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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

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: 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:

Test substance concentrations in test solution and test medium were analyzed by HPLC in each test group at the start of exposure, after 24 and 48 hours of exposure and at the end of exposure. 1.0 mL was collected from each of 6 test solutions or test cultures in each test group and mixed in a 10 mL glass sedimentation tube. This was used as the analysis sample at the start of exposure. After 24 and 48 hours of exposure and at the end of exposure, this was centrifuged* (3000 rpm, 10 min) to remove the algae and the supernatant was used as the analysis sample.

0.75 mL of standard solution prepared with acetonitrile was collected in a vial for measurement, mixed with an equal volume of purified water, and analyzed by HPLC.

0.75 mL of each analysis sample was collected in a vial for measurement, mixed with an equal volume of acetonitrile, and analyzed by HPLC

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
Medium for preparation of test solutions was placed in a water bath or incubator at 23±2°C before preparation of test solution.

Test substance stock solution and solvent stock solution were prepared and these were used to prepare the test solutions. Medium alone was used for the control group, and medium containing only the solvent (solvent concentration: 100 μL/L) was prepared for the solvent control group. Each stock solution was prepared at the time of use.

The appearance of the test solutions was recorded at the time of preparation


(1)Preparation of test substance stock solution I
Amount collected: 60 mg
Solvent: N,N-dimethylformamide
Final volume: 50 mL
Vessel: Measuring bottle
Concentration: 1200 mg/L
Mixing system: Manual invention (readily soluble), tightly stoppered

(2) Preparation of test substance stock solution II
Amount of stock solution I collected: 100 μL
Solvent: Test water (medium recommended in the Chemical Substances Control Law test guideline at 23±2°C)
Final volume: 1000 mL
Vessel: Measuring flask
Concentration: 0.120 mg/L
Solvent concentration: 100 μL/L
Mixing system: Stirrer, 5 min, tightly stoppered

(3) Preparation of solvent stock solution
Reagent: N,N-dimethylformamide
Amount collected: 100 μL
Solvent: Test water (medium recommended in the Chemical Substances Control Law test guideline at 23±2°C)
Final volume: 1000 mL
Vessel: Measuring flask
Reagent concentration: 100 μL/L
Mixing system: Stirrer, 5 min, tightly stoppered

Each stock solution of (2) and (3) was collected as shown in the table below to prepare test solutions.

Test solution (final volume): 0.10 L
Vessel: 500 mL stoppered glass Erlenmeyer flasks (Iwaki), tightly stoppered
Mixing system Manual inversion, tightly stoppered
Concentration ratio: -
Replicates for a concentration group: 6

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Single cell green algae
- Strain: ATCC226622
- Source (laboratory, culture collection): American Type Culture Collection

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

Refer to table 6

pH:

Refer to table 7

Nominal and measured concentrations:

Refer to table 1

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 mL glass stoppered Erlenmeyer flasks
- Headspace: in house measured value 490 mL
- Initial cells density: 5 x 10^3 cells/mL
- No. of vessels per concentration (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes/no
- Detailed composition if non-standard medium was used:

TEST MEDIUM / WATER PARAMETERS


OTHER TEST CONDITIONS
- Sterile test conditions: yes. The test vessels and other instruments used were sterilized as needed. Inoculation of algae was also done under sterile conditions.
- Adjustment of pH: pH of the test solutions was not adjusted
- Photoperiod: Continuous illumination
- Light intensity and quality: 65 µE/m^2/s (at the surface of the central flask)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Cell density measured at 24, 48 and 72 hours.
- Determination of cell concentrations: Cell density was measured with a particle counter after collecting 1.0 mL of test culture and mixing it with 9.0 mL of electrolyte solution for particle counter (Cellpack)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Test concentrations were set from the solubility of the test substance in the medium of 0.12 mg/L or less based on the results of the preliminary test.
- Preliminary study
- Test concentrations: 0.12, 0.060, 0.120 mg/L
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

other: ErC50

Effect conc.:

> 0.059 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

other: NOECr

Effect conc.:

> 0.059 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

other: EbC50

Effect conc.:

> 0.059 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

other: NOECb

Effect conc.:

> 0.059 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

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

Test Substance Concentration in Test Solutions and Test Cultures:
Test substance concentration was measured in test solution at the start of exposure and in test cultures after 24 and 48 hours of exposure and at the end of exposure. The results are shown in Table 1.
In the results of test substance concentration analysis, the ratio of the measured value to the nominal value was 85% for test solution at the start of exposure, and 23% for test culture at the end of exposure. From the results of the preliminary study, the main causes of decrease in concentration were considered to be volatilization of the test substance and migration to the algae.

Growth Curve:
Cell densities during the exposure period are shown in Table 2, growth rate in the control group in Table 3.
Cell density in the control group and solvent control group increased an average of 104-fold and 102-fold, respectively, in culturing for 72 hours. Since the coefficient of variation of repeated growth rate and coefficient of variation of daily growth rate did not exceed 15% and 35%, respectively, in the control group, growth was normal for the test conditions (closed system).

50% Growth Inhibitory Concentration (EC50) and No Observed Effect Concentration (NOEC):
Growth inhibition rates by concentration are shown in Table 4, 50% growth inhibitory concentration (EC50) and no observed effect concentration (NOEC) in Table 5,. The following conclusions were obtained from these results.

1) Inhibition concentration from comparison of growth rates
ErC50 (0-72h): >0.059 mg/L (95% confidence interval: could not be calculated)*
NOECr (0-72h): >0.059 mg/L


2) Inhibition concentration from comparison of area under growth curve
EbC50 (0-72h): >0.059 mg/L (95% confidence interval: could not be calculated)*
NOECb (0-72h): >0.059 mg/L

*:Since it was limit test of the maximum preparable concentration of test solution in the study protocol (0.120 mg/L, mean measured value: 0.059 mg/L) which obtained an inhibition rate of <50%, the result was “>test concentration

Environment of the Culture Apparatus, pH of Test Solution and Test Cultures, and Appearance of Test Solution:
The environment of the culture apparatus during the exposure period (temperature, light intensity, revolutions) are shown in Table 6, and pH of test solution at the start of exposure and test culture at the end of exposure are shown in Table 7.

The temperature of the culture apparatus was within the set range (23±2°C). pH was 8.1 at the start of exposure and 10.1-10.4 at the end of exposure. Fluctuation of pH by 1.5 or more was observed in all test groups, but if carbon dioxide assimilation and algal growth rate is high in closed conditions, increase of 1.5 or more is possible. Therefore, it was not considered to be a problem.

Appearance of test solution at preparation is shown in Table 8. No suspended solids, floating solids, precipitation or oily substances were observed and the solutions were colorless in any test group.

Results of Observation of Algae:
In the results of macroscopic observation of color of test cultures, a tendency toward increase in greenness was seen over time in all test groups.

In the results of microscopic observation of cell form at the end of exposure, no changes in cell form (such as contraction, distention, rupture) or cell agglutination were observed, and there were no differences from the control and solvent control groups

Reported statistics and error estimates:

The NOEC values were determined by students t-test or Aspin-Welch’s t-test, subsequent to F test for homogeneity of variances. Statistical analyses were performed using Yukms Statlight #3 software (Yukms Corp., Tokyo) and all test of significance were at α=0.05

Results of preliminary tests:

No.1 (open system: 300 mL glass Erlenmeyer flasks, air-permeable silicon stopper)

Concentration (mg/L)	Growth rate to solvent control group after 72 hours (%)	Analytic results at 0 hours (ratio to nominal)	Analytic result at 72 hours (ratio to nominal value, %)
			With algae	Without algae	Without algae, static
Control group	--	--	--	--	--
Solvent control group	100	--	--	--	--
0.012	133	92	--	--	--
0.0.60	143	--	--	--	--
0.120	111	85	< 2	< 2	78 (tightly stoppered)

No.2 (closed system: 500 mL stoppered glass Erlenmeyer flasks, tightly stoppered)

Concentration (mg/L)	Growth rate to solvent control group after 72 hours (%)	Analytic results at 0 hours (ratio to nominal)	Analytic result at 72 hours (ratio to nominal value, %)
			With algae	Without algae	Without algae, static
Control group	--	--	--	--	--
Solvent control group	100	--	--	--	--
0.012	100	--	--	--	--
0.0.60	99	--	--	--	--
0.120	103 [87]	83	19  [<2]	58 [2]	--

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

Nominal concentration (mg/L)	Measured concentration mg/L (percent of nominal)				Mean^ameasured concentration (mg/L)
	0 hour	24 hours	48 hours	72 hours
Control	<0.003	<0.003	<0.003	<0.003	--
Solvent Control	<0.003	<0.003	<0.003	<0.003	--
0.120	0.102 (85)	0.064 (53)	0.051 (43)	0.028 (23)	0.059 (49)

a: time weighted mean

Table 2: Cell densities of Pseudokirchneriella subcapitata during the 72 hour exposure

Nominal concentration (mean measured conc. mg/L)	Vessel No.	Cell densities (cells/mL)
		0 hour*	24 hours	48 hours	72 hours
Control	1 2 3 4 5 6 Average SD	5000 5000 5000 5000 5000 5000 5000 0	27300 25000 24800 26700 23900 26100 25600 1300	158500 156500 168500 165500 153500 152500 159200 6500	526800 521800 532800 537800 514800 487800 520300 17900
Solvent control	1 2 3 4 5 6 Average SD	5000 5000 5000 5000 5000 5000 5000 0	26800 26400 25500 23800 27100 25200 25800 1200	166500 179500 159500 154500 181500 165500 167800 10700	536800 495800 520800 511800 493800 503800 510500 16400
0.120 [0.059]	1 2 3 4 5 6 Average SD	5000 5000 5000 5000 5000 5000 5000 0	24700 26400 26100 25400 25000 23900 25300 900	150500 185500 149500 166500 155500 154500 160300 13700	489800 520800 483800 510800 461800 464800 488600 23800

a: time weighted men

SD: Standard deviation

*: Nominal initial densities

Table 3: Growth rate of control

Vessel No.	Growth rate
	µ (0-24h)	µ (24-48h)	µ (48-72h)
1	0.0707	0.0733	0.0500
2	0.0671	0.0764	0.0502
3	0.0667	0.0798	0.0480
4	0.0698	0.0760	0.0491
5	0.0652	0.0775	0.0504
6	0.0689	0.0736	0.0484	Average	SD	CV (%)
Average	0.0681	0.0761	0.0494	0.0645	0.0137	21.2
SD	0.0021	0.0024	0.0010
CV%	3.1	3.2	2.0

SD: Standard deviation

CV: Coefficient of variation

Table 4: Growth inhibition (%) of Pseudokirchneriella subcapitata

Nominal concentration [Mean^aMeasured Conc.]		Growth rate		Area under the growth curves
		Rate µ(0-72 h)	Inhibition (%)^*1 Iµ(0-72 h)	Area A (0-72 h)	Inhibition (%)^*1 IA(0-72h)
(mg/L)	Vessel No.
Control	1 2 3 4 5 6	0.0647 0.0646 0.0648 0.0650 0.0644 0.0636		10481000 10318000 10733000 10766000 10135000 9840000
	Average SD	0.0645 0.0005	-	10379000 358000	-
Solvent Control	1 2 3 4 5 6	0.0649 0.0638 0.0645 0.0643 0.0638 0.0641		10781000 10591000 10390000 10121000 10632000 10322000
	Average SD	0.0642 0.0004	-	10473000 240000	-
0.120 [0.059]	1 2 3 4 5 6	0.0637 0.0645 0.0635 0.0643 0.0629 0.0629		9782000 11035000 9720000 10435000 9574000 9559000
	Average SD	0.0636 0.0007	0.9	10018000 593000	4.3

a time weighted mean

*1 Values are the growth inhibition (%) relative to the solvent control.

* Indicates a significant difference (α = 0.05) from the solvent control. (There was no sign in this test)

** Indicates a significant difference (α = 0.01) from the solvent control (There was no sign in this test)

Table 5: Calculated EC50 and NOEC

ErC50 (0-72h) (mg/L)	95 percent confidence limits (mg/L)	NOECr (0-72 h) (mg/L)
>0.059	--	>0.059

Based on IA (0-72h) value (Areas under growth curve)

ErC50 (0-72h) (mg/L)	95 percent confidence limits (mg/L)	NOECr (0-72 h) (mg/L)
>0.059	--	>0.059

The EC50 values and associated 95% confidence limits could not be determined by the least squares linear regression analysis because the test was conducted at one concentration level.-- not calculated.

Table 6: Temperature, light intensity and revolutions in the incubation chamber

Exposure period (hours)	Temperature (°C)	Light intensity (µE/m2/s)	Revolutions (rpm)
2	21.5	60-61	100
24	21.0	60-62	100
48	21.6	61-63	100
72	21.9	63-64	100

Table 7: pH values of test cultures

Nominal concentration [Mean^aMeasured Conc.] (mg/L)	pH
	0 hour	72 hours (Vessel No.)
Control	8.1	10.3       (1)
Solvent control	8.1	10.4       (1)
0.120 [0.059]	8.1	10.1       (1)

a: time weighted mean

Table 8: Appearance of test solutions after preparation

Nominal concentration [Mean^aMeasured Conc.] (mg/L)	Suspended solids	Floating solids	Precipitation	Oily substances	Colour
Control	none	none	none	none	c-
Solvent control	none	none	none	none	c-
0.120 [0.059]	none	none	none	none	c-

a: time weighted mean

c-: colorless

Validity criteria fulfilled:

yes

Conclusions:

The 72 h ErC50 was >0.059 mg/L (maximum attainable concentration)

Description of key information

The 72 h ErC50 was >0.059 mg/L  (maximum attainable concentration)

Key value for chemical safety assessment

EC50 for freshwater algae:

0.059 mg/L

Additional information

Introduction

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

 

Method

1) Culture system: Semi-static (closed system), shaking culture (100 rpm)

2) Exposure period: 72 hours

3) Amount of test solution: 100 mL/vessel

4) Replicates: 6 vessels/test group

5) Initial cell density: Precultured algae 5´103 cells/mL

6) Test temperature: 23±2°C

7) Lighting: Continuous illumination of 65 μE/m2/s (at the surface of the central flask) (variation within the apparatus: within ±8%)

8) pH: pH of the test solutions was not adjusted.

9) Medium: The medium recommended in the Chemical Substances Control Law test guideline “Algal Growth Inhibition Test” was used in both preculturing and the test after filter sterilization (0.22 μm).

10) Test concentrations (nominal values): Control, solvent control, 0.120* mg/L (*Maximum attainable concentration of test solution in limit test)

 

Results

Calculated EC50 and NOEC

ErC50 (0-72h) (mg/L)	95 percent confidence limits (mg/L)	NOECr (0-72 h) (mg/L)
>0.059	--	>0.059

  

Based on IA (0-72h) value (Areas under growth curve)

ErC50 (0-72h) (mg/L)	95 percent confidence limits (mg/L)	NOECr (0-72 h) (mg/L)
>0.059	--	>0.059

 

The EC50 values and associated 95% confidence limits could not be determined by the least squares linear regression analysis because the test was conducted at one concentration level.-- not calculated.

 

Conclusion

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been assessed according to the Methods for Testing of New Chemical Substances, etc. <Algae Growth Inhibition Test, Daphnia Acute Immobilization Test and Fish Acute Toxicity Test> (Yakushokuhatsu No. 1121002, Heisei 15.11.13 Seikyoku No. 2, Kanpokihatsu No. 031121002, 2003). The 72 h ErC50 was >0.059 mg/L (maximum attainable concentration)