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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:
From 30 October 2015 To 09 May 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: CY50561001
- Expiration date of the lot/batch: 27 June 2017
- Purity: 65.4% (for the substance in solution)


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25°C, below 70 RH%)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: A stock solution with a concentration of 100 mg a.i./L (mg active ingredient/L) (nominal) was prepared with Test Item and test medium (ISO Medium). The test solutions were prepared by appropriate dilution of this stock solution just before introduction of the Daphnia (start of each renewal period).

Analytical monitoring:
yes
Details on sampling:
- Concentrations: All
- Sampling method: At each sampling occasion from all replicates, duplicate samples were taken into plastic tubes (approximately 2-2 mL).
- Sample storage conditions before analysis: Samples are stored at -20°C until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution with a concentration of 100 mg a.i./L (mg active ingredient/L) (nominal) was prepared with Test Item and test medium (ISO Medium). The test solutions were prepared by appropriate dilution of this stock solution just before introduction of the Daphnia (start of each renewal period).
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Source: Szent István University, Department of Aquaculture, 2100 Gödöllő, Páter Károly u. 1. – Hungary
- Feeding during test: No


ACCLIMATION
- Acclimation period: No

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
The reconstituted water (ISO medium) had a total hardness of 247 mg/L (as CaCO3).
Test temperature:
The water temperature was measured at the start and at the end of each renewal period in each test vessel. The test temperature was in the range of 20.3 – 20.7°C measured in the test vessels. The additionally measured temperature in the climate chamber was between 19.8 and 20.8°C.
pH:
The pH of the test solution was not adjusted and not varied by more than 1.5 units in any one test. The pH was measured at the start and at the end of each renewal period in each test vessel and was in the range of 7.11 – 7.78.
Dissolved oxygen:
The dissolved oxygen concentration was measured in each test vessel at the start and at the end of each renewal period and was in the range of 8.1 – 8.5 mg/L.
Nominal and measured concentrations:
Nominal concentrations: 6.25, 12.5, 25.0, 50.0 and 100.0 mg a.i./L (mg active ingredient/L)
Measured concentrations: The measured concentrations not deviated more than 20% from the nominal.
Details on test conditions:
TEST SYSTEM
- Test vessel: glass beaker
- Material, size, headspace, fill volume: at least 5 mL test solution/animal
- Renewal rate of test solution (frequency/flow rate): The frequency of the renewal periods were 24 hours.
- No. of organisms per vessel: 5 animals
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water (ISO medium, according to OECD 202) was used as dilution water for both the range finding and definitive tests. The same composition of reconstituted water was used for the tests and for breeding the test animals.

RANGE-FINDING STUDY
- Test concentrations: 0.1, 1, 10 and 100 [mg a.i./L]
- Results used to determine the conditions for the definitive study:
Untreated control: 10/0 (Number of treated/immobilized animals)
0.1 mg a.i./L: 10/0 (Number of treated/immobilized animals)
1 mg a.i./L: 10/0 (Number of treated/immobilized animals)
10 mg a.i./L: 10/0 (Number of treated/immobilized animals)
100 mg a.i./L: 10/5 (Number of treated/immobilized animals)
Reference substance (positive control):
no
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
84.77 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mobility
Details on results:
- Behavioural abnormalities: no abnormal behaviour or appearance of test animals was detected.
Results with reference substance (positive control):
For the evaluation of the quality of the Daphnia clone and the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
The date of the last study (Study Code: 16/281-023DA) with reference item Potassium dichromate (batch no.: A0345704) is: 14 - 15 September 2016.
The 24h EC50:0.93 mg/L, (95 % confidence limits: 0.86 – 0.99 mg/L).
Reported statistics and error estimates:
The 24h EC50 could not be determined due to the lack of sufficient immobilisation. The 48 hours EC50 value was calculated using Probit analysis by TOXSTAT software. The 48h EC100 value of the Test Item was determined from the raw data.
For the determination of the LOEC and NOEC, the immobilization at the test concentrations was tested on significant differences to the control values by Dunnett’s
Test using TOXSTAT software.
Validity criteria fulfilled:
yes
Conclusions:
The 48h EC50 value was 84.77 mg a.i./L (nominal) (95 % confidence limits: 76.02– 94.51 mg a.i./L).
Executive summary:

Acute toxicity of Diquat was assessed with Acute Immobilisation Test on Daphnia magna, over an exposure period of 48 hours in a semi-static system. Based on the performed preliminary range-finding test five test concentrations in a geometric series with a separation factor of 2.0 and one untreated control were tested in the main experiment.

The nominal concentrations of Test Item used in the main experiment were: 6.25, 12.5, 25.0, 50.0 and 100.0 mg a.i./L (mg active ingredient/L).

Test concentrations were analytically determined at the start and at the end of each renewal period. As the measured concentrations not deviated more than 20 per cent from the nominal in each case, the biological results are based on the nominal concentrations.

Twenty animals, divided into four groups (glass beaker) of five animals each were used at the test and for the control groups.

For the determination of the LOEC and NOEC, the immobilization at the test concentrations was tested on significant differences to the control values by Dunnett’s. Test using TOXSTAT software. The 24h EC50 could not be determined due to the lack of sufficient immobilisation. The 48 hours EC50 value was calculated using Probit analysis by TOXSTAT software. The 48h EC100 value of the Test Item was determined from the raw data.

All validity criteria were met during this study.

In conclusion, under the conditions of this Daphnia magna acute immobilisation study the calculated and observed endpoints for the effect of Diquat were the followings:

The 48h EC50 value:84.77 mg a.i./L (nominal) (95 % confidence limits: 76.02– 94.51 mg a.i./L)

The 48h EC100 value: >100 mg a.i./L (nominal)

The 48h No-Observed Effect Concentration (NOEC): 50 mg a.i./L (nominal)

The 48h Lowest Observed Effect Concentration (LOEC): 100 mg a.i./L (nominal)

Description of key information

Young Daphnia were exposed in a semi-static test (according to OECD 202 and EU Method C.2 and under GLP) to aqueous test medium containing the Test Item at different concentrations. The percentage of Daphnia being no longer capable of swimming at the end of the test or being dead was recorded for each test concentration.

No deviation from the guideline was observed during the test.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
84.77 mg/L

Additional information

Under the conditions of this study, the 48h EC50 value of Diquat is 84.77 mg a.i./L (nominal).