Hydrogen hydroxy[2-hydroxy-3-[(2-hydroxy-3-nitrobenzylidene)amino]-5-nitrobenzenesulphonato(3-)]chromate(1-), compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1)

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Hydrogen hydroxy[2-hydroxy-3-[(2-hydroxy-3-nitrobenzylidene)amino]-5-nitrobenzenesulphonato(3-)]chromate(1-), compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1)

EC Number:

287-267-9

EC Name:

Hydrogen hydroxy[2-hydroxy-3-[(2-hydroxy-3-nitrobenzylidene)amino]-5-nitrobenzenesulphonato(3-)]chromate(1-), compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1)

Cas Number:

85455-32-9

Molecular formula:

C13H7CrN3O10S.C11H25NO.H

IUPAC Name:

Hydrogen hydroxy[2-hydroxy-3-[(2-hydroxy-3-nitrobenzy-lidene)amino]-5-nitrobenzenesulphonato(3-)]chromate(1-), compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1)

Test material form:

solid: particulate/powder

Sampling and analysis

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 (48 hours). The 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 the 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 from additional replicates, which were prepared with test media, but without daphnids. These additional replicates were incubated under test conditions until sampling.

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

Test solutions

Vehicle:

no

Details on test solutions:

Preparation of the saturated solution
A saturated solution with a nominal concentration of 100 mg/L of the test item was prepared with dilution water two days prior to the start of the exposure (at -48 hours) in a glass flask. The test item was mixed with the dilution water by constant stirring with a magnetic stirrer at approximately 1100 rpm for 48 hours at room temperature. After completion of stirring, 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 for 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 test item solution, was used as the highest concentration level and as a stock solution for the preparation of further dilution levels by dilution with dilution water. During filtration, the filter was always kept covered.

Test concentrations
The undiluted saturated solution and five dilution levels prepared out of the saturated solution in a geometric series with a separation factor of 2.2 by dilution of the saturated solution with dilution water, were tested as follows: 1.94 - 4.27 - 9.39 - 20.7 - 45.5 - 100% of the saturated solution, which corresponds to geometric mean measured test item concentrations of: 1.28 – 2.85 – 6.20 – 13.7 – 29.5 – 64.9 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

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

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

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

Nominal and measured concentrations:

Nominal: 1.94 - 4.27 - 9.39 - 20.7 - 45.5 - 100% of the saturated solution
geometric mean measured test item concentrations of: 1.28 – 2.85 – 6.20 – 13.7 – 29.5 – 64.9 mg/L

Details on test conditions:

Test method
Based on the results of a preliminary range finding test, the study was performed under static conditions, since the measured concentrations remained stable over 48 hours under test 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

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EC10

Effect conc.:

4.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: CI: < 2:85 - 6:14

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

4.45 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: CI: 3:48 - 5.32

Effect concentrations 3

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

7.96 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: CI: 6.78 - 10.8

Effect concentrations 4

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

7.72 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: CI: 6:95 - 8.88

Effect concentrations 5

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

> 64.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 6

Duration:

24 h

Dose descriptor:

EC100

Effect conc.:

29.5 mg/L

Nominal / measured:

meas. (geom. mean)

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 observation data. The effect levels (EC10 / 50 / 100) given are based on the geometric mean measured concentrations of the test item.

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

Any other information on results incl. tables

Immobilization Ratesafter 24 and 48 hours of Exposure in the Definitive Test

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

Geometric mean measured test item concentration [mg/L]	IMMOBILIZATION [%]
	24 hours					48 hours
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
64.9	100	100	80	100	95	100	100	100	100	100
29.5	100	100	60	80	85	100	100	100	100	100
13.7	100	100	60	80	85	100	100	80	80	90
6.20	0	40	40	20	25	0	40	40	40	30
2.85	0	0	0	0	0	0	0	0	0	0
1.28	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 of Exposure in the Definitive Test

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

Geometric mean measured 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
64.9	5 / 5	5 / 5	4 / 5	5 / 5	19 / 20	5 / 5	5 / 5	5 / 5	5 / 5	20 / 20
29.5	5 / 5	5 / 5	3 / 5	4 / 5	17 / 20	5 / 5	5 / 5	5 / 5	5 / 5	20 / 20
13.7	5 / 5	5 / 5	3 / 5	4 / 5	17 / 20	5 / 5	5 / 5	4 / 5	4 / 5	18 / 20
6.20	0 / 5	2 / 5	2 / 5	1 / 5	5 / 20	0 / 5	2 / 5	2 / 5	2 / 5	6 / 20
2.85	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20
1.28	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 showed a concentration-related yellow color, from yellow (saturated solution) to light yellow (lowest concentration level), and were visually clear throughout the exposure period.

No immobility or any other sub-lethal effects were observed at the two lowest concentration levels 1.28 and 2.85 mg/L and in the control.

 

 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 concentrations of the test item Savinyl-Gelb 2GLS 01 trocken were analytically verified via HPLC-DAD in fresh media at the start (0 hours) and in old media at the end of the exposure (48 hours) in all concentration levels and in the control.

The measured concentrations of the test item at 48 hours were in the range of 99 to 103% of the initially measured concentrations in all concentration levels. The analytical results reflected the dilution factor of 2.2 between the concentration levels. The geometric mean measured concentrations were: 1.28 – 2.85 – 6.20 – 13.7 – 29.5 – 64.9 mg/L.

The measured test item concentrations remained stable within ± 20% of the initially measured concentrations over the test period. This indicates that the test item concentrations were successfully maintained for the duration of the test.

 

  Measured Concentrations of the Test Itemduring the Definitive Test

Sampling date	2017-04-26 Start of the exposure, 0 hours	2017-04-28 End of the exposure, 48 hours
Start of analysis	2017-04-26	2017-04-28
Dilution level of the saturated solution [%]	Savinyl-Gelb2GLS 01 trocken			Geometric mean measured test item concentration [mg/L]
	Meas. conc. [mg/L]	Meas. conc. [mg/L]	%
100*	65.4	64.5	99	64.9
45.5	29.6	29.3	99	29.5
20.7	13.8	13.7	99	13.7
9.39	6.21	6.19	100	6.20
4.27	2.84	2.86	101	2.85
1.94	1.26	1.30	103	1.28
Control	< LOQ	< LOQ

Meas. conc.= measured concentration of the test item, mean value of 2 injections, dilution factors taken into account

%                  = percent of the initially measured concentration of the test item

LOQ             = limit of quantification of the analytical method (0.2 mg/L of the test item)

*                    = saturated solution

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on the geometric mean measured concentrations of the test item , the 48-hour EC50 for Daphnia magna was 7.72 mg/L (95% confidence limits: 6.95 - 8.88 mg/L).

Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item were determined according to OECD 202 (2004) ..

The study was conducted under static conditions over a period of 48 hours with a saturated solution (prepared with a nominal concentration of100 mg/Lof the test item and further four dilution levels prepared in a geometric series with a separation factor of 2.2 (nominal concentrations of 1.94 to 100% of the saturated solution).

All test item concentrations showed a concentration-related yellow color, from yellow (saturated solution) to light yellow (lowest concentration level), and were visually clear 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 HPLC-DAD in fresh media at the start (0 hours) and in old media at the end of the exposure (48 hours) in all concentration levels and in the control.

The measured concentrations of the test item at 48 hours were in the range of 99 to 103% of the initially measured concentrations in all concentration levels. The analytical results reflected the dilution factor of 2.2 between the concentration levels. The geometric mean measured concentrations were: 1.28 – 2.85 – 6.20 – 13.7 – 29.5 – 64.9 mg/L.

The measured test item concentrations remained stable within ± 20% of the initially measured concentrations over the test period. This indicates that the test item concentrations were successfully maintained for the duration of the test.

The validity criteria of the test guideline were fulfilled.

 

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

(based on the geometric mean measured concentrations of the test item)

Effect levels	Test duration [hours]	Test Itemj Geometric mean measured test item concentrations [mg/L]
EC10 (with 95% confidence limits)	24	4.90	(Cl:≤2.85 – 6.14)
	48	4.45	(Cl: 3.48 – 5.32)
EC50 (with 95% confidence limits)	24	7.96	(Cl: 6.78 – 10.8)
	48	7.72	(Cl: 6.95 – 8.88)
EC100	24	> 64.9
	48	29.5