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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:
15 January 2019 - 1 April 2019
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)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
An analysis of the test solution was performed in the controls and 100 mg/L concentration at 0 hour and 72 hours of the test.
50 mL of sample was taken from each test solution and then analyzed after treatment with same method of recovery test.
Test solution at 72 hour was centrifuged (5000 rpm, 20 min) to achieve adequate separation of algae.



Vehicle:
no
Details on test solutions:
100 mg of the test item was added to 1 L volume of glass media bottle and 1000 mL dilution water was added up to give the 100 mg/L of test solution. The test solution was stirred for 10 minute using a magnetic stirrer at 500 rpm. A 100 mL portion of prepared 100 mg/L test solution was distributed to the 250 mL volume Erlenmeyer flasks with six replicates. Test solution was clear and there was no remarkable observation. The dilution water (algae culture medium) only was used for the control group.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Source
Gyeongnam Department of Environmental Toxicology and Chemistry, Korea Institute of Toxicology (KIT), originally purchased from American Type Culture Collection (ATCC), Manassas, VA, USA on May 2, 2017.

Pre-culture
Four days before the test, sterile OECD nutrient medium (OECD, 2006) was inoculated with about 5.0×103 cells/mL from a stock culture and incubated in an incubator under continuous illumination (shanking rate: about 100 times/min, and light intensity: 60-120 μE/m^2/s at 21-24 °C).

Culturing and dilution water
The algae were cultivated and tested in sterile OECD nutrient medium, prepared according to the OECD test guideline (2006).

Inoculum
The limit test was started (0 hour) by inoculating 5.0×10^3 cells/mL in 250 mL Erlenmeyer flasks each containing 100 mL of test solution under sterile conditions. These cells were taken from an exponentially growing pre-culture under the same conditions as for the test.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
22.1 ± 0.1 ºC
pH:
7.79-9.04
Nominal and measured concentrations:
Nominal concentration: 100 mg/L
Measured concentration: 96.1 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type (delete if not applicable): open
- Control end cells density: 5.0×10^3 cells/mL
- No. of vessels per concentration (replicates):
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Culture medium: The algae were cultivated and tested in sterile OECD nutrient medium, prepared according to the OECD test guideline (2006).

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: continuous illumination
- Light intensity and quality: 5644-5782 lux

Observation and measurement
During the test period, the temperature was recorded daily and continuously using a recorder. At the start of the test, the pH of the test solutions was measured in additional flasks specifically for pH measurement. At the end of the test, the pH was measured from R1 (Replicate 1) of each test concentrations. Light intensity was measured using a light meter (LI-250A light meter, quantum sensor) at the surface level of the test solutions every day. Samples (50 μL) were taken at 24, 48 and 72 hours and the cell densities were determined by an optical microscope.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell densitys: Growth was monitored daily by determining the cell density of each culture by direct counts using an optical microscope.

TEST CONCENTRATIONS
- Range finding study:
The range-finding test of this test item was conducted at the nominal solutions of 0.1, 1, 10 and 100 mg/L.
The cell density of 5.0×10^3 cells/mL was exposed to each test vessel without replication for 72-hour under sterile conditions.
Based on the results of the range-finding test, the limit test was performed in control and 100 mg/L test solution with six replicates per treatment
Reference substance (positive control):
no
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:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
yield inhibition
Key result
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:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
yield inhibition
Details on results:
All test and control cultures were inspected microscopically during 72 hours.
No negative effects on growth rate or yield of biomass were observed in any of the test cultures. Moreover, no abnormalities were detected in any of the cultures and there was no sign of contamination by foreign algal cells or protozoa. The derived (no) effect concentrations were: 72h-ErC50 > 100 mg/L, 72h-NOEC >= 100 mg/L.
Reported statistics and error estimates:
Plots of growth curves were produced using the measured cell concentrations in test culturesand controls together with the concentrations of the test item. The inhibition percentage of the growth rate for each treatment was calculated based on final population density (yield), and average specific growth rate.

Yield and average specific growth rate were analyzed by CETIS Version 1.8.7.15 (Tidepool Scientific Software, USA). Linear Interpolation methods were applied to obtain the effective concentrations, EC10 (and/or EC20) and EC50 for each approach. LOECs and NOECs were determined using the Dunnett Multiple Comparison Test.

Growth inhibition during the test period

Average growth rate

Nominal
concentration
Mean measuredconcentration Average
growth rate
Relative
inhibition
Mean relative
inhibition
(mg/L) (mg/L)   (%) (%)
Control NA 1.1425 NA NA
1.1247
1.1425
1.1511
1.1889
1.1511
100 88 1.1201 2.6 -0.8
1.1614 -1.0
1.1695 -1.7
1.1889 -3.4
1.1131 3.2
1.2018 -4.5

Measured concentrations of the test item in the test solution

Nominal concentration
(mg/L)
Measured concentration (mg/L)  Mean measured
concentration
(mg/L)

0

hours

72 hours
Control ND ND NA
100 85.7 90.3 88.0
(85.7) (90.3) (88.3)

ND: Not detected

NA: Not available

( ) : % of nominal concentration

Validity criteria fulfilled:
yes
Conclusions:
No negative effects on growth rate or yield of biomass were observed in any of the test cultures. The derived (no) effect concentrations were: 72h-ErC50 (Pseudokirchneriella subcapitata, growth rate) > 100 mg/L, 72h-NOErC (Pseudokirchneriella subcapitata, growth rate) >= 100 mg/L.
Executive summary:

The objective of this study was to assess the growth inhibition effect of 2,3-butanediol((2R,3S)-rich) to the Green Alga, Pseudokirchneriella subcapitata. The study was conducted in accordance with the OECD No. 201 and in compliance with GLP. This test item was very soluble in dilution water (algae culture media). Additional solubility test was not performed because it was possible to prepare 100 mg/L of test solution by direct addition of test item into the dilution water. The range-finding test was conducted at the nominal concentrations of 0.1, 1, 10 and 100 mg/L by static system.

There were no growth inhibition in all test solution by statistical analysis. Therefore, the limit test was performed with a control and a nominal concentration of 100 mg/L in six replicates. Cultures were incubated on a shaking incubator under continuous illumination at 22.1±0.1 °C for 72 hours. Growth was monitored daily by determining the cell density of each culture by direct counts using an optical microscope.

The analysis of test solution was conducted. As there was less than 20% deviation between measured and nominal test item concentrations, nominal concentrations were used for calculation of toxicity values.

No negative effects on growth rate or yield of biomass were observed in any of the test cultures. Moreover, no abnormalities were detected in any of the cultures and there was no sign of contamination by foreign algal cells or protozoa. The derived 72-hr EC50 was >100 mg/L for average growth rate and yield. The No Observed Effect Concentration (NOEC) and Lowest Observed Effect Concentration (LOEC) were >= 100 mg/L and >100 mg/L for average growth and yield.

The study fulfilled the all validity criteria of the guideline, and was considered adequate and reliable for the environmental hazard assessment.

Description of key information

72h-ErC50 (Pseudokirchneriella subcapitata, growth rate) > 100 mg/L (nominal, OECD 201)

72h-NOErC (Pseudokirchneriella subcapitata, growth rate) >= 100 mg/L (nominal, OECD 201)

Key value for chemical safety assessment

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

Additional information

The available key study investigated growth inhibition of 2,3 -butanediol((2R,3S)-rich) to the Green Alga, Pseudokirchneriella subcapitata. The study was conducted in accordance with the OECD No. 201 and in compliance with GLP. A limit test was performed at 100 mg/L test item under static test conditions, using 6 replicates for control and treatment group. Analysis of test solution was conducted. As there was less than 20% deviation between measured and nominal test item concentration, nominal concentrations were used for calculation of toxicity values.

No negative effects on growth rate or yield of biomass were observed in any of the test cultures. Moreover, no abnormalities were detected in any of the cultures and there was no sign of contamination by foreign algal cells or protozoa. The derived 72-hr EC50 was >100 mg/L for average growth rate and yield. The No Observed Effect Concentration (NOEC) and Lowest Observed Effect Concentration (LOEC) were >= 100 mg/L and >100 mg/L for average growth and yield.

The study fulfilled the all validity criteria of the guideline, and was considered adequate and reliable for the environmental hazard assessment.