[2-[[4-[(2-chloro-4-nitrophenyl)azo]phenyl]ethylamino]ethyl]trimethylammonium acetate

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:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From 2016-11-15 to 2016-11-25, with the definitive exposure phase from 2016-11-16 to 2016-11-23.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Version / remarks:

2006

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

All test item concentrations and the control were analytically verified via HPLC-DAD at the start (0 day, fresh medium) and at the end of the exposure (7 days, old medium).

Details on test solutions:

Stock solution
10.0 mg test item/L were prepared with dilution water.

Dispersion treatment
The stock solution was stirred for 23 hours (1100 rpm, room temperature).

Test concentrations
Based on the results of a preliminary range finding test 5 concentrations were tested with a dilution factor of 2: 0.625 - 1.25 - 2.50 - 5.00 - 10.0 mg/L.

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

Test organisms (species):

Lemna gibba

Details on test organisms:

Test organism
Duckweed, Lemna gibba, 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

Date of receipt
2008-02-26

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, see dilution water

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.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Hardness:

not measured

Dissolved oxygen:

not measured

Salinity:

not measured

Conductivity:

not measured

Nominal and measured concentrations:

Nominal: 0.625 - 1.25 - 2.50 - 5.00 - 10.0 mg/L
Geometric mean measured test item concentrations: 0.440 – 1.09 – 2.39 – 5.05 – 10.8 mg/L

Details on test conditions:

Test method
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.

Composition of Dilution water
Component Concentration in stock solution [g/L] Concentration in prepared medium [mg/L]
NaNO3 26 510
MgCl2  6 H2O 12 240
CaCl2  2 H2O 4.4 90
MgSO4  7 H2O 15 290
K2HPO4 · 3 H2O 1.4 30
NaHCO3 15 300
H3BO3 0.19 3.7
MnCl2  4 H2O 0.42 8.3
FeCl3  6 H2O 0.16 3.2
Na2-EDTA · 2 H2O 0.30 6.0
ZnCl2 3.3 mg/L 66 µg/L
CoCl2  6 H2O 1.4 mg/L 29 µg/L
Na2MoO4  2 H2O 7.3 mg/L 145 µg/L
CuCl2  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.

Application
Static with application of the test item at test start. 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

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.44 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: frond number growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6.45 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: frond number growth rate

Remarks on result:

other: CI: 5.09 - 7.88

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.44 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Frond number yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.42 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: frond number yield

Remarks on result:

other: CI: 1.98 - 2.97

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.09 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: dry weight growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.99 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: dry weight growth rate

Remarks on result:

other: CI: 2.48-3.66

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.44 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: dry weight yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.96 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: dry weight yield

Remarks on result:

other: CI: 1.71 - 2.23

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 SZBD168XV, purity 99.4%, CAS RN 591-35-5) to the monocotyledonous aquatic plant Lemna gibba was determined over a period of 7 days from 2016-10-12 to 2016-10-19 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 18.1 Chloride.

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 6.71 5.56 ± 2.83
95% confidence interval 5.42 – 8.00
Yield inhibition (number of fronds)
EyC50 4.50 4.59 ± 3.03
95% confidence interval 4.04 – 5.05
Growth rate inhibition (dry weight)
ErdwC50 6.76 5.50 ± 2.85
95% confidence interval 5.29 – 7.43
Yield inhibition (dry weight)
EydwC50 5.53 4.63 ± 2.47
95% confidence interval 4.56 – 6.50
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:

Sample size for 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, LOEC and statistical analysess
The NOEC and LOEC were determined by calculation of statistically significant differences of growth rates and yield.
The following statistical tests were conducted:

Shapiro-Wilk’s test on normal distribution was done with a significance level 0.01.
Levene’s test on variance homogeneity was done with a significance level 0.01.
Monotonicity of concentration/response was done by trend analysis by contrasts (significance level 0.05).
Multiple sequentially-rejective Welsh-t-test after Bonferroni was carried out with a significance level 0.05.
William’s multiple sequential t-test was carried out with a significance level 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, the standard error and the t-distribution with the software GraphPad Prism.

Frond Numbers

Geometric mean measured test item concentration [mg/L]		Repl. No.		Frond numbers per study day
			0 days*		2 days		5 days		7 days
10.8		1		12		21		21		21
		2		12		17		17		17
		3		12		18		18		18
		Mean		12		19		19		19
5.05		1		12		24		54		58
		2		12		23		46		56
		3		12		22		42		52
		Mean		12		23		47		55
2.39		1		12		23		45		61
		2		12		25		51		60
		3		12		21		40		54
		Mean		12		23		45		58
1.09		1		12		24		52		67
		2		12		24		52		72
		3		12		23		58		76
		Mean		12		24		54		72
0.440		1		12		25		63		99
		2		12		21		63		101
		3		12		23		64		97
		Mean		12		23		63		99
Control		1		12		25		73		118
		2		12		21		61		95
		3		12		25		76		115
		4		12		21		52		97
		5		12		25		66		113
		6		12		20		62		102
		Mean		12		23		65		107

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

Repl. No. = replicate number

Growth Rate and Yield Inhibition based on Frond number

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]
10.8		1				0.0799		74				9		91		8.67
		2				0.0498		84				5		95		13.9
		3				0.0579		81				6		94		12.0
		Mean		(+)		0.0630		80		(+)		7		93		11.5
5.05		1				0.225		28				46		51		3.08
		2				0.220		29				44		54		3.15
		3				0.209		33				40		58		3.31
		Mean		(+)		0.218		30		(+)		43		54		3.18
2.39		1				0.232		26				49		48		2.98
		2				0.230		26				48		49		3.01
		3				0.215		31				42		56		3.23
		Mean		(+)		0.226		28		(+)		46		51		3.07
1.09		1				0.246		21				55		42		2.82
		2				0.256		18				60		37		2.71
		3				0.264		15				64		32		2.63
		Mean		(+)		0.255		18		(+)		60		37		2.72
0.440		1				0.301		3				87		8		2.30
		2				0.304		2				89		6		2.28
		3				0.299		4				85		10		2.32
		Mean		(-)		0.301		3		(-)		87		8		2.30
Control		1				0.327						106				2.12
		2				0.296						83				2.35
		3				0.323						103				2.15
		4				0.299						85				2.32
		5				0.320						101				2.16
		6				0.306						90				2.27
		Mean				0.312						95				2.23

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 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   [%]
10.8	1	0.7		-0.109	100			-0.8	100
	2	0.5		-0.157	100			-1.0	100
	3	1.3		-0.020	100			-0.2	100
	Mean	0.8	(+)	-0.095	100	(+)		-0.7	100
5.05	1	4.3		0.150	50			2.8	74
	2	3.5		0.121	60			2.0	81
	3	3.4		0.117	61			1.9	82
	Mean	3.7	(+)	0.129	57	(+)		2.2	79
2.39	1	6.3		0.205	32			4.8	56
	2	6.5		0.209	30			5.0	54
	3	5.0		0.172	43			3.5	68
	Mean	5.9	(+)	0.195	35	(+)		4.4	59
1.09	1	6.8		0.216	28			5.3	51
	2	9.0		0.256	15			7.5	31
	3	9.2		0.259	14			7.7	29
	Mean	8.3	(-)	0.244	19	(+)		6.8	37
0.440	1	11.8		0.295	2			10.3	5
	2	12.6		0.304	-1			11.1	-3
	3	12.8		0.306	-2			11.3	-5
	Mean	12.4	(-)	0.302	-1	(-)		10.9	-1
Control	1	13.5		0.314			12.0
	2	11.5		0.291			10.0
	3	13.1		0.310			11.6
	4	11.5		0.291			10.0
	5	12.7		0.305			11.2
	6	11.4		0.290			9.9
	Mean	12.3		0.300			10.8

The initial biomass dry weight was 1.5 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
10.8		1		3		17
		2		3		14
		3		3		15
		Mean		3		15
5.05		1		3		12
		2		3		13
		3		3		10
		Mean		3		12
2.39		1		3		7
		2		3		6
		3		3		5
		Mean		3		6
1.09		1		3		6
		2		3		6
		3		3		7
		Mean		3		6
0.440		1		3		9
		2		3		9
		3		3		9
		Mean		3		9
Control		1		3		10
		2		3		9
		3		3		9
		4		3		8
		5		3		10
		6		3		10
		Mean		3		9

Further Observations on Days 2, 5 and 7

Geometric mean measured test item concentration [mg/L]		Observations on day
		2		5		7
10.8		2.4 + 3.3 + 4.1 ++		2.1 + 2.4 ++ 3.3 ++ 4.1 +++		2.1 + 2.4 ++ 3.2 ++ 3.3 ++ 4.1 +++
5.05		3.3 + 4.1 +		2.3 + 2.4 + 3.3 + 4.1 +		2.3 + 2.4 + 3.2 ++ 3.3 ++ 4.1 ++
2.39		3.3 +		3.3 +		3.3 ++
1.09		3.3 +		3.3 +		3.3 +
0.440		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

2.4   = discoloration of fronds

3.2   = loses of roots

3.3   = discoloration of roots

4.1   = break up of plants

+      = slight effects

++    = medium effects

+++  = strong effects

Validity criteria fulfilled:

yes

Conclusions:

ErC50 = 6.45 (5.09 – 7.88) mg/L based on frond number
ErC50 = 2.99 (2.48 – 3.66) mg/L based on dry weight

Executive summary:

Method

The effects of the test item on the growth of the monocotyledonous aquatic plant species Lemna gibba was determined according to the principles of OECD 221.

Lemna gibba was exposed to the test item for 7 days under static conditions. Based on a preliminary test, 5 nominal test item concentration levels were tested in a geometrical series with a dilution factor of 2: 0.625 - 1.25 - 2.50 - 5.00 - 10.0 mg/L, corresponding to the geometric mean measured test item concentrations: 0.440 – 1.09 – 2.39 – 5.05 – 10.8 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. The validity criteria of the test guideline were fulfilled.

The concentrations of the test item and the control were analysed via HPLC-DAD at the beginning and end of the exposure.

The measured concentrationsat the start and the end of the exposure were in the range of 98 to 106 % and < LOQ to 110 % of the nominal values, respectively. All effect values are given based on the geometric mean measured test item concentrations.

Results

NOEC-, LOEC-, EC-Values and 95 % Confidence Intervals after 7 Days of Exposure  (based on the geometric mean measured concentration of the test item [mg/L])

Frond number		Dry weight
Growth Rate Inhibition [mg/L]
NOEC	0.440	NOEC	1.09
LOEC	1.09	LOEC	2.39
ErC10	0.932 (0.440 – 1.65)	ErdwC10	0.786 (0.587 – 0.991)
ErC20	2.14 (1.36 – 2.98)	ErdwC20	1.10 (0.857 – 1.35)
ErC50	6.45 (5.09 – 7.88)	ErdwC50	2.99 (2.48 – 3.66)
Inhibition of Yield [mg/L]
NOEC	0.440	NOEC	0.440
LOEC	1.09	LOEC	1.09
EyC10	0.444 (0.440 – 0.604)	EydwC10	0.537 (0.440 – 0.654)
EyC20	0.678 (0.482 – 0.881)	EydwC20	0.742 (0.605 – 0.882)
EyC50	2.42 (1.98 – 2.97)	EydwC50	1.96 (1.71 – 2.23)

Description of key information

ErC50 =   6.45 (5.09 – 7.88) mg/l based on frond number

ErC50 = 2.99 (2.48 – 3.66) mg/l based on dry weight

Additional information

No studies on "Toxicity to aquatic plants other than algae" are available on the Target Substance in itself. However a study conducted on a Similar Substance has been used for the assessment.

Similar Substance 02 differ from the Target one only on the salification. More information are reported in section 13.

The effects of the test item on the growth of the monocotyledonous aquatic plant species Lemna gibba was determined according to the principles of OECD 221.

Lemna gibba was exposed to the test item for 7 days under static conditions. Based on a preliminary test, 5 nominal test item concentration levels were tested in a geometrical series with a dilution factor of 2: 0.625 - 1.25 - 2.50 - 5.00 - 10.0 mg/L, corresponding to the geometric mean measured test item concentrations: 0.440 – 1.09 – 2.39 – 5.05 – 10.8 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. The validity criteria of the test guideline were fulfilled.

The concentrations of the test itemand the controlwere analysed via HPLC-DAD at the beginning and end of the exposure.

The measured concentrations at the start and the end of the exposure were in the range of 98 to 106 % and < LOQ to 110 % of the nominal values, respectively. All effect values are given based on the geometric mean measured test item concentrations. The EC50 value based on growth rate calculated in this test are:

ErC50 =   6.45 (5.09 – 7.88) mg/L based on frond number

ErC50 = 2.99 (2.48 – 3.66) mg/L based on dry weight