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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:
2010-05-17 to 2010-06-09
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:
and EU Method C.2
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: All concentration levels and the control were analytically verified in the fresh media (0 h) and in the old media (48 h) by LC-MS/MS.
- Sampling method: For the old media, separate replicates without test organisms were prepared and incubated under test conditions. The method was validated according to SANCO 3029/99 rev.4 (2000-07-11).
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until start of analysis if necessary.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 100 mg/L was freshly prepared with dilution water. The stock solution was shaken with 20 rpm for 24 h (rotating shaker 3040, GFL) at room temperature.
- Eluate: Dilution water
- Differential loading: 6.25 - 12.5 - 25.0 - 50.0 - 100 mg/L
- Controls: Dilution water without test item tested under the same conditions as the test groups.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna (STRAUS)
- Strain: Clone 5
- Source: Institut für Wasser-, Boden- Lufthygiene (WaBoLu), further own breeding
- Age at study initiation (mean and range, SD): 2 to 24 h
- Method of breeding: In 2-3 L glass vessels with appr. 1.8 L culture medium, at 20 +/- 2 °C, in an incubator, 16 h illumination, illumination strength max. 20 µExm-2 x s-1
- Feeding during test: No feeding

ACCLIMATION
- Acclimation period: 2 h
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: No feeding
- Health during acclimation (any mortality observed): Healthy, no mortality observed


Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
No
Hardness:
Dilution water at test start: 267 (mg CaCO3/L)
Test temperature:
20.1 - 21.6 °C
pH:
Water Quality Parameters at Test Start (0 h, Fresh Media)
(measured in one replicate per concentration and control)

Nominal
test item
concentration
mg/L] pH-Value


100 7.65
50.0 7.64
25.0 7.64
12.5 7.66
6.25 7.55
Control 7.92

Water Quality Parameters at Test End (48 h, Old Media)
(measured in all replicates)

Nominal
test item
concentration
[mg/L] pH-Values
Replicates
1 2 3 4
100 7.56 7.57 7.54 7.55
50.0 7.58 7.20 7.34 7.48
25.0 7.60 7.58 7.59 7.60
12.5 7.59 7.38 7.57 7.59
6.25 7.57 7.56 7.57 7.60
Control 7.64 7.65 7.62 7.53
Dissolved oxygen:
Nominal
test item
concentration
mg/L] O2-Concentration

[mg/L]
100 8.65
50.0 8.65
25.0 8.66
12.5 8.68
6.25 8.68
Control 8.74

Water Quality Parameters at Test End (48 h, Old Media)
(measured in all replicates)

Nominal
test item
concentration
[mg/L] O2 -Concentration [mg/L]
Replicates
1 2 3 4
100 8.11 8.17 8.18 8.16
50.0 8.22 8.15 8.22 8.18
25.0 8.39 8.37 8.38 8.39
12.5 8.46 8.41 8.42 8.42
6.25 8.47 8.46 8.49 8.49
Control 8.62 8.53 8.54 8.52
Salinity:
Freshwater
Nominal and measured concentrations:
Please refer to "Any other information on materials and methods"
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass beakers, 50 mL
- Type (delete if not applicable): closed with parafilm
- Material, size, headspace, fill volume: Glass beakers (4 cm ID x 7 cm H), 50 mL
- Aeration: No
- Renewal rate of test solution (frequency/flow rate): One application at test start, static test
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): None


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Acc. to Directive 92/69/EEC Method C.2, Annex 1
- Conductivity: Dilution water at test start: 640 µS/cm
- Culture medium different from test medium: Elendt M4 acc. to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L
- Intervals of water quality measurement: At test start and test end


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16:8 light:dark cycle
- Light intensity: Diffuse light, illumination strength max. 20 µE x m-2 x s-1


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Immobility was assessed in all treatment levels after 24 h and 48 h. An animal was considered to be immobile, if it was not able to swim during a 15 second observation period following gentle agitation of the test vessel.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
Immobilization Rates of the Preliminary Test in [%]
(n = 20, divided into 2 replicates with 10 daphnids each)
Nominal
Test Item
Concentration
[mg/L] IMMOBILIZATION [%]
24 h 48 h
Replicates Replicates
1 2 MV 1 2 MV
100 60 60 60 100 100 100
10 0 0 0 0 0 0
Control 0 0 0 0 0 0


Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
70.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: (61.1-81.8)
Remarks:
Confidence interval
Details on results:
- Behavioural abnormalities: None
- Mortality of control: 0%
- Other adverse effects control: None
- Abnormal responses: None
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50/LC50: 1.70 (CI 1.51 - 1.97) mg/L
Reported statistics and error estimates:
The EC10- and the EC50-values were calculated by sigmoidal dose -response regression. The confidence intervals for the EC50-values were calculated from the best-fit values, the standard error and the t-distribution with the software GraphPad prism.

Immobilization Rates of the Definitive Test after 24 and 48 h of Exposure

                 (n = 20, divided into 4 replicates with 5 daphnids each)

Nominal Test Item Concentration

IMMOBILIZATION [%]

24 h

48 h

Replicates

Replicates

[mg/L]

1.

2.

3.

4.

MV

1.

2.

3.

4.

MV

100

40

60

60

60

55

60

100

100

100

90

     50.0

  0

  0

  0

  0

  0

  0

  40

    0

    0

10

     25.0

  0

  0

  0

  0

  0

  0

    0

    0

    0

  0

     12.5

  0

  0

  0

  0

  0

  0

    0

    0

    0

  0

        6.25

  0

  0

  0

  0

  0

  0

    0

    0

    0

  0

Control

  0

  0

  0

  0

  0

  0

    0

    0

    0

  0

Validity criteria fulfilled:
yes
Conclusions:
Based on the nominal concentrations of the test item the 48-h EC50 for Daphnia magna was estimated to be 70.7 mg/L (95 % confidence limits: 61.1 – 81.8 mg/L).
The NOEC after 48 h was determined to be 50.0 mg/L as the highest tested concentration with no biologically significant effect. The LOEC after 48 h was determined to be 100 mg/L as the lowest tested concentration with a biologically significant effect (Immobilization rates of > 10 % are seen to be biologically significant according to the guidelines).
Executive summary:

In the acute immobilization test with Daphnia magna(STRAUS) the effects of the test substance were determined according to OECD 202 (2004).

The study was conducted under static conditions with 5 concentration levels of the test item ranging from 6.25 to 100 mg/L set up in a geometrical series with a dilution factor of 2 over a period of 48 h. 20 test organisms were exposed to each test concentration and the control.
All concentration levels and the control were analysed by LC-MS/MS after 0 h and 48 h. Details of the analytical results are given in part 4.2.2.

The recoveries in the fresh (0 h) and old media (48 h) were in the range of 96 to 107 % of the nominal values, demonstrating that concentrations of the test item
were successfully maintained for the duration of the test. All effect values are given based on the nominal concentrations of the test item (Table 1).
Water quality parameters of pH-value and dissolved oxygen concentration, measured at 0 and 48 h, were determined to be within the acceptable limits. The validity criteria of the test guideline were fulfilled.

EC10-, EC50- (with Confidence Interval), EC100 - Values, NOEC and LOEC

              (based on the nominal concentrations of the test item)

Test Duration


[h]

Nominal Concentration
of the Test Item

[mg/L]

Confidence Interval
p = 95%

[mg/L]

EC10

24

       83.8

---

48

       50.0

---

EC50

24

       98.5

97.3 – 99.8

48

        70.7

61.1 – 81.8

EC100

24

> 100

---

48

> 100

---

NOEC

48

       50.0

---

LOEC

48

   100

---

Description of key information

Short term toxicity to invertebrates - OECD 202 – EC50: 70.7 mg/l, 48h - (PARAD consortium, NOACK, 2010)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Dose descriptor:
EC50
Effect concentration:
70.7 mg/L

Additional information

Short term toxicity to invertebrates - OECD 202 – EC50: 70.7 mg/l, 48h - (PARAD consortium, NOACK, 2010)


In an acute immobilization test with Daphnia magna(STRAUS) the effects of the test item (batch number:S700040031) were determined according to OECD 202 (2004) and Council Regulation (EC) No. 440/2008 Method C.2 (2008) from 2010 -05 -17 to 2010 -06-09 at Dr.U.Noack-Laboratorien, Käthe-Paulus-Straße 1, D-31157 Sarstedt, Germany.


The study was conducted under static conditions with 5 concentration levels of the test item ranging from 6.25 to 100 mg/L set up in a geometrical series with a dilution factor of 2 over a period of 48 h. 20 test organisms were exposed to each test concentration and the control.
All concentration levels and the control were analysed by LC-MS/MS after 0 h and 48 h. Details of the analytical results are given in part 4.2.2.
The recoveries in the fresh (0 h) and old media (48 h) were in the range of 96 to 107 % of the nominal values, demonstrating that concentrations of the test item were successfully maintained for the duration of the test. All effect values are given based on the nominal concentrations of the test item (Table 1). Water quality parameters of pH-value and dissolved oxygen concentration, measured at 0 and 48 h, were determined to be within the acceptable limits. The validity criteria of the test guideline were fulfilled.The EC50 after 48h was determined to be: 70.7 mg/l.


 


Evaluation: valid without restrictions, Klimisch-I , Key study


Assessment: The test item harmful to daphnia.

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