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Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 May 2003 to 19 September 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

April 1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

EC Comission Directive 1999/11/EC of 8 March 1999 (Official Journal No L 77/8)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Test concentrations were verified by chemical analysis. Triplicate 100 ml samples were taken from the control and each test concentration at 0 hours (main stock solutions) and after 48 hours (replicates pooled) for analysis.

Vehicle:

no

Details on test solutions:

The test substance, 200 mg, was dispersed in 2.0 litres of test medium and ultrasonicated for one hour with intermittent vigorous shaking to produce a stock dispersion of 100 mg/l. Serial dilutions of the stock dispersion were prepared with test medium to give the remaining nominal test concentrations (in descending order of concentration) of 46, 22, 10 and 4.6 mg/l.

Test organisms (species):

Daphnia magna

Details on test organisms:

Daphnia magna (Straus) used in this study were cultured in-house and were obtained from a strain originating from the Institute National de Recherche Chimique Applique (IRChA), France.
Stock cultures of Daphnia magna were maintained in glass vessels containing approximately 500 to 800 ml of Elendt M4 culture medium with a 16 hour light: 8 hour dark photoperiod at 19°C. Cultures were fed daily with a suspension of the unicellular green alga (Chiarella vulgaris). Culture conditions ensured that cultures reproduced 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.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

250 mg/l as CaCO3

Test temperature:

20 °C

pH:

6.8 - 7.2

Dissolved oxygen:

8.0 – 8.7 mgO2/l

Nominal and measured concentrations:

Nominal test concentrations: 4.6, 10, 22, 46 and 100 mg/L
Mean measured test concentrations (unfiltered): 4.3, 9.5, 21, 43 and 101 mg/L
Mean measured concentrations (filtered): 4.2, 9.7, 19, 39, 80 mg/l

The unfiltered samples ranged from 95.7 - 101 % of nominal (fresh medium; 0 hours) and from 91.6 - 101 % of nominal (expired medium; 48 hours). The filtered samples ranged from 71.1 – 90.6 % of nominal (fresh medium; 0 hours) and from 89.1 – 93.1 % of nominal (expired medium; 48 hours).

All results are expressed in terms of mean measured concentrations of unfiltered media. For details on the chemical analysis (measured concentrations of T-71) see Appendix 3 “T-71 – results daphnia immobilisation.pdf”).

Details on test conditions:

Experimental design: Four replicate vessels were prepared for the control and each test group. The test vessels were 250 ml capacity glass jars.
Five first instar Daphnia were placed without conscious bias into each glass jar, containing 100 ml of prepared test medium, or control medium, as appropriate, to give a loading of 20 ml test solution per organism. The jars were loosely covered to minimise evaporative losses.

Selection of test concentrations: Based on the findings of three range finding experiments, the concentration for the main test were determined (Nominal test concentrations: 4.6, 10, 22, 46 and 100 mg/L; Spacing factor 2.2). Nominal exposure concentrations quoted in this report refer to the test material as received; no allowance has been made for a purity of less than 100%.

Medium renewal: Daphnia were exposed to the test or control conditions for a period of 48 hours without renewal of test media.

Environmental conditions: Test vessels were maintained at 20°C, with a photoperiod of 16 hours light: 8 hours dark and without supplementary aeration or feeding during the 48 hour exposure period. The temperature, pH and dissolved oxygen levels were recorded in stock solutions at 0 hours and in each test vessel at 48 hours.

Criterion of effect: Observations of the condition of the test and control Daphnia were made following 24 and 48 hours exposure. Daphnia were considered to be immobile if they were unable to swim within approximately 15 seconds following gentle agitation of the test vessel.

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

42 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limits: 35- 52 mg/l

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

77 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95 % confident limits: 59 - >101 mg/l

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

9.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

There was a clear treatment related effect on the survival of the Daphnia, with no immobilisation at 4.3 and 9.5 mg/L, the two lowest concentrations, and 100% immobilisation at the highest test concentration of 101 mg/L. For details see attached document "T-71 - results daphnia immobilisation.pdf".

- Mortality of control: none
- Other adverse effects control: none
- Abnormal responses: none

Individual pH, temperature and dissolved oxygen values remained within acceptable limits during the study.

Results with reference substance (positive control):

Reference substances not reported.

Reported statistics and error estimates:

EC50 values were calculated using a logistic model (Ashton, 1972) for which 95% confidence limits were estimated by the likelihood ratio method (Williams, 1986).

Validity criteria fulfilled:

yes

Remarks:

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 > 3 mg/L in control and test vessels.

Description of key information

EC₅₀ (48h) of 42 mg/l (95% confidence limits: 35- 52 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:

EC50

Effect concentration:

42 mg/L