2,2'-[(3,3'-dichloro[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[N-(4-chloro-2,5-dimethoxyphenyl)-3-oxobutyramide]

Bioaccumulation: aquatic / sediment

Administrative data

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1999-12-17 - 2000-03-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 305 (Bioconcentration: Flow-through Fish Test)

Deviations:

yes

Remarks:

Only one test substance concentration (at least two concentrations recommended by OECD 305)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: "Method for Testing the Degree of Accumulation of Chemical Substances in Fish Body" stipulated in the "Testing Methods for New Chemical Substances" (July 13, 1974, Revised October 8, 1998, Kanpogyo No.5, Environment Agency, Japan)

GLP compliance:

yes

Remarks:

"OECD Principles of Good Laboratory Practice" (May 12, 1981)

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-[(3,3'-dichloro[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[N-(2,4-dimethylphenyl)-3-oxobutyramide]

EC Number:

225-822-9

EC Name:

2,2'-[(3,3'-dichloro[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[N-(2,4-dimethylphenyl)-3-oxobutyramide]

Cas Number:

5102-83-0

Molecular formula:

C36H34Cl2N6O4

IUPAC Name:

2,2'-[(3,3'-dichlorobiphenyl-4,4'-diyl)didiazene-2,1-diyl]bis[N-(2,4-dimethylphenyl)-3-oxobutanamide]

Test material form:

not specified

Radiolabelling:

no

Sampling and analysis

Details on sampling:

(1) Test water analysis
The test water was analyzed once before first analysis of test fish and at the same time of analysis of test fish thereafter. The number of each sample was one.
(2) Test fish analysis
The test fish was analyzed five times in duration of exposure (28d). Four fish were taken out at each sampling time and divided into two groups, then both were analyzed individually.
The control fish were analyzed before the initiation and at the termination of exposure. Four fish were taken out at each sampling time and divided into two groups, and then each was analyzed individually.
(3) Pretreatment for analysis
A) Test water
25 ml, of the test water was taken out from test tank, and pretreated for high-performance liquid chromatography (HPLC) analysis as follows :
- Addition of 8 g sodium chloride
- Addition of Chloroform/m-cresol (99/1 V/V) 20 mL
- Shaking for 5 min
- Phase separation, upper layer discarded, lower layer further processed:
- Filtration and dehydration (IPS filter paper)
- Filling up to 25 ml chloroform/m-cresol (99/1 V/V)
- Sample for HPLC analysis;
B) Test fish
- Measurement of weight and body length
- Chopping into pieces
- Refinement (polytron, 2 min. or more, an ice water)
==> Homogenate
- 1-5 g as sample for storage
- 5g of homogenate for analytical determination:
- Addition of 40 g anhydrous sodium sulfate: at room temperature till dryness
- Addition of chloroform/m-cresol (95/5 V/V) 80 mL
- Shaking for 5 min
- Vacuum filtration (Glass fiber filter, GA-200)
- Residue dicarded, proceded with filtrate:
- Filling up to 100 mL (chloroform / m-cresol (95/5 V/V)
- Taking out 2 mL for Column chromatography
==> Eluate
- Filling up to 10 mL (chloroform)
- Sample for HPLC analysis

Test solutions

Vehicle:

yes

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

(1) Preparation of stock solutions
Dispersant:
HCO-40 (Hydrogenated castor oil)
Sugar candy
Preparation:
Test substance and sugar candy (20 times amount of the test substance) were ground up. Then HCO-40 (20 times amount of the test substance) was added and the mixture was ground up again. An ion-exchanged water is added to the mixture to prepare 80 mg/L stock solution.
Control group: Sugar candy and HCO-40 were dissolved with ion-exchanged water to prepare 1600 mg/L stock solution, respectively.

(2) Test concentrations
Based on preliminary test results for 96-hour LC50 and analytical detection limits, test concentration of the test substance was set 0.1 mg/L. The control was set as a blank test.

Test organisms

Test organisms (species):

Cyprinus carpio

Details on test organisms:

Supplier
Sugishima fish farm (Address : 123-2 Gunchiku Ichibancho, Yatsushiro-shi, Kumamoto 866-0024, Japan)
Date received: November 11, 1999

Conditions for fish care before acclimatization
The fish were checked visually in the receiving and those demonstrating any abnormalities were removed. The fish were reared for 8 days in a flow through system following an external disinfection.

Conditions for acclimatization
After rearing, the fish were medicated to eliminate parasites and transferred to an acclimatizing aquarium. After the second external disinfection, they were acclimatized. The fish demonstrating any abnormalities during this period were removed and the remainder of the fish were reared for 27 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 16 days, following the external disinfection.

Length and Lot No at initial exposure
Length: average 8 ± 4 cm
Lot No.: TFC-991111

Age: Two yearings fish

Feeding
Feed: Feed for fry of carp
Composition: Proteins content > 43.0%
Lipid content: > 3.0%
Manufacturer: Nippon Formula Feed Mfg. Co., Ltd.
Feeding amount and interval:
Amount corresponding to about 2% total body weight was provided twice a day in halves.
The fish were starved for 24 hours before sampling.

Study design

Route of exposure:

aqueous

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

28 d

Total depuration duration:

0 d

Test conditions

Hardness:

Total hardness (Ca, Mg): 132 mg/L

Test temperature:

Test group: 24.8 - 25.8 °C
Control gorup: 25.3 - 25.8 °C

pH:

7.6

Dissolved oxygen:

Ca. 8 mg/L, continuously monitored during the study.

TOC:

1.3 mg/L

Details on test conditions:

Conditions of test and circumstances
(1) Supply of test water: Flow through system assembled at this laboratory was used.
(2) Test tank: 100-L glass tank
(3) Flow rate of test water: 2 mL/min for stock solution and 1600 mL/min for dilution water ; 2307 L/day for test water were supplied.
(4) Stock solution bottle: 25-L glass bottle (Frequency of renewal: 1- 2 times/week)
(5) Temperature of test water:
Test group 24.8 - 25.8°C
Control group 25.3 - 25.8°C
(6) Concentrations of dissolved oxygen in test water:
Test group: 7.9 - 8.0 mg/L
Control group 7.9 - 8.0 mg/L
(7) pH of test water
Test group: 7.5 - 7.6
Control group: 7.5 - 7.6
(8) Time of liradiation with light: Artificial light of white fluorescent lamp (14 hrs/day)
(9) Number of fish (at the beginning of exposure):
Test group: 24
Control group: 8
(10) Duration of exposure: 28 days.

Observation, measurement and cleaning of test tank
(1) Observation of test fish: Condition of test fish was observed visually twice a day.
(2) Flow rate of test water: Flow rate of stock solution and dilution water were measured with graduated cylinder and recorded once a day.
(3) Temperature of test water: Temperature of test water was measured with alcohol thermometer and recorded once a day.
(4) Concentration of dissolved oxygen in test water: Concentration of dissolved oxygen in test water was measured with dissolved oxygen probe and recorded twice a week.
(5) pH of test water: pH of test water was measured with pH meter once and more a week.
(6) Cleaning of test tank: In test period, excreta of carp, dirt on test tank, etc. were removed once about a day.

Nominal and measured concentrations:

Nominal: 0,1 mg/L
Mean measured: 0.0935 mg/L

Reference substance (positive control):

no

Details on estimation of bioconcentration:

As the measured concentrations of the test substance in test fish were less than the minimum determination limit of the test substance (0.58 µg/g) for all five measurements during the 28 day period (day 5, 8, 14, 21, 28), only the upper limit for possible bioconcentration as determined by the ratio of the mean measured concentration in water at steady state (0.0936 mg/L) and the limit of determination in fish (0,58 mg/kg) could be calculated: <= 6.2 ((0,58 mg/kg) / (0.0936 mg/kg)).
Therefore, a depuration period was not meaningful as well.

Results and discussion

Lipid content

Lipid content 1

Lipid content:

1.69 %

Time point:

start of exposure

Lipid content 2

Lipid content:

1.96 %

Time point:

end of exposure

Bioaccumulation factor

Key result

Type:

BCF

Value:

>= 0 - <= 6.2 dimensionless

Basis:

whole body w.w.

Calculation basis:

steady state

Remarks:

As no time of plateau could be determined, days of analysis 14, 21 and 28 were taken for the calculation of the steady state concentration in the test medium and the relation to the limit of determination in fish gave the upper limit of BCF of 6.2.

Remarks on result:

other: The concentration in none of the 5 treatment samples analyzed during the 28 d period was above the limit of determination. As such, no time of plateau could be determined.

Remarks:

Conc.in environment / dose:0.0935 mg/L

Depuration

Key result

Elimination:

not specified

Parameter:

other: The concentration in none of the 5 treatment samples analyzed during the 28 d period was above the limit of determination. As such, no analysis of depuration was possible.

Details on kinetic parameters:

As concentrations in all of the analyzed fish samples during the 28 day incubation period were below the limit of determination, no kinetic parameters could be determined.

Metabolites:

No data.

Details on results:

Results of test fish observation:
No abnormality in behavior or appearance was noted.
For further details on results see "Any other information on results incl. tables!

Any other information on results incl. tables

Concentration of test substance in test water:

The measured concentrations of the test substance in test water are shown in Table-1. Each concentration of the test substance was maintained at more than 91% of each nominated concentration. The variation of the concentrations of the test substance was within ± 20% of each other.

Table-1 Measured concentrations of the test substance in test water [mg/L]

After 1 days	After 5 days	After 8 days	After 14 days	After 21 days	After 28 days	Average (Standard deviation)
0.0949	0.0930	0.0926	0.0944	0.0952	0.0911	0.0935 (0.00161)

Bioconcentration factors

BCFs are shown in Table-2. BCFs of the test substance were < 6.2.

Table-2 BCFs

After 5 days	After 8 days	After 14 days	After 21 days	After 28 days
<6.2 <6.2	<6.2 <6.2	<6.2 <6.2	<6.2 <6.2	<6.2 <6.2

Calculation of BCFs in a steady-state (BCFss)

Because all of BCFs were less than 100, it was evaluated that a steady-state was reached at 28th day. BCFss was calculated an the basis of the results of analysis alter 14, 21 and 28 days from the initiation of exposure.

(1) Concentrations oftest substance in test water in a steady-state

The average concentrations of the test substance in test water in a steady-state are shown in Table-3. The average concentration was 93.6% of the nominated concentration.

Table-3 Concentrations of the test substance in test water in a steady-state (Unit : mg/L)

After 14 days	After 21 days	After 28 days	Average
0.0944	0.0952	0.0911	0.0936

(2) BCFs in a steady-state (BCFss) BCFss was calculated as <6.2.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In a test for bioconcentration according to OECD 305 and compliant to GLP (reliability category 1) no potential for bioconcentration of the submission substance in the fish Cyprinus carpio could be shown.
BCFss <= 6.2

Executive summary:

In a test for bioconcentration according to OECD 305 and compliant to GLP (reliability category 1) no potential for bioconcentration of the submission substance in the fish Cyprinus carpio could be shown. As all measured fish samples within the 28 days exposure period had concentrations below the limit of determination, the maximal possilbe BCFss is determined by the ratio of the limit of determination and the mean measured concentrations in the test medium:

BCFss <= 6.2