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Short-term toxicity to aquatic invertebrates

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Administrative data

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-12-16 to 2008-12-18
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Praepagen HY
- Molecular weight: 307 g/mol
- Physical state: liquid
- Analytical purity: 39.5 % (w/w, DIN EN ISO 2871-1)
- Lot/batch No.: DEG4036741
- Expiration date of the lot/batch: 2009-06-01
- Storage condition of test material: Room temperature, protected from moisture and light

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0.0625 - 0.125 - 0.250 - 0.500 - 1.00 mg/L
- Sampling method: The concentration levels 0.0625, 0.125 mg/L and the control were analysed directly. The samples of the concentrations levely 0.250 to 1.00 mg/L were diluted with dilution water prior to analysis.
- Sample storage conditions before analysis: All samples were stored at 6 +/- 2 °C if necessary.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The stock solutions (10.0 mg/L test substance were weighed out) were freshly prepared with dilution water prior to application and water renewal.
- Eluate: Dilution water
- Differential loading: 0.0625 - 0.125 - 0.250 - 0.500 - 1.00 mg/L
- Controls: Dilution water without test item tested under the same conditions as the test groups.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna STRAUS (Clone 5)
- Strain: Clone 5
- Source: Own breeding, source: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu)
- Age at study initiation (mean and range, SD): 2 - 24 h old
- Method of breeding: In glass vessels (2-3 L capacity) with approx. 1.8 L culture medium, at 20 +/- 2 °C, in an incubator, 16 h illumination, illumination strength max. 20 µE-m2*s-1. Culture medium Elendt M4 acc. to Elendt, modified to a total hardness of 160 to 180 mg CaCO3
- Feeding during test
- Food type: No feeding during the study



ACCLIMATION
- Acclimation period: At least 2 h in dilution water
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: No food
- Health during acclimation (any mortality observed): No mortality observed


Study design

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

Test conditions

Hardness:
Total hardness (Dilution water): 267 mg CaCO3/L (24 h); 263 mg CaCO3/L (48 h)
Test temperature:
Temperature: 19 - 20 °C
pH:
Water Parameters at 0 h and 24 h (New Media)
(measured in one additional replicate per concentration and control)
Nominal
Test Item Concentration
[mg/L] 0 h 24 h
pH-Value pH-Value
1.00 7.55 7.55
0.500 7.57 7.57
0.250 7.58 7.56
0.125 7.58 7.49
0.0625 7.56 7.51
Control 7.82 7.86

Water Parameters at 24 h (Old Media)
(measured in all replicates)
Nominal
Test Item Concentration
[mg/L] pH-Values
Replicates
1 2 3 4
1.00 7.38 7.45 7.44 7.44
0.500 7.40 7.39 7.41 7.41
0.250 7.39 7.41 7.40 7.40
0.125 7.42 7.41 7.40 7.39
0.0625 7.41 7.40 7.38 7.42
Control 7.35 7.36 7.34 7.33

Water Parameters at 48 h (Old Media)
(measured in all replicates)
Nominal
Test Item Concentration
[mg/L] pH-Values
Replicates
1 2 3 4
1.00 7.47 7.48 n.d. n.d.
0.500 7.47 7.47 7.48 7.47
0.250 7.46 7.48 7.49 7.48
0.125 7.46 7.47 7.49 7.46
0.0625 7.49 7.48 7.47 7.46
Control 7.45 7.52 7.52 7.48
Dissolved oxygen:
Water Parameters at 0 h and 24 h (New Media)
(measured in one additional replicate per concentration and control)
Nominal
Test Item Concentration
[mg/L] 0 h 24 h
O2-Concentration [mg/L] O2-Concentration [mg/L]
1.00 8.36 8.14
0.500 8.40 8.23
0.250 8.29 8.06
0.125 8.03 8.11
0.0625 8.30 8.15
Control 8.28 9.15

Water Parameters at 24 h (Old Media)
(measured in all replicates)
Nominal
Test Item Concentration
[mg/L] O2 -Concentration [mg/L]
Replicates
1 2 3 4
1.00 7.74 7.51 7.63 7.56
0.500 7.66 7.80 7.71 7.83
0.250 7.89 7.75 7.86 7.85
0.125 8.03 7.96 7.97 7.93
0.0625 7.95 7.88 8.01 7.92
Control 8.04 8.02 8.00 8.01

Water Parameters at 48 h (Old Media)
(measured in all replicates)
Nominal
Test Item Concentration
[mg/L] O2 -Concentration [mg/L]
Replicates
1 2 3 4
1.00 7.81 7.79 n.d. n.d.
0.500 8.10 8.09 7.96 7.93
0.250 8.07 8.00 8.15 8.17
0.125 7.94 8.20 8.13 8.12
0.0625 8.10 7.91 8.10 8.01
Control 8.02 8.17 8.13 8.10
Salinity:
Not measured, freshwater conditions
Nominal and measured concentrations:
nominal: 0.0625 - 0.125 - 0.250 - 0.500 - 1.00 mg/L
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, renewal after 24 h, semi-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: 656 µS/cm (24 h); 656 µS/cm (48 h)
- 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 determined in all groups after 24 and 48 h. A daphnid was considered to be immobile if it was not able to swim within 15 sec after gentle agitation of the test vessel.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: factor 2
- Range finding study
- Test concentrations: 1 - 10 - 100 mg/L
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.53 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: (0.52 - 0.55)
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.19 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mobility
Details on results:
-Mortality of control: 0 %
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:The tested concentration levels were visually clear i.e. completely dissolved throughout exposure.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50/LC50: 1.29 mg/L (CI 1.13 - 1.47 mg/L)
Reported statistics and error estimates:
EC100-values after 24 and 48 h were deduced directly from the dose-response-relationships without mathematical calculation.
EC10- and EC50-values were calculated by sigmoidal dose-response regression. Calculation of the confidence intervals for EC50 were carried out using standard procedures. The concentration effect relationships are shown graphically.

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)

Nominal
Test Item
Concentration

[mg/L]

IMMOBILIZATION [%]

24 h

48 h

Replicates

Replicates

1.

2.

3.

4.

MV

1.

2.

3.

4.

MV

1.00

80

60

100

100

85

100

100

100

100

100

0.500

  0

20

  20

    0

10

    0

  20

  40

  40

  25

0.250

  0

  0

    0

    0

  0

    0

    0

    0

    0

    0

0.125

  0

  0

    0

    0

  0

    0

    0

    0

    0

    0

0.0625

  0

  0

    0

    0

  0

    0

    0

    0

    0

    0

Control

  0

  0

    0

    0

  0

    0

    0

    0

    0

    0

Concentrations and Recovery Rates of Praepagen HY

Sampling

date

2008-12-16

0 h

new medium

2008-12-17

24 h

old medium

2008-12-17

24 h

new medium

2008-12-18

48 h

old medium

Start of

analysis

2008-12-16

2008-12-17

2008-12-17

2008-12-18

Nominal
Test Item
C
onc.

[mg/L]

Praepagen HY

without
daphnids

with
daphnids1)

without
daphnids2)

without daphnids

with
daphnids1)

without daphnids2)

Meas.

conc.

[mg/L]

RR

[%]

Meas.

conc.

[mg/L]

RR

[%]

Meas.

conc.

[mg/L]

RR

[%]

Meas.

conc.

[mg/L]

RR

[%]

Meas.

conc.

[mg/L]

RR

[%]

Meas.

conc.

[mg/L]

RR

[%]

1.00*

0.9783)

  98

1.00

100

1.01

101

0.990

  99

0.826

83

0.860

86

0.500*

0.4853)

  97

0.487

  97

0.509

102

0.525

105

0.389

78

0.354

71

0.250*

0.2423)

  97

0.266

106

0.264

106

0.242

  97

0.197

79

0.178

71

0.125

0.1203)

  96

0.135

108

0.1133)

  90

0.125

100

0.1103)

88

0.07193)

58

0.0625

0.06253)

100

0.0640

102

0.04533)

  72

0.06003)

  96

0.0437

70

0.04943)

79

Control

LOQ

LOQ

LOQ

LOQ

         Conc.               = Concentration
Meas. conc.
     = Measured concentration of test item, mean value of 2 injections

         RR                    = Recovery rate related to the nominal test item concentrations

         LOQ                 = Limit of quantification

         1)                       = Pooled samples from the test vessels per concentration level and the control

         2)                       = Separate replicates without daphnids incubated under test conditions

         3)                       = Repeated analysis (mean value of 2 replicates with 2 injections each)

         *                        = Biologically relevant concentration

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The recoveries of Praepagen HY in the fresh media (at 0 and 24 hours) at the biologically relevant test concentrations 0.250 to 1.00 mg/L were in the range of 97 to 105 % of the nominal values. The recovery rates of Praepagen HY of the old media (after 24 and 48 hours) at the biologically relevant test concentrations 0.250 to 1.00 mg/L determined in pooled samples from the test vessels were in the range of 78 to 106 % of the nominal values. The additionally prepared separate replicates at the biologically relevant test concentrations 0.250 to 1.00 mg/L (prepared and incubated without daphnids) gave recoveries in the range of 71 to 106 % of the test item in the old media (after 24 and 48 hours) which were comparable to the recoveries of the old media in the test vessels with daphnids.
As the recoveries in the old media with and without daphnids were comparable and only a small decrease of the recoveries in the old media after 24 h in comparison to those in the fresh media was observed, it can be concluded that sorption of the test item to the daphnids can be neglected for this Hydroxylethylquat.
Based on these facts a correction of the nominal concentrations due to the lowered recoveries in the old media after 24 h is not justified as the test item is not sorbed in an unacceptable degree to the glass wall or the daphnids and is partly dissolved and therefore available for exposure of the daphnids in the test medium. Nominal concentrations can be used for establishing of the EC-values on immobilization.
Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS) the effects of the test item Praepagen HY (batch number: DEG4036741) were determined according to OECD 202 (2004) from 2008-12-16 to 2008-12-18 at Dr.U.Noack-Laboratorien, D-31157 Sarstedt.

The study was conducted under semi-static conditions with 5 nominal test item concentrations ranging from 0.0625 to 1.00 mg/L set up in a geometric series with a dilution factor of 2 over a period of 48 hours. 20 test organisms were exposed to each test concentration and control.

Cationic surfactants are difficult to test as they exhibit different sorption mechanisms like classical van der Waals but also ionic and especially ion exchange. Cationic surfactants may sorb to negatively charged surfaces like glass and the surface of the daphnids but there is currently no mechanistic model to estimate the degree of sorption. Sorption may lead to low analytical recoveries of the test item during the test and it is therefore important to address sorption effects either to the daphnids and/or the glass surface of the test vessels.

The concentrations of Praepagen HY were determined at all concentration levels after 0 h and 24 h (fresh media) and after 24 h and 48 h (old media) via LC-MS/MS analysis. The recoveries of Praepagen HY in the fresh media (at 0 and 24 hours) at the biologically relevant test concentrations 0.250 to 1.00 mg/L were in the range of 97 to 105 % of the nominal values. The recovery rates of Praepagen HY of the old media (after 24 and 48 hours) at the biologically relevant test concentrations 0.250 to 1.00 mg/L determined in pooled samples from the test vessels were in the range of 78 to 106 % of the nominal values. The additionally prepared separate replicates at the biologically relevant test concentrations 0.250 to 1.00 mg/L (prepared and incubated without daphnia) gave recoveries in the range of 71 to 106 % of the test item in the old media (after 24 and 48 hours) which were comparable to the recoveries of the old media in the test vessels with daphnids.

As the recoveries in the old media with and without daphnids were comparable and only a small decrease of the recoveries in the old media after 24 h in comparison to those in the fresh media was observed, it can be concluded that sorption of the test item to the daphnids can be neglected for this Hydroxylethylquat.

Based on these facts a correction of the nominal concentrations due to the lowered recoveries in the old media after 24 h is not justified as the test item is not sorbed in an unacceptable degree to the glass wall or the daphnids and is partly dissolved and therefore available for exposure of the daphnids in the test medium. Nominal concentrations can be used for establishing of the EC-values on immobilization.

Water quality parameters of pH-value and dissolved oxygen concentration, measured at 0, 24 and 48 h, were determined to be within 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 Praepagen HY)

Test Duration


[h]

Nominal Concentration
of the Test Item
Praepagen HY

[mg/L]

Confidence Interval
p = 95%

[mg/L]

EC10

24

     0.500

---

48

     0.468

---

EC50

24

     0.737

0.663 – 0.818

48

     0.534

0.521 – 0.548

EC100

24

> 1.00

---

48

   1.00

---

NOEC

48

      0.250

---

LOEC

48

      0.500

---