2-[2-[4-[(2-cyanoethyl)ethylamino]-o-tolyl]vinyl]-1,3,3-trimethyl-3H-indolium dihydrogen phosphate

Toxicity to aquatic plants other than algae

Administrative data

Endpoint:

toxicity to aquatic plants other than algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2018-06-27 to 2018-07-05, with the definitive exposure phase from 2018-06-27 to 2018-07-04.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Version / remarks:

2006

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Council Regulation (EC) No. 761/2009 Method C.26

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-[2-[4-[(2-cyanoethyl)ethylamino]-o-tolyl]vinyl]-1,3,3-trimethyl-3H-indolium dihydrogen phosphate

EC Number:

276-470-8

EC Name:

2-[2-[4-[(2-cyanoethyl)ethylamino]-o-tolyl]vinyl]-1,3,3-trimethyl-3H-indolium dihydrogen phosphate

Cas Number:

72208-20-9

Molecular formula:

C25H30N3.H2O4P C25H32N3O4P

IUPAC Name:

2-[2-[4-[(2-cyanoethyl)ethylamino]-o-tolyl]vinyl]-1,3,3-trimethyl-3H-indolium dihydrogen phosphate

Test material form:

liquid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
All test item concentrations and the control were analytically verified via HPLC-DAD at the first interval. The control, the lowest and the highest test item concentration were analytically verified at the end of the exposure (7 days) and on every renewal day. Additional sampling was possible, but were only analysed as indicated. Freshly prepared and old media were analyzed. The samples were analyzed with an HPLC-DAD method. The method was implemented under non-GLP but documented in the raw data and validated. The method validation was not part of this GLP study.

Test solutions

Vehicle:

no

Details on test solutions:

Stock solution
The stock solution (100 mg test item/L were weighed out) were freshly prepared with dilution water prior to the start of the exposure (0 hours) and prior to the renewal of the test solutions (day 2 and 5). The solution was manually agitated.

Test concentrations
Five dilution levels were prepared out of the test item solution with dilution water in a geometrical series with a separation factor of 4 and tested as follows: 0.391 - 1.56 - 6.25 - 25.0 - 100 % of the stock solution, corresponding to 0.391 - 1.56 - 6.25 - 25.0 - 100 mg test item/L nominal concentration.
The concentrations are based on the results of a preliminary range finding test .

Control
Six replicates (without test item) were tested under the same test conditions as the test vessels.

Test organisms

Test organisms (species):

Lemna gibba

Details on test organisms:

Test organism
Duckweed, Lemna gibba G3, Lemnaceae, Arales, Arecidae, Monocotyledonae
Young, rapidly growing plants without visible lesions or discolouration (chlorosis) were used for the test.

Reason for the selection of the test organism
According to the guideline, Lemna gibba is a suitable species because it is a representative of temperate areas commonly used for toxicity tests.

Origin
EUROFINS-GAB GMBH, Eutinger Str. 24, 75223 Niefern-Öschelbronn, Germany

Cultivation at test facility
The species is cultured in the test facility. Density is kept low to prevent conglomerates of plants on the surface. At least once per week, plants are transferred to freshly prepared growth medium. Growth media and culturing vessels are autoclaved before use to enable the breeding of axenic cultures.

Breeding vessels
Crystallisation dishes of glass, vol. 900 mL, filled with ca. 500 mL growth medium, covered with glass tops

Medium
20X-AAP-medium (Algal Assay Procedure medium),
pH-value 7.5 ± 0.1

Composition of Dilution water
Component Concentration in stock solution [g/L] Concentration in prepared medium [mg/L]
NaNO3 26 510
MgCl2 x 6 H2O 12 240
CaCl2 x 2 H2O 4.4 90
MgSO4 x 7 H2O 15 290
K2HPO4 · 3 H2O 1.4 30
NaHCO3 15 300
H3BO3 0.19 3.7
MnCl2 x 4 H2O 0.42 8.3
FeCl3 x 6 H2O 0.16 3.2
Na2-EDTA · 2 H2O 0.30 6.0
ZnCl2 3.3 mg/L 66 µg/L
CoCl2 x 6 H2O 1.4 mg/L 29 µg/L
Na2MoO4 x 2 H2O 7.3 mg/L 145 µg/L
CuCl2 x 2 H2O 0.012 mg/L 0.24 µg/L
pH-value 7.5 ± 0.1
The pH of the test medium had to be 7.5 +/- 0.1 and was adjusted prior to testing with the addition of 1 N NaOH and HCl.

Temperature 24 ± 2 °C

Light regime
Continuous fluorescent light, 1100 – 4440 lux

Acclimatization of the test system
The test system (the test organism) was held for 7 days under test conditions to acclimatize. These acclimatized plants were used in the test.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test conditions

Hardness:

not measured

Test temperature:

Room temperature [°C]: min.: 22.2 max.: 23.6 mean value: 23.0

pH:

Nominal test item concentration
[mg/L] pH-value
0 h (Fresh medium) 2 d(Old medium) 2 d (Fresh medium) 5 d (Old medium) 5 d (Fresh medium) 7 d (Old medium)
100 7.24 8.16 7.40 8.39 7.42 8.13
25.0 7.53 8.27 7.49 8.39 7.48 8.20
6.25 7.61 8.28 7.54 8.41 7.50 8.32
1.56 7.60 8.25 7.56 8.41 7.48 8.42
0.391 7.62 8.24 7.57 8.42 7.48 8.45
Control 7.60 8.20 7.51 8.41 7.47 8.44

Dissolved oxygen:

not measured

Salinity:

not measured

Conductivity:

not measured

Nominal and measured concentrations:

Nominal: 0.391 - 1.56 - 6.25 - 25.0 - 100 mg/L

Details on test conditions:

Test method
Semi-tatic procedure

Test duration
7 days

Replicates
3 replicates per concentration level, 6 for the control.

Test vessels/test volumes
Crystallisation dishes with a volume of 500 mL, covered with glass tops and filled with 200 mL test solution were used in the test. The test vessels were placed on a black non-reflective surface to avoid stray light.
 
Dilution water
20X-AAP-medium according to the guideline.

Application
Semi-static, with renewal of the test solutions on day 2 and 5 (i.e. exposure to freshly prepared test and control solutions on two occasions during the test). The plants were placed into the test vessels, which already contain the test media.At the start of the exposure, 3 uniform, healthy plants (colonies of 4 fronds each), were introduced into each test vessel containing the test media. The initial frond number per test vessel was 12. The initial numbers of colonies and fronds were the same in each test vessel.



Temperature (Target)
24 ± 2 °C

Light regime (Target)
Continuous, fluorescent light, 6500 to 10000 lux on the surface of the test medium (difference of light intensity at any measured incubation place < 15 % from the mean value)

Placement of the test vessels
A randomised placement of the test vessels was carried out.

Type and frequency of measurements
The numbers of plants and fronds were determined at the start and
the end of the exposure. The number of fronds was determined every 2 - 3 days from each replicate of the control and the test concentrations. Every frond that visibly projected beyond the edge of a parent frond was counted as a separate frond. Fronds that lost their pigmentation were not counted.
Observations of frond size, appearance, indication of necrosis, chlorosis or gibbosity, colony break-up or loss of buoyancy, of root length and appearance, as well as of change in colour and destruction of roots, were made on every determination day and at the end of the exposure.

After 7 days, the determination of dry weight was carried out from 3 replicates per test concentration and 6 control replicates. Colonies from each test vessel were collected, rinsed with deionised water and then dried at 60 °C to a constant weight. Any root fragments were included. The dry weight was expressed to an accuracy of
0.1 mg.
The dry weight of the starting biomass was determined based on a sample of fronds (same number of fronds as in the test vessels) taken from the same batch used to inoculate the test vessels.

Physico-chemical Parameters
The pH-values were measured in the freshly prepared solutions before distribution into the replicates. The pH-values of the aged solution were measured from pooled replicates per concentration and control. The temperature of the medium in a surrogate vessel held under the same conditions in the growth room was recorded daily. The light intensity was measured prior to the start of the exposure at positions which had the same distance from the light source as the Lemna fronds.

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

0.391 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Frond number and dry weight

Remarks on result:

other: Growth Rate and Inhibition of Yield

Effect concentrations 2

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

< 0.391 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Frond number and dry weight

Remarks on result:

other: Growth Rate and Inhibition of Yield

Effect concentrations 3

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

1.04 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: frond number ihibition of yield

Remarks on result:

other: CI: 0.721 - 1.49

Effect concentrations 4

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

6.17 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Frond number Growth Rate Inhibition

Remarks on result:

other: CI: 5.51 - 7.27

Effect concentrations 5

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

0.786 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Dry weight Inhibition of yield

Remarks on result:

other: CI: 0.572 - 1.13

Effect concentrations 6

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

4.46 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: dry weight growth rate inhibition

Remarks on result:

other: CI: 3.55 - 5.44

Details on results:

The environmental conditions (pH-value, room temperature, light intensity) were determined to be within the acceptable limits.

Results with reference substance (positive control):

The acute toxicity of 3,5-Dichlorophenol (SIGMA-ALDRICH, batch number MKBZ0947V, purity 100.0 area %, CAS RN 591-35-5) to the monocotyledonous aquatic plant Lemna gibba was determined over a period of 7 days with exposure phase from 2018-04-04 to 2018-04-11 according to OECD Guideline 221. The plants used in the reference test were taken from the same laboratory culture as was used to determine the effects of Basic Red 23.

EC50-Values of the Reference Item based on the nominal concentrations [mg/L], (0-7 days)

Current Study Valid Range (average ± 3 x SD)
Growth rate inhibition (number of fronds)
ErC50 7.68 5.82 ± 3.18
95% confidence interval 7.31 to > 8.00
Yield inhibition (number of fronds)
EyC50 4.93 4.67 ± 2.87
95% confidence interval 4.42 to 5.53
Growth rate inhibition (dry weight)
ErdwC50 > 8.00 5.61 ± 2.76
95% confidence interval Not applicable
Yield inhibition (dry weight)
EydwC50 5.95 4.75 ± 2.49
95% confidence interval 5.18 to 6.75
SD = standard deviation

The observed responses to the reference item were within the valid range, confirming the normal sensitivity of the test system used in the study with the test item.

Reported statistics and error estimates:

Statistics
For the determination of NOEC, LOEC and EC-values, three replicates were included for the test concentrations and six replicates for the control.

NOEC and LOEC values
NOEC/LOEC was determined by calculation of the statistical significance of inhibition of growth rates and yield in comparison to the control: One Way Analysis of Variance (ANOVA) and DUNNETT’s test were used as a standard. A normality test and an equal variance test were done first.
The equal variance test for evaluation of NOEC/LOEC for growth rate on fronds failed. Therefore, the highest tested concentration with a growth rate inhibition of 100% was taken out for this specific evaluation. The SHAPIRO-WILK-Test was used to test for normally distributed populations. P-values for both normality and equal variance test were 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) was =0.05.

EC-values and statistical analyses
EC10-, EC20- and EC50-values (0 - 7 d) of the growth rate and yield (frond number and dry weight) inhibition were calculated by sigmoidal dose-response regression. Calculations of the confidence intervals of EC10-, EC20- and EC50-values were carried out from the best fit values, if possible, the standard error and the t-distribution with the software GraphPad Prism.


Software
The data for the tables in this report were computer-generated and rounded for presentation from the fully derived data. Consequently, if calculated manually based on the given data, minor deviations may occur from these figures.
Calculations were carried out using the following software:
- Excel, MICROSOFT CORPORATION
- SigmaPlot, SPSS INC.
- GraphPad Prism, GRAPHPAD SOFTWARE, INC.

Any other information on results incl. tables

Frond Numbers

Nominal test item concentration [mg/L]	Repl. No.	Frond numbers per study day
		0 days*	2 days	5 days	7 days
100	1	12	13	6	6
	2	12	15	4	4
	3	12	15	4	4
	Mean	12	14	5	5
25.0	1	12	20	22	25
	2	12	17	18	24
	3	12	20	20	29
	Mean	12	19	20	26
6.25	1	12	23	47	55
	2	12	24	39	52
	3	12	22	47	53
	Mean	12	23	44	53
1.56	1	12	28	75	122
	2	12	25	76	111
	3	12	25	64	101
	Mean	12	26	72	111
0.391	1	12	25	81	176
	2	12	25	89	169
	3	12	28	104	232
	Mean	12	26	91	192
Control	1	12	27	97	213
	2	12	26	105	240
	3	12	32	122	286
	4	12	26	110	305
	5	12	28	109	264
	6	12	29	131	219
	Mean	12	28	112	255

* = 3 colonies with 4 fronds each per replicate were inoculated at start of the exposure

Repl. No. = replicate number

  Growth Rate and Yield Inhibition based on Fronds after 7 d

            Statistically significant differences of growth rates and yield

            compared to control values are marked (+) and non-significant differences are marked (-).

                                               

Nominal test item concentration [mg/L]		Repl. No.		Average growth rate [d^-1]			Inhibition of average growth rate [%]		Yield [fronds]			Inhibition of yield [%]		Doubling time [d]
100		1				-0.099		100				-6		100		-7.00
		2				-0.157		100				-8		100		-4.42
		3				-0.157		100				-8		100		-4.42
		Mean		(+)		-0.138		100		(+)		-7		100		-5.28
25.0		1				0.105		76				13		95		6.61
		2				0.099		77				12		95		7.00
		3				0.126		71				17		93		5.50
		Mean		(+)		0.110		75		(+)		14		94		6.37
6.25		1				0.217		50				43		82		3.19
		2				0.209		52				40		84		3.31
		3				0.212		51				41		83		3.27
		Mean		(+)		0.213		51		(+)		41		83		3.25
1.56		1				0.331		24				110		55		2.09
		2				0.318		27				99		59		2.18
		3				0.304		30				89		63		2.28
		Mean		(+)		0.318		27		(+)		99		59		2.18
0.391		1				0.384		12				164		32		1.81
		2				0.378		13				157		35		1.83
		3				0.423		3				220		9		1.64
		Mean		(+)		0.395		9		(+)		180		26		1.76
Control		1				0.411						201				1.69
		2				0.428						228				1.62
		3				0.453						274				1.53
		4				0.462						293				1.50
		5				0.442						252				1.57
		6				0.415						207				1.67
		Mean				0.435						243				1.60

Repl. No. = replicate number

 

 

Specific Growth Rate and Yield Inhibition of Dry Weight after 7 d

                              Statistically significant differences of specific growth rates and yield compared

                               to control values are marked (+) and non-significant differences are marked (-).

 

Nominal test item concentration [mg/L]		Repl. No.		Dry weight [mg]		Specific dry weight growth rate [d^-1]			Inhibition of specific dry weight growth rate [%]		Yield of dry weight [mg]			Inhibition of yield dry weight   [%]
100		1		1.0				-0.113		100				-1.2		100
		2		1.2				-0.087		100				-1.0		100
		3		0.9				-0.128		100				-1.3		100
		Mean		1.0		(+)		-0.109		100		(+)		-1.2		100
25.0		1		2.5				0.018		96				0.3		99
		2		2.6				0.024		94				0.4		99
		3		2.5				0.018		96				0.3		99
		Mean		2.5		(+)		0.020		95		(+)		0.3		99
6.25		1		8.9				0.200		53				6.7		83
		2		7.5				0.175		59				5.3		87
		3		8.0				0.184		56				5.8		86
		Mean		8.1		(+)		0.186		56		(+)		5.9		85
1.56		1		18.7				0.306		28				16.5		59
		2		16.4				0.287		32				14.2		65
		3		14.9				0.273		35				12.7		69
		Mean		16.7		(+)		0.289		32		(+)		14.5		64
0.391		1		25.0				0.347		18				22.8		44
		2		25.1				0.348		18				22.9		43
		3		32.6				0.385		9				30.4		25
		Mean		27.6		(+)		0.360		15		(+)		25.4		37
Control		1		35.5				0.397						33.3
		2		40.6				0.416						38.4
		3		47.3				0.438						45.1
		4		48.7				0.442						46.5
		5		44.0				0.428						41.8
		6		39.6				0.413						37.4
		Mean		42.6				0.423						40.4

The initial biomass dry weight was 2.2 mg per replicate.

Repl. No. = replicate number

 

Colony Number (Plants) on Days 0 and 7

 

Nominal test item concentration [mg/L]		Replicate No.		Colony number
			Day 0		Day 7
100		1		3		12
		2		3		12
		3		3		13
		Mean		3		12
25.0		1		3		13
		2		3		13
		3		3		12
		Mean		3		13
6.25		1		3		7
		2		3		8
		3		3		7
		Mean		3		7
1.56		1		3		15
		2		3		12
		3		3		12
		Mean		3		13
0.391		1		3		19
		2		3		17
		3		3		25
		Mean		3		20
Control		1		3		19
		2		3		21
		3		3		27
		4		3		31
		5		3		24
		6		3		22
		Mean		3		24

 

 

Further Observations on Days 2, 5 and 7

Nominal test item concentration [mg/L]	Observations on day
	2	5	7
100	2.1+++ 2.4++ 2.5+ 3.2++ 3.3+ 4.1+++	2.1+++ 2.2+++ 2.3+ 2.4+++ 2.5++ 3.1++ 3.2++ 3.3+++ 4.1+++	2.1+++ 2.2+++ 2.3+ 2.4+++ 2.5++ 3.1++ 3.2++ 3.2+++ 4.1+++
25.0	2.3++ 2.5++ 3.1+ 3.2++	2.1+++ 2.2+ 2.3+++ 2.4++ 2.5+++ 3.1+++ 3.2+++ 3.3++ 4.1+++	2.1+++ 2.2+ 2.3+++ 2.4++ 2.5+++ 3.1+++ 3.2+++ 3.3++ 4.1+++
6.25	2.3+	2.1+ 2.3+++ 2.4+ 2.5+ 3.1+ 3.3+	2.1+ 2.3+++ 2.4+ 2.5+ 3.1+ 3.3+
1.56	1	2.1+ 2.3+ 2.5+	2.1+ 2.3+ 2.4+ 2.5+
0.391	1	1	1
Control	1	1	1

Observations were made compared to the appearance of control colonies (plants) and test media

 

1       = no observedeffects

2.1   = chlorosis

2.2   = necrosis

2.3   = gibbosity of the fronds

2.4   = discoloration of the fronds

2.5   = smaller frond

3.1   = shortened roots

3.2  = losses of roots

3.3   = discoloration of roots

4.1   = break up of plants

+      = slight effects

++    = medium effects

+++  = strong effects

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study, Basic Red 15 phosphate was found to inhibit the growth of the monocotyledonous aquatic plant Lemna gibba after 7-day exposure under semi-static conditions, with the following effect values (nominal test item concentrations): The EC50-values for inhibition of the specific growth rate (fronds and dry weight) (ErC50, ErdwC50) were 6.17 (5.21 to 7.27) and 4.46 (3.55 to 5.44) and yield (fronds and dry weight) (EyC50, EydwC50) were 1.04 (0.721 to 1.49) and 0.786 (0.572 to 1.13), respectively.

Executive summary:

The effects of the test item Basic Red 15 phosphate on the growth of the monocotyledonous aquatic plant speciesLemna gibba was determined according to the principles of OECD 221 at the test facility from2018-06-27 to 2018-07-05, with the definitive exposure phase from 2018-06-27 to 2018-07-04.

Lemna gibba was exposed to the test item for 7 days under semi-static conditions. Based on a preliminary test, 5 nominal test item concentration levels were tested in a geometrical series with a dilution factor of 4:0.391 - 1.56 - 6.25 - 25.0 - 100 mg/L. Three replicates were investigated for each test concentration and six for the control. Frond numbers were assessed on days 0, 2, 5 and 7. Environmental parameters (light, pH and temperature) were within the acceptable limits. All test solutions were clear and concentration-related red to pink coloured throughout the exposure.The validity criteria of the test guideline were fulfilled.

The concentrations of the test item were analytically verified via HPLC-DAD in fresh media at the start of the exposure and in old media (0 and 2 days) in all concentration levels. Additionally the test item were analyzed at day 2 and 5 in the fresh media and day 5 and 7 in old media in the highest and the lowest concentration level and the control.

At the start of the first interval the measured concentrations of Basic Red 15 phosphate liquid were in the range of 98 and 101% of the nominal values. At the end of the first interval the measured concentrations were between 79 and 96% of the nominal values.

 

NOEC-, LOEC-, EC-Values and 95% Confidence Intervals ofBasic Red 15phosphate liquidafter 7 Days of Exposure

                  (based on the nominal test item concentration [mg/L])

Frond number		Dry weight
Growth Rate Inhibition [mg/L]
NOEC	< 0.391	< 0.391
LOEC	0.391	0.391
ErC10	0.424 (<0.391 to 0.549)	< 0.391
ErC20	0.924 (0.740 to 1.17)	0.720 (< 0.391 to 1.23)
ErC50	6.17 (5.21 to 7.27)	4.46 (3.55 to 5.44)
Inhibition of Yield [mg/L]
NOEC	< 0.391	< 0.391
LOEC	0.391	0.391
EyC10	< 0.391	< 0.391
EyC20	< 0.391	< 0.391
EyC50	1.04 (0.721 to 1.49)	0.786 (0.572 to 1.13)