Asphalt, sulfonated, sodium salt

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:

2004-07-02 to 2004-07-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to Guideline ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum).

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum)

Deviations:

yes

Remarks:

: A re-growth study was not conducted due to a technical oversight.

Principles of method if other than guideline:

N/A

GLP compliance:

yes (incl. QA statement)

Remarks:

Department of Health of the Government of the United Kingdom

Test material

Test material information

Constituent 1

Reference substance name:

Asphalt, sulfonated, sodium salt

EC Number:

269-212-0

EC Name:

Asphalt, sulfonated, sodium salt

Cas Number:

68201-32-1

Molecular formula:

UVCB

IUPAC Name:

Asphalt, sulfonated, sodium salt

Details on test material:

- Name of test material (as cited in study report): Na+sulfonated asphalt
- Molecular formula (if other than submission substance): N/A
- Molecular weight (if other than submission substance): N/A
- Smiles notation (if other than submission substance): N/A
- InChl (if other than submission substance): N/A
- Structural formula attached as image file (if other than submission substance): N/A
- Substance type: active
- Physical state: black powder
- Analytical purity: N/A
- Impurities (identity and concentrations): N/A
- Composition of test material, percentage of components: N/A
- Isomers composition: N/A
- Purity test date: N/A
- Lot/batch No.: N/A
- Stability under test conditions: N/A
- Storage condition of test material: approximately 4 deg C, in the dark
- Other: Test material supplied by sponsor.

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

N/A

Test solutions

Vehicle:

no

Details on test solutions:

- 1000 mg of test material was dispersed in culture medium with the aid of ultrasonication for approx. 30 minutes and the volume adjusted to 500 mL to give a 2000 mg/L dispersion.
- Additional stock solutions were prepared by serial dilution.
- An aliquot of each of the stock dispersions was mixed with algal suspension (250 mL) to give the test concentrations: 62.5, 125, 250, 500 and 1000 mg/L.

Test organisms

Test organisms (species):

Skeletonema costatum

Details on test organisms:

TEST ORGANISM
- Common name: Marine algae
- Strain: CCAP 1077/5
- Source (laboratory, culture collection): cultures were obtained from the Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll
- Age of inoculum (at test initiation): N/A
- Method of cultivation: - Cultures were maintained by periodic replenishment of culture medium. Cultures were maintained at 20 +/- 1 deg C under continuous illumination of approx. 7000 lux and constant aeration.



ACCLIMATION
- Acclimation period: N/A
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: N/A

Study design

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

N/A

Test conditions

Hardness:

N/A

Test temperature:

20 +/- 1 deg C. The temperature within the incubator was recorded daily.

pH:

Range- 8.0-8.2. The pH of each control and test flask was determined at initiation of the test and after 72 hours.

Dissolved oxygen:

N/A

Salinity:

N/A

Nominal and measured concentrations:

- The test concentrations used in the definitive test were determined by a preliminary range-finding test.

- Test material (nominal concentrations): Control, 62.5, 125, 250, 500, and 1000 mg/L
- Positive control: 0.313, 0.625, 1.25, 2.5, and 5.0 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: conical flasks
- Type (delete if not applicable): closed- polyurethane foam bungs
- Material, size, headspace, fill volume: glass, 250 mL, 100 mL solution
- Aeration: no, but constant shaking at 150 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): N/A
- Renewal rate of test solution (frequency/flow rate): N/A
- Initial cells density: 1000 cells/mL
- Control end cells density: 2.75 x 10 E5
- No. of organisms per vessel: N/A
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): N/A


GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: N/A


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: standard marine algal medium
- Total organic carbon: N/A
- Particulate matter: N/A
- Metals: N/A
- Pesticides: N/A
- Chlorine: N/A
- Alkalinity: N/A
- Ca/mg ratio: N/A
- Conductivity: N/A
- Culture medium different from test medium: no
- Intervals of water quality measurement: N/A


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous light
- Light intensity and quality: approx. 7000 lux
- Salinity (for marine algae): N/A


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Cell counts at 0, 24, 48, and 72 h
- Determination of cell concentrations: haemocytometer and light microscope
- Chlorophyll measurement: no
- Other: N/A


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline: N/A
- Range finding study
- Test concentrations: 0.10, 1.0, 10, 100, and 1000 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate - Conducted under a separate protocol (exposure conditions were the same as those used in the definitive test).

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

other: EbC50

Effect conc.:

240 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

(area under growth curve)

Remarks on result:

other: 95% CL = 220 - 260 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

other: ErC50

Effect conc.:

390 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL = 350 - 430 mg/L

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

125 mg/L

Nominal / measured:

nominal

Basis for effect:

biomass

Remarks:

(area under growth curve)

Details on results:

GROWTH RESULTS:
- Both growth rate (r) and biomass (b) were affected by the presence of the test substance.
- For the area under the growth curve, there were no statistically significant differences between the control, 62.5, and 125 mg/L test concentrations ( P ≥ 0.05).
- The concentration of the control cultures increased by a factor of 71 (acceptance criteria is a factor of 16).
- The standard test method indicates that a re-growth study should be done to determine whether toxicity was due to the test substance or due to a reduction in light intensity. Due to a technical oversight, the re-growth study was not conducted. However, spectrophotometer readings indicated that the TS significantly absorbs light at 460 and 665 nm, the wavelengths required for photosynthesis, and therefore, it is probable that the observed toxicity of the TS was due to a reduction in light intensity and not the intrinsic toxic properties of the test material.

OBSERVATIONS ON CULTURES:
- All test and control cultures were observed microscopically at 72 h; misshapen cells were observed at 1000 mg/L.

OBSERVATIONS ON TEST MATERIAL SOLUBILITY:
- At test start, all control cultures were observed to be clear, colorless solutions; at 62.5 mg/L, the cultures were pale brown dispersions; at 125, 250, and 500 mg/L, the test cultures were observed to be brown dispersions, while the 1000 mg/L cultures were black/brown dispersions.
- After 72 h, the 1000 mg/L cultures were black/brown dispersions, and all other cultures were brown dispersions.

PHYSICO-CHEMICAL MEASUREMENTS:
- The temperature was maintained at 20 +/- 1 deg C.
- The pH range was 8.0-8.1 (determined at 0 and 72 h).

Results with reference substance (positive control):

POSITIVE CONTROL:
- EbC50 (72 h): 1.1 mg/L, 95% CI = 0.96 - 1.3 mg/L
- ErC50 (0-72 h) : 2.5 mg/L, 95% CI = 1.5 - 4.3 mg/L
- NOEC: 0.625 mg/L
- The results were within the normal range for this reference material given in the ISO Guideline.

Reported statistics and error estimates:

One way ANOVA incorporating Bartlett's test for homogeneity and Dunnett's multiple comparison procedure for comparing several treatments with a control was carried out on the area under the growth curve data at 72 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. EC50 values were determined from the equation for the fitted line in a plot of percent inhibition versus concentration.

Any other information on results incl. tables

Inhibition of Growth Rate and Biomass
Nominal Concentration (mg/L)	Area Under the Curve at 72 h	% Inhibition	Growth Rate (0 – 72 h)	% Inhibition
Control	8.62E+06	-	0.059	-
62.5	9.34E+06	[8]	0.062	[5]
125	8.87E+06	[3]	0.059	0
250	3.86E+06	55	0.048	19
500	3.61E+05	96	0.018	69
1000	-1.34E+05	102	0.002	97
[Increase in growth as compared to the controls]

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

More than the 16-fold increase in control density

Conclusions:

The 72 hr EbC50 of the test material to the growth of Skeletonema costatum has been found to to be 240 mg/L; 95% CI 220 - 260 mg/L and an ErC50 value of 390 mg/L; 95% CL 350 - 430 mg/L. The NOEC at 72 hours was 125 mg/L.

Executive summary:

N/A