Fatty acids, tall-oil, compds. with oleylamine

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

not required

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test substance is poorly soluble in water. At test initiation, nominal concentrations of the test substance were established by the addition of weights of the test substance to the respective vessel followed by the addition of RO (reverse osmosis) water (284 mL). Additions of synthetic sewage (16 mL) and microbial inoculums (200 mL) were made to give a final volume of 500 mL.
- Controls: Two controls without test material were tested in each test series, one at the start and one at the end.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Preparation of inoculum for exposure: A sample of activated sludge was obtained the day before the start of the test. The mixed liquor suspended solids (MLSS) content was determined, synthetic sewage (50 mL/L) was added and the mixture was aerated overnight.
- Pretreatment: none
- Initial biomass concentration: ca. 4.0 g MLSS/L

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20-21 °C

pH:

test start: 7.4-7.5
test end: 8.0

Dissolved oxygen:

6.0-7.6 mg O2/L (initial DO concentration in the cultures)

Nominal and measured concentrations:

10, 30, 100, 300, 1000 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: beakers (test bottles) and biological oxygen demand (BOD) bottles
- Aeration: oil-free compressed air at 1 L/min
- No. of vessels per concentration (replicates): 10-300 mg/L: 1; 1000 mg/L: 3
- No. of vessels per control (replicates): 2
- Biomass loading rate: 1.6 g/L (MLSS)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse osmosis (RO) water

OTHER TEST CONDITIONS
- Adjustment of pH: no

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : oxygen concentration after 3 hours contact time

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Duration:

3 h

Dose descriptor:

other: EC80

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50 (3 h): 6.9 mg/L (95% confidence limits, 5.3 and 8.8 mg/L)

Reported statistics and error estimates:

Since the permitted maximum difference for specific respiration rates in control beakers established at the beginning and end of the test period was 15%, this value was used to define the criteria for biologically significant levels of inhibition in mixtures containing the test substance.
The EC50 of the reference substance and 95% confidence limits were calculated using the SAFEstat curvefit programme (SAS Institute, 2002).

Respiration rates were decreased by, at most, 1% in one mixture at a concentration of 1000 mg/L.

Validity criteria fulfilled:

yes

Remarks:

Variations in respiration rates of controls <15%. The EC50 of 3,5-dichlorophenol was in the accepted range of 5-30 mg/L.

Reference 2

Endpoint:

toxicity to microorganisms

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

conducted in accredited testing laboratory however without QA-statement

Qualifier:

according to guideline

Guideline:

DIN 38412-8 (Pseudomonas Zellvermehrungshemmtest)

Deviations:

no

GLP compliance:

no

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 250 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: 400 mg/L
DOC-value of the test solution at test start: 5.3 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

Key result

Duration:

16 h

Dose descriptor:

IC50

Effect conc.:

>= 400 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

Duration:

16 h

Dose descriptor:

IC10

Effect conc.:

>= 400 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

Details on results:

In a saturated solution of the test material (a nominal loading rate 400 mg/L) no significant 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
400	342	348	362	351	4.7
Control cultures	366	369	377	368	0
	363	367

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

Validity criteria fulfilled:

not specified

Description of key information

Toxicity to microorganisms: not toxic to microorganisms: EC10 >1000 mg/L (OECD 209 EU C.11); EC10 > 400 mg/L (DIN 38412, Pseudomonas putida test)

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

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