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Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-06-27 to 2016-08-26 (experimental phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)
Version / remarks:
2014
Deviations:
no
Principles of method if other than guideline:
Test extended to 60 days and additional analysis of mono-ester constituents conducted using GC-MS.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Expiration date of the lot/batch: 2016-10-12

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from light, in the tightly closed original container.

OTHER SPECIFICS: Rosin consists mainly of an acidic fraction (85 – 95%) and a neutral fraction. The acidic fraction consists of diterpene acids (resin acids) and their dimerisation products. The neutral fraction comprises monoterpenes, sesquiterpenes, diterpenes and their oxygenated derivatives (e.g. alcohols, ketones etc.) and high molecular weight fractions (dimers, polymerics). The modification of rosin to produce the test item provides mono esters as main products (approx. 70%). Further ingredients are the corresponding di- and triglycerides (approx. 15%) and resin acids (approx. 12%).

Composition of test item (area determined by size exclusion chromatography):
6.2% Tri-ester of Glycerol
10.3% Di-ester of Glycerol
70.7% Mono-ester of Glycerol
12.1% Rosin acids
<0.1% Light ends

TOC of substance components (as a proportion of test item):
5.0% Tri-ester of Glycerol
8.1% Di-ester of Glycerol
51.9% Mono-ester of Glycerol
9.6% Rosin acids
0.0% Light ends
74.6% Total TOC (calculated)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Remarks:
Mixed inoculum of the aqueous phase of non-adapted activated sludge and pre-treated, non-adapted standard soil
Details on inoculum:
- Source of inoculum/activated sludge: The non-adapted activated sludge was sampled from the aeration tank of the Municipal sewage treatment plant, 31137 Hildesheim, Germany. This activated sludge receives predominantly domestic sewage and hardly any industrial chemical waste. The non-adapted standard soil was sampled at a depth of approx. 20 cm and sieved (2mm) and then delivered from LUFA Landwirtschaftliche Untersuchungs- und Forschungsanstalt Speyer, 67346 Speyer, Germany. Standard soil from LUFA at Speyer is certified and field-fresh soil established for biodegradation studies.

- Preparation of inoculum for exposure: The activated sludge was washed twice with chlorine free tap water. After the second washing, the settled sludge was resuspended in mineral salts medium and was maintained in an aerobic condition by aeration for 2 hours. Thereafter the sludge was homogenized with a blender. After sedimentation the supernatant was decanted and maintained in an aerobic condition by aeration with CO2-free air for 7 days. The soil was mixed with mineral salts medium according to OECD 310 and shaken on an overhead shaker overnight. Subsequently, the soil was allowed to settle for 4 hours and the supernatant was used as inoculum. The supernatant was maintained in an aerobic condition by aeration with CO2 free air for 6 days.

- Pretreatment: A stock solution of the test item in acetone with a concentration of 26.4 mg/mL was prepared. 100 µL of the stock solution was pipetted around the wall of the test vessel to produce a large surface area. The acetone was allowed to evaporate over 3 days. The test vessel for the solvent control was pre-treated with acetone in the same way.

- Concentration of sludge: 10 mL/L of activated sludge and 7 mL/L of soil were used to initiate inoculation.
- Initial cell/biomass concentration: 1.5 x 10^10 CFU/L for the inoculum of the aqueous phase of non-adapted activated sludge (corresponding to approximately. 1.5 x 10^8 CFU/L in the final test solution) and 1.1 x 10^10 CFU/L for the non-adapted standard soil (corresponding to approximately 1.1 x 10^8 CFU/L in the final test solution) (CFU = colony forming units).
Duration of test (contact time):
60 d
Initial conc.:
33 mg/L
Based on:
test mat.
Initial conc.:
24.6 mg/L
Based on:
TOC
Parameter followed for biodegradation estimation:
CO2 evolution
Remarks:
mineralisation
Parameter followed for biodegradation estimation:
test mat. analysis
Remarks:
primary degradation
Details on study design:
TEST CONDITIONS
- Composition of medium: Mineral salts medium according to OECD 310.
- Additional substrate: None.
- Solubilising agent: Acetone, evaporated to dryness before test start.
- Test temperature: 19.3 - 20.9°C
- pH: Not reported.
- pH adjusted: No.
- Aeration of dilution water: Not reported.
- Suspended solids concentration: Not reported.
- Continuous darkness: No, low light conditions.

TEST SYSTEM
- Culturing apparatus: 120 mL headspace flasks with 80 mL test medium, 1:2 headspace to liquid ratio.
- Number of culture flasks/concentration:
• three for the inoculum control (C1, C2, C3, plus C4, C5 at day 28)
• three for the solvent control (CS1, CS2, CS3, plus CS4, CS5 at days 28 and 60)
• one for the quality control (QC)
• three for the functional control (R1, R2, R3, plus R4, R5 at day 28)
• three for the test item concentration (P1, P2, P3, plus P4, P5 at days 28 and 60)
• three for the toxicity control (T1, T2, T3, plus T4, T5 at day 28)

To calculate the 95 % confidence interval for the mean percentage biodegradation 5 replicates of test item, reference item, toxicity control, inoculum control and solvent control were set up for day 28 and 5 replicates of test item and solvent control at test end (day 60).

- Method used to create aerobic conditions: Inoculum was maintained in aerobic condition by aeration before test start.
- Measuring equipment: Carbon analyser.
- Test performed in open system: No
- Details of trap for CO2 and volatile organics if used: Sodium hydroxide solution.
- Other: Agitation with a shaker (150 - 200 rpm).

SAMPLING
- Sampling frequency: Determination of CO2 was carried out at test start and on days 3, 7, 10, 14, 21, 28, 35, 42, 49, 56 and 60 for the test item and solvent control. The reference substance control and inoculum control was sampled at test start and on days 7, 14 and 28 and the toxicity control was sampled at test start and on days 14 and 28. The quality control was sampled at test start and on days 3, 7, 10, 14, 18, 21, 28 and 60.
- Sampling method: Sodium hydroxide solution was injected to each vessel sampled. The vessels were shaken on an overhead shaker for 1 hour at test temperature and analysed.
- Sterility check if applicable: None.
- Sample storage before analysis: All samples were stored in a refrigerator at 6 ± 2 °C until sample preparation.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes
- Abiotic sterile control: No
- Toxicity control: Yes, 33 mg/L test item + 30 mg/L reference item
- Other: Solvent control (mineral salts medium + inoculum; 100 µL acetone pipetted around the wall of the test vessel and allowed to evaporate), quality control (mineral salts medium + inoculum)

STATISTICAL METHODS:
The amount of TIC produced was calculated by correcting the results of the test item, reference item and toxicity control for endogenous TIC production of the control groups and the blind value of the sodium hydroxide solution. The biodegradation was calculated from the ratio theoretical TIC concentration at test start (ThIC = TOC) to net TIC production.

TIC = (mg C in the liquid + mg C in the headspace) = (VL x CL) + (VH x CH)

where:
VL = volume of liquid in the bottle (litre)
CL = concentration of IC in the liquid (mg/L as carbon)
VH = volume of the headspace (litre)
CH = concentration of IC in the headspace (mg/L as carbon)

%D = ((TICt - TICb) / TOC) x 100

where:
%D = biodegradation (%)
TICt = mg TIC produced in test bottle at time t
TICb = mean mg TIC produced in control bottles at time t
TOC = mg TOC added initially to the test vessel

The 95 % confidence interval for the mean percentage biodegradation was calculated using SigmaPlot (SPSS Corporation).
Reference substance:
benzoic acid, sodium salt
Preliminary study:
During preliminary investigations (non-GLP) different methods for test item application were trialled. For the main study, the test item was applied by preparing a stock solution in acetone with a concentration of 26.4 mg/mL. 100 µL of this was pipetted around the wall of the test vessel and the acetone allowed to evaporate over 3 days. It was observed in the preliminary study that when the test item was applied as described, the test item was forming a film on the glass wall surface (approximately two millimetres wide). This film detached from the wall after the test solutions were filled in and was floating in the solution. Therefore, this way of application was considered optimal, because the test item is in good contact with the test solutions.
Test performance:
In the toxicity control, biodegradation of 44% was determined after 14 days and it came to 50% (95% confidence interval: 48 - 52%) after 28 days. The test item in the toxicity control did not inhibit the biodegradation of the reference item.
Key result
Parameter:
% degradation (CO2 evolution)
Value:
34
Sampling time:
28 d
Remarks on result:
other: mineralisation
Remarks:
95% confidence interval: 32 - 36%
Key result
Parameter:
% degradation (CO2 evolution)
Value:
35
Sampling time:
60 d
Remarks on result:
other: mineralisation
Remarks:
95% confidence interval: 32 - 38%
Key result
Parameter:
% degradation (test mat. analysis)
Value:
89
Sampling time:
28 d
Remarks on result:
other: primary degradation (quantified results)
Key result
Parameter:
% degradation (test mat. analysis)
Value:
95
Sampling time:
60 d
Remarks on result:
other: primary degradation (quantified results)
Parameter:
% degradation (test mat. analysis)
Value:
93
Sampling time:
28 d
Remarks on result:
other: primary degradation
Remarks:
relative % degradation based on peak area at Day 28 compared to Day 0, from metabolite analysis report
Details on results:
A decline of resin acids was observed between test start and day 3 of the study and an increase of these acids was observed on day 7 of the study. This is in correlation with the start of the primary degradation (57% degradation) of the mono ester fraction between day 3 and day 7.Primary degradation of the mono ester fraction of dehydroabietic acid was observed to be faster than that of the hydrogenated mono ester fraction of resin acids. An approximate amount of 85% of the mono ester fraction of dehydroabietic acid was degraded by day 7, whereas only 35% of the mono ester fraction of the hydrogenated resin acid derivatives was degraded. By day 28, approximately 90% of both mono ester fractions were degraded. The amount of resin acids decreased between day 7 and day 10 and increased again between day 10 and day 21 but on a level lower than that on day 7. The amount decreased again between day 21 and day 28. The maximum amount of dehydroabietic acid was obtained on day 10 but then decreased steadily. Difficulties in fully interpreting the levels of resin acids over the course of the study are due to the concurrent formation and degradation.

The test item is classified as not readily biodegradable when considering the ultimate degradation results.. Nevertheless, the test item reveals a considerable potential for biodegradation, as the determination of the primary degradation of the monoester fraction as well as the determination of mineralisation shows.
Results with reference substance:
The percentage degradation of the reference substance exceeded the pass level of 60% within 7 days and came to 82% (95% confidence interval: 80 - 84%) after 28 days.

Table 1. Mean CO2 production and biodegradation

 Day

 Solvent control

 Inoculum control

 Reference substance      

 Test item      

 Toxicity control      

 

 Mean CO2 (mg C/L)

 Mean CO2 production (mg C/L)

 Mean CO2 (mg C/L)

 Mean CO2 production (mg C/L)

 Mean CO2 (mg C/L)

 Net mean CO2 production (mg C/L)  % degradation (95% confidence interval)  Mean CO2 (mg C/L)  Net mean CO2 production (mg C/L)  % degradation (95% confidence interval)  Mean CO2 (mg C/L)  Net mean CO2 production (mg C/L)  % degradation (95% confidence interval)

 0

 0.31

 -

 0.43

 -

 0.43

 -  -  0.25  -  -  0.35  -  -

 3

 0.85

 0.54

 nd

 nd

 nd

 nd  nd  0.92  0.13  1  nd  nd  nd
 7  0.81  0.50  0.95  0.52  14.74  13.79  79  3.71  2.96  12  nd  nd  nd
 10  1.14  0.83  nd  nd  nd  nd  nd  6.16  5.08  21  nd nd nd 
 14  1.15  0.84  1.28  0.85  15.50  14.22  81  7.24  6.15  25  19.72  18.53  44
 21  1.51  1.20  nd  nd  nd  nd  nd  9.25  7.80  32  nd  nd  nd
 28  1.90  1.59  2.60  2.17  16.94  14.34  82 (80 - 84)  10.25  8.41  34 (32 - 36)  23.02  21.08  50 (48 - 52)
 35  2.09  1.78  nd  nd  nd  nd  nd  11.82  9.79  40  nd  nd  nd
 42  1.75  1.44  nd  nd  nd  nd  nd  10.57  8.88  36  nd  nd  nd
 49  1.77  1.46  nd  nd  nd  nd  nd  10.96  9.25  38  nd  nd  nd
 56  1.16  0.85  nd  nd  nd  nd  nd  9.87  8.77  36  nd  nd  nd
 60  1.35  1.04  nd  nd  nd  nd  nd  9.80  8.51  35 (32 - 38)  nd  nd  nd

Mean CO2 values have been corrected for NaOH

nd = not determined

Table 2. Primary degradation of the test item

 Day  Toxicity control     Solvent control     Test item   
   Measured concentration of monoesters (mg/L)  % primary degradation  Measured concentrations of monoesters (mg/L)  % primary degradation  Measured concentrations of monoesters (mg/L)  % primary degradation
 0  24.0, 22.7  -  < LOQ  -  22.6, 20.8  7
 3  nd  nd  nd  nd  23.3, 24.3  0
 7  nd  nd  nd  nd  7.07, 5.20  74
 10  nd  nd  nd  nd  3.33, 3.90  84
 14  nd  nd  nd  nd  4.43, 3.66  82
 21  nd  nd  nd  nd  3.66, 2.36  87
 28  nd  nd  nd  nd  2.15, 3.22  89
 60  nd  nd  nd  nd  1.49, 1.26  94

Measured concentrations reported as replicates

nd = not determined

Table 3. Metabolite identification by GC-MS (taken from the metabolite identification report)

 Group  Day                  
   0  3  7  10  14  21  28
 Area - Group 1 (hydrogenated resin acids)  132523  75118  149314  27568  46132  49775  27585
 Area - Group 3 (dehydroabietic acid)  185282  11046  27738  24199  8193  7323  5317
 Area - Sum of Groups 1 and 3  317805  86164  177052  51767  54325  57098  32902
 % test item - Sum of Groups 1 and 3  3.56  0.97  1.99  0.58  0.61  0.64  0.37
               
 Area - Group 2 (monoester fraction of hydrogenated resin acids)  5244629  5651691  3377386  2705210  2005680  1658100  550585
 Area - Group 4 (mono ester fraction of dehydroabietic acid)  3674796  3818124  432561  493826  113708  60841  43329
 Area - Sum of Groups 2 and 4  8919425  9469815  3809947  3199036  2119388  1718941  593914
 % test item - Sum of Groups 2 and 4  100  106  43  36  24  19  7
Validity criteria fulfilled:
yes
Remarks:
Degradation of the reference item must be > 60% by day 14 (actual 81%) and the maximum amount of TIC in the inoculum controls must be < 3 mg C/L at the end of the test (actual 2.60 mg C/L)..
Interpretation of results:
not readily biodegradable
Remarks:
Nevertheless, the test item has a considerable potential for biodegradation, demonstrated by the primary degradation of the monoester fraction.
Conclusions:
The ultimate biodegradation of the test item was 34% after 28 days and 35% after 60 days, based on CO2 production. Primary biodegradation of the monoester fraction reached 74% after 7 days, 89% after 28 days and 95% after 60 days. Hydrogenated resin acids and dehydroabietic acid are early metabolites in the primary degradation of the mono ester fraction of the test item.
Executive summary:

The ready biodegradability of the test item Monoesters of hydrogenated rosin with glycerol was determined with a mixed inoculum of the aqueous phase of non-adapted activated sludge and pre-treated, non-adapted standard soil in the Headspace Test following OECD 310 (Noack Laboratorien 2017a). Biodegradation was assessed after 28 days, and the test was then extended to 60 days. Mineralisation of the test item was followed by TIC analyses to assess CO2 production. Primary degradation of the monoester fraction was determined by GC-MS analysis of representative monoester constituents.

The study was conducted using a nominal test item concentration of 33 mg/L, corresponding to a carbon content of 24.6 mg C/L. A stock solution of the test item in acetone was prepared and introduced to the test vessel by pipetting the stock solution around the wall of the test vessel to produce a big surface area. The acetone was allowed to evaporate over 3 days before the test start. CO2 was captured by sodium hydroxide solution and carbon concentrations were determined with a carbon analyser. The test included a reference item control, toxicity control, solvent control and inoculum control. All validity criteria for the study were met. This study is considered to be reliable without restriction (Klimisch 1).

The ultimate biodegradation of the test item was 34% after 28 days and 35% after 60 days, based on CO2 production. Primary biodegradation of the monoester fraction reached 74% after 7 days, 89% after 28 days and 95% after 60 days.

Additional analysis of extracts from the biodegradation study was conducted in order to identify degradation products (Noack Laboratorien 2017b). Resin acids are early metabolites in the primary degradation of the mono ester fraction of the test item, as demonstrated by an increase in these acids (based on analysis of hydrogenated resin acids and dehydroabietic acid) observed in parallel to a reduction of the mono ester fraction.

The test item is not readily biodegradable based on the ultimate degradation determined in the study. Nevertheless, the test item reveals a considerable potential for biodegradation, as the determination of the primary degradation of the monoester fraction as well as the determination of mineralisation shows.

Description of key information

In order to assess the degradation potential of monoester constituents, a ready biodegradation study was conducted with a test item enriched in monoesters (70.7%). The test item was not readily biodegradable (34% ultimate degradation after 28 days). However, primary degradation of monoester constituents was high (89% after 28 days, 95% after 60 days) and therefore the monoester fraction of the registered substance is not considered to be persistent or very persistent.

Key value for chemical safety assessment

Additional information

As rosin ester substances are UVCBs, a screening assessment was conducted in order to assess the potential for persistence of individual constituents. This was carried out using QSAR predictions for representative constituents conducted with the US EPA’s BIOWIN model (v 4.10). These predictions, combined with predictions for bioaccumulation potential, were used to determine if any constituents within the UVCB substances might lead to a PBT / vPvB concern. Full details of the screening assessment can be found in IUCLID Section 2.3. Based on this screening assessment, monoester constituents were considered to be borderline for both persistence and bioaccumulation, therefore it was not possible to conclude the PBT assessment for these constituents based on QSAR predictions alone. Two substances within the Rosin esters category, Resin acids and rosin acids, hydrogenated, esters with glycerol and Resin acids and rosin acids, hydrogenated, esters with pentaerythritol are going through the Substance Evaluation process. The Final Decision letters received for these substances requested a tiered testing approach be followed in order to assess the PBT concern, with studies conducted using the monoester fraction of the registered substances. The first study in the tiered testing approach, a ready biodegradation study conducted with the monoester fraction of Resin acids and rosin acids, hydrogenated, esters with glycerol, is now complete. Based on the results from this study, monoester constituents are not considered to be persistent or very persistent. The study is read across to the monoester fraction of Resin acids and rosin acids, hydrogenated, esters with pentaerythritol.

The ready biodegradability of the test item Monoesters of hydrogenated rosin with glycerol was determined with a mixed inoculum of the aqueous phase of non-adapted activated sludge and pre-treated, non-adapted standard soil in the Headspace Test following OECD 310 (Noack Laboratorien 2017a). Biodegradation was assessed after 28 days, and the test was then extended to 60 days. Mineralisation of the test item was followed by TIC analyses to assess CO2 production. Primary degradation of the monoester fraction was determined by GC-MS analysis of representative monoester constituents.

The study was conducted using a nominal test item concentration of 33 mg/L, corresponding to a carbon content of 24.6 mg C/L. A stock solution of the test item in acetone was prepared and introduced to the test vessel by pipetting the stock solution around the wall of the test vessel to produce a big surface area. The acetone was allowed to evaporate over 3 days before the test start. CO2 was captured by sodium hydroxide solution and carbon concentrations were determined with a carbon analyser. The test included a reference item control, toxicity control, solvent control and inoculum control. All validity criteria for the study were met. This study is considered to be reliable without restriction (Klimisch 1).

The ultimate biodegradation of the test item was 34% after 28 days and 35% after 60 days, based on CO2 production. Primary biodegradation of the monoester fraction reached 74% after 7 days, 89% after 28 days and 95% after 60 days.

Additional analysis of extracts from the biodegradation study was conducted in order to identify degradation products (Noack Laboratorien 2017b). Resin acids are early metabolites in the primary degradation of the mono ester fraction of the test item, as demonstrated by an increase in these acids (based on analysis of hydrogenated resin acids and dehydroabietic acid) observed in parallel to a reduction of the mono ester fraction.

The test item is not readily biodegradable based on the ultimate degradation determined in the study. Nevertheless, the test item reveals a considerable potential for biodegradation, as the determination of the primary degradation of the monoester fraction as well as the determination of mineralisation shows.