1,1'-(1,1-dimethyl-3-methylene-1,3-propanediyl)bisbenzene

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:

Start of study: 14 December 2001; End of study: 15 April 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study conducted according to GLP

Qualifier:

according to guideline

Guideline:

other: Test Method Relating to New Chemical Substances (Kanpogyo No. 5, Yakuhatsu No. 615, 49 Kikyoku No. 392, 1974; partially revised 1998)

GLP compliance:

yes

Radiolabelling:

no

Details on sampling:

Test water:
Samples were taken at the start (day 0) and days 7, 14, 21, 28, 35, 42, 49 and 60 of uptake, and test substance concentration was measured.

Fish:
After the start of uptake, 4 common carp were sampled on days 7, 14, 28 and 42 and 6 common carp on day 60, and test substance concentration was measured in 2 fish twice.

In addition, analysis by site was performed using the two remaining fish sampled at the end of uptake (day 60).

Because the concentration rate exceeded 1000, an elimination test was conducted using the fish remaining on day 60 of uptake. Four fish from each concentration were sampled on days 2, 4, 9 and 16 after start of elimination, and test substance concentration was measured by the same method as during the uptake period

Test organisms (species):

Cyprinus carpio

Details on test organisms:

TEST ORGANISM
- Common name: Common carp
- Source: Sankyou-Suisan Co. Ltd. (1-1 Ichigayata-machi, Shinjuku-ku, Tokyo)
- Age at study initiation: Within 1 year after hatching
- Length at study initiation: 8±4 cm
- Weight at study initiation: About 5 g

ACCLIMATION
- Acclimation period: At least 2 weeks
- Type and amount of food: Kyorin Baby Gold
- Feeding frequency: Approximately 2% of body weight every day
- Health during acclimation (any mortality observed): Mortality rate in the week before the test was less than 5%

Route of exposure:

feed

Total exposure / uptake duration:

60 d

Total depuration duration:

16 d

Test temperature:

24 ± 2 °C

pH:

6.0.8.5

Dissolved oxygen:

60% of saturated dissolved oxygen or more (5 mg/L or more at 24°C)

Details on test conditions:

TEST SYSTEM
- Test vessel: 56 L glass aqaurium
- Fill volume: 50 L
- Renewal rate of test solution (frequency/flow rate): 800 L/day (water exchanged 16 times/day)
- Biomass loading rate: High concentration 1: 44 fish/50L test water (1.0 g per liter/day or more)
Low concentration 2: 44 fish/50 L test water (1.0 g per liter/day or more)
Control: 24 fish/50 L test water (1.0 g per liter/day or more)

OTHER TEST CONDITIONS
- Photoperiod: About 16 hr/day
- Light intensity: Hf fluorescent lamps (wavelength 400 -700 nm)

RANGE-FINDING / PRELIMINARY STUDY
Acute toxicity test:
The 96 hr-LC50was measured according to the OECD Guideline for Testing of Chemicals Test No.203: Fish, Acute Toxicity Test. The result (LC50) was calculated from the nominal concentration without measuring the concentration of the test substance in the test solution.

Test Fish
Species: Medaka (Oryzias latipes, Cyprinodontidae)
Source: Sankyou-Suisan Co. Ltd.
(1-1 Ichigayata-machi, Shinjuku-ku,)
Date of receipt: 12 November 2001
Lot number: 01-H-1112
Preliminary treatment: The fish were visually inspected upon receipt, those with abnormalities were removed, and the rest were kept in flowing water.
Total length: 2±1 cm
Body weight: About 0.2 g

Acclimatization
Tank number:                   B-8
Water temperature:          24±2°C
Duration:                          At least 2 weeks
Type of feed:                   Tetrawerke  Tetramin
Amount of feed:               2% of body weight (every day, but not fed for 24 hours before the test)
Mortality rate in the week before the test:     Less than 5%

Test Conditions 
Test concentrations:                 0 (control), 1.0 mg/L
Rearing density:                       10 fish/concentration
Volume of test water:              3.0 L
Water temperature:                 24±2°C
Water exchange:                     No
Aeration:                                 Yes
Percent of dissolved oxygen:    ≥60% of saturated dissolved oxygen
Amount of feed:                      No
Test period:                             96 hours

Preparation of test water
Test group:
Approximately 500 mg of the test substance was dissolved in 40% (w/v) HCO-40/2 methoxyethanol solution and made up to 25 mL. 0.15 mL of this solution was collected and dispersed and dissolved in the dilution water while stirring to make 3 L (test substance concentration: 1.0 mg/L, HCO-40 concentration: 20 mg/L, 2 methoxyethanol concentration:»30 ppm (v/v)).

Control group:
0.15 mL of 40% (w/v) HCO-40/2 methoxyethanol solution was collected, dissolved in dilution water and made up to 3 L (HCO-40 concentration: 20 mg/L, 2 methoxyethanol concentration:»30 ppm (v/v)).

The 96 hr-LC50of the test substance was >1.0 mg/L. From the results of the acute toxicity test, the concentrations of test substance in the test water in the concentration test were set at 0.01 and 0.001 mg/L, which are less than 1/100 and 1/1000 of the 96 hr-LC50value

Nominal and measured concentrations:

High concentration 1: 0.01 mg/L (Auxiliary HCO-40: 0.2 mg/L, 2-methoxyethanol: ~25 ppm (v/v))
Low concentration 2: 0.001 mg/L (Auxiliary HCO-40: 0.02 mg/L, 2-methoxyethanol: ~25 ppm (v/v))
Control: 0 mg/L (Auxiliary HCO-40: 0.2 mg/L, 2-methoxyethanol: ~25 ppm (v/v))

Reference substance (positive control):

no

Details on estimation of bioconcentration:

Calculation of concentration rate:
The concentration rate at each measurement time was calculated by the following equation.

BCF = Cf at each measurement time / Cw up to each measurement time (mean)

If steady state was attained, the concentration rate at steady state was calculated by the following equation.

BCFss = Cf at steady state (mean) / Cw at steady state (mean)


BCF: Concentration rate at each measurement time
BCFss: Concentration rate at steady state
Cf: Concentration in fish (μg/g)
Cw: Concentration in water (mg/L)
Steady state: Steady state is attained when the variation of the concentration rate in three successive measurements in a period of 48 hours or more is 20% or less. The mean Cf and Cw are calculated from the final three successive measurements up to the end of uptake. However, if the concentration rate during the test period is less than 100, steady state is not considered to have been attained even if variation of the concentration rate is 20% or less. In that case, BCFss is not calculated.

Limit of concentration rate measurement
The test substance was not detected in the blank test on fish, and the detection limit was <0.012 μg/g. While it differs depending of fish body weight, the concentration rate can be measured when the concentration is about 2-fold or more at the nominal concentration in test water of 0.01 mg/L (high concentration 1) and about 12-fold or more at 0.001 mg/L (low concentration 2).

Lipid content:

6.1 %

Time point:

start of exposure

Remarks on result:

other:

Remarks:

The protocol stated that fish for measurement of lipid content would be collected at the start of uptake and the end of elimination test if an elimination test is conducted. Actually, the samples which should have been collected at the end of the elimination test were collected at the end of uptake. Mean lipid content at the end of uptake was within ±25% of that at the start. The elimination test period was 16 days, and variation of lipid content was probably small because it was shorter than the uptake period. Therefore, it was judged to have no effect on the test results.

Lipid content:

7.3 %

Time point:

end of exposure

Remarks on result:

other:

Remarks:

The protocol stated that fish for measurement of lipid content would be collected at the start of uptake and the end of elimination test if an elimination test is conducted. Actually, the samples which should have been collected at the end of the elimination test were collected at the end of uptake. Mean lipid content at the end of uptake was within ±25% of that at the start. The elimination test period was 16 days, and variation of lipid content was probably small because it was shorter than the uptake period. Therefore, it was judged to have no effect on the test results.

Key result

Conc. / dose:

0.01 mg/L

Temp.:

>= 22 - <= 26 °C

Type:

BCF

Value:

>= 427 - <= 3 330 dimensionless

Calculation basis:

steady state

Remarks:

Due to the variation in concentration rate in three successive measurements up to the end of uptake did not attain less than 20%, despite correction for 4% lipid concentration, the concentration rate at steady state (BCFss) was not calculated.

Key result

Conc. / dose:

0.001 mg/L

Temp.:

>= 22 - <= 26 °C

Type:

BCF

Value:

>= 423 - <= 4 410 dimensionless

Calculation basis:

steady state

Remarks:

Due to the variation in concentration rate in three successive measurements up to the end of uptake did not attain less than 20%, despite correction for 4% lipid concentration, the concentration rate at steady state (BCFss) was not calculated.

Details on results:

Environmental Factors which could Affect the Reliability of Test Results
There were no such events.

Management of Study Fish
During the experimental period, the percent of dissolved oxygen in all water tanks was 60% of the saturated dissolved oxygen and water temperature was 24±2°C, which both conformed to the test conditions. pH was within the appropriate range (6.0-8.5) of the environment for fish. Mortality and abnormality were <10% at each concentration during the experimental period. It was also confirmed that the fish were raised normally since there were no abnormalities in shape, swimming or feed intake in the surviving fish during the experimental period.

Lipid content (control group): Start: 6.1% (n=3, 6.0-6.2%) End: 7.3% (n=3, 6.5-8.2%)
(Mean lipid content at the end was within ±25% of that at the start.)

Lipid content in stock samples for measurement of lipid content:
Uptake period: High concentration 1: 5.5% (1.2-7.7%), Low concentration 2: 5.1% (3.0-6.7%)
Elimination period: High concentration 1: 2.9% (0.9-4.3%), Low concentration 2: 3.6% (2.4-5.8%)

Test Substance Concentration in Test Water
same as nominal values, 0.01048 mg/L for high concentration 1 and 0.000929 mg/L for low concentration 2. The coefficient of variation was 7.8% and 11.1%, respectively. Variation of test substance concentration in test water was within ±20% of the mean measured value.

Test Substance Concentration and Concentration Rate in Fish:
Concentrations in fish during the uptake period were 4.25-35.05 μg/g at high concentration 1 and 0.429-4.165 μg/g at low concentration 2. The concentration rate (BCF) was 427-3330 at high concentration 1 and 423-4410 at low concentration 2. Because variation in the concentration rate (mean) in three successive measurements up to the end of uptake exceeded 20% for high concentration 1 and low concentration 2, the concentration rate was calculated with correction for 4% lipid content.

Because the variation in the concentration rate (mean) in three successive measurements up to the end of uptake did not attained less than 20% despite correction for 4% lipid concentration, the defined level for steady state was not attained and the concentration rate at steady state (BCFss) was not calculated.

Concentration Rate by Site:
In order to examine the site of test substance concentration, the head, viscera, edible parts (muscle, bone) and outer skin (skin, fins) of two fish at each concentration were dissected and analyzed on day 60 after the start of uptake by the method of 5.5. At high concentration 1, the concentration rate in the head was relatively high compared to the other sites, and low in the edible parts. At low concentration 2, it was high in the viscera and low in the edible parts.

Elimination Test
An elimination test was conducted after the end of uptake since the concentration rate exceeded 1000. During the elimination test the concentration of test substance in the fish decreased over time, and the biological half-life (BHL) was calculated to be 4.5 days at high concentration 1 and 15.7 days at low concentration 2.

The results of measurement of concentration rate are shown below.

Uptake period		Day 7	Day 14	Day 28	Day 42	Day 60
High concentration 1	Mean concentration in water (mg/L)	0.00995	0.01007	0.01033	0.01054	0.01048
	Concentration rate               1	1610	957	1370	896	1080
	BCF       427~3330             2	427	1950	2810	3330	1250
Low concentration 2	Mean concentration in water (mg/L)	0.001013	0.000982	0.000943	0.000944	0.000929
	Concentration rate               1	469	1610	3730	4410	724
	BCF       423~4410             2	423	1740	2270	4120	2220

The concentration rates corrected for 4% lipid content are shown below

Uptake period		Day 7	Day 14	Day 28	Day 42	Day 60
High concentration1	Concentration rate               1	836	580	913	1330	960
	BCFL4        294~4170         2	294	1050	1840	1900	4170
High concentration 2	Concentration rate               1	323	1040	2930	3750	724
	BCFL4        323~3750         2	564	1740	1360	2460	2070

Concentration rates in each site were as follows:

Site	High concentration 1 (day 60)	Low concentration 2 (day 60)
Head Viscera Edible parts Outer skin	3660 2340 1510 1660	3340 5190 1610 2890

Validity criteria fulfilled:

not specified

Conclusions:

Based on the results of the study the test substance should be considered as bioaccumulative.

Description of key information

Based on the results of the study the test substance should be considered as bioaccumulative.

Key value for chemical safety assessment

BCF (aquatic species):

4 410 dimensionless

Additional information

Introduction

The bioaccumulative potential of the test item was assessed according to the test method relating to New Chemical Substances (Kanpogyo No. 5, Yakuhatsu No. 615, 49 Kikyoku No. 392, 1974; partially revised 1998).

 

Discussion

Concentrations in the fish during the uptake period were 4.25-35.05 μg/g at high concentration 1 and 0.429-4.165 μg/g at low concentration 2. The concentration rate (BCF) was 427-3330 at high concentration 1 and 423-4410 at low concentration 2. Concentration rates corrected for 4% lipid content were calculated because variation in the concentration rate (mean) exceeded 20% in three successive measurements up to the end of the uptake period at high concentration 1 and low concentration 2.

 

Despite correction for 4% lipid content, variation in the concentration rate (mean) in three successive measurements up to the end of the uptake period did not become 20% or less, and because the defined level for steady state was not attained, the concentration rate at the steady state (BCF_SS) was not calculated.

 

In order to examine the site of test substance concentration, the head, viscera, edible parts (muscle, bone) and outer skin (skin, fins) of two fish at each concentration were dissected and analyzed on day 60 after the start of uptake. At high concentration 1, the concentration rate in the head was relatively high compared to the other sites, and low in the edible parts. At low concentration 2, it was high in the viscera and low in the edible parts.

 

Concentration rates in each site were as follows.

 

Site	High concentration 1 (day 60)	Low concentration 2 (day 60)
Head	3660	3340
Viscera	2340	5190
Edible parts	1510	1610
Outer skin	1660	2890

 

An elimination test was conducted after the end of uptake since the concentration rate exceeded 1000. During the elimination test the concentration of test substance in the fish decreased over time, and the biological half-life (BHL) was calculated to be 4.5 days at high concentration 1 and 15.7 days at low concentration 2.

 

Results

The results of measurement of concentration rate are shown below.

Uptake period			Day 7	Day 14	Day 28	Day 42	Day 60
High concentration 1	Mean concentration in water (mg/L)		0.00995	0.01007	0.01033	0.01054	0.01048
	Concentration rate	1	1610	957	1370	896	1080
	BCF 427- 3330	2	427	1950	2810	3330	1250
Low concentration 2	Mean concentration in water (mg/L)		0.001013	0.000982	0.000943	0.000944	0.000929
	Concentration rate	1	469	1610	3730	4410	724
	BCF 423- 4410	2	423	1740	2270	4120	2220

 

The concentration rates corrected for 4% lipid content are shown below.

 

Uptake period			Day 7	Day 14	Day 28	Day 42	Day 60
High concentration 1	Concentration rate	1	836	580	913	1330	960
	BCFL4 294- 4170	2	294	1050	1840	1900	4170
Low concentration 2	Concentration rate	1	323	1040	2930	3750	724
	BCFL4 323- 3750	2	564	1740	1360	2460	2070

 

Based on the results of the study the test substance should be considered as bioaccumulative.