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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:
From 12 September 2000 to 17 November 2000
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
A real analytical method was not performed (TOC analysis only)
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
Part I (1984)
Deviations:
yes
Remarks:
A real analytical method was not performed (only TOC analysis)
GLP compliance:
yes (incl. certificate)
Remarks:
Inspected on April 03, 2000 / Signed on February 20, 2001
Specific details on test material used for the study:
No additional information
Analytical monitoring:
yes
Details on sampling:
Not applicable
Vehicle:
no
Details on test solutions:
Aqueous extracts of the test substance were tested. For this purpose, suspensions of the test substance were prepared in dilution water. For this purpose, the test substance was weighed into the glass vessels with the respective amount of water used for the preparation of the aqueous extracts. The test substance was introduced into the dilution water whilst shaking on a shaking machine (ca.130 rpm) for 24h at room temperature. It is assumed that during that time equilibration between the test substance and the water was achieved. Thereafter, the suspensions were filtered through filier 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. Prior to using in the test, the pH of the aqueous extracts was measured; pH was not adjusted. In the same way a Blank without any test substance
was prepared. As the test substance exhibits partial adsorptive properties the aqueous extracts of the test substance 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 substance, and losses due to adsorption were avoided. After that measure the test animals were introduced into the test solutions and the incubation was started.
Test organisms (species):
Daphnia magna
Details on test organisms:
Daphnia magna STRAUS of laboratory bred (originally derived from Dr. Knie, LWA Düsseldorf) were used for the test. In order to verify the sensitivity of the test system towards toxic substances the effect of potassium dichromate towards different animals of this bred is tested once a month. The value of the EC50 in this period of time was 0.9 mg/L potassium dichromate.
The animals used for the test were not older than 24h. The daphnia were cultivated in drinking water and fed with cells from the unicellular green alga Scenedesmus subspicatus according to SOF 5001 of Institut Fresenius Taunusstein. During the test the daphnia were not fed.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Remarks on exposure duration:
none
Post exposure observation period:
None
Hardness:
No data
Test temperature:
Temperature in the test solutions were checked at the beginning and the end of the test.
Comprised between 20.6 to 21.1°C
pH:
pH in the test solutions were checked at the beginning and the end of the test.
Comprised between 7.22 and 7.49
Dissolved oxygen:
Oxygen content in the test solutions were checked at the beginning and the end of the test.
Comprised between 7.7 and 8.4 mg O2/L
Salinity:
Not applicable
Conductivity:
No data
Nominal and measured concentrations:
- Nominal concentrations: 1, 2, 4, 8, 16 and 32 mg/L
- TOC analysis: see Tables 6.1.3/2 and 6.1.3/3 in "Any other information on results incl. tables".
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL-carboys
- Type (delete if not applicable): closed (glass-stoppers)
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
The dilution water (according to DIN 38412, part II) was prepared from four separate stock solutions:
- Solution 1: 0.22 g KCl were dissolved in 1L deionized water.
- Solution 2: 2.52g NaHCO3 were dissolved in 1L deionized water.
- Solution 3: 11.76g CaCl2.2H2O were dissolved in 1L deionized water.
- Solution 4: 4.93g MgSO4.7H2O were dissolved in 1L deionized water.
250mL of each solutions 1 to 4 were given into deionized water, the final volume was made up to 10L and the solution was stirred vigorously for about 30 min.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16h of light and 8h of darkness
- Light intensity: no data

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The effect of the test substance 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 sec were considered as immobile. Moving of antenna were not considered as swimming.

VEHICLE CONTROL PERFORMED: not applicable

RANGE-FINDING STUDY
- Test concentrations: 1.0 - 10 - 100 mg/L
- Results used to determine the conditions for the definitive study: After 48h of exposure, 0% immobilisation in the control and at 1 mg/L, 80% immobilisation at 10 mg/L and 90% immobilisation at 100 mg/L were observed.
Reference substance (positive control):
no
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
6.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: immobilisation
Details on results:
See Table 6.1.3/1 in "Any other information on results incl. tables".
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
No data

Table 6.1.3/1: Biological result of the main test

Concentration of the test substance (mg/L)

Ratio immobilised Daphnia (%)

24h

48h

Control (0)

0 [0/20]

0 [0/20]

1

0 [0/20]

0 [0/20]

2

0 [0/20]

10 [2/20]

4

0 [0/20]

15 [3/20]

8

15 [3/20]

60 [12/20]

16

45 [9/20]

95 [19/20]

32

100 [20/20]

100 [20/20]

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

Analytical part

The following TOC values were determined:

Table 6.1.3/2: TOC values

Nominal concentration (mg/L)

Measured values t0 (mg/L TOC)

Mean values of measured values t0 (mg/L TOC)

Mean values t0 measured values, net – Blank (mg/L TOC)

Measured values t48h (mg/L TOC)

Mean value of measured values t48h (mg/L TOC)

Mean values t48h measured values, net – Blank (mg/L TOC)

% recovery (mean value)

Blank

0.57

0.55

0

0.37

0.33

0

-

0.53

0.30

2

1.33

1.28

0.73

1.31

1.23

0.90

122.5

1.24

1.15

4

2.51

2.49

1.94

2.61

2.52

2.19

112.8

2.47

2.43

8

4.85

4.75

4.20

4.56

4.48

4.15

98.9

4.65

4.41

16

9.21

9.20

8.65

8.92

8.81

8.47

98.0

9.18

8.69

32

21.41

21.44

20.89

21.4

21.60

21.27

101.8

21.46

21.8

From these measured TOC-values and the known carbon content (56.02%) including the % purity of the test substance and the chemical formula of the test substance, the following concentrations and % recoveries on basis of the nominal concentrations were calculated:

Table 6.1.3/3: % recoveries of the nominal concentrations:

Nominal concentration (mg/L)

Mean calculated amount of test substance (mg/L) at t0

Mean calculated amount of test substance (mg/L) at t48h

% recovery at t0

% recovery at t48h

0

0.0

0.0

0

0

2

1.3

1.6

65.2*

80.3

4

3.5

3.9

86.6

97.7

8

7.5

7.4

93.7

92.6

16

15.4

15.1

96.5

94.5

32

37.3

38.0

116.5

118.7

* Possible outlier

Validity criteria fulfilled:
yes
Conclusions:
After 48 hours of exposure, immobilisations were 0% at 1 mg/L, 10% at 2 mg/L, 15% at 4 mg/L, 60% at 8 mg/L, 95% at 16 mg/L and 100% at 32 mg/L. Therefore, under the experimental conditions and based on the nominal test concentrations (analytically confirmed by TOC analysis), the 48-hour EC50 value was determined at 6.8 mg/L.
Executive summary:

The study was performed to assess the acute toxicity of the test substance to the freshwater invertebrate Daphnia magna. The method followed was designed to be compliant with OECD Guideline 202, Part I and GLP statement. In order to investigate the influence of the test substance towards the daphnia, the swimming behaviour of the animals was recorded. Following a preliminary range-finding test, twenty daphnids (four replicates, five daphnids per replicate) were exposed to the aqueous extracts from the test substance (as the test substance is considered poorly water soluble) over a range of nominal test concentrations of 0 (control), 1, 2, 4, 8, 16 and 32 mg/L. Under the conditions used for the test, immobilisation of the daphnia was observed at nominal concentrations >2 mg/L. Within TOC-analyses performed with the test solutions at t=0h and t=48h, it could be shown, that the organic matter of the test substance remained constant in the aqueous phase. The recoveries of the test substance on the basis of the nominal concentrations were observed to be in the range of >90%. For this reason, the results of this test are based an the nominal concentrations tested. After 48 hours of exposure, immobilisations were 0% at 1 mg/L, 10% at 2 mg/L, 15% at 4 mg/L, 60% at 8 mg/L, 95% at 16 mg/L and 100% at 32 mg/L. Therefore, under the experimental conditions and based on the nominal test concentrations, the 48-hour EC50 value was determined at 6.8 mg/L.

Description of key information

OECD guideline 202, key study, GLP, validity 2 :

48h-EC50 (Daphnia magna) = 6.8 mg/L based on nominal concentrations (analytically confirmed by TOC analysis)

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
6.8 mg/L

Additional information

One key study is available to assess the acute toxicity of the registered substance to the freshwater invertebrate Daphnia magna, under static conditions, according to the OECD Guideline 202 Part I, with GLP statement. Twenty daphnids (four replicates, five daphnids per replicate) were exposed to the aqueous extracts from the test substance over a range of nominal test concentrations of 0 (control), 1, 2, 4, 8, 16 and 32 mg/L. Under the conditions used for the test, immobilisation of the daphnia was observed at nominal concentrations >2 mg/L. Within TOC-analyses performed with the test solutions at t=0h and t=48h, it could be shown, that the organic matter of the test substance remained constant in the aqueous phase. The recoveries of the test substance on the basis of the nominal concentrations were observed to be in the range of >90%. For this reason, the results of this test are based an the nominal concentrations tested. After 48 hours of exposure, immobilisations were 0% at 1 mg/L, 10% at 2 mg/L, 15% at 4 mg/L, 60% at 8 mg/L, 95% at 16 mg/L and 100% at 32 mg/L. Therefore, under the experimental conditions and based on the nominal test concentrations, the 48-hour EC50 value was determined at 6.8 mg/L.