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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 December 2006 - 05 December 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
EU Comission Directive 88/302/EEC
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Test article name : LFC 2098
Chemservice sample code : AA26964
Declared purity (according to CoA) : 100%
Chemical class : Aminoester
Chemical formula : C23H31O4N3
Molecular weight : 413
Preparation date : 05/29/2006
Solubility in water : Insoluble
Density : Not available
Physical form at 20 °C : Solid (powder)
Colour : White
Storage conditions : Room temperature, protect from light
Safety precautions : Routinary hygienic procedures
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
The study was performed using aerobic activated sludge from ALSI (Alto Lambro Servizi ldrici) wastewater treatment plant treating mainly domestic wastewater. The sludge was washed by centrifugation and the supernatant liquid phase decanted. The washing procedure was not repeated, as any inhibiting substance effect has been found. A homogenized aliquot of the final sludge suspension has been weighted, thereafter dried and the ratio of wet to dry weight was calculated as 6.8%.

Based on this ratio, calculated amounts of wet sludge were suspended in the test water to get a concentration equivalent to 2.7 g dry material per litre. During holding, the sludge has been aerated at room temperature until use.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Post exposure observation period:
The respiration rate was measured after exactly three hours incubation with an oxygen electrode and was recorded every minute for ten minutes. The oxygen consumption (mgO2/L/minute) was determined from the linear part of the respiration curve.
Hardness:
Not measured
Test temperature:
20.8°C
pH:
7.76
Dissolved oxygen:
> 2.5 mg/L
Salinity:
Not measured
Conductivity:
Not measured
Nominal and measured concentrations:
Nominal : 1.0, 3.2, 10.0, 31.6 and 100.0 mg/L
Details on test conditions:
A deionised water stock solutions of 1 g/L was prepared by direct weighting. Given the low water solubility of the test item, the suspension was mixed by a magnetic stirrer for 24 hours to achieve the maximum solubilisation, then it was filtered by a filtering system. A properly volume of stock solution was added to the test water in each replicate to reach the desired concentration.

Under the same test conditions, 3,5-dichlorophenol was tested at three concentrations, 1.0, 15.0 and 30.0 mg/L.

A stock solution of 3.5-dichlorophenol at 0.5 g/L was prepared according to the description on OECD Guideline no. 209. 0.5 g of 3.5-dichlorophenol was dissolved in 10 ml 1M NaOH and diluted to about 30 ml with purified water. The final pH did not need any adjustment, being equal to 7.76.

One replicate per concentration and two for the negative control have been used.
Before the beginning of the test, 16 ml synthetic wastewater were added to the test beakers containing the previously stirred aqueous emulsion, obtained by adding an adequate volume of test item into tap water, to give a volume of 300 ml at each test concentration. Immediately prior to test start, 200 mL activated sludge inoculum with sludge concentration of 2.7 g/L (dry weight) has been added.

Two control beakers containing tap water and synthetic wastewater only, have been prepared at the beginning and at the end of test.

During the incubation period of exactly three hours the content of the test flasks were continuously aerated with compressed air (about 0.5-1 litre/minute).
The concentration of dissolved oxygen did not dropped below 2.5 mg/L during the incubation period and just prior to measurement of the respiratory rate the oxygen concentration was at least 6.5 mg/L.

The respiration rate was measured after exactly three hours incubation with an oxygen electrode and was recorded every minute for ten minutes. The oxygen consumption (mgO2/L/minute) was determined from the linear part of the respiration curve.

A deionised water stock solutions of 1 g/L was prepared by direct weighting. Given the low water solubility of the test item, the suspension was mixed by a magnetic stirrer for 24 hours to achieve the maximum solubilisation, then it was filtered by a filtering system. A properly volume of stock solution was added to the test water in each replicate to reach the desired concentration.

The pH was determined in the freshly prepared test medium of the control and at the highest test item concentration. At the end of the incubation period the pH values was determined in all test concentrations and controls.

The concentration of dissolved oxygen should not drop below 2.5 mg Oz/L.
The respiration rate of the two controls should not differ by more than 15%.
The 3-hours E050 of the test item 3,5—dichlorophenol should be in the range 5-30 mg/L.

The test item has been tested at five concentrations in a geometric series, namely 1.0, 3.2, 10.0, 31.6 and 100.0 mg/L.

Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Results with reference substance (positive control):
A full test to evaluate the toxicity of the test item LFC 2098 to activated sludge was performed in accordance with the EU Commission Directive 88/302/EEC.
The test item has been tested at five concentrations in a geometric series, namely 1.0, 3.2, 10.0, 31.6 and 100.0 mg/L.
The test concentrations were not measured during the test, therefore all reported results are related to the nominal concentrations of the test item.

The respiratory rate expressed as oxygen consumption has been measured after three hours of exposure. The measured oxygen concentrations in the test solutions and in the controls are reported in Table 1.
The reference substance 3,5-dichlorophenol has been tested at three nominal concentrations in parallel under the same test conditions to check the sludge activate batch. The measured oxygen concentrations in the test solutions are reported in Table 2.

The respiratory rates for the test concentrations and for the controls are reported in Table 3 together with the percentage inhibition of the respiration in the test concentrations compared to the controls.
The test meets the validity criteria indicated by the mentioned guidelines, since the concentration of dissolved oxygen did not drop below 2.5 mg 02/L, being always 2 6 mg/L just before the measurement of the respiration rates, and the respiration rate of the two controls differs for 3.8%.

The respiratory rate for the reference substance concentrations together with the percentage inhibition of the respiration in the test concentrations compared to the controls are reported in Table 4.
Reported statistics and error estimates:
For statistical analysis see appendix A appended to the attached background material

Table 1 - Oxygen consumption by activated sludge in presence of different concentrations of LPG 2098

Test item concentration (mg/L) Oxygen concentration (mg/L)
t1 t2 t3 t4 t5 t6 t7 t8 t9 t10
0 (control) 6.47 5.9 5.55 5.16 4.84 4.54 4 3.76 3.36 3.01
1 6.45 6.14 5.78 5.45 5.18 4.76 4.38 4.05 3.58 3.21
3.2 6.54 6.19 5.84 5.5 5.14 4.76 4.43 4.07 3.71 3.36
10 6.5 6.13 5.82 5.49 5.16 4.81 4.45 4.11 3.75 3.39
31.6 6.55 6.21 5.89 5.57 5.22 4.87 4.5 4.16 3.77 3.4
100 6.58 6.13 5.86 5.44 5.26 4.85 4.34 4.1 3.86 3.62
0 (control) 6.51 6.15 5.8 5.32 4.94 4.58 4.22 3.88 3.52 3.17

Table 2 -Oxygen consumption by activated sludge in presence of different concentrations of 3,5-dichlorophenol

Test item concentration (mg/L) Oxygen concentration (mg/L)
t1 t2 t3 t4 t5 t6 t7 t8 t9 t10
1 6.57 6.17 5.81 5.43 5.06 4.67 4.27 3.87 3.39 3.02
15 6.55 6.39 6.18 5.92 5.72 5.54 5.32 5.09 4.87 4.67
30 6.48 6.37 6.25 6.1 6.03 5.91 5.83 5.67 5.55 5.38

Table 3 - Respiratory rates for the test concentrations and for the controls and active sludge respiration inhibition caused by test item after 3-h contact

Test item concentration (mg/L) Rate of respiration (mg O2/L/h) Respiration inhibition (%)
0 (control) 20.8 N/A
1 19.4 4.9
3.2 19.1 6.4
10 18.7 8.3
31.6 18.9 7.4
100 17.8 12.7
0 (control) 20 N/A

Table 4 - Respiratory rates for the reference substance concentrations and active sludge respiration inhibition in comparison to controls after 3-h contact

Test item concentration (mg/L) Rate of respiration (mg O2/L/h) Respiration inhibition (%)
1 21.3 -4.4
15 11.3 44.6
30 6.6 67.6
Validity criteria fulfilled:
yes
Conclusions:
The test item LFC 2098 caused no significant toxic effect on aerobic treatment plant microorganisms after 3-hours contact, therefore the following endpoints have been assessed:

NOEC > 100 mg/L
LOEC > 100 mg/L
EC50 > 100 mg/L
Executive summary:

The toxicity of the test item LFC 2098 to activated sludge was assessed in a respiration inhibition study according to the EU Commission Directive 88/302/EEC, 0.11 (1988), and the OECD Guideline for Testing of Chemicals, No. 209 (1984).

For this purpose, aerobic activated sludge collected from the aeration tank of a civil wastewater treatment plant was submitted to a 3-hours incubation period in the presence of different concentrations of the test item under defined conditions.

The test item has been tested at five concentrations in a geometric series, namely 1.0, 3.2, 10.0, 31.6 and 100.0 mg/L. Besides the five concentrations, two negative controls were prepared.

To check the normal sensitivity of the batch of activate sludge 3,5-dichlorophenol has been used as a reference substance. For this purpose three nominal concentrations in the range 1-30 mg/L, has been tested.

The E050 of 3,5—dichlorophenol was found to be 18.4 mg/L, thus complying with the validity range indicated by the mentioned guidelines (5—30 mg/L).

The respiratory rate expressed as oxygen consumption has been measured after three hours of exposure.

The inhibitory effect of the test item is expressed as a percentage of the mean respiratory rate of two controls.

The test item is considered to be stable at the test conditions for a time corresponding to the exposure time. Therefore, all reported results are related to the nominal concentrations of the test item.

The respiration rate of the two controls was found to differ for 3.8%, thus complying with the validity criteria indicated by the mentioned guidelines (15%).

Based on the results, no significant toxic effects (3 15% inhibition) on sludge respiration rate has been found for the test item LFC 2098, therefore the E050 and the LOEC were estimated to be greater then 100 mg/L and the NOEC was determined to be 2100 mg/L.

At the beginning and the end of the test period, the pH values were in the range of 7.87-8.21 and the temperature was 20.8 °C.

Description of key information

Study conducted to recognised testing guidelines with GLP certification.

Key value for chemical safety assessment

Additional information

EC50 3 hour > 100 mg/L