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Bioaccumulation: aquatic / sediment

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bioaccumulation in aquatic species: fish
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
The study was conducted according to an appropriate OECD test guideline, and in compliance with GLP.
according to guideline
OECD Guideline 305 (Bioconcentration: Flow-through Fish Test)
GLP compliance:
Details on sampling:
- Sampling intervals/frequency for test organisms: Two fish collected from each vessel on days 0, 3, 7, 14, 21, 28 and 35 of the uptake phase and on days 1, 3, 7 and 10 of the depuration phase.

- Sampling intervals/frequency for test medium samples: Once prior to test, then on days 0, 3, 7, 14, 21, 28, 35 and 42 of the uptake phase and on days 1, 3, 7 and 10 of the depuration phase.

- Details on sampling and analysis of test organisms and test media samples (e.g. sample preparation, analytical methods):
Water samples were collected from mid-depth of each test vessel using a 10-ml volumetric pipette. Water samples were then transferred to 20-ml glass scintillation vials. All water samples were analysed as soon as possible after collection without storage.
Collected fish were euthanised by severing the spinal cord using a razor blade. The whole fish was then homogenised in a 20-ml scintillation vial using an ULTRA-TURRAX T-25 dispensing tool. Samples were combusted on day of collection.
Additional fish were collected for lipid analysis and metabolite characterisation. One fish was collected from each test vessel on Day 0 and Day 42 of the uptake phase and at Day 10 of the depuration phase for lipid analysis.
On Day 42 of the uptake phase, two fish were collected from each vessel for possible metabolite characterisation.
Details on preparation of test solutions, spiked fish food or sediment:
Approximately 1.71833 g of 14C-L3 was transferred to a 50 ml volumetric flask. The flask was brought to volume with dimethylformamide (DMF). LSC analysis indicated 2.38 E08 dpm/ml. Based on specific activity of the 14C-L3 (3.131 µCi/mg), the concentration of the primary stock solution was calculated to be 34.3 mg/ml.
Secondary stocks were prepared from the primary stock by dilution with DMF.
The secondary stock solutions were delivered to sealed mixing chambers where they were mixed with dilution water to achieve the desired test concentrations.
The concentration of DMF in the 14C-L3 treatment groups and the solvent control was 0.1 ml/l. All test solutions appeared clear and colourless throughout the test.
Test organisms (species):
Pimephales promelas
Details on test organisms:
- Common name: Fathead minnow

- Source: Thomas Fish Company, Anderson, California

- Length at study initiation (length definition, mean, range and SD): 58 mm (range 50-69 mm)

- Weight at study initiation (mean and range, SD): 1.32 g wet weight (range 0.798 - 2.39 g)

- Acclimation period: 14d

- Acclimation conditions (same as test or not): same

- Type and amount of food: Prime Tropical flake Red

- Health during acclimation (any mortality observed): No mortality observed
Route of exposure:
Test type:
Water / sediment media type:
natural water: freshwater
Total exposure / uptake duration:
42 d
Total depuration duration:
10 d
100 to 110 mg/L as CaCO3
Test temperature:
22 ± 2°C
7.7 - 8.2
Dissolved oxygen:
≥ 5.7 mg/l
Details on test conditions:
- Test vessel: 57 L polyethylene aquaria containing approximately 42-L of test solution.

- Type (delete if not applicable): Mixing chambers sealed to prevent volatilisation of test substance.

- Type of flow-through (e.g. peristaltic or proportional diluter): Continuous-flow diluter system

- Renewal rate of test solution (frequency/flow rate): 10 volume additions per day

- No. of organisms per vessel: 60

- No. of vessels per concentration (replicates): Each treatment group consisted of two replicate test chambers.

- Biomass loading rate: 1.9 g/L test water

- Source/preparation of dilution water: De-chlorinated municipal water from Bay City, Michigan.


- Photoperiod: 16 hours light; 8 hours dark

- Light intensity: 43 to 54 foot-candles
Nominal and measured concentrations:
Nominal: 3.4 and 34 µg/l
Mean measured: 1.7 and 21 µg/l
Reference substance (positive control):
Lipid content:
3 %
Time point:
other: average at test initiation
Lipid content:
1.4 %
Time point:
other: average at end of exposure
Lipid content:
1.3 %
Time point:
other: average at test termination
5 030 L/kg
whole body w.w.
Time of plateau:
14 d
Calculation basis:
steady state
Remarks on result:
other: environment / dose:1.7 µg/l
7 730 L/kg
whole body w.w.
Time of plateau:
21 d
Calculation basis:
steady state
Remarks on result:
other: environment / dose:21 µg/l
3 610 L/kg
whole body w.w.
Calculation basis:
Remarks on result:
other: environment / dose:1.7 µg/l
5 600 L/kg
whole body w.w.
Calculation basis:
Remarks on result:
other: environment / dose:21 µg/l
Depuration time (DT):
10 d
Details on kinetic parameters:
1.7 µg/l: Uptake rate (k1) = 1210; Depuration rate (k2) = 0.336; BCFk = 3610
21 µg/l: Uptake rate (k1) = 1040; Depuration rate (k2) = 0.186; BCFk = 5600
Metabolite characterisation: The percentage of radioactivity associated with L3 averaged 97.7% ± 1.6%. The percentage of radioactivity associated with unknown metabolite averaged 1.4% ± 1.9%. The percentage of radioactivity not extracted average 0.9% ± 0.3%.
Details on results:
All surviving fathead minnows in the test appeared normal and healthy throughout the test with no overt signs of toxicity, although occasional mortalities were observed. After 52 days in the diluter system, mortality in the solvent control, 1.7 and 21 µg/l treatment groups was 7.5, 8.3 and 6.7%, respectively. Consequently, the mortalities observed did not appear to be treatment-related.

There was no significant fish growth during the test in the control or treatment groups.

Table 1: Uptake phase


Duration of exposure (days)










Low concentration level

Concentration in the water (µg/l)

1.83; 1.73

1.58; 1.64

1.47; 1.52

1.80; 1.68

1.67; 1.65

1.63; 1.64

1.70; 1.68

2.20; 2.16

Mean measured: 1.7

Concentration infish (µg/kg)

169; 130; 120; <LOQ

5552; 4100; 2150; 3840

4544; 4482; 7118; 3242

10380; 13453; 5711; 3177

12738; 9096; 9722; 11329

9631; 9045; 3107; 5130

5329; 4694; 3271; 3018

4500; 3064; 3636; 3857

Steady-state: 8543 (average from days 14, 21, 28)








High concentration level

Concentration in the water (µg/l)

18.9; 21.0

17.7; 19.6

17.8; 20.0

19.5; 21.9

22.1; 23.6

20.1; 22.8

20.4; 21.9

19.4; 22.1

Mean measured: 21

Concentration infish (µg/kg)

829; 893; 1379; 659

29978; 41614; 28960; 46081

90966; 75402; 111957; 77535

146348; 107023; 141739; 87103

154362; 121214; 157552; 70150

234315; 136078; 201974; 225013

74898; 52501; 61638; 70620

59225; 215634; 164083; 207111

Steady-state: 162226 (average from days 21, 28, 42)









 Table 2: Depuration phase







Low concentration level

Concentration in the water (µg/l)





Concentration infish (µg/kg)

3062; 4197; 3320; 3079

1201; 2111; 1311; 1184

396; 501; 491; 469

123; 91.5; 124; 949

High concentration level

Concentration in the water (µg/l)

3.37; 3.71

2.63; 2.82

<LOQ; 1.58

<LOQ; 1.19

Concentration infish (µg/kg)

52835; 62826; 72766; 72986

25803; 41602; 36285; 43388

22456; 6643; 97873; 15930

6077; 19467; 4074; 17098


Validity criteria fulfilled:
Steady-state BCF values of 5030 l/kg (1.7 µg/l) and 7730 l/kg (21 µg/l) and kinetic BCF values of 3610 l/kg (1.7 µg/l) and 5600 l/kg (21 µg/l) were determined in a reliable study conducted according to an appropriate test protocol, and in compliance with GLP.

Description of key information


BCFss 5030 l/kg (1.7 µg/l); 7730 l/kg (21 µg/l). BCFk 3610 l/kg (1.7 µg/l); 5600 l/kg (21 µg/l). Lipid normalised (to 5%) values are: BCFss 18000 l/kg (1.7 µg/l);27600 l/kg (21 µg/l) and BCFk 12900 l/kg (1.7 µg/l); 20000 l/kg (21 µg/l).

A BCF value of 27600 is used in the exposure assessment as a worst case. BMF 0.38 (lipid-normalised steady-state);

BMF 0.45 (lipid-normalised, kinetic) A BMF value of 0.45 is used in the exposure assessment as a worst case. Depuration rate constants from BCF study: 0.336 d-1 (1.7 µg/l); 0.186 d-1 (21 µg/l).

Key value for chemical safety assessment

BCF (aquatic species):
27 600 dimensionless
BMF in fish (dimensionless):

Additional information

Steady-state BCF values of 5030l/kg(1.7 µg/l) and 7730l/kg(21 µg/l) and kinetic BCF values of 3610l/kg(1.7 µg/l) and 5600l/kg(21 µg/l) were determined in a reliable study conducted according to an appropriate test protocol, and in compliance with GLP. Lipid normalised (to 5%) values are: BCFss= 18 000 l/kg (1.7 µg/l) and 27 600 l/kg (21 µg/l) and BCFk= 12 900 l/kg (1.7 µg/l) and 20 000 l/kg (21 µg/l).

A fish feeding study is also available. A lipid-normalised steady-state BMF value of 0.38 and lipid-normalised kinetic BMF value of 0.45 were determined in a reliable study conducted in compliance with GLP. The growth corrected, lipid normalised kinetic BMF value was also calculated and reported to be 0.86; however, recent scientific discourse on the methodology to calculate growth corrected BCF and BMF values has revealed that these methods violate the rules of mass balance (Gobas et al., 2019). Therefore, the reported growth corrected values are not considered valid for the determination of bioaccumulation. The food in this study was very highly dosed (500 µg/g of14C-L3 nominal; 436 µg/g mean measured), which may limit the applicability of the values obtained.

Fish bioconcentration (BCF) studies are most validly applied to substances with log Kowvalues between 1.5 and 6. Practical experience suggests that if the aqueous solubility of the substance is low (i.e. below ~0.01 to 0.1 mg/l) (REACH Guidance R.11; ECHA, 2017), fish bioconcentration studies might not provide a reliable BCF value because it is very difficult to maintain exposure concentrations.Dietary bioaccumulation (BMF) tests are practically much easier to conduct for poorly water-soluble substances, because a higher and more constant exposure to the substance can be administered via the diet than via water.In addition, potential bioaccumulation for such substances may be expected to be predominantly from uptake via feed, as substances with low water solubility and high Kocwill usually partition from water to organic matter.

However, there are limitations with laboratory studies such as BCF and BMF studies with highly lipophilic and adsorbing substances. Such studies assess the partitioning from water or food to an organism within a certain timescale. The studies aim to achieve steady-state conditions, although for highly lipophilic and adsorbing substances such steady-state conditions are difficult to achieve. In addition, the nature of BCF and BMF values as ratio values, means that they are dependent on the concentration in the exposure media (water, food), which adds to uncertainty in the values obtained.

For highlylipophilic and adsorbingsubstances, both routes of uptake are likely to be significant in a BCF study, because the substance can be absorbed by food from the water.

Dual uptake routes can also occur in a BMF study, with exposure occurring via water due to desorption from food, and potential egestion of substance in the faeces and subsequent desorption to the water phase. Although such concentrations in water are likely to be low, they may result in significant uptake via water for highly lipophilic substances.

The OECD 305 advocates for calculating a growth dilution correction for kinetic BCF and BMF values, where the growth rate constant (i.e. kg) can be subtracted from the overall depuration rate constant (k2). In short, the uptake rate constant is divided by the growth-corrected depuration rate constant to give the growth corrected kinetic BCF or BMF value. However, recent scientific discourse on this topic has pointed out that correcting for growth in the depuration phase and not likewise accounting for the effects of lack of growth in the uptake phase (i.e. with regards to reduced feeding rate or respiration rate for a non-growing fish), results in an equation where the laws of mass balance are violated (Gobas et al., 2019). Essentially, the uptake parameters of the kinetic BCF or BMF calculation (i.e. k1) are those of a growing fish, but the depuration parameters are altered to reflect no growth (i.e. k2- kg). Based on this criticism of the growth dilution correction, these calculations are not considered best practice for the assessment of bioaccumulation (Gobas et al., 2019).

Gosset al. (2013) put forward the use of elimination half-life as a metric for the bioaccumulation potential of chemicals. Using the commonly accepted BMF and TMF threshold of 1, the authors derive a threshold value for keliminationof >0.01 d-1(half-life 70d) as indicative of a substance that does not bioaccumulate.

Depuration rates from BCF and BMF studies, being independent of exposure concentration and route of exposure, are considered to be a more reliable metric to assess bioaccumulation potential than the ratio BCF and BMF values obtained from such studies.

The depuration rate constant of 0.0378d-1from the BMF study for L3, may not be valid due to the very high loading of the food in this study potentially overloading metabolic/elimination pathways. This depuration rate is therefore not taken into account in the assessment of bioaccumulation.

The depuration rate constants of 0.336 d-1(1.7 µg/l)and 0.186 d-1(21 µg/l)obtained from the BCF study are considered to be valid and to carry most weight for bioaccumulation assessment. These rates are indicative of a substance which does not bioaccumulate.

Burkhardet al., 2012 has described fugacity ratios as a method to compare laboratory and field measured bioaccumulation endpoints. By converting data such as BCF and BSAF (biota-sediment accumulation factor) to dimensionless fugacity ratios, differences in numerical scales and unit are eliminated.

Fugacity is an equilibrium criterion and can be used to assess the relative thermodynamic status (chemical activity or chemical potential) of a system comprised of multiple phases or compartments (Burkhardet al., 2012). At thermodynamic equilibrium, the chemical fugacities in the different phases are equal. A fugacity ratio between an organism and a reference phase (e.g. water) that is greater than 1, indicates that the chemical in the organism is at a higher fugacity (or chemical activity) than the reference phase.

The fugacity of a chemical in a specific medium can be calculated from the measured chemical concentration by the following equation:

f = C/Z

Where f is the fugacity (Pa), C is concentration (mol/m3) and Z is the fugacity capacity (mol(m3.Pa)).

The relevant equation for calculating the biota-water fugacity ratio (Fbiota-water) is:

Fbiota-water= BCFWD/LW/ Klwx ρl/ ρB

where BCFWD/LWis ratio of the steady-state lipid-normalised chemical concentration in biota (µg-chemical/kg-lipid) to freely dissolved chemical concentration in water (µg-dissolved chemical/l-water), Klw is the lipid-water partition coefficient and ρlis the density of lipid and ρBis the density of biota.

A study to determine storage lipid-air partition coefficients of cVMS has been carried out (Dow Corning Corporation, 2015c). The conclusion from that study is that partitioning of cVMS compounds between storage lipids and air or water is reasonably similar, but not identical, to octanol. Kstorage lipid-airvalues for cVMS were systematically lower than Koctanol-airby 0.2 to 0.4 log units depending on temperature. Koctanol-watervalues may be expected to be similar.

The table below presents fugacity ratios calculated from the BCF data for L3, using both Kowfor Klwand Kow-0.4 as a worst case approximation.BMF values do not require adjustments because these values are already equivalent to fugacity-based values.

Table4.3.2Calculated biota-water fugacity ratios


Exposure concentration

BCF Value

Fbiota-waterusingKstorage lipid-water=Kow(6.6)

Fbiota-waterusingKstorage lipid-water=log Kow-0.4 (6.2)


1.7 µg/l





21 µg/l





1.7 µg/l





21 µg/l




The fugacity-based BCF directly reflect the thermodynamic equilibrium status of the chemical between the two media included in the ratio calculations. The fugacity ratios calculated are all below 1, indicating that the chemical in the organism tends to be at a lower fugacity (or chemical activity) than in the water. It should be noted however, that the BCF study may not have reached true steady-state in the timescale of the laboratory studies. The fugacity ratio indicates that uptake may be less than expected on thermodynamic grounds, suggesting that elimination is faster than might be expected on grounds of lipophilicity alone.

Collection and analysis (for L3) of surface water, sediments and select biota from aquatic food webs has been carried out in various studies by both industry and academia. In most cases, L3 was not present in quantifiable concentrations in biota.. See CSR Section 4.2.4 for further details.

Based on BMF, fugacity ratios and environmental monitoring data, Environment Canada (Environment Canada Health Canada March 2015 Final Screening Assessment for L3) concluded that L3 is not likely to/has a low potential to biomagnify through foodwebs.


Burkhard, L. P., Arnot, J. A., Embry, M. R., Farley, K. J., Hoke, R. A., Kitano, M., Leslie, H. A., Lotufo, G. R., Parkerton, T. F., Sappington, K. G., Tomy, G. T. and Woodburn, K. B. (2012). Comparing Laboratory and Field Measured Bioaccumulation Endpoints. Integrated Environmental Assessment and Management 8, 17-31.

Goss, K-U., Brown, T. N. and Endo, S. (2013). Elimination half-life as a metric for the bioaccumulation potential of chemicals in aquatic and terrestrial food chains. Environmental Toxicology and Chemistry 32, 1663-1671.

 Dow Corning Corporation (2015) Non-regulated study: Determination of storage lipid-to-air partition coefficients and their temperature dependence for Octamethylcyclotetrasiloxane (D4; CAS 556-67-2), Decamethylcyclopentasiloxane (D5; CAS 541-02-6) and Dodecamethylcyclohexasiloxane (D6; CAS 540-97-6). DOW CORNING CORPORATION HEALTH AND ENVIRONMENTAL SCIENCES (HES) TECHNICAL REPORT. HES Study No.: 17240-108. Report date: May 20, 2015.