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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:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Details on test solutions:
Direct dispersion in water. 500 mg test substance was dispersed in approximately 250 ml of water and subjected to ultrasonication for approximately 30 minutes. Synthetic sewage (16 ml), activated sewage sludge (200 ml) and water were added to a final volume of 500 ml to give a test concentration of 1000 mg/l.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
A mixed population of activated sewage sludge microorganisms was obtained on 7th April 1998 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Belper, Derbyshire, UK, which treats predominantly domestic sewage. The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of 21°C and was used on the day of collection. The pH of the sample was 7.3 and the suspended solids equal to 3.7g/l prior to use.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Post exposure observation period:
At 30 minute and 3 hour contact times, aliquotes were removed from the conical flask and poured into the measuring vessel (250 ml darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a Yellow Spring dissolved oxygen meter fiitted with a BOD probe. The contents of the measuring vessel were stirred constantly be magnetic stirrer. The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace for an approximate 10 minute period.
Test temperature:
21°C
Nominal and measured concentrations:
Based on the results from a range finding study, the definitive test was carried out as a limit test at a test concentration of 1000 mg/l.
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 ml conical flask

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Laboratory dechlorinated tap water

TEST CONCENTRATIONS
- Spacing factor for test concentrations: limit test at 1000 mg/l, in triplicate
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
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
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 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):
- Results with reference substance valid? Yes
- Relevant effect levels: EC50 5.2 mg/l

Table 1: Controls, reference substance (3, 5-dichlorophenol) and test substance: oxygen consumption and percentage inhibition of the respiration rate.

Flask

Nominal concentration (mg/l)

Oxygen consumption rate

(mg O2/l/min)

% Inhibition

respiration rate

Inoculum control

-

0.48

-

Inoculum control

-

0.40

-

 

Test substance

1000

0.42

5

Test substance

1000

0.42

5

Test substance

1000

0.46

5

 

Reference substance

32

0.05

89

Reference substance

10

0.11

75

Reference substance

3.2

0.31

30

 

Validity criteria fulfilled:
yes
Conclusions:
An ASRI 3hr EC50 of >1000 mg/l and NOEC of >= 1000 mg/l were determined in a reliable study conducted according to an appropriate test protocol, and in compliance with GLP.
Endpoint:
activated sludge respiration inhibition testing
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Please refer to the attached justification for grouping of substances provided in IUCLID Section 13.
Reason / purpose:
read-across source
Reason / purpose:
read-across source
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
Remarks on result:
other: Source: Safepharm Laboratories 1998
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: Source: Hüls 1993
Duration:
5.8 h
Dose descriptor:
EC10
Effect conc.:
1 115 other: µl/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: (Test 1)
Duration:
5.75 h
Dose descriptor:
EC10
Effect conc.:
1 421 other: µl/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: (Test 2)
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1993-09-28 to 1993-10-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Principles of method if other than guideline:
Method: Oxygen consumption test (Huls method)
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

- Method: A 5 l wide-necked bottle, with magnetic stirrer and 3520 ml distilled aqua, was covered with aluminum foil and autoclaved for 30 minutes at 120°C and 1 bar. After cooling down to room temperature, 200 ml of sterile stock solution and 90 ml seed suspension (liquid culture of the test organism, about 19 ​​hrs incubation, count 10 x 105 cells / ml) were stirred for 5 minutes.
The test substance was pipetted directly into the test bottles. Subsequently, the emulsifier was added. Then the flasks were completely filled with the inoculated culture solution and stoppered.
Emulsifier additive: Nonylphenol ethoxylated propoxylated (EO 10Mol PO 5Mol).

Test organisms (species):
Pseudomonas putida
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
5.8 h
Post exposure observation period:
At the end of the examination, 1 ml HgCl2 solution (final concentration about 3 mg HgCl2/l) was added to each flask to stop the biochemical reactions. Subsequently, the oxygen measurements were performed.
Test temperature:
Test 1: 24.7 - 25.0°C. Test 2: 24.8 - 25.1°C
Nominal and measured concentrations:
Test 1: 500, 1000, 1500, 2000 µl/L. Test 2: 1000, 1500, 1750, 2000 µl/L
Details on test conditions:
TEST SYSTEM

- Test vessel:

- Type (delete if not applicable): closed

- Material, size, headspace, fill volume: 100mL glass flasks with ground glass stopper

- No. of vessels per concentration (replicates): Four, two with HgCl2

- No. of vessels per blank (replicates): Nine, four with HgCl2


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : oxygen content of the test solutions measured using oxygen electrode.
Reference substance (positive control):
no
Duration:
5.8 h
Dose descriptor:
EC10
Effect conc.:
1 115 other: µl/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: (Test 1)
Duration:
5.75 h
Dose descriptor:
EC10
Effect conc.:
1 421 other: µl/l
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: (Test 2)
Details on results:
Mean bacteria-inhibiting concentration: 1268µl/l (1.3 ml/l to 2 significant figures)

Test 1:

 

Control

Test substance concentration:

500µl/L

1000µl/L

1500µl/L

2000µl/L

Oxygen concentration in flasks with HgCl2at start of incubation

8.44

 

8.28

8.63

8.65

8.55

8.57

8.52

8.43

8.54

8.32

8.47

8.43

Average value

8.45

8.60

8.44

8.52

8.48

Oxygen concentration in flasks without HgCl2at end of incubation

4.07

 

4.01

4.04

4.03

4.36

4.62

5.14

4.02

4.05

4.61

4.50

5.25

Average value

4.04

4.04

4.49

4.56

5.20

O2Consumption during incubation

4.41

4.56

3.95

3.96

3.28

Absolute difference during incubation

 

-0.14

0.46

0.45

1.13

% effect during incubation

 

-3.3

10.4

10.2

25.6

Test 2:

 

Control

Test substance concentration:

1000µl/L

1500µl/L

1750µl/L

2000µl/L

Oxygen concentration in flasks with HgCl2at start of incubation

8.22

 

7.89

8.05

8.23

8.11

8.10

8.08

8.16

8.26

8.30

8.26

8.12

Average value

8.08

8.25

8.21

8.18

8.10

Oxygen concentration in flasks without HgCl2at end of incubation

4.04

 

3.89

3.90

4.25

4.64

4.70

4.94

3.99

4.27

4.69

4.50

4.73

Average value

3.96

4.26

4.67

4.60

4.84

O2Consumption during incubation

4.13

3.99

3.54

3.58

3.27

Absolute difference during incubation

 

0.14

0.59

0.55

0.86

% effect during incubation

 

3.4

14.2

13.2

20.8

 

 

 

Conclusions:
A 5hr EC10 of 1.3 ml/l for toxicity to Pseudomonas putida was determined in a reliable study conducted according to an appropriate test protocol and in compliance with GLP.

Description of key information

Trimethoxy(2-methylpropyl)silane (CAS 18395-30-7): Toxicity to microorganisms: 5hr EC10 of 1.3 ml/l (1200 mg/l) for toxicity to Pseudomonas putida, RL1

Silane, trimethoxy(1,1,2-trimethylpropyl)- (CAS 142877-45-0): Limit test: 3-h EC50 > 1000 mg/l and NOEC ≥ 1000 mg/l; respiration rate (OECD, 209), RL1

Key value for chemical safety assessment

Additional information

There are no microorganism toxicity data available for trimethoxy(2,4,4-trimethylpentyl)silane (CAS 34396-03-7), therefore good quality data for the structurally-related substance trimethoxy(2-methylpropyl)silane (CAS 18395-30-7) and from Silane, trimethoxy(1,1,2-trimethylpropyl) - (CAS 142877-45-0), have been read across.

All substances share related hydrolysis products (2,4,4-trimethylpentyl)silanetriol, 2-methylpropyl)silanetriol and (1,1,2-trimethylpropyl)silanetriol; the other hydrolysis product is methanol. Methanol does not have adverse effects on microorganisms (OECD, 2004a and b). Both substances are expected to undergo minimal hydrolysis within the timescale of an ASRI test.

All substances are within a wider analogue group than the trimethoxy(2,4,4-trimethylpentyl)silane analogue group discussed in Section 1.4. Substances within this wider analogue group, in general, exhibit no evidence of significant toxicity to microorganisms.

It is therefore considered valid to read-across the results for trimethoxy(2-methylpropyl)silane (CAS 18395-30-7) and from Silane, trimethoxy(1,1,2-trimethylpropyl) - (CAS 142877-45-0) to fill the data gap for the registered substance.

This wider analogue group for the toxicity to microorganisms endpoint consists of a number of sub-classes of substances. However, read-across is carried out between substances with the same sub-class in most cases.

Table 7.4.2 presentsmicroorganism toxicity data available for substances within the sub-class of alkoxysilanes, silanols, acetoxysilanes and chlorosilanes, etc, where the Si part is of low-biological reactivity, once any hydrolysis is accounted for.

Table 7.4.2 Microorganism toxicity data available for substances within the relevant sub-class of chemicals

CAS

Name

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

Result: NOEC (or EC10/ EC20) (mg/l)

Guideline Number

Test method

Species

Duration

Reliability

001000-50-6

Butylchlorodimethylsilane

>340

 

88/302/EC

ASRI

 

3h

1a

001066-40-6

Hydroxytrimethylsilane

6670

 

OECD 209 and ISO 1892

ASRI

 

Uncertain

1a

001185-55-3

Trimethoxy(methyl)silane

>100

 

OECD 209

ASRI

 

3hr

1a

031795-24-1

Potassium methylsilanetriolate

>100

 

OECD 209

ASRI

 

3 h

1a

087135-01-1

1,6-Bis(trimethoxysilyl)hexane

>1000

 

OECD 209

ASRI

 

3h

1a

017980-47-1

Triethoxyisobutylsilane

>1000

≥1000

OECD 209

ASRI

 

3h

1a

005894-60-0

Trichloro(hexadecyl)silane

>1000

 

OECD 209

ASRI

 

3h

1a

016415-12-6

Hexadecyltrimethoxysilane

>1000

 

OECD 209

ASRI

 

3 hr

1a

002943-75-1

Triethoxyoctylsilane

>1000

 

OECD 209

ASRI

 

3 hr

1a

016068-37-4

4,4,7,7-tetraethoxy-3,8-dioxa-4,7-disiladecane

>8000

8000

DIN 38 412, Part 8 (Pseudomonas cell multiplication inhibition test)

growth inhibition test

P putida

16 h

1c

035435-21-3

Triethoxy(2,4,4-trimethylpentyl)silane

>100

 

OECD 209

ASRI

 

3hr

1a

018395-30-7

Trimethoxy(2-methylpropyl)silane

 

EC10 1.3 ml/l

Huls AG method

oxygen consumption

P putida

5.8 h

2c -don't use to derive PNEC

142877-45-0

Silane, trimethoxy(1,1,2-trimethylpropyl)-

>1000

1000

OECD 209

ASRI

 

3 hr

1a

013154-25-1

Chlorotri(3-methyl-propyl)silane

>1000

100

OECD 209

ASRI

 

3 hr

2b

126990-35-0

Dicyclopentyldimethoxysilane

>water solubility

 

OECD 209//EU C11/Huls AG method (WOE)

ASRI/ASRI/oxygen consumption P putida

 

3 hr

1a/1a/2c

139147-73-2

Silane, dichlorodicyclopentyl-

>100

 

OECD 209

ASRI

 

3 hr

1a

 

Additional information is given in a supporting report (PFA, 2013j) attached in Section 13 of the IUCLID dossier.

The influence of the test item on the respiration rate on the activated sludge was investigated in a 3 -h limit test test according to the OECD 209 and GLP (Safepharm, 1998). The concentration of the test item in the limit test was 1000 mg/l. Up to and including the concentration of 1000 mg/l, the test item had no significant inhibitory effect on the respiration rate of activated sludge after the incubation period of three hours. The EC50was at > 1000 mg/l and the NOEC at ≥1000 mg/l.

A second study with Pseudomonas putida was available (Hüls AG, 1993). A 5hr EC10 of 1.3 ml/l for toxicity to Pseudomonas putida was determined in a reliable study conducted according to an appropriate test protocol and in compliance with GLP.