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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:
2006-06-27 to 2006-07-31
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
equivalent or similar to guideline
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
adopted 2004-04-13
GLP compliance:
yes (incl. QA statement)
signed 2005-06-01
Analytical monitoring:
Details on sampling:
- Sampling method: Two samples (20 ml) were taken for analysis from the control and test media at 0 and 24 hours (fresh medium) and from the aged medium at 24 and 48 hours.
- Sample storage conditions before analysis: On each occasion, one of the samples from each batch was either analysed immediately or stored frozen for up to two days before analysis. The other samples remained frozen in case further analysis was required.

The temperature, pH and dissolved oxygen levels of control and test media were recorded at the start and at the end of the study. The total hardness and alkalinity of the dilution medium was measured before use.
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: On each occasion the test substance (400 mg) was dissolved in dilution medium (2 l) in a volumetric flask. The contents of the flask were shaken vigorously and then either used directly at the highest test concentration or diluted to provide the test media at the lower concentrations.
- Controls: Dilution medium control group was used.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM - Daphnia magna Straus
- Source: cultured in-house, strain originating from the Institute National de Recherche Chimique Appliqué (IRChA), France
- Method of breeding: Stock cultures of Daphnia magna were maintained in glass vessels containing approximately 0.8 litres of Elendt M4 culture medium in a temperature-controlled laboratory at nominally 20 ± 2°C. A photoperiod of 16 hours light:8 hours dark was maintained, with periods of subdued lighting at the beginning and end of each light phase. The culture medium was renewed three times each week.
Cultures were fed daily with a suspension of the unicellular green algae, Pseudokirchneriella subcapitata, to provide nominally 0.1 to 0.2 mg carbon per daphnid, per day, except during the initial three days when a slightly lower ration was given. Culture conditions ensure that the stock animals reproduce by parthenogenesis.
The day before the start of the study, all juvenile Daphnia were removed from the laboratory cultures. The following morning, juveniles produced by the gravid (egg-bearing) adult Daphnia were removed from the culture vessels and held in a separate holding vessel; these animals, which were less than 24 hours old, were used in the test. The test was conducted in Elendt M4 medium.
- Feeding during test No feed was given during the exposure period.
Test type:
Water media type:
Limit test:
Total exposure duration:
48 h
Hardness (Elendt M4 medium): 250 mg/L CaCO3
Alkalinity (Elendt M4 medium): 38 mg/L as CaCO3
Test temperature:
Control: Min. 21.3; Max. 21.5
12.5 mg/l: Min. 21.2; Max. 21.6
25 mg/l: Min. 21.3; Max. 21.6
50 mg/l: Min. 21.3; Max. 21.5
100 mg/l: Min. 21.4; Max. 21.7
200 mg/l: Min. 21.2; Max. 21.7
Continuous monitoring of an additional vessel containing dilution medium: 19.7 to 22.0°C.
Control: Min. 7.43; Max. 7.67
12.5 mg/l: Min. 7.94; Max. 8.05
25 mg/l: Min. 8.02; Max. 8.05
50 mg/l: Min. 8.02; Max. 8.04
100 mg/l: Min. 8.00; Max. 8.02
200 mg/l: Min. 6.67; Max. 7.65
Although the pH of the test medium at 200 mg/l was low (6.67) at the start of the test, this is not thought to have affected the integrity of the results as no
immobility was observed after 24 hours, and the pH of the test medium at this level was comparable to the control medium for the remainder of the test.
Dissolved oxygen:
Control: Min. 98 % ASV; Max. 99 % ASV
12.5 mg/l: Min. 99 % ASV; Max. 99 % ASV
25 mg/l: Min. 98 % ASV; Max. 100 % ASV
50 mg/l: Min. 98 %ASV; Max. 100 %ASV
100 mg/l: Min. 99 %ASV; Max. 100 %ASV
200 mg/l: Min. 98 %ASV; Max. 100 %ASV
No data
No data
Nominal and measured concentrations:
Nominal concentration: 0 (control), 12.5, 25, 50, 100 and 200 mg/l
Overall mean measured concentrations: 0 (control), 12.4, 23.6, 47.1, 92.7 and 197 mg/l
Details on test conditions:
The range finding test employed nominal test concentrations of 0 (control), 1, 10 and 100 mg/l. The definitive test concentrations, which were selected after discussion with the Sponsor and based on the results of the range finding test, were nominally 0 (control), 12.5, 25, 50, 100 and 200 mg/l.

- Test vessel: Glass dishes containing 100 ml of medium to give a loading of 20 ml medium per organism. The dishes were loosely covered.
- Aeration: No supplementary aeration was employed.
- Renewal rate of test medium (frequency/flow rate): Renewal of the test medium after 24 hours, as information provided by the Sponsor indicated that triethylmethylammonium tetrafluoroborate would hydrolyse.
- No. of organisms per vessel: Five animals
- No. of vessels per concentration (replicates): Four replicates
- No. of vessels per dilution medium control group (replicates): Four replicates

- Source/preparation of dilution water: The Elendt M4 medium was prepared in de-ionised water.
- Culture medium different from test medium: The test organisms were maintained and the tests conducted in Elendt M4 medium.

- Temperature: 19.7 to 22.0°C during the definitive test; Temperature was continuously monitored in an additional vessel containing
the same volume of dilution medium.
- Photoperiod: 16 hours light:8 hours dark; Periods of subdued lighting at the beginning and end of each light phase

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Daphnia were considered to be immobile if they were unable to swim within approximately 15 seconds following gentle agitation of the test vessel.
The numbers of mobile, immobile and floating Daphnia were counted approximately 24 and 48 hours after the start of the study.
Reference substance (positive control):
48 h
Dose descriptor:
Effect conc.:
106 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
triethylmethylammonium tetrafluoroborate
Basis for effect:
Remarks on result:
other: 95% CL: 86.1 and 133 mg/L (mean measured)
24 h
Dose descriptor:
Effect conc.:
> 197 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
triethylmethylammonium tetrafluoroborate
Basis for effect:
48 h
Dose descriptor:
Effect conc.:
23.6 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
triethylmethylammonium tetrafluoroborate
Basis for effect:
Details on results:
- Behavioural abnormalities: One Daphnia at 23.6 mg/l was trapped at the surface of the medium at 24 hours; it was re-submerged when transferred to the fresh medium and remained within the body of the medium for the remainder of the test.
After 48 hours, the highest concentration at which no immobilisation occurred was 23.6 mg/l; at the highest concentration tested (197 mg/l), 95% immobilisation occurred.
- Chemical analysis: The measured concentrations of triethylmethylammonium tetrafluoroborate in samples of test medium ranged between 89 and 104% of the nominal values during the test.
Results with reference substance (positive control):
Not applicable data
Reported statistics and error estimates:
Statistical analysis was performed using the SAFEStat LD50 application, SAS 8.2 (SAS INSTITUTE (1999) SAS OnlineDoc® Version Eight. SAS Institute Inc., Cary, NC, USA). Test results were expressed in terms of the mean measured concentrations.
The “no observed effect concentration” (NOEC) was derived by direct inspection of the data on the immobility of the animals. An incidence rate of more than 10% is considered to be significant.
Validity criteria fulfilled:
The effects of test item triethylmethylammonium tetrafluoroborate on freshwater caldocere Daphnia magna (neonates, < 24 h) were investigated in an acute immobilisation test according to OECD 202 (1984). Daphnids were exposed to the test item dissolved in Elendt M4 medium in a semi-static exposure system (media renewal after 24 hours). The measured concentrations of triethylmethylammonium tetrafluoroborate ranged from 89 and 104 % of nominal concentrations. The 48 h-EC50 and the NOEC were determined to be 106 mg/L (cl 95: 86.1 and 133 mg/L) and 23.6 mg/L triethylmethylammonium tetrafluoroborate (mean measured), respectively. These results are considered reliable since all validity criteria of the study were met.

Description of key information

In a reliable GLP- and guideline- conform key study of triethylmethylammonium tetrafluoroborate according to OECD 203, the 48-h EC50 for the immobilisation of Daphnia magna amounts to 106 mg/L (mean meaured, 95% CI: 86.1 - 133 mg/L) (Taylor, 2007) and is thus above the OECD test limit. Therefore, triethylmethylammonium tetrafluoroborate appears to have a low potential for toxicity to freshwater invertebrates.

Key value for chemical safety assessment

Additional information