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Bioaccumulation: aquatic / sediment

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Reference
Endpoint:
bioaccumulation in aquatic species: fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 26, 1994 to October 20, 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to GLP requirements and OECD method.
Qualifier:
according to
Guideline:
OECD Guideline 305 E (Bioaccumulation: Flow-through Fish Test)
Principles of method if other than guideline:
Under consideration of EPA 540/9-82-021: Subdivision N, Series 165-4
GLP compliance:
yes (incl. certificate)
Radiolabelling:
yes
Details on sampling:
Water samples (4 ml) were taken for radioactivity analyses from approximately the centre of the tanks in duplicate from all test concentrations on days 0, 3, 7, 14, 21, and 28 of accumulation phase and on days 1, 3, 7, 10, 14, 22, and 28 of depuration phase. At the same time intervals, 500 ml water samples were taken to test for the parent compound. Duplicate water samples (4 ml aliquots) also were taken from the control tank at 0, 14, and 28 days for radioactivity analyses. For each exposure tank: Fish (8 or 14) also were captured at random at the same sampling times during days 7-28 of the accumulation phase. Six fish were randomly captured during depuration days 3-14, 1 fish was removed on depuration day 22 showing fungal growth, and on depuration day 28 the remaining fish were removed. All the fish were rinsed with water, killed by cervical dislocation, and blotted dry. On accumulation days 3, 7, and 14, 2/6 fish were analysed whole and 4/6 were separated into edible (fillet and skin) and non-edible parts (head, fins, viscera and skeleton). Whole fish were weighed and solubilised and individual fish parts were pooled, weighed, and solubilised. Total radioactivity was measured. Control fish were treated in a similar manner. For accumulation days 21 and 28, fish edibles and non-edibles were analysed based on parent compound because of plateau levels. Due to small fish weight, edibles and non-edibles were extracted in pools of 12 fish rather than solubilised. Following extraction, fish parts were air-dried, homogenized, and remaining radioactivity was determined by solubilisation (non-extractables). Not stated how sampling was conducted for depuration phase other than 6/tank/time interval were analysed.
Vehicle:
yes
Details on preparation of test solutions, spiked fish food or sediment:
The application solutions were prepared by diluting stock solutions containing appropriate amounts of unlabelled and radiolabelled AHTN in dimethylformamide. Stock solutions were prepared and frozen at -20 C in 2 ml aliquots based on a flow-through of 300 l over 24 hours. Application solutions were prepared with the 2 ml aliquot mixed with 2.0 g Tween 80 and diluted to 2000 ml bidistilled water to yield the appropriate final test concentrations in 0.001% Tween 80 and 0.001% dimethylformamide. For initial level adjustments in the 75-litre tanks, portions of 0.5 ml of the stock solutions were mixed with 0.5 g Tween and added to the tanks.
Test organisms (species):
Lepomis macrochirus
Details on test organisms:
Bluegill sunfish weighing an average of 0.35 g at start of test; 101 fish at 1 ug/l; 97 fish at 10 ug/l; 20 fish in control.
Route of exposure:
aqueous
Test type:
flow-through
Water / sediment media type:
natural water: freshwater
Total exposure / uptake duration:
28 d
Total depuration duration:
28 d
Hardness:
no data
Test temperature:
20.0-23.6 C
pH:
7.8-8.1
Dissolved oxygen:
7.5-9.0 mg O2/l
TOC:
no data
Details on test conditions:
Test medium=tap water; light=16 hours/day, about 500 Lux; tanks containing 75 l test medium; fish fed daily with Kliba; loading ratio of 0.02, 0.12, and 0.11 g fish/litre test medium for 0 (control), 1, and 10 µg/l, respectively; application solution delivered at a rate of 2.0 l/24 hours via a Hamilton dispenser unit into a mixing flask; water flow into tanks=300 litres/24 hours.
Nominal and measured concentrations:
Nominal: 1 and 10 µg/l. Measured: 0.99+/-0.12 µg/l and 9.81+/-0.85 µg/l, respectively.
Reference substance (positive control):
no
Details on estimation of bioconcentration:
First-order kinetics was used to describe depuration curves of radioactivity in edibles, non-edibles, and whole fish by means of the best fitting exponential curve. Using the non-linear parameter estimation program MINSQ, plateau levels in fish and fish parts were calculated using the concentration of radioactivity in whole fish, edibles and non-edibles, average concentration of radioactivity in exposure water, accumulation time points, accumulation rate constant, and depuration rate constant based on accumulation data. BCF was calculated from the concentration of total radioactivity in parent equivalents in the fish or fish parts at plateau level related to the average concentration of total radioactivity in the water during exposure.
Type:
BCF
Value:
596
Basis:
whole body w.w.
Time of plateau:
3 d
Calculation basis:
kinetic
Remarks on result:
other: based on ratio of percentage parent compound in exposure water and fish
Remarks:
Conc.in environment / dose:9.81 ug/l
Type:
BCF
Value:
597
Basis:
whole body w.w.
Time of plateau:
7 d
Calculation basis:
kinetic
Remarks on result:
other: time of plateau is 3-7 days; based on ratio of percentage parent compound in exposure water and fish
Remarks:
Conc.in environment / dose:0.99 ug/l
Type:
BCF
Value:
527
Basis:
edible fraction
Time of plateau:
3 d
Calculation basis:
kinetic
Remarks on result:
other: based on ratio of total radioactivity in exposure water and fish
Remarks:
Conc.in environment / dose:9.81 ug/l
Type:
BCF
Value:
259
Basis:
edible fraction
Time of plateau:
7 d
Calculation basis:
kinetic
Remarks on result:
other: time of plateau is 3-7 days; based on ratio of percentage parent compound in exposure water and fish
Remarks:
Conc.in environment / dose:0.99 ug/l
Type:
BCF
Value:
825
Basis:
non-edible fraction
Time of plateau:
7 d
Calculation basis:
kinetic
Remarks on result:
other: time of plateau is 3-7 days; based on ratio of percentage parent compound in exposure water and fish
Remarks:
Conc.in environment / dose:0.99 ug/l
Type:
BCF
Value:
496
Basis:
edible fraction
Time of plateau:
7 d
Calculation basis:
kinetic
Remarks on result:
other: time of plateau is 3-7 days; based on ratio of total radioactivity in exposure water and fish
Remarks:
Conc.in environment / dose:0.99 ug/l
Type:
BCF
Value:
2 100
Basis:
non-edible fraction
Time of plateau:
3 d
Calculation basis:
kinetic
Remarks on result:
other: based on ratio of total radioactivity in exposure water and fish
Remarks:
Conc.in environment / dose:9.81 ug/l
Type:
BCF
Value:
2 136
Basis:
non-edible fraction
Time of plateau:
7 d
Calculation basis:
kinetic
Remarks on result:
other: time of plateau is 3-7 days; based on ratio of total radioactivity in exposure water and fish
Remarks:
Conc.in environment / dose:0.99 ug/l
Type:
BCF
Value:
1 325
Basis:
whole body w.w.
Time of plateau:
3 d
Calculation basis:
kinetic
Remarks on result:
other: based on ratio of total radioactivity in exposure water and fish
Remarks:
Conc.in environment / dose:9.81 ug/l
Type:
BCF
Value:
1 313
Basis:
whole body w.w.
Time of plateau:
7 d
Calculation basis:
kinetic
Remarks on result:
other: time of plateau is 3-7 days; based on ratio of total radioactivity in exposure water and fish
Remarks:
Conc.in environment / dose:0.99 ug/l
Elimination:
yes
Parameter:
DT50
Depuration time (DT):
2 d
Details on kinetic parameters:
Depuration rate constant calculated from accumulation data (k2±standard deviation) at 0.99 µg/l is 0.511±0.536, 0.406±0.201, and 0.671±0.432 for edibles, non-edibles, and whole fish, respectively. Depuration rate constant calculated from accumulation data (k2±standard deviation) at 9.81 µg/l is not determined, 1.252±0.240, and 1.065±1.949 for edibles, non-edibles, and whole fish, respectively. Depuration rate constant calculated from depuration data [k(dep)±standard deviation] at 0.99 µg/l is 0.450±0.095, 0.846±0.171, and 0.337±0.051 for edibles, non-edibles, and whole fish, respectively. Depuration rate constant calculated from depuration data [k(dep)±standard deviation] at 9.81 µg/l is 0.435±0.094, 0.717±0.260, and 0.577±0.164 for edibles, non-edibles, and whole fish, respectively. Accumulation rate constant calculated from BCF*k(dep) (k1) at 0.99 µg/l is 223, 1807, and 442 for edibles, non-edibles, and whole fish, respectively. Accumulation rate constant calculated from BCF*k(dep) (k1) at 9.81 µg/l is 229, 1506, and 765 for edibles, non-edibles, and whole fish, respectively.
Metabolites:
In water, one minor metabolite fraction W2 (2-2.5%), small amounts of metabolite fraction W3, but mostly parent compound (W1; 90-97%) were detected. In edibles and non-edibles after 21 and 28 days of accumulation, 40-50% of total radioactivity was parent compound with at least one metabolite fraction (F3; 33-36%), which corresponded to water metabolite fraction W3.
Results with reference substance (positive control):
No data
Details on results:
No mortality in controls. Mortality at 0.99 µg/l during accumulation and depuration phases was 7.9% and 2.0%, respectively. Mortality at 9.81 µg/l during accumulation and depuration phases was 7.2% and 1.0%, respectively.

Plateau residue levels calculated as parent equivalents according to total radioactivity and based on fresh weight were 0.49+/-0.08 mg/kg, 2.12+/-0.20 mg/kg, and 1.30+/-0.11 mg/kg for edibles, non-edibles, and whole fish, respectively, at 0.99 ug/l. At 9.81 ug/l, the plateau levels were 5.17+/-0.81 mg/kg, 20.60+/-0.16 mg/kg, and 12.99+/-1.40 mg/kg for edibles, non-edibles, and whole fish, respectively.

At 0.99 ug/l after 7 days depuration, 18.7, 20.1, and 20.4% of the radioactivity were detected in edibles, non-edibles, and whole fish, respectively, and after 28 days depuration, 5.4,4.8, and 3.8% of the radioactivity were detected in edibles, non-edibles, and whole fish, respectively.

At 9.81 ug/l after 7 days depuration, 29.5, 31.0, and 35.6% of the radioactivity were detected in edibles, non-edibles, and whole fish, respectively, and after 28 days depuration, 6.6, 6.2, and 10.5% of the radioactivity were detected in edibles, non-edibles, and whole fish, respectively.

BCF=864 at 9.81 ug/l (non-edible fraction) based on ratio of percentage parent compound in exposure water and fish (time of plateau=3 d).
BCF=257 at 9.81 ug/l (edible fraction) based on ratio of percentage parent compound in exposure water and fish (time of plateau=3 d).
Reported statistics:
No data
Validity criteria fulfilled:
not specified
Conclusions:
Exposure of Bluegill Sun fish to target concentrations of 1 or 10 µg AHTN/l for a 28-day period followed by a 28-day depuration period showed bioaccumulation. BCF values based on total radioactivity averaged 512±22, 2118±25, and 1320±8 for edibles, non-edibles, and whole fish, respectively, and BCF values based on average concentrations of parent compound averaged 258±1, 845±28, and 597±1 for edibles, non-edibles, and whole fish, respectively. Plateau levels were reached after 3-7 days and depuration from fish and fish parts had half-lives of about 1-2 days. At the end of the study, elimination of radioactivity was about 95% and still on-going. In water, one minor metabolite fraction W2 (2-2.5%), small amounts of metabolite fraction W3, but mostly parent compound (W1; 90-97%) were detected. In edibles and non-edibles after 21 and 28 days of accumulation, 40-50% of total radioactivity was parent compound with at least one metabolite fraction (F3; 33-36%), which corresponded to water metabolite fraction W3.
Executive summary:

14C-AHTN was tested for elimination and accumulation at target concentrations of 1 or 10 µg/l in Bluegill sunfish (~100 fish/concentration) over 28 days followed by a 28-day depuration period in a flow-through test system following OECD guideline 305E. Average 14C-AHTN concentrations in 0.001% Tween 80 and 0.001% dimethylformamide were determined to be 0.99 and 9.81 µg/l using liquid scintillation counting. Mortality in exposed fish was low. Plateau levels of 0.49±0.08 mg/kg, 2.12±0.20 mg/kg, and 1.30±0.11 mg/kg for edibles, non-edibles, and whole fish, respectively, at 0.99 µg/l and 5.17±0.81 mg/kg, 20.60±0.16 mg/kg, and 12.99±1.40 mg/kg for edibles, non-edibles, and whole fish, respectively, at 9.81 µg/l were reached after 3-7 days. Depuration from fish and fish parts had half-lives of about 1-2 days. BCF values based on total radioactivity expressed in mg parent equivalents per kg fish averaged 512±22, 2118±25, and 1320±8 for edibles, non-edibles, and whole fish, respectively, whereas BCF values based on concentrations of parent compound averaged 258±1, 845±28, and 597±1 for edibles, non-edibles, and whole fish, respectively. In both exposure water and fish and fish parts, analyses of radioactivity detected mostly parent compound plus one other metabolite. In water, another metabolite was detected in very small amounts. This study showed that plateau levels are rapidly reached following exposure to AHTN but uptake of AHTN is highly reversible.

Description of key information

The BCF of 597 L/kg ww was derived form a study on Bluegill Sunfish under GLP conditions and according to OECD TG 305E (Rel. 1, Van Dijk 1996) .

Key value for chemical safety assessment

BCF (aquatic species):
597 L/kg ww

Additional information

Several bioaccumulation studies on fish were performed. The EU Risk Assessment Report of AHTN selected a fish BCF of 597 l/kg that was reported in the study by Van Dijk (1996). It is performed on Bluegill Sunfish under GLP conditions and according to OECD TG 305E (Rel. 1).

This value is further supported by the results of Butte (1999), who conducted the study on Zebra fish according to the same OECD guideline. Moreover, the BCFBAF model form the EPISUITE BCFBAF module (US EPA) also calculated a similar BCF.