pentasodium 4-amino-3-[{4-[(2,4-disulfonatophenyl)diazenyl]-3-methylphenyl}diazenyl]-5-hydroxy-6-[(4-nitro-2-sulfonatophenyl)diazenyl]naphthalene-1,7-disulfonate

Toxicity to aquatic plants other than algae

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic plants other than algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24. Nov 2020 - 18. Feb 2021 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EU) No. 2016/266 amending Regulation (EC) No. 440/2008. Annex IV. Method C.26: “LEMNA SP. GROWTH INHIBITION TEST.”

Version / remarks:

adopted 01. March 2016

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Version / remarks:

adopted 23. March 2006

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

At the beginning and at the end of the experiments (after 7 days), the content of the test item in the test solution was determined using HPLC-UV-determination. On each sampling day (0d, 3d, 6d, 7d), validity of calibration was controlled by measuring QC samples (1.5 mg/L).

Test organisms (species):

Lemna minor

Test type:

static

Water media type:

freshwater

Total exposure duration:

7 d

Test temperature:

The temperature in the 2nd experiment was in the range 23.7 – 26.2 °C, and therefore
not in the desired range (22.0 - 26.0 °C). Because normal growth was observed in the
blank control, this can be stated as uncritical. The temperature in the 1st experiment was in the range 24.0 – 25.8 °C and thus in the desired range (22.0 - 26.0 °C).

pH:

In the test solutions and the blank control solution, the pH-value was measured at the start
and the end of the test and were in the range of 5.3 to 7.4.

Nominal and measured concentrations:

Nominal test concentrations
1st experiment test concentrations: 0.1 / 0.32 / 1 / 3.2 / 10 / 32 mg/L (a. s.)
2nd experiment test concentrations: 1 / 3.2 / 10 / 32 / 100 mg/L (a. s.)

Details on test conditions:

To each vessel 150 ± 5 mL of the respective test solution was added and plants containing 4 colonies with 12 fronds in total were given to each test vessel. Each test vessel contained the same number of fronds and colonies. The preparation of the test solutions and the insertion of the colonies was performed under sterile conditions. The test vessels were incubated covered with watch glasses for 7 days under continuous lighting. At the start, at incubation day 3 and 6 at the end, the frond number of each test vessel was determined. Additionally, the dry weight of biomass was determined. For this, the dry weight of one representative sample of 12 fronds from the pre-culture was determined at the start of the test. For this the fronds were dried at 60 °C for 24 h (1st experiment) and 23 h and 10 min. (2nd experiment) and were weighted. After this the fronds were dried again for 23 h and 40 min. (1st experiment) and 23.5 h (2nd experiment) and weighted again. At the end of the test, all colonies from one test vessel were collected and rinsed with demineralised water, dried at 60 °C for 23.75 h (1st experiment) and 23.5 h (2nd experiment) and then weighted. After this the fronds were dried again for 24 h and 10 min. (1st experiment) and 23 h and 25 min. (2nd experiment) and weighted again. This was performed for each test vessel. Before determination of dry weight at the end of the experiment, each test vessel was inspected in order to verify a normal and healthy appearance of the colonies.

Replicates
Treatments: 3 vessels, each filled with 150 ± 5 mL test solution and colonies
Blank Control: 3 vessels, each filled with 150 ± 5 mL STEINBERG medium
and colonies

Reference substance (positive control):

yes

Remarks:

A positive control (3,5-Dichlorophenol (1,3-Dichloro-5-hydroxybenzene, C6H4Cl2O, CAS-No. 591-35-5)) was tested in a separate study to assure that the test conditions are reliable. The EC50 value of the growth ratelay within the desired range of 1.7 - 5.7

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

11.46 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: value was extrapolated by the program

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

246.18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: value was extrapolated by the program

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

0.06 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: value was extrapolated by the program

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

0.64 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

0.06 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: value was extrapolated by the program

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

0.69 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

0.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

0.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

0.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

0.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Results with reference substance (positive control):

Positive Control - Biological Results of the Reference Study (202012R1011)

The study with the positive control 3,5-Dichlorophenol (1,3-Dichloro-5-hydroxybenzene, C6H4Cl2O, CAS-No. 591-35-5) was performed under GLP conditions in November 2020.

- Results with reference substance valid? yes
- Relevant effect levels: 7d ErC50 (frond number): 3.32 mg/L
7d EyC50 (frond number): 2.22 mg/L
7d ErC50 (dry mass): 4.58 mg/L
7d EyC50 (dry mass): 2.41 mg/L
- Other: none
- Results with reference substance valid? yes

Reported statistics and error estimates:

The 2nd experiment was used for the evaluation of the NOEC, LOEC and ECX values. Calculation of results and estimation of the biological data was accomplished using the software ToxRat® Professional, version 3.3.0. Two statistically determinations were conducted, because ToxRat could not be determine the EC10 values when all measured concentrations (a. s.) were taken into account. The first Determination was done with the three lowest concentrations 1 mg/L, 3.2 mg/L and 10 mg/L (a. s.) to determine the EC10 values. The details of calculation are stated in Annex
4: Calculations using ToxRat® Professional 3.3.0 for the Determination of EC10. The second Determination was done with all concentrations to determine the EC50, NOEC and LOEC.

Two valid experiments were performed.
The 1st experiment was performed using 6 concentrations ranging from 0.1 to 32 mg/L (nominal concentration active substance (a. s.)). Incubation time (test system Lemna minor) was 7 days. The frond number of each replicate was determined at the beginning, at day 3 and 6 during the test and at the end of the experiment. Additionally, the dry mass of 12 representative fronds was determined at the beginning of the experiment. At the end of the experiment the dry mass of each replicate was determined. In the 1st experiment too low inhibition values at the highest tested concentration of 32 mg/L (35.8 % for the endpoint frond number) were shown. Since no EC50 value could be determined,the 1st experiment was repeated with higher concentrations. Because of this, the 2nd experiment was performed using 5 concentrations ranging from 1 to 100 mg/L (nominal concentration a. s.). Incubation time (test system Lemna minor) was 7 days. The frond number of each replicate was determined at the beginning, at day 3 and during the test and at the end of the experiment. Additionally, the dry mass of 12 representative fronds was determined at the beginning of the experiment. At the end of the experiment the dry mass of each replicate was determined. The 2nd experiment was used for the evaluation of the NOEC, LOEC and ECX values.

Validity criteria fulfilled:

yes

Conclusions:

Two valid experiments were performed.
The 1st experiment was performed using 6 concentrations ranging from 0.1 to 32 mg/L (nominal concentration active substance (a. s.)). Incubation time (test system Lemna minor) was 7 days. The frond number of each replicate was determined at the beginning, at day 3 and 6 during the test and at the end of the experiment. Additionally, the dry mass of 12 representative fronds was determined at the beginning of the experiment. At the end of the experiment the dry mass of each replicate was determined. In the 1st experiment too low inhibition values at the highest tested concentration of 32 mg/L (35.8 % for the endpoint frond number) were shown. Since no EC50 value could be determined,the 1st experiment was repeated with higher concentrations. Because of this, the 2nd experiment was performed using 5 concentrations ranging from 1 to 100 mg/L (nominal concentration a. s.). Incubation time (test system Lemna minor) was 7 days. The frond number of each replicate was determined at the beginning, at day 3 and during the test and at the end of the experiment. Additionally, the dry mass of 12 representative fronds was determined at the beginning of the experiment. At the end of the experiment the dry mass of each replicate was determined. The 2nd experiment was used for the evaluation of the NOEC, LOEC and ECX values.

Executive summary:

In the 1st experiment too low inhibition values at the highest tested concentration of 32 mg/L (35.8 % for the endpoint frond number) were shown. Since no EC50 value could be determined, the 1st experiment was repeated with higher concentrations. Because of this, the 2nd experiment was performed using 5 concentrations ranging from 1to 100 mg/L (nominal concentration a. s.). Incubation time (test system Lemna minor) was 7 days. The frond number of each replicate was determined at the beginning, at day 3 and 6 during the test and at the end of the experiment. Additionally, the dry mass of 12 representative fronds was determined at the beginning of the experiment. At the end of the experiment the dry mass of each replicate was determined. The 2nd experiment was used for the evaluation of the NOEC, LOEC and ECX values. Growth rate μ and the yield were determined from the frond number and the drymass at the respective observation times.
Significant inhibition of plant growth was observed at the following nominal concentrations: 3.2 / 10 / 32 / 100 mg/L (active substance) for all endpoints.

Endpoint	NOEC	LOEC	EC10	EC50
Growth Rate frond number	0.20 mg/L	0.50 mg/L	0.69 mg/L	n. d.
Yield frond number	0.20 mg/L	0.50 mg/L	0.06 mg/L *	246.18mg/L *
Growth Rate dry mass	0.20 mg/L	0.50 mg/L	0.64 mg/L	n. d.
Yield dry mass	0.20 mg/L	0.50 mg/L	0.06 mg/L *	11.46 mg/L

n. d. = not determinable due to mathematical reasons or inappropriate data
* These values were extrapolated by the program.

Description of key information

Two valid experiments were performed.
The 1st experiment was performed using 6 concentrations ranging from 0.1 to 32 mg/L (nominal concentration active substance (a. s.)). Incubation time (test system Lemna minor) was 7 days. The frond number of each replicate was determined at the beginning, at day 3 and 6 during the test and at the end of the experiment. Additionally, the dry mass of 12 representative fronds was determined at the beginning of the experiment. At the end of the experiment the dry mass of each replicate was determined. In the 1st experiment too low inhibition values at the highest tested concentration of 32 mg/L (35.8 % for the endpoint frond number) were shown. Since no EC50 value could be determined,the 1st experiment was repeated with higher concentrations. Because of this, the 2nd experiment was performed using 5 concentrations ranging from 1 to 100 mg/L (nominal concentration a. s.). Incubation time (test system Lemna minor) was 7 days. The frond number of each replicate was determined at the beginning, at day 3 and during the test and at the end of the experiment. Additionally, the dry mass of 12 representative fronds was determined at the beginning of the experiment. At the end of the experiment the dry mass of each replicate was determined. The 2nd experiment was used for the evaluation of the NOEC, LOEC and ECX values.

 

Endpoint	NOEC	LOEC	EC10	EC50
Growth Rate frond number	0.20 mg/L	0.50 mg/L	0.69 mg/L	n. d.
Yield frond number	0.20 mg/L	0.50 mg/L	0.06 mg/L *	246.18mg/L *
Growth Rate dry mass	0.20 mg/L	0.50 mg/L	0.64 mg/L	n. d.
Yield dry mass	0.20 mg/L	0.50 mg/L	0.06 mg/L *	11.46 mg/L

n. d. = not determinable due to mathematical reasons or inappropriate data
* These values were extrapolated by the program.

Key value for chemical safety assessment

EC50 for freshwater plants:

11.46 mg/L

EC10 or NOEC for freshwater plants:

0.2 mg/L