2-Naphthalenol, heptyl-1-[2-[3-methyl-4-[2-(3-methylphenyl)diazenyl]phenyl]diazenyl]-

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 July 18 to August 7, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OCSPP 850.4500 “Algal Toxicity”, 2012

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: UNI EN ISO 8692:2012 “Fresh water algal growth inhibition test with unicellular green algae”.

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:

Reaction products of 3-Methylaniline with heptyl naphthalen-2-ol

Cas Number:

1619917-05-3

Molecular formula:

Not applicable (UVCB Substance)

IUPAC Name:

Reaction products of 3-Methylaniline with heptyl naphthalen-2-ol

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- batch No.of test material: 78-231-15
- Expiration date of the batch: December 01, 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, protected from light
- Stability in Solvent or Vehicle at RT: Stable in acetone

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

yes

Details on test solutions:

The test concentrations were 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, 0.031, 0.098, 0.313 and 1.0 mg test item/L.

The highest tested concentration of 1.0 mg/L corresponding to the maximum solubility of the test item obtained in the test medium.

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 0.1000 g of test item into 10.0 mL of acetone.
The resulting solution was observed to be dark red, 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:

not specified

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Test temperature:

24.0 – 24.1 °C

pH:

7.90 – 7.99 at the test start
8.70 – 8.98 at the test end

Nominal and measured concentrations:

Negative control (algal medium without test item), solvent control (algal medium without test item but with the same concentration of solvent as in test item concentrations tested) and 0.01, 0.031, 0.098, 0.313 and 1.0 mg test item/L.

From Stock Solvent Solution all the other stock solutions in acetone were prepared. In Table 1 details of preparation of test stock solutions used to prepare the test solutions are reported.

Table 1 Stock Solvent Solutions.

Stock Solvent Solutions Volume of stock solution used Final volume of acetone (mL) Nominal stock concentration (mg/L)
STS1 0.050 mL Stock Solution 5.0 100.0
STS2 0.155 mL Stock Solution 5.0 310.0
STS3 0.490 mL Stock Solution 5.0 980.0
STS4 1.565 mL Stock Solution 5.0 3130.0

These test item solutions were red, with no undissolved test item.

In order to prepare the test item concentrations in the culture medium, 20.0 μL of each Stock Solvent Solution was introduced into 200.0 mL of culture medium as reported in Table 2.

Table 2 Test solutions preparation.

Test
solution Nominal test item solution concentration
(mg/L) Final volume
(mL) Added volume
of stock solvent solution
(μL)
1 0.01 200 20.0 STS1
2 0.031 200 20.0 STS2
3 0.098 200 20.0 STS3
4 0.313 200 20.0 STS4
5 1.0 200 20.0 SS

The obtained solutions appeared clear and light red, with no undissolved test item; after pH check the test was start.

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, for the negative control and for the solvent control, 100 mL capacity conical glass flasks capped with air permeable stoppers were used.
Three replicates for each test concentrations, six for negative 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 transferring 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.

Temperature:
the incubation temperature was continuously monitored during the course of the study by a PT100 probe installed in the environmental test chamber. It was in the range 24.0– 24.1°C with a mean value of 24.0°C and a standard deviation of 0.02°C. The temperature range provided by OECD guideline is 24 ± 2°C. The temperature values during the test were within the provided range by guideline.
Light conditions:
continuous illumination; light intensity was ranged between 4486 and 4880 Lux, within the OECD recommended range (4440 - 8880 Lux).

Test duration:
72 hours

Test medium:
Reconstituted water according to OECD Guideline No. 201, 2011 (see Appendix D).
Test medium pH was measured with a portable pH meter at the beginning of the test and in each test flask at the end of the test. For fresh solutions the pH values were measured sampling an aliquot immediately after test solutions preparation, before pouring it in the test flasks. For spent solutions (after 72 hours), pH were measured in each test flask.
At the end of the test the pH value of solvent control replicates was between 8.87 and 8.90; (mean value 8.88) and it varied by a maximum of 1.00 units, in compliance with the values recommended by OECD guideline stating that the pH values should not increase more than 1.5 units during the test period.
The maximum variation of the pH for the negative control was 1.02, according to the OECD 201 (2011).

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Basis for effect:

other: growth rate and yield

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Basis for effect:

other: growth rate and yield

Details on results:

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.
Table 5 Effect of test item on the growth of Pseudokirchneriella subcapitata.

Nominal test item concentration (mg/L) replicate Cell density (n. cells/mL) (*)
24 h 48 h 72 h
0.0(negative control) A 63884 592767 4832867
B 61874 531167 4688067
C 70674 590667 4916667
D 65704 582667 4896867
E 61414 626067 4762867
F 66724 665467 4741867
Average value 65046 598133 4806533
0.0(solvent control) A 68004 575567 4682067
B 63034 661967 4853667
C 66654 537067 4672267
D 57314 698167 4676467
E 75274 660367 4816067
F 61154 619067 4788267
Average value 65239 625367 4748133
0.01 A 66074 696967 5087667
B 69954 639867 4655467
C 65704 609967 4792267
Average value 67244 648933 4845133
0.031 A 65804 668067 4759467
B 66804 652367 4702067
C 71974 668467 4902067
Average value 68194 662967 4787867
0.098 A 71414 627167 4765467
B 68254 636567 4782467
C 64174 703967 5027467
Average value 67948 655900 4858467
0.313 A 58104 550567 4762667
B 65364 588267 4687867
C 65634 638367 4760267
Average value 63034 592400 4736933
1.0 A 67594 488867 5169267
B 62854 641267 3855867
C 58824 610667 4457467
Average value 63091 580267 4494200
(*) Cell density at time 0 h: 104 cell/mL.

At the test end the cell density in the solvent control increased on average by a factor of 481. 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 17.2 % (maximum acceptable value 35%) and a coefficient of variation of average growth in solvent control replicates during the test period of 1.6 %(maximum acceptable value 7%).

Growth rate
In test item solutions, the algal growth rate (r) after 72 hours of exposure was inhibited by a value of 1.0 % for the concentration 1.0 mg test item/L compared to the solvent control.
For all test item concentrations no significant biological effect was shown in comparison to the solvent control for end-point growth rate after 72 hours.

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 0.2% for the concentration of 0.313 mg test item/L to a maximum value of 5.4% for the highest test item concentration of 1.0 mg/L, compared to the solvent control.
For all test item concentrations no significant biological effect was shown in comparison to the solvent control for end-point yield.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The toxicity of test item RED 2596 was tested on Pseudokirchneriella subcapitata.

The 72-hour EC10, EC20 and EC50 , 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 > 1.0 > 1.0 > 1.0 1.0 > 1.0
Yield > 1.0 > 1.0 > 1.0 1.0 > 1.0

The highest tested concentration of 1.0 mg/L corresponding to the maximum solubility of the test item obtained in the test medium.