Nonanoic acid

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

toxicity to microorganisms, other

Remarks:

Fresh water ciliate Tetrahymena pyriformis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Data on aliphatic compounds published 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well documented publication which meets basic scientific principles. Data from ciliate growth inhibition tests, preferably with the species Tetrahymena are also relevant for the risk assessment for STPs.

Qualifier:

no guideline followed

Principles of method if other than guideline:

TETRATOX assay: Tetrahymena pyriformis population growth impairment test

GLP compliance:

no

Specific details on test material used for the study:

The aliphatic chemicals used in the study were obtained from Aldrich Chemical Co., Milwaukee, WI and MTM Research Chemicals, or Lancaster Synthesis Inc., Windham, NH in the highest available purity at the time of purchase. The purity of n-nonanoic acid was >95%.

Analytical monitoring:

no

Vehicle:

yes

Remarks:

According to Schultz et al. (1997), for highly soluble compounds no solvent was used - others were used as stock solution in DMSO. Based on the limited solubility of n-nonanoic acid DMSO was probably used.

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- stock solutions were prepared by mixing test substance with sterile distilled water or with reagent-grade dimethyl sulfoxide (DMSO) just prior to use (within 1 hour). Concentration of DMSO in the test medium did not exceed 0.75% (max. 375 µL stock solution per 50 mL of medium). This concentration had no effect on the growth of the test organisms.

Test organisms (species):

Tetrahymena pyriformis

Details on inoculum:

- Laboratory culture: yes
- Test organism: Tetrahymena pyriformis (strain GL-c) in log-growth-phase
- Initial biomass concentration: 2500 cells/mL (i.e. 0.20 mL of a 48-h culture)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

40 h

Remarks on exposure duration:

Test duration allows for 8 to 9 cell cycles in controls.

Hardness:

See details on growth medium below.

Test temperature:

27 ± 1 °C

pH:

The Tetrahymena growth medium is TRIS-buffered at pH 7.4. Based on the limited buffer capacity at this pH towards the acidic range, more acidic pH values must be assumed for higher test item concentrations (see details on results for further information).

Dissolved oxygen:

Not reported

Salinity:

freshwater

Nominal and measured concentrations:

nominal concentrations; at least five graduate concentration levels, individual concentrations not reported

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): Erlenmeyer flasks, foam-stoppered
- Material, size, headspace, fill volume: glas, 250 mL, 150 mL, 50 mL
- Aeration: no data
- No. of organisms per vessel: 1.25 E5 (2500 cell/mL) in log-growth phase
- No. of vessels per concentration (replicates): 2 + 1 blank
- No. of tests per substance: 3 replicate independent tests;

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: sterile distilled water
- Culture medium different from test medium: no
- Test medium: Proteose peptone (5 g), D-glucose (5 g), Yeast extract (1 g,) Tris_HCl (1.2114 g) in 1000 mL distilled Water supplemented with 100 mL each of two salt solutions (Chlorides and Sulfates). Medium is sterilized. Details given in Schultz et al. (1997).

OTHER TEST CONDITIONS
- Adjustment of pH: non-neutralized (explicitly reported in database excerpt; Schultz, 2006; T05017)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Growth impairment: population density is measured spectrophotometrically (absorbance at 540 nm)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: no data
- Range finding study: preliminary to the definite test (3 independent replicate assays) a range finding assay was performed: one to two range-finding tests were performed before the actual testing, with determination of pH for lowest test item concentration resulting in complete inhibition of cell replication because of the known sensitivity of the system to pH (at pH <= 5.5 doubling time approaches infinity); no details reported for isobutyric acid.)

STATISTICAL EVALUATION
- results are given as 50 % inhibitory growth concentration in mM (IGC50)
- IGC50 values and the 95% fiducial intervals are determined by Probit analysis using the percent control-normalized absorbance as the dependent variable and the toxicant concentration in mg/L as the independent variable

Reference substance (positive control):

not specified

Key result

Duration:

40 h

Dose descriptor:

IC50

Effect conc.:

71 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth inhibition

Remarks on result:

other: original value reported: -log (IGC50) = 0.3509

Details on results:

The results are based on non-neutralized test item solutions (explicitly reported in database excerpt; Schultz, 2006; T05017). According to Schultz (1997), TRIS-buffered (pH 7.4) test medium is used, because of the known sensitivity of the system to pH (at pH <= 5.5 doubling time approaches infinity). However, it is also described that control cultures approach this pH after 40 to 44 h of growth. Accordingly, buffer capacity will be very limited in case of organic acids like n-nonanoic acid and it is likely that the observed toxicity is in part due to the acidic pH resulting from a concentration of 71 mg/L n-nonanoic acid, and not solely a result of intrinsic microorganism toxicity. Concluding, the IC50 (Tetrahymena pyriformis; 40 h; growth) of 71 mg/L used for chemical safety assessment of STP microorganisms must be considered as conservative.

Reported statistics and error estimates:

No details on statistics reported for n-nonanoic acid. Generally, IGC50 values and the 95% fiducial intervals are determined by Probit analysis using the percent control-normalized absorbance as the dependent variable and the toxicant concentration in mg/L as the independent variable. Statistical Analysis System (SAS) software was used for the evaluation.

Validity criteria fulfilled:

not applicable

Conclusions:

The concentration of n-nonanoic acid to inhibit the growth of Tetrahymena pyriformis by 50 % (40 h; spectrophotometric measurement of cell density) was determined to be 71 mg/L.

Executive summary:

In a valid 40 hour growth inhibition study, test cultures of the ciliate Tetrahymena pyriformis were exposed to n-nonanoic acid at five graduate concentrations under static conditions (individual concentrations not reported). The assay was performed in three independent replicate runs on the basis of a preceding range finding test. The IC50 value after 40 hours based on cell density was determined to be 71 mg/L, indicating low toxicity of n-nonanoic acid to aqueous microorganisms (ciliates) (Schultz et al., 2002/2006).

The results are based on non-neutralized test item solutions (explicitly reported in database excerpt; Schultz, 2006; T05017). According to Schultz (1997), TRIS-buffered (pH 7.4) test medium is used, because of the known sensitivity of the system to pH (at pH <= 5.5 doubling time approaches infinity). However, it is also described that control cultures approach this pH after 40 to 44 h of growth. Accordingly, buffer capacity will be very limited in case of organic acids like n-nonanoic acid and it is likely that the observed toxicity is in part due to the acidic pH resulting from a concentration of 71 mg/L n-nonanoic acid, and not solely a result of intrinsic microorganism toxicity. Concluding, the IC50 (Tetrahymena pyriformis; 40 h; growth) of 71 mg/L used for chemical safety assessment of STP microorganisms must be considered as conservative.

Description of key information

STP microorganism toxicity was assessed based on the ciliate Tetrahymena pyriformis: EC50 (40 h; cell density)= 71 mg/L

Key value for chemical safety assessment

EC50 for microorganisms:

71 mg/L

Additional information

In a valid 40 hour growth inhibition study, test cultures of the ciliate Tetrahymena pyriformis were exposed to n-nonanoic acid at five graduate concentrations under static conditions (individual concentrations not reported). The assay was performed in three independent replicate runs on the basis of a preceding range finding test. The IC50 value after 40 hours based on cell density was determined to be 71 mg/L, indicating low toxicity of n-nonanoic acid to aqueous microorganisms (ciliates) (Schultz et al., 2002/2006).

The results are based on non-neutralized test item solutions (explicitly reported in database excerpt; Schultz, 2006; T05017). According to Schultz (1997), TRIS-buffered (pH 7.4) test medium is used, because of the known sensitivity of the system to pH (at pH <= 5.5 doubling time approaches infinity). However, it is also described that control cultures approach this pH after 40 to 44 h of growth. Accordingly, buffer capacity will be very limited in case of organic acids like n-nonanoic acid and it is likely that the observed toxicity is in part due to the acidic pH resulting from a concentration of 71 mg/L n-nonanoic acid, and not solely a result of intrinsic microorganism toxicity. Concluding, the IC50 (Tetrahymena pyriformis; 40 h; growth) of 71 mg/L used for chemical safety assessment of STP microorganisms must be considered as conservative.

This is supported by the results of a study on ready biodegradability (OECD 301B; Celanese, 1999):

a) nonanoic acid is readily biodegradable,

b) disturbances in sewage treatment plants are not to be expected when nonanoic acid is released in appropriate amounts, and

c) inhibitory effects of nonanoic acid to sewage sludge bacteria was not observed at a concentration of 10 mg C/L = 14 mg nonanoic acid/L.

Higher test item concentrations had not been used in the ready biodegradability test.

This corroborates that using the result from the Tetrahymena pyriformis study for PNEC derivation (AF 10), the resulting PNEC of 7.1 mg/L will be protective also for other microorganisms.