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Environmental fate & pathways

Bioaccumulation: aquatic / sediment

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Administrative data

Endpoint:
bioaccumulation in aquatic species: fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 2019 to March 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 305 (Bioaccumulation in Fish: Aqueous and Dietary Exposure) -I: Aqueous Exposure Bioconcentration Fish Test
Deviations:
yes
Remarks:
The concentration of the test substance in some chambers was not maintained within +/- 20% of the mean of the measured values during the uptake phase. However, an evalaution of the deviation determined that it did not impact the results of the study.
GLP compliance:
yes (incl. QA statement)

Test material

1
Chemical structure
Reference substance name:
Diisopropylbenzene
EC Number:
246-835-6
EC Name:
Diisopropylbenzene
Cas Number:
25321-09-9
Molecular formula:
C12H18
IUPAC Name:
1,2-diisopropylbenzene
Constituent 1
Chemical structure
Reference substance name:
1,4-diisopropylbenzene
EC Number:
202-826-9
EC Name:
1,4-diisopropylbenzene
Cas Number:
100-18-5
Molecular formula:
C12H18
IUPAC Name:
1,4-di(propan-2-yl)benzene
Constituent 2
Chemical structure
Reference substance name:
1,3-diisopropylbenzene
EC Number:
202-773-1
EC Name:
1,3-diisopropylbenzene
Cas Number:
99-62-7
Molecular formula:
C12H18
IUPAC Name:
1,3-di(propan-2-yl)benzene
impurity 1
Chemical structure
Reference substance name:
1,2-bis(1-methylethyl)benzene
EC Number:
209-412-7
EC Name:
1,2-bis(1-methylethyl)benzene
Cas Number:
577-55-9
Molecular formula:
C12H18
IUPAC Name:
1,2-diisopropylbenzene
Test material form:
liquid
Specific details on test material used for the study:
The substance diisopropylbenzene / 1,2-diisopropylbenzene (CAS# 25321-09-9, EC#/ 246-835-6) is a mixture of isomers 1,3-diisopropylbenzene and 1,4-diisopropylbenzene.
Radiolabelling:
no

Sampling and analysis

Details on sampling:
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. At the start of the exposure, at the end of the uptake phase and at the end of depuration phase, additional fish were collected to determine lipid content. Moreover, supplementary fish were sampled from the rearing tank after the initiation of the test to enhance the robustness of the lipid analysis but were not taken into account for fish lipid content (Ln). After euthanasia, fish were rinsed with deionized water, blotted dry, weighed and measured before analytical procedure.

Test solutions

Vehicle:
yes
Details on preparation of test solutions, spiked fish food or sediment:
The water solubilities of m-diisopropylbenzene and p-diisopropylbenzene were determined to be 0.072 and 0.0405 mg.L-1 at 25°C, respectively. Since 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 and were as follows:
a. Control
b. Solvent Control (0.02 mL.L-1)
c. DIPB mixture at 10 μg.L-1 (nominal concentration) (DIPB10)
d. 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. 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. 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).

Test organisms

Test organisms (species):
Lepomis macrochirus
Details on test organisms:
The bluegill, Lepomis macrochirus, was used in the study. This species was obtained from Osage Catfisheries (1170 Nichols Road, Osage Beach, MO 65065, USA). All fish were on the same year class, undifferentiated sexually and came from the same source. Identification of the species was verified by the supplier. Fish arrived in the lab on 13/09/2019. They were acclimated in the test media from this date.

Study design

Route of exposure:
aqueous
Justification for method:
aqueous exposure method used for following reason: test substances were soluble at levels that were analytically measurable and appropriate for guideline
Test type:
flow-through
Water / sediment media type:
natural water: freshwater
Total exposure / uptake duration:
ca. 14 d
Total depuration duration:
ca. 15 d

Test conditions

Hardness:
Mean values for controls and exposure concentrations ranged from 10.8 to 11.1 dH.
Test temperature:
Mean values for controls and exposure concentrations ranged from 21.3 to 21.4 C.
pH:
Mean values for controls and exposure concentrations ranged from 7.6 to 7.7.
Dissolved oxygen:
Mean values for controls and exposure concentrations ranged from 84.3 to 87.7 % saturation.
Conductivity:
Mean values for controls and exposure concentrations ranged from 334.6 to 344.6 us.cm-1.
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass aquaria
- Material, size, headspace, fill volume: 50L
- Type of flow-through (e.g. peristaltic or proportional diluter): peristaltic
- Renewal rate of test solution (frequency/flow rate): ca. 180 mL/min
- 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 lab water system.
- 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.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours of light and 8 hours of dark

RANGE-FINDING / PRELIMINARY STUDY
- Test concentrations: nominal 10 and 100 ug/L for the mixed isomers
- Results used to determine the conditions for the definitive study:
• 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

Results and discussion

Lipid content
Lipid content:
ca. 5.052 %
Time point:
other: Mean lipid content over the depuration phase (i.e. the midpoint of the depuration phase calculated from the mean lipid values at the end of the uptake phase/start of depuration and the end of the depuration phase).
Bioaccumulation factoropen allclose all
Key result
Conc. / dose:
ca. 10 µg/L
Type:
BCF
Value:
1 628.2 L/kg
Basis:
normalised lipid fraction
Calculation basis:
kinetic
Key result
Conc. / dose:
ca. 100 µg/L
Type:
BCF
Value:
1 708.9 L/kg
Basis:
normalised lipid fraction
Calculation basis:
kinetic
Key result
Conc. / dose:
ca. 10 µg/L
Type:
BCF
Value:
1 726 L/kg
Basis:
normalised lipid fraction
Calculation basis:
steady state
Key result
Conc. / dose:
ca. 100 µg/L
Type:
BCF
Value:
1 892 L/kg
Basis:
normalised lipid fraction
Calculation basis:
steady state
Details on results:
- Mortality of test organisms: There were no observed mortalities in all conditions during the tests.
- Behavioural abnormalities: All fish appeared normal and healthy throughout the test.
-Growth measurements (length and total wet weight) for individual fish collected during uptake and depuration in each condition. 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.
-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. The choice of the best fit model was based on the normality of the residues (Q-Q plot and Shapiro-Wilk test). For each of the isomers and the mixture for both exposure concentrations, the Box-Cox transformed data was the best fit model.

Any other information on results incl. tables

Study Results for Individual Isomers and Mixture

      1,3DIPB isomer 1,4DIPB isomer         Mixed DIPB
   DIPB 10 ug/L   DIPB 100 ug/L   DIPB 10 ug/L   DIPB 100 ug/L   DIPB 10 ug/L   DIPB 100 ug/L
                   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  1701
 BCFKgL (L.kg-1)  1628.2  1708.9  1902.4  1919.4  1785.1  1828.7
 (CI 95%)  (1416.4 -1839.9)  (1483.3 -1934.4)  (1678.8 -2126)  (1673.4 -2165.5)  (1569.9 -2000.3)  (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  9.222  4.957  25.73  9.341  47.45
 BCFSS (L.kg-1)  1744  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                                   

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
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 were observed.

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 this study the BCFKgLbased 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 BCFKgLbetween 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 BCFKgLobtained in each condition confirmed as well the low impact of the noted deviation of the study plan. This deviation regarded 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. The BCFKgL(L.kg-1) for the 1,3DIPB isomer was between 1628.2 and 1708.9, and the BCFKgL(L.kg-1) for the 1,4DIPB isomer was between 1902.4 and 1919.4. These results suggest that the individual isomers as well as the mixture of isomers do not meet the Bioaccumulation (B) threshold of 2000 under a PBT/vPvB assessment.

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