Marine degradation and ecotoxicity of conventional, recycled and compostable plastic bags.

Plastic bags are currently a major component of marine litter, causing aesthetical nuisance, and undesirable effects on marine fauna that ingest them or are entangled. Plastic litter also rises concern on the ecotoxicological effects due to the potential toxicity of the chemical additives leached in aquatic environments. Conventional plastic bags are made of polyethylene, either from first use or recycled, but regulations restricting single-use plastics and limiting lightweight carrier bags (<50 µm thickness) have fostered the replacement of thin PE bags by compostable materials advertised as safer for the environment. In this study, we assess the degradation of commercially available plastic bags in marine conditions at two scales: aquariums (60 days) and outdoors flow-through mesocosm (120 days). Strength at break point and other tensile strength parameters were used as ecologically relevant endpoints to track mechanical degradation. Ecotoxicity has been assessed along the incubation period using the sensitive Paracentrotus lividus embryo test. Whereas PE bags did not substantially lose their mechanical properties within the 60 d aquarium exposures, compostable bags showed remarkable weight loss and tensile strength decay, some of them fragmenting in the aquarium after 3-4 weeks. Sediment pore water inoculum promoted a more rapid degradation of compostable bags, while nutrient addition pattern did not affect the degradation rate. Longer-term mesocosms exposures supported these findings, as well as pointed out the influence of the microbial processes on the degradation efficiency of compostable/bioplastic bags. Compostable materials, in contrast toPE, showed moderate toxicity on sea-urchin larvae, partially associated to degradation of these materials, but the environmental implications of these findings remain to be assessed. These methods proved to be useful to classify plastic materials, according to their degradability in marine conditions, in a remarkably shorter time than current standard tests and promote new materials safer for the marine fauna.

Effects of primary leachates of conventional and alternative plastics in Cyprinodon variegatus fish larvae: Endocrine disruption and toxicological responses.

The inclusion of hazardous substances in the formulation of plastics raises significant concerns, particularly, if those substances are released as primary leachates during plastic degradation and/or fragmentation. In this sense, the production of degradable plastics holding deleterious additives can increase the release of harmful substances into the environment. Additionally, the effects of primary leachates of "eco-friendly" materials remain unexplored. To address this, we performed exposures to primary leachates of alternative polymers, and commercial bags to verify possible responses associated with endocrine disruption and/or activation of the detoxification pathway in larvae of the marine fish model Cyprinodon variegatus. The chemical characterization evidenced a great number of additives in the formulation of the materials analyzed in this study. Those include, except for the PLA sample, relevant levels of the hazardous phthalates DEHP and DiBP. Regarding the effects on marine fish larvae, exposure to leachates from alternative polymers (10 g/L) PHB and PHBV produced remarkable mortality (100%). While the exposure to bag leachates of all tested materials (1 and 10 g/L) produced alterations in biomarkers for steroidogenic and detoxification pathways. To a lesser extent (10 g/L), three materials produced significant alterations in estrogenic biomarkers (Home-compostable bag 1, LDPE and Recycled PE bags). Although the alterations in gene expression were not directly correlated to the amount of DEHP or DiBP, we can conclude that primary leachates of "eco-friendly" bags are harmful to marine vertebrates.

UV Dosage Unveils Toxic Properties of Weathered Commercial Bioplastic Bags.

Previous studies indicated that weathered conventional plastics and bioplastics pose ecotoxicological risks. Here, the effects of artificial and natural weathering on the ecotoxicity of three compostable bags and a conventional polyethylene (PE) bag are investigated. With that aim, a 21-day artificial indoor weathering experiment featuring UV light, UV-filtered light, and darkness was run simultaneously to a 120-day outdoor littoral mesocosm exposure featuring natural light, UV-filtered light, and shaded conditions. Acute toxicity of so-weathered plastic specimens was tested in vivo using the sensitive Paracentrotus lividus sea-urchin embryo test. PE was nontoxic from the beginning and did not gain toxicity due to UV weathering. In contrast, for bioplastics, dry artificial UV weathering increased toxicity in comparison to the dark control. Weathering in outdoor mesocosm led to a rapid loss of toxic properties due to leaching in rainwater. With a higher UV dosage, a plastic-type-dependent regain of toxicity was observed, most likely driven by enhanced availability or transformation of functional additives or due to bioplastic degradation products. PE showed moderate UV absorbance, while bioplastics showed high UV absorbance. This study highlights the potential of biodegradable plastics to pose enhanced ecotoxicological risk due to weathering under environmentally relevant conditions.

A nationwide monitoring of atmospheric microplastic deposition.

Plastic production continues to increase every year, yet it is widely acknowledged that a significant portion of this material ends up in ecosystems as microplastics (MPs). Among all the environmental compartments affected by MPs, the atmosphere remains the least well-known. Here, we conducted a one-year simultaneous monitoring of atmospheric MPs deposition in ten urban areas, each with different population sizes, economic activities, and climates. The objective was to assess the role of the atmosphere in the fate of MPs by conducting a nationwide quantification of atmospheric MP deposition. To achieve this, we deployed collectors in ten different urban areas across continental Spain and the Canary Islands. We implemented a systematic sampling methodology with rigorous quality control/quality assurance, along with particle-oriented identification and quantification of anthropogenic particle deposition, which included MPs and industrially processed natural fibres. Among the sampled MPs, polyester fibres were the most abundant, followed by acrylic polymers, polypropylene, and alkyd resins. Their equivalent sizes ranged from 22 µm to 398 µm, with a median value of 71 µm. The particle size distribution of MPs showed fewer large particles than expected from a three-dimensional fractal fragmentation pattern, which was attributed to the higher mobility of small particles, especially fibres. The atmospheric deposition rate of MPs ranged from 5.6 to 78.6 MPs m-2 day-1, with the higher values observed in densely populated areas such as Barcelona and Madrid. Additionally, we detected natural polymers, mostly cellulosic fibres with evidence of industrial processing, with a deposition rate ranging from 6.4 to 58.6 particles m-2 day-1. There was a positive correlation was found between the population of the study area and the median of atmospheric MP deposition, supporting the hypothesis that urban areas act as sources of atmospheric MPs. Our study presents a systematic methodology for monitoring atmospheric MP deposition.

Occurrence and size distribution study of microplastics in household water from different cities in continental Spain and the Canary Islands.

The purpose of this study was to investigate the occurrence of microplastics (MPs) in drinking water in Spain by comparing tap water from different locations using common sampling and identification procedures. We sampled tap water from 24 points in 8 different locations from continental Spain and the Canary Islands by means of 25 µm opening size steel filters coupled to household connections. All particles were measured and spectroscopically characterized including not only MPs but also particles consisting of natural materials with evidence of industrial processing, such as dyed natural fibres, referred insofar as artificial particles (APs). The average concentration of MPs was 12.5 ± 4.9 MPs/m3 and that of anthropogenic particles 32.2 ± 12.5 APs/m3. The main synthetic polymers detected were polyamide, polyester, and polypropylene, with lower counts of other polymers including the biopolymer poly(lactic acid). Particle size and mass distributions were parameterized by means of power law distributions, which allowed performing estimations of the concentration of smaller particles provided the same scaling parameter of the power law applies. The calculated total mass concentration of the identified MPs was 45.5 ng/L. The observed size distribution of MPs allowed an estimation for the concentration of nanoplastics (< 1 µm) well below the ng/L range; higher concentrations are not consistent with scale invariant fractal fragmentation. Our findings showed that MPs in the drinking water sampled in this work do not represent a significant way of exposure to MPs and would probably pose a negligible risk for human health.

A protocol for lixiviation of micronized plastics for aquatic toxicity testing.

Plastics contain various types and amounts of additives that can leach into the water column when entering aquatic ecosystems. Some leached plastic additives are hazardous to marine biota at environmentally relevant concentrations. Disparate methodological approaches have been adopted for toxicity testing of plastic leachates, making comparison difficult. Here we propose a protocol to standardize the methodology to obtain leachates from microplastics (MPs) for aquatic toxicity testing. Literature reviewing and toxicity tests using marine model organisms and different types of MPs were conducted to define the main methodological aspects of the protocol. Acute exposure to leachates from the studied plastics caused negative effects on the early life stages of sea urchins and marine bacteria. We provide recommendations of key factors influencing lixiviation of MPs , such as particle size (<250 µm), solid-to-liquid ratio (1-10 g/L), mixing conditions (1-60 rpm), and lixiviation time (72 h). The proposed methodology was successful to determine the toxicity of leachates from different micronized plastics on marine biota. Our recommendations balance sensitivity, feasibility and environmental relevance, and their use would help ensure comparability amongst studies for a better assessment of the toxicity of plastic leachates on aquatic biota.

Filtration of biopolymer PHB particles loaded with synthetic musks does not cause significant bioaccumulation in marine mussels.

The role of the biopolymer polyhydroxybutyrate (PHB, <250 µm) as a vehicle of a synthetic musks mixture (celestolide, galaxolide, tonalide, musk xylene, musk moskene and musk ketone) to Mytilus galloprovincialis was investigated. For 30 days, virgin PHB, virgin PHB+musks (6.82 µg g-1) and weathered PHB+musks, were daily spiked into tanks containing mussels, followed by a 10-day depuration period. Water and tissues samples were collected to measure exposure concentrations and accumulation in tissues. Mussels were able to actively filter microplastics in suspension but the concentration of the musks found in tissues (celestolide, galaxolide, tonalide) were markedly lower than the spiked concentration. Estimated Trophic Transfer Factors suggest that PHB will only play a minor role on musks accumulation in marine mussels, even if our results suggest a slightly extended persistence in tissues of musks loaded to weathered PHB.

A Practical Tool for the Assessment of Polymer Biodegradability in Marine Environments Guides the Development of Truly Biodegradable Plastics.

Environmental persistence is one of the few shortcomings of plastic materials. As a consequence, alternative plastics labeled as compostable are replacing polyolefins in some commercial applications, such as food bags and trash bags. A rapid, high-throughput, and environmentally relevant method to assess the potential biodegradability in marine conditions is used to assess these materials already on the market, as well as novel bio-based polymers still in development. By fitting experimental data to a non-linear logistic model, ultimate biodegradability can be calculated without regard for incubation time. Whereas the commercial products show negligible or very low marine biodegradability, one of the novel materials exceeds the 20% biodegradation threshold relative to fully marine biodegradable PHB after 28 days. In addition, the sensitivity of the method can be enhanced and its duration reduced, at the expense of labor-demanding preconditioning of the microbial inoculum, by increasing the bacterial density in the incubation vessels. In contrast, pre-exposure of the inoculum to plastic, either in laboratory or field conditions, does not enhance the performance of the test.

Evaluating the alterations of the estrogen-responsive genes in Cyprinodon variegatus larvae for biomonitoring the impacts of estrogenic endocrine disruptors (EEDs).

Currently, endocrine disruptors (EDs) can be found in all the environmental compartments. To understand the effects of estrogenic EDs (EEDs), adults of Cyprinodon variegatus have been classically used as a marine model. However, it is during development that exposure to contaminants may generate permanent consequences. Thus, the aim of this study was to verify the effects produced by acute exposure to 17α-ethinylestradiol (EE2) in C. variegatus larvae. Quantitative PCR (qPCR) results revealed the induction of vtg and zp gene expression on exposure to 1000 ng/L EE2 and the induction of vtgc, zp2, zp3 and cyp19a2, and inhibition of vtgab, wap and cyp1a1 on exposure to 100 ng/L EE2. Lower concentrations inhibited the gene expression of vtgab and wap (50 ng/L), cyp1a1 (25 ng/L) and zp2 (12.5 ng/L). These alterations in gene expression allow us to affirm that larvae of C. variegatus are an efficient and sensitive model for biomonitoring EEDs.

Widespread alterations upon exposure to the estrogenic endocrine disruptor ethinyl estradiol in the liver proteome of the marine male fish Cyprinodon variegatus.

Quantitative proteomic changes in the liver of adult males of Sheepshead minnow (Cyprinodon variegatus) upon exposure to ethinyl estradiol (EE2) were assessed to provide an advanced understanding of the metabolic pathways affected by estrogenic endocrine disruption in marine fish, and to identify potential novel molecular biomarkers for the environmental exposure to estrogens. From a total of 3188 identified protein groups (hereafter proteins), 463 showed a statistically significant difference in their abundance between EE2 treatment and solvent control samples. The most affected biological processes upon EE2 exposure were related to ribosomal biogenesis, protein synthesis and transport of nascent proteins to endoplasmic reticulum, and nuclear mRNA catabolism. Within the group of upregulated proteins, a subset of 14 proteins, involved in egg production (Vitellogenin, Zona Pellucida), peptidase activity (Cathepsine E, peptidase S1, Serine/threonine-protein kinase PRP4 homolog, Isoaspartyl peptidase and Whey acidic protein), and nucleic acid binding (Poly [ADP-ribose] polymerase 14) were significantly upregulated with fold-change values higher than 3. In contrast, Collagen alpha-2, involved in the process of response to steroid hormones, among others, was significantly downregulated (fold change = 0.2). This pattern of alterations in the liver proteome of adult males of C. variegatus can be used to identify promising novel biomarkers for the characterization of exposure of marine fish to estrogens. The Whey acidic protein-like showed the highest upregulation in EE2-exposed individuals (21-fold over controls), suggesting the utility of abundance levels of this protein in male liver as a novel biomarker of xenoestrogen exposure.

Mesocosm trials reveal the potential toxic risk of degrading bioplastics to marine life.

If biodegradable plastics tackle the marine plastic pollution problem sufficiently remains questionable. To gain more insight in degradability, performance, and the impact of degradation on the toxicity, commercial bags made from two biodegradable plastics and one conventional plastic (PE) were exposed for 120 days in a mesocosm featuring benthic, pelagic, and littoral habitat simulations. Degradability was assessed as weight loss, and specimens were tested for toxicity using Paracentrotus lividus sea-urchin larvae after different exposure times. Both biodegradable bags showed degradation within 120 days, with the littoral simulation showing the highest and the pelagic simulation the lowest decay. Disregarding habitat, the home-compostable plastic showed higher marine degradation than the industrial-compostable material. The relevant initial toxicity of both biopolymers was lost within 7 days of exposure, pointing towards easily leachable chemical additives as its cause. Interestingly, littoral exposed specimens gained toxicity after 120 days, suggesting UV- induced modifications that increase biopolymer toxicity.

Is a compostable plastic biodegradable in the sea? A rapid standard protocol to test mineralization in marine conditions.

Due to environmental persistence, lack of a proper land-based waste management, and global circulation, marine ecosystems are especially threatened by plastics. The search for alternatives to conventional oil-based polymers gave rise to novel materials commercialized under different "green" labels based on compostability. However, current international standards are not effective in predicting actual biodegradability of plastic objects in natural scenarios, and degradation of these novel bioplastics in marine conditions is unwarranted. We present a simple and rapid standard protocol based on their biological oxygen demand, intended to support policy-makers and plastic industry in the search for truly marine-biodegradable plastics. Improvements include: development of an environmentally relevant nutrient formulation following Redfield ratio (106C:16 N:1P); use of a natural inoculum representative of marine habitats (sediment pore water); standardization of the test material by grinding to particles below 250 µm to shorten the incubation period, and selection of a truly biodegradable biopolymer (PHB), used as positive control. This protocol was successfully applied to show that commercial compostable plastics are not biodegradable in marine environments.

Acute toxicity of bioplastic leachates to Paracentrotus lividus sea urchin larvae.

In an attempt to ensure that bioplastics, progressively replacing petrochemical-derived plastics, do not release any harmful compound to the environment, the study assessed the toxic effects of three innovative bioplastic products: polyhydroxybutyrate resin (PHB), polylactic acid cups (PLA) and a polylactic acid/polyhydroxyalkanoate 3D printing filament (PLA/PHA), together with a synthetic polyvinyl chloride (PVC) toy in Paracentrotus lividus sea urchin larvae. PVC toy was the most toxic material, likely due to the added plasticizers; remarkably, even if PHB is conceived as a nontoxic polymer, it showed a slight toxicity and Gas Chromatography-Mass Spectometry analysis (GC-MS) revealed the presence of a wide range of additives. Conversely, PLA cups and PLA/PHA filament were innocuous for the larvae, a positive outcome for these renewable solutions. Proven that additives are also used in some bioplastic formulations, they should be carefully addressed to ensure that they are as safe as regarded.

The bisphenol A metabolite MBP causes proteome alterations in male Cyprinodon variegatus fish characteristic of estrogenic endocrine disruption.

The toxicological status of bisphenol A (BPA) is under strong debate. Whereas in vitro it is an agonist of the estrogen receptor with a potency ca. 105-fold lower than the natural female hormone estradiol, in vivo exposure causes only mild effects at concentration thresholds environmentally not relevant and inconsistent among species. By using a proteomic approach, shotgun liver proteome analysis, we show that 7-d exposure to 10 µg/L of the BPA metabolite, 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP), and not the same exposure to the parental molecule BPA, alters the liver proteome of male Cyprinodon variegatus fish. Different physiological and environmental conditions leading to biotransformation of BPA to MBP may partly explain the conflicting results so far reported for in vivo BPA exposures. The pattern of alteration induced by MBP is similar to that caused by estradiol, and indicative of estrogenic endocrine disruption. MBP enhanced ribosomal activity, protein synthesis and transport, with upregulation of 91% of the ribosome-related proteins, and 12 proteins whose expression is regulated by estrogen-responsive elements, including vitellogenin and zona pellucida. Whey acidic protein (WAP) was the protein most affected by MBP exposure (FC = 68). This result points at WAP as novel biomarker for xenoestrogens.

Assessment of Toxicity and Biodegradability of Poly(vinyl alcohol)-Based Materials in Marine Water.

Due to the continuous rise in conventional plastic production and the deficient management of plastic waste, industry is developing alternative plastic products made of biodegradable or biobased polymers. The challenge nowadays is to create a new product that combines the advantages of conventional plastics with environmentally friendly properties. This study focuses on the assessment of the potential impact that polyvinyl alcohol (PVA)-based polymers may have once they are released into the marine environment, in terms of biodegradation in seawater (assessed by the percentage of the Theoretical Oxygen Demand, or % ThOD, of each compound) and aquatic toxicity, according to the standard toxicity test using Paracentrotus lividus larvae. We have tested three different materials: two glycerol-containing PVA based ones, and another made from pure PVA. Biodegradation of PVA under marine conditions without an acclimated inoculum seems to be negligible, and it slightly improves when the polymer is combined with glycerol, with a 5.3 and 8.4% ThOD achieved after a period of 28 days. Toxicity of pure PVA was also negligible (<1 toxic units, TU), but slightly increases when the material included glycerol (2.2 and 2.3 TU). These results may contribute to a better assessment of the behavior of PVA-based polymers in marine environments. Given the low biodegradation rates obtained for the tested compounds, PVA polymers still require further study in order to develop materials that are truly degradable in real marine scenarios.

Towards standard methods for the classification of aquatic toxicity for biologically active household chemicals (BAHC) present in plastics, pharmaceuticals, and cosmetic products.

A standard method to test the aquatic toxicity of biologically active household chemicals (BAHC), including those with very low water solubility, is proposed. The method uses the common marine models Paracentrotus lividus embryos and Acartia clausi larvae, in order to advance towards derivation of water quality criteria for these emerging pollutants that currently lack environmental standards. Depending on the water solubility and octanol-water partition coefficient (Kow) of the substance, the protocol consists of testing the toxicity of the substances by serial dilutions of water stocks, dimethyl-sulfoxide stocks, or 100 mg/L lixiviates in seawater. When this method is applied to eleven model BAHC, the pharmaceutical fluoxetine, the antioxidant butylated hydroxytoluene, and the UV filters broadly present in cosmetics octocrylene and 4-methylbenzylidene camphor, are classified as very toxic to aquatic life, since their EC50 values are < 1 mg/L. In general, both biological models, P. lividus and A. clausi, yield the same classification of the substances tested, but variations in the classification of aquatic toxicity depending on methodological aspects are discussed. The use of A. clausi nauplii provides more protecting value to the toxicity parameters obtained by using this protocol.

Vitellogenin gene expression in marine mussels exposed to ethinylestradiol: No induction at the transcriptional level.

Vitellogenin (Vtg), a large multidomain protein precursor of egg-yolk proteins, is used as an endocrine disruption biomarker in fish, and in the last decades, its use has been extended to invertebrates like mollusks. However, it remains unclear whether invertebrate endocrine system produces Vtg in response to estrogens, like it occurs in oviparous vertebrates. In a previous study, no evidence of induction of Vtg expression at protein level was found in gonads of the marine mussel Mytilus galloprovincialis after exposure to the estrogenic chemical 17α-ethinylestradiol (EE2). In the present follow-up study, it was investigated whether there is any effect of EE2 on Vtg abundance at transcriptional level in M. galloprovincialis gonads. To this aim, RT-qPCR analysis targeting three different domains of Vtg transcript was performed on gonads of mussels that were exposed either 4 or 24 days to 100 ng/L EE2. In addition, several reference genes were analysed and a selection of these for potential use in further RT-qPCR analyses on mussel male and female gonads is provided. Results showed higher expression in females than in males for the three analysed Vtg domains, and no evidence of Vtg mRNA induction due to EE2 either in females or males. The present results, together with those obtained from previous analysis at protein level, support that Vtg is not an adequate biomarker for xenoestrogenicity in marine mussels. Additionally, nucleotide sequences of Vtg transcripts of three closely-related species from Mytilus edulis complex (M. galloprovincialis, M. edulis and M. trossulus) are provided and compared with Vtg sequences from other mollusk species to assess the level of conservation and evolutionary relationships among species.

Aquatic toxicity of chemically defined microplastics can be explained by functional additives.

A novel, systematic approach to relate plastic toxicity with chemical composition is undertaken. Using industrial methods, three petroleum-based polymers, low-density polyethylene (PE), polyvinyl chloride (PVC), and polyamide (PA), and the biopolymer polyhydroxybutyrate (PHB) were manufactured in different formularies including conventional and alternative additives, and microplastics of two sizes (<250 and <20 µm) were obtained with the aim to relate their composition with environmental impact in aquatic environments. Internationally accepted standard tests of regulatory use with marine organisms representative of microalgae (Tisochrysis lutea population growth), crustaceans (Acartia clausi larval survival), and echinoderms (Paracentrotus lividus sea-urchin embryo test) support the following conclusions. Aquatic toxicity of microplastics made from conventional oil-based polymers is due to leaching of chemical additives, and not to ingestion of microplastics. Use of alternative formulations based on natural rather than synthetic chemical additives did not consistently reduce aquatic toxicity except for the replacement of triclosan by the alternative biocide lawsone. In contrast, the biopolymer tested, PHB, seemed to impact marine plankton through different mechanisms associated to the higher abundance of plastic particles within the nanometric range found in this resin and absent in other materials.

Chemicals sorbed to environmental microplastics are toxic to early life stages of aquatic organisms.

Microplastics are ubiquitous in aquatic ecosystems, but little information is currently available on the dangers and risks to living organisms. In order to assess the ecotoxicity of environmental microplastics (MPs), samples were collected from the beaches of two islands in the Guadeloupe archipelago, Petit-Bourg (PB) located on the main island of Guadeloupe and Marie-Galante (MG) on the second island of the archipelago. These samples have a similar polymer composition with mainly polyethylene (PE) and polypropylene (PP). However, these two samples are very dissimilar with regard to their contamination profile and their toxicity. MPs from MG contain more lead, cadmium and organochlorine compounds while those from PB have higher levels of copper, zinc and hydrocarbons. The leachates of these two samples of MPs induced sublethal effects on the growth of sea urchins and on the pulsation frequency of jellyfish ephyrae but not on the development of zebrafish embryos. The toxic effects are much more marked for samples from the PB site than those from the MG site. This work demonstrates that MPs can contain high levels of potentially bioavailable toxic substances that may represent a significant ecotoxicological risk, particularly for the early life stages of aquatic animals.

Integrative assessment of ecological responses and chemical contamination of urban wastewater outfalls on soft bottom sediments of an estuarine system.

Sewage outfalls are one of the main anthropogenic impacts to coastal systems. Linking the chemical analysis in sediment, and changes in the ecological responses is one of the main tasks in ecosystem assessment for the protection of the marine environment. A three-tiered approach to evaluate the impact of the sewage of a recently built and obsolete Urban Waste Water Treatment Plants (UWWTPs) was performed. The sewage from each UWWTP differentially affected surrounding sediments. Macrobenthic community changes were influenced by the chemical composition in the sediments but also by grain size and organic matter. Abundance of the different families of the macrobenthic communities were correlated with most of the chemicals analyzed. On the contrary, toxicity bioassays were correlated with Zn and PCB concentrations but not with environmental variables. The toxicity tests linked the effect of chemical contamination on macrobenthic communities. A joint assessment of both chemical concentrations and their ecological effects is recommended.