Custom Yellow #2

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:

10-mar-2009 to 13-mar-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

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

- Substance type: Bright yellow powder
- Physical state: Solid
- Stability under test conditions: Unknown
- 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, t=24 h and t=72 h
Volume 1 ml
- 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 clear and colourless, except for the highest test concentration that was clear and slightly yellow.

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

Test organisms

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis 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 10E+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:

22.5 - 23.5°C

pH:

At t=0 h: 8.0
At t=72 h: 8.1 - 8.2

Dissolved oxygen:

Not determined.

Salinity:

Not applicable.

Nominal and measured concentrations:

Nominal concentrations: 0.10, 1.0, 10 and 100% of a 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 quantification, 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 Yellow #2 was used for the preparation of test concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 ml, all glass
- Type: open
- Material, size, headspace, fill volume: 100 ml, normal headspace, 50 ml
- Aeration: no
- Initial cells density: 10E+4 cells/ml
- Control end cells density: 1.269x10+6 cells/ml
- No. of vessels per concentration (replicates): 6 replicates of the highest concentration, 3 replicates for each lower concentration
- No. of vessels per control (replicates): 6
- 1 extra replicate of each test solution for sampling purposes, 2 extra replicates of the highest concentration without algae and 1 extra replicate of each lower test concentration without algae.

GROWTH MEDIUM
- Standard medium used: yes, M2
- Culture medium different from test medium: yes, for culturing M1 medium is used

OTHER TEST CONDITIONS
- Photoperiod: continuous
- Light intensity and quality: TLD-lamps of the type ‘Cool-white’ of 30 Watt, with a light intensity within the range of 89 to 98 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

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.:

> 0.05 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: EC50 > water solubility

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.05 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: NOEC = water solubility

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

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.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Due to the very low solubility of Custom Yellow #2 in test medium, concentration levels that might be toxic for algae could not be reached.

The EC50 for both growth rate reduction (ERC50: 0-72h) and yield inhibition (EYC50: 0-72h) was above 0.05 mg/l, which was the limit of quantification.

The NOEC for both growth rate reduction and yield inhibition was 0.05 mg/l.

In conclusion: Custom Yellow #2 is not considered to be ecotoxic based on the maximum soluble concentration and the absence of effects at this concentration.