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Endpoint:
toxicity to microorganisms, other
Remarks:
Inhibition of cell multiplication / Toxicity control from ready biodegradability study
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Refer to the Analogue Approach Justification document provided in Section 13
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Duration:
16 h
Dose descriptor:
EC50
Effect conc.:
8 062 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
WAF
Basis for effect:
other: inhibition of cell multiplication
Remarks on result:
other: Source: CAS 73296-86-3, monoester
Duration:
16 h
Dose descriptor:
EC50
Effect conc.:
9 411 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
WAF
Basis for effect:
other: inhibition of cell multiplication
Remarks on result:
other: Source: CAS 73296-86-3, diester
Duration:
14 d
Dose descriptor:
other: % degradation (CO2 evolution)
Effect conc.:
43.7 other: % degradation (CO2 evolution)
Basis for effect:
other: % degradation (CO2 evolution)
Remarks on result:
other: Source: Ready biodegradability study of CAS 73296-86-3, monoester
Endpoint:
toxicity to microorganisms, other
Remarks:
Toxicity control from biodegradation study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
13 May - 11 June 1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
GLP guideline study without analytics
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: OECD 301B: Ready biodegradability - CO2 evolution
Version / remarks:
17 July 1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Version / remarks:
December 1992
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
TEST CONDITIONS

- the Toxicity control contained 34.4 mg of the test substance, 80.6 mg of the reference substance sodium acetate and inoculum in 2 L of test medium.

- Composition of medium:
A) 8.50 g KH2PO4, 21.75 g K2HPO4, 67.20 g Na2HPO4 x 12 H2O, 0.50 g NH4Cl, dissolved in 1L MilliQ water, pH 7.4 +/- 0.2
B) 22.50 g MgSO4 x 7 H2O dissolved in 1L MilliQ water
C) 36.40 g CaCl2 x 2 H2O dissolved in 1L MilliQ water
D) 0.25 FeCl3 x 6 H2O dissolved in 1L MilliQ water
1 litre mineral medium contained 10 mL of solution A) and 1 mL of solutions B) to D) and MilliQ water
- Test temperature: 20-22°C
- pH (range between test start and end): 7.5- 8.0
- Preparation of test solutions: mineral components, MilliQ water (ca. 80 % total volume) and inoculum (1% final volume) were added to each bottle. This mixture was aerated with CO2-free air overnight to purge the system of CO2.

SAMPLING
- Sampling frequency: every second or third day during the first 10 days, and thereafter at least every fifth day until the 28th day
- Sampling method: Titration with Phenolphthalein as indicator; the CO2 produced in each test bottle reacted with the Ba(OH)2 in the gas scrubbing bottle and precipitated out as barium carbonate. The amount of CO2 produced was determined by titrating the remaining Ba(OH)2 with 0.05 M standardised HCl.


Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source of inoculum/activated sludge: freshly obtained from a municipal sewage treatment plant, Waterschap de Maaskant, s'Hertogenbusch, Netherlands
- Storage conditions: sludge was kept under continous aeration until further treatment
- Preparation of inoculum for exposure: Before use, the sludge was allowed to settle for at least 30 minutes and the liquid decanted.
- Concentration of sludge: 3.6 g/L of suspended solids
- Initial cell/biomass concentration: concentration of sludge supernatant: 1.44 x 10E5 cells/mL; the test system contained 1.44 x 10E3 cells/mL
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
14 d
Details on test conditions:
TEST SYSTEM
- Culturing apparatus: 2 litre all-glass brown coloured bottles
- Number of culture flasks/concentration: 1 for toxicity control
- Method used to create aerobic conditions: The test was started by bubbling CO2-free air through the solution (rate ca. 30-100 mL/min).
- Details of trap for CO2 and volatile organics if used: 4 g Ba(OH)2 x 8 H2O per L MilliQ water was filtered through filter paper and stored in a sealed vessel to prevent absorption of CO2 from air. Three CO2 absorbers (bottles filled with 100 mL 0.0125 M Ba(OH)2 were connected in series to the exit air line of each test bottle.
Reference substance (positive control):
yes
Remarks:
sodium acetate at a concentration of 40 mg/L, corresponding to TOC=12 mg/L
Duration:
14 d
Dose descriptor:
other: % degradation
Effect conc.:
43.7 other: % degradation
Basis for effect:
other: CO2 evolution
Details on results:
The toxicity control in the ready biodegradability study reached 43.7 % degradation (Pass level: 25%) within 14 days (based on ThCO2). Therefore, the test substance was assumed to be not inhibitory.
Results with reference substance (positive control):
The reference control containing sodium acetate reached the pass level of 60% biodegradation by day 14.

Table 1: Degradation of the toxicity control

Day No.

Degradation [%]

2

0.0

5

12.0

7

20.5

9

30.6

14

43.7

19

59.4

23

66.3

27

69.5

29

77.3

Table 2: Validity criteria

Criterion from the guideline

Outcome

Validity criterion fulfilled

Difference of extremes of replicate values of the removal of the test chemical at the plateau, at the end of the test or at the end of the 10-d window, as appropriate, is less than 20%.

 < 20%

 yes

Percentage degradation of the reference compound has reached the pass levels by day 14 (>60%).

 64.6%

 yes

The toxicity control should degrade to at least 35% (based on DOC) or at least 25% (based on ThOD or ThCO2) within 14 d.

 43.7%

 yes

The total CO2 evolution in the inoculum blank at the end of the test should not normally exceed 40 mg/L medium.

 28.1 mg/L

 yes

Endpoint:
toxicity to microorganisms, other
Remarks:
Inhibition of cell multiplication
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
16 April - 20 May 1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
GLP guideline study without analytical verification of the test item
Qualifier:
according to guideline
Guideline:
DIN 38412-8 (Pseudomonas Zellvermehrungshemmtest)
Version / remarks:
March 1991
Qualifier:
according to guideline
Guideline:
ISO 10712 (Water quality – Pseudomonas putida growth inhibition test (Pseudomonas cell multiplication inhibition test
Version / remarks:
January 1996
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preparation of Water Accomodated Fractions (WAFs); Due to the highly hydrophobic character of the test substance, the substane remained floating at the surface. Therefore, WAFs were prepared from a stock solution of a nominal loading rate of 12.5 g/L. The stock solution was stirred for 48 hours and left stabilising for another 26 hours. The water phase in the middle of the separating funnel was used for this test and identified as the WAF.
- Controls: Blank control and positive control
Test organisms (species):
Pseudomonas putida
Details on inoculum:
Details on Pseudomonas putida:
- Strain: P. putida MIGULA, Berlin 33/2 (DSM 50026)
- Source: BGA, Berlin, Germany
- Incubation temperature: 23 +/- 1 °C
- Incubation time: preliminary suspension cultures 6 +/- 1 hours, exposure 16 +/- 1 hours
- Age of bacteria stock culture at study initiation: max. 7 days
- Preliminary suspension cultures: a small amount of bacteria from the stock culture was inoculates in pre-culture medium (preparation see "Details on test conditions") in Erlenmeyer flasks. After incubation under continuous shaking in the darl, the final turbidity value of the bacterial suspension was adjusted up to FNU/436 nm = 50.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
16 h
Nominal and measured concentrations:
nominal: control, 310, 630, 1250, 2500, 5000 and 10000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL Erlenmeyer flasks, covered with Aluminium caps
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: nutrient medium: 18 g of agar dissolved in water, plus 50 mL of solution I, 125 mL of solution III and 100 mL of solution IV, made up to one L with water. For preparation of individual solutions, please see below. The nutrient medium is allowed to gel in portions of 6-10 mL at a slant.

Preparation of pre-culture medium: 25 mL of stock solutions I and III plus 50 mL of solution IV, made up to 1 L with water

Preparation of individual solutions:
Stock solution I: 10.0 g NaNO3, 2.4 g K2HPO4, 1.2 g KH2PO4, 1.0 g yeast extract, dissolved in 500 mL MilliQ water
Stock solution II: 10.0 g NaNO3, 2.4 g K2HPO4, 1.2 g KH2PO4, dissolved in 500 mL MilliQ water
Stock solution III: 40.0 g D (+) glucose monohydrate, dissolved in 500 mL MilliQ water
Stock solution IV: 0.01 g Fe (III) citrate, 4.0 g MgSO4 x 7 H2O, dissolved in 1 L MilliQ water

for further details on test solution preparation, please see Table 1 in section "Any other information on material and methods"

OTHER TEST CONDITIONS
- Adjustment of pH: The pH of the test substance stock solution was 6.5.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : After incubation for 16 hours, extinction values of the bacterial cell suspensions were measured and the cell multiplication inhibition for each test concentration was determined.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
16 h
Dose descriptor:
EC50
Effect conc.:
8 062 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
WAF
Basis for effect:
other: inhibition of cell multiplication
Duration:
16 h
Dose descriptor:
EC10
Effect conc.:
2 582 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
WAF
Basis for effect:
other: inhibition of cell multiplication
Details on results:
For further details on inhibition values, please see Table 2 in section "Any other information on results"
Results with reference substance (positive control):
EC50 = 22 mg/L (accepted range 10-30 mg/L)
Multiplication factor of the bacteria was > 60. Therefore it was concluded, that the test was valid.

Table 2: Extinction values of bacterial cell suspensions.

Concentration of test item [mg/L]

Mean extinction, corrected by turbidity control

[FNU 436 nm]

Inhibition [%]

310

242

-13.1

630

233

-8.9

1,250

239

-11.4

2,500

193

9.9

5,000

157

26.9

10,000

77

64.4

Table 3: Validity criteria

Criterion from the guideline

Outcome

Validity criterion fulfilled

The EC50 value of the reference substance 3,5 -dichlorophenol should be in the accepted range of 10 -30 mg/L.

 22 mg/L

 yes

The inoculum used in the control batch should be multiplied by a factor of at least 60 within the test period.

 factor of 179

 yes

Endpoint:
toxicity to microorganisms, other
Remarks:
Inhibition of cell multiplication
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
10 April - 20 May 1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
GLP guideline study without analytical verification of the test item
Qualifier:
according to guideline
Guideline:
DIN 38412-27 (Pseudomonas putida Zellvermehrungshemmtest)
Version / remarks:
March 1991
Qualifier:
according to guideline
Guideline:
ISO 10712 (Water quality – Pseudomonas putida growth inhibition test (Pseudomonas cell multiplication inhibition test
Version / remarks:
January 1996
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preparation of Water Accomodated Fractions (WAFs); Due to the highly hydrophobic character of the test substance, the substane remained floating at the surface. Therefore, WAFs were prepared from a stock solution of a nominal loading rate of 12.5 g/L. The stock solution was stirred for 48 hours and left stabilising for another 26 hours. The water phase in the middle of the separating funnel was used for this test and identified as the WAF.
- Controls: Blank control and positive control
Test organisms (species):
Pseudomonas putida
Details on inoculum:
Details on Pseudomonas putida:
- Strain: P. putida MIGULA, Berlin 33/2 (DSM 50026)
- Source: BGA, Berlin, Germany
- Incubation temperature: 23 +/- 1 °C
- Incubation time: preliminary suspension cultures 6 +/- 1 hours, exposure 16 +/- 1 hours
- Age of bacteria stock culture at study initiation: max. 7 days
- Preliminary suspension cultures: a small amount of bacteria from the stock culture was inoculates in pre-culture medium (preparation see "Details on test conditions") in Erlenmeyer flasks. After incubation under continuous shaking in the darl, the final turbidity value of the bacterial suspension was adjusted up to FNU/436 nm = 50.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
16 h
Nominal and measured concentrations:
nominal: control, 310, 630, 1250, 2500, 5000 and 10000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL Erlenmeyer flasks, covered with Aluminium caps
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: nutrient medium: 18 g of agar dissolved in water, plus 50 mL of solution I, 125 mL of solution III and 100 mL of solution IV, made up to one L with water. For preparation of individual solutions, please see below. The nutrient medium is allowed to gel in portions of 6-10 mL at a slant.

Preparation of pre-culture medium: 25 mL of stock solutions I and III plus 50 mL of solution IV, made up to 1 L with water

Preparation of individual solutions:
Stock solution I: 10.0 g NaNO3, 2.4 g K2HPO4, 1.2 g KH2PO4, 1.0 g yeast extract, dissolved in 500 mL MilliQ water
Stock solution II: 10.0 g NaNO3, 2.4 g K2HPO4, 1.2 g KH2PO4, dissolved in 500 mL MilliQ water
Stock solution III: 40.0 g D (+) glucose monohydrate, dissolved in 500 mL MilliQ water
Stock solution IV: 0.01 g Fe (III) citrate, 4.0 g MgSO4 x 7 H2O, dissolved in 1 L MilliQ water

for further details on test solution preparation, please see Table 1 in section "Any other information on material and methods"

OTHER TEST CONDITIONS
- Adjustment of pH: The pH of the test substance stock solution was 7.5.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
After incubation for 16 hours, extinction values of the bacterial cell suspensions were measured and the cell multiplication inhibition for each test concentration was determined.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Duration:
16 h
Dose descriptor:
EC50
Effect conc.:
9 411 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
WAF
Basis for effect:
other: inhibition of cell multiplication
Duration:
16 h
Dose descriptor:
EC10
Effect conc.:
4 792 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
WAF
Basis for effect:
other: inhibition of cell multiplication
Details on results:
For further details on inhibition values, please see Table 2 in section "Any other information on results"
Results with reference substance (positive control):
EC50 = 22 mg/L (accepted range 10-30 mg/L)
Multiplication factor of the bacteria was > 60. Therefore it was concluded that the test was valid.

Table 2: Extinction values of bacterial cell suspensions.

Concentration of test item [mg/L]

Mean extinction, corrected by turbidity control

[FNU 436 nm]

Inhibition [%]

310

207

3.7

630

251

-17.0

1,250

284

-32.8

2,500

251

-17.0

5,000

189

11.8

10,000

97

55.1

Table 3: Validity criteria

Criterion from the guideline

Outcome

Validity criterion fulfilled

The EC50 value of the reference substance 3,5 -dichlorophenol should be in the accepted range of 10 -30 mg/L.

 22 mg/L

 yes

The inoculum used in the control batch should be multiplied by a factor of at least 60 within the test period.

 factor of 179

 yes

Description of key information

Key value for chemical safety assessment

Additional information

Since no studies investigating the toxicity of Isooctadecanoic acid, mixed esters with oxybis[propanediol] to microorganisms are available, in accordance with Regulation (EC) No 1907/2006 Annex XI, 1.5, a read-across to the structurally related source substance Isooctadecanoic acid, ester with oxybis[propanediol] (CAS 73296-86-3) was conducted. The source substance is representative to evaluate the toxicity of the target substance to microorganisms.

The target substance Isooctadecanoic acid, mixed esters with oxybis[propanediol] exists as two different grades. Grade One is characterized by mainly triesters of C18 iso fatty acids of the alcohol component diglycerol. Grade Two is characterized by mainly tetraesters of C18 iso fatty acids of the alcohol component diglycerol.

The source substance Isooctadecanoic acid, ester with oxybis[propanediol] (CAS 73296-86-3) is characterized as a UVCB substance containing polyglycerol as alcohol component. Polyglycerol components are esterified to give mainly monoesters of C18 iso fatty acids.

Two studies are available for the source substance Isooctadecanoic acid, ester with oxybis[propanediol] (CAS 73296-86-3). Both studies investigated the effect of the test material on the inhibition of cell multiplication of Pseudomonas putida. Both were conducted in identical test systems. The studies were conducted according to ISO 10712 and DIN38412-8. The cells were exposed to nominal loading rates of 310, 630, 1250, 2500, 5000 and 10000 mg/ in a static freshwater system. After incubation for 16 hours, extinction values of the bacterial cell suspensions were measured and the cell multiplication inhibition for each test concentration was determined. An EC50 (16 h) of 8062 mg/L was calculated for the first study and an EC50 (16h) of 9411 mg/L for the second study.

In addition, for Isooctadecanoic acid, ester with oxybis[propanediol] (CAS 73296-86-3), a toxicity control from a ready biodegradability study is also available. This study supports the results from the cell multiplication-tests with Pseudomonas putida. If a compound degrades well in a ready biodegradability test, or does not inhibit the degradation of a positive control at a certain concentration, this concentration can be used as a NOEC value. This approach is in accordance with the Guidance on information requirements and chemical safety assessment (Chapter R.7b: Endpoint specific guidance, ECHA 2017).

A substance can be assumed to be not inhibitory to aquatic microorganisms, if in the toxicity control of a ready biodegradation test, more than 25% degradation based on oxygen demand (BOD/ThOD) occurred within 14 days (OECD guideline 301). The study for Isooctadecanoic acid, ester with oxybis[propanediol] (CAS 73296-86-3) was conducted according to OECD 301 B and EU Method C.4-C. Non-adapted, domestic activated sludge was exposed to 17.2 mg/L of the test material and 40.3 mg/L of the reference substance sodium acetate. The toxicity control attained 43.7 % degradation after 14 days of incubation. Hence, the substance is not toxic to aquatic microorganisms in the toxicity control and the test item concentration of 17.3 mg/L can be used as NOEC.

Based on the results for the structurally related read-across substance Isooctadecanoic acid, ester with oxybis[propanediol] (CAS 73296-86-3) (in accordance with Regulation (EC) No 1907/2006 Annex XI, 1.5) which is characterized by a similar ecotoxicological profile and comparable structure, it can be concluded that no toxicity of the target substance Isooctadecanoic acid, mixed esters with oxybis[propanediol] to microorganisms is expected.