Poly[oxy(methyl-1,2-ethanediyl)], .alpha.-[2-[[2,2-dimethyl-3-[(1-oxododecyl)oxy]propylidene]amino]methylethyl]-.omega.-[2-[[2,2-dimethyl-3-[(1-oxododecyl)oxy]propylidene]amino]methylethoxy]-

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005-03-08 to 2005-06-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

Oct. 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

July 31st 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Remarks:

Analysis of substance concentration technically not feasible because of the rapid hydrolysis of the test item and its degradation products.

Vehicle:

no

Details on test solutions:

Four experiments were performed.

First experiment:
As the solubility of the test substance lies below 100 mg/L, the water-accommodated fraction (WAF) was prepared for the test. This was done by weighing of the nominal load of 100 mg/L, adding the corresponding amount of demineralized water and shaking vigorously for 24 hours. The resulting solution was filtrated through 0.45 µm filter.

Second, third and fourth 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 demineralized 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.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
Unicellular freshwater green alga.

Genus, Species: Desmodesmus subspicatus
Strain: CHODAT
Family: Chlorophyceae
Order: Chlorococcales

The culture of Desmodesmus subspicatus was obtained in October 2004 by the "Collection of Alga", Institut für Pflanzenphysiologie of Universität Göttingen. The algae are kept as stock culture on solid agar at 8 °C.

ACCLIMATION
- Acclimation period: not applicable (after cultivation direct use for experiment)
- Culturing media and conditions: same as test conditions
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

post exposure observation not performed

Hardness:

not specified

Test temperature:

23 ± 2 °C

pH:

8.6 - 9.5

Dissolved oxygen:

no data

Salinity:

not applicable

Conductivity:

not specified

Nominal and measured concentrations:

nominal loading rates

first, second experiment: 100 mg/L
third experiment: 18, 46, 81, 220, 460 mg/L
fourth experiment: 2.2, 4.6, 10 mg/L

Details on test conditions:

The following experimental conditions apply to all experiments:

Vessels: screw cap cuvettes d=6mm
Duration: 72 hours
Temperature: 23 ± 2 °C
Lighting: 8000 ± 2000 Lux
Control: demineralized water with nutrient medium and algae
Treatments: test solution with nutrient medium and algae
Number of replicates: six replicates for the control, three replicates for each treatment

The mixture of the adjusted pre-culture and the concentrated nutrient solution (0.50 mL each, resulting in 1.00 mL) was added to the test vessels of each concentration. The test vessels were incubated open and stirred. Every 24 hours, the cell number/mL in each vessel was determined by photometric measurement at 440 nm. Before and after the test, the pH values of the control were measured.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EL50

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:

NOELR

Effect conc.:

18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

First experiment
An oily film on the surface was observed, which was assumed to be responsible for the inhibition, and potentially due to hydrolysis. Inhibitions of 15 % in growth rate, 33 % in AUC and 53 % in Yield were calculated.

Second experiment
The toxicity test was performed as in the first experiment. Inhibitions of 7 % in growth rate, 13 % in AUC and 29 % in Yield were calculated. No visible oily film on the surface could be observed.

Third experiment
The treatments showed inhibition values between 2 and 43 % in growth rate, 14 - 83 % in AUC and 11 - 92 % in Yield. The lowest concentration was determined as NOEC as no significant effect was observed. Probably caused by the test item properties, the deviation within the replicates was higher than in the control. To ensure the validity of the results, one additional experiment was performed with lower concentrations.

Fourth experiment
The treatments 10 and 2.2 mg/L didn't show a significant effect on the growth of the alga. The treatment 4.6 mg/L was not used for the evaluation as it became flocculent during the test, the alga were bound in lumps on the surface. Therefore the absorption could not be exactly measured.

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-lauroyloxy-propanal. But this product is not stable as well. 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 Sika Härter LJ were determined:

72h NOEC = 18 mg/L nominal concentration
72h LOEC = 46 mg/L nominal concentration
72h EC50 > 460 mg/L nominal concentration (concentration with 50% inhibition of the growth rate after 72 hours)
72h EC50 = 91 mg/L nominal concentration (concentration with 50% inhibition of the AUC after 72 hours)
72h EC50 = 85 mg/L nominal concentration (concentration with 50% inhibition of the Yield after 72 hours)

The reproducibility of the results is not very good as the biological effects are probably based on physical properties. This study was performed as worst-case as the limit of water solubility is calculated (following EPA) as lying well below 1 mg/L. As it can be seen from the thin oily film forming in the highest concentrations , the limit of solubility was definitely exceeded.

The validity criteria of the test guidelines were met.

Results with reference substance (positive control):

Reference control not performed.

Reported statistics and error estimates:

The estimation of the EC50s of the test item was accomplished using the software OriginTM. The calculated values for r resp. r2 are given in the graph. The data were evaluated using linear fit on a probability-logarithmic scale. The difference between the treatment 18 mg/L and the control can be considered as not significant (level of significance: 97.5 %) as the calculated t-values were smaller than the limit of significance. Therefore the concentration 18 mg/L is stated as NOEC. As the difference between the treatment 46 mg/L and the control can be considered as significant, the treatment 46 mg/L can be stated as LOEC.

Table 1: Growth Rate, AUC and Yield in the third experiment

Nominal Concentration in mg/L	Growth Rate [day-1]		AUC [Cell Concentration/mL*day]		Yield [Cell concentration/mL]
	Value	Mean (±SD)	Value	Mean (±SD)	Value	Mean (±SD)
0	1.95	1.91 (±0.03)	1770159	1597499 (±190811)	2134146	1917413 (±180563)
	1.92		1670607		1984818
	1.93		1767048		2003484
	1.92		1655052		1991040
	1.87		1399950		1704828
	1.87		1322175		1686162
18	1.80	1.87 (±0.06)	1060851	1368840 (±266948)	1362618	1708976 (±302669)
	1.91		1533723		1922598
	1.90		1511946		1841712
46	1.62	1.49 (±0.12)	631533	456280 (±152735)	802638	555832 (±215566)
	1.44		385764		460428
	1.40		351543		404430
81	1.64	1.68 (±0.08)	647088	719678 (±167773)	846192	974780 (±261349)
	1.62		600423		802638
	1.78		911523		1275510
220	1.44	1.58 (±0.12)	370209	50647 (±158752)	466650	740418 (±242259)
	1.63		612867		827526
	1.67		668865		927078
460	1.01	1.09 (±0.08)	267546	264435 (±23488)	124440	159698 (±37504)
	1.17		286212		199104
	1.09		239547		155550

SD = Standard Deviation

Table 2: Inhibition values in % for the third experiment

Nominal Concentration in mg/L	Mean Inhibition in %
	µ	AUC	Yield
0	0	0	0
18	2	14	11
46	22	71	71
81	12	55	49
220	17	66	61
460	43	83	92

Table 3: Growth Rate, AUC and Yield for the fourth experiment

Nominal Concentration in mg/L	Growth Rate [day-1]		AUC [Cell Concentration/mL*day]		Yield [Cell concentration/mL]
	Value	Mean (±SD)	Value	Mean (±SD)	Value	Mean (±SD)
0	1.94	1.95 (±0.01)	1739049	1773270 (±56343)	2109258	2142442 (±70357)
	1.94		1701717		2059482
	1.96		1854156		2239920
	1.96		1810602		2202588
	1.95		1795047		2159034
	1.94		1739049		2084370
2.2	1.95	1.94 (±0.01)	1717272	1721420 (±3592)	2140368	2094740 (±50292)
	1.93		1723494		2040816
	1.94		1723494		2103036
4.6*)	1.36	1.37 (±0.05)	335988	360876 (±92758)	360876	381616 (±64761)
	1.33		283101		329766
	1.43		463539		454206
10	1.94	1.94 (±0.01)	1711050	1764974 (±52917)	2078148	2119628 (±43702)
	1.94		1767048		2115480
	1.95		1816824		2165256

SD = Standard Deviation

Table 4: Inhibition values in % for the fourth experiment

Nominal Concentration in mg/L	Mean Inhibition in %
	µ	AUC	Yield
0	0	0	0
2.2	0.4	3	2
1.6	-	-	-
10	0.2	0.5	1

Validity criteria fulfilled:

yes

Conclusions:

Sika Härter LJ was tested for toxicity to aquatic freshwater algae in 72 h growth inhibition test according to OECD guideline 201 revealing an EC50 of > 460 mg/L and a NOEC of 18 mg/L.

Executive summary:

Sika Härter LJ was assessed in a GLP-compliant growth inhibition test according to 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 demineralized 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 the absorption of the cuvettes 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 15 % in growth rate, 33 % in AUC and 53 % 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 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 7 % in growth rate, 13 % in AUC and 29 % in Yield were calculated. No visible oily film on the surface could be observed. Based on the results of these experiments, the third experiment was performed in the same fashion using five concentrations between 18 and 460 mg/L. The treatments showed inhibition values between 2 and 43 % in growth rate, 14 - 83 % in AUC and 11 – 92 % in Yield. The lowest concentration was determined as NOEC as no significant effect was observed. Probably caused by the test item properties, the deviation within the replicates was higher than in the control. To ensure the validity of the results, one additional experiment was performed with lower concentrations.

The fourth experiment was performed in the same fashion using three concentrations between 2.2 and 10 mg/L. The treatments 10 and 2.2 mg/L didn’t show a significant effect on the growth of the alga. The treatment 4.6 mg/L was not used for the evaluation as it became flocculent during the test, the alga were bound in lumps on the surface. Therefore the absorption could not be exactly measured. 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 it can be seen from the tests with TOCdetermination, 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 72h EC50 based on the growth rate was determined greater than 460 mg/L. The overall NOEC was determined at 18 mg/L.

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 are definitely above the limit of water solubility and the “worst case” was tested. The effects were probably caused by physical effects of the unsolved parts of the test item.

Description of key information

Sika Härter LJ was tested for toxicity to aquatic freshwater algae in 72 h growth inhibition test according to OECD guideline 201 revealing an EC50 of > 460 mg/L and a NOEC of 18 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

18 mg/L

Additional information

Sika Härter LJ was assessed in an aquatic toxicity test to algae according to OECD guideline 201 and EU-method C.3. Four experiments were performed by preparing a water accomodated fraction. A nominal cocentration of 100 mg/L was tested in the first two experiments. In the main study five concentrations ranging from 18 to 460 mg/L were tested. In the last experiment three concentrations ranging from 2.2 to 10 mg/L were tested in order to ensure the validity of the main study. These two experiments were used for the evaluation of the results. The 72h EC50 based on the growth rate was determined greater than 460 mg/L. The overall NOEC was determined at 18 mg/L.