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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:
16 Mar 2018 - 28 Mar 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:
Commission Regulation (EC) No 440/2008, Annex, Part C, C.2.: "Daphnia sp. Acute Immobilisation Test", Official Journal of the European Union (EN), May 30, 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures",
Version / remarks:
2000
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: SANCO/3029/99 rev.4 11/07/00
Version / remarks:
Residues: Guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (part A; Section 4) and Annex III (part A; Section 5) of directive 91/414
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
March 2017
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Control and all test concentrations
- Sampling method: Duplicate samples at start of the test and at day 1 (freshly prepared media); duplicate samples from the aged test media of all test concentrations and the control at day 1 (after 24 hours of exposure) and at the end of the test by pooling the contents of the test beakers of each treatment.
- Sample storage conditions before analysis: Extracts were stored at 4 ± 4°C until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test item is not well soluble in test medium. To avoid physical effects of undissolved test item on the daphnids, no concentrations above the solubility limit of the test item in test water was tested.
Therefore, a defined amount of the test item was added directly to the test water for each test concentration and was carefully stirred for 24 hours in the dark to dissolve as much of the test item as possible. The highest test item nominal loading rate of 100 mg test item/L was prepared by mixing 104.9 mg test item into 1049 mL test water and 105.0 mg test item into 1050 mL test water, for the nominal loading rate of 45.5 mg test item/L, 47.74 mg test item were mixed into 1049.12 mL test water and 47.80 mg test item were mixed into 1050.4 mL test water, for the nominal loading rate of 20.7 mg test item/L, 21.70 mg were mixed into 1048.0 mL test water and 21.73 mg were mixed into 1049.5 mL test water. The nominal loading rate of 9.4 mg test item/L was prepared by mixing 23.0 mg into 2447 mL test water and 23.5 mg into 2500 mL test water. For the nominal loading rate of 4.3 mg test item/L, 25.0 mg were mixed into 5814 mL test water (two times). After cessation of mixing and a following period (1 hour) of settling to allow phase separation, the aqueous phase, i.e. the water accommodated fraction, was drawn off carefully and used as the test medium of the corresponding nominal test concentration. Each concentration was prepared separately.
The test media were prepared just before introduction of the daphnids (= start of the test) and test medium renewal on Day 1.
- Controls: test water without addition of the test item
- There were no remarkable observations in the test media (clear test medium) at any point during the test.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Straus, clone 5
- Source: in-house laboratory culture.
- Age at test start: 2 - 21.25 h old, not first brood progeny.


CULTIVATION
- Conditions: similar temperature and light conditions as used in the test.
- Medium: Elendt M4 medium
- Food type: green algae (Desmodesmus subspicatus)
- Frequency: at least on all working days
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
2.5 mmol/L (= 250 mg/L) as CaCO3
Test temperature:
20.3 - 21.0 °C in the freshly prepared media
19.7 - 20.2°C in the aged test media.
pH:
7.9 - 8.2 in the freshly prepared media
7.9 - 8.1 in the aged test media
Dissolved oxygen:
8.7 - 9.0 mg/L in the freshly prepared media
8.7 - 8.8 mg/L in the aged test media
Nominal and measured concentrations:
Nominal loading rates: 100, 45.5, 20.7, 9.4 and 4.3 mg test item/L (each prepared in independent WAF solutions)
Measured concentrations: 42.0, 20.0, 9.30, 3.17 and 1.42 mg test item/L (geometric mean measured concentrations).
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass beakers of approximately 110 mL [[or 60 mL?]] volume containing as much test medium as possible (i.e. the remaining head space was reduced to a technical possible minimum of some mL), kept closed during the whole period of the study with a conical glass stopper to avoid loss of the test item due to volatilisation.
- Renewal of test medium: on day 1.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Test was performed in a controlled environment room.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water (Elendt M4 medium), prepared with deionised water (conductivity <5 µScm^-1)
- Culture medium different from test medium: no
- Intervals of water quality measurement: The water temperature, pH-values and the dissolved oxygen concentrations were determined in all freshly prepared and aged test media of each treatment group.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light : 8 h dark
- Light intensity: 430 to 580 lux (measured once during the test)

EFFECT PARAMETERS MEASURED
- Mobility of the daphnids was determined by visual observation after 24 and 48 hours.

RANGE-FINDING STUDY
- Pre-experiments (non-GLP) were performed to determine a suitable concentration range and to establish suitable methods to prepare the test solutions.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate [[performed January 2017/2018?]]
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
5.43 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Details on results:
- After 48 hours of exposure no immobilisation of the test animals was observed in the control and up to and including the test item concentration of 3.17 mg test item/L. At the concentration of 9.30 mg test item/L and above, all animals were immobile.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- 48h-EC50: 1.02 mg/L
- 24h-EC50: 1.06 mg/L, indicating that the sensitivity of the daphnids was consistent with the level proposed by the OECD 202 guideline (EC50-24 h between 0.6 and 2.1 mg potassium dichromate/L).
Reported statistics and error estimates:
ECx values: Probit analyssi
NOEC determination: Step-down Cochran Armitage test.
Statistical analysis was performed with ToxRat Professional, Version 3.2.1, ToxRat(r) Solutions GmbH

Table 1: Summary of Analytical Results

Sample description

Nominal

Geometric mean1

concentration

% of

concentration2

[mg test item/L]

Nominal

[mg test item/L]

Control

<LOD

n.a.

4.3

33

1.42

9.4

34

3.17

20.7

45

9.30

45.5

44

20.0

100

42

42.0

1Calculated according to OECD Guidance Document No. 23, Annex 2
2The tabulated results represent results rounded to three significant digits

n.a.: not applicable

Table 2. Influence of Muguet Aldehyde on the Mobility of Daphnia magna

Geometric Mean Measured Concentration [mg test item/L]

No. of Daphnia tested

No. of immobilised Daphnia after

% of immobilised Daphnia after

24 h

48 h

24 h

48 h

Control

20

0

0

0

0

1.42

20

0

0

0

0

3.17

20

0

0

0

0

9.30

20

11

20

55

100

20.0

20

15

20

75

100

42.0

20

20

20

100

100

Validity criteria fulfilled:
yes
Remarks:
See 'Overall remarks' for details on validity criteria.
Conclusions:
The 48-hour NOEC was determined to be 3.17 mg test item/L and the 48-hour EC50 value was calculated to be 5.43 mg test item/L.
Executive summary:

The acute toxicity to aquatic invertebrates was examined in a study according to OECD TG 202 and in compliance with GLP criteria. In this study, groups of Daphnia magna (4 replicates of 5) were exposed to WAFs individually prepared at loading rates of 0 (control), 4.3, 9.4, 20.7, 45.5 and 100 mg/L for 48 hours under semi-static conditions. Nominal target concentrations were analytically verified, and geometric mean measured exposure concentrations determined to be 1.42, 3.17, 9.30, 20.0, and 42.0 mg/L, and effect concentrations expressed as such. Immobility of daphnids was recorded after 24 and 48 hours exposure. The test is valid. After 48 hours exposure, no effect on mobility was observed up to and including the test concentration of 3.17 mg/L. At test concentrations of 9.30 mg/L and higher, percentage immobility was 100%. Based on these findings, the 48-h EC50 value was determined to be 5.43 mg/L.

Description of key information

The acute toxicity to aquatic invertebrates was examined in a study according to OECD TG 202 and in compliance with GLP criteria. In this study, groups of Daphnia magna (4 replicates of 5) were exposed to WAFs individually prepared at loading rates of 0 (control), 4.3, 9.4, 20.7, 45.5 and 100 mg/L for 48 hours under semi-static conditions. Nominal target concentrations were analytically verified, and geometric mean measured exposure concentrations determined to be 1.42, 3.17, 9.30, 20.0, and 42.0 mg/L, and effect concentrations expressed as such. Immobility of daphnids was recorded after 24 and 48 hours exposure. The test is valid. After 48 hours exposure, no effect on mobility was observed up to and including the test concentration of 3.17 mg/L. At test concentrations of 9.30 mg/L and higher, percentage immobility was 100%. Based on these findings, the 48-h EC50 value was determined to be 5.43 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
5.43 mg/L

Additional information

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