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Ecotoxicological information

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:
06 Oct - 04 Nov 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Qualifier:
according to
Guideline:
other: EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
GLP compliance:
yes (incl. certificate)
Remarks:
Ministerium für Umwelt und Verkehr, Baden-Württemberg, Germany
Analytical monitoring:
not specified
Details on sampling:
SAMPLING
- Sampling frequency: Sampling was performed on Days 0, 7, 14, 21, and 28
- Sampling method: Removal of 3 bottles from each treatment group for analysis of O2 content. On Day 0 only 1 test bottle from each treatment group was measured.
- Other: Each 3-fold test assay (i.e. each test vessel) was measured twice on the day of sampling. On Day 0, only 1 test vessel was sampled and measured twice.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 3 bottles per treatment group: 0 mg/L test item + 0 mg/L reference item. The same amount of acetone as in the test solutions, but without the test substance was added to the test vessels and allowed to evaporate overnight, corresponding to the test item vessels.
- Reference item: 3 bottles per treatment group: 0 mg/L test item + 2 mg/L reference item
- Toxicity control: 3 bottles per treatment group: 1 mg/L test item + 1 mg/L reference item
Vehicle:
not specified
Details on test solutions:
PREPARATION OF TEST SOLUTION AND TEST BOTTLES
Due to the low solubility in water, the application of the test item was performed by means of an inorganic solvent (acetone). The test item was dissolved in acetone as a stock solution of 60.8 mg/20 mL.
One day prior to the test, the test item was transferred into each test vessel via stock solution in acetone giving the required test concetration of 2 mg/L. The acetone was allowed to evaporate over night. This procedure conforms to a direct application into the test medium.
A sufficient volume of ultra pure grade water was prepared in 5 L volumetric flasks. The flasks were filled at first to about three quarters of their volume with water. The water was strongly aerated for approx. 10 minutes to achieve oxygen saturation and allowed to stand for 24 h without aeration at test temperature. The O2 content was measured afterwards.
The individual salt stock solutions were added to the volumetric flasks (5 mL, each added to 5000 mL deionised water), and the respective reference item was added in the final concentration (2 mg/L for reference test and 1 mg/L for toxicity test). Subsequently, the test bottles were filled with oxygen saturated test medium, i.e. control bottles without test item and reference item, test item bottles and toxicity test bottles with test item and sodium-benzoate. Finally, each vessel was inoculated with 0.5 mL of inoculum.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source of inoculum/activated sludge: municipal sludge plant, Pforzheim, Germany
- Storage conditions: The effluent was kept under aerobic conditions in the period between sampling and application.
- Preparation of inoculum for exposure: The inoculum was filtered through a coarse filter (Macherey & Nagel) and the first 200 mL of the filtrate was discarded.
- Pretreatment: The inoculum was shaken during one week, for starvation. The initial number of microorgansims was determined by preparing dilution series of the inoculum in steps of 10E-01 NaCl solution and counting the bacterial colonies after incubation for at least 3 d in Petri dishes and yeast extract.
- Initial cell numbers: 7.97E+04 bacterial cells on average in each test vessel
Test type:
static
Total exposure duration:
28 d
Details on test conditions:
TEST CONDITIONS
- Composition of medium: Mineral medium, prepared from 4 stock solutions using ultra pure grade water.
- Solubilising agent: acetone
- Test temperature: 20 ± 2 °C
- Aeration of dilution water: A sufficient volume of ultra pure grade water was prepared in 5 L volumetric flasks. The flasks were filled at first to about three quarters of their volume with water. The water was strongly aerated for approx. 10 minutes to achieve oxygen saturation and allowed to stand for 24 h without aeration at test temperature. The O2 content was measured afterwards.
- Continuous darkness: Yes

TEST SYSTEM
- Culturing apparatus: BOD flasks with ground-in-glass stoppers.
- Number of culture flasks/concentration: 3 per measurement date.
- Method used to create aerobic conditions: Aeration of dilution water and inoculum prior to test.
- Measuring equipment: Oxygen concentrations were measured with a WTW Microprocessore Oximeter (OXI 340) and a calibrated electrode.
- Test performed in closed vessels due to significant volatility of test substance: The test was performed in closed bottles.
- Other: Each test vessel was inoculated with 0.5 mL inoculum.
Reference substance (positive control):
yes
Remarks:
Degradation of reference item (Benzoic Acid, Sodium salt) after 28 d: 88.0%.
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
2 mg/L

Tab 1: Degradation [%] of test item, reference and toxicity control

Time [d]

% degradation

8- Hydroxyquinoline

Na-Benzoate

Toxicity Control

7

1.2

64.4

20.0

14

-1.1

78.7

20.9

21

0.3

85.9

19.1

28

6.6

88.0

20.1

The degradation of the reference compound reached 60% by Day 14, confirming the activity of the inoculum.

The criterion for ready biodegradability of > 60% removal of the ThOD within the 28 d period was not reached. Therefore, the test substance cannot be considered as readily biodegradable.

The degradation of the toxicity control was < 25% (required pass level) after 14 d. Therefore, toxic effects of the test substance to the inoculum cannot be excluded. This is not unusual, as the test item acts as a fungicide and bactericide agent.

Validity criteria fulfilled:
yes
Conclusions:
Toxicity control: 20.1% degradation after 28 d.

Description of key information

Toxic effects on microorganisms cannot be excluded (domestic activated sludge, OECD 301 D)

Key value for chemical safety assessment

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

Additional information