(3-chloropropyl)triethoxysilane

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

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

Version / remarks:

adopted 22 July 2010

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Two tests were performed. An initial combined range-finding/limit test at 0, 10, 100 and 1000 mg/l. Inhibition at 1000 mg/l was observed therefore a dose response test at 5 test concentrations of 0, 10, 32, 100, 320 and 1000 mg/l was performed.
- Method: The test item was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 10 g/l. Therefore, weighed amounts (see Table 1 and Table 2 in "Any other information on materials and methods including tables") were added to amber coloured test vessels containing 200 mL Milli-RO water. Small watch glasses were used to transfer weighed amounts of test item to respective test vessels for the test concentrations up to 100 mg/l. For the highest concentrations of 320 mg/l and 1000 mg/l, counting vials were used to weigh the test items. The test item – Milli-RO water mixtures were magnetically stirred for approximately 15 minutes. Subsequently, 16 mL synthetic medium made up to 50 mL with Milli-RO water and 250 mL sludge were added, resulting in the required concentrations. Optimal contact between the test item and test organisms was ensured by applying continuous aeration and stirring.
- Controls: Combined Limit/Range-Finding Test: Test medium without test item, treated in the same way as the test item solutions. Abiotic control only test item without sludge.
Definitive dose response test: Test medium without test item, treated in the same way as the test item solutions.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): n/a
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): n/a
- Test concentration separation factor: 3.2
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): not reported

Test organisms (species):

activated sludge

Details on inoculum:

- Laboratory culture: No
- 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: Not reported
- Preparation of inoculum for exposure: The sludge was coarsely sieved (1 mm) and allowed to settle. The supernatant was removed and ISO-medium was added. The concentration of the suspended solids was determined (3.0 g/l, as used for the test). 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.
- Pretreatment: not reported
- Initial biomass concentration: The concentration of the suspended solids was determined (3.0 g/l, as used for the test). The amount of suspended solids in the final test mixture was 1.5 g/l

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

The temperature continuously measured in the temperature control vessels ranged between 20.0 and 22.8°C during the test.

pH:

Combined Limit/Range-Finding Test: pH 8.
Final definitive test: pH 7.9.
The pH in all test vessels, before addition of sludge, was between 7.2 and 7.3. After the 3-hour exposure period the pH was between 7.9 and 8.1.

Dissolved oxygen:

Dissolved oxygen concentration at the start was above 60-70% saturation (60% of air saturation is > 5 mg/l at 20°C

Salinity:

n/a

Conductivity:

not reported

Nominal and measured concentrations:

n/a

Details on test conditions:

TEST SYSTEM
Test system: Micro-organisms in activated sludge.
Source: Municipal sewage treatment plant: 'Waterschap Aa en Maas', 's -Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
Preparation of the sludge: The sludge was coarsely sieved (1 mm) and allowed to settle. The supernatant was removed and ISO-medium was added. The concentration of the suspended solids was determined (3.0 g/l, as used for the test). 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.
Adjusted ISO-medium, formulated using RO water (tap water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands) with the following composition:
CaCl2.2H2O 211.5 mg/l
MgSO4.7H2O 88.8 mg/l
NaHCO3 46.7 mg/l
KCl 4.2 mg/l

- Test vessel: All glass open vessels.
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: Glass, size and headspace not reported, fill volume 500 ml.
- Aeration: Yes: clean, oil-free air
- No. of vessels per concentration (replicates): Combined Limit/Range-Finding Test: Highest concentration in triplicate, lower concentrations with one replicate, the control with six replicates and an abiotic control with one replicate.
Definitive dose response test: 5 replicates per test group and 6 replicates for the control.
- No. of vessels per control (replicates): Combined Limit/Range-Finding Test: Highest concentration in triplicate, lower concentrations with one replicate, the control with six replicates and an abiotic control with one replicate.
Definitive dose response test: 5 replicates per test group and 6 replicates for the control.
- No. of vessels per vehicle control (replicates): n/a
- No. of vessels per abiotic control (replicates): 1
- Sludge concentration (weight of dry solids per volume): The concentration of the suspended solids was determined (3.0 g/l, as used for the test). The amount of suspended solids in the final test mixture was 1.5 g/l
- Weight of dry solids per volume of reaction mixture per unit of time: not reported

Test item solution (200 mL) and synthetic medium (16 mL) were mixed and made up to a final volume of 250 mL with Milli-RO water. Thereafter, 250 mL activated sludge was added. This was the start of the test.
After the 3-hour contact time the oxygen consumption was recorded for a period of approximately 10 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer.

- Nutrients provided for bacteria: Synthetic medium (sewage feed):
16 g peptone
11 g meat extract
3 g urea
0.7 g NaCl
0.4 g CaCl2.2H2O
0.2 g MgSO4.7H2O
2.8 g K2HPO4
Dissolved in Milli-RO water, made up to 1 litre and filtered. The pH was within 7.5 ± 0.5.
- Biomass loading rate: n/a

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO water: Tap water purified by reverse osmosis (Millipore Corp., Bedford, Mass., USA).
- Particulate matter: Not reported.
The pH was determined in the remaining part of the reaction mixture. This procedure was repeated for all test/reference item concentrations and controls.
The medium temperature was recorded continuously in temperature control vessels. The temperature control vessels were identically prepared compared to the control vessels. A temperature control vessel with a REES sensor was placed in each fume cupboard of the climate room.
Oxygen recording: Determination of oxygen was performed with multiple oxygen sensors connected to a BlueBox (GO-Systemelektronik GmbH, Germany), a multichannel measuring and controlling system.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: not reported
- Light intensity: not reported
- Details on termination of incubation: After the 3-hour contact time the oxygen consumption was recorded for a period of approximately 10 minutes.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): After the 3-hour contact time the oxygen consumption was recorded for a period of approximately 10 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer. Determination of oxygen was performed with multiple oxygen sensors connected to a BlueBox (GO-Systemelektronik GmbH, Germany), a multichannel measuring and controlling system.

TEST CONCENTRATIONS 0, 10, 32, 100, 320, 1000 mg/l.
- Spacing factor for test concentrations: 3.2
- Justification for using fewer concentrations than requested by guideline: n/a
- Range finding study
- Test concentrations: combined range-finder/limit test 0, 10, 100 and 1000 mg/l.
- Results used to determine the conditions for the definitive study: Inhibition at 1000 mg/l but not 100 mg/l.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

320 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

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.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: n/a
- Effect concentrations exceeding solubility of substance in test medium:
- Adsorption (e.g. of test material to the walls of the test container): not reported
- Blank controls oxygen uptake rate: mean: 54 mg O2/g.h
- Coefficient of variation of oxygen uptake rate in control replicates: 17 %

The temperature in one of temperature control vessels increased to 22.8°C and was, therefore, slightly above the range laid down in the Study Plan (20 ± 2°C). All validity criteria were met and the respiration rate in the controls was significantly higher than the minimum requirement. Therefore, this deviation does not impact the outcome or validity of the study.

Results with reference substance (positive control):

- Results with reference substance valid? Yes. The EC50 of 3,5-dichlorophenol was in the accepted range of 2 to 25 mg/l for total respiration.
- Relevant effect levels: EC50 6 mg/l.

Reported statistics and error estimates:

ECx
For the reference item, calculation of the EC50 value was based on a 3-parameter logistic cumulative distribution function (CDF) using non-linear regression analysis, with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the reference item.
For the test item, no EC50 value could be calculated because the test item proved to be non-toxic (EC50 > 1000 mg/l).
NOEC determination
An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the control revealed significant inhibition of the respiration rate (Dunnett’s Multiple t-test Procedure, a=0.05, one-sided, smaller).
Calculations were performed with ToxRat Professional v. 3.2.1 (ToxRat Solutions® GmbH, Germany).

Combined Limit/Range-finding Test

The combined limit/range-finding test showed no inhibition of the respiration rate at 10 and 100 mg/l and 29% at 1000 mg (3-chloropropyl)triethoxysilane per litre. Therefore, the EC₅₀ was above the highest concentration tested (1000 mg/l).

There was no oxygen uptake from abiotic processes.

Final Dose Response Test

No statistically significant inhibition of the respiration rate of the sludge was recorded up to 320 mg (3-chloropropyl)triethoxysilane per litre. At 1000 mg/l, the inhibitory effect of the test item on aerobic wastewater (activated sludge) bacteria showed 16% inhibition.

Table 3: Final Test Overview of Results

Treatment	Concentration (mg/l)	Mean respiration rate		% Inhibition of the respiration rate (mean value)
		mg O2/l.h	mg O2/g.h^1
Control		81.15	54.10
T1	10	68.52	45.68	15.56
T2	32	82.88	55.25	-2.13
T3	100	74.68	49.79	7.97
T4	320	82.52	55.01	-1.68
T5	1000	68.02	45.35*	16.18

¹) The amount of suspended solids in the final test mixture was 1.5 g/l.

* Statistically significantly different compared to control.

Validity criteria fulfilled:

yes

Conclusions:

An activated sludge respiration inhibition 3-hour EC50 value of >1000 mg/l and a NOEC value of 320 mg/l were determined for the registered substance, (3-chloropropyl)triethoxysilane (CAS 5089-70-3, EC 225-805-6). The study was conducted according to an appropriate test protocol (OECD 209), and in compliance with GLP and is considered reliable.

Description of key information

Toxicity to microorganisms: ASRI 3-hour EC₅₀ >1000 mg/l, NOEC 320 mg/l (loading rate) (OECD 209).

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

320 mg/L

Additional information

An activated sludge respiration inhibition 3-hour EC₅₀ value of >1000 mg/l and a NOEC value of 320 mg/l were determined for the registered substance, (3-chloropropyl)triethoxysilane (CAS 5089-70-3, EC 225-805-6) in a reliable study conducted according to an appropriate test protocol (OECD 209, adopted 22 July 2010), and in compliance with GLP (Charles River, 2022).

A supporting study reported an activated sludge respiration inhibition 5-hour EC₁₀ value of 2.0 ml/l (equivalent to 2.06 g/L based on a density of 1.03 g/cm3, Pseudomonas putida) based on oxygen consumption/respiration inhibition was determined for (3-chloropropyl)triethoxysilane (CAS 5089-70-3; EC No. 225-805-6) using a relevant test method and in compliance with GLP (Schoberl 1994). The result is considered to be reliable but is not used as key study because (i) the study is a 5-hour study instead of the recommended 16 hour growth inhibition type test recommended in ISO 10712 Test Method, (ii) the study uses an emulsifier (nonylphenol ethoxylate) to improve solubility of the test substance, and (iii) no reference substance was used during the study.