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

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-03-06 to 2018-03-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
All concentration levels and the control were analytically verified via HPLC-DAD in fresh media at the start of the exposure (0 hours) and in old media at the end of the exposure period (48 hours) as specified below. The analytical method was validated prior to this study according to SANCO 3029/99 rev.4 (2000).

Sampling for the analytical
At the start of the exposure (0 hours), samples were taken from all monitoring freshly prepared concentration levels and the control for analysis.
At the end of the exposure (48 hours), samples for the analyses of the old media were taken directly from the test vessels.

Criteria for the analytical monitoring (target)
Recoveries of the test item should be within ± 20% of the nominal or initially measured concentrations.

Vehicle:

no

Details on test solutions:

Stock solution
A stock solution (100 mg/L of the test item was weighed out) was freshly prepared with dilution water (see Table 2) prior to the start of the exposure (at 0 hours). The stock solution was mixed thoroughly by manual agitation.

Test concentrations
5 test item concentrations in a geometric series with a separation factor of √10, prepared by dilution of the stock solution 100 mg/L with dilution water were tested as follows: 0.0100 - 0.0316 - 0.100 - 0.316 - 1.00 mg/L
The test item concentrations were selected based on the results of a non-GLP preliminary range finding test.

Control
Dilution water without test item incubated under the same conditions as the test groups

Test organisms (species):

Daphnia magna

Details on test organisms:

Test system
Daphnia magna STRAUS (Clone 5).

Reason for the selection of the test system
Daphnia magna is the preferred species in accordance with the test guideline and is bred at the test facility.

Origin
Institut Dr. Nowak GmbH & Co. KG, Mayenbrook 1, 28870 Ottersberg, Germany

Breeder
Noack Laboratorien GmbH, Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany

Culture
In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20  2 °C, in an incubator, 16 hours illumination, light intensity of max. 1500 lx

Culture medium
Elendt M4, according to OECD 202, Annex 3 (2004) is used.

Composition of the Culture Medium Elendt M4 according to OECD 202, Annex 3 (2004)
Component Concentration [mg/L]
CaCl2 x 2 H2O 294
MgSO4 x 7 H2O 123
KCl 5.80
NaHCO3 64.8
Na2SiO3 x 5 H2O 7.47
NaNO3 0.274
KH2PO4 0.143
K2HPO4 0.184
Na2EDTA x 2 H2O 2.50
FeSO4 x 7 H2O 0.996
H3BO3 2.86
MnCl2 x 4 H2O 0.361
LiCl 0.306
SrCl2 x 6 H2O 0.152
RbCl 0.0710
NaBr 0.0160
Na2MoO4 x 2 H2O 0.0615
CuCl x 2 H2O 0.0168
ZnCl2 0.0130
CoCl2 x 6 H2O 0.0100
KI 0.00325
Na2SeO3 0.00219
NH4VO3 0.000575
Thiaminhydrochloride 0.075
Cyanocobalamin 0.0010
Biotin 0.00075
pH 8.2  0.8

Feeding of the culture stocks
The daphnids were fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus, with an algae cell density of > 106 cells/mL. The algae were cultured at the test facility.

Origin of the food algae
Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, 37073 Göttingen, Germany

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

Dilution water
0 hours:Total hardness [mg CaCO3/L]: 217

Test temperature:

18 - 22°C, constant within ± 1°C
19.5 - 20.5 °C

pH:

Water Quality Parameters in fresh Media at the Start of the Exposure (0 hours)
(measured in one additional replicate (without daphnids) per concentration level and control)
Nominal
test item concentration
[mg/L] pH-value Dissolved
O2 concentration [mg/L]
1.00 7.46 9.57
0.316 7.45 9.57
0.100 7.49 9.59
0.0316 7.41 9.58
0.0100 7.47 9.40
Control 7.51 9.41

Water Quality Parameters in old Media at the End of the Exposure (48 hours)
(measured in the replicate with the highest immobilization rate, with the exception of pH in the lowest concentration (containing daphnids) per concentration and control)
Nominal
test item concentration
[mg/L] 48 hours
pH-value Dissolved
O2 concentration
[mg/L] Replicate number
1.00 7.41 7.45 2
0.316 7.45 7.63 2
0.100 7.33 7.54 2
0.0316 7.31 8.00 2
0.0100 7.27 7.62 2pH + 4O2
Control 7.46 7.29 2

Water Quality Parameters of the Dilution Water at the Start of the Exposure (0 hours)
Dilution water dated: pH-Value
Dissolved
O2 concentration
[mg/L] Temperature

[°C] Conductivity

[µS/cm] Total hardness

[mg CaCO3/L]
2018-03-06 7.53 9.63 20.1 617 217

Dissolved oxygen:

see above

Conductivity:

Dilution water day 0: 617 [µS/cm]

Details on test conditions:

Test vessels
Glass beakers (4 (ID) x 7 (H) cm), 50 mL capacity, loosely covered with watch glasses

Test volume
20 mL

Dilution water
Same composition as the culture medium (see Table 2)
Number of daphnids and replicates
20 daphnids, divided into 4 replicates, each with 5 daphnids, were used per concentration level and control.

Age of the daphnids at the start of the exposure
Less than 24 hours old daphnids from a healthy stock were used for the study. Juvenile daphnids were removed from the culture vessels at the latest 24 hours before the start of the exposure and discarded. The juveniles born within the following period of max. 24 hours preceding the exposure were used for the test. No first brood progeny was used for the test.

Acclimatization
Acclimatization of the daphnids was not necessary, because the composition of the dilution water was equivalent to the culture medium.

Application
20 g test solution per replicate was weighed out into each test vessel. This corresponds to 20 mL per test vessel. The daphnids were inserted with a small amount of dilution water by a pipette.

Test temperature (target)
18 - 22 °C, constant within ± 1 °C
 
Illumination (target)
Diffuse light, light intensity of max. 1500 lx

Photoperiod (target)
16/8 hours light/dark cycle

Feeding
The daphnids were not fed during the study.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.096 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Results with reference substance (positive control):

A reference test was conducted as an acute immobilization test (acc. to AQS P 9/2 and OECD 202) in Elendt M4 medium (acc. to OECD 202 (2004), Annex 3) under static conditions with a test duration of 24 hours once per month in order to prove the validity of the test system and test conditions at the test facility.
Reference item Potassium dichromate p.a. (SIGMA)
Purity 99.0%
Batch No. MKBV0900V
Storage stability 2021-11-25
Test concentrations 1.00 – 2.00 – 4.00 mg/L
Ranges of validity EC50 (24 hours): 0.6 - 2.4 mg/L, according to AQS P 9/2 (clone 5)
EC50 (24 hours): 0.6 - 2.1 mg/L, according to OECD 202 (clone A)
Test duration 2018-03-13 to 2018-03-14

EC50-Value (with 95% confidence limits) of the Reference Item Potassium dichromate
based on nominal concentrations mg/L, (0 - 24 hours)
Current Study Valid Range
EC50 1.66 mg/L
0.6 - 2.4 mg/L, acc. to AQS P 9/2 (02/2000)
0.6 - 2.1 mg/L, acc. to OECD 202 (2004)

Reported statistics and error estimates:

Methods of evaluation
The EC0/100-values (after 24 and 48 hours) were empirically derived from the observation data.
All effect levels (EC10 / 50 / 100) given are based on the nominal test item concentrations. The concentration-effect relationships of the test item after 24 and 48 hours is shown graphically.

EC-values and statistical analyses
The EC10- and the EC50-values (after 24 and 48 hours) were calculated
by sigmoidal dose-response regression with the software GraphPad Prism. The respective 95% confidence limits were calculated from the standard error and the t-distribution. All calculations were carried out from the best-fit values with the software GraphPad Prism5. The concentration-effect relationships after 24 and 48 hours are shown graphically.
The EC50-value for the reference item and its 95% confidence limits were calculated accordingly.

Software
All data were computer-processed and rounded for presentation. Consequently, minor variations may occur from the original figures if manual calculations based on the original figures are made subsequently. Calculations were made using the following software:
- GraphPad Prism5, GRAPHPAD SOFTWARE, INC.
- Excel, MICROSOFT CORPORATION

Method Validation

Requirements of the method validation 

According to SANCO 3029/99 rev.4 (2000) using the following criteria.

 

Parameter, Acceptance Criteria and Results of the Method Validation

Parameter	Acceptance criteria	Result
Linearity	5 standard concentrations, r^2≥ 0.992	5.00 to 50.0 µg test item/L (n = 6), r^2≥ 0.992	x
Lowest calibration standard	S/N≥9	5.00 µg test item/L, S/N =14	x
Limit of Detection (LOD)	S/N of≥3	1.5 µg test item/L, S/N =5	x
Limit of Quantification (LOQ)	At least 20% above lowest calibration level after sample preparation	7.50 µg test item/L (1 x LOQ) 15.0 mg test item/L (2000 x LOQ)	x
Accuracy^1) (Fortified samples)	Mean recovery rate of 70-110% (ideally 80-100%) per fortification level (2 levels)	Lemnadilution water: 1 x LOQ: 93% (n = 5) 2000 x LOQ: 102% (n = 5) Daphnia dilution water^*): 1 x LOQ: 108% (n = 3) 2000 x LOQ: 102% (n = 3)	x
Precision^1)	Relative standard deviation≤20% per fortification level	Lemnadilution water: 1 x LOQ: 3.5% 2000 x LOQ: 1.3% Daphnia dilution water^*): 1 x LOQ: 0.71% 2000 x LOQ: 1.0%	x
Specificity (HPLC-DAD)	Comparison of spectra of sample peaks against spectra obtained from standard peaks	Spectra compared and test item verified	x
	Blank values < 30% of LOQ	Blank values < 30% of LOQ

 

x               = Criterion fulfilled

1)               = For details, see below

*)               = The most complex medium of all studies of aquatic toxicology was the medium for the lemna study (acc. to
   OECD 221), and was therefore selected exemplarily for the method validation of all study types.
   This method validation was checked by analysis of replicates prepared in thedaphniadilution water as
   described above.

 

Preparation of Fortified Samples

LOQ Level		Control		1		2000
Stock solution [mg test item/L]		-		1000
Medium		-		Water
Spiking solution		-		0.5 mg/L (Lemnadilution water or Daphniadilution water)		Stock solution
Replicates		1: 2 2: 1		1: 5 2: 3		1: 5 2: 3
Concentration of the LOQ [mg test item/L]		-		0.0075		15.0
Medium for preparation		Lemnadilution water containing 0.1% H3PO4or Daphniadilution water containing 0.1% H3PO4
Volume of spiking solution [mL]		-		0.06		0.06
Volume of medium [mL]		-		3.94		3.94
Dilution factor		-		-		500
Dilution medium		-		-		Dilution medium
Sample volume [mL]		Approx. 1		Approx. 1		0.05^1) 0.2^2)
Finale volume [mL]		Approx. 1		Approx. 1		5.0^1) 1.0^2)

¹⁾               first dilution step

²⁾                       second dilution step

1               in Lemna dilution medium

2               in Daphnia dilution medium

 

 

Measured Concentrations and Percent of Nominal Concentrations of the Fortified Samples of BasicBlue 159 Trichlorozinkate

               Fortified concentrations*: 0.00756 mg/L (1 x LOQ) and 15.1 mg/L (2000 x LOQ) of the test item

Replicate	BasicBlue159Trichlorozinkate
	Lemnadilution water				Daphniadilution water
	1 x LOQ		2000 x LOQ		1 x LOQ		2000 x LOQ
	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%
1	0.00736	97	15.6	103	0.00816	108	15.5	102
2	0.00685	91	15.2	100	0.00816	108	15.3	101
3	0.00675	89	15.3	101	0.00827	109	15.6	103
4	0.00716	95	15.5	103
5	0.00696	92	15.2	101
Mean	0.0070	93	15.4	102	0.00820	108	15.5	102
SD ±	0.0002		0.2		0.00006		0.2
CV [%]	3.5		1.3		0.71		1.0

Meas. conc.    = measured concentration of the test item, dilution factor taken into account

%                     = percent concentration of the fortified sample

*                       = weighing factor taken into account

 

The stability of prepared samples of the method validation for three replicates of the 1 x LOQ in Daphnia dilution water was shown for 7 days.

 

Validity criteria fulfilled:

yes

Conclusions:

Based on the nominal concentrations of the test item Basic Blue 159 Trichlorozinkate, the 48 h-EC50 for Daphnia magna was 0.0955 (0.0332 – 0.277) mg test item/L.

Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item Basic Blue 159 Trichlorozinkate were determined at the test facility according to OECD 202 (2004) from2018-03-06 to 2018-03-08.

The study was conducted under static conditions over a period of 48 hours with five concentration levels prepared by dilution from the stock solution of the test item (nominal concentration 100 mg/L). The tested concentration levels were0.0100 - 0.0316 - 0.100 - 0.3160 - 1.00 mg/L(separation factor√10).

The test item is a dark blue solid with a water solubility of 25.2 g/L (20ºC, OECD 105). The test item concentrations showed a concentration related blue colour and were visually clearly dissolved throughout the exposure period.

Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each concentration leveland the control.

The concentrationsof the test item were analytically verified via HPLC-DAD in fresh media at the start of the exposure (0 hours) and in old media at the end of the exposure period (48 hours) in all tested concentration levels and in the control.

The measured concentrations of the test item in fresh media at the start of the exposure (0 hours) were in the range of 85 to 93 % of the nominal values. At the end of the exposure period (48 hours), the measured test item concentrations in the old media were in the range of 84 to 89 % of the nominal values. Since the measured concentrations of the test item Basic Blue 159 Trichlorozinkate remainedstable within ± 20% of the nominal concentrations throughout the exposure period, the nominal concentrations were used for evaluation.

All effect concentrations (EC_{10 / 50 / 100}) are based on the nominal concentrations of the test item Basic Blue 159 Trichlorozinkate.

The validity criteria of the test guideline were fulfilled.

 

EC₁₀-, EC₅₀-and EC₁₀₀-Valuesand 95 % Confidence Limits (based on the nominal concentrations of the test item)

Basic Blue 159 Trichlorozinkate
Effect concentrations	Test duration [hours]	Nominal test item concentrations [mg test item/L]
EC10	24	0.0853 (<0.0100 – 0.267)
	48	0.0840 (<0.0100 – 0.268)
EC50	24	0.0957 (0.0334 – 0.276)
	48	0.0955 (0.0332 – 0.277)
EC100	24	0.316
	48	0.316

 

Description of key information

Based on the nominal concentrations of the structural analogue substance Basic Blue 159 trichlorozincate, the 48 h-EC50 for Daphnia magna was 0.0955 (0.0332 – 0.277) mg test item/L. Based on the analogue approach, Basic Blue 159 methyl sulfate is also considered acutely very toxic to aquatic invertebrates.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.096 mg/L