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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-10-16 to 2017-12-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
, signed 2016-05-31
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item was directly added on a weight basis due to a water solubility in the order of magnitude of the test concentration. The required mass to obtain accurate test concentrations was applied on pieces of Teflon, which were subsequently added to the dilution water. To achieve a maximum dilution of the test item, the assay was stirred for 20 ± 2 h.
- Controls: Inoculum blank (blank control), abiotic control, and reference assay (3,5-Dichlorophenol)
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Fresh samples of activated sludge are withdrawn on October 16th, 2017 (range finding test) and December 11th, 2017 (main test) from the sewage treatment plant Ruhrverband Kläranlage, Sunthelle 6, 57392 Schmallenberg, Germany, which is mainly fed with municipal wastewater.
- Preparation of inoculum for exposure: The sludge was left alone to settle and the supernatant was decanted. After a washing step with isotonic solution, a small amount of the sludge was weighed and dried. A stock solution of the washed activated sludge was prepared in chlorine-free tap water with a mixed liquor suspended solids level of about 3 – 4 g dry mass sludge/liter (main test 3.2 g/L).
- Pretreatment: The sludge stock solution was fed with synthetic sewage feed (25 ml/L) and was incubated and aerated overnight at 20 ± 2 °C in the dark.
- Initial biomass concentration: Activated sludge was adjusted to a mixed liquor suspended solids level of about 3 – 4 g dry mass sludge/liter (main test 3.8 g/L). The mixed liquor suspended solids level in the test vessel was nominally 1.5 g/L.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
test start: 20 °C
test end: 20 °C
pH:
pH at test end (3 h): 8.2 - 8.5
Nominal and measured concentrations:
Nominal test concentrations in the main test: 0 (control), 62.5, 125, 250, 500, 1000 mg test item/L
Details on test conditions:
TEST SYSTEM
- Test vessel: ! L Erlenmeyer flasks
- Material, size, headspace, fill volume: total volume 500 mL
- Aeration: stirred and aerated (air flow 0.5 to 1 L/min)
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6
- No. of vessels per reference control (replicates): 1
- No. of vessels per abiotic control (replicates): 1
- Sludge concentration (weight of dry solids per volume): main test 3.8 g/L
- Nutrients provided for bacteria: synthetic waste water according to OECD 209

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionized water, free from inhibitory concentrations of toxic substances (e.g. Cu2+)

EFFECT PARAMETERS MEASURED: respiration rate (total oxygen uptake including heterotrophic and nitrification uptake) after 3 h

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
- Test concentrations: 10, 100, 1000 mg/L
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
543 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: CL 95%: 293-782 mg/L
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
500 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
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
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
other: EC80
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
The mean total microbial respiration rates of the activated sludge incubated with 62.5, 125, 250, 500 and 1000 mg test item per liter, respectively, ranged from 23.1 to 28.2 mg O2/(g*h) after three hours. Starting at a loading of 1000 mg/L, m-toluic acid inhibited the respiration of the inoculum significantly. The NOEC was found to be 500 mg m-toluic acid per liter (nominal loading). The EC10 was found to be 543 mg m-toluic acid per liter (CL 95: 293 – 782 mg/L). The EC20, 50 and 80 were found to be higher than the highest concentration tested.
Results with reference substance (positive control):
An EC50 of the reference item 3,5-dichlorophenol for respiration inhibition between 2 and 25 mg/L was observed.
Reported statistics and error estimates:
For each endpoint, the NOEC, LOEC, and, if possible, the EC10, EC20, EC50 and EC80 were determined. A LOEC was calculated by using ANOVA followed by Williams’ test. For trend analysis, a Contrasts test (Monotonicity of Concentration/Response) for metric data and a Cochran-Armitage test for mortality were applied, respectively. Since the test results show a concentration-response relationship, they were statistically analysed to determine EC values together with 95% confidence intervals using Probit-analysis assuming log-normal distribution of the values.
The evaluation of the concentration-effect-relationships and the calculations of effect concentrations were based on the nominal test item concentrations.
For statistical calculations, the computer program ToxRat® was applied.

Mean inhibition (%) of the respiration rate by the test item.

TI = Test item; *: significant difference when compared with control (P < 0.05).

Test item concentration [mg TI/L]

62.5 mg/L

125 mg/L

250 mg/L

500 mg/L

1000 mg/L

18.2 %

1.4 %

6.3 %

9.0 %

15.8 %*

Validity criteria fulfilled:
yes
Conclusions:
Effects of m-toluic acid on the respiration inhibition of activated sewage sludge were determined in this GLP-conform 3 h respiration inhibition test according to OECD test guideline 209. The EC10 for the 3-h respiration inhibition of activated sludge by the test item m-toluic acid was estimated with 543 mg test item per litre (nominal). with a corresponding NOEC of 500 mg/L. The EC20, EC50 and EC80 were found to be higher than the highest tested concentration of 1000 mg/L.

Description of key information

In a GLP-conform OECD 209 respiration inhibition test on activated sludge, inhibiting effects of m-toluic acid on the respiration were observed; the EC10 for the 3-h respiration inhibition of activated sludge amounts to 543 mg/L (nominal). However, the corresponding EC50 is above the highest tested concentration of 1000 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
543 mg/L

Additional information