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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
16 July 2012 - 24 August 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., compds. with 2-propanamine
EC Number:
284-664-9
EC Name:
Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., compds. with 2-propanamine
Cas Number:
84961-74-0
Molecular formula:
N/A
IUPAC Name:
Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs.-, compd. with 2-propanamine (1:1)
Test material form:
liquid: viscous
Details on test material:
- Name of test material (as cited in study report): Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., compds. with 2-propanamine (1:1)
- Substance type: technical product (BIO-SOFT 411-E)
- Physical state: viscous liquid
- Analytical purity: 97% (communication with sponsor)
- Lot/batch No.: 50148
- Expiration date of the lot/batch: 30 June 2013
- Storage condition of test material: room temperature in the dark

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Samples were taken from the control (replicates R1 - R6 pooled) and each test group (replicates R1 - R3 pooled) at 0 and 72 hours for quantitative analysis. All samples were stored at approximately -20°C prior to analysis. Duplicate samples were taken at 0 and 72 hours and stored at approximately -20°C for further analysis if necessary. Only samples at the NOEC and above were procided for analysis.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 100 mg test material/L was prepared in the following way: an amount of test material (100 mg) was dissolved in culture medium with the aid of high shear mixing at approximately 7500 rpm for 5 minutes and the volume adjusted to 1 liter to give a 100 mg/L stock solution.
A series of dilutions was made from this stock solution to give further stock solutions of 32, 10, 3.2 and 1.0 mg/L. An aliquot (500 mL) of each stock solution was separately inoculated with algal suspension (10 mL) to give the required test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchnerella subcapitata
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland
- Age of inoculum (at test initiation): In log phase growth
- Method of cultivation: The master cultures were maintained in the laboratory by the periodic replenishment of culture medium (AAP-medium). The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1°C

ACCLIMATION
- Acclimation period: Prior to the start of the test sufficient master culture was added to appoximately 100 mL volumes of culture media (AAP) contained in conical flasks to give an initial cell density of approximately 10³ cell/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 - 150 rpm) and constant illumination at 24 ± 1°C until the algal cell density was approximately 10^4 - 10^5 cell/mL
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: Not checked at start of test. After 72 hours no abnormalities were detected in any of the control or test cultures.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Test temperature:
24 ± 1°C
pH:
At the start of the test: 7.3 - 7.9
At the end of the test: 7.6 - 7.9
Nominal and measured concentrations:
Test material:
Nominal: 1.0, 3.2, 10, 32 and 100 mg/L
Measured (for concentrations at NOEC (10 mg/L nominal) and higher): 7.5, 24 and 80 mg/L (geometric mean)
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Type (delete if not applicable): plugged with polyurethane foam bungs
- Material, size, headspace, fill volume: fill volume 100 mL
- Aeration: constant agitation by orbital shaker (150 rpm)
- Initial cells density: 5.07 x 10^3 cells/mL
- Control end cells density: 8.24 x 10^5 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (AAP)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: AAP medium prepared in reverse osmosis purified deionized water
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: approximately 7000 lux provided by warm white lighting (380 - 730 nm)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Coulter Multisizer Particle Counter, at 0, 23, 48 and 72 hours after start of exposure

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations: 0.10, 1.0, 10 and 100 mg test material/L.
- Results used to determine the conditions for the definitive study: no significant effect on growth at test concentrations of 0.10 and 1.0 mg test material/L, reduced growth at 10 and 100 mg test material/L (>50% growth reduction at 100 mg/L)
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
190 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: extrapolated value
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 80 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
160 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: extrapolated value
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
7.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
46 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
32 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
7.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Other: After the 72 hour test period, no abnormalities were detected in the control and test cultures at 1.0, 3.2, 10, 32 and 100 mg test material/L.
At the start of the test all control, 1.0 and 3.2 mg/L test cultures were observed to be clear colorless solutions, the 10, 32 and 100 mg/L test cultures were observed to be clear colorless solutions with foam at the surface. After the 72-hour test period all control, 1.0, 3.2, 10 and 32 mg test material/L test cultures were observed to be pale green dispersions whilst the 100 mg test material/L test cultures were observed to be very pale green dispersions.
- Any stimulation of growth found in any treatment: At the two lowest test material concentration (1.0 and 3.2mg/L), the average cell density was higher than that of the control after 48 and 72 hours.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: None
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50: ErC50 (0-72 h) = 1.4 mg potassium dichromate/L (95% confidence limits 1.2 - 1.7 mg/L); EyC50 (0-72 h) = 0.59 mg potassium dichromate/L (95% confidence limits 0.53 - 0.65 mg/L)
- Other: NOEC based on growth rate: 0.25 mg/L; NOEC based on yield: 0.25 mg/L
Reported statistics and error estimates:
One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) Students t-test was carried out on the growth 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. All statistical analyese were performed using the SAS computer software package (SAS, 1999-2001).
Both based on growth rate and based on yield, there were no statistically significant differences between the control, 1.0, 3.2 and 10mg test material/L test concentrations (P≥0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate or yield was 10 mg test material/L (nominal concentrions), equalling to 7.5 mg/L based on geometric mean measured concentrations.
The EC50 based on growth rate was higher than the highest tested concentration (> 100 mg/L), therefore an extrapolated EC50 value was determined using the XIFit Sigmoidal Dose-Response Model (A+((B-A/(1+((C/x)^D))))).

Any other information on results incl. tables

Table 1. Measured test material concentrations related to nominal concentrations.

Nominal

0 hours

72 hours

Geometric mean

mg/L

mg/L

% of nominal

mg/L

% of nominal

mg/L

% of nominal

Control

<LOQ

-

<LOQ

-

<LOQ

-

10

8.47

85

6.64

66

7.5

75

32

25.8

81

22.4

70

24

75

100

84.1

84

75.8

76

80

80

LOQ = Limit of quantitation

- = not applicable

Current regulatory advice is that in cases where a decline in measured concnetrations is observed, geometric mean measured concentrations should be used for calculating EC50 values. It was therefore considered justiable to base the results on the geometric mean measured test concentrations in order to give a 'worst case' analysis of the data.

 

Cell densities and pH values for the control and all tested concentrations are given in Table 2 which is attached in the background information section.

Inhibition of growth rate and yield in the definitive test for the control and all tested concentrations are given in Table 3 which is attached in the background information section.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The 72-hr NOEC for algae was 7.5 mg/L. The 72-hr EyC50 was 32 mg/L and the 72-hr ErC50 (extrapolated value) was 160 mg/L for algae.
Executive summary:

Pseudokirchneriella subspicatatawas exposed to concentrations of 0, 1.0, 3.2, 10, 32 and 100 mg/L of Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs.-, compd. with propanamine (1:1) for 72 hrs. The cell concentration was measured at 23, 48, and 72 hrs. Effect values were based on geometric mean measured concentrations as worst case estimate since the measured concentrations declined over the 72 hour test period from 81-85% of nominal to 66-76% of nominal. The 72-hr NOEC was 7.5 mg/L. The 72 -hr EyC50 was 32 mg/L, and the 72-hr ErC50 (extrapolated value) was 160 mg/L.