Nickel, [29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]-, chlorosulfonyl derivs., reaction products with 2-[(4-aminophenyl)sulfonyl]ethyl hydrogen sulfate monosodium salt, sodium salts

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 2017-12-06 to 2018-02-05, with the definitive exposure phase from 2018-01-26 to 2018-02-02

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

Constituent 1

Reference substance name:

Nickel, [29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]-, chlorosulfonyl derivs., reaction products with 2-[(4-aminophenyl)sulfonyl]ethyl hydrogen sulfate monosodium salt, sodium salts

EC Number:

305-644-9

EC Name:

Nickel, [29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]-, chlorosulfonyl derivs., reaction products with 2-[(4-aminophenyl)sulfonyl]ethyl hydrogen sulfate monosodium salt, sodium salts

Cas Number:

94891-43-7

Molecular formula:

C32 H16 N8 Ni . C8 H11 N O6 S2 . 2 Na

IUPAC Name:

Nickel, [29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]-, chlorosulfonyl derivs., reaction products with 2-[(4-aminophenyl)sulfonyl]ethyl hydrogen sulfate monosodium salt, sodium salts

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 and. The method validation was not part of this GLP study.

Test solutions

Vehicle:

no

Details on test solutions:

Preparation of the Test item solution
A stock solution with a nominal test item concentration of 100 mg/L was freshly prepared with dilution water (see Table 1) and agitated until the solution was visually clear.

Test concentrations
Based on the results of a preliminary range finding test 5 concentrations were tested with a dilution factor of √10: 1.00 - 3.16 - 10.0 - 31.6 - 100 mg/L, corresponding to the geometric mean measured test item concentrations: 0.691 – 2.64 – 8.07 – 26.4 – 87.3 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.: 23.5 max.: 25.5 mean value: 24.2

pH:

Geometric mean measured test item concentration
[mg/L] pH-value
0 h (Fresh medium) 7 d (Old medium)
87.3 7.50 8.25
26.4 7.50 8.25
8.07 7.51 8.21
2.64 7.54 8.19
0.691 7.59 8.18
Control 7.58 8.16

Dissolved oxygen:

not measured

Salinity:

not measured

Conductivity:

not measured

Nominal and measured concentrations:

Nominal: 1.00 - 3.16 - 10.0 - 31.6 - 100 mg/L
geometric mean measured test item concentrations: 0.691 – 2.64 – 8.07 – 26.4 – 87.3 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.

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

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

2.64 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Frond number

Remarks on result:

other: Growth Rate and Inhibition of Yield

Effect concentrations 2

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

0.691 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: dry weight growth rate inhibition and inhibition of yield

Effect concentrations 3

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.691 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Frond number

Remarks on result:

other: Growth Rate and Inhibition of Yield

Effect concentrations 4

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

< 0.691 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: dry weigt growth rate inhibition and inhibition of yield

Effect concentrations 5

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 87.3 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 Inhibition

Effect concentrations 6

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

17.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Dry weight Inhibition of yield

Remarks on result:

other: CI: 9.30 – 39.9

Effect concentrations 7

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

3.34 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: frond number ihibition of yield

Remarks on result:

other: CI: 2.81 - 5.30

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-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 with exposure phase 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 Reactive Violet 5 (Na).

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 determination of NOEC, LOEC and EC-values, three replicates were included for the test concentrations and six replicates for the control.

NOEC and LOEC values
NOEC/LOEC was determined by calculation of 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. Normality test failed for calculation of yield (fronds). Therefore, the data were transformed (Y=log(Y)).

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 ECx-values were carried out from the best fit values, the standard error and the t-distribution with the software GraphPad Prism.

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 confidence intervals of EC10-, EC20- and EC50-values were carried out from the best fit values, the standard error and the t-distribution with 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
87.3	1	12	22	30	38
	2	12	22	32	47
	3	12	23	33	43
	Mean	12	22	32	43
26.4	1	12	24	34	47
	2	12	27	33	41
	3	12	22	31	41
	Mean	12	24	33	43
8.07	1	12	25	37	47
	2	12	25	35	40
	3	12	23	36	46
	Mean	12	24	36	44
2.64	1	12	30	48	64
	2	12	30	45	60
	3	12	28	50	61
	Mean	12	29	48	62
0.691	1	12	30	52	84
	2	12	31	54	80
	3	12	31	58	88
	Mean	12	31	55	84
Control	1	12	31	60	92
	2	12	32	63	105
	3	12	30	58	98
	4	12	30	59	90
	5	12	33	57	88
	6	12	31	65	119
	Mean	12	31	60	99

* = 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]
87.3		1				0.165		45				26		70		4.21
		2				0.195		35				35		60		3.55
		3				0.182		39				31		64		3.80
		Mean		(+)		0.181		40		(+)		31		65		3.85
26.4		1				0.195		35				35		60		3.55
		2				0.176		41				29		67		3.95
		3				0.176		41				29		67		3.95
		Mean		(+)		0.182		39		(+)		31		64		3.82
8.07		1				0.195		35				35		60		3.55
		2				0.172		43				28		68		4.03
		3				0.192		36				34		61		3.61
		Mean		(+)		0.186		38		(+)		32		63		3.73
2.64		1				0.239		20				52		40		2.90
		2				0.230		23				48		45		3.01
		3				0.232		23				49		43		2.98
		Mean		(+)		0.234		22		(+)		50		43		2.97
0.691		1				0.278		7				72		17		2.49
		2				0.271		10				68		22		2.56
		3				0.285		5				76		12		2.44
		Mean		(-)		0.278		7		(-)		72		17		2.50
Control		1				0.291						80				2.38
		2				0.310						93				2.24
		3				0.300						86				2.31
		4				0.288						78				2.41
		5				0.285						76				2.44
		6				0.328						107				2.11
		Mean				0.300						87				2.31

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   [%]
87.3	1	6.2		0.162	40		4.2	63
	2	6.8		0.175	35		4.8	58
	3	5.8		0.152	44		3.8	66
	Mean	6.3	(+)	0.163	40	(+)	4.3	62
26.4	1	7.7		0.193	29		5.7	50
	2	7.9		0.196	27		5.9	48
	3	7.2		0.183	32		5.2	54
	Mean	7.6	(+)	0.191	29	(+)	5.6	50
8.07	1	8.3		0.203	25		6.3	44
	2	7.3		0.185	32		5.3	53
	3	8.1		0.200	26		6.1	46
	Mean	7.9	(+)	0.196	27	(+)	5.9	48
2.64	1	9.8		0.227	16		7.8	31
	2	9.0		0.215	20		7.0	38
	3	10.5		0.237	12		8.5	25
	Mean	9.8	(+)	0.226	16	(+)	7.8	31
0.691	1	11.1		0.245	9		9.1	19
	2	11.5		0.250	7		9.5	16
	3	12.2		0.258	4		10.2	10
	Mean	11.6	(+)	0.251	7	(+)	9.6	15
Control	1	12.6		0.263			10.6
	2	13.4		0.272			11.4
	3	13.7		0.275			11.7
	4	12.8		0.265			10.8
	5	12.5		0.262			10.5
	6	14.6		0.284			12.6
	Mean	13.3		0.270			11.3

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
87.3		1		3		4
		2		3		5
		3		3		5
		Mean		3		5
26.4		1		3		5
		2		3		5
		3		3		5
		Mean		3		5
8.07		1		3		5
		2		3		4
		3		3		4
		Mean		3		4
2.64		1		3		9
		2		3		7
		3		3		8
		Mean		3		8
0.691		1		3		10
		2		3		9
		3		3		8
		Mean		3		9
Control		1		3		11
		2		3		16
		3		3		11
		4		3		12
		5		3		9
		6		3		16
		Mean		3		13

 Further Observations on Days 2, 5 and 7

Geometric mean measured test item concentration [mg/L]		Observations on day
		2		5		7
87.3		1		1		3.3 +
26.4		1		1		3.3 +
8.07		1		1		1
2.64		1		1		1
0.691		1		1		1
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 the roots

+      = slight effects

++    = medium effects

+++  = strong effects

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study, Reactive Blue 38 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 > 87.3 mg/L. The EC50-values for inhibition of yield (fronds, EyC50) and dry weight inhibition of yield (EydwC50) were 3.34 (2.81 – 5.30) mg/L and 17.7 (9.30 – 39.9) mg/L, respectively.

Executive summary:

The effects of the test item Reactive Blue 38 on the growth of the monocotyledonous aquatic plant species Lemna gibba was determined according to the principles of OECD 221 and Council Regulation (EC) No. 761/2009 Method C. 26 at the test facility from 2017-12-06 to 2018-02-05, with the definitive exposure phase from 2018-01-26 to 2018-02-02.

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 mg/L, corresponding to the geometric mean measured test item concentrations: 0.691 – 2.64 – 8.07 – 26.4 – 87.3 mg/L. Three replicates were investigated for each test concentration and six 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 test item solutions were clear and concentration related blue coloured throughout the exposure period.

The concentrations of the test item Reactive Blue 38 and the control were analysed via HPLC-DAD at the beginning and end of the exposure.

The measured concentrations of Reactive Blue38 in the fresh media were in the range of 99 to 102% of the initial measured values. The measured concentrations in the old media were in the range of 48 to 75% of the initial measured values. All effect values are given based on the geometric mean measured test item concentrations.

 

NOEC-, LOEC-, EC-Values and 95% Confidence Intervals of Reactive Blue 38 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	0.691	NOEC	< 0.691
LOEC	2.64	LOEC	0.691
ErC10	1.24 (< 0.691 – 2.02)	ErdwC10	0.969 (< 0.691 – 1.59)
ErC20	2.40 (1.63 – 2.97)	ErdwC20	3.62 (1.91 – 6.44)
ErC50	> 87.3	ErdwC50	> 87.3
Inhibition of Yield [mg/L]
NOEC	0.691	NOEC	< 0.691
LOEC	2.64	LOEC	0.691
EyC10	< 0.691	EydwC10	< 0.691
EyC20	1.06 (< 0.691 – 1.88)	EydwC20	0.956 (< 0.691 – 1.41)
EyC50	3.34 (2.81 – 5.30)	EydwC50	17.7 (9.30 – 39.9)