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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:
01 July 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles
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
Qualifier:
according to guideline
Guideline:
ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Since Lowinox® AH25 was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 0.5 g/l, the test concentrations were prepared separately in Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA). Two 1-litre test bottles were filled with 250 ml of test substance mixtures with initial loading rates of 200 mg/l. These mixtures were stirred in closed dark brown glass bottles for 26½ hours. Subsequently, 16 ml synthetic sewage feed, 200 ml sludge and Milli-RO water up to 500 ml were added resulting in a loading rate of 100 mg/l. This loading rate was tested in duplicate. Optimal contact between the test substance and test organisms was ensured applying continuous aeration and stirring.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
The sludge was coarsely sieved, 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 (3.8 g/l of sludge, as used for the test). The pH was 8.0 on the day of testing. The batch of sludge was used one day after collection; therefore 50 ml of synthetic sewage feed 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:
Oxygen consumption was measured and recorded for approximately 10 minutes after the 3-hour exposure period.
Test temperature:
between 18.3 and 18.8°C.
pH:
8.2-8.3
Dissolved oxygen:
Oxygen consuption:
Control series: 42-44 mg O2/l/h
Lowinox® AH25 series: 35 mg O2/l/h
The oxygen concnetration at the start of the measurments was at least 7.0 mg/l
Nominal and measured concentrations:
Loading rate of 100 mg/l
Details on test conditions:
TEST SYSTEM
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: All glass, approximately 300 ml oxygen bottles (measuring) and
1 litre test bottles(exposure)
- Aeration:During exposure with clean, oil-free air
- No. of vessels per concentration (replicates):2
- No. of vessels per control (replicates):2/ series
- Biomass loading rate: 1.5 g/l suspended solids in final test mixture

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap-water purified by reverse osmosis (Milli-RO).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The respiration rate from each vessel, in mg O2/l/hr, was calculated from the linear part of the respiration curve, which was generally between 2.5 and 6.5 mg O2/l.

TEST CONCENTRATIONS
- Test concentrations: limit study at 100 mg/l
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
19% inhibition of respiration rate of the sludge was recorded at a loading rate of 100 mg Lowinox® AH25 per litre. The duplicate measurement confirmed the result of the first measurement. Therefore, no further testing was needed. Hence, the EC50 of Lowinox® AH25 exceeded a loading rate of 100 mg/l.
Results with reference substance (positive control):
Results with reference substance valid: Yes
The inhibitory effect of 3,5-dichlorophenol on aerobic waste water (activated sludge) bacteria increased with increased with increasing concentration , ranging from a 35 % inhibition at 5,0 mg/l to 75% at 30 mg/l.
Relevant effects levels: The EC50 of 3,5-dichlorophenol was: 9.3 mg/l
Reported statistics and error estimates:
For the reference substance the percentage inhibition was plotted against the logarithm of the concentrations and the EC50 was determined using linear regression analysis.
For Lowinox® AH25 no EC50 could be calculated because the test substance proved to be non-toxic (EC50 > 100 mg/l).
Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this present test, Lowinox® AH25 was slightly toxic to waste water (activated sludge) bacteria at a loading rate of 100 mg/l (19% inhibition of respiration rate of the sludge). The 3-hour EC50 of Lowinox® AH25 exceeded a loading rate of 100 mg/l.
Executive summary:

The influence of Lowinox® AH25 on the respiration rate of activated sludge was investigated after a contact time of 3 hours.

 

The study procedures described in this report were based on the OECD guideline No. 209, 1984. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No. 44012008 of 30 May 2008, Publication No. L142, Part Cl I and ISO Standard 81 92 (2007).

 

The batch of Lowinox® AH25 tested was a white to cream coloured powder with a purity of 95.9% (based on GC analysis).

 

Since Lowinox® AH25 was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 0.5 g/l, the test concentrations were prepared separately in Milli-RO water.

Two 1-litre test bottles were filled with 250 ml of test substance mixtures with initial loading rates of 200 mg/l. These mixtures were stirred in closed dark brown glass bottles for 26% hours.

Subsequently, 16 ml synthetic sewage feed, 200 ml sludge and Milli-RO water up to 500 ml were added resulting in a loading rate of 100 mg/l. This loading rate was tested in duplicate.

Two controls without test substance were tested in each test series. Optimal contact between the test substance and test medium was ensured applying continuous aeration and stirring during the 3-hour exposure period. Thereafter, oxygen consumption was measured and recorded for approximately 10 minutes.

 

19% inhibition of respiration rate of the sludge was recorded at a loading rate of 100 mg Lowinox® AH25 per litre. The duplicate measurement confirmed the result of the first measurement. Therefore, no further testing was needed. Hence, the EC50 of Lowinox® AH25 exceeded a loading rate of 100 mg/l.

 

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

 

Since all criteria for acceptability of the test were met, this study was considered to be valid.

 

In conclusion, Lowinox® AH25 was slightly toxic to waste water (activated sludge) bacteria at a loading rate of 100 mg/l (19% inhibition of respiration rate of the sludge, 3-hour EC50 > a loading rate of 100 mg/l).

Description of key information

3-hour EC50 > a loading rate of 100 mg/l

Key value for chemical safety assessment

EC50 for microorganisms:
100 mg/L

Additional information

The influence of Lowinox® AH25 on the respiration rate of activated sludge was investigated after a contact time of 3 hours. 

The batch of Lowinox® AH25 tested was a white to cream coloured powder with a purity of 95.9% (based on GC analysis).

 

Since Lowinox® AH25 was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 0.5 g/l, the test concentrations were prepared separately in Milli-RO water.

Two 1-litre test bottles were filled with 250 ml of test substance mixtures with initial loading rates of 200 mg/l. These mixtures were stirred in closed dark brown glass bottles for 26% hours.

Subsequently, 16 ml synthetic sewage feed, 200 ml sludge and Milli-RO water up to 500 ml were added resulting in a loading rate of 100 mg/l. This loading rate was tested in duplicate.

Two controls without test substance were tested in each test series. Optimal contact between the test substance and test medium was ensured applying continuous aeration and stirring during the 3-hour exposure period. Thereafter, oxygen consumption was measured and recorded for approximately 10 minutes.

 

19% inhibition of respiration rate of the sludge was recorded at a loading rate of 100 mg Lowinox® AH25 per litre. The duplicate measurement confirmed the result of the first measurement. Therefore, no further testing was needed. Hence, the EC50 of Lowinox® AH25 exceeded a loading rate of 100 mg/l.

In conclusion, Lowinox® AH25 was slightly toxic to waste water (activated sludge) bacteria at a loading rate of 100 mg/l (19% inhibition of respiration rate of the sludge, 3-hour EC50 > a loading rate of 100 mg/l).

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