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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:
08.01. – 26.01.2018
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:
The determination of the test item concentrations was carried out by HPLC method.
Preliminary test: The analytical determination of the test item concentrations were performed at the beginning of the test and at the end of the test. The samples for analysis were taken from the highest (100 mg·L-1) and the lowest test concentration (1 mg·L-1). 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 item was stable in dilution water at the conditions of the test.

Definitive test: The analytical determination of the test item concentrations were performed at the beginning of the test and at the end of the test. The samples for analysis were taken from the highest (100 mg·L-1) and the lowest (3 mg·L-1) 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 stock solutions of the test item were prepared in the dilution water. 50 mg of the test item was weighed into 500 mL of the dilution water for the preliminary test and 100 mg of the test item was weighed into 1000 mL of the dilution water for the definitive test.
The concentrations of solutions used in the preliminary and definitive test were obtained by dilution of the stock solution 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.

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:
yes
Total exposure duration:
48 h
Hardness:
2.53 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:
7.8
Dissolved oxygen:
8.9 mg·L-1
Conductivity:
1.13 µS·cm-1
Nominal and measured concentrations:
3, 5, 9, 17,31, 56, 100 mg/L + C(control)
Determined concentration of test substance
0 hours: 3.01 ; 100.9 mg·L-1
48 hours: 3.10 ; 105.0 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: 10
- No. of vessels per concentration (replicates): 2
- 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.:
50.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
20.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
EC0
Effect conc.:
3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC0
Effect conc.:
3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
EC100
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
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 nominal concentrations were used for all evaluation and results.

Test results:
24 hour – EC50 = 50.7 mg·L-1         (nominal concentration) (95% confidence limit: 34.2 – 92.3 mg·L-1)
48 hour – EC50 = 20.4 mg·L-1         (nominal concentration) (95% confidence limit: 14.3 – 29.9 mg·L-1)
Executive summary:

The test item, 5-aminotetrazole, 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.

The preliminary test was performed in a range of the test item nominal concentrations 1 – 100 mg·L-1. The test was performed as static.

Based on toxicity of the test item found in the preliminary test, the definitive test was performed in appropriate concentration range.

The analytical results showed, that the test item 5-aminotetrazole 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 item nominal concentrations 3 – 100 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 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 nominal concentrations were used for all evaluation and results.

Test results:

24 hour – EC50 = 50.7 mg·L-1         (nominal concentration)

(95% confidence limit: 34.2 – 92.3 mg·L-1)

48 hour – EC50 = 20.4 mg·L-1         (nominal concentration)

(95% confidence limit: 14.3 – 29.9 mg·L-1)

24 hour – EC0 = 3 mg·L-1                 (nominal concentration)

48 hour – EC0 = 3 mg·L-1                 (nominal concentration)

24 hour – EC100 > 100 mg·L-1       (nominal concentration)

48 hour – EC100 > 100 mg·L-1       (nominal concentration)

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 = 50.7 mg·L-1             (nominal concentration)

48 hour–EC50 = 20.4 mg·L-1              (nominal concentration)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
50.7 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, nominal concentrations were used.

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