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Administrative data

Endpoint:
bioaccumulation in aquatic species: fish
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
28 October 1997 to 06 February 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to OECD test guidance in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 305 C (Bioaccumulation: Test for the Degree of Bioconcentration in Fish)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Chemical Substances Control Law (Japanese law 117, 1974)
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Details on test material:
See below
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable.
Radiolabelling:
no

Sampling and analysis

Details on sampling:
Test water and fish were analysed on the following dates:
Day 0 Analysis of water
Day 7 Analysis of water and fish
Day 14 Analysis of water and fish
Day 21 Analysis of water and fish
Day 28 Analysis of water and fish
Day 35 Analysis of water
Day 42 Analysis of water and fish
Day 49 Analysis of water
Day 56 Analysis of water and fish
Analysis of control fish: Fish were collected and analysed at the beginning and the end of the exposure period (Days 0 and 56).

Test solutions

Vehicle:
yes
Details on preparation of test solutions, spiked fish food or sediment:
Dispersion method
Dispersing agent: FDPS (Naphthalene sulfonic acid - formaldehyde condensation product)
Procedure: The test substance (T-9601), 2.52463 g, and FDPS which is three times as much as the test substance were ground in a mortar with a pestle (An automatic apparatus was used (SOP/TNS/4.30)). Pure water was added drop wise into the mortar when the substances began to be coagulated. After the test substance was dispersed uniformly, the water dispersion was diluted with additional water to 2.5 L (Concentration of the test substance: 1000 ppm).
Temperature: 23.7±0.1°C
Test water: De-chlorinated water (prepared from the Tokyo metropolitan tap water)

Test organisms

Test organisms (species):
Cyprinus carpio
Details on test organisms:
Test Fish Carp (Cyprinus carpio L.)
Source; Saku-yoshoku Co., 3-5-14 Setagaya, Setagaya-ku, Tokyo, Japan
Date of purchase: September 17, 1997 (Lot No. 70917)
Acclimation: Carp of 20-40 g and about 10 cm were sorted out when received in Institute of Ecotoxicology, Gakushuin University (SOP 2.5), and they were acclimated in Aquarium No. 1 (SOP 2.5). On November 25, 1997, they were sorted out again and transferred to Aquarium No. 7. They were on medication (New Green F®, Shin Fuso Pharmaceutical Co., Ltd.) in water before the testing (SOP 2.3). Water temperature during the acclimation was gradually raised to 25°C by the start of the test (SOP 2.5).
Carp: Average body weight: 25.9 ± 4.5 g (SOP 2.8)
Average body length: 9.9 ± 0.6 cm (SOP 2.8)
Average fat content: 4.1 ± 0.5% (SOP 2.9 (2))
(measured by CHCl3-MeOH Extraction Method)

Study design

Route of exposure:
aqueous
Test type:
flow-through
Water / sediment media type:
natural water: freshwater
Total exposure / uptake duration:
8 wk

Test conditions

Hardness:
No data
Test temperature:
24.8 – 25.2°C
pH:
7.1 - 7.6
Dissolved oxygen:
7.0 - 7.6 ppm
TOC:
No data
Salinity:
Not applicable.
Details on test conditions:
Aquarium:
Aquarium for flow-through test. Made of glass, Test volume of 100l Flow rate: 300ml/min. (432L/day) Aquarium No, 8 was used for high exposure level, No. 9 for low exposure level, and No. 7 for control.
Water: De-chlorinated water (prepared from the Tokyo metropolitan tap water).
Number of fish: High and low exposure levels: 25 fish each; Control: 6 fish
Temperature 25.1±0.3°C
Concentrations: The test substance in water was set at concentrations as low as possible with consideration given to both the analytical sensitivity and the 43-hr LC5O value.
Concentration of the test substance Concentration of FDPS
High exposure level 0.1 ppm (w/v) 0.3 ppm (w/v)
Low exposure level 0.01 ppm (w/v) 0.03 ppm (w/v)
Control ---- 0.3 ppm (w/v)


Analysis of Test Fish
Pre-treatment
Three fish were collected with a dip net from high or low exposure level or control on their respective dates of analysis, and led to apparent death by hitting the back of the head, the surface of which was then wiped softly with a piece of gauze, and weighed in tenths of a gram with a top-loading balance. The body length, which is defined as a distance from the tip of mouth to the base of caudal fin, was measured in tenths of a centimetre. Two of three fish each were used for analysis, and the other was wrapped with a piece of aluminium foil, and then packed in a plastic bag and placed in a plastic case before stored in a freezer.
The individual test fish was cut into pieces and placed in a homogenizer cup, to which 150 ml of dichloromethane / acetone (2/1 (v/v)) was added, and homogenized at 10000 rpm for 10 minutes. The homogenate was transferred into a 250 ml centrifuge tube. The inside of the cup was washed with six 30 ml of dichloromethane / acetone (2/1 (v/v)) and the six washings were transferred into the centrifuge tube, which was then centrifuged at 8000 rpm for 15 minutes at 15 °C. The supernatant was filtered with Advantec No. 2 filter paper, of which the filtrate was collected in a 500 ml rounded-bottom flask. After that, the filtrate was evaporated to dryness under a reduced pressure with a rotary evaporator (water bath. 25°C). The residue was dissolved with divisions of 80 ml of n-hexane and 140 ml of acetonitrile successively, and its solutions were transferred into a 300 ml separatory funnel. The funnel was shaken for 15 minutes with a shaker and left stand for a while. The acetonitrile layer (lower layer) was transferred to a 500 ml rounded-bottom flask and evaporated to dryness under a reduced pressure with the rotary evaporator (water bath: 25°C).
This extract from fish was dissolved with 2 ml of n-hexane/ acetone (9/1) and the solution was injected into a Sep-Pack Silica® cartridge column (conditioned with 5 ml of n-hexane / acetone (9/1) in advance). The inside of the flask was washed with two 1 ml of the solvent mixture, and the two washings were also injected into the column (the test substance is adsorbed to the Sep-Pack Silica). Seven millilitres of n-hexane/ acetone (8/2) was gradually added to the column, and the eluate was pooled in a 50 ml rounded-bottom flask (the test substance is eluted in this fraction). After that, the eluate was evaporated to dryness under a reduced pressure with the rotary evaporator (water bath: 25°C).
The residue was dissolved with 2 ml of acetonitrile, and the solution was transferred into a 10 ml volumetric flask. Three millilitres of pure water was also added into the flask. The inside of the rounded-bottom flask was washed with several portions of acetonitrile, and each washings was transferred into the volumetric flask, which was then filled with acetonitrile (an acetonitrile solution containing 30% water). An aliquot of this final solution was filtered with LC disk 13 CR, and the filtrate was analysed by high performance liquid chromatography (HPLC).
Analysis of Test Water
Pre-treatment
About 500 ml (about 2000 ml) of test water of high exposure level was withdrawn from the middle of the aquarium with a conical flask. The supernatant water, 200 mL (800 ml) was measured with a graduated cylinder.
The supernatant measured was transferred into a 300 ml (1l) separatory funnel. The inside of the graduated cylinder was washed with a small amount of pure water and the washings was transferred into the funnel. Dichloromethane, 80 ml (120 ml), was added into the funnel, which was then shaken for 15 minutes with the shaker and left stand for a while. The lower layer was transferred into a 300 ml rounded-bottom flask, and concentrated under a reduced pressure with the rotary evaporator. This concentration was stopped immediately before dryness, and the residual solvent was removed by forced ventilation. The residue was dissolved in a small amount of acetonitrile, and the solution was transferred into a 10 ml (5 ml) volumetric flask, into which 3 ml (1.5 ml) of pure water was then added. The inside of the rounded-bottom flask was washed with several portions of acetonitrile, and each washings was transferred into the volumetric flask, which was then filled with acetonitriie (an acetonitrile solution containing 30% water). The final solution was filtered with LC disk 13 CR, and the filtrate was analyzed by HPLC.
Nominal and measured concentrations:
Concentration of the test substance Concentration of FDPS
High exposure level 0.1 ppm (w/v) 0.3 ppm (w/v)
Low exposure level 0.01 ppm (w/v) 0.03 ppm (w/v)
Control ---- 0.3 ppm (w/v)
Reference substance (positive control):
not required
Details on estimation of bioconcentration:
The 48-hr LC50 of the test substance to orange killifish was more than 186 ppm. Accordingly, the nominal concentrations to be used in the bioconcentration test were set at 0.1 ppm and 0.01 ppm. The exposure period of the test was set at 8 weeks. We obtained the bioconcentration factors (BCFs) ranging from 15 to 58 for the 0.1 ppm exposure level and from 62 to 124 for the 0.01 ppm exposure level.

Results and discussion

Bioaccumulation factoropen allclose all
Type:
BCF
Value:
> 15 - < 58 dimensionless
Basis:
whole body w.w.
Time of plateau:
56 d
Calculation basis:
steady state
Remarks on result:
other: Conc.in environment / dose:0.1 ppm (w/v)
Type:
BCF
Value:
> 62 - < 124 dimensionless
Basis:
whole body w.w.
Time of plateau:
56 d
Calculation basis:
steady state
Remarks on result:
other: Conc.in environment / dose:0.01 ppm (w/v)
Details on kinetic parameters:
Not calculated.
Metabolites:
Not assessed.
Results with reference substance (positive control):
Not applicable.
Details on results:
Further information in table form submitted under any other information
Reported statistics:
No data

Any other information on results incl. tables

Bioconcentration factors

Day

High exposure level

Low exposure level

A

BCFs

A

BCFs

B

B

7

A

25

A

114

B

15

B

82

14

A

43

A

77

B

29

B

88

21

A

42

A

62

B

37

B

105

28

A

31

A

11

B

33

B

104

42

A

40

A

98

B

44

B

96

56

A

51

A

121

B

58

B

124

(Note 1) Fish were given the letter A or B for identification when they were collected for analysis.

 

Analysis of Fish (High exposure level)

Sample fish

A

Final volume

 

 

 

(ml)

B

Peak area

C Concentration in final solution *

 

(ppm)

D

Mass detected in fish

 

 

(μg)

E

Fish weight

 

 

 

(g)

F Recovery

 

 

 

(%)

G

Concentration in fish

 

 

(ppm)

H Concentration in water (cumulative average) (ppm)

I

Bio-concentration factor

7

A

10

482191

4.7668

47.668

21.8

89.8

2.43

0.0965

25

7

B

10

282028

2.7921

27.921

21.0

89.8

1.48

0.0965

15

14

A

10#

384425

3.8375

76.750

20.0

89.8

4.27

0.0983

43

14

B

10#

339163

3.3871

67.742

26.4

89.8

2.86

0.0983

29

21

A

10#

530066

5.1702

103.404

27.4

89.8

4.20

0.0992

42

21

B

10#

404617

3.9523

79.046

24.1

89.8

3.65

0.0992

37

28

A

10#

364435

3.6158

72.316

26.1

89.8

3.09

0.0998

31

28

B

10#

385580

3.8250

76.500

25.7

89.8

3.31

0.0998

33

42

A

10#

501163

4.9150

98.300

27.4

89.8

4.00

0.101

40

42

B

10#

491192

4.8172

96.344

23.9

89.8

4.49

0.101

44

56

A

10#

725263

7.1585

143.170

31.2

89.8

5.11

0.101

51

56

B

10#

746819

7.3711

147.422

28.2

89.8

5.82

0.101

58

Calculation equations: Mass detected in fish: D=CXA (X2 (Dilution factor))#

                                  Concentration in fish: G=(DX100)/(EXF)

                                   Bioconcentration factor: I=G/H

*Based on two-point calibration

#On day 14 and after, we diluted the final solutions twofold to determine the test substance concentration within the assay range.

 

Analysis of Fish (Low exposure level)

Sample fish

A

Final volume

 

 

 

(ml)

B

Peak area

C Concentration in final solution*

 

(ppm)

D

Mass detection in fish

 

(μg)

E

Fish weight

 

 

 

(g)

F Recovery

 

 

 

(%)

G Concentration in fish

 

 

(ppm)

H Concentration in water (cumulative average) (ppm)

I

Bio-concentration factor

7

A

10

238201

2.3597

23.597

24.2

89.8

1.09

0.00952

114

7

B

10

146567

1.4557

14.557

20.7

89.8

0.783

0.00952

82

14

A

10

160313

1.6075

16.075

24.1

89.8

0.743

0.00959

77

14

B

10

161857

1.6228

16.228

21.3

89.8

0.848

0.00959

88

21

A

10

117668

1.1665

11.665

21.6

89.8

0.601

0.00966

62

21

B

10

234780

2.3035

23.035

25.4

89.8

1.01

0.00966

105

28

A

10

241177

2.3964

23.694

24.7

89.8

1.08

0.00974

111

28

B

10

204985

2.0383

20.383

22.5

89.8

1.01

0.00974

104

42

A

10

227145

2.2270

22.270

26.0

89.8

0.954

0.00971

98

42

B

10

224150

2.1976

21.976

26.2

89.8

0.934

0.00971

96

56

A

10

347790

3.4360

34.360

32.3

89.8

1.18

0.00976

121

56

B

10

330797

3.2685

32.685

30.0

89.8

1.21

0.00976

124

Calculation equations: Mass detected in fish: D=CXA

                                  Concentration in fish: G=(DX100) / (EXF)

                                  Bioconcentration factor: I=G/H

*Based on two-point calibration

 

Analysis of Fish (Control)

Sample fish

A

Final volume

 

 

 

(ml)

B

Peak area

C Concentration in final solution*

 

(ppm)

D

Mass detected in fish

 

 

(μg)

E

Fish weight

 

 

 

(g)

F Recovery

 

 

 

(%)

G Concentration in fish

 

 

(ppm)

H Concentration in water (cumulative average) (ppm)

I

Bio-concentration factor

0 – a

10

N.D.

---

---

21.6

---

---

0

---

0 – b

10

N.D.

---

---

24.9

---

---

0

---

56 – a

10

N.D.

---

---

38.1

---

---

0

---

56 – b

10

N.D.

---

---

37.7

---

---

0

---

(Note) Fish were given the letter “a” or “b” for identification when they were collected from the aquarium.

*Based on two-point calibration

 

Analysis of Water (High exposure level)

Exposure period (day)

A

Final volume

 

 

 

(ml)

B

Peak area

C

Concentration in final solution*

 

(ppm)

D

Absolute amount in final solution

 

(μg)

E

Volume of sample water

 

 

(g)

F

Recovery

 

 

 

(%)

G

Concentration in water (measured value)

(ppm)

H

Concentration in water (cumulative average) (ppm)

0

10

177926

1.77673

17.673

200

96.2

0.0919

---

7

10

195487

1.9432

19.432

200

26.2

0.101

0.0965

14

10

198990

1.9682

19.682

200

96.2

0.102

0.0983

21

10

199986

1.9657

19.657

200

96.2

0.102

0.0992

28

10

198010

1.9693

19.693

200

96.2

0.102

0.0998

35

10

195134

1.9409

19.409

200

96.2

0.101

0.100

42

10

203528

1.9953

19.953

200

96.2

0.104

0.101

49

10

204616

2.0041

20.041

200

96.2

0.104

0.101

56

10

201072

1.9635

19.635

200

96.2

0.102

0.101

Calculation equation: Absolute amount in final solution: D=CXA

                                Concentration in water: G=(DX100) /(EXF)

*Based on two-point calibration

 

Analysis of Water (Low exposure level)

Exposure period (day)

A

Final volume

 

 

 

(ml)

B

Peak area

C Concentration in final solution*

 

(ppm)

D

Absolute amount in final solution

 

(μg)

E

Volume of sample water

 

 

(g)

F Recovery

 

 

 

(%)

G Concentration in water (measured value)

(ppm)

H Concentration in water (cumulative average) (ppm)

0

5

141866

1.4102

7.0510

800

97.4

0.00905

---

7

5

156212

1.5552

7.7760

800

97.4

0.00998

0.00952

14

5

153464

1.5195

7.5975

800

97.4

0.00975

0.00959

21

5

155382

1.5327

7.6635

800

97.4

0.00984

0.00966

28

5

158286

1.5763

7.8815

800

87.4

0.0101

0.00974

35

5

153818

1.5321

7.6605

800

97.4

0.00983

0.00976

42

5

150251

1.4727

7.3635

800

97.4

0.00945

0.00971

49

5

159355

1.5601

7.8005

800

97.4

0.0100

0.00975

56

5

156923

1.5305

7.6525

800

97.4

0.00982

0.00976

Calculation equations: Absolute amount in final solution: D=CXA

                                  Concentration in water: G=(DX100) / (EXF)

*Based on two-point calibration

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
From the BCFs obtained, it can be concluded that the test substance, T-9601, is not bioaccumulative in carp.
Executive summary:

A bioconcentration study of the test substance, T-9601, with carp (Cyprinus carpio) was performed according to the standard method described in the governmental order, which prescribes the procedure of testing new chemical substances as required by the Chemical Substances Control Law (Japanese law 117, 1974). This method is also known as the "Bioaccumulation, Test for the Degree of Bioconcentration in Fish" in the OECD Guidelines for the Testing of Chemicals No.305C(1981). Study conducted in compliance with GLP.

The 48-hr LC50 of the test substance to orange killifish was more than 186 ppm. Accordingly, the nominal concentrations to be used in the bio-concentration test were set at 0.1 ppm and 0.01 ppm. The exposure period of the test was set at 8 weeks. We obtained the bio-concentration factors (BCFs) ranging from 15 to 58 for the 0.1 ppm exposure level and from 62 to 124 for the 0.01 ppm exposure level.

 

From the BCFs obtained, it can be concluded that the test substance, T-9601, is not bioaccumulative in carp.

 

On the basis of this data, the substance cannot be considered as bioaccumulative under environmentally relevant conditions. The substance is not considered to pose a chronic hazard to the aquatic environment on the basis of this data.

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