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Ecotoxicological information

Toxicity to microorganisms

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Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study primarily according to Robra test guideline with some minor deviations, non GLP, limited data on identity, purity, composition of test substance. No explanation on removal of outlier, no data on duration of exposure/incubation. No positive or negative control used. No reason for different no. of measurements between groups.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
other: Robra toxicity test to bacteria
Deviations:
yes
Remarks:
some minor differences in methods
Principles of method if other than guideline:
Deviations compared to guidance: low temperature batch culture, no data on density of bacteria used, washing procedure cell suspension shorter but at higher centrifugation. No actual concentration of stock determined. No data on exposure/incubation duration (guideline specifies 30 min at 20°C).
No positive or negative control used.
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Sample received from AkzoChemicals bv, Weert, The Netherlands
Name: dimethyl ether (DME)
Molecular formula: CH3OCH3
Molecular weight: 46
Cas no: 115-10-6
Purity: aerosol grade
Satbility: stable at test conditions
Solubility in water: 7.8 g/L (18C)
Pressure: 4.2 bar
Storage: at ambient conditions in the dark
CRL code: T89-8-1.1

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no
Details on test solutions:
100 ml of demineralized water was put in a 120 ml erlenmeyer flask closed with a miniert valve. Then 270 ml of test substance gas was injected through the valve into the erlenmeyer resulting in a nominal 5 g/L stock solution.

Test organisms

Test organisms (species):
Pseudomonas putida
Details on inoculum:
The P. putida strain DSM 50026 was obtained from the Deutsche Sammlung fur Mikroorganismen (Braunschweig, Germany)

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no

Test conditions

Hardness:
BOD water contained per litre demineralized water:
8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.3 mg Na2HPO4, 1.7 mg NH4Cl, 22.5 mg MgSO4(H2O)7, 27.5 mg CaCl2 and 0.25 mg FeCl3(H2O)6
Test temperature:
20°C
pH:
pH of maintenance medium and wash buffer: 7.5 and 7.2 respectively
Dissolved oxygen:
Oxygen is result parameter
Salinity:
BOD water contained per litre demineralized water :
8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.3 mg Na2HPO4, 1.7 mg NH4Cl, 22.5 mg MgSO4(H2O)7, 27.5 mg CaCl2 and 0.25 mg FeCl3(H2O)6
Nominal and measured concentrations:
Nominal concentrations:
100, 200, 400, 800, and 1600 mg/L
Details on test conditions:
Oxygen uptake rates were determined in a Biological Oxygen Monitor (BOM) (Yellow Springs Instruments, Ohio, USA) at 20°C. The BOM consists of a thermostated vessel (5 mL) with a magnetic stirrer and a Clark type oxygen electrode. The vessel is air closed tightly by this electrode. The reaction mixture in the BOM contained 0.1 mL of the washed cell suspension, 0.1 mL of a glucose solution (2.5 M), x ml of the test substance stock solution and 4.8-x mL of BOD water. To prevent the evaporation of the test substance from the mixture the test substance stock solution was injected directly into the closed vessel of the BOM containing the other constituents of the mixture.
Reference substance (positive control):
no
Remarks:
neither positive or negative

Results and discussion

Effect concentrations
Dose descriptor:
EC10
Effect conc.:
> 1 600 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
Measurements were carried out twice, but for the 100 mg/L dose, 4 times. No dose response could be observed. Under the test conditions, the highest attainable dose did not show a reduction > 10% compared to the control, based on nominal concentrations. In the 800 mg/L dose, an outlier was found (increased respiration rate), which was not considered in results.

Any other information on results incl. tables

Concentration

(mg/L)

% inhibition

Control

100

15

200

0

400

-3

800

-6

1600

9

Applicant's summary and conclusion

Validity criteria fulfilled:
not specified
Conclusions:
Based on nominal concentrations and a 10% inhibition of oxygen uptake at the highest tested concentration (1600 mg/L), the EC10 was estimated tp be > 1600 mg/L.
Executive summary:

In this toxicity test Pseudomonas putida cells were cultured under specified conditions. Subsequently the respiration rates of a washed cell suspension of these bacteria in the presence of various concentrations of the test substance were measured to determine the EC values. The toxicity test has been performed according to a modified ROBRA test guideline. The EC10 and the EC50 for Pseudomonas putida could not be determined due to the absence of oxygen respiration inhibition at the maximal attainable concentration tested (i.e. 1600 mg/litre). The EC10 can therefore be concluded > 1600 mg/L based on nominal concentration.