Reaction products of 2,4,6-trichloro-1,3,5-triazine copuled with 3-aminopropyldiethylamine, coupled with 3-acetamidoanilinium chloride, diazotised, coupled with 2,5-Xylidine, coupled with 3-amino-4-hydroxybenzenesulphonamide, further coupled with resorcinol and metallized with iron chloride.

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:

From May 22th to May 29th, 2019

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)

Version / remarks:

2006

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

UV/VIS spectrophotometric method

Details on sampling:

Three replicates samples were taken from each test concentration at the start, on day 5, and before and after sonication of the end samples. Six replicates were taken from the control (at the start, on day 5 and at the end before sonication).

Vehicle:

no

Details on test solutions:

- Formulation: the test solutions used in the test were prepared by mechanical dispersion without solubilizing agent. A test item stock solution was first prepared by suspending of 0.1402 g of test item in 1400 ml diluition water (20X AAP medium) in order to obtain the concentration of 100 mg test item/l. This stock solution was handled in ultrasonic bath for approximately 10 minutes. The test solutions of the subsequent lower test concentrations were prepared by appropriate dilution of this test item stock solution. The test solutions were freshly prepared in the testing laboratory just before introduction of the test organisms (at the start of the study).

- Untreated control: the dilution water (20X AAP Medium) was used as untreated control solution.

- Reference control (positive control): for the evaluation of the quality of the Lemna gibba cultures and the experimentl conditions, 3,5-dichlorophenol is tested at least twice a year to demonstrate satisfactory test conditions.

Test organisms (species):

Lemna gibba

Details on test organisms:

- Species and strain: Lemna gibba (G3)
- Source: Friedrich Schiller Universität, Institut für Allgemeine Botanik und Pflanzenphysiologie, Jena, Germany
- Justification for strain: Lemna gibba is a fast-growing species. the small size, the simple structure, vegetative reproduction and the short generation time makes it suitable and convenient for culturing and testing.
- Preculture: 7-10 days (7 days in this study) before testing, sufficient colonies are transferred from the stock culture aseptically into fresh sterile medium and cultured under the conditions of the test prior to beginning the test.
- Initial frond number: the initial frond number in the test culture was 11. The number of colonies and fronds was identical in each test vessel.
- Replicates and controls: the test included three replicates at test concentrations and six replicates for the control group.

Test type:

static

Water media type:

freshwater

Total exposure duration:

7 d

Test temperature:

The cultures were maintained at a temperature in the range of 24 ± 2 °C which was checked at the beginning of the study and every 24 hours (in a surrogate flask filled with water in the climate chamber). In addition, the temperature was continuosly measured (with a min/max thermometer) within the climate chamber during the experimental period.

pH:

The pH was checked at the start and at the end of the study in the test concentrations and the control. The pH of the control medium did not increase by more than 1.5 units during the test.

Nominal and measured concentrations:

Nominal concentrations of range-finding test: 0.1, 1.0, 10 and 100 mg/l
Nominal concentrations of definitive test: 6.25, 12.5, 25.0, 50.0 and 100.0 mg/l
-Test item concentrations in the definitive test: based on the results of the preliminary range-finding tests five test concentrations in a geometric series with a separation factor of 2.0 and one control were used in the main test.
The following nominal concentrations were tested: 6.25, 12.5, 25.0, 50.0 and 100.0 mg/l. The measured concentrations deviated no more than ± 20 % from the nominal during the experiment, thus biological results and endpoints are based on nominal concentrations.

Details on test conditions:

-Light intensity: test vessels were placed randomly on a tray illuminated continuously at a light intensity between 6500-10000 lux using fluorescent light tubes (with a spectral range of 400-700 nm). The light intensity was checked and recorded at the start of the test at the position occupied by test containers. The differences in light intensity between the measurements points (i.e. position of fronds) did not exceed ± 15 % and thus provided equal conditions for each test culture.
-Test units: all-glass beakers (total capcity of 400 ml) were used and were covered by glass petri dishes. The volume of the test liquid in the vessels was 210 ml. Each test unit was uniquely identified with study code, test item concentration (and control) and replicate.
-Test water: 20X AAP medium was used for maintaining Lemna cultures and for use as the diluent in the preparations of test solutions. An appropriate amount of each stock solutions (A-C) was added to deionised water to obtain the appropriate concentrations in the final medium. The pH was adjusted to 7.5 ± 0.1 N HCl. The medium was then filtered through a o.22 µm membrane filter into steril container. The medium was prepared at least one day before use to allow the pH to stabilize. The pH of the medium was checked prior to use.

Reference substance (positive control):

yes

Remarks:

3,5- dichlorophenol

Duration:

7 d

Dose descriptor:

EC10

Remarks:

based on frond number

Effect conc.:

13.587 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

EC20

Remarks:

based on frond number

Effect conc.:

20.938 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

EC50

Remarks:

based on frond number

Effect conc.:

47.886 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

NOEC

Remarks:

based on frond number

Effect conc.:

6.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

LOEC

Remarks:

based on frond number

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

EC10

Remarks:

based on frond number

Effect conc.:

8.884 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

7 d

Dose descriptor:

EC20

Remarks:

based on frond number

Effect conc.:

12.868 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

7 d

Dose descriptor:

EC50

Remarks:

based on frond number

Effect conc.:

26.142 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

7 d

Dose descriptor:

NOEC

Remarks:

based on frond number

Effect conc.:

6.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

7 d

Dose descriptor:

LOEC

Remarks:

based on frond number

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

7 d

Dose descriptor:

EC10

Remarks:

based on dry weight

Effect conc.:

6.609 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

EC20

Remarks:

based on dry weight

Effect conc.:

9.796 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

EC50

Remarks:

based on dry weight

Effect conc.:

26.142 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

NOEC

Remarks:

based on dry weight

Effect conc.:

< 6.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

LOEC

Remarks:

based on dry weight

Effect conc.:

6.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

EC10

Remarks:

based on dry weight

Effect conc.:

3.243 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

7 d

Dose descriptor:

EC20

Remarks:

based on dry weight

Effect conc.:

4.997 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

7 d

Dose descriptor:

EC50

Remarks:

based on dry weight

Effect conc.:

11.423 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

7 d

Dose descriptor:

NOEC

Remarks:

based on dry weight

Effect conc.:

< 6.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

7 d

Dose descriptor:

LOEC

Remarks:

based on dry weight

Effect conc.:

6.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Details on results:

Validity of the test: the doubling time of frond number in the control was 2.03 days (less than 2.5 days). The validity criterion was within acceptable limit and therefore the study can be considered as valid.

Results with reference substance (positive control):

-The average specific growth rate (based on frond number) was statistically significantly different from the control group at the concentrations of 12.5, 25, 50 and 100 mg/l (Jonckheere-Terpstra Test).
-The average specific growth rate (based on dry weight) was statistically significantly different from the control group at every test concentration (Jonckheere-Terpstra Test).
-Yield (based on frond number) was statistically significantly different from the control group at the concentrations of 12.5, 25, 50 and 100 mg/l (Jonckheere-Terpstra Test).
-Yield (based on dry weight) was statistically significantly different from the control group at every test concentration (Jonckheere-Terpstra Test ).

Measured concentrations during the study

Nominal concentration	Mean of the measured concentrations (mg/l)
(mg/l)	(n = 3)
	Start of the study	Day 5	End of the study
			Before sonication	After sonication
6.25	5.87	5.37	5.66	5.58
12.5	11.6	11.7	11.6	12.8
25	23.9	22.5	23.1	24.4
50	48.1	43.4	46.5	48.9
100	100	99	103	106
Control (n = 6)	<LOD	<LOD	<LOD	-*

LOD = limit of detection

 *= no data

Percentage of the Measured Concentrations in comparison to the Nominal Concentrations

Nominal concentration	Mean of the measured concentrations (mg/l)
(mg/l)	(n = 3)
	Start of the study	Day 5	End of the study
			Before sonication	After sonication
6.25	93.92	85.92	90.56	89.28
12.5	92.8	93.6	92.8	102.4
25	95.6	90	92.4	97.6
50	96.2	86.8	93	97.8
100	100	99	103	106
Control (n = 6)	<LOD	<LOD	<LOD	-*

LOD = limit of detection

 *= no data

Growth Rates (µ) and Percentage Inhibition of µ based on Frond Number

Nominal concentration	Growth rate (r) and % inhibition of r
[mg/l]	0–168 h(based on frond number)
	µ	% Iµ
Control	0.341	-
6.25	0.336	1.43
12.5	0.321*	5.91
25	0.244*	28.54
50	0.148*	56.77
100	0.092*	73.06

Growth rates (µ) and percentage inhibition of µ based on dry weight

Nominal concentration	Growth rate (r) and % inhibition of r
[mg/l]	0–168 h(based on dry weight)
	µ	% Iµ
Control	0.3301	-
6.25	0.2792*	15.41
12.5	0.2266*	24.21
25	0.1702*	48.45
50	0.0519*	84.29
100	-0.0062^#*	101.88

#: negative value indicates less dry weight than the initial dry weight

*: statistically significantly different compared to the control values (Jonckheere-Terpstra Test)

Yield (y) and percentage inhibition of yield based on frond number

Concentration	Yield (y) and % inhibition of yield
[mg/l]	0–168 h(based on frond number)
	y	% Iy
Control	109	-
6.25	105	3.67
12.5	93.33*	14.37
25	50.00*	54.13
50	20.00*	81.65
100	10.00*	90.83

Yield (y) and percentage inhibition of yield based on dry weight

Concentration	Yield (y) and % inhibition of yield
[mg/l]	0–168 h(based on dry weight)
	y	% Iy
Control	0.00578	-
6.25	0.00386*	33.26
12.5	0.00306*	47.15
25	0.00148*	74.47
50	0.00028*	95.16
100	-0.00003^#*	100.46

#: negative value indicates less dry weight than the initial dry weight

*: statistically significantly different compared to the control values (Jonckheere-Terpstra Test)

Symptoms, changes of lemna gibba plants observed during the test

nominal conc. mg/l	3rd day		5th day		7th day
	symptoms	degree of change	symptoms	degree of change	symptoms	degree of change
control	-	-	-	-	-	-
6.25^1	-	-	-	-	grouping	*
12.5^1	-	-	-	-	frond size root length	* *
25^1	frond size	*	frond size root length	* *	frond size root length chlorosis	* * *
50^1	frond size root length	* *	frond size root length necrosis	* ** *	frond size root length chlorosis necrosis	** ** * *
100^1	frond size root length necrosis	* * *	frond size root length necrosis	** ** **	frond size root length chlorosis necrosis	** ** * **

1: The test item was adsorbed to the roots observed after 3, 5 and 7 days.

Legend:

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

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

Abbreviations:

Grouping: grouping of fronds (plants)

Frond size: decrease of frond size

Root length: decrease of the root length

Necrosis: death of fronds

Chlorosis: chlorosis of fronds

Summary of the biological endpoints

Endpoints for response variables (0-7 d)		Concentration(mg/l)
		based on nominal concentrations
Growth rate	ErfnC10	13.587
(based on frond number)	95 % conf. limits	11.922 – 15.211
	ErfnC20	20.938
	95 % conf. limits	18.976 – 22.871
	ErfnC50	47.886
	95 % conf. limits	44.279 – 52.040
	NOEC	6.25
	LOEC	12.5
Yield	EyfnC10	8.884
(based on frond number)	95 % conf. limits	6.997 – 10.703
	EyfnC20	12.868
	95 % conf. limits	10.680 – 14.978
	EyfnC50	26.142
	95 % conf. limits	23.039 – 29.654
	NOEC	6.25
	LOEC	12.5
Growth rate	ErdwC10	6.609
(based on dry weight)	95 % conf. limits	4.831 – 8.321
	ErdwC20	9.796
	95 % conf. limits	7.695 – 11.801
	ErdwC50	20.795
	95 % conf. limits	17.822 – 24.223
	NOEC	<6.25
	LOEC	6.25
Yield	EydwC10	3.243
(based on dry weight)	95 % conf. limits	2.271 – 4.213
	EydwC20	4.997
	95 % conf. limits	3.786 – 6.155
	EydwC50	11.423
	95 % conf. limits	9.775 – 13.093
	NOEC	<6.25
	LOEC	6.25

Validity criteria fulfilled:

yes

Conclusions:

EC50 (7d) based on frond number growth rate is 47.886 mg/l
The analytically measured test item concentrations remained within ± 20 % of the nominal during the test period, thus all biological results are based on the nominal concentrations.

Executive summary:

Basic Brown 022 was tested according to OECD 221 using Duckweed (Lemna gibba); preculture was prepared 7 days before the test; healthy colonies with 3-4 fronds were used. 20X AAP medium was used to prepare test solutions.

Based on the results of the preliminary experiments, nominal concentrations of 6.25, 12.5, 25.0, 50.0 and 100 mg/l were tested in the main study. The measured concentrations deviated less than 20 % from the nominal during the experiment in each concentration level.

Two colonies with four fronds and one colony with three fronds were added to each vessel for a 7-day exposure in testing solutions.A static test was run, using 5 test concentrations with three replicates and the control group with six replicates; assessment of growth and morphological changes was done on days 3, 5 and 7.

For determination of the test item concentrations, samples were taken from each concentration level and control at the start, on day 5 and at the end of the test. At the end of the test, samples were taken and analyzed prior and after the ultrasonic treatment of the sample preparation procedure. Samples were analysed by UV/VIS spectrophotometric method.

ECx, NOEC and LOEC were determined for both response variables (i.e. growth rate and yield) based on frond number and dry weight.

For the determination of the LOEC and NOEC, Jonckheere-Terpstra Test was used by SPSS PC+ software. For determination of the ECx values with 95 % confidence limits, Probit analysis was performed using SPSS PC+software.

As for frond number growth rate:

EC10 = 13.587 mg/l

EC20 = 20.938 mg/l

EC50 = 47.886 mg/l

NOEC = 6.25 mg/l

LOEC = 12.5 mg/l

Description of key information

EC50 (7d) based on frond number growth rate = 47.886 mg/l

Key value for chemical safety assessment

EC50 for freshwater plants:

47.886 mg/L

EC10 or NOEC for freshwater plants:

6.25 mg/L

Additional information

Basic Brown 022 was tested according to OECD guideline 221.

Two preliminary tests were run at concentartions of 0.1, 1, 10 and 100 mg/l starting from a stock solution at 1000 mg/l. In the first test, precipitation was seen, thus it was repeated. In the second test, stock solution was filtrated prior use; precipitation was seen, however, concentrations remained within ± 20 % of nominal values at 10 and 100 mg/l.

A stock solution at 100 mg/l was prepared for the main test and handled in ultrasonic bath for 10 minutes, before use. The main tests was run using test concentrations of: 0, 6.25, 12.5, 25, 50 and 100 mg/l, with 3 replicates per test concentration and 6 replicates for the control group; stock solution was diluted with 20X AAP medium; two Lemna gibba colonies with four, and one colony with three fronds were added to each vessel; assessment of growth and morphological changes on days 3, 5 and 7.

Evaluated endpoints were ECx, NOEC and LOEC, for both response variables (i.e. growth rate and yield) based on frond number and dry weight.

All validity criteria were met and therefore the study can be considered as valid.

The analytically measured test item concentrations remained within ± 20 % of the nominal during the test period therefore all biological results are based on the nominal concentrations. Adsorption of test item on roots of plants was seen at all test concentrations.