3-trimethoxysilylpropyl methacrylate
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activated sludge respiration inhibition testing
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Directly weighed into the test flasks, tap water added and solution stirred intensely for two minutes at room temperature.
activated sludge of a predominantly domestic sewage
Details on inoculum:
The study was performed with aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Fullinsdorf, Switzerland) treating predominantly domestic wastewater. The sludge was washed once with tap water by centrifugation and the supertant liquid phase was decanted. A homogenised aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated.
Based on this ratio, an aliquot of washed sludge was suspended in tap water to obtain a concentration equivalent to 3 g dry material/Litre. During the holding of two days prior to use, the sludge was fed daily with 50 mL synthetic wastewater/Litre and kept at room temperature under continuous aeration until use. Immediately before use, the dry weight of the activated sludge was measured again in the inoculum used in the test. The pH of the activated sludge inoculum was adjusted from 8.2 to 7.0 with a diluted sulfuric acid solution.
Determined in one control at the start and at the end of the 3-hour incubation period, and was continuously monitored in all test media and controls during measurement of the respiration rate.
7.3 to 8.4. Determined in all test media and controls at the start and at the end of the 3-hour incubation period. At the start of the test, the pH values in the test media and the control ranged from 7.3 to 7.7. At the end of the test, pH values between 8.2 and 8.4 were measured.
7.4 to 8.9 mg O2/l. Determined in all test media and controls at the start and at the end of the 3-hour incubation period. Oxygen concentration (mg O2/l) in the test media and the control ranged from 8.5 to 8.9, and at the end of the test was measured between 7.4 and 8.4.
10, 32, 100, 320 and 1000 mg/l (nominal).
- Test vessel: 1000 ml glass flasks. The test flasks were labelled with the necessary information to assure unmistakable identification
At the start of the test, 200 mL activated sludge inoculum with a sludge concentration of 3.7 g dry weight/L (corresponding to about 1.5 g dry material/L test medium). The sludge was added in time intervals of 15 minutes (an arbitrary but convenient interval) first to a control, then to the test solutions of the reference item, then to the test solutions of the test item and finally to the second control.
During the incubation period of exactly 3 hours, all test media and the controls were continuously aerated with compressed air at a flow of approximately one litre pre minute. The concentration of dissolved oxygen did not drop below 2.5 mg/L during the incubation period and just before measurement of the respiration rates, the dissolved concentrations were at least 7.4 mg/L. The temperature in the test media, measured in one control, was 19°C at the start and at the end of the incubation period.
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water
Measurement of respiration rate was carried out on a well-mixed sample of each test medium poured into a BOD-flask after 3 hours incubation. Dissolved oxygen concentration was then measured with an oxygen electrode continuously for about ten to fifteen minutes. During measurement samples were stirred continuously. The oxygen consumption rate in each vessel (in mg O2 /L/min) was determined from the linear part of the respiration curve in the range of at least 6.5-2.5 mg O2/L. In the case of very rapid oxygen consumption, the range used was below the limits indicated above but always within the linear part of the respiration curve. In case of low oxygen consumption the rate was determined over a period of at least 10 minutes.
>= 1 000 mg/L
> 1 000 mg/L
The two controls had a difference in respiration rate of 11%, 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 12 mg/l, which is within the accepted range of 5 to 30 mg/l required by the guideline for a valid study.
Toxicity of test item:
All test concentrations were clear solutions.
The test item had no significant inhibitory effect (<15%) on the respiration rate of activated sludge after the incubation period of 3 hours up to and including the highest test item concentration of 1000 mg/L. Thus, the 3 -hour NOEC of the test item to activated sludge microorganisms was at least 1000 mg/L. This value might be even higher, but concentrations exceeding 1000 mg/L were not tested. The 3 -hour EC20, EC50 and EC80 could not be calculated, but were clearly higher than 1000 mg/L. The results are reported in Table 1.
The oxygen consumption rates of the two controls (run at the start and at the end of the test) differed by 11%.
Table 1: Controls, reference substance (3, 5-dichlorophenol) and test substance (trimethoxysilylpropyl methacrylate): oxygen consumption and percentage inhibition of the respiration rate.
Nominal concentration (mg/l)
(mg O2/l/min)
% Inhibition
respiration rate
Control (Flask 1)
Control (Flask 10)
Test substance (Flask 5)
Reference substance (Flask 2)
Toxicity of 3,5-dichlorophenol (positive control)
The results are presented in Table 1.
The 3-hour EC50 of the reference substance was calculated to be 12 mg/L (the 95% confidence limits were not calculated. The 3-hour EC50 is within the guideline recommended range of 5-30 mg/L), confirming suitability of the activated sludge used.
An activated sludge respiration inhibition 3 hour EC50 value of >1000 mg/l and NOEC value of =1000 mg/l (nominal) were 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 nominal concentration of 1000 mg/l.
The inhibitory effect of the test substance on the respiration rate of aerobic wastewater microorganisms of activated sludge was investigated in a 3 -hour respiration inhibition test according to EU Commission Directive 87/302/EEC and the OECD Test Guideline 209.
The following nominal concentrations of the test substance were tested: 10, 32, 100, 320 and 1000 mg/L. Concentrations exceeding 1000 mg/L were not tested.
In addition, two controls and three different concentrations of the reference item 3,5 -dichlorophenol (5, 16 and 50 mg/L) were tested in parallel. The results of these treatments confirmed suitability of the activated sludge and the method used.
Up to and including the concentration of nominal 1000 mg/L, the test substance has no inhibitory effect on the respiration rate of activated sludge after the incubation period of 3 hours. Thus, the 3-hour NOEC of the test substance was at least 1000 mg/L. This value might even be higher, but concentrations exceeding 1000 mg/L were not tested. The 3-hour EC20, EC50 and EC80 could not be calculated but were clearly higher than 1000 mg/L.
Toxicity to micro-organisms: Activated sludge respiration inhibition 3 hour EC50 > 1000 mg/L; NOEC = 1000 mg/L (nominal) (OECD 209)
EC10 or NOEC for microorganisms:
1 000 mg/L
An activated sludge respiration inhibition 3 hour EC50 value of >1000 mg/L and NOEC value of =1000 mg/L (nominal) were 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 wastewater (activated sludge) bacteria at a nominal concentration of 1000 mg/L. It can therefore be concluded that at the highest concentration of 1000 mg/L tested, no effect was observed.
3-Trimethoxysilylpropyl methacrylate hydrolyses rapidly to form 3-(trihydroxysilyl)propyl methacrylate and methanol.
Methanol does not have adverse effects on micro-organisms (OECD 2004a).
A second high reliability Pseudomonas putida 7 hour growth inhibition study is available (Hüls Corporation, 1995). An EC50 was not reached, but results can be interpolated using regression analysis to give EC50 of 5548 mg/L, EC10 of 2164 mg/L and EC90 of 14 222 mg/L. These results were determined in a reliable study conducted according to an appropriate test protocol, and in compliance with GLP. The report states that test concentrations 2500 and 5000 mg/L showed turbidity and precipitation which may be attributable to hydrolysis of the parent substance and condensation reactions of the silanol hydrolysis product.
Only these two highest concentrations exhibited significant inhibition of respiration rate (11.2% and 46.3%, respectively). Therefore, the inhibitory effects could be related to physical effects.
A third study is also available (Union Carbide, 1993), which gives a sewage micro-organisms growth-inhibition 16 hour EC10 of >2500 mg/L. The original reference was not available for review and the reliability of this result is not assignable.
OECD (2004a): SIDS Initial Assessment Report for SIAM 19, Berlin, Germany, 18-20 October 2004, Methanol, CAS 67-56-1.