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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:
1994-04-19 to 1994-04-20
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The test procedure was described in sufficient detail and was in accordance with generally accepted scientific standards, with acceptable restrictions. The restrictions were that a reference substance was not used to check the sensitivity of the microorganisms. The study was conducted in compliance with GLP.
Principles of method if other than guideline:
Method: Oxygen consumption test (Huls method)
GLP compliance:
yes
Vehicle:
no
Details on test solutions:
Test substance was dissolved in water to give a stock solution of 50 mg/ml. This was pipetted in graded concentrations directly in the test flasks.
Test organisms (species):
Pseudomonas putida
Details on inoculum:
The bacterial culture was originally from the Institute for Water, Soil, and Air Hygiene of the BGA (Berlin).
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
5.75 h
Test temperature:
24.5-26.1 °C
Nominal and measured concentrations:
12.5, 25, 37.5 and 50 mg/l
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 ml BOD bottles

- Type (delete if not applicable): closed

- No. of vessels per concentration (replicates): 4 with test substance (2 sterilised with HgCl2).

- No. of vessels per control (replicates): 4 without test substance and sterilised with HgCl2. 5 flasks without test substance or HgCl2.

- Biomass loading rate: 10 E+05 bacteria count/ml

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Oxygen concentration measured at end of test.

TEST CONCENTRATIONS
- Range finding study was used to determine the concentrations used in the main study
Reference substance (positive control):
no
Key result
Duration:
5.75 h
Dose descriptor:
EC10
Effect conc.:
13 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Key result
Duration:
5.75 h
Dose descriptor:
EC50
Effect conc.:
43 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Results with reference substance (positive control):
A positive control was not included.
Reported statistics and error estimates:
Regression analysis performed on results

Table 1Effect of test substance on oxygen consumption

 

Conditions

Concentration of test substance (mg/ml)

0

12.5

25

37.5

50

Test substance with HgCl2

Initial value*

8.24

8.28

8.22

8.14

8.17

Difference**

 

-0.04

0.02

0.11

0.08

Test substance without HgCl2

Final value*

4.25

4.60

5.52

6.01

6.30

Oxygen consumption after incubation

 

3.99

3.69

2.7

2.15

1.86

Absolute difference after incubation

 

 

0.30

 

1.29

1.84

2.31

% effect

 

 

7.6

32.3

46.1

53.5

* Average of 2 (or four for control) cultures

** Difference between control and test values

Linear regression after Probit transformation

Measure value mg/ml

% effect

log 10

Probit

Expected Probit

12.5

7.6

1.0969

3.6245

3.7

25

32.3

1.3979

4.5442

4.4

37.5

46.1

1.6740

4.9024

4.9

50

53.5

1.6990

5.0876

5.2

 

Regression analysis: Log Concentration = a; Probit = y; y=a+bx

a = 0.98

b= 2.47

n= 4

r =0.9898

Conclusions:
A pseudomonas putida respiration inhibition 5.75 hour EC50 of 43 mg/l and EC10 of 13 mg/l were determined in a reliable study described in sufficient detail and conducted in accordance with generally accepted scientific standards, with acceptable restrictions. The restrictions were that a reference substance was not used to check the sensitivity of the microorganisms. The study was conducted in compliance with GLP.

Description of key information

Toxicity to microorganisms: 5.75 hour EC50 43 mg/L, EC10 13 mg/L (Oxygen consumption test).

Key value for chemical safety assessment

EC50 for microorganisms:
43 mg/L
EC10 or NOEC for microorganisms:
13 mg/L

Additional information

A respiration inhibition test in a single bacterial strain is available for 3-aminopropyl(triethoxy)silane (Schoberl, 1994). In this study, a Pseudomonas putida respiration inhibition 5.75-hour EC50 of 43 mg/L and EC10 of 13 mg/L were determined for the registered substance. The study was reliable and conducted in accordance with generally accepted scientific standards and described in sufficient detail and in compliance with GLP with acceptable restrictions. The restrictions were that a reference substance was not used to check the sensitivity of the microorganisms.

To support and/or confirm this result, a second study with an analogous substance, triethoxy(3-isocyanatopropyl)silane (CAS 24801-88-5), is read across. Both substances hydrolyse rapidly and share the same silanol hydrolysis product, 3-aminopropylsilanetriol. The isocyanate in the read-across substance, triethoxy(3-isocyanatatopropyl)silane will undergo very rapid hydrolysis (half-life of about 5 minutes) to form the registered substance, 3-aminopropyl(triethoxy)silane, CAS 919-30-2 and carbon dioxide. 3-Aminopropyl(triethoxy)silane then undergoes rapid hydrolysis to form 3-aminopropylsilanetriol and ethanol. This study reports an activated sludge respiration inhibition 3-hour NOEC value of 10 mg/L and EC10 of 17 mg/L.

In view of the transit time in the sewage treatment plant, STP (about 12 -15 hours); it is considered that the STP microorganisms will be exposed to both the silanol hydrolysis product and the parent substance, 3-aminopropyl(triethoxy)silane.

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

Carbon dioxide which is the by-product of the isocyanate hydrolysis, is exempt from registration under REACH Article 2(7) (a) because it is a substance with sufficiently known information and it is considered to cause minimum risk due to its intrinsic properties. Carbon dioxide is therefore not assessed further.

3-Aminopropyl(triethoxy)silane (CAS 919-30-2) and triethoxy(3-isocyanatopropyl)silane (CAS 24801-88-5) are within an analogue group of substances within which there is some evidence of toxicity to microorganisms, i.e. an effect concentration (EC) could be derived and was <1000 mg/L. Data are available for two substances, one of these substances indicates toxicity, the other substance does not. As a worst-case, data will be read across from the substance that indicates toxicity.

Table 7.6 presents microorganism toxicity data available for substances relevant to 3-aminopropyl(triethoxy)silane. It is considered valid to read-across the results for triethoxy(3-isocyanatopropyl)silane (CAS 24801-88-5) 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 the supporting reports (PFA, 2013ai and PFA, 2013l) attached in Section 13.

Table 7.6. Microorganism toxicity data for relevant substances

CAS

Chemical Name (CAS Name)

Guideline Number

Test method

Klimisch code

Year

Duration

Result: E(I)C50 (mg/L)

Result: NOEC (Or EC10 /EC20) (mg/L)

406679-89-8

Silane, (isocyanatomethyl)dimethoxymethyl-

OECD 209

ASRI

1

2003

3 h

>1000

24801-88-5

Triethoxy(3-isocyanatopropyl)silane

OECD 209

ASRI

1

2013

3 h

180

NOEC 10, EC10 17, EC20 31

Heterotrophic inhibition of the respiration rate was comparable to the total inhibition

In the study with triethoxy(3-isocyanatopropyl)silane (CAS 24801-88-5), an activated sludge respiration inhibition 3 hour NOEC value of 10 mg/L and EC10 of 17 mg/L were reported (Desmares-Koopmans, 2013).

An IC50>5000 mg/L was reported in secondary literature (handbook, Chandra 1997). The original reference was not available for review and no further information is available. The reliability of this result is not assignable.

References:

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

PFA (2013ah). Peter Fisk Associates, STP Microorganism toxicity III-18 Analogue Group report, PFA.300.003.011

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