Development of a LC-QTOF-MS based dilute-and-shoot approach for the botanical discrimination of honeys.

Honeys of particular botanical origins can be associated with premium market prices, a trait which also makes them susceptible to fraud. Currently available authenticity testing methods for botanical classification of honeys are either time-consuming or only target a few "known" types of markers. Simple and effective methods are therefore needed to monitor and guarantee the authenticity of honey. In this study, a 'dilute-and-shoot' approach using liquid chromatography (LC) coupled to quadrupole time-of-flight-mass spectrometry (QTOF-MS) was applied to the non-targeted fingerprinting of honeys of different floral origin (buckwheat, clover and blueberry). This work investigated for the first time the impact of different instrumental conditions such as the column type, the mobile phase composition, the chromatographic gradient, and the MS fragmentor voltage (in-source collision-induced dissociation) on the botanical classification of honeys as well as the data quality. Results indicated that the data sets obtained for the various LC-QTOF-MS conditions tested were all suitable to discriminate the three honeys of different floral origin regardless of the mathematical model applied (random forest, partial least squares-discriminant analysis, soft independent modelling by class analogy and linear discriminant analysis). The present study investigated different LC-QTOF-MS conditions in a "dilute and shoot" method for honey analysis, in order to establish a relatively fast, simple and reliable analytical method to record the chemical fingerprints of honey. This approach is suitable for marker discovery and will be used for the future development of advanced predictive models for honey botanical origin.

Biomonitoring of bisphenol A (BPA) and bisphenol analogues in human milk from South Africa and Canada using a modified QuEChERS extraction method.

A sensitive modified QuEChERS extraction method was developed to assess the levels of free and conjugated bisphenols (BPs) in human milk collected between 2018 and 2019 from two regions of South Africa (the Limpopo Province Vhembe district, n = 194; Pretoria, n = 193) and Canada (Montreal, n = 207). Total BPA (free and conjugated) and BPS were the predominant bisphenols detected in samples from Vhembe and Pretoria, whereas total BPS was the predominant bisphenol detected in Montreal samples. The levels of total BPA in samples from Vhembe and Pretoria ranged between < MDL-18.61 and

Simultaneous targeted and non-targeted analysis of plastic-related contaminants in e-waste impacted soil in Agbogbloshie, Ghana.

An LC-MS based analytical method was developed and validated for the simultaneous targeted analysis and suspect screening of plastic-related contaminants in e-waste impacted soils. Satisfactory recoveries (97 ± 13 %) were achieved using ultrasound-assisted extraction for 14/15 of the targeted analytes (7 bisphenols and 8 plasticizers) in a range of agricultural and non-agricultural soils. The method was applied to 53 soil samples collected in May 2015 in the region of Agbogbloshie (Ghana) at e-waste facilities (incl. Dump, trade, and burn sites), neighboring non-agricultural (incl. upstream, downstream, and community) and agricultural fields, and at two control agricultural sites away from e-waste recycling facilities. Bisphenol A (BPA) and bis(2-ethylhexyl) phthalate (DEHP) were the two dominant contaminants in e-waste soil (with concentrations up to 48.7 and 184 µg g-1, respectively), especially at the trade site, where e-waste was sorted and dismantled. The non-targeted workflow was successfully applied to identify additional plastic-related contaminants previously unreported in e-waste impacted soils, including bis(2-propylheptyl) phthalate, diisononyl phthalate, trioctyl trimellitate, 4-dodecylbenzenesulfonic acid, perfluorooctanesulfonic acid, perfluorobutanesulfonic acid, diphenyl phosphate, and triethylene glycol monobutyl ether. The agricultural soils surrounding the e-waste sites were also contaminated by plastic-related chemicals (especially DEHP), highlighting the impact of e-waste activities on the surrounding agricultural system.

Development of a method based on ATR-FTIR spectroscopy to detect maple syrup adulterated with added syrups.

BACKGROUND: Food adulteration is a global concern, whether it takes place intentionally or incidentally. In Canada, maple syrup is susceptible to being adulterated with cheaper syrups such as corn, beet, cane syrups, and many more due to its high price and economic importance. RESULTS: In this study, the use of attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy was investigated to detect maple syrups adulterated with 15 different sugar syrups at different concentration levels. The spectra were collected in the range of 4000-650 cm-1 in the absorbance unit. These spectra were used to build six libraries and three models. A method that is capable of performing a qualitative library search using a similarity search, which is based on the first derivative correlation search algorithm, was developed. This method was further evaluated and proved to be able to capture adulterated and reject non-adulterated maple syrups, belonging to the color grades golden and amber maple syrups, with an accuracy of 93.9% and 92.3%, respectively. However, for the maple syrup belonging to the dark color grade, this method demonstrated low specificity of 33.3%, and for this reason it was only able to adequately detect adulterated samples from the non-adulterated ones with an accuracy of 81.4%. CONCLUSION: This simple and rapid method has strong potential for implementation in different stages of the maple syrup supply chain for early adulteration detection, particularly for golden and amber samples. Further evaluation and improvements are required for the dark color grade. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Targeted and non-targeted screening of flame retardants in rural and urban honey.

Flame retardants (FRs) are often added to commercial products to achieve flammability resistance, but they are not chemically bonded to the materials, so, they can be easily released into the environment during the production and disposal processes. When honeybees travel to collect nectar during the pollination process, they are prone to be contaminated by chemicals in the air. Therefore, honey contamination has been proposed as an indicator of the pollution status in a particular region. To date, the occurrence of flame retardants in urban honey has yet to be explored. In this study, a direct injection method was used, coupled with LC-QTOF-MS, to analyze honey samples. This method was applied to urban (n = 100) and rural (n = 100) honey samples from the Quebec province (Canada), and the levels of flame retardants in urban and rural honey samples were not significantly different. In the targeted approach, two of the target FRs, tris(2-butoxyethyl) phosphate (TBOEP) and triphenyl phosphate (TPHP), were detected and confirmed at an average trace concentration (<1 ng mL-1). Additionally, a non-targeted screening workflow with an in-house-built library was developed and validated to screen for flame retardants in honey. Tris (2-chloropropyl) phosphate (TCIPP) was identified in honey using the non-targeted screening workflow and confirmed using a pure analytical standard, but there are other compounds detected in the non-targeted analysis that have yet to be validated. This study was the first to report FR compounds based on a direct injection method, coupled with a non-targeted screening workflow, at a trace level in a honey matrix. It also showed that a non-targeted workflow was effective to detect and identify unknown compounds present in the honey sample; hence, this provided a novel angle for the occurrence of FRs in air, with honey as a bio-indicator.

Effects of weathering on the properties and fate of secondary microplastics from a polystyrene single-use cup.

In this work, we probed the changes to some physicochemical properties of polystyrene microplastics generated from a disposable cup as a result of UV-weathering, using a range of spectroscopy, microscopy, and profilometry techniques. Thereafter, we aimed to understand how these physicochemical changes affect the microplastic transport potential and contaminant sorption ability in model freshwaters. Exposure to UV led to measured changes in microplastic hydrophobicity (20-23 % decrease), density (3% increase), carbonyl index (up to 746 % increase), and microscale roughness (24-86 % increase). The settling velocity of the microplastics increased by 53 % after weathering which suggests that UV aging can increase microplastic deposition to sediments. This impact of aging was greater than the effect of the water temperature. Weathered microplastics exhibited reduced sorption capacity (up to 52 % decrease) to a model hydrophobic contaminant (triclosan) compared to unaged ones. The adsorption of triclosan to both microplastics was slightly reversible with notable desorption hysteresis. These combined effects of weathering could potentially increase the transport potential while decreasing the contaminant transport abilities of microplastics. This work provides new insights on the sorption capacity and mobility of a secondary microplastic, advances our knowledge about their risks in aquatic environments, and the need to use environmentally relevant microplastics.

Effect of nanopesticides (azoxystrobin and bifenthrin) on the phenolic content and metabolic profiles of strawberries (Fragaria × ananassa).

BACKGROUND: Nanoencapsulation has opened promising fields of innovation for pesticides. Conventional pesticides can cause side effects on plant metabolism. To date, the effect of nanoencapsulated pesticides on plant phenolic contents has not been reported. RESULTS: In this study, a comparative evaluation of the phenolic contents and metabolic profiles of strawberries was performed for plants grown under controlled field conditions and treated with two separate active ingredients, azoxystrobin and bifenthrin, loaded into two different types of nanocarriers (Allosperse® polymeric nanoparticles and SiO2 nanoparticles). There were small but significant decreases of the total phenolic content (9%) and pelargonidin 3-glucoside content (6%) in strawberries treated with the nanopesticides. An increase of 31% to 125% was observed in the levels of gallic acid, quercetin, and kaempferol in the strawberries treated with the nanoencapsulated pesticides compared with the conventional treatments. The effects of the nanocarriers on the metabolite and phenolic profiles was identified by principal component analysis. CONCLUSION: Overall, even though the effects of nanopesticides on the phenological parameters of strawberry plants were not obvious, there were significant changes to the plants at a molecular level. In particular, nanocarriers had some subtle effects on plant health and fruit quality through variations in total and individual phenolics in the fruits. Further research will be needed to assess the impact of diverse nanopesticides on other groups of plant metabolites. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Food Thermal Labels are a Source of Dietary Exposure to Bisphenol S and Other Color Developers.

To test the hypothesis that migration from the thermal labels on plastic film packaging is a major source of exposure to bisphenols and alternative color developers in food, we analyzed 140 packaging materials from packaged fresh food purchased in North America. No bisphenol A (BPA) was detected in either the packaging samples or thermal labels. However, significant amounts of bisphenol S (BPS) and alternative color developers (up to 214 µg/cm2) were present in thermal labels; their relative occurrence varied among stores. In a controlled experiment, we wrapped fish in film with a thermal label for 5 days at 4 °C. The fish in contact with the label contained BPS (≤1140 ng/g wet weight [ww]), 4-hydroxyphenyl 4-isoprooxyphenylsulfone (D-8) (≤230 ng/g ww), bis(2-chloroethyl)ether-4,4'-dihydroxydiphenyl sulfone monomer (D-90) (≤3.41 ng/g ww), and/or Pergafast-201 (≤1.87 ng/g ww). The corresponding film samples were then tested using migration cells for 10 days; significantly higher BPS migration was observed systematically from the films with thermal labels compared to plain films. This study provides evidence, for the first time, that BPS and alternative thermal label color developers migrate from packaging materials into food. Further, BPS migration significantly exceeded the European Union Specific Migration Limit (50 ng/g ww), suggesting that further risk assessment studies are warranted.

Baseline distribution of petroleum hydrocarbon contamination in the marine environment around the coastline of Qatar.

Levels of organic contaminants (TPHs, PAHs) were simultaneously determined in both abiotic (sediments, seawater) and biotic (Pinctada radiata oysters) samples at four sites along the coastline of Qatar (Arabian Gulf) in 2017-2018. TPHs and PAHs were more frequently detected in oyster tissues than sediment and seawater samples collected from the same areas. While levels of TPHs and PAHs in seawater and sediments were lower than previous local studies and worldwide studies, PAHs levels observed in pearl oyster tissue (25.9-2240 µg/kg) were relatively higher than in previous studies in Qatar. In general, eight PAHs compounds were detected in oyster tissue, with benzo(a)pyrene displaying the highest concentration. The coast of Qatar could be affected by seasonal patterns of pollutants, where TPHs and PAHs levels increased in winter compared to summer. These results provide key information on the use of the pearl oyster as a bioindicator species and Qatar's marine environment.

Characterization of different contaminants and current knowledge for defining chemical mixtures in human milk: A review.

Hundreds of xenobiotics, with very diverse origins, have been detected in human milk, including contaminants of emerging concern, personal care products and other current-use substances reflecting lifestyle. The routes of exposure to these chemicals include dermal absorption, ingestion and inhalation. Specific families of chemicals are dominant among human milk monitoring studies (e.g., organochlorine pesticides, bisphenol A, dioxins), even though other understudied families may be equally toxicologically relevant (e.g., food-processing chemicals, current-use plasticizers and flame retardants, mycotoxins). Importantly, the lack of reliable human milk monitoring data for some individual chemicals and, especially, for complex mixtures, is a major factor hindering risk assessment. Non-targeted screening can be used as an effective tool to identify unknown contaminants of concern in human milk. This approach, in combination with novel methods to conduct risk assessments on the chemical mixtures detected in human milk, will assist in elucidating exposures that may have adverse effects on the development of breastfeeding infants.

Exploring the synergistic effects of essential oil and plant extract combinations to extend the shelf life and the sensory acceptance of meat products: multi-antioxidant systems.

To develop multi-antioxidant systems for the preservation of meat products, mixtures of essential oils or plant extracts were investigated for their antioxidant interactions. The combinatorial study revealed that the chemical diversity of both major and minor components of these ingredients contributed to the antioxidant interactions. A shift from antagonistic or additive interaction to synergistic one was achieved by modulating the ratio of mono-components of multi-antioxidant systems. Mixtures containing oregano/thyme (25/50 of IC50), thyme/clove (25/100) and thyme/cinnamon (50/25) oils as well as cranberry/rosemary (25/25), cranberry/green tea (25/25), cranberry/apple (25/25), rosemary/apple (50/25) and grapeseed/cranberry (50/50) extracts have shown synergistic antioxidant effects. Among the investigated systems, thyme/clove oils and oregano/thyme oils/grape-seed extract systems have extended, in situ, the shelf-life of chicken and ground pork products stored at 4 °C by 2 to 4 folds. The sensory acceptability of treated samples was rated to be moderately better than control. This study lays the ground for the development of efficient natural multi-antioxidant systems. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-022-05653-4.

Development of quantitative structure-retention relationship models to improve the identification of leachables in food packaging using non-targeted analysis.

Quantitative structure-retention relationship (QSRR) models can be used to predict the chromatographic retention time of chemicals and facilitate the identification of unknown compounds, notably with non-targeted analysis. In this study, QSRR models were developed from the data obtained for 178 pure chemical standards and four types of analytical columns (C18, phenylhexyl, pentafluorophenyl, cyano) in liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS). First, different data partitioning ratios and feature selection methods [random forest (RF) and support vector machine (SVM)] were tested to build models to predict chromatographic retention times based on 2D molecular descriptors. The internal and external performances of the non-linear (RF) and corresponding linear predictive models were systematically compared, and RF models resulted in better predictive capacities [p < 0.05, with an average PVE (proportion of variance explained) value of 0.89 ± 0.02] than linear models (0.79 ± 0.03). For each column, the resulting model was applied to identify leachables from actual plastic packaging samples. An in-depth investigation of the top 20 most intense molecular features revealed that all false-positives could be identified as outliers in the QSRR models (outside of the 95% prediction bands). Furthermore, analyzing a sample on multiple chromatographic columns and applying the associated QSRR models increased the capacity to filter false positives. Such an approach will contribute to a more effective identification of unknown or unexpected leachables in plastics (e.g. non-intended added substances), therefore refining our understanding of the chemical risks associated with food contact materials.

Chemical contaminants in canned food and can-packaged food: a review.

Canning, as a preservation technique, is widely used to extend the shelf life as well as to maintain the quality of perishable foods. During the canning process, most of the microorganisms are killed, reducing their impact on food quality and safety. However, the presence of a range of undesirable chemical contaminants has been reported in canned foods and in relation to the canning process. The present review provides an overview of these chemical contaminants, including metals, polymeric contaminants and biogenic amine contaminants. They have various origins, including migration from the can materials, formation during the canning process, or contamination during steps required prior to canning (e.g. the disinfection step). Some other can-packaged foods (e.g. beverages or milk powder), which are not canned foods by definition, were also discussed in this review, as they have been frequently studied simultaneously with canned foods in terms of contamination. The occurrence of these contaminants, the analytical techniques involved, and the factors influencing the presence of these contaminants in canned food and can-packaged food are summarized and discussed.

A lesion-mimic mutant of Catharanthus roseus accumulates the opioid agonist, akuammicine.

Catharanthus roseus is a medicinal plant that produces an abundance of monoterpenoid indole alkaloids (MIAs), notably including the anticancer compounds vinblastine and vincristine. While the canonical pathway leading to these drugs has been resolved, the regulatory and catalytic mechanisms controlling many lateral branches of MIA biosynthesis remain largely unknown. Here, we describe an ethyl methanesulfonate (EMS) C. roseus mutant (M2-117523) that accumulates high levels of MIAs. The mutant exhibited stunted growth, partially chlorotic leaves, with deficiencies in chlorophyll biosynthesis, and a lesion-mimic phenotype. The lesions were sporadic and spontaneous, appearing after the first true bifoliate and continuing throughout development. The lesions are also the site of high concentrations of akuammicine, a minor constituent of wild type C. roseus leaves. In addition to akuammicine, the lesions were enriched in 25 other MIAs, resulting, in part, from a higher metabolic flux through the pathway. The unique metabolic shift was associated with significant upregulation of biosynthetic and regulatory genes involved in the MIA pathway, including the transcription factors WRKY1, CrMYC2, and ORCA2, and the biosynthetic genes STR, GO, and Redox1. Following the lesion-mimic mutant (LMM) phenotype, the accumulation of akuammicine is jasmonate (JA)-inducible, suggesting a role in plant defence response. Akuammicine is medicinally significant, as a weak opioid agonist, with a preference for the κ-opioid receptor, and a potential anti-diabetic. Further study of akuammicine biosynthesis and regulation can guide plant and heterologous engineering for medicinal uses.

Application of Nontarget Analysis and High-Resolution Mass Spectrometry for the Identification of Thermal Transformation Products of Oxytetracycline in Pacific White Shrimp.

ABSTRACT: Oxytetracycline (OTC) is an antibiotic authorized for use in aquaculture; it is often detected in seafood products, especially shrimp. Previous studies investigating the fate of OTC in shrimp tissues after cooking were limited to quantification of parent compound residues and did not describe any potential transformation products formed. Hence, the main objective of this study was to apply a nontarget analysis workflow to study the fate of OTC in shrimp muscle. Furthermore, "water" and "spiked" models were evaluated for their suitability to track the transformation of OTC in incurred muscle and to determine whether the matrix plays a role in the transformation pathway. First, four different extraction methods were compared for the determination of OTC in muscle. Second, raw and cooked samples were then extracted using a suitable method (acidified water-methanol-acetonitrile, with cleanup of samples achieved using freezing) and were analyzed by high-performance liquid chromatography quadrupole time-of-flight mass spectrometry. OTC levels were reduced by 75 and 87% in muscle and water, respectively. Identification of thermal transformation products was limited to formula generation, but results showed that different compounds were identified in spiked and incurred muscle.

Occurrence of legacy and replacement plasticizers, bisphenols, and flame retardants in potable water in Montreal and South Africa.

The occurrence of thirty-nine contaminants including plasticizers, bisphenols, and flame retardants in potable water from Montreal and South Africa was analyzed to determine their presence and concentrations in different water sources. In Montreal, five bottled water (BW) brands and three drinking water treatment plants (DWTP) were included. In South Africa, water was sampled from one urban DWTP located in Pretoria, Gauteng, and one rural DWTP located in Vhembe, along with water from the same rural DWTP which had been stored in small and large plastic containers. A combination of legacy compounds, typically with proven toxic effects, and replacement compounds was investigated. Bisphenols, Dechlorane-602, Dechlorane-603, and s-dechlorane plus (s-DP) were not detected in any water samples, and a-dechlorane plus (a-DP) was only detected in one sample from Pretoria at a concentration of 1.09 ng/L. Lower brominated polybrominated diphenyl ethers (PBDE)s were detected more frequently than higher brominated PBDEs, always at low concentrations of <2 ng/L, and total PBDE levels were statistically higher in South Africa than in Montreal. Replacement flame retardants, organophosphate esters (OPEs), were detected at statistically higher concentrations in Montreal's BW (68.56 ng/L), drinking water (DW) (421.45 ng/L) and Vhembe (198.33 ng/L) than legacy PBDEs. Total OPE concentrations did not demonstrate any geographical trend; however, levels were statistically higher in Montreal's DW than Montreal's BW. Plasticizers were frequently detected in all samples, with legacy compounds DEHP, DBP, and replacement DINCH being detected in 100 % of samples with average concentrations ranging from 6.89 ng/L for DEHP in Pretoria to 175.04 ng/L for DINCH in Montreal's DW. Total plasticizer concentrations were higher in Montreal than in South Africa. The replacement plasticizers (DINCH, DINP, DIDA, and DEHA) were detected at similar frequencies and concentrations as legacy plasticizers (DEHP, DEP, DBP, MEHP). For the compounds reported in earlier studies, the concentrations detected in the present study were similar to other locations. These compounds are not currently regulated in drinking water but their frequent detection, especially OPEs and plasticizers, and the presence of replacement compounds at similar or higher levels than their legacy compounds demonstrate the importance of further investigating the prevalence and the ecological or human health effects of these compounds.

Uptake and Translocation of a Silica Nanocarrier and an Encapsulated Organic Pesticide Following Foliar Application in Tomato Plants.

Pesticide nanoencapsulation and its foliar application are promising approaches for improving the efficiency of current pesticide application practices, whose losses can reach 99%. Here, we investigated the uptake and translocation of azoxystrobin, a systemic pesticide, encapsulated within porous hollow silica nanoparticles (PHSNs) of a mean diameter of 253 ± 73 nm, following foliar application on tomato plants. The PHSNs had 67% loading efficiency for azoxystrobin and enabled its controlled release over several days. Thus, the nanoencapsulated pesticide was taken up and distributed more slowly than the nonencapsulated pesticide. A total of 8.7 ± 1.3 µg of the azoxystrobin was quantified in different plant parts, 4 days after 20 µg of nanoencapsulated pesticide application on a single leaf of each plant. In parallel, the uptake and translocation of the PHSNs (as total Si and particulate SiO2) in the plant were characterized. The total Si translocated after 4 days was 15.5 ± 1.6 µg, and the uptake rate and translocation patterns for PHSNs were different from their pesticide load. Notably, PHSNs were translocated throughout the plant, although they were much larger than known size-exclusion limits (reportedly below 50 nm) in plant tissues, which points to knowledge gaps in the translocation mechanisms of nanoparticles in plants. The translocation patterns of azoxystrobin vary significantly following foliar uptake of the nanosilica-encapsulated and nonencapsulated pesticide formulations.

Targeted screening of 11 bisphenols and 7 plasticizers in food composites from Canada and South Africa.

A sensitive method based on ultrasound-assisted liquid extraction coupled with liquid chromatography was applied to screen 18 plastic-related contaminants in 168 food composites (namely fish fillets, chicken breast, canned tuna, leafy vegetables, bread and butter) collected in Montreal (Canada), Pretoria and Vhembe (South Africa). Bisphenol A (BPA), bisphenol S (BPS) and seven plasticizers (di-n-butyl phthalate (DBP), diethyl phthalate (DEP), (2-ethylhexyl) phthalate (DEHP), di-(2-ethylhexyl) adipate (DEHA), di-isononyl phthalate (DINP), di-(isononyl)-cyclohexane-1,2-dicarboxylate (DINCH)) were detected in different foods from both countries. DBP and DEP were the most frequently detected contaminants in food collected in Montreal (75% for both) and DINP was the most frequently detected contaminant in food from South Africa (67%). DEHA concentration in packaged fish were significantly higher than the values for non-packaged fish (p < 0.01) suggesting that the packaging film can be one source of DEHA in fish.

Evaluation of different extractions for the metabolite identification of malachite green in brook trout and shrimp.

Applications of mass spectrometry-based metabolomics in food science have developed fast in the last decade. Sample preparation and data processing are critical in non-target/metabolomic workflows but there is currently no standardized protocol for the development of these methods. The impact of data processing parameters or the inclusion of a different matrix is not often taken into account during the selection of an extraction. Thus, this study aimed to investigate the impact of different extractions, e.g., QuEChERS, and data processing on the determination of malachite green metabolites in two different organisms, brook trout and shrimp. The results obtained confirm the need for a harmonized approach for the validation of non-target workflows, as depending on the comparison criteria, the matrix, the mode of ionization or data processing, a different extraction could be chosen. This study also identified for the first time des-methylated leucomalachite green as another metabolite in the two organisms.

Weathering pathways and protocols for environmentally relevant microplastics and nanoplastics: What are we missing?

To date, most studies of microplastics have been carried out with pristine particles. However, most plastics in the environment will be aged to some extent; hence, understanding the effects of weathering and accurately mimicking weathering processes are crucial. By using microplastics that lack environmental relevance, we are unable to fully assess the risks associated with microplastic pollution in the environment. Emerging studies advocate for harmonization of experimental methods, however, the subject of reliable weathering protocols for realistic assessment has not been addressed. In this work, we critically analysed the current knowledge regarding protocols used for generating environmentally relevant microplastics and leachates for effects studies. We present the expected and overlooked weathering pathways that plastics will undergo throughout their lifecycle. International standard weathering protocols developed for polymers were critically analysed for their appropriateness for use in microplastics research. We show that most studies using weathered microplastics involve sorption experiments followed by toxicity assays. The most frequently reported weathered plastic types in the literature are polystyrene>polyethylene>polypropylene>polyvinyl chloride, which does not reflect the global plastic production and plastic types detected globally. Only ~10% of published effect studies have used aged microplastics and of these, only 12 use aged nanoplastics. This highlights the need to embrace the use of environmentally relevant microplastics and to pay critical attention to the appropriateness of the weathering methods adopted moving forward. We advocate for quality reporting of weathering protocols and characterisation for harmonization and reproducibility across different research efforts.