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Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
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))
Version / remarks:
adopted: 22 July 2010
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: (EC) No 440/2008, Method C.11
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Date of receipt : 2016-04-05
Expiry date : 2017-09-08
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Test item concentration/s : 100 mg/L, 3 replicates; 100 mg/L with ATU, 2 replicates
Control : 6 replicates; 4 replicates with ATU
Test item concentration in physico-chemical oxygen consumption control : 100 mg/L
Concentration of reference compound 3,5-Dichlorophenol : 2.5, 5, 10, 20 and 40 mg/L
The test item and reference compound concentrations were not confirmed by analytical methods, they were based on nominal concentrations.
Test organisms (species):
activated sludge, domestic
Details on inoculum:
Type : mixed population of aquatic microorganisms (activated sludge)
Origin : aeration tank of a domestic waste water treatment plant (Municipal WWTP Cologne-Stammheim)
Date of collection : 2017-03-13
Microbial inoculum : The sludge was settled and the supernatant was decanted. After centrifuging the sludge (15 min at 3500 rpm and 20°C) the supernatant was decanted again. Approximately 1 g of the wet sludge was dried in order to calculate the amount of wet sludge to achieve a concentration of activated sludge of 3 g/L (dry weight) suspended solids. The calculated amount of sludge was dissolved in synthetic medium and then filled up to a defined end volume with deionised water.
Storage of sludge : aeration of the activated sludge at 20 ± 2 °C, daily fed with synthetic medium
pH of the suspension before application : 7.7
Test type:
not specified
Water media type:
other: deionised water
Limit test:
yes
Total exposure duration:
3 h
Remarks on exposure duration:
Stirring period of the test item before start of incubation time : 1 hour
Test temperature:
20 ± 2 °C
pH:
7.3 - 8.4
Nominal and measured concentrations:
100 mg/L
Details on test conditions:
To measure the oxygen consumption, 250 mL of sludge with the test item (or control or reference compound) was incubated for 3 h in 300 mL closed Erlenmeyer flasks (with air inlet and outlet) and aerated through a glass tube at 50-100 L/h with clean oil-free air. For the measurement, the content of the Erlenmeyer flasks was completely transferred to 250 mL BOD bottles and oxygen content was measured with an oxygen meter (redox electrode).
Six controls without the test item were included in the test design, three at the start and the others at the end of the test series.
A limit test was performed with 3 replicates with a test item concentration of 100 mg/L. Each batch of activated sludge was checked using 5 concentrations in the range of 2.5 – 40 mg/L of 3,5-Dichlorophenol as a reference compound.
Reference substance (positive control):
yes
Remarks:
Reference compound : 3,5-Dichlorophenol (Acros Organics) Coding number : Ökotox_1/04 Purity : 99.9 % Batch number : A0357150 Expiry date : 2020-03-24
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
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
Details on results:
RFR 6634 showed 1.4 % respiration inhibition of activated sludge at the highest test item concentration of 100 mg/L.
RFR 6634 showed no statistical significant difference of respiration inhibition of activated sludge between control and a limit test item concentration of 100 mg/L.
Results with reference substance (positive control):
EC50 of the reference compound was 12.147 mg/L.
Reported statistics and error estimates:
As no significant inhibitory effect was measured at a limit test item concentration of 100 mg/L no statistical analysis was required to determine the EC50.
The No Observed Effect Concentration was calculated according to STUDENT-t test for Homogeneous Variances using the statistics programme ToxRatPro Version 2.10 (released 2010-09-10). The EC50 value for the reference substance was calculated from the respiration rates at different test item concentrations using the same statistics programme mentioned above.
Validity criteria fulfilled:
yes
Remarks:
In the present oxygen uptake rate is 38.464 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour, the coefficient of variation of oxygen uptake was 4.6 %, EC50 of reference was 12.147 mg/L; thus the test is valid.
Conclusions:
RFR 6634 showed 1.4% respiration inhibition of activated sludge at a test item concentration of 100 mg/L. The EC50 is higher than 100 mg/L and the NOEC is equal or higher than 100 mg/L after 3h..
Executive summary:

A study was performed to assess the toxicity of RFR 6634 to bacteria. The purpose of this test was to provide a rapid screening method whereby substances which may adversely affect aerobic microbial treatment plants can be identified, and to indicate suitable non-inhibitory concentrations of test items to be used in biodegradability tests. The study was conducted in accordance with OECD Guideline 209. The activated sludge was exposed to RFR 6634 at a limit test item concentration of 100 mg/L. The respiration rate of each mixture was determined after aeration periods of 3 hours. RFR 6634 showed 1.4% respiration inhibition of activated sludge at a test item concentration of 100 mg/L. The EC50 is higher than 100 mg/L. The NOEC is equal or higher than 100 mg/L. The effect value relates to a nominal concentration, since no analytical monitoring was performed.

Description of key information

A study was performed to assess the toxicity of RFR 6634 to bacteria. The purpose of this test was to provide a rapid screening method whereby substances which may adversely affect aerobic microbial treatment plants can be identified, and to indicate suitable non-inhibitory concentrations of test items to be used in biodegradability tests. The study was conducted in accordance with OECD Guideline 209. The activated sludge was exposed to RFR 6634 at a limit test item concentration of 100 mg/L. The respiration rate of each mixture was determined after aeration periods of 3 hours. RFR 6634 showed 1.4% respiration inhibition of activated sludge at a test item concentration of 100 mg/L. The EC50 is higher than 100 mg/L. The NOEC is equal or higher than 100 mg/L. The effect value relates to a nominal concentration, since no analytical monitoring was performed.

Key value for chemical safety assessment

EC50 for microorganisms:
100 mg/L
EC10 or NOEC for microorganisms:
100 mg/L

Additional information

Results should read "EC 50 > 100 mg/L and NOEC ≥ 100"