N-[2,5-dichloro-4-(1,1,2,3,3,3-hexafluoropropoxy)-phenyl-aminocarbonyl]-2,6-difluorobenzamide

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:

23 Jul 2003 to 20 Nov 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

other: Proposal for Updating Guideline 305, Bioconcentration: Flow-through Fish Test

Version / remarks:

June 1996; Paris, France.

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

yes

Details on sampling:

SAMPLING INTERVALS
- Weight of the fish: The total weight of fish in each tank was determined by bulk weighing (total wet weight) each population at 14 day intervals. After each fish sampling occasion (a maximum of every 7 days), an adjustment was then be made to the quantity of food dispensed.
- Analysis of test solutions: Day -2, -1, 0,1, 2, 7, 14, 21, 28, 35, 42, 49, 56, 60, 61, 120
- Analysis of fish samples: Day 7, 14, 21, 28, 35, 42, 49, 52, 54, 56, 58, 60, 61, 63, 68, 75, 82, 89, 96, 103, 117, 120 (lipid analysis only)
On days 56, 60, 110 and 120 additional fish were sampled and retained should further metabolite analysis be required.

SAMPLING AND PREPARATION
- Test solution: The analytical methods employed to measure the concentrations of [14C]-labelled test substance in the test solutions in this study were based on scintillation counting (LSC) to determine total [14C] residues, and reverse phase liquid chromatography (LC) to characterise the [14C] residues. All results based on LSC are quoted as μg/L equivalents of [14C]-labelled test substance. Water samples for LSC were taken from below the surface film and away from the sides and bottom of the test vessels, by pipetting 10 mL of water directly into scintillation vials. Specific analysis for 14C-lablled test substance was carried out by liquid extraction followed by LSC of the hexane extract and reverse phase LC on exposure days 0, 14, 28, 47 and 60. A sample of approximately 1 L of sub-surface water from each tank was collected in a separating funnel. Due to the expected adsorption of the test substance to glassware, no sub-samples were removed for analysis by LSC at this point. A 25 mL aliquot of hexane was added to the separating funnel which was stoppered and shaken for approximately 2 minutes. The two phases were then left to separate for at least 30 minutes, after which 3 x 1L ml of the aqueous phase was removed for LSC analysis. The remainder of the aqueous layer was discarded after the volume was recorded. 3 x 100 µL of the hexane was analysed by LSC before being blown down to dryness and re—dissolved in methanol (2 mL) for reverse phase LC analysis.
- Fish tissues by combustion: On each sampling occasion, 4 fish were randomly selected from each test vessel. The fish were killed, dissected and frozen until required for analysis. The tissues were then dried for approximately 1 hour at 60 ˚C before being combusted in a sample oxidiser.
- Analysis of fish tissue by TLC: Analysis of fish tissue residues by TLC was performed using solvent extracts of homogenised fish sampled on exposure days 56, 60, 110 and 120. The fish were sampled from the test vessels at the designated time and stored in a deep freeze for subsequent analysis. Each batch of fish sampled (4 fish per vessel per sampling occasion) was removed from the freezer, defrosted and weighed. The fish were then frozen in liquid nitrogen and homogenised to a powder using a pestle and mortar. Duplicate 1 g portions were then extracted twice with 5 mL acetonitrile and twice with 5 mL acetonitrile:water (50:50) using a roller/shaking device After each extraction the mixture was centrifuged (10395 G for 10 minutes). The acetonitrile and acetonitrile:water supernatants were transferred to separate 10 mL volumetric flasks and made up to volume, if required, with deionised water. An 8.2 mL sub-sample from each extract was removed and analysed by LSC. The remaining 1.8 mL was reserved for TLC. After the final solvent extraction, the residual fish tissue was sub-sampled in triplicate for combustion.
- Lipid analysis of the fish tissues: The lipid analysis was carried out on fish sampled on exposure day 0 (8 excess fish from stock supply) and day 120 (4 fish from each treatment vessel). 4 males and 4 females were sampled on day 0, 1 male and 3 females from each of the solvent control and 10 µg/L [14C]- labelled test substance concentration and 2 males and 2 females were taken from the 1.0 µg/L [14C]- labelled test substance on day 120. On each occasion the sacrificed fish were weighed prior to being stored frozen. On 17 February 2004, the fish were defrosted and the total weight of the pooled fish determined. The fish were then frozen in liquid nitrogen and homogenised to a powder using a pestle and mortar. Lipid analysis was then performed between the dates 23 March and 29 April 2004. Depending upon the weight of fish, triplicate 2 - 4 g samples of the homogenate were solvent extracted with 80 mL of chloroform : methanol (50:50) on a roller-shaker overnight. The homogenate was filtered through a filter paper and the filtrate collected in a pre-dried (50°C for 24 hours) and pre-weighed 250 mL round bottomed flask. The extraction vessel and filter were rinsed with a further 80 mL chloroform : methanol (50:50) mixture. The solvent mixture was then removed by rotary evaporation (40°C and under vacuum). The round bottom flask was placed in an oven at 50°C. The flask was removed periodically and weighed until the flask and its lipid contents had reached a constant weight.

Vehicle:

yes

Remarks:

Triethylene Glycol (TEG)

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

- Preparation of stock concentrates and stock solutions: Nominal [14C]-labelled test substance concentrations of 1.0 μg/L, 10 μg/L and a solvent control were used in the study. The [14C]-labelled test substance was continuously dosed into the test vessels from stock solutions made up in triethylene glycol (TEG).
- Nominal 1.0 µg/L concentrate and stocks: The test substance was mixed in 25 mL acetone and the specific activity of the resulting concentrate was measured to be 898 MBq/g. The total radioactivity of this solution was determined to be 59.8 MBq and this is equated to 67 mg of the test substance.
- Nominal 10 µg/L concentrate and stocks: The test substance was mixed in 25 mL acetone and the specific activity of the resulting concentrate was measured to be 90 MBq/g. The total radioactivity of this solution was determined to be 58.1 MBq and this is equated to 646 mg of the test substance. Known volumes of the concentrate were evaporated and re-dissolved in TEG to give 100 mg/L of the test substance stocks. Sub-samples (3 x 0.1 g) of each stock solution were weighed into scintillation vials and analysed by LSC.
- Dosing of stocks: Dosing stocks were replenished every 14 to 17 days. Similarly 1.5 L of TEG were added to a glass jar to act as the solvent control stock solution. A magnetic follower was added to each jar. The jars were then weighed and placed on the test rig and the magnetic stirrers started. The stock solutions were delivered at a nominal flow rate of 0.070 mL/min to the mixing chambers. The flow rates of the stock solutions and of the dilution water were measured on day 0 and thereafter three times per week. The test and control tanks were dosed at 100 µL/L of TEG.
- Preparation of additional stocks: For the nominal 1.0 µg/L concentration a second mixed concentrate was prepared on exposure day 56 (using unlabelled test substance) with a specific activity of 876 MBq/g. The total radioactivity of this solution was determined to be 1.9 MBq and this equated to 2 mg test substance in 25 mL acetone. For the nominal 10 µg/L concentration a second mixed concentrate was also prepared on exposure day 50 (using unlabelled lufenuron) with a specific activity of 91 MBq/g The total radioactivity of this solution was determined to be 10.3 MBq and this equated to 113 mg lufenuron in 21 mL acetone. These mixed concentrates were then dosed following the same procedure as for the original.

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: Fathead minnow
- Source: Bred stocks from the test facility that had hatched between 14 and 15 October 2003
- Batch No.: Ref 47/02
- Length at study initiation: 41 - 60 mm
- Weight at study initiation: 2.1 - 6.3 g
- Feeding: The proprietary fish food is described by the suppliers as containing 60% protein, 16% fat, 7% ash and 0.5% fibre.

ACCLIMATION
- Acclimation period: From 15 Oct. 2002 until use
- Acclimation conditions: In fibreglass aquaria under artificial lighting. No medication was given to the fish prior to the study.
- Health during acclimation: The fish were considered to be in good condition as no mortalities were observed in the stock population over the 48 hours prior to the start of the test and only 0.2% mortality had occurred during the previous 7 days in the batch of fish used in this study.

Route of exposure:

aqueous

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

60 d

Total depuration duration:

60 d

Hardness:

40.7 – 55.0 mg/L as CaCO3

Test temperature:

25 ± 1°C

pH:

7.0 - 8.0

Dissolved oxygen:

4.8 - 8.2 mg O2/L

Conductivity:

241 ± 22.5 µS/cm at 25 ˚C

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass vessels (external dimensions; 915 mm x 305 mm x 465 mm (length x width x height)), with a maximum capacity of 120 L.
- Fill volume: 105 L
- Type of flow-through: Peristaltic diluter
- Renewal rate of test solution: The test solutions were renewed at a nominal rate of 700 mL to produce 9.6 volume additions per 24 hours
- No. of organisms per vessel: 136
- No. of vessels per concentration (replicates): 1
- No. of vessels per control / vehicle control (replicates): 1
- Biomass loading rate: The fish loading at the start of the exposure phase (day 0) was 0.46 g/L/day based on a flow rate of 700 mL/min and the solvent control mean bulk weighing.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water was dechlorinated tap water, which had been passed through activated carbon, coarsely filtered to remove particulate material and dechlorinated with sodium thiosulphate. Salts were added, as required, to maintain minimum hardness levels, and the treated water was then passed through an ultraviolet steriliser to a second set of 20 and 10 µm filters. The supply was then delivered to a temperature controlled header tank in the test laboratory set to the nominal test temperature of 25 ± 1°C and finally filtered to 5 µm before use. The dechlorinated water supply is also monitored periodically for total ammonia, chemical oxygen demand, total organic carbon, total suspended solids, a range of cations and anions, trace metals, pesticides and other hydrocarbons. It is considered that none of the contaminants were present in sufficient quantity to have adversely affected the quality of the study.
- Test procedure: The test system was prepared on 9 July 2003 when the randomised vessels were filled with dilution water for the first time. [14C]-labelled test substance was first dosed into the test system on exposure day -2 (21 July 2003) to allow time for test solutions to reach equilibrium before fish exposure. The exposure phase started on exposure day 0 (23 July 2003), by placing the required number of fathead minnow into each vessel.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light:8 hours dark with 20 minute dawn and dusk transition periods
- Light intensity: 610 — 660 lux

Nominal and measured concentrations:

Nominal concentrations: 0 (solvent control), 1.0, 10 µg/L
Measured concentrations: 0 (solvent control), 0.9, 8.7 µg/L, respectively. (see Table 1 - Table 2 in 'Any other information on results incl. tables')

Reference substance (positive control):

no

Details on estimation of bioconcentration:

The kinetic bioconcentration factor, BCFk, was calculated using the KINETICS program as the ratio of the uptake rate constant to the depuration rate constant. 95%confidence limits for the kinetic bioconcentration factor were calculated using the Bootstrap method.

Lipid content:

10.98 %

Time point:

other: throughout study

Remarks on result:

other: 1.0 µg/L treatment

Lipid content:

8.3 %

Time point:

other: throughout study

Remarks on result:

other: 10 µg/L treatment

Key result

Conc. / dose:

10 µg/L

Temp.:

25 °C

pH:

7.5

Type:

BCF

Value:

16 265 L/kg

Basis:

whole body w.w.

Time of plateau:

120 d

Calculation basis:

kinetic

Remarks on result:

other: 5% lipid normalised; 10 µg/L treatment

Conc. / dose:

1 µg/L

Temp.:

25 °C

pH:

7.5

Type:

BCF

Value:

12 756 L/kg

Basis:

whole body w.w.

Time of plateau:

120 d

Calculation basis:

kinetic

Remarks on result:

other: 5% lipid normalised; 1 µg/L treatment

Conc. / dose:

1 µg/L

Temp.:

25 °C

pH:

7.5

Type:

BCF

Value:

28 000 L/kg

Basis:

whole body w.w.

Time of plateau:

120 d

Calculation basis:

kinetic

Remarks on result:

other: 1 µg/L treatment

Conc. / dose:

10 µg/L

Temp.:

25 °C

pH:

7.5

Type:

BCF

Value:

27 000 L/kg

Basis:

whole body w.w.

Time of plateau:

120 d

Calculation basis:

kinetic

Remarks on result:

other: 10 µg/L treatment

Conc. / dose:

1 µg/L

Temp.:

25 °C

pH:

7.5

Type:

BCF

Value:

21 000 L/kg

Basis:

whole body w.w.

Time of plateau:

60 d

Calculation basis:

steady state

Remarks on result:

other: 1 µg/L treatment

Conc. / dose:

10 µg/L

Temp.:

25 °C

pH:

7.5

Type:

BCF

Value:

19 000 L/kg

Basis:

whole body w.w.

Time of plateau:

60 d

Calculation basis:

steady state

Remarks on result:

other: 10 µg/L treatment

Remarks on result:

other: > 25% depuration after 29 days; > 79% depuration within 60 days.

Rate constant:

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

Value:

938

Remarks on result:

other: 1.0 µg/L treatment

Rate constant:

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

Value:

644

Remarks on result:

other: 10 µg/L treatment

Rate constant:

overall depuration rate constant (d-1)

Value:

0.034

Remarks on result:

other: 1.0 µg/L treatment

Rate constant:

overall depuration rate constant (d-1)

Value:

0.024

Remarks on result:

other: 10 µg/L treatment

Details on kinetic parameters:

ACCUMULATION PHASE
- 1.0 µg/L treatment: The uptake rate constant, K1, based on the predicted curve from the KINETICS program was calculated be 938/day with 95% confidence limits (736, 1170).
- 10 µg/L treatment: The uptake rate constant, K1, based on the predicted curve from the KINETICS program was calculated to be 644/day with 95% confidence limits (542, 768).

DEPURATION PHASE
The depuration rate constants, K2, based on the predicted curves from the KINETICS program were calculated to be 0.0335/day with 95% confidence limits (0.0256, 0.0440) for the nominal 1.0 µg/L and 0.0239/day with 95% confidence limits (0.0184, 0.0313) for the nominal 10 µg/L treatments.

Metabolites:

- Analytical results - fish tissues by Thin Layer Chromatography (TLC): Fish extracts analysed by TLC at the end of the accumulation and depuration phases confirmed that the residues in the fish tissues were predominately the test substance, therefore no metabolite characterisation was required. For the 1.0 and 10 µg/L treatments, 92 - 96% and 91 - 96% of the 14C residues respectively were characterised as the parent substance.

Details on results:

An overview of the results is provided in Table 1 - Table 7 in 'Any other information on results incl. tables'.

- Analytical results- test solutions: The measured concentrations in the 1.0 µg/L tank during the exposure period ranged from 0.50 to 1.7 µg/L with a time weighted mean of 0.90 µg/L 14C-labelled test substance equivalents, representing 90% of the nominal value. The measured concentration in the 10 µg/L tank similarly ranged from 4.9 to 20 µg/L with a time weighted mean of 8.7 µg/L 14C-labelled test substance equivalents, representing 87% of the nominal value. Time weighted means are used for calculation of the results. Characterisation of the radioactivity in the methanol samples by HPLC confirmed that it was predominately the test substance. The range measured between exposure day 0 and day 60 was 94 - 100% in the 1.0 µg/L test substance treated vessels and 97 - 99% in the 10 µg/L test substance treated vessels.

- Analytical results- fish tissues by combustion: Due to an oversight in the sampling schedule, measurement of radioactivity in the individual tissues as described in the method was not performed on exposure days 56, 60, l 10 and 120. In order to obtain data for these occasions, data from the specific analysis has been used to calculate whole body concentrations.

- Exposure (accumulation) phase: The lipid content in the fish used for calculation of the lipid BCF for [14C]l-labelled test substance was 9.6% (w/w), the mean value of all lipid analyses. The radioactivity expressed as 14C-labelled test substance equivalents was found to accumulate within the tissues. Although a clear plateau is not apparent, measured concentrations do not increase consistently over the last four whole body concentrations. Therefore, BCF at the end of exposure has been calculated to give an effective “plateau” (whole body concentration of 18630 µg 14C-labelled test substance equivalents/kg) for the nominal 1.0 µg/L treatment. The mean concentrations of 14C-labelled test substance equivalents determined in the various tissues and in the whole body during the plateau stage of the exposure phase are summarised with the respective, calculated BCF values for the nominal 1.0 µg/L treatment in the data below. The data below are the means of the individual concentrations recorded at exposure days 54 and 59 viscera, flesh and carcass and 54, 56, 58 and 60 for whole body. Standard deviations are calculated for these means.
1.0 µg/L 14C-labelled test substance
Viscera (wet weight): 26538 ± 12887 µg/kg; BCF = 29000L/kg
Flesh (wet weight): 11988 ± 8786 µg/kg; BCF = 13000L/kg
Carcass (wet weight): 14625 ± 3147 µg/kg; BCF = 16000L/kg
Whole body (wet weight): 18630 ± 8280 µg/kg; BCF = 21000L/kg
Whole body (lipid content): 194063 ± 86250 µg/kg; BCF = 220000L/kg

Similarly, for the nominal 10 µg/L treatment, the radioactivity expressed as 14C- labelled test substance equivalents was found to accumulate within the tissues and reached an effective “plateau” (whole body concentration of 168540 µg 14C- labelled test substance equivalents/kg) at the end of exposure. The values below are the means of the individual concentrations determined at exposure days 54 and 58 for viscera, flesh and carcass and 54, 56, 58 and 60 for whole body. Standard deviations are calculated for these means.
10 µg/L 14C-labelled test substance
Viscera (wet weight): 402713 ± 84552 µg/kg; BCF = 46000L/kg
Flesh (wet weight): 130225 ± 103379 µg/kg; BCF = 15000L/kg
Carcass (wet weight): 129963 ± 3553 µg/kg; BCF = 16000L/kg
Whole body (wet weight): 168540 ± 37271 µg/kg; BCF = 19000L/kg
Whole body (lipid content): 1755625 ± 388240 µg/kg; BCF = 200000L/kg

- Depuration phase: During the 60 days depuration phase the levels of [14C]-labelled test substance equivalents in the whole fish decreased steadily. Within 29 days of transfer to clean water the whole body residues in fish had dropped from the mean plateau phase concentrations by 49 and 25% in the nominal 1.0 and 10 μg/L treatments, respectively. After 60 days, 83 and 79% depuration had been achieved for the nominal 1.0 and 10 μg/L treatments, respectively. After 60 days of depuration > 79% of the [14C]-labelled test substance equivalents had been eliminated from the fish. These values are based on the mean values obtained for the accumulation plateau.

- Kinetic bioconcentration factor: The kinetic bioconcentration factor (BCFk) was calculated as the ratio of the uptake rate constant to the depuration rate constant (K1/K2). For the nominal 1.0 and 10 µg/L treatments, these were 28000 with 95% confidence limits (25000, 31000) and 27000 with 95% confidence limits (24000, 31000), respectively.

- Lipid content of the fish tissues: Samples showed a difference in lipid content between male and female fish and > 25% variation between male fish lipid was observed between the start and end of the study. This is considered to be due to changes in the sexual maturity of the males as the study progressed and is not considered to affect the validity of the study. As the difference was not consistent between the sexes, the mean lipid content was used for lipid normalisation of the BCF. The mean lipid contents on days 0 and at the end of the depuration phase were 8.8 and 9.8% respectively. The overall mean lipid content was 9.6%(w/w).

- Mortalities: Less than 5% mortality was observed in the population tested, which is considered acceptable for this type of study.

- Fish length and weight: The mean weights of the fish sampled as above at day 0 was 4.0 g with a range of weights from 2.1 to 6.3 g. The mean weight of fish sampled on day 120 from the solvent control was 5.0 g with a range of weights 2.4 to 12.1g.

Table 1. Water analysis- LSC results

Study day	Solvent control	1.0 µg/L	10 µg/L
-1	<0.5	0.76	8.1
0	<0.5	0.86	9.6
1	<0.5	0.56	5.3
2	<0.5	0.75	4.9
5	<0.5	0.50	5.0
7	<0.5	0.53	5.4
14	<0.5	0.73	5.3
21	<0.5	1.1	10
28	<0.5	0.84	5.4
37	<0.5	1.1	11
42	<0.5	0.85	20
47	<0.5	0.99	17
49	<0.5	1.7	13
56	<0.5	1.0	11
60	<0.5	1.1	7.6
61	<0.5	0.19	1.4
120	<0.5	<0.50	<0.50
Time weighted mean	<0.5	0.90	8.7
Percentage of nominal	-	90	87

a. Time weighted mean excludes data from days -1, 61 and 120

 

Table 2. Water analysis- HPLC results

Study day	Treatment (µg/L)	Measured conc. of the test substance equivalents by LSC (µg/L)	Percentage of radioactivity recovered in total procedure	Percentage of test substance determined in methanol sample by HPLC
0	1.0	0.86	97	100
	10	9.6	95	99
14	1.0	0.73	90	94
	10	5.3	94	97
28	1.0	0.84	95	99
	10	5.4	90	99
47	1.0	0.99	91	97
	10	17	96	98
60	1.0	1.1	88	98
	10	7.6	96	98

 

Table 3. Measured concentration of 14C-labelled test substance equivalents in fish tissues (exposed at 1.0 µg/L; expressed as µg/kg)

A) Accumulation phase

Fish no.#	Exposure days	7	14	21	28	35	42	49	52	54	56	58	60
1	Viscera	4100	10800	35600	40600	36300	34800	64500	30400	16800	-	28100	-
	Flesh	500	2000	3900	5200	3200	2900	13100	6800	29300	-	4000	-
	Carcass	1000	5400	8500	14100	6300	9400	28800	12200	20300	-	7800	-
	Whole body	1100	6200	14400	20600	8100	11200	34000	14000	20700	24500	8700	30200
2	Viscera	7000	16800	28700	37700	62400	50700	53900	91000	35300	-	43500	-
	Flesh	2100	4200	6300	2100	4500	11100	10100	9000	6000	-	5400	-
	Carcass	3600	9000	4500	13500	16300	23100	19900	18300	3200	-	24200	-
	Whole body	4100	10100	8300	15500	21700	30200	26700	27400	7000	-	27000	-
3	Viscera	11500	11200	34500	59300	34400	18000	27200	52200	9500	-	35500	-
	Flesh	2400	2600	5800	3700	7200	4700	8400	10600	21100	-	7100	-
	Carcass	3800	5600	7100	8900	18400	9700	17400	16900	4600	-	19300	-
	Whole body	5100	6700	13100	15500	21400	11500	19500	24700	7500	-	22000	-
4	Viscera	9400	19800	27200	26600	33800	41500	47700	65600	8100	-	35500	-
	Flesh	1900	2000	3400	5400	8400	5300	5900	16300	16400	-	6600	-
	Carcass	1600	5200	8800	10300	16000	22100	8200	20300	21500	-	16100	-
	Whole body	3200	6800	12200	14400	20000	26200	11100	32600	17900	-	20800	-
Mean	Viscera	8000	14650	31500	41050	41725	36250	48325	59800	17425	-	35650	-
	Flesh	1725	2700	4850	4100	5825	6000	9375	10675	18200	-	5775	-
	Carcass	2500	6300	7225	11700	14250	16075	18575	16925	12400	-	16850	-
	Whole body	3375	7450	12000	16500	17800	19775	22825	24675	13275	24500	19625	30200
BCF(min)	Whole body	1100	6200	8300	14400	8100	11200	11100	14000	7000	-	8700	-
BCF(max)	Whole body	5100	10100	14400	20600	21700	30200	34000	32600	20700	-	24000	-
BCF	Whole body	3750	8278	13333	18333	19778	21972	25361	27417	14750	27222	21806	33556
Bioaccumulation Plateau Phase Mean BCF*										20700

  

B) Depuration phase

Fish no. #	Study days	61	63	68	75	82	89	96	103	110	117	120
	Depuration days	1	3	8	15	22	29	36	43	50	57	60
1	Viscera	51600	77300	47100	600	29300	20900	9900	NR.	-	26800	-
	Flesh	5500	9100	5900	100	5700	2800	1300	6400	-	11600	-
	Carcass	10500	17200	20700	100	8800	6400	3000	5800	-	4600	-
	Whole body	19500	22000	24600	100	13600	9500	3300	NR	3700	7700	3200
2	Viscera	NR	68100	49700	41300	11500	27400	18500	18300	-	2000	-
	Flesh	NR	12000	11500	12900	3200	3300	4200	2700	-	300	-
	Carcass	NR	20900	19900	12100	4500	9000	5200	6700	-	600	-
	Whole body	NR	28300	26100	19800	6400	13200	8200	9300	-	600	-
3	Viscera	42600	35500	38600	1600	29300	NR	38300	4900	-	3900	-
	Flesh	6800	8500	8000	200	2600	3600	1900	800	-	1400	-
	Carcass	21100	18700	18100	200	5800	4900	5700	1600	-	2000	-
	Whole body	25000	22200	22000	300	7700	NR	9300	2200	-	2400	-
4	Viscera	69400	37500	4600	4500	24100	11400	5800	3400	-	6500	-
	Flesh	7700	5700	900	1100	6600	2600	600	500	-	2200	-
	Carcass	22000	18200	900	1900	10600	4100	1500	1500	-	5600	-
	Whole body	31000	22100	1100	2600	13700	5600	1700	2000	-	5700	-
Mean	Viscera	54533	54600	35000	12000	23550	19900	18125	8867	-	9800	-
	Flesh	6667	8825	6575	3575	4525	3075	2000	2600	-	3875	-
	Carcass	17867	18750	14900	3575	7425	6100	3850	3900	-	3200	-
	Whole body	25167	23650	18450	5700	10350	9433	5625	4500	3700	4100	3200
(min)	Whole body	19500	22000	1100	100	6400	5600	1700	2000	-	600	-
(max)	Whole body	31000	28300	26100	19800	13700	13200	9300	9300	-	7700	-
%Depuration from Plateau phase (mean whole body)^		-38	-27	1	69	44	49	70	76	80	78	83

NR: No result, due to incomplete combustion of tissue by sample oxidiser, so replicate data not included in Mean calculations and “whole body” calculations not possible

- Individual tissue measurements not available, mean whole body concentration calculated from specific analysis

* Mean plateau BCF (Whole body) over the period 54 to 60 days was 20700. This figure has been calculated using whole body concentrations during the stable plateau phase (study days 54 to 60) and the time weighted mean measured concentration of 14C-labelled test substance equivalents in water during the exposure period

# Fish number refers to replicate number of each batch of 4 fish sampled at each time point

^ Mean whole body plateau concentration is 18630

Table 4. Measured concentration of 14C-labelled test substance equivalents in fish tissues (exposed at 10 µg/L; expressed as µg/kg)

A) Accumulation phase

Fish no.#	Study days	7	14	21	28	35	42	49	52	54	56	58	60
1	Viscera	98500	116800	251400	NR	444600	482400	405200	241600	100000	-	765100	-
	Flesh	12900	21900	21800	21500	39300	39500	45200	40300	651600	-	55600	-
	Carcass	22700	60800	51100	53900	77900	114900	91800	88100	30200	-	125700	-
	Whole body	11900	71700	62100	NR	106700	141200	110700	98500	81100	173000	192500	181000
2	Viscera	99800	151500	370300	340900	380700	170300	668400	603400	334300	-	337800	-
	Flesh	17100	38900	25700	74600	42000	43900	84500	33500	49100	-	84800	-
	Carcass	27800	43100	67600	81400	95700	91500	NR.	119800	190800	-	146900	-
	Whole body	37500	57800	100800	141600	119600	104900	NR.	156500	221400	-	193600	-
3	Viscera	104500	191600	157500	286700	348200	419100	343400	419200	463600	-	273500	-
	Flesh	17000	27400	41600	30500	80500	55300	85600	539400	51200	-	43400	-
	Carcass	41300	70300	91800	69700	150700	162100	129300	135800	141200	-	134500	-
	Whole body	49300	88000	102000	86400	199500	216200	174200	221400	162800	-	166200	-
4	Viscera	102500	124600	374500	325200	287100	443500	408200	451800	473800	-	473600	-
	Flesh	13600	35500	32800	56300	44500	106900	49800	62300	61400	-	44700	-
	Carcass	34600	55800	84500	116700	89400	156300	78700	176500	147600	-	122800	-
	Whole body	36100	68800	104600	156800	98400	207600	103800	219700	170600	-	143200	-
Mean	Viscera	101325	146125	288425	317600	365150	378825	456300	429000	342925	-	462500	-
	Flesh	15150	30925	30475	45725	51575	61400	66275	168875	203325	-	57125	-
	Carcass	31600	57500	73750	80425	103425	131200	99933	130050	127450	-	132475	-
	Whole body	33700	71575	92375	128267	131050	167475	129567	174025	158975	17300	173875	181000
BCF(min)	Whole body	11900	57800	62100	86400	98400	104900	103800	98500	81100	-	143200	-
BCF(max)	Whole body	49300	88000	104600	156800	199500	216200	174200	221400	221400	-	193600	-
BCF	Whole body	3874	8227	10618	14743	15063	19250	14893	20003	18273	19885	19986	20805
Bioaccumulation Plateau Phase Mean BCF*										19373

 

B) Depuration phase

Fish no.#	Study days	61	63	68	75	82	89	96	103	110	117	120
	Depuration days	1	3	8	15	22	29	36	43	50	57	60
1	Viscera	465100	594300	311800	14700	360300	100100	7700	49200	-	70800	-
	Flesh	44600	59000	31800	3500	48100	25400	1400	13400	-	8700	-
	Carcass	116500	134800	62300	4200	75200	53800	2100	25000	-	22500	-
	Whole body	142100	164000	82100	4800	106100	64600	2500	31100	44000	25500
2	Viscera	353600	433900	337200	437900	272200	254600	99000	158300	-	190800	-
	Flesh	50300	103800	50700	80800	NR	85500	28700	20800	-	55100	-
	Carcass	101500	201300	122300	131600	92000	116800	45500	38600	-	43500	-
	Whole body	121200	248700	157900	160200	NR	155100	58700	48500	-	56500	-
3	Viscera	641900	545300	NR	367000	51000	548000	238800	6000	-	248000	-
	Flesh	64700	158900	30400	186900	14900	39400	59700	1700	-	62500	-
	Carcass	212200	136900	130400	166000	27300	87100	98200	2100	-	51200	-
	Whole body	286500	227700	NR	221400	33100	125500	134200	2300	-	86900	-
4	Viscera	NR	761000	578600	423000	58800	379400	384800	13100	-	31920	-
	Flesh	NR	103900	87600	112000	17100	42700	26100	3900	-	11175	-
	Carcass	NR	142800	141600	105800	22400	108100	69400	6700	-	20950	-
	Whole body	NR	220100	199400	168400	31600	162500	89400	6900	-	22870	-
Mean	Viscera	486867	583625	409200	310650	185575	320525	182575	56650	-	135380	-
	Flesh	53200	1 06400	50125	95800	26700	48250	28975	9950	-	34369	-
	Carcass	143400	153950	114150	101900	54225	91450	53800	18100	-	34538	-
	Whole body	183267	215125	146467	138700	56933	126925	71200	22200	30200	47943	36000
(min)	Whole body	121200	164000	82100	4800	31600	64600	2500	2300	-	22870	-
(max)	Whole body	286500	248700	199400	221400	106100	162500	134200	48500	-	86900	-
%Depuration from Plateau phase (mean whole body)^		-9	-28	13	18	66	25	58	87	74	72	79

NR: No result, due to incomplete combustion of tissue by sample oxidiser, so replicate data not included in Mean calculations and “whole body” calculations not possible

- Individual tissue measurements not available, mean whole body concentration calculated from specific analysis

* Mean plateau BCF (Whole body) over the period 54 to 60 days was 19373. This figure has been calculated using whole body concentrations during the stable plateau phase (study days 54 to 60) and the time weighted mean measured concentration of 14C-labelled test substance equivalents in water during the exposure period

# Fish number refers to replicate number of each batch of 4 fish sampled at each time point

^ Mean whole body plateau concentration is 168540

Table 5. Analysis of fish tissues expressed as mean concentration in lipid

A) accumulation phase

		1.0µg/L treatment (µg/kg in lipid)
	Study days	7	14	21	28	35	42	49	52	54	56	58	60
Mean	Whole body	35156	77604	125000	171875	185417	205990	237760	257031	138281	255208	204427	314583
BCF^	Whole body	39062	86227	138889	190972	206019	228878	264178	285590	1 53646	283564	227141	349537
Mean plateau BCF (Whole body)*										215625
		10µg/L treatment (µg/kg in lipid)
	Study days	7	14	21	28	35	42	49	52	54	56	58	60
Mean	Whole body	351042	745573	962240	1336115	1365104	1744531	1349656	1813021	1655990	1802083	1811198	1885417
BCF	Whole body	40350	85698	110602	153576	156909	200521	155133	208363	190344	2071 36	208 1 84	216715
Mean plateau BCF (Whole body)*										201796

 

B) Depuration phase

		1.0µg/L treatment (µg/kg in lipid)
Study days		61	63	68	75	82	89	96	103	110	117	120
Depuration days		1	3	8	15	22	29	36	43	50	57	60
Mean^	Whole body	268750	246354	192188	59375	107813	98260	58594	46875	38542	75729	33333
		10µg/L treatment (µg/kg in lipid)
Study days		61	63	68	75	82	89	96	103	110	117	120
Depuration days		1	3	8	15	22	29	36	43	50	57	60
Mean	Whole body	1909031	2240885	1525698	1444792	593052	1322135	741667	231250	458333	499406	375000

^ Based on mean % lipid content of 9.6% (w/w)

* This figure has been calculated using whole body concentrations during the stable plateau phase (study days 54 to 60 for the 1.0 and 10µg/L) and the mean measured concentration of 14C-labelled test substance equivalents in water during the exposure period

Table 6. TLC analysis- Fish

Study day	Treatment (µg/L)	Mean recovery of radioactivity (%)	% of extractable characterised as the test substance
56	1.0	106	92
56	10	95	91
60	1.0	96	94
60	10	101	95
110	1.0	82	96
110	10	95	95
120	1.0	85	92
120	10	85	96

Table 7. Lipid analysis

Sample identity	replicate	Lipid content (% w/w)	Mean lipid content (% w/w)	SD	Mean lipid content throughout study (% w/w)
Study day 0 excess fish at start male fish	1 2 3	5.6 5.7 5.3	5.5*^	0.21	9.6
Study day 0 excess fish at start female fish	1 2 3	10.0 - 13.9	12.0*^	2.76
Study day 120 solvent control male fish	1 2 3	4.3 4.4 -	4.4	0.07
Study day 120 solvent control female fish	1 2 3	12.3 12.6 12.6	12.5	0.17
Study day 120 1.0µg/L male fish	1 2 3	11.8 11.6 11.2	11.5*	0.15
Study day 120 1.0µg/L female fish	1 2 3	16.4 13.4 -	14.9*	0.62
Study day 120 10µg/L male fish	1 2 3	3.5 3.3 3.6	3.5^	0.31
Study day 120 10µg/L female fish	1 2 3	12.0 12.9 11.7	12.2^	2.12

- Sample lost

a. >25% variation from start to end of test, not considered to affect the validity of the study

  * Used in calculating the mean lipid content for 1.0µg/L, = 10.98 % (w/w)

^ Used in calculating the mean lipid content for 10µg/L, = 8.30 % (w/w)

 

 Table 8. kinetic bioconcentration factors (BCFk)

	1.0µg/L treatment	10µg/L treatment
Uptake rate constant (K1)	938/day	644/day
Depuration rate constant (K2)	0.00335/day	0.0239/day
BCFk	28000	27000

 

 

Validity criteria fulfilled:

yes

Conclusions:

In a bioaccumulation study in Fathead minnow, following OECD TG 305 and under GLP, the kinetic bioconcentration factor (BCFk) were calculated to be 28000 and 27000 L/kg for 1.0µg/L and 10µg/L, respectively. After 5% lipid content normalisation, the BCFk values were 12756 and 16265 L/kg for 1.0 and 10 µg/L treatments, respectively.

Executive summary:

The bioaccumulation potential of the 14C- Dichlorophenyl-ring-labelled test substance was investigated in a flow-through study in accordance with OECD TG 305 and in compliance with GLP criteria. In this study, Fathead minnow (Pimephales promelas) were exposed to the radiolabelled test substance at nominal concentrations of 1 and 10µg/L (measured time weighted concentrations: 0.9 and 8.7µg/L, respectively) for 60-day uptake phase (136 fish per concentration). Afterwards, the fish were transferred to aquaria without the test substance for a 60-day depuration phase. In addition, a solvent control (acetone) was included as well and tested under the same condition. Specific activities were measured and confirmed by using LSC, TLC and HPLC. The test conditions were as following: pH 7.0 – 8.0, 25 ± 1 °C, 16 hours light -8 hours darkness cycle with 20 minute dawn and dusk transition periods (610 – 660 Lux). The water conditions were 40.7 – 55.0 mg/L total hardness (as CaCO3), 241 ± 22.5 µS/cm conductivity (at 25 ˚C) and 4.8 - 8.2 mg O2/L.

The results show that the test substance predominated the radioactivity in the analysed samples. The range measured test substance (between day 0 and day 60) was 94 -100% and 97 - 99% in the 1.0 and 10µg/L treatments, respectively. The radioactivity expressed as 14C-labelled test substance equivalents was found to accumulate within the tissues. Although a clear plateau is not apparent, measured concentrations do not increase consistently over the last four whole body concentrations. Therefore, BCF at the end of exposure has been calculated to give an effective “plateau” (whole body concentration of 18630 µg 14C-labelled test substance equivalents/kg) for the nominal 1.0 µg/L treatment. Similarly, for the nominal 10 µg/L treatment, the radioactivity expressed as 14C- labelled test substance equivalents was found to accumulate within the tissues and reached an effective “plateau” (whole body concentration of 168540 µg 14C- labelled test substance equivalents/kg) at the end of exposure. The mean values of lipid content were 10.98% (w/w) and 8.30% (w/w) for the 1.0 and 10 µg/L treatments, respectively. The depuration of accumulated residues from the whole body was steady with approximately > 25% depuration after 29 days and > 79% depuration within 60 days. Based on these findings, the update rate constants (K1) were calculated to be 938/day for the 1.0µg/L treatment and 644/day for the 10 µg/L treatment. The depuration rate constants (K2) were calculated to be 0.0335/day for the 1.0µg/L treatment and 0.0239/day for the 10 µg/L treatment. The kinetic bioconcentration factor (BCFk) were calculated to be 28000 and 27000 L/kg for 1.0 µg/L and 10 µg/L, respectively. After 5% lipid content normalisation, the BCFk values were 12756 and 16265 L/kg for 1.0 and 10 µg/L treatments, respectively.

Description of key information

All available data was assessed and the studies representing the worst-case effects were included as key study. Other studies are included as supporting information.

BCF = 16265 L/kg (5% lipid normalised; 10 µg/L treatment), whole fish, Pimephales promela, aqueous, freshwater, OECD proposal for updating OECD TG 305, Maynard 2004

Key value for chemical safety assessment

BCF (aquatic species):

16 265 dimensionless

Additional information

Five studies are available for this endpoint. Three of them followed standard test guideline and complied with GLP (one Reliability 1 and two Reliability 2 studies). The study representing the worst-case effect (i.e. showed highest BCF value) was included as key study. The effect value (BCF = 16265) from the key study was selected in CSA. In this study (Maynard 2004), Fathead minnow (Pimephales promelas) were exposed to the 14C-Dichlorophenyl-ring-labelled test substance at nominal concentrations of 1 and 10µg/L (measured time weighted concentrations: 0.9 and 8.7µg/L, respectively) for 60-day uptake phase (136 fish per concentration). Afterwards, the fish were transferred to aquaria without the test substance for a 60-day depuration phase. In addition, a solvent control (triethylene glycol) was included as well and tested under the same condition. The test conditions were as following: pH 7.0 – 8.0, 25 ± 1 °C, 16 hours light -8 hours darkness cycle with 20 minute dawn and dusk transition periods (610 – 660 Lux). The water conditions were 40.7 – 55.0 mg/L total hardness (as CaCO3), 241 ± 22.5 µS/cm conductivity (at 25 ˚C) and 4.8 - 8.2 mg O2/L. The kinetic bioconcentration factor (BCFk) were calculated to be 28000 and 27000 L/kg for 1.0µg/L and 10µg/L, respectively. After 5% lipid content normalisation, the BCFk values were 12756 and 16265 L/kg for 1.0 and 10µg/L treatments, respectively.

One of the supporting study is a 190-d water/sediment outdoor microcosm study. The maximum BAFs (on Day 7) of the test substance were determined to be 294 and 360 in whole fish (bluegill sunfish, Lepomis macrochirus) for 0.5 mg/L and 5.0 µg/L treatments, respectively (Volz 2003). One supporting study is a 77-d laboratory study on bluegill sunfish (Lepomis macrochirus). The kinetic bioconcentration factor (BCFk) were calculated to be 5300 L/kg for the whole fish (Forbis 1986). The other two supporting studies did not follow standard test guideline but were well documented, and met generally accepted scientific principles. They were in compliance with GLP criteria. One study spiked via the water phase. Four species of aquatic invertebrate (Daphnia magna, Gammarus pulex, Chironomus riparius and Lumbriculus variegatus) were exposed separately to the 14C-dichlorophenyl-labelled test substance at a nominal concentration of 0.4 μg/L in water-sediment systems. The BAF values were determined to be 12, 32, 56 and 210 L/kg for Lumbriculus variegatus, Daphnia magna, Gammarus pulex and Chironomus riparius, respectively (Thompson 2003a). The last supporting study spiked via the sediment phases and used the same test organisms. These organisms were exposed to the 14C-dichlorophenyl-labelled test substance at a nominal sediment concentration of 10 μg/kg dry weight. The BAF values were determined to be 1.5, < 4.6, 2.6 and 8.0 for Lumbriculus variegatus, Daphnia magna, Gammarus pulex and Chironomus riparius, respectively.