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Toxicity to microorganisms

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Reference
Endpoint:
toxicity to microorganisms
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Data is from peer reviewed journal
Justification for type of information:
Data is from peer reviewed journal
Qualifier:
according to
Guideline:
other: as below
Principles of method if other than guideline:
Using analogous methods of the cell multiplication inhibition test, the toxicity threshold (TT) of test material was determined for flagellate protozoa (Entosiphon sulcatum).
GLP compliance:
not specified
Analytical monitoring:
not specified
Details on sampling:
No data
Vehicle:
not specified
Details on test solutions:
No data
Test organisms (species):
Entosiphon sulcatum
Details on inoculum:
To prepare feeding of the Entosiphon stock or preliminary cultures with bacteria, decant nutrient solution and suspend the bacterial centrifugate with a quantity of a 50% solution of stock solution I in sterile double-distilled water until the initial volume is reached again. Centrifuge and decant again this suspension as described above. Take up the centrifugate with a 50°/0 solution of stock solution I in sterile double-distilled water.
Test type:
static
Water media type:
freshwater
Total exposure duration:
72 h
Remarks on exposure duration:
No data
Post exposure observation period:
No data
Hardness:
No data
Test temperature:
25°C
pH:
6.9
Dissolved oxygen:
No data
Salinity:
No data
Conductivity:
No data
Nominal and measured concentrations:
No data
Details on test conditions:
For maintenance of the test strain of Entosiphon sulcatum continuously inoculate the expected required number of stock cultures at 72 h or 96 h intervals. For this, mix 8 ml of stock solution I and 8 ml of sterile double-distilled water in 300-ml Erlenmeyer flasks stoppered with metal caps and add 2 ml of a stock culture and 2 ml of the adjusted bacterial suspension for Entosiphon stock cultures. Store stock cultures at 25°C. Before inoculation control the flasks containing the stock cultures by means of an inverse microscope. Pretreatment of the bacteria used for feeding preliminary cultures of Entosiphon sulcatum does not differ from that of bacteria used for feeding the stock cultures. Keep preliminary cultures at 25°C over 72 h before using them for inoculation of the test cultures. Before inoculation control each flask containing the preliminary cultures by means of an inverse microscope.
Reference substance (positive control):
not specified
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
236 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Remarks:
Multiplication of the cells was inhibited at this concentration.
Details on results:
No data
Results with reference substance (positive control):
No data
Reported statistics and error estimates:
No data
Validity criteria fulfilled:
not specified
Conclusions:
Based on the growth inhibition of microorganisms, LOEC was 236 mg/l for test material in Entosiphon sulcatum when it was exposed for 72 h.
Executive summary:

In toxicity to microorganism study for test material the biological effect was evaluated by using analogous methods of the cell multiplication inhibition test. For maintenance of the test strain of Entosiphon sulcatum continuously inoculate the expected required number of stock cultures at 72 h or 96 h intervals. For this, mix 8 ml of stock solution I and 8 ml of sterile double-distilled water in 300-ml Erlenmeyer flasks stoppered with metal caps and add 2 ml of a stock culture and 2 ml of the adjusted bacterial suspension for Entosiphon stock cultures. Store stock cultures at 25°C. Before inoculation control the flasks containing the stock cultures by means of an inverse microscope. Pretreatment of the bacteria used for feeding preliminary cultures of Entosiphon sulcatum does not differ from that of bacteria used for feeding the stock cultures. Keep preliminary cultures at 25°C over 72 h before using them for inoculation of the test cultures. Before inoculation control each flask containing the preliminary cultures by means of an inverse microscope. The toxicity threshold (TT) of test material was determined to be 236 mg/l for Entosiphon sulcatum. Therefore LOEC was considered to be 236mg/l for 72h in Entosiphon sulcatum.

Description of key information

Toxicity to microorganism:

In toxicity to microorganism study for test material the biological effect was evaluated by using analogous methods of the cell multiplication inhibition test. For maintenance of the test strain of Entosiphon sulcatum continuously inoculate the expected required number of stock cultures at 72 h or 96 h intervals. For this, mix 8 ml of stock solution I and 8 ml of sterile double-distilled water in 300-ml Erlenmeyer flasks stoppered with metal caps and add 2 ml of a stock culture and 2 ml of the adjusted bacterial suspension for Entosiphon stock cultures. Store stock cultures at 25°C. Before inoculation control the flasks containing the stock cultures by means of an inverse microscope. Pretreatment of the bacteria used for feeding preliminary cultures of Entosiphon sulcatum does not differ from that of bacteria used for feeding the stock cultures. Keep preliminary cultures at 25°C over 72 h before using them for inoculation of the test cultures. Before inoculation control each flask containing the preliminary cultures by means of an inverse microscope. The toxicity threshold (TT) of test material was determined to be 236 mg/l for Entosiphon sulcatum. Therefore LOEC was considered to be 236mg/l for 72h in Entosiphon sulcatum.

Key value for chemical safety assessment

Additional information

Toxicity to microorganism:

Two experimental studies was used to explain the effect of test material on microorganisms:

In toxicity to microorganism study for test material the biological effect was evaluated by using analogous methods of the cell multiplication inhibition test. For maintenance of the test strain of Entosiphon sulcatum continuously inoculate the expected required number of stock cultures at 72 h or 96 h intervals. For this, mix 8 ml of stock solution I and 8 ml of sterile double-distilled water in 300-ml Erlenmeyer flasks stoppered with metal caps and add 2 ml of a stock culture and 2 ml of the adjusted bacterial suspension for Entosiphon stock cultures. Store stock cultures at 25°C. Before inoculation control the flasks containing the stock cultures by means of an inverse microscope. Pretreatment of the bacteria used for feeding preliminary cultures of Entosiphon sulcatum does not differ from that of bacteria used for feeding the stock cultures. Keep preliminary cultures at 25°C over 72 h before using them for inoculation of the test cultures. Before inoculation control each flask containing the preliminary cultures by means of an inverse microscope. The toxicity threshold (TT) of test material was determined to be 236 mg/l for Entosiphon sulcatum. Therefore LOEC was considered to be 236mg/l for 72h in Entosiphon sulcatum.

Similarly in Toxicity to microorganisms study for test chemical, the biological effect was evaluated. By using analogous methods of the cell multiplication inhibition test. Prior to the preparation of test cultures neutralize the Ethyl butyrate of a known content in sterile double-distilled water to be tested, select the concentration of the acid or alkaline solution to be added in such a way that the volume added is kept as small as possible. Do not adjust pH where the eft'oct of the pH of the pollutant solution to be studied is to be included in the test. Prepare from the pollutant solution prepared as described or the sewage and sterile double-distilled water, four parallel dilution series in 300-ml Erlenmeyer flasks, stoppered with cotton-lined plastic caps. Each of the dilutions contains 1 part r/v of the pollutant solution in 20 to 214 parts 0/v of mixture. Prepare the dilution series as follows: the first flask of each series contains 160ml of pollutant solution at the start. Starting from this flask prepare the subsequent dilution stein at a constant dilution ratio by consistently mixing 80ml of preliminary pollutant dilution and 80ml double distilled water. Consequently, each flask contains 80 ml of culture liquid at the start. Make up each flask of the three dilution series to be inoculated to 100ml by adding 5 ml each of stock solution I, 5 ml each of stock solution II and I0 ml each of the prepared bacterial suspension from the preliminary culture having a known adjusted extinction value

Keep stock cultures of the test strain, Pseudoraonas putida, on the nutrient for stock and preliminary cultures in agar slant tubes. Prepare, for onward culturing of the test strain, new stock cultures at intervals of 1 week each. Incubate the inoculated stock cultures at 25"C for 24 h and keep in stock. If needed, prepare preliminary cultures from stock cultures on the above mentioned nutrient medium in agar slant tubes and incubate at 25°C for 24 h. Then, wash off the cell material with sterile saline. Determine the extinction of the monochromatic radiation at 436nm for a 10ram layer of the bacterial suspension by photoelectric measurement. On the basis of the values thus measured, adjust the final turbidity value of the bacterial suspension by means of sterile saline in such a way that the extinction value for a measuring sample " that has been subject to onward dilution 1 + 9 with saline will correspond to the extinction value of a Formazin standard suspension TE/F/436 nm= 10.*

The toxicity threshold (TT) of test chemical was determined to be 140mg/l in  bacteria Pseudomonas putida. Therefore LOEC was considered to be 140mg/l for 16 h for Pseudomonas putida.