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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:
2016-11-01 to 2016-12-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: "Algal Growth.Inhibition Test" stipulated in the "Testing Methods for New Chemical Substances"
Version / remarks:
(March 31, 2011, No. 0331-7, Phannaceutical and Food Safety Bureau, Ministry ofHealth, Labour and Welfare; March29, 2011, No. 5, Manufacturing Industries Bureau, Ministry ofEconomy, Trade and Industry; No. 110331009, Environmental Policy Bureau, Ministry of the Environment, Japan; latest revision, December 21, 2015, No. 1221- 1, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare; December 9, 2015, No. 1, Manufacturing Industries Bureau, Ministry ofEconomy, Trade and Industry; No. 1512211, Environmental Policy Bureau, Ministry of the Environment., Japan)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- Chemical name: Reaction product of 3-dimethylaminopropylamine and propylene oxide with 1,1'-[(3-dimethylaminopropyl)imino]-bis-2-propanol and 1-[(3-dimethylaminopropyl)(2-hydroxy-1-methylethyl)amino]-2-propanol as main component
- Name of test material (as cited in study report): DMAPA-2PO
- CAS number: CAS 63469-23-8 (1,1'-[(3-(dimethylaminopropyl)imino)]-bis-2-propanol)
- Molecular formula: C11H26N2O2 (main component)
- Molecular weight: 218.3 (main component)
- Substance type: yellow liquid
- Physical state: liquid
- Analytical purity: 100 %
- Impurities (identity and concentrations): not reported
- Lot/batch No.: 5Y1024
- Expiration date of the lot/batch: NA
- Stability under test conditions: stable
Analytical monitoring:
yes
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The weighed test sample of 100 mg and medium of 1000 mL were mixed, and stirred until being dissolved to prepare the stock solution of 100 mg/L.
Required volume of the stock solution and medium were mixed and stirred to prepare the test solution in the preparation container. The test solution was divided into each test vessel.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): final test solutions were clear and colourless
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: ATCC 22662
- Source (laboratory, culture collection): American Type Culture Collection
- Pre-culture: The algae were counted in pre-culture incubated under the same conditions as the test for 3 days (from November 4 to November 7, 2016) as inoculums, and were added to test vessels to bring the initial cell concentration of 0.75x1E+04 cells/mL.

ACCLIMATION
- Acclimation period: not relevant (except pre-culture 3 days before start of the test)
Test type:
other: Rotary shaking (100 rpm)
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
not specified
Test temperature:
22.3-22.5 °C
pH:
t=0h: 7.9-9.5
t=72h: 8.3-8.6
Dissolved oxygen:
not relevant
Salinity:
not relevant
Nominal and measured concentrations:
Preliminary study:
nominal test concentrations: 1.0, 10 and 100 mg/L (100 mg/L analysed)
measured test concentration t = 0 h: 94.5 (pH unadjusted), 100 (pH adjusted) mg/L
measured test concentration t = 72 h: 102 (pH unadjusted), 100 (no algae, pH unadjusted), 107 (pH adjusted), 103 (no algae, pH adjusted) mg/L

Final test:
nominal test concentrations: control, 6.25, 12.5, 25.0, 50.0 and 100 mg/L
measured test concentration t = 0 h: n.d., n.d., 10.9, 22.4, 50.8, 98.1 mg/L
measured test concentration t = 72 h: n.d., n.d., 10.7, 20.9, 50.0, 103 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Sterilized Erlemneyer flask (with gas-permeable Silicosen ®)
- Material, size, headspace, fill volume: 300 mL all-glass flasks filled with 100 mL test solution
- Initial cells density: 7,500 cells/mL
- Control end cells density: 130 x 10,000 cells/mL (mean)
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: At the pre-culture and algae growth inhibition test, the OECD medium (OECD TG 201; March 23, 2006) prepared with puriified water was used.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2, according to the OECD 201 Guideline, formulated using Milli-RO water.
- Culture medium different from test medium: Yes (M1 versus M2). Three days before the start of the test the algal stock culture (culture in M1) was inoculated in the same culture medium (M2) used in the test. The culture was maintained under the same conditions as used in the test.
- Intervals of measurements: pH was measured at the beginning and at the end of the test. Temperature was continuously measured in a control vessel. At the end of the final test microscopic observations were performed on all test concentrations to observe for any abnormal appearance of the algae.

OTHER TEST CONDITIONS
- Sterile test conditions: no information
- Adjustment of pH:
- Photoperiod: continuous illumination
- Light intensity and quality: Nominal 90 μmol·m-2·s-1 ± 20% (within ± 15% from the average light intensity). Continuous illumination provided by fluorescent lights with wavelength range of 400-700 nm

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]: Particle counter; Model COULTER Z2 (Beckman Coulter, Inc.), System biological microscope; Model BX41 (Olympus Corporation)
- Effect calculated parameters: specific growth rate and yield

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: yes
- Test concentrations: 1.0, 10 and 100 mg/L (setup as preliminary test)
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
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
Remarks on result:
other: analytically confirmed
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
25 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
- Any stimulation of growth found in any treatment: no
Reported statistics and error estimates:
The EC50 was estimated as "larger than the highest test concentration" since no less than 50% of inhibition rate was not obtained within the exposure levels. The EC50 was denoted as EC50 based on growth rate.
Regarding the growth rate, Bartlett's test was done to determine the homogeneity of variance for the data. Then one-way analysis of variance and Dunnett's multiple comparison test was used to determine the significant difference between the control level and exposure levels. The statistical analysis was conducted using computer program (running on Microsoft software "Excel") constructed by our laboratory. NOEC was determined by the results of statistical analysis and cell condition.
Validity criteria fulfilled:
yes
Conclusions:
A 72-h growth inhibition test with the unicellular green alga Pseudokirchneriella subcapitata was performed with the test substance DMAPA-2PO according to the OECD guideline 201 (GLP conditions). It can be concluded that the test item had no significant inhibitory effect on the growth of Pseudokirchneriella subcapitata. The analytically confirmed nominal EC50 for growth rate inhibition (72h-ErC50) was >100 mg/L, 72-h NOErC was 25 mg/L. The results of the test can be considered reliable without restrictions.

Description of key information

Two studies were performed with the test item.

In the first study from Yoshikawa (2016), which was considered as key study, the acute toxicity of the test substance to the unicellular green alga Pseudokirchneriella subcapitata was performed according to the OECD guideline 201 (GLP conditions). The test item had no significant inhibitory effect on the growth of Pseudokirchneriella subcapitata. The analytically confirmed nominal EC50 for growth rate inhibition (72h-ErC50) was > 100 mg/L.

The second study from Migchielsen (2002) was a full test performed according to OECD TG 201. A 72-h growth inhibition test with the unicellular green alga Selenastrum capricornutum (Pseudokirchneriella subcapitata) yielded a 72-h NOErC of 22 mg/L and a 72-h ErC50 of 56 mg/L, based on nominal concentrations. The study was considered as supporting as no analytical measurements were performed.

Measurements and analytical determinations are an OECD requirement. While analytical measurements were included in the study of Yoshikawa (2016), this was not the case for the study of Migchielsen (2002), in the latter study the results were expressed as nominal concentrations. As the newer study of Yoshikawa (2016) follows the OECD 201 guideline for algae testing, the 72-h EC50 value for growth inhibition of > 100 mg/L is considered more reliable than the EC50 of 56 mg/L obtained in the study of Migchielsen (2002). The 72-h ErC50 > 100 mg/L is considered as key value for the PNEC derivation.


Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L
EC10 or NOEC for freshwater algae:
25 mg/L

Additional information

EC50 for freshwater algae is > 100 mg/L.