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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 Sep. - 05 Dec. 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
March 2006
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: soluble

OTHER SPECIFICS: White Powder
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 100 mg/L test substance, Negative Control (NC)
- Sampling method: Samples of 1.0 mL were taken from the additional vessels of treatment A and NC at 0 h, 24 h, 48 h and 72 h and filled into 1.5 mL Eppendorf vials. At 72 h additionally samples of the test solution were taken. The samples were stored in the freezer at ≤ -18 °C.
- Sample storage conditions before analysis: Storage at -20 °C. Additional retain samples were stored in the freezer at ≤ -18 °C.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: 55.5 mg of the test item are dissolved in 500 ml OECD TG 201 medium by stirring with a magnetic stirrer for about 5 minutes. The methodical dilution by a factor of 1.11 results in a start concentration of 100 mg/L. For the accompanying chemical analysis two vessels including algae were prepared (one for each, the control and the treatment).
- Eluate: Algal growth medium
- Controls: yes
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata (previous name: Raphidocelis subcapitata)
- Strain: No. 61.81 SAG
- Source (laboratory, culture collection): Culture Collection of Algae at the University of Goettingen, subcultured at Hydrotox GmbH since October 2015
- Age of inoculum (at test initiation): 4 days (82.78 × 10^5 cells/mL; to obtain a nominal inoculum concentration of 7 × 10^4 cells/mL, 1.27 mL pre-culture was diluted into 150 mL OECD TG 201 medium).
- Method of cultivation: Suspension of culture in Holm-Hansen medium under axenic conditions.

ACCLIMATION
- Acclimation period: 4d
- Culturing media and conditions (same as test or not): culturing - Holm-Hansen medium, test - OECD TG 201 medium; other conditions are the same (23.2 - 23.3 °C and 73.2 μE m-² s-1 ± 5.6 % continuous lighting)
- Any deformed or abnormal cells observed: no
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Hardness:
no data
Test temperature:
23.3 - 23.4 °C
pH:
control: 7.7 - 7.8
test item treatment: 7.6 - 7.7
Dissolved oxygen:
n.a.
Salinity:
n.a.
Conductivity:
no data
Nominal and measured concentrations:
Nominal Concentration: 100 mg/L
Recovery rates of 1-Benzyl-3-carbamoyl-pyridinium, chloride in the test item treatment ranged between 101 and 103 % of the nominal concentrations. The concentrations of 1-Benzyl-3-carbamoyl-pyridinium, chloride in the control were below the limit of quantification.
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer glass flasks 100 mL, Schott, Mainz
- Type (delete if not applicable): closed (sealed with cellulose stoppers)
- Fill volume: 50 mL
- Aeration: no
- Initial cells density: 0.070x10^5 cells/mL
- Control end cells density: 6.174 x10^5
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (OECD TG 201 medium)

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: Holm-Hansen medium

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continous lighting
- Light intensity and quality: 73.2 μE m-² s-1 ± 5.6 %

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Measurement of chlorophyll fluorescence to be converted into cell counts per ml by means of a correlation factor. The correlation factor is based on cell counts with a Coulter Counter and the corresponding fluorescence is measured with a microplate fluorescence reader.
- Chlorophyll measurement: Chlorophyll fluorescence

TEST CONCENTRATIONS
- Range finding study: yes, not GLP-conform
* Test concentrations (in range finding test): 10 and 100 mg/L
* Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
no
Remarks:
Quality assurance takes place regularly by testing the test organisms to potassium dichromate. The recent quality testing was performed in June 2016 which resulted in an ErC50 of 0.99 mg/L (CL 95 %: 0.89 – 1.1 mg/L). The EyC50 was 0.52 mg/L.
Key result
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:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
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.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
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 - the morphology of the algal cells in the test loading rates as well as in the control showed no obvious abnormality.
- Unusual cell shape: no
- Colour differences: not reported
- Flocculation: not reported
- Adherence to test vessels: not reported
- Aggregation of algal cells: not reported

- Any stimulation of growth found in any treatment: no
- 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: no
Results with reference substance (positive control):
n.a.
Reported statistics and error estimates:
ECx and NOEC/LOEC values were determined with the statistical software ToxRat 3.2.1 (ToxRat Solutions GmbH, Alsdorf), as far as mathematically possible.

The inhibition of yield in the test item treatments, in relation to the control, is presented in Table 2. Respectively, the inhibition of the specific growth rate in the test item treatments, in relation to the control, is presented in Table 3. Both evaluations are illustrated as graphs and shown in the following illustration part of this documentation (figure 1 and figure 2). The effect concentrations after 72 h exposure are calculated with the statistical software ToxRat Professional 3.2.1 (see Table 4).

Table 2: Inhibition of yield after 24 h, 48 h and 72 h exposure

    Nominal test item loading rate [mg/L]  Inhibition of yield [%]      
 24 h  48 h  72 h
 NC  -- --  -- 
 100 15.3  20.5  24.4 

Table 3: Inhibition of growth rate after 24 h, 48 h and 72 h exposure

    Nominal test item loading rate [mg/L]  Inhibition of growth rate [%]      
 24 h  48 h  72 h
 NC  -- --  -- 
 100 12.2  10.9 10.8

Table 4: Effect concentrations for yield and growth rate at the end of the Test (72 h)

 (Lowest/No Observed) Effect Concentration      Nominal test item concentration [mg/L]
Yield   Growth rate
LOEC >100   <100
NOEC >=100  >=100 
EC50   >100 >100 
Validity criteria fulfilled:
yes
Conclusions:
The 72-h ErC50 of 1-Benzyl-3-carbamoyl-pyridinium, chloride to Pseudokirchneriella subcapitata (formerly known as Raphidocelis subcapitata) is ≥ 100 mg/L.
Executive summary:

In a 72 h growth inhibition test, cultures of Raphidocelis subcapitata (Strain No. 61.81 SAG, now classified as Pseudokirchneriella subcapitata) were exposed to 1-Benzyl-3-carbamoylpyridinium, chloride at a nominal concentrations of 0 (control) and 100 mg/L under static conditions in accordance with the OECD Guideline 201 (March 2006) and under GLP. The test design was chosen as limit test, as a pre-test (not GLP conform) with 10 and 100 mg/L nominal concentrations showed no growth inhibition.

Within the limit test, the test item concentration of 100 mg/L resulted in an inhibition of yield of 24.4 % (after 72 h) and an inhibition of the growth rate of 10.8 % (after 72 h) which is statistically not a significant difference to the control. As no effect was observed the EC50 and LOEC for both, growth rate and yield, must exceed 100 mg/L. The NOEC, for yield and growth rate, exceeds or equals 100 mg/L.

Based on the results of this study,1-Benzyl-3-carbamoyl-pyridinium, chloride would not be classified as toxic to R. subcapitata (now classified as P. subcapitata) in accordance with the classification system of CLP. This toxicity study is classified as acceptable and satisfies the guideline requirement for an aquatic toxicity study to algae.

Results synopsis:

Test organism: Raphidocelis subcapitata (now classified as Pseudokirchneriella subcapitata)

Test type: static

72 h ErC50:  > 100 mg/L

72 h NOEC:  >= 100 mg/L

72 h EYC50:  > 100 mg/L

72 h NOEC:  >= 100 mg/L

Endpoint(s) effected:  Growth rate and yield

Description of key information

A limit test in accordance with the OECD Test Guideline 201 (23 March 2006) didn't show a significant inhibition of yield (24.4 %) or inhibition of growth (10.8 %) caused by 1-Benzyl-carbamoyl-pyridinium, chloride on the algae Raphidocelis subcapitata (Strain No. 61.81 SAG, now classified as Pseudokirchneriella subcapitata). The results based on the growth rate are as follows: ErC50 100 mg/L, NOErC 100 mg/L, LOErC > 100 mg/L. The results on yield are similar:  EbC50 ≥ 100 mg/L, NOEbC 100 mg/L, LOEbC > 100 mg/L. The recovery rate of the measured concentrations was for all treatment samples between 100 % and 103 %.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
100 mg/L

Additional information