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Environmental fate & pathways

Bioaccumulation: aquatic / sediment

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bioaccumulation in aquatic species: fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 May 2020 - 17 Feb 2023
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 305 (Bioaccumulation in Fish: Aqueous and Dietary Exposure) -III: Dietary Exposure Bioaccumulation Fish Test
Version / remarks:
2 October 2012
GLP compliance:
yes (incl. QA statement)
Details on sampling:
- Sampling intervals/frequency for test organisms: day 0, 14 (uptake phase), 15, 18, 21, 28, 35, 42 (depuration phase)
- Sampling intervals/frequency for test medium samples: day 0, 14 (uptake phase)
- Sample storage conditions before analysis: chilled conditions
- Details on sampling and analysis of test organisms and test media samples: Food samples: extraction with tetrahydrofuran, vortex mixing for 1 min, sonication for 15 min, centrifugation at 3000 rpm for 15 min. Fish samples: homogenization by mincing with scissors, extraction with tetrahydrofuran by mixing with a blender for 5 min, sonication for 5 min. centrifugation at 2500 rpm for 15 min.
corn oil
Details on preparation of test solutions, spiked fish food or sediment:
- Method: evaporation to dryness and solution in corn oil
- Controls: corn oil without test substance
- Concentration of vehicle in test medium: Test substance: 2.49 mL corn oil in 120.06 g food; Control: 2.4 mL corn oil in 120.16 g food
- Evidence of undissolved material: none reported

- Details on fish food: Inicio 917 1.1 mm trout pellets (Biomar, Denmark) (50% protein and 16% fat content)
- Details of spiking: test substance dissolved in corn oil
- Quantity of corn oil vehicle per unit mass of fish food: Test substance: 2.49 mL corn oil in 120.06 g food; Control: 2.4 mL corn oil in 120.16 g food
- Method of mixing: manual shaking followed by rotational agitation for 5 min
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
- Common name: rain bow trout
- Source: Commcercial supplier, UK
- Length at study initiation: mean 8.13 cm
- Weight at study initiation: mean 5.37 g
- Weight at termination (mean and range, SD): 5.28 - 9.66 g
- Lipid content at test initiation: 4.26% (tissue weight)
- Health status: no abnormalities reported
- Description of housing/holding area: same as test, acclimatization for at least 2 weeks
- Feeding during test
- Food type: Inicio 917 1.1 mm trout pellets (Biomar, Denmark)
- Amount: 2% of body weight (on day 27 2.7% were fed in error)
- Frequency: daily

- Acclimation period: at least 2 weeks
- Acclimation conditions: same as test
- Type and amount of food: Inicio 917 1.1 mm trout pellets (Biomar, Denmark)
- Feeding frequency: daily
- Health during acclimation: no mortality reported
Route of exposure:
Justification for method:
dietary exposure method used because stable, measurable water concentrations cannot be maintained
Test type:
Water / sediment media type:
natural water: freshwater
Total exposure / uptake duration:
14 d
Total depuration duration:
28 d
174 - 176 mg CaCO3
Test temperature:
13.9 - 14.3°C
7.6 - 8.1
Dissolved oxygen:
72 - 95% air saturation
<1.0 - 1.3 mg/L
Details on test conditions:
- Test vessel: aquaria
- Material, fill volume: glass, 100 L
- Aeration: continuous aeration
- Renewal rate of test solution: nominal 800 mL/min equal to approximately 1152 L per d
- No. of organisms per vessel: 50
- No. of vessels per concentration: 1
- No. of vessels per control / vehicle control: 1

- Source/preparation of dilution water: tap water, dechlorinated by activated carbon, softened to the desired water hardness
- Metals: none
- Pesticides: none
- Chlorine: none
- Holding medium different from test medium: no
- Intervals of water quality measurement: pH, oxygen, temperature daily

- Photoperiod: 16 h light/ 8 h dark, with 20 min dawn and dusk transition periods
- Overall daily feeding rate used in the study: 2 % body weight, adjusted after every sampling interval (on day 27 erroneously 2.7% was fed)
- Number of feeds per day: 1

The solubility in the vehicle corn oil was determined with nominal test substance concentrations of 10, 100, 1000 µg/g. The 1000 µg/g concentration contained undissolved material, while the lower concentrations were observed to be clear.

The homogeneity of the test substance on food was tested with the concentrations 10, 100 µg/g. The test substance dissolved in corn oil was administered to fish food same as in the test five times. The test substance concentrations were in the range of ±15%.

The palability of treated food was determined for 7 days. The feeding was considered to be normal. With a test substance concentration of 100 µg/g, radioactivity in fish was in the range of 1.05 - 1.91 µg equivalents/g after 7 d. This was determined to be sufficient for measuring a 95% reduction in body burden. Therefore, the concentration of 100 µg/g food was chosen for the test.
Nominal and measured concentrations:
Nominal 0 (control), 100 µg test substance/g food
Measured at d0: 0 (control), 112 µg/g
Measured at d14: 0 (control), 104 µg/g
Reference substance (positive control):
Lipid content:
5.33 %
Time point:
other: end of depuration
Lipid content:
4.65 %
Time point:
end of exposure
Remarks on result:
other: end of uptake/start of depuration
Lipid content:
4.26 %
Time point:
start of exposure
Conc. / dose:
100 µg/g food
0.386 dimensionless
whole body w.w.
Calculation basis:
Conc. / dose:
100 µg/g food
0.582 dimensionless
whole body w.w.
Calculation basis:
other: Lipid normalized
Conc. / dose:
100 µg/g food
0.15 dimensionless
whole body w.w.
Calculation basis:
kinetic, corrected for growth
Depuration time (DT):
7 d
Rate constant:
growth-corrected depuration rate constant (d-1)
Rate constant:
growth-corrected half-life (d)
Details on kinetic parameters:
- Uptake rate constant k(f): 0.00176 kg food / kg fish / day
- Depuration rate constant k(e): 0.0455
- Indication of bi- or multiphasic kinetics: none
2 metabolites could be identified:
M1: Palmitic acid, C16H32O2
M2: C32H63O3N
Details on results:
- Mortality of test organisms: None
- Behavioural abnormalities: None
- Observations on feeding behavior: Normal feeding bevaior
- Observations on body length and weight: No abnormalities
- Mortality and/or behavioural abnormalities of control: None
- Loss of test substance during test period: None
Reported statistics:
Data was analysed using GraphPad Prism (ver. 9.0.1) and R software with package OECD-TG305 bcmfR (v0.4-18). Three optimization models were tested against the data. Linear regression, a Box-Cox transformation and a Natural Log transformation. Box-Cox showed optimum results and was ultimately used for evaluation.

Table 1: Validity criteria for OECD 305-III

Criterion from the guideline


Validity criterion fulfilled

The temperature variation is less than ± 2 °C

13.9 – 14.3°C


The concentration of dissolved oxygen does not fall below 60% saturation



The concentration of the test substance in fish food before and at the end of the uptake phase is within a range of ± 20% (based on at least three samples at both time points)

104 – 112 % recovery


The homogeneity of test item in feed is confirmed to be less than ±15 % from the

mean (by analysis at least triplicate portions)



Concentrations of test substance are not detected, or are present only at typical trace levels, in un-spiked food or control fish tissues relative to treated samples

No test substance in untreated food or control fish reported


The mortality or other adverse effects/disease in both control and treated fish is less than 10% at the end of the test; where the test is extended over several weeks or months, death or other adverse effects in both sets of fish should be less than 5% per month and not exceed 30% in all.

No mortality or adverse effects reported



Validity criteria fulfilled:
see Table 1 in "Any other information on results incl. tables" for further details

Description of key information

BMF: 0.582 kg food / kg fish (lipid normalised, OECD 305, dietary exposure)

Key value for chemical safety assessment

Additional information

Experimental data on bioaccumulation of (Z)-N-Octadec-9-enylhexadecan-1-amide (CAS No. 16260-09-6) is available from an OECD 305 GLP-study by dietary route. 

Radio-labeled substance was administered to rainbow trout (Oncorhynchus mykiss) via food over a period of 14 days, followed by a 24 days depuration phase. The concentration of the test substance in food during the uptake phase was 100 µg/g.

The mean concentration of total radioactivity in fish tissue was 2.54 µg equivalents/g after 14 days of exposure, but declined to 0.514 µg equivalents/g. C hromatographic analysis of the extracts showed that the test substance had been metabolized, accounting for 42.0% sample radioactivity (1.07 µg/g) after 14 days of exposure. After 28 days depuration, crodamide 203 accounted for 5.8% sample radioactivity (0.03 µg/g).
The test substance was metabolized to palmitic acid (≤22.6% sample radioactivity), Met 5 (≤14.3%), Met 7 (≤56.8%), polar material (≤3.3%) and up to eight low level, unidentified metabolites (each comprising ≤4.5% sample radioactivity).

The calculation of effect levels and kinetic parameters showed a BMF of 0.582 kg food/kg fish (lipid normalized) and a growth corrected half-life of 59.2 d.