Copper, [29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]-, [[3-(cyclohexylamino)propyl]amino]sulfonyl derivs.

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 2018-06-14 to 2018-10-08

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)

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]-, [[3-(cyclohexylamino)propyl]amino]sulfonyl derivs.

EC Number:

309-627-7

EC Name:

Copper, [29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]-, [[3-(cyclohexylamino)propyl]amino]sulfonyl derivs.

Cas Number:

100545-46-8

Molecular formula:

C32H16-τN8Cu.(SO3)m.(SO3H)p.(C9H19N2SO2)O.(C9H20N2SO2)n

IUPAC Name:

Copper, [29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]-, [[3-(cyclohexylamino)propyl]amino]sulfonyl derivs.

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 18-25°C, dark, in tightly closed original container, in evacuated desiccator

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
* 0 mg/L of test item (control)
* 100 mg/L of test item (limit test)

- Sampling method: At the start of the exposure and at the renewal (0 and 24 hours), monitoring samples of the fresh media were taken after preparation of the WSF and analyzed. Sampling of the old media was not carried out, since the test item concentration was below the LOD in the fresh media.

- Preparation of samples: The control and the sample were analysed undiluted.

- Sample storage conditions before analysis: 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.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Water Accommodated Fraction: In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996 and OECD 2000), is to expose the organisms to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture, an UVCB substance and is poorly soluble in water and in the permitted solvents. Using this approach, aqueous media were prepared by mixing the test item with dilution water for a prolonged period sufficient to ensure equilibration between the test item and the water phase.

A water soluble fraction (WSF) was prepared 24 ± 1 hour prior to the start of the exposure (day -1) as well as prior to the renewal of the test solutions (day 0). For the preparation of the WSF, an appropriate amount of the test item was weighed and placed onto a glass slide. The glass slide with the test item was inserted in a brown glass flask filled with an appropriate amount of dilution water. This dispersion was shaken for 24 hours with 20 rpm at room temperature. Undissolved particles were removed by membrane filtration (membrane filter 0.20 µm, RC, MACHEREY-NAGEL). The filter was saturated in order to avoid adsorption during the filtration. The first 25 mL of the filtrate were discarded. The filtration was interrupted for 15 minutes to allow adsorption and saturation of the filter material with dissolved test item. Thereafter, the filtration was continued. The next 25 mL were discarded. The following filtrate, i.e. the water soluble fraction (WSF) was used in the test. During filtration, the filter was always kept covered with the test solution. The WSF was checked via laser beam (Tyndall effect) for undissolved test item. The Tyndall effect was negative.

- Controls: Dilution water without test item incubated under the same conditions as the test group.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: STRAUS
- Source: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany
- Age of parental stock (mean and range, SD): less than 24 hours (at test initiation)
- Feeding during test: no

ACCLIMATION
- Acclimation period: Acclimatization of the daphnids was not necessary, because the composition of the dilution water was equivalent to the culture medium.

Study design

Test type:

semi-static

Water media type:

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

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Hardness:

Dilution Water at the Start of the Exposure (0h): 211 mg CaCO3/L
Dilution Water at the Renewal (24 hours): 215 mg CaCO3/L

Test temperature:

18 - 22 °C, ± 1 °C

pH:

Fresh media at start of exposure (0 hours): 8.08 - 7.97 (100 mg/L - control)
fresh Media at the Renewal (24 hours): 7.70 - 7.95 (100 mg/L - control)

24-hours old Media at the Renewal (24 hours): 7.67 - 7.73 (100 mg/L - control)
at the End of the Exposure (48 hours) : 7.76 - 7.79 (100 mg/L - control)

Dilution Water at the Start of the Exposure (0 hours): 7.97
Dilution Water at Renewal (24 hours): 7.95

Dissolved oxygen:

Fresh media at start of exposure (0 hours): 8.23 mg/L - 9.25 mg/L (100 mg/L - control)
fresh Media at the Renewal (24 hours): 8.45 mg/L - 9.13 mg/L (100 mg/L - control)

24-hours old Media at the Renewal (24 hours): 8.10 mg/L - 8.33 mg/L (100 mg/L - control)
at the End of the Exposure (48 hours) : 8.91 mg/L - 8.72 mg/L (100 mg/L - control)

Dilution Water at the Start of the Exposure (0 hours): 9.25 mg/L
Dilution Water at Renewal (24 hours): 9.13 mg/L

Conductivity:

Dilution Water at the Start of the Exposure (0h) : 522 µS/cm
Dilution Water at Renewal (24h) : 554 µS/cm

Nominal and measured concentrations:

Nominal concentration : 100 mg/L of test item
Measured concentration: measured concentration of the test item in the fresh media at the start of the exposure (0 hours) and at the renewal (24 hours) was below the limit of detection (LOD = 0.2 mg/L), the analytical monitoring at the end of the exposure was waived and the test item concentration was considered to be below the LOD in the 24-hours old media.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers (4 (ID) x 7 (H) cm), 50 mL capacity, loosely covered with watch glasses
- Type : closed with watch glasses
- Material, size, headspace, fill volume: 50 mL glass beaker, filled at 20 mL
- Aeration: No
- Renewal rate of test solution: The test solutions were renewed after 24 hours. For this purpose, a second set of preconditioned test vessels was filled with the freshly prepared test solutions and the daphnids were be transferred by pipette
- No. of organisms per vessel: 5 daphnids
- No. of vessels per concentration (replicates): 4 replicates
- No. of vessels per control (replicates): 4 replicates


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16/8 hours light/dark cycle
- Light intensity: Diffuse light, light intensity of max. 1500 lx


RANGE-FINDING STUDY
- Test concentrations:
* 0 mg/l of test item (control)
* 100 mg/l of test item

- Results used to determine the conditions for the definitive study: A preliminary range finding test was conducted at the test facility under semi-static conditions over a period of 48 hours with three WSFs with nominal test item loadings of 1, 10 and 100 mg/L prepared with dilution water.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate p.a. (SIGMA)

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EL10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: nominal test item loading

Effect concentrations 2

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: nominal test item loading

Effect concentrations 3

Duration:

24 h

Dose descriptor:

EL100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: nominal test item loading

Effect concentrations 4

Duration:

48 h

Dose descriptor:

EL10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: nominal test item loading

Effect concentrations 5

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: nominal test item loading

Effect concentrations 6

Duration:

48 h

Dose descriptor:

EL100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: nominal test item loading

Details on results:

- Behavioural abnormalities: non observed
- Mortality of control:non observed

Results with reference substance (positive control):

- Results with reference substance valid: Yes
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)

Reported statistics and error estimates:

An EC50-value was calculated for the reference item by sigmoidal dose-response regression. The respective confidence limits for the EC50 were empirically derived from the observation data as follows: The highest concentration level without any effect (EC0) and the lowest concentration level causing 100% immobilization (EC100) were used as confidence limits, since calculation was not possible by the software. All calculations were carried out from the best-fit values with the software GraphPad Prism.