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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:
08.02. – 26.02.2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
Council Regulation (EC) No. 440/2008, published in O.J.L 142, 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
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 (100 mg.L-1) and lowest (1 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 analyzed 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.
Analytical results of the concentrations of the test substance are presented in Table 5. The analytical results showed that the test substance was stable but poorly soluble in dilution water under current test conditions.

Full 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 (100 mg.L-1) and lowest (4 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:
Since the test substance is poorly soluble in dilution water at concentrations needed for the test, the stock saturated solution (nominal concentration 100 mg·L-1) of the test substance in dilution water was prepared for the preliminary and the full test.
The stock saturated solution was prepared as follows: the suspension of 100 mg of the test substance in 1000 mL of the dilution water was ultrasonicated for 30 minutes and stirred for suitable period (72 hours) on a shaft stirrer and subsequently filtered through 0.45 μm filter. This solution was used for testing. The concentrations of solutions used in the preliminary and the full test were obtained by dilution of the stock saturated solution with dilution water.
Test organisms (species):
Daphnia magna
Details on test organisms:
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
- Acclimation period and conditions:
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.
- Type and amount of food: a mixture of algae
- Health during acclimation: no mortality observed
The sensitivity of the test species is periodically monitored by testing with the reference substance, potassium dichromate.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
The content of sum Ca + Mg ions should have the value of 2.50 +/- 0.10 mmol/L, values measured in this study were 2.55 mmol/L in the preliminary test and 2.53 mmol·L-1 in the full test. Measured values meet the requirements.
Test temperature:
20.0 ± 2.0 °C; with a variation of ± 1°C at each individual test
pH:
values measured were 7.9 in the preliminary and the full test
Dissolved oxygen:
higher than 3 mg/L
7.7 mg·L-1 within test
Conductivity:
1.65 µS·cm-1
Nominal and measured concentrations:
100, 59, 35, 21, 12, 7 and 4 mg·L-1
Details on test conditions:
TEST SYSTEM
- Test vessel: glass beakers 50ml
- Type (delete if not applicable): open
- Aeration: no
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- Biomass loading rate: 50 mL per 10 individuals

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 TG 202).
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 1 000 mL of deionized water.
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

OTHER TEST CONDITIONS
- Adjustment of pH: The dilution water should have of pH 7.8 +/- 0.2, values measured in this study were 7.9 in the preliminary and the full test.
- Photoperiod: natural variation of daily light and dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
immobilisation of daphnia after 24 and 48 hours
Daphnia is considered as immobile (incapable of swimming) when exhibit no motion during 15 seconds after a gentle agitation of the test container. The values of 24hour-EC50 and 48hour-EC50 are obtained by calculation. The values of EC0 and EC100 are identified by direct observation.

RANGE-FINDING STUDY
- Test concentrations: 100, 50, 10, 5 and 1 mg·L-1 + C(control)
- Results used to determine the conditions for the definitive study:
Immobilisation % (48 h) 90, 85, 10, 5, 0, 0 %
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 0.165 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.:
>= 0.069 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
EC0
Effect conc.:
0.035 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC0
Effect conc.:
0.02 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
EC100
Effect conc.:
> 0.165 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
> 0.165 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 substance, potassium dichromate.
The results of the verification test with K2Cr2O7, carried out in period from 11.11. to 13.11. 2015 are the following:
24 hour – EC50 = 0.83 mg.L-1(95% confidence limit: 0.69 – 1.01 mg.L-1)
48 hour – EC50 = 0.86 mg.L-1(95% confidence limit: 0.44 – 0.58 mg.L-1)

Interlaboratory test data
48 hour – EC50 = 0.46 – 1.06 mg·L-1

The immobilisation of the test organism by the reference substance (48hour – EC50) obtained from our last reference test meets the calculated range from the interlaboratory test.
Validity criteria fulfilled:
yes
Remarks:
1. Immobilisation in the controls < 10 % at the end of the test. 2. The dissolved oxygen concentrations (throughout the test) > 3 mg·L-1.
Conclusions:
The value of EC50 and the confidence intervals of the EC50 were calculated using the software ToxRat Professional Version 3.2.1.
The EC0 and EC100 values were identified by direct observation. The initial measured and calculated concentrations were used for all evaluations and results.
The highest immobilisation observed at 24 hours was 35% in the full test. Therefore the EC50 value could not be calculated.
24 hour–EC50 ˃ 0.165 mg·L-1
48 hour–EC50 = 0.069 mg·L-1
(95% confidence limit: 0.059 – 0.082 mg·L-1)
Executive summary:

The test substance, 2-(3´,5´-Dichloro-2´-hydroxyphenyl)-4-quinazolinone, 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 full 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 range of the test substance nominal concentrations 1 – 100 mg·L-1. Based on toxicity of the test substance found in preliminary test, the full test was performed in appropriate concentration range.

The analytical results showed that the test substance 2-(3´,5´-Dichloro-2´-hydroxyphenyl)-4-quinazolinone was sufficiently stable in dilution water at conditions of the test; therefore the full test was performed as static one.

The full test was performed in range of the test substance nominal concentrations 4 - 100 mg·L-1. Samples for analytical determination were taken at the beginning and at the end of the test.

The analytical results showed that the test substance is very poorly soluble substance.

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

Test results:

24 hour–EC50 ˃ 0.165 mg·L-1

48 hour–EC50 = 0.069 mg·L-1

(95% confidence limit: 0.059 – 0.082 mg·L-1)

24 hour–EC0 = 0.035 mg·L-1

48 hour–EC0 = 0.020 mg·L-1

 

24 hour–EC100 ˃ 0.165 mg·L-1

48 hour–EC100 ˃ 0.165 mg·L-1

Description of key information

The value of EC50 and the confidence intervals of the EC50 were calculated using the software ToxRat Professional Version 3.2.1.

The EC0 and EC100 values were identified by direct observation. The initial measured and calculated concentrations were used for all evaluations and results.

The highest immobilisation observed at 24 hours was 35% in the full test. Therefore the EC50 value could not be calculated.

24 hour–EC50>0.165 mg·L-1               

48 hour–EC50= 0.069 mg·L-1    

(95% confidence limit: 0.059 – 0.082 mg·L-1)

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
0.069 mg/L

Additional information