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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:
2004-10-08 to 2005-08-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
Deviations:
no
Principles of method if other than guideline:
NA
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Details on sampling:
NA
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- First experiment: As solubility lies below 100 mg/L, the water-accommodated fraction was prepared for the test. This was done by weighing of the nominal load of 100 mg/L, adding the corresponding amount of dilution water and shaking vigorously for 24 hours. The resulting solution was filtrated through 0.45 μm filter.

- Second experiment: The water-accommodated fraction was prepared for the test. This was done by weighing of the nominal load of 100 mg/L, adding the corresponding amount of dilution water and stirring slowly for 24 hours. The solution was left to stand for about 15 minutes. Then the lower phase was used for the test.

- Third experiment: The water-accommodated fraction was prepared for the test. This was done by weighing of each nominal load, adding the corresponding amount of dilution water and stirring slowly for 24 hours. The solutions were left to stand for about 15 minutes. Then the lower phase was used for the test.

- Fourth experiment: The water-accommodated fraction was prepared for the test. This was done by weighing of each nominal load, adding the corresponding amount of dilution water and stirring slowly for 24 hours. The solutions were left to stand for about 15 minutes. Then the lower phase was used for the test.

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Strain/clone: Berlin
- Source: Umweltbundesamt Berlin
- Age of parental stock: between 0 and 24 hours
- Feeding during test: no

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
24 hours before the start of the test, the adult animals were separated from the young. 23 hours later, the adults were caught with the help of a glass tube, and the new-born daphnia, aged between 0 and 23 hours, were sieved from the medium and immediately placed into a 250 mL-beaker containing dilution water. After a settling-in period of 30 minutes, animals which showed no apparent damage were used for the test.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
NA
Hardness:
250 mg CaCO3/L
Test temperature:
20 +/- 2 °C
pH:
7.8 - 8.3 (first experiment)
7.8 - 8.1 (second experiment)
7.7 - 8.2 (third and fourth experiment)
Dissolved oxygen:
8.3 - 9.1 mg/L (first experiment)
7.9 - 8.8 mg/L (second experiment)
7.3 - 8.9 mg/L (third experiment)
7.6 - 8.0 mg/L (fourth experiment)
Salinity:
NA
Nominal and measured concentrations:
First and Second Experiment:
Concentration (nominal): 100 mg/L

Third Experiment:
Concentration (nominal): 22, 46, 100, 220 and 460 mg/L

Fourth Experiment:
Concentration (nominal): 0.46, 1.0, 2.2, 4.6 and 10 mg/L

Details on test conditions:
TEST SYSTEM
- Test vessel: glass beakers (tall shape)
- Material, size, fill volume: glass, 50 mL, 20 mL
- Aeration: After preparation, the dilution water was aerated.
- Renewal rate of test solution (frequency/flow rate): after 24 hours
- No. of organisms per vessel: 5 daphnids
- No. of vessels per concentration (replicates): two (first and second experiment), four (third and fourth experiment)
- No. of vessels per control (replicates): two (first and second experiment), four (third and fourth experiment)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to Guideline

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Light intensity: none

EFFECT PARAMETERS MEASURED:
- After 24 and 48 hours, the immobilized daphnia were counted.
- The pH, the concentration of dissolved oxygen in the test vessels were measured at the beginning, after 24 hours (old and new test solution) and at the end of the test.


Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
59 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
115 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
FIRST EXPERIMENT
- Immobility: 100 mg/L: 80% and 100% after 24 hours and 48 hours, respectively
- Immobilisation of the daphnia was probably caused by the oily film which could be seen on the surface. During the shaking period for the preparation of the WAF, an emulsion was produced. This emulsion couldn’t be separated by membrane filtration and during the test, the unsolved part of the test item rose to the surface. The effect on the daphnia can be stated as a physical effect. Therefore the experiment was repeated, but the test solution was prepared by stirring instead of shaking.

SECOND EXPERIMENT
- Immobility: 100 mg/L: 20% after 48 hours
- The daphnia were immobilised caused by a thin oily film on the surface. Concerning the observed immobility values, a difference could be noticed between 24 and 48 hours. After 24 hours the medium was renewed. Based on these results, the third experiment was performed under the same conditions with five concentrations in a geometric series.
The TOC measurements gave results between 0 and 3.0 mg/L TOC in the test solutions 30 minutes and 24 hours after stirring. This is barely above the limit of detection. Therefore this non-specific analytical method is not sensitive enough for the determination of the content of the test item in the solutions.

THIRD EXPERIMENT
- Immobility: 45%, 25%, 65%, 45% and 75% at 22 mg/L, 46 mg/L, 100 mg/L, 220 mg/L and 460 mg/L, respectively, after 24 hours; 65%, 30%, 80%, 50% and 90% at 22 mg/L, 46 mg/L, 100 mg/L, 220 mg/L and 460 mg/L, respectively, after 48 hours

- In all treatments, a thin oily film on the surface could be observed. This film was probably responsible for the immobilisation. As in the lowest concentration immobilisation was observed, a further experiment with lower concentrations was performed.

FOURTH EXPERIMENT
- Immobility: 5% at 2.2 mg/L after 48 hours
- Less than 10 % immobilisation was noted at the concentration of 2.2 mg/L after 48 hours. As (according to OECD-Guideline) immobilisation of 5 % may also occur in the control without invalidating the test, this effect was stated as not significant.

CONTROL
- Mortality/behavioural abnormalities of control: none (in all four experiments)

Results with reference substance (positive control):
Biological Results of the Reference Item
Parameter Value 95%-confidence interval
NOEC 24 h 1.0 mg/L n.d.
24h EC50i 1.9 mg/L 1.8 – 2.0 mg/L
24h EC100i 3.0 mg/L n.d.
n. d. = not determinable
VALIDITY
The 24h-EC50i of K2Cr2O7 should lie between 0.6 and 2.1 mg/L.
The 24h-EC50i of K2Cr2O7 was determined as 1.9 mg/L.
Immobilisation in the controls may not exceed 10 %.
Immobilisation in the controls was 0 %.
Reported statistics and error estimates:
NA

The biological results are presented in the following table:

Table: Biological Results Test Item

Parameter

Value (Nominal Concentration)

95%-confidence interval

NOEC 24h

10 mg/L

n. d.

24h EC50i

115 mg/L

n. d.

24h EC100i

> 460 mg/L

n. d.

NOEC 48h

10 mg/L

n. d.

48h EC50i

59 mg/L

n. d.

48h EC100i

> 460 mg/L

n. d.

Validity criteria fulfilled:
yes
Conclusions:
Sika Hardener LH was tested for toxicity to aquatic freshwater invertebrates in a 48 h-static test according to EU method C.2.
The following results could be determined for the test item Sika Hardener LH (VP)(species: Daphnia magna).

24h NOEC = 10 mg/L nominal concentration
48h NOEC = 10 mg/L nominal concentration
24h EC50i = 115 mg/L nominal concentration
48h EC50i = 59 mg/L nominal concentration

Executive summary:

Sika Hardener LH was assessed in a short-term toxicity to aquatic invertebrates study according to EU-method C.2 and OECD guideline 202. Four experiments were performed. As the test item is poorly water soluble, the “water-accommodated fraction” (WAF) was used in the first experiment. This was done by weighing of the nominal load 100 mg/L, adding the corresponding amount of dilution water and shaking vigorously for 24 hours. The resulting solution was filtrated through 0.45 μm filters. Ten daphnia were exposed to the test item for 48 hours in a semi-static test system with medium renewal after 24 hours. 80 % of the daphnia were immobilised on the surface after 24 hours. After 48 hours, 100 % were immobilised. Immediately after the beginning of the test, an oily film on the surface was observed, which was assumed to be responsible for the immobilisation, and potentially due to hydrolysis.

In the second experiment, the WAF was prepared by weighing of the nominal load 100 mg/L, adding the corresponding amount of dilution water and stirring for 24 hours instead of shaking. The resulting solution was left to stand for 15 minutes, then the lower phase was taken for the test. Ten daphnia were exposed to the test item for 48 hours in a semi-static test system with medium renewal after 24 hours. After 24 hours, none of the daphnia were immobilised on the surface, after 48 hours 20 % of the daphnia were immobilised. The TOC of the test solution was measured after 30 minutes of stirring and after the complete stirring period of 24 hours. The TOC lay in a range between 0 and 3.0 mg/L.

Based on the results of these experiments, a third experiment was performed in the same fashion as the second experiment, using five concentrations between 22 and 460 mg/L. Twenty daphnia per treatment were exposed to the test item for 48 hours in a semi-static test system with medium renewal after 24 hours. After 24 and 48 hours, the immobilized daphnia were counted. The concentrations showed toxicity between 30 and 90 % immobilisation. No animals were immobilised in the control.

Therefore a further experiment was performed using five concentrations between 0.46 and 10 mg/L. Twenty daphnia per treatment were exposed to the test item for 48 hours in a semi-static test system with medium renewal after 24 hours. After 24 and 48 hours, the immobilised daphnia were counted. No animals were immobilised in the control and in the treatments.

As no analytical method exists for the test item and because of the immediate hydrolysis of the test item, it was planned to analyse the product of hydrolysis 2,2-Dimethyl-3-lauroyloxypropanal. But this product is not stable as well. As can be seen from the tests with TOC determination, the soluble part is too low to be determined as TOC. Therefore no analytical determination of the test item in the test solutions was possible and the biological results were based on the nominal concentrations. The following results could be determined for the test item Sika Hardener LH(species: Daphnia magna).

 

24h NOEC = 10 mg/L nominal concentration

48h NOEC = 10 mg/L nominal concentration

24h EC50i = 115 mg/L nominal concentration

48h EC50i = 59 mg/L nominal concentration

 

Because of the immediate hydrolysis, no water solubility could be determined. The calculated value (following EPA-calculation) is much smaller than 1 mg/L. The water solubility of the product of hydrolysis is below 1 mg/L. Therefore it can be stated that the determined effects definitely occur above the limit of water solubility and the “worst case” was tested. The effects were probably caused by physical effects of the undissolved parts of the test item.

Description of key information

SIKA Hardener LH was assessed in a short-term toxicity to aquatic invertebrates study according to EU method C.2 and OECD guideline 202. A water accommodated fraction (WAF) of 100 mg/L was prepared and nominal concentrations tested. Young Daphnia were exposed in a semi-static test to test item at a nominal concentration of 100 mg/L. The 48 h-EC50 was determined to be 59 mg/L and the 48 h-NOEC to be 10 mg/L.
24h NOEC = 10 mg/L nominal concentration
48h NOEC = 10 mg/L nominal concentration
24h EC50i = 115 mg/L nominal concentration
48h EC50i = 59 mg/L nominal concentration

 

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
59 mg/L

Additional information

Sika Hardener LH was assessed in a short-term toxicity to aquatic invertebrates study according to EU-method C.2 and OECD guideline 202. Four experiments were performed. As the test item is poorly water soluble, the “water-accommodated fraction” (WAF) was used in the first experiment. This was done by weighing of the nominal load 100 mg/L, adding the corresponding amount of dilution water and shaking vigorously for 24 hours. The resulting solution was filtrated through 0.45 μm filters. Ten daphnia were exposed to the test item for 48 hours in a semi-static test system with medium renewal after 24 hours. 80 % of the daphnia were immobilised on the surface after 24 hours. After 48 hours, 100 % were immobilised. Immediately after the beginning of the test, an oily film on the surface was observed, which was assumed to be responsible for the immobilisation, and potentially due to hydrolysis.

In the second experiment, the WAF was prepared by weighing of the nominal load 100 mg/L, adding the corresponding amount of dilution water and stirring for 24 hours instead of shaking. The resulting solution was left to stand for 15 minutes, then the lower phase was taken for the test. Ten daphnia were exposed to the test item for 48 hours in a semi-static test system with medium renewal after 24 hours. After 24 hours, none of the daphnia were immobilised on the surface, after 48 hours 20 % of the daphnia were immobilised. The TOC of the test solution was measured after 30 minutes of stirring and after the complete stirring period of 24 hours. The TOC lay in a range between 0 and 3.0 mg/L.

Based on the results of these experiments, a third experiment was performed in the same fashion as the second experiment, using five concentrations between 22 and 460 mg/L. Twenty daphnia per treatment were exposed to the test item for 48 hours in a semi-static test system with medium renewal after 24 hours. After 24 and 48 hours, the immobilized daphnia were counted. The concentrations showed toxicity between 30 and 90 % immobilisation. No animals were immobilised in the control.

Therefore a further experiment was performed using five concentrations between 0.46 and 10 mg/L. Twenty daphnia per treatment were exposed to the test item for 48 hours in a semi-static test system with medium renewal after 24 hours. After 24 and 48 hours, the immobilised daphnia were counted. No animals were immobilised in the control and in the treatments.

As no analytical method exists for the test item and because of the immediate hydrolysis of the test item, it was planned to analyse the product of hydrolysis 2,2-Dimethyl-3-lauroyloxypropanal. But this product is not stable as well. As can be seen from the tests with TOC determination, the soluble part is too low to be determined as TOC. Therefore no analytical determination of the test item in the test solutions was possible and the biological results were based on the nominal concentrations. The following results could be determined for the test item Sika Härter LH(species: Daphnia magna).

 

24h NOEC = 10 mg/L nominal concentration

48h NOEC = 10 mg/L nominal concentration

24h EC50i = 115 mg/L nominal concentration

48h EC50i = 59 mg/L nominal concentration

 

Because of the immediate hydrolysis, no water solubility could be determined. The calculated value (following EPA-calculation) is much smaller than 1 mg/L. The water solubility of the product of hydrolysis is below 1 mg/L. Therefore it can be stated that the determined effects definitely occur above the limit of water solubility and the “worst case” was tested. The effects were probably caused by physical effects of the undissolved parts of the test item.