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Environmental fate & pathways

Bioaccumulation: aquatic / sediment

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Reference
Endpoint:
bioaccumulation in aquatic species: fish
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
March 4, 2002 to May 27, 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with Japanese test guidelines in compliance with GLP.
Qualifier:
according to
Guideline:
other: "Test method relating to the new chemical substances" (Kanpogyo No. 5, Yakuhatsu No. 615, 49 Kikyoku No. 392, 1974; partly revised in 1998)
Deviations:
not specified
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
None specified
Radiolabelling:
no
Details on sampling:
Test solution was sampled from the control water and analyzed twice according to the analytical method for second concentration level. The measured values were < 0.001 mg/L as the concentration in test water and less than the detection limit.

The analysis was carried out according to the following procedure:

First concentration level -> Test solution was diluted to 10 folds with purified water -> About 1.0 mL was sampled into vial for auto sampler -> LC/MS measurement

Second centration level -> About 1.0 mL of test was sampled into vial for auto sampler -> LC/MS measurement
Vehicle:
yes
Details on preparation of test solutions, spiked fish food or sediment:
[First concentration level]
Test substance 1.6g (40% [w/v] HCO-40/tetrahydrofuran solution 160 mL; Tetrahydrofuran) -> Diluted to the constant volume 200 mL (Concentration of test substance: 8000 mg/L; Concentration of HCO-40: 320000 mg/L)

[Second concentration level]
Feed stock solution for the first concentration level 20 mL (Tetrahydrofuran) -> Diluted to the constant volume 200 mL (Concentration of test substance: 800 mg/L; Concentration of HCO-40: 32000 mg/L)

[Control]
40% (w/v) HCO-40/tetrahydrofuran solution 800 mL (Tetrahydrofuran) -> Diluted to the constant volume 1000 mL (Concentration of HCO-40: 320000 mg/L)

Frequency of preparation: Approximately every 7 days, for test group only.

Preparation of test water
Feed stock solution prepared as shown in above was supplied to mixing tube using metering pump for feed stock solution, mixed with dilution water that was supplied using metering pump for dilution water to prepare the test solution at the designated concentration. This solution was supplied to the test vessel.
Test organisms (species):
Cyprinus carpio
Details on test organisms:
Species: Carp (Cyprinus carpio, carp family)
Supplier: Sankyo Suisan (1-1, Ichigaya-tamachi, Shinjuku-ku Tokyo)
Received date: November 12, 2001
Lot number: 01-K-1112
Pre-treatment: Visual observation was made upon receipt and abnormal fish were removed. The remaining normal fish were maintained in an aquarium in flow-through water. During the acclimation period, fish were kept without medicated bathing.
Total length: 8+/-4cm
Body weight: Approximately 5 g
Age: About 1 year after hatching

Acclimation
Aquarium number: C-2
Water temperature: 24+/-2°C
Period: 2 weeks or more
Type of food: Babygold, Kyorin Co., Ltd.
Feeding: The food was given in an amount about 2% of body weight of fish (daily).
Mortality during a week before the test: < 5%
Route of exposure:
feed
Test type:
flow-through
Water / sediment media type:
natural water: freshwater
Total exposure / uptake duration:
28 d
Hardness:
Not specified
Test temperature:
Water temperature: 24+/-2°C
pH:
pH: 6.0-8.5
Dissolved oxygen:
During the experimental period, the dissolved oxygen concentrations in all test vessels were > 60% of the saturated dissolved oxygen concentration.
TOC:
Not specified
Salinity:
Not specified
Details on test conditions:
Supplied amount of dilution water: 800 L/day (water exchange: 16 times/day)
Supplied amount of feed stock solution: 20 mL/day
Number of fish (at the start of exposure period)
First concentration level: 28 fish/50 L test water (1.0 g/L/day or more)
Second concentration level: 28 fish/50 L test water (1.0 g/L/day or more)
Control: 24 fish/50 L test water (1.0 g/L/day or more)
Water temperature: 24+/-2 °C
Dissolved oxygen concentration: 60% or more of the saturated dissolved oxygen concentration (5 mg/L or more at 24 °C)
Aeration: Yes
pH: 6.0-8.5
Lighting period, type: Approximately 16hr/day, Hf fluorescent lamp (wavelength 400-700 nm)
Exposure period: 28 days

Introduction and administration of fish
The bioaccumulation test system was operated without fish for more than 2 days to confirm that the test concentration was kept at the designated level. Then fish were introduced into the test vessels and the test was started. During exposure period, the following items were observed and measured.
Feeding: Babygold, Kyorin Co., Ltd., was given in an amount about 2% of the body weight of fish once a day (except holidays).
Condition of fish: Appearance, swimming and food intake of fish were observed.
Dissolved oxygen and water temperature: The dissolved oxygen and temperature were measured when test water was sampled.
Desktop dissolved oxygen meter: DOL-10 (No. 1), Denki Kagaku Keiki Co., Ltd.
Mobile thermometer: 2455 02 (No. 4), Yokogawa Electric Corp.
Lipid content: Lipid content was measured using control fish at the start and the end of exposure period. (n = 3 respectively)
pH: pH was measured at the start and the end of exposure period.
Desktop pH meter: HM-40V (No. 1), Toa Denpa Kogyo Kaisha (Toa DKK)

Measurement of test substance concentration
Test water: At the start (day 0) and on days 4, 7, 14, 21 and 28 of the exposure period, the test substance concentration of each test water was measured.
Fish: At the start (day 0) and on days 4, 7, 14, 21 and 28 of the exposure period, 4 fish were sampled respectively and the test substance concentration was measured twice using 2 fish at each measurement.
Nominal and measured concentrations:
Concentration level (nominal concentration)
First concentration level: 0.2 mg/L (concentration of auxiliary agent; 8 mg/L for HCO-40, approximately equal to 17 ppm (v/v) for tetrahydrofuran)
Second concentration level: 0.02 mg/L (concentration of auxiliary agent; 0.8 mg/L for HCO-40, approximately equal to 24 ppm (v/v) for tetrahydrofuran)
Control: 0 mg/L (concentration of auxiliary agent; 8 mg/L for HCO-40, approximately equal to 17 ppm (v/v) for tetrahydrofuran)
Reference substance (positive control):
not specified
Details on estimation of bioconcentration:
Bioconcentration factor
Calculation of bioconcentration factor
Bioconcentration at each measurement time is calculated by the following equation:
BCF = Cf at each measurement time / Cw over the days until each measurement (mean)
If the steady-state was attained the bioconcentration factor at steady-state (BCFss) was also calculated by the following equations.
BCFss = Cf at steady-state (mean) / Cw at steady-state (mean)
Where
BCF: Bioconcentration factor at each measurement time
BCFss: Bioconcentration factor at steady-state
Cf: Concentration of the test substance in test fish (μg/g)
Cw: Concentration of the test substance in test water (mg/L)
Steady-state: Steady-state is decided to be reached, when three successive BCF at intervals of 48 hours or longer are within 20% of each other. Mean value of Cf and Cw are calculated from the data of the last three successive measurements leading up to the end of exposure period. If BCF values are smaller than 100 in all points, it is considered that steady-state is reached even if the variation of BCF values are 20% or more. In this case BCFss is not calculated.

Determination limit of bioconcentration factor
As the results of the fish blank test, the test substance was not detected and the detection limit concentration was < 1.3 μg/g. BCF can be obtained when the bioconcentraion level of the test substance is greater than about 7-folds at 0.2 mg/L (First concentration level), and greater than about 65-folds at 0.02 mg/L (Second concentration level) of the nominal concentration of test water, respectively, although it depends on the body weight of fish
Lipid content:
4.5 - 7.1 %
Time point:
start of exposure
Lipid content:
4.3 - 6.5 %
Time point:
end of exposure
Type:
BCF
Value:
< 7 other: fold
Basis:
not specified
Time of plateau:
28 d
Calculation basis:
steady state
Remarks on result:
other: Conc.in environment / dose:0.200 mg/L
Type:
BCF
Value:
< 84 other: fold
Basis:
not specified
Time of plateau:
28 d
Calculation basis:
steady state
Remarks on result:
other: Conc.in environment / dose:0.0182 mg/L
Details on kinetic parameters:
Not specified
Metabolites:
Not specified
Results with reference substance (positive control):
Reference substance not utilised in this study
Details on results:
Environmental factor that may have affected the reliability of the test results: There was no factor that may have affected the reliability of the test results.

Maintenance of test fish
During the experimental period, the dissolved oxygen concentrations in all test vessels were > 60% of the saturated dissolved oxygen concentration, and the water temperature was 24+/-2 °C. These met the requirements for the valid test conditions. The values of pH were within the adequate range (6.0 - 8.5) for the fish cultivation environment. During the experimental period, the frequency of death and abnormality in fish was < 10 % in every concentration level. No abnormality was observed in appearance, swimming and food intake of the surviving fish during the test period, and it was confirmed fish were maintained normally.
Lipid content:
At the start of exposure period 5.5 % (n=3, 4.5 – 7.1 %)
At the end of exposure period 5.4 % (n=3, 4.3 – 6.5 %)
(The mean value of lipid content at the end was within +/-25 % of that at the start.)

Concentration of test substance in test water
The mean concentration of the test substance in test solution during 28 days of the exposure period was 0.200 mg/L for the first concentration level and 0.0182 mg/L for the second concentration level, and these were sufficiently close to the nominal values. The coefficient of variation for the concentrations was 4.0 % and 5.6 % for the first concentration level and second concentration level, respectively. The variation of the test substance concentrations in test solutions were maintained within +/-20 % of the mean measured values.

Concentration of test substance and bioconcentration factor (BCF) in test fish
The test substance concentration in test fish was < 1.27 μg/g for the first concentration level and < 1.50 μg/g for the second concentration level. The bioconcentration factor (BCF) was < 7 folds for the first concentration level and < 84 folds for the second concentration level.

Based on above results, it is considered that the bioaccumulation level of the test substance in fish is low.
Reported statistics:
Not specified

The results of bioconcentration measurement are shown in below table:

Exposure period

Day 4

Day 7

Day 14

Day 21

Day 28

First conc. level

Mean concentration in test water (mg/L)

0.194

0.195

0.195

0.199

0.200

BCF                       1

BCFss < 7              2

< 6

< 6

< 6

< 6

< 7

< 7

< 6

< 7

< 6

< 6

Second conc. level

Mean concentration in test water (mg/L)

0.0187

0.0184

0.0181

0.0180

0.0182

BCF                        1

BCFss < 84            2

< 53

< 52

< 73

< 59

< 77

< 78

< 67

< 84

< 56

< 58

 

Validity criteria fulfilled:
not specified
Conclusions:
The test substance concentration in test fish was < 1.27 μg/g for the first concentration level and < 1.50 μg/g for the second concentration level. The bioconcentration factor (BCF) was < 7 folds for the first concentration level and < 84 folds for the second concentration level.

Based on above results, it is considered that the bioaccumulation level of the test substance in fish is low.
Executive summary:

Study title: Bioaccumulation study of K-1594 in carp

Study number: A010430

Study period: From March 4, 2002 to May 27, 2002

Test method: The study was conducted in accordance with "Test method relating to new chemical substances" (Kanpogyo No. 5, Yakuhatsu No. 615, 49 Kikyoku No. 392, 1974; partly revised in 1998).

- Supply of test water: Flow-through system (800L/day)

- Concentration of the test substance in test water (nominal concentration):

First concentration level: 0.2 mg/L

Second concentration level: 0.02 mg/L

Control: 0 mg/L

- Concentration of auxiliary agent in test water (nominal concentration):

First concentration level: 8 mg/L for HCO-40, approximately equal to 17 ppm (v/v) for tetrahydrofuran

Second concentration level: 0.8 mg/L for HCO-40, approximately equal to 24 ppm (v/v) for tetrahydrofuran

Control: 8 mg/L for HCO-40, approximately equal to 17 ppm (v/v) for tetrahydrofuran

- Exposure period: 28 days

- Analytical method: Analysis was conducted by high performance liquid chromatograph-mass spectrometer after pre-treatment.

- 96hr LC50 to killifish: > 20 mg/L

- Lipid content in fish body:

At the start of exposure period: 5.5 % (n=3, 4.5 – 7.1 %)

At the end of exposure period: 5.4 % (n=3, 4.3 – 6.5 %)

 

Concentration of test substance in test water

The mean concentration of the test substance in test solution during 28 days of the exposure period was 0.200 mg/L for the first concentration level and 0.0182 mg/L for the second concentration level, and these were sufficiently close to the nominal values. The coefficient of variation for the concentrations was 4.0 % and 5.6 % for the first concentration level and second concentration level, respectively. The variation of the test substance concentrations in test solutions were maintained within +/-20 % of the mean measured values.

 

Concentration of test substance and bioconcentration factor (BCF) in test fish

The test substance concentration in test fish was < 1.27 μg/g for the first concentration level and < 1.50 μg/g for the second concentration level. The bioconcentration factor (BCF) was < 7 folds for the first concentration level and < 84 folds for the second concentration level.

 

Based on above results, it is considered that the bioaccumulation level of the test substance in fish is low.

Description of key information

Key value determined in laboratory study performed to recognised Japanese test guidelines in compliance with GLP.

Key value for chemical safety assessment

BCF (aquatic species):
7 dimensionless

Additional information

The test substance concentration in test fish was < 1.27 μg/g for the first concentration level and < 1.50 μg/g for the second concentration level. The bioconcentration factor (BCF) was < 7 folds for the first concentration level and < 84 folds for the second concentration level.

 

Based on above results, it is considered that the bioaccumulation level of the test substance in fish is low.