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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 April 1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Read across on similar substance. Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Justification for type of information:
See read-across justification in Section 13.
Reason / purpose:
other: Target substance
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Qualifier:
according to
Guideline:
other: EEC Official Journal L133 (30 May 1988)
Deviations:
no
GLP compliance:
yes
Vehicle:
no
Details on test solutions:
Direct dispersion in water.
It was considered unrealistic and unnecessary to test at concentrations in excess of 1000 mg/L. However, due to the low water solubility of the test material, the test vessels contained a fine dispersion of the test material in water and not a true solution.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Inoculum
Type: A mixed population of activated sewage sludge micro-organisms.
Source: The aeration stage of the Severn Trent Water Authority sewage treatment plant at Belper, Derbyshire, treating predominantly domestic sewage.
Date of Collection:: 20th April 1989
Preparation: The sample was maintained at 21 ± 1 °C with continuous aeration and used on the day of collection. The pH was buffered to pH 7.6 with sodium hydroxide solution and the suspended solids level adjusted to 3.5 g/L prior to use.
Dilution water: Laboratory tap water, dechlorinated by sodium thiosulphate (hardness approximately 50 mg/L as CaCO3, pH 6.0 - 8.5).
Synthetic Sewage
Type: - dissolved in 1 litre tap water
16 g peptone
11 g meat extract
3 g urea
0.7 g NaCI
0.4 g CaCl2.2H20
0.2 g MgSO4.7H20
2.8 g K2HP04
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Hardness:
Hardness approximately 50 mg/L as CaCO3
Test temperature:
21 ± 1 °C
pH:
pH 6.0 - 8.5
Nominal and measured concentrations:
Test Concentrations: Test material 1000 mg/L, 4 replicates.
Details on test conditions:
Exposure Conditions
Contact time: 30 minutes and 3 hours.
Vessels: 500 mL conical flasks.
Aeration: Compressed air via narrow bore glass tubes
Measuring Apparatus : 250 mL darkened glass BOD bottle fitted with a Yellow Springs BOD probe, (Model 54) and a magnetic stirrer.
Test Concentrations: Test material 1000 mg/L, 4 replicates.
Reference Substance : 3.2, 10 and 32 mg/L (3,5-dichlorophenol)
Temperature: 21 ± 1 °C.
Photo Period: Normal laboratory lighting.
Control: Inoculated medium without test or reference substance.

Procedure: At time "0" 16 mL of synthetic sewage was made up to 300 mL with water in a 500 mL conical flask. 200 mL inoculum were added and the mixture aerated at approximately 0.5 - 1 litre per minute. Thereafter, at 15 minute intervals the procedure was repeated with appropriate amounts of the test or reference substance being added.
As each vessel reached 30 minutes contact time the contents were poured in to the measuring vessel and the rate of respiration measured. The procedure was repeated after a 3-hour contact period.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
30 min
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:
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
Results with reference substance (positive control):
3,5 dichlorophenol EC50 (30 minutes): 20 mg/L
Variation in respiration rates of Controls 1 and 2: ± 3 %
3,5 dichlorophenol EC50 (3 hours): 12 mg/L
Variation in respiration rates of Controls 1and 2: ± 3 %

 The rate of respiration for each culture was measured for a 10-minute period (between approximately 6.5 mg O2/L and 2.5 mg O2/L) after 30 minutes and 3 hours contact time.

The oxygen consumption rates for the control, test and reference cultures after 30 minutes and 3 hours contact time are given below, together with percentage inhibition values as calculated from the equation:

% inhibition = 1 – [2RS / (Rc1+ Rc2)] x 100

Where:

RS = oxygen consumption rate for test and reference samples.

Rc1+ Rc2= Oxygen consumption rates for Controls 1 and 2.

Percent inhibition is plotted against concentration and the EC50values derived by inspection for 30 minutes and 3 hours contact time.

Oxygen consumption rates and percentage inhibition for 30 minutes contact time

Concentration

(mg/L)

O2consumption rates

(mgO2/L/min)

% inhibition

Control

R1

R2

 

Test

1000R1

1000R2

1000R3

1000R4

 

Reference

3.2

10

32

 

0.40

0.38

 

 

0.39

0.38

0.40

0.39

 

 

0.36

0.31

0.13

 

-

-

 

 

0

3

<3>

0

 

 

8

21

67

R1– R4: Replicates 1, 2, 3 and 4

<increase>

Oxygen consumption rates and percentage inhibition for 3 hours contact time

Concentration

(mg/L)

O2consumption rates

(mgO2/L/min)

% inhibition

Control

R1

R2

 

Test

1000R1

1000R2

1000R3

1000R4

 

Reference

3.2

10

32

 

0.38

0.40

 

 

0.38

0.40

0.38

0.41

 

 

0.33

0.29

0.12

 

-

-

 

 

3

<3>

3

<5>

 

 

15

26

69

R1– R4: Replicates 1, 2, 3 and 4

<increase>

Validity criteria fulfilled:
yes
Conclusions:
EC50 30 minutes: >1000 mg/L
EC50 3 hours: >1000 mg/L
Executive summary:

EC50 30 minutes: >1000 mg/L and EC50 3 hours: >1000 mg/L were found in an activated sludge, respiration inhibition test. The test was conducted in accordance with OECD Guideline No 209 and EEC Official Journal L133 (30 May 1988).

It was considered unrealistic and unnecessary to test at concentrations in excess of 1000 mg/L. However, due to the low water solubility of the test material, the test vessels contained a fine dispersion of the test material in water and not a true solution.

Description of key information

A 3 h EC50 of >1000 mg/L was obtained in an activated sludge respiration inhibition test using the read-across substance Zinc Hydroxystannate.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L

Additional information

The short term toxicity of the read-across substance Zinc Hydroxystannate to activated sludge in a respiration inhibition test was investigated in accordance with GLP and the standardised guidelines OECD Guideline No 209 and EEC Official Journal L133 (30 May 1988).

An EC50 3 h of >1000 mg/L was found under the conditions of this study. It was considered unrealistic and unnecessary to test at concentrations in excess of 1000 mg/L. However, due to the low water solubility of the test material, the test vessels contained a fine dispersion of the test material in water and not a true solution.