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

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Start of study: 8 April 2005. End of study: 25 July 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to guideline and GLP.

Qualifier:

according to guideline

Guideline:

other: Methods for Testing of New Chemical Substances, etc. (Yakushokuhatsu No. 1121002, Heisei 15.11.13 Seikyoku No. 2, Kanpokihatsu No. 031121002, 2003).

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Two sets were analyzed during the exposure period (the start of exposure and before water renewal after 24 hours, and after water renewal after 48 hours and before water renewal after 72 hours). 0.75 mL of test solution was collected from each test vessel, and after adding and mixing an equal amount of acetonitrile, it was analyzed by high performance liquid chromatography (HPLC). The test substance concentration in each test solution was quantified from the ratio the peak area of the standard solution

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The method of preparing test solutions is shown below. Test substance stock solution II was prepared at the time of use.
Dilution water only for the control group and water containing only the solvent (concentration: 100 μL/L) for the solvent control group were prepared.
Test substance stock solution I was prepared at the start of the exposure period, and stored in a refrigerator during the exposure period. The stock solution I was confirmed to be stable refrigerated for 96 hours or more.

(1) Preparation of test substance stock solution I
Amount collected:35 mg
Solvent: N,N-dimethylformamide
Final volume: 25 mL
Vessel: Measuring bottle
Concentration: 1400 mg/L
Mixing system: Manual invention

(2) Preparation of test substance stock solution II
Amount of stock solution I collected: 520 μL
Solvent: Test water = dilution water
Final volume: 5200 mL x 3
Vessel: Measuring flask
Concentration: 0.140 mg/L
Solvent concentration: 100 μL/L
Mixing system Stirrer: 2 min

3) Preparation of solvent stock solution
Reagent: N,N-dimethylformamide

Preparation of test solution:
Each stock solution of (2) and (3) was collected to prepare test solutions (see table 1 in materials and methods)
Test solution (final volume):5.0L
Vessel: 5.0L glass aqualium
Mixing system: stirred with glass stick
Concentration ratio: 1.4

Test organisms (species):

Oryzias latipes

Details on test organisms:

TEST ORGANISM
- Common name: Japanese Medaka
- Source: Tsunashima Fishing (5-18-1 Tsunashimanishi, Kohoku-ku, Yokohama-shi, Kanagawa)
- Age at study initiation: Within 1 year after hatching
- Length at study initiation: Mean 2.16 cm (1.94-2.56 cm), n=10
- Weight at study initiation: Mean 0.075 g (0.056-0.135 g), n=10
- Feeding during test: Not fed

ACCLIMATION
-Water: Dilution water
-Method: Flow-through system
-Water temperature: 24±1°C
-Percent of dissolved oxygen: 80% or more of saturated concentration
-pH: 6.5-8.5
-Light: Room lighting, 16 hours (1000 lux or less) / 8 hours dark
-Type of Feed: Tetrawerke Tetramin
-Amount of feed: About 2% of body weight/day (not fed for 24 hours before start of exposure)

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

Minimum: 23.3
Maximum: 24.3
Refer to table 7 in results section

pH:

Minimum: 7.2
Maximum7.9
Refer to table 9 in results section

Dissolved oxygen:

Minimum:5.7
Maximum: 8.4
Refer to table 8 in results section

Nominal and measured concentrations:

Refer to table 1 in results section

Details on test conditions:

TEST SYSTEM
- Test vessel: 5.0 L capacity glass water tank (water surface covered with Tephlon lid)
- Renewal rate of test solution: Total exchange of test solution every 24 hours
- No. of organisms per vessel: 10
- No. of vessels per concentration: 1
- No. of vessels per control: 1

TEST MEDIUM / WATER PARAMETERS
The dilution water used was dechlorinated Yokohama city tap water with residual chlorine removed by activated carbon, which was sufficiently aerated and temperature controlled at 24±2°C. A total of 35 measurement items indicated in the marine product water standard (1983) are measured in the dechlorinated tap water periodically (every 6 months). Hardness is ordinarily 30-100 mg/L (CaCO3 conversion) and pH is 6.5-8.5. The absence of residual chlorine was verified at the time of use. The water quality was judged to be appropriate for the fish.

OTHER TEST CONDITIONS
- Adjustment of pH: Test solution pH was not regulated
- Photoperiod: 16 hours light/ 8 hours dark
- Light intensity: Room lighting, 1000 lux or less

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
The number of mortalities was recorded 24, 48, 72 and 96 hours after start of exposure, and any signs of toxicity and abnormalities observed were recorded. Symptoms commonly described and their definitions are shown below*. Dead fish were quickly removed upon discovery to avoid degrading water quality.

* Common symptoms and their definitions
Mortality: The fish does not react to stimulation.

Abnormal respiration: Movement of the operculum is abnormal compared to the control group fish.

Abnormal swimming: Swimming is clearly different from the control group fish, such as sluggish movement, hyper reaction, convulsion, inversion.

Inability to swim: Although moving at the bottom or at the surface of water, the fish is unable to swim in the water. Includes overturning and apparent death.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: Test concentrations were set with a solubility of the test substnace in the dilution water of 0.14 mg/L (in-house measurement) or less.
- Range finding study
- Test concentrations: 0.014, 0.044, 0.140 mg/L
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 0.092 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% confidence intervals could not be determined

Details on results:

Test substance concentration in test solutions:
Results of analysis of test substance concentration in test solutions are shown in Table 1. For results of analysis of test solutions, the ratio of the measured value to the nominal value was 87-93% at the time of preparation of test solution, and 42-53% after 24 hours. The main reason for decrease in concentration was thought to be sublimation of the test substance.

Median lethal concentration (LC50):
Cumulative mortality at each time is shown in Table 2. 96-hour mortality in the control group was 10% and 0% in the solvent control group, which fulfilled the validity conditions of the test.

The median lethal concentration (LC50) based on the above results is shown in Table 3 and below.

96-hour LC50: >0.092 mg/L (95% confidence interval could not be determined)

Highest 0% mortlaity concentration and lowest 100% mortality concentration
The highest concentration with 0% mortality and the lowest concentration with 100% mortality at each time are shown in Table 4, and the results calculated for 96 hours are shown below.

Highest concentration causing 0% mortality for 96 hours: >0.092 mg/L
Lowest concentration causing 100% mortality for 96 hours: >0.092 mg/L

Toxicological symptoms:
Observed toxicological symptoms are shown in Table 5.

No abnormalities were observed during the exposure period in the control, solvent control, 0.040, 0.055, 0.075, 0.100 mg/L group. Internal hemorrhage and surfacing were observed in the 0.140 mg/L group.

Appearance, temperature, percent of dissolved oxygen and pH of test solutions:
Appearance of test solutions is shown in Table 6, temperature in Table 7, percent of dissolved oxygen in Table 8 and pH in Table 9.

The appearance of test solutions was colorless throughout the exposure period in all test groups.

The water temperature was 24±1°C in all test groups, the percent of dissolved oxygen was 60% or more of the saturated dissolved oxygen (saturated dissolved oxygen at 24.0°C: 8.25 mg/L), which all fulfilled the test standards. pH was within the appropriate range (6.0-8.5) of environment for Medaka

Sublethal observations / clinical signs:

Table 1: Measured concentration of the test substance in test water

Nominal concentration (mg/L)	Measured concentration mg/L (percent of nominal)				Mean^ameasured concentration (mg/L)
	0 hour (new)	24 hours (old)	48 hours (new)	72 hours (old)
Control	<0.003	<0.003	<0.003	<0.003	---
Solvent Control	<0.003	<0.003	<0.003	<0.003	---
0.040	0.036 (90)	0.018 (45)	0.035 (88)	0.017 (43)	0.025 (63)
0.055	0.050 (91)	0.025 (45)	0.048 (87)	0.029 (53)	0.037 (67)
0.075	0.066 (88)	0.036 (48)	0.068 (91)	0.037 (49)	0.050 (67)
0.100	0.092 (92)	0.042 (42)	0.090 (90)	0.043 (43)	0.064 (64)
0.140	0.130 (93)	0.060 (43)	0.127 (91)	0.067 (48)	0.092 (66)

^a: time weighted mean

new: freshly prepared test solutions

old: test solutions on 24 hours after preparation of new solutions

Table 2: Mortality of the Medaka exposed to the test substance

Nominal concentration (mg/L)	Mean^ameasured concentration (mg/L)	Cumulative mortality (percent mortality)
		24 hours	48 hours	72 hours	96 hours
Control	---	0 (0)	0 (0)	0 (0)	1 (10)
Solvent control	---	0( 0)	0( 0)	0( 0)	0( 0)
0.040	0.025	0 (0)	0 (0)	0 (0)	1 (10)
0.055	0.037	0( 0)	0( 0)	0( 0)	0( 0)
0.075	0.050	0 (0)	0 (0)	0 (0)	0 (0)
0.100	0.064	0( 0)	0( 0)	0( 0)	0( 0)
0.140^b	0.092	0 (0)	0 (0)	0 (0)	0 (0)

^a: time weighted mean

^b: The maximum attainable concentration under the present test conditions and preparation methods

Table 3: Calculated LC50 values

Exposure period (hours)	LC50 (mg/L)	95 percent confidence limits (mg/L)	Statistical method
24	>0.092	--	--
48	>0.092	--	--
72	>0.092	--	--
96	>0.092	--	--

--: Could not be determined

The LC50 value and its 95% confidence limits could not be determined by statistical method because the mortality of Medaka at the maximum concentration level was less than 50%

Table 4: Highest concentration in 0% mortality and the lowest concentration in 100% mortality

Exposure period (hours)	Highest concentration in 0% mortality (mg/L)	Lowest concentration in 100% mortality (mg/L)
24	>0.092	>0.092
48	>0.092	>0.092
72	>0.092	>0.092
96	>0.092	>0.092

Table 5: Observed toxicological symptoms

Nominal concentration (mg/L)	Mean^ameasured concentration (mg/L)	Symptoms (Symptom-number of fish)
		24 hours	48 hours	72 hours	96 hours
Control	---	N	N	N	N
Solvent control	---	N	N	N	N
0.040	0.025	N	N	N	N
0.055	0.037	N	N	N	N
0.075	0.050	N	N	N	N
0.100	0.064	N	N	N	N
0.140^b	0.092	N	Ihi-1	SUR-2(Ihi-1)	SUR-2(Ihi-1)

a: time weighted mean

N: No toxicological symptom was observed

Ihi: integument internal hemorrhage

SUR: surfacing

Table 6: Appearance of test solutions

Nominal concentration (mg/L)	Mean^ameasured concentration (mg/L)	Appearance of test solutions
		0 hours	24 hours		48 hours		72 hours		96 hours
		new	old	new	old	new	old	new	old
Control	---	C-	C-	C-	C-	C-	C-	C-	C-
Solvent control	---	C	C-	C-	C-	C-	C-	C-	C-
0.040	0.025	C-	C-	C-	C-	C-	C-	C-	C-
0.055	0.037	C-	C-	C-	C-	C-	C-	C-	C-
0.075	0.050	C-	C-	C-	C-	C-	C-	C-	C-
0.100	0.064	C-	C-	C-	C-	C-	C-	C-	C-
0.140	0.092	C-	C-	C-	C-	C-	C-	C-	C-

^a: time weighted mean

new: freshly prepared test solutions

old: test solutions on 24 hours after preparation of new solutions

C-: Colorless

Table 7: Temperature of test solutions

Nominal concentration (mg/L)	Mean^ameasured concentration (mg/L)	Temperature (°C)
		0 hours	24 hours		48 hours		72 hours		96 hours
		new	old	new	old	new	old	new	old
Control	---	23.5	23.9	23.8	23.8	23.8	24.1	23.9	24.0
Solvent control	---	23.5	23.9	23.8	23.9	23.9	24.2	24.0	24.2
0.040	0.025	23.6	24.0	23.8	24.0	23.8	24.2	23.9	24.2
0.055	0.037	23.6	24.0	23.8	24.0	23.9	24.2	24.0	24.3
0.075	0.050	23.6	24.0	23.8	24.0	23.8	24.2	23.8	24.2
0.100	0.064	23.3	24.0	23.7	24.0	23.7	24.2	23.8	24.2
0.140	0.092	23.5	24.0	23.7	24.0	23.7	24.2	23.9	24.2

^a: time weighted mean

new: freshly prepared test solutions

old: test solutions on 24 hours after preparation of new solutions

Table 8: Dissolved oxygen concentrations in test solution

Nominal concentration (mg/L)	Mean^ameasured concentration (mg/L)	Dissolved oxygen concentration (mg/L)
		0 hours	24 hours		48 hours		72 hours		96 hours
		new	old	new	old	new	old	new	old
Control	---	8.4	6.2	8.3	6.0	8.3	6.0	8.1	5.8
Solvent control	---	8.3	6.7	8.3	6.4	8.3	6.4	8.1	6.3
0.040	0.025	8.3	6.2	8.3	6.0	8.3	6.0	8.2	5.7
0.055	0.037	8.3	6.6	8.3	6.4	8.3	6.6	8.1	6.4
0.075	0.050	8.3	6.5	8.3	6.4	8.3	6.5	8.1	6.6
0.100	0.064	8.3	5.7	8.3	5.7	8.3	5.8	8.1	5.8
0.140	0.092	8.3	5.9	8.2	5.7	8.3	6.0	8.1	5.7

^a: time weighted mean

new: freshly prepared test solutions

old: test solutions on 24 hours after preparation of new solutions

Table 9: pH values of test solutions

Nominal concentration (mg/L)	Mean^ameasured concentration (mg/L)	Dissolved oxygen concentration (mg/L)
		0 hours	24 hours		48 hours		72 hours		96 hours
		new	old	new	old	new	old	new	old
Control	---	7.7	7.3	7.9	7.2	7.8	7.3	7.7	7.2
Solvent control	---	7.7	7.4	7.9	7.3	7.8	7.3	7.8	7.3
0.040	0.025	7.7	7.3	7.9	7.3	7.8	7.3	7.8	7.2
0.055	0.037	7.7	7.3	7.9	7.3	7.8	7.4	7.8	7.3
0.075	0.050	7.7	7.3	7.9	7.3	7.8	7.3	7.8	7.4
0.100	0.064	7.8	7.3	7.9	7.2	7.8	7.3	7.8	7.3
0.140	0.092	7.8	7.2	7.9	7.2	7.8	7.3	7.8	7.3

^a: time weighted mean

new: freshly prepared test solutions

old: test solutions on 24 hours after preparation of new solutions

Validity criteria fulfilled:

yes

Remarks:

96 hour mortality in the control group was 10% and 0% in the solvent control group, which fulfilled the validity conditions of the test.

Conclusions:

The LC50 after 96 h exposure based on time weighted mean was >0.092 mg/L.

Description of key information

The LC50 after 96 h exposure based on time weighted mean was >0.092 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

0.092 mg/L

Additional information

Summary

1)   Exposure method: Semi-static (total exchange of test solution every 24 hours). Water surface covered with Teflon sheet

2)   Exposure time: 96 hours

3)   Test concentrations: Control group, solvent control group, 0.040, 0.055, 0.075, 0.100, 0.140 mg/L (Maximum preparable concentration of test solution) (Nominal value)

Common ratio

Solvent concentration: 100μL/L (N,N-dimethylformamide)

4)  Test solution volume:  5.0 L/vessel

5)   Replicates: 1 vessel/test group

6)   Number of animals: 10 fish/test group 

7)   Test temperature: 24 ± 1°C

8)   Lighting: Room lighting, 16 hours (1000 lux or less) / 8 hours dark

9)   Analysis: High performance liquid chromatography (HPLC)

 

Test Results:

1)   Test Substance Concentration in Test Solutions:

For results of analysis of test solutions, the ratio of the measured value to the nominal value was 87-93% at the time of preparation of test solution, and 42-53% after 24 hours. The main reason for decrease in concentration was thought to be volatilization of the test substance.

2)   Median lethal concentration (LC50) after 96-hr exposure: >0.092 mg/L (95% confidence interval could not be determined

3)   Highest concentration causing 0% mortality after 96-hr exposure: >0.092 mg/L

4)   Lowest concentration causing 100% mortality after 96-hr exposure:    >0.092 mg/L