1-[2-[[4-[(2-chloro-4-nitrophenyl)azo]phenyl]ethylamino]ethyl]pyridinium acetate

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From April 09 to 12, 2017. Definitive exposure phase from April 10 to 12, 2017.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted on a structural analogue. the original reliability of the study was 1. The complete RA justification is attached at section 13

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
All concentration levels and the control were analytically verified via LC-MS/MS 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 monitoring
At the start of the exposure (0 hours), samples were taken from all 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 active substances should be within ± 20% of the nominal or initially measured concentrations.

Vehicle:

no

Details on test solutions:

Preparation of the stock solution
A stock solution (1.00 mg/L of the test item was weighed out) was prepared with dilution water on day prior to the start of the exposure period (at -24 hours). The test item was mixed with the dilution water by constant stirring with a magnetic stirrer at approximately 1100 rpm for 24 hours at room temperature. After completion of stirring, the stock solution was checked via laser beam (Tyndall effect) for undissolved test item. No Tyndall effect was present in the stock solution. Therefore, the solution was used as the highest concentration level and for the preparation of further concentration levels by dilution with dilution water to get the definitive test concentrations.

Test concentrations
5 test item concentrations in a geometric series with a separation factor of 2, prepared by dilution of the stock solution 1.00 mg/L with dilution water, were tested as follows:
0.0625 - 0.125 - 0.250 - 0.500 - 1.00 mg/L
The test 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 für Wasser-, Boden- und Lufthygiene (WaBoLu),
14195 Berlin, 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), modified to a total hardness of 160 to 180 mg CaCO3/L, is used.

Culture feeding
The culture 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:

no

Total exposure duration:

48 h

Hardness:

see any other information on material and methods incl. tables

Test temperature:

see any other information on material and methods incl. tables

pH:

see any other information on material and methods incl. tables

Dissolved oxygen:

see any other information on material and methods incl. tables

Conductivity:

see any other information on material and methods incl. tables

Details on test conditions:

Test method
The study was performed under static conditions.

Test duration
48 hours

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

Test volume
20 mL in each test replicate (see ‘Application’)

Dilution water
Same as culture medium

Number of daphnids and replicates
20 daphnids, divided into 4 replicates, each with 5 daphnids, were used for each loading level and the control.

Age of the daphnids
Less than 24 hours old daphnids from a healthy stock were used for at the start of the exposure 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 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

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

0.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: CI: 0.250 - 1.00

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.266 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: CI: 0.227 - 0.319

Duration:

24 h

Dose descriptor:

EC10

Effect conc.:

0.287 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: CI: 0.250 - 1.00

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

0.145 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: CI: <0.0625 - 0.195

Duration:

24 h

Dose descriptor:

EC100

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Results with reference substance (positive control):

The percentage of immobility for the reference item potassium dichromate (SIGMA-ALDRICH, batch number MKBV0900V, purity 99.0%, CAS RN 7778-50-9) was determined after 24 hours from 2017 04-06 to 2017-04-07.

EC50-Value (with 95% confidents limits) of the Reference Item Potassium dichromate
based on nominal concentrations mg/L, (0 - 24 hours)
Current Study Valid Range
EC50 1.72 mg/L
0.6 - 2.4 mg/L, acc. to AQS P 9/2 (02/2000); clone 5
0.6 - 2.1 mg/L, acc. to OECD 202 (2004); clone A
95% confidence limits 1.34 - 1.93 mg/L

Reported statistics and error estimates:

Methods of evaluation The EC100-values (after 24 and 48 hours) were empirically derived from the observed immobilization rates.
The effect concentrations (EC10 / 50 / 100) were based on the nominal concentrations of the test item, since the test item concentrations were within ± 20% of the nominal concentrations.

EC-values and statistical analyses
The EC10 / 50-values after 24 hours were calculated by linear dose-response regression and the EC10 / 50-values after 48 hours were calculated by sigmoidal dose-response regression. Since only one partial effect was observed after 24 hours, the highest concentration level resulting in 0% immobilization and the lowest concentration level causing 100% immobilization were used as 95% confidence limits for the EC10 / 50-values after 24 hours. The respective 95% confidence limits for the EC10 / 50-values after 48 hours 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 are shown graphically.
The EC50-value with 95% confidence limits for the reference item was calculated accordingly, by sigmoidal dose-response regression.

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

Immobilization Ratesafter 24 and 48 hours ofExposure in the Definitive Test

             (n = 20, divided into 4 replicates with 5 daphnids each)

Nominal test item concentration [mg/L]	IMMOBILIZATION [%]
	24 hours					48 hours
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
1.00	100	100	100	100	100	100% mortality after 24 hours
0.500	20	60	60	60	50	60	100	100	100	90
0.250	0	0	0	0	0	40	60	60	20	45
0.125	0	0	0	0	0	0	0	20	0	5
0.0625	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

 

Absolute Numbers of immobileDaphnids after 24 and 48 hours ofExposure in the Definitive Test

             (n = 20, divided into 4 replicates with 5 daphnids each)

Nominal test item concentration [mg/L]	Number of immobile Daphnids / Total number of Daphnids
	24 h					48 h
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
1.00	5 / 5	5 / 5	5 / 5	5 / 5	20 / 20	5 / 5	5 / 5	5 / 5	5 / 5	20 / 20
0.500	1 / 5	3 / 5	3 / 5	3 / 5	10 / 20	3 / 5	5 / 5	5 / 5	5 / 5	18 / 20
0.250	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	2 / 5	3 / 5	3 / 5	1 / 5	9 / 20
0.125	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	1 / 5	0 / 5	1 / 20
0.0625	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20
Control	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20

 

 

Additional Observations during the Definitive Test

 

All test item concentrations were visually clear throughout the exposure period. The concentration levels 0.250 to 1.00 mg/L showed a concentration-related strong to light orange colour. The concentration level of 0.125 mg/L was slightly orange in fresh medium and colorless in old medium. The orange colour was clearer in fresh media (0 hours) than in old media (48 hours) at the respective concentration levels. The concentration level 0.0625 mg/L was colourless throughout the exposure period. Since colour of the test solutions is observed visually without measurement of wavelength, differences between Range Finder and Main Study (red – orange) are considered to be subjective and not test item related.

  Water Quality Parameters

 

The measured water quality parameters(i.e. pH-value, dissolved oxygen concentration, total water hardness and water temperature) were within the acceptable limits.

During the test period, the temperature in the incubator was 19 - 20 °C.

 

 

Measured Exposure Concentrations during the Definitive Test

 

The concentrationsof the test item were analytically verified via LC-MS/MS 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 105 to 111% of the nominal values in all test item concentrations. In old media at the end of the exposure period (48 hours), the measured test itemconcentrations were in the range of 97 to 104% of the nominal values of the test concentrations.The measured concentrations of the test itemBasic Red 18.1 Chloride remained stable within ± 20% of the nominal concentrations throughout the exposure period, which indicates that the test item concentrations were successfully maintained for the duration of the test.

 

Measured Concentrations of the Test ItemBasic Red 18.1 Chlorideduring the Definitive Test

Sampling date	2017-04-10 Start of the exposure, 0 hours		2017-04-12 End of the exposure, 48 hours
Start of analysis	2017-04-10		2017-04-12
Nominal test item concentration [mg/L]	Basic Red 18.1 Chloride
	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%
1.00	1.11	111	1.04	104
0.500	0.527	105	0.503	101
0.250	0.271^1)	108	0.249	99
0.125	0.139^1)	111	0.126	101
0.0625	0.0688^1)	110	0.0604	97
Control	< LOQ		< LOQ

Meas. conc.    = measured concentration of the test item, mean value of two injections, dilution factors taken into account

%                     = percent of the nominal concentration of the test item

LOQ                = limit of quantification of the analytical method (0.005 mg/L of thetest item)

¹⁾                      = reanalyzed on 2017-04-12, mean value of 2 replicates with 2 injections each

Validity criteria fulfilled:

yes

Conclusions:

48 hour EC50 was 0.266 mg/L (95% confidence limits: 0.227 – 0.319 mg/L).

Executive summary:

Method

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item were determined at the test facility according to OECD 202 (2004) from 2017-04-09 to 2014-04-12, with the definitive exposure phase from 2017-04-10 to 2017-04-12.

The study was conducted under static conditions over a period of 48 hours with five nominal concentrations of the test item ranging from 0.0625 to 1.00 mg/l in a geometric series with a separation factor of 2.

The test item is a red powder, which is dispersible with water. All test item concentrations were visually clear throughout the exposure period. The concentration levels 0.250 to 1.00 mg/l showed a concentration-related strong to light orange colour. The concentration level of 0.125 mg/l was slightly orange in fresh medium and colorless in old medium. The orange colour was clearer in fresh media (0 hours) than in old media (48 hours) at the respective concentration levels. The concentration level 0.0625 mg/L was colourless throughout the exposure period.Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each concentration leveland the control.

The concentrations of the test item were analytically verified via LC-MS/MS 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.

Results

The measured concentrations of the test item in fresh media at the start of the exposure (0 hours) were in the range of 105 to 111 % of the nominal values in all test item concentrations. In old media at the end of the exposure period (48 hours), the measured test item concentrations were in the range of 97 to 104 % of the nominal values of the test concentrations. The measured concentrations of the test item remained stable within ± 20 % of the nominal concentrations throughout the exposure period and no hint for possible degradation products was observed in the respective chromatograms, which indicates that the test item concentrations were successfully maintained for the duration of the test.

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

The validity criteria of the test guideline were fulfilled.

 

EC₁₀-, EC₅₀- (with 95 % Confidence Limits) and EC₁₀₀-Values

(based on the nominal concentrations of the test item)

Basic Red 18.1 Chloride
Effect concentrations	Test duration [hours]	Toxicity endpoint based on the nominal concentrations of the test item [mg/L]
EC10 (with 95% confidence limits)	24	0.287	(Cl: 0.250 – 1.00)
	48	0.145	(Cl:≤0.0625 – 0.195)
EC50 (with 95% confidence limits)	24	0.500	(Cl: 0.250 – 1.00)
	48	0.266	(Cl: 0.227 – 0.319)
EC100	24	1.00
	48	1.00

Description of key information

EC50 (48h)= 0.266 mg/l

Additional information

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item were determined at the test facility according to OECD 202 (2004) from 2017-04-09 to 2014-04-12, with the definitive exposure phase from 2017-04-10 to 2017-04-12.

The study was conducted under static conditions over a period of 48 hours with five nominal concentrations of the test item ranging from 0.0625 to 1.00 mg/l in a geometric series with a separation factor of 2.

The test itemis a red powder, which is dispersible with water.All test item concentrations were visually clear throughout the exposure period. The concentration levels 0.250 to 1.00 mg/l showed a concentration-related strong to light orange colour. The concentration level of 0.125 mg/l was slightly orange in fresh medium and colorless in old medium. The orange colour was clearer in fresh media (0 hours) than in old media (48 hours) at the respective concentration levels. The concentration level 0.0625 mg/L was colourless throughout the exposure period.Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each concentration leveland the control.

The concentrations of the test itemwereanalytically verified via LC-MS/MS 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 105 to 111 % of the nominal values in all test item concentrations. In old media at the end of the exposure period (48 hours), the measured test itemconcentrations were in the range of 97 to 104 % of the nominal values of the test concentrations.The measured concentrations of the test item remained stable within ± 20 % of the nominal concentrations throughout the exposure period and no hint for possible degradation products was observed in the respective chromatograms, which indicates that the test item concentrations were successfully maintained for the duration of the test.

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

The validity criteria of the test guideline were fulfilled.

 

EC₁₀-, EC₅₀- (with 95 % Confidence Limits) and EC₁₀₀-Values

(based on the nominal concentrations of the test item)

BasicRed 18.1 Chloride
Effect concentrations	Test duration [hours]	Toxicity endpoint based on the nominal concentrations of the test item [mg/L]
EC10 (with 95% confidence limits)	24	0.287	(Cl: 0.250 – 1.00)
	48	0.145	(Cl:≤0.0625 – 0.195)
EC50 (with 95% confidence limits)	24	0.500	(Cl: 0.250 – 1.00)
	48	0.266	(Cl: 0.227 – 0.319)
EC100	24	1.00
	48	1.00