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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:
key study
Study period:
11 September 1998 to 24 December 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to
Guideline:
other: Method for Testing the Degree of Accumulation of Chemical Substances in Fish Body stipulated in the Testing Methods for New Chemical Substances
Version / remarks:
July 13, 1974, Kanpogyo No.5, Planning and Coordination Bureau, Environment Agency, Yakuhatu No.615, Pharmaceutical Affairs Bureau, Ministry of Health and Welfare, and 49 Kikyoku No.392, Basic Industries Bureau, Ministry of International Trade and Industry, Japan
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 305 C (Bioaccumulation: Test for the Degree of Bioconcentration in Fish)
Version / remarks:
1981
GLP compliance:
yes
Radiolabelling:
not specified
Details on sampling:
- The concentrations of the test material in the test water for both level 1 and 2 were analysed 16 times; twice a week intervals (n=1). The concentrations in the test fish for both levels were analysed 4 times; in weeks 2, 4, 6 and 8 (n=2). The control fish were analysed at the initiation and the termination of exposure (n=2).
Vehicle:
yes
Remarks:
HCO-40 (Hydrogenated castor oil)
Details on preparation of test solutions, spiked fish food or sediment:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Test material supplied by the sponsor and HCO-40 (40 times amount of the test material) were dissolved with acetone. After acetone was evaporated from the solution, ion-exchanged water was added to the mixture to prepare 1000 mg/L stock solution.
- For level 1 a 400 mg/L stock solution was prepared
- For level 2 a 40 mg/L stock solution was prepared
- Control: HCO-40 was dissolved in ion-exchanged water to prepare 16 g/L stock solution of each concentration.
Test organisms (species):
Cyprinus carpio
Details on test organisms:
TEST ORGANISM
- Common name: Carp
- Length at study initiation: average 9.8 cm
- Weight at study initiation: average 22.9 g
- Lipid content at test initiation: average 4.2%
- Health status: The fish were checked visually in the receiving and those demonstrating any abnormalities were removed. The fish were reared for 6 days in a flow through system following an external disinfection.
- Feeding during test
- Food type: Pelleted feed for carp.
- Amount: An amount corresponding to about 2% of the total body weight of the test fish was fed twice a day in halves. On the day of fish sampling no feed was given.

ACCLIMATION
- Acclimation period: After rearing, the fish were medicated to eliminate parasites and transferred to an acclimatising aquarium. After the second external disinfection, they were acclimatised. The fish those demonstrating any abnormalities during this period were removed and reared for 40 days in a flow through system at the temperature of 25 + 2°C. The fish were then transferred to test tanks and reared at the same temperature in the flow through system for another 26 days, following the external disinfection.
Route of exposure:
aqueous
Justification for method:
aqueous exposure method used for following reason: The test material was stable during the exposure period, as shown by the finding that IR spectra of the test material before the initiation and after the termination of the exposure, were identical under the storage conditions.
Test type:
flow-through
Water / sediment media type:
natural water
Remarks:
Underground water from the premises of the testing laboratory.
Total exposure / uptake duration:
8 wk
Hardness:
117 mg/L (Ca, Mg)
Test temperature:
25 ± 2°C
pH:
At initial exposure: 7.9
At termination of exposure: 7.6
Dissolved oxygen:
Level 1: 6.8 - 7.6 mg/L
Level 2: 7.1 - 7.9 mg/L
Control: 7.2 - 7.9 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 L glass tank
- Type of flow-through: A flow through system assembled at the testing laboratory was used.
- Renewal rate of test solution (frequency/flow rate): 2 mL/min for stock solution and 800 mL/min for dilution water; 1155 litres/day for test water were supplied.
- No. of organisms per concentration: 11
- No. of organisms per control: 5

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilution water for the test was underground water from the premises of the testing laboratory. The dilution water for test was taken out on 3 August 1998, and it was analysed and measured (once every six months in the testing laboratory). It was confirmed that the dilution water met the ministerial ordinance of the Ministry of Health and Welfare (December 21, 1992), water quality criteria for fisheries (Shadanhozin Nihon Suisansigen Hogokyokai, March 1983), and OECD Guidelines for Testing of Chemicals 210, Fish, Early-life Stage Toxicity Test (July 17, 1992).

TEST CONCENTRATIONS
- Based on preliminary test results for 48-hour LC50 and analytical detection limits, test concentrations of the test material for the main study were decided on as follows. The control was set as a blank test.
Level 1: 1 mg/L (as the test material)
Level 2: 0.1 mg/L (as the test material)

OBSERVATIONS
- Observation of test fish: Condition of test fish was observed visually twice a day.
- Flow rate of test water: Flow rate of stock solution and dilution water were measured with graduated cylinder and recorded once a day.
- Temperature of test water: Temperature of test water was measured with alcohol thermometer and recorded once a day.
- Concentration of dissolved oxygen in test tank: Concentration of dissolved oxygen in test tank was measured with dissolved oxygen probe and recorded twice a week.
- In the test period, excreta of carp, dirt on test tank, etc. were removed once about a day.
Nominal and measured concentrations:
Nominal: 1 and 0.1 mg/L
Measured: See table 1. Each average concentration of the test material in test water was maintained at more than 95% of each the nominated concentration.
Reference substance (positive control):
not specified
Details on estimation of bioconcentration:
Calculation of bioconcentration factors (BCFs)

Level 1 and Level 2: K = (P x A (t) / A (std) / B x D x C / G –E) /F x 100
A: Peak area (µV·sec) [A(std): Standard solution, A(t): Sample]
B: Ratio of portion used for analysis 1-150
C: Final volume (25 mL)
D: Dilution factor
E: Average concentration of blank in analysis of control 0 µg/g
F: Recovery rate 61.5 and 88.9 %
G: Weight of test fish (g)
K: Concentration of test material in test fish (µg/g)
H: Average concentration of test material in test water (mg/L)
J: BCF J = K/H
P: Concentration of test material in standard solution 0.40 mg/L

Control: E = P x A (t) / A (std) / B x C
A: Peak area (µV·sec) [A(std): Standard solution, A(t): Sample]
B: Ratio of portion used for analysis 1/150
C: Final volume (25 mL)
G: Weight of test fish (g)
I: Concentration of blank in test fish (µg/g) I = E / G
P: Concentration of test material in standard solution 0.40 mg/L


- From the minimum limit of determination of the test material BCFs could be obtained for cases of a BCF exceeding the following:
Level 1: Peak A: 0.8, Peak B: 5.5
Level 2: Peak A: 8.0, Peak B: 54
Key result
Conc. / dose:
1 mg/L
Temp.:
25 °C
pH:
7.9
Type:
BCF
Basis:
not specified
Remarks on result:
other: Peak A ≤ 0.8 – 74
Key result
Conc. / dose:
0.1 mg/L
Temp.:
25 °C
pH:
7.9
Type:
BCF
Basis:
not specified
Remarks on result:
other: Peak A ≤ 8.0 – 74
Key result
Conc. / dose:
1 mg/L
Temp.:
25 °C
pH:
7.9
Type:
BCF
Basis:
not specified
Remarks on result:
other: Peak B ≤ 5.5 – 74
Key result
Conc. / dose:
0.1 mg/L
Temp.:
25 °C
pH:
7.9
Type:
BCF
Basis:
not specified
Remarks on result:
other: Peak B ≤ 54 – 130
Details on results:
TEST MATERIAL CONCENTRATIONS
- Each average concentration of the test material in test water was maintained at more than 95% of each the nominated concentration.

BIOCONCENTRATION FACTORS
Peak A: Level 1 ≤ 0.8 – 74 Level 2 ≤ 8.0 – 74
Peak B: Level 1 ≤ 5.5 – 74 Level 2 ≤ 54 – 130

OBSERVATIONS
- Test fish observations: No abnormality in behaviour or appearance was noted.

DISCUSSION
- Recovery of test material from test fish: The average recovery rate of component 1 (Peak A) from test fish was 61.5% (n=2). The information provide by the sponsor suggested that the component 1 is changed to converted product C and converted product D. An unknown peak was detected on the chromatogram of HPLC analysis in the recovery test for the test fish analysis. This unknown peak was analysed by liquid chromatography-mass spectrometry (LC-MS) and the result suggests that it is converted product C. Therefore, it is considered that a part of the component 1 (Peak A) was changed to converted product C by the contact with the fish homogenate and this change made the recovery rate low.
- Bioconcentration factors of hydrolysed reaction product: It was suggested that the component 1 of the test material was changed to converted product C and converted product D. Therefore, the test fish at 8 weeks were also analysed and the chromatograms obtained were compared with that of the authentic sample of the converted products. As a result, the peak that had the same retention time as converted product D in the authentic sample was noticed. The mass spectrum by LC-MS of this peak was the same as that of the converted product D. When bioconcentration factors for Peak D were determined by a comparison of the peak area of component 1 (Peak A) on the chromatogram of the standard solution, they were 1.0 and 0.8 at Level 1 and 5.7 and 5.8 at Level 2.

Table 1: Measured concentrations of test material in test water (average value at times from start of exposure)

Peak

Level

Measured test material concentration (mg/L)

2 weeks

4 weeks

6 weeks

8 weeks

A

1

1.00

1.03

1.02

1.01

2

0.0976

0.0997

0.0992

0.101

B

1

0.984

0.965

0.980

0.990

2

0.0960

0.0999

0.100

0.100

Table 2: BCFs

Peak

Level

BCFs

2 weeks

4 weeks

6 weeks

8 weeks

A

1

≤ 0.8

≤ 0.8

7.0

74

≤ 0.8

≤ 0.8

≤ 0.8

≤ 0.8

2

≤ 8.0

≤ 8.0

31

74

8.0

8.0

8.0

8.0

B

1

5.5

5.5

16

74

5.5

5.5

5.5

5.5

2

54

54

54

130

54

54

54

54

 

Validity criteria fulfilled:
not specified
Conclusions:
Under the conditions of this study, the bioconcentration factors of the test material in peak A were: level 1: ≤ 0.8 – 74 and level 2: ≤ 8.0 – 74 and in peak B were: level 1: ≤ 5.5 – 74 and level 2: ≤ 54 – 130.
Executive summary:

The bioaccumulation potential of the test material in fish was investigated in accordance with Japanese guidelines and in a study similar to that of OECD 305C. The test was performed under GLP conditions.

During the study Cyprinus carpio were exposed to the test material for 8 weeks at concentrations of 1 and 0.1 mg/L (nominal) in a continuous flow through system. The test material was confirmed to be stable under storage and test conditions.

The average recovery rate of component 1 (Peak A) from test fish was 61.5% (n=2). The information provided by the sponsor suggested that the component 1 is changed to converted product C and converted product D. An unknown peak was detected on the chromatogram of HPLC analysis in the recovery test for the test fish analysis. This unknown peak was analysed by liquid chromatography-mass spectrometry (LC-MS) and the result suggests that it is converted product C. Therefore, it is considered that a part of the component 1 (Peak A) was changed to converted product C by the contact with the fish homogenate and this change made the recovery rate low. Since it was suggested that the component 1 of the test material was changed to converted product C and converted product D, the test fish at 8 weeks were also analysed and the chromatograms obtained were compared with that of the authentic sample of the converted products. As a result, the peak that had the same retention time as converted product D in the authentic sample was noticed. The mass spectrum by LC-MS of this peak was the same as that of the converted product D. When bioconcentration factors for Peak D were determined by a comparison of the peak area of component 1 (Peak A) on the chromatogram of the standard solution, they were 1.0 and 0.8 at Level 1 and 5.7 and 5.8 at Level 2.

Under the conditions of this study, the bioconcentration factors of the test material in peak A were: level 1: ≤ 0.8 – 74 and level 2: ≤ 8.0 – 74 and in peak B were: level 1: ≤ 5.5 – 74 and level 2: ≤ 54 – 130.

Description of key information

Under the conditions of the study, the bioconcentration factors of the test material in peak A were: level 1: ≤ 0.8 – 74 and level 2: ≤ 8.0 – 74 and in peak B were: level 1: ≤ 5.5 – 74 and level 2: ≤ 54 – 130.

Key value for chemical safety assessment

Additional information

The bioaccumulation potential of the test material in fish was investigated in accordance with Japanese guidelines and in a study similar to that of OECD 305C. The test was performed under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

During the study Cyprinus carpio were exposed to the test material for 8 weeks at concentrations of 1 and 0.1 mg/L (nominal) in a continuous flow through system. The test material was confirmed to be stable under storage and test conditions.

The average recovery rate of component 1 (Peak A) from test fish was 61.5% (n=2). The information provided by the sponsor suggested that the component 1 is changed to converted product C and converted product D. An unknown peak was detected on the chromatogram of HPLC analysis in the recovery test for the test fish analysis. This unknown peak was analysed by liquid chromatography-mass spectrometry (LC-MS) and the result suggests that it is converted product C. Therefore, it is considered that a part of the component 1 (Peak A) was changed to converted product C by the contact with the fish homogenate and this change made the recovery rate low. Since it was suggested that the component 1 of the test material was changed to converted product C and converted product D, the test fish at 8 weeks were also analysed and the chromatograms obtained were compared with that of the authentic sample of the converted products. As a result, the peak that had the same retention time as converted product D in the authentic sample was noticed. The mass spectrum by LC-MS of this peak was the same as that of the converted product D. When bioconcentration factors for Peak D were determined by a comparison of the peak area of component 1 (Peak A) on the chromatogram of the standard solution, they were 1.0 and 0.8 at Level 1 and 5.7 and 5.8 at Level 2.

Under the conditions of the study, the bioconcentration factors of the test material in peak A were: level 1: ≤ 0.8 – 74 and level 2: ≤ 8.0 – 74 and in peak B were: level 1: ≤ 5.5 – 74 and level 2: ≤ 54 – 130.