1H-Benz[de]isoquinoline-1,3(2H)-dione, 6-amino-, N,N'-bis[mixed 2-ethylhexyl and 3-[(2-ethylhexyl)oxy]propyl] derivs

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from May 02 to May 16, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: - OECD Series on Testing and Assessment No 23 - Guidance Document On Aquatic Toxicity Testing Of Difficult Substances And Mixtures ENV/JM/MONO(2000)6

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Raction product of 2-Ethyl-1-hexylamine and 3-(2-Ethylhexyloxy)propylamine with 4-bromo-1,8 naphthalic anhydride

Cas Number:

1971906-58-7

Molecular formula:

Not applicable (UVCB substance)

IUPAC Name:

Raction product of 2-Ethyl-1-hexylamine and 3-(2-Ethylhexyloxy)propylamine with 4-bromo-1,8 naphthalic anhydride

Test material form:

liquid: viscous

Specific details on test material used for the study:

Test item identification: YELLOW 2747
Batch number: 78-242-1
Manufacturing date: February 01, 2017 *
Expiry date : February 01, 2018
Chemical name: 1H-Benz[de]isoquinoline-1,3(2H)-dione, 6-amino-,N,N’-bis[mixed 2-ethylhexyl and 3-[(2-ethylhexyl)oxy]propyl] derivs
C.A.S. number: 1971906-58-7
The test item was stored at room temperature in the dark

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

yes

Remarks:

acetone

Details on test solutions:

The test concentration was chosen in accordance with the results of a no GLP pre-test (range-finding test), whose relevant Raw Data were archived under the code of the present study.
Five concentrations of test item in a geometric series, differing by a constant equal to 3.2, were tested; tested concentrations were 0.01, 0.03, 0.10, 0.31 and 1.0 mg test item/L.

Since the test item is insoluble in water, it was added to the algal medium by the means of a solvent vehicle (acetone). Therefore a solvent control (algal medium whit the same concentration of acetone used as carrier) was added in order to verify the absence of toxic effect due to the solvent; moreover a negative control (algal medium without test item) was prepared.
The test medium was prepared as follows:
the day of the test start, a 10000 mg/L Stock Solvent Solution (SS) was prepared by direct weighing 0.0500 g of test item into 5.0 mL of acetone.
The resulting solution was observed to be yellow, with no undissolved test item.

Test organisms

Test organisms (species):

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

Details on test organisms:

The study was performed with the unicellular, fresh-water green algae Pseudokirchneriella subcapitata strain (formerly known as Selenastrum capricornutum), cultured in the laboratories of the Test Facility and originally purchased from the Institute of Plant Physiology of the University of Göttingen, Germany.
The algae were cultured in a climatic chamber at 24 °C ± 2 °C under continuous uniform illumination in the range 4440 - 8880 Lux in the spectral range 400-700 nm. The culture medium was the same of the test medium; the stock cultures were weekly transferred to fresh medium and maintained in continuous shaking to ensure the necessary amount of CO2 and to keep algae in suspension. Cultures containing deformed or abnormal cells were discarded. Only exponentially growing algal cultures have been used to start the test

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

23.9 – 24.0 °C

pH:

8.07 – 8.13 at the test start
7.60 – 7.70 at the test end

Nominal and measured concentrations:

0.01, 0.03, 0.10, 0.31 and 1.0 mg test item/L
The biological results were referred to nominal concentrations of test item.

Details on test conditions:

The treatment vessels were identified by experiment number, group and replicate number.
All the described procedures were conducted under a laminar flow bench to ensure the sterility of the algal cultures.
For the test concentrations and for the negative control, 100 mL capacity conical glass flasks capped with air permeable stoppers were used.
Three replicates for each test concentrations, six for negative control and solvent control were prepared.

Since the test item is coloured, to avoid adsorption of photosynthetically active light that could limit growth of algal cultures during the exposure, the reduction of the light path by reducing the volume of test solution was adopted as recommended in the OECD 23 (2000).

At test start, 45 mL of test solutions were poured into a test flask and inoculated with algae to obtain a starting cell density of about 104 cells/mL. Two further flasks for each group were prepared from the first one transferred 15 mL of test solutions.
The algal inoculum was taken from a pre-culture, started 3 days before the beginning of the test in order to ensure that the assay was performed with exponentially growing algal population. The cell density of the inoculum was around 106 cells/mL.

The test flasks were incubated in a climatic chamber, under continuous illumination and constant shaking. The flasks were randomly distributed under the light and were replaced every day during the test.
Light conditions:
continuous illumination; light intensity was ranged between 5082 and 5210 Lux, within the OECD recommended range (4440 - 8880 Lux).

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.64 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Effective concentration calculated by Statistical Software greater than the highest tested concentration (1.0 mg/L)Effective concentration calculated by Statistical Software greater than the highest tested concentration (1.0 mg/L)

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.33 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.03 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Results with reference substance (positive control):

No statistically significant difference between negative control and solvent control was observed; therefore the solvent control was considered as comparative control in the statistical analysis.
At the test end the cell density in the solvent control increased on average by a factor of 372. This value complies with the validity criterion of the test, according to the mentioned guidelines, which indicates a minimum increase factor of 16.
The solvent control also met the other validity criterion, with a coefficient of variation of daily growth rates of 18.6 % (maximum acceptable value 35%) and a coefficient of variation of average growth in solvent control replicates during the test period of 1.3 %(maximum acceptable value 7%).

Any other information on results incl. tables

Growth rate

In test item solutions, the algal growth rate (r) after 72 hours of exposure was inhibited by a minimum value of 2.5 % for the concentration 0.03 mg test item/L to a maximum value of 34.9 % for the highest tested concentration, 1.0 mg test item/L, compared to the solvent control.

 

A significant biological effect was shown from test item nominal concentration 0.10 mg/L in comparison to the solvent control for end-point growth rate after 72 hours.

 

The percentage of growth inhibition for the tested concentrations is reported in Table 6.

 

Table6 Algal growth rate and inhibition caused by the test item over 72 hours of exposure.

 

Nominal test item concentration (mg/L)	0-24 h		0-48 h		0-72 h
	Mean growth rate	Mean inhibition %	Mean growth rate	Mean inhibition %	Mean growth rate	Mean inhibition %
0.0 (negative control)	1.5646	-	1.8381	-	1.9700	-
0.0 (solvent control)	1.5544	0.6	1.8593	-1.2*	1.9723	-0.1*
0.01	1.5389	1.0	1.8461	0.7	1.9740	-0.1*
0.03	1.4351	7.7	1.8449	0.8	1.9225	2.5
0.1	1.3088	15.8	1.7813	4.2	1.8929	4.0
0.31	1.3058	16.0	1.7709	4.8	1.7780	9.9
1.0	0.8745	43.7	1.0297	44.6	1.2844	34.9

*Growth valuewasslightly higher than the negative control

 

The E_rC₁₀, the E_rC₂₀and the E_rC₅₀values, the NOEC and LOEC values,in terms of nominal test item concentrations, for growth rate end-point, are reported in the following table:

 

Time (h)	ErC10(mg/L)	ErC20(mg/L)	ErC50(mg/L)	NOEC (mg/L)	LOEC (mg/L)
0 - 24	0.05 (N/A – 0.133)	0.39 (N/A – 0.54)	>1.0 (N/A)	0.03	0.1
0 - 48	0.45 (0.24 – 0.59)	0.63 (0.49 – 0.76)	1.10* (1.00 – 1.22)	0.31	1.0
0 - 72	0.31 (0.22 – 0.39)	0.57 (0.49 – 0.66)	1.64* (1.33 – 2.02)	0.1	0.31

*Effective concentration calculated by Statistical Software greater than the highest tested concentration (1.0 mg/L)

Yield

 

After 72 hours of exposure, the yield (Y), as difference of algal density between the end and the beginning of the test, was inhibited by a minimum value of 13.9 % for the concentration of 0.03 mg test item/L to a maximum value of 86.8% for the highest test item concentration of 1.0 mg/L, compared to the solvent control.

Until the 0.03 mg/L test item concentration no significant biological effect was shown in comparison to the solvent control for end-point yield.

 

The percentage of inhibition in terms of biomass for the tested concentrations is reported in Table 7.

 

Table7 Algal yield inhibition caused by the test item over 72 hours of exposure.

 

Nominal test item concentration (mg/L)	Mean yield at 72h (cell/mL)	Mean yield inhibition %
0.0 (negative control)	3690867	--
0.0 (solvent control)	3711267	-0.6*
0.01	3744733	-0.9*
0.03	3195733	13.9
0.10	2918800	21.4
0.31	2112533	43.1
1.0	490733	86.8

* Biomass value higher than the negative control or solvent control.

The E_yC₁₀, the E_yC₂₀and the E_yC₅₀valueswith confidence limits (LCL, Lower Confidence Level and UCL, Upper Confidence Level), the NOEC and LOEC valuesat 72 hours, in terms of nominal test item concentrations, for yield end-point, are reported below:

 

Time (h)	EyC10(mg/L)	EyC20(mg/L)	EyC50(mg/L)	NOEC (mg/L)	LOEC (mg/L)
0 - 72	0.06 (0.02 – 0.10)	0.11 (0.07 – 0.17)	0.33 (0.25 – 0.43)	0.03	0.1

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The toxicity of test item YELLOW 2747 was tested on Pseudokirchneriella subcapitata.

The 72-hour EC10, EC20 and EC50 with their 95% confidence limits (LCL, Lower Confidence Level and UCL, Upper Confidence Level), the NOEC and LOEC, calculated in terms of the nominal concentrations of test item, for the two end-points, are reported in the following table:


Endpoint 0 - 72 h EC10 (mg/L) 0 - 72 h EC20(mg/L) 0 - 72 h EC50(mg/L) 0 - 72 h NOEC(mg/L) 0 - 72 h LOEC(mg/L)
Growth rate 0.31(0.22 – 0.39) 0.57(0.49 – 0.66) 1.64*(1.33 – 2.02) 0.1 0.31
Yield 0.06(0.02 – 0.10) 0.11(0.07 – 0.17) 0.33(0.25 – 0.43) 0.03 0.1
*Effective concentration calculated by Statistical Software greater than the highest tested concentration (1.0 mg/L)