Copper, [29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]-, [[2-[(4-chloro-6-methoxy-1,3,5-triazin-2-yl)amino]ethyl]amino]sulfonyl sulfo derivs., sodium salts

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:

2018-05-23 to 2018-05-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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 (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Copper, [29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]-, [[2-[(4-chloro-6-methoxy-1,3,5-triazin-2-yl)amino]ethyl]amino]sulfonyl sulfo derivs., sodium salts

EC Number:

275-060-6

EC Name:

Copper, [29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]-, [[2-[(4-chloro-6-methoxy-1,3,5-triazin-2-yl)amino]ethyl]amino]sulfonyl sulfo derivs., sodium salts

Cas Number:

70955-64-5

Molecular formula:

C36.2H18.9Cl0.7CuN11.5O10.2S3.4Na2.7

IUPAC Name:

Copper, [29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]-, [[2-[(4-chloro-6-methoxy-1,3,5-triazin-2-yl)amino]ethyl]amino]sulfonyl sulfo derivs., sodium salts

Test material form:

solid: particulate/powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
The limit concentration and the control were analytically verified via HPLC-DAD in the fresh media at the start of the exposure (0 hours) and in the 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 the test replicates containing daphnids.


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

Test solutions

Vehicle:

no

Details on test solutions:

Limit concentration
A limit concentration of 100 mg/L of the test item was tested. The limit concentration was selected based on the results of a non-GLP preliminary range finding test.

Preparation of the limit concentration
A limit concentration (100 mg/L of the test item were weighed out) was freshly prepared with dilution water prior to the start of the exposure (at 0 hours). The limit concentration was mixed thoroughly by manual agitation.

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, with an algae cell density of > 106 cells/mL. The algae were cultured at the test facility.

Study design

Test type:

static

Water media type:

freshwater

Remarks:

Elendt M4, according to OECD 202, Annex 3 (2004)

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Hardness:

Dilution water
0 hours: Total hardness [mg CaCO3/L]: 256

Test temperature:

target: 18 - 22°C, constant within ± 1°C
measured: 20 °C

pH:

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

Nominal pH-value Dissolved O2 concentration
test item [mg/L]
concentration
[mg/L]
100 7.55 8.65
Control 7.40 8.72

Water Quality Parameters in old Media at the End of the Exposure (48 hours)
(measured in one replicate (containing daphnids) of the limit concentration and the control)

Nominal pH-value Dissolved O2 concentration Replicate number
test item [mg/L]
concentration
[mg/L]
100 7.48 7.55 1
Control 7.47 8.24 1

Water Quality Parameters of the Dilution Water at the Start of the Exposure (0 hours)
Dilution water pH-value Dissolved Temperature Conductivity Total hardness
dated: O2 concentration [mg/L] [°C] [µS/cm] [mg CaCO3/L]
2018-05-23 7.74 9.02 20.6 693 256

Dissolved oxygen:

see above

Salinity:

Not applicable, freshwater

Conductivity:

Dilution water day 0: 693 [µS/cm]

Nominal and measured concentrations:

Nominal test item concentration: 100 mg/L
Measured test item concentrations: 96.1 mg/L (0 hours) - 94.3 mg/L (48 hours)

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 Same composition as the culture medium

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

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 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.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 5

Duration:

24 h

Dose descriptor:

EC100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 6

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

> 100 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 05-03 to 2018-05-04. For results of the most recent of the monthly performed reference tests, see the table below.

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 2.07 mg/L 0.6 - 2.4 mg/L, acc. to AQS P 9/2 (02/2000); clone 5
95% confidence limits 1.22 – 3.66 mg/L 0.6 - 2.1 mg/L, acc. to OECD 202 (2004); clone A

Reported statistics and error estimates:

Methods of evaluation
The EC10 / 50 / 100-values after 24 and 48 hours for the test item were empirically derived from the observation data. Since no effects on Daphnia magna were observed in the limit concentration of the test item, no calculations were made. All effect concentrations (EC10 / 50 / 100) given were based on the nominal concentration of the test item.

EC-values and statistical analyses for reference item
An EC50-value was calculated for the reference item by sigmoidal dose-response regression from the best-fit values with the software GraphPad Prism. The respective 95 % confidence limits were calculated from the standard error and the t-distribution.

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

In the limit concentration 100 mg/L of the test item Reactive Blue 71, no effects on Daphnia magna were observed.

Immobilization Rates after 24 and 48 hours of Exposure in the Definitive Test

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

ReactiveBlue 71 Nominal test item concentration [mg/L]	IMMOBILIZATION [%]
	24 hours					48 hours
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
100	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

 

Absolute Numbers of immobile daphnids after 24 and 48 hours of Exposure in the Definitive Test

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

Reactive Blue 71 Nominal test item concentration [mg/L]	Number of immobile Daphnids / Total number of Daphnids
	24 hours					48 hours
	Replicates					Replicates
	1	2	3	4	Sum	1	2	3	4	Sum
100	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

The limit concentration of 100 mg test item/L was blue and visually clear throughout the exposure, while the control was colourless and visually clear throughout the exposure.

The concentration of the test item Reactive Blue 71 was analytically verified via HPLC-DAD in the fresh media at the start of the exposure (0 hours) and in the old media at the end of the exposure (48 hours) in the limit concentration and in the control.
The measured concentration of the test item in fresh media at the start of the exposure (0 hours) was 96% of the nominal value of the limit concentration. At the end of the exposure period (48 hours), the measured test item concentration in the old media was 94% of the nominal value of the limit concentration. Since the measured concentration of the test item Reactive Blue 71 did remain stable within ±20% of the nominal concentration throughout the exposure period, the nominal concentration was used for evaluation. The analytical results are presented in the table below.

Measured Concentrations of the Test Itemduring the Definitive Test

Sampling date	Fresh Media, 0 hours		Old Media, 48 hours
Nominal test item concentration [mg/L]	Reactive Blue 71
	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%
100	96.1	96	94.3	94
Control	< LOQ		< LOQ

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

%                  = percent of the nominal concentration of the test item

LOQ             = limit of quantification of the analytical method (0.500 mg/L of the test item)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In the limit concentration of 100 mg/L of the test item Reactive Blue 71, no effects on Daphnia magna were observed.
Based on the nominal concentrations of the test item Reactive Blue 71, the 48 hours-EC10 / 50 / 100 for Daphnia magna was > 100 mg/L.

Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item Reactive Blue 71 were determined at the test facility according to OECD 202 (2004) from 2018-05-23 to 2018-05-25.

A concentration of100 mg/L was tested in a limit test under static conditions over a period of 48 hours. The limit concentration of 100 mgtest item/L was blue and visually clearly dissolved throughout the exposure, while the control was colourless and visually clear throughout the exposure.

Twenty daphnids, divided into 4 replicates with 5 daphnids each, were exposed to the limit concentration and the control.

The concentration of the test item Reactive Blue 71 was analytically verified via HPLC-DAD in the fresh media at the start of the exposure (0 hours) and in the old media at the end of the exposure (48 hours) in the limit concentration and in the control.

The measured concentration of the test item in fresh media at the start of the exposure (0 hours) was 96% of the nominal value of the limit concentration. At the end of the exposure period (48 hours), the measured test item concentration in the old media was 94% of the nominal value of the limit concentration. Since the measured concentration of the test item Reactive Blue 71 did remain stable within ±20% of the nominal concentration throughout the exposure period, the nominal concentration was used for evaluation.

The effect concentrations given in the table below are based on the nominal concentration of the test item Reactive Blue 71.

No effects were found at the limit concentration of 100 mg/L of the test item in Elendt M4 medium.

The validity criteria of the test guideline were fulfilled.

 

Table1:   EC_{10 / 50 / 100}-Values

(based on the nominal concentration of the test item)

Effect concentrations	Test duration [hours]	Reactive Blue 71 Nominal concentration of the test item [mg/L]
EC10 / 50 / 100	24	> 100
	48	> 100