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Toxicity to microorganisms

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toxicity to microorganisms, other
Toxicity check from the ready biodegradation study.
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 November 2016 to 06 December 2016
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
according to
other: OECD 301F
Version / remarks:
Adopted 17.07.92
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
Mineral medium
Details on test solutions:
- Method:The solutions of the test and reference items in test flasks were prepared, in separate batches, in mineral medium 100 mg/L of test item and 100 mg /l of reference item.
0.2500 g of reference item was weighed into 250 ml measuring flask and made up to the mark giving a concentration of 1 g of reference item in 1 liter.
The test subtance was added directly to the mineral medium at this stage on a weight basis.16.4 mg of the test item was weighed into each of triplicate test flasks when needed. 16.4 ml of stock solution of the reference item was introduced into each of the replicate right flasks.

- Eluate: Mineral medium
concentration of elements in mineral medium (mg/L)
P 116 mg/L
N 1,3 mg/L
Na 86 mg/L
K 122 mg/L
Mg 2.2 mg/L
Ca 9.9 mg/L
Fe 0.05–0.1 mg/L
pH 7.4 ± 0.2

- Controls:
Reference item plus inoculum
Inoculum blank.

- Chemical name of vehicle: Mineral medium
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Sewage Treatment Plant ”Czajka” , Warsaw.
- Preparation of inoculum for exposure: The coarse particles were removed by settling and the supernatant was discarded. The sludge was washed in the mineral medium. The concentrated sludge was suspended in mineral medium to yield a concentration of 3-5 g suspended solids/l and it was aerated for 5 days, at the test temperature of 22 ° C, up to application. A sample was withdrawn just before use for the determination of the dry weight of the suspended solids.
- Initial biomass concentration: 30 mg/L SS
Test type:
Water media type:
other: Mineral medium
Limit test:
Total exposure duration:
28 d
Test temperature:
22º C
7.19 - 8.33
Nominal and measured concentrations:
Nominal: 100 mg/L
Details on test conditions:
- Test vessel: Respirometer flask
- Type: closed
- No. of vessels per concentration (replicates): Two.
- No. of vessels per control (replicates): Three.
- No. of vessels per abiotic control (replicates): One
- Sludge concentration (weight of dry solids per volume): 30 mg/L suspended solids.
- Nutrients provided for bacteria: Reference item and test item.

- Source/preparation of dilution water: The double-distilled water was taken from redistillation set. It must contain no more than 10% of the organic carbon content introduced by the test material. There is 100 mg/L of test item what gives 56.4 mg/L of organic carbon. Thus, the used water must contain less than 5.64 mg/L of organic carbon. This was checked by DOC analysis. The measured value was about 4 mg/L of organic carbon

- Adjustment of pH: No
- Details on termination of incubation: At the end of incubation (28 days) the pH of the contents of the flasks was measured.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The oxygen uptake from the readings taken at regular and frequent intervals was calculated, using the methods given by the manufacturer of the equipment. The data were read out every 112 min during the 28 day test (40 320 min that is 360 readings).

- Test concentrations: 100 mg/L.
Reference substance (positive control):
sodium acetate anhydrous/ acetic acid, sodium salt
Key result
28 d
Dose descriptor:
other: % degradation
Test item plus reference item
Effect conc.:
74.1 other: % biodegradation
Nominal / measured:
not specified
Conc. based on:
test mat.
Basis for effect:
other: Oxygen uptake
Key result
14 d
Dose descriptor:
other: % biodegradation
Effect conc.:
57 other: % biodegradation
Nominal / measured:
not specified
Conc. based on:
test mat.
Basis for effect:
other: Oxygen uptake
Details on results:
According to the OECD guidelone 301 F, if in a toxicity test, containing both the reference item and the test substance, less than 25 % degradation based on COD occurred within 14 days, the test substance can be assumed to be inhibitory.
The toxicity test resulted in 57 % degradation in 14 days, therefore the substance is not considered inhibitory.
Results with reference substance (positive control):
- Results with reference substance valid
- Relevant effect levels: The reference item has reached the pass level (60%) on day 6 (the limit is by day 14).
Validity criteria fulfilled:
The test item is not inhibitory to activated sludge at a concentration of 100 mg/L.
Executive summary:

A screening test was conducted to determine the ready biodegradability of the test item according to OECD method 301F, Manometric respirometry test. The study was conducted under GLP conditions. Inoculum blanks, procedure control with reference susbtance, toxicity control and abiotic sterile control were concurrently conducted. The toxicity control consisted of the reference item at 100 mg/L(acetic acid, sodium salt) plus the test item at a concentration of 100mg/L and the inoculum at a concentration of 30 mg/L SS. The toxicity test resulted in 57 % degradation by day 14 and 74.1 % degradation by day 28. Therefore, in accordance to the above mentioned guideline and the Guidance on Information Requirements and Chemical Safety Assessment, Chapter 7b (see overall remarks), the test item can be assumed to be non-inhibitory to activated sludge and the concentration of 100 mg/L can be used as a NOEC.

Description of key information

Key study: Test method according to OECD 301F. GLP study. The test item did not inhibit the degradation of the positive control at a concentration of 100 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
100 mg/L

Additional information

In accordance to the Guidance on Information Requirements and Chemical Safety Assessment, Chapter 7b, version 3.0 February 2016, page 170, the absesnce of microbial toxicity was inferred from the biodegradation study OECD 301F. The test item does not inhibit the degradation of the positive control at a concentration of 100 mg/L, therefore this concentration can be used as a NOEC value.