Registration Dossier

Diss Factsheets

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 January to 20 February 2016
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The test was conducted according to an appropriate guideline under GLP conditions. It has been assigned reliability 2 due to the non-specific analysis (TOC) of the test substance concentrations.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0.32, 1.0, 3.2, 10, 32 and 100 mg/L.
- Sampling method: 4 replicates of each test concentration without algae were made for sampling purposes. Specific analyses proved not possible and, after consultation with the sponsor, it was decided to analyse the Total Organic Carbon (TOC) concentration as a surrogate measure.
Singular samples for TOC-analysis were taken from all test concentrations without algae and the control without algae according to the schedule below:
Frequency at t=0 h, t=24 h and t=72 h
Volume 40 mL
Additionally, singular reserve samples of 40 mL were taken from all test solutions without algae for possible analysis. If not already used, these samples were stored in a refrigerator for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.
- Sample storage conditions before analysis: Storage Samples were stored in a refrigerator until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The batch of CF-73 tested was a clear light yellow liquid and completely soluble in test medium at the tested concentrations. No correction was made for the purity/composition of the test item.
Preparation of test solutions started with the highest test concentration, i.e. 100 mg/L. No special treatment other than careful mixing was required to dissolve the test item in medium. After adjusting the pH of the resulting solution from 9.4 (combined limit/range-finding test) or 9.7 (final test) to 8.1 with 1N HCl (Merck, Darmstadt, Germany), lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All final test solutions were clear and colourless.
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: Test medium without test item or other additives.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): All final test solutions were clear and colourless.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis 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. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- 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.

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions (same as test or not): Yes
- Any deformed or abnormal cells observed: None reported
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
0.24 mmol/L (24 mg CaCO3/L)
Test temperature:
22-23°C
pH:
9.7 in the 100 mg/l stock solution, adjusted to 8.1 before dilution of lower test concentrations.
pH ranged from 8.0-8.2 during the test.
Dissolved oxygen:
Not measured
Nominal and measured concentrations:
Nominal: 0.32, 1.0, 3.2, 10, 32 and 100 mg/L.
TOC measured: Initial: 0.18, 0.56, 1.8, 5.6, 18, 56 mg/l;
After 72 h: 0.73, 0.89, 1.5, 5.1, 16, 53 mg/l.
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL, all-glass, containing 50 mL of test solution
- Type (delete if not applicable): open / closed: capped
- Material, size, headspace, fill volume: glass, 100 ml, 50 ml, 50 ml
- Aeration: No
- Initial cells density: 1 x 10⁴ cells/mL.
- Control end cells density: 172.2 x 10⁴ cells/mL.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes: M2 medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO water
- Culture medium different from test medium: no
- Intervals of water quality measurement: pH measured at the beginning and end of the test. Temperature was measured continuously.

OTHER TEST CONDITIONS
- Sterile test conditions: yes/no: Not reported
- Adjustment of pH: Yes
- Photoperiod: Continuous
- Light intensity and quality: TLD-lamps with a light intensity within the range of 86 to 92 μE/m2/s.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Cell density at the beginning of the test and at 24, 48 and 72 hours.
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]: 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 680 nm using a spectrophotometer with immersion probe (path length = 20 mm). Algal medium was used as blank. In order to correct for turbidity, one control per concentration without algae was measured at each time interval.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: approximately 3.2
- Justification for using less concentrations than requested by guideline:
- Range finding study: Yes
- Test concentrations: 1, 10 and 100 mg/l
- Results used to determine the conditions for the definitive study: growth rate inhibition: 2.7 %, 4.4 % and 24.3 % at 1, 10 and 100 mg/l respectively.
Inhibition of yield: 13.2, 20.3 and 70.5 % at 1, 10 and 100 mg/l respectively.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
11 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval ranging from 9.7 to 12 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
3.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield inhibition
Remarks on result:
other: 95% confidence interval ranging from 2.4 to 3.9 mg/L.
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield reduction
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
3.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % CL 2.8-4.8 mg/l
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.6
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CL 0.31-0.90
Details on results:
- Exponential growth in the control (for algal test): yes
- Any stimulation of growth found in any treatment: no
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
- Results with reference substance valid?Yes
- EC50: 1.5 mg/l with a 95% confidence interval ranging from 1.4 to 1.6 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. Hence, the 72h-ERC50 for the algal culture tested corresponds with this range.
- Other: The EC50 for yield decrease (72h-EYC50) was 0.51 mg/L with a 95% confidence interval ranging from 0.49 to 0.53 mg/L. The historical ranges for yield decrease lie between 0.43 and 1.1 mg/L. Hence, the 72h-EYC50 for the algal culture tested corresponds with this range.
Reported statistics and error estimates:
Shapiro-Wilk´s Test on Normal Distribution.
Levene´s Test on Variance Homogeneity (with Residuals).
Determination of NOEC:
Williams Multiple Sequential t-test Procedure
Validity criteria fulfilled:
yes
Conclusions:
A 72-h EC50 of 11 mg/l, an EC10 of 3.9 mg/l and a NOEC of 1 mg/l (nominal concentration) have been determined for the effects of the submission substance on growth rate of the green algae Pseudokirchneriella subcapitata. Each constituent of the substance hydrolyses rapidly in water, therefore it is likely that the algae were exposed to the hydrolysis products of the test substance.

Description of key information

Toxicity to freshwater algae: 72 hour EC50 11 mg/L, EC10 3.9 mg/l and NOEC 1 mg/L (nominal), for the effects of the test substance on growth rate of Pseudokirchneriella subcapitata, in accordance with guideline OECD 201. The rapid hydrolysis rates of the constituents of the test substance mean that the algae were predominantly exposed to the hydrolysis products of the test substance.

Key value for chemical safety assessment

EC50 for freshwater algae:
11 mg/L
EC10 or NOEC for freshwater algae:
3.9 mg/L

Additional information

A 72-h EC50 of 11 mg/L, an EC10 of 3.9 mg/L and a NOEC of 1 mg/L (nominal concentration) have been determined for the effects of the submission substance on growth rate of the green algae Pseudokirchneriella subcapitata. Each constituent of the submission substance hydrolyses rapidly or very rapidly in contact with water, therefore it is likely that the algae were exposed to the hydrolysis products of the test substance.

Verification of test concentrations was carried out by Total Organic Carbon (TOC) analysis. At nominal test item concentrations of 3.2 mg/L and lower, the measured TOC concentrations fluctuated during the exposure period (69-83% of initial after 72 hours), which may be attributed to these concentrations nearing the detection limit of 1.0 mg C/L of the TOC analyser. Higher concentrations remained stable during the exposure (82-95% of initial). Since TOC-analysis is a non-specific method, results are reported in terms of the nominal concentration expressed as mg test item per litre.

Categories Display