4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

27 September 2005 to 22 December 2005

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

No analytical measurements have been performed; assessment as per ECHA's comments.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

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

Analytical monitoring:

no

Details on sampling:

The range-finding test for each sample of the test substance DUSANTOX® 86 (a total of five samples: no. E 382001/05 through no. E 382005/05) involved three concentrations of the test substance in a graded series: from 10 mg/l to 100 mg/l and the controls (control and control A).In the limit test (based on the results of the range-finding test), only the highest tested concentration of the test substance DUSANTOX® 86 = 100 mg/l and the controls (control and control A) were used for two samples (no. E 382002/05 and no. 382005/05).In the definitive test (based on the results of the range finding test), 5 (five) concentrations of the test substance DUSANTOX® 86 in a graded series: from 25 mg/l to 100 mg/l were used for a further three samples (no. E 382001/05, no. E 382003/05 and no. E 382004/05), and the controls (control and control A).In the control test (the condition of the algal culture used and the suitability of the test conditions are continuously checked), five concentrations of the reference substance K2Cr2O7 from 0.40 mg/l to 1.50 mg/l and the control were used.

Vehicle:

yes

Details on test solutions:

The test substance - DUSANTOX 86, (five samples) - was provided by the Sponsor of the study, who was responsible for ensuring the homogeneity, stability and purity of the test substance.Given that the test substance is almost insoluble in water, an auxiliary solvent - acetone - which is of low toxicity to algae, was used for the preparation of the stock solutions.The following test solutions were used to test the growth inhibition of freshwater algae Selenastrum capricornutum:1. stock solutions of the test substance (5 samples); prepared by dissolving the test substance (each sample) in acetone, to which further acetone was added to obtain a volume representing the required concentrations, i.e. 100 g/l2. other solutions of the test substance (5 samples); prepared by adding the corresponding amounts of the relevant stock solution into the necessary volume (at least 200 ml) of the nutrient medium, then thoroughly homogenising the mixture using a homogeniser. These test solutions were placed into test vessels immediately after preparation and subsequently inoculated with algal culture3. control; nutrient solution (nutrient medium) with no test substance added4. control A; nutrient solution (nutrient medium) containing the auxiliary substance (acetone) at a concentration of 1 ml/l, this control contained no test substance either.

Test organisms (species):

other: Selenastrum capricornutum Printz

Details on test organisms:

Selenastrum capricornutum Printz (ATCC 22662 or 61.81 SAG) planktonic freshwater algae, strain Skulberg 1959/1, originating from the collection of phototrophic microorganism cultures of the Institute of Botany at the Slovak Academy of Sciences, Dúbravská cesta 14, Bratislava.Stock cultureThe stock solutions of the algal culture for the tests were cultivated in Erlenmeyer flasks (250 ml), with approx. 100 ml of the nutrient solution (nutrient medium), under continuous lighting, at laboratory temperature, in a fluorescent-lit cultivation box. The nutrient solution (nutrient medium) was prepared by mixing unit of the concentrated nutrient solution and 8 units of deionised water. The algal stock culture was transferred to a fresh nutrient solution (nutrient medium) once a week.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

No post exposure observation period specified within the report.

Hardness:

Not specified.

Test temperature:

21°C to 25°C, constant temperature for each test ± 2°C

pH:

Not specified.

Dissolved oxygen:

Not specified.

Salinity:

Not applicable (freshwater test)

Nominal and measured concentrations:

Not specified.

Details on test conditions:

temperature: 21°C to 25°C, constant temperature for each test ± 2°C (OECD); 23°C ± 2°C (STN EN ISO 8692)lighting conditions: continuous (fluorescent-lit box); 6000 lux to 10,000 lux, value recorded = 7,800 luxtest vessels: 150 ml Erlenmeyer flasks with air-permeable stoppersquantity of test solution: 50 mlnumber of test organisms: 104 cells per ml-1other conditions: no aeration, algal suspension stirred at least 3 times a dayperiod of exposure: 72 h; assessment of the growth of the algal culture once per 24 hAll of the water used for the preparation of the nutrient solution (nutrient medium) and test solutions was deionised (conductivity = 0.90 µS.cm-1 to 0.96 µS.cm-1 < 5 µS.cm-1).

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

69 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Prepared by dissolving the test substance in acetone, to which further acetone was added to obtain a volume representing the required concentrations.

Basis for effect:

cell number

Remarks on result:

other: Sample 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

116 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Prepared by dissolving the test substance in acetone, to which further acetone was added to obtain a volume representing the required concentrations.

Basis for effect:

growth rate

Remarks on result:

other: Sample 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Prepared by dissolving the test substance in acetone, to which further acetone was added to obtain a volume representing the required concentrations.

Basis for effect:

cell number

Remarks on result:

other: Sample 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Prepared by dissolving the test substance in acetone, to which further acetone was added to obtain a volume representing the required concentrations.

Basis for effect:

growth rate

Remarks on result:

other: Sample 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

85 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Prepared by dissolving the test substance in acetone, to which further acetone was added to obtain a volume representing the required concentrations.

Basis for effect:

cell number

Remarks on result:

other: Sample 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

125 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Prepared by dissolving the test substance in acetone, to which further acetone was added to obtain a volume representing the required concentrations.

Basis for effect:

growth rate

Remarks on result:

other: Sample 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

78 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Prepared by dissolving the test substance in acetone, to which further acetone was added to obtain a volume representing the required concentrations.

Basis for effect:

cell number

Remarks on result:

other: Sample 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

168 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Prepared by dissolving the test substance in acetone, to which further acetone was added to obtain a volume representing the required concentrations.

Basis for effect:

growth rate

Remarks on result:

other: Sample 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Prepared by dissolving the test substance in acetone, to which further acetone was added to obtain a volume representing the required concentrations.

Basis for effect:

cell number

Remarks on result:

other: Sample 5

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Prepared by dissolving the test substance in acetone, to which further acetone was added to obtain a volume representing the required concentrations.

Basis for effect:

growth rate

Remarks on result:

other: Sample 5

Details on results:

Evaluation of the range-finding tests carried out: at a concentration of 10 mg/l , the test substance DUSANTOX® 86 caused almost no growth inhibition of freshwater algae Selenastrum capricornutum at 72 h in the five samples (samples no. 382001/05 to no. E 382005/05) compared with the control (Iµ = -0.2% to 0.6%, or IA = -1.2% to 6.6%); at a concentration of 100 mg/l , in samples no. E 382002/05 and no. E 382005/05, it caused approx. 33% and 23% inhibition of biomass growth respectively, and growth rate inhibition of 5.7% and 8.5% respectively; however, for the other samples tested - no. E 382001/05, no. E 382003/05 and no. E 3820004/05 - the inhibition of biomass growth observed reached 86%, approx. 74% and approx. 48% respectively, and growth rate inhibition reached 42%, 34% and approx. 21% respectively, compared with the control.In the control, the algal cell density increased over 72 h at least 111 x > 67 x, and the growth rate reached µ = 1.57 d-1 > 1.4 d-1 (thus meeting a condition of validity of the test). pH changes in the controls did not exceed 1.38 units in the course of the test, which is < 1.5 units (thus meeting another condition of validity of the test).Based on the results of the range-finding tests, the concentrations of the test substance for the definitive tests (samples no. E 382001/05, no. E 382003/05 and no. E 3820004/05) were chosen in the range of 25 mg/l to 100 mg/l , and the limit tests (samples no. E 382002/05 and no. E 382005/05), for which the maximum concentration of the test substance 100 mg/l was used, were conducted.During all of the tests conducted (both the definitive and limit tests) with the test substance DUSANTOX® 86, at 72 h the algal cell density in the control had increased by at least 103 x > 67 x and the minimum growth rate had reached µ = 1.54 d-1 > 1.4 d-1 (thus meeting a condition of validity of the test). pH changes in the controls did not exceed 1.37 units in the course of the test, which is < 1.5 units (thus meeting another condition of validity of the test).Observed effects:No significant morphological changes in the algal culture cells were observed in the test solutions of any of the five test samples of the test substance DUSANTOX® 86 compared with the control cultures.As stated, all conditions of the test were met during the growth inhibition tests conducted on freshwater algae Selenastrum capricornutum. In the tests conducted on all controls (both the controls and ‘control A’s), the algal cell density (number of cells per ml) increased at least 103 x and the growth rate reached µ > 1.54 d-1; at the same time, pH changes did not exceed 1.38 units. Equally, the 72 h EbC(IbC)50 and 72 h ErC(IrC)50 values established for the reference substance - potassium dichromate (K2Cr2O7) comply with the required values, therefore, the results of the growth inhibition tests in freshwater algae Selenastrum capricornutum can be considered valid.Given the fact that at test concentrations ≥ 25 mg/l , the test substance gradually separated from the solution during the tests despite the application of an auxiliary substance (acetone p.a.), it can be assumed that the values for the growth inhibition of algae Selenastrum capricornutum for the individual concentrations do not correspond with the actual concentration of the test substance DUSANTOX® 86 in the solution, therefore, the 72 h EbC(IbC)50 and 72 h ErC(IrC)50 values may be inaccurate; in any case, the 72 h EbC(IbC)50 and 72 h ErC(IrC)50 values for the test substance DUSANTOX® 86 (for all five samples) are > 10 mg/l .

Results with reference substance (positive control):

During the control test, the algal cell density in the control increased 120 x, i.e. > 67 x, and the growth rate reached µ = 1.60 d-1, i.e. > 1.4 d-1(thus meeting a condition of validity of the test). pH changes in the control reached 1.35 units in the course the test, which is < 1.5 units (thus meeting another condition of validity of the test).The 72 h EbC(IbC)50 and 72 h ErC(IrC)50 values were established for the reference substance - potassium dichromate K2Cr2O7 - on the basis of the control test conducted on freshwater green algae Selenastrum capricornutum.

Reported statistics and error estimates:

The recorded data (the algal culture density), together with the calculated values of algal growth inhibition are summarised in the form of tables.For each test concentration, algal growth inhibition was calculated in relation to the control based on the area under the growth curves (IA) or based on the growth rate (Iµ). Standard statistical methods were used to calculate the EC(IC)50 for a specific time period. The green algae growth inhibition values (%) for each definitive test and the control test were processed, in relation to the test concentrations, using a statistical method - a logarithmic linear function - and the 72 h EbC(IbC)50 and 72 h ErC(IrC)50 were calculated, together with 95% confidence intervals.The results of the tests were assessed on a PC using the TOXICITY 3.01 software program.

TOXICITY OF DUSANTOX 86

Sample no.:	72h EbC(IbC)50	72h ErC(IrC)50
	[mg.l^-1]
E 382001/05	69(66 – 72)	116(113 – 120)
E 382002/05	>100	>100
E 382003/05	85(81 – 92)	125(106 – 224)
E 382004/05	78(72 – 87)	168(145 – 206)
E 382005/05	>100	>100

( ) – 95% confidence intervals.

 

REFERENCE SUBSTANCE TOXICITY

TOXICITY		95% confidence interval [mg.l^-1]
EC(IC)50	[mg.l^-1]
72 h EbC(IbC)50	0.73	0.67 – 0.76
72 h ErC(IrC)50	1.31	1.26 – 1.37

 

Validity criteria fulfilled:

yes

Conclusions:

72 h EbC(IbC)50 and 72 h ErC(IrC)50 values for the test substance DUSANTOX® 86 (for all five samples) are reported as > 10 mg/l. In accordance with ECHA Guidance document, Chapter R.7b – Endpoint Specific Guidance, “Figure R. 7.8-5: Considerations for difficult substances” the limit value of 100 mg/l is taken as the definitive value for hazard classification purposes. The substance is not considered to be harmful to algae at the limit of concentration in water.

Executive summary:

The study ETX-A-017/2005, “DUSANTOX® 86, Growth Inhibition Test in Freshwater Algae” was conducted (carried out) in compliance with the principles of GLP and in accordance with the following test methods:

C.3: Algal Growth Inhibition, Part C of Annex no. 5 to Decree of the Ministry of Economy of the Slovak Republic no. 2/2002 Coll. of 27 March 2002 implementing Act No. 163/2001 Coll. on Chemical Substances and Chemical Preparations

OECD 201: OECD Guidelines for the Testing of Chemicals, "Alga, Growth Inhibition Test"

STN EN ISO 8692 (75 7740): Water quality -- Freshwater algal growth inhibition test with unicellular green algae

The growth inhibition test in freshwater algae is intended to determine the toxic effects of chemical substances on the growth of different types of planktonic freshwater algae.

The test involves observing changes in the growth of the algal culture in a defined nutrient solution (medium) in relation to the concentrations of the test substance, compared with the control. Exponentially growing cultures of algae are exposed to various concentrations of the test substance over several generations under defined conditions. The inhibition is measured as a reduction in the growth or growth rate of algae compared with the growth or growth rate of control cultures cultivated under the same conditions. The growth rate of algal populations is regularly observed and recorded after 24 h, 48 h and 72 h of cultivation, using the Bürker chamber counting method, and the 72 h EC(IC) is established, where possible.

As stated, all conditions of the test were met during the growth inhibition tests conducted on freshwater algaeSelenastrum capricornutum. In the tests conducted on all controls(both the controls and ‘control A’s), the algal cell density (number of cells per ml) increased at least 103 x and the growth rate reached µ > 1.54 d-1; at the same time, pH changes did not exceed 1.38 units. Equally, the 72 h EbC(IbC)50 and 72 h ErC(IrC)50 values established for the reference substance - potassium dichromate (K2Cr2O7) comply with the required values, therefore, the results of the growth inhibition tests in freshwater algaeSelenastrum capricornutum can be considered valid.

Given the fact that at test concentrations ≥ 25 mg/l , the test substance gradually separated from the solution during the tests despite the application of an auxiliary substance(acetone p.a.), it can be assumed that the values for the growth inhibition of algae Selenastrum capricornutum for the individual concentrations do not correspond with the actual concentration of the test substance DUSANTOX® 86 in the solution, therefore, the 72 h EbC(IbC)50 and 72 h ErC(IrC)50 values may be inaccurate; in any case, the 72 h EbC(IbC)50 and 72 h ErC(IrC)50 values for the test substance DUSANTOX® 86 (for all five samples) are > 10 mg/l.

 

In accordance with ECHA Guidance document, Chapter R.7b – Endpoint Specific Guidance, “Figure R. 7.8-5: Considerations for difficult substances” the limit value of 100 mg/l is taken as the definitive value for hazard classification purposes. The substance is not considered to be harmful to algae at the limit of concentration in water.

 

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 – 29 October 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Version / remarks:

US Environmental Protection Agency, Office for Chemical Safety and Pollution Prevention, Ecological Effects Test Guideline OCSPP 850.4500 Algal Toxicity. January 2012.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Official Journal of the European Communities, L 383 A, Part C.3, Algal inhibition test. 29 December 1992.

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD Guidelines for the Testing of Chemicals. Test Guideline 201, Freshwater Alga and Cyanobacteria, Growth Inhibition Test. Adopted 23 March 2006.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

No further details specified in the study report

Analytical monitoring:

yes

Details on sampling:

At the start of the test, samples were taken from excess test solutions and at the end of the test from a single replicate from each test concentration (containing algae). Samples taken for analysis at 72 hours were analysed post-centrifugation, all other samples were analysed directly.

Vehicle:

no

Details on test solutions:

This study was run with a control, solvent control and nominal exposure concentrations of 0.489, 1.57, 5.00, 16.0 and 51.3 μg/L.
A primary stock concentrate (PS1) of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline, with a nominal concentration of 0.513 g/L, was prepared by adding a nominal 0.01026 g of test substance and made up to 20 mL with the solvent (dimethylformamide (DMF)) in a volumetric flask. The stock was inverted to mix and observed to be a clear and colourless solution.
Intermediate stock concentrates (IS1-IS4) with nominal concentrations of 0.16, 0.05, 0.0157 and 0.00489 g/L respectively, were prepared by addition of an appropriate volume of PS1 or by serial dilution to 10 mL DMF in a volumetric flask. The stocks were inverted to mix and observed to be clear and colourless solutions.
The primary and intermediate stocks were used to prepare the test solutions by direct addition of the appropriate volume, using a microliter syringe, to AAP medium followed by approximately 5 minutes of stirring. The solvent control was prepared in the same way using solvent only. The concentration of solvent was the same in all test concentrations (0.1 mL/L) with the exception of the control which contained AAP medium only. After stirring, all test solutions were observed as being clear and colourless.
The appropriate test solution (100 mL volume) was dispensed into each test and blank vessel.

Test organisms (species):

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

Details on test organisms:

The test species was the unicellular green alga Pseudokirchneriella subcapitata strain CCAP 278/4 from laboratory cultures maintained under axenic conditions. A 3-day old culture of the alga in the exponential growth phase was used as inoculum for the test. The culture was grown in the medium, and under the environmental conditions, described for the test.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Remarks on exposure duration:

As per guideline.

Post exposure observation period:

No post exposure observation specified in the study report

Hardness:

Not specified

Test temperature:

21-24°C

pH:

7.48 to 7.99

Dissolved oxygen:

Not specified

Salinity:

Not applicable

Conductivity:

Not specified

Nominal and measured concentrations:

This study was run with a control, solvent control and nominal exposure concentrations of 0.489, 1.57, 5.00, 16.0 and 51.3 μg/L.

Details on test conditions:

.Test procedure and apparatus
The test vessels were glass conical flasks of 250 mL nominal capacity closed with foam bungs. Each flask contained 100 mL of test solution. The cultures were incubated at 21-24°C controlled at ± 2°C (the nominal test temperature), under continuous "cool-white" illumination of approximately 6000 lux, with nominal orbital shaking at 160 rpm.
Six replicates of the control and solvent control and triplicates of each concentration of the test substance were employed. The position of each test replicate vessel in the incubator was randomised daily. One blank vessel (without algal inoculum) was incubated concurrently for each control, solvent control and test concentration sampling occasion. Blanks were not randomised daily.
The algal cell densities of the inoculum and test cultures were determined by electronic particle counting, using a Coulter counter and counting between a lower and upper threshold equivalent spherical diameter of approximately 2.3 and 5.0 μm respectively. Each replicate test vessel was inoculated with 1.028 mL of the inoculum culture to give a nominal cell density of 0.5 × 104 cells/mL. One 100 mL volume of Coulter electrolyte, inoculated in the same manner, had a cell density of 0.559 × 104 cells/mL. This value was used for growth calculations.
After 24, 48, and 72 hours, samples were removed from each test and blank vessel. The appropriate blank particle count was subtracted from that of the test culture to obtain the cell density.
At the end of the test, microscopic observations were made on a sample taken from a single test replicate of each concentration.

Preparation of test solutions
This study was run with a control, solvent control and nominal exposure concentrations of 0.489, 1.57, 5.00, 16.0 and 51.3 μg/L.
A primary stock concentrate (PS1) of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline, with a nominal concentration of 0.513 g/L, was prepared by adding a nominal 0.01026 g of test substance and made up to 20 mL with the solvent (dimethylformamide (DMF)) in a volumetric flask. The stock was inverted to mix and observed to be a clear and colourless solution.
Intermediate stock concentrates (IS1-IS4) with nominal concentrations of 0.16, 0.05, 0.0157 and 0.00489 g/L respectively, were prepared by addition of an appropriate volume of PS1 or by serial dilution to 10 mL DMF in a volumetric flask. The stocks were inverted to mix and observed to be clear and colourless solutions.
The primary and intermediate stocks were used to prepare the test solutions by direct addition of the appropriate volume, using a microliter syringe, to AAP medium followed by approximately 5 minutes of stirring. The solvent control was prepared in the same way using solvent only. The concentration of solvent was the same in all test concentrations (0.1 mL/L) with the exception of the control which contained AAP medium only. After stirring, all test solutions were observed as being clear and colourless.
The appropriate test solution (100 mL volume) was dispensed into each test and blank vessel.

Analytical Method
To establish what concentrations were achieved, analysis for 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline in the test solutions was undertaken using the validated liquid chromatography - mass spectrometer (LC-MS/MS) method detailed in Appendix 4. Method validation was completed in a separate study.
At the start of the test, samples were taken from excess test solutions and at the end of the test from a single replicate from each test concentration (containing algae). Samples taken for analysis at 72 hours were analysed post-centrifugation, all other samples were analysed directly.

Physical and chemical parameters
The pH of the control and test solutions was measured at the start of the test with a calibrated pH meter, using the excess remaining after filling the test vessels. At the end of the test the pH of a single replicate from each test concentration (containing algae) was determined.
Temperature values were continually recorded using a Max/Min thermometer checked against a liquid-in-glass thermometer. Current, maximum and minimum temperatures were recorded daily in an additional test vessel (without algae). The light intensity was measured once during the study, in each of four representative positions, using a photometer reading in lux (cosine).

Statistical analysis
The data for cell counts obtained at 24, 48 and 72 hours was entered into electronic data files and the 0-72 hour data statistically analysed using CETIS . A statistical test was performed to check for significant differences between the control and solvent control for each response variable.
Further statistical procedures were applied to test for significant differences (p <0.05) between the solvent control and test concentrations and to determine EC50, EC20 and EC10 values along with the associated 95% confidence intervals, where appropriate, for yield and average specific growth rate. The individual statistical procedures applied to each set of data are detailed within the results.
Lowest Observed Effect Concentration (LOEC) is defined as the lowest tested concentration at which the substance is observed to have a statistically significant effect on growth (p <0.05) when compared with the controls, within a given exposure time. However, all test concentrations above the LOEC must have a harmful effect equal to or greater than those observed at the LOEC. The No Observed Effect Concentration (NOEC) is defined as the test concentration immediately below the LOEC.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

13 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 13 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

13 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 13 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Analytical data
The concentrations of the test solutions ranged between 81-93% of nominal at the beginning of the test and 4-19% at the end of the test with two concentrations measured as Analytical calibrations were constructed using a minimum of 5 calibration levels, with a minimum R2 value of 0.995. The maximum percentage difference from nominal concentration for standards at the LOQ was less than 20% and less than 15% at levels greater than the LOQ.
On the basis of the analytical data, geometric mean measured concentrations were used for the calculation and reporting of results. Where the 72 hour analytical results were
Biological data
Algal cell particle densities (cell particles per unit volume) were measured as a surrogate for biomass.

Growth rates
The growth rates for the entire test duration (0 to 72 hours) and section-by-section (0-24 hours, 24-48 hours and 48-72 hours) were calculated for each replicate culture.
The 0-72 hour growth rates were examined by Equal Variance Two Sample t-test to identify significant differences (p <0.05) between the control and solvent control. There was no significant difference between the control and solvent control. The solvent control was therefore compared to the treatments by one-way analysis of variance and Bonferroni Adjusted t-tests to identify significant differences (p <0.05).
The ErC50, ErC20 and ErC10 values were determined to be outside of the concentration range tested using the nonlinear regression method.

Yield
This response is defined as the biomass at the end of the test minus the starting biomass. For the purposes of calculation, the cell particle density count (cell particles per unit volume) has been used as an acceptable surrogate for biomass.
The percentage inhibition in yield compared to the solvent control was calculated for each treatment group.
The cell particle densities were examined by Equal Variance Two Sample t-test to identify significant differences (p <0.05) between the control and solvent control. There was no significant difference between the control and solvent control. The solvent control was therefore compared to the treatments by one-way analysis of variance and Bonferroni Adjusted t-tests to identify significant differences (p <0.05).
The EyC10 value with its associated confidence intervals was subsequently calculated using the nonlinear regression method. The EyC20 and EyC50 values were determined to be outside of the concentration range tested.

Additional biological data
The microscopic observations, made at the end of the test, showed that compared to the control the algal cells sampled from the solvent control and each test concentration appeared normal.

Results with reference substance (positive control):

Not required

Reported statistics and error estimates:

(i) To achieve ≥16 fold exponential increase in biomass in the control replicates within the 72 hour test period. In this test cell particle density increase (measured as a surrogate for biomass) was 110 times for the control and 117 times for the solvent control over the 72 hour exposure period.
(ii) The mean coefficients of variation for control replicate sectional (daily) specific growth rates must not exceed 35% and in this test was determined to be 20.7% for the control and 19.8% for the solvent control.
(iii) The replicate coefficient of variation of average specific growth rates during the whole test period in the control replicate cultures must not exceed 7% and was calculated as 2.3% for the control and 3.0% for the solvent control.

Analytical Results

Nominal concentration of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline (µg/L)	Measured concentration of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline
	0 hour		72 hour		Geometric mean measured concentration
	(µg/L)	% of nominal	(µg/L)	% of nominal	(µg/L)	% of nominal
Control	<LOQ	-	<LOQ	-	<LOQ	-
Solvent control	<LOQ	-	<LOQ	-	<LOQ	-
0.489	0.398	81	<LOQ	<LOQ	0.158^*	32
1.57	1.32	84	<LOQ	<LOQ	0.287^*	18
5.00	4.31	86	0.223^Ϯ	4	0.980	20
16.0	14.7	92	3.11	19	6.76	42
51.3	47.8	93	3.54	7	13.0	25

^*Where 72 hour results were <LOQ, a value of LOQ/2 (0.0625 µg/L) was used to calculate the geometric mean.

^Ϯ The 72 hour analysis for the nominal 5.00 µg/L concentration was initially measured as <LOQ and was reanalysed using a lower dilution factor to bring it into calibration range.

All measured are quoted to 3 significant figures. Percentages calculated unrounded value and quoted to the nearest integer.

The limit of quantification (LOQ) in this study was 0.125 µg/L taken from the method validation study.

 

Algal cell particle density

Geometric mean measured concentration of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline (µg/L)	Replicate	Algal cell particle density (10^4cells/mL)
		24 hours	48 hours	72 hours
Control	A	3.49	12.7	65.2
	B	3.88	12.4	60.7
	C	3.66	13.0	60.8
	D	3.53	12.3	61.9
	E	3.56	11.9	51.0
	F	3.95	13.9	70.4
	Mean	3.68	12.7	61.7
Solvent control	A	3.68	12.7	68.4
	B	3.52	12.6	70.7
	C	3.56	14.7	49.1
	D	3.43	12.7	65.3
	E	3.66	12.9	72.7
	F	3.50	11.6	65.6
	Mean	3.56	12.9	65.3
0.158	A	3.54	13.7	71.4
	B	3.51	11.3	66.8
	C	3.73	13.3	67.2
	Mean	3.59	12.8	68.5
0.287	A	4.00	13.7	70.5
	B	3.33	12.4	61.1
	C	3.31	12.4	63.2
	Mean	3.55	12.8	64.9
0.980	A	3.43	13.3	64.9
	B	3.96	13.8	71.0
	C	3.76	13.6	70.7
	Mean	3.72	13.6	68.9
6.76	A	3.56	16.0	68.2
	B	3.85	13.6	55.1
	C	3.50	14.6	56.3
	Mean	3.64	14.7	59.9
13.0	A	3.51	14.7	53.0
	B	3.23	15.5	57.4
	C	3.50	14.2	62.3
	Mean	3.41	14.8	57.6

Inoculum (Day 0) cell density = 0.559 x 10⁴ cells/mL

Algal cell particle density values are quoted to 3 significant figures.

 

Algal growth rate

Geometric mean measured concentration of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline (µg/L)	Replicate	0-72 hours Growth rate/day	0-24 hours Growth rate/day	24-48 hours Growth rate/day	48-72 hours Growth rate/day
Control	A	1.586	1.831	1.289	1.639
	B	1.562	1.938	1.161	1.588
	C	1.563	1.880	1.269	1.540
	D	1.569	1.841	1.246	1.618
	E	1.504	1.852	1.203	1.458
	F	1.612	1.955	1.256	1.625
	Mean	1.566	1.883	1.237	1.578
Solvent control	A	1.602	1.884	1.240	1.684
	B	1.613	1.839	1.279	1.722
	C	1.492	1.852	1.417	1.207
	D	1.587	1.815	1.310	1.634
	E	1.623	1.878	1.257	1.733
	F	1.588	1.835	1.198	1.732
	Mean	1.584	1.851	1.284	1.619
0.158	A	1.617	1.847	1.350	1.653
	B	1.594	1.837	1.168	1.778
	C	1.596	1.898	1.270	1.621
	Mean	1.602	1.861	1.263	1.684
0.287	A	1.612	1.967	1.235	1.635
	B	1.564	1.783	1.317	1.594
	C	1.576	1.778	1.325	1.626
	Mean	1.584	1.843	1.292	1.618
0.980	A	1.585	1.813	1.357	1.585
	B	1.614	1.957	1.248	1.638
	C	1.613	1.906	1.284	1.651
	Mean	1.604	1.892	1.296	1.625
6.76	A	1.601	1.850	1.505	1.449
	B	1.530	1.930	1.258	1.402
	C	1.537	1.833	1.430	1.348
	Mean	1.556	1.871	1.398	1.400
13.0	A	1.517	1.837	1.429	1.285
	B	1.544	1.754	1.570	1.308
	C	1.571	1.835	1.400	1.478
	Mean	1.544	1.809	1.466	1.357

Growth rate values are quoted to 3 decimal places.

 

Mean growth rates over the test period

Geometric mean measured concentration of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline (µg/L)	Mean growth rate/day (0-72 hours)	0-72 hours mean growth rate/day percentage inhibition from solvent control (%)	Mean growth rate/day (0-24 hours)	Mean growth rate/day (24-48 hours)	Mean growth rate/day (48-72 hours)
Control	1.566 (1.529-1.603)	1	1.883 (1.841-1.925)	1.237 (1.199-1.275)	1.578 (1.523-1.633)
Solvent control	1.584 (1.535-1.634)	-	1.851 (1.830-1.872)	1.284 (1.224-1.344)	1.619 (1.455-1.783)
0.158	1.602 (1.572-1.633)	-1	1.861 (1.824-1.898)	1.263 (1.160-1.366)	1.684 (1.590-1.778)
0.287	1.584 (1.522-1.646)	0	1.843 (1.721-1.965)	1.292 (1.236-1.348)	1.618 (1.594-1.642)
0.980	1.604 (1.563-1.646)	-1	1.892 (1.809-1.975)	1.296 (1.233-1.359)	1.625 (1.585-1.665)
6.76	1.556 (1.459-1.654)	2	1.871 (1.812-1.930)	1.398 (1.255-1.541)	1.400 (1.342-1.457)
13.0	1.544 (1.477-1.611)	3	1.809 (1.755-1.863)	1.466 (1.363-1.569)	1.357 (1.238-1.476)

95% confidence limits are shown in parentheses

No significant differences (p<0.05) from the solvent control detected. Only the 0-72 hour growth rates were statistically analysed. The 0-24, 24-48 and 48-72 hour growth rates were not statistically analysed.

All biological measurements are quoted to 3 decimal places. Percentages quoted to the nearest integer.

 

Algal yield

Geometric mean measured concentration of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline (µg/L)	Replicate	Yield (10^4cells/mL)
		24 hours	48 hours	72 hours
Control	A	2.93	12.1	64.6
	B	3.32	11.8	60.1
	C	3.10	12.4	60.2
	D	2.97	11.7	61.3
	E	3.00	11.3	50.4
	F	3.39	13.3	69.8
	Mean	3.12	12.1	61.1
Solvent control	A	3.12	12.1	67.8
	B	2.96	12.0	70.1
	C	3.00	14.1	48.5
	D	2.87	12.1	64.7
	E	3.10	12.3	72.1
	F	2.94	11.0	65.0
	Mean	3.00	12.3	64.7
0.158	A	2.98	13.1	70.8
	B	2.95	10.7	66.2
	C	3.17	12.7	66.6
	Mean	3.03	12.2	64.3
0.287	A	3.44	13.1	69.9
	B	2.77	11.8	60.5
	C	2.75	11.8	62.6
	Mean	2.99	12.2	64.3
0.980	A	2.87	12.7	64.3
	B	3.40	13.2	70.4
	C	3.20	13.0	70.1
	Mean	3.16	13.0	68.3
6.76	A	3.00	15.4	67.6
	B	3.29	13.0	54.5
	C	2.94	14.0	55.7
	Mean	3.08	14.4	59.3
13.0	A	2.95	14.1	52.4
	B	2.67	14.9	56.8
	C	2.94	13.6	61.7
	Mean	2.85	14.2	57.0

Yield values are quoted to 3 significant figures.

 

Mean yields over the test period

Geometric mean measured concentration of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline (µg/L)	Mean yield (0-72 hours) (10^4 cells/mL)	Mean yield 95% Cl (10^4 cells/mL)	Percentage inhibition from solvent control (%)
Control	61.1	54.4-67.8	6
Solvent control	64.7	55.8-73.6	-
0.158	67.9	61.5-74.2	-5
0.287	64.3	52.1-76.6	1
0.980	68.3	59.7-76.8	-6
6.76	59.3	41.3-77.3	8
13.0	57.0	45.4-68.5	12

No significant differences (p <0.05) from the solvent control detected

 

Test solution pH measurements

Geometric mean measured concentration of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline (µg/L)	pH
	0 hours	72 hours
Control	7.48	8.28
Solvent control	7.56	8.15
0.158	7.58	8.34
0.287	7.697	8.22
0.980	7.71	8.22
6.76	7.76	8.06
13.0	7.99	7.90

 

Test incubator temperature

Time (hours)	Current Temperature (°C)	Maximum Temperature (°C)	Minimum Temperature (°C)
0	21.7	-	-
24	21.5	21.7	21.4
48	21.4	21.7	21.4
72	21.4	21.6	21.4

Max/Min thermometer checked against a mercury thermometer (± 0.2°C)

Validity criteria fulfilled:

yes

Conclusions:

Not harmful at the limit of solubility in water.

Executive summary:

Subject: Determination of toxicity to the green alga Pseudokirchneriella subcapitata

Guideline followed: OECD 201

Test species: Green alga Pseudokitchneriella subcapitata

Media: AAP media

Test concentrations: Control, solvent control and nominal concentrations of 0.489, 1.57, 5.00, 16.0 and 51.3 µg/L.

Control, solvent control and geometric mean measured concentrations of 0.158*, 0.278*, 0.890, 6.7 and 13.0 µg/L

*At 72 hours, the lowest two concentrations were measured as <LOQ. Where required, a value of LOQ/2 has been used to calculated the geometric mean.

Length of test: 72 hours

Nominal test temperature: 21-24°C controlled at ± 2°C

 

Results based on geometric mean measured concentrations of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline

Based on 0-72 hour growth rate, compared to the solvent control, the results obtained were:

	4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline (µg/L)	95% confidence limits (µg/L)
NOEC	13.0	-
LOEC	>13.0	-
ErC50	>13.0	N/A
ErC20	>13.0	N/A
ErC10	>13.0	N/A

 

Based on 0-72 hour yield compared to the solvent control, the results obtained were:

	4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline (µg/L)	95% confidence limits (µg/L)
NOEC	13.0	-
LOEC	>13.0	-
ErC50	>13.0	N/A
ErC20	>13.0	N/A
ErC10	8.66	2.86-14.9

 

Description of key information

0-72 hour growth rate and yield: NOEC 13.0 µg/LL.  No effects noted at the limit of solubility in water. 

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

Key study:

Subject: Determination of toxicity to the green alga Pseudokirchneriella subcapitata

Guideline followed: OECD 201

Test species: Green alga Pseudokitchneriella subcapitata

Media: AAP media

Test concentrations: Control, solvent control and nominal concentrations of 0.489, 1.57, 5.00, 16.0 and 51.3 µg/L.

Control, solvent control and geometric mean measured concentrations of 0.158*, 0.278*, 0.890, 6.7 and 13.0 µg/L

*At 72 hours, the lowest two concentrations were measured as <LOQ. Where required, a value of LOQ/2 has been used to calculated the geometric mean.

Length of test: 72 hours

Nominal test temperature: 21-24°C controlled at ± 2°C

Results based on geometric mean measured concentrations of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline

Based on 0-72 hour growth rate, compared to the solvent control, the results obtained were:

	4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline (µg/L)	95% confidence limits (µg/L)
NOEC	13.0	-
LOEC	>13.0	-
ErC50	>13.0	N/A
ErC20	>13.0	N/A
ErC10	>13.0	N/A

 

Based on 0-72 hour yield compared to the solvent control, the results obtained were:

	4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline (µg/L)	95% confidence limits (µg/L)
NOEC	13.0	-
LOEC	>13.0	-
ErC50	>13.0	N/A
ErC20	>13.0	N/A
ErC10	8.66	2.86-14.9

 No effects were noted at the limit of solubility in water. In accordance with ECHA Guidance document, Chapter R.7b – Endpoint Specific Guidance, “Figure R. 7.8-5: Considerations for difficult substances” the limit value of 100 mg/l is taken as the definitive value for hazard classification purposes. The substance is not considered to be harmful to algae at the limit of concentration in water. No classification is applicable.

 

Supporting study:

A supporting study in Algae was rejected due to no analytical measurements.  In this study, all other conditions of the test were met during the growth inhibition tests conducted on freshwater algae Selenastrum capricornutum. In the tests conducted on all controls(both the controls and ‘control A’s), the algal cell density (number of cells per ml) increased at least 103 x and the growth rate reached µ > 1.54 d-1; at the same time, pH changes did not exceed 1.38 units. Equally, the 72 h EbC(IbC)50 and 72 h ErC(IrC)50 values established for the reference substance - potassium dichromate (K2Cr2O7) comply with the required values, therefore, the results of the growth inhibition tests in freshwater algae Selenastrum capricornutum can be considered valid.

Given the fact that at test concentrations ≥ 25 mg/l , the test substance gradually separated from the solution during the tests despite the application of an auxiliary substance(acetone p.a.), it can be assumed that the values for the growth inhibition of algae Selenastrum capricornutum for the individual concentrations do not correspond with the actual concentration of the test substance DUSANTOX® 86 in the solution, therefore, the 72 h EbC(IbC)50 and 72 h ErC(IrC)50 values may be inaccurate; in any case, the 72 h EbC(IbC)50 and 72 h ErC(IrC)50 values for the test substance DUSANTOX® 86 (for all five samples) are > 10 mg/l.