Sodium [3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulphonato(3-)]hydroxychromate(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:

2019-01-09 to 2019-01-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

according to OECD and GLP guidline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Sodium [3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulphonato(3-)]hydroxychromate(1-)

EC Number:

233-357-8

EC Name:

Sodium [3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulphonato(3-)]hydroxychromate(1-)

Cas Number:

10127-27-2

Molecular formula:

C16H11CrN5O8S.Na

IUPAC Name:

sodium [3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulphonato(3-)]hydroxychromate(1-)

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 samples were analysed with a HPLC-DAD method, implemented under non-GLP and documented finally in the GLP raw data. The method was validated.

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 test replicates containing daphnids.

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

Test solutions

Vehicle:

no

Details on test solutions:

Stock solution, media preparation
150 mg test item/L were freshly prepared with dilution water. The dispersion was stirred with a magnetic stirrer for 1 hour (1100 rpm, room temperature).

Test concentrations
The stock solution and four dilution levels were prepared with dilution water in a geometrical series with a separation factor of 2.2 and tested as follows:
6.40 - 14.1 - 31.0 - 68.2 - 150 mg test item/L, corresponding to the geometric mean measured concentrations 6.38 – 15.6 - 34.8 - 79.3 - 171 mg/L.

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, obtained from continuous laboratory cultures.

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

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), is used. The composition of the culture medium is presented in the table below.

 

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 to 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. The algae were cultured at the test facility.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

Dilution water
0 hours: Total hardness [mg CaCO3/L]: 283

Test temperature:

target: 18 - 22°C, constant within ± 1°C
measured: 19 - 19.5 °C

pH:

Water Quality Parameters at the Start of the Exposure (0 hours)
(measured in one additional replicate (without daphnids) per concentration level and control)

Geometric mean pH-value Dissolved
measured test item O2 concentration
concentration
[mg/L] [mg/L]
171 7.85 8.84
79.3 7.80 8.93
34.8 7.82 8.95
15.6 7.82 8.95
6.38 7.86 8.96
Control 7.74 9.04


Water Quality Parameters at the End of the Exposure (48 hours)
(measured in one replicate per concentration level containing daphnids)

Geometric mean pH-value Dissolved Replicate number
measured test item O2 concentration
concentration
[mg/L] [mg/L]
171 7.61 8.35 4
79.3 7.56 8.54 4
34.8 7.58 8.70 4
15.6 7.58 8.61 4
6.38 7.60 8.59 4
Control 7.65 8.51 4


Water Quality Parameters of the Dilution Water at the Start of the Exposure (0 hours)
Dilution water pH-value Dissolved O2 concentration Temperature Conductivity Total hardness
dated: [mg/L] [°C] [µS/cm] [mg CaCO3/L]
2019-01-09 7.74 9.04 19.1 626 283

Dissolved oxygen:

see above

Salinity:

Not applicable, freshwater

Conductivity:

Dilution water day 0: 626 [µS/cm]

Nominal and measured concentrations:

Sampling date Fresh Media, 0 hours Old Media, 48 hours Geometric mean measured test item concentration [mg/L]
Nominal test item concentration
[mg/L] Meas. conc. [mg/L] % Meas. conc. [mg/L] %
150 170 113 172 114 171
68.2 78.3 115 80.3 118 79.3
31.0 35.3 114 34.3 111 34.8
14.1 15.4 109 15.9 113 15.6
6.40 6.10 95 6.68 104 6.38
Control < LOQ < LOQ < LOQ

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

Dilution water ISO water according to OECD 202, Annex 3 was used.
Component Concentration [mg/L]
KCl 5.75
NaHCO3 64.8
CaCl2 x 2 H2O 294
MgSO4 x 7 H2O 123


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

Age of the daphnidsat the start of the exposure
2 to 24 hours old daphnids from a healthy stock were used for the study. Juvenile daphnids were removed from the culture vessels at the latest 22 hours before the start of the exposure and discarded. The juveniles born within this period of max. 22 hours preceding the exposure were used for the test after an acclimatization phase of at least 2 hours in the dilution water. No first brood progeny was used for the test.

Acclimatization At least 2 hours in the dilution water (see above)

Application
20 g test solution per replicate was weighed 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

Key result

Duration:

24 h

Dose descriptor:

EC10

Effect conc.:

171 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: confidence limits: 44.4 - >171 mg/L

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

79.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: confidence limits: 70.0 - 92.7 mg/L

Effect concentrations 3

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 171 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 4

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

116 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: confidence limits: 105 - 129 mg/L

Effect concentrations 5

Key result

Duration:

24 h

Dose descriptor:

EC100

Effect conc.:

> 171 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 6

Key result

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

> 171 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 2018 12-04 to 2018-12-05. For results of the most recent of the monthly performed reference tests, see the table below.

EC50-Value (with Confidence Limits) of the Reference Item Potassium dichromate
based on nominal concentrations [mg/L], (0 - 24 hours)
Current Study EC50 Confidence limits Valid Range
2.03 mg/L 1.00 - 4.00 mg/L 0.6 - 2.1 mg/L, acc. to OECD 202 (2004); clone A

Reported statistics and error estimates:

Methods of evaluation
The EC100-values after 24 and 48 hours and the EC50-value after 24 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-value after 24 hours and the EC10- and the EC50-values after 48 hours were calculated by sigmoidal dose-response regression. The concentrations enclosed to the EC10-value after 24 hours were chosen as lower and upper confidence limits. The respective 95% confidence limits for the EC10- and the EC50-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 Prism. The concentration-effect relationships of the test item are shown graphically.

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 Prism, GRAPHPAD SOFTWARE, INC.
- Excel, MICROSOFT CORPORATION

Any other information on results incl. tables

The percentage of immobility, determined in all concentration levels of the test item and in the control after 24 and 48 hours is given in the table below.

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
171	0	40	0	0	10	80	100	80	100	90
79.3	0	0	0	0	0	20	0	0	20	10
34.8	0	0	0	0	0	0	0	0	0	0
15.6	0	0	0	0	0	0	0	0	0	0
6.38	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

The absolute numbers of immobile daphnids are presented in the table below.

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	Sum	1	2	3	4	Sum
171	0 / 5	2 / 5	0 / 5	0 / 5	2 / 20	4 / 5	5 / 5	4 / 5	5 / 5	18 / 20
79.3	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	1 / 5	0 / 5	0 / 5	1 / 5	2 / 20
34.8	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20
15.6	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20
6.38	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 and concentration-related yellow to orange throughout the exposure period. After 24 hours of the exposure as well as at the end of exposure a concentration-related sedimentation of the test item was observed in the three highest concentration levels (34.8 – 79.3 – 171 mg/L). The control was colorless and visually clear throughout the exposure.

 

 

 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 – 19.5 °C.

 

 

 Measured Exposure Concentrations during the Definitive Test

 

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 were in the range of 95 to 115% of the nominal test item concentrations at 0 hours and at 48 hours in the range of 104 to 118% of the nominal test item concentrations.

The measured test item concentrations remained stable within ± 20% of the nominal 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 Item during the Definitive Test

Sampling date	Fresh Media, 0 hours		Old Media, 48 hours		Geometric mean measured test item concentration [mg/L]
Nominal test item concentration [mg/L]	AluminiumOrange G
	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%
150	170	113	172	114	171
68.2	78.3	115	80.3	118	79.3
31.0	35.3	114	34.3	111	34.8
14.1	15.4	109	15.9	113	15.6
6.40	6.10	95	6.68	104	6.38
Control	< LOQ		< LOQ		< LOQ

Meas. conc.= measured concentration of the test item

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

LOQ             = limit of quantification of the analytical method (2 mg/L of the test item)

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 116 mg/L (confidence limits: 105 – 129 mg/L).

Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item (batch number: CHM8004219) were determined at the test facility according to OECD 202 (2004) from 2019-01-09 to 2019-01-11. The study was conducted under static conditions over a period of 48 hours with a test item solution with a nominal concentration of 150 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 6.40 to 150 mg/L).

All test item concentrations were visually clear and concentration-related yellow to orange throughout the exposure period. After 24 hours of the exposure as well as at the end of exposure a concentration-related sedimentation of the test item was observed in the three highest concentration levels (31.0 – 68.2 – 150 mg/L). The control was colorless and visually clear throughout the exposure.

Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each concentration level and 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 test start were in the range of 95 to 115% of the nominal values. At 48 hours, the measured concentrations were in the range of 104 to 118% of the nominal values. The geometric mean measured concentrations were 6.38 – 15.6 - 34.8 - 79.3 - 171 mg/L.

The measured test item concentrations remained stable within ± 20% of the nominal concentrations over the test period. This indicates that the test item concentrations were successfully maintained for the duration of the test. The effect concentrations given are based on the geometric mean measured test item concentrations of the test item.

The validity criteria of the test guideline were fulfilled.

 

Table1:   EC₁₀-, EC₅₀- (with Confidence Limits) and EC₁₀₀-Values

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

Effect levels		Test duration [hours]		test item Geometric mean measured test item concentrations [mg/L]
EC10 (with confidence limits)		24		171 (44.4 - > 171)
		48		79.3 (70.0 – 92.7)
EC50 (with confidence limits)		24		> 171
		48		116 (105 - 129)
EC100		24		> 171
		48		> 171