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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:
28/5/2009-29/07/2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
According to the OECD Guideline 209 and it is GLP.
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
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
The final nominal concentrations tested were 10, 32, 100, 320 and 1000 mg/L.
Based on the results of a pre-test, no stock solution could be prepared due to the insufficient water solubility of the test item under the pre-test conditions.
Therefore, test item amounts of 5.09, 16.24, 50.69, 160.34 and 500.48 mg were weighed onto glass slides by means of an analytical balance and transferred into the designated test vessels. Then, 284 mL tap water was added to each vessel.
The test item was mixed into the tap water by intense stirring for 40 minutes at room temperature to dissolve a maximum amount of the test item and/or disperse it as homogeneously as possible. No emulsifiers or solvents were used.
After stirring, the appearance of the test media was evaluated, and the test concentrations ≥100 mg/L were clearly above the water solubility limit of the test item under the test conditions. Then, 16 mL synthetic sewage feed and 200 mL activated sludge inoculum were added.
Additionally, two controls containing only tap water, synthetic sewage feed and inoculum were tested in parallel to the five concentrations of the test item under identical test conditions.
The reference item 3,5-dichlorophenol was tested in parallel under identical test conditions, and functioned as a positive control. The nominal concentrations tested were 5, 16 and 50 mg/L. A stock solution of 3,5-dichlorophenol was prepared according to the test guidelines: 0.5 g 3,5-dichlorophenol was dissolved in 10 mL 1 M 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. Thereafter, the mixture was made up to one liter with purified water. The pH was 7.7. The final concentration of 3,5-dichlorophenol amounted to 500 mg/L. Aliquots of this 3,5-dichlorophenol stock solution were mixed with synthetic sewage feed and tap water in the respective test vessels to obtain the desired concentrations.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
The study was performed with aerobic activated sludge from a wastewater treatment plant treating predominantly domestic water (ARA Ergolz II, Füllinsdorf / Switzerland).

The sludge was washed twice with tap water by centrifugation and the 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.
Based on this ratio, an aliquot of washed sludge was suspended in tap water to obtain a concentration equivalent to 3 g dry material per liter. During the holding period of three days prior to use, the sludge was fed daily with 50 mL synthetic sewage feed* per liter and was kept at room temperature under continuous aeration until use. Before use, the dry weight of the activated sludge was measured again in the inoculum used for the test. The pH of the activated sludge inoculum was 7.5.

* Synthetic sewage feed (filled up to a final volume of 1 liter with deionized water):
16 g peptone
11 g meat extract
3 g urea
0.7 g NaCl
0.4 g CaCl2 × 2H2O
0.2 g MgSO4 × 7H2O
2.8 g K2HPO4
Test type:
not specified
Water media type:
not specified
Limit test:
no
Total exposure duration:
3 h
pH:
8.4
Dissolved oxygen:
9.9 mg O2/L
Nominal and measured concentrations:
Nominal: 10, 32, 100, 320 and 1000 mg/L
Details on test conditions:
The test was performed in 2000-mL glass beakers. The test vessels were labeled with the necessary information to ensure unmistakable identification.
At the start of the test (after the stirring period of 40 minutes), synthetic sewage feed and activated sludge inoculum (see Sections 3.3 and 3.4.1) were added. The inoculum had a sludge concentration of 2.3 g/L dry weight (corresponding to about 0.9 g dry material per liter test medium). The sludge was added in time intervals of 15 minutes (an arbitrary but convenient interval) first to a control, secondly to the test solutions of the reference item, thirdly to the test solutions/dispersions of the test item, and finally to the second control.
During the incubation period of 3 hours all test media and the controls were continuously aerated by intense stirring on magnetic stirrers to avoid possible foaming and/or stripping of the test item. The concentration of dissolved oxygen did not drop below 2.5 mg/L during the incubation period. Just before measurement of the respiration rates the dissolved oxygen concentrations were at least 8.7 mg/L. The temperature in the test media, measured in one control, was 21 °C at the start and at the end of the incubation period.
Reference substance (positive control):
yes
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
>= 1 000 mg/L
Details on results:
At the test concentrations ≥100 mg/L, the test item was not completely dissolved in the test media. Thus, the test concentrations ≥100 mg/L were clearly above the water solubility limit of the test item under the test conditions.
Up to and including the highest test concentration of 1000 mg/L, the test item had no significant inhibitory effect (≤10%) on the respiration rate of activated sludge after the incubation period of three hours. Based on the physical appearance of the test solutions/dispersions, the saturation concentration (water solubility limit of the test item under the test conditions) was <100 mg/L and resulted in less than 10% inhibition after three hours contact time. Thus, the 3-hour NOEC of the test item to activated sludge microorganisms was at the water solubility limit of the test item under the test conditions, or at 1000 mg/L. The 3-hour EC20, EC50 and EC80 were clearly higher than the water solubility limit of the test item 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 9% (guideline-recommendation: the two control respiration rates should be within 15% of each other).
Results with reference substance (positive control):
The 3-hour EC50 of the reference item 3,5-dichlorophenol (positive control) was calculated to be 18 mg/L (the 95% confidence limits could not be calculated due to mathematical reasons). The 3-hour EC50 is within the guideline-recommended range of 5-30 mg/L, confirming suitability of the activated sludge used.

Vessel nº Nominal concentration of test chemical (mg/L) Oxygen consumption rate (mg O2 L-1 min -1) Inhibition (%)
Control
A 0 1,45
B 0 1,583
Mean Deviation (%) 1,517
9,2
Test item
5 10 1,56 -2,9
6 32 1,649 -8,7
7 100 1,833 -20,9
8 320 1,744 -15
9 1000 2,048 -35
Reference item
1 5 1,478 2,6
2 16 0,8 47,3
3 50 0,185 87,8
Validity criteria fulfilled:
yes
Conclusions:
The 3-hour NOEC of the test item to activated sludge microorganism was at the water solubility limit of the substance under the test conditions, or at 1000 mg/L. The 3 -hour EC20, EC50 and EC80 were clearly higher than the water solubility limit of the test item under the test conditions, or higher than 1000 mg/L.
Executive summary:

The inhibitory effect of the test item 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, Commission Regulation (EC) No. 440/2008, Part C.11 and the OECD Guideline for Testing of Chemicals, No. 209. The study was performed under GLP compliance.

The following nominal concentrations of the substance were tested: 10, 32, 100, 320 and 1000 mg/L. In addition, two controls and three different concnetrations of the reference item 3,5 -dichlorophenol (5, 16 and 50 mg/L) were tested in parallel. The results of these treatments confirmed the suitability of the activated sludge and the method used.

Up tp and including the highest test concentration of 1000 mg/L, the test item had no significant inhibitory effect ( ≤10%) on the respiration rate of activated sludge after the incubation period of three hours. Based on the physical appearance of the test solution/dispersions, the saturation concentration (water solubility limit of the substance under the test conditions) was <100mg/L and resulted in less than 10% inhibition after three hours contact time. Thus, the 3 -hour NOEC of the test item to activated sludge microorganism was at the water solubility limit of the substance under the test conditions, or at 1000 mg/L. The 3 -hour EC20, EC50 and EC80 were clearly higher than the water solubility limit of the test item under the test conditions, or higher than 1000 mg/L.

Description of key information

A reliable study was available to assess the inhibitory effect of the test item on the respiration rate of aerobic wastewater microorganisms of activated sludge in a 3-hour respiration inhibition test according to the OECD 209 Guideline and under GLP compliance.

Up to and including the highest test concentration of 1000 mg/L, the test item had no significant inhibitory effect ( ≤10%) on the respiration rate of activated sludge after the incubation period of three hours. Based on the physical appearance of the test solution/dispersions, the saturation concentration (water solubility limit of the substance under the test conditions) was <100mg/L and resulted in less than 10% inhibition after three hours contact time. Thus, the 3 -hour NOEC of the test item to activated sludge microorganism was at the water solubility limit of the substance under the test conditions, or at 1000 mg/L. The 3 -hour EC20, EC50 and EC80 were clearly higher than the water solubility limit of the test item under the test conditions, or higher than 1000 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L
EC10 or NOEC for microorganisms:
1 000 mg/L

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