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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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Qualifier:
according to guideline
Guideline:
DIN 38412-8 (Pseudomonas Zellvermehrungshemmtest)
Deviations:
no
GLP compliance:
yes
Remarks:
Quality Assurance Programme/GLP standards not specified
Specific details on test material used for the study:
- Lot/batch No.: UG-181-96
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: water accommodated fraction (WAF)
A saturated stock solution of the test material with an initial weight of 240 mg was prepared in 500 ml of water for injection and stirred overnight at 20 °C to 25 °C. After a filtration step over a sterile membrane filter (Minisart NML, Sartorius, Göttingen, Germany, pore size 0.2 µm) the solution was used in the test.
- Controls: water for injection
Test organisms (species):
Pseudomonas putida
Details on inoculum:
- Laboratory culture: Pseudomonas putida DSM 50026, Stamm Berlin 33/2 obtained from the German collection of microorganisms (DSM, 38124 Braunschweig, Germany)
- Method of cultivation: Stock cultures were stored in a freezer at -80 °C in test tubes with Casein soya bean digest broth containing 7.5 % DMSO. The working culture of the strain Pseudomonas putida was stored in test tubes in solid nutrient medium. For the preservation of the test strain new strain cultures were made at one week intervals. These cultures were incubated and stored at 25 °C.
- Preparation of inoculum for exposure: The material of inoculation for the precultures was taken from a working culture up to 7 days old. One day prior to the start of the experiment the inoculation was multiplied in liquid medium. This grown preculture was diluted with medium in a way that a defined turbidity of FAU (Formazine Attenuation Units) = 10 was yielded arithmetically, e. g. by addition of 10 ml of a bacterial suspension with a turbidity of FAU = 100 to 90 ml of medium. The preculture was incubated for 7 hours at 21 °C ± 1 °C. After the incubation period the bacterial suspension was diluted with culture medium so that a defined turbidity of FAU = 50 was yielded arithmetically.
- Initial biomass concentration: FAU = 5
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
16 h
Test temperature:
21°C +/- 1 °C
Nominal and measured concentrations:
nominal loading rate: 380 mg/L
DOC-value of the test solution at test start: 2.0 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 300 mL Erlenmeyer flasks, fill volume 100 mL
- Bacterial starting concentration: FAU = 5
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 5

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: nutrient solution containing dextrose (2 g/L)
- Culture medium different from test medium: The culture medium contained supplemental yeast extract (50 mg/L)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): growth inhibition measured at test end
Reference substance (positive control):
no
Duration:
16 h
Dose descriptor:
IC50
Effect conc.:
>= 380 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Remarks on result:
other: results are expressed in terms of loading rates
Key result
Duration:
16 h
Dose descriptor:
IC10
Effect conc.:
>= 380 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Remarks on result:
other: results are expressed in terms of loading rate
Details on results:
In a saturated solution of the test material (a nominal loading rate 380 mg/L) no inhibition effects on the test organisms were observed.

Table 1: Formazine Attenuation Units (FAU) at test end (16 ± 1 h)

Nominal loading rate

(mg/l)

Formazine Attenuation Units (FAU/436 nm)

Inhibition

(%)

Replicate

Mean value

1

2

3

380

492

499

486

492

-1.7

Control cultures

487

481

484

484

0

491

477

starting value of the biomass in the control (t0): 8

Validity criteria fulfilled:
not specified
Conclusions:
The results indicate that the test material is not toxic to microorganisms up to the limits of its water solubility and a NOEC and thus a PNEC cannot be determined.

Description of key information

Toxicity to microorganisms: not toxic to microorganisms: inhibition of growth < 10% (DIN 38412, part 8, Pseudomonas cell multiplication inhibition test) at a nominal loading rate of 380 mg/L.

Key value for chemical safety assessment

Additional information

EC10 or NOEC for microorganisms > water solubility


The results indicate that the test material is not toxic to microorganisms up to the limits of its water solubility and a NOEC and thus a PNEC cannot be determined.