2-[2-[4-(diethylamino)phenyl]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:

May 2018

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-(diethylamino)phenyl]vinyl]-1,3,3-trimethyl-3H-indolium dihydrogen phosphate

EC Number:

278-248-6

EC Name:

2-[2-[4-(diethylamino)phenyl]vinyl]-1,3,3-trimethyl-3H-indolium dihydrogen phosphate

Cas Number:

75535-16-9

Molecular formula:

C23H29N2.H2O4P

IUPAC Name:

2-[2-[4-(diethylamino)phenyl]vinyl]-1,3,3-trimethyl-3H-indolium dihydrogen phosphate

Test material form:

solid: particulate/powder

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. 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:

Preparation of the Test item solution
A stock solution with a nominal test item concentration of 10.0 mg/L was freshly prepared with dilution water and agitated until the solution was visually clear. The test item solution was clear and purple coloured.

Test concentrations
Based on the results of a preliminary range finding test. 5 concentrations were tested with a dilution factor of 4: 0.0156 - 0.0625 - 0.250 - 1.00 - 4.00 mg/L, corresponding to the geometric mean measured test item concentrations: 0.0137 - 0.0535 – 0.240 – 0.899 – 3.72 mg/L.

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 9 days under test conditions to acclimatize. These acclimatized plants were used in the test.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test conditions

Hardness:

not measured

Test temperature:

Room temperature [°C]: min.: 23.4 max.: 24.3 mean value: 23.9

pH:

Geometric mean measured test item concentration
[mg/L] pH-value
0 h (Fresh medium) 7 d (Old medium)
3.72 7.57 8.38
0.899 7.58 8.38
0.240 7.58 8.37
0.0535 7.58 8.36
0.0137 7.59 8.35
Control 7.58 8.30

Dissolved oxygen:

not measured

Salinity:

not measured

Conductivity:

not measured

Details on test conditions:

Test method
Semi-static 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). 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:

NOEC

Effect conc.:

0.014 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Frond number and Dry weight

Remarks on result:

other: Growth Rate Ihibition and Inhibition of Yield

Effect concentrations 2

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

0.053 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Frond number and Dry weight

Remarks on result:

other: Growth Rate Inhibition and Inhibition of Yield

Effect concentrations 3

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

0.581 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Frond number Growth Rate Inhibition

Remarks on result:

other: CI: 0.510 - 0.667

Effect concentrations 4

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

0.528 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: dry weight growth rate inhibition

Remarks on result:

other: CI: 0.477 - 0.588

Effect concentrations 5

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

0.204 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Frond number Inhibition of yiled

Remarks on result:

other: CI: 0.182 - 0.230

Effect concentrations 6

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

0.151 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: dry weight inhibition of yiled

Remarks on result:

other: CI: 0.129 - 0.177

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, 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 from 2017-10-06 to 2017-10-13 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 Acid Red 299.

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 6.76 5.71 ± 2.95
95% confidence interval 4.62 – 7.87
Yield inhibition (number of fronds)
EyC50 5.80 4.65 ± 2.96
95% confidence interval 4.50 – 7.05
Growth rate inhibition (dry weight)
ErdwC50 6.36 5.61 ± 2.76
95% confidence interval 5.14 – 7.12
Yield inhibition (dry weight)
EydwC50 5.39 4.67 ± 2.37
95% confidence interval 4.86 – 6.07
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 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. Normality test failed for calculation of yield (fronds). Therefore, the data were transformed (Y=sqrt(Y)).

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, 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

Geometric mean measured test item concentration [mg/L]	Repl. No.	Frond numbers per study day
		0 days*	3 days	5 days	7 days
3.72	1	12	12	16	17
	2	12	13	15	18
	3	12	14	14	15
	Mean	12	13	15	17
0.899	1	12	19	24	29
	2	12	20	21	26
	3	12	19	25	30
	Mean	12	19	23	28
0.240	1	12	20	37	55
	2	12	21	38	59
	3	12	21	37	62
	Mean	12	21	37	59
0.0535	1	12	23	47	93
	2	12	23	45	89
	3	12	24	54	92
	Mean	12	23	49	91
0.0137	1	12	25	53	115
	2	12	25	55	113
	3	12	28	54	117
	Mean	12	26	54	115
Control	1	12	26	56	116
	2	12	29	51	118
	3	12	23	50	95
	4	12	24	47	111
	5	12	27	43	114
	6	12	25	47	101
	Mean	12	26	49	109

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

Repl. No. = replicate number

Table4:      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 (-).

                                               

Geometric mean measured 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]
3.72		1				0.0498		84				5		95		13.9
		2				0.0579		82				6		94		12.0
		3				0.0319		90				3		97		21.7
		Mean		(+)		0.0470		85		(+)		5		95		15.9
0.899		1				0.126		60				17		83		5.5
		2				0.110		65				14		86		6.3
		3				0.131		58				18		81		5.3
		Mean		(+)		0.122		61		(+)		16		83		5.7
0.240		1				0.217		31				43		56		3.2
		2				0.228		28				47		52		3.1
		3				0.235		26				50		49		3.0
		Mean		(+)		0.227		28		(+)		47		52		3.1
0.0535		1				0.293		7				81		17		2.4
		2				0.286		9				77		21		2.4
		3				0.291		8				80		18		2.4
		Mean		(+)		0.290		8		(+)		79		18		2.4
0.0137		1				0.323		-3				103		-6		2.2
		2				0.320		-2				101		-4		2.2
		3				0.325		-3				105		-8		2.1
		Mean		(-)		0.323		-2		(-)		103		-6		2.2
Control		1				0.324						104				2.1
		2				0.327						106				2.1
		3				0.296						83				2.4
		4				0.318						99				2.2
		5				0.322						102				2.2
		6				0.304						89				2.3
		Mean				0.315						97				2.2

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 (-).

 

Geometric mean measured 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   [%]
3.72		1		1.7				0.009		98				0.1		99
		2		1.6				0.000		100				0.0		100
		3		1.6				0.000		100				0.0		100
		Mean		1.6		(+)		0.003		99		(+)		0.0		100
0.899		1		3.9				0.127		64				2.3		87
		2		3.9				0.127		64				2.3		87
		3		4.5				0.148		59				2.9		84
		Mean		4.1		(+)		0.134		62		(+)		2.5		86
0.240		1		8.4				0.237		33				6.8		62
		2		7.8				0.226		36				6.2		65
		3		8.5				0.239		33				6.9		61
		Mean		8.2		(+)		0.234		34		(+)		6.6		63
0.0535		1		14.9				0.319		10				13.3		25
		2		14.8				0.318		11				13.2		25
		3		15.9				0.328		8				14.3		19
		Mean		15.2		(+)		0.322		10		(+)		13.6		23
0.0137		1		19.4				0.356		0				17.8		-1
		2		17.8				0.344		3				16.2		8
		3		20.6				0.365		-3				19		-7
		Mean		19.3		(-)		0.355		0		(-)		17.7		0
Control		1		20.6				0.365						19.00
		2		21.5				0.371						19.90
		3		16.9				0.337						15.30
		4		18.5				0.350						16.90
		5		18.9				0.353						17.30
		6		19.7				0.359						18.10
		Mean		19.3				0.356						17.70

The initial biomass dry weight was 1.6 mg per replicate.

Repl. No. = replicate number

 

 Colony Number (Plants) on Days 0 and 7

 

Geometric mean measured test item concentration [mg/L]		Replicate No.		Colony number
			Day 0		Day 7
3.72		1		3		8
		2		3		10
		3		3		12
		Mean		3		10
0.899		1		3		3
		2		3		3
		3		3		3
		Mean		3		3
0.240		1		3		6
		2		3		6
		3		3		6
		Mean		3		6
0.0535		1		3		12
		2		3		9
		3		3		10
		Mean		3		10
0.0137		1		3		10
		2		3		9
		3		3		9
		Mean		3		9
Control		1		3		11
		2		3		13
		3		3		11
		4		3		11
		5		3		11
		6		3		10
		Mean		3		11

 

 

Further Observations on Days 3, 5 and 7

Geometric mean measured test item concentration [mg/L]	Observations on day
	3	5	7
3.72	2.1+ 2.4+	2.1+ 3.2++ 3.3++ 4.1++	2.1++ 2.4++ 3.2+++ 3.3+++ 4.1++
0.899	2.3+	2.3+ 3.3+	2.1+ 2.3++ 2.4+ 3.3++
0.240	1	2.3+ 3.3+	2.3+ 3.3+
0.0535	1	1	1
0.0137	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

2.3   = gibbosity of the fronds

2.4   = discoloration of the fronds

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 Violet 16 (dihydrogenphosphate) was found to inhibit the growth of the monocotyledonous aquatic plant Lemna gibba after 7-day exposure under static conditions, with the following effect values (geometric mean measured test item concentrations): The EC50-values for inhibition of the specific growth rate (fronds and dry weight) (ErC50, ErdwC50) were 0.581 (0.510 – 0.667) mg/L and 0.528 (0.477 – 0.588) mg/L and yield (fronds and dry weight) (EyC50, EydwC50) were 0.204 (0.182 - 0.230) mg/L and 0.151 (0.129 – 0.177) mg/L, respectively.

Executive summary:

The effects of the test item Basic Violet 16 (dihydrogenphosphate) on the growth of the monocotyledonous aquatic plant species Lemna gibba was determined according to the principles of OECD 221 at the test facility from 2018-02-14 to 2018-03-19, with the definitive exposure phase from 2018-03-09 to 2018-03-16.

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 4:0.0156 - 0.0625 - 0.250 - 1.00 - 4.00 mg/L, corresponding to the geometric mean measured test item concentrations: 0.0137 - 0.0535 – 0.240 – 0.899 – 3.72 mg/L. Three replicates were investigated for each test concentration and six for the control. Frond numbers were assessed on days 0, 3, 5 and 7. Environmental parameters (light, pH and temperature) were within the acceptable limits. All test solutions were clear and concentration-related purple coloured throughout the exposure.The validity criteria of the test guideline were fulfilled.

The concentrations ofthe test item Basic Violet 16 (dihydrogenphosphate) and the control were analysed 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 and on every renewal day.

At the start of the first interval the measured concentrations of Basic Violet 16 (dihydrogenphosphate) were in the range of 95 and 116% of the nominal values. At the end of the first interval the measured concentrations were between 68 and 88% of the nominal values. All effect values are given based on the geometric mean measured test item concentrations.

 

NOEC-, LOEC-, EC-Values and 95% Confidence Intervals of Basic Violet 16 (dihydrogenphosphate) after 7 Days of Exposure

                  (based on the geometric mean measured test item concentration [mg/L])

Frond number		Dry weight
Growth Rate Inhibition [mg/L]
NOEC	0.0137	0.0137
LOEC	0.0535	0.0535
ErC10	0.0755 (0.0587 – 0.0969)	0.0497 (0.0401 – 0.0617)
ErC20	0.149 (0.122 – 0.178)	0.110 (0.0915 – 0.131)
ErC50	0.581 (0.510 – 0.667)	0.528 (0.477 – 0.588)
Inhibition of Yield [mg/L]
NOEC	0.0137	0.0137
LOEC	0.0535	0.0535
EyC10	0.0379 (0.0324 – 0.0447)	0.0273 (0.0214 – 0.0343)
EyC20	0.0611 (0.0525 – 0.0709)	0.0453 (0.0369 – 0.0560)
EyC50	0.204 (0.182 – 0.230)	0.151 (0.129 – 0.177)