Reaction mass of disodium 4-amino-3-[[4-[(2,4-diaminophenyl)azo]phenyl]azo]-5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate and disodium 4-amino-3-[[4-[(2-amino-4-hydroxyphenyl)azo]phenyl]azo]-5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate

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 2018-04-24 to 2018-05-03, with the definitive exposure phase from 2018-04-25 to 2018-05-02.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

according to OECD and GLP guidelines

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

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 4-amino-3-[[4-[(2-amino-4-hydroxyphenyl)azo]phenyl]azo]-5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate

EC Number:

270-629-5

EC Name:

Disodium 4-amino-3-[[4-[(2-amino-4-hydroxyphenyl)azo]phenyl]azo]-5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate

Cas Number:

68460-07-1

Molecular formula:

C28H22N8O8S2.2Na

IUPAC Name:

disodium 4-amino-3-({4-[(2-amino-4-hydroxyphenyl)diazenyl]phenyl}diazenyl)-5-hydroxy-6-(phenyldiazenyl)naphthalene-2,7-disulfonate

Constituent 2

Reference substance name:

Disodium 4-amino-3-[[4-[(2,4-diaminophenyl)azo]phenyl]azo]-5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate

EC Number:

272-559-0

EC Name:

Disodium 4-amino-3-[[4-[(2,4-diaminophenyl)azo]phenyl]azo]-5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate

Cas Number:

68877-33-8

Molecular formula:

C28H23N9O7S2.2Na

IUPAC Name:

disodium 4-amino-3-({4-[(2,4-diaminophenyl)diazenyl]phenyl}diazenyl)-5-hydroxy-6-(phenyldiazenyl)naphthalene-2,7-disulfonate

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 beginning and at the end of the exposure and at 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.

Test solutions

Details on test solutions:

Preparation of the test item solution
A stock solution with a nominal test item concentration of 150 mg/L
was freshly prepared with demineralized water one day prior to the start of exposure (0 hours) and prior to the renewal of the test solutions (24 hours). The solution was stirred for 24 hours at approx. 1100 rpm at room temperature. Prior to testing, the nutrient salts for the medium were added to obtain the final stock solution.

 
Test concentrations
The stock solution and four dilution levels were prepared with dilution water in a geometrical series with a separation factor of 2 and tested as follows: 9.38 - 18.8 - 37.5 - 75.0 - 150 mg/L, corresponding to the geometric mean measured test item concentrations of 5.82 – 13.1 – 28.0 – 62.9 – 140 mg/L.
The test concentrations were 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.: 24.3 max.: 25.7 mean value: 24.7

pH:

Geometric mean measured 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)
140 7.64 8.46 7.65 8.42 7.66 8.52
62.9 7.65 8.46 7.65 8.42 7.65 8.49
28.0 7.63 8.45 7.61 8.41 7.66 8.50
13.1 7.62 8.42 7.60 8.42 7.67 8.56
5.82 7.61 8.37 7.54 8.41 7.68 8.61
Control 7.59 8.30 7.57 8.35 7.64 8.84

Dissolved oxygen:

not measured

Salinity:

not measured

Conductivity:

not measured

Details on test conditions:

Test method Semi-static procedure with media renewal every 2-3 days

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

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.Random repositioning of the test vessels when observation were made was carried out.

Biological Parameters

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.:

< 5.82 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 and Inhibition of Yield

Effect concentrations 2

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

5.82 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 and Inhibition of Yield

Effect concentrations 3

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

< 5.82 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 and Inhibition of Yield

Effect concentrations 4

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

< 5.82 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 and Inhibition of Yield

Effect concentrations 5

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 140 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Frond number

Remarks on result:

other: Growth Rate Inhibition

Effect concentrations 6

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

85.3

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Dry weight

Remarks on result:

other: Growth Rate Inhibition

Remarks:

CI (62.0 - > 140)

Effect concentrations 7

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

< 5.82 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: Inhibition of Yield

Details on results:

The environmental conditions (pH-value, room temperature, light intensity) were determined to be within the acceptable limits. The test media showed a concentration-related black colouring..

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 from 2018-04-04 to 2018-04-12, with the definitive exposure phase from 2018-04-04 to 2018-04-11 according to OECD Guideline 221.

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 – > 8.00
Yield inhibition (number of fronds)
EyC50 4.93 4.67 ± 2.87
95% confidence interval 4.42 – 5.53
Growth rate inhibition (dry weight)
ErdwC50 > 8.00 5.61 ± 2.76
95% confidence interval -
Yield inhibition (dry weight)
EydwC50 5.95 4.75 ± 2.49
95% confidence interval 5.18 – 6.75
SD = standard deviation

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, LOEC and statistical analyses
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 of 0.01.

Levene’s test on variance homogeneity was done with a significance level of 0.01.

Monotonicity of concentration/response was done by trend analysis by contrasts (significance level 0.05).

William’s multiple sequential t-test was carried out with a significance level of 0.05.

Step-down Jonckheere-Terpstra test was done with a significance level of 0.05.


EC-values and statistical analyses
EC10-, EC20- and EC50-values (0 - 7 d) of the growth rate and yield (frond number) inhibition and dry weight yield as well as the EC10- and EC20-values of dry weight growth rate were calculated by empirical analysis of the inhibition values. The EC50 of fry weight growth was calculated by sigmoidal dose-response regression. Calculations of the confidence intervals 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.
- ToxRat Version 3.2.1, ToxRat Solutions GmbH

Any other information on results incl. tables

Biological Data

 

Frond numbers, evaluations of average specific growth rate inhibition, yield inhibition and dry weight inhibition are given in the tables below. Colony number and further observations are listed in the tables below.All effect values are given based on the geometric mean measured test itemconcentrationof the test item.

 

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
140	1	12	24	37	59
	2	12	21	36	56
	3	12	21	37	55
	Mean	12	22	37	57
62.9	1	12	24	45	65
	2	12	25	43	61
	3	12	24	40	61
	Mean	12	24	43	62
28.0	1	12	25	44	73
	2	12	24	46	80
	3	12	21	45	68
	Mean	12	23	45	74
13.1	1	12	27	55	113
	2	12	24	54	100
	3	12	26	57	112
	Mean	12	26	55	108
5.82	1	12	27	68	123
	2	12	23	69	134
	3	12	24	57	115
	Mean	12	25	65	124
Control	1	12	22	99	228
	2	12	28	106	272
	3	12	29	106	262
	4	12	29	110	248
	5	12	29	122	320
	6	12	31	95	236
	Mean	12	28	106	261

* = 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 (-).

                                               

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]
140		1				0.228		48				47		81		3.05
		2				0.220		50				44		82		3.15
		3				0.217		50				43		83		3.19
		Mean		(+)		0.222		50		(+)		45		82		3.13
62.9		1				0.241		45				53		79		2.87
		2				0.232		47				49		80		2.98
		3				0.232		47				49		80		2.98
		Mean		(+)		0.235		46		(+)		50		80		2.95
28.0		1				0.258		41				61		76		2.69
		2				0.271		38				68		73		2.56
		3				0.248		44				56		78		2.80
		Mean		(+)		0.259		41		(+)		62		75		2.68
13.1		1				0.320		27				101		59		2.16
		2				0.303		31				88		65		2.29
		3				0.319		27				100		60		2.17
		Mean		(+)		0.314		28		(+)		96		61		2.21
5.82		1				0.332		24				111		55		2.08
		2				0.345		21				122		51		2.01
		3				0.323		26				103		59		2.15
		Mean		(+)		0.333		24		(+)		112		55		2.08
Control		1				0.421						216				1.65
		2				0.446						260				1.55
		3				0.440						250				1.57
		4				0.433						236				1.60
		5				0.469						308				1.48
		6				0.426						224				1.63
		Mean				0.439						249				1.58

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   [%]
140		1		8.2				0.202		51				6.2		82
		2		7.6				0.191		54				5.6		84
		3		8.4				0.205		50				6.4		81
		Mean		8.1		(+)		0.199		52		(+)		6.1		82
62.9		1		8.9				0.213		48				6.9		80
		2		9.4				0.221		46				7.4		78
		3		8.9				0.213		48				6.9		80
		Mean		9.1		(+)		0.216		48		(+)		7.1		79
28.0		1		10.7				0.240		42				8.7		75
		2		10.7				0.240		42				8.7		75
		3		10.1				0.231		44				8.1		76
		Mean		10.5		(+)		0.237		43		(+)		8.5		75
13.1		1		14.0				0.278		33				12.0		65
		2		13.5				0.273		34				11.5		66
		3		14.1				0.279		32				12.1		65
		Mean		13.9		(+)		0.277		33		(+)		11.9		65
5.82		1		15.8				0.295		29				13.8		60
		2		17.2				0.307		26				15.2		56
		3		15.5				0.293		29				13.5		61
		Mean		16.2		(+)		0.298		28		(+)		14.2		59
Control		1		29.5				0.384						27.5
		2		38.9				0.424						36.9
		3		36.6				0.415						34.6
		4		37.1				0.417						35.1
		5		43.8				0.441						41.8
		6		31.4				0.393						29.4
		Mean		36.2				0.413						34.2

The initial biomass dry weight was 2.0 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
140		1		3		6
		2		3		8
		3		3		8
		Mean		3		7
62.9		1		3		8
		2		3		7
		3		3		8
		Mean		3		8
28.0		1		3		7
		2		3		6
		3		3		6
		Mean		3		6
13.1		1		3		8
		2		3		6
		3		3		9
		Mean		3		8
5.82		1		3		10
		2		3		11
		3		3		10
		Mean		3		10
Control		1		3		28
		2		3		30
		3		3		29
		4		3		35
		5		3		43
		6		3		30
		Mean		3		33

 

 

Further Observations on Days 2, 5 and 7

Geometric mean measured test item concentration [mg/L]	Observations on day
	2	5	7
140	3.3 +++ 5.2 ++	2.4 + 3.3 +++ 5.2 ++	2.4 ++ 3.3 +++ 5.2 ++
62.9	3.3 +++ 5.2 ++	2.4 + 3.3 +++ 5.2 ++	2.4 ++ 3.3 +++ 5.2 ++
28.0	2.4 + 3.3 +++ 5.2 ++	2.4 + 3.3 +++ 5.2 ++	2.4 + 3.3 +++ 5.2 ++
13.1	3.3 +++ 5.2 ++	2.4 + 3.3 +++ 5.2 ++	2.4 + 3.3 +++ 5.2 ++
5.82	3.3 +++ 5.2 ++	3.3 +++ 5.2 ++	3.3 +++ 5.2 ++
Control	1	1	1

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

 

1       = no observedeffects

2.4    = discoloration of the fronds

3.3   = discoloration of roots

5.2    = sedimentation of the test item

+      = slight effects

++    = medium effects

+++         = strong effects

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study, the test item 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 (geometric mean measured test item concentrations): The EC50-values for inhibition of the specific growth rate (fronds and dry weight) (ErC50, ErdwC50) were > 140 mg/L and 85.3 (62.0 - > 140) mg/L, respectively.

Executive summary:

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 atthe test facility from 2018-04-24 to 2018-05-03, with the definitive exposure phase from 2018-04-25 to 2018-05-02.

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 2:9.38 - 18.8 - 37.5 - 75.0 - 150 mg/L, corresponding to the geometric mean measured test item concentrations of 5.82 – 13.1 – 28.0 – 62.9 – 140 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 homogenous and concentration-related black colored throughout the exposure.The validity criteria of the test guideline were fulfilled.

The concentrations of the test item and the controlwere analysed via HPLC-DAD at the beginning and end of the exposure and at every renewal day. Freshly prepared and old media were analysed.

The measured concentrations of the test item in the fresh and old test media were in the range of 83 to 106% and 38 to 92% of the nominal values, respectively. All effect values are given based on the geometric mean measured test item concentrations.

 

NOEC-, LOEC-, EC-Values and 95% Confidence Intervals of the test item 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	< 5.82	< 5.82
LOEC	5.82	5.82
ErC10	< 5.82	< 5.82
ErC20	< 5.82	< 5.82
ErC50	> 140	85.3 (62.0 - > 140)
Inhibition of Yield [mg/L]
NOEC	< 5.82	< 5.82
LOEC	5.82	5.82
EyC10	< 5.82	< 5.82
EyC20	< 5.82	< 5.82
EyC50	< 5.82	< 5.82