Reaction products of Sodium 1-amino-4-bromo-9,10-dioxoanthracene-2-sulphonate coupled with Aniline, subsequently coupled with p-(1,1-dimethylpropyl)phenol, subsequently coupled with Ethyl benzoylacetate, subsequently coupled with Fuming Sulfuric acid, sodium salts.

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:

May from 04 to 12, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Version / remarks:

adopted 23rd March, 2006

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

For determination of the test item concentrations, samples were taken from each concentration level and control at the start and at the end of the test.

Vehicle:

no

Details on test solutions:

For preparation of test solutions a stock solution was first prepared by dissolving an amount of 0.1287 g test item in 1002 ml dilution water (20X AAP medium), resulting a stock solution of 128.4 mg/l nominal concentration. The test solutions of the chosen test concentrations were prepared by appropriate dilution of the stock solution.

Test organisms (species):

Lemna gibba

Details on test organisms:

TEST ORGANISM
- Common name: duckweed.
- Source: Friedrich Schiller Universität, Institut für Allgemeine Botanik und Pflanzenphysiologie, Jena, Germany. Organisms were in-house cultureed and kept under axenic conditions; preculture was prepared 8 days before the tests.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test temperature:

22.7 – 23.2 °C in the test vessels

pH:

8.11 - 8.71

Nominal and measured concentrations:

4.0, 8.0, 16.0, 32.1, 64.2 and 128.4 mg/l nominal (corresponding to 3.125, 6.25, 12.5, 25.0, 50.0 and 100 mg active ingredient/l)

Details on test conditions:

TEST SYSTEM
- Test vessel: 400 ml Petri dishes-covered glass beakers were filled with 160 ml testing solutions.
- No. of colonies per vessel: three colonies.
- No. of fronds per colony: three colonies with three and one colony with two fronds.
- No. of vessels per concentration: three replicates.
- No. of vessels per control: six replicates.

GROWTH MEDIUM
- Standard medium used: 20X AAP Medium.

OTHER TEST CONDITIONS
- Photoperiod: the test vessels were placed randomly on a tray, illuminated continuously.
- Light intensity and quality: between 6500-10000 lux (7105 ± 166 lux as mean value) using fluorescent light tubes (with a spectral range of 400-700 nm).

EFFECT PARAMETERS MEASURED
- Determination of frond number: at the end of the test fronds were counted in each test vessel.
- Dry and fresh weight determination: every colonies, fronds and root fragments of each test vessel were collected and the dry weight and fresh weights measured in the same way as at the start of the study.
- Observations: assessment of growth and morphological changes on days 3, 5 and 7.

RANGE-FINDING STUDY
- Test concentrations: 0.1, 1.0, 10 and 100 mg/l, nominal.
- No. of vessels per concentration: two replicates (containing 11 fronds in total per test vessel).
- No. of vessels per control: three replicates.
- Results used to determine the conditions for the definitive study: the percentages recorded for growth rate inhibition were 6.33, 6.92, 18.95 and 22.97 at 0.1, 1.0, 10 and 100 mg/l, respectively.

ACCEPTANCE CRITERIA
For the test to be valid, the doubling time of frond number in the control must be less than 2.5 days (60 h), corresponding to approximately a 7-fold increase in 7 days and an average specific growth rate of 0.275 d-1.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 128.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (corresponding to 100 mg active ingredient/l)

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 128.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: (corresponding to 100 mg active ingredient/l)

Details on results:

GROWTH RATE
Growth rates based on frond number
NOErC (7d): 4.0 mg/l, nominal; LOErC (7d): 8.0 mg/l, nominal
ErC10 (7d): 14.77 mg/l, nominal
ErC20 (7d): 98.25 mg/l, nominal
ErC50 (7d) > 128.4 mg/l, nominal

Growth rates based on dry weight
NOErC (7d): 4.0 mg/l, nominal; LOErC (7d): 8.0 mg/l, nominal
ErC10 (7d): 6.19 mg/l, nominal
ErC20 (7d): 35.13 mg/l, nominal
ErC50 (7d) > 128.4 mg/l, nominal

Growth rates based on fresh weight
NOErC (7d): 4.0 mg/l, nominal; LOErC (7d): 8.0 mg/l, nominal
ErC10 (7d): 9.61 mg/l, nominal
ErC20 (7d): 38.38 mg/l, nominal
ErC50 (7d) > 128.4 mg/l, nominal

YIELD
Yeld based on frond number
NOEyC (7d): 4.0 mg/l, nominal; LOEyC (7d): 8.0 mg/l, nominal
EyC10 (7d): 2.15 mg/l, nominal
EyC20 (7d): 10.47 mg/l, nominal
EyC50 (7d) > 128.4 mg/l, nominal

Yeld based on dry weight
NOEyC (7d): 4.0 mg/l, nominal; LOEyC (7d): 8.0 mg/l, nominal
EyC10 (7d): 0.98 mg/l, nominal
EyC20 (7d): 4.27 mg/l, nominal
EyC50 (7d): 73.24 mg/l, nominal

Yeld based on fresh weight
NOEyC (7d): 4.0 mg/l, nominal; LOEyC (7d): 8.0 mg/l, nominal
EyC10 (7d): 2.13 mg/l, nominal
EyC20 (7d): 6.63 mg/l, nominal
EyC50 (7d): 58.23 mg/l, nominal

MEASURED CONCENTRATIONS
The measured concentrations did not deviated more than 20 % from the nominal during the experiment therefore the biological results and endpoints are based on the nominal concentrations.

Results with reference substance (positive control):

ErC50 (7d): 7.831 mg/l, based on frond numbers
ErC50 (7d): 7.722 mg/l, based on dry weight
EyC50 (7d): 5.000 mg/l, based on frond numbers
EyC50 (7d): 5.609 mg/l, based on dry weight

Growth rates (µ) and percentage inhibition of µ

Concentration[mg/l]	Growth rate (r) and % inhibition of r, based on frond number		Growth rate (r) and % inhibition of r, based on dry weight		Growth rate (r) and % inhibition of r, based on fresh weight
	r	% Ir	r	% Ir	r	% Ir
Control	0.326	0.00	0.32769	0.00	0.36977	0.00
4	0.319	2.21	0.31293	4.50	0.36166	2.19
8	0.287*	11.80	0.27546*	15.94	0.32062*	13.29
16	0.287*	11.87	0.27271*	16.78	0.30880*	16.49
32.1	0.283*	13.12	0.26932*	17.81	0.30924*	16.37
64.2	0.276*	15.17	0.25523*	22.11	0.29006*	21.56
128.4	0.254*	22.21	0.22464*	31.45	0.24446*	33.89

*: statistically significantly different compared to the control values (Dunnett t-Test; α = 0.05)

Yield rates (y) and percentage inhibition of y

Concentration[mg/l]	Yield and % inhibition of yield, based on frond number		Yield and % inhibition of yield, based on dry weight		Yield and % inhibition of yield, based on fresh weight
	y	% Iy	y	% Iy	y	% Iy
Control	97.33	0.0	0.00509	0.00	0.07239	0.00
4	91.67	5.8	0.00447	12.03	0.06803	6.03
8	71.33*	26.7	0.00332*	34.78	0.04942*	31.73
16	71.33*	26.7	0.00325*	36.02	0.04508*	37.72
32.1	69.00*	29.1	0.00316*	37.92	0.04519*	37.58
64.2	65.33*	32.9	0.00280*	44.87	0.03885*	46.34
128.4	54.00*	44.5	0.00216*	57.59	0.02663*	63.22

*: statistically significantly different compared to the control values (Dunnett t-Test; α = 0.05)

Observations during the Test - Symptoms, changes of Lemna gibba plants observed during the test

Concentration mg/l	3^rdday of the experiment		5^thday of the experiment		7^thday of the experiment
	Symptoms	Degree of change	Symptoms	Degree of change	Symptoms	Degree of change
Control	–	–	–	–	–	–
4	–	–	–	–	–	–
8	–	–	Root length	*	Root length	*
16	–	–	Root length Frond size	* *	Root length Frond size	* *
32.1	–	–	Root length Frond size	* *	Root length Frond size	* *
64.2	–	–	Root length Frond size	** *	Root length Frond size	** *
128.4	frond size	*	Root length Frond size	** *	Root length Frond size	** *

"–": the plants were healthy, there were not any symptoms observed

*: slight, **: medium, ***: strong change

Abbreviations

Root length: decrease of the root length

Frond size: decrease of frond size

MEASURED CONCENTRATIONS

Nominal conc. mg/l	Measured conc. in % of the nominal
	(at the start)	(at the end)
128.4	105	103
64.2	106	106
32.1	98	103
16	98	102
8	96	102
4	100	102
Control	not detected	not detected

Validity criteria fulfilled:

yes

Remarks:

doubling time of frond number in the control was 2.13 days

Conclusions:

ErC50 (7d) > 128.4 mg/l (nominal), based on frond number
EyC50 (7d) > 128.4 mg/l (nominal), based on frond number

Executive summary:

The phytotoxicity potential of test item was investigated on the freshwater aquatic plant Lemna gibba.

Exponentially growing cultures of Lemna gibba were exposed to the concentrations of 4.0, 8.0, 16.0, 32.1, 64.2 and 128.4 mg/l nominal (corresponding to 3.125, 6.25, 12.5, 25.0, 50.0 and 100 mg active ingredient/l), over a period of 7 days, in a static system under defined conditions. For determination of the test item concentrations, samples were taken from each concentration level and control at the start and at the end of the testb and were determined using HPLC method with VIS detection. The measured concentrations did not deviated more than 20 % from the nominal during the experiment.

The effects of the substance compared to the control plant development were demonstrated by the reduction of average specific growth rates and yield (both calculated on the basis of frond number, fresh weight and dry weight).

The 7-days NOEC related to both growth rate and yield (based on frond number, dry weight and fresh weight) was determined to be 4.0 mg/l and the LOEC was determined to be 8.0 mg/l. On the basis of the frond number, the 7-days EC50 resulted to be greater than 128.4 mg/l, nominal, for both growth rate and yield.

Conclusion

ErC50 (7d) > 128.4 mg/l (nominal), based on frond number

EyC50 (7d) > 128.4 mg/l (nominal), based on frond number

Description of key information

ErC50 (7d) > 128.4 mg/l (nominal), based on frond number

EyC50 (7d) > 128.4 mg/l (nominal), based on frond number

Key value for chemical safety assessment

EC10 or NOEC for freshwater plants:

4 mg/L

Additional information

The phytotoxicity potential of test item was investigated on the freshwater aquatic plant Lemna gibba. Exponentially growing cultures of Lemna gibba were exposed to the concentrations of 4.0, 8.0, 16.0, 32.1, 64.2 and 128.4 mg/l nominal (corresponding to 3.125, 6.25, 12.5, 25.0, 50.0 and 100 mg active ingredient/l), over a period of 7 days, in a static system under defined conditions. For determination of the test item concentrations, samples were taken from each concentration level and control at the start and at the end of the testb and were determined using HPLC method with VIS detection. The measured concentrations did not deviated more than 20 % from the nominal during the experiment.

The effects of the substance compared to the control plant development were demonstrated by the reduction of average specific growth rates and yield (both calculated on the basis of frond number, fresh weight and dry weight).

The 7-days NOEC related to both growth rate and yield (based on frond number, dry weight and fresh weight) was determined to be 4.0 mg/l and the LOEC was determined to be 8.0 mg/l. On the basis of the frond number, the 7-days EC50 resulted to be greater than 128.4 mg/l, nominal, for both growth rate and yield.