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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:
August 06, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
- To the test mixtures (250 mL) containing deionised water, synthetic sewage feed (16 mL) and the appropriate test/reference concentration and in one set ATU (2.5 mL), the inoculum of activated sludge (250 mL) was added to the final volume of 500 mL. All test substances were weighed directly into the test vessels and were dispersed by intense aeration.

- The reference assay with the concentrations 0.2, 1, 5, and 25 mg/L was set up by dilution of a stock solution. The blank control was set up in the same way as the test substance concentrations but without test substance.

- The abiotic control was prepared with the highest test substance concentration, water and synthetic sewage feed.

- The nitrification controls were prepared with ATU (N-Allylthiourea, 99.6 % w/w pure, batch no. S6971358) using 2.5 mL of a stock solution for each nitrification control. These controls were performed for each concentration of the test/reference substance assay (except abiotic control).
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Microbial Inoculum:
Activated sludge from the municipal waste water treatment plant of 75172 Pforzheim, Germany, collected from the aeration tank, with an dry substance of 3.77 g/L was used as the microbial inoculum for the test. This plant was predominantly treating domestic sewage. The sludge (6.37 L) was used one day after collection. The sludge was settled and the upper layer with finer solids was decanted and discarded. The remaining sludge was washed three times with tap water by centrifugation (10 min at 3,000 rpm). After centrifuging, the supernatant was decanted and discarded and the sludge was resuspended in chlorine free tap water (8 L). This resulted in a concentration of 3.0 g/L (dry substance). A part of the activated sludge was replaced with synthetic sewage feed (~ 5 %). The activated sludge was continuously aerated at room temperature, the solids did not settle down. This sludge was mixed homogenously and was used as microbial inoculum for all flasks.

- Synthetic Medium:
The test was performed in a synthetic sewage feed that was prepared with the following amounts of substances in 1 litre of deionised water: 16 g peptone, 11 g meat extract, 3 g urea, 0.7 g NaCl, 0.4 g CaCl2 × 2 H2O, 0.2 g MgSO4 × 7 H2O, 2.8 g K2HPO4. The solution was sterilized prior to storage. If the preparation was not used immediately it was stored in the dark at a temperature of between 0 – 10ºC for up to one week.
Test type:
other: The test assays were aerated during the duration of exposure (3 h).
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Test temperature:
20.0 – 22.5 °C (measured during O2-determination at 3 h after start of respiration)
pH:
7.46 – 7.55
Nominal and measured concentrations:
Nominal: 10, 100 and 1,000 mg/L
Details on test conditions:
Duration: 3 h
Test vessels: 1 litre glass beakers
Test volume: 500 mL
Nutrient solution: 16 mL synthetic sewage feed
Reference substance (positive control):
yes
Remarks:
3,5 dichlorophenol (DCP), 99.4 % pure, batch no. 10157809
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: for the total oxygen uptake, for the heterotrophic oxygen uptake and for the oxygen uptake due to nitrification
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: for the total oxygen uptake, for the heterotrophic oxygen uptake and for the oxygen uptake due to nitrification
Results with reference substance (positive control):
The EC50 of the total oxygen uptake for DCP was 4.24 mg/L after 3 h. This is within the accepted range of 2 to 25 mg/L for total respiration. For the heterotrophic oxygen uptake for DCP, the EC50 was 11.7 mg/L after 3 h. This is within the recommended range of 5 to 40 mg/L. For the oxygen uptake due to nitrification the EC50 was determined at 1.04 mg/L after 3 h. This value was also within the recommended range of 0.1 to 10 mg/L.
Reported statistics and error estimates:
The statistical evaluation was performed for specific respiration rate of the control and all test substance concentrations using SAS® (2002–2010). The calculation of the DCP EC50 values for the total oxygen uptake, heterotrophe oxygen uptake and oxygen uptake due to nitrifiers was calculated following normal distribution. Since a stimulation effect was observed at 1,000 mg/L, no statistical evaluation for NOEC or LOEC was conducted.

All validity criteria of OECD Guideline 209 were fulfilled:

The mean oxygen uptake rate of the blank control were determined to be 21 mg O2/(g x h) dry matter after 3 h. Hence, the rate was not less than 20 mg oxygen uptake per g of activated sludge (dry weight of suspended solids) per hour.

The coefficients of variation of the control for total oxygen uptake were 6% and for the heterotrophic oxygen uptake 10% after 3 h and did not exceed 30% at the end of the test. All the values with reference substance were within recommended range.

Validity criteria fulfilled:
yes
Conclusions:
The EC50 for the test substance for the total oxygen uptake, for the heterotrophic oxygen uptake and for the oxygen uptake due to nitrification was > 1,000 mg/L . Based on these results the NOEC was determined to be 1,000 mg/L.
Executive summary:

A study was conducted to determine the toxicity of the test substance on microorganisms according to OECD Guideline 209, in compliance with GLP. The test system was activated sludge from a municipal wastewater treatment plant. The microorganisms were exposed at 0, 10, 100 and 1,000 mg/L (nominal). Three replicates each were used for the control and the highest dose and one each for the other concentrations. Additionally, to evaluate the effects on nitrifying bacteria, each concentration of the test substance and the control was set up in parallel with N-allylthiourea (ATU). Four concentrations of 3,5-dichlorophenol (DCP) as toxic reference substance were tested in parallel to demonstrate the sensitivity of the system. Oxygen uptake rate was determined after 3 h. This test fulfilled all the validity criteria. After 3 h, no inhibition effects were observed up to the highest dose level of 1,000 mg/L for total oxygen uptake, heterotrophic oxygen uptake and for oxygen uptake due to nitrification. A stimulating effect was observed at 1,000 mg/L. Hence, under the study conditions, the EC50 was > 1,000 mg/L (nominal). The NOEC was 1,000 mg/L (nominal) (Falk S, 2015).

Description of key information

Key value for chemical safety assessment

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

Additional information

A study was conducted to determine the toxicity of the test substance on microorganisms according to OECD Guideline 209, in compliance with GLP. The test system was activated sludge from a municipal wastewater treatment plant. The microorganisms were exposed at 0, 10, 100 and 1,000 mg/L (nominal). Three replicates each were used for the control and the highest dose and one each for the other concentrations. Additionally, to evaluate the effects on nitrifying bacteria, each concentration of the test substance and the control was set up in parallel with N-allylthiourea (ATU). Four concentrations of 3,5-dichlorophenol (DCP) as toxic reference substance were tested in parallel to demonstrate the sensitivity of the system. Oxygen uptake rate was determined after 3 h. This test fulfilled all the validity criteria. After 3 h, no inhibition effects were observed up to the highest dose level of 1,000 mg/L for total oxygen uptake, heterotrophic oxygen uptake and for oxygen uptake due to nitrification. A stimulating effect was observed at 1,000 mg/L. Hence, under the study conditions, the EC50 was > 1,000 mg/L (nominal). The NOEC was 1,000 mg/L (nominal) (Falk S, 2015).