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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
effects on growth of green algae
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2021
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)
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch/Lot no.: 202102001
- Purity: 99.5 wt.%
- Expiry data: February 28, 2022

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, Protect from moisture
- Solubility: Test item was soluble in dilution water and it was possible to prepare 100 mg/L of test solution by direct addition of test item.
- Stability: Stability test was performed at 50 mg/L of test solution under test condition (without test organisms). The concentration decreased to 32.8% of initial concentration after 72 hours in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis.
Analytical monitoring:
yes
Vehicle:
no
Details on test solutions:
101 mg of the test item (purity: 99.5%) was dissolved in 1000 mL dilution water to prepare 100 mg/L of test solution (stock test solution). 0.35, 1.05, 3.1, 9.3, 27.9, 83.5 and 250 mL of 100 mg/L
of test solution was mixed with proper amount of dilution water to prepare 0.07-50 mg/L of test solution. A 100 mL portion of prepared each test solution was distributed to the 250 mL volume
Erlenmeyer flasks with three replicates. The dilution water (algae culture medium) only was used for the control group. All test solution was clear and no remarkable phenomenon was observed.

Based on the results of the range-finding test, the definitive test was performed in control and nominal concentration of: 0.07, 0.21, 0.62, 1.86, 5.57, 16.7 and 50 mg/L test solutions with three replicates per treatment.

Mean measured concentration (mg/L): Control (0), 0.025, 0.060, 0.275, 0.912, 2.95, 8.74, 24.6
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain No. ATCC 22662
- Source (laboratory, culture collection): Gyeongnam Branch Institute, Korea Institute of toxicology (KIT), originally purchased from American Type Culture Collection (ATCC), Manassas, VA, USA on December 17, 2018.
- Age of inoculum (at test initiation): Three days before the test, sterile OECD nutrient medium (OECD, 2006) was inoculated with about 5.0×103cells/mL from and incubated in an incubator under continuous illumination (shanking rate: about 100 times/min, and light intensity: 60-120 μE/m2/s at 21-24 °C). These cells were taken from an exponentially growing pre-culture under the same conditions as for the test.
- Method of cultivation: The algae were cultivated and tested in sterile OECD nutrient medium.

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22.1 ± 0.2 ºC
pH:
7.54-8.99
Nominal and measured concentrations:
Based on the results of the range-finding test, the definitive test was performed in control and 0.07, 0.21, 0.62, 1.86, 5.57, 16.7 and 50 mg/L test solutions with three replicates per treatment. Mean measured concentration (mg/L): Control (0), 0.025, 0.060, 0.275, 0.912, 2.95, 8.74, 24.6
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL volume Erlenmeyer flasks
- Initial cells density: The definitive test was started (0 hour) by inoculating 5.0×103 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. The inoculated flasks were then placed in a shaking incubator (DAIHAN Scientific, Korea).
The cultures were incubated, without media renewal, for 72 hours under continuous illumination.

- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes (OECD test guideline 2006)

TEST CONDITIONS
- Culture medium different from test medium: no
- Photoperiod: continuous illumination
- Light intensity and quality: 5911-5927 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Samples (50 µL) were taken at 24, 48 and 72 hours and the cell densities were determined by an optical microscope.

The temperature of shaking incubator was maintained 22.1 ± 0.2 ºC and light intensity
was 5911-5927 lux. pH of test solution was 7.54-8.99 during the test.

TEST CONCENTRATIONS
- Range finding study: A 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 5.0×103 cells/mL was exposed to each test vessel without replication for 72-hour under sterile conditions.

- Results used to determine the conditions for the definitive study: 5% inhibition (of growth rate and yield) was observed in 0.07-1.18 mg/L and 75% inhibition (of growth rate and yield) was observed in 9.79-52.9 mg/L as nominal concentration by statistical analysis using range-finding test result (TABLE 1) in results
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.912 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (total fraction)
Basis for effect:
yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.275 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (total fraction)
Basis for effect:
yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
3.07 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (total fraction)
Basis for effect:
yield
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.912 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (total fraction)
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.912 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (total fraction)
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
10.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (total fraction)
Basis for effect:
growth rate
Details on results:
Observation of abnormalities (for algal test): . No abnormalities were detected in any of the cultures and there was no sign of contamination by foreign algal cells or protozoa

The limit of quantitation (LOQ) of test item was 0.05 mg/L and the concentration was not
detected in lowest test solution of 0.07 mg/L (because all samples were analyzed after dilution
2 time). Also, the concentrations were not detected in 0.21 mg/L of test solution after 48 hours
by degradation of test item. For those cases, half of LOQ value was applied for ND (not
detected) samples to calculate mean measured concentration in this study.
The measured concentrations of test solution were 0.025, 0.205, 0.562, 1.93, 6.02, 17.4 and
50.5 mg/L at 0 hour and 0.025, 0.100, 0.285, 1.00, 3.26, 10.2 and 28.5 mg/L at 24 hours for
nominal concentrations of 0.07, 0.21, 0.63, 1.86, 5.57, 16.7 and 50 mg/L. The measured
concentrations were 0.025, 0.025, 0.656, 2.37, 7.02 and 18.9 mg/L at 48 hours and 0.025, 0.025,
0.170, 0.546, 1.62, 4.69 and 13.4 mg/L at 72 hours.
The geometric mean measured concentrations during test period were 0.025, 0.060, 0.275,
0.912, 2.95, 8.74 and 24.6 mg/L (28.6-53.0% of nominal concentration) and this result was
used for toxicity value (EC50) evaluation (Table 2).

VALIDITY OF THE TEST:
- Increase of biomass in the control cultures within the 72-hour test period: 96-fold (criteria: ≥16-fold)
- Mean coefficient of variation for section-by-section specific growth rate in the control cultures: 13.6% (criteria: ≤35%)
- Coefficient of variation of average specific growth rates in replicate control cultures during the whole test period: ≤7% (criteria: 1.1%)
- Change of pH in control medium during the test period: 1.06 (criteria: ≤1.5)
Reported statistics and error estimates:
STATISTICAL ANALYSIS AND EXPRESSION OF THE RESULTS:
Plots of growth curves were produced using the measured cell concentrations in test cultures
and 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.

TABLE 1 - Range-finding study



































Nominal concentration (mg/L)% Inhibition - Average Growth Rate% Inhibition - Yield
control00
0.11.647.25
13.6215.4
1030.475.7
10092.699.6

 


TABLE 2 - The 72-hour ECx values (95% confidence limit), LOECs and NOECs of the test item in terms
of the mean measured concentrations were as follows



































Estimates of ToxicityAverage growth rateYield
72-hr EC10
(95% confidence limits)
1.61
(0.81-2.16)
0.36
(0.00-0.50)
72-hr EC20
(95% confidence limits)
3.70
(3.20-4.07)
0.67
(0.30-0.97)
72-hr EC50
(95% confidence limits)
10.2
(9.67-10.5)
3.07
(1.94-3.74)
NOEC0.2750.275
LOEC0.9120.912
Validity criteria fulfilled:
yes
Conclusions:
The growth inhibition effects of Toluene-4-sulphonohydrazide was assessed according to OECD Guideline No. 201. The test resulted in 72-hr EC50 values (95% confidence limits) expressed in terms of the geometric mean measured concentration of Toluene-4-sulphonohydrazide to Pseudokirchneriella subcapitata were 10.2 mg/L (9.67-10.5 mg/L) for average growth rate and 3.07 mg/L (1.94-3.74 mg/L) for yield.
The No Observed Effect Concentration (NOEC) and Lowest Observed Effect Concentration
(LOEC) values expressed in terms of the mean measured concentration were 0.275 mg/L and
0.912 mg/L for average growth rate and yield.
Executive summary:

This study was conducted to assess the growth inhibition effect of Toluene-4-sulphonohydrazide to the Green
Alga, Pseudokirchneriella subcapitata. The toxicity was expressed as the media effective concentration (EC50).
The study was conducted in accordance with the OECD Guideline for Testing of Chemicals, Section 2, Effects on Biotic Systems, No. 201 “Freshwater Alga and Green Alga, Growth Inhibition Test” (July 28, 2011) and Guidelines for the testing of chemicals, Section 3.1 “Freshwater Alga, Growth Inhibition Test”, Public Notice No. 2020-46, issued by the National Institute of
Environmental Research (NIER), Korea on November 03, 2020.


Range-finding test was conducted at the nominal concentrations of 0.1, 1, 10 and 100 mg/L by static system. 5% inhibition (of growth rate and yield) was observed in 0.07-1.18 mg/L and 75% inhibition (of growth rate and yield) was observed in 9.79-52.9 mg/L as nominal concentration by statistical analysis using range-finding test result. Therefore, the definitive test was performed with a control and a nominal concentration of 0.07, 0.21, 0.62, 1.86, 5.57, 16.7, and 50 mg/L in three replicates. Cultures were incubated on a shaking incubator under continuous
illumination at 22.1 ±0.2°C for 72 hours. Growth was monitored daily by determining the cell density of each culture by direct counts using an optical microscope. The measured concentrations were 0.025, 0.060, 0.275, 0.912, 2.95, 8.74 and 24.6 mg/L (35.7, 28.6, 44.4, 49.0, 53.0, 52.3, 49.2% of nominal concentration) for nominal concentrations of 0.07, 0.21, 0.62, 1.86, 5.57, 16.7, and 50 mg/L and these measured concentrations were used for calculation of toxicity values.
The 72-hr EC50 values (95% confidence limits) expressed in terms of the geometric mean measured concentration of Toluene-4-sulphonohydrazide to Pseudokirchneriella subcapitata were 10.2 mg/L (9.67-10.5 mg/L) for average growth rate and 3.07 mg/L (1.94-3.74 mg/L) for yield.
The No Observed Effect Concentration (NOEC) and Lowest Observed Effect Concentration (LOEC) values expressed in terms of the mean measured concentration were 0.275 mg/L and 0.912 mg/L for average growth rate and yield.

Description of key information

The growth inhibition effects of Toluene-4-sulphonohydrazide was assessed according to OECD Guideline No. 201. The test resulted in 72-hr EC50 values (95% confidence limits) expressed in terms of the geometric mean measured concentration of Toluene-4-sulphonohydrazide to Pseudokirchneriella subcapitata were 10.2 mg/L (9.67-10.5 mg/L) for average growth rate and 3.07 mg/L (1.94-3.74 mg/L) for yield. 
The No Observed Effect Concentration (NOEC) and Lowest Observed Effect Concentration (LOEC) values expressed in terms of the mean measured concentration were 0.275 mg/L and 0.912 mg/L for average growth rate and yield.

Key value for chemical safety assessment

EC50 for freshwater algae:
10.2 mg/L
EC10 or NOEC for freshwater algae:
1.61 mg/L

Additional information

The growth inhibition effects of Toluene-4-sulphonohydrazide was assessed according to OECD Guideline No. 201. The test resulted in 72-hr EC50 values (95% confidence limits) expressed in terms of the geometric mean measured concentration of Toluene-4-sulphonohydrazide to Pseudokirchneriella subcapitata were 10.2 mg/L (9.67-10.5 mg/L) for average growth rate and 3.07 mg/L (1.94-3.74 mg/L) for yield.
The No Observed Effect Concentration (NOEC) and Lowest Observed Effect Concentration (LOEC) values expressed in terms of the mean measured  concentration were 0.275 mg/L and 0.912 mg/L for average growth rate and yield.

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