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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:
12 May 2003 to 17 December 2003
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
April 1984
according to guideline
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
Official Journal of the European Communities, L 383, 172 - 178, 29 December 1992
GLP compliance:
Analytical monitoring:
Details on sampling:
Duplicate samples for possible analysis were taken from all concentrations and the blank-control.
Sampling: Frequency: At t= 0 h and at t= 48 h.
Volume: 10 ml from the approximate centre of the test vessels
Storage: Samples were stored in a freezer until transportation to TNO Nutrition and Food Research for analysis.

Additionally, singular reserve samples of 10 ml were taken. These samples were stored in a freezer for possible analysis for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

After termination of the toxicity study the frozen samples were transported to the analytical facility. After receipt on 18 August 2003 the samples were stored at < -18°C until analysis on 21 August 2003.
Details on test solutions:
Preparation of stock solutions was performed by weighing 500 mg (range-finding test) or 100 mg (final test) of test substance and adding it to 5 litre or 1 litre, respectively, of test medium in a silanised volumetric flask. Preparation was conducted under dimmed light conditions. After magnetical stirring for 12 minutes under dark conditions, the resulting solutions were clear and colourless. Lower test concentrations were prepared by subsequent dilution of the stock. The separation factor of the test concentration was 1.8. Since the pyrophosphate part of the test substance caused a drop in the pH of the test medium, pH of the test medium was adjusted with ortho-Phosphoric acid (85%) to 6.0 in the final test.
Test organisms (species):
Daphnia magna
Details on test organisms:
- Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by acyclical parthenogenesis under specified breeding conditions
- Source: in-house laboratory culture with a known history
- Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality > 20%, presence of males, ephippia or discoloured animals and there was be no delay in the production of the first brood.
- Characteristics: for the test selection of young daphnia with an age of < 24 hours, from parental daphnids of more than two weeks old

- Start of each batch: with newbom daphnids, i.e. less than 3 days old, by placing about 250 of them into 10 litres of medium in an all-glass culture vessel
- Maximum age of the cultures: 4 weeks
- Renewal of the cultures: after 7 days of cultivation half of the medium twice a week
- Feeding: daily, a suspension of fresh water algae
- Medium: M7, as prescribed by Dr. Elendt-Schneider (1990)
- Temperature of medium: 18 – 22°C
Test type:
Water media type:
Limit test:
Total exposure duration:
48 h
250 mg/l CaCO3
Test temperature:
20.4 and 21.4°C
5.6 - 6.7
The pH in the 100 mg/l concentration was just below 6.0 (the lower limit
prescribed by the protocol) throughout the test. Also the pH in the blank-control
was just below 6.0 in the freshly prepared medium at t= 72 h. Since the given pH
range in the protocol describes the optimum pH range for Zebra-fish (6.0 - 8.5) this
was not considered to have affected the study. For details on pH values see attachment "T-1063FM - results immobilisation daphnia.pdf".
Dissolved oxygen:
8.2 - 9.0 mg/l
Nominal and measured concentrations:
Nominal test concentrations: 10, 18, 32, 56 and 100 mg/L
Analysis of the samples taken at the start (t=0) of the final test showed mean measured concentrations of 9.3 mg/l (93 %), 29.7 mg/l (93 %) and 95.1 mg/l (95 %) at the nominal concentrations of 10, 32 and 100 mg/l, respectively. After 48 hours of exposure the mean test substance concentrations measured remained constant (84 - 107 % of nominal). For details see attachment "T-1073FM - results immobilisation daphnia.pdf"). Therefore results were based on nominal concentrations.
Details on test conditions:
- Test vessel: 100 ml, all-glass with synthetic cover lids
- Volume of solution: ca. 80 ml
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Introduction of daphnia: Within 40 minutes after preparation of the test solutions
- Biomass loading rate: 16 ml of test solution per organism

- Medium: ISO, prepared in Milli-RO water [ISO International Standard 6341]
- Source/preparation of dilution water: freshly prepared ultra-pure water (tap water)purified by reverse osmosis (milli-RO); Millipore Corp., Bedford, Mass., USA)
- Culture medium different from test medium: yes
- Temperature of medium: Continuously in a temperature control vessel, beginning at the start of the test.
- pH and dissolved oxygen: At the beginning and at the end of the test, for all concentrations and the control.

- Adjustment of pH: with ortho-Phosphoric acid (85%) to 6.0 (final test)
- Light: No light
- Feeding: No feeding

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Immobility (including mortality) at 24 and 48 hours


- Test concentrations: range of 0.1 to 100 mg/l increasing by a factor of 10 and a blank-control
- Test conditions: similar to those applied in the final test with the following exceptions: Ten daphnia per concentration (in duplicate, 5 per vessel). Dissolved oxygen concentrations and pH were only measured in the blank-control and the highest test concentration. No sampling for determination of actual test concentrations was performed.
- Results used to determine the conditions for the definitive study: After 48 hours of exposure 90 % of the daphnids exposed to 100 mg/l were immobilised. No immobilisation was observed in any of the lower test concentrations at both observation points. For details see attachment "T-1063FM - results immobilisation daphnia.pdf". Therefore, the 48h-EC50 was expected to be between 10 and 100 mg/l.
Reference substance (positive control):
potassium dichromate
Key result
48 h
Dose descriptor:
Effect conc.:
42 mg/L
Nominal / measured:
Conc. based on:
Basis for effect:
Remarks on result:
other: 95% confidence interval between 36 and 53 mg/l
Details on results:
In the control, no daphnia were immobilised or trapped at the surface of the water. T-1063FM did not induce significant acute immobilisation of Daphnia magna at 18 mg/l after 48 hours of exposure (NOEC).The 24h-EC50 was 69 mg/l (95% confidence interval between 56 and 91 mg/l).
For details of exact immobilisation values, see attachment "T-1063FM - results immobilisation daphnia.pdf".

Observations on the exposure media: Test concentrations at 10 and 18 mg/l were completely clear throughout the test period. In the vessels at 32 mg/l a floating layer was observed and in the vessels at 56 and 100 mg/l both a floating layer and a test substance precipitate was observed. Immobilised daphnids were observed under a microscope and no test substance was observed to be attached to their bodies. Therefore, daphnids were not mechanically hampered by the test substance.
Results with reference substance (positive control):
The reference test was carried out to check the sensitivity of the test system as used by NOTOX. Daphnia were exposed for a maximum of 48 hours to K2Cr2O7 concentrations of 0.10, 0.18, 0.32, 0.56, 1.0 and 1.8 mg/l and to a blank control.
Ten daphnia were exposed per concentration.
The reference substance, potassium dichromate (K2Cr2O7, art. 4864, batch no. K28974764) was obtained from Merck, Darmstadt, Germany.
The actual responses in this reference test with K2Cr2O7 are within the ranges of the expected responses at the different concentrations. Hence, the sensitivity of this batch of D. magna was in agreement with the historical data collected at NOTOX.
The 24h-EC50 was 1.2 mg/l with a 95% confidence interval between 1.0 and 1.5 mg/l.
The 48h-EC50 was 0.60 mg/l with a 95% confidence interval between 0.52 and 0.76 mg/l.
For details see attachment "T-1063FM - results immobilisation daphnia.pdf".

The 24h-EC50 (based on the initial concentration) of potassium dichromate was within the range 0.6 mg/l to 1.7 mg/l.
Validity criteria fulfilled:
In the control, no daphnids have been immobilised or showed other sub-lethal effects; the dissolved oxygen concentration at the end of the test was > 5 mg/L in control and test vessels.

Description of key information

EC50 (48h) of 42 mg/l (95% confidence limits: 36-53 mg/l) for Daphnia magna (EU Method C.2, OECD guideline 202)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Dose descriptor:
Effect concentration:
42 mg/L

Additional information