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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:
12 September 2000 to 17 November 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: All concentrations
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test materials)
Suspensions of the test material were prepared in dilution water. The test material was weighed into the glass vessels with the respective amount of water used for the preparation of the aqueous extracts. The test material was introduced into the dilution water whilst shaking on a shaking machine (~130 rpm for 24 hours at room temperature). It is assumed that during that time equilibration between the test material and the water was achieved.
Thereafter, the suspensions were filtered through filter paper which was previously rinsed thoroughly with ultrapure water (Seral, „Purelab Plus") in order to eliminate possible soluble impurities from the filter material. The filtered extracts were used directly for the test without any further dilution steps.
As the test material exhibits partial adsorptive properties, the aqueous extracts of the test material were transferred into the respective test vessels, and after approx. 1 hour, the aqueous extracts were replaced by a second part of the aqueous extract. By that measure the surfaces of the glass walls were saturated with the test material, and losses due to adsorption were avoided.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Strain: Daphnia magna STRAUS
- Age at study initiation: The animals used for the test were not older than 24 hours
- Feeding during test: No

ACCLIMATION
- Acclimation conditions (same as test or not): Yes. Daphnia were laboratory bred and cultivated in drinking water
- Type and amount of food: Fed with cells from the unicellular green alga Scenedesmus subspicatus
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Test temperature:
20 ± 1°C
pH:
7.13 to 7.51
Dissolved oxygen:
7.9 to 8.9 mg O2/L
Nominal and measured concentrations:
- Nominal concentrations: 0 (blank), 1, 2, 4, 8, 16 and 32 mg/L
- Mean values of measured concentrations (after 48 hours) : 0.0, 1.9, 3.8, 7.8, 160 and 25.4 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL-carboys made of glass
- Type: closed with glass stoppers as the test material contains volatile components
- Material, size, headspace, fill volume: The carboys were filled up to the neck in order to avoid any gas phase
- Aeration: The test material was introduced into the dilution water whilst shaking on a shaking machine. No aeration of the test vessels took place as the oxygen content of the test solutions was sufficient for the whole incubation period.
- 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: The test material was dissolved in dilution water according to DIN 38412, part 11, as physiological conditions for the daphnia are necessary after the preparation of the aqueous extract. The dilution water was prepared from 4 separate stock solutions; in these solutions, the following substances were dissolved in 1 L of deionised water: Solution 1 0.22 g KCl; Solution 2 2.52 g NaHCO3; Solution 3 11.76 g CaCl2.2H2O; and Solution 4 4.93 g MgSO4.7H2O. 250 mL of each of solution was added to deionised water, the final volume was made up to 10 000 mL and the solution was stirred vigorously for about 30 min.
- Culture medium different from test medium: No
- Intervals of water quality measurement: pH, oxygen content and temperature in the test solutions were checked at the beginning and the end of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: No adjustment
- Photoperiod: 16 hours of light and 8 hours of darkness

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
The effect of the test material to the animals was examined by the determination of the swimming behaviour of the daphnia. For this purpose the respective test vessel was gently agitated. Daphnia showing no swimming within 15 seconds were considered as immobile. The movement of antenna was not considered as swimming.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: Yes. A screening-test was performed in order to find out the concentration range of the test material for the main test.
- Test concentrations: 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: Yes. There was no immobilisation at 1 mg/L with 100 % immobilisation observed after 24 and 48 hours at 100 mg/L. For the intermediate dose of 10 mg/L, no immobilisation was observed at 24 hours and 70 % immobilisation was observed after 48 hours.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
24 h
Dose descriptor:
EC0
Effect conc.:
5.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 49 % (95 % CL)
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
17 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 39 % (95 % CL)
Duration:
24 h
Dose descriptor:
EC100
Effect conc.:
54 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 78 % (95 % CL)
Duration:
48 h
Dose descriptor:
EC0
Effect conc.:
2.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 93 % (95 % CL)
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
8.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 49 % (95 % CL)
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
24 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 63 % (95 % CL)
Details on results:
The EC-values were calculated on the basis of the nominal concentrations being tested. Within TOC-analyses performed with the test solutions at t0 and t48 hours, it could be shown that the organic matter of the test material remained constant in the aqueous phase. The recoveries of the test material on the basis of the nominal concentrations were observed to be in the range of >80 %. For this reason, the results of this test are based on the on the nominal concentrations tested.

VALIDITY OF THE TEST
In the control without any test substance no immobilisation of the animals was observed. In the test solutions the oxygen concentration did not drop below a value of 60% of the saturation value (~5 mg/L).
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50: 0.9 mg/L
- Other: In order to verify the sensitivity of the test system towards toxic substances the effect of potassium dichromate towards different animals of this breed is tested once a month.
Reported statistics and error estimates:
Numbers of immobilised daphnia were plotted against the respective concentration, and, using a validated computer program on the Basis of EXCEL 5.0, the EC50 value was determined as well as the EC100 and the EC0 together with the 95 percent confidence limits. These calculations are done by logarithmic data transformation and following linear regression calculations. The validation of the computer program had been done using test data from Draper and Smith, 1966.

Table 1: Main Test Results

Concentration of the test material

Ratio immobilised

Daphnia [%]*

pH-Value

O2-Content

[mg O2/L]

 

Temperature [°C]

(mg/L)

24 h

48 h

t 0

48 h

t 0

48 h

t 0

48 h

Control (0)

0 [0/20]

0 [0/20]

7.22

7.47

8.2

7.9

20.7

20.8

1

0 [0/20]

0 [0/20]

7.48

7.45

8.8

8.4

20.6

21.1

2

0 [0/20]

0 [0/20]

7.47

7.46

8.5

8.1

20.6

21.1

4

0 [0/20]

0 [0/20]

7.52

7.51

8.6

8.2

20.5

21.1

8

0 [0/20]

65 [13/20]

7.49

7.47

8.7

8.1

20.6

21.1

16

45 [9/20]

95 [19/20]

7.51

7.47

8.9

8.4

20.6

21.1

32

85 [17/20]

100 [20/20]

7.29

7.13

8.5

8.1

20.6

21.0

*Numbers in brackets give the absolute numbers of immobilised animals at each concentration.

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this study, immobilisation of the daphnia was observed at nominal concentrations of 8 mg/L and greater. The 48 h EC50 was 8.3 mg/L.
Executive summary:

The potential of the test material to cause acute toxicity to Daphnia magna was investigated in a study conducted accordance with the standardised guideline OECD 202 under GLP conditions.

Daphnia magna were exposed to the test material for 48 hours in freshwater under static conditions at nominal loading rates of 0 (blank), 2, 4, 8, 16 and 32 mg/L. In order to investigate the influence of the test material towards the daphnia, the swimming behaviour of the animals was recorded after 24 and 48 hours. Suspensions of the test material were prepared in dilution water. The test material was introduced into the dilution water whilst shaking on a shaking machine (~130 rpm for 24 hours at room temperature). Thereafter, the suspensions were filtered through filter paper which was previously rinsed thoroughly with ultrapure water. The filtered extracts were used directly for the test without any further dilution steps.

As the test material exhibits partial adsorptive properties, prior to the addition of the test organisms, the aqueous extracts of the test material were transferred into the respective test vessels and after approx. 1 hour the aqueous extracts were replaced by a second part of the aqueous extract. By that measure the surfaces of the glass walls were saturated with the test material, and losses due to adsorption were avoided.

After 24 hours, no immobilisation was observed in the 0, 1, 2, 4 and 8 mg/L test concentrations, with 45 and 85 % immobilisation being observed at the 16 and 32 mg/L concentrations, respectively. After 48 hours, , no immobilisation was observed in the 0, 1, 2 and 4 mg/L test concentrations, with 65, 95 and 100 % immobilisation being observed at the 8, 16 and 32 mg/L concentrations, respectively.

The EC-values were calculated on the basis of the nominal concentrations being tested. Within TOC-analyses performed with the test solutions at t0 and t48 hours, it could be shown that the organic matter of the test material remained constant in the aqueous phase. The recoveries of the test material on the basis of the nominal concentrations were observed to be in the range of >80 %. For this reason, the results of this test are based on the on the nominal concentrations tested.

Under the conditions of this study, immobilisation of the daphnia was observed at nominal concentrations of 8 mg/L and greater. The 48 h EC50 was 8.3 mg/L.

Description of key information

Immobilisation of the daphnia was observed at nominal concentrations of 8 mg/L and greater. The 48 h EC50 was 8.3 mg/L.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
8.3 mg/L

Additional information

The potential of the test material to cause acute toxicity to Daphnia magna was investigated in a study conducted accordance with the standardised guideline OECD 202 under GLP conditions. The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

Daphnia magna were exposed to the test material for 48 hours in freshwater under static conditions at nominal loading rates of 0 (blank), 2, 4, 8, 16 and 32 mg/L. In order to investigate the influence of the test material towards the daphnia, the swimming behaviour of the animals was recorded after 24 and 48 hours.

Suspensions of the test material were prepared in dilution water. The test material was introduced into the dilution water whilst shaking on a shaking machine (~130 rpm for 24 hours at room temperature). Thereafter, the suspensions were filtered through filter paper which was previously rinsed thoroughly with ultrapure water. The filtered extracts were used directly for the test without any further dilution steps.

As the test material exhibits partial adsorptive properties, prior to the addition of the test organisms, the aqueous extracts of the test material were transferred into the respective test vessels and after approx. 1 hour the aqueous extracts were replaced by a second part of the aqueous extract. By that measure the surfaces of the glass walls were saturated with the test material, and losses due to adsorption were avoided.

After 24 hours, no immobilisation was observed in the 0, 1, 2, 4 and 8 mg/L test concentrations, with 45 and 85 % immobilisation being observed at the 16 and 32 mg/L concentrations, respectively. After 48 hours, , no immobilisation was observed in the 0, 1, 2 and 4 mg/L test concentrations, with 65, 95 and 100 % immobilisation being observed at the 8, 16 and 32 mg/L concentrations, respectively.

The EC-values were calculated on the basis of the nominal concentrations being tested. Within TOC-analyses performed with the test solutions at t0 and t48 hours, it could be shown that the organic matter of the test material remained constant in the aqueous phase. The recoveries of the test material on the basis of the nominal concentrations were observed to be in the range of >80 %. For this reason, the results of this test are based on the on the nominal concentrations tested.

Under the conditions of this study, immobilisation of the daphnia was observed at nominal concentrations of 8 mg/L and greater. The 48 h EC50 was 8.3 mg/L.