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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004-10-06 to 2005-08-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
1984 (Proposal for updating Guideline 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
- Method:
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 deionised 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 deionised 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 deionised 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 deionised 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):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Green algae
- Strain: CHODAT
- Source (laboratory, culture collection): “Collection of Alga” Institut für Pflanzenphysiologie of Universität Göttingen
- Method of cultivation: Four days before the start of each test, an aliquot of the stock culture containing a few cells was brought into 50 mL pre-culture medium and incubated for 96 hours. The resulting culture grew exponentially. After adjustment to a cell concentration of about 5*104/mL by photometric measurement and addition of pre-culture medium, the culture was usable for the test.


Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
NA
Hardness:
NA
Test temperature:
23 +/- 2 °C
pH:
8.6 - 9.6
Dissolved oxygen:
NA
Salinity:
NA
Nominal and measured concentrations:
First, second and third experiment:
Nominal concentrations: 100 mg/L

Fourth experiment:
Nominal concentrations: 0, 4.4, 10, 22, 46, 100, 220, 460 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: screw cap cuvettes d=6mm
- Fill volume: 4 mL
- Type: open
- Initial cells density: 6222 cells/mL (first, second and third experiment); 12444 cells/mL (fourth experiment)
- Control end cells density: 875228 cells/mL (first exp.); 1200846 cells/mL (second exp.); 1923635 cells/mL (third exp.); 1956819 cells/mL (fourth exp.)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

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

OTHER TEST CONDITIONS
- Adjustment of pH: none
- Light intensity and quality: 8000 +/- 2000 Lux

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: spectrophotometer (measured at the beginning (0 h) and after 24, 48 and 72 hours)
- Other: Temperature and light intensity of the control was measured at the beginning (0 h) and after 24, 48 and 72 hours; pH value of the control was measured at the beginning (0 h) and after 72 hours

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ~ 2.2 (fourth experiment)
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 460 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
46 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control: yes
- Colour differences: The measured absorptions were corrected by the colour of the test solutions.
- Observation of abnormalities (for algal test):
FIRST EXPERIMENT
- Inhibition in %: 21 (growth rate) at 102 mg/L
Inhibition of the algal growth 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 alga 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
- Inhibition in %: 0 (growth rate) at 100 mg/L
No oily film could be observed on the surface. Based on these results, the third experiment was performed under the same conditions as a limit test.
The TOC measurements gave results of 1.02 and 2.86 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
- Inhibition in %: 37 (growth rate) at 101 mg/L

FOURTH EXPERIMENT
- Inhibition in %: 0, -1, 0, 5, 5, 8 and 11 (growth rate) at 4.6, 10, 22, 46, 100, 220 and 460 mg/L, respectively
A thin oily film could be observed on the surface of the treatments 220 and 460 mg/L. The biological results were determined based on the results of this experiment.

Results with reference substance (positive control):
NA
Reported statistics and error estimates:
NA

Biological Results

The estimation of the EyC50 of the test item was accomplished using the software OriginTM. The calculated values for r resp. r2are given in the graph.

The data were evaluated using linear fit on a probability-logarithmic scale.

 Equation:                      Y = A + B * X

 The following values were determined:

Table: Biological Results of the Test Item

Parameter

Value

95%-confidence-interval

NOEC 72 h

22 mg/L

n. d.

LOEC 72 h

46 mg/L

n. d.

72h ErC50

> 460 mg/L

n. d.

72h EbC50

> 460 mg/L

n.d.

72h EyC50

440 mg/L

> 110 mg/L

72h-ErC100

> 460 mg/L

n. d.

72h-EbC100

> 460 mg/L

n. d.

72h-EyC100

> 460 mg/L

n. d.

n. d. = not detectable

Validity criteria fulfilled:
yes
Conclusions:
Sika Hardener LH was tested for toxicity to aquatic freshwater algae in a 72 h-growth inhibition test according to EU method C.3.
The following results could be determined for the test item Sika Härter LH (VP)(species: Desmodesmus subspicatus).

72h NOEC = 22 mg/L nominal concentration
72h LOEC = 46 mg/L nominal concentration
72h ErC50 > 460 mg/L nominal concentration
72h EbC50 > 460 mg/L nominal concentration
72h EyC50 = 440 mg/L nominal concentration

Executive summary:
Sika Hardener LH was assessed in an aquatic toxicty test to algae according EU-method C.3 and OECD guideline 201. 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 deionised water and shaking vigorously for 24 hours. The resulting solution was filtrated through 0.45 μm filters.

The treatments were used to incubate the unicellular freshwater green alga Desmodesmus subspicatus for a period of 72 hours. The cell concentration of each replicate was determined by measuring absorption at 440 nm every 24 hours with a spectral photometer. With these measured values, the number of cells was calculated (linear correlation between cell concentration and absorption given). Then the growth rate, the area under the growth curve (AUC1) and the yield2 could be determined. Inhibitions of 21 % in growth rate, 37 % in AUC and 66 % in yield were calculated. An oily film on the surface was observed, which was assumed to be responsible for the inhibition, and potentially due to hydrolysis.

In the second experiment, the WAF (water-accommodated fraction) 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. The toxicity test was performed as in the first experiment. Inhibitions of 0 % in growth rate, 1 % in AUC and 2 % in yield were calculated. No visible oily film on the surface was observed. The TOC of the test solution was measured 30 minutes and 24 hours after the begin of the stirring period. The TOC lay in a range between 1 and 2.9 mg/L.

Based on the results of these experiments, the third experiment was performed in the same fashion using the treatment 100 mg/L. The treatment showed inhibition values of 37 % in growth rate, 83 % in AUC and 87 % in yield. The deviation within the results of the second and the third experiment were probably caused by the test item properties. In order to reach reliable results, an additional experiment was performed using seven concentrations in a geometric series.

The fourth experiment was performed in the same fashion using seven concentrations ranging from 4.6 to 460 mg/L. Inhibitions between 0 and 11 % in growth rate, 0 - 25 % in AUC and 0 - 43 % in yield were calculated. In the treatments 220 and 460 mg/L, a thin oily film on the surface was observed. This experiment was used for the evaluation of the results.

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 results of the 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 for the test item Sika Hardener LH (VP) (species Desmodesmus subspicatus) were determined:

 

72h NOEC = 22 mg/L nominal concentration

72h LOEC = 46 mg/L nominal concentration

72h ErC503 > 460 mg/L nominal concentration

72h EbC504 > 460 mg/L nominal concentration

72h EyC505 = 440 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 occur definitely 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 an aquatic toxicty test to algae according EU-method C.3 and OECD guideline 201. A water accommodated fraction (WAF) of 100 mg/L was prepared and nominal concentrations of up to 100 mg/L were tested. An additional test was performed with seven concentrations ranging from 4.6 to 460 mg/L. This experiment was used for the evaluation of the results. The 72 h-EC50 based on growth rate was determined greater than 460 mg/L. The 72 h-EC50 based on yield was 440 mg/L. The overall NOEC was determined at 22.0 mg/L.

 

Key value for chemical safety assessment

EC50 for freshwater algae:
460 mg/L
EC10 or NOEC for freshwater algae:
22 mg/L

Additional information

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 deionised water and shaking vigorously for 24 hours. The resulting solution was filtrated through 0.45 μm filters.

The treatments were used to incubate the unicellular freshwater green alga Desmodesmus subspicatus for a period of 72 hours. The cell concentration of each replicate was determined by measuring absorption at 440 nm every 24 hours with a spectral photometer. With these measured values, the number of cells was calculated (linear correlation between cell concentration and absorption given). Then the growth rate, the area under the growth curve (AUC1) and the yield2 could be determined. Inhibitions of 21 % in growth rate, 37 % in AUC and 66 % in yield were calculated. An oily film on the surface was observed, which was assumed to be responsible for the inhibition, and potentially due to hydrolysis.

In the second experiment, the WAF (water-accommodated fraction) 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. The toxicity test was performed as in the first experiment. Inhibitions of 0 % in growth rate, 1 % in AUC and 2 % in yield were calculated. No visible oily film on the surface was observed. The TOC of the test solution was measured 30 minutes and 24 hours after the begin of the stirring period. The TOC lay in a range between 1 and 2.9 mg/L.

Based on the results of these experiments, the third experiment was performed in the same fashion using the treatment 100 mg/L. The treatment showed inhibition values of 37 % in growth rate, 83 % in AUC and 87 % in yield. The deviation within the results of the second and the third experiment were probably caused by the test item properties. In order to reach reliable results, an additional experiment was performed using seven concentrations in a geometric series.

The fourth experiment was performed in the same fashion using seven concentrations ranging from 4.6 to 460 mg/L. Inhibitions between 0 and 11 % in growth rate, 0 - 25 % in AUC and 0 - 43 % in yield were calculated. In the treatments 220 and 460 mg/L, a thin oily film on the surface was observed. This experiment was used for the evaluation of the results.

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 results of the 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 for the test item Sika Härter LH (VP) (species Desmodesmus subspicatus) were determined:

 

72h NOEC = 22 mg/L nominal concentration

72h LOEC = 46 mg/L nominal concentration

72h ErC503 > 460 mg/L nominal concentration

72h EbC504 > 460 mg/L nominal concentration

72h EyC505 = 440 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 occur definitely 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.