Fatty acids, C6-12, mixed tetraesters with heptanoic acid, pentaerythritol, 3,5,5-trimethylhexanoic acid and valeric acid

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

Study period:

03 Mar 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - Guideline study, tested with the source substance Reaction product of pentaerythritol and trimethylolpropane with n-pentanoic acid, 2-methylbutyric acid, n-heptanoic acid, 3,5,5-trimethylhexanoic acid, n-octanoic acid and n-decanoic acid. According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (ECHA, 2012)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom, date of inspection 22 Jan 1996

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: To prepare the test series amounts of test material (50 and 500 mg) were each separately dispersed in water (approximately 250 mL) with the aid of ultrasonication for a period of 30 min. Synthetic sewage (16 mL), activated sewage sludge (200 mL) and water, to a final volume of 500 mL, were added to each giving the test concentrations of 100 and 1000 mg/L. Based on the results of the range-finding study a "limit test' was conducted for the definitive study at a test concentration of 1000 mg/L (three replicate vessels) to confirm that at this concentration no effect on respiration of the activated sewage sludge was observed.

Test organisms (species):

activated sludge, domestic

Details on inoculum:

- Laboratory culture: obtained from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Belper, Derbyshire, UK
- Method of cultivation: Activated sludge 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 7.3 and the suspended solids equal to 4.0 g/L prior to use.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test temperature:

21 °C

pH:

7.3

Nominal and measured concentrations:

1000 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Material, size, headspace, fill volume: 300 mL with water and 200 mL of inoculum in 500 mL conical flask
- Aeration: aerated with compressed air
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 2


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: laboratory tap water dechlorinated by passage through an activated carbon filter

OTHER TEST CONDITIONS
- Light intensity: normal laboratory lighting

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Rate of respiration was measured after 30 min and 3 h using a Yellow Springs dissolved oxygen meter (over the linear portion of the oxygen consumption trace, i.e. between approximately 8.3 mgO2/L and 0.5 mgO2/L)

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 any of the test concentrations employed.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

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:

The No Observed Effect Concentration (NOEC) after 3 h was greater than or equal to 1000 mg/L.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50, 16 mg/L (30 min) and 14 mg/L (3 h)

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

Concentration (mg/L)	O2 consumption rates (mg O2/L/min)	% Inhibition
Control
R1	0.59	-
R2	0.60	-
Test substance
1000 R1	0.64	[8]
1000 R2	0.64	[8]
1000 R3	0.67	[13]
Reference substacne
3.2	0.57	4
10	0.46	23
32	0.08	87

[increase in respiration rate as compared to controls]

R1 -R3=Replicates

Reference 2

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 November 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD & EU test guidelines in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Analytical monitoring:

no

Details on sampling:

A nominal concentration of 100 mg/L was tested in duplicate.

Vehicle:

no

Details on test solutions:

Since HATCOL 3331 was hardly soluble in water, the test substance was quantitatively added to the test vessels.

Test organisms (species):

activated sludge

Details on inoculum:

Test System: Micro-organisms in activated sludge
Source: Municipal sewage treatment plant: 'Waterschap de Maaskant, 's-Hertogenbosch, the Netherlands.
Number of micro-organisms: Number of micro-organisms was determined as the amount of Mixed Liquor Suspended Solids (MLSS) per litre test medium.
Preparation of the sludge: The sludge was coarsely sieved and washed with tap water. A small amount of the sludge was weighed and dried at ca. 105°C to determine the amount of suspended solids (3.5 g/L of sludge, as used for the test). Before use the pH was checked (measured value: 7.8).
The batch of sludge was used on the subsequent day, therefore 50 ml of synthetic sewage feed was added to each litre of activated sludge at the end of each working day. The sludge was kept aerated at test temperature until use.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

30 min

Post exposure observation period:

No post exposure observation period specified in the study report.

Hardness:

Not specified in the study report.

Test temperature:

20.6°C.

pH:

7.6 - 7.8

Dissolved oxygen:

Not applicable - respiration inhibition test.

Salinity:

Not applicable - freshwater study

Nominal and measured concentrations:

Nominal concentration of 100 mg/l

Details on test conditions:

Contact time: 30 minutes, during which aeration and stirring took place.
Vessels: All glass, 300 ml oxygen bottles and 1 L test bottles.
Milli-RO / MiIi-Q water: Tap-water purified by reverse osmosis (Mill-RO) and subsequently passed over activated carbon and ion-exchange cartridges (MiIi-Q) (Milipore Corp., Bedford, Mass., USA).
Synthetic sewage feed: 16 g peptone
11 g meat extract
3 9 urea
0.7 g NaCI
0.4 9 CaCI2.2H2O
0.2 9 MgSO4 .7H2O
2.8 g K2HPO4
Dissolved in 1 L MiIi-Q water and filtered.
The pH was 7.1.
Air supply: Clean, oil-free air.
Oxygen meter: WT inolab Oxi Level 2 supplied with a WT CeliOx 325 oxygen electrode, electrolyte type EL Y IG.
Recorder: Flatbed recorder SE 102 (Kipp & Zonen).

Performance of the test: The synthetic sewage feed (16 ml) and an adequate amount of the test substance were mixed and made up to 300 ml with MiIi-RO water. Activated sludge (200 ml) was added and the mixture was aerated in a 1 L bottle during the contact time, using a pipette as an aeration device.
Then a well mixed sample of the contents was poured into a 300 ml oxygen bottle, and the flask was sealed with an oxygen electrode connected to a recorder, forcing the air out of the vessel. Oxygen consumption was measured and recorded for approximately 10 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer.
The pH was determined in the remaining part of the reaction mixture.
This procedure was repeated for the duplicate concentration. In each test series two controls without test substance were tested, one at the start and one at the end.
Each batch of activated sludge was checked for sensitivity by testing the reference substance 3,5-dichlorophenol.
The flasks were filled up to 500 ml final volume.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

dissolved

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

No significant inhibition of respiration rate of the sludge was recorded at 100 mg HATCOL 3331 per litre. The duplicate measurement confirmed the result of the first measurement. Therefore no further testing was needed.

Results with reference substance (positive control):

The EC50 of 3,5-dichlorophenol was 11 mg/L (regression line: Y = 66.93 X -20.50, Y = % inhibition and X = log concentration (mg/L)).

Reported statistics and error estimates:

Not specified in the study report.

Table 1: pH, oxygen concentration at the start of measurement and the influence of 3,5-dichlorophenol and HATCOL 3331 on the oxygen consumption of microbes in activated sludge and percentage inhibition of respiration rate.

Flask	Concentration reference/test substance (mg/l)	Oxygen conc. at the start (= mg O2/l)	Oxygen consumption mg O2/l/hr	Inhibition %	pH
C1	-	7.4	32	-	7.7
C2	-	8.5	28	-	7.8
Mean C1 + C2			30 (∆13%)
R1	3.2	7.5	27	10	7.6
R2	10	7.9	14	53	7.6
R3	32	8.4	7	77	7.6
T1	100	7.4	30	0	7.6
T2	100	7.4	28	7	7.6

C: Control

R: Reference substance, 3,5-dichlorophenol

T: Test substance, HATCOL 3331

 

Table 2: Determination of the EC50 value for 3,5-dichlorophenol

Parameter: % inhibition

Concentration (mg/l)	X Log conc. (mg/l)	Y Inhibition (%)
3.2	0.505	10
10	1.000	53
32	1.505	77

Prediction of X values based on known Y values

Known Y Inhibition (%)	10^Xreg(mg/l)	10^X95%-(mg/l)	10^X95%+(mg/l)
50	11.3	0.2	658.2

Regression line: Y = 66.93 X -20.50

Slope: 66.9324

Intercept: -20.4955

Multiple R: 0.9859

n = number of observations: 3

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this present test, HATCOL 3331 was not toxic to waste water (activated sludge) bacteria at a concentration of 100 mg/L.

Executive summary:

The influence of HATCOL 3331 on the respiration rate of activated sludge was investigated after a contact time of 30 minutes.

The study procedure was based on OECD Guideline No. 209, adopted April 4, 1984 and EEC Directive 67/548 amended November 18, 1987 (87/302), Part C, Publication No. L 133, adoptedMay 30, 1988.

Since HATCOL 3331 was hardly soluble in water, the test substance was quantiatively added to the test vessels. A nominal concentration of 100 mg/L was tested in duplicate. The concentration was approved by the study director in the study files.

No significant inhibition of respiration rate of the sludge was recorded at 100 mg HATCOL 3331 per litre. The duplicate measurement confirmed the result of the first measurement. Therefore no further testing was needed.

The respiration rates of the controls were within 15% of each other.

The EC50 of the reference substance, 3,5-dichlorophenòl, was 11 mg/L.

Therefore, the test was considered to be valid.

In conclusion, under the conditions of this present test, HATCOL 3331 was not toxic to wastewater (activated sludge) bacteria at a concentration of 100 mg/L.

Reference 3

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 November 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD & EU test guidelines in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Analytical monitoring:

no

Details on sampling:

Since HATCOL 5236 was hardly soluble in water, the test substance was quantiatively added to the test vessels. A nominal concentrtion of 100 mg/l was tested in duplicate.

Vehicle:

no

Details on test solutions:

Since HATCOL 5236 was hardly soluble in water, the test substance was quantiatively added to the test vessels. A nominal concentrtion of 100 mg/L was tested in duplicate.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Test System: Micro-organisms in activated sludge.
Source: Municipal sewage treatment plant: 'Waterschap de Maaskant', 's-Hertogenbosch, the Netherlands.
Number of micro-organisms: Number of micro-organisms was determined as the amount of Mixed Liquor Suspended Solids (MLSS) per litre test medium.
Preparation of the sludge: The sludge was coarsely sieved, washed and diluted with tap-water. A small amount of the sludge was weighed and dried at ca. 105°C to determine the amount of suspended solids (2.5 g/l of sludge, as used for the test). Before use the pH was checked (measured value: 7.6).
The batch of sludge was used on the subsequent day, therefore 50 ml of synthetic sewage feed was added to each lire of activated sludge at the end of each working day. The sludge was kept aerated at test temperature until use.
Rationale: Recognized by international guidelines as the recommended test system.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

30 min

Post exposure observation period:

No post exposure observation period specified in the study report.

Hardness:

Not specified in the study report.

Test temperature:

The temperature of the test medIum was 21.6°C.

pH:

7.8 - 8.1

Dissolved oxygen:

Not applicable - respiration inhibition study

Salinity:

Not applicable - freshwater study

Nominal and measured concentrations:

Nominal concentrations.

Details on test conditions:

TEST PROCEDURE AND CONDITIONS
Contact time: 30 minutes, during which aeration and stirring took place.
Vessels: All glass, 300 ml oxygen bottles and 1 L test bottles.
Milli-RO / Milli-Q water: Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon and ¡on-exchange cartridges (Milli-Q) (Millipore Corp., Bedford, Mass., USA).
Synthetic sewage feed: 16 g peptone; 11 g meat extract; 3 g urea; 0.7 g NaCI; 0.4 g CaCI2.2H20; 0.2 g MgSO4 .7H2O; 2.8 g K2HPO4. Dissolved in 1 L Milli-Q water and filtered. The pH was 7.1.
Air supply: Clean, oil-free air.
Oxygen meter: WTW inolab Oxi Level 2 supplied with a WT CellOx 325 oxygen electrode, electrolyte type ELY/G.
Recorder: Flatbed recorder SE 102 (Kipp & Zonen).
Performance of the test: The synthetic sewage feed (16 ml) and an adequate amount of the test substance were mixed and made up to 300 ml with Milli-RO water. Activated sludge (200 ml) was added and the mixture was aerated in a 1 L bottle during the contact time, using a pipette as an aeration device.
Then a well mixed sample of the contents was poured Into a 300 ml oxygen bottle, and the flask was sealed with an oxygen electrode connected to a recorder, forcing the air out of the vessel. Oxygen consumption was measured and recorded for approximately 10 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer. The pH was determined In the remaining part of the reaction mixture.
This procedure was repeated for the duplicate concentration. In each test series two controls without test substance were tested, one at the start and one at the end.
Each batch of activated sludge was checked for sensitivity by testing the reference substance 3.5-dichlorophenol.
The flasks were filled up to 500 ml final volume.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol.

Key result

Duration:

30 min

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

Toxicity of HATCOL 5236
No inhibition of respiration rate of the sludge was recorded at 100 mg HATCOL 5236 per litre.
The duplicate measurement confirmed the result of the first measurement.
Therefore no further testing was needed.

Experimental conditions
The temperature of the test medium was 21.6°C.

Acceptability of the test
The mean respiration rate of control 1 and 2 was 31 mg O2/l/hr. The difference between the controls was 3%.

Since all criteria for acceptability of the test were met, this study was considered to be valid.

Results with reference substance (positive control):

The EC50 of 3,5-dichlorophenol was 12 mg/l (regression line: Y = 56.01 X -1 0.87, Y = % inhibition and X = log concentration (mg/l)).

Reported statistics and error estimates:

Not specified in the study report.

pH, oxygen concentration at the start of measurement and the influence of 3,5-dichlorophenol and HATCOL 5236 on the oxygen consumption of microbes in activated sludge and percentage inhibition of respiration rate

Flask	Concentration reference/test substance (mg/l)	Oxygen conc. at the start (= mg O2/l)	Oxygen consumption mg O2/l/hr	Inhibition %	pH
C1	-	8.3	30	-	8.1
C2	-	8.2	31	-	8.1
Mean C1 + C2			31 (∆3%)
R1	3.2	8.0	25	18	8.1
R2	10	8.4	17	44	8.1
R3	32	8.5	8	74	8.1
T1	100	7.0	33	-8^1	7.8
T2	100	6.5	33	-8^1	7.9

C: Control; R: Reference substance; 3,5-dichlorophenol; T: Test substance, HATCOL 5236;¹: Negative values indicate simulation of respiration rate of the sludge. These values are considered as not significant.

 

Determination of the EC50-value for 3,5-dichlorophenol

Parameter: % inhibition

Concentration (mg/l)	X Log conc. (mg/l)	Y Inhibition (%)
3.2	0.505	18
10	1.000	44
32	1.505	74

 

Prediction of X values based on known Y values

Known Y Inhibition (%)	10^Xreg(mg/l)	10^X95%(mg/l)	10^X95%+(mg/l)
50	12.2	5.2	28.5

 

Validity criteria fulfilled:

yes

Conclusions:

In conclusion. under the conditions of the test, HATCOL 5236 was not toxic to waste water (activated sludge) bacteria at a concentration of 100 mg/l.

Executive summary:

The Influence of HATCOL 5236 on the respiration rate of activated sludge was investigated after a contact time of 30 minutes.

The study procedure was based on OECD Guideline No. 209, adopted April4, 1984 and EEC Directive 67/548 amended November 18,1987 (87/302), Part C, Publication No. L133, adopted May 30, 1988.

Since HATCOL 5236 was hardly soluble in water, the test substance was quantitatively added to the test vessels. A nominal concentration of 100 mg/l was tested in duplicate. The concentration was approved by the study director in the study files.

No Inhibition of respiration rate of the sludge was recorded at 100 mg HATCOL 5236 per lire.

The duplicate measurement confirmed the result of the first measurement. Therefore no further testing was needed.

The respiration rates of the controls were within 15% of each other.

The EC50 of the reference substance, 3,5 dichlorophenol. was 12 mg/l.

Therefore, the test was considered to be valid.

In conclusion under the conditions of this present test, HATCOL 5236 was not toxic to waste water (activated sludge) bacteria at a concentration of 100 mg/l.

Description of key information

The read across for substance, CAS: 156558-98-4; EC: 451-190-0; is based upon the analogous substances to which basic form, degree of substitution of functional groups is not considered to effect the proposed read across for the endpoint of toxicity to microorganisms. The NOEC for the substance based on the mean of the information available is deemed to be 400 mg/L, to which no adverse effects were noted upto the limit of solubility.

Key value for chemical safety assessment

EC50 for microorganisms:

100 mg/L

Additional information

HATCOL 3331

The influence of HATCOL 3331 on the respiration rate of activated sludge was investigated after a contact time of 30 minutes.

Since HATCOL 3331 was hardly soluble in water, the test substance was quantitatively added to the test vessels. A nominal concentration of 100 mg/L was tested in duplicate. The concentration was approved by the study director in the study files.

No significant inhibition of respiration rate of the sludge was recorded at 100 mg HATCOL 3331 per litre. The duplicate measurement confirmed the result of the first measurement. Therefore no further testing was needed.

The respiration rates of the controls were within 15% of each other.

The EC50 of the reference substance, 3,5-dichlorophenòl, was 11 mg/l.

Therefore, the test was considered to be valid.

In conclusion, under the conditions of this present test, HATCOL 3331 was not toxic to wastewater (activated sludge) bacteria at a concentration of 100 mg/l.

 

HATCOL 5236

The Influence of HATCOL 5236 on the respiration rate of activated sludge was investigated after a contact time of 30 minutes.

Since HATCOL 5236 was hardly soluble in water, the test substance was quantitatively added to the test vessels. A nominal concentration of 100 mg/l was tested in duplicate. The concentration was approved by the study director in the study files.

No Inhibition of respiration rate of the sludge was recorded at 100 mg HATCOL 5236 per lire.

The duplicate measurement confirmed the result of the first measurement. Therefore no further testing was needed.

The respiration rates of the controls were within 15% of each other.

The EC50 of the reference substance, 3,5 dichlorophenol was 12 mg/l.

Therefore, the test was considered to be valid.

In conclusion under the conditions of this present test, HATCOL 5236 was not toxic to waste water (activated sludge) bacteria at a concentration of 100 mg/l.

 

The toxicity of the source substance PE/TMP tetra/tri C5, i-C5, C7, C8, i-C9, C10 ester to activated sludge microorganisms was investigated according to OECD 209 under GLP conditions (Mead, 1998). A test concentration of 1000 mg/L (nominal) did not inhibit the respiration rate of microorganisms from a sewage treatment plant. Therefore, the test substance is not toxic to activated sludge microorganisms and a NOEC (3 h) of ≥ 1000 mg/L resulted.

Based on the results from a structurally similar source substance (in accordance to Regulation (EC) No 1907/2006 Annex XI, 1.5) it can be concluded that Pentaerythritol tetraesters of n-C5, n-C7, n-C8, i-C9 and n-C10 fatty acids with pentaerythritol will not show effects to activated sludge microorganisms and it is not expected that the degradation process in sewage treatment plants will be inhibited.