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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:
21 September 2012 - 01 November 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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:
The batch of 3-Isocyanatopropyltriethoxysilane tested was a clear, colourless liquid with a purity of 96.5%. No correction was made for the purity/composition of the test substance. The test substance was pipetted below the surface of the Milli-RO-water and stirred briefly to mix (5 minutes). 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 by 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.

Preparation of the sludge: 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 (2.9 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:
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 approximately 10-11 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:
Between 19 and 22°C
pH:
The pH in all test series, before addition of sludge was between 7.2 and 7.5. After the 3-hour exposure period the pH was between 7.7 and 8.2.
Dissolved oxygen:
The respiration rate 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.

Mean respiration rates:
Control: 28 mg O2/l/h
T 1: loadingrate of 10 mg/l: 26 mg O2/l/h
T2: loading rate of 32 mg/l: 22 mg O2/l/h
T3 loading rate of 100 mg/l: 22 mg O2/l/h
T4: loading rate of 320 mg/l: 11 mg O2/l/h
T5: loading rate of 1000 mg/l: 3 mg O2/l/h
Nominal and measured concentrations:
Nominal concentrations at a loading rate of 10, 32, 100, 320 and 1000 mg/l and a control
Details on test conditions:
Vessels: All glass open bottles/vessels
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.
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).
The amount of suspended solids in the final test mixture was 1.45 g/l.
The final test was a standard test that did not include a nitrification inhibitor or abiotic conditions.
Numbers replicates loading rates for the control in the final test: 6
Numbers replicates for the test substance loading rates in the final test: 5
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
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
Key result
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
17 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: (3.5 - 84 mg/l)
Key result
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
31 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: (6.6 - 140 mg/l)
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
180 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: (41 - 760 mg/l)
Details on results:
No statistically significant inhibition of the respiration rate of the sludge was recorded at loading rate of 10 mg 3-Isocyanatopropyltriethoxysilane / litre (Bonferroni t-Test: α=0.05 Toxstat). At higher loading rates the inhibitory effect of 3-Isocyanatopropyltriethoxysilane on aerobic waste water (activated sludge) bacteria increased with increasing loading rates, ranging from 21% inhibition at a loading rate of 32 mg/l to 89% at a loading rate of 1000 mg/l.
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 in the combined limit/range-finding and final test, respectively.
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 were compared with those obtained for the control using TOXSTAT Release 3.5, 1996, D.D. Gulley, A.M. Boelter, H.L. Bergman.
Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this present test 3-Isocyanatopropyltriethoxysilane was not toxic to waste water (activated sludge) bacteria at or below a loading rate of 10 mg/l (NOEC). This was determined in a reliable study conducted according to an appropriate test protocol, and in compliance with GLP.

The EC10 was at a loading rate of 17 mg/l (95% confidence interval: 3.5 – 84 mg/l).
The EC20 was at a loading rate of 31 mg/l (95% confidence interval: 6.6 – 140 mg/l).
The EC50 was at a loading rate of 180 mg/l (95% confidence interval: 41 – 760 mg/l).

Description of key information

Toxicity to microorganisms: 3-hour NOEC 10 mg/L, EC10 17 mg/L (OECD 209; ASRI)

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
17 mg/L

Additional information

The toxicity of triethoxy(3-isocyanatopropyl)silane has been determined in an activated sludge respiration inhibition study in accordance with OECD Test Guideline 209 and in compliance with GLP. A 3-hour EC50 value of 180 mg/L, NOEC value of 10 mg/L and EC10 value of 17 mg/L were obtained for the registered substance.

Under the conditions of this present test, the registration substance, triethoxy(3-isocyanatopropyl)silane, was not toxic to wastewater (activated sludge) bacteria at or below loading rates of 10 mg/L (NOEC) and 17 mg/L (EC10). The results are considered to be reliable.

The registered substance hydrolyses very rapidly to form an ultimate silanol hydrolysis product, 3-aminopropylsilanetriol. In view of the very rapid/rapid hydrolysis, it is the silanol hydrolysis product that is relevant for environmental risk assessment.

The other hydrolysis products are carbon dioxide and ethanol.

Ethanol does not have adverse effects on microorganisms (OECD 2004).

Reference:

OECD (2004): SIDS Initial Assessment Report for SIAM 19, Berlin, Germany, 19-22 October 2004, Ethanol, CAS 64-17-5

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