Fatty acids, C5-10, esters with pentaerythritol

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 Feb 1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study with acceptable restrictions. No analytical monitoring of the test substance is performed. The analytical purity is not stated.

Principles of method if other than guideline:

Cell multiplication inhibition test according to Bringman and Kuehn (1980), Water Research 14

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A 10 mg/l (nominal concentration) of the test substance in acetone was prepared and used for the test.
- Chemical name of vehicle: Acetone
- Concentration of vehicle in test medium (final test solution(s) including control(s)): 0.1% v/v

Test organisms (species):

Pseudomonas putida

Details on inoculum:

- Laboratory culture: Pseudomonas putida, strain NCIMB9494, obtained as a freeze-dried culture from National Collections of Industrial and Marine
Bacteria Ltd, Aberdeen, UK
- Method of cultivation: The freeze-dried culture was rehydrated in 0.5 ml of nutrient broth (Oxoid Ltd). A loop of this suspension was streaked onto a nutrient agar (Oxoid Ltd) slope in a universal bottle. This was incubated at 25 °C for 24 hours, and then stored at laboratory temperature, until use
as stock culture.
- Preparation of inoculum for exposure: 18-20 hours before the start of the test 4 ml of test medium concentrate were added to 46 ml of deionised
water in a sterile conical flask. A loop of Pseudomonas putida stock culture was added to this growth medium solution, and then incubated overnight at 25 °C on an orbital shaker (150 rpm).
- Pretreatment: After incubation the cells were diluted by addition of fresh growth medium solution at 25 °C to an optical density which gave an
absorbance of 0.8 (± 0.05) at 600 nm (4 cm cells). This was used as the test inoculum.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

6 h

Nominal and measured concentrations:

Nominal: 10 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: conical flasks
- Fill volume: 50 ml volume
- No. of vessels per concentration: 3
- No. of vessels per control: 3
- No. of vessels per vehicle control: 3

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Basalt salt solution was prepared dissolved in 1 L of deionised water. This solution was autoclaved at 121 °C for 15 minutes. A glucose solution was prepared containing 6.25 g of glucose dissolved in 1 L of deionised water. This solution was autoclaved at 121 °C for 15 minutes. Equal volumes of the salt solution and the glucose solution were mixed together with a sterile flask to form a growth mediumconcentrate.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Growth inhibition after 6 hours by measurements of the optical density of
each flask content at 600 nm

Reference substance (positive control):

yes

Remarks:

3,5 dichlorophenol

Duration:

6 h

Dose descriptor:

EC10

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Duration:

6 h

Dose descriptor:

EC50

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Details on results:

- Any observations that might cause a difference between measured and nominal values: The test material did not completely
dissolve in the test system.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: 96% inhibition of growth at 18 mg/L solution of 3,5 dichlorophenol

The nominal 10 mg/l solution of the test substance gave no inhibition of growth. The EC10 and EC50 (6h) for the test substance to Pseudomonas putida was > 10 mg/l (nominal) test material.

Table 1: Optical density results

Flasknumber	Contents	Mean of optical density at 600 nm (4 cm cells)	Minus value for uninoculated solution	% inhibition
1-3	Control	0.602	0.569
4-6	18 mg/l 3,5 dichlorophenol	0.025		96
7-9	0.1% Acetone	0.589	0.579
13-15	10 mg/l PentaerythritolC7-C10 tetra ester	0.630	0.614	-
16	Blank uninoculated	0.033
17	0.1% Acetone uninoculated	0.010
19	10 mg/l PentaerythritolC7-C10 tetra ester uninoculated	0.016

 

Reference 2

Endpoint:

activated sludge respiration inhibition testing

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

supporting study

Study period:

14 May 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study with acceptable restrictions: No analytical purity is given. , no analytical monitoring)

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Test substanz was emulsified using Ultra-Turrax.

Test organisms (species):

activated sludge, domestic

Details on inoculum:

- Laboratory culture: Activated sludge from sewage plant Bempflingen, Germany
- Preparation of inoculum for exposure: Activated sludge was washed with tap water twice, afterwards centrifuged and the dry residue was
determined. The amount of the sludge was weighed per liter, which was corresponded to 4 g dry mass.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Hardness:

not stated

Test temperature:

20 ± 2 °C

pH:

not stated

Dissolved oxygen:

> 6.5 mg/L

Nominal and measured concentrations:

Nominal: 10000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: beaker
- Material: glass; Size: 1 L; Fill volume: 500 mL
- Aeration: continously to reach an oxygen content > 6.5 mg/L
- No. of vessels per concentration: 1
- No. of vessels per control: 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dechlorinated tap water

EFFECT PARAMETERS MEASURED: Respiration inhibition by decrease of oxygen content was measured in 15-minutes-pulses over a test period of 3 h using an OXI-meter.

Reference substance (positive control):

yes

Remarks:

3,5 dichlorphenol

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 10 000 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:

EC10

Effect conc.:

> 10 000 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, result is in the range of 5 to 30 mg/L
- Relevant effect levels: EC50 = 5 mg/L

An increase about 43% of respiration rate could be observed at the nominal tested concentration of 10000 mg/L in comparison to the control.

Table 1: Respiration rate of control, reference and test substance and inhibition of the respiration rate of test and reference substance (*A negative sign means an increase of the respiration)

	Replicates	Concentration [mg/L]	Respiration rate [mg O2/L x h]	Inhibition [%]
Control	1		24.0 25.0	-
Referenz	1 2 3	2.5 10 30	22.0 11.0 5.0	10.2 55.1 79.6
Test substance	1	10000	35.0	-43.0*

 *: negative value indicate an increase of respiration

Reference 3

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

28 - 29 Oct 2008

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP Guideline study with acceptable restrictions: No monitioring of the test substance is performed. Although the test substance is considered poorly soluble in water no information about undissolved test material is given and no effort is being made to remove undissolved test material. However, as no adverse effects are detected this deficiencies are considered to be negligible.

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

yes

Remarks:

No monitioring of the test substance is performed. Although the test substance is considered poorly soluble in water no information about undissolved test material is given and no effort is being made to remove undissolved test material.

GLP compliance:

yes (incl. QA statement)

Remarks:

Department of Health, UK

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Information provided by the Sponsor indicated that the test substance is not sufficiently soluble to prepare an aqueous solution. Therefore, at test inition weights were established by the addition of appropriate volumes (based on specific gravity, 0.99) of test substance to the test vessels containing RO water, synthetic sewage and microbial inoculum (see Table 1).

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 from Worlingworth Sewage Treatment Works, which treats predominantely domestic waste. In the laboratory, the sample was maintained under aerobic conditions until required. One day one of collection, an aliquot (10 mL) of the activated sludge was filtered through a dried, preweighed Whatman GF/C filter paper, which was then dried again at ~ 105 °C for at least one hour, allowed to cool in a desiccator before being reweighed. The mixed liquor suspended solids content of the activated sludge was then calculated. Synthetic sewage (50 mL/L) was added to the stock of activated sludge. The mixture was aerated overnight. The mixed liquor suspended solids content of the activated sludge was adjusted to 4 g/L by addition of tap water. Aliquots (200 mL) were added to the test vessels to give a final suspended solids concentration of 1.6 g/L.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

test start: 19.5 - 19.6 °C
test end: 19.3 - 19.4 °C

pH:

test start: 7.3 - 7.4
test end: 7.6 - 7.9

Nominal and measured concentrations:

Nominal: 10, 100 and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: beaker
- Type: closed (loosely covered with aluminium foil)
- Aeration: using a glass aerator connected to a laboratory supply of oil-free compressed air (~ 1 L/min.)
- No. of vessels per concentration: 1 (10, 100 mg/L); 3 (1000 mg/L)
- No. of vessels per control: 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse osmosis (RO) water
- Culture medium different from test medium: no, according to OECD guideline 209
- Intervals of water quality measurement: The pH and temperature were measured at the start and end of the test.

EFFECT PARAMETERS MEASURED: The respiration rate was measured after a test period of 3 h.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

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:

EC10

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

The test substance showed no inhibitory effect on the respiration rate of activated sludge at any of the concentrations employed in the test. Respiration rates of mixtures containing the test substance showed an increase in respiration rate above that of the controls.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50 (3h) = 6.4 mg/L (4.1 - 9.6 mg/L)

Table 1: Preparation of test solutions

Treatment	Test substance [µL] or reference substance [mL]	Synthetic sewage [mL]	RO water [mL]	Microbial Inoculum [mL]
Control 1	0	16	284	200
Control 2	0	16	284	200
Test substance [mg/L]
10	5.1	16	284	200
100	50	16	284	200
1000	505	16	284	200
Reference substance [mg/L]
3	3	16	281	200
10	10	16	274	200
32	32	16	252	200

Table 2: Dissolved oxygen concentration and measurement times

Treatment	Initial dissolved oxygen conc. in culture [mg O2/L]	Initial measured dissolved oxygen conc. [mg O2/L]	Final measured dissolved oxygen conc. [mg O2/L]	Measurement time [min]
Control 1	6.9	6.5	2.5	8.5
Control 2	6.9	6.5	2.5	9.0
Test substance [mg/L]
10	4.5	4.2	1.5	5.3
100	5.0	4.8	1.5	6.3
1000	5.8	5.5	2.5	5.7
1000	4.9	4.6	1.5	5.8
1000	5.5	4.9	2.0	5.8
Reference substance [mg/L]
3	7.0	6.5	3.2	9.5
10	8.0	7.8	5.8	9.9
32	7.4	8.1	7.7	8.1

Table 3: Measurement of respiration rate

Treatment	Specific respiration rate [mg O2/g/h]	% inhibition or stimulation ()
Control 1	17.6	-
Control 2	16.7	-
Test substance [mg/L]
10	19.1	0 (11)
100	19.6	0 (14)
1000	19.7	0 (15)
1000	20.0	0 (17)
1000	18.8	0 (9)
Reference substance [mg/L]
3	13.0	24
10	7.6	56
32	1.9	89

Description of key information

EC50 (6h) > 10 mg/L (nominal)
EC50 (3 h) > 1000 mg/L for the respiration of activated sludge microorganisms (OECD 209); read-across

Additional information

One study, investigating the toxicity to Pseudomonas putida, was available for CAS 68424-31-7. This cell multiplication inhibition test (Comber and Coleman, 1991) according to Bringman and Kuehn (1980) determined EC10 and EC50 (6h) > 10 mg/L (nominal). No inhibition on the growth of Pseudomonas putidawas observed at the tested concentration (10 mg/L nominal) Furthermore, the biodegradation tests exhibit the readily biodegradability of the test substance. Therefore, the sensitive single species as well as the activated sludge test shows no inhibition and a disturbance in the biodegradation process of a sewage treatment plant is not anticipated.

In accordance to Regulation (EC) No. 1907/2006 Annex XI, 1.5 a read across to two structurally related category members decanoic acid, mixed esters with heptanoic acid, octanoic acid, pentaerythritol and valeric acid (71010-76-9), and fatty acids, C8-10 (even numbered), di-and triesters with propylidynetrimethanol (CAS 11138-60-6) was conducted to support the above mentioned data on CAS 68424-31-7. This read-across is justified in detail in the overall summary (IUCLID chapter 6.1) and within the category justification in IUCLID Section 13. In this case of read-across, the best suited (highest degree of structural similarity, nearest physico-chemical properties) read-across substances were used for the assessment. The fatty acid chain length of C5 – C10 of the first read-across substance is similar to that of the target substance and both substances contain the same alcohol (pentaerythritol) and are tetra-esters. The remaining polyol ester category member has a different alcohol component (TMP) but chain lengths between C8 and C10.

The first read across study investigating toxicity to aquatic microorganisms was conducted with decanoic acid, mixed esters with heptanoic acid, octanoic acid, pentaerythritol and valeric acid (CAS No. 71010-76-9) under static conditions according to OECD 209 (Dickinson, 2008). In this study non-adapted activated sludge obtained from a domestic sewage treatment plant was used as inoculum. Test concentrations of 10, 100 and 1000 mg/L were prepared. Test substance was added directly to the test vessels. The test substance did not inhibit the respiration rate of activated sludge microorganisms at any of the concentrations employed in the test. Hence, the 3 h-EC50 was determined to be > 1000 mg/L based on the nominal test concentration. Therefore, it can be concluded that the test substance will not exhibit effects on the respiration rate of microorganisms.

The second read across study with fatty acids, C8-10 (even numbered), di-and triesters with propylidynetrimethanol (CAS 11138-60-6) was conducted according to OECD 209 investigating aquatic microorganisms (Kuttler, 1998). In this study activated sludge obtained from a domestic sewage treatment plant was used as inoculum. A test concentration of 10000 mg/L was prepared and was added directly to the test vessels. The test substance did not inhibit the respiration rate of activated sludge microorganisms, the 3 h-EC50 was therefore determined to be > 10000 mg/L based on the nominal test concentration. Therefore it can be concluded that the test substance will not exhibit effects on the respiration rate of microorganisms.

Based on these results from structurally related read-across substances which are characterized by a similar ecotoxicological profile, it can be concluded that pentaerythritol tetraesters of n-decanoic, n-heptanoic, n-octanoic and n-valeric acids (CAS 68424-31-7) will not have adverse effects on the respiration rate of microorganisms, therefore, a disturbance in the biodegradation process of a sewage treatment plant is not anticipated. As it can be seen in the data matrix of the category justification in section 13 and the overall endpoint summary IUCLID 6.1, all reliable data in the polyol esters category support this hazard assessment by showing a consistent pattern of results, i.e. no microbial inhibition and no toxic effects were observed to aquatic organisms up to the limit of water solubility.