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Ecotoxicological information

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:
October-December 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report Date:
2020

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
GLP compliance:
yes
Remarks:
GLP compliance statement is included in attached full study report

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
Batch #: 191016
Expiration Date: October 15, 2020
0.9% (9306 mg/L) mineral complexed organic matter (based on total organic carbon content of 0.51%)
1124 mg/L Fe
3333 mg/L Na
2187 mg/L total inorganic carbon
pH=7.28
Density: 1.011 g/mL at 25 degrees C

Sampling and analysis

Analytical monitoring:
yes

Test solutions

Vehicle:
no
Details on test solutions:
Water Solubility Test
The water solubility of the test substance was determined prior to the beginning of the study. The test substance was determined to be water-soluble.

Range-finding Test
A preliminary range-finding test was conducted to determine the concentrations needed for the definitive test. The five concentrations used were 1 mg/L, 5 mg/L, 10 mg/L, 50 mg/L and 100 mg/L. There was one flask of test medium for each concentration. The algae were introduced to each concentration at an initial cell density of 10,000 cells/mL, and at 72 hours, the terminal density was measured. The range-finder indicated an EC50 (Median Effective Concentration) of >100 mg/L, so a limit test was conducted in lieu of a definitive.

Test Substance, Control and Positive Control Preparation
The limit test stock solution was prepared by dissolving 0.16 g of test substance in 800 mL of OECD medium and mixing for 15 minutes. A test substance control solution was made using only dilution water. A positive control solution was prepared using zinc chloride (Mfg: Acros, Lot: A0371487, Exp: 27 Feb 2022). Solutions for the limit test were prepared from the stock solution. All concentrations were double the desired final concentration until media containing algae cells was added in equal volume to prepared test concentration.

Limit Test
A limit test was conducted using a concentration of 100 mg/L of the test substance. The test cultures were divided into 250 mL flasks containing the appropriate concentration of test substance (four flasks for test concentration and six flasks for the control). A positive control group consisted of three flasks treated with 10 mg/L zinc chloride. The flasks were set on a shaker table for constant agitation at approximately 100 cycles/minute. At 24, 48, and 72 hours following dosing, observations of inhibition were made for each test container by measuring the mean cell density with a hemocytometer.

Test Validity
The test was considered valid if the 72-hour terminal control increased by a factor of at least 16 times. Additionally, the mean coefficient of variation (C.V.) among control replicates at 24, 48, and 72 hours should be less than 35%.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Experimental Organism
Species: Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum)
Justification for Species: One of the freshwater alga species specified in the regulations
Source: STILLMEADOW, Inc. cultures
Age: Mass culture initiated 3-7 ± 1 days prior to dosing
Density: Initial cell density was 10,000 cells/mL

OECD Media Preparation
Stock Solutions I-IV are prepared with the listed concentrations in deionized water, sterilized by membrane filtration (0.22 μm pore size), and stored in the dark at ~4°C for up to 6 months. The OECD Medium was prepared by combining 20 mL of Stock Solution I and 2 mL each of Stock Solutions II, III and IV for every 1000 mL of sterile deionized water.

Cultivation Method
Pseudokirchneriella subcapitata parent and daughter cultures are continuously maintained at STILLMEADOW, Inc. Prior to testing, 800 mL of sterile OECD medium was inoculated with 5-day old organisms. Autoclaved media was adjusted to a pH of 8.1 ±0.1 with 0.1 mL of 0.1 N sodium hydroxide (Mfg: Fluka, Lot: H053B, Exp: 05 Aug 2020). All procedures were conducted aseptically.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h

Test conditions

Hardness:
N/A
Test temperature:
24-26 degrees C
pH:
8.1 (plus or minus 0.1)
Dissolved oxygen:
Test containers received constant agitation on shaker table set at approximately 100 cycles/minute.
Salinity:
N/A
Conductivity:
N/A
Details on test conditions:
Organism Husbandry
Test Room: Environmentally controlled chamber (Chamber A)
Test Chambers: 250 mL sterile Erlenmeyer flasks with foam stoppers; filled with 100 mL of the appropriate solution
Dissolved Oxygen Concentrations: Test containers received constant agitation on shaker table set at approximately 100 cycles/minute
Test/Nutrient Medium: OECD media with a pH of 8.1 ± 0.1.
Environmental Controls
Set to Maintain: Temperature of 24 ± 2°C
Continuous lighting (4300 ± 645 lux)
Actual Temperature: Range-finding: 23-26ºC
Limit: 24-26ºC
Actual Light Intensity: Range-finding: 3680-4650 lux
Limit: 3940-4810 lux
Reference substance (positive control):
yes
Remarks:
A positive control solution was prepared using zinc chloride (Mfg: Acros, Lot: A0371487, Exp: 27 Feb 2022).

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
ca. 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
and cell density
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:
and cell density
Details on results:
Test Validity
The test was considered valid because the 72 hour terminal control increased by a factor of at least 16 times. The mean coefficient of variation was 16.845% at 72 hours.

Chemical and Physical Monitoring
Chemical and physical monitoring data (chamber temperature and light intensity) of test chambers for the limit test are presented in Appendix C of the attached report.

Limit Test
Initial cell density was 10,000 cells/mL. The only observation recorded was that the cells in the test substance group were clumping at 48 and 72 hours. Results presented are based on nominal concentrations. Daily cell counts, terminal pH and average growth rate are presented in Table 2 below and in the attached report.

Evaluation of Results
At 72 hours, the test concentration of FBS Transit that resulted in cell density and growth rate EC50 was determined to be >100 mg/L. At 72 hours, the cell density and growth rate NOEC was determined to be 100 mg/L. Results presented are based on nominal concentrations. Statistics are presented in Appendix D.
Results with reference substance (positive control):
See Table 2 below and in the attached report.
Reported statistics and error estimates:
The mean cell counts of the test concentration containers were compared to the control counts to determine the concentration of the test substance which resulted in a Median Effective Concentration (EC50) and the No Observed Effect Concentration (NOEC). The NOEC and EC50 were determined by a commercially available statistical program (ToxCalc Version 5.0™). Linear Interpolation was used for EC50 determination. See Appendix D in the attached report.

Any other information on results incl. tables

Table 2a: Limit Test Average Growth Rate Results





















































































Test Conc.Rep Cell Density x 10^4 cells/mL Initial pHTerminal pHAverage Growth Rate
mg/L 24 hr48 hr72 hr   
 A61878 7.20.061
 B4124 a 7.1NA
ControlC616808.17.20.061
 D41213 a 7.1NA
 E62782 8.30.061
 F42086 8.30.062

 


Table 2b- Limit Test Average Growth Rate Results
































































































Test Conc.


Rep Cell Density x 10^4 cells/mL Initial pHTerminal pHAverage Growth Rate
mg/L 24 hr48 hr72 hr   
 A518 b80 b 8.20.061
100B430 b86 b8.18.30.062
 C420 b70 b 8.30.059
 D624 b79 b 8.20.061
C2A120 7.80.000
10 mg/LB1117.87.90.000
ZnCl2C111 7.90.000

a- Replicate was considered an outlier and was not included in any statistical analysis.


b- Cells clumping


NA- not applicable as replicate was an outlier


Note: all organisms had no observable abnormalities unless otherwise noted.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
FBS Transit does not inhibit algal growth.