Benzeneacetic acid, 2-(6-carboxy-2,3-dimethylphenoxy)-

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:

24-nov-2008 -- 18-dec-2008

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:

2009

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

Reference substance name:

2-[2-(carboxymethyl)phenoxy]-3,4-dimethylbenzoic acid

EC Number:

601-485-7

Cas Number:

117570-93-1

Molecular formula:

C17 H16 O5

IUPAC Name:

2-[2-(carboxymethyl)phenoxy]-3,4-dimethylbenzoic acid

Details on test material:

- Name of test material (as cited in study report): ASA404 C7
- Substance type: Off white powder
- Physical state: Solid
- Analytical purity: 99.9%
- Lot/batch No.: 072301
- Expiration date of the lot/batch: 18 March 2009
- Stability under test 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 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)
- Method: Preparation of test solutions started with a loading rate of 100 mg/l applying a 10-minute treatment period with ultrasonic waves followed by 49 hours of magnetic stirring to accelerate the dissolving of the test substance in the test medium. Obtained solutions contained undissolved material dispersed through and floating on the surface. The dispersion was consequently filtered through a 0.45 µm membrane filter (Whatman, RC55) to remove the non-dissolved material. The lower test concentrations were prepared by subsequent dilutions of the filtrate in test medium. . The final test solutions were all clear and colourless. Note that the control received similar treatment to prevent for any possible treatment related effects.

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 104 cells/ml.

- Controls: blank control and reference substance

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): 3 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 104 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

Test conditions

Hardness:

0.24 mmol/l (24 mg CaCO3/l)

Test temperature:

22.0 - 23.7°C.

pH:

At t=0 h: 7.9 - 8.1
At t=72 h: 6.1 - 8.3

Nominal and measured concentrations:

Nominal concentrations: 0.1, 1.0, 10 and 100 mg/l
Measured concentrations:
At t=0 h: 99% of nominal
At t=72 h: 106% of initial

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 ml, all-glass,
- Type: open
- Material, size, headspace, fill volume: glass, 150 ml, normal headspace, 50 ml
- Aeration: no
- Initial cells density: 10000 cells/ml
- Control end cells density: ca 257000 cells/m;.
- No. of vessels per concentration (replicates): 6 replicates of the highest concentration, 3 replicates for in-between concentrations.
- No. of vessels per control (replicates): 1 replicate of each test concentration without algae.
- No. of vessels per vehicle 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: 60 to 120 µE/m2/s using TLD-lamps of the type ‘Cool-white’ of 30 Watt



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 immersion probe (pathlength =20 mm). Algal medium was used as blank and the extra replicates as background for the treated solutions.
72 h NOErC, 72 h NOEbC, 72 H ErC50, 72 h EbC50


TEST CONCENTRATIONS
- 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

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

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 1.7 mg/l with a 95 % confidence interval ranging from 1.2 to 2.5 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 0.58 mg/l with a 95 % confidence interval ranging from 0.36 to 0.92 mg/l.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

It is concluded that this test is valid and that Pseudokirchneriella subcapitata, ASA404 C7 did not reduce growth rate at any of the concentrations tested. The yield was significantly reduced at 100 mg/l.

The EC50 for growth rate reduction (ERC50: 0-72h) and the EC50 for yield inhibition (EYC50: 0-72h) were beyond the range tested, i.e. exceeded 100 mg/l.

The NOEC for growth rate was 100 mg/l.


The EC50 for growth rate reduction (ERC50: 0-72h) and the EC50 for yield inhibition (EYC50: 0-72h) were beyond the range tested, i.e. exceeded 100 mg/l.

The NOEC for growth rate was 100 mg/l.