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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:
29.01. – 16.02.2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
Daphnia sp. Acute Immobilisation Test, Council Regulation (EC) No. 440/2008, published in O.J.L 142, 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Preliminary test: The analytical determination of the test substance concentrations was performed at the beginning and at the end of the test. The samples for analysis were taken from the highest concentration and from the lowest test concentration. The samples for analysis (0 hours) were prepared at the beginning of the test and immediately delivered in transport box to analytical laboratory. The samples were analysed on the day of delivery. The samples for analysis at the end of the test (48 hours) were delivered to analytical laboratory immediately after the end of testing. The samples were analysed on the day of delivery. All samples were stored at laboratory temperature.
The analytical results showed, that the test substance was stable in dilution water at the conditions of the test.

Definitive test: The analytical determination of the test substance concentrations was performed at the beginning and at the end of the test. The samples for analysis were taken from the highest and the lowest test concentrations. The samples for analysis (0 hours) were prepared at the beginning of the test and immediately delivered in transport box to analytical laboratory. The samples were analysed on the day of delivery. The samples for analysis at the end of the test (48 hours) were delivered to analytical laboratory immediately after the end of testing. The samples were analysed on the day of delivery. All samples were stored at laboratory temperature.
Details on test solutions:
The test substance is poorly soluble in dilution water at concentrations needed for the test. For this reason the preliminary experiment was performed for finding suitable period for achieving the highest solubility.
For the preliminary experiment the solutions were prepared as follows: 100 mg of the test substance in 1000 mL of dilution water was ultrasonicated for 30 minutes and stirred for period 3, 24, 48 and 72 hours on a shaft stirrer, subsequently filtered through 0.45 μm filter and the concentrations of the test substance in resulted solutions were determined.
On the basis of the measured concentrations of the test substance, the period of 72 hours stirring was chosen.
Final procedure for the preparation of the test solutions was then set as follows:
The saturated solutions of the test substance were prepared in the dilution water. The saturated solutions were ultrasonicated for 30 minutes and stirred for period 72 hours and subsequently filtered through 0.45 μm filter.
The concentrations of test solutions used in the preliminary and definitive test were obtained by dilution of the saturated solutions with dilution water.
Test organisms (species):
Daphnia magna
Details on test organisms:
Daphnia magna Straus were obtained by aperiodic parthenogenesis from own laboratory breeding. Daphnia were fed by a mixture of algae. The sensitivity of the test species is periodically monitored by testing with the reference substance, potassium dichromate.The grow-up gravid females are separated into dilution water. Young daphnia born in 24 hours are segregated to the handling container from which they entered the experiment. At handling a care should be taken of not to penetrate the air under animal’s shell.
Because culturing and test medium was different we included pre-test acclimation period: brood daphnids were maintained in dilution water for 48 hours prior to the start of the test.

TEST ORGANISM
- Common name: Daphnia Magna
- Strain: Straus
- Source: own laboratory breeding
- Age at study initiation: young daphnia born in 24 hours
- Method of breeding: aperiodic parthenogenesis
- Feeding during test: no

ACCLIMATION
The grow-up gravid females are separated into dilution water. Young daphnia born in 24 hours are segregated to the handling container from which they entered the experiment.
- Type and amount of food: a mixture of algae
- Health during acclimation: no mortality observed
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
2.54 mmol/L
The content of sum Ca + Mg ions should have the value of 2.50 +/- 0.10 mmol/L.
Test temperature:
20.0 ± 2.0 °C; with a variation of ± 1°C at each individual test
pH:
8.0
Dissolved oxygen:
9.2 mg·L-1
Conductivity:
2.49 µS·cm-1
Nominal and measured concentrations:
1000, 710, 510, 360, 260, 190 and 140 mL·L-1 + C(control)
67.4, 47.9, 34.4, 24.3, 17.5, 12.8, 9.6 mg·L-1 + C(control)
Determined concentration of test substance
0 hours: 9.56 ; 67.4 mg·L-1
48 hours: 9.97 ; 70.24 mg·L-1
Details on test conditions:
TEST SYSTEM
- Test vessel: glass beakers 50ml
- Type (delete if not applicable): open
- Aeration: no
- Lighting: daylight
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 2
- Biomass loading rate: at least 2 ml per individual

For holding and culturing of the daphnia the M4 medium was used (according to Annex 2 of the EU method C.2 and according to Annex 3 of the OECD TG202).
For test was used dilution water (according to Annex 2 of the EU method C.2 and according to Annex 3 of the OECD TG 202).

Dilution water was prepared just before the test by admixing 2.5 mL of stored solutions A, B, C and D into 1000 mL of deionized water.
Because culturing and test medium was different we included pre-test acclimation period: brood daphnids were maintained in dilution water for 48 hours prior to the start of the test.
A. 117.6 g CaCl2.2H20 in 1 L of deionized water
B. 49.3 g MgSO4.7H2O in 1 L of deionized water
C. 25.9 g NaHCO3 in 1 L of deionized water
D. 2.3 g KCl in 1 L of deionized water

Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
49.4 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
39.5 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
EC0
Effect conc.:
9.6 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC0
Effect conc.:
9.6 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
EC100
Effect conc.:
> 67.4 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
> 67.4 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Results with reference substance (positive control):
Reference test
The sensitivity of the test species and correctness of test performance is periodically verified in six-month period by testing with the reference item, potassium dichromate. The results of the verification test with K2Cr2O7, carried out in period from 06.12. to 08.12.2017 are the following:

24 hour – EC50 = 1.10 mg·L-1 (95% confidence limit: 0.96 – 1.28 mg·L-1)
48 hour – EC50 = 0.77 mg·L-1 (95% confidence limit: 0.69 – 0.86 mg·L-1)

Validity criteria fulfilled:
yes
Conclusions:
There is stated in the guideline that, if evidence is available to demonstrate that the concentration of the test item has been satisfactorily maintained within ± 20 percent of the nominal or measured initial concentration throughout the test, the results can be based on nominal or measured initial values.
The initial measured concentrations were used for all evaluations and results.

Test results:
24 hour – EC50 = 49.4 mg·L-1        (95% confidence limit: 40.0 – 68.5 mg·L-1)
48 hour – EC50 = 39.5 mg·L-1        (95% confidence limit: 32.3 – 52.0 mg·L-1)
Executive summary:

The test item, Propatyl nitrate, was tested in acute immobilisation test on Daphnia magna.

The test was performed according to method C.2 – Daphnia sp. Acute Immobilisation Test, Council Regulation (EC) No. 440/2008, published in O.J.L 142, 2008.

Since the test substance is poorly soluble in dilution water at concentrations needed for the test, the saturated solutions of the test substance in dilution water were prepared for preliminary and definitive test. The saturated solutions were prepared by adding measured amounts of the test substance to the dilution water. The saturated solutions were ultrasonicated for 30 minutes and stirred for period 72 hours on a shaft stirrer and subsequently filtered through 0.45 μm filter.

The preliminary test was performed in a range of the test substance measured concentrations 0.6 – 60.7 mg·L-1. Based on toxicity of the test substance found in the preliminary test, the definitive test was performed in appropriate concentration range.

The analytical results showed, that the test substance Propatyl nitrate was sufficiently stable in dilution water in the conditions of the test, therefore the definitive test was performed as static one.

The definitive test was performed in a range of the test substance measured concentrations 9.6 – 67.4 mg·L-1. Samples for analytical determination were taken at the beginning and at the end of the test.

There is stated in the guideline that, if evidence is available to demonstrate that the concentration of the test substance has been satisfactorily maintained within ± 20 percent of the nominal or measured initial concentration throughout the test, the results can be based on nominal or measured initial values.

The initial measured concentrations were used for all evaluations and results.

Test results:

24 hour – EC50 = 49.4 mg·L-1        (95% confidence limit: 40.0 – 68.5 mg·L-1)

48 hour – EC50 = 39.5 mg·L-1        (95% confidence limit: 32.3 – 52.0 mg·L-1)

24 hour – EC0 = 9.6 mg·L-1                

48 hour – EC0 = 9.6 mg·L-1                 

24 hour – EC100 > 67.4 mg·L-1       

48 hour – EC100 > 67.4 mg·L-1       

Description of key information

The test substance was tested in acute immobilisation test on Daphnia magna according to method C.2 – Daphnia sp. Acute Immobilisation Test (GLP study). The test was performed as static.

24 hour–EC50 = 49.4 mg·L-1             (95% confidence limit: 40.0 – 68.5 mg·L-1)

48 hour–EC50 = 39.5 mg·L-1              (95% confidence limit: 32.3 – 52.0 mg·L-1)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
39.5 mg/L

Additional information

Because concentrations of the test substance have been satisfactorily maintained within ± 20 percent of the nominal or measured initial concentration throughout the test,measured initial concentrations were used.