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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:
Study initiation: 2012.2.9 Study completion: 2012.3.29
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)
GLP compliance:
yes
Specific details on test material used for the study:
Substance name: ESA
Chemical name: 1,3,2-Dioxathiolane, 2,2- dioxide
Receipt date: 2011.12.26
Term of validity: 2013.10
Purity: 99.1%
Appearance: Lemon yellow solid
Storage condition: Room temperature
Analytical monitoring:
yes
Details on sampling:
Analysis of the test substance in the test solution was performed at the test start (0 hour). 24 hours, 48 hours and test end (72 hours) using HPLC-Triple
Quad LC/MS (Agilent Technologies).

Vehicle:
yes
Remarks:
methyl alcohol
Details on test solutions:
Test concentrations
Test concentration was set up to 10.0 mg/l. The maximum test concentration was set up on the basis of maximum solubility (100,000 mg/L) of test substance for organic solvent (methyl alcohol).
Nominal concentration of the test was set up to 4.0, 5.0, 6.3, 7 .9 and 10.0 mg/l (geometric series 1.26). Nominal concentration was set up on the basis of results of preliminary test carried out for active ingredients of test substance (0.001 , 0.01, 0.1, 1.0, 5.0 and 10.0 mg/ L).

Preparation of test solutions
Stock solution (100,000 mg/ L, based on 99.1 % of active ingredients) was prepared by putting 1,009.1 mg of test substance into volumetric flask of 10ml after measurement with analytical balance (sartorius CPA224S) and adding methyl alcohol up to marked line. After that, stock solution (1,000 mg/L) was prepared by putting the prepared 1.0 ml of stock solution (100,000 mg/ L) into volumetric flask of 100 ml. and adding growth medium up to marked line. Test solution was prepared by adding growth medium up to marked line after putting 2.00. 2.50, 3.15, 3.950 and 5.00 ml of stock solution (1,000 mg/ L) into volumetric flask of 500 ml respectively. In test solution, 100 ml was put into erienmeyer flask by making replicates per each nominal concentration (nominal concentrations were 4.0, 5.0, 6.3, 7.9 and 10.0 mg/L). Stock solution and test solution were prepared on the day that the test started.

Control
OECD-recommended growth medium was used for control group. Methyl Alcohol was used for solvent control.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Supplier
American Type Culture Collection (ATCC : P.O.Box 1549, Manassas, VA 20108 USA)
Storage method: Storage after division of algae purchased from place of procurement, in ultra low temperature storage tank (Air Liquide, ARPEGE40, -196 °C)

Selection of test species
Algae (Pseudokirchneriella subcapitata) is test species recommended by OECD guidelines for the testing of chemicals, Test No. 201 : Freshwater Alga and Cyanobacteria, Growth Inhibition Test (Adopted : 23 March 2006) and National Institute of Environmental Research Notification No. 2010-29 (16 August 2010).
This species was selected as test system because of the large amount of toxicological test history data existing for use of this species.

Culture conditions
Culture environment
Algae was cultured in erlenmeyer flask of capacity 250 ml, by using shaking incubator (J-SCR, JISICO). Setup of shaking incubator was 21 - 24 ºC of temperature, 24 hours of light condition, 4,440-8,880 Lux of illumination, and 100 rpm of revolutions per minute.

Monitoring of culture conditions
Temperature of incubator was measured every day, by using thermometer that calibration was carried out from external professional institute. Light intensity inside incubator was measured every day, by using ANAF-12 digital lux meter (Tokyo Koden, Japan). In the results of measuring culture conditions, there was no abnormality that could have an affect on test.

Culture medium
Culture medium was used, which was.prepared according to OECD guidelines for the testing of chemicals, Test No.201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test, (Adopted : 23 March 2006). pH of prepared growth media was measured just after preparation of culture medium.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22.5 °C (min 22.4 - max 22.5)
pH:
7.70 (min 7.28 - max 8.31)
Nominal and measured concentrations:
Nominal concentration was 4.0, 5.0. 6.3, 7.9 and 10.0 mg/L, and analysis was carried out for concentration of test substance in test solution at 0 hour, 24 hours,
48 hours and 72 hours after start of test.
As the result of analysis at the start of the test (0 hour), the concentrations of ESA in test solutions were 3.570, 4.656, 5.515, 7.234 and 8.970 mg/l (89.3, 93.1, 87.5, 91.6 and 89.7 % of nominal concentrations).
At 24 hours. the concentrations of ESA in test solutions were 2.995, 3.978, 5.119, 6.644 and 8.394 mg/ L (74.9, 79.6, 81.2, 84.1 and 83.9 % of nominal concentrations).
At 48 hours, the concentrations of ESA in test solutions were 3.307, 4.253, 5.484, 7.087 and 8.865 mg/l (82.7, 85.1, 87.0, 89.7 and 88.7 % of nominal concentrations).
At 72 hours, the concentrations of ESA in test solutions were 3.834, 4.893, 6.286 8.207 and 10.131 mg/ L (95.8, 97.9, 99.8, 103.9 and 101.3 % of nominal concentrations).
The geometric means of concentrations during the test period (0-72 hours) were 3.412, 4.431, 5.585, 7.272 and 9.068 mg/L(85.3, 88.6, 88.7, 92.0 and 90.7 % of nominal concentrations).
Details on test conditions:
Exposure condition
Algae that reached exponential growth phase after preculture for 4 days was used. Algae was injected into erlenmeyer flask to be 5 x 10^3~10^4 cells/ml, and treatment was repeatedly carried out 3 times. 100 ml of test solution were put into erlenmeyer flask of capacity 250 ml. The same exposure condition as culture condition was maintained, and exposure was carried out in static system that test solution was not replaced for 72 hours.
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 9.068 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
4.43 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
5.59 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.:
> 9.068 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
3.41 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Details on results:
Percent inhibition and Abnormal appearances
Inhibition rate for average specific growth rate, and inhibition rate for yield was calculated in 4.0, 5.0, 6.3, 7.9 and 10.0 mg/L of test substance treatment group.
At 72 hours after start of test, percent inhibition for average specific growth rate was 3.07, 4.13, 4.46, 4.83 and 8.10 % respectively.
At 72 hours after start of test, percent inhibition for yield was 9.61, 14.05, 15.39, 16.86 and 28.82 % respectively.
During test period, abnormal appearances were not observed in control group, solvent control group and test substance treatment group.

Validation of test
Number of cells in control group increased 10^9 times during test period(OECD standard : 16 times and over).
Average coefficient of variation of specific growth rate at each section (μ0-24, μ24-48, μ48-72) of control group replicates was 22.2, 22.1 and 30.1 % respectively, and average coefficient of variation of specific growth rate at each section of
control group average was 24.8 % (OECD standard: within 35 %), and coefficient of variation of 72-hour average specific growth rate in control group replicates was 1.9 % (OECD standard: within 7 %).
Change (between the max and the min) of control group pH was 0.61 (standard: within 1.5)
Validity criteria fulfilled:
yes
Conclusions:
Growth inhibition test for algae by ESA was carried out in static system (72 hours). 72-hour ErC50 of percent inhibition for average specific growth rate was more than 9.068 mg/ L, and No Observed Effect Concentration (NOEC) was 4.43 mg/L, and Lowest Observed Effect Concentration (LOEC) was 5.59 mg/L. 72-hour EyC50 of percent inhibition for yield was more than 9.068 mg/ L, and Lowest Observed Effect Concentration(LOEC) was 3.41 mg/ L. Determination of EC50 was based on measured concentrations. During the test period there was no disturbance which influenced test results.
Executive summary:

Summary

Results of growth inhibition test that observe and investigate growth inhibition rate and abnormal behaviours, by exposing algae (Pseudokirchneriella subcapitata) to test substance ESA under static system for 72 hours are as follows.

 

The test was carried out according to OECD guidelines for the testing of chemicals Test No. 201, "Freshwater Algae and Cyanobacteria, Growth Inhibition test" (Adopted 23 March 2006) and National Institute of Environmental Research Notification No. 2010-29 (16 August 2010) Regulations on designation of test institute for examination of toxicity of chemicals, etc. annex 5 chapter 2 clause 1 Algae growth inhibition test.

 

The test was performed using a control, a solvent control and concentrations of 4.0, 5.0, 6.3, 7 .9 and 10.0 mg/ L(nominal concentration, geometric series 1.26). Each geometric mean of test substance during the test period (0-72 hours) was 3.412, 4.431, 5.585, 7.272 and 9.068 mg/L (85.3, 88.6, 88.7, 92.0 and 90.7 % of nominal concentrations)

 

Observation time

LC50 (mg/L)1

95 % confidence limits

NOEC2(mg/L)

LOEC3(mg/L)

72 hours

ErC50

> 9.068

-

4.43

5.59

EyC50

> 9.068

-

-

3.41

1. Median Effect Concentration, Based on Measured Concentration

2. No Observed Effect Concentration, Based on Measured Concentration

3. Lowest Observed Effect Concentration, Based on Measured Concentration

 

Abnormal appearances were not observed in negative control group, solvent control group and test substance treatment group during the test period.

The mean of temperature was 22.5 ± 0.1 °C (min 22.4 - max 22.5).

The mean of pH was 7.70 ± 0.23 (min 7.28 - max 8.31).

The average light intensity of incubator was 6.186 ± 495 Lux (min 5,560 – max 6,930).

Description of key information

Summary

Results of growth inhibition test that observe and investigate growth inhibition rate and abnormal behaviours, by exposing algae (Pseudokirchneriella subcapitata) to test substance ESA under static system for 72 hours are as follows.

 

The test was carried out according to OECD guidelines for the testing of chemicals Test No. 201, "Freshwater Algae and Cyanobacteria, Growth Inhibition test" (Adopted 23 March 2006) and National Institute of Environmental Research Notification No. 2010-29 (16 August 2010) Regulations on designation of test institute for examination of toxicity of chemicals, etc. annex 5 chapter 2 clause 1 Algae growth inhibition test.

 

The test was performed using a control, a solvent control and concentrations of 4.0, 5.0, 6.3, 7 .9 and 10.0 mg/ L(nominal concentration, geometric series 1.26). Each geometric mean of test substance during the test period (0-72 hours) was 3.412, 4.431, 5.585, 7.272 and 9.068 mg/L (85.3, 88.6, 88.7, 92.0 and 90.7 % of nominal concentrations)

 

Observation time

LC50 (mg/L)1

95 % confidence limits

NOEC2(mg/L)

LOEC3(mg/L)

72 hours

ErC50

> 9.068

-

4.43

5.59

EyC50

> 9.068

-

-

3.41

1. Median Effect Concentration, Based on Measured Concentration

2. No Observed Effect Concentration, Based on Measured Concentration

3. Lowest Observed Effect Concentration, Based on Measured Concentration

 

Abnormal appearances were not observed in negative control group, solvent control group and test substance treatment group during the test period.

The mean of temperature was 22.5 ± 0.1 °C (min 22.4 - max 22.5).

The mean of pH was 7.70 ± 0.23 (min 7.28 - max 8.31).

The average light intensity of incubator was 6.186 ± 495 Lux (min 5,560 – max 6,930).

Key value for chemical safety assessment

EC50 for freshwater algae:
9.07 mg/L
EC10 or NOEC for freshwater algae:
4.43 mg/L

Additional information