Sodium 4-[[3-(acetylamino)phenyl]amino]-1-amino-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate

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:

experimental study

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:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

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).

Details on test solutions:

Preparation of the Saturated solution
A saturated solution with a nominal loading 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 glass slide. The glass slide with the test item was inserted into a glass bottle with an appropriate amount of dilution water. The saturated 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 saturated solution, was used in the test. During filtration, the filter was always be kept covered.
The saturated solution was checked via laser beam (Tyndall effect) for undissolved test item, which was negative.

Test concentrations
Based on the results of a preliminary range finding test (see section 8.1) 5 concentrations were tested with a dilution factor of √10: 1.00 - 3.16 - 10.0 - 31.6 - 100% of the saturated solution, corresponding to the geometric mean measured test item concentration of: 0.208 – 0.642 – 2.14 – 7.14 – 21.7 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

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

Nominal and measured concentrations:

Nominal: 2.00 - 6.32 - 20.0 - 63.2 - 200 mg/L
Geometric mean measured test item concentrations: 0.218 – 0.661 – 2.11 – 7.07 – 22.1 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, 4 uniform, healthy plants (colonies of 3 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.:

0.208 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.:

> 21.7 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:

NOEC

Effect conc.:

< 0.208 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.:

0.689 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: frond number yield

Remarks on result:

other: CI: 0.271 - 1.85

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 0.208 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.:

> 21.7 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:

NOEC

Effect conc.:

< 0.208 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.:

2.86 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: dry weight yield

Remarks on result:

other: CI: 1.28 - 21.7

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 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 tested by trend analysis by contrasts (significance level 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 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.
- ToxRat Version 3.2.1, ToxRat Solutions GmbH

All effect values are given based on the nominal andgeometric mean measured concentrationof the test itemAcid Blue324.

 

 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
21.7		1		12		16		34		40
		2		12		19		34		42
		3		12		19		35		43
		Mean		12		18		34		42
7.14		1		12		18		34		41
		2		12		20		36		41
		3		12		18		30		36
		Mean		12		19		33		39
2.14		1		12		17		33		41
		2		12		18		32		43
		3		12		16		31		39
		Mean		12		17		32		41
0.642		1		12		22		39		64
		2		12		18		36		55
		3		12		19		38		51
		Mean		12		20		38		57
0.208		1		12		17		39		63
		2		12		20		48		92
		3		12		19		43		74
		Mean		12		19		43		76
Control		1		12		19		58		96
		2		12		20		45		92
		3		12		18		60		102
		4		12		19		46		83
		5		12		20		47		80
		6		12		21		63		129
		Mean		12		20		53		97

* = 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 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]
21.7		1				0.172		42				28		67		4.03
		2				0.179		40				30		65		3.87
		3				0.182		39				31		64		3.80
		Mean		(+)		0.178		40		(+)		30		65		3.90
7.14		1				0.176		41				29		66		3.95
		2				0.176		41				29		66		3.95
		3				0.157		47				24		72		4.42
		Mean		(+)		0.169		43		(+)		27		68		4.10
2.14		1				0.176		41				29		66		3.95
		2				0.182		39				31		64		3.80
		3				0.168		43				27		68		4.12
		Mean		(+)		0.175		41		(+)		29		66		3.96
0.642		1				0.239		19				52		39		2.90
		2				0.217		27				43		49		3.19
		3				0.207		30				39		54		3.35
		Mean		(+)		0.221		26		(+)		45		47		3.15
0.208		1				0.237		20				51		40		2.93
		2				0.291		2				80		6		2.38
		3				0.260		13				62		27		2.67
		Mean		(-)		0.263		12		(-)		64		24		2.66
Control		1				0.297						84				2.33
		2				0.291						80				2.38
		3				0.306						90				2.27
		4				0.276						71				2.51
		5				0.271						68				2.56
		6				0.339						117				2.04
		Mean				0.297						85				2.35

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   [%]
21.7	1	6.2		0.203	34		4.7	59
	2	7.2		0.224	27		5.7	51
	3	6.9		0.218	29		5.4	53
	Mean	6.8	(+)	0.215	30	(+)	5.3	55
7.14	1	7.4		0.228	26		5.9	49
	2	7.3		0.226	27		5.8	50
	3	7.2		0.224	27		5.7	51
	Mean	7.3	(+)	0.226	27	(+)	5.8	50
2.14	1	7.5		0.230	26		6.0	48
	2	7.9		0.237	23		6.4	45
	3	7.2		0.224	27		5.7	51
	Mean	7.5	(+)	0.230	25	(+)	6.0	48
0.642	1	9.8		0.268	13		8.3	28
	2	10.2		0.274	11		8.7	25
	3	9.2		0.259	16		7.7	34
	Mean	9.7	(+)	0.267	14	(+)	8.2	29
0.208	1	9.1		0.258	17		7.6	34
	2	11.7		0.293	5		10.2	12
	3	10.1		0.272	12		8.6	26
	Mean	10.3	(+)	0.274	11	(+)	8.8	24
Control	1	12.8		0.306			11.3
	2	12.1		0.298			10.6
	3	13.9		0.318			12.4
	4	10.8		0.282			9.3
	5	13.1		0.310			11.6
	6	15.9		0.337			14.4
	Mean	13.1		0.309			11.6

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
21.7		1		4		6
		2		4		4
		3		4		7
		Mean		4		6
7.14		1		4		4
		2		4		5
		3		4		4
		Mean		4		4
2.14		1		4		5
		2		4		4
		3		4		4
		Mean		4		4
0.642		1		4		8
		2		4		7
		3		4		5
		Mean		4		7
0.208		1		4		9
		2		4		12
		3		4		9
		Mean		4		10
Control		1		4		12
		2		4		12
		3		4		11
		4		4		9
		5		4		12
		6		4		17
		Mean		4		12

 Further Observations on Days 2, 5 and 7

Geometric mean measured test item concentration [mg/L]		Observations on day
		2		5		7
21.7		1		1		1
7.14		1		1		1
2.14		1		1		1
0.642		1		1		1
0.208		1		1		1
Control		1		1		1

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

 

1      = no observedeffects

+      = slight effects

++    = medium effects

+++  = strong effects

Validity criteria fulfilled:

yes

Conclusions:

In this study, Acid Blue 324 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 > 21.7 mg/L, respectively. The EC50-values for inhibition of yield (fronds, EyC50) and dry weight inhibition of yield (EydwC50) with 95% confidence intervals were 0.689 (0.271 – 1.85) mg/L and 2.86 (1.28 – 21.7) mg/L, respectively.

Executive summary:

The effects of the test item Acid Blue324 on the growth of the monocotyledonous aquatic plant species Lemna gibba was determined according to the principles of OECD 221 atthe test facility from2016-11-15 to 2016-11-25, with the definitive exposure phase from2016-11-16 to 2016-11-23.

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 the saturated solution, corresponding to the geometric mean measured test item concentrations: 0.208 – 0.642 – 2.14 – 7.14 – 21.7 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 ofthe test item Acid Blue324 and the controlwere analysed via HPLC-DAD at the beginning and end of the exposure.

The measured initial concentrations of Acid Blue324 were 0.218 – 0.661 – 2.11 – 7.07 – 22.1 mg/L and reflect the dilution factor of √10. The measured concentrations in the old media were in the range of 91 to 103% of the initially measured values.

 

Table1:   NOEC-, LOEC-, EC-Values and 95 % Confidence Intervals of Acid Blue324 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	0.208	NOEC	< 0.208
LOEC	0.642	LOEC	0.208
ErC10	< 0.208	ErdwC10	< 0.208
ErC20	0.510 (0.254 – 1.32)	ErdwC20	1.26 (0.753 – 1.99)
ErC50	> 21.7	ErdwC50	> 21.7
Inhibition of Yield [mg/L]
NOEC	< 0.208	NOEC	< 0.208
LOEC	0.208	LOEC	0.208
EyC10	< 0.208	EydwC10	< 0.208
EyC20	< 0.208	EydwC20	< 0.208
EyC50	0.689 (0.271 – 1.85)	EydwC50	2.86 (1.28 – 21.7)

 

Description of key information

Acid Blue 324 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 7-d ErC50 (frond number) was > 21.7 mg/L, corresponding to the solubility limit in the test medium. The 7-d NoErC (frond number) was found at 0.208 mg/L. The substance is not acutely toxic to aquatic plants up to to the limit of solubility (ErC50 > 100 mg/L nominal). The substance showed effects, so that it can be regarded as chronically harmful to aquatic plants.

Key value for chemical safety assessment

EC10 or NOEC for freshwater plants:

0.208 mg/L