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

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2018-06-11 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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition 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: Room temperature, protected from light, in the tightly closed original container, since 2017-11-02 additionally in evacuated desiccator.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
* 0 mg/l (control)
* 1.00 mg/L of test item
* 3.16 mg/L of test item
* 10.0 mg/L of test item
* 31.6 mg/L of test item
* 100 mg/L of test item

- Sampling method:
Each loading level and the control will be analytically verified via HPLC-DAD at least at the start of the exposure (0 hours).
At the start of exposure (0 hours), sampling was carried out after preparation of the loading levels.
At test start the measured test item concentrations were below the LOD in all loading levels. Therefore no analytical monitoring was carried out at the end of exposure (72 hours), all test loadings can be considered to be below the LOD in the aged media too and the method was not validated.

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

- Sample storage conditions before analysis: all samples were stored at 6 +/- 2°C until the start of the analysis, if necessary. Pepared 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 will be prepared by mixing the test item with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase.

*Preparation of the WSF: Five water accommodated fractions (WAFs) were prepared with a nominal loading of the test item of 1.00 - 3.16 - 10.0 - 31.6 - 100 mg/L(spacing factor √10). The loading levels are based on the results of a preliminary range finding test. For each loading level, an appropriate amount of the test item was weighed out. The test item was applied onto a glass slide. The glass slide with the test item was inserted in a glass flask with an appropriate amount of demineralized water. These dispersions will be shaken at 20 rpm for 24 hours at room temperature. Undissolved particles will be 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 will be 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 water soluble fractions (WSFs) were used in the test. During filtration, the filter was always be kept covered. The solutions were checked via laser beam (Tyndall effect) for undissolved test item.The Tyndall effect was negative. The components of the dilution water were added to the WSFs.

Replicates: 3 replicates per loading level.

- Controls: 6 replicates (without test item) will be incubated under the same test conditions as the test item replicates.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokierchneriella subcapitata
- Strain: SAG 61.81
- Source (laboratory, culture collection): Sammlung von Algenkulturen (SAG) Pflanzenphysiologis Institut der Universität Göttingen Nikolausberger Weg 18, D-37073 Göttingen
- Age of inoculum (at test initiation): Four days old
- Method of cultivation:
* Fresh stocks are prepared every month on Z-Agar.
* Light intensity amounts 2567 - 5130 lux for 24 hours per day.
* Nutrient medium Z according to LÜTTGE et al. (1994)
* Culture apparatus: Climate room

ACCLIMATION
- Acclimation period: NON INDIQUE - A COMPLETER
- Culturing media and conditions (same as test or not): A COMPLETER
- Any deformed or abnormal cells observed: no

Study design

Test type:

static

Water media type:

other: OECD TG 201 medium

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

0.24 mmol Ca+Mg/L

Test temperature:

21 - 24°C, controlled at ± 2°C

pH:

8.1 ± 0.2

Nominal and measured concentrations:

Nominal concentration : 1.00 - 3.16 - 10.0 - 31.6 - 100 mg/L
Measured concentration: < LOD

Details on test conditions:

TEST SYSTEM
- Test vessel: sterile Erlenmeyer flasks (250mL) with cotton wool plugs
- Type: cloed with cotton wool plugs
- Material, size, headspace, fill volume: 250 mL sterile glass Erlenmeyer flasks

- Initial cells density: 5523 cells/mL
- Control end cells density: 1826992 cells/mL
- No. of organisms per vessel: 5523 cells/mL
- No. of vessels per concentration (replicates): 3 replicates
- No. of vessels per control (replicates): 6 replicates


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD TG 201 medium
- Culture medium different from test medium: no
- Intervals of water quality measurement: : 0 and 72h

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24hours/day light
- Light intensity and quality: approximately 4440 to 8880 lux, corresponding to 60 to 120 µE*m-2*s-1


EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations:
*Chlorophyll a-fluorescence: The cell density was measured daily via chlorophyll a- fluorescence, excitationat 436 nm, emission at 685 nm. Fluometer: Microplate Reader chameleon V (HIDEX) with software Micro Win 4.41 (MIKROTEC LABORSYSTEME GMBH) No self-fluorescence was found in the preliminary range finding test at the loading level of 100 mg/L.

- Microscopic evaluation: Microscopic evaluation of the cells at the start and the end of exposure was carried out. The cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: √10
- Range finding study: yes
- Preliminary test concentrations:
1.00 mg/L -10.0 mg/L - 100 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (K2Cr2O7)

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

other: NOEL

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: nominal test item loading

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

other: LOEL

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: nominal test item loading

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: nominal test item loading

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: nominal test item loading

Basis for effect:

growth rate

Effect concentrations 5

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: nominal test item loading

Basis for effect:

growth rate

Effect concentrations 6

Key result

Duration:

72 h

Dose descriptor:

other: NOEL

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: nominal test item loading

Basis for effect:

biomass

Effect concentrations 7

Duration:

72 h

Dose descriptor:

other: LOEL

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: nominal test item loading

Basis for effect:

biomass

Effect concentrations 8

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: nominal test item loading

Basis for effect:

biomass

Effect concentrations 9

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: nominal test item loading

Basis for effect:

biomass

Effect concentrations 10

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: nominal test item loading

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid: yes
- EC50: 0.574 mg/L CI: 0.549 - 0.596 mg/L (Growth rate)
- EC50: 0.337 mg/L CI: 0.313 - 0.345 mg/L (Biomass)

Reported statistics and error estimates:

One way Analysis of variance (NOEL/LOEL) :
Growth rate : Normality test (Shapiro-wilk) Passed (P=0.120)
Yield: Normality test (Shapiro-wilk) Passed (P=0.151)

The program states no significant difference for all nominal test item loadings compared to the control. Therefore, the NOEL is > or = 100 mg/l

Applicant's summary and conclusion

Validity criteria fulfilled:

yes