Sodium 3-[[4-amino-9,10-dihydro-3-[2-(2-methoxyethoxy)ethoxy]-9,10-dioxo-1-anthryl]amino]-2,4,6-trimethylbenzenesulphonate

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:

2017-08-30 to 2017-12-22

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.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 3-[[4-amino-9,10-dihydro-3-[2-(2-methoxyethoxy)ethoxy]-9,10-dioxo-1-anthryl]amino]-2,4,6-trimethylbenzenesulphonate

EC Number:

274-366-7

EC Name:

Sodium 3-[[4-amino-9,10-dihydro-3-[2-(2-methoxyethoxy)ethoxy]-9,10-dioxo-1-anthryl]amino]-2,4,6-trimethylbenzenesulphonate

Cas Number:

70179-77-0

Molecular formula:

C28H30N2O8S.Na

IUPAC Name:

sodium 3-[[4-amino-9,10-dihydro-3-[2-(2-methoxyethoxy)ethoxy]-9,10-dioxo-1-anthryl]amino]-2,4,6-trimethylbenzenesulphonate

Test material form:

solid: particulate/powder

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 at the end of the test.
The samples were diluted by a factor of 2 with acetonitrile. Additional samples of the control blank and the dilution solvent were taken at test start and test end without any sample treatment.

Test solutions

Vehicle:

no

Details on test solutions:

Test Concentrations: 131, 42.0, 13.1, 4.20 and 1.31 mg test item/L (referring to 100, 32, 10, 3.2 and 1.0 mg active ingredient/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 131 mg test item/L was prepared by dissolving 132.2, 131.2 and 131.8 mg test item into 1009.16, 1001.53 and 1006.11 mL test water by intense stirring for 15 to 40 minutes. Adequate volumes of this stock solution were diluted with test water to prepare the test media of the 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: In the freshly prepared and aged test medium of the highest nominal concentration of 131 mg test item/L (100 mg active ingredient/L) were observed to be dark violet. The lower the concentration, the lighter the test medium was coloured ranging from dark violet (42.0 mg test item/L (32 mg active ingredient/L)) to violet (4.20 and 13.1 mg test item/L (3.2 and 10 mg active ingredient/L)) to light violet (1.31 mg test item/L (1.0 mg active ingredient/L)). Additionally, the test item was observed to lie at the bottom of the test beaker in the aged test media at test concentration of 42.0 and 131 mg test item/L (32 and 100 mg active ingredient/L).

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 our 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:

23 to 24 °C

pH:

7.4 to 7.6 in the freshly prepared test media;
8.2 to 8.6 in the aged test media

Nominal and measured concentrations:

131, 42.0, 13.1, 4.20 and 1.31 mg test item/L (referring to 100, 32, 10, 3.2 and 1.0 mg active ingredient/L) and a control.

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.
23 to 24 °C
pH-Values: The pH-values were measured in all freshly prepared and aged test media.
7.4 to 7.6 in the freshly prepared test media;
8.2 to 8.6 in the aged test media
Light Regime: Continuous illumination
Light Intensity: 7657 Lux (mean value) with a range of 7030 to 8160 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:

act. ingr.

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:

act. ingr.

Basis for effect:

biomass

Effect concentrations 3

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

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:

act. ingr.

Basis for effect:

biomass

Effect concentrations 5

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

50.8 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

frond number

Effect concentrations 6

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

88.3 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Effect concentrations 7

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

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:

act. ingr.

Basis for effect:

biomass

Effect concentrations 9

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

21.6 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

frond number

Effect concentrations 10

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

26.9 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Effect concentrations 11

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

64.3 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Effect concentrations 12

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Effect concentrations 13

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

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:

act. ingr.

Basis for effect:

biomass

Effect concentrations 15

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

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:

act. ingr.

Basis for effect:

biomass

Effect concentrations 17

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

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:

act. ingr.

Basis for effect:

biomass

Effect concentrations 19

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Effect concentrations 20

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Details on results:

The 7-day EyC50 was calculated to be > 131 mg test item/L (> 100 mg active ingredient/L) for frond number and dry weight, respectively.
The 7-day ErC50 was calculated to be > 131 mg test item/L (> 100 mg active ingredient/L) for frond number and dry weight, respectively.
The 7-day EyC10 was calculated to be 28.4 and 35.3 mg test item/L (21.6 and 26.9 mg active ingredient/L) for frond number and dry weight, respectively.
The 7-day ErC10 was calculated to be 84.5 and > 131 mg test item/L (64.3 and > 100 mg active ingredient/L) for frond number and dry weight, respectively.
The 7-day NOEyC and the LOEyC were determined to be 13.1 and 42.0 mg test item/L (10 and 32 mg active ingredient/L) for frond number and 42.0 and 131 mg test item/L (32 and 100 mg active ingredient/L) for dry weight, respectively.
The 7-day NOErC and the LOErC were determined to be 13.1 and 42.0 mg test item/L (10 and 32 mg active ingredient/L) for frond number and 42.0 and 131 mg test item/L (32 and 100 mg active ingredient/L) for dry weight, respectively

Reported statistics and error estimates:

The ErC50 and the EyC50 (see Definitions), the corresponding EC20 and EC10 values and where possible 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.
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. The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, 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 > 131 mg test item/L (> 100 mg active ingredient/L) for frond number and dry weight, respectively.
The 7-day ErC50 was calculated to be > 131 mg test item/L (> 100 mg active ingredient/L) for frond number and dry weight, respectively.
The 7-day NOEyC and the LOEyC were determined to be 13.1 and 42.0 mg test item/L (10 and 32 mg active ingredient/L) for frond number and 42.0 and 131 mg test item/L (32 and 100 mg active ingredient/L) for dry weight, respectively.
The 7-day NOErC and the LOErC were determined to be 13.1 and 42.0 mg test item/L (10 and 32 mg active ingredient/L) for frond number and 42.0 and 131 mg test item/L (32 and 100 mg active ingredient/L) for dry weight, respectively.

Executive summary:

Title: Acid Violet 126: Toxicity to the Aquatic Plant Lemna gibba in a Semi-Static Growth Inhibition Test

Purpose: The purpose of this study was to determine the inhibitory effect of the test item Acid Violet 126 on the growth of the freshwater aquatic plant Lemna gibba. For this purpose, cultures of Lemna gibba were exposed in a semi-static test to various concentrations under defined conditions. The inhibition of growth in relation to control cultures was determined over a test period of 7 days. The test method of application and the test system are recommended by the test guidelines and Lemna gibba is the recommended test species. The purpose of the analytical part of this study was to verify the concentrations of the test item in the test medium.

Guidelines/Recommendations:

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

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

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

Material and Methods

Test Item: Acid Violet 126; batch no.: PQD21/1609023; purity: 76.1 %, according to certificate of analysis.

Test Species: Lemna gibba G 3

Test Design: This study encompassed 7 treatment groups (6 dose rates of the test item and a control) with three replicates per test concentration and control.

At test start 12 fronds were introduced in each replicate and incubated for 7 days under semi-static conditions. The frond numbers were determined on day 2, 5 and 7. The dry weight of each replicate was determined at test termination.

Endpoints: Yield and growth rate based on frond number and dry weight.

Test Concentrations: 131, 42.0, 13.1, 4.20 and 1.31 mg test item/L (referring to 100, 32, 10, 3.2 and 1.0 mg active ingredient/L) and a control.

Test Conditions: Water temperature: 23 to 24 °C; pH values in the freshly prepared test media 7.4 to 7.6, in the aged test media 8.2 to 8.6; continuous illumination; mean light intensity: 7657 lux (7030 to 8160 lux).

Conclusion: The influence of Acid Violet 126 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 > 131 mg test item/L (> 100 mg active ingredient/L) for frond number and dry weight, respectively. The 7-day ErC50 was calculated to be > 131 mg test item/L (> 100 mg active ingredient/L) for frond number and dry weight, respectively. The 7-day NOEyC and the LOEyC were determined to be 13.1 and 42.0 mg test item/L (10 and 32 mg active ingredient/L) for frond number and 42.0 and 131 mg test item/L (32 and 100 mg active ingredient/L) for dry weight, respectively. The 7-day NOErC and the LOErC were determined to be 13.1 and 42.0 mg test item/L (10 and 32 mg active ingredient/L) for frond number and 42.0 and 131 mg test item/L (32 and 100 mg active ingredient/L) for dry weight, respectively The quantification of the test item Acid Violet 126 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 114% of the nominal test concentrations were found (average of all test concentrations). In the aged test media after 48 or 72 hours test duration, 85% of the nominal value was determined (average of all test concentrations). All reported results refer to nominal concentrations because the variation of the initial test concentrations is rather high and therefore not considered as a solid basis for calculations. This study is classified acceptable and satisfies the guideline requirements for aquatic plant studies.