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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
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 performed according to the OECD 202 guideline and under GLP compliance.
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
April 13, 2004
Deviations:
no
Principles of method if other than guideline:
N/A
GLP compliance:
yes
Specific details on test material used for the study:
See test material information above
Analytical monitoring:
yes
Vehicle:
no
Details on test solutions:
PREPARATION OF TEST SOLUTION
The test was dispensed by elutriating with test water. The purity of test substance (content of Active ingredients) was calculated 98 % based on the test material information chart and COA (Annex 1, Annex 2).
When dispensing test substance, which was 1000 mg/L of stock solution, the precipitation occurred, but the test solution was processed to fit the set concentration over stirring and dilution to verify the toxic values. To analyze toxic value and test concentration, definitive test was carried out after stock solution was dispensed inevitably.
Test organisms (species):
Daphnia magna
Details on test organisms:
SOURCE
Invertebrates Breeding Room in the Health Care Research Laboratory Korea Testing & Research Institute (KTR), Hwasun-gun, Jeollanam-do, Korea

CULTURING METHODS
Growth stage : Juveniles less than 24-hour-old (female)
Water : Elendt M4 medium
Vessel : 1 L glass beaker
pH : 6.0 - 9.0
Dissolve oxygen : 3 mg/L or more
Hardness : 140 mg/L - 250 mg/L
Light : 16-hour light (700 lux or less) / 8-hour dark
Water temperature : (18 - 22) ℃
Culture period : 28 days or less
Reference substance : Potassium dichromate, SIGMA-ALDRICH
Feed : Chlorella sp.
Offspring removal : Removed on the day offspring were produced
Production of first brood : within 12 days

Gravid adults were isolated 24 hours prior to initation of the test. Young daphnids produced overnight(〈 24 hours old) were used for testing.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Remarks on exposure duration:
None
Post exposure observation period:
No data
Hardness:
140 - 250 mg/L
Test temperature:
20±1 ℃
pH:
6.0 – 9.0 (Not adjusted)
Dissolved oxygen:
3 mg/L or more
Salinity:
No data
Conductivity:
No data
Nominal and measured concentrations:
- Nominal concentration (mg/L): control, 0.5, 1, 2, 4, 8, 16 (geometric series 2.0)
- Measured concentration (mg/L): control, 0.40, 0.84, 1.66, 3.24, 5.93, 11.58
Details on test conditions:
TEST CONDITIONS
Room : Invertebrates Test Room
Test species : Dapnia magna
Duration of exposure : 48 hours
Exposure procedure : Static
Volume of test solution : 100 mL (65 mm H. × 57 mm φ)
Number of vessels : 4 vessel / test group
Number of loading daphnids : 20 daphnids / test group (5 daphnids / vessel)
Temperature : 20±1 ℃
D.O. : 3 mg/L or more
pH : 6.0 – 9.0 (Not adjusted)
Hardness : 140 mg/L - 250 mg/L
Light : 16-hour light (08:00 - 24:00), 8-hour dark (24:00 - 08:00)
Feeding : None

CULTURING AND DILUTION WATER
Elendt M4 medium was used as culturing and dilution water, and was prepared according to the method of OECD Guideline for Testing of Chemicals No. 202 "Daphnia sp., Acute Immobilisation Test" (2004), was aerated sufficiently and adjusted to 20±1 ℃, and then it was used as dilution water. The medium was strongly aerated for more than 2 hours before use and the hardness was measured with HI 96735 (Hanna Instruments, USA).

ENVIRONMENTAL CONDITIONS
pH, dissolved oxygen and water temperature were recorded daily for all treatments.
pH, dissolved oxygen (DO) and water temperature, during the exposure period were maintained as follows:
- pH : 8.09 (7.84 – 8.32)
- Dissolved oxygen : 7.37 mg/L (7.04 – 7.78)mg/L 81.5 % (77.9 - 86.1)% of ASV
- Temperature : 20.2 ℃ (20.0 ℃ – 20.4 ℃)
No food and air was provided during the test period.

ENVIRONMENTAL MONITORING
Water temperature : Water thermometer (PC 2400, Sato, Japan) 1 times / week
pH and DO : Water thermometer (PC 2400, Sato, Japan) 1 times / week
Light : Lux meter (ANA-F11, TOKYO Photoelectric Co., Ltd, Japan) and Recorder (KR100, Konics, Korea) 1 time / 3 month
As the result of the monitoring, there was no observed an alteration affecting the test.

OBSERVATION :
The daphnids were inspected at test start 24 and 48 hours. Those animals that were not able to swim within 15 seconds, after gentle agitation of the test vessel were considered to be immobilised (even if they can still move their antennae). In addition to immobility, any abnormal behavior or appearance were reported.

RANGE-FINDING STUDY
The range-finding test for this test substance was taken under the set of static system in the test solution of 0.1, 1.0, 10, 100 mg/L. Two groups of five in repeated processes (a total of 10 Daphnia magna) were exposed and swimming inhibition objects were also observed for next 48 hours.
Results after 48h: 0% immobility in control, 0.1 and 1.0 mg/L test concentrations. 100% immobility at 10 and 100 mg/L.

DEFINITIVE TEST:
From preliminary experimental results, 100 % swimming inhibition in a setting density of 10 mg/L got shown and swimming inhibition at 0.1 mg/L wasn’t monitored.
Therefore we would conduct definitive test in the concentration of settings; 0.5, 1, 2, 4, 8, and 16 mg/L in the difference between test groups and control.
After we put 100mL test solution to a 120 mL glass beaker, a total of 20 daphnia magna by randomly putting 5 for 4 in test vessel with repeated were exposed.

STORAGE STABILITY TEST:
The analysis on the storage stability test substance was conducted according to the APHA Standard Method: 2012 test method by Environmental Health Assessment Team in the Korea Testing & Research Institute, the test solution was prepared at a set concentration of 0.02 mg/L and subjected to a storage stability test for 10 days for 16 hours under the light condition, 8 hours under the dark condition (19-21)°C was maintained at 81.3% of the initial concentration. Thus, it was estimated under the storage conditions of the test material in test water M4 media).

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 10mg/L and subjected to a concentration of test material for 96 hours, was maintained at (100.0 - 101.7) % of the initial concentration. Thus, the definitive test was conducted with static system without replacing the test solution during the test period.
Reference substance (positive control):
yes
Remarks:
Positive control : The test was performed in control, 0.2, 0.4, 0.8, 1.6, 3.2 mg/L (geometric series 2.0, nominal concentration) as active ingredient of Potassium dichromate
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
4.788 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
3.351 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
NOEC
Effect conc.:
1.66 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
1.66 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
ENVIRONMENTAL CONDITIONS
During test period, the water quality change in the test solution was measured as follows:
- pH : 8.09 (7.84 - 8.32)
- Dissolved oxygen : 7.37 mg/L (7.04 – 7.78) mg/L, 81.5 % (77.9 – 86.1) % of ASV
- Temperature : 20.2 ℃ (20.0 – 20.4) ℃
- Hardness : 211 mg/L

ANALYSIS OF TEST SOLUTIONS
- Storage stability test:
As a result of test material stability test for 10 days in the test solution of nominal concentration 0.02 mg/L, it showed up at 106.3 % of initial concentration after 1 day elapsed, 100.0 % of initial concentration after 3 days elapsed, 68.8 % of initial
concentration after 7 days elapsed, and 81.3 % of initial concentration after 10 days elapsed.
- Stability test:
As a result of test material stability test for 48 hours in the test solution of nominal concentration 10 mg/L, it showed up at 100.0 % of initial concentration after 24 hours and 101.7 % of initial concentration after 48 hours.
- Analysis of the test substance in the test solution:
The average of measurement test concentration is 0.40, 0.84, 1.66, 3.24, 5.93, 11.58 mg/L.
Reported statistics and error estimates:
EC50 and 95 % confidence limit was calculated by Probit method (EPA600/4-85/13,1985) based on the result of the observation of immobilisation at the time of 24 and 48 hours after finishing test material processing.
NOEC does not have any toxic symptoms, and it indicated maximum test concentration (measured concentration) that mortality hasn’t occurred.

See the study report attached

Validity criteria fulfilled:
yes
Remarks:
- The rate of immobilisation of control is 0%, it is suitable for the validation criteria (less than 10%) of the test. - Dissolved oxygen of test solution is at minimum of 7.04 mg/L, and it is suitable for validation criteria (≥ 3 mg/L).
Conclusions:
Under the test conditions, the test item zinc cyanide exhibits a 48h-EC50 of 3.351 mg/L [95% CL 2.837 - 3.920 mg/L], based on mean measured concentrations.
Executive summary:

The study was conducted to assess the acute toxicity of zinc cyanide to Daphnia magna under static system. The study was conducted in accordance with the OECD 202 guideline and under GLP conditions.

The test concentration of this test was conducted with a set concentration of 0.5, 1, 2, 4, 8, 16 mg/L (the average measured concentration: 0.40, 0.84, 1.66, 3.24, 5.93, 11.58 mg/L), and exposed 20 Daphnia magna for each test concentration.

The result of this exponential acute toxicity test in static system for the Daphnia magna of Zinc cyanide is as follows, and EC50 and NOEC were designated by measured concentration.

24h-EC50 is 4.788 mg/L, and 95 % of confidence interval is (3.981–5.776) mg/L

48h-EC50 is 3.351 mg/L, and 95 % of confidence interval is (2.837–3.920) mg/L

24h-NOEC is 1.66 mg/L

48h-NOEC is 1.66 mg/L

Description of key information

For that endpoint, a reliable experimental study was available and was conducted in accordance with the OECD 202 guideline and under GLP conditions.

The study was conducted to assess the acute toxicity of zinc cyanide to Daphnia magna under static system.

Under the test conditions, the test item zinc cyanide exhibits a 48h-EC50 of 3.351 mg/L [95% CL 2.837 - 3.920 mg/L], based on mean measured concentrations.

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

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
3.351 mg/L

Additional information