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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:
2012-06-08
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
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
CYANOX® 1790 Antioxidant
IUPAC nomenclature - Tris(4-tert-butyl-3-hydroxy-2,6-dimethylbenzyl) isocyanurate
Synonym - [tris(4-t-butyl-3-hydroxy-2,6-dimethyl-benzyl)-s-triazine-2,4,6-(1H,3H,5H) trione]
Lot WP1031901
Appearance - White powder, odorless
CAS No. 40601-76-1
Molecular Formula - C42H57N3O6
Molecular Weight - 699.92 g/mole
Purity > 99%
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
The batch of CYANOX® 1790 Antioxidant tested was an off white powder with a purity >96%. 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 approximately 24 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 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 (2.93 g/l of sludge, as used for the test). The pH was 7.7 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 14. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer. The pH and temperature were determined in the remaining part of the reaction mixture.
Test temperature:
Between 18.2 and 19.8°C
pH:
The pH in all test series, before addition of sludge was between 7.3 and 7.4.
After the 3 hour exposure period the pH was between 7.2 and 8.2.
Nominal and measured concentrations:
Nominal, loading rates, 10, 100 and 1000 mg/l.
Details on test conditions:
TEST SYSTEM
- Type : open
- Material, size, headspace, fill volume: All glass
- Aeration:During exposure with clean, oil-free air
- No. of vessels per concentration (replicates): 1 for 10 mg/l, 1 for 100 mg/l and 3 for 1000 mg/l.
- No. of vessels per control (replicates): 2
- 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 and 7 mg O2/l.

TEST CONCENTRATIONS
- Test concentrations: combined limit/range-finding test: 10, 100 and 1000 mg/l (loading rates).
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
(loading rate)
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
(loading rate)
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
In the combined limit/range-finding test no inhibition of the respiration rate of the sludge was recorded at any of the loading rates tested.

There was no significant oxygen uptake from abiotic processes and the results at 1000 mg/l with a nitrification inhibitor showed very slight heterotrophic inhibition (12%) of the respiration rate.
Results with reference substance (positive control):
The EC50 of 3,5-dichlorophenol was in the accepted range of 2 to 25 mg/l for total respiration (7.7 mg/l).
Reported statistics and error estimates:
ECx
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 CYANOX® 1790 Antioxidant no EC50 could be calculated because the test substance proved to be non-toxic (EC50 > a loading rate of 1000 mg/l).

The respiration rate (R) from each vessel, in mg O2/l.h, was calculated or interpolated from the linear part of the respiration curve, which was generally between 2 and 7 mg O2/l.

Oxygen consumption:

Controls                                                            :  30 mg O2/l h (average value)

CYANOX® 1790 Antioxidant at 10 mg/l      :  31 mg O2/l h

CYANOX® 1790 Antioxidant at 100 mg/l    :  30 mg O2/l h

CYANOX® 1790 Antioxidant  at 1000 mg/l :  32, 33 and 36 mg O2/l h

Validity criteria fulfilled:
yes
Conclusions:
The test material was not toxic to waste water (activated sludge) bacteria at or below a loading rate of 1000 mg/l (NOEC).
Executive summary:

In a study conducted according to OECD TG 309 and under GLP conditions, no inhibition of the respiration rate of the sludge was recorded at any of the loading rates tested. There was no significant oxygen uptake from abiotic processes and the results at 1000 mg/l with a nitrification inhibitor showed very slight heterotrophic inhibition (14%) of the respiration rate. The EC50 exceeded a loading rate of 1000 mg/L.

Description of key information

In an OECD Guideline 209 sludge respiration inhibition test, the test material was not toxic to waste water (activated sludge microorganisms) up to a maximum nominal concentration of 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

Additional information

In a 3 -h sludge respiration inhibition test conducted according to OECD Guideline 209, the test substance was not toxic to waste water treatment microorganisms (activated sludge) at the maximum nominal limit concentration of 1000 mg/L.