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Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January 02, 2006 to February 09, 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Guideline study performed in compliance with GLP, available as unpublished report, no restrictions, fully adequate for assessment.
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Identity: TBzTD
- Aggregate state/physical form at room temperature: solid
- Color: white to yellowish
- Solubility in water: insoluble
- Sotrage: at room temperature at about 20°C, away from direct sunlight
- Batch: 2041 0003
- Expiration date: October 31, 2006
- Purity: > 90%
Analytical monitoring:
no
Details on sampling:
The start amount of substance was directly weighed into the designated test vessel.
Vehicle:
no
Details on test solutions:
Test substance solutions:
- 1000 mg/L
- 503.1 mg of the test item was transferred to vessel with 284 mL tap water
- mixed into tap water by ultrasonic treatment (15 min) (no solvents or emulsifiers)
- intense stirring for 24h at RT in the dark

Controls:
- 2 containing only tap water, synthetic wastewater and inoculum

Positive control:
- 3.5-dichlorophenol
- nominal conc tested were 5, 16 and 50 mg/L
- 0.5g of the substance was dissolved in 10 mL 1M NaOH and diluted to about 30 mL with purified water. Excess NaOH was neutralized with 0.5 mol/L H2SO4 to the point of incipient precipitation.
- this mixture was made up to one liter with purified water, pH was 7.7
- the final conc amounted to 500 mg/L
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- aerobic activated sludge from a wastewater treatment plant treating predominantly domestic wastewater: ARA Ergolz II, Füllinsdorf, Switzerland
- sludge was washed twice with tap water and by centrifugation
- supernatant liquid phase was decanted
- a homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated
- suspension with final conc of 3g dry material per liter was made
- during holding period (3 days) prior to use, the sludge was fed daily with 50 mL synthetic wastewater per liter and was kept at room temperature under continuous aeration until use.
- before use dry weight was determined again and diluted to 4g dry material per liter tap water
- pH was adjusted from 8.4 to 7.2

Synthetic waste water:
- peptone (16g)
- meat extract (11g)
- urea (3g)
- NaCl (0.7g)
- CaCl2.2H20 (0.4g)
- MgSO4.7H20 (0.2g)
- K2HPO4 (2.8g)
filled up to a final volume of 1 liter with deionized water
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Hardness:
no data
Test temperature:
19 °C (both at start and end)
pH:
- the pH of the activated sludge inoculum was adjusted from 8.4 to 7.2
Dissolved oxygen:
- did not drop below 2.5 mg/L during the incubation period
- just before measurements of the respiration rates DO conc were at least 8.2 mg/L
Salinity:
-
Nominal and measured concentrations:
Only nominal concentrations based on the original amount of substance weighed.
Details on test conditions:
- Test vessels: 2000 ML glass beakers
- Inoculum sludge conc: 4g/L dry weight
- sludge added in intervals of 15 min
- Incubation period: 3h
- Aeration: intense stirring on magnetic stirrers
Reference substance (positive control):
yes
Remarks:
3.5-dichlorophenol (purity 98.8%)
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/L
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
At the limit test concentration of 1000 mg/L, TBzTD was unevenly suspended in the test medium, and partially floated on the surface of the test medium. Thus, the limit test concentration was above the water solubility limit of TBzTD under the test conditions.

The test item TBzTD had no significant inhibitory effect «15%) on the respiration rate of activated sludge after the incubation period of three hours at the limit test concentration of 1000 mg/L. Based on the physical appearance of the test suspension, the saturation concentration (water solubility limit of TBzTD under the test conditions) was <1000 mg/L, and resulted in less than 15% inhibition after three hours contact time. Thus, the 3-hour NOEC of TBzTD to activated sludge microorganisms was at the limit of water solubility of TBzTD under the test conditions, or at 1000mg/L. This value might even be higher, but concentrations exceeding 1000 mg/L were not tested. The 3-hour EC20, EC50 and EC8O were clearly higher than the limit of water solubility of TBzTD under the test conditions, or higher than 1000 mg/L.

The oxygen consumption rates of the two controls (run at the start and at the end of the test) differed by 14% (guideline-recommended maximum variation: 15%).
Results with reference substance (positive control):
The 3-hour EC50 of the reference item 3,5-dichlorophenol (positive control) was calculated to be 17 mg/L (the 95% confidence limits were not calculable). The 3-hour EC50 is within the guideline-recommended range of 5 - 30 mg/L, thus confirming suitability of the activated sludge used.

Table: influence of TBzTD and 3.5 -dichlorophenol on the oxygen consumption of activated sludge

 Vessel no.  Test chemical  Nominal conc of test chem (mg/L)  Oxygen consumption rate (mgO2/L min-1)  Inhibition (%)  pH Values  Oxygen conc (mg O2/L)
 1  control  0  0.832    8.5 -8.6  8.4 -8.2
 10  control  0  0.716    8.4 -8.8  8.5 -8.9
 Mean deviation (%)     0.799 (13.9)       
 2  3.5 -DCP  5  0.691  10.8  8.4 -8.7  8.3 -8.7
3  3.5 -DCP  16  0.71  52.0  8.4 -8.9  8.9 -8.8
 4  3.5 -DCP  50  0.126  83.7  8.2 -8.7  8.9 -9.1
 5  TBzTD  1000  0.800  -3.3  8.3 -8.8  8.3 -8.5
Validity criteria fulfilled:
yes
Conclusions:
Thus, the 3-hour NOEC of TBzTD to activated sludge microorganisms was at the limit of water solubility of TBzTD under the test conditions, or at 1000mg/L.
Executive summary:

The inhibitory effect of the test item TBzTD on the respiration rate of aerobic wastewater microorganisms of activated sludge was investigated in a 3-hour respiration inhibition test according to the EU Commission Directive 88/302/EEC, Part C.11, and the OECD Guideline for Testing of Chemicals, No. 209.

In accordance with the EU Commission Directive 92/69/EEC (C.1, C.2, and C.3) and the OECD Series on Testing and Assessment No. 23 (Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000), a limit test was performed with one concentration of TBzTD of nominal 1000 mg/L, to demonstrate that the test item has no toxic effect on activated sludge microorganisms up to and including this concentration.

In addition, two controls and three different concentrations of the reference item 3,5-dichlorophenol (5, 16, and 50 mg/L) were tested in parallel. The results of these treatments confirmed suitability of the activated sludge and the method used.

The test item TBzTD had no significant inhibitory effect (<15%) on the respiration rate of activated sludge after the incubation period of three hours at the limit test concentration of 1000 mg/L. Based on the physical appearance of the test suspension, the saturation concentration (water solubility limit of TBzTD under the test conditions) was <1000 mg/L, and resulted in less than 15% inhibition after three hours contact time. Thus, the 3-hour NOEC of TBzTD to activated sludge microorganisms was at the limit of water solubility of TBzTD under the test conditions, or at 1000mg/L. This value might even be higher, but concentrations exceeding 1000 mg/L were not tested. The 3-hour EC20, EC50 and EC80 were clearly higher than the limit of water solubility of TBzTD under the test conditions, or higher than 1000 mg/L.

Description of key information

The 3-hour NOEC of TBzTD to activated sludge microorganisms was at the limit of water solubility of TBzTD under the test conditions, or at 1000mg/L. 

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L

Additional information

The inhibitory effect of the test item TBzTD on the respiration rate of aerobic wastewater microorganisms of activated sludge was investigated in a 3-hour respiration inhibition test according to the EU Commission Directive 88/302/EEC, Part C.11, and the OECD Guideline for Testing of Chemicals, No. 209.

In accordance with the EU Commission Directive 92/69/EEC (C.1, C.2, and C.3) and the OECD Series on Testing and Assessment No. 23 (Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000), a limit test was performed with one concentration of TBzTD of nominal 1000 mg/L, to demonstrate that the test item has no toxic effect on activated sludge microorganisms up to and including this concentration.

In addition, two controls and three different concentrations of the reference item 3,5-dichlorophenol (5, 16, and 50 mg/L) were tested in parallel. The results of these treatments confirmed suitability of the activated sludge and the method used.

The test item TBzTD had no significant inhibitory effect (<15%) on the respiration rate of activated sludge after the incubation period of three hours at the limit test concentration of 1000 mg/L. Based on the physical appearance of the test suspension, the saturation concentration (water solubility limit of TBzTD under the test conditions) was <1000 mg/L, and resulted in less than 15% inhibition after three hours contact time. Thus, the 3-hour NOEC of TBzTD to activated sludge microorganisms was at the limit of water solubility of TBzTD under the test conditions, or at 1000mg/L. This value might even be higher, but concentrations exceeding 1000 mg/L were not tested. The 3-hour EC20, EC50 and EC80 were clearly higher than the limit of water solubility of TBzTD under the test conditions, or higher than 1000 mg/L.

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