Sodium m-[[4-[[p-[bis(2-hydroxyethyl)amino]phenyl]azo]-1-naphthyl]azo]benzenesulphonate

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 2018-02-20 to 2018-03-01, with the definitive exposure phase from 2018-02-21 to 2018-02-28

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

1

Reference substance name:

Sodium m-[[4-[[p-[bis(2-hydroxyethyl)amino]phenyl]azo]-1-naphthyl]azo]benzenesulphonate

EC Number:

266-867-4

EC Name:

Sodium m-[[4-[[p-[bis(2-hydroxyethyl)amino]phenyl]azo]-1-naphthyl]azo]benzenesulphonate

Cas Number:

67674-28-6

Molecular formula:

C26H25N5O5S.Na

IUPAC Name:

sodium m-[[4-[[p-[bis(2-hydroxyethyl)amino]phenyl]azo]-1-naphthyl]azo]benzenesulphonate

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 start (0 day, fresh medium) and at the end of the exposure (7 days, old medium). The samples were analysed with an HPLC-DAD method. The method was implemented under non-GLP but documented in the raw data and validated

Test solutions

Vehicle:

no

Details on test solutions:

Preparation of the Test item solution
A test item solution of 100 mg test item/L was prepared once 24 ± 1 hour prior to the start of the exposure. An appropriate amount of the test item was weighed out. The test item was applied onto a piece of inert plastic material (Parafilm). The inert plastic peace with the test item was inserted into a glass bottle with an appropriate amount of dilution water (see Table 2). The test item solution was stirred for 24 ± 1 hours (1100 rpm, room temperature) with a magnetic stirrer. Undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC, Macherey-Nagel). The filter was saturated in order to avoid adsorption during the filtration. The first 25 mL of the filtrate were discarded. The filtration was interrupted for 15 minutes to allow adsorption and saturation of the filter material with dissolved test item. Thereafter, the filtration was continued. The next 25 mL were discarded. The following filtrate, i.e. the test item solution, was used in the test. During filtration, the filter was always kept covered.
The test item solution was checked via laser beam (Tyndall effect) for undissolved test item. The Tyndall effect was positive.

Test concentrations
Based on the results of a preliminary range finding test (see Annex II) 5 concentrations were tested with a dilution factor of √10: 1.00 - 3.16 - 10.0 - 31.6 - 100% of the test item solution, corresponding to the geometric mean measured test item concentrations: 0.0120 – 0.0354 – 0.108 – 0.392 – 1.38 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 7 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.: 22.5 max.: 25.7 mean value: 24.4

pH:

Geometric mean measured test item concentration
[mg/L] pH-value
0 h
(Fresh medium) 7 d
(Old medium)
1.38 7.74 8.38
0.392 7.71 8.38
0.108 7.74 8.37
0.0354 7.76 8.38
0.0120 7.80 8.36
Control 7.89 8.36

Dissolved oxygen:

not measured

Salinity:

not measured

Conductivity:

not measured

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.

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

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

1.38 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.:

> 1.38 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.:

> 1.38 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.:

> 1.38 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.:

> 1.38 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

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, LOEC and statistical analyses
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.

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 and third order polynomial 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*	2 days	5 days	7 days
1.38	1	12	25	67	110
	2	12	29	73	108
	3	12	24	80	119
	Mean	12	26	73	112
0.392	1	12	25	75	114
	2	12	28	87	128
	3	12	28	69	111
	Mean	12	27	77	118
0.108	1	12	26	72	123
	2	12	33	88	138
	3	12	27	94	140
	Mean	12	29	85	134
0.0354	1	12	28	69	111
	2	12	29	94	126
	3	12	28	82	124
	Mean	12	28	82	120
0.0120	1	12	20	57	94
	2	12	19	68	102
	3	12	18	46	75
	Mean	12	19	57	90
Control	1	12	19	53	98
	2	12	18	46	93
	3	12	18	46	81
	4	12	17	42	72
	5	12	21	52	97
	6	12	19	49	91
	Mean	12	19	48	89

* = 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]
1.38	1		0.317	-11		98	-28	2.19
	2		0.314	-10		96	-25	2.21
	3		0.328	-15		107	-40	2.11
	Mean	(+)*	0.319	-12	(+)*	100	-31	2.17
0.392	1		0.322	-13		102	-33	2.16
	2		0.338	-19		116	-51	2.05
	3		0.318	-12		99	-29	2.18
	Mean	(+)*	0.326	-14	(+)*	106	-38	2.13
0.108	1		0.332	-17		111	-45	2.08
	2		0.349	-22		126	-64	1.99
	3		0.351	-23		128	-67	1.97
	Mean	(+)*	0.344	-21	(+)*	122	-59	2.02
0.0354	1		0.318	-12		99	-29	2.18
	2		0.336	-18		114	-49	2.06
	3		0.334	-17		112	-46	2.08
	Mean	(+)*	0.329	-15	(+)*	108	-41	2.11
0.0120	1		0.294	-3		82	-7	2.36
	2		0.306	-7		90	-17	2.27
	3		0.262	8		63	18	2.65
	Mean	(-)	0.287	-1	(-)	78	-2	2.42
Control	1		0.300			86		2.31
	2		0.293			81		2.37
	3		0.273			69		2.54
	4		0.256			60		2.71
	5		0.299			85		2.32
	6		0.289			79		2.39
	Mean		0.285			77		2.44

    

Repl. No. = replicate number

  * Statistically significance is caused by growth stimulation

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

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   [%]
1.38	1	14.6		0.383	3		13.6	8
	2	15.2		0.389	1		14.2	4
	3	16.4		0.400	-1		15.4	-4
	Mean	15.4	(-)	0.390	1	(-)	14.4	3
0.392	1	17.0		0.405	-3		16.0	-8
	2	20.5		0.431	-10		19.5	-32
	3	16.6		0.401	-2		15.6	-5
	Mean	18.0	(-)	0.413	-5	(-)	17.0	-15
0.108	1	18.7		0.418	-6		17.7	-20
	2	21.3		0.437	-11		20.3	-37
	3	21.7		0.440	-12		20.7	-40
	Mean	20.6	(+)*	0.432	-10	(+)*	19.6	-32
0.0354	1	17.1		0.406	-3		16.1	-9
	2	20.5		0.431	-10		19.5	-32
	3	19.0		0.421	-7		18.0	-22
	Mean	18.9	(-)	0.419	-6	(-)	17.9	-21
0.0120	1	17.9		0.412	-5		16.9	-14
	2	19.5		0.424	-8		18.5	-25
	3	15.1		0.388	2		14.1	5
	Mean	17.5	(-)	0.408	-4	(-)	16.5	-11
Control	1	17.4		0.408			16.4
	2	16.0		0.396			15.0
	3	15.3		0.390			14.3
	4	13.2		0.369			12.2
	5	17.1		0.406			16.1
	6	15.7		0.393			14.7
	Mean	15.8		0.394			14.8

The mean initial biomass dry weight was 1.0 mg per replicate.

Repl. No. = replicate number

* Statistically significance is caused by growth stimulation

Colony Number (Plants) on Days 0 and 7

 

Geometric mean measured test item concentration [mg/L]	Replicate No.	Colony number
		Day 0	Day 7
1.38	1	4	11
	2	4	12
	3	4	13
	Mean	4	12
0.392	1	4	13
	2	4	14
	3	4	12
	Mean	4	13
0.108	1	4	11
	2	4	15
	3	4	12
	Mean	4	13
0.0354	1	4	9
	2	4	14
	3	4	12
	Mean	4	12
0.0120	1	4	8
	2	4	9
	3	4	7
	Mean	4	8
Control	1	4	5
	2	4	5
	3	4	4
	4	4	6
	5	4	6
	6	4	5
	Mean	4	5

Further Observations on Days 2, 5 and 7

Geometric mean measured test item concentration [mg/L]	Observations on day
	2	5	7
1.38	2.5 +	2.5 +	2.5 +
0.392	2.5 +	2.5 +	2.5 +
0.108	2.5 +	2.5 +	2.5 +
0.0354	2.5 +	2.5 +	2.5 +
0.0120	1	1	1
Control	1	1	1

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

 

1      = no observedeffects

2.5  = fronds are smaller, compared to the control

+      = slight effects

++    = medium effects

+++    = strong effects

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study, Acid Red 299 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 both inhibition of the specific growth rate of fronds (ErC50) and dry weight (ErdwC50) were > 1.38 mg/L. The EC50-values for both inhibition of yield (fronds, EyC50) and dry weight inhibition of yield (EydwC50) were > 1.38 mg/L (limit of solubility in test medium).

Executive summary:

The effects of the test item Acid Red 299 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 2018-02-20 to 2018-03-01, with the definitive exposure phase from 2018-02-21 to 2018-02-28.

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√10:1.00 - 3.16 - 10.0 - 31.6 - 100% of a saturated solution, corresponding to the geometric mean measured test item concentrations: 0.0120 – 0.0354 – 0.108 – 0.392 – 1.38 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 test item solutions were clear and concentration related violet coloured throughout the exposure period.

The concentrations of the test item Acid Red 299 and the control were analysed via HPLC-DAD at the beginning and end of the exposure.

The measured initial concentrations of Acid Red 299 in the fresh media were 0.0144 – 0.0431 – 0.149 – 0.449 – 1.54 mg/L. The measured concentrations in the old media were in the range of 53 to 80% of the initial measured values.

No toxic effects were found, but slight stimulating effects were observed.

 

NOEC-, LOEC-, EC-Values and 95% Confidence Intervals of Acid Red 299
after 7 Days of Exposure

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

Frond number		Dry weight
Growth Rate Inhibition [mg/L]
NOEC	1.38	NOEC	1.38
LOEC	> 1.38	LOEC	> 1.38
ErC10	> 1.38	ErdwC10	> 1.38
ErC20	> 1.38	ErdwC20	> 1.38
ErC50	> 1.38	ErdwC50	> 1.38
Inhibition of Yield [mg/L]
NOEC	1.38	NOEC	1.38
LOEC	> 1.38	LOEC	> 1.38
EyC10	> 1.38	EydwC10	> 1.38
EyC20	> 1.38	EydwC20	> 1.38
EyC50	> 1.38	EydwC50	> 1.38