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

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:

23-mar-2009 to 01-may-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 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000

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: Volume 2 ml, at t=0 h, t=24 h and t=72 h
- 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)
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 up to 32% of the filtrate were clear and colourless while the undiluted filtrate was clear and light yellow.

After preparation, volumes of 50 ml were added to each replicate of the respective test concentration. Subsequently, 1 ml of an algal suspension was added to each replicate providing a cell density of 10^4 cells/ml.

- Controls: blank control and reference substances

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture
- Age of inoculum (at test initiation): 4 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/ml.
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None.

Test conditions

Hardness:

0.24 mmol/l (24 mg CaCO3/l)

Test temperature:

between 22.0 and 22.9°C

pH:

At t=0 h: 7.5 - 8.1
At t=72 h: 7.6 - 7.9

Dissolved oxygen:

Not determined.

Salinity:

Not applicable.

Nominal and measured concentrations:

Final study:
Nominal concentrations: 1.0, 3.2, 10, 32 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 0.08 mg/l, from the start of the test. Therefore, these results indicated that medium solubility was below 0.08 mg/l. Analysis of the filter residue showed that Custom Red #3 was used for the preparation of test concentrations. Based on these results the effect concentrations were expressed as percentages of a 0.45 µm filtered solution prepared at a loading rate of 100 mg/l.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 ml, all glass
- Type: open
- Material, size, headspace, fill volume: 150 ml, normal headspace, 50 ml
- Aeration: no
- Initial cells density: 10000 cells/ml
- Control end cells density: 1343000 cells/ml
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6


GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: TLD-lamps of the type ‘Cool-white’ of 30 Watt, with a light intensity within the range of 90 to 94 µE/m2/s

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter cell densities were determined by spectrophotometric measurement of samples at 720 nm using a Varian Cary 50 single beam spectrophotometer with cuvettes (pathlength =10 mm). Algal medium was used as blank and the extra replicates without algae as background for the treated solutions.]
- Other: 72 h NOErC, 72 h NOEyC, 72 h ErC50, 72 h EyC50

TEST CONCENTRATIONS
Limit study:
- Spacing factor for test concentrations: 10
- Test concentrations: 0.1, 1.0, 10 and 100% of a 0.45 µm filtered solution prepared at a loading rate of 100 mg/l.
Main study
- Spacing factor for test concentrations: 3.2
- 1.0, 3.2, 10, 32 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

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.9 other: % of the filtrate

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 2.3-6.7 % of the filtrate. As all measured concentrations were below the LOD (0.08 mg/L), the EC50 is also < 0.08 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 other: % of the filtrate

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: As all measured concentrations were below the LOD (0.08 mg/L), the NOEC is also < 0.08 mg/L

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Any stimulation of growth found in any treatment: no

Growth rates were in the range of the controls in a solution containing 1.0% of the filtrate, whereas the growth rate of algae exposed to higher concentrations were strongly reduced. Statistically significant reduction of growth rate was found at test concentrations containing 3.2% of the filtrate and higher (Bonferroni t-test, α = 0.05).

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50:
The EC50 for growth rate reduction (ERC50: 0-72h) was 1.6 mg/l with a 95 % confidence interval ranging from 1.2 to 2.1 mg/l. The historical ranges for growth rate reduction lie between 0.82 and 2.3 mg/l. Hence, the ERC50: 0-72h for the present batch corresponds with this range.

The EC50 for yield inhibition (EYC50: 0-72h) was 0.73 mg/l with a 95 % confidence interval ranging from 0.51 to 1.1 mg/l. Historical ranges are not yet available.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study with Pseudokirchneriella subcapitata, Custom Red #3 reduced growth rate and inhibited the yield of this fresh water algae species significantly in a solution containing 3.2% of the 0.45 µm filtered solution prepared at a loading rate of 100 mg/l and higher.

The EC50 for growth rate reduction (ERC50: 0-72h) was 3.9% with a 95% confidence interval ranging from 2.3 to 6.7% of the filtrate.

The NOEC for growth rate reduction was 1.0% of the filtrate.

As all measured concentrations were below the limit of detection (0.08 mg/L) of the analytical method, both the NOEC and EC50 are < 0.08 mg/L.