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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-02-13 to 2018-04-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 1731200025
- Expiration date of the lot/batch: November 10, 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored in original container; keep original container closed and store in a well-ventilated area at 13–29 °C.
- Stability under test conditions: Stable under recommended storage conditions (SDS)
Analytical monitoring:
not required
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Due to unknown water solubility of the test item, the liquid test item was added directly. The required mass to obtain the accurate test concentrations was applied on pieces of Teflon, which were subsequently added to the dilution water. To achieve a maximum dissolution of the test item, the assay was stirred for 20 ± 2 h (following the indications for poorly water soluble substances in the "Leitfaden für Meldungen neuer Stoffe nach dem Chemikaliengesetz").
Test organisms (species):
activated sludge, domestic
Details on inoculum:
Fresh samples of activated sludge were withdrawn on February 12th, 2018 from the sewage treatment plant Ruhrverband Kläranlage, Sunthelle 6, 57392 Schmallenberg, Germany, which is mainly fed with municipal wastewater. On return to the laboratory the sludge was left alone to settle and the supernatant was decanted. The sludge was washed one time 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 3.9 g dry mass sludge/liter. The sludge stock solution was fed with synthetic sewage feed (see chapter 4.3) and was incubated and aerated overnight at 20 ± 2 °C in the dark. The amount of synthetic sewage applied was 25 mL/L. After this preincubation, the activated sludge was adjusted to a mixed liquor suspended solids level of about 3.8 g dry mass sludge/liter. 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
Post exposure observation period:
None
Test temperature:
20 ± 2 °C
pH:
7.5 ± 0.5
Nominal and measured concentrations:
Nominal test concentrations: 10.0, 100.0. 1000.0 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 1 L Erlenmeyer flasks
- Type (delete if not applicable): Open
- Material, size, headspace, fill volume: 500 mL for set-up
- Aeration: Stirred and aerated for 180 min at 0.5-1 L/min
- Test vessels:
- Inoculum blank: 6 vessels containing only inoculum (3 replicates were measured at the beginning and 3 at the end of the test assay series.
- Test assay A: 1 vessel containing test item (10 mg/L) and inoculum.
- Test assay B: 1 vessel containing test item (100 mg/L) and inoculum.
- Test assay C: 3 vessels containing test item (1000 mg/L) and inoculum.
- Abiotic control: 1 vessel containing test item (1000 mg/L) only.
- Reference assay A: 1 vessel containing reference item (1 mg/L) and inoculum.
- Reference assay B: 1 vessel containing reference item (6 mg/L) and inoculum.
- Reference assay C: 1 vessel containing reference item (36 mg/L) and inoculum.
- Sludge concentration (weight of dry solids per volume): 3.8 g dry mass sludge/liter. A final concentration of 1.5 g dw/L was used in the test.
- Nutrients provided for bacteria:
- For the preparation of the synthetic sewage for sludge stock feeding the following reagents (analytical grade) were used:
- Peptone 160 g/L (16.0 g/100 mL)
- Meat extract 110 g/L (11.0 g/100 mL)
- Urea 30 g/L (3.0 g/100 mL)
- K2HPO4 28 g/L (2.8 g/100 mL)
- NaCl 7 g/L (0.7 g/100 mL)
- CaCl2 x 2H2O 4 g/L (0.4 g/100 mL)
- MgSO4 x 7H2O 2 g/L (0.2 g/100 mL)

- For the preparation of the synthetic sewage for test assay feeding the following reagents (analytical grade) were used:
- Peptone 16.0 g/L (8.0 g/500 mL)
- Meat extract 11.0 g/L (5.5 g/500 mL)
- Urea 3.0 g/L (1.5 g/500 mL)
- K2HPO4 4.8 g/L (2.4 g/500 mL)
- NaCl 0.7 g/L (0.35 g/500 mL)
- CaCl2 x 2H2O 0.4 g/L (0.20 g/500 mL)
- MgSO4 x 7H2O 0.2 g/L (0.10 g/500 mL)

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

OTHER TEST CONDITIONS
- Adjustment of pH: No

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Respiration rate (total oxygen uptake including heterotrophic and nitrification uptake)

TEST CONCENTRATIONS
A range-finding test was conducted to check if any adverse effect can be observed up to and including a nominal loading of 1000 mg/L test item.
- Test concentrations: 10, 100, and 1000 mg/L
- Results used to determine the conditions for the definitive study:
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
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Key result
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
Results with reference substance (positive control):
The EC50 for the reference item was found to be 19.5 mg/L and is therefore within the accepted range of the OECD 209 guideline of 2 to 25 mg/L

Table 1. Mean inhibition (%) of the respiration rate by the test item

Test item concentration [mg TI/L]

10 mg/l

100 mg/L

1000 mg/L

-5.30%

-18.30%

-358.90%

TI = Test item; No significant difference when compared with control (P < 0.05).

Considering the observed stimulation of microbial respiration hence the absence of an adverse effect up to the limit concentration, EC-values are reported as EC10 ≥ 1000 mg/L, EC20 ≥ 1000 mg/L, EC50 ≥ 1000 mg/L, and EC80 ≥ 1000 mg/L, respectively (nominal concentrations).

A significant abiotic oxygen demand by the test item was not observed during the 3 h test period.

The test is considered valid, as:

- with 38 mg O2/g activated sludge (dry weight of suspended solids) in an hour, the oxygen uptake rate in blank controls was ≥ 20 mg O2/g in an hour,

- with 8 %, the coefficient of variation of oxygen uptake rate in control replicates is ≤ 30 %,

- with 19.5 mg/L, the EC50 (3 hours) of the reference substance 3,5-dichlorophenol is in the accepted range of 2 to 25 mg/L.

Validity criteria fulfilled:
yes
Conclusions:
There was no significant inhibition of bacterial respiration by the test item up to and including a nominal concentration of 1000 mg per liter. An activated sludge respiration inhibition test according to OECD 209 resulted in an EC50 > 1000 mg/L and a NOEC ≥ 1000 mg/L.
Executive summary:

To investigate potential harmful effects of the test item Sodium-Isostearoyl-Lactylate on microorganisms, an activated sludge respiration inhibition test was performed according to OECD 209 and under GLP.

A definitive test with the nominal test concentrations of 10, 100, and1000 mg /L was performed. The pH in the test vessels was 7.5 ± 0.5. No inhibition was observed in the test vessels up to and including a nominal concentration of 1000 mg/L test concentration (-358.9 to -5.3%)

The respiration rates in the test vessels were not significantly lower than the respiration rate of the controls (Williams' Multiple Sequential t-test).

The results are therefore:

EC50 > 1000 mg/L

NOEC ≥ 1000 mg/L

The test is considered valid, as:

- with 38 mg O2/g activated sludge (dry weight of suspended solids) in an hour, the oxygen uptake rate in blank controls was ≥ 20 mg O2/g in an hour,

- with 8 %, the coefficient of variation of oxygen uptake rate in control replicates is ≤ 30 %,

- with 19.5 mg/L, the EC50 (3 hours) of the reference substance 3,5-dichlorophenol is in the accepted range of 2 to 25 mg/L.

Description of key information

There was no significant inhibition of bacterial respiration by the test item up to and including a nominal concentration of 1000 mg per litre. An activated sludge respiration inhibition test according to OECD 209 resulted in an EC50 > 1000 mg/L and a NOEC ≥ 1000 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

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