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Short-term toxicity to aquatic invertebrates

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short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 to 17 October 2009
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
GLP compliance:
Analytical monitoring:
Details on sampling:
- Concentrations: Dilution water control, solvent control (dimethylformamide 0.1 mg/L) and a nominal concentrations of 40, 55, 70, 85 and 100 mg/L of the test substance
- Sampling method: Analytical samples taken at 0 and 48 hours and analysed by HPLC-UV instrumentation
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: This study was run with a dilution water control (DWC), solvent control and nominal concentrations of 40, 55, 70, 85 and 100 mg/L. A nominal 1000 g/L primary stock solution of the test substance was prepared by the addition of 5.0 g (nominal) of the test substance to a 5 ml volumetric flask which was made up to the mark with dimethylformamide. 200 µL of this concentrated stock solution was then added to 2000 mL of culture medium to produce a top concentration of 100 mg/L of DMF. This stock solution was stirred vigorously at approximately 30°C to aid the solubility of the test substance. The solution was then cooled to 20°C after which it was filtered through a 1.2 µm GFC filter paper and on all occasions observed to be a clear and colourless solution. The highest test concentration used this solution without any further alteration.

The remaining test solutions were prepared from the 100 mg/L solution by the addition of appropriate volumes to 500 ml of dilution water and stirred thoroughly. A solvent control was similarly prepared by the addition of 100 µl/L of DMF to 500 mL of dilution water to give a solvent concentration of 200 µl/L in each exposure vessel. All solvent additions were made sub-surface, gradually, by microliter syringe, into solutions vigorously stirred by magnetic follower. The resultant solutions were clear and colourless.
Equalising additions of DMF resulted in a solvent concentration of 200 µl/L, however since no symptoms of toxicity were observed in the control, solvent control or test concentrations at either 24 or 48 hours, it is considered that this did not impact the validity of the study.
Test organisms (species):
Daphnia magna
Details on test organisms:
- Common name: Daphnia Magna
- Age at study initiation (mean and range, SD): < 24 hrs old obtained from a single culture vessel.
- Source: obtained from continuous laboratory cultures held at Brixham Environmental Laboratory
- Age of parental stock (mean and range, SD): Parent animals were 22 days old and had been maintained with a twice weekly renewal of reconstituted water medium since birth. The test organisms and the cultre from which they were obtained showed no evidence of disease before the test period.

- Acclimation conditions (same as test or not): yes
- Type and amount of food: fed on a mixed algae diet of Chlorella vulgaris, CCAP 211/12 and Pseudokirchneriella subcapitata, strain CCAP 278/4. The algal cultures were harvested, centrifuged and measured for cell density using a Coulter Counter. The algal cells were then re-suspended in D. magna culture medium (reconstituted water), stored in a refrigerator, in the dark, and used within two weeks. The algal stocks, once re-suspended, were analysed for total organic carbon content. The feeding was designed so that each vessel receive a nominal 0.06 mg C/day of each algal suspension.
- Feeding frequency: fed daily ad libitum
- Health during acclimation (any mortality observed): no mortality observed

Test type:
Water media type:
Limit test:
Total exposure duration:
48 h
Post exposure observation period:
48 hours
217.3 mg/L CaCO3
Test temperature:
Temperature values taken at 0, 24 and 48 hours were 20.0, 20.7 and 20.1°C, with the values recorded automatically over the test period remaining within 20.0 ± 1°C
pH values ranged from 7.56 to 7.83
Dissolved oxygen:
Dissolved oxygen concentrations ranged from 6.0 to 9.0 mg/L
The conductivity was 645 uS cm-1
645 µS/cm
Nominal and measured concentrations:
Nominal concentrations: Dilution water control, solvent control, 40, 55, 70, 85, 100 mg/L
Measured concentrations: Dilution water control, solvent control, 36, 50, 64, 78, 92 mg/L

The mean measured concentrations ranged from 90 to 92% of the nominal concentrations.
On the basis of the analytical data the nominal concentrations were used for the calculation and reporting of results
Details on test conditions:
- Test vessel: glass beakers
- Type (delete if not applicable): covered with loose fitting glass lids
- Material, size, headspace, fill volume: 80 ml of test solution providing a depth of approx. 50 mm
- Aeration: no
- No. of organisms per vessel: 5 impartially selected
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): not reported
- No. of vessels per vehicle control (replicates): not reported
- Biomass loading rate: 62.5 daphnia/L

- Source/preparation of dilution water: Elendt's M4 D. magna medium.
- pH: 8.1
- Ammoniacal Nitrogen as N: < 0.002 mg/L
- Solid Susptended at 105°C: < 3.00 mg/L
- Dissolved Organic Carbon: 0.70 mg/L
- COD: < 12 mg/L
- Organochlorine pesticides: < 0.03 µg/L
- Organophosphorus pesticides: < 0.01 µg/L
- PCBs: < 0.001 µg/L
- Alkalinity: 37 mg CaCO3/L
- Hardness: 217.3 mg/L as CaCO3
- Culture medium different from test medium: no
- Intervals of water quality measurement:

Final nominal concentrations in the media:
- NaHCO3: 64,8 mg/L
- CaCl2. 2H2O: 293.8 mg/L
- MgSO4.7H2O: 123.3 mg/L
- KCl: 5.8 mg/L
- Na2SiO3.5H2O: 0.747 mg/L
- NaNO3: 0.02274 mg/L
- KH2PO4: 0.0143 mg/L
- K2HPO4: 0.0184 mg/L

- Adjustment of pH: no
- Photoperiod: 16 hours light: 8 hours dark with 20 minute dawn and dusk transition periods
- Light intensity: 11.6 µ Einsteins m2/s

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Observations were made 24 and 48 hours after the commencement of the test. Each daphnia was viewed by eye and was defined as affected if showing no whole body movement, relative to the water, within a period of 15 seconds even if movement of individual appendages was visible. Daphnia so affected were termed immobille. Any overt symptoms of toxicity were also recorded.

Reference substance (positive control):
Key result
48 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
48 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Details on results:
- Mortality of control: none
- there was no immobility observed in the dilution water control or solvent control. No other symptoms of toxicity were observed.
Validity criteria fulfilled:
The acute toxicity of the test substance was determined in a 48 static test according to the OECD guideline 202. Under the conditions of the present study, The 48-h EC50 exceeded the nominal concentration of 100 mg/L. There was no mobility observed within the period of the test. The results of the test can be considered reliable without restriction.

Description of key information

The 48 hour EC50 to Daphnia magna was reported as >100 mg/l (nominal).

Key value for chemical safety assessment

Additional information

Acute toxicity (48 hour) to aquatic invertebrates has been investigated with Daphnia magna following procedures as laid out in OECD Guideline 202. The 48 hour EC50 and the 48 hour NOEC were reported as >100 mg/l (nominal).

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