[Name confidential or not available]

Toxicity to microorganisms

Link to relevant study record(s)

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.

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.