Reaction product of mixed triesters of heptanoic acid (n-C7), nonanoic acid (n-C9) and trimethylolpropane (i-C7)

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:

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

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

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 2

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

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

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.

Reference 3

Endpoint:

toxicity to microorganisms

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Data based on the inhibition control of a ready biodegradability study. This approach is in accordance with the Guidance on information requirements and chemical safety assessment (Chapter R.7b: Endpoint specific guidance, ECHA 2012).

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

GLP compliance:

no

Test organisms (species):

activated sludge, domestic

Details on inoculum:

- Source of inoculum/activated sludge: Activated sludge from the aeration basin of the domestic sewage treatment plant in Lachen-Speyerdorf, Germany (2008-05-08)- Preparation of inoculum for exposure: The sludge was aerated continually, washed with and re-suspended in mineral medium.- Initial biomass concentration: 25 mg suspended solids/L

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

14 d

Test temperature:

22 ± 1°C

Nominal and measured concentrations:

Nominal test substance concentration:30 ± 2 mg/L

Details on test conditions:

TEST SYSTEM- Test vessel: 2000 mL-SCHOTT-flasks - Fill volume: 1500 mL- Aeration: with purified, CO2-scribbed, moistened airTEST MEDIUM / WATER PARAMETERS- Source/preparation of dilution water: Mineral medium according to OECD guidelineEFFECT PARAMETERS MEASURED:CO2 evolution on day 0, 2, 4, 7, 9, 11 and 14

Reference substance (positive control):

yes

Remarks:

Anilin

Key result

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

30.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Result of toxicity control from ready biodegradability test

Results with reference substance (positive control):

The reference material (Anilin) was degraded by 3.5% after 7 days, 60% pass level was reached.

Degradation (%) the toxicity control in ready biodegradability study following OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Day	Toxicity control
2	1.3
4	41.8
7	65.9
9	70.4
11	74.4
14	80.4
17	77.2
23	84.3
29	87.9

Reference 4

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

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: 2TEST MEDIUM / WATER PARAMETERS- Source/preparation of dilution water: dechlorinated tap waterEFFECT 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

Key result

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

Key result

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 5

Endpoint:

toxicity to microorganisms

Type of information:

experimental study

Adequacy of study:

supporting 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

Key result

Duration:

6 h

Dose descriptor:

EC10

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Key result

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 Pentaerythritol C7-C10 tetra ester	0.630	0.614	-
16	Blank uninoculated	0.033
17	0.1% Acetone uninoculated	0.010
19	10 mg/l Pentaerythritol C7-C10 tetra ester uninoculated	0.016

 

Reference 6

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

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:

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

Key value determined by experiment to OCED guideline 209 and EU test standard C11.

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.

CAS 11138 -60 -6

The study was conducted with CAS 11138-60-6 using activated sludge (domestic) according to OECD 209. It showed an increase about 43% of respiration rate at the nominal tested concentration of 10000 mg/L in comparison to the control. No inhibition of respiration rate was observed. Therefore the EC50 and EC10 (3h) can be stated as > 10000 mg/L (nominal).

CAS 78 -16 -0

The study with CAS 78-16-0 was based on the inhibition control of a ready biodegradability study. Since more than 80.4% degradation occurred in the toxicity control, the substance is with a high probability not toxic to aquatic microorganisms. The test item concentration in the toxicity control of 30.4 mg/L can be used as NOEC value for the toxicity to aquatic microorganisms.

HATCOL 1765

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.

 

CAS 71010-76-9

The 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.