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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:
2000-03-06 till 2000-03-16
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:
adopted April 4, 1984
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
December 29, 1992
Deviations:
not applicable
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
For the analytical measurement a sample from the freshly prepared test medium from the concentration of the closed system was taken at the start of the study. For the determination of the maintenance of the test material concentration during the exposure period a sample was taken from the medium at the end of the exposure period.
Vehicle:
no
Details on test solutions:
A stock preparation with a test material concentration of 0.1 g/L was prepared Therefore, the calibrated flask with the test material and the vehicle, reconstituted water, was treated with an Ultra Thurrax and than in an ultrasonic device for 60 minutes. The stock preparation was diluted with reconstituted water to the different test medium concentrations.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: waterglea
- Strain: Straus
- Age at study initiation: not older than 24 h
- Source: Merck KGaA, Darmstadt, Institute of Toxicology
- Age of parental stock (mean and range, SD):
- Feeding during test : no

ACCLIMATION
- Acclimation period: no
- Breeding conditions: same as test
- Type and amount of food: suspension of fresh water algae once a week
- Health during acclimation: no mortality observed
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
252 mg CaCO3/L
Test temperature:
20 to 21°C
pH:
7.65 - 7.95
Dissolved oxygen:
93.7 - 97.6 %
Nominal and measured concentrations:
Nominal concentrations: 0, 10, 18, 32, 56, 100 mg/L
Measured concentrations: see details in Table 1
Details on test conditions:
TEST SYSTEM
- Test vessel: glass vessel
- Type: closed with parafilm
- Material, size: glass, 25 mL
- Volume of solution: 30 mL
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Preparation of dilution water: The water was composited according to ELENDT M4 (1990)

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light - 8 hours dark regime

EFFECT PARAMETERS MEASURED
The mobility was determined by visual control and recorded after 24 and 48 hours.

RANGE-FINDING STUDY
- Test concentrations: 1, 10, 100 mg/L
- Results used to determine the conditions for the definitive study: yes, at 100 mg/L Daphnia showed immobilization
Reference substance (positive control):
no
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
16 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
45 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
8.1 mg/L
Nominal / measured:
meas. (geom. 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: no
- Abnormal responses: no
- Any observations that might cause a difference between measured and nominal values: no

In the high test material concentration, the analytically determined test material concentration at the start of the study was 60 % of the nominal value. The analytical test material concentration at nominally 0.010 and 0.032 g/L were below the limit of detection already at the start of the study. Therefore instead of these two concentrations the nominal concentration of 0.056 g/L was analysed at the end of the study. For this analytical concentration of 0.025 g/L was determined. At the end of the study the test material concentrations were 45 % of the nominal value (75 % of the initial concentration).
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 #.

Table 1: Measured concentrations after 0 and 48h

Nominal concentration [mg/L]

Analytical concentration [mg/L]

After 0 h

After 48 h

10

< 5

< 5

32

< 5

<5

56

25

45%

100

60

60%

45

45%

Table 2: Immobilized Daphnia after 24 and 48 h 

Nominal concentration [mg/L]

Immobilized/ exposed Daphnia magna

24 hours

48 hours

Control

0/20

0/20

10

0/20

0/20

18

0/20

0/20

32

14/20

8/20

56

20/20

19/20

100

20/20

20/20

Validity criteria fulfilled:
yes
Conclusions:
The test item showed acute toxicity towards Daphnia according to OECD 202 (NOEC=8 mg/L, EC50=16 mg/L mean measured concentration).
Executive summary:

The objective of the study was to determine the acute toxicity of the test item using Daphnia magna according to OECD 202 and EU Method C.2. For this purpose, young Daphnia magna were exposed to aqueous test material concentrations under defined conditions. 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 immobilization up to 48 hours. Due to the high volatilization and recovery of the test material it was necessary to include some changes of the regular guidelines in this study and to use a closed test system. The study was started with the exposure of Daphnia to test material concentrations of nominal 10, 18, 32, 56 and 100 mg/L in a static system. Analytical controls were carried out with 10, 32, and 100 mg/L. Initially, the lower concentrations were below the limit of quantification and the recovery of the high concentrations was 60 %. At the end of the study, the recovery of the high concentration was 45% of the nominal concentration. This amounts to 75 % of the initial analytical concentration of 0.06 g/L. Thus the test material concentration could not be maintained at > 80 % of the initial concentration throughout the experimental part.24 hours after start of the experimental part more Daphnia magna were immobilized than at the end. So, the EC50 values changed to a higher value. The analytically determined test material concentration after about 48 hours was about 45 % of the nominal value. The calculated analytical 48 h EC50 was 16 mg/L.

Description of key information

The test item showed acute toxicity towards Daphnia according to OECD 202 (NOEC=8 mg/L, LC50=16 mg/L mean measured concentration) (reference 6.1.3 -1).

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
16 mg/L

Additional information

The objective of the study was to determine the acute toxicity of the test item using Daphnia magna according to OECD 202 and EU Method C.2.For this purpose, young Daphnia magna were exposed to aqueous test material concentrations under defined conditions. 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 immobilization up to 48 hours. Due to the high volatilization and recovery of the test material it was necessary to include some changes of the regular guidelines in this study and to use a closed test system. The study was started with the exposure of Daphnia to test material concentrations of nominal 10, 18, 32, 56 and 100 mg/L in a static system. Analytical controls were carried out with 10, 32, and 100 mg/L. Initially, the lower concentrations were below the limit of quantification and the recovery of the high concentrations was 60 %. At the end of the study, the recovery of the high concentration was 45% of the nominal concentration. This amounts to 75 % of the initial analytical concentration of 0.06 g/L. Thus the test material concentration could not be maintained at > 80 % of the initial concentration throughout the experimental part.24 hours after start of the experimental part more Daphnia magna were immobilized than at the end. So, the EC50 values changed to a higher value. The analytically determined test material concentration after about 48 hours was about 45 % of the nominal value. The calculated analytical 48 h EC50 was 16 mg/L (reference 6.1.3 -1).