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Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Study initiation: 2012.2.09 Study completion: 2012.3.29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity 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, 72 hours and test end(96 hours) using HPLC-Triple Quad LC/ MS(Agilent Technologies).
Vehicle:
yes
Remarks:
methyl alcohol
Details on test solutions:
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 10 ml after measurement with analytical balance (sartorius CPA224S), and adding methyl alcohol up to marked line.
In solvent control group, 5,000 ml was prepared by adding test water after putting 0.5 ml of methyl alcohol in water tank of 7L. 5,000 ml of test solution were prepared by adding test water after putting 0.05, 0.09, 0.16, 0.28 and 0.50 ml of stock solution into water tank of 7L respectively (nominal concentrations of each water tank were 1.0, 1.8, 3.2, 5.6 and 10.0 mg/ L).
Stock solution and test solution were prepared on the day that the test started and test solution was replaced (at 24 hours, 48 hours and 72 hours).

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 1.0, 1.8, 3.2, 5.6 and 10.0 mg/ L (geometric series 1. 78). Nominal concentration was set up on the basis of results of range-finding test carried out for active ingredients of test substance (0.1 , 1.0, 5.0 and 10.0 mg/ L). Dead fish was not observed in control group, solvent control group and test substance treatment group 0.1, 1.0, 5.0 and 10.0 mg/L for range-finding test.

Control
The tap water used for control was dechlorinated by an activated reverse osmosis (RO) filter and aerated for more than 24 hours. Methyl Alcohol was used for solvent control.

Test organisms (species):
Oryzias latipes
Details on test organisms:
Supplier
Obtain: National Institute of Environmental Research
Obtain Address: Environmental Research Complex, Kyungseo- dong, Seo-gu, lncheon, Korea
Supply: Green Chemistry Team, Korea Environment Corporation
Supply Address: Environmental Research Complex, Kyungseo-dong, Seo-gu, lncheon, Korea

Total length of Oryzias Latipes is 1 - 3 cm

Selection of test species
Oryzias latipes are a recommended species for use by OECD Test Guideline 203 (1992), U.S. EPA Toxic Substances Control Act Test Guideline (27 September 1985) and National Institute of Environmental Research Notification No. 2010-29 (16 August 2010). Oryzias latipes was selected as test system because of the large amount of toxicological test history data existing for use of this species.

Acclimatization and fasting
Eighty one Oryzias latipes were placed in 5 water tanks (13 liter) and fish were acclimatized to test conditions for seven days. Dead fish did not occur, and health condition was good during period of acclimatization. Feeding was stopped approximately 24 hours prior to the start of the definitive test.

Breeding conditions
Breeding environment
Oryzias latipes were bred in 80 liter water tanks. Those were bred in the surrounding conditions set up to 21 ºC - 25 ºC of water temperature. 16 hours (08:00 - 24:00) of light condition, 8 hours (24:00 - 08:00 in the next day) of dark condition. 400 - 600 Lux of illumination. Feed of Oryzias latipes was supplied in the amount that could be eaten up within 5 minutes (approximately 3% of fish weight), 2 times a day (morning and afternoon).

Monitoring of breeding conditions
The temperature of the breeding ropm and breeding water were adjusted by air-conditioning system and were record each hour automatically. As evidenced by monitoring results, there were no temperature excursion impacting the test.

Breeding water
The tap water in the laboratory used for breeding water was dechlorinated by an activated reverse osmosis (RO) filter and aerated for more than 24 hours. The quality of the breeding water at our facility is measured annually. There were no breeding water excursions impacting the test.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
68 mg CaCO3 / L (at 0 hour, 24 hours, 48 hours and 72 hours : 67, 68, 68 and 69 mg CaCO3 / L respectively).
Test temperature:
22.28 ± 0.11 °C (min 22.1 - max 22.5).
pH:
7.70 ± 0.21 (min 7.13 - max 8.10).
Dissolved oxygen:
91.58 ± 2.86 % (min 85.3 - max 95.8)
Nominal and measured concentrations:
Nominal concentrations were 1.0, 1 .8, 3.2, 5.6 and 10.0 mg/L.
As the result of analysis at the start of the test (0 hour), the concentrations of ESA in test solutions were 2.054, 3.057, 4.900, 8.006 and 13.623 mg/ L (205.4,
169.8, 153.1 , 143.0 and 136.2 % of nominal concentrations)
The concentrations at 24 hours (before replacement of the new test solutions) were 2.080, 3.231, 5.484, 8.687 and 14.336 mg/ L (208.0, 179.5, 171.4, 155.1 and
143.4 % of nominal concentrations).
The concentrations at 24 hours (after replacement of the new test solutions) were 1.473, 2. 151, 3.599, 6.391 and 11 .159 mg/L (147.3, 119.5, 112.5, 114.1 and
111.6 % of nominal concentrations).
The concentrations at 48 hours (before replacement of the new test solutions) were 1.724, 2.439, 4.261, 6.631 and 10.730 mg/L (172.4, 135.5, 132.2, 118.4 and
107.3 % of nominal concentrations).
The concentrations at 48 hours (after replacement of the new test solutions) were 1.504, 2.390, 4.856, 8.254 and 15.659 mg/L (150.4, 132.8, 151.7, 147.4 and
156.6 % of nominal concentrations).
The concentrations at 72 hours(before replacement of the new test solutions) were 2.788, 4.248, 7.347, 11.305 and 20.271 mg/L (278.8, 236.0, 229.6, 201.9 and
202.7 % of nominal concentrations).
The concentrations at 72 hours(after replacement of the new test solutions) were 2.346, 3.208, 4.778, 7.641 and 12.706 mg/L (234.6, 178.2, 149.3, 136.5 and
127.1 % of nominal concentrations).
The concentrations at the end of the test (96 hours) were 2.551 , 3.886, 6.489, 13.961 and 26.128 mg/ L (255.1, 215.9, 202.8, 249.3 and 261.3 % of nominal
concentrations).
The geometric mean of concentrations during the test period (0 - 96 hours) were 2.039, 3.038, 5.160, 8. 709 and 15.238 mg/L (203.9, 168.8, 161.3, 155.5 and
152.4 % of nominal concentrations).
Details on test conditions:
Test water (dechlorinated tap water)
The tap water was dechlorinated by an activated reverse osmosis (RO) filter and aerated for more than 24 hours.

Exposure condition
5 L of test solution was placed in each 7 L test vessel (W24 x L 13 x H23 cm). 10 fish were added to each 7 L test and control vessel. The same exposure condition as breeding condition was maintained. No food was provided to the fish during the 96-hour exposure to the test substance under the semi-static (24 hours) system.

Observation and Measurement
Mortality and abnormal behaviors
After 3, 24, 48, 72 and 96 hours, any mortalities or sub-lethal effects of exposure were determined by visual inspection of the test fish. Test fish were considered to be dead when there was no gill movement or no reaction when the fish was touched with a glass bar.

Analysis of water characteristics
Dissolved oxygen (Orion Star Series DO meter, Thermo Scientific) and pH (Orion Star Series pH meter, Thermo Scientific) were measured every 24 hours during the test, and temperature of test solutions was measured daily.

Calculation of LC50 and NOEC
LC50 and confidence limits were statistically calculated using probit analysis method (EPA600/4-85/ 13, 1985) based on a mortality value of 96 hours. NOEC was determined by the highest concentration causing no mortality. Determination of LC50 and NOEC were based on measured concentration.
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 15.238 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
15.238 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
Mortality and abnormal behaviors
During the definitive test period, there were no mortalities or abnormal behaviors in the control group, solvent control group and the treatment group.

Weight and Length of Test system
For the test systems used in this study, the control weight mean was 0.054 ± 0.017 g (min 0.029 - max 0.081) and the treatment group weight mean was 0.056 ± 0.029 g(min 0.016 - max 0.127).
The control length mean was 19.12 ± 1.96 mm (min 16.60 - max 22.80) and the treatment group length mean was 19.21 ± 3.14 mm(min 12.40 - max 25.90).
Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity (96-Hour LC50) of the test materials (ESA) for Oryzias latipes has been determined to be higher than 15.238 mg/L and the NOEC (No Observed Effect Concentration) was 15.238 mg/L. Determination of LC50 and NOEC was based on measured concentrations. During the test period there was no disturbance which influenced test results.
Executive summary:

Summary

This Acute toxicity test of ESA was performed using Oryzias latipes for 96 hours under the semi-static (24-hour) system. Observation were made including documenting any abnormal behaviors of Oryzias latipes and total number of mortalities.

The method followed that described in the OECD Guidelines for the Testing of Chemicals Test No. 203 (Adopted 17 July 1992) and National Institute of Environmental Research Notification No. 2010-29 (16 August 2010).

The toxicity test was performed using a control, a solvent control and concentrations of 1.0, 1.8, 3.2, 5.6 and 10.0 mg/L (nominal concentration, geometric series 1.78).

Each geometric mean of test substance during the test period (0 - 96 hours) was 2.039, 3.038, 5.160, 8.709 and 15.23 mg/L (203.9, 168.8, 161.3, 155.5 and 152.4 % of nominal concentrations).

The test results were as follows:

Observation time

LC50 (mg/L)1

95 % confidence limits

NOEC2(mg/L)

96 hours

> 15.238

-

15.238

1. Median Effect Concentration, Based on Measured Concentration

2. No Observed Effect Concentration, Based on Measured Concentration

Dead fish and abnormal behaviors were not observed in control group, solvent control group and test substance treatment group during the test period.

The mean of temperature was 22.28 ± 0.11 °C (min 22.1 - max 22.5).

The mean of pH was 7.70 ± 0.21 (min 7.13 - max 8.10).

The mean of DO was 91.58 ± 2.86 % (min 85.3 - max 95.8).

The mean of test system control weight was 0.054 ± 0.017 g (min 0.029 - max 0.081 ). The mean of treatment group weight was 0.056 ± 0.029 g (min 0.016 - max 0.127).

The mean of test system control length was 19.12 ± 1.96 mm (min 16.60 - max 22.80). The mean of treatment group length was 19.21 ± 3.14 mm(min 12.40 - max 25.90).

Description of key information

Summary

This Acute toxicity test of ESA was performed using Oryzias latipes for 96 hours under the semi-static (24-hour) system. Observation were made including documenting any abnormal behaviors of Oryzias latipes and total number of mortalities.

The method followed that described in the OECD Guidelines for the Testing of Chemicals Test No. 203 (Adopted 17 July 1992) and National Institute of Environmental Research Notification No. 2010-29 (16 August 2010).

The toxicity test was performed using a control, a solvent control and concentrations of 1.0, 1.8, 3.2, 5.6 and 10.0 mg/L (nominal concentration, geometric series 1.78).

Each geometric mean of test substance during the test period (0 - 96 hours) was 2.039, 3.038, 5.160, 8.709 and 15.23 mg/L (203.9, 168.8, 161.3, 155.5 and 152.4 % of nominal concentrations).

The test results were as follows:

Observation time

LC50 (mg/L)1

95 % confidence limits

NOEC2(mg/L)

96 hours

> 15.238

-

15.238

1. Median Effect Concentration, Based on Measured Concentration

2. No Observed Effect Concentration, Based on Measured Concentration

Dead fish and abnormal behaviors were not observed in control group, solvent control group and test substance treatment group during the test period.

The mean of temperature was 22.28 ± 0.11 °C (min 22.1 - max 22.5).

The mean of pH was 7.70 ± 0.21 (min 7.13 - max 8.10).

The mean of DO was 91.58 ± 2.86 % (min 85.3 - max 95.8).

The mean of test system control weight was 0.054 ± 0.017 g (min 0.029 - max 0.081 ). The mean of treatment group weight was 0.056 ± 0.029 g (min 0.016 - max 0.127).

The mean of test system control length was 19.12 ± 1.96 mm (min 16.60 - max 22.80). The mean of treatment group length was 19.21 ± 3.14 mm(min 12.40 - max 25.90).

Key value for chemical safety assessment

LC50 for freshwater fish:
15.24 mg/L

Additional information