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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:
other: Study well conducted. Missing some details expected of a GLP report. Purity information and analytical certificate present.
Qualifier:
according to guideline
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 (26.8 g/L) 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 wastewater. 50 mL of synthetic sewage was added to 1 L of the activated sludge. This was then aerated overnight at 20 °C and was kept aerated during the day of the test (1989.04.20).
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:
20 °C
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/L (Nominal)
Reference substance (positive control):
yes
Remarks:
2,4,5-trichlorophenol
Duration:
30 min
Dose descriptor:
other: EC20
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: Approx
Duration:
30 min
Dose descriptor:
EC50
Effect conc.:
800 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: Approx
Duration:
30 min
Dose descriptor:
EC10
Effect conc.:
42.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
The validity of the test is shown by two criteria. Firstly, the start control respiration rates are within 14% of each other and secondly, the EC50 of the reference compound is within the described range (5-30 mg/L).
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.

Method

The activated sludge concentration in the incubation vessels was 0.62 g dry weight/L. Prior to use the activated sludge was homogenized for two minutes in a lab blender 400. The homogenized activated sludge was incubated in a waterbath (100 Hz, 20°C) (Gallenkamp) for 30 minutes with various concentrations of the test compound and synthetic sewage. The stock solution of the test compound (25.0 g/L) was titrated to a neutral pH. The test compound concentrations of the reference compound were spaced by a factor two. The pH of the reaction mixture before the respiration measurement varied from 7.0 to 7.8. The respiration rates were measured in a Biological -Oxygen Monitor YSI 5300 (Yellow Springs Instruments, Ohio, USA). The BOM consisted of a thermostated vessel with a magnetic stirrer and an oxygen electrode. The vessel is tightly closed by the electrode to measure the oxygen depletion. The volume of the vessel was 10 ml and the temperature in the vessel was 20°C.

Validity criteria fulfilled:
yes
Conclusions:
Study is missing some information in methods, reference substance used was not the guideline reccomended chemical but was structurally related, and study is not carried out to OECD but to EEC guidelines. This does not affect the reliability of the study. A well distributed dose response curve was not achieved. However the critical validity criteria (see above) were met and the reference substance did fall within the reccomended 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. Triethylenetetramine showed inhibition (3-48%) of the respiration rates at the concentrations tested and due to this result the log-normal distribution of the effects cannot be used. However it can be concluded from the figure that the EC20 (the concentration with 20% inhibition of the respiration in comparison to the control) and EC50 of triethylenetetramine are about 60 and 800 mg/dm , respectively. The results are presented in detail in the table above. The test can be considered reliable with restrictions.

The EC10 was determined by the reviewer with the use of the test data provided in the report and toxcalc v 5.0 software to be 42.5 mg/L

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:
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.
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 26.76 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 werecultured 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)2SO4, 714 mg NaP4. 12 H2O and 91.6 ml NaOH (50%). The CO2 flow was 150 mL/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 O2/l.min or between 1.4 and 2.5 mg O2 /(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:
1.25, 6.25, 12.5, 25, 50 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):
no
Duration:
2 h
Dose descriptor:
EC50
Effect conc.:
15.7 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 12.5 - 18.9 mg/L
Duration:
2 h
Dose descriptor:
NOEC
Effect conc.:
1.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
At 1.3 mg/L respiration was not significantly inhibited. Almost complete inhibition was observed at 50 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 triethylenetetramine on the respiration activity of nitrifying bacteria was determined. The respiratation activity of bacteria exposed to the test substance during two 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 15.7 mg/L, with 95% confidence limits of 12.5 and 18.9 mg/L.

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
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 growht 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, 12.5, 25, 50, 100, 200, 400 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.:
5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Duration:
17 h
Dose descriptor:
EC50
Effect conc.:
137 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) [%]

12.5

6

25

25

50

41

100

51

200

61

400

73

Description of key information

EC50(2 h) of 15.7 mg/L

NOEC(2 h) was 1.3 mg/L

Key value for chemical safety assessment

EC50 for microorganisms:
15.7 mg/L
EC10 or NOEC for microorganisms:
1.3 mg/L

Additional information

The toxicity of the substance to STP microorganisms was tested in three 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 1.25, 6.25, 12.5, 25 and 50 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(2h) of 15.7 mg/L (nominal) based on the respiration rate was determined. The determined NOEC(2 h) was 1.3 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(30min) value was 1600 mg/L (nominal). Based on the raw data an EC10 (30 min) of 42.5 mg/L was recalculated using the software toxcalc v 5.0.

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: 12.5, 25, 50, 100, 200, 400 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 137 mg/L (nominal) was determined.