Table 2.

Summarization of numerous methods to assess the biodegradation of polymers in various experimental settings, together with their advantages and limitations.

No.	Test Material	Brief Description, Aim, and Degradation Condition	Advantages or Limitation	Ref.
Physical methods
1	PHBHHx/PBAT (80/20)	TEM: To analyse the cross-section of the sheet. SEM: To analyse the surface morphology of the melt blend sheet. Ruthenium tetroxide (RuO4) is used as a staining agent. Condition: Seawater for 28 days with 42% biodegradation.	TEM: The PBAT region darkens owing to the lower permeability of the electron beam through the attachment of RuO4 to the phenyl group in the PBAT as compared with the PHBHHx region, allowing for clear observation.	[60]
2	PHB, P(HB-co-12%-HHx)	SEM: To analyse the surface morphology. Conditions: Activated sludge for 18 days with a 0.65% recovery weight.	SEM: Able to observe the porous and rough surface, indicating fast degradation, also contributing to the low degree of crystallinity of HHx.	[62]
Chromatographic methods
3	P(HB-co-HHx) films.	Thermally assisted hydrolysis and methylation–gas chromatography (THM-GC): Rapid analysis of the copolymer composition during biodegradation. Condition: Farm soil pH 5.3 for 28 days at 34 °C with 1.91% recovery weight.	THM-GC: Able to observe the local biodegradation behaviour of the degraded films based on the changes in the copolymer composition.	[44]
4	Intracellular P(HB-co-HV) in Ralstonia eutropha and recombinant Escherichia coli.	Py-GC-MS: Rapid analysis of PHA contents and their monomer compositions accumulated intracellularly	Py-GC-MS: Promising tool used to rapidly screen PHA-containing strains based on polymer contents, along with their monomer compositions. The data obtained by this method indicate results similar to those of conventional GC-FID.	[66]
5	Whole bacterial cells of Cupriavidus necator accumulating P(HB-co-HV).	THM-GC in the presence of TMAH: Rapid and direct compositional analysis of P(HB-co-HV) in whole cells.	THM-GC in the presence of TMAH: Chromatograms clearly indicate peaks derived from the HB and HV units of the polymer chains without any interference by the bacterial matrix components and no cumbersome sample pre-treatment required. The data obtained by this method indicate results similar to those of conventional GC-FID.	[67]
Spectroscopic methods
6	Extracted PHAs from bacteria cells such as E. coli and Pseudomonas sp.	NMR: To characterize PHAs. FTIR: To investigate functional groups of the PHAs.	FTIR data indicate the presence of hydroxyoctaoate, medium-chain-length PHAs and hydroxydecanoate with strong bands at 1631 cm−1, 1548 cm−1, and 1409 cm−1. NMR spectra show the presence of interconnection functional groups of HC=CH bonds at 3.363 ppm and CH2O-COOH bonds at 2.548 ppm. Both methods require a sample extraction process prior to each analysis.	[68]
7	Bioplastics from starch/chitosan reinforced with PP.	FTIR: To investigate functional groups of the bioplastics. The spectra portray main bonds of O-H hydrogen bonds (carboxylic acid), C-H alkanes, C=C alkenes, and C-O alcohols.	FTIR spectrum indicates that the functional groups of bioplastics have similarity with their constituent components. Sample extraction process is required.	[69]
8	Bioplastics from starch/chitosan reinforced with PP.	XRD: To analyse the degree of crystallinity.	The XRD data evince that the biopolymer had an amorphous crystalline structure, with the major wide peaks located between 18° and 30°.	[69]
9	Single-use bioplastics (SUPs).	XRF: To determine the elemental composition and concentration in the SUPs.	Most of the SUPs exceed the standard values, and the highest concentrations of Cu, Cr, Mo, Zn, Fe, and Pb were 1898 mg/kg, 1586 mg/kg, 95 mg/kg, 1492 mg/kg, 1900 mg/kg, and 7528 mg/kg. XRF is a non-destructive analytical method which enables the same sample to be used again for further analysis.	[70]
Respirometric methods
10	PHBHHx/PBAT (80/20).	BOD tester: To measure biodegradation induced by the microbial metabolism. Condition: Seawater for 28 days with 42% biodegradation.	The seawater was placed together with samples in a fermenter, and the amount of consumed oxygen (O2) gas was measured. This method is an indirect measurement of O2 that has been utilized for complete sample degradation, which may carry some errors. It is preferable to cross-check the results with the weight loss data.	[60]
Meta-analysis
11	PHAs	Statistical analysis: To estimate the PHA mean biodegradation rate and lifetime. Condition: Seawater	The statistical analysis enables the estimation of the biodegradation of PHA in the seawater as 0.04–0.09 mg/day/cm.	[71]
Thermal methods
12	Poly(butylene succinate-co-butylene adipate) film sample	DSC: To determine the degree of crystallinity.	The obtained degree of crystallinity of the original and heated film samples were 46.1 and 42.4%, respectively, evinced the crystallinity of the PBSA film, which was considerably lower for the heat-treated film.	[48]
13	PHAs accumulated in Pseudomonas putida	DSC and TGA: To analyse the thermal properties.	Thermal properties indicate that the accumulated PHA is semi-crystalline, with a good thermal stability, Td of 264.6 to 318.8 °C, Tm of 43 °C, Tg of −1.0 °C, and ΔHf of 100.9 J/g. Requires sample pre-treatment of PHA extraction.	[72]