Registration Dossier

Ecotoxicological information

Toxicity to microorganisms

Currently viewing:

Administrative data

activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Test conducted to GLP. Sufficient substance information was provided (without analysis certificate). No clear guideline was used and no quality criteria were reported and no reference substance was tested. Reliable with restrictions mentioned.

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
no guideline followed
Principles of method if other than guideline:
Test Principles,
In the nitrification inhibition test, the effect of a test substance on the respiration activity of nitrifying bacteria is determined. Nitrifying bacteria are exposed to a range of concentrations of the test substance and the respiratation activity is compared to a control. Without test substance. From the relation between the concentration and the inhibition, the concentration causing an inhibition of 50% (EC50) may be determined.
Standard procedure/ test method used : Internal SOP: T16, T33, 47
GLP compliance:

Test material

Test material form:
other: liquid
Details on test material:
Name: tetraethylenepentamine (TEPA)
-Molecular Weight: 189
-Molecular Formular:H2N-(C2H4NH)4H (linear Fraction)
CAS reg. No:- 112-57-2
-Fraction Content:97.3%
-Identified impurities : Teta Tepa
-Appearance:Pale Yellow Liquid
-Stability: Stable at test conditions
-Solubility in water: Soluble at test concentrations 20ºc = miscible
-Vapour pressure:< 0.01 mbar
-Storage: 20-40ºc
CRL code: T88-10.2.9

Sampling and analysis

Analytical monitoring:

Test solutions

Details on test solutions:
A stock solution of 25 g/l was prepared by dissolving the test substance in demineralized water containing concentrated HCL.
The presence of the test substance in the medium caused a change of the pH, which was outside the range that can be supported by the test organism. Therefore, the pH was neutralized in the stock solution to a value between 7.0 and 7.3. The chosen test concentrations were prepared by dilution of the stock solution with the suspension of bacteria.

Test organisms

Test organisms (species):
other: Primarily nitrifying bacteria.
Details on inoculum:
Nitrifying bacteria were kept in a continuous culture, according to Blok (1981) and CRL Internal Standard Operation Procedure T 16. The bacteria werecultured in a wuppertaler tank with a total volume of approximately 120 liter. Nitrifying bacteria are chemo-autotroph; they may use CO2 HC03- as a carbon source.

The culture medium contained per liter 50 g (NH4)2S04, 714 mg Nap4. 12 H20 and 91.6 ml NaOH (50%). The C02 flow was 150ml/min.

The bacteria contained a high cell concentration of nitrifying bacteria and had a dry weight between 1.1 and 1.4 d.s./l. The maximum respiration activity varied between 1.6 and 3.5 mg O/l.min or between 1.4 and 2.5 mg 0 /(g d.s).min in March and September, respectively.

The optimal pH for the reactions is 8.2, with limits of 7.5 and 8.5.

origin : ARLA
dry weight: 1.44 g d.s./l

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
2 h
Post exposure observation period:
No post exposure period.

Test conditions

Not Reported
Test temperature:
During the exposition period, the vessels were held at room temperature, around 20°C
Measured see results table below
Dissolved oxygen:
Measured to determine respiration activity see table below
Not Reported
Nominal and measured concentrations:
77,140,250,450 mg/l (Nominal)
Details on test conditions:
Various concentrations of the test substance were added to nitrifying bacteria. The suspension was mixed (aerated) during 2 hours. After 2 hours the respiration activity was measured and compared to the activity in a control without test substance. Next, the pH was measured. During the exposition period, the vessels were held at room temperature, around 20°C. The respiration activity was measured at 20 ± 1°C.
Reference substance (positive control):

Results and discussion

Effect concentrations
2 h
Dose descriptor:
Effect conc.:
97.3 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
respiration rate
Remarks on result:
other: 95% CL
Details on results:
At 77 mg/l respiration was significantly inhibited. Almost complete inhibition was observed at 250 mg/l.
Results with reference substance (positive control):
No reference substance tested.
Reported statistics and error estimates:
For each test concentration, the respiration activity and the inhibition as compared to the control were calculated. The inhibition percentages were plotted against the test concentrations and the EC50 and its 95% confidence limits, if possible, were determined by Probit Analysis using a SAS procedure.
(SAS, 1985).

Any other information on results incl. tables

General Commments

No clear guideline was used and no quality criteria were reported and no reference substance was tested. Internal SOPs were referred to and procedure appears to be standardised and reliably conducted. GLP accreditation was given and suitable substance information (without analysis certificate) was given.

Applicant's summary and conclusion

Validity criteria fulfilled:
with restrictions
Although no official guideline was followed this study was carried out to laboratory specific SOP's and was given GLP accreditation. Lack of reference substance and quality criteria do restrict the reliability of this data. However the basic methodology for the study was sound and substance data was
sufficient. This study can therefore be considered reliable with the restrictions mentioned.
Executive summary:

Lacking guideline, Reference substance and clear quality criteria. However a standard method was followed from SOP's and the study was given GLP accreditation.

The effect of tetraethylenepentamine (TEPA) on the respiration activity of nitrifying bacteria was determined. The respiratation activity of bacteria exposed to the test substance during three hours was compared to a control without test substance and the inhibition was calculated. From the relation between the concentration and the inhibition percentages, the EC50 was determined. The EC50 was 97.3 mg TEPA per liter, with 95% confidence limits of 88.7 and 105.8 mg / l . The EC10 was 46 mg/l and was calculated with toxcalc.. The respiration activity was inhibited almost completely by a concentration of 250 mg TEPA per liter.