9-(2-carboxyphenyl)-3,6-bis(diethylamino)xanthylium bis[3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-4-hydroxy-N-3-(isopropoxypropyl)benzenesulphonamidato(2-)]cobaltate(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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

9-(2-carboxyphenyl)-3,6-bis(diethylamino)xanthylium bis[3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-4-hydroxy-N-3-(isopropoxypropyl)benzenesulphonamidato(2-)]cobaltate(1-)

EC Number:

275-640-9

EC Name:

9-(2-carboxyphenyl)-3,6-bis(diethylamino)xanthylium bis[3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-4-hydroxy-N-3-(isopropoxypropyl)benzenesulphonamidato(2-)]cobaltate(1-)

Cas Number:

71566-55-7

Molecular formula:

C44H50CoN10O10S2.C28H31N2O3

IUPAC Name:

9-(2-carboxyphenyl)-3,6-bis(diethylamino)xanthylium bis[3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-4-hydroxy-N-3-(isopropoxypropyl)benzenesulphonamidato(2-)]cobaltate(1-)

Test material form:

solid

Details on test material:

- Physical state / Appearance: Solid / red
- Total Organic Carbon: 612 mg/g

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient temperature in a tightly sealed container without exposure to light

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples of each test item loading rate and the control collected at exposure initiation and termination were analysed.

Test solutions

Vehicle:

no

Details on test solutions:

- The preliminary test (non-GLP):
The preliminary test was performed in a static test design. Four test item concentrations: 100-fold diluted filtrate of a loading of 10 mg/L, 10-fold diluted filtrate of a loading of 10 mg/L, filtrate of a loading of 10 mg/L, filtrate of a loading of 100 mg/L plus the control, were used for exposure.
Appropriate amounts of the test item were weighed separately in glass flasks with ISO test water and mixed with appropriate volumes of the ISO test water [SOP/W/7]. The mixture of a loading of 10 mg/L and the mixture of a loading of 100 mg/L were both visually heterogeneous (with visible undissolved particles) and colored. The mixtures and the control (1500 mL ISO test water) were left for mechanical shaking for 48 hours (30ºC, 90 rpm, darkness). After 48 hours of mechanical shaking, the mixtures (visually heterogeneous) and the control were left at room temperature for a settling phase (no shaking). After 24 hours of settling, the mixtures were visually heterogeneous. All mixtures and the control were filtrated, first through a conditioned paper filter (qualitative cellulose filter) and next through a conditioned nitrocellulose membrane filter (HAWG filter, 0.22 Vm, Millipore), [SOP/W/37]. Filtration was chosen as the separation method since centrifugation was not possible with the large volumes of test solution. Each filtrate was visually homogeneous (without any visible undissolved
particles). The filtrate of a loading of 10 mg/L was diluted in a volumetric ratio 1:9 with ISO test water (i.e. 50 mL of the filtrate was mixed with 450 mL of ISO test water). From each, the filtrate of a loading of 10 mg/L, the filtrate of a loading of 100 mg/L and the control, two samples were collected for chemical determinations (50 mL each). One was transferred to chemical analyses at exposure initiation, the second one was stored under test conditions [SOP/W/83].
The procedure of diluting the loading filtrates to get the test media as performed in the preliminary test was changed in the definitive test to the preparation of individual loadings without dilution of the filtrates as recommended in OECD Guidance Document No. 23 and taking into account the results of the water solubility study.

- The definitive test:
The definitive test was performed as a static test. The total period of exposure was 48 hours. Based on the results of the preliminary test, the definitive test was performed using five test item concentrations: filtrate of a loading of 100, 50, 25, 12.5, and 6.25 mg/L plus the control.
Appropriate amounts of the test item were weighed separately in glass flasks with ISO test water and mixed with appropriate volumes of the ISO test water [SOP/W/7]. The mixtures were visually heterogeneous (with visible undissolved particles) and colored. The mixtures and the control (1500 mL ISO test water) were left for mechanical shaking for 48 hours (30ºC, 90 rpm, darkness). After 48 hours of mechanical shaking, the mixtures (visually heterogeneous) and the control were left at room temperature for a settling phase (no shaking). After 24 hours of settling, the mixtures were visually heterogeneous. All mixtures and the control were filtrated, first through a conditioned paper filter (qualitative cellulose filter) and next through a conditioned nitrocellulose membrane filter (HAWG filter,
0.22 Vm, Millipore), [SOP/W/37]. Filtration was chosen as the separation method since centrifugation was not possible with the large volumes of test solution. Each filtrate was visually homogeneous (without any visible undissolved particles). From each filtrate and the control, two samples were collected for chemical determinations (50 mL each). One was transferred to chemical analyses at exposure initiation, the second one was stored under test conditions [SOP/W/83].

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain/clone: Daphnia magna Straus
- Source: Originated from the standard laboratory culture cultivated at the Institute of Industrial Organic Chemistry, Branch Pszczyna, Department of Ecotoxicology
- Feeding during test: None
- Culturing: Daphnia magna was cultured in glass beakers with a capacity of 150 mL (one parent per vessel) containing 100 mL of ISO test water at room temperature. The daily light cycle was 16 h light : 8 h dark (KANLUX electronic time programmer, Poland).
The culture was maintained in the ISO test water [SOP/W/67]. The Daphnia magna were fed with a suspension of algae; mixture of two species Pseudokirchneriella subcapitata : Desmodesmus subspicatus (in 2:1 ratio) originating from separate cultures in the Laboratory of Aquatic Toxicology [SOP/W/65]. Group B vitamins and micronutrients necessary for proper growth were supplied with the lyophilized suspension of Spirulina sp.. The Daphnia magna used in the tests were less than 24 h old.
- Culture medium: The ISO test water recommended by the OECD Guideline No. 202 (2004) was used to culture the test organisms and as a diluent/solvent of the test item. The medium was prepared on the basis of deionised water [SOP/W/71] by adding stock solutions of reagent-grade chemicals. The stock solutions for culturing Daphnia magna were renewed on a regular basis [SOP/W/18, SOP/W/67]. The alkalinity determined for ISO test water was 67.65 mg/L as NaHCO3. The hardness determined for ISO test water was 233.6 mg/L as CaCO3 [SOP/W48, SOP/W/82].

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

233.6 mg/L as CaCO3

Test temperature:

19.4 - 20.2 ºC

pH:

7.29 - 7.50

Dissolved oxygen:

8.4 - 8.5 mg/L

Nominal and measured concentrations:

100, 50, 25, 12.5, and 6.25 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 mL capacity, containing 100 mL of a given test item concentration or the control. The beakers were covered with transparent lids in order to minimize evaporation and to prevent accidental contamination
- Aeration: No
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: 20 mL of a given treatment per Daphnia magna

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO test water
- Alkalinity: 67.65 mg/L as NaHCO3
- Intervals of water quality measurement: The pH values and the dissolved oxygen concentrations were measured at exposure initiation, i.e. before splitting up into replicates, and at exposure termination in pooled replicates.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light : 8 h dark
- Light intensity: Fluorescent lighting was used

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Daphnia magna were observed for immobilization after 24 and 48 h of exposure. The Daphnia magna were considered immobile if they showed no ability to swim within 15 seconds after swirling the test vessel.


RANGE-FINDING STUDY
In the preliminary test, the pH values measured at exposure initiation were in the range of 7.31 – 7.38, and in the range of 7.36 – 7.45 at exposure termination. The dissolved oxygen concentrations measured at exposure initiation were in the range of 7.9 – 8.2 mg/L, and in the range of 7.7 – 7.8 mg/L at exposure termination. The temperature was in the range of 19.3 – 20.4°C. At exposure termination, the immobilization of Daphnia magna was 65% in the test item concentration: filtrate of a loading of 100 mg/L. In the remaining test item concentrations and the control, no immobilization of Daphnia magna was observed during exposure.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

76.8 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate

Basis for effect:

mortality

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EL10

Effect conc.:

39.68 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate

Basis for effect:

mortality

Effect concentrations 3

Duration:

48 h

Dose descriptor:

LOELR

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate

Basis for effect:

mortality

Effect concentrations 4

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate

Basis for effect:

mortality

Details on results:

The pH values measured at exposure initiation were in the range of 7.34 – 7.50, and in the range of 7.29 – 7.47 at exposure termination. The dissolved oxygen concentrations measured at exposure initiation were in the range of 8.3 – 8.6 mg/L, and in the range of 8.2 – 8.5 mg/L at exposure termination.
The conductivity of the test medium measured at exposure initiation in the control, before split up into replicates, was 586 μS/cm.
The recorded temperature was in the range of 19.4 – 20.2ºC with a variation of up to 0.8 ºC. This is compliant with the allowed variation during exposure of ± 1.0 ºC.
During exposure, no immobilization was observed in the control and in the filtrate of a loading of 12 and 6.25 mg/L. In the filtrate of a loading of 25, 50 and 100 mg/L, the immobilization was 5, 10 and 75%, respectively.

Results with reference substance (positive control):

The test with the reference material potassium dichromate was performed from December 01, 2016 to December 03, 2016. The purity of the reagent used in the test was 99.8%. The reagent (batch no.: 1403/02/16) was provided by Avantor (supplier of reagents). Five concentrations of the reference material ranging from 0.32 to 3.2 mg/L and a control were used. There were four replicates of each reference material concentration and the control. Five young Daphnia magna were introduced into each replicate.
The EC50 after 48 h was determined to be 0.63 mg/L (further results see below)

Any other information on results incl. tables

Table 2. Endpoint values based on the nominal concentrations of the reference material [mg/L]

Endpont value	Time of exposure
	24 h	48 h
EC50	0.86 (0.70 – 1.05)	0.63 (0.53 – 0.75)
EC20	0.53 (0.38 – 0.65)	0.44 (0.34 – 0.53)
EC10	0.41 (0.27 – 0.53)	0.37 (0.26 – 0.45)
LOEC	0.56	0.56
NOEC	0.32	0.32

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The test substance is acutely harmful for aquatic organisms.