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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:
2008
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)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 100 mg test substance / L.
- Sampling method: Samples of the test medium for determination of the actual test substance concentrations were taken immediately before introduction of the Daphnia and after 48 hours. Actual concentrations of the test substance in the test medium were determined in the surplus of the test medium at the beginning of the test and in the pooled test medium of the replicates at the end of the test.
One sample each was taken, diluted appropriately and analysed in duplicate by HPLC on the same day.
- Sample storage conditions before analysis: The samples were analysed on the day of sampling.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test solution was prepared by addition of 25.0 mg the test substance to 250 mL reconstituted water and ultrasonication for about 15 minutes.
- Eluate: no.
- Differential loading: no.
- Controls: For the negative control group only reconstituted water according to ISO 6341 was used.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): none.
- Concentration of vehicle in test medium (stock solution and final test solution): not applicable.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Visual observations were made at the same time as the animal observation. The control medium and the test medium were clear and colourless throughout the test period.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Neonates of Daphnia magna (Crustacea, Cladocera), , were obtained by hatching ephippia, supplied in the microbiotest "Daphtoxkit F TM " (Daphnia magna; Micro BioTests Inc; Kleimoer 15, B-9030 Mariakerke (Gent); BELGIUM). .
Batch No. of the ephippia in the test: DM021007.

- Strain: /
- Source: Ephippia were supplied in the microbiotest "Daphtoxkit F TM " (Daphnia magna; Fa. Micro BioTests Inc; Kleimoer 15, B-9030 Mariakerke (Gent); BELGIUM).
- Age at study initiation): less than 24 hours old.
- Weight at study initiation: /
- Length at study initiation: /
- Valve height at study initiation, for shell deposition study: not applicable.
- Peripheral shell growth removed prior to test initiation: not applicable.
- Method of breeding: Neonates were obtained by hatching ephippia. The ephippia were hatched in the laboratory in a petri dish containing reconstituted water in a temperature controlled room at approx. 20-22 °C under continuous illumination of approximately 6 000 Lux. According to MicroBioTests Inc. the largest hatching occurs between 72 and 80 hours and the Daphnia must be collected at the latest 90 hours after initiation of hatching. The neonates were collected for this test at approximately 87 hours after initiation of hatching.
- Feeding during test: none.
- Food type: not applicable.
- Amount: not applicable.
- Frequency: not applicable.


ACCLIMATION
- Acclimation period: <24 h.
- Acclimation conditions (same as test or not): same.
- Type and amount of food: Approximately 2 hours before the start of the test the neonates were fed with a suspension of Spirulina powder, as also in traditional Daphnia cultures the neonates have the opportunity to take up some feed particles. .
- Feeding frequency: once.
- Health during acclimation: not applicable.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
None.
Hardness:
250 mg CaCO3 / L.
Test temperature:
21 °C.
pH:
6.6 in the test substance medium and 7.3 in the control medium.
Dissolved oxygen:
Actual result: at least 8.7 mg/L.
Salinity:
Not applicable.
Nominal and measured concentrations:
Nominal: 0 and 100 mg/L.
Actual, at time 0 h: 95.7 mg/L.
Actual, at time 48 h: 105.2 mg/L.
Geometric mean of the actual concentrations: 100.3 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: beaker
- Type: covered with a transparent glass lid.
- Material, size, headspace, fill volume: 250 mL glass beaker, filled with 125 mL medium.
- Aeration: No extra aeration, but slight circulation of air is still possible when the beakers are closed with the transparent glass lid.
- Type of flow-through: /
- Renewal rate of test solution (frequency/flow rate): not applicable.
- No. of organisms per vessel: 5.
- No. of vessels per concentration (replicates): 4.
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): not applicable.
- Biomass loading rate: /


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The stock solutions for preparation of the reconstituted water according to ISO 6341 were obtained by MicroBioTests Inc. (Batch No. ISOD191007). The water was aerated and the pH, the dissolved oxygen concentration and the water hardness were checked before using.
- Culture medium different from test medium: no.
- Intervals of water quality measurement: Temperature daily. Oxygen and pH at the start and the end of the test. Total hardness before the start.


OTHER TEST CONDITIONS
- Adjustment of pH: no.
- Photoperiod: 4 a.m. to 8 p.m.
- Light intensity: /


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
24 and 48 hours after the start of the exposure to the test substance the neonates were observed for their swimming behaviour. Animals not able to swim within approximately 15 s after gentle agitation of the test container were evaluated as immobilised. Animals trapped at the surface were counted, to evaluate a possible influence of the test substance to the water surface tension.


TEST CONCENTRATIONS
0 and 100 mg/l in the main study.
- Spacing factor for test concentrations: not applicable.
- Justification for using less concentrations than requested by guideline: not applicable.
- Range finding study
- Test concentrations: 1, 10 and 100 mg/L.
- Results used to determine the conditions for the definitive study: No immobilisation was detected in any of the concentrations tested.
Reference substance (positive control):
no
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC0
Effect conc.:
> 100 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
No daphnia was immobilised, neither in the test substance group nor in the control group.

- Behavioural abnormalities: none.
- Observations on body length and weight: not applicable.
- Other biological observations: no.
- Mortality of control: no immobilisation in the negative control group.
- Other adverse effects control: no.
- Abnormal responses: no.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no.
- Effect concentrations exceeding solubility of substance in test medium: not applicable.
Results with reference substance (positive control):
- Results with reference substance valid?: yes. The batch of ephippia is provided together with a specification sheet with batch number and date of expiry. Additionally for confirmation of the sensitivity of the test animals of the used batch the results of a reference test with potassium dichromate are provided. 24h EC50 of potassium dichromate: 1.12 mg/L for the batch used. The acceptable range of the EC50 24h according to the OECD guideline 202 is 0.6 mg/L to 2.1 mg/L.
Reported statistics and error estimates:
not applicable.

Quality criteria

·        Constant conditions were maintained (temperature, pH).

·        There was no immobilisation in the negative control group (guidelines: maximum of 10 %).

·        There were no control Daphnia trapped at the water surface.

·        The dissolved oxygen concentration was higher than 3 mg/L at each determination.

·        The test substance concentration has been satisfactorily maintained to within 80 % of the initial concentration throughout the duration of the test.

Validity criteria fulfilled:
yes
Conclusions:
EC50,48h > 100 mg/L.
EC0,48h ≥100 mg/L.
NOEC48h ≥100 mg/L.
Executive summary:

The acute toxicity of "Tazobactam Acid" to Daphnia magna was determined according to EC-methods.

A limit-test with one concentration of nominal 100 mg test substance per litre was performed. The test solution was prepared by addition of 25.0 mg the test substance to 250 mL reconstituted water and ultrasonication for 15 minutes. Only reconstituted water was used for the negative control group. The exposure of the Daphnia was for 48 h in a static test.

Results:

EC50,48h > 100 mg/L.

EC0,48h ≥100 mg/L.

NOEC48h ≥100 mg/L.

Description of key information

Key study. Test method according to EU C.2. GLP study. A limit test with a nominal concentration of 100 mg/L revealed a 48h NOAEC ≥100 mg/L and EC50,48h > 100 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
100 mg/L

Additional information