Sulfonation products of (N-phenyl-4-[[4-(phenylamino)phenyl][4-(phenylimino)cyclohexa-2,5-dien-1-ylidene]methyl]aniline, monohydrochloride and 4,4'-[(4-iminocyclohexa-2,5-dien-1-ylidene)methylene]bis[N-phenylaniline], monohydrochloride), sodium salts

Toxicity to aquatic plants other than algae

Administrative data

Endpoint:

toxicity to aquatic plants other than algae

Remarks:

Toxicity to the Aquatic Plant Lemna gibba

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-03-06 to 2017-05-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD, EU guideline, GLP

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 221 (Lemna sp. Growth Inhibition Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 761/2009, C.26.

Test guideline 3

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", December 15, 2000

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

pentasodium 4-({4-[(4-aminophenyl)({4-[(4-sulfonatophenyl)amino]cyclohexa-2,5-dien-1-ylidene})methyl]phenyl}amino)benzene-1-sulfonate 4-{[4-({4-[(4-sulfonatophenyl)amino]cyclohexa-2,5-dien-1-ylidene}({4-[(4-sulfonatophenyl)amino]phenyl})methyl)phenyl]amino}benzene-1-sulfonate

EC Number:

944-086-6

IUPAC Name:

pentasodium 4-({4-[(4-aminophenyl)({4-[(4-sulfonatophenyl)amino]cyclohexa-2,5-dien-1-ylidene})methyl]phenyl}amino)benzene-1-sulfonate 4-{[4-({4-[(4-sulfonatophenyl)amino]cyclohexa-2,5-dien-1-ylidene}({4-[(4-sulfonatophenyl)amino]phenyl})methyl)phenyl]amino}benzene-1-sulfonate

Test material form:

solid

Details on test material:

UVCB

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Duplicate samples were taken at the start and at each test medium renewal from the freshly prepared test media of all test concentrations and from the control.
For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the renewal periods, duplicate samples of all aged test media and control were sampled at each test medium renewal and at the end of the test.

Test solutions

Vehicle:

no

Details on test solutions:

Test Concentrations: 100, 32, 10, 3.2 and 1.0 mg test item/L and a control.
Control: In the control, test medium was used without addition of the test item.
Dosage of Test Item: The test medium of the highest test concentration of nominal 100 mg test item/L was prepared by dissolving 106.1, 106.8 and 102.4 mg test item into 1061, 1068 and 1024 mL test water by intense stirring for 10 minutes and ultrasonic treatment for 5 minutes, respectively. Adequate volumes of this test medium were diluted with test water to prepare the test media of the other desired test concentrations.
The test media were prepared just before introduction of the Lemna (= start of the test) and each test medium renewal.
Appearance of the Test Item in Test Medium:
The test medium was blue coloured by the test item ranging from strong colouration (100 mg/L) to medium (10 and 32 mg/L) to slight colouration (3.2 mg/L). The lowest concentration of 1.0 mg/L was not coloured. In the aged test media, slighter colouration was observed.

Test organisms

Test organisms (species):

Lemna gibba

Details on test organisms:

Species: Lemna gibba G 3
Origin: The fronds introduced in the test were taken from ibacon's in-house laboratory culture.
Breeding Conditions: The plants are cultured in the laboratories of ibacon under standardised conditions according to the test guidelines.
Pre-Culture: For at least 7 days under test conditions with weekly media exchange.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test conditions

Test temperature:

22.1 to 23.7 °C

pH:

7.4 to 7.7 in the freshly prepared test media;
8.2 to 8.5 in the aged test media

Nominal and measured concentrations:

nominal: 100, 32, 10, 3.2 and 1.0 mg test item/L and a control.
In the freshly prepared test media at the start of the test and at the renewal of the test media 96% of the nominal test concentrations were found (average of all test concentrations). In the aged test media at day 2, day 5 and day 7 of the test duration, 90% of the nominal value was determined (average of all test concentrations).
During the test the test organism were exposed to a mean of 93% of nominal. Therefore, all reported results refer to nominal concentrations.

Details on test conditions:

Test Environment: Controlled environment room
Water Temperature: The temperature was measured daily in a test vessel filled with water and incubated under the same conditions as the test vessels.
22.1 to 23.7 °C
pH-Values: The pH-values were measured in all freshly prepared and aged test media.
7.4 to 7.7 in the freshly prepared test media;
8.2 to 8.5 in the aged test media
Light Regime: Continuous illumination
Light Intensity: 7445 Lux (mean value) with a range of 7030 to 7890 lux.
The illumination was achieved by fluorescent tubes, installed above the test vessels. Measurements were performed once during the test at 6 places distributed over the experimental area at the surface of the test media. Differences in light intensity over the test area did not exceed ± 15 %.

Reference substance (positive control):

yes

Remarks:

For the evaluation of the quality of the Lemna gibba and the experimental conditions the substance 3,5-dichlorophenol is tested at least twice a year to demonstrate satisfactory test conditions.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Effect concentrations 2

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 3

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 5

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Effect concentrations 6

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

55.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 7

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 8

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 9

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

51.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Effect concentrations 10

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

25.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 11

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 12

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

72.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 13

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Effect concentrations 14

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 15

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 16

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 17

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Effect concentrations 18

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 19

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 20

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

The 7-day EyC50 was calculated to be > 100 mg test item/L for frond number and dry weight, respectively.
The 7-day ErC50 was calculated to be > 100 mg test item/L for frond number and dry weight, respectively.
The 7-day EyC10 was calculated to be 51.6 and 25.9 mg test item/L for frond number and dry weight, respectively.
The 7-day ErC10 was calculated to be > 100 and 72.1 mg test item/L for frond number and dry weight, respectively.
The 7-day NOEyC and the LOEyC were determined to be 32 and 100 mg test item/L for frond number and dry weight, respectively.
The 7-day NOErC and the LOErC were determined to be 32 and 100 mg test item/L for frond number and dry weight, respectively.
The shape of fronds and colonies after the test period of 7 days was not different to those in the control up to and including the nominal test concentration of 32 mg test item/L.
At the highest test item concentration of 100 mg test item/L the roots were shortened.
Analytical results:
In the freshly prepared test media at the start of the test and at the renewal of the test media 96% of the nominal test concentrations were found (average of all test concentrations). In the aged test media at day 2, day 5 and day 7 of the test duration, 90% of the nominal value was determined (average of all test concentrations).
During the test the test organism were exposed to a mean of 93% of nominal. Therefore, all reported results refer to nominal concentrations.

Reported statistics and error estimates:

Statistical Analysis:
The EC50/20/10 (the concentrations of the test item corresponding to 50, 20 and 10 % inhibition of growth rate (frond number and dry weight) and yield (frond number and dry weight) compared to the control), and their 95 %-confidence limits were calculated by probit analysis.
For the determination of the 7-day LOEyC and NOEyC values significant differences at the test concentrations compared to the control values were tested by the Williams t-test (frond number) and the Dunnett's t-test (dry weight).
For the determination of the 7-day LOErC and NOErC values significant differences at the test concentrations compared to the control values were tested by the Williams t-test (frond number) and the Dunnett's t-test (dry weight).
The software used to perform the statistical analysis was ToxRat Professional, Version 2.10.05, ToxRat Solutions GmbH.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

Doubling Time of Frond Number in Control: 1.9 days, validity criterion was met.

Conclusions:

The influence of the test item on the growth of the freshwater plant Lemna gibba was assessed in a semi-static concentration-response test.
The 7-day EyC50 was calculated to be > 100 mg test item/L for frond number and dry weight, respectively.
The 7-day ErC50 was calculated to be > 100 mg test item/L for frond number and dry weight, respectively.
The 7-day NOEyC and the LOEyC were determined to be 32 and 100 mg test item/L for frond number and dry weight, respectively.
The 7-day NOErC and the LOErC were determined to be 32 and 100 mg test item/L for frond number and dry weight, respectively.
The quantification of the test item Duasyn-Tintenblau SLK in the test samples was performed using liquid chromatography with UV detection.
In the freshly prepared test media at the start of the test and at the renewal of the test media 96% of the nominal test concentrations were found (average of all test concentrations). In the aged test media at day 2, day 5 and day 7 of the test duration, 90% of the nominal value was determined (average of all test concentrations).
During the test the test organism were exposed to a mean of 93% of nominal. Therefore, all reported results refer to nominal concentrations.

Executive summary:

Title: The test item: Toxicity to the Aquatic Plant Lemna gibba in a Semi-Static Growth Inhibition Test

Commission Regulation (EC) No 761/2009, Annex, Part C, C.26.: "Lemna sp. Growth Inhibition Test", Official Journal of the European Union (EN), dated August 24, 2009

OECD Guideline 221: "Lemna sp. Growth Inhibition Test", adopted March 23, 2006

SANCO/3029/99 rev.4 11/07/00: Residues: Guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (part A; Section 4) and Annex III (part A; Section 5) of directive 91/414

OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures", December 15, 2000

Conclusion:

The influence of the test item on the growth of the freshwater plant Lemna gibba was assessed in a semi-static concentration-response test.

The 7-day EyC50 was calculated to be > 100 mg test item/L for frond number and dry weight, respectively.

The 7-day ErC50 was calculated to be > 100 mg test item/L for frond number and dry weight, respectively.

The 7-day NOEyC and the LOEyC were determined to be 32 and 100 mg test item/L for frond number and dry weight, respectively.

The 7-day NOErC and the LOErC were determined to be 32 and 100 mg test item/L for frond number and dry weight, respectively.

The quantification of the test item in the test samples was performed using liquid chromatography with UV detection. In the freshly prepared test media at the start of the test and at the renewal of the test media 96% of the nominal test concentrations were found (average of all test concentrations). In the aged test media at day 2, day 5 and day 7 of the test duration, 90% of the nominal value was determined (average of all test concentrations). During the test the test organism were exposed to a mean of 93% of nominal. Therefore, all reported results refer to nominal concentrations.

This study is classified acceptable and satisfies the guideline requirements for aquatic plant studies.