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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:
2015-11-02 to 2016-10-31
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Experimental study on the registered substance performed according to the OECD 201 guideline and under GLP conditions.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
March 23, 2006
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
See test material information
Analytical monitoring:
yes
Vehicle:
no
Details on test solutions:
PREPARATION OF TEST SOLUTION
This test substance was prepared by suspending in test water. The purity of the test substance (active ingredient content) was calculated as 98% with the reference to the test substance information sheet and the COA. Although the precipitate was formed when 1000 mg/L stock solution in test material was prepared, the test material was diluted while stirring in order to mix the test material uniformly (dispersion) and the test solution was treated according to the set concentration.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Test species : Pseudokirchneriella subcapitata
Originally source : American Type Culture Collection (ATCC No. 22662)
Freshwater algae Culturing Room
Incubator : Shaking Incubator
Vessel : 250 mL Erlenmeyer flask
Light : continuous illumination (24 hours light condition)
Temperature : (21 - 24) ℃
Light intensity : (4440 - 8880) Lux
Revolution : approximately 100 rpm
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
No remark
Post exposure observation period:
No data
Hardness:
No data
Test temperature:
The mean temperature in the incubator was 22.4 ℃ (22.4 ℃ - 22.6 ℃)
pH:
The mean pH was 8.13 (7.72 – 9.17)
Dissolved oxygen:
No data
Salinity:
No data
Conductivity:
No data
Nominal and measured concentrations:
- Nominal test concentration (mg/L): Control, 0.1, 0.3, 0.9, 2.7, 8.1, 24.3
- Mean measured test concentration (mg/L): Control, 0.09, 0.27, 0.79, 2.21, 6.55, 19.13
Details on test conditions:
MEDIUM
OECD nutrient medium was used as culturing and dilution water, and was prepared according to the method of OECD Guideline for Testing of Chemicals No. 201 "Freshwater Alga and Cyanobacteria, Growth Inhibition Test"

ENVIRONMENTAL CONDITIONS
Temperature : Thermo-recorder (TR-71U, TND, T&D Co., Japan), 1 time / hour
Light : Lux meter (ANA-F11, TOKYO Photoelectric Co., Ltd, Japan), 1 time / week
As the result of the monitoring, there was no observed an alteration affecting the test.


PRE-CULTURE
- Culture room
Freshwater alga Culturing Room, Health Care Research Laboratory, KTR
- Culture conditions
Two to four days before the test, sterile OECD nutrient medium was inoculated about 1.0×10^5cells/mL from a stock culture and incubated in a shaking incubator under continuous illumination (shaking rate: about 100 times/min, and light intensity : 4440 ~
8880 lux)at 21-24℃ to give an algal suspension in log phase growth.
Freshwater algae that reached the exponential growth period were used for the test, no abnormal shape was observed by monitoring the test freshwater algae.

TEST CONDITIONS
Room : Freshwater algae Test Room
Test species : Pseudokirchneriella subcapitata
Duration of exposure : 72 hours
Vessel : 250 mL Erlenmeyer flask
Volume of test solution : 100 mL (140 mm H. × 30 mm Neck I.D.)
Incubator : Shaking Incubator
Light : continuous illumination (24 hours light condition)
Temperature : (21 - 24) ℃
Light intensity : (4440 - 8880) Lux
Revolution : approximately 100 rpm

STORAGE STABILITY TEST
The test substance was analyzed by following the test method KS M 0032 standardized by KOLAS according to the Convergence-Integration Material Assessment Team from Korea Testing & Research Institute. Without controlling the available component content of the Pb in the test substance, the test solution with the established concentration of 10, 100mg/L was dispensed and examined in the shaking incubator (100 rpm/min) with the light condition of (25±2)℃ for 10 days in order to verify the storage stability, resulting in its concentration stabilized at 107.7% of the initial concentration. Therefore, the stability is confirmed in the tested storage water condition

STABILITY TEST
The analysis on the test substance was conducted according the KOLAS standard KS M 0032 test method by Convergence-Integration Material Assessment Team in the Korea Testing & Research Institute, as a result of the test solution, which was prepared at a set concentration of 10 mg/L and subjected to a concentration of test material for 72 hours, was maintained at 100.0 % of the initial concentration. Thus, the definitive test was conducted with static system without replacing the test solution during the test period.

RANGE-FINDING TEST
The range-finding test of this test substance was conducted at the nominal concentrations of 0.1, 1, 10, and 100 mg/L. The cell density of 1.0×10^4cells/mL was exposed to the each test vessel without replication for 72-hour under sterile conditions.

DEFINITIVE TEST
With a preliminary test result, it was observed that the inhibition rates for the average growth rate at the set concentrations of 0.1, 1, 10 and 100 mg/L were 0.6, 44.3, 96.8 and 105.7% and the inhibition rates on yield were 2.3, 82.2, 99.7 and 100.5%. So this test was conducted in 3 times repeatedly at the set concentrations of 0.1, 0.3, 0.9, 2.7, 8.1, 24.3 mg/L in the test group and the control group.

INOCULATION
The test was started (0 hour) by inoculating 1.0×104 cells/mL in 250 mL conical 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.

CULTURE CONDITIONS
The inoculated flasks were then placed in a shaking incubator (Jisico Scientific, Korea). The cultures were incubated, without media renewal, for 72 hours under continuous illumination. The gaseous exchange and suspension of the algal cells were ensured by the action of the orbital shaker oscillating at 100 cycles per minute.

OBSERVATION AND ENVIRONMENT CONDITIONS
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 (Replicate1) of each test concentrations. At the start and end of the test, light intensity was measured using a lightmeter at the surface level of the test solutions.
During the exposure period, pH, light intensity and temperature were maintained as follows:
- The mean pH was 8.13
- The mean light intensity in the incubator was 6061 Lux (5900 Lux - 6380 Lux).
- The mean temperature in the incubator was 22.4 ℃

MEASUREMENTS
Samples were taken at 0, 24, 48 and 72 hours and the cell densities were determined by direct counts using a microscope and hemacytometer.
- Crop plot calculation: Number of cells in test solution (mL-1)
= Number of counted cells x 1/4 x 10000.
- Calculation of the mid-point: Number of cells in test solution(mL-1)
= Counted number of cells × 5 × 10000.
Growth curves were plotted for each test concentration and the mean biomass of the control group for each measurement time, and the inhibition rate of freshwater algae growth by treatment concentration was calculated by calculating the average specific growth rate or yield.

STATISTICAL ANALYSIS METHOD
Inhibition rate against average growth rate and calculation of inhibition rate against yield
The average specific growth rate and yield were calculated by the number of cells measured by hourly observation. We also used the calculated average specific growth rate and yield to calculate inhibition rates (Table 2, Table 3).
EC50 and 95 % confidence limits were statistically calculated by using probit method (EPA600/4-85, 1985).
No observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) were calculated using Analysis of variance (ANOVA) techniques (SPSS, ver. 19.0.0.1). The mean for each concentration was compared with the control mean using Dunnett's test.
As the results of the Dunnett's test, the NOEC was determined as the highest test concentration which no statistically significant inhibition in yield relative to the control was observed. (p>0.05) The LOEC was determined as the lowest test concentration
which a statistically significant inhibition in yield relative to the control was observed.
(p<0.05)
Determinations of ECx, NOEC and LOEC were based on measured concentration.
Reference substance (positive control):
yes
Remarks:
The test result (0.10, 0.18, 0.32, 0.58, 1.0 mg/L (established concentration, common ratio 1.8) was applied to the reference substance test setting Potassium dichromate.
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.878 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.322 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.09 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: average growth rate and yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.27 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: average growth rate and yield
Details on results:
ENVIRONMENTAL CONDITIONS
- The mean pH was 8.13 (7.72 – 9.17).
- The mean light intensity in the incubator was 6061 Lux (5900 Lux - 6380 Lux).
- The mean temperature in the incubator was 22.4 ℃ (22.4 ℃ - 22.6 ℃).

ANALYSIS OF TEST SOLUTIONS
- Storage stability test
After carrying out the stability test of the test substance for 10 days in the test solution with the setting concentration of 0.02 mg/L, the initial concentration was 115.4 % at 1 day, 107.7 % at 3 days, 84.6 % at 7 days, and 107.7 % at 10 days (Table 9).
- Stability test
After carrying out the stability test of the test substance for 72 hours in the test solution with the setting concentration of 10 mg/L, the initial concentration was 100.0 % after 24 hours, 100.0 % after 48 hours, and 100.0 % after 72 hours.
- Analysis of the test substance in the test solution
The mean measured test concentrations were 0.09, 0.27, 0.79, 2.21, 6.55, and 19.13 mg/L.

See the attached study report

Validity criteria fulfilled:
yes
Conclusions:
Under the test conditions, the 72hr-EC50 values expressed in terms of the mean measured concentration of zinc cyanide to the algae Pseudokirchneriella subcapitata were determined as 0.878 mg/L (95% confidence limits 0.744 - 1.035 mg/L) for average growth rate, and 0.322 mg/L (0.191 - 0.515 mg/L) for yield. 72 hours-LOEC was 0.27 mg/L, and 72 hours-NOEC was 0.09 mg/L.
Executive summary:

This study was performed to assess the inhibitory effect of zinc cyanide on the growth of the unicellular green alga, Pseudokirchneriella subcapitata. This study was conducted in accordance with the OECD 201 guideline and under GLP conditions.

- The toxicity test was performed with control and nominal concentrations of 0.1, 0.3, 0.9, 2.7, 8.1, 24.3 mg/L each in triplicates, with mean measured concentrations of 0.09, 0.27, 0.79, 2.21, 6.55, 19.13 mg/L, respectively.

- The cultivation was carried out for 72 hours with continuous agitation under illumination in succession. Numbers of cell in freshwater algae were monitored daily, using a scientific microscope.

Under the test conditions, the 72hr-EC50 values expressed in terms of the mean measured concentration of zinc cyanide to the algae Pseudokirchneriella subcapitata were determined as 0.878 mg/L (95% confidence limits 0.744 - 1.035 mg/L) for average growth rate, and 0.322 mg/L (0.191 - 0.515 mg/L) for yield. 72 hours-LOEC was 0.27 mg/L, and 72 hours-NOEC was 0.09 mg/L.

Description of key information

For that endpoint, a reliable experimental study was available and was conducted in accordance with the OECD 201 guideline and under GLP conditions.

The study was performed to assess the inhibitory effect of zinc cyanide on the growth of the unicellular green alga, Pseudokirchneriella subcapitata.

Under the test conditions, the 72hr-EC50 values expressed in terms of the mean measured concentration of zinc cyanide to the algae Pseudokirchneriella subcapitata were determined as 0.878 mg/L (95% confidence limits 0.744 - 1.035 mg/L) for average growth rate, and 0.322 mg/L (0.191 - 0.515 mg/L) for yield. 72 hours-LOEC was 0.27 mg/L, and 72 hours-NOEC was 0.09 mg/L.

All validty criteria of the study were met, as stated in the test guideline. Therefore, this study can be considered acceptable for that endpoint.

Key value for chemical safety assessment

EC50 for freshwater algae:
0.878 mg/L
EC10 or NOEC for freshwater algae:
0.09 mg/L

Additional information