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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:
29 March 2012 - 18 April 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Also complies with OECD GLP regulations. Deviations were limited, listed below, and are believed not to impact the reliability of the test result.
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:
- Name of test material (as cited in study report): S-10793
- IUPAC nomenclature - Sodium diisobutyldithiophosphinate
- Lot S-20227-170B
- Appearance - White powder with lumps
- CAS No. 13360-78-6
- Molecular Formula - C8H18PS2.Na
- Molecular Weight - 232 g/mole
- Purity 93-94%
- Expiration date of the lot/batch: 16 December 2013
- Storage condition of test material: At room temperature in the dark
Analytical monitoring:
no
Vehicle:
no
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 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:
- Name and location of sewage treatment plant where inoculum was collected: municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Method of cultivation: 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 (2.9 g/l of sludge, as used for the test). The pH was 7.9 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.
- Initial biomass concentration: Number of micro-organisms was determined as the amount of Mixed Liquor Suspended Solids (MLSS) per litre test
medium.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
After the 3-hour contact time, the oxygen consumption was recorded for a period of 9 to 12 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:
18.1 - 21.1 °C
pH:
The pH in all test series, before addition of sludge was between 7.2 and 7.6.
After the 3-hour exposure period the pH was between 7.9 and 8.4
Dissolved oxygen:
Start of measurement: 6.4 - 8.8 mg/L
Nominal and measured concentrations:
Nominal loading rates: 10, 32, 100, 320 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): 5
- No. of vessels per control (replicates): 6 (blanks)
- Biomass loading rate: 1.45 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:
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: full test, 10, 32, 100, 320 and 1000 mg/l (loading rates)
The final test was a standard test that did not include a nitrification inhibitor or abiotic conditions.

Range finding study
- Test concentrations: 10, 100 and 1000 mg/l (3 replicates for the highest loading rate, 1 for lower loading rates, 2 replicates of blank control, 1 abiotic control and 1 highest loading rate with nitrification inhibitor)
- Results used to determine the conditions for the definitive study:
* The combined limit/range-finding test showed -1, 19 and an average of 59% inhibition of the respiration rate at a loading rate of 10, 100 and 1000 mg/l, respectively. Therefore, the expected NOEC was below a loading rate of 100 mg/l and the expected EC50 between a loading rate of 100 and 1000 mg/l.
* 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 slightly lower than the total inhibition. Therefore, N-allylthiourea was not used in the final test.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
10 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:
EC10
Effect conc.:
23 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
(loading rate)
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: (4.2-130 mg/l 95% CL)
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
53 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
(loading rate)
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: (10-270 mg/l 95% CL)
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
640 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
(loading rate)
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: estimated value
Duration:
3 h
Dose descriptor:
other: EC80
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:
No statistically significant inhibition of the respiration rate of the sludge was recorded at a loading rate of 10 mg / litre (Bonferroni t-Test: α=0.05). At higher loading rates the inhibitory effect of S-10793 on aerobic waste water (activated sludge) bacteria increased with increasing loading rate, ranging from an average of 13% inhibition at a loading rate of 32 mg/l to an average of 61% at a loading rate of 1000 mg/l.

Oxygen consumption (average values) - % Inhibition relative to the control value
Controls : 48 mg O2/l h - -
10 mg/l : 46 mg O2/l h - 5
32 mg/l : 42 mg O2/l h - 13*
100 mg/l : 39 mg O2/l h - 19*
320 mg/l : 29 mg O2/l h - 39*
1000 mg/l : 19 mg O2/l h - 61*
* Statistically significant (Bonferroni t-Test: alpha 0.05)
Results with reference substance (positive control):
The EC50 of 3,5-dichlorophenol was 5.3 and 4.6 mg/l, in the range-finding and final test respectively. (within the accepted range of 2 to 25 mg/l for total respiration)
Reported statistics and error estimates:
ECx
The percentage inhibition was plotted against the logarithm of the loading rates. The EC50, and if possible the EC10, EC20 and EC80 for the test substance, were determined using linear regression analysis. If available, the 95% confidence limits of the EC values were given.

NOEC estimation
The NOEC was based on statistical analysis of the data. Data obtained for the test concentrations TA.M. Boelter, H.L. Bergman.
Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this present test S-10793 was not toxic to waste water (activated sludge) bacteria at or below a loading rate of 10 mg/l (NOEC).

The EC10 was at a loading rate of 23 mg/l (95% confidence interval: 4.2 - 130 mg/l).
The EC20 was at a loading rate of 53 mg/l (95% confidence interval: 10 - 270 mg/l).
The EC50 was estimated to be at a loading rate of 640 mg/l.
The EC80 was above the highest loading rate tested (> 1000 mg/l).
Executive summary:

The influence of the test matrial on the respiration rate of activated sludge was investigated after a contact time of 3 hours following OECD TG 209 and under GLP conditions.

The final test was performed based on the result of a preceding combined limit/range-finding test. Five loading rates, ranging from 10 to 1000 mg/l, increasing with a factor 3.2 were tested. Five replicates per loading rate and six replicates for an untreated control group were tested.

No statistically significant inhibition of the respiration rate of the sludge was recorded at a loading rate of 10 mg/l. At higher loading rates the inhibitory effect of the test material on aerobic waste water (activated sludge) bacteria increased with increasing loading rate, ranging from an average of 13% inhibition at a loading rate of 32 mg/l to an average of 61% at a loading rate of 1000 mg/l.

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

Description of key information

Under the conditions of this study, the test material was not toxic to waste water (activated sludge) bacteria at or below a loading rate of 10 mg/l (NOEC). 

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
10 mg/L

Additional information

The influence of the test matrial on the respiration rate of activated sludge was investigated after a contact time of 3 hours following OECD TG 209 and under GLP conditions.

The final test was performed based on the result of a preceding combined limit/range-finding test. Five loading rates, ranging from 10 to 1000 mg/l, increasing with a factor 3.2 were tested. Five replicates per loading rate and six replicates for an untreated control group were tested.

No statistically significant inhibition of the respiration rate of the sludge was recorded at a loading rate of 10 mg/l. At higher loading rates the inhibitory effect of the test material on aerobic waste water (activated sludge) bacteria increased with increasing loading rate, ranging from an average of 13% inhibition at a loading rate of 32 mg/l to an average of 61% at a loading rate of 1000 mg/l.

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