bis({9-[2-(carboxy-κO)phenyl]-6-(bis(tallow alkyl hydrogenated)amino)-3H-polyheterocycl-3-ylidene}-3-bis(tallow alkyl hydrogenated)iminium) zinc(2+) dichloride

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:

23-mar-2009 to 16-apr-2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Details on test material:

Substance type: dark purple waxy solid
- Physical state: solid
- Stability under storage conditions: stable
- Storage condition of test material: at room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all test concentrations and the control
- Sampling method:
Frequency: at t=0 h and t=48 h
Volume: 2 ml from the approximate centre of the test vessels
- Sample storage conditions before analysis: Samples were stored in a freezer until analysis

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preparation of test solutions started with a loading rate of 100 mg/l applying 1 day of magnetic stirring to reach maximum solubility of the test substance in the test medium. The resulting aqueous mixture was filtered through a membrane filter (Whatman, 0.45 µm) where after the filtrate was used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the filtrate in test medium. The final test solutions were all clear and colourless.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Source: In-house laboratory culture
- Age at study initiation (mean and range, SD): < 24 hours
- Method of breeding: With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium in an all-glass culture vessel.
- Feeding during test: No

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

180 mg/l expressed as CaCO3

Test temperature:

between 19.4 and 20.2°C

pH:

At t=0 h: 7.6 - 7.8
At t= 48 h: 7.6 - 7.9

Dissolved oxygen:

At t=0 h: 8.7 - 9.0
At t= 48 h: 9.0

Salinity:

Not applicable.

Nominal and measured concentrations:

Final test
Nominal concentrations: 10, 18, 32, 56 and 100% of a 0.45 µm filtered solution prepared at a loading rate of 100 mg/l.

Measured concentrations: Analytical results showed that the actual test concentrations were below the limit of detection, i.e. below 0.05 mg/l, from the start of the test. Therefore, these results indicated that medium solubility was below 0.05 mg/l. Analysis of the filter residue showed that Custom Red #3 was used for the preparation of test concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 ml, all glass
- Type (delete if not applicable): open - ~80 ml, normal headspace
- Aeration: no
- 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: Adjusted ISO medium
- Culture medium different from test medium: yes, for culturing M7 medium is used, pH 7.7 +/- 0.3

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours photoperiod daily

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : EC50 daily

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Test concentrations: 10, 18, 32, 56 and 100% of a 0.45 µm filtered solution prepared at a loading rate of 100 mg/l

Reference substance (positive control):

yes

Remarks:

potassium dichromate (K2Cr2O7)

Results and discussion

Effect concentrations

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: % of the 0.45 µm filtered solution prepared at a loading rate of 100 mg/l.

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: The EC50 exceeded the maximum solubility in test medium; measured concentrations were below the limit of detection.

Details on results:

- Behavioural abnormalities: no
- Mortality of control: no
- Other adverse effects control: no

No significant immobilisation was observed in the solution containing 56% of the 0.45 µm filtered solution prepared at a loading rate of 100 mg/l (NOEC) or in the lower concentrations. In the undiluted filtrate a maximum response of 25% immobilisation was observed, which confirmed the result of the combined limit/range-finding test: the 48h-EC50 exceeding the maximum solubility in test medium.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50/LC50:
The 24h-EC50 was 0.47 mg/l with a 95% confidence interval between 0.44 and 0.53 mg/l.

The 48h-EC50 was 0.28 mg/l with a 95% confidence interval between 0.25 and 0.31 mg/l.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Custom Red #3 did not induce acute immobilisation of Daphnia magna at a concentration containing 56% of the 0.45 µm filtered solution prepared at a loading rate of 100 mg/l after 48 hours of exposure (NOEC).

The 48h-EC50 exceeded the solubility in test medium, i.e. exceeded a 0.45 µm filtered solution prepared at a loading rate of 100 mg/l. The measured concentration in this solution was below the detection limit of the analytical method (0.05 mg/l).