4,4'-isopropylidenebis[2-allylphenol]

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Testing was conducted between 3rd August 2014 and 13th October 2014.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

Principles of method if other than guideline:

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC, 1996 and OECD, 2000), is to expose organisms to a saturated solution of the test item in cases where the test item is of high purity and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, a saturated solution was prepared by stirring an excess (50 mg/L) of test item in culture medium for a period of 24 hours prior to removing any undissolved test item present by filtration (0.2 µm Sartorius Sartopore, first approximate 2 liters discarded in order to pre-condition the filter) to give a saturated solution of the test item.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-isopropylidenebis[2-allylphenol]

EC Number:

217-121-1

EC Name:

4,4'-isopropylidenebis[2-allylphenol]

Cas Number:

1745-89-7

Molecular formula:

C21H24O2

IUPAC Name:

4-{1-[4-hydroxy-3-(prop-2-en-1-yl)phenyl]propyl}-2-(prop-2-en-1-yl)phenol

Test material form:

liquid: viscous

Details on test material:

Identification: 4,4'-isopropylidenebis[2-allylphenol] (TK 11907)
Huntsman description: Liquid
Harlan description: Straw colored viscous liquid
Batch: AG16A55043
Purity: 89%
Expiry date: 11 June 2016
Storage conditions: Room temperature in the dark

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Water samples were taken from the control and all surviving test groups from fresh preparations at 0 and 72 hours and from old solutions at 24 and 96 hours for quantitative analysis. The samples were stored frozen prior to analysis.

Duplicate samples and samples at 24 (fresh media), 48 (old and fresh media), and 72 hours (old media) were taken and stored frozen for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

Test Water
The test water used for both the range-finding and definitive tests was the same as that used to maintain the stock fish.

Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 140 mg/L as CaCO3. After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature.

Procedure
Preliminary Media Preparation Trial
Information provided by the Sponsor indicated the test item was practically insoluble in water.

Preliminary solubility work conducted indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing.

Based on this information the test item was categorized as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test item under test conditions.

Test organisms

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

Test Species
The test was carried out using juvenile rainbow trout (Oncorhynchus mykiss). Fish were obtained from Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK and maintained in house since 03 September 2014. Fish were maintained in a glass fiber tank with a "single pass" water renewal system. Fish were acclimatized to test conditions from
16 September 2014 to 23 September 2014. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.

The water temperature was controlled at approximately 14 °C with a dissolved oxygen content of greater than or equal to 8.4 mg O2/L. These parameters were recorded daily. The stock fish were fed commercial trout pellets which was discontinued approximately 24 hours prior to the start of the definitive test. There was less than 1% mortality in the 7 days prior to the start of the test and the fish had a mean standard length of 5.1 cm (sd = 0.2) and a mean weight of 1.60 g
(sd = 0.17) at the end of the definitive test. Based on the mean weight value this gave a loading rate of 0.56 g bodyweight/liter (static volume).

The diet and diluent water are considered not to contain any contaminant that would affect the integrity and outcome of the study.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

None

Test conditions

Hardness:

No data

Test temperature:

Temperature was maintained at approximately 14 °C throughout the test

pH:

The pH was 7.8 at with fresh media and 8.1 - 8.3 with old media. There were no treatment related differences.

Dissolved oxygen:

dissolved oxygen was 9.1 to 10.2 mg O2 / L throughout the study. There were no treatment related differences.

Salinity:

Not applicable as a fresh water study was conducted

Nominal and measured concentrations:

Initial test nominal concentration: 2.5 % v/v saturated solution.
Definitive test nominal concentration: 0.625, 1.25, 2.5, 5.0 and 10% v/v saturated solution.

Details on test conditions:

Initial Test
In accordance with the recommendations of REACh, an initial test was conducted according to the threshold approach recommended by ECHA. Using this approach the lowest EC50 value from either the Algal Growth Inhibition study or Acute Toxicity to Daphnia magna study is set as the threshold concentration and a “Limit test” is conducted at this threshold concentration. If no mortalities are observed this indicates that fish are not the most sensitive species and that the LC50 is greater than the threshold concentration. The EC50 value obtained from the Acute Toxicity to Daphnia magna (Harlan Study Number 41401337) was the lowest of these two EC50 values and hence the test was conducted at a single concentration of 2.5 % v/v saturated solution.

A nominal amount of test item (1125 mg) was dispersed in 22.5 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours in the dark. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A dilution was made from this saturated solution to give a test solution of 2.5% v/v saturated solution.

In the initial test seven fish were added to each 20 liter test and control vessel and maintained at approximately 14 C in a temperature controlled room in the dark for a period of 96 hours under semi-static test conditions. A semi-static test regime was employed in the test involving a daily renewal of the test preparations to prevent the build up of nitrogenous waste products. The control group was maintained under identical conditions but not exposed to the test item.

Each vessel was covered to reduce evaporation. After 3, 6, 24, 48 and 70 hours any mortalities or sub-lethal effects of exposure were determined by visual inspection of the test fish.

A sample of each control and test concentration was taken for chemical analysis at 0, 24 and
48 hours and stored frozen, however as mortalities were observed in the test that resulted in the test being terminated chemical analysis of these samples was not conducted.

Definitive Test
Based on the results of the initial test the following test concentrations were assigned to the definitive test: 0.625, 1.25, 2.5, 5.0 and 10% v/v saturated solution.


Experimental Preparation
A nominal amount of test item (1125 mg) was weighed out under non-actinic light and dispersed in 22.5 liters of test water with the aid of propeller stirring at approximately 1500 rpm for
24 hours in the dark. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give test solutions of 0.625, 1.25, 2.5, 5.0 and 10% v/v saturated solution.

Each test solution was mixed with a flat bladed stirrer for one minute to ensure adequate mixing and homogeneity.

The concentration and stability of the test item in the test preparations were verified by chemical analysis of the fresh media at 0 and 72 hours and of the old media at 24 and 96 hours.


Exposure Conditions
As in the initial test, 25-30 liter glass exposure vessels were used for each test concentration. At the start of the test seven fish were placed in each test vessel at random, in the test preparations. The test vessels were then covered to reduce evaporation and maintained at approximately 14 C in a temperature controlled room in the dark for a period of 96 hours. The test vessels were aerated via narrow bore glass tubes. The fish were not individually identified and received no food during exposure.

The control group was maintained under identical conditions but not exposed to the test item.

A semi-static test regime was employed in the test involving a daily renewal of the test preparations to prevent the build up of nitrogenous waste products.

Evaluations
Test Organism Observations
Any mortalities and sub-lethal effects of exposure were recorded at 3, 6, 24, 48, 72 and 96 hours after the start of exposure. The criteria of death were taken to be the absence of both respiratory movement and response to physical stimulation.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

0.21 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% confidence limits (0.18 - 0.25)

Effect concentrations 2

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.15 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 3

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

0.36 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

Mortality Data
Analysis of the mortality data by the Linear Interpolation method at 24, 48 and 72 hours and by trimmed Spearman-Karber method (Hamilton et al, 1977) at 96 hours based on the time-weighted mean measured test concentrations gave the following results:

The results of the definitive test showed the highest test concentration resulting in 0% mortality to be 0.092 mg/L, the lowest test concentration resulting in 100% mortality to be 0.36 mg/L. The Lowest Observed Effect Concentration (LOEC) was considered to be 0.36 mg/L. The No Observed Effect Concentration (NOEC) was 0.15 mg/L.

Sub-Lethal Effects
Sub-lethal effects of exposure were observed at test concentrations of 0.15 mg/L and above. These responses were moribund, moribund with swollen abdomen, swimming at bottom, darkened pigmentation and exaggerated gill movement.

After approximately 6 hours exposure 7 out of 7 fish at 2.2 mg/L were observed to be moribund. Due to animal welfare implications (Animals (Scientific Procedures) Act 1986) these fish were killed and classed as mortalities for the following observational time point.

After approximately 24 hours exposure 7 out of 7 fish at 0.79 mg/L were observed to be moribund. Due to animal welfare implications (Animals (Scientific Procedures) Act 1986) these fish were killed and classed as mortalities for the following observational time point.

After approximately 47 hours exposure 4 out of 4 fish at 0.36 mg/L were observed to be moribund. Due to animal welfare implications (Animals (Scientific Procedures) Act 1986) these fish were killed and classed as mortalities for the following observational time point.

After approximately 96 hours exposure 1 out of 7 fish at 0.15 mg/L were observed to be moribund. Due to animal welfare implications (Animals (Scientific Procedures) Act 1986) this fish was killed and classed as mortalities for the following observational time point.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

The LC50 values and associated confidence limits at 24, 48 and 72 hours were calculated by the Linear Interpolation method using the ToxCalc computer software package (ToxCalc, 1999) and at 96 hours by the trimmed Spearman-Karber method (Hamilton et al, 1977) using the ToxCalc computer software package (ToxCalc, 1999) and at 6 hours the LC50 value was calculated using the geometric mean method as follows:

LC50 value = √C1 x √C2

Where:

C1 = concentration showing 0% mortality
C2 = concentration showing 100% mortality

When only one partial response is shown the trimmed Spearman-Karber method is appropriate.

If there are no test concentrations showing mortalities between 0% and 100%, then the Linear Interpolation method is used. The concentrations resulting in 0% and 100% mortality will be the 95% confidence limits.


Time-Weighted Mean Measured Test Concentrations
The time-weighted mean measured test concentrations (OECD, 2008) were calculated as follows:

Area = ((C0 – C1) / (1n(C0) – 1n(c1))) x days

TWM = (total area / total number of days of the test

Area = area under the exponential curve for each renewal period
C0 = measured concentration at the start of each renewal period (mg/L)
C1 = measured concentration at the end of each renewal period (mg/L)
Days = number of days in the renewal period
TWM = time-weighted mean measured test concentration (mg/L)

Any other information on results incl. tables

Sublethal observations / clinical signs:

Initial Test Results

After approximately 70 hours exposure 3 out of 7 fish at 2.5% v/v saturated solution were observed dead, 3 out of 7 were found to be moribund and 1 out of 7 fish was observed swimming abnormally. Due to animal welfare implications (Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012) these fish were killed as the severity limit for the test type had been exceeded.

 

Based on this information test concentrations of 0.625, 1.25, 2.5, 5.0 and 10% v/v saturated solution were selected for the definitive test.

Verification of Test Concentrations

Analysis of the fresh test preparations at 0 and 72 hours showed measured test concentrations to range from 0.0636 to 2.35 mg/L. A decline in measured test concentration was observed in the old media at 24 and 96 hours to between 0.0483 and 2.01 mg/L and hence it was considered appropriate to calculate the results based on the time-weighted mean measured test concentrations only in order to give a “worst case” analysis of the data.

 

The time-weighted mean measured test concentrations were determined to be:

 

Nominal Test Concentration (% v/v Saturated Solution)	Time-Weighted Mean Measured Test Concentration (mg/L)
0.625	0.092
1.25	0.15
2.5	0.36
5.0	0.79
10	2.2

 

Mortality Data

Time (h)	LC50(mg/L)	95% Confidence limits (mg/L)
3	>2.2	-
6	1.3	0.79 – 2.2*
24	0.58	0.36 – 0.79*
48	0.26	0.15 – 0.36*
72	0.26	0.15 – 0.36*
96	0.21	0.18 – 0.25

* Concentrations resulting in 0% and 100% mortalities respectively

Validation Criteria

The test was considered to be valid given that none of the control fish died or showed signs of stress during the test and that the oxygen concentration at the end of the test was ≥60% of ASV (6.1 mg O₂/L) in the control and test vessels.

 

Water Quality Criteria

Temperature was maintained at approximately 14 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

 

Observations on Test Item Solubility

The test item preparations were observed to be clear colorless solutions throughout the test.

CumulativeMortality Data in the Initial Test

Nominal Concentration (% v/v Saturated Solution)	Cumulative Mortality (Initial Population = 7)
	3 Hours	6 Hours	24 Hours	48 Hours	70 Hours
Control	0	0	0	0	0
2.5	0	0	0	0	7*

*   4 fish killed in extremis at approximately70 hours, and classed as mortalities.

Sub-lethal Effects of Exposure in the Initial Test

Nominal Concentration (% v/v Saturated Solution)	Sub-lethal Effects	Time (Hours)
		3	6	24	48	70
Control	No abnormalities detected	7	7	7	7	7
2.5	No abnormalities detected	7	7	7	7	0
	Moribund	0	0	0	0	3
	Abnormal swimming	0	0	0	0	1

 

Cumulative Mortality Data in the DefinitiveTest

Nominal Concentration (% v/v Saturated Solution)	Time-weighted Mean Measured Test Concentration (mg/L)	Cumulative Mortality (Initial Population = 7)						% Mortality
		3 Hours	6 Hours	24 Hours	48 Hours	72 Hours	96 Hours	96 Hours
Control		0	0	0	0	0	0	0
0.625	0.092	0	0	0	0	0	0	0
1.25	0.15	0	0	0	0	0	1^****	14
2.5	0.36	0	0	0	7^***	7	7	100
5.0	0.79	0	0	7^**	7	7	7	100
10	2.2	0	7*	7	7	7	7	100

*7 fish killed in extremis at approximately 6 hours, classed as mortality at6hours

^**7 fish killed in extremis at approximately 24 hours, classed as mortality at 24 hours

^***4 fish killed in extremis at approximately 47 hours, classed as mortality at 48 hours

^****1 fish killed in extremis at approximately 96hours, classed as mortality at 96 hours

Sub-lethal Effects of Exposure in the DefinitiveTest

Nominal Concentration (% v/v Saturated Solution)	Sub-lethal Effects	Time (Hours)
		3	6	24	48	72	96
Control	No abnormalities detected	7	7	7	7	7	7
0.625	No abnormalities detected	7	7	7	7	7	7
1.25	No abnormalities detected	7	7	7	7	7	6
	Moribund	0	0	0	0	0	1^**
2.5	No abnormalities detected	7	7	7	0	A/D	A/D
	Moribund with swollen abdomen	0	0	0	4^*
5.0	No abnormalities detected	7	7	0	A/D	A/D	A/D
	Moribund	0	0	7
10	Swimming at bottom	7	0	A/D	A/D	A/D	A/D
	Darkened pigmentation	4	0
	Exaggerated gill movement	7	0
	Moribund	0	7

^*Observation of sub-lethal effects recorded at approximately 47 hours, 4 fish from 2.5% v/v saturated solution killedin extremis, classed as mortality at 48 hours.

^**Observation of sub-lethal effects recorded at approximately 96 hours, 1 fish from 1.25% v/v saturated solution killedin extremis, classed as mortality at 96 hours

A/D = All fish dead

Water Quality Measurements

Nominal Concentration (% v/v Saturated Solution)	Time (Hours)
	0 Hours (Fresh Media)			24 Hours (Old Media)
	pH	mg O2/L	T ºC	pH	mg O2/L	T°C
Control	7.8	9.3	14	8.1	10.0	14
0.625	7.8	9.5	14	8.1	10.0	14
1.25	7.8	9.6	14	8.1	10.0	14
2.5	7.8	9.6	14	8.2	9.9	14
5.0	7.8	9.6	15	8.1	9.8	14
10	7.8	9.5	14	8.1	10.0	15

 

Nominal Concentration (% v/v Saturated Solution)	Time (Hours)
	24 Hours (Fresh Media)			48 Hours (Old Media)
	pH	mg O2/L	T ºC	pH	mg O2/L	T°C
Control	7.8	9.2	14	8.3	10.1	14
0.625	7.8	9.5	14	8.2	10.0	14
1.25	7.8	9.3	14	8.2	9.9	14
2.5	7.8	9.4	14	8.2	10.2	14
5.0	A/D			A/D
10	A/D			A/D

 

Nominal Concentration (% v/v Saturated Solution)	Time (Hours)
	48 Hours (Fresh Media)			72 Hours (Old Media)
	pH	mg O2/L	T ºC	pH	mg O2/L	T°C
Control	7.8	9.3	14	8.3	10.1	14
0.625	7.8	9.4	14	8.2	9.8	14
1.25	7.9	9.4	14	8.2	10.0	14
2.5	A/D			A/D
5.0	A/D			A/D
10	A/D			A/D

 

Nominal Concentration (% v/v Saturated Solution)	Time (Hours)
	72 Hours (Fresh Media)			96 Hours (Old Media)
	pH	mg O2/L	T ºC	pH	mg O2/L	T°C
Control	7.8	9.2	15	8.3	10.0	14
0.625	7.8	9.1	14	8.3	9.8	14
1.25	7.8	9.2	14	8.2	9.8	14
2.5	A/D			A/D
5.0	A/D			A/D
10	A/D			A/D

A/D= All fish dead

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Exposure of rainbow trout (Oncorhynchus mykiss) to the test item has been investigated and based on the time-weighted mean measured test concentrations gave the following results:
The 96 h LC50 was 0.21 mg/L with 95 % confidence limits of 0.18 - 0.25 mg/L.
The 96 h NOEC was 0.15 mg/L and the 96 h LOEC was 0.36 mg/L.

Executive summary:

Introduction:

A study was performed to assess the acute toxicity of the test item to rainbow trout (Oncorhynchus mykiss). The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No 203, "Fish, Acute Toxicity Test" referenced as Method C.1 of Commission Regulation (EC) No. 440/2008.

 

Methods:

Information provided by the Sponsor indicated the test item was practically insoluble in water.

 

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.

 

A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 7.7 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this item under test conditions.

 

Following a failed threshold approach limit test at a nominal concentration of 2.5% v/v saturated solution, fish were exposed, in groups of seven, to an aqueous solution of the test item at nominal concentrations of 0.625, 1.25, 2.5, 5.0 and 10% v/v saturated solution for a period of 96 hours at a temperature of approximately14 °C under semi-static test conditions. The test item solution was prepared by stirring an excess (50 mg/L) of test item in test water using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration (0.2 µm Sartorius Sartopore filter, first approximate 2 liters discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the required test concentrations. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

 

Results:

Analysis of the fresh test preparations at 0 and 72 hours showed measured test concentrations to range from 0.0636 to 2.35 mg/L. A decline in measured test concentration was observed in the old media at 24 and 96 hours to between 0.0483 and 2.01 mg/L and hence it was considered appropriate to calculate the results based on the time-weighted mean measured test concentrations only in order to give a “worst case” analysis of the data.

Exposure of rainbow trout (Oncorhynchus mykiss) to the test item gave the following results based on the time-weighted mean measured test concentrations:

 

Time Point (Hours)	LC50 (mg/L)	95% Confidence Limits (mg/L)			No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
96	0.21	0.18	-	0.25	0.15	0.36