1,6-Bis(methoxybenzoyloxy)hexane

Bioaccumulation: aquatic / sediment

Link to relevant study record(s)

Reference

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 January 2010 - 31 March 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study includes data generated according to generally valid and internationally accepted testing guidelines and performed according to GLP. The study was based on the OECD Test Guideline 305.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Radiolabelling:

no

Details on sampling:

- Sampling intervals/frequency for test organisms:
For both Level 1 concentration and Level 2 concentration: 4 fish each on 4, 7, 14, 21 and 28 days afte
r the start of exposure and analyzed on 2 test fish in a lump.
- Sampling intervals/frequency for test medium samples:
Level 1 concentration: twice a week with one replicate
Level 1 concentration: twice a week with one replicate

Vehicle:

yes

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

- Preparation of the solution of the test substance for stock solution:
(1) For level 1 concentration:
The test substance (1.00 g: calculated from the purity) dissolved in acetone to make exactly 500 ml, as the 2000 mg/L acetone solution of the test substance wasprepared.

(2) For level 2 concentration:
Collect 50 ml of this 2000 mg/L solution, added acetone to make exactly 500 mL, as the 200 mg/L ac etone solution of the test substance was prepared.

These solutions of the test substance for stock solution were prepared at the time of preparation of the first and fifth stock solution.

- Preparation of stock solution:
(1) Level 1 concentration:
Collect 60 ml of the test substance for stock solution ( for level 1 concentration) , added 60 g of dispersant agent (HCO-40) and then diluted by water to prepare the stock solution (10L) of 12mg/L as the test substance concentration.

(2) Level 2 concentration:
Collect 60 ml of the test substance for stock solution ( for level 2 concentration) , added 60 g of dispersant agent (HCO-40) and then diluted by water to prepare the stock solution (10L) of l.2mg/L as the test substance concentration.

(3) Control group:
60ml of acetone, added 60 g of dispersant agent (HCO-40) was dissolved into water to prepare control water (10L) without containing the test substance.

The above solutions were supplied to the respective test chambers from 10L volume glass container. The stock solution were prepared once a week and changed.

- Test concentrations:

Based on 96 hours LC50 value and analytical sensitivity, following test concentrations to be used in bioaccumulation study were decided.

Level 1 concentration: 0.0080 mg/L
Level 2 concentration: 0.00080mg/L

Concurrently, the control group was set as a blank test.

Test organisms (species):

Cyprinus carpio

Details on test organisms:

TEST ORGANISM
- Common name: Carp
- Strain: Cyprinus carpio
- Source: Kitamura Y ourijoh, 388 Guntiku·12bancho, Yatsushiro, Kumamoto, Japan
- Age at study initiation (mean and range, SD): New born fish
- Length at study initiation (lenght definition, mean, range and SD): (6.9 - 7.6cm, before the exposure starts), Average 7 .2 cm
- Weight at study initiation (mean and range, SD): (4.2 - 5.4g, before the exposure starts), Average 4.8 g
- Weight at termination (mean and range, SD): (6.1 - 8.2g, before the exposure starts), Average 7.0 g
- Method of breeding: The fish were reared in a static water chamber (with circulating water cleaning
filtering system) from November 17,2009 to January 19,2010 following an external disinfection.
- Health status: Neither death nor abnormality such as disease was observed in Level 1 concentration, Level 2 concentration and control group.
- Description of housing/holding area: Static water chamber (with circulating water cleaning filtering system)
- Feeding during test: Yes. Feeding was stopped within 10 hours before sampling of the test fish.
- Food type:
The formula feed for carp [manufactured by Nihon Nosan Kogyo Co., Ltd.: crude protein of above 39. 0%, crude fat of above 3.0%] spike with animal fat and oil for cultivated aquatic animals [Riken field oil Q , manufactured by Riken vitamin Co., Ltd ] at 5% (w/w)

- Amount: Amount corresponding to about 1 - 2 % of body weight of test fish twice a day

- Frequency: Fed daily, twice a day

ACCLIMATION
- Acclimation period: From January 19,2010 to February 5,2010. (more than 16 days)
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: The formula feed for carp [manufactured by Nihon Nosan Kogyo Co., Ltd.: crude protein of above 39.0%, crude fat of above 3.0%] spike with animal fat and oil for cultivated aquatic animals [Riken field oil Q , manufactured by Riken vitamin Co., Ltd ] at 5% (w/w)

- Feeding frequency: Fed daily, twice a day

- Health during acclimation (any mortality observed): No mortality observed. The fish demonstrating any abnormality by visual observation were removed

Route of exposure:

aqueous

Test type:

flow-through

Water / sediment media type:

not specified

Total exposure / uptake duration:

28 d

Hardness:

78 mg/L

Test temperature:

25 ± 2 °C

pH:

7.87

Dissolved oxygen:

8. 0 mg/L

TOC:

1. 0 mg/L

Salinity:

Not specified

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 L Glass Water Tank were used (three pieces)
- Type of flow-through : A flow through bioaccumulation test system (the fourth affiliation) assembled by this laboratory was used.
- Volume of test water: 900 ml/min (flow volume of stock solution: 36 ml/ hour)
- Temperature of test water: 25 ± 2°C
- Duration of exposure: 28 days
- Irradiation period : 16 hours /day (commercial fluorescent light)
- No. of organisms per Test vessel: 64 fish each (Level 1 and 2 concentrations) at the exposure start
- No. of organisms per Control vessel: 25 fish at the exposure start

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Tap water was filtered through PF Filter to eliminate the suspending materials, and then it let rung through activated charcoal filter for dechlorination and filtered through again with PF Filter.
- Total dissolved solid: 149 mg/L
- Dissolved oxygen concentration : More than 60% of the saturating concentration (4.9mg/L at 25°C)

- Metals:
Fe: < 0.03 mg/L
Ni: < 0.001 mg/L
As: < 0.001 mg/L
Hg < 0.00005mg/L
Cd: < 0.001 mg/L
Pb : < 0.001 mg/L
Cr: 0.005 mg/L
- Total nitrogen: 0. 53 mg/L
- Chlorine: < 0. 01 mg/L

- Holding medium different from test medium:
Same

- Intervals of water quality measurement:
More than once a year

- Intervals of test medium replacement:
The stock solutions were prepared once a week and changed.

- Fat (Lipid) analysis:
The test fish (5 each) were collected from the acclimatization chamber 2 days before the start of exposure and from the control chamber 3 days after the end of exposure to determine the lipid content.

PRELIMINARY STUDY: ACUTE TOXICITY TEST
-Test fish
Species: Orange-red killifish (Oryzia latipes)

- Test conditions:
Test chamber: 16 Liter Round Shape Water Tank made by Stainless Steel
Volume of test water: 10 Liter per a concentration level
Number of fish: 10 fish per a concentration level
Temperature of test water: 25±2°C
Irradiation time: 16 hours/day (Commercial fluorescent light)
Dissolved oxygen concentration and pH: These values are measured at the initiation and termination of exposure, and at the immediately before and after the change of test water.

Duration of exposure : 96 hours
Exposure method: Semi static system (change the test water for every 24 hours)

- Test concentrations: 10mg/L and control

- Results used to determine the conditions for the Bioaccummulation study:

Concentration of
Test Substance Mortality Number of fish/amount of test water
24h 48h 72h 96h
10 mg/L 0 0 0 0 10/10L
Control 0 0 0 0 10/10L


96 hour LC50 value of test substance was more than 10 mg/L.

Based on 96 hours LC50 value and analytical sensitivity, following test concentrations to be used in bioaccumulation study were decided:
Level 1 concentration: 0.0080 mg/L
Level 2 concentration: 0.00080mg/L

Concurrently, the control group was set as a blank test.

Nominal and measured concentrations:

Level 1 concentration: 0.0080 mg/L
Level 2 concentration: 0.00080mg/L

Reference substance (positive control):

no

Details on estimation of bioconcentration:

BASIS FOR CALCULATION OF BCF
Bioconcentration factors (BCF) of test substance in test fish were calculated using the equations below . Based on the calculated detection limit concentration of test substance in fish body, if exceeded, following magnifications, BCFs could be calculated.

Equation for the concentration in fish body and recovery rate:

Concentration in fish body (J) = G'/F (μg/g)

Average concentration in fish body in a steady state ( J') =(J1,··· ,J6)/6 (μg/g)
Recovery rate (R) = (GR'/M) x 100 (%)
(GR') = GR - BL (μg)
(GR) = (B x Wt x E x U x S) / (C x T) (μg)
Mean recovery rate (R') = [R(1)+R(2)]/2 (%)

Absolute volume in fish body sampled after deducting blank
(G') = G -GB' (μg)
(GB') = (GB1'+· · ·+GB4')/4
(GB) = (Bx Wt x E x Ux S) / [C x (R'/100) x T] (μg)

Bioaccumulation factor (L) = J/K
Bioaccumulation factor in a steady state (L') = J'/K'

Lipid content:

3.9 %

Time point:

other: 2 days before the start of exposure

Lipid content:

3.2 %

Time point:

other: 3 days after the end of exposure

Type:

BCF

Value:

> 6.9 - < 8.2 other: fold

Basis:

whole body w.w.

Time of plateau:

28 d

Calculation basis:

steady state

Remarks on result:

other: Based on the detection limit concentration of test substance in fish body Conc.in environment / dose: Level 1 concentration 0.0080 mg/L

Type:

BCF

Value:

> 67 - < 73 other: fold

Basis:

whole body w.w.

Time of plateau:

28 d

Calculation basis:

steady state

Remarks on result:

other: Based on the detection limit concentration of test substance in fish body Conc.in environment / dose: Level 2 concentration 0.00080 mg/L

Details on results:

- External change and feeding conditions oftest fish:
Change of the appearance of test fish, the feeding status and so on were visually observed in every working day. As a result, there was no abnormality.

- pH in the diluted water tank and test water tank
The pH in the diluted water tank and the test water tank were measured twice a week using a pH meter.
The diluted water tank 7.6 - 7.7
Level 1 water tank 7.5 - 7.7
Level 2 water tank 7.5 - 7.7
Control water tank 7.6 - 7.7

- Test substance concentration in test water:
Level 1concentration:0.00610 - 0.00653 mg/L (Average: 0.00637mg/L)
Level 2 concentration: 0.000587 - 0.000677 mg/L (Average: 0.000650mg/L)

- Abnormality of test fish
Neither death nor abnormality such as disease was observed in Level 1 concentration, Level 2 concentration and control group.

- Bioconcentration factors
Because the bioconcentration factors in the determination on 14, 21 and 28 days after the start of exposure were also less than 100 fold in the Level 1 Level 1 concentration and the Level2 concentration, bioconcentration factor in the steady state (BCFss) could not be confirmed.

The bioconcentration factors (BCF) of the test substance in the last three determinations (6points/ test group) was less than 6.9 fold to 8.2 fold for Level 1 concentration and less than 67 fold to less than 73 fold for Level 2 concentration.

- Lipid Content:
As a result, the lipid contents 2 days before the start of exposure and 3 days after the end of exposure were 3.9 % (w/w, mean) and 3.2% (w/w, mean), respectively.

Validity criteria fulfilled:

yes

Conclusions:

The bioconcentration factors (BCF) of the test substance in the last three determinations (6points/ test group) was less than 6.9 fold to 8.2 fold for Level 1 concentration and less than 67 fold to less than 73 fold for Level 2 concentration.

As conclusion, the degree ofbioconcentration of the test substance in carp was low.

Executive summary:

The purpose of the study was to examine the degree of bioaccumulation to the fish of Setafix Z1077 based on the OECD Guideline 305 (Bioconcentration: Flow-through Fish Test).

PRELIMINARY STUDY: Acute toxicity test

Test fish: Orange·red killifish (Oryzias laptipes)

Duration of exposure: 96 hours

Exposure method: Semi static system (Renewal of test water at every 24 hours)

96 hour LC50 value of test substance was more than 10 mg/L.

Based on the 96 hours LC50 value and analytical sensitivity, test concentrations to be used in

bioaccumulation test were decided.

MAIN STUDY: Bioaccumulation test

Test fish: Carp ( Cyprin us carpio)

Test concentrations:

Level 1 concentration 0.0080 mg/L

Level 2 concentration 0.00080 mg/L

Duration of exposure: 28 days

Exposure method: Continuous flow system

Analytical method: High performance liquid chromatography(HPLC)

RESULTS

Bioconcentration factors (Steady State): For less than 100 fold could not be confirmed for both Level 1 concentration and Level 2 concentration

Bioconcentration factors: [The bioconcentration factors (BCF) of the test substance in the last three determinations (6points/ test group)]

Level 1 concentration: less than 6.9 fold - 8.2 fold

Level 2 concentration: less than 67 fold - less than 73 fold

Conclusion

As conclusion, the degree of bioconcentration of the test substance in carp was low.

Description of key information

The bioconcentration factors (BCF) of the test substance in the last three determinations (6 points/ test group) was less than 6.9 fold to 8.2 fold for Level 1 concentration and less than 67 fold to less than 73 fold for Level 2 concentration.

As conclusion, the degree of bioconcentration of the test substance in carp was low.

Additional information

The purpose of the study was to examine the degree of bioaccumulation to the fish of Setafix Z1077 based on the OECD Guideline 305 (Bioconcentration: Flow-through Fish Test).

PRELIMINARY STUDY: Acute toxicity test

Test fish: Orange·red killifish (Oryzias laptipes)

Duration of exposure: 96 hours

Exposure method: Semi static system (Renewal of test water at every 24 hours)

96 hour LC50 value of test substance was more than 10 mg/L.

Based on the 96 hours LC50 value and analytical sensitivity, test concentrations to be used in

bioaccumulation test were decided.

MAIN STUDY: Bioaccumulation test

Test fish: Carp ( Cyprin us carpio)

Test concentrations:

Level 1 concentration 0.0080 mg/L

Level 2 concentration 0.00080 mg/L

Duration of exposure: 28 days

Exposure method: Continuous flow system

Analytical method: High performance liquid chromatography(HPLC)

RESULTS

Bioconcentration factors (Steady State): For less than 100 fold could not be confirmed for both Level 1 concentration and Level 2 concentration

Bioconcentration factors: [The bioconcentration factors (BCF) of the test substance in the last three determinations (6points/ test group)]

Level 1 concentration: less than 6.9 fold - 8.2 fold

Level 2 concentration: less than 67 fold - less than 73 fold

Conclusion

As conclusion, the degree of bioconcentration of the test substance in carp was low.