5-(diisopropylamino)-2-[[4-(dimethylamino)phenyl]azo]-3-methyl-1,3,4-thiadiazolium methyl sulphate

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 2018-01-31 to 2018-02-08, with the definitive exposure phase from 2018-01-31 to 2018-02-07.

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

Qualifier:

according to guideline

Guideline:

other: Council Regulation (EC) No. 761/2009 Method C.26

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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.

Details on test solutions:

Preparation of the A stock solution with a nominal concentration of 20.0 mg test item/L
Test item solution was freshly prepared with dilution water and agitated until the solution was visually clear.

Test concentrations
Out of the stock solution six dilution levels were prepared in a geometrical series with a separation factor of 4 and tested as follows: 0.0156 - 0.0625 - 0.250 - 1.00 - 4.00 - 16.0 mg/L, corresponding to the geometric mean measured test item concentration of: 0.00759 – 0.0149 – 0.0476 – 0.490 – 2.93 – 14.0 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

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

Test temperature:

see any other information on materials and methods

pH:

see any other information on materials and methods

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. 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 3 replicates per test concentration and 6 control replicates. Colonies from each test vessel were collected, rinsed with deionised water and then dried at about 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:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.015 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Frond number and Dry weight

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

0.028 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Frond number and Dry Weight

Remarks:

CI (0.0194 - 0.0504)

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

3.52 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Frond number and Dry Weight

Remarks:

CI (1.87 - 5.56)

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.015 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Inhibition of Yield

Remarks on result:

other: Frond number and Dry Weight

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

0.026 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Inhibition of Yield

Remarks on result:

other: Frond number and Dry Weight

Remarks:

CI (0.0180 - 0.0381)

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

0.169 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Inhibition of Yield

Remarks on result:

other: Frond number and Dry Weight

Remarks:

CI (0.0956 - 0.370)

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 Basic Blue 159 Trichlorozinkate.

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 and LOEC values
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. Normality test failed for calculation of yield (fronds). Therefore, the data were transformed (Y=Y(Log)).

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.

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
14.0	1	12	18	22	22
	2	12	18	20	21
	3	12	18	20	20
	Mean	12	18	21	21
2.93	1	12	22	37	43
	2	12	21	33	40
	3	12	21	36	46
	Mean	12	21	35	43
0.490	1	12	20	38	47
	2	12	19	38	44
	3	12	20	35	40
	Mean	12	20	37	44
0.0476	1	12	23	50	68
	2	12	20	51	76
	3	12	23	54	80
	Mean	12	22	52	75
0.0149	1	12	25	70	122
	2	12	21	68	120
	3	12	26	70	113
	Mean	12	24	69	118
0.00759	1	12	22	67	116
	2	12	24	76	122
	3	12	24	69	134
	Mean	12	23	71	124
Control	1	12	23	68	121
	2	12	20	59	85
	3	12	23	68	106
	4	12	22	72	104
	5	12	20	64	106
	6	12	22	61	106
	Mean	12	22	65	105

* = 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 (-).

    

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]
14.0	1		0.0866	72		10	89	8.00
	2		0.0799	74		9	90	8.67
	3		0.0730	76		8	91	9.50
	Mean	(+)	0.0800	74	(+)	9	90	8.72
2.93	1		0.182	41		31	67	3.80
	2		0.172	44		28	70	4.03
	3		0.192	38		34	63	3.61
	Mean	(+)	0.182	41	(+)	31	67	3.81
0.490	1		0.195	37		35	62	3.55
	2		0.186	40		32	65	3.73
	3		0.172	44		28	70	4.03
	Mean	(+)	0.184	40	(+)	32	66	3.77
0.0476	1		0.248	20		56	40	2.80
	2		0.264	15		64	31	2.63
	3		0.271	12		68	27	2.56
	Mean	(+)	0.261	16	(+)	63	32	2.66
0.0149	1		0.331	-7		110	-19	2.09
	2		0.329	-6		108	-17	2.11
	3		0.320	-4		101	-9	2.16
	Mean	(-)	0.327	-6	(-)	106	-15	2.12
0.00759	1		0.324	-5		104	-12	2.14
	2		0.331	-7		110	-19	2.09
	3		0.345	-12		122	-32	2.01
	Mean	(-)	0.333	-8	(-)	112	-21	2.08
Control	1		0.330			109		2.10
	2		0.280			73		2.48
	3		0.311			94		2.23
	4		0.308			92		2.25
	5		0.311			94		2.23
	6		0.311			94		2.23
	Mean		0.309			93		2.25

                                           

Repl. No. = replicate number

  negative value = 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   [%]
14.0	1	3.1		0.148	60		2.0	85
	2	3.2		0.153	59		2.1	85
	3	3.3		0.157	58		2.2	84
	Mean	3.2	(+)	0.153	59	(+)	2.1	85
2.93	1	6.1		0.245	34		5.0	64
	2	6.3		0.249	33		5.2	62
	3	7.0		0.264	29		5.9	57
	Mean	6.5	(+)	0.253	32	(+)	5.4	61
0.490	1	8.5		0.292	21		7.4	46
	2	8.8		0.297	20		7.7	44
	3	8.0		0.283	24		6.9	50
	Mean	8.4	(+)	0.291	22	(+)	7.3	46
0.0476	1	11.2		0.332	11		10.1	26
	2	13.1		0.354	5		12.0	12
	3	13.2		0.355	4		12.1	12
	Mean	12.5	(+)	0.347	7	(+)	11.4	17
0.0149	1	16.2		0.384	-4		15.1	-10
	2	17.2		0.393	-6		16.1	-18
	3	15.4		0.377	-2		14.3	-4
	Mean	16.3	(-)	0.385	-4	(-)	15.2	-11
0.00759	1	16.6		0.388	-5		15.5	-13
	2	18.6		0.404	-9		17.5	-28
	3	18.1		0.400	-8		17.0	-24
	Mean	17.8	(+)*	0.397	-7	(+)*	16.7	-22
Control	1	16.9		0.390			15.8
	2	14.0		0.363			12.9
	3	14.7		0.370			13.6
	4	14.8		0.371			13.7
	5	14.3		0.366			13.2
	6	14.3		0.366			13.2
	Mean	14.8		0.371			13.7

The mean initial biomass dry weight was 1.1 mg per replicate.

Repl. No. = replicate number

negative value = growth stimulation

* = significant difference based on 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
14.0	1	4	15
	2	4	18
	3	4	18
	Mean	4	17
2.93	1	4	7
	2	4	5
	3	4	6
	Mean	4	6
0.490	1	4	4
	2	4	4
	3	4	4
	Mean	4	4
0.0476	1	4	9
	2	4	9
	3	4	8
	Mean	4	9
0.0149	1	4	16
	2	4	13
	3	4	15
	Mean	4	15
0.00759	1	4	12
	2	4	15
	3	4	16
	Mean	4	14
Control	1	4	12
	2	4	10
	3	4	12
	4	4	14
	5	4	14
	6	4	11
	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
14.0	3.2 + 3.3 +	2.4 ++ 3.2 ++ 3.3 ++ 4.1 ++	2.4 ++ 3.2 ++ 3.3 ++ 4.1 ++
2.93	3.3 +	2.3 + 3.3 +	2.3 ++ 3.1 ++ 3.2 + 3.3 +
0.490	1	1	2.3 ++
0.0476	1	1	2.3 +
0.0149	1	1	1
0.00759	1	1	1
Control	1	1	1

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

 

1      = no observedeffects

2.3   = gibbosity of the fronds

2.4   = discoloration of the fronds

3.1  =roots shortened

3.2  = losses of roots

3.3   = discoloration of roots

4.1   = Break up of plants

+      = slight effects

++    = medium effects

+++         = strong effects

Validity criteria fulfilled:

yes

Conclusions:

In this study, Basic Blue 159 Trichlorozinkate 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 inhibition of the specific growth rate of fronds (ErC50) and dry weight (ErdwC50) were 3.52 (1.87 – 5.56) and 9.01 (7.68 – 10.7) mg/L, respectively. The EC50-values for inhibition of yield (fronds, EyC50) and dry weight inhibition of yield (EydwC50) with 95% confidence intervals were 0.169 (0.0956 – 0.370) mg/L and 0.838 (0.494 – 1.50) mg/L, respectively.

Executive summary:

The effects of the test item Basic Blue159 Trichlorozinkate on the growth of the monocotyledonous aquatic plant species Lemna gibba was determined according to the principles of OECD 221 and Council Regulation No. 761/2009 Method C.26 at the test facility from 2018-01-31 to 2018-02-08, with the definitive exposure phase from 2018-01-31 to 2018-02-07.

Lemna gibba was exposed to the test item for 7 days under static conditions. Based on a preliminary test, 6 nominal test item concentration levels were tested in a geometrical series with a dilution factor of 4:0.0156 - 0.0625 - 0.250 - 1.00 - 4.00 - 16.0 mg/L, corresponding to the geometric mean measured test item concentrations: 0.00759 – 0.0149 – 0.0476 – 0.490 - 2.93 – 14.0 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 test solutions were clear and concentration related blue coloured throughout exposure.The validity criteria of the test guideline were fulfilled.

The concentrations ofthe test item Basic Blue159 Trichlorozinkate and the controlwere analysed via HPLC-DAD at the beginning and end of the exposure.

The measured concentrations at the start of the exposure of Basic Blue159 Trichlorozinkate were in the range of 92 and 99% of the nominal values.At the end of the exposure the measured concentrations were between < LOQ and 83% of the nominal concentrations. All effect values are given based on the geometric mean measured test item concentrations.

 

NOEC-, LOEC-, EC-Values and 95 % Confidence Intervals of Basic Blue159 Trichlorozinkate 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	0.0149	NOEC	0.0149
LOEC	0.0476	LOEC	0.0476
ErC10	0.0283 (0.0194 – 0.0504)	ErdwC10	0.0852 (0.0496 – 0.149)
ErC20	0.0718 (0.0380 – 0.161)	ErdwC20	0.555 (0.353 - 0845)
ErC50	3.52 (1.87 – 5.56)	ErdwC50	9.01 (7.68 – 10.7)
Inhibition of Yield [mg/L]
NOEC	0.0149	NOEC	0.0149
LOEC	0.0476	LOEC	0.0476
EyC10	0.0256 (0.0180 – 0.0381)	EydwC10	0.0342 (0.0260 – 0.0480)
EyC20	0.0379 (0.0254 – 0.0579)	EydwC20	0.0627 (0.0437 – 0.0941)
EyC50	0.169 (0.0956 – 0.370)	EydwC50	0.838 (0.494 – 1.50)

Description of key information

The source substance Basic Blue 159 trichloro zincate 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 was 3.52 mg/L. The 7-d ErC10 was 0.0283 mg/L. The target substance is considered acutely and chronically very toxic to aquatic plants.

Key value for chemical safety assessment

EC50 for freshwater plants:

3.52 mg/L

EC10 or NOEC for freshwater plants:

0.028 mg/L