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

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:

This study was conducted between 20 December 2016 and 03 February 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese notification, Yakushokuhatsu 0331 No.7, Heisei 23.03.29 Seikyoku No.5, Kanpokihatsu No.110331009, March 31, 2011; the latest revision, December 21, 2015

Deviations:

no

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

Analytical monitoring:

yes

Details on sampling:

Frequency: Two sets (One set consists of a new sample of test solution at the time of preparation and an old sample of the same test solution immediately prior to renewal or at the end of exposure.)
Collection: (i) At the time of preparation: Collected from the middle layer of the vessel for measuring the test conditions of each test group.
(ii) At the time immediately prior to renewal and at the end of exposure: Collected from the middle layer of one vessel of each test group.

Vehicle:

no

Details on test solutions:

Preparation of Test Solutions

A water-soluble fraction* (WSF) was used to expose the test species to the test substance because it was considered that this multi-component test substance was partially soluble in water.

Test concentrations:

Test group Loading rate*1 (mg/L)
Control –
Conc. 1 10
Conc. 2 18
Conc. 3 32
Conc. 4 56
Conc. 5*2 100

Separation factor: 1.8
*1: Ratio of test substance to water (in mg/L) used in the preparation of the WSF.
*2: The loading rate was the maximum concentration given in the Test Guideline.

Test organisms (species):

Daphnia magna

Details on test organisms:

1) Common name: Daphnia magna
2) Scientific name: Daphnia magna
3) Source: National Institute for Environmental Studies, Japan
4) Date of receipt: July 18, 1995 The daphnids have been maintained at the testing facility.
5) Growth stage: Juveniles less than 24-hour-old (female)
6) Culture conditions of the parent daphnids that produced the test juveniles:
Water: Cultured in dechlorinated water for 48 hours or more after rearing in Elendt M4 medium, a reconstituted water recommended in the Test Guideline.
Biomass loading: 1 daphnid or less/80 mL (25 daphnids or less/2 L)
Vessel: 2-L glass beaker
Temperature: 20±1℃
Dissolved oxygen concentration (D.O.): 60% or more of saturation
pH: 6.0-9.0
Light: Fluorescent light, 16-hour light (800 lux or less) / 8-hour dark
Culture period: 28 days or less
Feed: Chlorella vulgaris (unicellular green algae)
Feeding amount: 6 mg C (organic carbon)/2 L / day
Renewal of water in the culture vessel: The first renewal was performed within 5 days after the start of culturing; subsequently, 3 times per week.
Offspring removal: Removed on the day offspring were produced.
7) Procedure of collection of the loading daphnids (Juveniles less than 24-hour-old): Juveniles less than 24-hour-old (female) were used for the test. At the start of exposure, it was confirmed that the parent daphnids fulfilled the following conditions:
Week age: 2-4 weeks (Culture period: January 17, 2017 to February 1, 2017)
Mortality of parent daphnids: 20% or less during the 2-week period prior to the start of exposure
Abnormality: Not observed
Production of first brood: within 12 days

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

≤ 250 mg CaCO3/L

Test temperature:

20 ± 1℃

pH:

6.0 - 9.0

Dissolved oxygen:

>3 mg/L

Nominal and measured concentrations:

Nominal (loading rate): 10, 18, 32, 56, 100 mg/L
Measured: 0.192, 0.326, 0.384, 0.630, 1.45 mg/L

Details on test conditions:

Test Conditions
1) Duration: 48 hours
2) Exposure procedure: Semi-static (batchwise renewal of the test solutions at 24-hour exposure)
3) Volume of test solutions: 100 mL/vessel
4) Number of vessels: 4 vessels/test group
5) Number of loading daphnids: 20 daphnids/test group (5 daphnids/vessel)
6) Temperature: 20 ± 1°C
7) D.O.: 3 mg/L or more, not aerated
8) pH: 6.0–9.0 (variation range <1.5), not adjusted
9) Light: Fluorescent light, 16-hour light (800 lux or less) / 8-hour dark
10) Feeding: None

Results of Range-finding Test
A range-finding test was conducted at loading rates*1 below the upper limit concentration (100 mg/L) given in the Test Guideline. A water-accommodated fraction*2 (WAF) was used as a test solution because the test substance was multi-component test substance and partially soluble in water. The results are given below.
*1: Ratio of test substance to water (in mg/L) used in the preparation of the WAF.
*2: An aqueous fraction containing the dissolved and/or suspended and/or emulsified fraction of a multi-component substance or mixture.

Results of the range-finding test
Loading rate (mg/L) Immobility after 48-hour exposure (%) Concentration of test substance in test solutions (mg/L)
At the start of exposure 24 hours after the start of exposure
Control 0 - -
100 10 6.08 1.78

Number of loading daphnids: 10 daphnids/test group (5 daphnids/vessel, 2 replicates/test group)

Results of First Exposure (rejected)
Based on the results of the range-finding test, the definitive test was conducted. The loading rate and the results of the definitive test are given below.

Test group Loading rate (mg/L) Immobility after 48-hour exposure (%) The time-weighted mean of measured concentration of test substance in test solutions (mg/L)
Control - 0 -
Conc. 1*1 100 15 4.28
*1: Limit test at the loading rate which was the maximum concentration given in the Test Guideline

In Conc. 1, the percentage of immobilisation was 15%. Therefore, this exposure was not adopted. Re-exposure was conducted with the WSF because there was a possibility that the insoluble fraction physically affected the test species.

Test Procedure
The loaded daphnids were introduced into each test vessel using a glass pipette at the start of exposure. The water surface was covered with a Teflon sheet to prevent evaporation of the test solutions and volatilization of the test substance. After 24-hour exposure, daphnids were transferred to the newly prepared test solutions using a glass pipette (renewal). The test ended at 48-hour exposure. During the exposure, observations of the loaded daphnids, measurements of the test conditions and observations of test solutions were performed according to the following procedure:

1) Observation of the loaded daphnids
Frequency: 24-hour (before renewal) and 48-hour exposure.
Items: number of immobilised daphnids *1, other abnormal symptoms*2.

*1: Individuals that were unable to swim within 15 seconds after gentle agitation of the test vessel were considered immobile.
*2: Daphnids showing abnormal behavior or appearance (such as discoloration) and floating daphnids on the surface of the water were recorded, if any.

2) Measurement of test conditions and observation of test solutions
Frequency: Two sets
Samples: (i) At the time of preparation: The test solution in the vessel for measuring the test condition of each test group.
(ii) At the time immediately prior to renewal and at the end of exposure: The test solution in one vessel of each test group.
Items: Temperature, D.O., pH, and appearance of the test solution.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

0.63 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EL0

Effect conc.:

56 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 1.45 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Details on results:

Measurements of Test Conditions and Observations of Test Solutions

In all the test groups, the temperature of the test solutions was in the range of 20 ± 1°C, the D.O. values were 3 mg/L or more, and the pH values were 6.0–9.0 of which the variation was within the range of 1.5, fulfilling the test conditions. In the appearance of test solutions of all the test groups, no suspended substances, no floating substances and no precipitations were observed, and the color was colorless during exposure.

Observation of Loaded Daphnids
In the control, the percentage of immobilisation was 0% at the end of exposure, and no daphnids were observed floating on the surface of the water.
In Conc. 1-5, the percentage of immobilisation was 0, 5, 0, 0, and 5%, respectively, at the end of exposure. The immobilised daphnid in Conc. 2 was judged not to be affected by the test substance, because there was not a correlation between concentration and the response. Based on the results of the range-finding test and the rejected exposure, it was considered that the immobilised daphnid in Conc. 5 was affected by the test substance.

Median Effect Concentration (EC50) and Median Effect Loading Rate (EL50)
The 48-hour EC50 and EL50 are also given below.

48-hour EC50: >1.45 mg/L (95% confidence limits: not available)
48-hour EL50: >100 mg/L (95% confidence limits: not available)

Highest Concentration with 0% Immobility (EC0) and Highest Loading Rate with 0% Immobility (EL0), Lowest Concentration with 100% Immobility (EC100) and Lowest Loading Rate with 100% Immobility (EL100)
The EC0, EL0, EC100 and EL100 values at the end of exposure are given below.
48-hour EC0: 0.630 mg/L
48-hour EL0: 56 mg/L
48-hour EC100: not observed
48-hour EL100: not observed

Validity of the Test
This test is judged valid because the results satisfied the validity criteria.

Results with reference substance (positive control):

Acute immobilisation tests of the reference substance (potassium dichromate, guaranteed reagent) have been conducted periodically (approximately every 6 months).
The 48-hour median effect concentration (EC50) values are described below.

Latest result:
0.75 mg/L (95% confidence limits: 0.67–0.83 mg/L)
Exposure period: January 12, 2017 to January 14, 2017

The EC50 of reference substance fell within the standard range defined by the testing facility (average of EC50 of reference substance in the past ± 5 times of the standard deviation).
Result since June 1998:
Average ± standard deviation: 0.72 ± 0.12 mg/L, n=38
(Minimum–maximum: 0.49–1.02 mg/L)

 Table 1 The Number of Immobilised Daphnia magna (Percent Immobility)

Test Group	Loading^a Rate	Mean^b Measured Concentration	Cumulative Number of ImmobilisedDaphnia (PercentImmobility)
	(mg/L)	(mg/L)	24 Hours		48 Hours
Control	--	--	0	(0)	0	(0)
Conc.1	10	0.192	0	(0)	0	(0)
Conc.2	18	0.326	1	(5)	1	(5)
Conc.3	32	0.384	0	(0)	0	(0)
Conc.4	56	0.630	1	(5)	0	(0)
Conc.5	100	1.45	0	(0)	1	(5)

a:      The ratio of test substance to water (in mg/L) used in the preparation of the WSF

b:       Time-weighted mean

Table 2          Median Effect Concentration (EC50) and Median Effect Loading Rate (EL50)

(1) EC50

Exposure Period (Hours)	EC50 (mg/L)	95-Percent Confidence Limits (mg/L)			Statistical Method
24	>1.45^*1		--		--
48	>1.45^*1		--		--

*1:  The EC50 value and its 95% confidence limits could not be determined by statistical method because the immobility ofDaphniaat the maximum concentration level was less than 50%. Therefore, the EC50 value was given as an estimated concentration range.

(2) EL50

Exposure Period (Hours)	EL50 (mg/L)	95-Percent Confidence Limits (mg/L)			Statistical Method
24	>100^*2		--		--
48	>100^*2		--		--

*2:      The EL50 value and its 95% confidence limits could not be determined by statistical method because the immobility ofDaphniaat the maximum concentration level was less than 50%. Therefore, the EL50 value was given as an estimated concentration range

Table 3   Highest concentration with 0% immobility (EC0) and highest loading rate with 0% immobility (EL0), lowest concentration with 100% immobility (EC100) and lowest loading rate with 100% immobility (EL100)

 

(1) EC0 and EC100 

Exposure Period (Hours)	EC0 (mg/L)	EC100 (mg/L)
48	0.630	--

-- :  Not observed

 (2) EL0 and EL100 

Exposure Period (Hours)	EL0 (mg/L)	EL100 (mg/L)
48	56	--

-- :  Not observed

 

Validity criteria fulfilled:

yes

Conclusions:

The result given below was calculated from the time-weighted mean of measured concentrations and loading rate. 
   48-hour median effect concentration (EC50): >1.45mg/L (95% confidence limits: not available)
   48-hour median effect loading rate (EL50): >100mg/L (95% confidence limits: not available)

Executive summary:

The study was conducted in accordance with the test method relating to new chemical substances "Alga growth inhibition test, Daphniasp.acute immobilisation test, and fish acute toxicity test" (Japanese notification, Yakushokuhatsu 0331 No.7, Heisei 23.03.29 Seikyoku No.5, Kanpokihatsu No.110331009, March 31, 2011; the latest revision, December 21, 2015) and the OECD Guideline for Testing of Chemicals No.202 “Daphniasp.,Acute Immobilisation Test” (2004).

 

The test was conducted with Daphnia magna over a 48 -hour exposure period.

Based on the results of the preliminary test, the definitive test was carried out at loading rate of 10, 18, 32, 56 and 100 mg/L. A concurrent blank control was performed in parallel. 4 replicates were used with 5 daphnia for each replicate. The test was performed with a semi- static system over 48 hours.

Results

The result given below was calculated from the time-weighted mean of measured concentrations and loading rate. 

   48-hour median effect concentration (EC50): >1.45mg/L (95% confidence limits: not available)

   48-hour median effect loading rate (EL50): >100mg/L (95% confidence limits: not available)

Description of key information

Alga growth inhibition test was conducted based OECD 202.

The result given below was calculated from the time-weighted mean of measured concentrations and loading rate. 

   48-hour median effect concentration (EC50): >1.45 mg/L (95% confidence limits: not available)

   48-hour median effect loading rate (EL50): >100 mg/L (95% confidence limits: not available)

Less than 50% immobilisation occured at loading levels up to 100 mg/L therefore an accurate EL50 could not determined however the EL50 must be greater than 100 mg/L (measured concentration 1.45 mg/L).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L

Additional information

The registered substance is insoluble with water solubility of 1mg/L at 20℃. The test results showed 48h EL50>100mg/L which indicating no hazard identified at highest loading rate. Therefore LC50=100mg/L is used for CSR for worst cases.