[[4-[[2-(4-cyclohexylphenoxy)ethyl]ethylamino]-2-methylphenyl]methylene]malononitrile

Bioaccumulation: aquatic / sediment

Link to relevant study record(s)

Reference

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Justification for type of information:

Data is from experimental study report

Qualifier:

according to guideline

Guideline:

OECD Guideline 305 (Bioaccumulation in Fish: Aqueous and Dietary Exposure) -III: Dietary Exposure Bioaccumulation Fish Test

Qualifier:

equivalent or similar to guideline

Guideline:

other: equals EU Council Regulation 440/2008 Method C.13 (2008) Background Document to the fish Dietary Study-Protocol FISH Dietary Bioaccumulation Study – Basic Protocol

Principles of method if other than guideline:

To determine bioaccumulation factor an experiment was conducted with food as exposure route. This study consists of two phases the exposure phase and post exposure phase(depuration).During the uptake phase, separate groups of fish of one species are fed with a special diet of fish food mixed with at least two concentrations (100 and 1000 μg/g fish food) of the test item. As a reference compound hexachlorobenzene is also added to the fish diet at a nominal concentration of 100 μg/g. The uptake phase is run for 20 days. The concentration of the test item in control and test diets are analysed one day prior to the uptake phase, at the beginning of the uptake phase and 10 days thereafter. The test item and reference compound concentration in fish is followed by HPLC analysis and UV/VIS-detection 10 days after beginning of the uptake phase and at the end of the uptake phase. A tissue specific analysis was also carried out on dissected fish and the separated guts of the fish at the end of the uptake phase. The test dilution water was measured one day prior to the beginning of the uptake phase and on day 10 and 20 of the uptake phase for the control and both test item concentrations.After 20 days the depuration period is begun by feeding the fish with a diet free from the test item and the reference compound. During the depuration phase fish samples were taken at several times for analysis. Fish for tissue specific analysis were also taken, the guts were removed and both, dissected fish and guts were analysed separately. After a period of 28 days, the concentration of test item in fish was below the LOQ of Macrolex Yellow 6G of 0.401 μg/g fish wet weight and that was the last data point to be included in the regression to estimate the depuration rate constant.

GLP compliance:

not specified

Radiolabelling:

no

Details on sampling:

- Sampling intervals/frequency for test organisms: During the depuration phase fish samples for each test item concentration and control were taken on day 2, 4, 7, 14, 28 and 32 after the beginning of the depuration phase.

Details on preparation of test solutions, spiked fish food or sediment:

PREPARATION OF SPIKED FISH FOOD
- Details on fish food (source, fat content as supplied, etc):

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

Name : Zebra fish (Danio rerio)
Source : Aqua KlöGer (Germany)
Date of birth : October 2008
Date supplied : 2009-01-12
Mortalities during acclimation period : < 5 %
Medication : none
Mean standard length and mean wet weight (sample from keeping tanks at the beginning of the study) (n = 26) : 3.2 cm (S.D. = 0.17 cm) 325.2 mg (S.D. = 56.4 mg)
Fish were fed throughout on a sufficient diet (Tetra Min Mini Granules, Tetra GmbH) at least once a day, occasionally supplemented by live daphnids and frozen bloodworms (Chironomus larvae).

Route of exposure:

feed

Test type:

flow-through

Water / sediment media type:

other: synthetic freshwater

Total exposure / uptake duration:

20 d

Total depuration duration:

32 d

Test temperature:

mean < ± 2 °C (in the temperature range 20 - 25 °C)

Details on test conditions:

PRETREATMENT OF TEST ITEM:
To produce the test diet (see p. 22 for details) the reference compound hexachlorobenzene and the test item Macrolex Yellow 6G were dissolved in a mixture of solubilisers (45% 1,4-Dioxan, 45% Tetrahydrofuran, 10% Acetone). The solution was mixed with the fish food. The solubilisers were allowed to evaporate for the next 24 hours at room temperature.

TEST SYSTEM
- Test vessel: glass aquaria holding 40 L of test medium
- Experimental design : 2 test concentration plus 1 control
90 animals per test concentration and 30 animals for the control, flow-through system (frequency of water renewal: about 6 times per day)
Method of application : oral incorporation of test item via spiked fish food
Loading : 0.1 - 1 g fish (wet weight) per litre test medium
Temperature : mean < ± 2 °C (in the temperature range 20 - 25 °C)
Aeration : gentle aeration via narrow glass tubes
Test item concentration/s in fish diet : 100 μg/g and 1000 μg/

OTHER TEST CONDITIONs
- Photoperiod: 16 h light: 8 hrs dark

Nominal and measured concentrations:

100 ug/g and 1000 ug/g

Reference substance (positive control):

yes

Details on estimation of bioconcentration:

BASIS INFORMATION
- Measured/calculated logPow: 7.88

Lipid content:

8.5 %

Time point:

start of exposure

Remarks on result:

other: at 100 ug/g test material concentration

Lipid content:

7.6 %

Time point:

end of exposure

Remarks on result:

other: at 100 ug/g test material concentration

Lipid content:

13.1 %

Time point:

end of exposure

Remarks on result:

other: at 1000 ug/g test material concentration

Key result

Conc. / dose:

1 000 µg/g food

Temp.:

>= 20 - <= 25 °C

Type:

BMF

Value:

0.003 dimensionless

Basis:

total lipid content

Time of plateau:

20 d

Remarks on result:

other: other details not available

Parameter:

DT50

Depuration time (DT):

9.706 d

Remarks on result:

other: other details not available

Details on kinetic parameters:

- Uptake rate constant k(s):520

Validity criteria fulfilled:

yes

Conclusions:

The observed BMF was 0.0026 of test chemical on fish Danio rerio in 20 days. On the basis of this BMF value it is concluded that this test chemical is non bioaccumuative in nature.

Executive summary:

For determination bioaccumulation factor an experiment was conducted with food instead of water as the exposure route. Apart from that, the general provisions of OECD TG 305 – Bioconcentration: Flow-through Fish Test (June 1996) which is equivalent to the Council regulation (EG) 440/2008 EU method C.13, Bioconcentration: Flow-Through Fish Test (2008) were used to conduct the study. The “Background document to the Fish Dietary Study-Protocol” and “Fish Dietary Bioaccumulation Study - Basic Protocol” was used as guidance. This study consists of two phases the exposure phase and post exposure phase(depuration).During the uptake phase, separate groups of fish of one species are fed with a special diet of fish food mixed with at least two concentrations (100 and 1000 μg/g fish food) of the test item. As a reference compound hexachlorobenzene is also added to the fish diet at a nominal concentration of 100 μg/g. The uptake phase is run for 20 days. The concentration of the test item in control and test diets are analysed one day prior to the uptake phase, at the beginning of the uptake phase and 10 days thereafter. The test item and reference compound concentration in fish is followed by HPLC analysis and UV/VIS-detection 10 days after beginning of the uptake phase and at the end of the uptake phase. A tissue specific analysis was also carried out on dissected fish and the separated guts of the fish at the end of the uptake phase. The test dilution water was measured one day prior to the beginning of the uptake phase and on day 10 and 20 of the uptake phase for the control and both test item concentrations. After 20 days the depuration period is begun by feeding the fish with a diet free from the test item and the reference compound. During the depuration phase fish samples were taken at several times for analysis. Fish for tissue specific analysis were also taken, the guts were removed and both, dissected fish and guts were analysed separately. After a period of 28 days, the concentration of test item in fish was below the LOQ of of test chemical of 0.401 μg/g fish wet weight and that was the last data point to be included in the regression to estimate the depuration rate constant. The lipid cocntent of fish at start of exposure was 8.5 % and at end of exposure was 13.1 %. The test item was shown to be stable in spiked fish food with recoveries ranging from 97.5 to 99.8% at the test item concentration 100 μg/g and 99.2 to 105.2% at the test item concentration 1000 μg/g (3 repeat determinations during uptake phase). The recoveries for the reference compound hexachlorobenzene in spiked fish food ranged from 86.9 to 97.1% at the test item concentration 100 μg/g and from 70.3 to 106.7% at the test item concentration 1000 μg/g, respectively. The uptake efficiency (α) for HCB was 0.548 (54.8%) at a HCB concentration of 100 μg/g (and a test item concentration 1000 μg/g) indicating suitability of the test system. For the test item an uptake efficiency of 0.063 (6.3%) for the test item concentration 1000 μg/g was calculated. Because of limited uptake, at a test item concentration of 100 μg/g the concentration in fish tissue after 20 days uptake was below the LOQ and therefore no assimilation efficiency could be determined. The mean recovery of test item from spiked fish tissue was 97.5%, while the mean recovery of the reference compound hexachlorobenzene was 93.2%. Some fish were dissected and fish tissue and gut samples were analysed separately. From the data, correcting for fish growth during the test, a BMF of 0.0026 was determined for the test item concentration 1000 μg/g. After 28 days of depuration (i.e. feeding unspiked food), no test chemical could be determined any more. At a concentration of 100 μg/g the bioaccumulation was too low to allow quantification. The calculated half life of the test item from the above data was 9.706 days. The observed BMF was 0.0026 of test chemical on fish Danio rerio  in 20 days. On the basis of this BMF value it is concluded that this test chemical is non bioaccumuative in nature.

Description of key information

The observed BMF was 0.0026 of test chemical on fish Danio rerio  in 20 days. On the basis of this BMF value it is concluded that this test chemical is non bioaccumuative in nature.

Key value for chemical safety assessment

BMF in fish (dimensionless):

0.003

Additional information

For determination bioaccumulation factor an experiment was conducted with food instead of water as the exposure route (Experimental study report, 2009). Apart from that, the general provisions of OECD TG 305 – Bioconcentration: Flow-through Fish Test (June 1996) which is equivalent to the Council regulation (EG) 440/2008 EU method C.13, Bioconcentration: Flow-Through Fish Test (2008) were used to conduct the study. The “Background document to the Fish Dietary Study-Protocol” and “Fish Dietary Bioaccumulation Study - Basic Protocol” was used as guidance. This study consists of two phases the exposure phase and post exposure phase(depuration).During the uptake phase, separate groups of fish of one species are fed with a special diet of fish food mixed with at least two concentrations (100 and 1000 μg/g fish food) of the test item. As a reference compound hexachlorobenzene is also added to the fish diet at a nominal concentration of 100 μg/g. The uptake phase is run for 20 days. The concentration of the test item in control and test diets are analysed one day prior to the uptake phase, at the beginning of the uptake phase and 10 days thereafter. The test item and reference compound concentration in fish is followed by HPLC analysis and UV/VIS-detection 10 days after beginning of the uptake phase and at the end of the uptake phase. A tissue specific analysis was also carried out on dissected fish and the separated guts of the fish at the end of the uptake phase. The test dilution water was measured one day prior to the beginning of the uptake phase and on day 10 and 20 of the uptake phase for the control and both test item concentrations. After 20 days the depuration period is begun by feeding the fish with a diet free from the test item and the reference compound. During the depuration phase fish samples were taken at several times for analysis. Fish for tissue specific analysis were also taken, the guts were removed and both, dissected fish and guts were analysed separately. After a period of 28 days, the concentration of test item in fish was below the LOQ of of test chemical of 0.401 μg/g fish wet weight and that was the last data point to be included in the regression to estimate the depuration rate constant. The lipid cocntent of fish at start of exposure was 8.5 % and at end of exposure was 13.1 %. The test item was shown to be stable in spiked fish food with recoveries ranging from 97.5 to 99.8% at the test item concentration 100 μg/g and 99.2 to 105.2% at the test item concentration 1000 μg/g (3 repeat determinations during uptake phase). The recoveries for the reference compound hexachlorobenzene in spiked fish food ranged from 86.9 to 97.1% at the test item concentration 100 μg/g and from 70.3 to 106.7% at the test item concentration 1000 μg/g, respectively. The uptake efficiency (α) for HCB was 0.548 (54.8%) at a HCB concentration of 100 μg/g (and a test item concentration 1000 μg/g) indicating suitability of the test system. For the test item an uptake efficiency of 0.063 (6.3%) for the test item concentration 1000 μg/g was calculated. Because of limited uptake, at a test item concentration of 100 μg/g the concentration in fish tissue after 20 days uptake was below the LOQ and therefore no assimilation efficiency could be determined. The mean recovery of test item from spiked fish tissue was 97.5%, while the mean recovery of the reference compound hexachlorobenzene was 93.2%. Some fish were dissected and fish tissue and gut samples were analysed separately. From the data, correcting for fish growth during the test, a BMF of 0.0026 was determined for the test item concentration 1000 μg/g. After 28 days of depuration (i.e. feeding unspiked food), no test chemical could be determined any more. At a concentration of 100 μg/g the bioaccumulation was too low to allow quantification. The calculated half life of the test item from the above data was 9.706 days. The observed BMF was 0.0026 of test chemical on fish Danio rerio  in 20 days. On the basis of this BMF value it is concluded that this test chemical is non bioaccumuative in nature.