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Environmental fate & pathways

Bioaccumulation: aquatic / sediment

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Reference
Endpoint:
bioaccumulation in aquatic species: fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2019-11 to 2020-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 305 (Bioaccumulation in Fish: Aqueous and Dietary Exposure) -I: Aqueous Exposure Bioconcentration Fish Test
GLP compliance:
yes (incl. certificate)
Radiolabelling:
no
Details on sampling:
- Sampling intervals/frequency for test organisms:
Uptake phase: T0.2, T1, T2, T3, T4, T7, T9, T11, T14 (days)
Depuration phase: T14.2, T15, T17, T18, T21, T25, T29 (days)
- Sampling intervals/frequency for test medium samples:
Uptake phase: T0, T0.2, T1, T1.2, T2, T2.2, T3, T3.2, T4, T4.2, T7, T7.2, T8, T8,2, T9, T9.2, T10, T10.2, T11, T11.2, T14 (days)
Depuration phase: T0.2, T1, T11, T15 (days)
- Sample storage conditions before analysis: analysis was performed immediately after sampling.
- Details on sampling and analysis of test organisms and test media samples (e.g. sample preparation, analytical methods):
At each sampling time, a sufficient number of fish were collected to provide 4 replicates samples from the controls, treatments (DIPB10 and 100).
Fish were randomly removed from the test chamber and were euthanized in order to carry out the measurements of weight and length and chemical analysis. For this, fish were placed for about five minutes in a beaker containing MS222 (CAS number 886-86-2) at a lethal concentration of approximately 300 mg.L-1. After euthanasia, fish were rinsed with deionized water, blotted dry, weighed and measured before analytical procedure. After euthanasia and weighing, Lepomis macrochirus were introduced in a 50 mL centrifuge tubes. Each organism was sliced in approximately 4 to 5 parts with scissors and 15 stainless steel beads were introduced in each centrifuge tube. A first grinding step on the GenoGrinder was performed (20 min at 1000 rpm). Then, 10 mL of methanol and 10 mL of hexane were added. Samples were stirred on the GenoGrinder for 5 min at 1000 rpm and centrifuged for 5 min at 4696 rpm. Supernatant were transferred in a new 50 mL centrifuge tube and 10 mL of ultrapurewater were added in this new tube. Samples were stirred on the GenoGrinder for 5 min at 1000 rpm and centrifuged for 5 min at 4696 rpm. 1 mL of hexane extract (supernatant) is pipetted into a 1.5 mL amber vial or diluted in hexane before liquid injection on a GC-MS/MS system.
The lipid quantification in whole fish tissue was performed according to the “Smedes method” (Smedes, 1999) . Fish lipids were extracted by cyclohexane and isopropanol, transfer of lipids to the cyclohexane phase was realized by addition of water, the phases separation by centrifugation, and lipid determination by weighing after solvent evaporation.
The principle of the method for water sample analysis is:
A volume of 2 mL of aqueous sample was dispensed in a 20 mL glass amber vial closed as quickly as possible. Then, sample was placed on the rack unit (room temperature) of the SPME-GC-MS/MS system for analysis. Automated SPME-GC-MS/MS was performed with successive phases of incubation, extraction, thermal desorption in the split-splitless (SSL) injector, GC-MS/MS analysis and re-conditioning of the fiber on the cooled injection system (CIS) inlet.

Vehicle:
yes
Remarks:
Since test item was poorly soluble under the test condition, stock solutions were prepared in Dimethylformamide, considered as an appropriate solvent. Solvent concentration did not exceed 0.02 mL.L-1 during the test in all test conditions.
Details on preparation of test solutions, spiked fish food or sediment:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: stock solutions were prepared in Dimethylformamide, considered as an appropriate solvent. The test solutions were prepared by dilution of the stock solutions with housing water in a mixing chamber and with vigorous stirring to aid dispersion.
- Controls: Control and Solvent Control (0.02 mL.L-1)
- 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)): 0.02 mL.L-1
- Evidence of undissolved material (e.g. precipitate, surface film, etc): none
Test organisms (species):
Lepomis macrochirus
Details on test organisms:
TEST ORGANISM
- Common name: bluegill
- Source: obtained from Osage Catfisheries (1170 Nichols Road, Osage Beach, MO 65065, USA). Fish arrived in the lab on 13/09/2019. They were acclimated in the test media from this date.
- Age at study initiation (mean and range, SD): All fish were on the same year class, undifferentiated sexually and came from the same source.
- Length at study initiation (length definition, mean, range and SD): 5.2931cm (SD: 0.081
- Weight at study initiation (mean and range, SD): 1.726 g (SD: 0.216)
- Weight at termination (mean and range, SD): 3.124 g (SD: 0.471)
- Method of breeding: The water used for holding and testing was freshwater obtained from a mixture of deionized water and tap water issued from our water system. The water quality is suitable to rear fish species in the laboratory.
- Lipid content at test initiation (mean and range, SD): mean: 2.0194% (SD: 0.648)
- Health status: not specified, but expected to be good.
- Description of housing/holding area: not specified
- Feeding during test : yes
- Food type: Fish were fed with small granular product supplied by Special Diet Services (www.sdsdiets.fr).
During both phases, the fish were fed with the appropriate diet daily at approximately 1.5 % of body weight (wet weight). The initial feed rate was based on weight measurements of a sample of fish collected from the rearing tanks on the first day of the test.
The amount of feed was adjusted based on the wet weights of sampled fish at each interval during testing period to account for growth during the test. Any uneaten food as well feces were removed from the different test chambers shortly after feeding.

ACCLIMATION
- Acclimation period: Fish arrived in the lab on 13/09/2019. They were acclimated in the test media from this date (day 0 of the final bioaccumulation study: 9/12/2019)
- Acclimation conditions (same as test or not): yes
- Type and amount of food / Feeding frequency: not specified
- Health during acclimation (any mortality observed): not specified
Route of exposure:
aqueous
Test type:
flow-through
Water / sediment media type:
natural water: freshwater
Total exposure / uptake duration:
14 d
Total depuration duration:
15 d
Hardness:
9.22-11.8 °dH (164.6-212.4 mgCaCO3/L)
Test temperature:
21.08-21.5°C
pH:
7.4-7.9
Dissolved oxygen:
78.3-91.9%
Conductivity:
328.6-351.5 µS/cm
Details on test conditions:
TEST SYSTEM
- Test vessel:/Material, size, headspace, fill volume: 70-L glass aquaria and filled with approximately 50 L of housing water
- Type (delete if not applicable): open
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): Stock solutions and housing water were dispensed to the different mixing chambers with a multichannel syringe pump (set at 200 µL.hr-1) and peristaltic pumps (set at ca. 180 mL.min-1), respectively.
- Renewal rate of test solution (frequency/flow rate): Since test chambers used were filled with 50 L of test media, at least 5 volumes replacements were performed per day (180mL× 60 min × 24 hours =259,2L.day-1)
- No. of organisms per vessel: 80
- No. of vessels per concentration (replicates): 1
- No. of vessels per control / vehicle control (replicates): 1
- Biomass loading rate: Loading rates at the start of the test were 0.65, 0.65, 0.65 and 0.67 g.L-1.day-1 for the control, solvent control, DIPB10 and DIPB100 groups, respectively.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The water used for holding and testing was freshwater obtained from a mixture of deionized water and tap water issued from our water system.
- Particulate matter: <2 mg/L (suspended matter)
- Metals: Al <5; As<0.2; Cd<0.2; Cr0.2; Co<0.2 Cu 5.5; Fe<5; Hg<0.015; Ni<0.2; Pb0.2; Zn7 µg/L
- Pesticides: - Chlorine: 3.6 mg/L
- Ca/mg ratio: 44.9/14

- Holding medium different from test medium: no
- Intervals of water quality measurement: Water Temperature was measured in all test chambers at the beginning and at the end of the test and at approximately weekly intervals during the test. Temperature was also monitored continuously only in the control test chamber.
Dissolved oxygen, pH and conductivity measurements have been performed in each test chamber at the beginning and at the end of the test and at least once a week during the uptake and depuration phases.
Hardness and alkalinity were measured in the control test chambers at the beginning and at the end of the uptake phase and at the end of the depuration phase.
Intervals of test medium replacement: see above

OTHER TEST CONDITIONS
- Adjustment of pH: No. The pH measurements were in all conditions within a range of ± 0.5. Water did not differ by more than 2°C in treatments or controls groups.
- Photoperiod: 16 hours of light and 8 hours of dark
- Light intensity: At the start of the uptake phase, light intensity at the surface of the water was 200 lux.

RANGE-FINDING / PRELIMINARY STUDY
- Test concentrations: 10 and 100 µg/L (nominal)
- Results used to determine the conditions for the definitive study: yes
• DIPB isomers were quite stable in water during the test in both conditions (DIPB10/DIPB100)
• A steady-state seemed to be reached quickly during the uptake phase
• The depuration phase was very fast
• For both conditions, BCF values estimation were < 2000 L.kg-1 and in the same order of magnitude for both conditions
Nominal and measured concentrations:
Nominal: 10 and 100 µg/L DIPB mixture
Measured: 5.166 and 23.54 µg/L DIPB mixture (corresponding to 2.203µg/L 1,3-DIPB and 2,663µg/L 1,4-DIPB, and 9.922µg/L 1,3-DIPB and 12.25 µg/L 1,4-DIPB respectively)
Reference substance (positive control):
no
Details on estimation of bioconcentration:
BASIS FOR CALCULATION OF BCF
- Estimation software: estimation of the parameters k1 and k2 were performed by using mathematical models and statistical methods made available as an R-package named “bcmfR” (latest version 0.4-18). This package is distributed via OECD website :https://www.oecd.org/chemicalsafety/testing/section-3-environmental-fate-behaviour-software-tg-305.htm.
Lipid content:
5.052 %
Time point:
other: mean lipid values at the end of the uptake phase/start of depuration and the end of the depuration phase
Key result
Conc. / dose:
10 µg/L
Temp.:
21.4 °C
pH:
7.7
Type:
BCF
Value:
1 628.2 L/kg
Basis:
other: lipid normalised, growth corrected kinetic bioconcentration factor
Calculation basis:
kinetic, corrected for growth
Remarks on result:
other: 1,3-DIPB
Remarks:
CI95%: 1416.4-1839.9
Key result
Conc. / dose:
10 µg/L
Temp.:
21.4 °C
pH:
7.7
Type:
BCF
Value:
1 902.4 L/kg
Basis:
other: lipid normalised, growth corrected kinetic bioconcentration factor
Calculation basis:
kinetic, corrected for growth
Remarks on result:
other: 1,4-DIPB
Remarks:
CI95%: 1678.8-2126
Key result
Conc. / dose:
10 µg/L
Temp.:
21.4 °C
pH:
7.7
Type:
BCF
Value:
1 785.1 L/kg
Basis:
other: lipid normalised, growth corrected kinetic bioconcentration factor
Calculation basis:
kinetic, corrected for growth
Remarks on result:
other: DIPB mixture
Remarks:
CI95%: 1569.9-2000.3
Key result
Conc. / dose:
100 µg/L
Temp.:
21.4 °C
pH:
7.6
Type:
BCF
Value:
1 708.9 L/kg
Basis:
other: lipid normalised, growth corrected kinetic bioconcentration factor
Calculation basis:
kinetic, corrected for growth
Remarks on result:
other: 1,3-DIPB
Remarks:
CI95%: 1483.3-1934.4
Key result
Conc. / dose:
100 µg/L
Temp.:
21.4 °C
pH:
7.6
Type:
BCF
Value:
1 919.4 L/kg
Basis:
other: lipid normalised, growth corrected kinetic bioconcentration factor
Calculation basis:
kinetic, corrected for growth
Remarks on result:
other: 1,4-DIPB
Remarks:
CI95%: 1673.4-2165.5
Key result
Conc. / dose:
100 µg/L
Temp.:
21.4 °C
pH:
7.6
Type:
BCF
Value:
1 828.7 L/kg
Basis:
other: lipid normalised, growth corrected kinetic bioconcentration factor
Calculation basis:
kinetic, corrected for growth
Remarks on result:
other: DIPB mixture
Remarks:
CI95%: 1593.9-2063.5
Details on kinetic parameters:
- Uptake rate constant k1:
1,3DIPB isomer: DIPB10 condition: 575.3 L.kg-1.day-1; DIPB100 condition: 662.25 L.kg-1.day-1
1,4DIPB isomer: DIPB10 condition: 597.29 L.kg-1.day-1; DIPB100 condition: 685.31 L.kg-1.day-1
DIPB mixture: DIPB10 condition: 582.17 L.kg-1.day-1; DIPB100 condition: 672.62 L.kg-1.day-1
- Depuration rate constant k2g:
1,3DIPB isomer: DIPB10 condition: 0.3497 day-1; DIPB100 condition: 0.3835 day-1
1,4DIPB isomer: DIPB10 condition: 0.3107 day-1; DIPB100 condition: 0.3534 day-1
DIPB mixture: DIPB10 condition: 0.3228 day-1; DIPB100 condition: 0.364 day-1
Details on results:
- Mortality of test organisms: There were no observed mortalities in all conditions during the tests.
- Behavioural abnormalities: none. All fish appeared normal and healthy throughout the test.
- Observations on feeding behavior: no abnormalities
- Observations on body length and weight: Growth measurements (length and total wet weight) for individual fish collected during uptake and depuration in each condition were determined. A linear least squares correlation was calculated for the ln(fish weight) vs. day for each condition for the whole study, uptake and depuration phases. An analysis of the covariance was performed and highlighted that there was no significative difference (p<0.05) of the overall fish growth between phase (uptake/depuration) and between all conditions. In this context, fish weight from all conditions were pooled and overall growth rate constant (kg) was calculated by performing a linear least squares correlation on the individual data of the control, solvent control, DIPB10 and DIPB 100 conditions. The growth rate constants kg was determined to be 0.0206 day-1
- Reproduction during test period: no
- Other biological observations: no
- Organ specific bioaccumulation: not determined
- Bound residues forming a plateau:
- Mortality and/or behavioural abnormalities of control: none
- Loss of test substance during test period: yes, see reported nominal and measured (TWA) concentrations above.
- Non-eliminated residues (NER) at the end of elimination phase: none, at the end of the depuration phase the substances concentrations measured are - Results with vehicle control: the substances concentrations measured are
Reported statistics:
In order to calculate a kinetic BCF for each isomer and the mixture, estimation of the parameters k1 and k2 were performed by using mathematical models and statistical methods made available as an R-package named “bcmfR” (latest version 0.4-18). This package is distributed via OECD website :https://www.oecd.org/chemicalsafety/testing/section-3-environmental-fate-behaviour-software-tg-305.htm.
The choice of the best fit model was based on the normality of the residues (Q-Q plot and Shapiro-Wilk test).

Water concentration of the test item during all the test period (uptake and depuration phase)    

 

DIPB10 condition

Analytical measurements of both isomers 1,3 DIPB and 1,4DIPB were made in water.

Due to technical problems (e.g.syringe leaking, power shutdown) some of the sampling points were out of the range of the expected value during the uptake phase (day 1-3). Until the rest of the uptake phase the concentration of the test item remained stable.

As the concentrations of the test item were not maintained within ±20% of the mean of the measured concentrations values during the uptake phase and as a flow-through exposure system was used, a time-weighted arithmetic mean (TWAM) of the exposure concentration was calculated.

This TWAM was calculated according to the procedure described OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD, 2019).

 

A summary of the results is given in the table below:

Time-weighted average (TWA) concentration (µg.L-1) in DIPB10 condition during the uptake phase

 

 

 

-20%

+20%

Cw(µg.L-1)

1,3DIPB

2.203

1.763

2.644

1,4DIPB

2.663

2.130

3.196

Mixture

5.166

4.133

6.199

 

DIPB100 condition

Analytical measurements of both isomers 1,3 DIPB and 1,4DIPB were made in water.

Similarly to DIPB10 condition, at this highest concentration condition (DIPB100 condition) some of the sampling points were out of the range of the expected value during the uptake phase. This could be explained as well by some technical limit but also by the solubility of the test item in this condition. Indeed, the concentration of the test item in water tend to increase during the exposition.

In this way, as the concentrations of the test item were not maintained within ±20% of the mean of the measured concentrations values during the uptake phase and as a flow-through exposure system was used, a time-weighted arithmetic mean (TWAM) of the exposure concentration was calculated for both isomers and the mixture.

A summary of the results is given in the table below:

 

Time-weighted average (TWA) concentration (µg.L-1) in DIPB100 condition

 

 

 

-20%

+20%

Cw(µg.L-1)

1,3DIPB

9.922

7.938

11.906

1,4DIPB

12.253

9.802

14.704

Mixture

23.540

18.832

28.248

 

 

Fish tissues Concentration of test item

 

DIPB10 condition

Analytical measurement of both isomers 1,3 DIPB and 1,4DIPB were made in fish tissues.

Even if the concentrations of the test item in water were not maintained within ±20% during all the test, it occurred only between day 1 and day 3 and remained stable after. A steady state is then reached for the last three sampling points (from day 9 to 14) of the uptake phase (three successive analysis made on samples at intervals of at least 2 days are within ±20% of each other). Results of the last three points are given in the table below.

Days of the uptake phase

1,3DIPB (µg.g-1)

1,4DIPB (µg.g-1)

 

Mixture DIPB (µg.g-1)

 

 

 

Mean (µg.g-1)

 

Mean (µg.g-1)

 

Mean (µg.g-1)

9

4.413

3.881

6.791

5.336

11,894

9.785

9

4.257

5.850

10,729

9

2.444

3.629

6,447

9

4.409

5.075

10,068

11

3.127

3.656

4.479

4.647

8,074

8.814

11

2.638

3.922

6,964

11

6.113

6.358

13,239

11

2.746

3.830

6,981

14

3.712

3.991

4.747

4.887

8,980

9.425

14

5.638

6.547

12,935

14

3.800

4.840

9,172

14

2.815

3.414

6,613

 

 

 

 

 

 

 

mean

3.843

 

4.957

 

9,341

-20%

3.074

 

3.965

 

7,473

+20%

4.611

 

5.948

 

11,210

 

In this context, the concentration of the test item and both isomers in the fish tissue at steady-state is summarised in the table below:

 

1,3 DIPB

1,4 DIPB

mixture DIPB

Cf(µg.g-1)

3.843

4.957

9.341

 


 

DIPB100 condition

Analytical measurement of both isomers 1,3 DIPB and 1,4DIPB were made in fish tissues.

In this DIPB100 condition, the concentrations of the test item in water were not maintained within ±20% during all the test probably due to its low solubility in water and the fact that this concentration tend to increase during the exposure.

As in the DIPB10 condition the steady state was reached from days 9 to 11, the steady state is, in the DIPB100 condition, therefore empirically considered to be reached for the last four sampling points of the uptake phase. It was performed, in order to smooth the oscillation of the test item concentration in fish that occurred. The results of the last four points are given in the table below.

 

Days of the uptake phase

1,3DIPB (µg.g-1)

1,4DIPB (µg.g-1)

Mixture DIPB (µg.g-1)

 

 

Mean (µg.g-1)

 

Mean (µg.g-1)

 

Mean (µg.g-1)

7

19.864

16.607

26.936

20.955

49,682

39.875

7

9.332

10.904

21,482

7

16.276

23.335

42,050

7

20.957

22.646

46,288

9

20.126

13.474

29.194

19.301

52,357

34.793

9

8.073

11.629

20,915

9

9.644

14.596

25,732

9

16.053

21.784

40,167

11

31.809

25.430

36.84

34.306

72,876

63.414

11

29.36

40.091

73,727

11

31

44.386

80,028

11

9.552

15.907

27,027

14

19.901

20.369

28.694

28.369

51,587

51.739

14

17.218

23.425

43,145

14

26.275

36.415

66,550

14

18.083

24.942

45,674

 

 

 

 

 

 

 

mean

18.970

 

25.733

 

47,455

-20%

15.176

 

20.586

 

37,964

+20%

22.764

 

30.879

 

56,946

 

In this context, the concentration of the test item and both isomers in the fish tissue at steady-state is summarised in the table below:

 

1,3 DIPB

1,4 DIPB

mixture DIPB

Cf(µg.g-1)

18.970

25.733

47.455

 

 

 

A summary of the different parameters estimated and subsequent calculations obtained for all conditions is given in the table below:

 

1,3DIPB isomer

1,4DIPB isomer

Mixture DIPB

DIPB10 condition

DIPB100 condition

DIPB10 condition

DIPB100 condition

DIPB10 condition

DIPB100 condition

Kinetic BCF

 

 

 

 

 

 

kg( day-1)

0.0206

0.0206

0.0206

0.0206

0.0206

0.0206

Ln(%)

5.052

5.052

5.052

5.052

5.052

5.052

k1(L.kg-1.day-1)

575.3

662.25

597.29

685.31

582.17

672.62

k2(day-1)

0.3703

0.4041

0.3313

0.374

0.3434

0.3846

k2g(day-1)

0.3497

0.3835

0.3107

0.3534

0.3228

0.364

BCFK(L.kg-1)

1553.6

1638.6

1802.7

1832.6

1695.5

1718.7

BCFKg(L.kg-1)

1645.1

1726.7

1922.2

1939.4

1803.7

1847.7

BCFKL(L.kg-1)

1537.6

1621.7

1784.1

1813.7

1678.0

1701.0

BCFKgL(L.kg-1)

(CI 95%)

1628.2

(1416.4-1839.9)

1708.9

(1483.3-1934.4)

1902.4

(1678.8-2126)

1919.4

(1673.4-2165.5)

1785.1

(1569.9-2000.3)

1828.7

(1593.9-2063.5)

t1/2g(days)

1.9821

1.8072

2.2307

1.9616

2.1475

1.9041

Steady-state BCF

 

 

 

 

 

 

Cf(mg.kg-1)

3.843

18.97

4.957

25.73

9.341

47.45

Cw(mg.L-1)

2.203

9.922

2.663

12.253

5.166

23.540

BCFSS(L.kg-1)

1744.0

1912*

1861

2100*

1790

2016*

BCFSSL(L.kg-1)

1726

1892*

1842

2078*

1808

1995*

* These data have to be taken with care because the steady state was empirically considered to be reached for the last four sampling points of the uptake phase.

 

Validity criteria fulfilled:
yes
Conclusions:
The BCFKgL (L.kg-1) obtained in this study for the test item (mixture of isomers DIPB) was between 1785.1 and 1828.7.
Executive summary:

The aim of this study was to obtain laboratory data to determine the bioconcentration factor (BCF) of the test item at two different concentrations in the bluegill (Lepomis macrochirus), through aqueous exposure based on the OECD TG 305 (2012). The test item, diisopropylbenzene, was a mixture of isomers 1,3-diisopropylbenzene (1,3DIPB) and 1,4-diisopropylbenzene (1,4DIPB). Since the test item was poorly soluble under the test condition, stock solutions were prepared in Dimethylformamide, considered as an appropriate solvent. Four different conditions were used during the test: (i) Control, (ii) Solvent Control, (iii) DIPB mixture at 10 μg.L-1 (nominal concentration) (DIPB10) and (iv) DIPB mixture at 100 μg.L-1 (nominal concentration) (DIPB100). The test solutions were prepared by dilution of the stock solutions with housing water in a mixing chamber and with vigorous stirring to aid dispersion. The test was divided into two phases, the uptake phase which lasted 14 days followed by a depuration phase. The total duration of the test lasted 29 days. No toxic effect of the test item and/or the solvent control have been highlighted.

According to the Guidance on Information Requirements and Chemical Safety Assessment (Chapter R.11: PBT/vPvB Assessment, ECHA; Version 3.0 – June 2017), the resulting BCF that is preferred for a comparison with the bioaccumulation criteria is the kinetic growth corrected and lipid normalised (to 5% lipids) BCF value (BCFKgL). Moreover, it has to be compared to the BCFSSL. In our study the BCFKgL based on first order kinetics is in the same order of magnitude than the BCFSSL, indicating clearly that a steady-state has been attained in the uptake phase.

By comparing BCFKgL between conditions (DIPB10 and DIPB100) for both isomers (and consequently for the mixture), it is important to note as well that the difference is not clearly marked with 5% and 1% of difference, respectively. This calculation was made according to the guidance document on aspects of OECD TG 305 on fish bioaccumulation (2017; §2.5.1).

This lack of clear difference between BCFKgL obtained in each condition confirmed as well the low impact of the deviation made to the study plan. This deviation concerned regarding the fact that the concentration of the test substance in the test chambers was not maintained within +/- 20% of the mean of the measured values during the uptake phase.

By consequence, the BCFKgL (L.kg-1) obtained in this study for the test item (mixture of isomers DIPB) was between 1785.1 and 1828.7.

Description of key information

In a study conducted according to OECD Guideline 305 (Bioaccumulation: Flow-through Fish Test) Pimephales promelas were exposed to m-/p-diisopropylbenzene mixture concentrations of 10 and 100 µg/L (nominal) for 29 d.

The lipid normalised, growth corrected kinetic bioconcentration factor (BCFKgL) obtained in this study for the test item (mixture of isomers DIPB) was between 1785.1 and 1828.7 L.kg-1.

Key value for chemical safety assessment

BCF (aquatic species):
1 828.7 L/kg ww

Additional information

The aim of this study was to obtain laboratory data to determine the bioconcentration factor (BCF) of the test item at two different concentrations in the bluegill (Lepomis macrochirus), through aqueous exposure based on the OECD TG 305 (2012). The test item, diisopropylbenzene, was a mixture of isomers 1,3-diisopropylbenzene (1,3DIPB) and 1,4-diisopropylbenzene (1,4DIPB). Since the test item was poorly soluble under the test condition, stock solutions were prepared in Dimethylformamide, considered as an appropriate solvent. Four different conditions were used during the test: (i) Control, (ii) Solvent Control, (iii) DIPB mixture at 10 μg.L-1 (nominal concentration) (DIPB10) and (iv) DIPB mixture at 100 μg.L-1 (nominal concentration) (DIPB100). The test solutions were prepared by dilution of the stock solutions with housing water in a mixing chamber and with vigorous stirring to aid dispersion. The test was divided into two phases, the uptake phase which lasted 14 days followed by a depuration phase. The total duration of the test lasted 29 days. No toxic effect of the test item and/or the solvent control have been highlighted.

According to the Guidance on Information Requirements and Chemical Safety Assessment (Chapter R.11: PBT/vPvB Assessment, ECHA; Version 3.0 – June 2017), the resulting BCF that is preferred for a comparison with the bioaccumulation criteria is the kinetic growth corrected and lipid normalised (to 5% lipids) BCF value (BCFKgL). Moreover, it has to be compared to the BCFSSL. In our study the BCFKgL based on first order kinetics is in the same order of magnitude than the BCFSSL, indicating clearly that a steady-state has been attained in the uptake phase.

By comparing BCFKgL between conditions (DIPB10 and DIPB100) for both isomers (and consequently for the mixture), it is important to note as well that the difference is not clearly marked with 5% and 1% of difference, respectively. This calculation was made according to the guidance document on aspects of OECD TG 305 on fish bioaccumulation (2017; §2.5.1).

This lack of clear difference between BCFKgL obtained in each condition confirmed as well the low impact of the deviation made to the study plan. This deviation concerned regarding the fact that the concentration of the test substance in the test chambers was not maintained within +/- 20% of the mean of the measured values during the uptake phase.

By consequence, the BCFKgL (L.kg-1) obtained in this study for the test item (mixture of isomers DIPB) was between 1785.1 and 1828.7.