4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with [(dimethylamino)methyl]phenol and piperazine

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:

9th December 2016 - 7th March 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Alga growth inhibition test, Daphnia sp. acute immobilisation test, and fish acute toxicity test (Japanese notification, Yakushokuhatsu 0331 No.7, Heisei 23.03.29 Seikyoku No.5, Kanpokihatsu No.110331009)

Version / remarks:

The latest revision, December 21, 2015

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name of the new chemical substance (by IUPAC nomenclature): 4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with [(dimethylamino)methyl]phenol and piperazine
Alternate name: THEMIS
CAS RN: 159034-96-5
Molecular weight: Mn:3,000，Mw:11,000
Purity (%) of the new chemical substance subjected to the study: 100.0
Lot number of the new chemical substance subjected to the study: 160715
Melting point: 120 to 140°C
Appearance at room temperature: powder, pale pink
Stability: Swell on exposure to water; Soften at greater than or equal to 100°C
Source: 1) Supplier: Ajinomoto Fine-Techno Co., Inc.; 2) Expiration date: July 20, 2017
Storage condition: room temperature, shaded, air-tight
Storage area: desiccator

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The concentrations of the test substance in all test solutions (10, 18, 32, 56, 100 mg/L) and test cultures were measured at the beginning of exposure and at 24, 48 and 72 hours after the beginning of exposure by Liquid Chromatography/Mass Spectrometry (LC/MS).
- Sampling method: Collect 1.0 mL of each test solution from the middle layer of all test vessels and mix

Vehicle:

no

Details on test solutions:

Preparation of Test Solutions
Appropriate amount of sample was collected into media bottle，and the dilution water was added and mixed using the method of stirred with a magnetic stirrer for 48 hours. And then filtrated with membrane filter HA 0.45 µm.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Unicellular green algae
- Strain: ATCC22662
- Source (laboratory, culture collection): American Type Culture Collection
- Age of inoculum (at test initiation): not specified.

ACCLIMATION
Preculture period: From January 27 to 30, 2017
In order to adapt the algae to the test conditions and ensure that the algae were in the exponential growth phase when used to inoculate the test solutions, the algae were precultured under the same conditions as the test culture. Neither modified nor unusual cells were confirmed.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22°C, variation range over the exposure period is within ±2°C

pH:

At the beginning of exposure:7.9 to 8.0
72 hours after the beginning of exposure: 7.8

Nominal and measured concentrations:

Nominal (loading rate): 10, 18, 32, 56, 100 mg/L
Measured: 0.236, 0.253, 2.00, 1.31, 2.16 mg/L

Details on test conditions:

Test Conditions:
Test vessels and other apparatus were sterilized as necessary. Algae were introduced into culture medium by an aseptic technique.
1) Exposure procedure:Static, open system, and shaking (100 rpm)
2) Duration: 72 hours
3) Volume of test solution:100 mL/vessel, (99 mL/vessel when algae were inoculated to the test solution. 1 mL/vessel was used for analysis before inoculation)
4) Number of vessels: 6 vessels/control group, 3 vessels/concentration group
5) Initial biomass:5 E+03 cells/mL, algae of exponential growth phase (dried weight of algae at the exponential growth phase: 1.8 E-8 mg/cell, n=23)
6) Temperature: 22°C, variation range over the exposure period is within ±2°C
7) Light: 65 to 75 µE/m2/s, continuous illumination with white fluorescent lamp (on the surface of test culture)
8) pH: not adjusted

Results of Range-finding Test
- Test concentrations: 100 mg/L
- Results used to determine the conditions for the definitive study: Inhibition Iμ(0-72h): 1.9%.
The concentration of the test substance was decreased at 72 hours after the beginning of exposure. Although the details were unknown, the causes for the concentration decrease were considered that the test substance was difficult to be dissolved stably in the test solution because the test solution was the WAF. In addition, transfer of the test substance to algae and the effect of pretreatment for analysis (centrifugation operation) were also considered as the causes for concentration decrease.

Results of First Exposure (rejected)
- Test concentrations: 100 mg/L
- Results: Since the biomass of 100 mg/L was obviously less than that of control at 24 hours after the beginning of exposure and the growth inhibition was observed. Re-exposure was conducted with the WSF because there was a possibility that the insoluble fraction physically affected the test organisms.

Test Concentrations
- Spacing factor for test concentrations: 1.8

Effect Parameters Measured
- Measurement of biomass: The biomass of algae in each test vessel was measured at 24, 48 and 72 hours after the beginning of exposure. One milliliter of test culture (0.5 mL at 72 hours) was suspended in 9.0 mL of electrolyte (9.5 mL at 72 hours) to count the algal cells by electronic particle counter.
- Color observation of test culture and microscopic observation of algae: The color of the test cultures was observed by comparison with the extra vessel of each test group at 0, 24, 48 and 72 hours after the beginning of exposure. At 72 hours after the beginning of exposure, appearance of algae was observed through the microscope.
- Measurement of test conditions: The pH values of the test solutions and the test cultures were measured at the beginning of exposure and at 72 hours after the beginning of exposure. At the beginning of exposure, a part of the test solution in the extra vessel was collected and pH was measured. At 72 hours after the beginning of exposure, pH of the test culture was measured in one vessel (No.1) in each test group. During the exposure period, temperatures, light intensities, and revolutions in the culturing apparatus were measured at least once a day.

Reference substance (positive control):

yes

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 2.16 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.16 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Details on results:

Conditions in the Culturing Apparatus, pH of Test Solutions and Test Cultures, and Appearance of Test Solutions
The temperature in the culturing apparatus during exposure period ranged within 22±2°C, nominal value. The pH values of test solutions at the beginning of exposure were 7.9 to 8.0 and test cultures at 72 hours after the beginning of exposure were all 7.8. Test solutions before inoculation were colorless, and showed no suspended solids, no floating solids, and no precipitates in all test groups.

Concentrations of Test Substance in Test Solutions and Test Cultures
Measured concentration of the concentration group 1 to 5 at the beginning of exposure were 0.235, 0.242, 3.03, 0.861 and 3.23 mg/L, respectively and the measured values of concentration group 3 and 4 were inverse to the order of the loading rate. Although the details were unknown, it was considered that the dissolved state of each component was changed due to a slight difference in the stirring state at the preparation, which might have affected the inverse of concentrations.
The time weighted means of measured concentrations of the test substance in concentration group 1 to 5 were 0.236, 0.253, 2.00, 1.31 and 2.16 mg/L, respectively.

50% Growth Inhibition Concentration (EC50), 50% Growth Inhibition Loading Rate (EL50), No Observed Effect Concentration (NOEC), and No Observed Effect Loading Rate (NOELR)
The growth rate and percent inhibition of the growth rate are shown in Table 2. Fifty percent Growth Inhibition Concentration (EC50), 50% Growth Inhibition Loading Rate (EL50), No Observed Effect Concentration (NOEC), and No Observed Effect Loading Rate (NOELR) are shown in Table 3. The ErC50, ErL50, NOECr, and NOELRr values are shown below.

ErC50 (0-72h): >2.16 mg/L (95% confidence limits: not available)
NOECr (0-72h): 2.16 mg/L

ErL50 (0-72h): >100 mg/L (95% confidence limits: not available)
NOELRr (0-72h): 100 mg/L

The highest concentration of the test was the maximum test concentration (loading rate: 100 mg/L, time weighted mean of measured concentrations: 2.16 mg/L) given in the Test Guideline. As the inhibition rate at the highest concentration was less than 50%, the ErC50 and the ErL50 values were shown as the range, “> highest concentration”.
The NOECr and the NOELRr values were determined by an analysis of variance (ANOVA), Williams test, subsequent to Bartlett test for homogeneity of variances. All tests of significance level were set at α=0.05, except Bartlett test, which was at α=0.01.

Observation of Algae
In all test groups, the color of the test cultures (as observed with the naked eye) showed a tendency to get more greenish during the exposure period.
By microscopic observation at the end of exposure, in all concentration groups neither unusual cell shape of algae (contraction, expansion, damaged cell etc.) nor agglutination was observed, and the algae looked normal compared with those in the control group.

Validity of the Test
The biomass in the control cultures increased exponentially above 16 fold during 72-hour culture. The mean coefficient of variation for section by section specific growth rates in the control cultures and the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 35% and 7%, respectively.
This test is valid because these results met the test validity criteria

Results with reference substance (positive control):

Growth inhibition test of a reference substance (potassium dichromate, guaranteed reagent) has been conducted periodically (approximately every six months) and 72-hour medium growth inhibition concentrations were estimated by comparison of growth rates (ErC50). The latest value of ErC50 was follows.
ErC50 = 0.745 mg/L (95% confidence limits: 0.722-0.768 mg/L, Exposure period: From December 6 to 9, 2016)
The ErC50 of reference substance fell within the standard range defined by the testing facility (average of ErC50 of reference substance in the past ± 5 times of the standard deviation).
The average values of ErC50 since June, 2000 were as follows.
Average ErC50 standard deviation = 0.811 ± 0.0711 mg/L, n=34, (Minimum-maximum = 0.687-0.965 mg/L)

Reported statistics and error estimates:

TheErC50 and ErL50values and associated 95% confidence limits could not be determined by least squares linear regression analysis because the growth inhibition (%) at the maximum concentration level was less than 50%. Therefore, the ErC50and ErL50values were given in estimated concentration range.
 
The NOECr and NOELRr values were determined by an analysis of variance (ANOVA),Williamstest, subsequent to Bartlett test for homogeneity of variances. Statistical analyses were performed using Yukms Statlight #4 software (Yukms Corp., Tokyo) and all tests of significance level were set at α=0.05, except Bartlett test, which was set at α=0.01.

Table 1          Measured Concentration of the Test Substance in Test solutions andTest Cultures

Test Group	Loading Rate (mg/L)	Measured Concentration (mg/L)				Mean^aMeasured Concentration (mg/L)
		0 Hour	24 Hours	48 Hours	72 Hours
Control	--	--	--	--	--	----
Conc.1	10	0.235	0.286	0.205	0.208	0.236
Conc.2	18	0.242	0.294	0.239	0.212	0.253
Conc.3	32	3.03	1.90	1.78	1.68	2.00
Conc.4	56	0.861	1.57	1.62	0.784	1.31
Conc.5	100	3.23	1.91	2.24	1.59	2.16

a         : Time weighted mean

Table 2           Growth Inhibitions (%) of Pseudokirchneriella subcapitata

Test Group	Loading Rate [Mean^aMeasured Concentration] (mg/L)	Vessel No.	Growth Rate
			Rate	Inhibition(%)^*1
			μ(0-72h)	Iμ(0-72h)
		1	0.0803
		2	0.0773
		3	0.0808
Control	--	4	0.0796
		5	0.0796
		6	0.0785
		Average	0.0794	-
		SD	0.0013
		1	0.0778	2.0
	10	2	0.0807	-1.6
Conc.1	[0.236]	3	0.0813	-2.4
		Average	0.0799	-0.7
		SD	0.0019
		1	0.0818	-3.0
	18	2	0.0808	-1.8
Conc.2	[0.253]	3	0.0798	-0.5
		Average	0.0808	-1.8
		SD	0.0010
		1	0.0805	-1.4
	32	2	0.0826	-4.0
Conc.3	[2.00]	3	0.0799	-0.6
		Average	0.0810	-2.0
		SD	0.0014
		1	0.0804	-1.3
	56	2	0.0788	0.8
Conc.4	[1.31]	3	0.0788	0.8
		Average	0.0793	0.1
		SD	0.0009
		1	0.0810	-2.0
	100	2	0.0779	1.9
Conc.5	[2.16]	3	0.0786	1.0
		Average	0.0792	0.3
		SD	0.0016

a     : Time weighted mean

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

SD : Standard deviation

There is no significant difference (α<0.05)between the concentration group and the control.

Table 3     EC50 and NOEC                     

                   Based onI_μ(0-72h) values (Growth rates)

ErC50 (0-72h) (mg/L)	95-Percent Confidence Limits (mg/L)	NOECr (0-72h) (mg/L)
>2.16	--	2.16

--　Not calculated

Table 4     EL50 and NOELR                     

                   Based onI_μ(0-72h) values (Growth rates)

ErL50 (0-72h) (mg/L)	95-Percent Confidence Limits (mg/L)	NOELRr (0-72h) (mg/L)
>100	--	100

--　Not calculated

 

Validity criteria fulfilled:

yes

Conclusions:

50% growth inhibition concentration, ErC50 (0-72h) was determined to be >2.16 mg/L (95% confidence limits: not available) and No observed effect concentration, NOECr (0-72h) was 2.16 mg/L.
50% growth inhibition loading rate, ErL50 (0-72h) was determined to be >100 mg/L (95% confidence limits: not available), and No observed effect loading rate, NOELRr (0-72h) was 100 mg/L.

Executive summary:

The inhibition of test item to the growth of Pseudokirchneriella subcapitata, Unicellular green algae was assessed based on OECD 201. A static test was conducted and the test organisms were exposed to the test substance at loading rate of 10, 18, 32, 56, 100 mg/L for 72 hours.

The time weighted means of measured concentrations of the test substance were 0.236, 0.253, 2.00, 1.31 and 2.16 mg/L, respectively.

By microscopic observation at the end of exposure, in the concentration groups neither unusual cell shape of algae (contraction, expansion, damaged cell etc.) nor agglutination was observed, and the algae looked normal compared with that in the control group.

 

The Inhibition results were calculated from the time weighted mean of measured concentrations. 

50% growth inhibition concentration, ErC50 (0-72h) was determined to be >2.16 mg/L (95% confidence limits: not available) and No observed effect concentration, NOECr (0-72h) was 2.16 mg/L.

50% growth inhibition loading rate, ErL50 (0-72h) was determined to be >100 mg/L (95% confidence limits: not available), and No observed effect loading rate, NOELRr (0-72h) was 100 mg/L.

Description of key information

The inhibition of test item to the growth of Pseudokirchneriella subcapitata, Unicellular green algae was assessed based on OECD 201.

 

50% growth inhibition concentration, ErC50 (0-72h) was determined to be >2.16 mg/L (95% confidence limits: not available) and No observed effect concentration, NOECr (0-72h) was 2.16 mg/L.

50% growth inhibition loading rate, ErL50 (0-72h) was determined to be >100 mg/L (95% confidence limits: not available), and No observed effect loading rate, NOELRr (0-72h) was 100 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

The registered substance is insoluble with water solubility of 1mg/L at 20℃. The test results showed 72h ErL50>100mg/L and NOELRr=100mg/L, which indicating no hazard identified at highest loading rate. Therefore ErC50=100mg/L, and NOEC = 100mg/L is used for CSR for worst cases.