Fragrance Encapsulates: Effect of Polymeric Shell Purification Method on the Accuracy of Biodegradability Testing

Fragrance encapsulates are widely used in consumer care applications such as fabric softeners or other liquid laundry products; they provide multiple benefits, from fragrance protection in the commercial product to a controlled release and improved sensorial experience for the consumers. Polymeric fragrance encapsulates are in the scope of the EU regulation restricting the use of intentionally added microplastic particles, and industry is actively working on innovation programs to find biodegradable alternatives. However, particular attention needs to be paid to claims that a fragrance encapsulation system is biodegradable, because biodegradation test results can vary considerably depending on how a test material is prepared, which can even lead to false‐positive biodegradation test results, as shown in our study. We demonstrate the importance of the sample preparation phase of the process. We show how the biodegradation level can fluctuate from 0% to 91%, depending on how the test material is isolated from a given microcapsule slurry system, and we present a method that can be used to obtain trustworthy biodegradation results. Environ Toxicol Chem 2024;43:1242–1249. © 2024 Givaudan France SAS. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.


INTRODUCTION
Fragrance encapsulates are widely used in consumer care applications, such as fabric softeners or other liquid laundry products; they provide multiple benefits, from fragrance protection in the commercial product to a controlled release mechanism and improved sensorial experience for consumers.Encapsulates adhere to fabric and release scent long after the wash cycle, when the polymer membranes are ruptured by the action of friction (Zhao et al., 2019).In addition, because encapsulation is far more efficient at deposition than traditional perfume oil, it allows lower fragrance dosages to be used, already making it a more sustainable way to use fragrance in such products (Bône et al., 2011;Bruyninckx & Dusselier, 2019).It is crucial to underline that intentionally added microplastics from fragrance encapsulates represent an extremely small contribution to overall microplastics emissions.Based on figures included in the European Chemicals Agency (ECHA) background document, microplastics from fragrance encapsulates are estimated to contribute <0.5% of the total "intentionally added" microplastic release in the European Union (EU) and <0.001% of all microplastic releases, including secondary microplastics formed from plastic waste (Final Background Document, 2020).
At the European level, intentionally added synthetic polymer microparticles ("microplastics") in products are subject to a restriction under the EU chemical legislation Registration, Evaluation, Authorisation, and Restriction of Chemicals (REACH; EU, 2023).The regulation, which came into force on October 17, 2023, prohibits the commercial use of any solid polymer that is contained in microparticles or that forms a continuous surface coating on microparticles.Such a polymer can not be used singly or in a mixture, in a concentration equal to or greater than 0.01% by weight in products, when its release into the environment is considered unavoidable.The regulation excludes degradable or water-soluble polymers and natural polymers that have not been chemically modified, because they do not possess the same long-term persistence and therefore do not contribute to the identified risk.For encapsulation of fragrances, a transition period of 6 years (i.e., until October 17, 2029) has been granted to provide sufficient time to comply with the regulation and transition to suitable alternatives, for example, biodegradable polymers.
The challenge for the fragrance industry is to develop biodegradable technologies that deliver the same performance of cross-linked polymer encapsulates for all applications (e.g., laundry detergents, fabric softeners, personal care products) that are stable during the product shelf-life, but that biodegrade when released to the environment (Bruyninckx & Dusselier, 2019;Mehlhase, 2020).The ECHA has proposed a framework of standardized test methods and pass criteria to identify degradability for the purpose of the restriction (Final Background Document, 2020;Commission Regulation [EU], 2023).The permitted test methods are organized into five groups, on the basis of their design and underlying rationale.There are also specific requirements for the test material to be used in degradation tests.The test material should be comparable in terms of composition, form, size, and surface area to the polymer particles present in the product or, if not technically feasible, to the polymer particles that are disposed of or released to the environment.Polymers used for encapsulation may be tested (1) in the form placed on the market, (2) in the form of isolated coating, or (3) in the form placed on the market, with the organic core of the material replaced by an inert material such as glass.The test material shall be of comparable thickness to the solid polymer coating of the particle placed on the market.
For fragrance encapsulation, testing the form placed on the market is not appropriate.Typically, the commercial product is a complex mixture consisting of an aqueous slurry containing the fragrance encapsulates, some free fragrance oil, and additives such as suspending agents or preservatives.In standard tests, biodegradation is followed by the measurement of sum parameters such as oxygen consumption or CO 2 production, which do not provide information on individual constituents.Even intact capsules isolated from the slurry are not suitable for biodegradation testing because the fragrance within the capsule will contain biodegradable ingredients, leading to false-positive biodegradation test results.Also, it is extremely complicated, if not impossible, to make a "fragrance" encapsulate around an inert material such as a glass bead.In some cases, depending on the encapsulation technology, it may be possible to prepare a form containing a nonbiodegradable model perfume or single nonbiodegradable fragrance ingredient.However, the "inert" core material can significantly impact the biodegradation test because the polymer coating (or capsule wall material) is a much smaller portion of the total mass of the capsule.Thus, to overcome the background noise of the test system (i.e., to be able to measure CO 2 evolution or O 2 consumption from biodegradation of the polymer), excessively high concentrations of test material would be needed, which can lead to unwanted side effects such as toxicity to the microorganisms caused by the excessively high concentration of any nonbiodegradable fragrance ingredient present.Therefore, the most appropriate test material for biodegradation testing is the isolated polymer coating.This has an additional benefit in that it represents the particulate material released to the environment (i.e., the spent capsule shell after it has been ruptured and the fragrance released).As mentioned previously, the composition, form, size, and surface area of the test material has to be comparable to the polymer particles put on the market and/or released to the environment.
To obtain an accurate assessment of the biodegradability of the polymeric shell, it is crucial that a "pure" sample of the isolated polymer is tested.Any unreacted materials, additives, or free fragrance present in the slurry, as well as the fragrance core of the capsule, should be systematically removed during the preparation of samples for biodegradation testing.The isolated sample should only consist of polymeric material that is part of the microcapsule shell.Any residuals present should be at levels that are low enough not to contribute to the biodegradation observed during testing.A level of <10% by weight of readily biodegradable nonpolymeric organic additives (or other constituents) in the test material was considered by the ECHA Risk Assessment Committee unlikely to confound the results of the Organisation for Economic Co-operation and Development (OECD) screening tests (ECHA Committee for Risk Assessment, 2020) with additional measures required to demonstrate biodegradability of the polymer itself when concentrations are higher than 10% w/w (Commission Regulation [EU], 2023).
To date, there are very few data available in the scientific literature on the biodegradability of fragrance microcapsules using standard biodegradation tests such as OECD test guideline 301F (OECD, 1992).Gasparini et al. (2020) have presented a quantification method based on pyrolysis gas chromatographymass spectrometry (GC-MS) for residual volatiles to validate sample quality prior to biodegradation testing and have demonstrated the confounding impact of residual volatiles on the estimated biodegradability of fragrance encapsulates using an adaptation of OECD test guideline 301F (OECD, 1992).Our research builds on this and highlights the importance of removing all materials that are not bound to the capsule shell, including any residual water-soluble and insoluble nonvolatile materials.
Attempts to obtain sustainable fragrance microcapsules concentrate on the microcapsule shell material, the encapsulation method, and of course the biodegradability.Baiocco et al. (2021) describe the encapsulation of a fragrance ingredient using biosourced ingredients for the shell in an attempt to use environmentally friendly encapsulation.Biodegradation testing of synthesized microcapsules using standard OECD test methods has the disadvantages of long test duration (up to 60 days) and high cost.Thus, methods to prescreen biodegradable features of the microcapsules at a laboratory level have recently been used, such as 1 H nuclear magnetic resonance (Kim et al., 2021).Xiao et al. (2018) have explored an organic solvent-free interfacial polymerization process to obtain biodegradable microcapsules.The biodegradability is tested in phosphate-buffered saline at 37 °C, over 30 days.Scanning electron microscopy after the testing showed that most of the shell was destroyed, providing an insight into the environmental fate of the microcapsules.Although these alternative methods may provide some useful insights into the expected biodegradation of microcapsules and could be used to screen for potential biodegradable technologies, the biodegradability of the microcapsule polymer must ultimately be proved in accordance with the rules laid down in Appendix 15 of the EU "microplastic" restriction, namely, the permitted test methods and the pass criteria for those methods (Commission Regulation [EU], 2023).
In the present study we demonstrate the importance of the sample preparation phase and the effect that inappropriate methods have on the results of biodegradation testing.All samples were tested in our accredited facilities at Givaudan in an OECD test guideline 301F ready biodegradability test, prolonged for up to 60 days.This is one of the permitted methods in groups 1 and 2 of the EU restriction regulation, in which meeting the pass criteria of 60% mineralization (over 28 and 60 days, respectively) as consumed O 2 demonstrates biodegradation of the polymer test material, excluding it from the scope of the EU restriction.

Microcapsules
We studied five fragrance microcapsules of different shell composition, provided by Givaudan unless otherwise noted.

Purification methodologies for capsule shell preparation before biodegradation testing
For the purpose of preparing samples for biodegradation testing, the starting encapsulate dispersion, or slurry, did not contain any additives, such as suspension aids or preservative systems, to avoid interference with the purification processes and to prevent false-positive or -negative biodegradation test results.
The purification processes required to isolate the polymer microcapsule shell from the slurry should include the removal of both water-soluble components (e.g., unreacted polymer starting materials) and water-insoluble components (e.g., unreacted polymer starting materials) in the slurry, the separation of the solid capsules from the aqueous slurry, removal of the fragrance from the capsule as well as any residual fragrance entrapped inside the capsule shell itself, and finally the drying of the isolated polymer material.However, each step needs to be rigorous to ensure that the final sample for biodegradation testing does not contain significant quantities of organic material that are not part of the capsule shell.
To demonstrate the importance of these steps, five different methodologies were used: methods A, B, C, D, and E. Method A investigates the impact of removing neither any residues from the aqueous phase nor the fragrance core within the shell.The other four methods, B-E, all extract the fragrance core but have different degrees of tolerance toward residual watersoluble and water-insoluble components in the slurry and noncovalently bonded constituents of the polymeric shell.
Table 1 summarizes the differences among the five methods in terms of removal of (1) the fragrance core, (2) the solubles, and (3) the insolubles in the aqueous phase.
Each method is described below and presented schematically in Figure 1.
Method A. Solubles, insolubles, and core oil kept.(1) The slurry was lyophilized.(2) The resulting solid was homogenized using a ball mill.

Method B. Solubles and insolubles kept.
(1) The slurry was added to ethanol and left to equilibrate for 1 h.The system was centrifuged for 30 min at 7142 g.The ethanol phase (clear) was emptied and was replaced by fresh ethanol.The procedure was repeated four times.(2) After the last wash, the powder was dried at 80 °C and ground using a ball mill.If >1% fragrance could be detected in the isolated powder by GC-flame ionization detection (FID), a further ethanol wash was performed, and then the powder was dried at 80 °C and ground again.

Method C. Nonattached insolubles kept (filtration). (1)
The slurry was lyophilized.The resulting solid was homogenized using a ball mill until a paste was obtained.(2) The resulting paste containing fragrance oil and polymeric shells was suspended in ethyl acetate, and the mixture was stirred for 1 h at room temperature.(3) The solid was collected by filtration.The solid was then recovered and dried under vacuum (10 mbar) at 50 °C.The extraction step was repeated a total of five times.(4) The powder was suspended in Milli-Q water (0.5% w/ w) and stirred for 24 h.The solid was recovered by centrifugation and washed twice.(5) The product was dried for 2.5 days at room temperature, and then under vacuum (10 mbar) at 50 °C.The solid was homogenized by blade milling (5 min at 20,100 rpm; IKA Tube Mill).( 6) The solid obtained from water extraction was extracted an additional five times with ethyl acetate as just described.It was dried under vacuum (10 mbar) at 50 °C overnight.
This method is very close to the Gasparini et al. (2020) method.
(1) The slurry was centrifuged for 45 min at 1438 g.The water phase was discarded.The centrifugation tube containing the capsules cake was filled with deionized water and dispersed under agitation for 10 min.The mixture was centrifuged for 45 min at 1438 g.The water phase was discarded.The upper phase (cake) as well as any sedimentation phase was kept.
The procedure was repeated twice.
(2) The tube containing the cake and the sediment was filled with ethanol and left to equilibrate for 1 h at room temperature.It was then centrifuged for 30 min at 7142 g.The ethanol phase (clear) was discarded.The procedure was repeated four times.
(3) After the last wash the powder was dried at 80 °C and ground in a ball mill.If the fragrance content remaining in the sample was found to still be >1% by GC-FID, another ethanol wash was performed, and the powder was dried at 80 °C and ground again.
Method E. Complete purification.
(1) The slurry was centrifuged for 45 min at 1438 g.The water phase was discarded.The centrifugation tube containing the capsules cake was filled with deionized water and dispersed by stirring for 10 min.The mixture was centrifuged for 45 min at 1438 g.The water phase was discarded as well as any sedimentation phase.Only the upper phase (cake) of the centrifugation tube was kept.The procedure was repeated twice.
(2) The tube containing the capsule cake was filled with ethanol and left to equilibrate for 1 h at room temperature.It was then centrifuged for 30 min at 7142 g.The ethanol phase (clear) was discarded.The procedure was repeated four times.(3) After the last wash the powder was dried at 80 °C and ground in a ball mill.If the fragrance content remaining in the sample was found to still be >1% by GC-FID, another ethanol wash was performed, and the powder was dried at 80 °C and ground again.
Extractions are done by means of centrifugation unless mentioned otherwise.Water extraction steps lead to removal of water-soluble residuals and, as explained in the next paragraph, the possibility of removing insoluble (dispersible) residuals of the aqueous phase.Ethanol or ethyl acetate extraction steps permit the quantitative removal of perfumery raw materials.
When undergoing centrifugation, the fragrance microcapsules of the aqueous slurries we studied separate as an upper "cake" phase.Any water-soluble residuals remain in the middle aqueous phase, whereas any water-insoluble (dispersible) residuals sediment and separate as the bottom phase (Figure 2).The material retained in the centrifugation tube for the next purification step is the key difference between methods D and E, with the latter only keeping the upper "cake" containing the fragrance capsules.
The detection and quantification of residual fragrance in the isolated and dried polymer powder was conducted by GC-FID using an internal standard analysis.The target criteria of the fragrance industry is <5 mass% residual fragrance before submission to biodegradation testing (International Association for Soaps, Detergents and Maintenance Products/International Fragrance Association Europe, 2019).We confirm nearly quantitative removal of the fragrance (residual levels less than 1 mass% of the solid) for all the samples studied.Method A is an exception, because the core fragrance is deliberately kept inside the polymeric shell.The dry isolated polymer is subjected to elemental analysis, and the resulting elemental composition is used to calculate the theoretical oxygen demand of the sample, which in turn is used to determine the % biodegradation achieved in OECD test guideline 301F (OECD, 1992).

BIODEGRADATION TESTING
The isolated shell material was subjected in an OECD test guideline 301F ready biodegradability test, prolonged up to 60 days, using an OxiTop Control System.Sewage sludge from a sewage treatment plant treating predominantly domestic wastewater was used as the inoculum, at a concentration of 30 mg/L dry matter.The test material concentration was 50 mg/L, which in function of the exact elemental composition of each individual sample, gives theoretical oxygen demand (ThOD) in the 50 to 100 mgO 2 /L range, in a total test volume of 255 mL.All samples were tested in duplicate.The samples were stirred for the duration of the study in a closed flask at a constant temperature of 22 °C (±1 °C), and the oxygen consumption was determined by measuring the pressure drop in the respirometer flask.The Biochemical Oxygen Demand (BOD), which is the amount of oxygen taken up by the microbial population during biodegradation of the test chemical (corrected for uptake by the blank inoculum, duplicate flasks run in parallel) was expressed as a percentage of ThOD (calculated from the elemental composition).For test materials containing nitrogen, the concentration of nitrate at test end was determined using a nitrate ion selective electrode.The net amount of nitrate in test flasks was obtained by subtracting the average amount of nitrate found in the inoculum blanks.A weighted ThOD was then calculated from the ThOD (ammonium) and ThOD (nitrate).
Experiments were run in several series, with most experiments starting between May 03, 2022 and February 16, 2023.Alongside every series of test materials, sodium benzoate (NaB) was tested as a reference material, in duplicate, at a test concentration of 50 mg/L, giving a ThOD of 83.5 mgO 2 /L.Biodegradation of NaB was in the range of 88.1 ± 0.5% to 93.1 ± 0.8% on day 28 of the test, and 90.6 ± 3.3% to 94.1 ± 3.2% on day 60 of the test.
The average particle size (d50) of the Microcapsule 2 sample powder submitted to the biodegradability test was 6 µm (measured using a Malvern Mastersizer laser diffraction particle size analyzer).This result is consistent with the treatment that the capsules undergo during the sample preparation, including crushing/milling.The d50 of the capsules in the initial commercial slurry is 31 µm, with a shell thickness less than that of the powder submitted to the biodegradability test.Therefore, the powder sample prepared and used in the biodegradability test is suitable, and is representative of the commercial microcapsules.

RESULTS AND DISCUSSION
Biodegradation results are shown in Table 2 for the five microcapsule types treated according to the various preparation methods described.Now we discuss the influence of the preparation methods applied to the level of biodegradability of the microcapsules in the listed order, from Microcapsule 1 to Microcapsule 5.
FIGURE 2: Images of the initial capsule slurry (i), the different phases formed after centrifugation of the slurry (ii), and the final isolated shell powder (iii).In (ii), tube A contains three phases: the uppermicrocapsules cake, the middle-aqueous phase, and the bottomsediment phase (Method D).The tube B contains two phases: the upper-microcapsules cake and the lower-aqueous phase (Method E).

Microcapsule 1
This was the only capsule subjected to all preparation methods and therefore served as a reference for all subsequent methods.Applying the various preparation methods to Microcapsule 1 resulted in a very broad and inconsistent range of biodegradability levels.The results ranged from as high as 91% down to 0% (see Table 2), and the following provides an interpretation for each method used: Method A. The high level of biodegradability is a consequence of removing only the water from the capsule slurry.All remaining materials, including those that may themselves be biodegradable and independently of whether they are chemically bound to the capsule shell or not, are subjected to the biodegradation test.Method B. Only the fragrance oil phase is removed, and the other materials that remain can biodegrade, again independently of whether they are bound or not to the capsule shell.Using this method, a large decrease in the level of biodegradability was observed, indicating that the fragrance oil phase included in method A makes a significant contribution to the measured level of biodegradation seen.Methods and D. When both the soluble materials and the fragrance oil core are removed, there are inconsistent values reported.Method C resulted in similar biodegradation levels to method B. Differences between Methods C and D consist in the freeze-drying initial step in Method C, in employing filtration rather than centrifugation, and in the order and number of aqueous/organic extraction steps, which perhaps are not so efficient in removing soluble residuals.The biodegradation level observed for the sample obtained using Method D was, as expected, lower than that using Method B, indicating the further removal of biodegradable soluble materials.Method E. In this method, all materials that are not bound to the capsule shell have been removed through various washings, and the amount of residual fragrance is less than 1 mass% of the solid.In this case, the measured level of biodegradability is 0. This is considered to reflect the true biodegradability of the polymer capsule shell material.

Microcapsule 2
As with Microcapsule 1, we observe a clear variation in the level of biodegradability seen as a function of the preparation method (Table 2).
Method A. Again, when only the water is removed, we observe the highest level of biodegradability.Methods B, C, and E. As the level of washing increases, the biodegradability continues to decrease as more nonbound materials are removed.The lowest value is observed for the most stringent method, E. However, the level of 62% BOD observed is higher than the OECD test guideline 301F pass criteria of 60%, indicating that this capsule shell meets the OECD biodegradability standard irrespective of the sample preparation method.Method D. This method was removed from the testing sequence.
To further validate our methodology, we applied further testing to Microcapsules 3, 4, and 5.

Microcapsule 3
This is a capsule type used typically for fragrance encapsulation: one formed primarily from melamine cross-linked with formaldehyde, which is generally considered to be nonbiodegradable.
Method A. When only the water was removed through freezedrying, a level of 56% biodegradation was observed, indicating that the biodegradation occurs through the retention of residual materials.Method E. This confirms the result for method A above, with method E involving the complete removal of all residues and a correspondingly significant decrease in the biodegradation potential.
We also evaluated two supplied capsules which are claimed to be biodegradable by the suppliers.The results in Table 2 clearly indicate that if the capsules are subjected to the stringent preparation Method E, the level of biodegradability at 60 days is significantly reduced, indicating that we would not be able to substantiate the biodegradable claim for these capsules.
Figure 3 illustrates the impact of not removing any residuals nor the fragrance core (Method A) on the different microcapsules tested.Only Microcapsule 3 does not seem biodegradable; all the other capsules give scores well above 60%.However, when all residuals are removed as well as the fragrance core (Method E), only Microcapsule 2 is seen to satisfy the requirements for being described as biodegradable.Note the significant score gap for Microcapsules 1, 3, 4, and 5 between the two methods.
In summary, our results show that inappropriate sample preparation will lead to misleading biodegradation results.The impact of not adequately removing the fragrance core and other residuals (e.g., Methods A-D) can lead to false-positive results, with several samples tested in our study exceeding the 60% pass criteria of the OECD test guideline 301F and the ECHA microplastic restriction regulation.However, when all residuals are removed as well as the fragrance core (Method E), only Microcapsule 2 is indeed shown to be biodegradable.
Of all the sample preparation methods evaluated, we therefore recommend using Method E to obtain trustworthy biodegradation results.This sample preparation method gives the lowest biodegradation value for all the microcapsules tested because it adequately removes material not associated with the capsule polymeric shell.The latter was confirmed via analytical methods, whose details are not provided in our study.
In summary, to demonstrate the efficiency of the aqueous washing procedure, two samples of Microcapsule 2, were treated with four aqueous wash steps.The aqueous phases collected were submitted to thermogravimetric analysis and size exclusion chromatography (SEC-MALS).According to both methods, the amount of the residues in the wash liquors decreased rapidly with each additional wash step (Figure 4).We found that after two aqueous wash steps, more than 95% of the total residues were removed, while an additional third and fourth wash contributed only marginally to the overall efficiency of the aqueous extraction procedure.To evaluate the impact of the number of wash steps on the biodegradation results, we tested samples that had been washed two, three, and four times.The increased number of wash steps did not significantly impact the result of the test; the variations in the results (a difference of < 5% biodegradation) were within the variability of the biodegradation test.Therefore, we adopted two aqueous washes as the standard in our Method E. The efficiency of the removal of the fragrance by the solvent extraction steps was confirmed, as mentioned previously, by GC-FID with internal standard analysis showing residual perfume in the final powder of <1 mass% of the solid.

CONCLUSIONS
In the present study we demonstrate the importance of the sample preparation protocol on the biodegradability test result obtained for fragrance microcapsules.The polymeric shell material needs to be thoroughly purified from any nonbound materials as well as from the encapsulated fragrance oil.When this aspect is not respected, misleading false-positive biodegradation results are obtained, depending on the biodegradability level of the impurities that remain in the sample.
Thus, particular attention needs to be paid when a fragrance encapsulation system is claimed to be biodegradable.As shown in our study, the biodegradation level for a given sample can fluctuate, for example, from 0% to 91% (see Table 2, Microcapsule 1), because it dramatically depends on how the test material is isolated from a given microcapsule slurry system.
From the five different encapsulates tested here, only Microcapsule 2 passes the biodegradation test criteria of >60% with the stringent Method E.
We thus recommend use of preparation Method E of the present study or equivalent, to obtain trustworthy biodegradation results.
Adaptation of the sample preparation method can be required, depending on the microcapsule system.Validation via analytical methods of the initial aqueous phase of the slurry (and in the following washing steps) as well as of the isolated microcapsule shell is also key in providing correct biodegradation test results.

FIGURE 1 :
FIGURE 1: Schematic illustration of the polymeric shell extraction Methods A-E.When not indicated otherwise, the separation after an aqueous or organic extraction occurs by centrifugation.

FIGURE 3 :
FIGURE 3: Biodegradation score with OECD test guideline 301F of Microcapsules 1-5 using Methods A and E as described in the text.Presented as an average of two replicates with error bars showing standard deviation.The line at 60% represents the biodegradation pass criteria.

FIGURE 4 :
FIGURE 4:The amounts of residues found in wash liquor samples by thermogravimetric analysis (TGA) and size exclusion chromatographymulti-angle light scattering (SEC-MALS).

TABLE 1 :
Information on the polymeric shell extraction methods, A-E, relative to purification of the fragrance oil and the soluble and insoluble ingredients of the slurry aqueous phase

TABLE 2 :
Biodegradation% over 60 days, according to OECD test guideline 301F, for the five samples tested and the different sample preparation methods employed a Biodegradation% claimed by the supplier.The result is the average of two replicates with ± showing standard deviation.Negative biodegradation values represent no biodegradation.Abbreviation: NM = not measured.