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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:
09 November 2012 - 16 November 2012
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
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
The batch of F-D3 tested was a white solid with a purity of 99.8%. No correction was made for the purity/composition of the test substance. 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 with initial loading rates of 2.5 times the final loading rate. These mixtures were stirred in closed dark brown bottles for approximately 25.5 hours in the first experiment and approximately 24 hours in the second experiment. 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:
Source:Municipal sewage treatment plant: 'Waterschap Aa en Maas', Heeswijk-Dinther, The Netherlands, receiving predominantly domestic sewage.
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 Experiments 1 and 2). The pH was 7.6 for Experiment 1 and 7.2 for Experiment 2 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 10-13 minutes. 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:
Experiment 1: between 19 and 21°C
Experiment 2: between 20 and 21°C
pH:
Experiment 1:
The pH in all test series before addition of sludge was 7.5.
After the 3 hour exposure period the pH was between 7.3 and 8.1.
Experiment 2:
The pH in all test series before addition of sludge was between 7.5 and 7.6.
After the 3 hour exposure period the pH was between 7.6 and 7.7.



Dissolved oxygen:
Mean respiration rates in (mg O2/l h) :
Experiment 1:
C1(control):38
C2(control):35
C3(control):41
C4(control):37
C5(control):36
C6(control):34
T1 (loading rate of 10 mg F-D3/l):34
T2 (loading rate of 100 mg F-D3/l):35
T3a (loading rate of 1000 mg F-D3/l ) :35
T3b ( loading rate of 1000 mg F-D3/l): No value
T3c ( loading rate of 1000 mg F-D3/l):40

Experiment 2:
C1 (control):28
C2 (control):32
T3a (a loading rate of 1000 mg F-D3/l):29
T3b (a loading rate of 1000 mg F-D3/l):35
T3c (a loading rate of 1000 mg F-D3/l):29
Nominal and measured concentrations:
nominal concentrations:
experiment 1: control, 10, 100, 1000 mg/l
experiment 2: control and 1000 mg/l
Details on test conditions:
Vessels: All glass open bottles/vessels.
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.
Biomass loading rate: 1.5 g/l suspended solids in final test mixture.
Source/preparation of dilution water: Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges (Milli-Q)

Experiment 1:
nr replicates loading rates of 10 and 100 mg/l:1
nr of replicates loading rate of 1000 mg/l:3 (1 replicated : no value)
nr of replicates of control: 6
Abiotic controle:1
the highest loading rate with a nitrification inhibitor:1
Test concentrations: loading rates of 10, 100, 1000 mg/l

Experiment 2:
nr of replicates per concentrations: 3
nr of replicates per control:2
Concentrations: loading rates of 1000 mg/l


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:
Experiment 1 showed 8%, 5% and no inhibition (average of 2 values) of the respiration rate at a loading rates of 10, 100 and 1000 mg/l, respectively.
Since one of the respiration curves at the highest loading rate (1000 mg/l) could not be used for reliable determination of the respiration rate, a limit test at a loading rate of 1000 mg/l was repeated.

In the limit test, Experiment 2, no inhibition of the respiration rate of the sludge (average value) was recorded at a loading rate of 1000 mg F-D3 per litre (NOEC). Thus, the EC50 was above the highest loading rate tested (1000 mg/l).

There was no 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.
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 (11 and 8.9 mg/l, for Experiments 1 and 2 respectively.
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 F-D3 no EC50 could be calculated because the test substance proved to be non-toxic (EC50 > a loading rate of 1000 mg/l).

NOEC estimation
No effect on the respiration inhibition was observed at the highest concentration tested, therefore this was considerate to be the NOEC.
Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this present test, F-D3 was not toxic to waste water (activated sludge) bacteria at or below a loading rate of 1000 mg/l (NOEC).
The EC50 exceeded a loading rate of 1000 mg/l.

Description of key information

Toxicity to microorganisms: ASRI 3hr EC50 >1000; NOEC ≥1000 mg/l (OECD 209)

Key value for chemical safety assessment

Additional information

Under the conditions of this present test, F-D3 was not toxic to waste water (activated sludge) bacteria at or below a loading rate of 1000 mg/l (NOEC).

The EC50exceeded a loading rate of 1000 mg/l. The exposure may however be limited by the solubility of the test substance in solution.