Hydrogen [[[(2-ethylhexyl)amino]sulphonyl][[(3-methoxypropyl)amino]sulphonyl]-29H,31H-phthalocyaninesulphonato(3-)-N29,N30,N31,N32]cuprate(1-), compound with N,N'-di(o-tolyl)guanidine (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:

2014 - 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well performed OECD study with GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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 [[[(2-ethylhexyl)amino]sulphonyl][[(3-methoxypropyl)amino]sulphonyl]-29H,31H-phthalocyaninesulphonato(3-)-N29,N30,N31,N32]cuprate(1-), compound with N,N'-di(o-tolyl)guanidine (1:1)

EC Number:

276-657-4

EC Name:

Hydrogen [[[(2-ethylhexyl)amino]sulphonyl][[(3-methoxypropyl)amino]sulphonyl]-29H,31H-phthalocyaninesulphonato(3-)-N29,N30,N31,N32]cuprate(1-), compound with N,N'-di(o-tolyl)guanidine (1:1)

Cas Number:

72428-99-0

Molecular formula:

C32H13CuN8O4S2.(SO3H)m.(C4H11NO)n.(C8H18N) (3-m-n).C15H17N3

IUPAC Name:

hydrogen [[[(2-ethylhexyl)amino]sulphonyl][[(3-methoxypropyl)amino]sulphonyl]-29H,31H-phthalocyaninesulphonato(3-)-N29,N30,N31,N32]cuprate(1-), compound with N,N'-di(o-tolyl)guanidine (1:1)

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The effects of the saturated solution of the test item were tested in a limit test.
- Sampling method: The saturated solution and the control were analytically verified at the start of the exposure (0 h) and at the end of the exposure
(48 h) as specified below. At the start of the exposure (0 h), samples were taken immediately after preparation of the saturated solution and analysed.
At the end of the exposure (48 h), samples were taken directly from the test vessels. The method was validated according to SANCO 3029/99 rev.4 (2000-07-11).

- Sample storage conditions before analysis: The original and diluted samples were stored at room temperature until sample preparation and until analysis, if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The saturated solution (5.00 mg/L test item was weighed out) was prepared with dilution water two days prior to the start of the exposure
(0 h). The test item was dispersed in the dilution water by stirring with approximately 1100 rpm for 48 h at room temperature. After completion of stirring, undissolved test item was removed by centrifugation for 10 minutes at 18533 g to achieve the saturated solution.

- Eluate: ISO Test water, according to OECD 202, Annex 3
- Differential loading: Limit test with the saturated solution
- Controls: Dilution water without test item tested under the same conditions as the test groups
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): None
- Concentration of vehicle in test medium (stock solution and final test solution): None
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain: Clone 5
- Source:
Origin: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu)
Breeder: DR.U.NOACK-LABORATORIEN, Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany
- Age at study initiation (mean and range, SD): 2 to 24 h old daphnids from a healthy stock were used for the study
- Method of breeding:
Culture: In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20 +- 2°C, in an incubator, 16 h illumination; light intensity
of max. 20 µE x m-2 x s-1
Culture medium: Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L
Cultur feeding: 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
- Feeding during test: No feeding

ACCLIMATION
- Acclimation period: At least 2 h in dilution water.
- Acclimation conditions (same as test or not): Same as test conditions.
- Health during acclimation (any mortality observed): No mortality observed.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Hardness:

249 mgCaCO3/L

Test temperature:

18 - 22°C, constant within ± 1°C
Measured: 22.0

pH:

pH at the Start of the Exposure (0 h)
(measured in one additional replicate of the saturated solution and control)

Geometric mean measured
concentration
[mg/L] pH-value
1.17 7.29
(Saturated solution)
Control 7.64

pH at the End of the Exposure (48 h)
(measured in all replicates)

Geometric mean measured
concentration pH-values
[mg/L] Replicates
1 2 3 4
1.17 7.00 7.03 7.06 7.10
(Saturated solution)
Control 6.85 6.83 6.90 6.96

Dissolved oxygen:

O2-concentration at the Start of the Exposure (0 h)
(measured in one additional replicate of the saturated solution and control)

Geometric mean measured
concentration
[mg/L] O2-concentration [mg/L]
1.17 8.74
(Saturated solution)
Control 8.61

O2-concentration at the End of the Exposure (48 h)
(measured in all replicates)

Geometric mean measured
concentration O2-concentration [mg/L]
[mg/L] Replicates
1 2 3 4
1.17 8.49 8.54 8.49 8.48
(Saturated solution)
Control 7.57 8.22 8.37 8.38

Nominal and measured concentrations:

Saturated solution (geometric mean measured concentration of 1.17 mg/L)

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers (4 cm ID x 7 cm H), 50 mL capacity, loosely covered with watch glasses
- Test volume: 20 mL
- Aeration: None
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO Test water, according to OECD 202, Annex 3
Component Concentration [mg/L]
KCl 5.76
NaHCO3 64.8
CaCl2 x 2 H2O 294
MgSO4 x 7 H2O 123

- Water Quality Parameters of the Dilution Water prior to the Start of the Exposure (0 h):
pH-value DissolvedO2-concentration[mg/L] Temperature[°C] Conductivity[µS/cm] Total hardness [mg CaCO3/L]
7.64 8.61 22.0 654 249

- Culture medium different from test medium: Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L
- Intervals of water quality measurement: test start (0h) and test end (48h)

OTHER TEST CONDITIONS
- Photoperiod: 16/8 h light/dark cycle
- Light intensity: Diffuse light, light intensity of max. 20 µE x m-2 x s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Immobilisation, 24 h interval

TEST CONCENTRATIONS
- Test concentrations: Saturated solution (Geometric mean measured concentration of 1.17 mg/L)

- Range finding study: None

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EL10

Effect conc.:

> 1.17 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 1.17 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Duration:

48 h

Dose descriptor:

EL100

Effect conc.:

> 1.17 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 4

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

> 1.17 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 5

Duration:

48 h

Dose descriptor:

LOELR

Effect conc.:

> 1.17 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

See below

Results with reference substance (positive control):

The percentage immobility for the reference item was determined after 24 h. The EC50-value with 95 % confidence interval (CI) was calculated by sigmoidal dose-response regression. The EC50-value for the most recent of the monthly performed reference tests was:
EC50: 2.1 mg/L (CI 1.8 - 2.4 mg/L)
The EC50-value of the reference item potassium dichromate after 24 h is within the prescribed concentration range of 0.6 - 2.4 mg/L of quality criteria according to AQS P 9/2 (02/2000) for daphnids clone 5 cultured in Elendt M4 medium. The EC50-value of the reference item is also within the recommended range of 0.6 - 2.1 mg/L according to OECD-Guideline 202.

Any other information on results incl. tables

During the test, the incubator temperature was 19°C.

The water quality parameterswere within the acceptable limits.

Biological Data

The percentage immobility, determined in all test and control groups after 24 and 48 h under static conditions, is given in the table below.

 

Immobilisation Rates after 24 and 48 h of Exposure in the Definitive Test

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

	IMMOBILISATION [%]
Geometric mean measured concentration   [mg/L]	24 h					48 h
	Replicates					Replicates
	1.	2.	3.	4.	MV	1.	2.	3.	4.	MV
1.17         (Saturated solution)	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

MV = mean value

 

The saturated solution wasslightly blue and clear throughout exposure. At the end of the exposure (48 h), a slight sedimentation of the test item was observed.

Measured Exposure Concentrations during the Definitive Test

The saturated solution of the test itemand the control were analytically verified by HPLC-DAD at the start of the exposure (0 h) and at the end of the exposure (48 h).
The measured concentration of the test item was 1.16 mg/L at the start of the exposure (0 h) and 1.18 mg/L at the end of the exposure (48 h). The analytical results are presented in the tables below.

Measured Concentrations of the Test Item during the Definitive Test

Sampling date	2014-04-09 Start of the exposure, 0 h	2014-04-11 End of the exposure, 48 h
Date of analysis	2014-04-09	2014-04-11
Nominal loading of the test item [mg/L]
	Meas. conc.  [mg/L]	Meas. conc. [mg/L]	%
5.00 (Saturated solution)	1.16	1.18	102
Control	< LOQ	< LOQ

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

%                  = percent, related to the initially measured concentration of the test item

LOQ             = limit of quantification of the analytical method (0.5 mg/L of the test item)

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

At the saturated solution with a geometric mean measured concentration of 1.17 mg/L of the test item , no effects were observed on Daphnia magna.

Executive summary:

In the acute immobilisation test with Daphnia magna (STRAUS), the effects of the saturated solution* with 5 mg/L of the test item was determined according to OECD 202 (2004). The limit test was conducted under static conditions over a period of 48 h .

A saturated solution was prepared by stirring with approximately 1100 rpm for 48 h at room temperature. After completion of stirring, undissolved test item was removed by centrifugation for 10 minutes at 18533 g. Twenty daphnids were exposed to the saturated solution concentration and the control.

The saturated solution of the test item and the control were analytically verified by HPLC-DAD at the start of the exposure (0 h) and at the end of the exposure (48 h).
The measured concentration of the test item was 1.16 mg/L at the start of the exposure (0 h) and 1.18 mg/L at the end of the exposure (48 h). The geometric mean measured test item concentration was calculated to be 1.17 mg/L.

 

The effect values given are based on the geometric mean measured concentrations of the test item .

 

The water quality parameters (i.e. pH-value and dissolved oxygen concentration), measured at the start (0 h) and at the end of the exposure (48 h), were within the acceptable limits. The validity criteria of the test guideline were fulfilled.

 

  

 

At the saturated solution* with a geometric mean measured concentration of

1.17 mg/L of the test item ,

no effects were observed on Daphnia magna.

 

 

EL₁₀-, EL₅₀- and EL₁₀₀-Values, NOEL and LOEL

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

Endpoints	Test duration [h]
		Geometric mean measured concentration [mg/L]
EL10	24	> 1.17
	48	> 1.17
EL50	24	> 1.17
	48	> 1.17
EL100	24	> 1.17
	48	> 1.17
NOEL	48	1.17
LOEL	48	> 1.17