between 21 - 29% of the TRR. Parent compound accounted for 11 - 16% of the TRR. The two other metabolites identified (CPT-glucuronide and [trade name]-hydroxy) were <3% of the TRR in all samples.

The metabolic reactions of the test item found in fish were:
• Hydrolytic cleavage to CPT
• Conjugation of CPT with glucuronic acid
• Hydroxylation of the cyclohexyl ring to [trade name]-hydroxy
• Conjugation of [trade name]-hydroxy with glucuronic acid to [trade name]-glucuronide and with sulfuric acid to [trade name]-sulfate

For details on the results of the metabolism investigations see Table 2 under "any other information on results incl. tables" below

Details on results:

- Mortality of test organisms: The fish showed no mortalities throughout the test in all test vessels.
- Behavioural abnormalities: The fish showed no abnormal behaviour throughout the test in all test vessels.

Reported statistics:

The uptake rate constant (Ku) and the depuration rate constant (Kd) were determined by the OriginTM 6.0 computer programme (OriginTM 6.0 by Microcal Software, Inc. MicrocalTM). This is a non-linear kinetic modelling programme which provides optional parameter estimates of rate constants by utilising the actual (observed) bioconcentration study data. Preliminary values for Ku and Kd were calculated according to OECD guideline 305 (Annex 4). The bioconcentration factor at steady-state, the time to reach 95% of steady-state (3.0/kd) for total [14C]-residues in edible parts of fish, non-edible parts of fish and in whole fish, and the time to reach ½ of test compound clearance (ln2/kd) were also calculated. A measure of the variability of the estimated parameters were provided by the standard deviation of each estimate.

Overall results

The test item accumulated in bluegill sunfish with a total residue kinetic bioconcentration factor of about 275.5 to 289.8 for whole fish (sum of radio labelled compounds, parent, metabolites and mineralization products). When exposure ceases, the residues were depurated with a half-life of about 0.42 to 0.55 days. After 14 days in uncontaminated water 100% of the mean plateau radioactivities were depurated from whole fish in both test levels.

The Origin(TM) calculated kinetic BCF(TRR) values for edible parts and whole fish corresponded well with the respective average steady-state (days 7 - 28) bioconcentration factors 158.5 X (edible parts) and 326.8 X (whole fish) for 0.005 mg [14C]-test item/L and of 108.0 X (edible parts) and 291.6 X (whole fish) for 0.05 mg [14C]-test item/L, respectively. These values correspond to the calculated steady-state total residue levels of 0.601 mg/kg edible parts and 1.24 mg/kg whole fish for 0.005 mg [14C]-test item/L and of 5.03 mg/kg edible parts and 13.6 mg/kg whole fish for 0.05 mg [14C]-test item/L, respectively. Taking into account that in the test level of 0.05 mg/L about 30% parent compound were detected in edible parts and about 16% parent compound in non-edible (viscera) parts after 15 days of exposure the steady-state-BCF for parent (based on whole fish, wet weight) is about 71, the steady-state-BCF for parent (normalised to 6% lipid content) is about 54.

Table 1: Optimal parameters determined for uptake rate constant (Ku), depuration rate constant (Kd), time to reach 95% of steady-state, the time for half clearance and the kinetic bioconcentration factors

(based on TRR)

Origin Calculation Results	Nominal test level of 0.005 mg [14C]-test item/L			Nominal test level of 0.05 mg [14C]-test item/L
Parameter (based on TRR)	Edible Parts	Viscera	Whole Fish	Edible Parts	Viscera	Whole Fish
Kinetic Bioconcentration Factor BCF(TRR)	153.6	530.1	289.8	116.9	520.1	275.5
Time to Reach 95% of Steady-State (days)	3.2	1.8	2.4	4.0	1.5	1.8
t(1/2) for Clearance (days)	0.73	0.43	0.55	0.93	0.35	0.42
Uptake Rate Constant (Ku) (1/day)	146.2 (± 8.63)	860.3 (± 68.5)	363.0 (± 14.9)	86.8 (± 8.02)	1027.5 (± 112)	452.3 (± 31.8)
Depuration Rate Constant (Kd) (1/day)	0.952 (± 0.10)	1.62 (± 0.37)	1.25 (± 0.23)	0.742 (± 0.22)	1.98 (± 0.53)	1.64 (± 0.45)

Table 2: Results of the metabolism investigations in edible and viscera tissues

	edibles (day 7)		edibles (day 15)		viscera (day 7)		viscera (day 15)
	% TRR	mg/kg	% TRR	mg/kg	% TRR	mg/kg	% TRR	mg/kg
TRR in extract	98.6	3.76	99.2	5.98	98.9	17.02	98.6	26.52
compound
CPT-glucuronide	n.d.	n.d.	0.3	0.02	1.3	0.22	0.9	0.24
CPT	60.6	2.31	55.9	3.37	20.7	3.56	25.3	6.81
[trade name]-sulfate	n.d	n.d.	6.4	0.39	29.0	4.99	25.4	6.83
[trade name]-glucuronide	1.6	0.06	2.9	0.18	27.9	4.81	21.2	5.72
[trade name]-hydroxy	0.8	0.03	1.2	0.07	2.6	0.45	2.5	0.68
Test item	33.5	1.28	29.8	1.80	11.0	1.90	16.3	4.37
TRR in sample	100.0	3.81	100.0	6.03	100.0	17.21	100.0	26.91
TRR analysed (in extract)	98.6	3.76	99.2	5.98	98.9	17.02	98.6	26.52
solids	1.4	0.05	0.8	0.05	1.1	0.19	1.4	0.39
identified	96.6	3.68	96.5	5.82	92.5	15.92	91.6	24.64
characterised	2.1	0.08	2.7	0.16	6.4	1.10	7.0	1.88
number of characterised peaks	1		6		13		13
largest peak characterised	2.1	0.08	0.8	0.05	0.9	0.16	0.9	0.23
smallest peak characterised	2.1	0.08	0.2	0.01	0.2	0.04	0.2	0.07

n.d. = not detected or below limit of quantitation (<LOQ)

Table 3: Mean tissue residues (given in mg/kg wet weight) at steady state

	Nominal test level of 0.005 mg [14C]-test item/L	Nominal test level of 0.05 mg [14C]-test item/L
Edible Parts:	0.601 mg/kg (based on TRR)	5.03 mg/kg (based on TRR)
Non Edible Parts:	2.41 mg/kg (based on TRR)	26.9 mg/kg (based on TRR)
Whole Fish:	1.24 mg/kg (based on TRR)	13.6 mg/kg (based on TRR)

Table 4: Water quality parameters

Study Day	pH-values				Dissolved oxygen in %
	Control	0.005 mg/L	0.05 mg/L	0.05 mg/L	Control	0.005 mg/L	0.05 mg/L	0.05 mg/L
	A*	B*	C*	D*	A*	B*	C*	D*
0	6.9	6.9	6.9	6.9	84	84	90	72
7	6.7	6.9	6.7	6.8	90	94	87	94
15	6.7	6.8	6.7	6.8	79	89	83	94
21	6.9	7.0	6.9	+	87	94	89	+
27	6.7	6.9	6.7	+	95	94	89	+
34	7.0	7.0	7.0	+	96	97	98	+
42	7.0	7.0	7.0	+	98	99	99	+
Mean	6.8	6.9	6.8	6.8	90	93	91	87
SD	0.1	0.1	0.1	0.1	7.0	5.0	5.8	12.7
Maximum	7.0	7.0	7.0	6.9	98	99	99	94
Minimum	6.7	6.8	6.7	6.8	79	84	83	72

* Aquaria (Part 1 of study: A: control; B, C: uptake/depuration; Part 2 of study: D: biotransformation part)

Table 5: Temperature measurements

Study Day	Test Temperature Measurements in °C
	Control	0.005 mg/L	0.05 mg/L	0.05 mg/L
	A*	B*	C*	D*
0	25.3	25.3	25.3	25.3
7	24.9	24.8	25.0	25.0
15	24.8	24.8	25.1	24.9
21	24.1	24.1	24.1	+
27	23.8	23.6	23.6	+
35	24.2	24.2	24.3	+
42	23.9	23.9	23.9	+
Mean	24.4	24.4	24.5	25.1
SD	0.6	0.6	0.7	0.2
Maximum	25.3	25.3	25.3	25.3
Minimum	23.8	23.6	23.6	24.9

+: no measurements, all fish have been sampled after 15 days

* Aquaria (Part 1 of study: A: control; B, C: uptake/depuration; Part 2 of study: D: biotransformation part)

 

Table 6: Determination of Total Organic Carbon (TOC) in mg/L in Test Water

	Control	0.005 mg/L	0.05 mg/L	0.05 mg/L
	A*	B*	C*	D*
day -4**	< 2	<2	<2	<2
day 0	43.7	31.4	38.2	37.6
day 8	43.5	42.4	39.8	44.5
day 16	39.0	41.0	39.4	+
day 21	40.5	44.2	42.7	+
day 29	< 2	< 2	< 2	+
day 37	< 2	< 2	< 2	+
day 42	< 2	< 2	< 2	+

* Aquaria (Part 1 of study: A: control; B, C: uptake/depuration; Part 2 of study: D: biotransformation part)

** without solvent and test item

+: no measurements, all fish were sampled after 15 days

Table 7: Lipid content (whole fish) in g on Day 0, on Day 28 and on Day 42

Sample	Lipid content (g)	Lipid content (g)	Lipid content (g)
	Day 0	Day 28	Day 42
A1	0.21	0.53	1.21
A2	0.28	0.71	0.79
A3	0.21	0.38	0.81
A4	0.15	0.33	0.87
B1	0.25	0.66	0.47
B2	0.22	0.49	0.49
B3	0.23	0.48	0.99
B4	0.31	0.51	0.77
C1	0.25	0.32	0.62
C2	0.31	0.49	0.79
C3	0.28	0.34	0.71
C4	0.21	0.62	0.79
Mean	0.24	0.49	0.78

A: control fish, B: treated fish (low conc.), C: treated fish (high conc.)

1 - 4 : # of individual fish sampled from the respective group

 

Table 8: Lipid content (whole fish) on Day 0, on Day 28 and on Day 42 (g/kg wet weight; %)

Sample	Day 0		Day 28		Day42
	g/kg ww	%	g/kg ww	%	g/kg ww	%
A1	78.4	7.84	84.4	8.44	125.1	12.51
A2	52.4	5.24	108.6	10.86	93..7	9.37
A3	51.5	5.15	82.6	8.26	125.6	12.56
A4	63.3	6.33	97.9	9.79	104.3	10.43
B1	61.0	6.10	65.3	6.53	120.8	12.08
B2	65.3	6.53	95.1	9.51	126.6	12.66
B3	77.7	7.77	95.0	9.50	103.3	10.33
B4	67.5	6.75	99.0	9.90	83.8	8.38
C1	68.3	6.83	100.9	10.09	96.3	9.63
C2	70.3	7.03	94.0	9.40	129.7	12.97
C3	94.9	9.49	103.3	10.33	117.9	11.79
C4	66.0	6.60	61.8	6.18	110.2	11.02
Mean		6.81		9.07		11.15
Overall mean		7.94%*

A: control fish, B: treated fish (low conc.), C: treated fish (high conc.)

1 - 4 : # of individual fish sampled from the respective group

* Overall mean (day 0 + 28) is used for BCF normalisation to 6% lipid content

Table 9: Uptake and Depuration of Radioactivity

Sampling Day	Fish Edible Part		Fish Viscera Part		Whole Fish (Calculated from Edible Part and Viscera)
	% Radioactivity relative to the maximum reached value during steady state (wet weight)
	0.005 mg/L	0.05 mg/L	0.005 mg/L	0.05 mg/L	0.005 mg/L	0.05 mg/L
1	47.5%	46.4%	43.7%	42.8%	51.0%	47.5%
3	89.1%	100.0%	58.4%	53.7%	77.9%	75.3%
7	57.4%	60.5%	86.1%	81.7%	80.1%	88.4%
10	80.5%	76.5%	98.4%	100.0%	100.0%	100.0%
15	67.6%	84.7%	96.4%	93.1%	94.5%	91.0%
21	76.2%	70.4%	100.0%	49.1%	94.5%	61.3%
28	100.0%	56.1%	79.5%	77.8%	93.0%	77.3%
29	25.2%	22.1%	17.0%	10.4%	23.1%	14.4%
31	10.4%	23.7%	16.0%	13.3%	15.4%	16.7%
35	0.4%	0.6%	1.0%	0.5%	0.9%	0.6%
38	0.5%	0.2%	0.7%	0.2%	0.7%	0.2%
42	0.0 %	0.1%	0.4%	0.1%	0.3%	0.1%

Analysed concentrations of test item in water:

During the 28-day bioconcentration (uptake) phase water concentrations ranged from 0.0029 mg/L to 0.0056 mg/L for the nominal concentration of 0.005 mg/L and from 0.041 mg/L to 0.050 mg/L for the nominal concentration of 0.05 mg/L. The average water concentration during the uptake phase was 0.0041 (SD: 0.001) mg/L for the nominal concentration of 0.005 mg/L and 0.047 (SD: 0.002) mg/L for the nominal concentration of 0.05 mg/L.

Analysed stock solutions:

The analysis of stock solutions of the test compound all tests revealed that the test item was not completely stable in stock solutions with dimethylformamide in the time range between 7 and 28 days. The test compound degraded by ca. 2% within 15 days and ca. 3 - 8% within 28 days. CPT, also know as a metabolite of the test item, was identified by HPLC co-chromatography as the degradation product

in the stock solution.

Table 10: Validity criteria for OECD 305

Criterion from the guideline	Outcome	Validity criterion fulfilled
The water temperature variation is less than ± 2°C	24.4-25.1 °C	yes
The concentration of dissolved oxygen does not fall below 60% saturation	72%-99%	yes
The concentration of the test substance in the chambers is maintained within ± 20% of the mean of the measured values during the uptake phase	0.005 mg/L (nominal); 0.0041 mg/L (average measured); -18% of nominal 0.05 mg/L (nominal); 0.047 mg/L (average measured); -6% of nominal	yes
The concentration of the test substance is below its limit of solubility in water	Water solubility: 2.5 mg/L	yes
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	yes

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Description of key information

BCF (whole fish, wet weight) = 71 (OECD 305 and further guidelines, Lepomis macrochirus)

BCF (whole fish, normalized to 6% lipid content) = 54 (OECD 305 and further guidelines, Lepomis macrochirus)

Key value for chemical safety assessment

BCF (aquatic species):

54 dimensionless

Additional information

One study is available investigating the potential for bioaccumulation of the test substance using the bluegill sunfish (Lepomis macrochirus) as test organism (2007). The study was conducted according to GLP and followed international standard guidelines (OECD 305, EPA OPPTS 850.1730, EPA OPP 72-6, EPA OPP 165-4).

The objective of the study was to measure uptake, metabolism and depuration 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 metabolites in fish and water. Radiolabeled test substance was used. A dosing system was employed to maintain mean water concentrations of 0.005 mg and 0.05 mg test substance/L for the bioconcentration part and 0.05 mg test substance/L for the biotransformation part of the study. 60 fish were exposed for the bioconcentration part of the study (one control, two treatment levels) for 28 days. After the exposure of 28 days remaining test fish were placed in clean water for further 14 days in order to determine the depuration of the test item. 30 fish were exposed for a period of 7 and 15 days respectively in order to investigate the biotransformation of the test substance in part 2 of the study. Lipids were extracted from four additional fish (part 1) on days 0, 28 and 42 in order to quantify the mean lipid content in fish.

Throughout the test in all test vessels the fish showed no mortalities or abnormal behaviour. The test substance accumulated in bluegill sunfish with a total residue kinetic bioconcentration factor of about 275.5 to 289.8 for whole fish (sum of radio labelled compounds, parent substance, metabolites and mineralization products). When exposure ceases, the residues were depurated with a half-life of about 0.42 to 0.55 days. After 14 days in uncontaminated water 100% of the mean plateau radioactivities were depurated from whole fish in both test levels. The uptake rate constant (Ku), depuration rate constant (Kd), time to reach 95% of steady-state, the time for half clearance and the kinetic bioconcentration factors (based on TRR) for edible parts, viscera and for whole fish were determined using the Origin^TM non-linear kinetic modeling computer programme. The optimal parameter estimates were determined to be:

Origin Calculation Results	Nominal test level of 0.005 mg [14C]-test item/L			Nominal test level of 0.05 mg [14C]-test item/L
Parameter (based on TRR)	Edible Parts	Viscera	Whole Fish	Edible Parts	Viscera	Whole Fish
Kinetic Bioconcentration Factor BCF(TRR)	153.6	530.1	289.8	116.9	520.1	275.5
Time to Reach 95% of Steady-State (days)	3.2	1.8	2.4	4.0	1.5	1.8
t(1/2) for Clearance (days)	0.73	0.43	0.55	0.93	0.35	0.42
Uptake Rate Constant (Ku) (1/day)	146.2 (± 8.63)	860.3 (± 68.5)	363.0 (± 14.9)	86.8 (± 8.02)	1027.5 (± 112)	452.3 (± 31.8)
Depuration Rate Constant (Kd) (1/day)	0.952 (± 0.10)	1.62 (± 0.37)	1.25 (± 0.23)	0.742 (± 0.22)	1.98 (± 0.53)	1.64 (± 0.45)

 

A mean lipid content (day 0-28) of 7.94% for the batch used was found. The analysis of stock solutions of the test compound from all tests revealed that the test substance was not completely stable in stock solutions (dimethylformamide) in the time range between 7 and 28 days. The test compound degraded by ca. 2% within 15 days and ca. 3 – 8% within 28 days. CPT, also known as a metabolite from the test item, was identified by HPLC co-chromatography as the degradation product in the stock solution. The parent compound accounted for 77 – 96% of the radioactivity in the profile of all water samples after extraction and concentration of the organic phase. In the samples collected during the exposure phase of fish, two metabolites were detected with ca. 1 – 12% of the total radioactive residue (TRR). The parent compound and metabolite parent compound-sulfate were identified by HPLC co-chromatography. Metabolite CPT was assigned by its retention time and chromatographic comparison. These metabolites are probably mainly formed by metabolism in fish since they were not present at the beginning (day 0) of the exposure phase. Between 92% and 97% of the TRR in edibles and viscera could be identified by LC-MS/MS and 1H-NMR spectroscopy. 2 - 7% of the TRR were characterised by their behaviour during extraction and RP-HPLC analysis. The metabolites characterised accounted for 1 – 13 peaks in the HPLC profiles. No single peak not identified was >3% of the TRR. The major compound detected in edible fish samples was the metabolite CPT followed by the parent compound. CPT accounted for ca. 56 – 61% of the TRR in edibles. Ca. 30 – 34% were represented by the parent compound. In viscera, the three metabolites CPT, parent compound-sulfate and parent compound-glucuronide were all in the range between 21 – 29% of the TRR. Parent compound accounted for 11 – 16% of the TRR. The two other metabolites identified (CPT-glucuronide and parent compound-hydroxy) were <3% of the TRR in all samples.

The metabolic reactions of the test substance found in fish were:

• Hydrolytic cleavage to CPT

• Conjugation of CPT with glucuronic acid

• Hydroxylation of the cyclohexyl ring to parent compound-hydroxy

• Conjugation of parent compound-hydroxy with glucuronic acid to parent compound-glucuronide and with sulfuric acid to parent compound-sulfate

A figure of the proposed metabolic pathway based on these results is attached to the respective endpoint study record.

Taking into account that about 30% parent compound were detected in edible parts and about 16% parent compound in non-edible (viscera) parts after 15 days of exposure the steady-state-BCF for parent (based on whole fish, wet weight) is about 71, the steady-state-BCF for parent (normalised to 6% lipid content) is about 54. A normalization to 5% lipid content would result in an even lower value.