Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., calcium salts

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2009

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2006 (corrected 2001)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Duplicate samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. All samples were stored frozen prior to analysis.

Vehicle:

no

Details on test solutions:

- Method: water accommodated fractions (WAFs)
Nominal amounts of test item (20, 32, 20, 64 and 200 mg) were each separately added to the surface of 20, 10, 2, 2 and 2 liters of culture medium to give the 1.0, 3.2, 10, 32 and 100 mg/L loading rates respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. Visual observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2-4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth sipho ning (the first 75-100 mL discarded). Microscopic observations of the WAFs were performed after filtering and showed there to be no micro-dispersions of test item present. An aliquot (1 litre) of each of the loading rate WAFs was separately inoculated with algal suspension (5.7 mL) to give the required test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAF.
- Controls: test water (AAP medium)

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS
Research Services Ltd, Scottish Marine Institute, Ohan, Argyll, Scotland.
- Age of inoculum (at test initiation): not reported. Prior to the start of the test master culture was adde
d to culture media to give an initial cell density of approximately 10E+03 cells/mL. The flasks were
kept under constant agitation by orbital shaker (100 - 150 rpm) and constant illumination at 24 ± 1 °C
until the algal cell density was approximately 10E+04 - 10E+05 cells/mL.
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenish
ment of culture medium under constant aeration and constant illumination at 21 ± 1 °C.
ACCLIMATION
- Acclimation period: see above "Age of inoculum"
- Culturing media and conditions (same as test or not): same

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.15 mM (15 mg CaCO3/L)

Test temperature:

24 ± 1 °C

pH:

control
test start (0 h): 7.8
test end (72 h): 8.2
treatments
test start (0 h): 7.6 - 7.9
test end (72 h): 8.0 - 8.4

Nominal and measured concentrations:

nominal loading rates: 1.0, 3.2, 10, 32, and 100 mg/L as WAF
measured test concentrations:
test start (0 h): 0.68 – 71 mg/L
test end (48 h): 74 – 99 % of the initially measured values
The biological results are based on the loading rate of the test item.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks containing 100 mL test medium, plugged with polyurethane foam bungs
- Aeration: continuous shaking at about 150 rpm
Initial cells density: 6 x 10E+03 cells/mL
- Control end cells density: 5.35 x 10E+05 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
GROWTH MEDIUM
- Standard medium used: yes (AAP Medium)
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse osmosis purified deionized water
- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH was measured in each treatment at the start and end of the test. The temperature in the incubator was recorded daily.
OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: approximately 7000 lux, warm white light (380 – 730 nm)
EFFECT PARAMETERS MEASURED (with observation intervals if applicable): daily determination of the algal cell density
- Determination of cell concentrations: electronic particle counter; triplicate counts
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations: nominal 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: 47 % inhibition of the growth rate at 100 mg/L.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

nominal loading rates

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

17 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

nominal loading rates

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

nominal loading rates

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no abnormalities in the control and at loading rates of 1.0 to 32 mg/L; cell debris and few intact cells were observed in the 100 mg/L test cultures.
- Any stimulation of growth found in any treatment: slight stimulation of growth (cell density) at loading rates of 1.0 and 3.2 mg/L (20 % and 12 % after 72 h)
- Effect concentrations exceeding solubility of substance in test medium: no

For details see attached document "Tables Cell densities and inhibition of growth.pdf"

Results with reference substance (positive control):

- Results with reference substance valid? yes
- 72-hour ErC50: 1.0 mg/L 95%, confidence limits 0.90 - 1.2 mg/L (within the range 0.9 - 1.5 mg/L as recommended by the guidelines)

Reported statistics and error estimates:

Determination of ELx Values
Software used: Xlfit software package (ID Business Solution Ltd.)
For each individual test vessel (mean values for yield), percentage inhibition (arithmetic axis) was plotted against test concentration (logarithmic axis) and a line fitted by computerized interpolation. ELx values were then determined from the equation for the fitted line.
Where appropriate 95% confidence limits for the EL5o values were calculated, using the simplified method of evaluating dose-effect experiments of Litchfield and Wilcoxon (1949).

Statistical analyses
Software used: SAS computer software package (SAS, 1999 - 2001).
One way analysis of variance incorporating Bartlett's test for homogeneity of variance and Dunnett's multiple comparison procedure for comparing several treatments with a control was carried out on the growth rate and yield data after 72 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups.
y

Validity criteria fulfilled:

yes

Remarks:

Increase of biomass >16 after 72 hours; mean coefficient of variation for section-by-section specific growth rate <35%; coefficient of variation for average specific growth rate <7%.

Description of key information

effect values for Pseudokirchneriella subcapitata, based on loading rates (OECD 201, EU C.3):

0-72 h average specific growth rates:

EL50: > 100 mg/L, EL10: 17 mg/L, NOELR: 10 mg/L

Additional information

ELr50 for freshwater algae: > 100 mg/L

ECr10 for freshwater algae: 17 mg/L

NOELR for freshwater algae: 10 mg/L