reaction product of: saturated, monounsaturated and multiple unsaturated long-chained partly estrified alcohols of vegetable origin (Brassica napus L., Brassica rapa L., Helianthus annuus L., Glycine hispida, Gossypium hirsutum L., Cocos nucifera L., Elaeis guineensis) with O,O-diisobutyldithiophosphate and 2-ethylhexylamine and hydrogen peroxide

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 17,1998 to January 20,1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted in compliance with agreed test protocols following guidance.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Principles of method if other than guideline:

not applicable.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

428-630-5

EC Name:

-

Molecular formula:

Not applicable

IUPAC Name:

reaction product of Z-9-octadecen-1-ol and O,O-diisobutyl hydrogen dithiophosphate

Details on test material:

- Substance type: orange-yellow, clear liquid
- Physical state: orange-yellow, clear liquid
- Lot/batch No.: 12447
- Storage condition of test material: in a well closed container and protected from light at room temperature

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
none

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

The nominal concentration of the test group was 100 mg/l. 100 mg test substance and 1 1 test medium were stirred for 24 hours and centrifbged at 3,500 rpm for 10 minutes in order to separate the unsolved substance. The clear aqueous phase was used for the test. The amount of the test substance dissolved in test medium was checked by DOC analysis (Dissolved Organic Carbon) at the beginning of the test and after 72 hours.

Test solutions

Vehicle:

no

Details on test solutions:

The nominal concentration of the test group was 100 mg/l. 100 mg test substance and 1 1 test medium were stirred for 24 hours and centrifbged at 3,500 rpm for 10 minutes in order to separate the unsolved substance. The clear aqueous phase was used for the test.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: algae

- Source (laboratory, culture collection): SAG - Sammlung von Algenkulturen, University Giittingen

- Age of inoculum (at test initiation): no data

- Method of cultivation: The stock culture growth up in conical flasks containing nutrient solution. The algae were shaken permanently and were incubated at 22-24 oC with continuous lighting.n.

- Culturing media and conditions:

Culture medium:
NH4Cl 15.0 mg/l
MgCl2.6H2O 12.0 mg/l
CaCl2.2H2O 18.0 mg/l
MgSO4.7H2O 15.0 mg/l
KH2PO4 1.6 mg/l
FeCl3.6H2O 0.08 mg/l
Na2EDTA.2H2O 0.10 mg/l
H3BO3 0.185 mg/l
MnCl2.4H2O 0.415 mg/l
ZnCl2 0.003 mg/l
CoCl2.6H2O 0.0015 mg/l
CuCl2.2H2O 0.00001 mg/l
Na2MoO4.2H2O 0.007 mg/l
NaHCO3 50.0 mg/l


- Any deformed or abnormal cells observed: no data

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

none.

Test conditions

Hardness:

no data

Test temperature:

22.5-23.4 oC

pH:

6.9 - 8.4

Dissolved oxygen:

no data

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal concentratins: 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks, 100 ml
- Test volume: 30 ml
- Aeration: yes
- Initial cells density (test): 1.0 x 10^4 cells/mL
- Initial cells density (control): 1.00 x 10^4 cells/mL
- Control end cells density: 34.3x 10^4 cells/mL



GROWTH MEDIUM
- Standard medium used: no
- Detailed composition if non-standard medium was used:
NH4Cl 15.0 mg/l
MgCl2.6H2O 12.0 mg/l
CaCl2.2H2O 18.0 mg/l
MgSO4.7H2O 15.0 mg/l
KH2PO4 1.6 mg/l
FeCl3.6H2O 0.08 mg/l
Na2EDTA.2H2O 0.10 mg/l
H3BO3 0.185 mg/l
MnCl2.4H2O 0.415 mg/l
ZnCl2 0.003 mg/l
CoCl2.6H2O 0.0015 mg/l
CuCl2.2H2O 0.00001 mg/l
Na2MoO4.2H2O 0.007 mg/l
NaHCO3 50.0 mg/l

OTHER TEST CONDITIONS

- Adjustment of pH: no
- Photoperiod: continuous
- Salinity (for marine algae): not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: counting chamber


TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline: limit test

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Duration:

72 h

Dose descriptor:

IC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

none

Results with reference substance (positive control):

not applicable.

Reported statistics and error estimates:

none.

Any other information on results incl. tables

Determination of the Cell Concentration

 

The cell concentration in each flask was determined at 24, 48 and 72 hours after start of the test by counting the cells in a counting chamber. Four samples of each test vessel were counted. The average values were calculated. Results are shown in table 1.

 

Table1:Average values of cell concentration of the replicates at24,48and72hours after start of test

 

nominal concentration of test substance (mg/l)	24hours x10^4cells/ml	48hours x10^4cells/m	72hours x10^4cells/m
0	4.86	21.38	34.27
100	4.69	20.11	33.29

 

At the nomind concentration of100 mg/l test substanceno inhibition of cell growth was determined.

The cell concentrationinthe control cultures should have increased by a factor of at least16within three days. This condition was fulfilled.

 

Determination of the Concentration of the Test Substance

 

The amount of the test substance dissolved in test medium was checked by DOC analysis (Dissolved Organic Carbon) at the beginning of the test and after 72 hours. The result of analysis was compared with the carbon content of the test substance of 61.9 per cent (Table 2).

 

Table 2: DOC concentration during the test

	concentration ofDOC (correctedfor theDOCcontent in the control) mg/l	calculated concentration of test substance mg/l
0 hours	2.87	4.6
72hours	2.57	4.2

 

 

The concentration of test substance in test water, measured as DOC, was 4.6 mg/l at the start of the test. After 72 hours the concentration was 4.2 mg/l. 

 

Conclusion

 

The IC50 (72 hours) alga of the test substance is greater than 100 mg/l (nominal concentration).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The IC50 (72 hours) alga of the test substance is greater than 100 mg/l (nominal concentration).

Executive summary:

Methods:

A study was performed to assess the effect of the test material on the growth of the algaScenedesmus subspicatus. The method followed that described in Method C.3 of Commission Directive 92/69/EEC.

Procedure:

The nominal concentration of the test group was 100 mg/l. 100 mg test substance and 1L test medium were stirred for 24 hours and centrifuged at 3,500 rpm for 10 minutes in order to separate the unsolved substance. The clear aqueous phase was used for the test. Stock culture of algae was added to get an initial cell concentration of 10^4 cell/ml.

Results:

Determination of the Cell Concentration

The cell concentration in each flask was determined at 24, 48 and 72 hours after start of the test by counting the cells in a counting chamber. Four samples of each test vessel were counted. The average values were calculated. Results are shown in table 1.

 

Table1:Average values of cell concentration of the replicates at24,48and72hours after start of test

 

nominal concentration of test substance (mg/l)	24hours x10^4cells/ml	48hours x10^4cells/m	72hours x10^4cells/m
0	4.86	21.38	34.27
100	4.69	20.11	33.29

 

At the nominal concentration of100 mg/l test substanceno inhibition of cell growth was determined.

The cell concentrationinthe control cultures should have increased by a factor of at least16within three days. This condition was fulfilled.

 

Determination of the Concentration of the Test Substance

The amount of the test substance dissolved in test medium was checked by DOC analysis (Dissolved Organic Carbon) at the beginning of the test and after 72 hours. The result of analysis was compared with the carbon content of the test substance of 61.9 per cent (Table 2).

 

Table 2: DOC concentration during the test

	concentration ofDOC (correctedfor theDOCcontent in the control) mg/l	calculated concentration of test substance mg/l
0 hours	2.87	4.6
72hours	2.57	4.2

  

The concentration of test substance in test water, measured as DOC, was 4.6 mg/l at the start of the test. After 72 hours the concentration was 4.2 mg/l. 

 

Conclusion

The IC50 (72 hours) alga of the test substance is greater than 100 mg/l (nominal concentration).