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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:
2002-04-04 to 2003-06-24
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
Part I, 1984-04-04
GLP compliance:
yes (incl. QA statement)
Remarks:
2003-04-29
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
at ambient temp., dark, dry and in original container
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 27%, 37%, 53%, 72% and 100% saturated solutions starting from a 1000 mg/L stock solution
Vehicle:
no
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna STRAUSS (Berlin)
- Age at study initiation: ≤ 24 h
- Method of breeding: In M4-medium at 20 ± 2°C in a 2 L beaker under a 16 h light/8 h dark cycle and medium renewal twice per week.
- Source: Umweltbundesamt Berlin
- Age of parental stock: Sep. 2001 (pretest), 2003-04-02 (main test)
- Feeding during test: No
- Food type: Finely ground fish feed Tetra Animin®, plus boiled and in M4-medium suspended single celled fresh water green algae (Desmodesmus subspicatus, Pseudokirchneriella subcapitata).

ACCLIMATION
- Acclimation period: 30 min in the water used in the preparation of the dilutions of the test substance.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Test temperature:
21 ± 1°C
pH:
7.8 - 7.9 (t= 0 h) and 8.2 - 8.3 (t= 48 h)
Dissolved oxygen:
8.2 - 8.4 mg/L (t= 0 h) and 7.8 - 8.1 mg/L (t= 48 h)
Salinity:
CaCl2*2H2O: 179.0 mg/L
MgSO4*7H2O: 69.5 mg/L
NaCl: 16.1 mg/L
KCl: 0.65 mg/L
Nominal and measured concentrations:
Pre-test: 0, 10, 100 and 1000 mg/L (nominal concentration)
Main-test: 0, 27, 37, 52, 72 and 100% (nominal saturation in respect to a 1000 mg/L stock solution)
Details on test conditions:
TEST SYSTEM
- Test vessel: 50 mL glass beaker
- Volume of solution: 20 mL
- 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: Neustader tap water with the addition of 179. 0 mg/L CaCl2*2H2O, 69.5 mg/L MgSO4*7H2O, 16.1 mg/L NaCl and 0.65 mg/L KCl to satisfy the criteria for minerals of the ISO 6341 and OECD No. 202 guidelines.
- Culture medium different from test medium: The culture medium was M4-medium, while the tests were conducted in supplemented Neustader tap water.

OTHER TEST CONDITIONS
- Light intensity: The tests were conducted without light.

EFFECT PARAMETERS MEASURED: Number of immobilized Daphnia after 24 and 48 h.

RANGE-FINDING STUDY
- Test concentrations: 0, 10, 100 and 1000 mg/L (nominal concentration)
Reference substance (positive control):
yes
Remarks:
The positive control was performed with potassium dichromate (K2Cr2O7) at 7 different concentrations (3.0, 2.5, 1.5, 1.25, 1.0 and 0.625 mg/L) for 24 h at 21 ± 1°C. The Number of replicates und used Daphnia were the same as in the main test.
Key result
Duration:
48 h
Dose descriptor:
EC50
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Remarks on result:
other: EC(50) > solubility of test material in test medium
Duration:
48 h
Dose descriptor:
NOEC
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Remarks on result:
other: NOEC >= solubility of test material in test medium
Details on results:
- Immobilisation of control: 0
- Effect concentrations exceeding solubility of substance in test water: Yes
Results with reference substance (positive control):
- Results with reference substance valid: Yes
-24 h EC50: 0.625 mg/L
-NOEC 48 h: 0.652 mg/L

Reported statistics and error estimates:
only for the test with reference material needed:
Program: Origin TM

 Table 1: pH and O2-concentration

Saturation

[%]

pH

[t = 0 h]

pH

[t = 48 h]

O2-concentration

[mg/L, t = 0 h]

O2-concentration

[mg/L, t = 48 h]

0

7.9

8.2

8.3

7.8

100

7.8

8.2

8.4

7.9

72

7.8

8.3

8.2

8.0

52

7.8

8.3

8.2

8.0

37

7.8

8.3

8.2

8.0

27

7.8

8.3

8.3

8.1

 

Table 2: Results of the reference test

Conc.

[mg/L]

Immobilization

[absolute num.]

Immobilization

[%]

pH

[t = 0 h]

pH

[t = 48 h]

O2-conc.

[mg/L, t = 0 h]

O2-conc.

[mg/L, t = 48 h]

0

0

0

0

0

0

7.8

8.0

8.6

8.6

3.0

5

5

5

5

100

7.7

7.8

8.8

8.6

2.5

5

5

5

5

100

7.7

7.8

8.8

8.6

2.0

5

5

5

5

100

7.7

7.8

8.9

8.5

1.5

5

5

5

4

95

7.8

7.8

8.8

8.7

1.25

2

4

0

4

50

7.8

7.8

8.8

8.6

1.0

0

0

3

5

40

7.8

7.7

8.7

8.8

0.625

0

0

0

1

5

7.8

7.7

8.5

8.8

 

Table 3: Endpoints determined form the reference test

Endpoint

Value [mg/L]

Error

NOEC 48 h

0.625

n.a.

24 h EC50i

1.1

n.a.

24 h EC100i

2.0

n.a.

Table 4: Results of the pre-test

Conc.

[mg/L]

Immobilization

[absolute num.]

Immobilization

[%]

pH

[t = 0 h]

pH

[t = 48 h]

O2-conc.

[mg/L, t = 0 h]

O2-conc.

[mg/L, t = 48 h]

0

0

0

0

0

0

7.9

8.1

8.3

8.2

1000

2

1

2

1

30

7.9

8.0

8.4

8.2

100

0

0

0

0

0

7.9

8.0

8.1

8.3

10

0

0

0

0

0

7.9

8.1

8.0

8.2

Table 5: Results of the main test

Conc.

[mg/L]

Immobilization

[absolute num., t = 24 h]

Immobilization

[%, t = 24 h]

Immobilization

[absolute num., t = 48 h]

Immobilization

[%, t = 48 h]

0

0

0

0

0

0

0

0

0

0

0

100

0

0

0

0

0

0

0

0

0

0

72

0

1

0

0

5

1

1

0

0

10

52

0

0

1

0

5

0

0

1

0

5

37

1

0

0

0

5

1

0

0

0

5

27

0

0

0

0

0

0

0

0

0

0

Table 6: Endpoints determined from the main test

Endpoint

Value

95 % confidence interval

NOEC 48 h

100 % saturation

n.a.

48 h EC50i

>100 % saturation

n.a.

48 h EC100i

>>100 % saturation

n.a.

Table 7: Results of the DOC-analysis

Saturation [%]

DOC -conc.

[mg/L]

DOC

[%]

0 h

48 h

48 h

 0

5.11 

4.56

89 

 100

3.58 

3.86 

108 

 72

2.77 

3.15 

114 

 52

2.35 

3.36 

143 

 37

2.05 

2.72 

133 

 27

2.50 

3.41 

136 

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test substance on Daphnia magna (age: <= 24 h) was assessed in a 48 h acute toxicity test conducted in freshwater in accordance with EN ISO 6341, following OECD 202 (1984). 5 concentrations (between 100% saturated solution and 27%) were tested resulting in an EC50 (48h) of > solubility of the test item in test medium (dissolved test item).
Executive summary:

In this study, the water accommodated fraction of the test substance, was evaluated under laboratory conditions according to the OECD 202, for its potential acute toxicity on the aquatic invertebrate Daphnia magna. Following a pretest with nominal concentrations of 0, 10, 100 and 1000 mg/L, the main test was conducted with a saturated test solution (100%) and diluted samples of 72, 52, 37, 27 % test substance. The stock solution used was set up with an in-weight of 1000 mg/L. After 2 h of shaking the stock solution was filtered (0.45 µm) and used for testing.

Both tests were carried out under the same conditions: For 48 h at 21°C in a test volume of 20 mL without light and 4 replicas with each 5 Daphnia per concentration. Because a direct analytical verification method of the test substance was not available an unspecific DOC-analysis was performed as an alternative method (LOQ: 1 mg/L). In addition, a positive control test with the reference substance potassium dichromat was performed. Based on the number of immobilized Daphnia, the effect concentrations (48 h EC50) and the no-observed-effect concentration (NOEC) were determined. The conducted reference test with potassium dichromate as positive control resulted in a 24 h EC50 of 1.1 mg/L, which is in the range (0.6-2.4 mg/L) of the test requirements. In case of the main test, no immobilized Daphnia were observed in the negative control nor at nominal test substance concentrations of 27% and 100% after 48 h. At concentrations of 37% and 52% each a single individuum showed immobilization effects whereas and at 72% two individuals in different replicates were immobilized. The estimated immobilization values (5-10%) at these concentrations are in the uncritical range of the test criteria and is considered to be not significant. Because no effects were observed at the nominal test substance concentration of 1000 mg/L, the key effect concentration after 48 h (48 h EC50) was estimated to be above the tested maximum concentration. The corresponding NOEC is stated to be 100% (solubility of the test item in test medium.

Thus, under the condition of the test the EC(50) is > solubility of the test item in test medium.

Description of key information

The effect of the test substance on Daphnia magna (age: <= 24 h) was assessed in a 48 h acute toxicity test conducted in freshwater in accordance with EN ISO 6341, following OECD 202 (1984). 5 concentrations (between 100% saturated solution and 27%) were tested resulting in an EC50 (48h) of > solubility of the test item in test medium (dissolved test item).

Key value for chemical safety assessment

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