Fatty acids, C16-18 and C18-unsatd., esters with propylene glycol

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:

1 Aug - 22 Sept 1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

GLP Guideline study with acceptable restrictions (no analytical purity of the test substance).

Qualifier:

according to guideline

Guideline:

other: ISO 10712 "Determination of the inhibitory effect of water constituents on bacteria (Pseudomonas cell multiplication inhibition test)

Qualifier:

according to guideline

Guideline:

other: German Water Hazard Classification Scheme (Bewertung Wassergefährdender Stoffe LTWS - Nr 10)

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the government of the United Kingdom

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: 12.5 g of test material was placed on the surface of 1 L of sterile reverse osmosis water to give a loading rate of 12500 mg/L which was stirred for 23 h. Stirring was stopped after 23 h and the contents allowed to stand for 1 h prior to removal of the aqueous phase of WSF (Water soluble fraction). The WSF was sterilised by filtration through 0.45 µm filters prior to testing. To an aliquot (80 mL) of the 12500 mg/L loading rate WSF, nutrient stock solutions and bacterial suspension were added to give the required concentration of 10000 mg/L loading rate WSF.
- Eluate: no
- Differential loading: no
- Controls: yes, nutrient medium with bacterial suspension without test material

Test organisms (species):

Pseudomonas putida

Details on inoculum:

- Laboratory culture: yes, Pseudomonas putida strain NCIMB 8248
- Method of cultivation: Freeze dried cultures were obtained from the National Collections of Industrial and Marine Bacteria, Aberdeen, Scotland. Cultures were maintained in the laboratory by routine sub-culturing onto fresh agar slopes approximately once per week. Cultures were maintained in the laboratory at a temperature of 25 ± 1 °C.
- Preparation of inoculum for exposure: 17 h prior to commencing the test, an aqueous suspension of P. putida was produced by adding pre-culture medium to a stock culture of the bacterium and gently shaking in order to wash the bacterial cells off the solid medium. The resultant suspension was dispersed into a sterile flask plugged with sterile non-absorbant cotton wool and incubated at 25 ± 1 °C. After the initial incubation period of 17 h, the bacterial suspension was diluted using pre-culture medium to give a turbidity of approx. 100 Formazine Turbidity Units (FTU). An aliquot of 50 mL of the 100 FTU bacterial suspension was added to 450 mL of pre-culture medium and incubated at 25 ± 1 °C. After incubation for approx. 8 h the bacterial suspension had a turbidity of approx. 50 FTU.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

16 h

Test temperature:

25 ± 1 °C

Nominal and measured concentrations:

nominal: control, 10000 mg/L

Details on test conditions:

TEST SYSTEM
- Material, size, headspace, fill volume: glass conical flasks, 250 mL, headspace: 150 mL, fill volume: 100 mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: sterile reverse osmosis water was used to prepare the nutrient medium according to the guideline.
- Culture medium different from test medium: no

EFFECT PARAMETERS MEASURED: Inhibitory effect on cell multiplication was measured after 16 h.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: Control, 1000, 10000 mg/L loading rate WSF
- Results used to determine the conditions for the definitive study: No inhibitory effect on cell multiplication was observed.

Reference substance (positive control):

no

Key result

Duration:

16 h

Dose descriptor:

NOEC

Effect conc.:

>= 10 000 mg/L

Nominal / measured:

nominal

Conc. based on:

other: filtered test solution

Basis for effect:

growth inhibition

Key result

Duration:

16 h

Dose descriptor:

EC10

Effect conc.:

> 10 000 mg/L

Nominal / measured:

nominal

Conc. based on:

other: filtered test solution

Basis for effect:

growth inhibition

Key result

Duration:

16 h

Dose descriptor:

EC50

Effect conc.:

> 10 000 mg/L

Nominal / measured:

nominal

Conc. based on:

other: filtered test solution

Basis for effect:

growth inhibition

Details on results:

After 16 hours the EC50 was determined to be >= 10 000 mg/L.

Reported statistics and error estimates:

A Students t-test was carried out to determine any statistically significant differences between the test and control groups.

Table 1: Results from validation of mixing procedure

Time [h]	DOC [mg C/L]
24	3.534
48	< Control
72	0.218
96	0.343
120	0.485

Conclusions:

After 16 hours the EC50 was determined to be >= 10 000 mg/L.

Reference 2

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 18 Jul 1997 to 27 Jul 1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

GLP Guideline study. In accordance to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, 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:

EU Method C.11 (Biodegradation: 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: The test substance was added directly to the test vessels containing 100 mL deionised water. Approx. 100 mL of drinking water was added to 16 mL of synthetic sewage. 200 mL of inoculum was added additionally. This mixture was added to the test vessels.
- Eluate: no
- Differential loading: yes
- Controls: yes, activated sludge + synthetic sewage control

Test organisms (species):

activated sludge, domestic

Details on inoculum:

- Laboratory culture: no
- Pretreatment: Sludge was allowed to settle on day of collection and washed with 1 – 2 L drinking water. 200 mL was synthetic sewage was added and filled up to 4 L with drinking water. This solution was stirred and aerated overnight.
- Preparation of inoculum for exposure: On the day of testing, the sludge was washed again with 2 L drinking water and added to 5.3 L drinking water.
- Other: Activated sludge was obtained from sewage treatment plant Marl-West, Marl, Germany.
- Initial biomass concentration: 1515 mg/L dry weight

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

18 - 21 °C

pH:

8.1 - 8.5

Nominal and measured concentrations:

nominal: 0, 45.8, 91.6, 183.2, 366.4, 1099 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: graduated cylinders
- Size, fill volume: 500 mL, fill volume: 500 mL, headspace: 0 mL
- Aeration: continuously
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Synthetic sewage was prepared according to the guideline 88/302/EEC C.11

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : O2 consumption after 3 h

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2 - 3

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

> 1 099 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.:

> 1 099 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
- Relevant effect levels: EC50: 8.7 mg/L

Validity criteria fulfilled:

yes

Conclusions:

After 3 hours the EC50 was determined to be > 1099 mg/L (EU Method C.11, activated sludge).

Reference 3

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:

from 18 Jul 1997 to 27 Jul 1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

GLP Guideline study. In accordance to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Justification for type of information:

Please refer section 13 for read across justification.

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: 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: The test substance was added directly to the test vessels containing 100 mL deionised water. Approx. 100 mL of drinking water was added to 16 mL of synthetic sewage. 200 mL of inoculum was added additionally. This mixture was added to the test vessels.
- Eluate: no
- Differential loading: yes
- Controls: yes, activated sludge + synthetic sewage control

Test organisms (species):

activated sludge, domestic

Details on inoculum:

- Laboratory culture: no
- Pretreatment: Sludge was allowed to settle on day of collection and washed with 1 – 2 L drinking water. 200 mL was synthetic sewage was added and filled up to 4 L with drinking water. This solution was stirred and aerated overnight.
- Preparation of inoculum for exposure: On the day of testing, the sludge was washed again with 2 L drinking water and added to 5.3 L drinking water.
- Other: Activated sludge was obtained from sewage treatment plant Marl-West, Marl, Germany.
- Initial biomass concentration: 1515 mg/L dry weight

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

18 - 21 °C

pH:

8.1 - 8.5

Nominal and measured concentrations:

nominal: 0, 45.8, 91.6, 183.2, 366.4, 1099 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: graduated cylinders
- Size, fill volume: 500 mL, fill volume: 500 mL, headspace: 0 mL
- Aeration: continuously
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Synthetic sewage was prepared according to the guideline 88/302/EEC C.11

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : O2 consumption after 3 h

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2 - 3

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

> 1 099 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.:

> 1 099 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
- Relevant effect levels: EC50: 8.7 mg/L

Validity criteria fulfilled:

yes

Conclusions:

After 3 hours the EC50 was determined to be > 1099 mg/L (EU Method C.11, activated sludge).

Reference 4

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:

1 Aug - 22 Sept 1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

GLP Guideline study with acceptable restrictions (no analytical purity of the test substance).

Justification for type of information:

Please refer section 13 for read across justification.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: ISO 10712 "Determination of the inhibitory effect of water constituents on bacteria (Pseudomonas cell multiplication inhibition test)

Qualifier:

according to guideline

Guideline:

other: German Water Hazard Classification Scheme (Bewertung Wassergefährdender Stoffe LTWS - Nr 10)

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the government of the United Kingdom

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: 12.5 g of test material was placed on the surface of 1 L of sterile reverse osmosis water to give a loading rate of 12500 mg/L which was stirred for 23 h. Stirring was stopped after 23 h and the contents allowed to stand for 1 h prior to removal of the aqueous phase of WSF (Water soluble fraction). The WSF was sterilised by filtration through 0.45 µm filters prior to testing. To an aliquot (80 mL) of the 12500 mg/L loading rate WSF, nutrient stock solutions and bacterial suspension were added to give the required concentration of 10000 mg/L loading rate WSF.
- Eluate: no
- Differential loading: no
- Controls: yes, nutrient medium with bacterial suspension without test material

Test organisms (species):

Pseudomonas putida

Details on inoculum:

- Laboratory culture: yes, Pseudomonas putida strain NCIMB 8248
- Method of cultivation: Freeze dried cultures were obtained from the National Collections of Industrial and Marine Bacteria, Aberdeen, Scotland. Cultures were maintained in the laboratory by routine sub-culturing onto fresh agar slopes approximately once per week. Cultures were maintained in the laboratory at a temperature of 25 ± 1 °C.
- Preparation of inoculum for exposure: 17 h prior to commencing the test, an aqueous suspension of P. putida was produced by adding pre-culture medium to a stock culture of the bacterium and gently shaking in order to wash the bacterial cells off the solid medium. The resultant suspension was dispersed into a sterile flask plugged with sterile non-absorbant cotton wool and incubated at 25 ± 1 °C. After the initial incubation period of 17 h, the bacterial suspension was diluted using pre-culture medium to give a turbidity of approx. 100 Formazine Turbidity Units (FTU). An aliquot of 50 mL of the 100 FTU bacterial suspension was added to 450 mL of pre-culture medium and incubated at 25 ± 1 °C. After incubation for approx. 8 h the bacterial suspension had a turbidity of approx. 50 FTU.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

16 h

Test temperature:

25 ± 1 °C

Nominal and measured concentrations:

nominal: control, 10000 mg/L

Details on test conditions:

TEST SYSTEM
- Material, size, headspace, fill volume: glass conical flasks, 250 mL, headspace: 150 mL, fill volume: 100 mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: sterile reverse osmosis water was used to prepare the nutrient medium according to the guideline.
- Culture medium different from test medium: no

EFFECT PARAMETERS MEASURED: Inhibitory effect on cell multiplication was measured after 16 h.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: Control, 1000, 10000 mg/L loading rate WSF
- Results used to determine the conditions for the definitive study: No inhibitory effect on cell multiplication was observed.

Reference substance (positive control):

no

Key result

Duration:

16 h

Dose descriptor:

NOEC

Effect conc.:

>= 10 000 mg/L

Nominal / measured:

nominal

Conc. based on:

other: filtered test solution

Basis for effect:

growth inhibition

Key result

Duration:

16 h

Dose descriptor:

EC10

Effect conc.:

> 10 000 mg/L

Nominal / measured:

nominal

Conc. based on:

other: filtered test solution

Basis for effect:

growth inhibition

Key result

Duration:

16 h

Dose descriptor:

EC50

Effect conc.:

> 10 000 mg/L

Nominal / measured:

nominal

Conc. based on:

other: filtered test solution

Basis for effect:

growth inhibition

Details on results:

After 16 hours the EC50 was determined to be >= 10 000 mg/L.

Reported statistics and error estimates:

A Students t-test was carried out to determine any statistically significant differences between the test and control groups.

Table 1: Results from validation of mixing procedure

Time [h]	DOC [mg C/L]
24	3.534
48	< Control
72	0.218
96	0.343
120	0.485

Conclusions:

After 16 hours the EC50 was determined to be >= 10 000 mg/L.

Description of key information

EC10 (16 h) > 10000 mg/L (ISO 10712, Pseudomonas putida)
EC10 (3 h) > 1099 mg/L (EU Method C.11, activated sludge)

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 099 mg/L

Additional information

Since no studies investigating the toxicity to microorganisms of the test substance are available, in accordance to Regulation (EC) No. 1907/2006 Annex XI, 1.5 a read across conducted to the two structurally related substances propylene glycol diisostearate (CAS 68958-54-3) and butylene glycol dicaprylate / dicaprate (CAS 853947-59-8). This read-across is justified in detail in the overall summary (IUCLID chapter 6.1) and within the analogue justification in IUCLID Section 13.

The study with propylene glycol diisostearate was performed according to ISO 10712 under GLP conditions with Pseudomonas putida (Mead, 1997). A filtered test substance concentration of nominal 10000 mg/L was tested in this study. No inhibition of growth was observed after an incubation of 16 h resulting in an EC10 of > 10000 mg/L (nominal). The influence of the read-across substance butylene glycol dicaprylate / dicaprate to activated sludge microorganisms was investigated in a GLP study according to EU Method C.11 (Diefenbach, 1997). A test concentration of nominal 1099 mg/L did not result in an inhibition of the respiration rate after 3 hours. Thus, an EC10 of > 1099 mg/L was derived.

Based on the results from structurally related read-across substances (in accordance to Regulation (EC) No 1907/2006 Annex XI, 1.5) which are characterized by an equal ecotoxicological profile, it can be concluded that the test substance will not exhibit toxic effects to microorganisms.