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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 Feb – 14 Mar 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
yes (incl. certificate)
Remarks:
Landesanstalt für Umwelt, Messungen und Naturschutz Baden-Würtemberg, Germany
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: To the test mixtures (500 mL), which contained deionised water (234 mL), synthetic sewage feed (16 mL) and the appropriate test / reference item concentration, the inoculum of activated sludge (250 mL) was added. The test item was prepared by directly weighing 50 mg of the test item into 500 mL. All components were dispersed by intense aeration.
A stock solution of 3,5-dichlorophenol was prepared by dissolving 200 mg DCP in 200 mL water. The reference item was dosed by dissolving defined volumes of the stock solution in the test solutions.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source: Municipal wastewater treatment plant Pforzheim, Germany, collected from the aeration tank
- Preparation of inoculum for exposure: The sludge was used one day after collection. It was settled and the upper layer with finer solids was decanted and discarded. Before starting the test, the remaining sludge was washed two times with tap water by centrifugation (10 minutes at 3000 rpm). After centrifuging, the supernatant was decanted and discarded and the sludge was resuspended in chlorine free tap water. This procedure was repeated once. The mixed liquor suspended solids (MLSS) in the sludge inoculum were adjusted to a concentration of 3.0 g/L (± 10%). The activated sludge was continuously aerated at the test temperature, the solids did not settle down.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Test temperature:
19.3 – 20.9 °C
pH:
7.21 - 7.25 (control)
6.38 -6.88 (pH of test item concentration and abiotic control)
Nominal and measured concentrations:
control, 100 mg/L (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 1 litre glass beakers filled with 500 mL test solution
- Aeration: yes
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per abiotic control (replicates): 3

TEST MEDIUM / WATER PARAMETERS
- Synthetic medium: The test was performed in a synthetic sewage feed that was prepared with the following amounts of substances in 1 litre of deionised water: 16 g peptone, 11 g meat extract, 3 g urea, 0.7 g NaCl, 0.4 g CaCl2 × 2 H2O, 0.2 g MgSO4 × 7 H2O, 2.8 g K2HPO4.
The solution was sterilized prior to storage. If the preparation was not used immediately it was stored in the dark at a temperature of between 0 – 10 ºC for 4 days.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Respiration rate measurements were conducted 3 hours after test start

TEST CONCENTRATIONS
- Range finding study
Test concentrations: control, 10.0, 100 and 1000 mg/L
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
100 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):
The EC50 of the total oxygen uptake for the reference item 3,5-dichlorophenol (DCP) was estimated to be between 5 and 25 mg/L after 3 hours. This is within the recommended range of 2 to 25 mg/L.
Reported statistics and error estimates:
The statistical evaluation was performed for specific respiration rate of the control and the test item concentrations using SAS® (2002–2010). The
calculation was performed using SAS Software service pack 9.3. The NOEC was determined using the t-Test pooled. P-values below 0.05 showed statistically significant differences to the control.

Table: Results of the range-finding test, specific respiration rates

Test assay nominal [mg/L] 

Specific respiration rates after 3 h [mg O2/(g × h)]

Control (1) 

24.13

Control (2) 

24.58

Control (3) 

23.93

10.0 

23.71

100 

24.06

1000 (1) 

0.88

1000 (2) 

0.27

1000 (3) 

0.63

DCP 0.2 

23.44

DCP 1 

18.35

DCP 5 

14.94

DCP 25 

3.47

Abiotic control 

-0.26

Table: Results of the range-finding test, inhibition

Test assay nominal [mg/L] 

Inhibition after 3 h [%]

10.0 

2.1

100 

0.6

1000 (mean) 

97.6

DCP 0.2 

3.2

DCP 1 

24.2

DCP 5 

38.3

DCP 25 

85.7

Table: Specific respiration rates in the main test

Test assay nominal [mg/L] 

Specific respiration rates after 3 h [mg O2/(g × h)]

Control (1) 

24.11

Control (2) 

25.04

Control (3) 

18.53

Mean

22.56

Stdev

3.52

CV (%)

16

100 (1) 

22.57

100 (2) 

24.65

100 (3) 

26.07

Mean

24.43

Stdev

1.76

DCP 0.2 

23.11

DCP 1 

18.50

DCP 5 

17.09

DCP 25 

2.73

Abiotic control (1)

-0.19

Abiotic control (2)

0.35

Abiotic control (3)

0.01

Table: Inhibition of total oxygen uptake in the main test

Test assay nominal [mg/L] 

Mean inhibition after 3 h [%]

100

-8.3

DCP 0.2 

-2.4

DCP 1 

18.0

DCP 5 

24.2

DCP 25 

87.9

Description of key information

NOEC (3h) = 100 mg/L with activated sludge (nominal, OECD 209)

Key value for chemical safety assessment

Additional information

One study investigating the toxicity of Reaction mass of ammonium diaqua[bis(oxalate)]oxoniobate(1-) hydrate and ammonium hydrogen oxalate oxalic acid (1:1:1) dihydrate to microorganisms is available. The limit test at 100 mg/L test substance concentration was performed with activated sludge in a static test design according to OECD 209 (CBMM Europe BV, Key, 2014, Activated sludge). Mean specific respiration rates in the control and in the 100 mg/L test item concentration were 22.56 and 24.43 mg O2/(g x h), respectively. No significant inhibition occurred, resulting in a NOEC of 100 mg/L.