Tripotassium/trisodium 5-(4-chloro-6-(N-(4-(4-chloro-6-(5-hydroxy-2,7-disulfonato-6-(2-sulfonatophenylazo)-4-naphthylamino)-1,3,5-triazin-2-yl-(N-methyl)amino)phenyl)amino)-1,3,5-triazin-2-ylamino)-4-hydroxy-3-(2-sulfonatophenylazo)naphthalene-2,7-disulfonate

Toxicity to aquatic plants other than algae

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic plants other than algae

Remarks:

lemna

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 November to 01 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

other: EC C.26

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

At the beginning and at the end of the experiment, the content of the test item in the test solution was analytically determined.
For elimination of any plant parts before analytical determination, samples were filtered through PFTE syringe filters.

Vehicle:

no

Test organisms (species):

Lemna minor

Details on test organisms:

TEST ORGANISM
- Common name: duckweed
- Strain: Lemna minor
- Source: Laboratory culture obtained from Umweltbundesamt (Federal Environmental Agency) Berlin in February 2017

ACCLIMATION
- Acclimation period: 7 days in Steinberg medium
- Any deformed or abnormal cells observed: none
- colonies kept as a permanent culture under conditions comparable to the test conditions

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test temperature:

22.5 – 23.8 °C

pH:

at start: 5.6 ; at study end: 6.9-7.4

Nominal and measured concentrations:

nominal: 0, 1.4, 4.5, 14, 45 and 141 mg/L (0, 1,3, 10, 30 and 100 mg/L as a.i.)
measured (geometric mean): 0, not calculable, 3.1, 11.8, 46.6 and 135.9 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:glass beaker of 250 mL
- Type: closed with watch glass
- Fiill volume: 150 mL
- Aeration: no
- Agitation: no
- No. of colonies per vessel: 4
- No. of fronds per colony: 3
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes, Steinberg medium

TEST MEDIUM / WATER PARAMETERS: Steinberg medium according to the guideline

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous light
- Light intensity: 7000 lux

EFFECT PARAMETERS MEASURED
- Determination of frond number: manual counting on day 0, 3, 6 and 7
- Determination of biomass: dry weight on day 0 and 7

RANGE-FINDING STUDY
- Test concentrations: 0 and 141 mg/L (nominal)
- Results used to determine the conditions for the definitive study: 55% toxicity at 141 mg/L

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Duration:

7 d

Dose descriptor:

EC50

Remarks:

ErC50

Effect conc.:

> 135.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

frond number

Duration:

7 d

Dose descriptor:

EC50

Remarks:

ErC50

Effect conc.:

> 135.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

7 d

Dose descriptor:

EC50

Remarks:

EyC50

Effect conc.:

98.13 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

frond number

Duration:

7 d

Dose descriptor:

EC50

Remarks:

EyC50

Effect conc.:

74.09 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

11.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

frond number

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

11.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

All fronds look healthy and normal at day 7

Parameter Validity criteria Observed value Assessment
Doubling time of frond number in days Less than 2.5 1.9 Valid
Increase factor of fronds at least 7 13.4 Valid
Average specific growth rate per day at least 0.275 0.370 Valid

Results with reference substance (positive control):

Biological Results of C6H4Cl2O based on frond numbers
Parameter Value 95% confidence interval
7d ErC50 (frond number) 3.21 mg/L 2.6 – 3.9 mg/L
7d EyC50 (frond number) 2.21 mg/L 1.9 – 2.7 mg/L

Biological Results of C6H4Cl2O based on dry mass
Parameter Value 95% confidence interval
7d ErC50 (dry mass) 2.62 mg/L n.d.
7d EyC50 (dry mass) 2.49 mg/L n.d.
n.d. not determinable due to mathematical reasons or inappropriate data

Frond numbers at the end of the test (initial 12)

Nominal Concentration	Mean Frond Number			CV of Frond Number
mg/L	3 d	6 d	7 d	7 d
Blank control	33.7	115.7	161.0	9.40%
1.4	36.0	111.0	180.0	23.50%
4.5	35.0	120.3	193.7	20.03%
14	34.3	115.3	192.7	12.46%
45	33.3	91.7	122.3	15.60%
141	25.7	60.0	86.0	19.14%

CV = Coefficient of variation

Inhibition Values based on frond number

Nominal concentration in mg/L	% Inhibition (frond number)
	Growth Rate (0 - 7d)	Yield (0 - 7d)
Blank control	0	0
4.5	-6.7	-21.9
14	-6.8	-21.3
45	10.8	26.0
141	24.6	50.3

Negative inhibition values indicate a stimulation of plant growth compared to the blank control.

 

Dry Mass at the end of the test (initial 1.4 mg)

Nominal Concentration	Replicate	Dry Mass in mg	Mean Dry Mass in mg	CV of Dry Mass
mg/L		7 d	7 d	7 d
Blank control	a	25.5	26.7	9.45 %
	b	29.6
	c	25.0
1.4	a	21.0	23.8	25.46 %
	b	30.8
	c	19.7
4.5	a	27.3	25.2	15.58 %
	b	27.7
	c	20.7
14	a	23.4	24.8	10.84 %
	b	27.9
	c	23.1
45	a	14.3	14.9	12.28 %
	b	17.0
	c	13.5
141	a	12.6	11.6	11.93 %
	b	12.1
	c	10.0

CV = Coefficient of variation

Inhibition Values based on dry mass

Nominal concentration in mg/L	% Inhibition (dry mass)
	Growth Rate (0 - 7d)	Yield (0 - 7d)
Blank control	0	0
4.5	2.1	5.8
14	2.5	7.5
45	19.8	46.5
141	28.5	59.8

Validity criteria fulfilled:

yes

Conclusions:

The ErC50 of the substance is > 135.9 mg/L (100 mg/L nominal a.i.). The NOEC is 11.8 mg/L (14 mg/L nominal a.i.)

Executive summary:

A study with Lemna minor was performed using 5 concentrations ranging from 1.4 to 141 mg/L ( (1.3 -100 mg/L as a.i.) The measured concentrations are (geometric mean): ND, 3.1, 11.8, 46.6 and 135.9 mg/L. Incubation time was 7 days. Significant inhibition of plant growth was observed in the 2 highest concentrations. All validity criteria were fulfilled. The ErC50 of the substance is > 135.9 mg/L (>100 mg/L nominal a.i.). The NOEC is 11.8 mg/L (14 mg/L nominal a.i.) based on measured concentrations.

 

Description of key information

A study with Lemna minor was performed using 5 concentrations ranging from 1.4 to 141 mg/L ( (1.3 -100 mg/L as a.i.) The measured concentrations are (geometric mean): ND, 3.1, 11.8, 46.6 and 135.9 mg/L. Incubation time was 7 days. Significant inhibition of plant growth was observed in the 2 highest concentrations. All validity criteria were fulfilled. The ErC50 of the substance is > 135.9 mg/L (>100 mg/L nominal a.i.). The NOEC is 11.8 mg/L (14 mg/L nominal a.i.) based on measured concentrations (Laus 2018).

In a study according to OECD 219 Lemna gibba fronds were exposed to a formulation of the substance at 0.01, 1, 50 and 100 mg/L during 7 days. Effects on growth rate, biomass and yield were reported at all concentrations above 1 mg/L. No toxic symptoms and change in appearance of the fronds were observed. Based on active ingredient the EC50 is ≥ 35 mg/L and the NOEC for growth rate is 1 mg/L. The publication contains only limited information and it is not clear whether other components of the formulation may have influenced this result (Darsana 2015).

Key value for chemical safety assessment

EC50 for freshwater plants:

100 mg/L

EC10 or NOEC for freshwater plants:

14 mg/L