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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:
06 - 29 Jun 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Staatliches Gewerbeaufsichtsamt Hildesheim, Hildesheim, Germany (03.01.2017)
Analytical monitoring:
yes
Remarks:
GC-MS
Details on sampling:
- Concentrations: All concentration levels and the control in fresh media (at the start of exposure and at renewal at 0 and 24 h) and in aged media (at renewal and at the end of exposure at 24, and 48 h).
- Sampling method: At the start of the exposure and at renewal (0 and 24 h), samples of the fresh media were taken after preparation of all test item concentrations. At renewal and at the end of exposure (24 and 48 h), samples of the 24-h old media were taken directly from the test vessels.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until the start of analysis, if necessary. Prepared samples were stored in the autosampler at room temperature until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A saturated solution with a nominal concentration of 2.0 mg/L was prepared by adding 2.22 µL/L test item (relative density accounted for) to a brown glass flask filled with 40 g glass beads (4 mm diameter, VWR) and dilution water (without headspace), closing the flask with a screw cap with septum, and shaking for 24 h at 20 rpm at room temperature. After shaking, the dispersion was allowed to stand for approx. 1 h for the separation of undissolved test item before removing the saturated solution from the approximate center of the water body. The saturated solutions were prepared 1 d before the start of the exposure and 1 d before the renewal of test solutions. The saturated solution was used as the highest concentration level and as a stock solution for the preparation of further dilution levels by dilution with dilution water in a geometric series with a dilution factor of 2.
- Controls: Dilution water without test item, incubated under the same conditions as the treatments. The control solution was prepared the same way as the saturated solution but without addtion of test item.
- Evidence of undissolved material: No Tyndall effect was observed in any test solution.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Strain/clone: Clone 5
- Age of daphnids at test start: < 24 h
- Culture: In 2 - 3 L glass vessels with approx. 1.8 L culture medium, at 20 ± 2 °C, in an incubator with 16 h illumination (max. 1500 lx) using culture medium Elendt M4 (according to OECD 202, Annex 3) with a modified total hardness of 160 - 180 mg CaCO3/L.
- Feeding (culture): At least 5 times per week ad libitum with a mix of > 10^6 cells/mL of unicellular green algae sourced from the test facility (e.g. P. subcapitata and D. subspicatus)
- Feeding during test: No feeding during the test.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Juvenile daphnids were removed from the culture vessels the latest 24 h before the start of the exposure and discarded. The juveniles born within the following period of max. 24 h preceding the exposure were used for the test. No first brood progeny was used for the test.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
165 - 174 mg CaCO3/L
Test temperature:
20.0 - 20.1 °C
pH:
7.46 - 7.96 (fresh media)
7.62 - 8.17 (aged media)
Dissolved oxygen:
8.14 - 9.17 mg/L O2 (fresh media)
4.74 - 8.43 mg/L O2 (aged media)
Conductivity:
423 - 435 µS/cm
Nominal and measured concentrations:
Control, 6.25, 12.5, 15.0, 50.0, and 100% of the saturated solution of nominal 2.0 mg/L
Control, 0.0760, 0.165, 0.321, 0.613, and 1.16 mg/L (geom. mean measured)
Details on test conditions:
TEST SYSTEM
- Test vessel: 130 mL sealed glass flasks (4.5 (ID) x 9.5 (H) cm) with screw cap filled to the top with test solution
- Renewal rate of test solution: 24 h
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: Culture medium same as test medium
- Intervals of water quality measurement: The water quality parameters (pH, dissolved oxygen, temperature, conductivity and total hardness) were measured at the start of the exposure (0 h) and at the renewal of the test solutions (24 h) in fresh and aged media.

OTHER TEST CONDITIONS
- Photoperiod: 16 h light, 8 h dark
- Light intensity:max. 1500 lx

EFFECT PARAMETERS MEASURED:
- Immobilization: after 24 and 48 h

RANGE-FINDING STUDY
- Test concentrations: Control, 10, and 100% dilution level of the saturated solution with a nominal loading of 2.0 mg/L
- Results used to determine the conditions for the definitive study: 45% Immobilization after 48 h exposure to the saturated solution (100% dilution level)
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.696 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Remarks on result:
other: 95% confidence interval: 0.611 - 0.839 mg/L
Details on results:
- Mortality of control: 0%
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: All tested concentration levels were visually clear throughout the exposure period. No Tyndall effect was observed in the saturated solution (observations directly after preparation at 0 and 24 h).
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
- Results with reference substance valid? A reference test is conducted once a month at the test facility and the most recent test dated from 01 - 02 Jun 2017. The results fell within the required range of 0.6 - 2.1 mg/L set out by the OECD guideline 202.
- EC50 (24 h): 2.05 mg/L potassium dichromate
Reported statistics and error estimates:
The EC100 values were empirically derived from the data. The EC10 and EC50 values were calculated by sigmoida dose-response regression with the software GraphPad Prism. The respective 95% confidence limits were calculated from the standard error and the t-distribution.
FITTING RESULTS OF THE EVALUATION OF THE EC10 AND EC50 VALUES AFTER 48 HOURS
Equation: Sigmoidal dose-response, variable slope
y = bottom + (top - bottom)/(1 + 10^((log EC50 - x) * HillSlope))
BEST FIT VALUES:
Bottom: -1.051; Top: 145.1; log EC50: -0.04911; HillSlope: 2.490; EC10 [mg/L]: 0.3267; EC50 [mg/L]: 0.6957
GOODNESS OF FIT:
Degrees of freedom: 16; R square: 0.9453; Absolute sum of squares: 1508; Sy.x:9.708

VALIDITY CRITERIA

 The study fulfilled the validity criteria set out by the OECD guideline 202 (Table 1).

 

Table 1: Validity criteria for OECD 202.

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.

In the control, no daphnids were immobilized or showed any signs of disease or stress.

Yes

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

The dissolved O2 concentration in the 24 h old media was ≥ 4.74 mg/L in all concentration levels and the control.

Yes

 

 

ANALYTICAL RESULTS

The measured concentrations of the test item in fresh media at the start of exposure and at renewal (0 and 24 h) were in the range of 0.0803 to 1.50 mg/L. The measured concentrations in aged media at renewal and at the end of the test (24 and 48 h) ranged from 69 – 88% of the initially measured concentrations. The measured concentrations and the calculated geometric mean measured concentrations are provided in Table 2.

  

Table 2. Measured concentrations of the test item during the definitive test. 

 

0 h

Start of the exposure interval

24 h

End of the exposure interval

24 h start of the exposure interval

48 h end of the exposure interval

 

 

 

 

Geometric mean measured test item concentration [mg/L]

Dilution level of the sat. solution [%]

Meas. conc. [mg/L]

meas. conc.

[mg/L]

 

%

meas. conc.

[mg/L]

meas. conc.

[mg/L]

 

%

100*

1.50

1.08

72

1.27

0.8812)

69

1.16

50.0

0.725

0.554

76

0.657

0.535

81

0.613

25.0

0.380

0.298

78

0.359

0.261

73

0.321

12.5

0.186

0.146

79

0.176

0.155

88

0.165

6.25

0.08031)

0.0681

85

0.0914

0.0667

73

0.0760

Control

< LOQ

< LOQ

< LOQ

< LOQ

 

Meas. conc.      = measured concentration of the test item

%                         = percentage of the initially measured concentration of the test item

LOQ                     = limit of quantification of the analytical method (0.002 mg test item/L)

1)                         = reanalyzed

2)                         = mean value of two samples, which were analyzed with different dilution factors

*                          = saturated solution

 

 

BIOLOGICAL RESULTS

 

Table 3. Immobilization Rates after 48 hours of exposure in the definitive test (n = 20, divided into 4 replicates with 5 daphnids each).

Geometric mean measured concentration of the test item [mg/L]

Immobilization

[%[

replicates

1

2

3

4

MV

1.16

80

100

100

100

95

0.613

60

40

20

40

40

0.321

0

20

20

0

10

0.165

0

0

0

0

0

0.0760

0

0

0

0

0

Control

0

0

0

0

0

 

Validity criteria fulfilled:
yes
Remarks:
For further details please refer to “Any other information on results incl. tables”.
Conclusions:
The test resulted in an EC50 (48 h) of 0.696 mg/L (geom. mean measured, OECD 202, D. magna).
Executive summary:

There is one study, in which the short-term toxicity of test item to aquatic invertebrates was assessed according to OECD guideline 202 and GLP.

In a semi-static test with a 24 h medium renewal rate, Daphnia magna was exposed to five concentration levels in a closed system for 48 h. The test media were prepared from a saturated solution with a nominal loading concentration of 2.0 mg test item/L and four further dilution levels prepared from the saturated solution in a geometric series with a dilution factor of 2 (nominal 6.25 – 50%). Test item concentrations were analytically verified by GC-MS in fresh media at the start of exposure and at renewal (0 and 24 h) and in aged media at renewal and at the end of exposure (24 and 48 h) in all concentration levels and the control.

The measured concentrations of the test item in fresh media ranged from 0.0803 – 1.50 mg/L. The measured concentrations in aged media were 69 – 88% of the initially measured concentrations. Since test item concentrations were not within 80 – 120% of nominal concentrations, effect concentrations were based on the calculated geometric mean measured concentrations, which were 0.076, 0.165, 0.321, 0.613, and 1.16 mg/L. After 48 h of exposure, immobility was observed, except at the two lowest concentration levels and in the control. The derived EC50 (48 h) was 0.696 mg/L.

Description of key information

EC50 (48 h) = 0.696 mg/L (geom. mean measured, OECD 202, D. magna)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
0.696 mg/L

Additional information

There is one study, in which the short-term toxicity of test item to aquatic invertebrates was assessed according to OECD guideline 202 and GLP.

In a semi-static test with a 24 h medium renewal rate, Daphnia magna was exposed to five concentration levels in a closed system for 48 h. The test media were prepared from a saturated solution with a nominal loading concentration of 2.0 mg test item/L and four further dilution levels prepared from the saturated solution in a geometric series with a dilution factor of 2 (nominal 6.25 – 50%). Test item concentrations were analytically verified by GC-MS in fresh media at the start of exposure and at renewal (0 and 24 h) and in aged media at renewal and at the end of exposure (24 and 48 h) in all concentration levels and the control.

The measured concentrations of the test item in fresh media ranged from 0.0803 – 1.50 mg/L. The measured concentrations in aged media were 69 – 88% of the initially measured concentrations. Since test item concentrations were not within 80 – 120% of nominal concentrations, effect concentrations were based on the calculated geometric mean measured concentrations, which were 0.076, 0.165, 0.321, 0.613, and 1.16 mg/L. After 48 h of exposure, immobility was observed, except at the two lowest concentration levels and in the control. The derived EC50 (48 h) was 0.696 mg/L.

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