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

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Start of study: 1 April 2005. End of study: 27 June 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to GLP and guideline

Qualifier:

according to guideline

Guideline:

other: Standards for Testing Facilities Conducting Tests on New Chemical Substances, etc. (Yakushokuhatsu No. 1121003, Heisei 15.11.17 Seikyoku No. 3, Kanpokihatsu No. 031121004, 2003)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

0.75 mL was collected from each test group and mixed with equivalent amount of acetonitrile and then used for HPLC analysis

0.75 mL of standard solution prepared with acetonitrile was collected in a vial for measurement, mixed with an equal volume of purified water (JIS K0557 A4 grade water) and analyzed by HPLC

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
Test substance stock solution I was prepared as shown, and test solutions were prepared by diluting and mixing with the dilution water. Dilution water only for the control group and water containing only the solvent (concentration: 100 μL/L) for the solvent control group were prepared. 100 mL of the prepared test solution was put into each of 4 test vessels for each test group. Stock solution I was prepared at the start of exposure and stored refrigerated and protected from light (stable for 48 hours or more under these conditions).

(1) Preparation of test substance stock solution I
Amount collected: 120 mg
Solvent: N,N-dimethylformamide
Final volume: 100 mL
Vessel: Measuring bottle
Concentration: 1200 mg/L
Mixing system: Ultrasonication, 2 min, tightly stoppered

(2) Preparation of test substance stock solution II
Amount of stock solution I collected: 200 μL
Solvent: Dilution water
(Elendt M4 solution sufficiently aerated at 20±1°C)
Final volume: 2000 mL
Vessel: Media bottle
Concentration: 0.120 mg/L
Solvent concentration: 100 μL/L
Mixing system Stirrer, 10 min, tightly stoppered

(3) Preparation of solvent stock solution
Reagent: N,N-dimethylformamide
Amount collected: 1000 μL
Solvent: Dilution water
(Elendt M4 solution sufficiently aerated at 20±1°C)
Final volume: 100 mL
Vessel: Measuring bottle
Reagent concentration: 10000 μL/L
Mixing system Manual inversion (readily soluble)

The stock solutions of (2) and (3) were collected according to the following table and diluted with dilution water to prepare test solutions.
Dilution water (final volume): 0.50 L
Vessel: Measuring flask
Mixing system: Manual inversion, tightly stoppered
Concentration ratio: 1.41

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Source: National Institute for Environmental Studies
- Age at study initiation: Female juveniles (age 24 hours or less)
- Feeding during test: Not fed


ACCLIMATION
- Type and amount of food: Chlorella vulgaris (single cell green algae)
- Feeding frequency: 0.2 mg C (organic carbon content) / animal / day
- Health during acclimation (any mortality observed): Parental mortality in 2 weeks before start of exposure: <5%

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

Refer to table 6

pH:

Refer to table 8

Dissolved oxygen:

Refer to table 7

Nominal and measured concentrations:

Refer to table 1

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL glass beakers (The test vessels were lidded and the water surface was covered with a Teflon sheet to prevent vaporization of the test solution and volatilization of the test substance.)
- Renewal rate of test solution: Total exchange of test solution after 24 hours
- No. of organisms per vessel: 5
- No. of vessels per concentration : 4
- No. of vessels per control: 4


TEST MEDIUM / WATER PARAMETERS
The medium (Elendt M4) described in the OECD chemical testing guideline No.202 Daphnia sp., Acute Immobilization Test (2004) was used. Its composition is shown in Attachment 2. Theoretical hardness is 250 mg/L (CaCO3 conversion). It was confirmed that hardness (250 mg/L or less, CaCO3 conversion) and pH (6.0-9.0) were within the appropriate range at the start of exposure

OTHER TEST CONDITIONS
- Adjustment of pH: Test solution pH was not regulated
- Photoperiod: 16 hours light/8 hours dark
- Light intensity: Room lighting (800 lux or less)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Immobility at 24 and 48 h. Animals unable to swim within 15 seconds after gently agitating the test vessel were considered to be immobilized.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Test concentrations were set with solubility of the test substance in the dilution water of 0.12 mg/L or less (in-house measurement).
- Preliminary study
- Test concentrations: 0.0120, 0.0380, 0.120 mg/L
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

no

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

0.091 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limits 0.082 - 0.106 mg/L

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.057 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limits 0.052 - 0.063 mg/L

Details on results:

Test substance concentrations in test solutions
Results of analysis of test substance concentration in test solutions are shown in Table 1.

In the results of analysis, the ratio of the measured value to the nominal value was 82-93% at the time of test solution preparation and 62-74% 24 hours later. In a preliminary test, decrease in concentration was more remarkable in test solution containing the organism than in test solution not containing the organism. Therefore, the main reason for the decrease in test substance concentration was considered to be migration to the organism

Median Effective Concentration (EC50) for Immobility
The immobility rate at each time and the concentration-immobility curve are shown in Table 2 and Fig 1 respectively.

The immobility rate during the exposure period was 0% in both the control group and solvent control group. There were no Daphnia floating on the surface in either the control group or solvent control group, and the conditions for the validity of the test were met.

From the results, the median effective concentration (EC50) for immobility is shown in Table 3 and below,

24-hour EC50: 0.091 mg/L (95% confidence interval: 0.082-0.106 mg/L)
48-hour EC50: 0.057 mg/L (95% confidence interval: 0.052-0.063 mg/L)

Highest Concentration causing 0% Immobility and Lowest Concentration causing 100% Immobility:
The highest concentration causing 0% immobility and lowest Concentration causing 100% Immobility are shown in Table 4 and below.

24-hour highest concentration causing 0% immobility: 0.046 mg/L
48-hour highest concentration causing 0% immobility: 0.031 mg/L
24-hour lowest concentration causing 100% immobility: >0.096 mg/L
48-hour lowest concentration causing 100% immobility: 0.096 mg/L

Appearance, Temperature, Dissolved Oxygen Concentration and pH of Test Solutions:
Appearance of test solutions is shown in Table 5, temperature in Table 6, dissolved oxygen concentration in Table 7 and pH in Table 8. The water temperature was 20±1°C and the dissolved oxygen concentration was 60% or more of the saturated dissolved oxygen concentration (saturated dissolved oxygen concentration 20.0°C: 8.8 mg/L) in all test groups, all of which fulfilled the test standards. pH was within the appropriate range (6.0-9.0) of environment for Daphnia.

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) (Percent of Nominal, %)
	0 hour (new)	24 hours (old)	24 hours (new)	48 hours (old)
Control	<0.003	<0.003	<0.003	<0.003	--
Solvent Control	<0.003	<0.003	<0.003	<0.003	--
0.0300	0.0028 (93)	0.019 (63)	0.027 (90)	0.020 (67)	0.023  (77)
0.0420	0.036 (86)	0.026 (62)	0.035 (83)	0.029 (69)	0.031  (74)
0.0600	0.052 (87)	0.041 (68)	0.050 (83)	0.043  (72)	0.046  (77)
0.0850	0.070 (82)	0.057 (67)	0.070  (82)	0.060 (71)	0.064  (75)
0.120	0.108 (90)	0.084 (70)	0.106  (88)	0.089 (74)	0.096 (80)

a: Time weighted mean

New: freshly prepared test solutions

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

Table 2: The number of immobilized Daphnia magna (Percent immobility)

Nominal concentration (mg/L)	Mean^ameasured concentration (mg/L)	Cumulative mortality (percent mortality)
		24 hours	48 hours
Control	---	0 (0)	0 (0)
Solvent control	---	0( 0)	0( 0)
0.0300	0.023	0( 0)	0( 0)
0.0420	0.031	0( 0)	0( 0)
0.0600	0.046	0( 0)	4( 20)
0.0850	0.064	1 (5)	13 (65)
0.120	0.096	12 (60)	20 (100)

a: Time weighted mean

Table 3: Calculated EC50 values

Exposure period (hours)	EC50 (mg/L)	95 percent confidence limits (mg/L)	Statistical method
24	0.091	0.082 – 0.106	Probit^*1
48	0.057	0.052 – 0.063	Probit^*1

*1: Using the concentrations of 0.023 – 0.096 mg/L

Table 4: Highest concentration in 0% immobility and lowest concentration in 100% immobility

Exposure period (hours)	Highest concentration in 0% immobility (mg/L)	Lowest concentration in 100% immobility (mg/L)
24	0.046	>0.096
48	0.031	0.096

Table 5: Appearance of test solutions

Nominal concentration (mg/L)	Mean measured concentration (mg/L)	Appearance of test solutions
		0 hours	24 hours		48 hours
		new	old	new	old
Control	---	C-	C-	C-	C-
Solvent control	---	C	C-	C-	C-
0.0300	0.023	C-	C-	C-	C-
0.0420	0.031	C-	C-	C-	C-
0.0600	0.046	C-	C-	C-	C-
0.0850	0.064	C-	C-	C-	C-
0.120	0.096	C-	C-	C-	C-

New: freshly prepared test solutions

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

Colour: C-; Colourless Table 6: Temperature of test solutions

Nominal concentration (mg/L)	Mean^ameasured concentration (mg/L)	Temperature (°C)
		0 hour new	24 hours old	24 hours new	48 hours old
		new	old	new	old
Control	---	20.0	19.8	19.9	20.0
Solvent control	---	20.1	19.9	19.9	20.0
0.0300	0.023	20.3	19.9	20.0	20.0
0.0420	0.031	20.3	20.0	20.0	20.0
0.0600	0.046	20.3	20.0	20.1	20.1
0.0850	0.064	20.3	20.0	20.1	20.1
0.120	0.096	20.3	20.0	20.1	20.0

 a: time weighted mean

New: freshly prepared test solutions

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

Table 7: Dissolved oxygen concentrations in test solutions

Nominal concentration (mg/L)	Mean^ameasured concentration (mg/L)	Dissolved oxygen concentration (mg/L)
		0 hour new	24 hours old	24 hours new	48 hours old
		new	old	new	old
Control	---	8.8	8.7	8.8	8.8
Solvent control	---	8.7	8.6	8.8	8.8
0.0300	0.023	8.7	8.7	8.8	8.7
0.0420	0.031	8.6	8.7	8.7	8.7
0.0600	0.046	8.7	8.7	8.8	8.8
0.0850	0.064	8.6	8.6	8.7	8.7
0.120	0.096	8.7	8.7	8.8	8.7

a: time weighted mean

New: freshly prepared test solutions

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

Table 8: pH values of test solutions

Nominal concentration (mg/L)	Mean^ameasured concentration (mg/L)	pH
		0 hour new	24 hours old	24 hours new	48 hours old
		new	old	new	old
Control	---	8.2	8.2	8.3	8.3
Solvent control	---	8.3	8.3	8.3	8.3
0.0300	0.023	8.4	8.2	8.4	8.3
0.0420	0.031	8.3	8.2	8.4	8.3
0.0600	0.046	8.3	8.2	8.3	8.3
0.0850	0.064	8.3	8.2	8.4	8.3
0.120	0.096	8.4	8.3	8.4	8.4

a: time weighted mean

New: freshly prepared test solutions

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

Conclusions:

The 48 h EC50 for Daphnia magna was 0.057 mg/L (95% confidence interval: 0.052 - 0.063 mg/L).

Description of key information

The 48 h EC50 for Daphnia magna was 0.057 mg/L (95% confidence interval: 0.052 - 0.063 mg/L).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.057 mg/L

Additional information

Introduction

This study was conducted in accordance with Method for Testing of New Chemical Substances, etc. <Algae Growth Inhibition Test, Daphnia Immobilization Test and Fish Acute Toxicity Test> (Yakushokuhatsu No. 1121002, Heisei 15.11.13 Seikyoku No. 2, Kanpokihatsu No. 031121002, 2003).

 

Method

1)   Exposure method: Semi-static (total exchange of test solution after 24 hours)

2)   Exposure period: 48 hours

3)   Test concentrations (nominal values): Control group, solvent control group, 0.0300, 0.0420, 0.0600, 0.0850, 0.120 mg/L (0.120 mg/L was the maximum preparable concentration of test solution)

Common ratio: 1.4

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

4)   Test solution volume: 100 mL/vessel

5)   Replicates: 4 vessels/test group

6)   Number of test organisms: 20/test group (5/vessel)

7)   Test temperature: 20±1°C

8)   Lighting: Room lighting, 16 hours light (800 lux or less) / 8 hours dark

9)   Analysis: High performance liquid chromatography (HPLC)

Results

1)   Test substance concentration in test solution

In the results of analysis of test substance concentration in test solutions, the ratio of the measured value to the nominal value was 82 -93% at the time of test solution preparation and 62-74% 24 hours later.

In a preliminary test, decrease in concentration was more remarkable in test solution containing the organism than in test solution not containing the organism. Therefore, the main reason for the decrease in test substance concentration was considered to be migration to the organism.

2)   Results of 24-hour exposure

Median effective concentration (EC50) for immobility: 0.091 mg/L
(95% confidence interval: 0.082-0.106 mg/L)

Highest concentration causing 0% immobility: 0.046 mg/L

Lowest concentration causing 0% immobility: >0.096 mg/L

 

3)   Results of 48-hour exposure

Median effective concentration (EC50) for immobility: 0.057 mg/L (95% confidence interval: 0.052-0.063 mg/L)

Highest concentration causing 0% immobility: 0.031 mg/L

Lowest concentration causing 0% immobility: 0.096 mg/L 

Conclusion

The acute toxicity of the test item to Daphnia magna was assessed according to the Method for Testing of New Chemical Substances, etc. <Algae Growth Inhibition Test, Daphnia Immobilization Test and Fish Acute Toxicity Test> (Yakushokuhatsu No. 1121002, Heisei 15.11.13 Seikyoku No. 2, Kanpokihatsu No. 031121002, 2003). The 48 h EC50 for Daphnia magna was 0.057 mg/L (95% confidence interval: 0.052 - 0.063 mg/L).