Disodium [5-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-4-hydroxybenzene-1,3-disulphonato(4-)]cuprate(2-)

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:

from 2017-09-26 to 2017-09-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

according to OECD 202 and GLP

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:

Disodium [5-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-4-hydroxybenzene-1,3-disulphonato(4-)]cuprate(2-)

EC Number:

276-541-3

EC Name:

Disodium [5-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-4-hydroxybenzene-1,3-disulphonato(4-)]cuprate(2-)

Cas Number:

72252-61-0

Molecular formula:

C16H10CuN4O8S2.2Na

IUPAC Name:

disodium [5-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-4-hydroxybenzene-1,3-disulphonato(4-)]cuprate(2-)

Test material form:

solid: nanoform

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

The control and the lowest concentrated sample (7.68 mg/L) were
analyzed without dilution. The other samples were diluted with HPLC
water. For dilution steps, see Table 15.
Table 15: Dilution Steps
Nominal test item
concentration
[mg/L]
Dilution
factor
Sample
volume
[mL]
Final
volume
[mL]
180 50 0.1 5.0
81.8 20 0.05 1.0
37.2 8 0.125 1.0
16.9 4 0.25 1.0

Sample storage
All samples were stored at 6 ± 2 °C until the start of the analysis, if
necessary. Prepared samples were stored in the autosampler at
room temperature until analysis.
Evaluation
Quantification of the standards was calculated by peak area based
an the external standard.

Test solutions

Vehicle:

no

Details on test solutions:

Stock solution
The stock solution (180 mg test item/L, corresponding to 145 mg a.s./L
of the test item was weighed out) was freshly prepared with dilution
water (see Table 2) prior to the start of the exposure (0 hours) and
prior to the renewal of the test solutions (24 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 2.2, prepared by dilution of the stock solution of 180 mg test
item/L with dilution water, were tested as follows:
7.68 - 16.9 - 37.2 - 81.8 - 180 mg test item/L, corresponding to
6.18 - 13.6 - 29.9 - 65.8 - 145 mg a.s./L
The test concentrations were selected based an the results of a nonGLP preliminary range finding test. For results, see section 7.1.
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. 20 µEm-2  s-1

Culture medium
Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L, was used. The composition of the culture medium is presented in Table 2.

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


Composition of the Culture Medium according to ELENDT (1990)
Component Concentration [mg/L]
CaCl2 x 2 H2O 147
MgSO4 x 7 H2O 123
KCl 5.80
NaHCO3 64.8
Na2SiO3 4.30
NaNO3 0.27
KH2PO4 0.14
K2HPO4 0.18
Na2EDTA x 2 H2O 5.00
FeSO4 x 7 H2O 1.99
H3BO3 0.29
MnCl2 x 4 H2O 0.36
LiCl 0.30
SrCl2 x 6 H2O 0.15
RbCl 0.071
NaBr 0.016
Na2MoO4 x 2 H2O 0.063
CuCl x 2 H2O 0.017
ZnCl2 0.013
CoCl2 x 6 H2O 0.010
KJ 0.00325
Na2SeO3 0.00219
NH4VO3 0.000575
Thiaminhydrochloride 0.075
Cyanocobalamine 0.0010
Biotin 0.00075
pH 8.2  0.8

Study design

Test type:

semi-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]: 167
24 hours:Total hardness [mg CaCO3/L]: 167

Test temperature:

18 - 22°C, constant within ± 1°C
20.9 °C

pH:

ater Quality Parameters in fresh Media at the Start of the Exposure and at Renewal
(0 and 24 hours)
(measured in one additional replicate (without daphnids) per concentration, level and control)
Nominal
main component
concentration
[mg/L]
0 hours 24 hours
pH-value
Dissolved
02 concentration
[mg/L]
pH-value
Dissolved
02 concentration
[mg/L]
145 7.60 8.86 7.93 8.91
65.8 7.84 8.84 8.21 8.87
29.9 7.93 8.83 8.33 8.87
13.6 7.93 8.78 8.47 8.88
6.18 7.97 8.72 8.44 8.84
Control 7.82 8.84 8.55 8.85
Table 10: Water Quality Parameters in 24-hours old Media at Renewal (24 hours)
(measured in one replicate (containing daphnids) per concentration level and control)
Nominal
main component
concentration
[m g/L] 1
pH-values
Replicates
2 3 4
Dissolved
1 2
02 concentration
Replicates
3
[mg/L1
4
145 7.66 - - - 8.44 - - -
65.8 7.68 - - - 8.75 - -
29.9 7.65 - - - 8.69
-
•
- - -
13.6 7.67 - - - 8.59 - - -
6.18 7.63 - - - 8.71 - - -
Control 7.65 - - ' - 8.60 - - -

Water Quality Parameters in 24-hours old Media at the End of the Exposure (48 hours)
(measured in one replicate (containing daphnids) per concentration level and control)
Nominal
main component
concentration
[mg/L] 1
pH-values
Replicates
2 3 4
Dissolved
1 2
02 concentration
Replicates
3
[mg/L]
4
145 7.72 - - .- 8.79 - - -
65.8 7.72 - - - 8.85 - - -
29.9 . 7.76 - - - 8.77 - - -
13.6 7.74 - - - 8.65 - - -
6.18 7.72 - - - 8.52 - - -
Control 7.80 - - - 8.77 - - -
Table 12: Water Quality Parameters of the Dilution Water at the Start of the Exposure and at
Renewal (0 and 24 hours)
Dilution water
dated:
pH-Value Dissolved
02 concentration
[mg/L]
Temperature
[°C]
Conductivity
[pS/cm]
Total hardness
fing CaCO3/L1
0 hours: 2017-09-26 7.82 8.84 20.9 490 167
24 hours: 2017-09-27 8.55 8.85 20.9 434 167

Dissolved oxygen:

see above

Conductivity:

Dilution water day 0: 490 [µS/cm]
Dilution water day 1: 434 [µS/cm]

Details on test conditions:

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 daphnidsat 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 was not necessary, because the dilution water was equivalent to the culture medium.

Application
20 g test solution per replicate were 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 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

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EC10

Effect conc.:

> 180 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Nominal test item concentration

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

>= 180 mg/L

Nominal / measured:

nominal

Conc. based on:

other: nominal test item concentrations

Basis for effect:

mobility

Effect concentrations 3

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 180 mg/L

Nominal / measured:

nominal

Conc. based on:

other: nominal t4est item concentrations

Basis for effect:

mobility

Effect concentrations 4

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

>= 180 mg/L

Nominal / measured:

nominal

Conc. based on:

other: nominal test item concentrations

Basis for effect:

mobility

Effect concentrations 5

Duration:

24 h

Dose descriptor:

EC100

Effect conc.:

> 180 mg/L

Nominal / measured:

nominal

Conc. based on:

other: nominal test item concentrations

Basis for effect:

mobility

Effect concentrations 6

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

> 180 mg/L

Nominal / measured:

nominal

Conc. based on:

other: nominal test item concentrations

Basis for effect:

mobility

Effect concentrations 7

Duration:

24 h

Dose descriptor:

EC10

Effect conc.:

> 145 mg/L

Nominal / measured:

nominal

Conc. based on:

other: nominal main component concentrations

Basis for effect:

mobility

Effect concentrations 8

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

>= 145 mg/L

Nominal / measured:

nominal

Conc. based on:

other: nominal main component concentrations

Basis for effect:

mobility

Effect concentrations 9

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 145 mg/L

Nominal / measured:

nominal

Conc. based on:

other: nominal main component concentrations

Basis for effect:

mobility

Effect concentrations 10

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 145 mg/L

Nominal / measured:

nominal

Conc. based on:

other: nominal main component concentrations

Basis for effect:

mobility

Effect concentrations 11

Duration:

24 h

Dose descriptor:

EC100

Effect conc.:

> 145 mg/L

Nominal / measured:

nominal

Conc. based on:

other: nominal main component concentrations

Basis for effect:

mobility

Effect concentrations 12

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

> 145 mg/L

Nominal / measured:

nominal

Conc. based on:

other: nominal main component concentrations

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 (Table 2) 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. The results of the most recent test are presented.

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 2017-09-12 to 2017-09-13. For results of the most recent of the monthly performed reference tests

Reference item Potassium dichromate p.a. (SIGMA)

Purity 99.0%

Batch number MKBV0900V

Expiry date 2021-11-25

Test concentrations 0.500 - 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)

Exposure phase 2017-09-12 to 2017-09-13

Reported statistics and error estimates:

Methods of evaluation
The EC10-, EC50- and EC100-values (after 24 and 48 hours) were empirically derived from the observation data (lowest concentration level resulting in x% immobilization). Only immobilization of 10% occurred in the highest test concentration, which is considered not biologically relevant.
All effect concentrations (EC10 / 50 / 100) given are based on the nominal concentrations, since the measured test item concentrations were within the range of ± 20% of the nominal concentrations. The concentration-effect relationships of the test item after 24 and 48 hours are shown graphically.


EC50-value for the reference item
An EC50-value was calculated for the reference item by sigmoidal
dose-response regression.
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.

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

Requirements of the method validation according to SANCO 3029/99 rev.4 (2000) using following criteria.                    

The results in Table 16 and Table 17demonstrate the validity of the analytical method.

 

Table16:    Parameter, Acceptance Criteria and Results of the Method Validation

Parameter	Acceptance criteria	Result
Linearity	5 standard concentrations, r^2≥ 0.992	0.4 to 8.0 mg test item/L (n = 6), r^2≥ 0.992	ü
Lowest calibration standard	S/N≥9	0.4 mg test item/L, S/N = 551	ü
Limit of Detection (LOD)	S/N≥3	Not necessary, as S/N of lowest calibration standard >30	-
Limit of Quantification (LOQ)	At least 20% above lowest calibration level after sample preparation	1.0 mg test item/L (1 x LOQ) 10 mg test item/L (10 x LOQ)	ü
Accuracy^1) (Fortified samples)	Mean recovery rate of 70-110% (ideally 80-100%) per fortification level (2 levels)	1 x LOQ: 92% (n = 5) 10 x LOQ: 96% (n = 5)	ü
Precision^1)	Relative standard deviation≤20% per fortification level inAlgae dilution water	1 x LOQ: 1.7% 10 x LOQ: 1.0%	ü
Specificity (HPLC-DAD)	Comparison of spectra of sample peaks against spectra obtained from standard peaks	Spectra compared and test item verified	ü
	Blank values < 30% of LOQ	Blank values < 30% of LOQ

ü     =Criterion fulfilled

¹⁾      =For details, seeTable 17.

 

Measured Concentrations and Percent of Nominal Concentrations of the Fortified Samples of the test item

          Fortified concentrations*: 1.01 mg/L (1 x LOQ) and 10.1 mg/L (10 x LOQ)

Replicate		test item
		1 x LOQ			10 x LOQ
		Meas. conc. [mg/L]		%		Meas. conc. [mg/L]		%
1		0.947		93		9.56		94
2		0.960		95		9.76		96
3		0.928		92		9.79		97
4		0.928		92		9.72		96
5		0.922		91		9.79		97
Mean		0.94		92		9.7		96
SD ±		0.02				0.1
CV [%]		1.7				1.0

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

%                     = percent of nominal concentration of the fortified concentration

*                       = weighing factor taken into account

SD                   = Standard deviation

CV                   = Coefficient of variation

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on the nominal concentrations of the test item, the 48 hour-EC50 for Daphnia magna was > 180 mg test item/L, corresponding to > 145 mg main component/L. The observed maximum immobilization of 10% is considered biologically not relevant.

Executive summary:

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) from2017-09-26 to 2017-09-28.

The study was conducted under semi-static conditions over a period of 48 hours with 5 concentrations of the test item in the range of7.68 to 180 mg test item/L, corresponding to 6.18 – 145 mg a.s./L, prepared in a geometric series with a separation factor of 2.2.

All tested concentration levels were visually clear and showed a concentration-related brown to yellow colouring throughout the exposure.

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 of all concentration levels and in the control at the start of the exposure and at renewal (0 and 24 hours) and in 24-hours old media at renewal and at the end of the test (24 and 48 hours).

The measured concentrations of the test item in fresh media at the start of the exposure and at renewal (0 and 24 hours) were in the range of 91 to 101% of the nominal values, and 91 to 100% in old media at renewal and at the end of the test (24 and 48 hours).The analytical results are presented in Table 7.

The measured test item concentrations were all within ± 20% of the nominal concentrations. This indicates that the test item concentrations were successfully maintained for the duration of the test. Therefore, all effect concentrations given inTable 1are based on the nominal test item and main component concentrations of the test item.

The validity criteria of the test guidelines were fulfilled.

 

Table1:   EC₁₀-, EC₅₀-and EC₁₀₀-Values

(based on the nominal concentrations of the test item)

test item
Effect concentrations	Test duration [hours]	Nominal test item concentrations [mg test item/L]		Nominal main component concentrations [mg/L]
EC10	24	> 180		> 145
	48	≥180		≥145
EC50	24	> 180		> 145
	48	> 180		> 145
EC100	24	> 180		> 145
	48	> 180		> 145