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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:
03 JUL 2003 - 22 OCT 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
OECD Guideline for Testing of Chemicals, No. 202: "Daphnia sp., Acute Immobilisation Test and Reproduction Test", adopted April 4, 1984.
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
EEC, 1992 Annex to Commission Directive 92/69/EEC of 31" July 1992 adapting to technical progress for the seventeenth time Council Directive 67/548/EEC on the approximation of laws, regulations and administrative provisions relating to the classification, packing and labeling of dangerous substances (Official Journal No. L 383 A, December 29th, 1992) Part C.2. "Acute toxicity for Daphnia"
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: nominal 0, 1, 2, 5, 11 and 23 mg/L
- Sampling method: Samples were taken from all concentrations. Directly after preparation in the laboratory the test media were given to analysis. The maintenance of the test material concentrations was confirmed by determination of further samples after a period of 48 hours.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock preparation with a test material concentration of 0.1 g/L was prepared. Therefore, the calibrated flask with test material and reconstituted water was treated in an ultrasonic device for 1 hour. Subsequently, the preparation was stirred with a magnetic stirrer for further 23 hours. After that the preparation was passed through a separator/ funnel for 6 hours. The separated stock preparation was diluted with reconstituted water to the different test media concentrations and was used for the study.
- Controls: Reconstituted water
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna (Straus)
- Strain: Straus
- Source: in house
- Age at study initiation (mean and range, SD): not older than 24 hours
- Feeding during test: The Daphnia magna were not fed during the test.
- Food type: In the breeding and before test start, the Daphnia were fed with a suspension of fresh water algae.
- Amount: sufficient
- Frequency: once a week

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS:
The strain with the parent generation was bred and maintained in vessels containing Daphnia magna of different groups. Preparing a new study, juvenile Daphnia magna were separated and placed in 60 ml of reconstituted water. The water was renewed and the Daphnia magna were fed with a suspension of fresh water algae once a week. Newborn animals were separated and allocated to the different dose groups.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
250 mg CaCO3/L
Test temperature:
21 - 23 °C
pH:
at test start: 7.9
at test end: 7.8 - 7.9
Dissolved oxygen:
at test start: 95.7 - 96.5 %
at test end: 94.5 - 96.8 %
Nominal and measured concentrations:
nominal: 0, 1, 2, 5, 11 and 23 mg/L
mean measured: 0, 0.17, 0.40, 0.98, 1.99 and 3.71 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 50 mL all-glass vessels
- Type (delete if not applicable): The vessels were sealed with ground stoppers.
- Volume of solution: 50 mL reconstituted water with or without test material
- Aeration: The test medium was not aerated during the test.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water according to ELENDT (1990) was used as dilution water.
Macro nutrients (mg/L):
CaCI2 x 7 H2O: 293.80
MgSO4 x 7 H2O: 123.30
NaHCO3: 64.80
KCI: 5.80
Na2SiO3 x 9H2O: 10.00
NaNO3: 0.27
KH2PO4: 0.14
K2HPO4: 0.18

Trace elements (mg/L):
B: 0.5000
Fe: 0.2000
Mn: 0.1000
Li, Rb and Sr: 0.0500
Mo: 0.0250
Br: 0.0125
Cu and Zn: 0.0063
Co and I: 0.0025
Se: 0.0010
V: 0.0003

Macro nutrients (mg/L):
Na2EDTA x 2H2O: 2.50

Vitamins (µg/L):
Thiamine: 75.00
B12: 1.00
Biotin: 0.75

After preparation, the reconstituded water was aerated for 24 hours.
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: not specified.
- Photoperiod: 16 hours light - 8 hours dark regime controlled by a timer
- Other: The study was located in an air-conditioned room in the Institute of Toxicology.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): The mobility was determined by visual control and recorded after 24 and 48 hours.

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
- Test concentrations: A range-finding test was carried out with concentrations of 100, 50, 10, and 1 mg/L test material.
- Results used to determine the conditions for the definitive study: Daphnia magna exposed to test material concentrations of 100 and 50 mg/L showed immobilization after 48 hours.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
1.8 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95 % confidence intervals: 1.5 - 2.1 mg/L
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
1.2 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95 % confidence intervals: 1.0 - 1.4 mg/L
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.4 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Behavioural abnormalities: no
- Observations on body length and weight: no
- Other biological observations: no
- Mortality of control: no
- Other adverse effects control: no
- Immobilisation of control: 0 %
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No remarkable observations were made concerning the appearance of the test media All test media were clear solutions and remained clear throughout the entire test period.
Reported statistics and error estimates:
The EC50 was calculated as a logit analysis according the procedure of Unkelbach and Wolf (1985) using the PC-program 511.
In case that due to some extraordinary constellation of the study results - as often is the case, if small numbers of animals are used - the usual logit analysis cannot be applied. A modification of the logit analysis applying the so-called "l/(2n)-rule" (cf Cobb and Church, 1983) is performed. (I.e. at the highest dose with 0 % responders the mortality rate is replaced by l/(2n), and at the lowest dose with 100 % responders the rate is replaced by l-l/(2n), n being the number of animals used.) All estimates in which this procedure was employed are marked by #.
Validity criteria fulfilled:
yes
Conclusions:
The study was conducted under GLP according to OECD Guideline 202 and EU Method C.2 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or deviations from the guidelines, the validity criteria were met. Hence, the results can be considered as reliable to assess the toxicity of the test substance towards daphnids.
Daphnia magna were exposed to five test material concentrations of nominal 1 - 23 mg/L beside a control with reconstituted water only for 48 hours under defined conditions. In this closed static test system, daphniae exposed to concentrations of 5 to 23 mg/L showed immobilisation, whereas the concentrations of 1 and 2 mg/L revealed no biological effect.
The limit of quantification of the analytical method (HPLC-UV) was about 0.02 mg/L. At the start, the analytically determined concentrations were about 16 - 20 % of the nominal concentrations. At the end of the experimental part, the analytically determined test material concentrations were 17-21 % of the nominal concentrations. During the experimental phase of the study, the test material concentrations could be maintained at > 80 % of the initial.
Based on mean measured concentrations of the test item, the EC50 values with the 95 % confidence intervals and the no observed effect concentration (NOEC) were determined to be: 24h EC50 = 1.8 (1.5 - 2.1) mg/L, 48h EC50 = 1.2 (1.0 - 1.4) mg/L, NOEC = 0.4 mg/L.
Executive summary:

The objective of this study was to determine the acute toxicity of the test material using Daphnia magna. The GLP compliant study was performed according to OECD TG 202 and EU Method C.2.

Young Daphnia magna were exposed to aqueous test material concentrations under defined conditions. Due to the poor recovery of the test material after 48 in an open test system, a closed static test system was chosen.

The study comprised of four vessels per concentration containing five Daphnia magna, i.e., 20 Daphnia per concentration (test medium group). The Daphnia were observed for immobilisation up to 48 hours. Test material concentrations of nominal 1, 2, 5, 11, and 23 mg/L were used.


The limit of quantification of the analytical method was about 0.02 mg/L. At the start, the analytically determined concentrations were about 16 - 20 % of the nominal concentrations. At the end of the experimental part, the analytically determined test material concentrations were 17 - 21 % of the nominal concentrations. During the experimental phase of the study, the test material concentrations could be maintained at > 80 % of the initial.

The EC50 values with the 95 % confidence intervals and the no observed effect concentration (NOEC) based on mean measured concentrations of the test material are given below.
24h EC50 = 1.8 mg/L (95 % confidence intervals: 1.5 - 2.1 mg/L)
48h EC50 = 1.2 mg/L (95 % confidence intervals: 1.0 - 1.4 mg/L)
NOEC = 0.4 mg/L

Concluding the test material, solved in reconstituted water, was tested in a closed static test system. Under the given conditions of this study the 48 h EC50 value for the nominal and analytical concentration was in the range of 1.0 to 10.0 mg/L.

Description of key information

Short-term toxicity to aquatic invertebrates: EC50(48h) = 1.2 mg/L for Daphnia magna based on immobilisation (static, closed, OECD 202, GLP)

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
1.2 mg/L

Additional information

The objective of this study was to determine the acute toxicity of the test material using Daphnia magna. For this purpose, young Daphnia magna were exposed to aqueous test material concentrations under defined conditions. Due to the poor recovery of the test material after 48 in an open test system, a closed static test system was chosen.

The study comprised of four vessels per concentration containing five Daphnia magna, i.e., 20 Daphnia per concentration (test medium group). The Daphnia were observed for immobilisation up to 48 hours. Test material concentrations of nominal 1, 2, 5, 11, and 23 mg/L were used.

The GLP compliant study was performed according to OECD TG 202 and EU Method C.2.

The limit of quantification of the analytical method was about 0.02 mg/L. At the start, the analytically determined concentrations were about 16 - 20 % of the nominal concentrations. At the end of the experimental part, the analytically determined test material concentrations were 17 - 21 % of the nominal concentrations. During the experimental phase of the study, the test material concentrations could be maintained at > 80 % of the initial.

The EC50values with the 95 % confidence intervals and the no observed effect concentration (NOEC) based on mean measured concentrations of the test material are given below.
24h EC50 = 1.8 (1.5 - 2.1) mg/L
48h EC50 = 1.2 (1.0 - 1.4) mg/L
NOEC = 0.4 mg/L

Concluding the test material, solved in reconstituted water, was tested in a closed static test system. Under the given conditions of this study the 48 h EC50 value for the nominal and analytical concentration was in the range of 1.0 to 10.0 mg/L.