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Endpoint:
water solubility
Type of information:
experimental study
Adequacy of study:
key study
Study period:
5 May - 5 October 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The saturation concentration of the test item Matrilox LP101M in ISO medium used for Fish early Life Stage tests (OECD 210) as well as Daphnia reproduction tests (OECD 211) was to be determined as a basis for aquatic toxicity testing. ISO medium according to OECD 203 is a low salt medium and closely mimics natural waters in this respect. Results of this test are therefore generally relevant with regard to solubility in water.
For testing the saturation concentration in water it was very important to avoid any formation of micro-droplets or colloidal suspensions. This especially applies in the present case as the test item has a low surface tension (29.6 mN/m) indicating surface activity. The most appropriate method for this substance type (high hydrophobicity; tendency of to form micro-droplets or colloidal suspensions) is the slow-stir flask method as demonstrated e.g. by Letinski et al. (2002; 2016).
Unfortunately, no OECD or other technical guideline is yet available for this method, while the most recent draft of OECD Series on Testing and Assessment No. 23 (Guidance document on aquatic toxicity testing of difficult test chemicals and mixtures; draft 2015) explicitly proposes this method to determine the saturation concentration in water. The applied methodology therefore considered OECD 123 (Partition Coefficient (1-Octanol/Water): Slow-Stirring Method), OECD Guideline No.105 ("Water Solubility", flask method) and Letinski et al. (2002). While according to OECD 123 a vortex of a depth between 0.5 and at maximum 2.5 cm would be acceptable, this relates to the n-Octanol - water interface and cannot be transferred 1:1 to the water solubility experiment (water-air interface). As such, Letinski et al. state that stirring produced “little or no visible vortex”. To avoid formation of micro-droplets or colloidal suspensions the latter approach was followed.

References:
Letinski, D.J.; Connelly, M.J.; Peterson, D.R.; Parkerton, T.F. (2002)
Slow-stir water solubility measurements of selected alcohols and diesters
Chemosphere, 48, 257-265

Letinski, D.J.; Parkerton, T.F.; Redman, A.D.; Connelly, M.J.; Peterson, B. (2016)
Water solubility of selected C9-C18 alkanes using a slow-stir technique: Comparison to structure - property models
Chemosphere, 150, 416-423
Qualifier:
equivalent or similar to guideline
Guideline:
other: OECD Guideline for Testing of Chemicals, Guideline No.123: "Partition coefficient (1-octanol/water): Slow-stirring method"
Version / remarks:
adopted March 23, 2006
Qualifier:
equivalent or similar to guideline
Guideline:
other: EU method A.23, "PARTITION COEFFICIENT (1- OCTANOL/WATER): SLOW-STIRRING METHOD
Version / remarks:
Commission Regulation (EC) No.440/2008, Annex, Part A, Methods for the determination of physico-chemical properties, 2014
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 105 (Water Solubility)
Version / remarks:
adopted July 27, 1995
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method A.6 (Water Solubility)
Version / remarks:
Commission Regulation (EC) No 440/2008, Annex, Part A, Methods for the determination of physico-chemicals properties, 2014
Guideline:
other: OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures"
Version / remarks:
December 15, 2000
Guideline:
other: OECD Guideline for Testing of Chemicals, Section 2, No. 203 "Fish, Acute Toxicity Test"
Version / remarks:
adopted July 17, 1992
Principles of method if other than guideline:
A slow stirring approach in a glass bottle was performed, i.e. the test item was carefully added onto the surface of the water phase (ISO medium) in excess, such that droplets swimming on the surface were visible (density lower than water). Stirring was kept to a minimum (no visible vortex) to avoid formation of colloidal suspension or micro-droplets. Test item concentrations were monitored by analysis of the test item in the water phase via LC (HPLC)-MS/MS.
GLP compliance:
yes (incl. QA statement)
Type of method:
other: Flask Method Slow Stirring Approach
Specific details on test material used for the study:
Partition Coefficient N-Octanol/Water (LogKow):
OECD 117: >6.2, see IUCLID section 4.7;
QSAR (KOWWIN): 12.1 (1);
QSAR (ACDLabs): 11.75 (2);
QSAR (ChemAxon): 10.4 (3);

(1) KOWWIN v. 1.68; © U.S. Environmental Protection Agency
(2) ACD ToxSuite 2.95.1 LogD – identical to log Kow due to constant behaviour over pH range 1 to 9; Danish (Q)SAR Database, Division of Diet, Disease Prevention and Toxicology, National Food Institute, Technical University of Denmark, http://qsar.food.dtu.dk
(3) ChemAxon, Calculator Plugin 17.10.0 (2017-05-30), https://www.chemaxon.com/

Density: 0.948 g/mL (see IUCLID section 4.4)
Surface Tension: 29.6 mN/m @20°C (see IUCLID section 4.10)
Physical state: liquid
Key result
Water solubility:
0.078 µg/L
Conc. based on:
test mat.
Loading of aqueous phase:
100 mg/L
Incubation duration:
21 d
Temp.:
22 °C
pH:
8
Remarks on result:
other: Test water: ISO medium according to OECD 203
Details on results:
Please see secton "Overall remarks, attachments".

Please see secton "Overall remarks, attachments".

Conclusions:
Water solubility (flask method, slow stir; GLP): 0.078 µg/L (ISO medium according to OECD 203)
Executive summary:

The purpose of this study was the determination of the solubility of the test item Matrilox LP101M in ISO medium (according to OECD 203) that can be used for Fish Early-Life Stage Toxicity tests as well as Daphnia magna Reproduction tests (OECD Guideline for Testing of Chemicals, Section 2, No. 210 and 211, respectively) based on analytical determination by Liquid Chromatography (HPLC) with MS/MS-detection. ISO medium according to OECD 203 is a low salt medium and closely mimics natural waters in this respect. Results of this test are therefore generally relevant with regard to solubility in water.

Because of the chemical properties of the test item (low surface tension (29.6 mN/m) indicating surface activity; high hydrophobicity) it was very important to avoid any formation of micro-droplets or colloidal suspensions while determining the saturation concentration in water. The most appropriate method for this substance type is the slow-stir flask method as demonstrated e.g. by Letinski et al. (2002; 2016).

Up to now, no OECD or other technical guideline is yet available for this method, while the most recent draft of OECD Series on Testing and Assessment No. 23 (Guidance document on aquatic toxicity testing of difficult test chemicals and mixtures; draft 2015) explicitly proposes this method to determine the saturation concentration in water. The applied methodology therefore considered beyond others OECD 123 (Partition Coefficient (1-Octanol/Water): Slow-Stirring Method), OECD Guideline No.105 ("Water Solubility", flask method) and  Letinski et al. (2002).

A Slow stirring approach in a glass bottle was performed, i.e. a distinct volume of the liquid test item was added to a distinct volume of ISO medium. The whole volume was stirred with no visible vortex formation to avoid formation of colloidal suspension or microdroplets until saturation of test item concentration in the ISO medium was reached. The test was performed under sterile conditions due to the biodegradability of the test item. Test item concentrations were monitored by analysis of the test item via analytical determination by Liquid Chromatography (HPLC) with MS/MS-detection in several samples taken after distinct time intervals.

Test item concentration of 100 mg/L was stirred in 3 replicates for 44 days without or only minimal vortex formation. Solubilisation kinetics in ISO medium was fast and saturation was likely achieved already at day 2. Fluctuations in concentration over time seem to be immanent to the experimental system, such that no typical saturation curve was obtained. It is therefore appropriate to use the arithmetic mean over all valid samples as the most representative value for the true water solubility. Thus, for the true solubility of the test item in water (ISO medium) a value of 0.078 µg/L was determined based on the arithmetic mean value over valid samples determined from solvent extraction (mean value for all flasks day 0 – 21),  corrected for recovery of the lowest fortification sample. This value is strongly supported by the solubility determined via direct measurement in ISO medium of 0.075 µg/L. Consideration of water solubility results determined via slow stir flask method for similar compounds corroborate the determined water solubility for trimethylolpropane tripelargonate.

The validity criteria for the analytical method of the direct test item determination in the aqueous samples have been met according to SANCO 3029.

The validity criteria for the analytical method of the test item enrichment by organic solvent extraction are not in full accordance to SANCO 3029 with respect to a lower recovery value than 70% in the lowest fortification level of 0.01 µg test item/L. However, SANCO 3029 states that lower recoveries are acceptable for difficult substances which clearly applies for the test item (high logKow, low concentration range). Therefore, the achieved recovery of 60% is acceptable.

Endpoint:
water solubility
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study has been performed according to the current guidelines (OECD 105 and GLP).
Qualifier:
according to guideline
Guideline:
OECD Guideline 105 (Water Solubility)
Deviations:
yes
Remarks:
As all measured DOC concentrations during equilibration laby below the LOQ, it was not possible to evaluate the measurements regarding differences during equilibration. This deviation was stated as uncritical. For more details see further in the RSS.
Qualifier:
according to guideline
Guideline:
EU Method A.6 (Water Solubility)
GLP compliance:
no
Type of method:
flask method
Key result
Water solubility:
< 0.08 mg/L
Conc. based on:
test mat.
Temp.:
20 °C
pH:
ca. 6
Remarks on result:
not determinable
Details on results:
- A preliminary test was performed in which the measured concentration lay below 5 mg/L. No preliminary test evaluating the temparature dependency was performed.
- During the final test, all values measured at the plateau stage were indicative of concentrations below the LOQ of 0.08 mg/L. A standard deviation is thus not calculable. The plateau was considered to be reached after 8 days.

 For the final test, 8μL – 100 μL (equivalent to 7.6 – 94.8 mg) TMP pelargonate was used to prepare a saturated solution as the water solubility detected in the pre-test measured conc lay below 5 mg/L. To determine the dependency of the water solubility on the amount of the test item added, four different loading rates were chosen.

 

Flasks:

No 1: 37.9 mg/L

No 2: 37.9 mg/L

No 3: 94.8 mg/L

No 4: 189.6 mg/L

No 5: 474.0 mg/L

 

Flask 1A was used for the sampling point 24h, flask 1B for the sampling point 48h and flask 1C for the sampling point 72h.

 

Performance of the test:

 

4 vessels (1C & 2 to 5) were set onto magnetic stirrers immediately and slow stirring was started. After 24+/- 2 hrs, flask 1B (sampling point 48h) and further after 24 +/-2 hrs, flask 1A (sampling point 24h) were set on the stirrer and all flasks were stirred further for 24 +/- 2 hrs at room temperature (20 +/- 0.5°C).

Then flasks 1A-C were taken from the stirrer and stored at 20+/- 0.5°C for equilibration. Samples were taken, as shaking of the test vessels was continued.

As all samples were clear, no centrifugation had to be performed. Immediately before measurement the clear samples were prepared for measurement by dilution with acetonitrile 1/1.

After 3 days signals below LOQ were measured only. As slow stirring was used and longer equilibration times were expected, the stirring time was extended and samples were taken after 8 days (total stirring time) again.

No turbidity was noticed in the samples taken again. After sample prep, the samples were measured with HPLC-MS-MS.

The final measurement was performed as determination from flask 2-5. All filtrated test solutions showed no Tyndall effect.

Conclusions:
Interpretation of results (migrated information): insoluble (< 0.1 mg/L)
Solubility of the test item was below 0.08 mg/L according to the LOQ of 0.04 mg/L and a dilution factor of 2 (dilution water/acetonitrile of 1/1). No dependency of solubility on amount of test item (nominal load 37.9 - 474 mg/L) and no temperature dependency of water solubility were evaluable.
Executive summary:

The solubility of the test substance in water was determined from the measured concentrations of the test item using LC-MS-MS in the clear test solutions.

 

In a preliminary study, solubility of the test item in water could be estimated as below 5 mg/L. The flask method was used for the determination of the solubility of the test item in water. No test for temperature dependency of the water solubility was performed as all measured concentrations of the main test lay below 0.08 mg/L.

 

In the main study, 8μL – 100 μL TMP pelargonate was pipetted onto the surface of 200 ml water in seven individual vessels (equivalent to 37.9 - 474 mg/L). Five vessels (flasks 1C (for sampling point 72h) and 2-6) were set onto the magnetic stirrer immediately. After 24+/- 2 hrs, flask 1B (sampling point 48h) and further after 24 +/-2 hrs, flask 1A (sampling point 24h) were set on the stirrer and all flasks were stirred further for 24 +/- 2 hrs at room temperature (20 +/- 0.5°C). Then flasks 1A-C were taken from the stirrer and stored at 20+/- 0.5°C for equilibration.

 

Samples were taken from the aqueous phase with a Pasteur pipette, examined for turbidity, diluted with acetonitrile 1/1 and analysed using LC-MS-MS while stirring of the test vessels was continued.

After 3 days only signals below LOQ were measured. As slow stirring was used and longer equilibration times were expected, the stirring time was extended and samples were taken again after 8 days (total stirring time).

No test item concentrations above 0.08 mg/L (LOQ) were measured. As no concentrations of DOC on day 8 were detected, vessels 2-5 were sampled and analysed in the same fashion. Dependency of solubility on the amount of the test item (nominal load) could not be detected. All measured concentrations lay below 0.08 mg/L. At the plateau, the concentration of trimetilolpropano tripelargonato is stated as: < 0.08 mg/L at 20.0 +/- 0.5°C for the following nominal concentration range of the test item in water: 37.9 – 474 mg/L.

Description of key information

Water solubility (flask method, slow stir; GLP): 0.078 µg/L (ISO medium according to OECD 203)

Key value for chemical safety assessment

Water solubility:
0.078 µg/L
at the temperature of:
22 °C

Additional information

The purpose of the reliable key study (Sonntag, 2017; GLP; RL1) was the determination of the solubility of the test item Matrilox LP101M (Trimethylolpropane trinonanoate) in ISO medium (according to OECD 203) that can be used for Fish Early-Life Stage Toxicity tests as well as Daphnia magna Reproduction tests (OECD Guideline for Testing of Chemicals, Section 2, No. 210 and 211, respectively) based on analytical determination by Liquid Chromatography (HPLC) with MS/MS-detection. ISO medium according to OECD 203 is a low salt medium and closely mimics natural waters in this respect. Results of this test are therefore generally relevant with regard to solubility in water.

Because of the chemical properties of the test item (low surface tension (29.6 mN/m) indicating surface activity; high hydrophobicity) it was very important to avoid any formation of micro-droplets or colloidal suspensions while determining the saturation concentration in water. The most appropriate method for this substance type is the slow-stir flask method as demonstrated e.g. by Letinski et al. (2002; 2016).

Up to now, no OECD or other technical guideline is yet available for this method, while the most recent draft of OECD Series on Testing and Assessment No. 23 (Guidance document on aquatic toxicity testing of difficult test chemicals and mixtures; draft 2015) explicitly proposes this method to determine the saturation concentration in water. The applied methodology therefore considered beyond others OECD 123 (Partition Coefficient (1 -Octanol/Water): Slow-Stirring Method), OECD Guideline No.105 ("Water Solubility", flask method) and  Letinski et al. (2002).

A Slow stirring approach in a glass bottle was performed, i.e. a distinct volume of the liquid test item was added to a distinct volume of ISO medium. The whole volume was stirred with no visible vortex formation to avoid formation of colloidal suspension or microdroplets until saturation of test item concentration in the ISO medium was reached. The test was performed under sterile conditions due to the biodegradability of the test item. Test item concentrations were monitored by analysis of the test item via analytical determination by Liquid Chromatography (HPLC) with MS/MS-detection in several samples taken after distinct time intervals.

Test item concentration of 100 mg/L was stirred in 3 replicates for 44 days without or only minimal vortex formation. Solubilisation kinetics in ISO medium was fast and saturation was likely achieved already at day 2. Fluctuations in concentration over time seem to be immanent to the experimental system, such that no typical saturation curve was obtained. It is therefore appropriate to use the arithmetic mean over all valid samples as the most representative value for the true water solubility. Thus, for the true solubility of the test item in water (ISO medium) a value of 0.078 µg/L was determined based on the arithmetic mean value over valid samples determined from solvent extraction (mean value for all flasks day 0 – 21),  corrected for recovery of the lowest fortification sample. This value is strongly supported by the solubility determined via direct measurement in ISO medium of 0.075 µg/L. Consideration of water solubility results determined via slow stir flask method for similar compounds corroborate the determined water solubility for trimethylolpropane tripelargonate.

The validity criteria for the analytical method of the direct test item determination in the aqueous samples have been met according to SANCO 3029.

The validity criteria for the analytical method of the test item enrichment by organic solvent extraction are not in full accordance to SANCO 3029 with respect to a lower recovery value than 70% in the lowest fortification level of 0.01 µg test item/L. However, SANCO 3029 states that lower recoveries are acceptable for difficult substances which clearly applies for the test item (high logKow, low concentration range). Therefore, the achieved recovery of 60% is acceptable.

A further (supporting) study is available (Johannes, 2013). Also this study was performed according to a slow stir flask method, and nominal concentrations applied onto the aqueous surface was between 37.9 - 474 mg test item/L. Sampling was performed after 3 days and 8 days of slow stirring. LC-MS/MS was used for analytical determination of the test item in the aquatic phase. The LOQ was at 80 µg test item/L, and all measured samples, independently from the applied nominal test item concentration or incubation time were below LOQ. The overall result for water solubility of trimethylolpropane trinonanoate from this study was:

Water solubility (20°C) < 80 µg/L.

This study is in full agreement with the key study on water solubility. Due to the high LOQ of the analytical method (80 µg/L) in relation to the actual solubility of the test item in water (0.078 µg/L, see key study above), the test item could not be determined in the aqueous phase in this study. The supporting study therefore confirms the very low water solubility of the submission substance.