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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:
2003-05-16 till 2003-11-11
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
according to guideline
OECD Guideline 305 E (Bioaccumulation: Flow-through Fish Test)
Version / remarks:
Principles of method if other than guideline:
not applicable
GLP compliance:
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material: not applicable
Details on sampling:
- Concentrations: The first was 2 µg/L and the second was 0.2 µg/L of 2,2'-methylenebis(6-tert-butyl-4-methylphenol).
The test water analysis was perfomed twice a week (n=1).
The fish analysis was performed every two weeks (n=2).
The control fish analysis was performed before the initiation and the termination of exposure.
Details on preparation of test solutions, spiked fish food or sediment:
- Test solution: 2,2-methylenebis (6-tert-butyl-4-methylphenol) was dissolved in 2-methoxy ethanol of the 5-fold amount of castor oil (HCO-40). The sock solution was prepared as a concentration of 1000 mg/L.
- Controls: blank and without 2,2'-methylenebis(6-tert-butyl-4-methylphenol)
- Chemical name of vehicles: castor oil (HCO-40) and 2-methoxy ethanol
- Concentrations of vehicle in final test solution: first = 200 mg/L and second = 20 mg/L
It was 25 L prepared and changed 2 times per week.
Test organisms (species):
Cyprinus carpio
Details on test organisms:
- Supplier: Kitamura fish farm (Yatsushiro City, Kumatoto Prefecture, Japan)
- Conditions for fish keeping: At reception, fish showing visible abnormal signs were removed. Fouthy-six fish for the test and twelve fish for control were reared according to flow through sytem for 1-2 months after external disinfeciton, which was performed according to static condition for 24 h in an aqueous solution containing 50 mg/L Terramycin and 7 g/L sodium chloride.
- Condition of acclimation: Fish were reared in an acclimation tank according to flow through system at temperature of 25 ± 2 °C for about 60 days. During the period, abnormal fish were removed. Then the fish were transfered to test tanks and reared again at the same temperature according to flow through system for about one month.
- Length and lipid content at the initiation of exposure: length 6.3 ~ 10.0 cm, lipid content 3.45 - 4.93 % at the beginning and the end of the test.
- Feeding amount and interval: amount corresponding to about 2 % of the total body weight of test fish was fed twice a day by halves. On the day of fish sampling feed was not supplied.
Route of exposure:
Test type:
Water / sediment media type:
natural water: freshwater
Total exposure / uptake duration:
60 d
not reported
Test temperature:
25.1 °C - 25.8 °C meaured at each test vessel at the beginning and the end of the test
7.9 ~ 8.2 measured at each test vessel at the beginning and the end of the test
Dissolved oxygen:
7.5 ~ 8.1 mg/L measured at each test vessel at the beginning and at the end of the test
not reported
not reported
Details on test conditions:
- Test water: Groundwater that has been pumped into the Kurume branch. 20 µL Protoperithecien/test waser and minute; 1600 mg/L/minute; 2304 L /day.
- Temperature: 25 ± 2 °C; Temperature controlled test water was supplied continuously into the flow through system (Aquatron).
- Test tank: Glass tank of 100 liters in volume, exchange frequency 1-2 times per month
- Flow rate of test water: At a rate of 1158 mL/day of test water was supplied.
- Number of fish (at the initiation of exposure): 25 fish/Glass tank
- During the exposure: 60 days
- Feeding: Food was shared, 2 times per day, 2 % of fish body weight
- Photoperiod: 14 h light / 10 h dark
Nominal and measured concentrations:
nominal concentrations: first = 2 and second = 0.2 µg/L
Reference substance (positive control):
Details on estimation of bioconcentration:
Known amount of the test substance was added into test water and fish homogenate, respectively, then analyzed according to the procedures which were established previously. Blank test was also performed in exactly same way without the test substance. These tests were carried out in duplicate. Recovery rates were used as correction factors fot the determination of concentration of the test substance in analyzing samples.
Calculation of bioconcentration factor (BCF):
BCF = concentration of test substance in fish / concentration of test substance in water
Lipid content:
3.45 %
Time point:
start of exposure
Lipid content:
4.93 %
Time point:
end of exposure
400 - 840 L/kg
total lipid content
Time of plateau:
60 d
Calculation basis:
steady state
Remarks on result:
other: environment / dose:2.0 µg/L
320 - 780 L/kg
total lipid content
Time of plateau:
60 d
Calculation basis:
steady state
Remarks on result:
other: environment / dose:0.2 µg/L
Details on results:
- Behavioural abnormalities: none
- Mortality and/or behavioural abnormalities of control: none
- Loss of test substance during test period: no

no remarks

Validity criteria fulfilled:
(- The recovery rate of the test substance did not exceed 20 % by the end of the test. The dissolved oxygen concentration remained above 60 % of the air-saturation value throughout the exposure period. - The pH did not vary by more than 1 unit.)
The measured BCF at the tested concentrations were:
400 ~ 840 L/kg (2.0 µg/L)
320 ~ 780 L/kg (0.2 µg/L)
Executive summary:

The bioconcentration factor (BCF) of 2,2'-methylenebis(6 -tert-butyl-4 -methylphenol) was determined according OECD guideline 305 E "Bioaccumulation: Degree of Bioconcentration in Fish" (1996 -06 -14).

The exposurer concentrations were verified by analytical measurement during the test (60 days) and were found to be maintained with 87.1 - 93.4 % of the nominal concentrations throughout the test. The BCF value obtained was 400 ~ 840 L/kg at the 2.0 µg/L exposure level and 320 ~ 780 L/kg at the 0.2 µg/L exposure level.

Description of key information

The bioconcentration potential of DBMC in fish was tested in a 60-days-test with carp. The resulting BCF value is 840.

Key value for chemical safety assessment

BCF (aquatic species):
840 dimensionless

Additional information

MITI (2003) performed a bioconcentration test performed according to OECD 305. The substance was investigated in a 60 -days flow-through test with carp at nominal concentrations of 2.0 µg/L and 0.2 µg/L. The BCF values obtained were 400 -840 (at 2.0 µg/L) and 320 -780 (at 0.2 µ/L). The bioconcentration in this test was assessed as low. The test concentrations (0.2 and 2.0 µg/L) are below the value for water solubility of 7 µg/L.

This test has been evaluatied by the European PBT working group (2009), who found the results to be the most suitable. The BCF value of 840 was the highest reported value.

In an earlier study by MITI (1992) the bioconcentration of DBMC in fish was tested in an 8 -week flow-through-test according to the MITI method (1974), which is equivalent to the OECD 305 C (1981) guideline. The nominal concentrations in the test were 1.0 mg/L and 0.1 mg/L. The derived BCF values were in a range of 23 -125. The data were described in the OECD-SIDS report (2000), which was approved by the Japanese government. The PBT working group evaluated these data in the PBT report (2009) and stated that the results should be treated with caution.