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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to OECD 202 and GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: 25.0 - 50.0 - 100 - 200 - 400 mg/L
- Sampling method: All concentration levels, the pH control and the control after 0 h (fresh media) and 48 h (old media) were analytically verified via HPLC. For the old media, separate replicates without test organisms were prepared and incubated under test conditions.
- Sample storage conditions before analysis:All samples were stored at 6 ± 2 °C until start of analysis if necessary.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution (400 mg/L) was freshly prepared, dispersion treatment was agitation.
- Eluate: Dilution water
- Differential loading: 25.0 - 50.0 - 100 - 200 - 400 mg/L
- Controls: Dilution water without test item tested under the same conditions as the test groups

Test organisms

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


Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
No

Test conditions

Hardness:
Dilution water at test start: 274 (mg CaCO3/L)
Test temperature:
21.8 °C
pH:
Water Quality Parameters at Test Start (0 hours, Fresh Media)
(measured in one additional replicate per concentration level, pH control and control)

Nominal
test item
concentration
mg/L] pH-Value


400 (pH-control)* 7.14
400 3.47
200 4.89
100 6.47
50.0 6.88
25.0 7.11
Control 7.77

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

Nominal
test item
concentration
[mg/L] pH-Values
Replicates
1 2 3 4
400 (pH-control)* 7.03 7.11 7.06 7.13
400 3.38 3.36 3.29 3.29
200 5.51 5.33 5.49 5.88
100 7.20 6.95 6.92 6.89
50.0 7.27 7.14 7.21 7.21
25.0 7.38 7.35 7.31 7.30
Control 7.34 7.34 7.50 7.48
Dissolved oxygen:
Water Quality Parameters at Test Start (0 hours, Fresh Media)
(measured in one additional replicate per concentration level, pH control and control)

Nominal
test item
concentration
mg/L] O2-Concentration

[mg/L]
400 (pH-control)* 7.87
400 7.84
200 7.93
100 7.95
50.0 7.97
25.0 8.04
Control 8.16

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

Nominal
test item
concentration
[mg/L] O2 -Concentration [mg/L]
Replicates
1 2 3 4
400 (pH-control)* 6.45 6.93 7.07 7.12
400 8.03 8.09 7.99 7.97
200 7.72 7.80 7.65 6.53
100 7.69 7.52 7.82 7.62
50.0 7.75 7.69 7.70 7.72
25.0 7.84 7.80 7.78 7.69
Control 7.93 8.01 8.00 7.93
Salinity:
Freshwater
Nominal and measured concentrations:
Concentrations and Recovery Rates of the Test Item Pyrazolsäure-4, trocken

Sampling date 2009-08-11
Test start (0 h) 2009-08-13
Test end (48 h)
Start of analysis 2009-08-11 2009-08-13
Pyrazolsäure-4, trocken
Nominal
test item
concentration
[mg/L] Measured
concentration1)

[mg/L] RR


[%] Measured
concentration1)

[mg/L] RR


[%]
400 (pH-control)* 399 100 381 95
400 400 100 407 102
200 205 103 205 103
100 99.4 99 97.7 98
50.0 49.6 99 42.9 86
25.0 24.8 99 16.22) 65
Control < LOQ -- < LOQ --
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) :


TEST CONCENTRATIONS
- Range finding study
Immobilization Rates of the Preliminary Range Finding Test
(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
800 100 100 100 100 100 100
400 100 100 100 100 100 100
50 0 0 0 0 0 0
Control 0 0 0 0 0 0
800 (pH-control)* 30 30 30 100 100 100

Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrations
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
141 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: (100 - 200)
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: 1.50 (CI 1.39 - 1.63) mg/L
Reported statistics and error estimates:
The EC10-values after 24 and 48 h, the EC50-value after 24 h and the EC50 for the of the reference test 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.
Since only 0 % and 100 % immobilization occurred after 48 h, the EC50-value was determined by calculating the geometric mean of the highest concentration causing no immobilization and the lowest concentration causing 100 % immobilization. As confidence limits for the EC50 the highest concentration causing no immobility and the lowest concentration causing 100 % immobility were used, because only 0% and 100 % mortality occurred within the tested concentration range.


Any other information on results incl. tables

Percentage of Daphnids Incapable of Swimming after 24 and 48 h of Exposure

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

pyrazolic acid 4

IMMOBILIZATION [%]

Nominal

Test Item

Concentration

24 h

48 h

Replicates

Replicates

[mg/L]

1.

2.

3.

4.

MV

1.

2.

3.

4.

MV

400 (pH-control)*

  60

  40

  20

  20

  35

100

  60

100

  80

  85

400

100

100

100

100

100

100

100

100

100

100

200

  40

100

  80

  80

  75

100

100

100

100

100

100

    0

    0

    0

    0

    0

    0

    0

    0

    0

    0

    50.0

    0

    0

    0

    0

    0

    0

    0

    0

    0

    0

    25.0

    0

    0

    0

    0

    0

    0

    0

    0

    0

    0

Control

    0

    0

    0

    0

    0

    0

    0

    0

    0

    0

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In this study the test substance was found to be toxic to Daphnia magna after 48 h at concentrations of 200 mg/L (LOEC48 h) and higher. The NOEC after 48 hours was 100 mg/L. The EC50 (48 h) was determined to be 141 mg/L (CI: 100 - 200 mg/L). All effect values given are based on the nominal concentrations of the test item
Executive summary:

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

The study was conducted under static conditions with 5test item concentration levels ranging from 25.0 to 400 mg/L set up in a geometrical series with a dilution factor of 2over a period of 48 hours. 20 test organisms were exposed to each test concentration and the control.
All concentration levels and the control were analysed via HPLC after 0 h and 48 h.

The recoveries were in the range of 98 to 103 % of the nominal values at the biologically relevant test item concentrations of 100 to 400 mg/L, demonstrating that concentrations of the test substance
were successfully maintained for the duration of the test. All effect values given are based on the nominal concentrations of the test item (Table 1).
The w
ater quality parameters (pH-value and dissolved oxygen concentration), measured at 0 h and 48 h, were determined to be within the acceptable limits. The validity criteria of the test guideline were fulfilled.

Table 1:    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
pyrazolic acid 4

[mg/L]

Confidence Interval
p = 95%

[mg/L]

EC10

24

162

---

48

127

---

EC50

24

187

178 – 196

48

141

100 – 200

EC100

24

400

---

48

200

---

NOEC

48

100

---

LOEC

48

200

---