Bontron E-108

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:

17 December 2001 to 20 December 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): S178207
- Physical state: Powder
- Analytical purity: 91.4% w/w total ligand, 25.9% w/w free ligand and 69.1% w/w for the strength of S178207
- Lot/batch No.: NBZ 0155/44
- Stability under test conditions: stable
- Storage condition of test material: in the dark at ambient temperature

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 1.0 ml/l nominal concentration of S178207.

- Sampling method: Aqueous samples were diluted with tetrahydrofuran and analysed by HPLC using a UV/Vis detector. The samples were quantified against standards of S178207.

- Sample storage conditions before analysis: not stated in report

Test solutions

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock concentrate of S178207 was prepared by dispersing 0.1 g of S178207 in 10 ml od dimethylformamide. The test solution was prepared from this stock solution by addition of an appropriate volume to 1 litre of culture medium and stirring thoroughly; thus giving a final dimethylformamide concentration of 0.10 ml/l in each exposure vessel. Additions were made sub-surface, gradually, by microlitre syringe, into solutions vigorously stirred by magnetic follower. the resultant solutions were clear and colourless.

- Eluate: same as culture media

- Differential loading: no

- Controls: a solvent control was prepared by the addition of 100 µl of dimethylformaide to 1 litre of culture medium, to give a solvent concentration of 0.1 ml/l. Additions were made sub-surface, gradually, by microlitre syringe, into solutions vigorously stirred by magnetic follower. the resultant solutions were clear and colourless.

The (negative) control consisted of culture medium only.

Control groups was maintained under identical conditions but not exposed to the test material.

- Chemical name of vehicle (organic solvent, emulsifier or dispersant): dimethylformamide

- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 1.0 mg/l

- Evidence of undissolved material (e.g. precipitate, surface film, etc): none stated in report

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: ATCC 22662
- Source (laboratory, culture collection): not stated in report
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: the culture was grown in the medium and under the same environmental conditions, described for the test.


ACCLIMATION
- Acclimation period: not stated in report
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: not applicable to algae study

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

All test and control cultures were inspected microscopically at 72 hours.

Test conditions

Hardness:

Not stated in report.

Test temperature:

The daily temperature measurements recorded, by thermometer, in the incubator ranged from 24 to 24.1°C. The hourly temperature measurements, recorded automatically, remained within 24 ± 2°C.

pH:

At the start of the test the pH ranged from 7.43 to 7.44 and at the end of the test the range was from 9.61 to 10.30. A maximum increase of 2.86 pH units was observed over the test duration.

Dissolved oxygen:

Not stated in report

Salinity:

Not stated in report

Nominal and measured concentrations:

Single nominal concentration of 1.0 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 ml borosilicate glass conical flask
- Type: closed, with polyurethane foam bungs
- Material, size, headspace, fill volume: 250 ml borosilicate glass conical flasks each containing 100 ml of test solution
- Aeration: none
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable as static test conditions
- Renewal rate of test solution (frequency/flow rate): not applicable as static test conditions
- Initial cells density: 1.05E+04 cells/ml
- Control end cells density:
- No. of organisms per vessel: not applicable
- No. of vessels per concentration (replicates): six
- No. of vessels per control (replicates): six
- No. of vessels per vehicle control (replicates): six


GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: not applicable


TEST MEDIUM / WATER PARAMETERS
To approximately 900 ml of distilled water, was added 1 ml solutions of A1, A2, A3 and B. These were made up to 1 litre with distilled water. These solutions were autoclaved at 121°C for 15 minutes and allowed to cool. 1 ml of solution C was added using aseptic techniques. All solutions were stored under refrigeration.

Solution A1:
12.75 g of NaNO3, 6.082 g of MgCl2.6H2O, 2.205 g of CaCl2.2H2O and 500 ml of distilled water

Solution A2:
7.35 g of MgSO4.7H2O and 500 ml of distilled water

solution A3:
0.684 g K2HPO4.3H2O and 500 ml distilled water

Solution B (Micronutrients):
0.093 g of H3BO3, 0.208 g of MnCl2.4H2O, 0.080 g of FeCl3.6H2O, 0.150 g of Na2EDTA.2H2O, 1.64 mg of ZnCl2, 0.714 mg of CoCl2.6H2O, 3.36 mg Na2MoO4.2H2O, 0.006 mg CuCl2.2H2O and 500 ml of distilled water

Solution C
7.50 g NaHCO3 and 500 ml of distilled water


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: any change in pH was not adjusted
- Photoperiod: continuous illumination
- Light intensity and quality: Light intensity, measured once during the study, was 7500 lux (by cosine receptor). This was also measured in terms of quantum response and was 90.9 ╢ Einsteins per m2 per second
- Salinity (for marine algae): not applicable


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Determined by electronic particle counting, using a coulter model ZM, counting at a lower threshold equivalent spherical diameter of approximately 2.3 µm.
- Chlorophyll measurement: no
- Other: not applicable


TEST CONCENTRATIONS
- Spacing factor for test concentrations: not applicable, as only one test concentration used

- Justification for using less concentrations than requested by guideline: not stated in report

- Range finding study
-no range finding study conducted

Reference substance (positive control):

not specified

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

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): There were no abnormalities detected in any of the control or test cultures at 72 hours.
- Any stimulation of growth found in any treatment: yes
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

Not stated in report.

Reported statistics and error estimates:

Comparison of biomass integral and gowth rate were examined by one-way analysis of variance and dunnett's procedure was used to identify significant differences (P = 0.05) from the solvent control.

Any other information on results incl. tables

The pH shift was considered to be a function of the very high growth factors observed (mean culture medium control 72 hour cell density = 223 x 0 hour initial inoculum density). The pH shift occurred despite a high orbital shaking rate of 160 rpm to improve mass transfer of carbon dioxide into the test solutions.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on area under the growth curve and the logarithmic growth rate over the test period, the results obtained were:

No observed effect concentration (NOEC) = 1.0 mg/l
Lowest observed effect concentration (LOEC) = >1.0 mg/l
Median effective concentration, both biomass (EbC50) and growth rate (ErC50) = >1.0 mg/l

Executive summary:

A study was performed to assess the effect of the test material on the growth of the green algae Selenastrum capricornutum. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test".

Based on area under the growth curve and the logarithmic growth rate over the test period, the results obtained were:

No observed effect concentration (NOEC) = 1.0 mg/l

Lowest observed effect concentration (LOEC) = >1.0 mg/l

Median effective concentration, both biomass (EbC50) and growth rate (ErC50) = >1.0 mg/l