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 microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11/08/1993 to 11/23/1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP study conducted in compliance with agreed test protocols and guidance; restriction because the procedures were similar but different from OECD 209, such as test organisms exposed to test sample for prolonged exposure period.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

991

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Version / remarks:

The test was performed according to the guideline DIN 38412, part L8 (March 1991).

Deviations:

no

Principles of method if other than guideline:

not applicable.

GLP compliance:

yes

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

Test material form:

other: liquid

Details on test material:

- Physical state: liquid
- Storage condition of test material: room temperature, protected from light

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:

To reach a maximum nominal concentration of 10 g/l in the test, a stock emulsion containing 12.5 g/l of the test article was p~pared.A stock emulsion containing 1 g/l of the test article was prepared in the presence of the emulgator Tween 80. Following stirring overnight at room temperature, homogenous emulsions were obtained. These were filte d through paper filters and samples were analysed by DOC measurement. The Doc was determined as the result from subtracting the values for TOC (total organic carbon) and IC (inorganic carbon). Analysis was performed at least in duplicate. The DOC content of the stock emulsion containing 1 g/l of the test article in the presence of Tween 80 (100 mg/l) was corrected by the DOC of purified water, containing Tween 80 (100 mg/l).

Test solutions

Vehicle:

no

Details on test solutions:

The main test was performed with a series of five nominal concentrations of the two stock emulsions prepared with the test article, each concentration being tested in triplicate. The highest concentration represented nominal concentrations of 10 g/l and the lowest of 50 mg/l of the test article. The nominal test concentrations were obtained by adding appropriate volumes of the test article stock emulsions, containing 12.5 g/l and 1 g/l (in the presence of Tween 80, 100 mg/l) of the test article.

Test organisms

Test organisms (species):

Pseudomonas putida

Details on inoculum:

Pseudomonas purida, DSM 50026 (lyophilisate, obtained March 1993)

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

16 h

Post exposure observation period:

none

Test conditions

Hardness:

no data

Test temperature:

21 +/- 1°C

pH:

The pH-value of the cultures containing the highest nominal concentrations of the test article (10000mg/l) was 7.3.

Dissolved oxygen:

no data

Salinity:

not applicable

Nominal and measured concentrations:

Aqueous stock emulsion containing 1.0 g/l (nominal) test article and with TWEEN 80 (100 m/l): 800, 400, 200, 100, 50 mg/l
Aqueous stock emulsion containing 12.5 g/l (nominal) test article: 10, 5.0, 2.5, 1225, 0.6 g/l
Aqueous stock emulsion containing 0.1 g/l (nominal) test article: 0.05, 0.1, 0.2, 0.4, 0.8 g/l

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 ml Erlenmeyer flasks
- No. of organisms per vessel: no data
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 3
- Biomass loading rate: no data

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: in the dark


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol (97% pure)

Results and discussion

Effect concentrations

Duration:

16 h

Dose descriptor:

IC50

Effect conc.:

> 10 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Details on results:

Control cultures were treated accordingly with the exception that they contained appropriate volumes of purified water (or purified water in the presence of 100 mg/l of the emulsifier) instead of the test article. The cell multiplication of the concurrent control cultures met validity criteria

In order to confm the functional integrity of the test system, the reference substance 3,5-dichlorophenol was used in control tests performed on a regular basis. The EC50 and EC10 values obtained were comparable to those stated in DIN 38412 L8. Thus, in terms of sensitivity, the experimental system meets the requirements of the guideline.

With an aqueous emulsion of the test article in the absence of Tween 80, values of EC10 and EC50 could not be calculated, because maximum inhibition of 24% was obtained with the highest nominal concentration tested (10.0 g/l).

The EC10 was determined to be 0.08 g/l, and the EC50 was determined to be 0.15 g/l of the test article, when emulsified in the presence of Tween 80, which supported solubility / bioavailability of test article.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50(16h) = 17.3 mg/L; EC10(16h) = 6.5 mg/L;
According to the guideline DIN 38412 L8, the mpective values for this substance obtained in a series of interlaboratory comparison experiments were 21.4 +/- 4.91 mg/l and 13.7 +/- 4.36 mg/l.

Reported statistics and error estimates:

none

Any other information on results incl. tables

The turbidity of the test culturesforeach concentration of the test articleandthat of the control cultureswithin the preliminary test arepresented in Table1.Inhibitory effects were observed at nominal concentrations >100mg/l.

 

The turbidity of the test culturesforeach concentration ofthetest articleandthat of the control cultureswithin the main test arepresented in Table2.

 

Thecorresponding values fortheinhibition of cell multiplication compiled in Table 3.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

With an aqueous emulsion of the test article in the absence of Tween 80, it may be stated that the EC50 is achieved at a concentration > 10.0 g/l.

Executive summary:

Guidance

Theaimofthisstudy was toassessthe potential inhibitory effect of the test article on thegrowthofPseudonroMs putida:MIGULA (DSM50026). Test wasperformed according tothe guideline DIN38412,partL8 (March 1991).

 

Material and Method

Adescending range of concentrations of the test article Folpan was assayed for potential inhibitory effects on thegrowthof Pseudomonasputida.Accordingtothe recommendations of the "Kommission Bewertung Wassergef Wnder Stoffe", stock emulsions of12.5g/land 100 mg/l were prepared. Additionally,astock emulsion of 1.0g/lwas prepared in the presence ofanemulsifier Tween80, 100mg/l).

 

 

RESULTS

Within the preliminary test, inhibitory effects were observed at or above nominal concentrations of5.0g/lusing the filtered phases of the stock emulsions of12.5g/l.However, inhibitory effects were observed at or above concentrations of200mg/lwhen using a stock solution of1 g/lin the presence of the emulsifier Tween80.

Negative inhibition values indicateanenhancement ofcellmultiplication as compared to control cultures, whilst positive values indicateanimbibition of growth relativetocontrol cultures.

 

Theresults of thepreliminarytest were confirmed by the main test,performedwith descending concentrations ofthefiltered phases of thestockemulsions containing nominal concentrations of 12.5g/land1g/lin the presence of the emulsifier Tween80.A maximum inhibition of 24% was obtained with the highest nominal concentrationtested(10.0 g/l) whereas clear inhibitory effects were observed atorabove concentrations of0.05mg/l when using astocksolution of 1g/l,prepared inthe presence of the emulsifier Tween80.This effect demonstrates the influence of Tween80,i.e. in supporting solubility bioavailability of potential toxic compounds.

The EC10wasdetermined to be 0.04g/l,and the EC50wasdetermined to be 0.15g/lof the test article, when emulsified in the presence of Tween80.

Withanaqueous emulsion of the test article in the absence of Tween80,it maybestated that the EC50 is achieved ataconcentration > 10.0g/l.

 

The pH-value of the culture containing the highest nominal concentration of the test article(10000mg/l) was7.3at initiation of the test.

 

Aqueous stock emulsions of12.5g/land 1g/l(in presence of the emulsifier Tween 80, 100mg/l)of the test article contained values of 920 mgC/land67mgC/l, respectively. The DOC of the stock emulsion containing Tween 80 was correctedforthe DOC of purified water, containing Tween 80(100mg/l).

 

The mean biomass of the strain Pseudomonasputida,determined from the turbidity of theculturesat the beginning and end of the 16 +/-1 h- incubation periodofthe maintest,increased by more than 100-fold from 1.9to194. Therefore, the criterionofvalidityof thistest was fulfilled.

 

Within the last representative referencetest(performed between the 21St and 23dofJuly,

1993) with 3-dichlorophenol,testedunder the same conditions, pronounced inhibitory effects on cellgrowthwas documented, whichresultedinEC50andEC10values of 17.3 and6.5mg/l,respectively.Accordingtothe guideline DIN 38412L8,the respective values forthis substanceobtained in a seriesof interlaboratorycomparison experiments were 21.4 +/-4.91 mg/ and 13.7 +/-4.36 mg/l.

 

CONCLUSIONS

With an aqueous emulsion of the test article in the absence of Tween80,it maybestated that the EC50 is achieved ataconcentration > 10.0g/l.