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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 Mar - 26 Mar 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Sampling method: Samples for analysis were taken from all test concentrations and the control. In addition, filter containing undissolved residue was retained for possible analysis. Samples were taken at t=0 h and t=48 h at a volume of 2.0 ml from the approximate center of the test vessels. At the end of the exposure period, the replicates were pooled at each concentration before sampling.
- Sample storage conditions before analysis: Samples were stored in a freezer until analysis.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Preparation of test solutions started with a loading rate of 100 mg/l applying a 2-day period of magnetic stirring followed by filtration through a 0.45 μm membrane filter (rc55, Whatman) to remove undissolved fraction. The obtained Water Soluble Fraction was used as the highest concentration. The lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium.
- Eluate:
no
- Differential loading:
no
- Controls:
blank control, reference control prepared with test medium
- Evidence of undissolved material: The final test solutions were all clear and colourless.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Freshwater flea
- Source: In-house laboratory culture
- Age of parental stock: > 2 weeks old
- Breeding conditions: About 250 newborn daphnids (less than 3 days old) were placed into 5 L of medium in an all-glass culture vessel. The daphnids were bred in M7 medium at 18-22 °C and were fed daily with a suspension of fresh water algae. After 7 days of cultivation half of the medium was renewed twice a week. The maximum age of the cultures was 4 weeks. The hardness of the medium was 180 mg CaCO3 per L and the pH was 7.7 ± 0.3.
- Health during breeding: no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and no delay in the production of the first brood

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h

Test conditions

Hardness:
180 mg CaCO3 per L
Test temperature:
19 - 20 °C
pH:
Control: 7.9 - 8.2
Treatment: 7.8 - 8.2
Dissolved oxygen:
Control: 9.1 - 9.5
Treatment: 9.0 - 9.5
Nominal and measured concentrations:
Nominal: Control; Solutions containing 10, 18, 32, 56 and 100% of the WSF prepared at a loading rate of 100 mg/l.
Measured: not detected (Control), 32, 57 and 102 mg/l in solutions containing 32, 56 and 100% of WSF, respectively
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 ml, all-glass, containing 80 ml of test solution
- Aeration: No aeration of the test solutions.
- 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: M7 medium; preparation according to guideline
- Intervals of water quality measurement: The pH, dissolved oxygen concentration and temperature was measured at the beginning and at the end of the test, for all concentrations and the control. In addition after 24 hours, immediately after counting the immobilized daphnids oxygen levels were measured in the test container with the solution of lowest concentration at which all the Daphnia magna were immobilized. Temperature was measured continuously in a temperature control vessel beginning at the start of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light / 8 h darkness

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Immobility of the daphnids was recorded at 24 hours and at 48 hours.

RANGE-FINDING STUDY
- Test concentrations: 20 daphnids each exposed to control and nominal loading rate of 100 mg/L; 10 daphnids each exposed to solutions of 1.0 and 10 mg/L
- Results used to determine the conditions for the definitive study: No immobility was observed at any of the test concentrations besides the highest where all daphnids were immobilised. Microscopic observation performed at the end of the test revealed that the immobilised daphnids were still alive but were covered with test substance and stuck with each other. Therefore, the observed effect was considered to be of mechanical nature. Therefore, the expected EC50 was between 10 and 100 mg/l.
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrations
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
89 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CI: 76 - 104 mg/L
Remarks:
nominal concentrations were analytically confirmed
Details on results:
- Behavioural abnormalities: no abnormalities reported
- Immobility of control: No immobilisation of the control was observed after 48 h.
Results with reference substance (positive control):
- Results with reference substance valid?
yes
- Concentrations: control, 0.10, 0.18, 0.32, 0.56, 1.0 and 1.8 mg/L
- EC50 (48 h) = 0.29 mg/L (95% CI: 0.25 - 0.34 mg/L)
Reported statistics and error estimates:
The 24 and 48h-EC50-value was calculated from the webits of the percentages of affected daphnids and the logarithms of the corresponding test substance concentrations (initially measured) using the maximum likelihood estimation method. ToxRat Professional v 2.10.05 (ToxRat Solutions® GmbH, Germany) was used to perform the analyses.

Any other information on results incl. tables

Table 1: Number of introduced daphnids and incidence of immobility in the final test

Time (h)

Replicate

Nominal concentration of the test substance (mg/L)

Control

10

18

32

56

100

0

A

5

5

5

5

5

5

B

5

5

5

5

5

5

C

5

5

5

5

5

5

D

5

5

5

5

5

5

Total introduced

20

20

20

20

20

20

24

A

0

0

0

0

0

0

B

0

0

0

0

0

0

C

0

0

0

0

0

0

D

0

0

0

0

0

0

Total immobilised

0

0

0

0

0

0

Effect %

0

0

0

0

0

0

48

A

0

0

0

0

2

5

B

0

0

0

0

0

3

C

0

0

0

0

0

2

D

0

0

0

0

1

3

Total immobilised

0

0

0

0

3

13

Effect %

0

0

0

0

15

65

Table 2: Concentrations of the test substance in test medium - final test

Time of sampling (h)

% of water-soluble fraction (1)

Analysed concentration (mg/L)

Relative to initial

0

0

n.d.

-

32

32.4

-

56

56.9

-

100

102

-

48

0

n.d.

n.a.

32

32.3

100

56

55.8

98

100

101

99

(1) Percentage of a water soluble fraction (WSF) prepared at a loading rate of 100 mg/l.

n.d. Not detected.

n.a. Not applicable.

Table 3: Validity criteria

Criterion from the guideline

Outcome

Validity criterion fulfilled

In the control, including the control containing the solubilising agent, not more than 10% of the daphnids should have been immobilized.

 0%

 yes

The dissolved oxygen concentration at the end of the test should be ≥ 3 mg/L in control and test vessels.

 8.1 - 8.2 mg/L

 yes

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
For further details please refer to “Any other information on results incl. tables”.
Conclusions:
The EC50 (48 h) was reported as 89 mg/L (95% CI: 76 - 104 mg/L).