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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:
guideline study with acceptable restrictions
Remarks:
Study reliably conducted. Missing some detail expected of a GLP report. Purity information and analytical certificate present.
Qualifier:
according to
Guideline:
other: EEC L133 1988 p 118-122
Deviations:
yes
Remarks:
Alternative reference compound used.
Principles of method if other than guideline:
- Instead of 3,5-dichlorophenol, 2,4,5-trichlorophenol was used as a reference compound because 3,5 -dichlorophenol was not commercially available. 2,4,5 Trichlorophenol was chosen for its structural resemblance and is expected to give the same resultas 3,5-dichlorophenol.
GLP compliance:
yes
Analytical monitoring:
no
Details on sampling:
No Sampling
Vehicle:
not specified
Details on test solutions:
The stock solution of the test compound (25.2 g/dm3) was titrated to a neutral pH. No further information on stock preparation.
Test organisms (species):
activated sludge, domestic
Details on inoculum:
Secondary activated sludge was collected from the RZWI Nieuwgraaf in Duiven (1989.04.19). This activated sludge plant predominantly treats domestic waste water. 50 cm3 of synthetic sewage was added to 1 dm3 of the activated sludge. This was then aerated overnight at 20 °C and was kept aerated during the day of the test (1989.04.19).
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
30 min
Post exposure observation period:
No Data
Hardness:
No Data
Test temperature:
20ºc
pH:
See Results Table Below
Dissolved oxygen:
See oxygen consumption in results table below
Salinity:
No Data
Nominal and measured concentrations:
0, 20, 60, 180, 540, 1620 mg/dm³ (Nominal)
Details on test conditions:
See method discription below.
Reference substance (positive control):
yes
Remarks:
2,4,5-trichlorophenol
Duration:
30 min
Dose descriptor:
other: EC 20
Effect conc.:
100 other: mg/dm3
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
30 min
Dose descriptor:
EC50
Effect conc.:
1 600 other: mg/dm3
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
30 min
Dose descriptor:
EC10
Effect conc.:
60 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: calculated
Details on results:
The validity of the test is shown by two criteria. Firstly, the start control respiration rates are within 9% of each other and secondly, the EC50 of the reference compound is within the described range (5-30 mg/dm3).Pentaethylenehexamine does not show a significant inhibition of the respiration rate at the concentrations tested, and due to this result the log-normal distribution of the effects cannot calculated. The EC values are therefore estimated.
Results with reference substance (positive control):
See results table Below
Reported statistics and error estimates:
EC 20 and EC 50 results could be estimated. However due to the fact that only the higher concentrations in the testing range had effect on the respiration of the sludge not all of the EC concentrations could be calculated.
Validity criteria fulfilled:
yes
Conclusions:
Study is missing some information on methods, reference substance used was not the recommended one, and study is not carried out to OECD guidelines this does not validility however. A well distributed dose response curve was also not achieved. However the critical validity criteria (see above) were met and the reference substance did fall within the recommended limits. The study can therefore be considered reliable with the minor restrictions mentioned.
Executive summary:

Study was reliably conducted to EEC guidelines. Critical validity criteria were met. Substance ID was sufficient and the test was carried out to GLP.

Tetraethylenepentamine showed inhibition (6 -52%) of the respiration rate at the concentrations tested. A dose response curve could not be calculated. However the endpoints EC20 and EC50 were estimated.

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
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.
Qualifier:
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:
yes
Analytical monitoring:
no
Vehicle:
yes
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 (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 were cultured in a wuppertaler tank with a total volume of approximately 120 liter. Nitrifying bacteria are chemo-autotroph; they may use CO2 HCO3- 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
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
2 h
Post exposure observation period:
No post exposure period.
Hardness:
Not Reported
Test temperature:
During the exposition period, the vessels were held at room temperature, around 20 °C
pH:
Measured see results table below
Dissolved oxygen:
Measured to determine respiration activity see table below
Salinity:
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) for 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):
no
Duration:
2 h
Dose descriptor:
EC50
Effect conc.:
97.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% CL: 88.7 - 105.8 mg/L
Duration:
2 h
Dose descriptor:
EC10
Effect conc.:
46 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
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).

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.

Validity criteria fulfilled:
yes
Remarks:
with restrictions
Conclusions:
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.

Endpoint:
toxicity to microorganisms, other
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to
Guideline:
other: ISO/TC 147/SC5/WG 1
Version / remarks:
Inhibition of cell multiplication
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test substance was dissolved in the growth medium using concentrated stock solutions (10 and 25 g/dm³). these solutions were titrated to a neutral pH using a 1 M H2SO4 solution.
Test organisms (species):
Pseudomonas putida
Details on inoculum:
- Laboratory culture: Pseudomonas putida strain was obtained from the Agricultural University Wageningen , Dept. of Microbiology. The strain was maintained on yeast/glucose slants which contained per dm³ demiwater: 15 g agar, 5 g glucose and 1 g yeast extract.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
17 h
Test temperature:
25°C
Nominal and measured concentrations:
Nominal test substance concentrations: control, 31.3, 62.5, 125, 250, 500 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: test medium: 1.55 g/L K2HPO4, 0.85 g/L NaH2PO4, 0.5 g/L NH4Cl, 0.1 g MgSO4(H2O)7, 0.1 cm² trace element solution, 2 g/L glucose, 0.1 g/L yeast extract

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The turbidity was determined photometriclly (wavelenth 435 nm) after an incubation period of 17 h.
Duration:
17 h
Dose descriptor:
EC10
Effect conc.:
14 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Duration:
17 h
Dose descriptor:
EC50
Effect conc.:
186 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Reported statistics and error estimates:
Probit analysis with a SAS procedure.

Table 1: Percentage of inhibition of the growth of P. putida.

Test substance concentration [mg/L]

Inhibition of multiplication (H) [%]

31.3

15

62.5

33

125

43

250

52

500

63

1000

89

Description of key information

EC10 (2 h): 46 mg/L (respiration inhibition of nitrifying bacteria).

Key value for chemical safety assessment

EC50 for microorganisms:
97.3 mg/L
EC10 or NOEC for microorganisms:
46 mg/L

Additional information

The toxicity of the substance to STP microorganisms was tested in two different studies.

One of the available studies investigated the effect of the substance on the respiration activity of nitrifying bacteria. A guideline is not stated in the report. In the test nitrifying bacteria were exposed to nominal substance concentrations of 77, 140, 250 and 450 mg/L for two hours. The respiration activity was determined in an open respirometer according to Blok (1974) by measuring the concentration of dissolved oxygen. The respiration activity was compared to the respiration of nitrifying bacteria in an untreated control. An EC50 (2 h) of 97.3 mg/L (nominal) based on the respiration rate was determined. The determined EC10 (2 h) was 46 mg/L.

The second available study followed EEC guideline L133 (1988). The tested substance concentrations were 20, 60, 180, 540, 1620 mg/L (nominal). The respiration of activated sludge was followed for 30 minutes. The estimated EC50 (30 min) value was 1600 mg/L (nominal). 

A study investigating the growth inhibition of Pseudomonas putida was conducted according to guideline ISO/TC 147/SC5/WG 1. The inhibition of bacterial growth was tested at following test concentrations: 31.3, 62.5, 125, 250, 500, 1000 mg/L. After an incubation period of 17 h the growth inhibition was tetermined by measruing the turbitity of the test solutions. An EC50(17 h) of 186 mg/L (nominal) was determined.