Calcium di(octanoate)

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

Study period:

15 Mar - 16 Mar 1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

ISO 10712 (Water quality – Pseudomonas putida growth inhibition test (Pseudomonas cell multiplication inhibition test

Version / remarks:

Please "Principles of Method if other than Guideline" below for details.

Principles of method if other than guideline:

Pseudomonas putida, an aerobic gram-negative bacterial species from the genus Pseudomonas, is a natural habitant of surface waters. This bacterium is a model organism involved in biological self-purification. The multiplication of bacterial cells of Pseudomonas putida is inhibited by dissolved toxic water ingredients. After a certain period, the increase in the number of cells in a test culture free from toxic influence and with a defined standardized quantity of nutrients will exceed that observed in a test culture containing dissolved toxic substances and kept under identical conditions.
The concentration of the bacterial suspension is measured turbidi-metrically; it is expressed as the extinction of the primary light of the monochromatic radiation at 436 nm for a layer thickness of 10 nm. The concentration at which the inhibitory action of a test substance starts, will be present in that step of a dilution series of the test substance having an extinction value at the end of the test period that is equal or > 10% below the mean extinction value of the control cultures. This toxicity threshold (TT) of the test substance for Pseudomonas putida will be determined graphically.

- Bewertung Wassergefahrdender Stoffe, iii Bestimmung der akuten Bakterientoxizitat, Ad-hoc-Asbeitsgruppe 1 (Obmann Dr. Niemitz), LTwS, Nr. 10, September 1979
- NEN 6509, Water - Determination of the impact of toxic substances on the growth of a pure culture of bacteria, June 1980.
- NPR 6508, Water - Necessaries, method and recipes for the breeding of Pseudomonas fluorescens bacteria, November 1979.
- Bringman, g., Kuhn, R., Comparison of the toxicity thresholds of water pollutants to bacteria, algae and protozoa in the cell multiplication inhibition test, Water Research, 14, 231-241 (1980)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Chemical name: Caprylic acid
Appearance: liquid
CAS: 124-07-2
Batch no.: 27-1-88
Purity: C6: 0.5%, C8: 99%, C10: 0.5%
Storage: At ambient temperature in the dark, dry
Stability: Stable at storage conditions

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
A test solution of 680 mg/l in sterile Milli-Q water (Millipore Corp., Bedford, Mass., U.S.A.) was prepared. Also an over saturated test substance solution of 1360 mg/l was prepared. Both the test substance solution and the over saturated test substance solution were diluted 1.25 in sterile Milli-Q water and mixed for 10 minutes on a magnetic stirrer. The pH of the test substance solution and the over saturated test substance solution was adjusted 7.0 +/- 0.2 using an appropriate NaOH solution to ensure minimal increase of volume.

Test organisms (species):

Pseudomonas putida

Details on inoculum:

Stock cultures of Pseudomonas putida, obtained from RIVN, Bilthoven The Netherlands, were kept on the nutrient used for stock and preliminary cultures in agar plant tubes. New stock cultures were started at intervals of 1 week. The inoculated stock cultures were incubated at 25 Deg C.

Preliminary cultures of Pseudomonas putida
Using aseptic techniques, small amounts of bacteria from a 7-day old stock culture of Pseudomonas putida were inoculated in fluid medium in Erlenmeyer flasks. The preliminary cultures were incubated at 25 Deg C for 18 + 2 hours. Subsequently the extinction of the monochromatic radiation at 436 nm for a 10 nm layer of the bacterial suspension was determined by photoelectric measurement. On the basis of the values measured, the final turbidity value of the bacterial suspension was adjusted by means of sterile saline in such a way that the extinction value for a sample that has been subject to onward dilution 1 + 9 with saline corresponded with the extinction value of a Formazin standard suspension TU/F/436 nm = 10. These preliminary culture of bacterial suspensions were used for inoculation of the test flasks.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

18 h

pH:

6.8 - 7.2, adjusted with NaOH

Nominal and measured concentrations:

nominal: 0.266, 0.531, 1.063, 2.125, 4.25, 8.50, 17.00, 34.00, 68.00, 136.00, 272.00, 544.00, 1088.00 mg/L

Details on test conditions:

TEST SYSTEM
Four parallel dilution series in 300 ml Erlenmeyer flasks stoppered with aluminium caps were prepared from the prepared test substance solution and sterile Milli-Q water. Each of the dilutions contained 1 part v/v of the test substance solution in 2 to 2 parts v/v of mixture. The dilution series were prepared as follows: the first flask of each series contained 160 ml of test substance solution, from this flask the subsequent dilution steps at a constant dilution ratio were prepared by consistently mixing 80 ml of preliminary test substance dilution and 80 ml sterile Milli-Q water. Consequently, each flask contained 80 ml of test substance solution at the start. Each flask of the three inoculated dilution series (I, II, III) was made up to a final volume of 100 ml by adding 5 ml of stock solution I, 5 ml of a stock solution II and 10 ml of the prepared bacterial suspension from the preliminary culture having a known adjusted extinction value (see any other information on materials and methods below for details on stock solutions).
Following inoculation, the extinction value at 436 nm will correspond to the extinction value of the Formazin standard suspension TU/F/436 nm = 10. The flasks of the dilution series that were not inoculated (series t), were made up to 100 ml by adding 5 ml of stock solution I, 5 ml of stock solution II and 10 ml of saline. Ten control culture flasks (series B) with 80 ml sterile Milli-Q water, 5 ml stock solution 1, 5 ml stock solution II and 10 ml prepared bacterial solution were also included.
Both inoculated and non-inoculated dilution series as well as the control culture flasks were left at 25 Deg C for 18 +/- 2 hours. After termination of the test period the cell suspensions were homogenized and the extinction of the monochromatic radiation at 436 nm in a 10 nm layer in the four dilution series and the control flasks was measured. If after termination of the test period, colouration or turbidity occurred in the dilution series for chemical-physical reasons, the analogous steps of dilution of the non-inoculated series will be used as photometric blank values for turbidity of the inoculated dilution series.

Calculations and interpretation of results
The following values were calculated:
- mean extinction value of the control cultures (series B)
- mean extinction values of each concentration of the inoculated dilution series (series I, II and III)
- mean extinction value of the control cultures, reduced by the 10% extinction-difference value
The mean extinction values of each dilution step were plotted against the logarithm of the mean values of the concentration of the test substance. The mean value of the control culture, reduced by 10% extinction-difference value, was plotted as a horizontal reference.

Reference substance (positive control):

yes

Remarks:

Methanol

Key result

Duration:

18 h

Dose descriptor:

EC10

Effect conc.:

912 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mean extinction value

Details on results:

None of the concentrations in the dilution series of the test material showed colouration or turbidity; therefore no correction for the extinction values of the dilution series was made.
For the performance of the acute bacteria toxicity testing of the test material, the solubility of the test material must be available. The solubility of the test material in water was determined in analogy to the known solubilities in water of three different fatty acids. The fatty acids each vary in the length of the carbon-hydrogen chain; C8, Caprylic acid, solubility in water at 20 oC is 680 mg/l; C14, Myristic acid, solubility in water at 20 oC is 20 mg/l; C18, Stearic acid, solubility in water at 20 oC is 3.0 mg/l. According to the chain length and the corresponding solubility value, the solubility of the test material (C8) was adjusted to 680 mg/l. Also an excess of test substance (1360 mg/l) was dissolved in Milli-Q water and exposed to the bacteria. The real solubility of this over saturated test substance solution is therefore unknown. The extra test flasks with the over saturated test substance solution were prepared as follows: in addition to the test procedure for the preparation dilution series, four Erlenmeyer flasks were filled with 80 ml over saturated test substance solution, 5 ml stock solution I, 5 ml stock solution II and 10 ml bacterial suspension (except t, which was not inoculated but filled with 10 ml sterile saline).
A range finding experiment was carried out. All the extinction values measured in the range finding experiment lie above the horizontal reference line, so no toxicity threshold (TT) value of the test material for Pseudomonas putida can be determined. However in the main experiment, it seemed that the test material is toxic at concentrations above the estimated saturated concentration of the test substance solution. If the concentration at which inhibitory action of the inhibition starts is soluble in water than a TT value of 912 mg/l can be determined. This value corresponds to an assessment value for bacterio-toxicity of 3.1, however due to the estimated solubility, the assessment value for bacterio-toxicity will be < 3.3.
The TT value of methanol determined for Pseudomonas putida was 21502 mg/l, 13873 mg/l.

Results with reference substance (positive control):

- Results with reference substance valid? Yes

Reported statistics and error estimates:

The toxicity threshold (TT) is determined graphically. The mean extinction values of each dilution step were plotted against the logarithm of the mean values of the test substance concentration.

Table 1: Extinction of the test substances solutions after 16 - 20 h

Concentration   [mg prod./L]	Extinction at 436 nm
	Replicate I	Replicate II	Replicate III	Arithm. mean of replicates	Replicate without inoculum
0.226	0.490	0.500	0.470	0.487	0.000
0.531	0.471	0.480	0.469	0.473	0.000
1.063	0.463	0.460	0.456	0.460	0.000
2.125	0.452	0.450	0.460	0.454	0.000
4.250	0.458	0.430	0.466	0.451	0.000
8.500	0.447	0.450	0.459	0.452	0.000
17.000	0.469	0.434	0.463	0.455	0.000
34.000	0.458	0.450	0.470	0.459	0.000
68.000	0.525	0.512	0.497	0.511	0.000
136.000	0.588	0.580	0.570	0.579	0.000
272.000	0.710	0.740	0.745	0.732	0.000
544.000	0.985	0.979	0.990	0.985	0.000
1088.000	0.198	0.212	0.220	0.210	0.000

Table 2: Extinction after 16 - 20 h in the reference substance and the blank control

Concentration   [mg prod./L]	Extinction at 436 nm
	Reference substance	Blank controls
4937.500	0.488	0.449
9875.000	0.440	0.440
19750.00	0.415	0.452
39500.00	0.270	0.459
79000.00	0.155	0.458
		0.449
		0.433
		0.439
		0.440
		0.438
Arithmetic mean		0.446

With respect to the historical test with the reference substance methanol, it can be concluded that the test conditions were optimal and the results are valid.

Validity criteria fulfilled:

yes

Remarks:

With respect to the known TT value of methanol (6600 mg/l) and considering the existing differences in definitions for the TT value in the literature concerned, it can be concluded that the test conditions were optimal and results obtained are valid.

Conclusions:

The test material was investigated for its ability to inhibit the cell multiplication of the bacteria species Pseudomonas putida. Cultures of the bacteria were exposed to concentrations ranging from 0.266 to 544.000 mg/l and to an oversaturated concentration of the test material. Based on the solubility of the test material in water, no toxicity threshold value of the test material for Pseudomonas putida could be determined. The assessment figure or value for bacteriotoxicity is therefore < 3.3 (Bewertung Wassergefahrdender Stoffe, 1979).

Description of key information

A study investigating the toxicity of the substance to Pseudomonas putida was conducted on octanoic acid. This cell multiplication inhibition test (1988) was conducted similarly to ISO 10712. The test organism was exposed to a wide range of nominal concentrations (0.266 - 1088.00 mg/L). The study determined an EC10 (18h) of 912 mg/L (nominal).

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

912 mg/L