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activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-09-03 to 2009-09-03
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
See endpoint summary for justification of read-across
according to guideline
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Known volume of test substance, Milli-RO water, synthetic sewage feed (16 ml) and activated sludge (200 ml) were mixed and made up to 500 ml with Milli-RO water in a 1 litre bottle.

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:

- Preparation of inoculum for exposure: The sludge was coarsely sieved then washed.

- Initial biomass concentration: 3.8 g/l
Test type:
Water media type:
Limit test:
Total exposure duration:
3 h
Post exposure observation period:
The oxygen consumption was measured and recorded for approximately 10 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer.
Test temperature:
Nominal and measured concentrations:
100 mg/l (nominal)
Details on test conditions:
- Test vessel:

- Material, size, headspace, fill volume: glass, 300ml oxygen bottles and 1L test bottles

- Aeration: continuous

- No. of vessels per concentration (replicates): two

- No. of vessels per control (replicates): two

- Biomass loading rate: 100 mg/L


- 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)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
After the 3-hour contact time, a well mixed sample of the contents of the reaction mixture was poured into a 300 ml oxygen bottle, and the flask was sealed with an oxygen electrode connected to a recorder, forcing the air out of the vessel. WTW inolab Oxi 730 supplied with a WTW CellOx 325 oxygen electrode, electrolyte type ELY/G.


- Spacing factor for test concentrations: 100 mg/L only

- Justification for using less concentrations than requested by guideline: No significant inhibition of respiration rate of the sludge was recorded at 100mg Trichloro(vinyl)silane per litre. Therefore no further testing was required.
Reference substance (positive control):
3,5 - Dichlorophenol
Key result
3 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
The two controls had a difference in respiration rate of 7%, which is less than the 15% limit required by the guideline for a valid study.
Results with reference substance (positive control):
A positive control test with 3, 5-Dichlorophenol yielded an EC50 of 8.7 mg/L, which is within the accepted range of 5 to 30 mg/l required by the guideline for a valid study.

Table 1: Results of the activated sludge respiration inhibition test

Test sample

Loading rate (mg/L)

Oxygen conc. at start (mg O2/L)

Oxygen consumption (mg O2/L/h)

% Inhibition

respiration rate




Initial control






Final control






Trichloro(vinyl)silane replicate (1)






Trichloro(vinyl)silane replicate (2)
























Validity criteria fulfilled:
An activated sludge respiration inhibition 3hr EC10 value of >100 mg/L (loading rate) was determined in a reliable study conducted according to an appropriate test protocol, and in compliance with GLP. This indicates that the test substance was not toxic to waste water (activated sludge) bacteria at a loading rate of 100 mg/l.

Description of key information

Toxicity to microorganisms: ASRI 3-hour EC50 >100 mg/L (loading rate) (OECD 209), read-across from a structurally-related substance.

Key value for chemical safety assessment

EC50 for microorganisms:
100 mg/L

Additional information

An activated sludge respiration inhibition 5-hour EC10 value of 1.1 ml/L (equivalent to 1.03 g/L based on a density of 0.94 g/cm3, Pseudomonas putida) based on oxygen consumption/respiration inhibition was determined for trimethoxy(vinyl)silane (CAS 2768-02-7; EC No. 220-449-8) using a relevant test method and in compliance with GLP (Evonik 1993). 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.


Other results are available for the registered substance in secondary sources to which reliability cannot be assigned; bacterial respiration inhibition values of >2500 mg/L (Chandra, 1997) and 3-h NOEC of =1000 mg/L were reported for the substance (Bennet, 2001).


Therefore, good quality data for the analogous substance trichloro(vinyl)silane (CAS 75-94-5; EC No. 200-917-8), have been read across. The submission substance, trimethoxy(vinyl)silane, hydrolyses very rapidly in contact with water to form vinylsilanetriol (CAS 143-48-6) and methanol (CAS 67-56-1; EC No. 200-659-6). The read-across substance, trichloro(vinyl)silane, also hydrolyses very rapidly in contact with water to form vinylsilanetriol and hydrogen chloride, which rapidly dissolves in water to form hydrochloric acid (CAS 7647-01-0; EC No. 231-595-7). Both substances therefore hydrolyse very rapidly and form the same silanol hydrolysis product; vinylsilanetriol. In view of the very rapid hydrolysis of the registered substance, it is the silanol hydrolysis product that is relevant for environmental risk assessment.


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


Hydrochloric acid is not expected to have adverse effects on microorganisms in a sewage treatment plant, where the pH is maintained within a favourable range.


Trimethoxy(vinyl)silane (CAS 2768-02-7) and trichloro(vinyl)silane (CAS 75-94-5) are within an analogue group of substances within which in general, there is no evidence of significant toxicity to microorganisms.


This group consists of substances containing a number of different functional groups but specific read-across is between substances with similar functionality. In this case, read-across from a substance with different functional group i.e. trichlorosilanes (Si-Cl) is used for the registered substance, trimethoxy(vinyl)silane. This is because data available for the trimethoxysilanes functional group does not contain any substances with a vinyl side-chain and it was considered important to cover this structural feature. Both trichloro(vinyl)silane and trimethoxy(vinyl)silane hydrolyse very rapidly to form the same silanol hydrolysis product, vinylsilanetriol.


It is therefore considered valid to read-across the results for trichloro(vinyl)silane (CAS 75-94-5) to fill the data gap for the registered substance since the source and target substances generate the same silanol hydrolysis product. Additional information is given in a supporting report (PFA 2013j) attached in Section 13.


The table below presents microorganism toxicity data for substances relevant to trimethoxy(vinyl)silane (CAS 2768-02-7).


Table: Microorganism toxicity data available for relevant substances

 CAS No. Chemical Name (CAS Name)  Main code  Guideline Number  Test method  PFA Klimisch code  Year  Duration (hours)  Result: E(I)C50 (mg/L)  Result: NOEC (or EC10/EC20) (mg/L)  Study ID Code [1] 
 58942-60-0 Trichloro(hexadecyl)silane  I-2 -T-C  OECD 209  ASRI    >1000   =1000
 75-94-5  Trichloro(vinyl)silane  I-2 -T-C  OECD 209  ASRI  1  2009  3  >100
 87135-01-1  1,6 -Bis(trimethoxysilyl)hexane  I-2 -T-Me-H  OECD 209 ASRI   1  2009  3  >1000
 16415-12-6  Hexadecyltrimethoxysilane  I-2 -T-Me-H  OECD 209  ASRI  1    3  >1000


[1]This is a unique internal ID code which can be used to identify the reference for the study.


In the study with trichloro(vinyl)silane (CAS 75-94-5), an activated sludge respiration inhibition 3-hour EC50 value of >100 mg/L was determined in a study conducted in accordance with OECD Test Guideline 209 and in compliance with GLP.





OECD (2004): SIDS Initial Assessment Report for SIAM 19, Berlin, Germany, 18-20 October 2004, Methanol, CAS 67-56-1


PFA (2013j). Peter Fisk Associates, STP Microorganism toxicity Main Analogue Group report, PFA.300.003.006

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