4,4'-(9H-fluoren-9-ylidene)bis(2-chloroaniline)

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:

03 Jun - 07 Oct 2016

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:

2012

Deviations:

yes

Remarks:

The measured concentrations in water at the target concentrations of 10 and 100 ng/L varied within ± 30 and ± 33%, respectively, of the mean concentration. See section "Any other information on material and method incl. tables" for explanation.

Qualifier:

according to guideline

Guideline:

EU Method C.13 (Bioconcentration: Flow-through fish test)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes

Radiolabelling:

no

Details on sampling:

- Sampling intervals/frequency for test organisms and test medium: See Table 1 in "Any other information on material and methods incl. tables".
- Sample storage conditions before analysis: Samples were analysed on the day of sampling
- Details on sampling and analysis of test organisms and test media samples:
Test medium:
Samples were taken from the control and both concentrations and analyzed before introduction of the fish to check if the actual test concentrations were in agreement with the expected concentrations. A singular sample was taken from the control, while triplicate samples were taken from both concentrations (bottom, middle, top) in order to determine, next to stability, the homogeneity of the concentrations; Singular sub-samples of 2 mL each were taken from the 500 mL-sample for analysis.
Fish sample:
Fish were sampled with a small fishing net, rinsed quickly with untreated water, blotted dry and then instantly killed. Each replicate was homogenized (1:1) in Milli-RO water using a blender (Turrax). A sub-sample of 400 mg homogenizate (containing approximately 200 mg fish) was taken and used for concentration analysis; one fish sampled per replicate

Vehicle:

yes

Remarks:

Dimethylformamide

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

PREPARATION AND APPLICATION OF TEST SOLUTION:
- Pre-conditioning: Mixing- and test vessels were pre-conditioned with the test concentrations, i.e. test medium containing the test concentrations was led through the vessels for four days, using the flow-through system, before the start of the BCF study
- Method: During the uptake phase, the stock solutions were dosed via a computer-controlled system (DaVinci) consisting of micro-dispensers (Gilson). Via this system the dosed volume from the stock entered a mixing flask separately from the medium supply. The medium was supplied via a flow meter and the flow rate was 16 L/h, allowing approximately six volume replacements (64 litres) through each aquarium each day. In the mixing flask the dosed stock volume and the medium were mixed under continuous stirring at a ratio of 1 : 10,000.
During the depuration phase the medium was supplied directly via the same flow meters at a rate of 16 L/h without the dosing of stock solution.
- Controls: Test medium without test item but including DMF in the same amount as used in the treated solutions.
- Chemical name of vehicle: dimethylformamide
- Concentration of vehicle in test medium: 0.1 mL/L

Test organisms (species):

Cyprinus carpio

Details on test organisms:

TEST ORGANISM
- Common name: Carp
- Source: Zodiac, proefacc, "De Haar Vissen", Wageningen University and Research Centre, The Netherlands
- Length at study initiation: 5.2 ± 0.3 cm, based on measurement of all fish used for this test
- Weight at study initiation: 4.53 ± 0.61 g, based on weighing of all fish used for this test
- Weight at termination (mean and range, SD): 6.56 ± 0.85 g
- Lipid content at test initiation (mean and range, SD): 4% (control group at test start)
Feeding during test:
- Food type and amount: Pelleted fish food: Rough protein: 45%; Rough fat: 10%; Cellulose: 1.3%; Rough ashes: 8.6%; Phosphor: 1.1%; Copper: 5 mg/kg; Vitamin A: 22,500 IU/kg; Vitamin C: 300 mg/kg; Vitamin D3: 2,500 IU/kg; Vitamin E: 200 mg/kg
- Frequency: ration was 2% of body weight per day. Recalculation of the feeding rate was performed after each sampling point based on mean fish weights

ACCLIMATION
- Acclimation period: At least 12 days after delivery; Fish were introduced into the test vessels four days after the start of the flow-through system, i.e. test concentrations were allowed to stabilise for four days in the vessels before the exposure of the fish was initiated.
- Acclimation conditions (same as test or not): Adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands)
- Type and amount of food: pelleted fish food (Cyprico Crumble Excellent (300-500 µm), Coppens International bv, Helmond, The Netherlands)
- Feeding frequency: Daily
- Health during acclimation: In the batch of fish used for the test, mortality during seven days prior to the start of the test was less than 5%

Route of exposure:

aqueous

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

35 d

Total depuration duration:

21 d

Hardness:

196 to 232 mg/L (Control)
214 mg/L (10 and 100 ng/L)

Test temperature:

21.6 - 23.1 °C (Control)
21.4 - 22.8 °C (10 ng/L)
21.3 - 22.7 °C (100 ng/L)

pH:

7.5 - 8.0 (Control; 10 and 100 ng/L)

Dissolved oxygen:

7.0 - 9.2 mg/L (Control)

TOC:

0.16 - 0.37 mg/L (ISO)
44 to 66 mg/L (Control ISO + DMF)

Details on test conditions:

TEST SYSTEM
Test vessel:
- Type: open (covered by a removable Perspex plate)
- Material, size, headspace, fill volume: 64 litres (40x40x40 cm) consisting of stainless steel
- Aeration: continuously
- Type of flow-through: computer-controlled system consisting of micro-dispensers into a mixing flask (Da Vinci Europe, Schiedam, The Netherlands)
- Renewal rate of test solution (frequency/flow rate): test item is dosed every 1-60 minutes and mixed with a continuous flow of dilution water to provide continuous renewal of the test solutions
- No. of organisms per vessel: 111 (total number of fish)
- No. of vessels per concentration (replicates): 44
- No. of vessels per control / vehicle control (replicates): 23
- Biomass loading rate: 0.52 g fish (wet weight)/litre/day
- Pre-conditioning: Mixing- and test vessels were pre-conditioned with the test concentrations, i.e. test medium containing the test concentrations was led through the vessels for four days, using the flow-through system, before the start of the BCF study.
- Cleaning: Uneaten food and fecal debris were siphoned daily from the test chambers about an hour after feeding

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands); CaCl2.2H2O: 211.5 mg/L; MgSO4.7H2O: 88.8 mg/L; NaHCO3: 46.7 mg/L; KCl: 4.2 mg/L
- Holding medium different from test medium: yes
- Intervals of water quality measurement: Conductivity, pH, nitrate, nitrite and ammonia concentration: once a week. Temperature: continuous. In addition, pH and temperature were measured before transferring the fish to the test system.
- Intervals of test medium replacement: six volume replacements (64 litres) through each aquarium each day

OTHER TEST CONDITIONS
- Euthanasia: The fish sampled were instantly killed by a strike to the head followed by cervical incision. At the end of the test the surviving fish were rapidly killed by exposing them to ca. 1.2% ethylene glycol monophenylether in water
- Adjustment of pH: Adjusted ISO medium with a hardness of 180 mg CaCO3 per litre and a pH of 7.7 ± 0.3.
- Photoperiod: 16 hours photoperiod daily

RANGE-FINDING / PRELIMINARY STUDY
- Test concentrations:10 and 100 ng/L
- Results used to determine the conditions for the definitive study: Based on the results of the pre-test a main test was conducted.

Nominal and measured concentrations:

Nominal: Control, 10 and 100 ng/L
Measured:
10 ng/L: 79% mean recovery with a coefficient of variation of 20%
100 ng/L: 85% mean recovery with a coefficient of variation of 19%

Reference substance (positive control):

no

Details on estimation of bioconcentration:

Evaluation of the results started with obtaining the uptake curve by plotting the measured concentrations in/on fish against time on arithmetic scales. If the curve reached a 'plateau', the steady state bioconcentration factor, BCFss, was calculated from:

BCFss = Cf,ss / Cw,ss

Where: Cf,ss = concentration in fish at steady state
Cw,ss = concentration in water at steady state

The mean BCFss was based on the average of the concentrations measured at the various sampling points during the exposure period.
The kinetic BCF (BCFk) was calculated as the ratio ku/kd, the two first-order kinetic constants. The kd (depuration rate constant) was calculated first and then used for the calculation of the ku (uptake rate constant).

kd

Cf = Cf,0 * e^-Kdt

Where: Cf = concentration in fish
Cf,0 = concentration in fish at the start of the depuration phase
kd = depuration rate constant
t = time
ku

Cf/Cw = ku/kd * (1 - e^-kdt)

Where: Cf = concentration in fish
Cw = concentration in water
ku = uptake rate constant
kd = depuration rate constant
t = time

Single First Order (SFO) kinetics were fitted to the data. Optimisations were performed using non-linear regression with the program ModelMaker (AP Benson, Wallingford, Oxfordshire, UK, 2004).
The half-life (DT50) was calculated as:

DT50 = - ln0.5/ kd

Normalisation for lipid content was performed for both the BCFss and the BCFk. First the concentration in the fish was corrected for the lipid content at each sampling point, using the formula below. Then the BCFss and the BCFk were re-calculated.

Cf,L = 0.05/L * Cf

Where: Cf,L = lipid-normalized concentration in fish (mg/kg)
L = lipid fraction
Cf = concentration of test item in fish (mg/kg)

Lipid content:

4.3 %

Time point:

start of exposure

Remarks on result:

other: Control group

Lipid content:

>= 2 - <= 4 %

Time point:

end of exposure

Remarks on result:

other: Control group

Key result

Conc. / dose:

10 ng/L

Temp.:

>= 21.4 - <= 22.8 °C

pH:

7.8

Type:

BCF

Value:

9 883 L/kg

Basis:

whole body w.w.

Remarks:

Results after Lipid normalisation

Calculation basis:

kinetic

Key result

Conc. / dose:

100 ng/L

Temp.:

>= 21.3 - <= 22.7 °C

pH:

7.8

Type:

BCF

Value:

> 9 986 L/kg

Basis:

whole body w.w.

Remarks:

with lipid correction

Calculation basis:

kinetic

Key result

Conc. / dose:

10 ng/L

Temp.:

>= 21.4 - <= 22.8 °C

pH:

7.8

Type:

BCF

Value:

4 779 L/kg

Basis:

whole body w.w.

Remarks:

without lipid correction

Calculation basis:

kinetic

Key result

Conc. / dose:

100 ng/L

Temp.:

>= 21.3 - <= 22.7 °C

pH:

7.8

Type:

BCF

Value:

5 039 L/kg

Basis:

whole body w.w.

Remarks:

before lipid correction

Calculation basis:

kinetic

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

2.7 d

Remarks on result:

other: 10 ng/L

Remarks:

before lipid correction

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

4.5 d

Remarks on result:

other: 100 ng/L

Remarks:

before lipid correction

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

4.3 d

Remarks on result:

other: 10 ng/L

Remarks:

after lipid correction

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

4 d

Remarks on result:

other: 100 ng/L

Remarks:

after lipid correction

Rate constant:

other: uptake rate constant, per day

Value:

1 214

Remarks on result:

other: 10 ng/L

Rate constant:

other: uptake rate constant, per day

Value:

773

Remarks on result:

other: 100 ng/L

Rate constant:

overall depuration rate constant (d-1)

Value:

0.25

Remarks on result:

other: 10 ng/L

Rate constant:

overall depuration rate constant (d-1)

Value:

0.15

Remarks on result:

other: 100 ng/L

Details on kinetic parameters:

- Kinetic Bioconcentration factor: 10 ng/L: 4779 L/kg; 100 ng/L: 5039 L/kg
- Lipid normalized kinetic bioconcentration factor (BCFkL): 10 ng/L: 9883 L/kg and 100 ng/L: 9986 L/kg

Details on results:

- Mortality of test organisms: Mortality or other adverse effects in the control and both target concentrations was less than 10% at the end of the test

Acceptability of the test

The water temperature variation was ± 2 °C for both test concentrations and the control.

The oxygen concentration was maintained at least at 60% of the air saturation value throughout the test (>5 mg/L at 22 °C).

The analytical data showed with coefficients of variation of 20-19% respectively for the nominal concentrations of 10 ng/L and 100 ng/L, that the test concentrations were relatively well maintained. However, due to very low test concentrations, the impact of adsorption of feed and fish in the chambers may be not be disregardable. Furthermore, the fluctuating test concentration revealed in fact a variability of  > 20% of the mean of the measured values, but did not show a concentration decline. Thus, this is considered to present an acceptable deviation.” 

Test concentrations were below the limit of solubility.

Mortality or other adverse effects in the control and both target concentrations was less than 10% at the end of the test.

Biological Results:

No steady state concentration in fish was reached for both target concentrations during the 35-day uptake phase. Therefore, no BCFss could be calculated but the BCF at the end of the exposure period was >5,000 L/kg at both target concentrations. The kinetic bioconcentration factors, BCFk, of 4,4’-(9H-fluoren-9-ylidene)bis(2-chloroaniline) at target concentrations of 10 and 100 ng/L were 4779 and 5039 L/kg, respectively. When corrected for lipid still no steady state was reached within the exposure period. Therefore, no BCFssL could be calculated but the BCFL at the end of the exposure period was >5,000 L/kg at both target concentrations. The BCFkL values at target concentrations of 10 and 100 ng/L were 9,883 and 9,986 L/kg, respectively. The DT50 value for depuration, before and after lipid normalisation, was between 3 and 5 days for both target concentrations. Thus showing, that 4,4’-(9H-fluoren-9-ylidene)bis(2-chloroaniline) was cleared rapidly from fish tissues. A test item is considered to be bioaccumulative or very bioaccumulative when the BCF is >2,000 or >5,000, respectively. The BCFk obtained, before and after lipid normalisation, with 4,4’-(9H-fluoren-9-ylidene)bis(2-chloroaniline) were ≥5,000 and therefore the test item is considered to be very bioaccumulative in fish.

Analytical Results:

Analysis of test medium

Water concentrations could not be kept within ± 20% of the mean concentration. This was due to the very low test concentrations in combination with the tendency of the test item to adsorb. However, the mean percentages of recovery were 79 and 85% for the 10 and 100 ng/L concentrations, respectively, with coefficients of variation of 20 and 19%, respectively. These data evidence that the test concentrations were relatively well maintained. The average concentrations of 4,4’-(9H-fluoren-9-ylidene)bis(2-chloroaniline) during the 35-day uptake phase were 7.9 ± 1.6 ng/L and 85 ± 16 ng/L at target concentrations of 10 and 100 ng/L, respectively. Samples of the test medium were also taken during the depuration phase, i.e. on days 37, 42 and 56 of the study. Fish were transferred to clean vessels containing water without the test item for the start of the depuration phase and the flow-through of water was 16 L/h. However, trace concentrations of the test item were measured on days 37 and 42. This was most probably related to the high accumulation of test item in/on the fish during the uptake phase that was released from the fish during the depuration phase.

Analysis of fish

On day 35, the remaining fish were transferred into dilution medium without, 4,4’-(9H-fluoren-9-ylidene)bis(2-chloroaniline) and without DMF to start the depuration phase. After 21 days of depuration, 4,4’-(9H-fluoren-9-ylidene)bis(2-chloroaniline) concentrations in fish previously exposed to target concentrations of 10 and 100 ng/L were reduced to 3 and 2% of the concentration at the start of the depuration phase, respectively.

Table 1: Biological Results: Effect parameters

Target conc. (ng/L)	BCFss (L/kg)	BCFk (L/kg)	DT50 (d)	BCFssL (L/kg)	BCFkL (L/kg)	DT50L (d)
10	-	4.779	2.7	-	9.883	4.3
100	-	5.039	4.5	-	9.986	4.0

- Steady state was not reached during the 35-day exposure period

Table 2: Analytical Results: Average concentration in water during the uptake phase

Time of measurement (days)	Average 4,4’-(9H-fluoren-9-ylidene)bis(2-chloroaniline) concentration ± SD (ng/L)
	Target concentration 10 ng/L	Target concentration 100 ng/L
0-1	8.0 ± 2.5	78 ± 23
0-3	7.7 ± 2.1	76 ± 19
0-7	7.4 ± 1.8	79 ± 17
0-14	7.7 ± 1.8	80 ± 15
0-21	7.8 ± 1.6	79 ± 14
0-28	8.0 ± 1.6	82 ± 16
0-35	7.9 ± 1.6	85 ± 16

 

Table 3: Analytical Results: Average concentrations in fish

Time (days)		Target concentration: 10 ng/L		Target concentration: 100 ng/L
		Concentration (ng/kg fish)	BCF at day x (L/kg)	Concentration (ng/kg fish)	BCF at day x (L/kg)
Uptake phase	1	3.707 ± 906	464 ± 113	58.086 ± 3.486	746 ± 45
	3	11.572 ± 1.122	1.510 ± 146	132.509 ± 5.637	1.749 ± 74
	7	25.344 ± 1.550	3.436 ± 210	252.418 ± 9.389	3.204 ± 119
	14	39.401 ± 11.350	5.087 ± 1.465	484.060 ± 64.018	6.045 ± 799
	21	41.438 ± 8.887	5.342 ± 1.146	390.891 ± 97.396	4.948 ± 1.233
	28	28.349 ± 5.036	3.545 ± 630	260.470 ± 60.502	3.167 ± 736
	35	50.336 ± 14.327	6.379 ± 1.816	458.695 ± 69.446	5.396 ± 817
Depuration phase	37	26.446 ± 4.599		424.671 ± 61.471
	42	11.302 ± 5.318		133.311 ± 35.932
	56	1.480 ± 537		9.167 ± 8.606

Validity criteria fulfilled:

yes

Conclusions:

The bioaccumulation test conducted with 4,4'-(9H-fluoren-9-ylidene)bis(2-chloroaniline) was considered as valiid without restriction. The kinetic BCF lipid-normalised values obtained at 10 and 100 ng/L were 9883 and 9986 L/kg respectively. Before lipid correction the results were 4779 and 5039 L/kg at 10 and 100 ng/L respectively.
However, the half lives of 4,4'-(9H-fluoren-9-ylidene)bis(2-chloroaniline) during the depuration phase was calculated to be between 3 and 5 days for both target concentrations. the substance is then found to be cleared rapidely from fish tissues.

Executive summary:

The bioaccumulation test with 4,4'-(9H-fluoren-9-ylidene)bis(2-chloroaniline) was conducted under flow-through conditions at 2 targeted concentrations: 10 and 100 ng/L. Despite the low solubility of the substance and its tendency to adsorb, the test concentrations were maintained relatively stable during the test (coefficient of variations were 20 and 19% at 10 and 100 ng/L respectively). Hence to reduce the loss of the test item that could be linked with adsorption, the mixing- and test vessels were pre-conditioned with the test item. However due to the very low targeted concentrations, water concentrations could not be kept within 20% of the mean concentration. Considering all above pre-treatment applied and the difficult properties of the test item for such a study, this deviation was considered as acceptable.

Stock solutions of the test item were prepared in dimethylformamide (DMF) and dosed via a computer-controlled system. At the start of the experiment 44 fish were exposed to each target concentration and 23 fish were exposed to a solvent control. No steady state concentration in fish was reached at both concentration until 35 days of uptake phase. The depuration phase lasted 21 days. Because the steady state concentrations were not reached in the fish, kinetic bioconcentratrion factors were calculated. The kinetic BCF were calculated to be 4779 and 5039 L/kg at 10 and 100 ng/L respectively. After lipid normalisation, the values are 9883 and 9986 L/kg respectively. the half lives during the depuration phase were between 3 and 5 days demonstrating a rapid clearance from fish tissues.

With kinetic BCF values above 5000 L/kg, the test items needs to be considered as very bioaccumulable.

Description of key information

BCF_kL = 9883 L/kg at 10 ng/L targeted concentration

BCF _kL = 9986 L/kg at 100 ng/L targeted concentration

Before lipid normalisation, the BCF were around 5000 L/kg for both targeted concentration.

The half-life of the test item in the fish during the depuration phase was between 3 and 5 days, demonstrating a rapid capacity for clearance by the fish.

Key value for chemical safety assessment

BCF (aquatic species):

9 900 L/kg ww

Additional information

A bioconcentration test according to OECD 305 and GLP was performed with 4,4’-(9H-fluoren-9-ylidene)bis(2-chloroaniline) as test substance (Charles River Laboratories, 2017). As the test substance was found to be highly toxic under chronic conditions for aquatic invertebrates, a pre-test was performed as an initial step in order to determine the toxicity of the chosen target concentrations to fish. Ten fish were exposed to test concentrations of 10 and 100 ng/L for a total test period of 10 days under flow-through conditions. There were no adverse effects observed in the fish at both concentrations. Therefore, these concentrations were choosen for the performance of the main test.

For pre- and main test, stock solutions for the bioconcentration test were prepared in dimethylformamide (DMF) and dosed via a computer-controlled system consisting of micro-dispensers into a mixing flask separately from the dilution medium supply (ISO medium). Medium was supplied via a flow meter at a rate of 16 L/h. In the mixing flask, the dosed spike solution volume and the dilution medium at a ratio of 1:10,000 were mixed with continuous stirring. The final target concentrations were 10 and 100 ng/L.

At the start of the exposure period, 44 fish were exposed to each target concentration and 23 fish were exposed to a solvent control. The uptake phase and the depuration phase of the study lasted for 35 and 21 days, respectively. During both phases test medium samples and fish samples were taken for chemical analysis.

Water concentrations could not be kept within ± 20% of the mean concentration. This was due to the very low test concentrations in combination with the tendency of the test item to adsorb. The average concentrations of 4,4’-(9H-fluoren-9-ylidene)bis(2-chloroaniline) during the 35-day uptake phase were 7.9 ± 1.6 ng/L and 85 ± 16 ng/L at target concentrations of 10 and 100 ng/L, respectively.

No steady state concentration in fish was reached for both target concentrations during the 35-day uptake phase. Therefore, no BCFss could be calculated but the BCF at the end of the exposure period was >5000 L/kg at both target concentrations. The kinetic bioconcentration factors, BCF_k, of 4,4’-(9H-fluoren-9-ylidene)bis(2-chloroaniline) at target concentrations of 10 and 100 ng/L were 4779 and 5039 L/kg, respectively.

When corrected for lipid still no steady state was reached within the exposure period. Therefore, no BCF_ssL could be calculated but the BCF_L at the end of the exposure period was >5000 L/kg at both target concentrations. The BCF_kL values at target concentrations of 10 and 100 ng/L were 9883 and 9986 L/kg, respectively.

The DT50 value for depuration, before and after lipid normalisation, was between 3 and 5 days for both target concentrations. Thus showing, that 4,4’-(9H-fluoren-9-ylidene)bis(2-chloroaniline) was cleared rapidly from fish tissues.

 

Effect parameters obtained in this study are summarized in table below.

Target conc. (ng/L)	BCFss (L/kg)*	BCFk (L/kg)	DT50 (d)	BCFssL (L/kg)	BCFkL (L/kg)	DT50L (d)
10	-	4779	2.7	-	9883	4.3
100	-	5039	4.5	-	9986	4.0

*Steady state was not reached during the 35-day exposure period