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Ecotoxicological information

Toxicity to microorganisms

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activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-01-19 to 2012-02-16
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The result was sourced from a toxicity control test in a ready biodegradation study
according to guideline
other: OECD 301B
Principles of method if other than guideline:
The result was obtained in a toxicity control test conducted as part of a ready biodegradation study
GLP compliance:
Analytical monitoring:
Details on sampling:
On test days 1, 4, 7,10, 13, 18, 22 and 25, a 7 mL sample was removed from the first KOH CO2 trap on each test system and analysed for CO2 evolution. on Day 28, 1 mL of concentrated HCl was added to each test vessel following pH measurements to terminate biological activity.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
The activated sludge was obtained from the Wareham Wastewater Treatment Plant, Wareham, Massachussets, which receives primarily domestic waste. Approximately 8 L of activated sludge was collected on 18 January 2012, and transported to the laboratory. Upon arrival at the laboratory, the sludge was passed through a 2 mm sieve, recombined and centrifuged at 1000 rpm for 10 minutes. The supernatant was discarded, the sludge was washed with mineral media (1000 mL) and the contents were centrifuged at least once again and the supernatant discarded. The moisture content of the activated sludge was determined, using an automated moisture analyser (Sartorius MA-150) to be 94.90% and the percent solid determined to be 5.1%. A 15 mg solids/mL inoculum solution was prepared (1.5 g dry weight sludge brought to 100 mL with mineral media) and aerated until used. The test substance flasks, the blank flasks, the procedural control flask and the toxicity control flask all received 6 mL of the inoculum to produce an activated sludge concentration of 30 mg/L.

In addition, a 50 g aliquot of fresh soil, a 25 g aliquot of Weweantic River sediment and a 25 g aliquot of Taunton River sediment were collected near the laboratory. The soil and sediment were suspended in 1.0 L of Weweantic River water. The suspension was filtered through glass wool prior to use. A 3.0 mL aliquot of the soil/sediment filtrate was added to each test vessel containing 2991 mL of mineral medium and 6.0 mL of activated sludge as shown in Table 2.
Test type:
Water media type:
Total exposure duration:
28 d
Test temperature:
The pH of the test medium was measured to be 7.42 on day 1 and ranged from 7.19 to 7.32 on day 28.
Nominal and measured concentrations:
20 mg C/L
Reference substance (positive control):
Sodium benzoate
Key result
28 d
Dose descriptor:
Effect conc.:
20 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
respiration rate
Details on results:
The test cumulative net CO2 evolved from the toxicity control was 51.60% on day 13 and therefore the test substance was not considered toxic to the inoculum based on the OECD 301B Guideline (i.e.>25% CO2 evolution by day 14 is not considered toxic).
Validity criteria fulfilled:
51.6% net CO2 was evolved on a day 13 from the toxicity control test in a ready biodegradation study in accordance with OECD 301B. The result is considered to be reliable.

Description of key information

The toxicity to microorganisms of the substance is assessed based on a toxicity control study in an OECD 301B; at 20 mg/L test concentration, the substance was considered not inhibitive to microorganisms.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
20 mg/L

Additional information

The toxicity of the substance to microorganisms was assessed based on a toxicity control test in a ready biodegradation study in accordance with OECD 301B and in compliance with GLP. The toxicity control vessel was fortified with 0.0552 g of the test substance and 3.0 mL of the 10 mg C/mL of the reference substance, sodium benzoate. The toxicity control vessel has a total concentration of 20 mg C/L (test and reference substance).

The test evolved a cumulative CO2 of 51.6% on day 13 in the toxicity control test, therefore the substance was not considered to be toxic to the inoculum based on the OECD 301B Test Guideline. Therefore, at the test concentration used during the test at which no inhibition to microorganisms was observed was therefore set as the NOEC value; i.e NOEC = 20 mg/l.