Reaction mass of 1,3-dioxane-5,5-dimethanol and 1,3-Propanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]-

Biodegradation in water and sediment: simulation tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: simulation testing on ultimate degradation in surface water

Type of information:

experimental study

Remarks:

Final draft report

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 309 (Aerobic Mineralisation in Surface Water - Simulation Biodegradation Test)

Version / remarks:

2004

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and batch number of test material: Eurofins Selcia Ltd. - 12530SXD012-5
- Purity: 99.9 %

RADIOLABELLING INFORMATION
- Radiochemical purity: 98.5 %
- Specific activity: 6.91 MBq/mg
- Locations of the label: Central Carbon
- Expiration date of radiochemical substance: Not reported. The radiochemical purity of the test item was assessed prior and post application to test system to confirm its stability throughout the dosing periods.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Frozen (- 80 °C)
- An overall mean radiochemical purity (average over pre and post application) of 99.39 % was determined by HPLC analysis of the application solution. This indicates the stability and purity of the test item application solution throughout the application process.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- The provided test item solution (in acetonitrile) was diluted with HPLC grade water. No additional treatment was necessary.

Radiolabelling:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

natural water: freshwater

Details on source and properties of surface water:

- Details on collection: Source (LRA Labsoil)
Location (Calwich Abbey Lake, Staffordshire, England)
Sampling depth (30 - 40 cm - lake edge -)
Sampling date/time (24/01/2022 11:25 am – 12:00 pm)
Visual quality (clear, colourless)
- Storage conditions: 4 ± 2 °C
- Storage length: Not reported
- Temperature at time of collection: 5.0 °C
- pH at time of collection: 8.34
- Electrical conductivity (at sampling depth): 241 ppm (481 µS/cm)
- Redox potential initial/final: Not reported
- Oxygen concentration (at sampling depth): 85.6 %
- Hardness: Not reported
- Inorganic Carbon (IC): 49.6 mg/L
- Ammonium: 0.062 mg/L
- Nitrite: 0.024 mg/L
- Nitrate: 3.43 mg/L
- Total Phosphorous: 0.092 mg/L
- Dissolved Orthophosphate: < 0.05 mg/L
- Total Nitrogen: 7.03 mg/L
- Biological Oxygen Demand (BOD): 2.7 mg/L
- Total Organic Carbon (TOC): 5.14 mg/L
- Dissolved Organic Carbon (DOC): 4.66 mg/L
- Chlorophyll-a concentration (algal biomass): 0.185 µg/L
- Particulate matter: 24.67 mg/L
- Suspended solids: 15 mg/L
- Water filtered: yes
- Type and size of filter used: 100 µm filter bag

Duration of test (contact time):

90 d

Initial conc.:

10 µg/L

Based on:

test mat.

Initial conc.:

100 µg/L

Based on:

test mat.

Initial conc.:

10 µg/L

Based on:

other: reference substance

Initial conc.:

100 µg/L

Based on:

test mat.

Remarks:

sterile control

Parameter followed for biodegradation estimation:

radiochem. meas.

Details on study design:

TEST CONDITIONS
- Volume of test solution/treatment: 0.10 mL of application solution in 300 mL of surface water or in 500 mL of sterile surface water
- Test temperature: 12 ± 2 °C
- pH: the pH of the matrix characterisation sample (90 ± 2 DAT untreated control unit) was measured every 7 ± 2 days.
- pH adjusted: Not reported
- Aeration of dilution water: aeration was ensured with continuous agitation (approximately 100 rpm), and connection to a flow-through apparatus (only treated samples)
- Continuous darkness: yes
- Any indication of the test material adsorbing to the walls of the test apparatus: Not reported

TEST SYSTEM
- Method used to create aerobic conditions: continuous agitation (approximately 100 rpm) and flow-through apparatus (untreated control samples were not connected to flow-through apparatus)
- Method used to control oxygen conditions: the oxygen concentration of the matrix characterisation sample (90 ± 2 DAT untreated control unit) was measured every 7 ± 2 days.

- Test performed in open system: yes (flow-through apparatus)
- Details of trap for CO2 and volatile organics if used: The flow-through apparatus included three different volatile trapping mediums (one trap containing ethylene glycol, one trap containing 0.05 M sulphuric acid in water, and two traps containing 2 M sodium hydroxide in water) for non-sterile samples and one different trapping medium (two traps containing 2 M sodium hydroxide in water) for sterile and reference control samples. The flow-through apparatus was designed to pull any evolved gases from the incubated samples through the connected trapping solutions. As the gases pass through the solution any volatile components react with the solutions and are ‘trapped’. Trapping solutions were collected every 7 ± 2 days. Trapping solutions (ca. 25 mL in volume) were decanted into pre-weighed plastic pots before being replenished with ca. 25 mL of fresh tapping solution. Duplicate weighted ca. 0.4 – 0.5 mL aliquots were removed from each collected trapping solution and analysed for radioactivity via LSC.

SAMPLING
- Sampling frequency:
-Non-Sterile Aerobic Degradation = samples were collected at 0, 1, 3, 7, 14, 28, 60, and 90 DAT (Days After Treatment)
-Aerobic Sterile Controls = samples were collected at 7, 28, 60, and 90 DAT
-Aerobic Reference Controls = samples were collected at 90 DAT
-Capture and Assessment of Volatile Components & Confirmation of Mineralization = trapping solutions were collected every 7 ± 2 days.
- Sampling method used per analysis type:
Immediately following application, 0 DAT surface water samples (not attached to the flow-through apparatus) were decanted into pre-weighed pots (AQ1). At all other timepoints, samples were removed from the flow-through apparatus after the required incubation period and extracted prior to analysis. Sample trapping solutions were emptied into pre-weighed pots (pre-acid traps) and the two NaOH (2 M) trapping vessels were refilled with fresh NaOH (2 M) solution. Approximately half (ca. 150 mL) of each surface water sample was decanted from the sample vessel into pre-weighed pots (AQ1). The remaining volume (ca. 150 mL) of surface water in the sample vessel was acidified to pH 2 - 3 by adding ca. 3 - 5 drops of concentrated 12 M HCl. The sample was returned to the flow-through apparatus connected to the refilled traps (post-acid traps) to enable the capture of any dissolved organic carbon (14CO2) within the sample.

Following 1 ± 0.25 hours of agitation and aeration, the acidified surface water samples and NaOH (2 M) traps were decanted into additional pre-weighed pots (labelled AQ2 and post-acid traps, respectively). The empty sample vessels for all timepoints were then rinsed with ca. 300 mL acetonitrile and transferred into further pre-weighed pots (VR1). Empty sample vessels for samples with a mass balance < 95 % (including reference control samples) were further rinsed with ca. 300 mL acetone and transferred into pre-weighed pots (VR2). Reference control sample vessels were also further rinsed a third time with ca. 300 mL acetone and transferred into pre-weighed pots (VR3).

Each aqueous phase (AQ1 and AQ2) and vessel rinse (VR1, VR2 and VR3) for all samples were weighed and analysed via LSC for radioactivity on the day of extraction. All aqueous phases (AQ1 and AQ2) and vessel rinses (VR1, VR2 and VR3) were stored frozen (- 20 ± 10 °C) following each timepoint. Pre- and post-acid trapping solutions for all samples were also weighed and analysed via LSC for radioactivity. AQ1 samples from treated non-sterile experimental vessels at 100 µg/L were also analysed by HPLC-βRAM.

Recovery of radioactivity from the aqueous phases (AQ1 and AQ2), vessel rinse (VR1) and volatile traps was calculated (as a % of the Total Applied Radioactivity (TAR)) and summarised together within a mass balance recovery table to ascertain the mass balance for each sample treated with [14C]-Polyol PX and the reference control samples treated with [14C]-sodium benzoate.

- Sterility check if applicable: Glassware and surface water used to generate sterile control units were sterilized via autoclave for at least 20 minutes at a temperature ≥ 121 °C prior to dispensing. Duplicate samples were prepared by dispensing approximately 300 mL of the sterilized water into sterilized sample vessels using aseptic techniques in a laminar flow cabinet. Application of the test item was also performed using aseptic techniques in a laminar flow cabinet.
- Sample storage before analysis: Frozen (- 20 ± 10 °C)

CONTROL AND BLANK SYSTEM
- Untreated control: Untreated control samples were prepared (one per sampling timepoint) without the addition of the test item but with the addition of the application ‘vehicle’ used (water : acetonitrile = 1 : 0.08 ).
- Abiotic sterile control: Sterile control samples were established at the high dose concentration (100 µg/L) only.
- Reference control: Duplicate reference control samples treated with [14C]-sodium benzoate were established at a nominal dose concentration of 10 µg/L.

STATISTICAL METHODS: The kinetic modelling software KinGUII (version 2.1) was used to extrapolate the degradation kinetics of [14C]-Polyol PX in sterile and non-sterile surface water. The % TAR data was determined for parent [14C] Polyol PX was plotted against time in a logarithmic plot.

Using the guidance of Boesten et al. (2014), the parent data was fitted to kinetic model Single First Order (SFO) and the goodness of fit was assessed visually and statistically. A χ2 value < 15 % is considered to be a good statistical fit (Boesten et al., 2014). A t-test probability value of < 0.05 indicates that the slope of the determined regression line was significantly different from zero.

Where % TAR values fell below the LOD, values were corrected to half the LOD as recommended in the SANCO10058 guidance (Boesten et al., 2014), pages 76 and 131.

Reference substance:

other: [14C]-sodium benzoate

Key result

Compartment:

natural water: freshwater

DT50:

23.01 d

Type:

(pseudo-)first order (= half-life)

Temp.:

12 °C

Remarks on result:

other: SFO (Single First-Order kinetics)

Transformation products:

not specified

Remarks:

One major unidentified degradation product, assigned Unknown-1, was detected at ≥ 5 % of applied radioactivity from 7 days after application onwards, reaching a maximum percentage of 95.46 % of the applied radioactivity at 42 days after application.

Details on transformation products:

TRANSFORMATION PRODUCTS WERE DETECTED AND QUANTIFIED (BUT NOT IDENTIFIED) WITH THE SAME METHOD USED FOR THE PARENT COMPOUND.

IDENTIFICATION OF THE TRANSFORMATION PRODUCTS IS PLANNED TO BE PERFORMED WITH A NON-GLP HIGH RESOLUTION HPLC STUDY.

Evaporation of parent compound:

yes

Remarks:

Measured as total volatiles' radioactivity

Volatile metabolites:

yes

Residues:

yes

Details on results:

TEST CONDITIONS
- Aerobicity, moisture, temperature and other experimental conditions maintained throughout the study: Yes
- Anomalies or problems encountered (deviations from test conditions):
Trough out the incubation period, there were occasions where samples were found not connected to the flow through apparatus, lights were left on, and/or the shaker was turned off for a maximum period of 96 hours. A number of temperature deviations occurred during the incubation period. The temperature did not deviate by more than 0.2 °C for more than a 12-hour period. --> All samples achieved a mass balance > 90 % (> 88.28 % for reference control samples) and no significant difference in degradation was observed between timepoints sampled before and after the deviation. Temperature deviations were minor. Overall, there was no significant impact the integrity of the samples or study.

A mass balance of 88.81 % was achieved for one of the reference control replicate samples --> No significant impact. The purpose of reference control samples is to confirm the presence of an active microbial population and demonstrate the ability of the surface water to mineralise the test item throughout the incubation period. The ability of the surface water to mineralise the test item was demonstrated by the significant generation of volatiles (collected in trapping solutions) throughout the incubation period.

Specific Activity Check (SPAC) samples were prepared for application solutions 1 and 2 at a maximum of 26 hours prior to dosing in error.
Due to a suspected error in the dilution of post dosing SPAC samples for application solution 1, SPAC samples were prepared on two further occasions. This meant that analysis of application solution 1 was actually carried out within 26 hours post dosing. --> No impact.
Application solutions were still assessed within a reasonable time period prior to and post dosing. All applications solutions met the required criteria and were deemed suitable for use based on a determined purity ≥ 95 % and homogeneity confirmed by a calculated RSD of ≤ 3 % between triplicate replicates of the application solution pre and post dosing.

MAJOR TRANSFORMATION PRODUCTS
- Range of maximum concentrations in % of the applied amount and day(s) of incubation when observed: one major unidentified degradation product (Unknown-1) ranged from its lowest concentration at 1 DAT (0.35%), reaching a maximum percentage (94.58 - 96.34%) of the applied radioactivity at 42 DAT.
- Range of maximum concentrations in % of the applied amount at end of study period: 90.91 - 95.28%

MINOR TRANSFORMATION PRODUCTS
- Range of maximum concentrations in % of the applied amount and day(s) of incubation when observed: minor degradation products were detected since day 0 (1.18-1.47%), reaching a maximum at 42 DAT (2.13%).
- Range of maximum concentrations in % of the applied amount at end of study period: 0.30-0.32%

TOTAL UNIDENTIFIED RADIOACTIVITY (RANGE) OF APPLIED AMOUNT: One major unidentified degradation product, assigned Unknown-1, was detected at ≥ 5 % of applied radioactivity from 7 days after application onwards, reaching a maximum. Sum of Unknowns Detected at < 10 % TAR ranged from 0.30% at 90 DAT to 2.13% at 42 DAT.

EXTRACTABLE RESIDUES
- Average % of Total applied radioactivity (TAR) at day 0: 101.55% (10 µg/L), 99.27% (100 µg/L)
- Average % of Total applied radioactivity (TAR) at end of study period: 95.51% (10 µg/L), 97.38% (100 µg/L)

NON-EXTRACTABLE RESIDUES
- Average % of Total applied radioactivity (TAR) at day 0: 0.80% (10 µg/L), 0.62% (100 µg/L)
- Average % of Total applied radioactivity (TAR) at end of study period: 0.68% (10 µg/L), 0.62% (100 µg/L)

MINERALISATION
Analysis of the mineralization of [14C]-Polyol PX was performed using the 2 M sodium hydroxide traps associated to individual incubation samples from 60 DAT (non-sterile, 10 µg/L), 90 DAT (non-sterile, 100 µg/L) and 90 DAT reference control, using a barium chloride precipitation method. The method confirmed that the radioactivity was present as [14C]-carbonate (i.e., from 14CO2). After barium chloride precipitation 0.00 % was detected in the pooled trapping solutions.
- % of applied radioactivity present as CO2 prior to precipitation at 60 DAT (10 µg/L): 9.12%
- % of applied radioactivity present as CO2 prior to precipitation at 90 DAT (100 µg/L): 3.91%
- % of applied radioactivity present as CO2 prior to precipitation at 90 DAT (reference control): 55.51%

VOLATILIZATION
- % of the applied radioactivity present as volatile organics at end of study: 14.40% (10 µg/L), 3.93% (100 µg/L)

STERILE TREATMENTS
- Transformation of the parent compound: The major transformation product (Unknown 1) was not detected in any of the sterile samples. Only minor transformation products (< 10% TAR) were detected.
- Formation of transformation products: average sum of unknowns detected at < 10 % TAR ranged from 1.33% at 7 DAT to 1.19% at 90 DAT
- Formation of extractable and non-extractable residues:
Extractable residues
- Average % of Total applied radioactivity (TAR) at end of study period: 99.80%
Non-extractable residues
- Average % of Total applied radioactivity (TAR) at end of study period: 0.45%
- Volatilization: Average replicate total volatiles generated remained < 1 % (% TAR) for all sterile samples throughout the experimental period.

Results with reference substance:

The data obtained with the reference substance demonstrates that the Calwich Abbey surface water collected was microbially active and able to mineralise the reference material [14C]-sodium benzoate. The average replicate recovery (% TAR) over the 90 DAT period was marginally below 90 % (89.53 % TAR).

Conclusions:

A mass balance of 90 – 110 % was achieved in all samples treated with [14C]-Polyol PX and significant degradation of the reference material [14C]-sodium benzoate was observed throughout the test confirming the validity of the test as per OECD309.

DegT50 of [14C]-Polyol PX in non-sterile surface water is 23.01 days. [14C]-Polyol PX is moderately persistent (DegT50 between 22 and 60 days) (Hollis, 1991) in Calwich Abbey surface water. Biotic degradation into reoccurring transformation product/s Unknown-1 was observed in non-sterile samples. A decline phase was not observed for Unknown-1 and therefore a DegT50 > 1000 days was generated suggesting persistence of this transformation product/s.

Executive summary:

The biotransformation of [14C]-Polyol PX was studied in filtered lake water (pH 8.34, dissolved organic carbon 4.66 mg/L) from Calwich Abbey Lake (Staffordshire, England) for 90 d under aerobic conditions in dark at 12 ± 2 ºC. [14C]-Polyol PX was applied at the rates of 10 and 100 µg/L. The experiment was conducted in accordance with OECD Guideline for the Testing of Chemicals, Number 309, Aerobic Mineralisation in Surface Water – Simulation Biodegradation Test (OECD 2004), and in compliance with the OECD-GLP standards. The test system consisted of ca. 300 mL glass mineralisation vessels (ca. 500 mL for sterile samples), attached with traps for the collection of CO2 and volatile organic compounds. Samples were analysed at 0, 1, 3, 7, 14, 28, 60, and 90 DAT (Days After Treatment) (Non-Sterile Aerobic Degradation); 7, 28, 60, and 90 DAT (Aerobic Sterile Controls); 90 DAT (Aerobic Reference Controls) and every 7 ± 2 days (Trapping solutions). The water samples were extracted with two decantation steps and further rinsing of the vessels with acetonitrile and acetone. The [14C]-Polyol PX residues were analysed by HPLC-βRAM.

 

A mass balance of 90 % - 110 % of applied radioactivity was achieved for all sterile and non-sterile timepoints. The concentration of parent compound decreased from 97.3 % at day 0 to 0% of the applied amount already at 42 days after application, mantaining this rate until the end of the study period. The half-life/DT50 (50% decline time) of [14C]-Polyol PX in aerobic water was 23.01 days. One major unidentified degradation product (Unknown-1) was detected at ≥ 5 % of applied radioactivity from 7 days after application onwards, reaching a maximum percentage of 95.46 % of the applied radioactivity at 42 days after application.

 

The major transformation product detected is not yet identified (Unknown-1), with maximum concentration of 96.34 % of the applied amount, observed on the 42nd day of incubation. The corresponding concentration at the end of the study period was between 90.91 and 95.28 % of the applied amount. The minor transformation products were detected but not identified, and formed at a maximum of 2.13 % of the applied amount. At study termination, evolved 14CO2 and volatile organic compounds accounted for 3.91% (100 µg/L) and 14.40% (10 µg/L)/3.93% (100 µg/L) of the applied amount, respectively.

 

In sterile treatments the concentration of [14C]-Polyol PX remained between 98.53 and 90.3%, observed at 7 and 90 DAT, respectively. The major transformation product (Unknown 1) was not detected in any of the sterile samples. Only minor transformation products (< 10% TAR) were detected with a maximum of 0.44% at 60 DAT.

Reference 2

Endpoint:

biodegradation in water: sediment simulation testing

Data waiving:

other justification

Justification for data waiving:

other:

Qualifier:

according to guideline

Guideline:

OECD Guideline 308 (Aerobic and Anaerobic Transformation in Aquatic Sediment Systems)

Description of key information

A GLP OECD 309 study (aerobic mineralization in surface water) has been performed, and resulted in a DegT50 of 23.01 days in water, based on radiochemical measures.

A GLP OECD 308 study (biodegradation in water - sediment simulation testing) is currently being performed, no available studies neither GLP or non-GLP are available to cover the information requirements. The RSS with the results will be made available with the next dossier submission (see waiver with attached document).

Key value for chemical safety assessment

Half-life in freshwater:

23.01 d

at the temperature of:

12 °C