Fatty acids, C9, hexaesters with dipentaerythritol

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

activated sludge respiration inhibition testing

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

11 Feb 1999

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

No analytical purity given. Read across study

Justification for type of information:

Based on its composition and physico chemical properties, fatty acids, C5-9, hexaesters with dipentaerythritol is considered to be a suitable read across substance to address the toxicity to microorganisms endpoint of the test substance, fatty acids, C9, hexaesters with dipentaerythritol.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)

Qualifier:

according to guideline

Guideline:

other: EEC Commission Directive 87/302/EEC

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom (date of inspection 23 Mar 1998)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Test material was prepared by direct dispersion in water. An amount of test material (500 mg) was dispersed in approx. 250 mL of water and subjected to ultrasonication for approx. 30 min. Synthetic sewage (16 mL), activated sewage sludge (200 mL) and water were added to a final volume of 500 mL to give the test concentration of 1000 mg/L. Synthetic sewage was prepared according to the OECD guideline.
- Differential loading: yes
- Controls: The control group was maintained under identical conditions but not exposed to the test material.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Source of inoculum: Activated sludge was obtained from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Belper, Derbyshire (UK), which predominantely treats domestic sewage.
- Method of cultivation: The sample was maintained on continuous aeration in the laboratory at a temperature of 21 °C and was used on the day of collection. The pH of the sample was 7.5 and the suspended solids equal to 3.9 g/L prior to use.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test temperature:

21 °C

Nominal and measured concentrations:

Nominal: 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 mL conical flasks
- No. of vessels per concentration: 3
- No. of vessels per control: 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: laboratory tap water dechlorinated by passage through an activated carbon filter with a total hardness of approx. 100 mg/L as CaCO3.

EFFECT PARAMETERS MEASURED: The respiration rate was recorded after 30 min and 3 h test period.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: No significant effect on respiration was observed at all test concentrations employed.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

30 min

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

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

Key result

Duration:

3 h

Dose descriptor:

NOEC

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:

- Effect concentrations exceeding solubility of substance in test medium: The effect concentration exceeded the water solubility of the test substance.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50 (30 min): 12 mg/L, EC50 (3 h): 11 mg/L

The study resulted in no effects on the respiration rate of an activated sludge microorganism population (EC50 (3 h): > 1000 mg/L; NOEC ≥ 1000 mg/L).

Table 1: Oxygen consumption rates and percentage inhibition values in the definitive study after 3 h contact time

(- : increase in respiration rate as compared to controls)

Nominal concentration [mg/L]	Initial O2 reading [mg O2/L]	Measurement period [min]	Final O2 reading [mg O2/L]	O2 Consumption rates [mg O2/L/min]	% Inhibition
Control R1 R2	6.3	9	0.7	0.62	-
	7.2	10	2.0	0.52	-
Test substance 1000 R1 1000 R2 1000 R3	6.5	9	1.3	0.58	-2
	6.9	9	1.8	0.57	0
	6.7	10	1.4	0.53	7
3,5-dichlorophenol 3.2 10 32	6.1	8	1.9	0.53	7
	7.4	10	4.6	0.28	51
	8.1	10	7.1	0.10	82

Conclusions:

Under the study conditions, the nominal 3 h EC50 was > 1000 mg/L and the 3 h NOEC was 1000 mg/L.

Executive summary:

A study was conducted to determine the toxicity of the read across substance, fatty acids, C5-9, hexaesters with dipentaerythritol, to activated sludge according to OECD Guideline 209, in compliance with GLP. Activated sludge from a predominately domestic sewage treatment plant was exposed to a limit test concentration of 1000 mg/L for 3 h. No effects on respiration were observed. Under the study conditions, the nominal 3 h EC50 was therefore > 1000 mg/L and the 3 h NOEC was 1000 mg/L (Mead, 1999).

Reference 2

Endpoint:

toxicity to microorganisms

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

6 Jan 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Justification for type of information:

Based on its composition and physico chemical properties, isononanoic acid, mixed esters with dipentaerythritol, heptanoic acid and pentaerythritol is considered to be a suitable read across substance to address the toxicity to microorganisms endpoint of the test substance, fatty acids, C9, hexaesters with dipentaerythritol.

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

other: ISO 10712 (Water quality - Pseudomonas putida growth inhibition test)

Deviations:

yes

Remarks:

the test period is 6 h instead of 16 h

Principles of method if other than guideline:

The method is originally described by Bringman and Kuehn which has been modified by Slabbert. This procedure measures the degree of inhibition of growth of a pure culture of Pseudomonas putida during a 6 h period when the cells are in the logarithmic growth phase.

Bringman, G. and Kuehn, R. (1980): Comparison of the toxicity thresholds of water pollutants to bacteria, algae and protozoa in the cell multiplication inhibition test. Water Research 14, 231 – 241.

Slabbert, J.L. (1986): Improved bacterial growth test for rapid water toxicity screening. Bull. Environ. Contam. Toxicol. 37, 565 – 569.

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A 50 mg/L stock solution was prepared in water by weighing 0.0196 g of test substance onto a glass slip which was then added to 392 mL of sterile growth medium and stirred overnight. After allowing the stirred solution to settle, 49 mL were removed from below the surface of the undissolved material and transferred, aseptically to each of the flasks (Water-Accommodated Fraction).
- Eluate: no
- Differential loading: no
- Controls: yes / containing inoculated growth medium

Test organisms (species):

Pseudomonas putida

Details on inoculum:

- Laboratory culture: Pseudomonas putida (strain NCIMB9494) was obtained as a freeze-dried culture from National Collections of Industrial and Marine Bacteria Ltd, Aberdeen, UK and stored at 4 °C until use.
- 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 h and then stored at laboratory temperature, until use as the stock culture.
- Preparation of inoculum for exposure: 18 – 20 h before the start of the test, 4 mL of test medium concentrate was 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 the incubated overnight at 25 °C on an orbital shaker (150 rpm). After this period the cells were diluted by addition of fresh growth medium solution at 25 °C to an optical density which gave an absorbance of 0.85 ± 0.05 at 600 nm.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

6 h

Test temperature:

25 ± 0.5 °C

Nominal and measured concentrations:

Nominal: 50 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: conical flasks
- Fill volume: 50 mL
- Aeration: constantly shaken at 150 rpm on an incubator shaker
- No. of vessels per concentration: 3
- No. of vessels per control: 3

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionised water
- Culture medium different from test medium: no / Preparation: 26.25 g dipotassium hydrogen phosphate, 11.25 g potassium dihydrogen phosphate, 1.175 g trisodium citrate dehydrate, 2.5 g ammonium sulphate and 0.25 g magnesium sulphate heptahydrate were dissolved in 1 L of deionised water and autoclaved at 121 °C for 15 min, and then stored at 4 °C. The glucose solution was prepared containing 6.25 g of glucose dissolved in 1 L deionised water. This solution was autoclaved at 121 °C for 15 min, and then stored at 4 °C. Prior to use, equal volumes of each solution were mixed together in a sterile flask.

EFFECT PARAMETERS MEASURED: Growth inhibition was recorded after 6 h of incubation.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: limit test

Reference substance (positive control):

yes

Remarks:

3,5 dichlorophenol at 18 mg/L

Key result

Duration:

6 h

Dose descriptor:

other: EL10

Effect conc.:

> 50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

(WAF)

Basis for effect:

growth inhibition

Key result

Duration:

6 h

Dose descriptor:

other: EL50

Effect conc.:

> 50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

(WAF)

Basis for effect:

growth inhibition

Results with reference substance (positive control):

- Relevant effect levels: 95% inhibition at a nominal concentration of 18 mg/L after 6 h

Table 1: Optical density and inhibition results

Flask number	Nominal concentration	Mean of optical density at 600 nm	Corrected value for uninoculated solution	% inhibition
1-3	Control	0.368	0.366	0
4-6	Reference substance 18 mg/L	0.020	0.018	95
10-12	50 mg/L test substance	0.514	0.511	0
13	blank	0.002	-	-
15	50 mg/L test substance without inoculum	0.003	-	-

Conclusions:

Under the study conditions, the nominal 6 h EL10 was determined to be > 50 mg/L based on growth inhibition

Executive summary:

A study was conducted to determine the toxicity of the read across substance, isononanoic acid, mixed esters with dipentaerythritol, heptanoic acid and pentaerythritol, to aquatic microorganisms (Pseudomonas putida) according to a method similar to ISO 10712, in compliance with GLP. A Water Accommodated Fraction (WAF) at a nominal concentration of 50 mg/L was used. No inhibitory effect was observed after 6 h. Under the study conditions, the nominal 6 h EL10 was therefore determined to be > 50 mg/L based on growth inhibition (Comber, 1993).

Description of key information

Based on testing with structurally comparable read across substances, no toxicity of the test substance to aquatic microorganisms at saturation is expected.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

Additional information

Study 1:

A study was conducted to determine the toxicity of the read across substance, fatty acids, C5-9, hexaesters with dipentaerythritol, to activated sludge according to OECD Guideline 209, in compliance with GLP. Activated sludge from a predominately domestic sewage treatment plant was exposed to a limit test concentration of 1000 mg/L for 3 h. No effects on respiration were observed. Under the study conditions, the nominal 3 h EC50 was therefore > 1000 mg/L and the 3 h NOEC was 1000 mg/L (Mead, 1999).

Study 2:

A study was conducted to determine the toxicity of the read across substance, isononanoic acid, mixed esters with dipentaerythritol, heptanoic acid and pentaerythritol, to aquatic microorganisms (Pseudomonas putida) according to a method similar to ISO 10712, in compliance with GLP. A Water Accommodated Fraction (WAF) at a nominal concentration of 50 mg/L was used. No inhibitory effect was observed after 6 h. Under the study conditions, the nominal 6 h EL10 was therefore determined to be > 50 mg/L based on growth inhibition (Comber, 1993).