Trisodium 5,5'-[(2-sulphonato-1,4-phenylene)bis(azo)]bis(salicylate)

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 2018-01-12 to 2018-01-22, with the definitive exposure phase from 2018-01-12 to 2018-01-19

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:

Trisodium 5,5'-[(2-sulphonato-1,4-phenylene)bis(azo)]bis(salicylate)

EC Number:

276-501-5

EC Name:

Trisodium 5,5'-[(2-sulphonato-1,4-phenylene)bis(azo)]bis(salicylate)

Cas Number:

72230-95-6

Molecular formula:

C20H14N4O9S.3Na

IUPAC Name:

trisodium 3,3'-[(2-sulfonato-1,4-phenylene)didiazene-2,1-diyl]bis(6-hydroxybenzoate)

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
All concentration levels and the control were analytically verified via HPLC-DAD at the beginning and at the end of the exposure and on every renewal day. Freshly prepared and old media were analysed.

Test solutions

Details on test solutions:

Stock solution, media preparation 120 mg test item/L were freshly prepared with dilution water. The dispersion was treated with ultrasound for 5 minutes at room temperature.

Test concentrations
One limit concentration was tested. The limit concentration (120 mg/L) is based on the results of a preliminary range finding test (non-GLP). For results of the range finding test, see Annex II.

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, 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, 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 9 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:

yes

Total exposure duration:

7 d

Test conditions

Hardness:

not measured

Test temperature:

see any other information on materials and methods

pH:

see any other information on materials and methods

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
Six replicates for the limit concentration and 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
Semi-static, with renewal of the test solutions on day 3 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.


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 6 replicates of the limit 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.:

120 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Frond number and Dry weight

Effect concentrations 2

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

> 120 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Frond number and Dry weight

Effect concentrations 3

Duration:

7 d

Dose descriptor:

other: EC10, EC20 and EC50

Effect conc.:

> 120 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Frond number and Dry weight

Effect concentrations 4

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

120 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Inhibition of Yield

Remarks on result:

other: Frond number and Dry weight

Effect concentrations 5

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

> 120 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Inhibition of Yield

Remarks on result:

other: Frond number and Dry weight

Effect concentrations 6

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

< 120 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Inhibition of Yield

Remarks on result:

other: Frond number and Dry weight

Effect concentrations 7

Duration:

7 d

Dose descriptor:

other: EC20 and EC50

Effect conc.:

> 120 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Inhibition of Yield

Remarks on result:

other: Frond number and Dry weight

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 337.

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:

NOEC, LOEC and statistical analyses NOEC/LOEC were determined by calculation of statistical significance of growth rates and yield. As a standard, a t-test was used for NOEC/LOEC calculations. When running a t-test, a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance test are 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is =0.05. The Equal Variance test failed for calculation of statistical significance of growth rate (dry weight). Therefore, the data were transformed (Y squared).

EC-values and statistical analyses EC-values were estimated empirically based on the results of the single treatment level (limit test design).

Software
All data were computer generated and rounded for presentation from the fully derived data. Consequently, if calculated manually based on the given data minor variations may occur from these figures.
Calculations and graphics will be carried out using 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]	Replicate No.	Frond numbers per study day
		0 days*	3 days	5 days	7 days
120	1	12	33	67	118
	2	12	36	69	139
	3	12	29	65	108
	4	12	37	76	121
	5	12	33	64	123
	6	12	35	76	136
	Mean	12	34	70	124
Control	1	12	29	57	98
	2	12	33	69	119
	3	12	33	79	104
	4	12	35	71	137
	5	12	33	71	98
	6	12	28	58	101
	Mean	12	32	68	110

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

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]
120	1		0.327	-4		106	-9	2.12
	2		0.350	-11		127	-30	1.98
	3		0.314	0		96	2	2.21
	4		0.330	-5		109	-12	2.10
	5		0.332	-6		111	-14	2.08
	6		0.347	-10		124	-27	2.00
	Mean	(-)	0.333	-6	(-)	112	-15	2.08
Control	1		0.300			86		2.31
	2		0.328			107		2.11
	3		0.308			92		2.25
	4		0.348			125		1.99
	5		0.300			86		2.31
	6		0.304			89		2.28
	Mean		0.315			98		2.21

Repl. No. = replicate number

 

 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 (-).

 

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   [%]
120	1	24.7		0.374	2			22.9	6
	2	28.1		0.393	-3			26.3	-8
	3	18.9		0.336	12			17.1	30
	4	20.7		0.349	8			18.9	22
	5	18.9		0.336	12			17.1	30
	6	24.0		0.370	3			22.2	9
	Mean	22.6	(-)	0.360	6	(-)		20.8	15
Control	1	22.6		0.361			20.8
	2	26.1		0.382			24.3
	3	27.4		0.389			25.6
	4	26.6		0.385			24.8
	5	28.2		0.393			26.4
	6	25.4		0.378			23.6
	Mean	26.1		0.381			24.3

The initial biomass dry weight was 1.8 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
120		1		3		15
		2		3		13
		3		3		18
		4		3		16
		5		3		14
		6		3		17
		Mean		3		16
Control		1		3		8
		2		3		11
		3		3		10
		4		3		10
		5		3		8
		6		3		9
		Mean		3		9

 

 

Further Observations on Days 3, 5 and 7

Nominal test item concentration [mg/L]		Observations on day
		3		5		7
120		3.3 +++		3.3 +++		3.3 +++
Control		1		1		1

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

 

1      = no observedeffects

3.3  = discoloration of roots

+      = slight effects

++    = moderate effects

+++         = strong effects

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study, the test item was found not 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 concentrations): The EC50-values for inhibition of the specific growth rate and yield of fronds (ErC50) and dry weight (ErdwC50) were > 120 mg/L, respectively. The NOEC-values for specific growth rate and yield of fronds and dry weight were 120 mg/L, respectively.

Executive summary:

The effect of the test item 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 2018-01-12 to 2018-01-22, with the definitive exposure phase from 2018-01-12 to 2018-01-19.

 

Lemna gibba was exposed to the test item for 7 days under semi-static conditions. Based on a preliminary test, a limit test item concentration of 120 mg/L was tested. Six replicates were investigated for the limit concentration and 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.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 in the end of the exposure and on 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 104 to 108% and 105 to 111% of the nominal values, respectively. All effect values are given based on the nominal test item concentrations.

 

NOEC-, LOEC-, EC-Values and 95% Confidence Intervals of the test item after 7 days of exposure

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

Frond number		Dry weight
Growth Rate Inhibition [mg/L]
NOEC	120	NOEC	120
LOEC	> 120	LOEC	> 120
ErC10	> 120	ErdwC10	> 120
ErC20	> 120	ErdwC20	> 120
ErC50	> 120	ErdwC50	> 120
Inhibition of Yield [mg/L]
NOEC	120	NOEC	120
LOEC	> 120	LOEC	> 120
EyC10	> 120	EydwC10	< 120
EyC20	> 120	EydwC20	> 120
EyC50	> 120	EydwC50	> 120