Philippa Kaur

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SubjectsThe study comprised 34 men (19–26 years, mean 23 years; body mass index 19–31 kg/m2, mean 24 kg/m2) with no physical disabilities or mental disorders, including color blindness and claustrophobia30,31. All subjects were informed of the aims, the study procedure, and the financial compensation for participation, and were asked to follow the rules of the study. Prior to each experiment, subjects underwent short-term starvation31,54 (18–20 h; no food except pure water after lunch (12–1 pm) or dinner (6–7 pm), no later than 1 pm or 7 pm, respectively, one the day before the test), no medical treatments, and normal sleep (at least 6 h, between 10 pm the day before the test day to 8 am on the test day)31. Prior to starting each experiment, subjects were stabilized on a chair for ~ 10 min in a room next to the testing room. Based on an assessment with a pre-experiment questionnaire and the first blood glucose level, measured before starting the experiment (see “Geomagnetic orientation assay” section below), subjects who had not followed these rules were not allowed to take the test on the day and the test was postponed. The study was approved by the Institutional Review Board of Kyungpook National University and all the procedures followed the regulations for human subject research. Informed consent was obtained from all subjects.Modulation of GMFThe ambient GMF in the testing room had a total intensity 45.0 μT, inclination 53°, and declination − 7° (Daegu city, Republic of Korea); the total intensity of 50.0 μT in our previous study31 was changed due to a reconstruction of the building; 45.0 μT was maintained throughout the period of this study. To provide the subjects with various GMF-like magnetic fields (i.e., by modulating of total intensity, inclination, or direction of magnetic north), the coil system from our previous studies6,7,31 was used. It comprised three double-wrapped, orthogonal, rectangular Helmholtz coils (1.890 × 1.890 m, 1.890 × 1.800 m, and 1.980 × 1.980 m for the north–south, east–west, and vertical axes, respectively), electrically-grounded with copper mesh shielding. The subject was seated on a rotatable plastic chair with no metal components, at the center of the three-dimensional coils with his head positioned in the middle space of the vertical axis of the coils. To modulate the geomagnetic north, each pair of coils was supplied with direct current from a power supply (MK3003P; MKPOWER, Republic of Korea). The magnetic field was measured using a 3-axis magnetometer (MGM 3AXIS; ALPHALAB, USA); the field homogeneity at the position of the subject’s head was found to be 95%. The testing room was shielded by a six-sided Faraday cage comprising 10 mm thick aluminum plates, and was grounded during the entire experiment40. Background electromagnetic noise was measured inside the coils at the start and the end of each experimental day. It was attenuated by the Faraday cage more than 200-fold over the range from 500 Hz to 100 MHz as described in detail in our previous study31. The 60 Hz power frequency magnetic field was no more than 2 nT (3D NF Analyzer NFA 1000; Gigahertz Solutions, Germany). All electronic devices were placed outside the Faraday cage during the experiments, with the exception of the switch button module for GMF modulation and the antenna for generating the oscillating magnetic fields. The temperature experienced by the subjects was maintained at 25 ± 0.5 °C (Data logger 98,581; MIC Meter Industrial, Taiwan) by air circulation through the honeycomb on the ceiling of the Faraday cage31.Geomagnetic orientation assayAdopting a two-alternative forced choice (2-AFC) paradigm33,34, a geomagnetic orientation assay was conducted similar to our previous study31. Experiments were performed at 09:30–11:30 am or 1:00–5:00 pm (local time, UTC + 09:00) (each experiment: 50 min–1 h 10 min; mean ≈ 1 h, which was shorter by approximately 30 min than that in the previous study: 1 h 20 min–1 h 40 min; mean ≈ 1 h 30 min). Depending on the experiment, starved or unstarved subjects were tested individually. Prior to each experiment, the subjects were asked to remain with their heads facing the front, with eyes closed and earmuffs on during the experiment. In particular, they were asked to concentrate on sensing, if they could, the ambient geomagnetic north during the association phase, and to use the sensed information, depending on the experiment, to orient toward one of the two modulated magnetic norths (0°/180° for magnetic north–south axis or 90°/270° for magnetic east–west axis, rotated clockwise with respect to the ambient geomagnetic north) during the test phase. Subjects were instructed to avoid distracting thoughts and to think immediately “which direction is modulated magnetic north?” whenever they were distracted during the test phase, or felt they were being biased by experiences from earlier experiments. While seated on the rotatable chair, the subject’s blood glucose level was measured shortly before the first session and immediately after each session with eyes open except in the ‘dark’ experiment (Accu-Chek Guide; Roche, Germany)31. If the determined value before the first session varied by more than 15% relative to the mean (Table S2)31, the experiment was postponed and repeated at a later date (approximately 2% of experiments). The subjects were stabilized with eyes closed for 2 min before the first trial in the absence of visual, auditory, olfactory, and haptic sensory cues. For the ‘dark’ experiment (light intensity ≈ 0 lx), subjects wore home-made ‘blind’ goggles and were stabilized with eyes closed for 5 min55,56, and then asked whether they could see any light. If they could, the goggles were adjusted to prevent leakage of light, and the subject then had another 5 min of stabilization with eyes closed before starting the experiment. The subjects were illuminated with light from a filtered/non-filtered diffused light-emitting diode, depending on the experiment (Table S1). The home-made filter goggles were worn throughout the experiment, including the association and test phase, when required. The goggles contained glass filters (Tae Young Optics, Republic of Korea) to provide the eyes with particular wavelengths of light (Spectrometer USB4000-UV-VIS, Ocean Optics, USA) (Fig. S1). Each experiment consisted of 16 sequential trials for ‘no-association’ and ‘food-association’. For the food-association, a subject facing toward the ambient geomagnetic north was gently provided with a chocolate chip31 on his right palm by an experimenter, and given 30 s to eat it, while during no-association trials, food was not provided during the association phase. After a subsequent 5 s interval, the experimenter gently touched the subject’s right thenar area using a paper rod, as the cue to start the test. One of the two modulated magnetic north directions, depending on the experiment, was randomly provided 3 s before the cue for the test. Each of the modulated magnetic north directions was provided eight times for the no-association and food-association sessions. Subjects were informed of the nearly equal probability for each of the modulated magnetic north directions before each experiment. With the touch cue, subjects were asked to rotate freely toward any direction (clockwise or counterclockwise) by themselves (1–4 cycles of two-thirds rotation) and try to sense the direction of the modulated magnetic north during a 1 min period. Rotation was allowed within the rotation angle (− 30° to 210° for the magnetic north–south axis or − 120° to 120° for the east–west axis, depending on experiments, with respect to the ambient magnetic north), which was confined by the plastic stool (Fig. 1A) touching the left or right ankle of the subjects. When subjects determined the direction of the magnetic north, they stopped rotating to face toward the direction and lifted their right hand to indicate the direction to the experimenter. The direction was measured by the experimenter at 10° intervals using the scale on the walls of the Faraday cage31. A prerequisite for correct orientation was that the subject indicated the direction within the range of 30° to the both sides with respect to the magnetic cardinal directions, which was instructed to the subjects before each experiment. When the direction the subjects indicated was out of the 30° range, the trial was not included in the data and was repeated (approximately 0.63% of trials). Before the subsequent trial, the subject was gently rotated to face toward the ambient geomagnetic north and then rested for 5 s. For the ‘dark’ experiment, subjects were asked whether they could see any leaked light immediately after the last measurement of blood glucose level at the end of experiment. If the subject could see leaked light, the experiment was nullified and repeated later on (approximately 3% of experiments; 2/68). All experiments were performed in a double-blind fashion. The experimenter who conducted the orientation assay knew whether a subject was starved or not, wearing filter goggles, and food-associated or not, but did not know the random magnetic north sequences that were controlled by the personal computer (PC) system. Another experimenter responsible for analyzing the data did not know whether the subject was starved or not, the experimental conditions, including light wavelengths, or whether an oscillating magnetic field had been provided to the subjects. Thus, none of the experimenters were aware of all the subject information and data during the experiments and data analysis. The correct orientation rate was calculated by (the number of correct orientation trials/total number of trials) (raw data, Appendix S3). All the subjects participated in all the experiments performed in random order with an interval of at least 3 days between experiments. After each experiment, the subjects were asked to answer a post-experiment questionnaire about whether they closed their eyes when required during the entire period of the experiment. In cases when a subject did not maintain closed eyes, the experiment was repeated (approximately 1% of experiments). For each subject, a preliminary experiment on the “magnetic north–south axis” was conducted twice (unstarved and starved for each) with no goggles for adaption to the experimental procedure. These data were not included in the results.Experiments with oscillating magnetic fieldsExperiments with oscillating magnetic fields were performed using the standard geomagnetic orientation assay described above. To produce the oscillating magnetic fields, oscillating currents from a function generator (AFG3021; Tektronix, USA. For each magnetic field, sweep of 500 ms; interval of 1 ms. See Fig. S6A) were amplified (ENI 2100L RF power amplifier; Bell Electronics, USA) and fed into a calibrated coil antenna (30 cm diameter, 6509 loop antenna; ETS-LINDGREN, USA) mounted on a wooden frame, comprised of a single winding of coaxial cable. The oscillating magnetic fields were measured daily, before the first and after the last experiment of the day, using a spectrum analyzer (SPA-921TG; Com-Power, USA) with a calibrated loop antenna (48 cm diameter, AL-130R; Com-Power, USA) and a calibrated magnetometer (Probe HF 3061, NBM-550; Narda, Germany). Magnetic field intensities were measured on the glabella of the subjects; variations in intensity between subjects due to different seating heights were less than 10% of the average values (Table S3). The function generator and amplifier were placed outside the Faraday cage, and switched on during the dummy load control experiments with no signal from the PC system. The band widths of the monochromatic magnetic fields, i.e., 1.260 and 1.890 MHz were 0.020 and 0.019 MHz (“average”, √3 kHz), respectively, at the bottoms of the peaks. During the test phase, the maximum values of magnetic noise on the glabella of subjects including the dummy load did not exceed the following values: (1) 5 Hz–9 kHz; 2 nT/√ 2 kHz of “average” and 8 nT/√ 9 kHz of “max-hold” (0.05 nT/√ 2 kHz of “average” and 5 nT/√ 9 kHz of “max-hold” in the dummy load) (3D NF Analyzer NFA 1000; Gigahertz Solutions, Germany); (2) 9 kHz–500 kHz; 5 nT/√ 3 kHz of “average” and 8 nT/√ 3 kHz of “max-hold” (≈ 0 nT/√ 3 kHz of “average” and ≈ 1 nT/√ 3 kHz of “max-hold” in the dummy load) (the AL-130R antenna) (Fig. S6C); and (3) 500 kHz–30 MHz; 0.006 nT of 3.780 MHz harmonic in the 1.260 MHz, 0.03 nT of 5.670 MHz harmonic in the 1.890 MHz, and ≈ 0 nT in the dummy load (/√ 10 kHz of “average”) (Fig. S6B), and 0.15 nT/√ 10 kHz of “max-hold” at the same frequencies above and ≈ 0 nT in the dummy load (the AL-130R antenna).Statistical analysisTo determine the significance of orientation data from the 2-AFC paradigm, a one-sample t-test (test mean: 0.5), paired sample t-test, or two-sample t-test was performed using Origin software 11 (Origin, USA). 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Choice of biowaste treatment plants and sample identifiersCompost samples were collected from four central municipal biowaste treatment plants (denominated as #1 to #4) in Baden-Wurttemberg, Germany (Table 1). All plants used a state-of-the-art two-stage biowaste treatment process comprising of (a) anaerobic digestion/biogas production and (b) subsequent composting of the solid digestate to produce a high-quality mature compost sold for direct use as fertilizer in agriculture. The composts were regularly analyzed by an independent laboratory for quality and residual contamination and consistently fulfilled the quality requirements of the label RAL-GZ 251 Gütezeichen Kompost of the German Bundesgütegemeinschaft Kompost e.V. (www.gz-kompost.de). Plants #1 and #3 produce in addition a liquid fertilizer, which is separated from the solid digestate at the end of stage a) by press filtration and which is also intended for direct use on agricultural soil (replacement of liquid manure). In case of plants #1, #3, and #4 up to 25 wt% of shrub/tree cuttings were added to the solid digestate for composting. All plants used sieving (typically with a 12 or a 20 mm mesh) at the end of the process to assure the necessary purity of their finished composts. Whenever technically possible, we as well took samples of the pre-compost immediately before this final sieving step to evaluate its contribution to the removal of residual BPD fragments. For analysis, composts were passed consecutively through two sieves with mesh sizes of 5 mm and 1 mm, yielding two fragment preparations for IR-analysis namely a > 5 mm fraction corresponding to the contamination by residual “macroplastic” (5 mm is a commonly used upper size limit for “microplastic”, anything larger is macroplastic) and a 1–5 mm fraction corresponding to the regulatory relevant residual contamination by microplastic. The lower limit of 1 mm rather than 2 mm was chosen in anticipation of the expected changes in regulation, where the replacement of the 2 mm limit by a 1 mm limit is imminent.Table 1 Technical data of the investigated plants and incidence of BDP fragments in the sampled composts.Full size tableOccurrence of plastic fragments  > 1 mm in the sampled compostsComposting times of 5–9 weeks were used in the investigated plants (Table 1), which is shorter than the 12 weeks indicated in EN 13432 for the 90% disintegration of a compostable plastic material, but a realistic time span for state-of-the-art technical waste treatment. Since we were not in a position to estimate the quantity of BDP entering the plants, since for technical reasons we were unable to obtain a representative sample, we cannot say, whether any residual BDP detected by us in the finished composts was due to a yet incomplete disintegration process or whether it corresponds to the 10% material still permissible by EN 13432 even after the full composting step. However, in 7 out of the 12 sampled composts and pre-composts fragments with chemical signatures corresponding to the BDPs poly (lactic acid) (PLA) and poly (butylene-adipate-co-terephthalate) (PBAT) were identified in the > 5 mm and/or the 1–5 mm sieving fractions using FTIR analysis3 (Fig. 1; Table 1). All recovered fragments appeared to stem from foils, bags or packaging, since they were thin compared to their length and width (see Suppl Figure S1 for typical examples). Fragments with overlapping signatures, most likely PBAT/PLA mixtures or blends, were also found (see Suppl Figure S2 for the interpretation of the spectra). In addition, the recorded BDP fragment spectra (Fig. 1A) showed high similarity to the FTIR spectra of commercial compostable bags sold in the vicinity of the biowaste treatment plants (Fig. 1B), which together with the geometry of the recovered fragments led us to assuming that the majority of the BDP entered the biowaste in the form of such bags.Figure 1FTIR spectra of BDP fragments from composts and commercial bags. (A) BDP fragments recovered from the composts and (B) the commercial compostable bags. Fragments were coded as follows: p or f for pre-compost or finished compost, followed by the plant number (#1 to #4), an indication of the size fraction ( > 5 mm or 1–5 mm) in which the fragment was found, and finally, the fragment number. Fragment F#1_5mm_4 therefore represents the 4th fragment collected in the  > 5 mm size fraction from the finished compost of plant number 1. Bags were arbitrarily numbered 1–10, see Suppl Table S1 for supplier information. The spectra (in grey) of the reference materials for PLA and PBAT are given as basis for the interpretation. Spectra in red refer to test samples consisting only of PBAT, while those in blue indicate samples composed of PBAT/PLA mixtures.Full size imageThe BDP fragments were found alongside fragments of commodity plastics (mostly PE) in all cases. Finished composts tended to contain fewer and smaller fragments than the corresponding pre-composts. The final sieving of the pre-composts to prepare the finished composts hence appears to be quite effective in removing such fragments, in particular those from the > 5 mm size fraction (Table 1) and for that reason has become state-of-the-art in preparing quality composts (contamination by plastic fragments > 2 mm of less than 0.1 wt%). Given that the size of the fragments is a crucial factor regarding ecological risk, we analyzed the sizes (length Î width) of the BDP fragments in comparison to that of the plastic fragments with signatures of commodity plastics such as PE (Fig. 2). BDP fragments found in a given compost sample tended to be smaller than the fragments stemming from non-BDP materials, which may indicate that BDPs degrade faster or tend to disintegrate into tinier particles than commodity plastics. This may also explain why in the compost from plant #2, no BDP fragments were found in the particle fraction retained by the 5 mm sieve ( > 5 mm fraction), while 19 such particles were found in the fraction then retained by the 1 mm sieve (1–5 mm fraction). Interestingly, plant #2 is the only one included in our study that uses no mechanical breakdown of the incoming biowaste. This reduces the mechanical stress on the incoming material. Mechanical stress can alter the properties of plastic foils such as the crystallinity whereby crystallinity has been shown to influence the biological degradation of BDP such as PLA7.Figure 2Size distribution of plastic fragments  > 1 mm. (A) Fragments found in the finished compost from plant #1, (B) in the finished compost from plant #2, and (C) in the pre-compost from plant #3. For reasons of statistical relevance, only samples containing more than 20 BDP fragments per kg of compost were included in the analysis.Full size imageMaterial characteristics of BDP fragments in comparison to those of commercial biodegradable bagsIn order to verify whether the BDP fragments recovered from the composts differed from the compostable bags in any parameter with possible relevance for biodegradation and environmental impact16, the physico-chemical properties of bags and fragments were studied in detail. Since we wanted to have a maximum of information of the BDP fragments, size/weight was a limiting factor in selecting fragments for analysis. Fragments of at least 1 mg were required for the FT-IR analysis. 5 mg-fragments could be analyzed in addition by 1H-NMR, while the full set of analytics (FT-IR, 1H-NMR, and DSC) required at least 10 mg of sample.For insight into the chemical composition, 1H-NMR spectra of the commercial bags and all suitable BDP fragments were compared (Fig. 3). In case of material mixtures and blends, the 1H-NMR analysis allows quantification of the PBAT/PLA weight ratio in the materials and also of the ratio of the butylene terephthalate (BT) and butylene adipate (BA) units in the involved PBAT polyesters.Figure 31H NMR spectra of BDP fragments from composts and commercial bags. (A) BDP fragments recovered from the composts and (B) the commercial compostable bags. Fragments were coded as follows: p or f for pre-compost or finished compost, followed by the plant number (#1 to #4), an indication of the size fraction ( > 5 mm or 1–5 mm) in which the fragment was found, and finally, the fragment number. Bags were arbitrarily numbered 1–10, see Suppl Table S1 for supplier information. The spectra (in grey) of the reference materials for PLA and PBAT are given as basis for the interpretation. Spectra in red refer to test samples consisting only of PBAT, while those in blue indicate samples composed of PBAT/PLA mixtures. (C) Chemical structures of PLA and PBAT, chemical shifts of the protons are assigned as indicated in the reference spectra in (B).Full size imageThe 1H-NMR spectra corroborate the FTIR measurements in that all investigated commercial bags were made from PBAT/PLA mixtures of varied composition (Table 2). By comparison, some of the fragments, for instance, f#1_5mm_4, appeared to consist of only PBAT. Other fragments, e.g., f#1_1mm_9, were mixtures of PLA and PBAT (Table 2). However, even in the case of PBAT/PLA mixtures, the average PBAT content tended to be higher in the fragments than in the bags, while the BT/BA monomer ratio in the respective PBATs, was also significantly higher in the fragments than in the bags. If we assume the fragments to stem from similar compostable bags as the ones included in our comparison, this would mean that during composting of such a bag, the PLA degrades more quickly than the PBAT, whereas within a given PBAT polyester, the BA unit is more easily degraded than the BT unit. Evidence can indeed be found in the pertinent literature that PLA has faster biodegradation kinetics than PBAT, while BT is more resistant to mineralization than BA17,18.Table 2 Composition of commercial compostable bags and BDP fragments recovered from the composts as analyzed by 1H-NMR.Full size tableNext, differential scanning calorimetry (DSC) was used to analyze fragments compared to commercial bags in regard to the presence of amorphous vs. crystalline domains, a parameter expected to affect biodegradation kinetics and therefore the putative environmental impact of the produced microplastic16 upon release into the environment with the composts. Whereas amorphous domains show glass transition, crystalline domains show melting, both of which can be discerned by the respective phase transition enthalpy in the DSC curves (Fig. 4).Figure 4DSC curves of BDP fragments and compostable bags #1 and #7. Curves for the reference materials (in grey) for PLA and PBAT are given for comparison. Curves were recorded during the first heating run (temperature range: − 50 °C to 200 °C, heating rate: 10 °C min−1). (A) and (B) curves in red refer to test samples consisting only of PBAT, while those in blue indicate samples composed of PBAT/PLA mixtures. Fragments were coded as follows: p or f for pre-compost or finished compost, followed by the plant number (#1 to #4), an indication of the size fraction ( > 5 mm or 1–5 mm) in which the fragment was found, and finally, the fragment number.Full size imageThe curve for the reference PBAT shows a glass transition temperature (Tg) of − 29 °C and a broad melting range between 100 and 140 °C for the crystalline domains, while that of the PLA reference shows a glass transition temperature of 58 °C and a narrower melting peak between 144 °C and 162 °C. The curve for commercial bag #1, which had a comparatively high PLA content, shows a pronounced melting peak in the expected range; the same is the case for fragment p#3_5mm_1 and to a lesser extent for fragment p#3_5mm_9, two fragments, which also have high PLA contents. The DSC curves of the other fragments and bag #1 are undefined in comparison, which is due to their high PBAT content. According to the DSC curves, most of the investigated materials are semicrystalline, i.e., contain both amorphous (glass transition) and crystalline (melting) domains. However, the DCS data alone allow only a qualitative discussion of the differences between fragments and bags.To obtain quantitative data on the crystallinity differences, wide angle X-ray scattering (WAXS) spectra were recorded. WAXS requires fragments at least 3 cm long, which restricted the number of fragment samples to three, all of which were found in pre-compost samples. The corresponding curves are shown in Fig. 5A–C. The spectra of the commercial biodegradable bags are shown in Suppl Figure S3. Foils were in addition prepared by heat pressing from the reference materials for PLA and PBAT in order to include them into the WAXS measurements (Fig. 5D). While the foils produced from the PBAT reference material produced crystallinity peaks at 16.2°, 17.3°, 20.4°, 23.2°, and 24.8°, the foil prepared from the PLA reference material showed only an amorphous halo at 15.5° and 31.5°, which is in accordance with values published in the literature19. A more pronounced crystallinity peak was obtained in the case of an additionally annealed PLA foil.Figure 5WAXS curves with Lorenz fitting for (A) fragment p#3_5mm_1, (B) fragment p#3_5mm_9, and (C) fragment p#4_5mm_2. (D) WAXS curves for foils produced from the PBAT and PLA reference materials; the percent values indicate the crystallinity. The dash lines are the fitting peak curves for the XRD spectrum. Crystallinity can be obtained by dividing the integration area of the fitted peaks by the integration area of the entire spectrum. Fragments were coded as follows: p or f for pre-compost or finished compost, followed by the plant number (#1 to #4), an indication of the size fraction ( > 5 mm or 1–5 mm) in which the fragment was found, and finally, the fragment number.Full size imageIn case of the fragments and bags, the peaks of PLA and PBAT overlapped to some extent in the WAXS spectra, but by conducting Lorenz fitting using Origin software, the overall crystallinity could be calculated as follows:$$chi = { 1}00% , *{text{ Aa}}/left( {{text{Aa }} + {text{ Ac}}} right)$$where χ is the crystallinity and Aa and Ac represent the areas of the amorphous and crystalline peaks.Using this equation, crystallinities of 55% (fragments p#3_5mm_1), 34% (p#3_5mm_9), and 34% (p#4_5mm_2) were calculated for the fragments. The foils prepared in house for the reference materials had similar crystallinities (43% in case of the annealed PLA foil and 26% of the PBAT foil), while the simple PLA foil was amorphous. By comparison, for eight of the commercial bags, crystallinities in the range from 1% to 7% were calculated, whereas these values were 14% and 15% for the remaining two bag types (Suppl Figure S3).The high crystallinity of the larger fragments recovered from the pre-compost samples suggests that crystalline domains of BDP materials may indeed disintegrate more slowly than the amorphous ones, as prior studies on microbial biodegradation have suggested7,8. Admittedly, such large fragments per se would not enter the environment, since the final sieving step used to prepare the finished composts is quite efficient at removing them. However, it is tempting to extrapolate that residual BDP in general are remnants of the more crystal domains of the original material, even though experimental proof of this assumption is at present not possible. 10 wt% of a BDP bag is allowed to remain after standard composting. It is usually assumed that any such residues continue to degrade with comparable speed. However, should these residues correspond to the more crystalline domains, rather than degrading with similar speed as the bulk material, the more crystalline fragments can be expected to persist for a much longer and at present unpredictable length of time in the environment, e.g. when applied to the soil with the composts; in particular, when they are also enriched in PBAT and BT units as suggested by our analysis of the chemical composition. Data from the use of biodegradable foils in agriculture show that the degradation in the environment may take years20. Altogether this may have unforeseen economic and environmental consequences, especially when considering the high fraction of BDP fragments < 5 mm. Putative consequences include changes in soil properties, the soil microbiome and therefore in plant performance21, a factor indispensable for worldwide nutrition.Residues of BDP fragments  1 mm were found in the collected LF samples. This is hardly surprising, given that the LF is produced by press filtration of the digestate after the anaerobic stage. Such a filtration step can be expected to retain fragments > 1 mm in the produced filter cake, which goes into the composting step, leaving the filtrate, i.e. the LF, essentially free of such particles. Anaerobic digestion is currently not assumed to contribute significantly to the degradation of BDP17,22, but the process conditions (mixing, pumping) may promote breakdown of larger fragments, particularly when additives such as plasticizers23 leach out of the material.Since the residual solids content of the LF is low (plant #1: 8.6 wt%, plant #3: 5.8 wt%), a combination of enzymatic-oxidative treatment and µFTIR imaging originally developed for environmental samples from aqueous systems24,25 could be adapted for the analysis (size and chemical signature) of particles in the LF down to a size of 10 µm. The corresponding data are compiled in Table 3. In all cases, residual fragments from PBAT-based polymers represented the dominant plastic fraction in the investigated samples; i.e. approximately 53% of all plastic particles in the LF from plant #1 (11,520 BDP particles per liter) and 65% in the case of plant #3 (12,480 BDP particles per liter). Liquid manure is applied several times a year to fields at a concentration of 2–3 L m−2. According to our analysis > 20,000 BDP microparticles of a size ranging from 10 µm to 500 µm enter each m2 of agricultural soil whenever LF is applied on agricultural surfaces.Table 3 Microplastic fragments (BDP/all) found per liter of liquid fertilizer.Full size tableDue to the complexity of the matrix, a similar analysis of individual plastic fragments  1 mm. Six compost samples representing the more contaminated ones based on the content of fragments > 1 mm, namely, f#1, f#2, p#3, f#3, p#4 and f#4 (nomenclature: f or p for finished or pre-compost, followed by plant number), were extracted with a 90/10 vol% chloroform/methanol mixture. The amounts of PBAT and PLA in the obtained extracts were then quantified via 1H-NMR (Table 4). Briefly, the intensity of characteristic signals in the extract spectra of the compost samples (see Suppl Figure S4) were compared to peak intensities produced by calibration standards of the pure polymer dissolved at a known concentration in the chloroform/methanol. All samples and standards were normalized using the 1,2-dichloroethan signal at 3.73 ppm as internal standard. See also Suppl Figure S5 for an exemplification of the quantification of the PBAT/PLA ratios. Based on the amounts of PBAT and PLA extracted from a known amount of compost, the total mass concentration (wt% dry weight) of these polymers in the composts was calculated.Table 4 Evidence of PBAT and PLA residues caused by fragments  2 mm. Moreover, residues of PBAT and PLA were found in all investigated compost samples, including the finished compost from plant #4, which had shown no contamination by larger BPD fragments (Table 1). The pre-compost from that plant had shown a few contaminating BDP fragments in the > 5 mm fraction. However, in regard to the fragments More