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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:
23 August 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study has been conducted according to OECD guideline in compliance with GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
yes
Details on test solutions:
The test substance was not completely soluble in test medium at the loading rates initially prepared. Therefore, 1-Litre test bottles were filled with 200 ml of test substance mixtures in Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with initial loading rates of 2.5 times the final loading rate. These mixtures were stirred in closed dark brown bottles for 24 hours, except for the abiotic control which was stirred for 25.5 hours. Subsequently, 16 ml synthetic medium, 250 ml sludge and Milli-RO water up to 500 ml were added resulting in the required loading rates. Optimal contact between the test substance and test organisms was ensured applying continuous aeration and stirring.
Test organisms (species):
activated sludge
Details on inoculum:
The sludge was coarsely sieved (1 mm), washed and diluted with ISO-medium. A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids (3.0 g/l of sludge, as used for the test). The pH was 7.3 on the day of testing. The batch of sludge was used one day after collection; therefore 50 ml of synthetic medium was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Post exposure observation period:
After the 3-hour contact time, the oxygen consumption was recorded for a period of 10 to 15 minutes. The pH and temperature were determined in the remaining part of the reaction mixture. This procedure was repeated for all test/reference substance concentrations/loading rates and controls.
Hardness:
211.5 mg/l CaCl2.2H2O
Test temperature:
between 18.2 and 19.5°C
pH:
7.5 ± 0.5
Dissolved oxygen:
The aeration was adjusted in such a way that the dissolved oxygen concentration at the start was above 60-70% saturation (60% of air saturation is > 5 mg/l at 20°C) and to maintain the sludge flocs in suspension.
Nominal and measured concentrations:
1000 mg/l
Details on test conditions:
Vessels: All glass open bottles/vessels.

Milli-RO / Milli-Q water Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges (Milli-Q) (Millipore Corp., Bedford, Mass., USA).

Synthetic medium (=sewage feed)
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
Dissolved in Milli-Q water, made up to 1 litre and filtered. The pH was within 7.5 ± 0.5.

Inhibitor of nitrification: A 2.32 g/l solution of N-allylthiourea (ATU, Merck Schuchardt OHG, Hohenbrunn, Germany) was prepared. 2.5 ml of this solution was added to 500 ml final test medium (final ATU concentration: 11.6 mg/l).

Air supply: Clean, oil-free air.

Aeration: The aeration was adjusted in such a way that the dissolved oxygen concentration at the start was above 60-70% saturation (60% of air saturation is > 5 mg/l at 20°C) and to maintain the sludge flocs in suspension.

Reference substance (positive control):
yes
Remarks:
1, 3,5-dichlorophenol (5.0 mg/l)
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:
NOEC
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this present test 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol was not toxic to waste water (activated sludge) bacteria at or below a loading rate of 1000 mg/l (NOEC).
Executive summary:

The study procedures described in this report were based onthe OECD guideline No. 209, 2010.In addition, the procedures were designed to meet the test methods of theCouncilRegulation (EC) No. 440/2008 of, Publication No. L142, Part C11 and ISO Standard 8192 (2007).

In a combined limit/range-finding test loading rates of 10, 100 and 1000 mg/l were tested. The highest loading rate was tested in triplicate, lower concentrations consisted of one replicate. In addition, blank-controls and a nitrification control of the sludge were included. Furthermore, an abiotic control and the highest concentration with a nitrification inhibitor were tested.

 

In the combined limit/range-finding test no statistically significant inhibition of the respiration rate of the sludge was recordedat a loading rate of 1000 mg 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol per litre (Two Sample t-Test : α=0.05 Toxstat).

 

There was no significant oxygen uptake from abiotic processes and the results at 1000 mg/l with a nitrification inhibitor showed that the heterotrophic inhibition of the respiration rate was comparable to the total inhibition.

The batch of activated sludge was checked for sensitivity by testing the reference substance 3,5-dichlorophenol, which showed normal sensitivity.

 

The study met the acceptability criteria prescribed by the protocol and was considered valid.

 

In conclusion, 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol was not toxic to waste water (activated sludge) bacteria at or below a loading rate of 1000 mg/l (NOEC).

Description of key information

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

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