Interactions of airborne graphene oxides with the sexual reproduction of a model plant: When production impurities matter.

The increasing use of graphene-related materials (GRMs) in everyday-life products raises concerns for their possible release into the environment and consequent impact on organisms. GRMs have widely varying effects on plants and, according to recent evidences, graphene oxide (GO) has the potential to interfere with the sexual reproduction owing to its acidic properties and production residues. Here, stigmas of the model plant Cucurbita pepo (summer squash) were subjected to simulated dry depositions of GO and GO purified from production residues (PGO). Stigmas were then hand-pollinated and GRM deposition was checked by ESEM and confocal microscopy. Analysis of stigma integrity, pH homeostasis and pollen-stigma interactions did not reveal negative effects. Fruit and seed production were not affected, but GO depositions of 22.1 ± 7.2 ng mm-2 affected the normal development of seeds, decreasing seed dimensions, seed germination and germination speed. The elemental analysis revealed that GO has significant quantities of production residues, such as strong acids and oxidants, while PGO has only traces, which justifies the differences observed in the effects caused by the two materials. Our results show that GO depositions of up to 11.1 ± 3.6 ng mm-2, which fall within the variation range of total dry particulate matter depositions reported in the literature, are safe for reproduction of C. pepo. This is the first "safety" limit ever recorded for depositions of "out-of-the-box" GO concerning the reproduction of a seed plant. If confirmed for wind-pollinated species, it might be considered for policymaking of GRMs emissions in the air.

Is airborne graphene oxide a possible hazard for the sexual reproduction of wind-pollinated plants?

Products containing graphene-related materials (GRMs) are becoming increasingly common, allowing GRM nanoparticles (NPs) to enter the environment during their life cycle. Thanks to their lightness and bidimensional geometry, GRM NPs can be easily dispersed in the air and travel very long distances. The flowers of wind-pollinated plants may be exposed to airborne GRMs, being apt to intercept pollen from the air and, inevitably, other airborne particles. Here, stigmas of four wind-pollinated plants (Corylus avellana, common hazel; Juglans regia, walnut; Quercus ilex, holm oak; Zea mays, maize) were exposed to airborne graphene oxide (GO) and GO purified from production residues (PGO) at a concentration of 3.7 ng m-3. Subsequently, the stigmas were pollinated and the adhesion of GOs and their effects on stigma integrity and pollen-stigma interaction were examined. The effect of GO NPs in presence of liquid water on the stigma of C. avellana was also investigated. GOs NPs were intercepted by all species, but their effect varied among them. GO reduced pollen adhesion in J. regia and Q. ilex, whereas pollen germination was unaffected in all four species. The presence of a film of water neither completely removed GO NPs from the stigma, nor it enhanced the toxic effect of GO acidity. PGO never affected pollen-stigma interaction, indicating that the phytotoxic substances used for the production of GO, still in traces in commercial GO, are the main cause of GO toxicity. These results reconfirm the need to verify GRMs effects also on key biological processes beside single model organisms.

Graphene environmental biodegradation: Wood degrading and saprotrophic fungi oxidize few-layer graphene.

The environmental biodegradability profile of graphene related materials (GRMs) is important to know in order to predict whether these materials will accumulate in soil or will be transformed by primary decomposers. In this study, few-layer graphene (FLG) was exposed to living and devitalized axenic cultures of two white-rot basidiomycetes (Bjerkandera adusta and Phanerochaete chrysosporium) and one soil saprotrophic ascomycete (Morchella esculenta) with or without lignin, for a period of four months. Over this time, the increase of fungal biomass and presence of H2O2 and oxidizing enzymes [laccase/peroxidase and lignin peroxidase (LiP)] in growth media was assessed by gravimetric and spectrophotometric measurements, respectively. Raman spectroscopy and transmission electron microscopy (TEM) were used to compare the structure of FLG before and after incubation. All of the test fungi decreased pH in growth media and released H2O2 and laccase/peroxidase, but only basidiomycetes released LiP. Independent of growth media composition all fungi were found to be capable to oxidize FLG to a graphene oxide-like material, including M. esculenta, which released only laccase/peroxidase, i.e. the most common enzymes among primary decomposers. These findings suggest that FLG involuntarily released into terrestrial environments would likely be oxidized by soil microflora.

Effects of Few-Layer Graphene on the Sexual Reproduction of Seed Plants: An In Vivo Study with Cucurbita pepo L.

Products containing graphene-related materials (GRMs) are becoming quite common, raising concerns for environmental safety. GRMs have varying effects on plants, but their impact on the sexual reproduction process is largely unknown. In this study, the effects of few-layer graphene (FLG) and a similarly layered phyllosilicate, muscovite mica (MICA), were tested in vivo on the reproductive structures, i.e., pollen and stigma, of Cucurbita pepo L. ssp. pepo 'greyzini' (summer squash, zucchini). Pollen was exposed to FLG or MICA, after careful physical-chemical characterization, at concentrations of 0.5 and 2 mg of nanomaterial (NM) per g of pollen for up to six hours. Following this, pollen viability was tested. Stigmas were exposed to FLG or MICA for three hours and then analyzed by environmental scanning electron microscopy to verify possible alterations to their surface. Stigmas were then hand-pollinated to verify the effects of the two NMs on pollen adhesion and in vivo pollen germination. FLG and MICA altered neither pollen viability nor the stigmatic surface. However, both NMs equivalently decreased pollen adhesion and in vivo germination compared with untreated stigmas. These effects deserve further attention as they could impact on production of fruits and seeds. Importantly, it was shown that FLG is as safe as a naturally occurring nanomaterial.

Desiccation tolerance and lichenization: a case study with the aeroterrestrial microalga Trebouxia sp. (Chlorophyta).

MAIN CONCLUSIONS: A comparative study of isolated vs. lichenized Trebouxia sp. showed that lichenization does not influence the survival capability of the alga to the photo-oxidative stress derived from prolonged desiccation. Coccoid algae in the Trebouxia genus are the most common photobionts of chlorolichens but are only sporadically found in soil or bark outside of a lichen. They all appear to be desiccation tolerant, i.e. they can survive drying to water contents of below 10%. However, little is known about their longevity in the dry state and to which extent lichenization can influence it. Here, we studied the longevity in the dry state of the lichenized alga (LT) Trebouxia sp. in the lichen Parmotrema perlatum, in comparison with axenically grown cultures (CT) isolated from the same lichen. We report on chlorophyll fluorescence emission and reactive oxygen species (ROS) production before desiccation, after 15-45 days in the dry state under different combinations of light and air humidity and after recovery for 1 or 3 days in fully hydrated conditions. Both the CT and the LT were able to withstand desiccation under high light (120 µmol photons m(-2) s(-1) for 14 h per day), but upon recovery after 45 days in the dry state the performance of the CT was better than that of the LT. By contrast, the quenching of excess light energy was more efficient in the LT, at high relative humidities especially. ROS production in the LT was influenced mostly by light exposure, whereas the CT showed an oxidative burst independent of the light conditions. Although lichenization provides benefits that are essential for the survival of the photobiont in high-light habitats, Trebouxia sp. can withstand protracted periods of photo-oxidative stress even outside of a lichen thallus.