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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:
2020-06-03 to 2020-06-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
adopted 13 April 2004
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
May 30, 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)
Version / remarks:
October 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Sampling method: For determination of the test item concentrations in the test solutions, all four test vessel were analysed at the start and at the end of the test. In case of control samples, all four test vessel were analysed at the start and at the end of the test.
- Sample storage conditions before analysis: no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: stock solution (100 mg/L in nominal) prepared by dilution of 0.0506 g test item in 506 mL dilution water (ISO Medium) using mechanical dispersion without any solubilising agent. Test solutions of subsequent lower concentrations were prepared by appropriate dilution of this stock solution.
- Controls: negative control, reference control
- Evidence of undissolved material: no
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Age at study initiation: less than 24 hours old
- Method of breeding: Daphnia were bred in the Laboratory of TOXI-COOP ZRT. under similar temperature and light conditions as in the test, and in reconstituted water of a similar quality regarding to pH, components of the main ions and total hardness as the test water used in the test. Daphnia were fed with centrifuged green alga suspension.
- Source: Laboratory of Hydrobiology (Central Agricultural Office, Directorate of Plant-, and Soil Protection) 2100 Gödöllő, Kotlán S. u. 3. Hungary
- Sex: female
- Feeding during test : no
- Food type: The Daphnia are fed with concentrated algal suspension of Raphidocelis subcapitata during the holding. Test animals are not fed during the exposure.

ACCLIMATION
Test animals were bred under similar conditions as that used during the exposure period, therefore additional acclimatisation before the test was not necessary.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
None
Hardness:
The reconstituted water (ISO medium) had an approximate theoretical total hardness of 249 mg/L (as CaCO3).
Test temperature:
20.7 – 20.9°C
pH:
7.49 – 9.45
Dissolved oxygen:
8.10 – 8.33 mg/L
Salinity:
not applicable
Conductivity:
not specified
Nominal and measured concentrations:
nominal: 0 (control), 6.25, 12.5, 25, 50 and 100 mg/L
measured: (control not measured), 5.715, 12.43, 25.93, 50.37 and 102.66 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: beaker
- Type: open
- Material, size, fill volume: glass; volume app. 50 mL; with ~40 mL test medium
- Volume of solution: 50 mL
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration: 4
- No. of vessels per control: 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO-Medium prepared using ultrapurified water

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light (artificial illumination) and 8 hours darkness
- Light intensity: 611 lux

EFFECT PARAMETERS MEASURED: immobilisation after 24 and 48 hours of exposure

RANGE-FINDING STUDY
- Test concentrations: (control), 0.1, 1, 10, 100, 100 (pH adjusted)
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (K2Cr2O7)
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95 % conf. limits: (91.4 – 331.8 calculated)
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
66.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95 % conf. limits: 18.7 – 82.5 mg/L
Duration:
48 h
Dose descriptor:
other: EC20
Effect conc.:
79.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95 % conf. limits: 42.6 – 100.7 mg/L
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Behavioural abnormalities: no
- Observations on body length and weight: no
- Mortality of control: no
- Other adverse effects control: no
- Immobilisation of control: no
- Abnormal responses: no
- Any observations that might cause a difference between measured and nominal values: no
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: 24h EC50 = 1.12 mg/L (95 % confidence limits: 1.00–1.76 mg/L)
- Limit test: no
Reported statistics and error estimates:
The 48-h ECx values were calculated by Probit analysis using SPSS software.
The 48-h NOEC, LOEC values were determined directly from the raw data.
Analysis was based on the nominal test item concentrations.

Table 1: Immobilisation of the test animals (summary table)

Concentration
[mg/L]

Number of
Daphnia
tested

Number of immobilised
Daphnia after

% of immobilised
Daphnia after

24 h

48 h

24 h

48 h

Control

20

0

0

0

0

6.25

20

0

0

0

0

12.5

20

0

0

0

0

25

20

0

0

0

0

50

20

0

0

0

0

100*

20

3

8

15

40

*Remark: precipitate was observed at the bottom of test vessels in the concentration of 100 mg/L

Table 2: Concentration of Test Item measured in the Test solutions

Nominal concentration mg/L

Mean of the measured concentrations

Start (June 03, 2020)

End (June 05, 2020)

Concentration (mg/L) 

% of the nominal

Concentration (mg/L) 

% of the nominal

Control

not detected

-

not detected

-

6.25

5.937

95

5.715

91

12.5

11.87

95

12.43

99

25

24.93

100

25.93

104

50

48.53

97

50.37

101

100

99.47

99

102.66

103

Validity criteria fulfilled:
yes
Conclusions:
In a GLP-compliant Acute Immobilisation Test according to OECD TG 202 with Daphnia magna the 48-hour EC50 of the test item was determined to be >100 mg/L (nominal).
Executive summary:

To evaluate the influence of the test item on the mobility and survival of Daphnia magna an Acute Immobilisation Test according to OECD TG 202, EU Method C.2. and US EPA OCSPP 850.1010 was carried out under static conditions and in compliance with the GLP principles. Five animals in four replicates were exposed for 48 hours to nominal test item concentrations of 6.25, 12.5, 25, 50 and 100 mg/L. In addition, a blank control including the test medium but without the test item was performed. The test item concentrations were analytically verified by using HPLC-UV to be 5.715, 12.43, 25.93, 50.37 and 102.66 mg/L after 48 hours. The test item concentration was thus 91 – 104 % of the nominal at the end of the experimental phase (at the startof the test: 95 –100 of the nominal). Therefore, all biological results are based on nominal concentrations.The suitability of the test system was confirmed by a positive control with the reference substance potassium dichromate (24h EC50 = 1.12 mg/L). All validity criteria of the test guidelines were fulfilled. The mobility of the test animals was recorded after 24 and 48 hours of exposure. As a result, the 48-hour EC50 of the test item was determined to be >100 mg/L in nominal (95 % conf. limits: 91.4 – 331.8 calculated). The 48-hour EC10 was found to be 66.1 mg/L in nominal ( 95 % conf. limits: 18.7 – 82.5 mg/L).

Description of key information

In a GLP-compliant Acute Immobilisation Test according to OECD TG 202 with Daphnia magna the 48-hour EC50 of the test item was determined to be >100 mg/L (nominal).

Key value for chemical safety assessment

Additional information

To evaluate the influence of the test item on the mobility and survival of Daphnia magna an Acute Immobilisation Test according to OECD TG 202, EU Method C.2. and US EPA OCSPP 850.1010 was carried out under static conditions and in compliance with the GLP principles. Five animals in four replicates were exposed for 48 hours to nominal test item concentrations of 6.25, 12.5, 25, 50 and 100 mg/L. In addition, a blank control including the test medium but without the test item was performed. The test item concentrations were analytically verified by using HPLC-UV to be 5.715, 12.43, 25.93, 50.37 and 102.66 mg/L after 48 hours. The test item concentration was thus 91 – 104 % of the nominal at the end of the experimental phase (at the startof the test: 95 –100 of the nominal). Therefore, all biological results are based on nominal concentrations.The suitability of the test system was confirmed by a positive control with the reference substance potassium dichromate (24h EC50 = 1.12 mg/L). All validity criteria of the test guidelines were fulfilled. The mobility of the test animals was recorded after 24 and 48 hours of exposure. As a result, the 48-hour EC50 of the test item was determined to be >100 mg/L in nominal (95 % conf. limits: 91.4 – 331.8 calculated). The 48-hour EC10 was found to be 66.1 mg/L in nominal (95 % conf. limits: 18.7 – 82.5 mg/L).