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Review

Towards Understanding the Factors behind the Limited Integration of Multispecies Ecotoxicity Assessment in Environmental Risk Characterisation of Graphene-Family Materials—A Bibliometric Review

by
Ildikó Fekete-Kertész
1,
Krisztina László
2,* and
Mónika Molnár
1
1
Department of Applied Biotechnology and Food Science, Faculty of Chemical Technology and Biotechnology, Budapest University of Technology and Economics, Műegyetem rkp. 3, H-1111 Budapest, Hungary
2
Department of Physical Chemistry and Materials Science, Faculty of Chemical Technology and Biotechnology, Budapest University of Technology and Economics, Műegyetem rkp. 3, H-1111 Budapest, Hungary
*
Author to whom correspondence should be addressed.
C 2023, 9(4), 90; https://doi.org/10.3390/c9040090
Submission received: 2 August 2023 / Revised: 14 September 2023 / Accepted: 18 September 2023 / Published: 25 September 2023
(This article belongs to the Special Issue Carbons for Health and Environmental Protection)
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Abstract

:
Even though graphene-family materials (GFMs) hold great promise for various applications, there are still significant knowledge gaps in ecotoxicology and environmental risk assessment associated with their potential environmental impacts. Here, we provide a critical perspective on published ecotoxicity studies of GFMs based on meticulous bibliometric research. Based on the results of our review paper, in order to fill in the current critical knowledge gaps, the following issues are recommended for consideration: performing more studies on GFMs’ effects at environmentally relevant concentrations and more field and laboratory studies with marine and terrestrial organisms. It is also recommended to assess the ecotoxicity of GFMs in more environmentally relevant conditions, such as in trophic chain transfer studies and by multispecies exposure in micro- or mesocosms, as well as gaining insights into the interactive effects between GFMs and environmental pollutants. It was also revealed that despite their widespread detection in different environmental compartments the potential impacts of GFMs in complex test systems where hierarchical trophic organisation or trophic transfer studies are significantly under-represented. One of the main causes was identified as the difficulties in the physicochemical characterisation of GFMs in complex terrestrial test systems or aquatic micro- and mesocosm studies containing a sediment phase. The lack of tools for adequate characterisation of GFMs in these complex test systems may discourage researchers from conducting experiments under environmentally relevant test conditions. In the coming years, fundamental research about these complex test systems will continue to better understand the mechanism behind GFM toxicity affecting organisms in different environmental compartments and to ensure their safe and sustainable use in the future.

Graphical Abstract

1. Introduction

Protection and preservation of the natural environment have become one of the major scientific challenges that humanity is facing today. The occurrence and accumulation of carbon-based nanomaterials in the aquatic environment are undeniable; however, most researchers and innovation advisors do not perceive graphene as posing a risk to the environment based on the “just carbon” approach [1]. Due to industrial and domestic applications of synthetic nanoparticles, they are released into aquatic and terrestrial ecosystems, and their release is expected to constantly increase as their production and application rate grow steadily [2].
In the 21st century, graphene-family materials (GFMs) have been hailed as ‘miracle’ materials and a group of revolutionary two-dimensional carbon-based nanomaterials of exciting and unique characteristics [3]. Graphene-family materials are a group of two-dimensional materials that are composed of sp2-bonded carbon atoms arranged in a hexagonal lattice structure. The most well-known material in this family is graphene, which is a single layer of graphite [4,5]. Graphene has remarkable properties, such as high mechanical strength, excellent thermal and electrical conductivity, and unique optical properties, making it a promising material for various applications, ranging from electronics to energy storage and beyond [6].
Graphene can be produced using several methods, including mechanical exfoliation, chemical vapour deposition, epitaxial growth, and reduction of graphene oxide [6,7]. By today, a wide choice of mass-produced graphene in terms of size, quality, and price is available for any particular application [8]. Apart from graphene, other members of the graphene-family materials include few-layer graphene (FLG), graphene oxide (GO), reduced graphene oxide (rGO), and graphene derivatives with various functional groups. Few-layer graphene refers to a stack of a few graphene layers, typically less than 10 layers [9]. Graphene oxide, a material with a non-stoichiometric composition, is generally obtained with the wet oxidative exfoliation of graphite [10,11,12,13]. The introduction of the oxygen-containing functional groups makes it hydrophilic and easier to process. Reduced graphene oxide is obtained by the physical (most often thermal [14]) or chemical reduction of graphene oxide, which restores some of the graphene-like properties [9]. Graphene derivatives with functional groups, such as graphene with nitrogen [15,16], sulphur [17], or fluorine atoms [18] attached to the carbon lattice, are also part of the graphene-family materials [6]. Heteroatom-doped graphenic materials can be obtained either in a direct one-step method or in two steps when a post-treatment is applied. The typical characterization methods include atomic force microscopy (AFM) [19], scanning and high-resolution transmission microscopies (SEM and (HR)TEM, respectively) as well as powder X-ray diffraction (XRD) for morphology. Temperature-programmed desorption (TPD) [20], X-ray photoelectron spectroscopy (XPS) [21], or determination of the zeta potential [22] are the most widely used techniques to reveal the chemical properties. Interestingly, Raman spectroscopy simultaneously provides morphological and chemical information [23].
The graphene industry is growing due to the rise in demand for various applications of graphene globally. The global graphene market size was valued at USD 620 million in 2020 and is projected to reach USD 1479 by 2025 [24]. To reach the goals of the 2030 Agenda for Sustainable Development of the United Nations [25] on sustainable development and environmental safety, current knowledge gaps have to be identified and need to be further investigated to better understand the environmental impacts of engineered nanomaterials (ENMs), in particular, GFMs [26].
Graphene-based materials (GBMs) and their fragments can be released into the environment through various pathways during different stages, including manufacturing, use, and end-of-life. Graphene-based materials can enter the environment through intentional applications, such as in environmental remediation, agricultural practices, and consumer products, as well as unintentional releases during production, use, and disposal [27]. Although GFM-based products are mostly used in the form of polymer composites, they are likely to degrade in the aquatic environment due to abiotic and biotic degradation processes, resulting in the release of GFMs [28]. Based on the probabilistic material flow analysis of GFMs in Europe from 2004 to 2030, both in the consumption and end-of-life phases, it is estimated that over 50% will be incinerated and oxidised in waste plants, while 16% will be sent to landfills, and 12% will be exported from Europe. Additionally, approximately 1.4% of annual GBM production is predicted to end up in the environment. Projections also indicate that the expected release concentrations in 2030 will be 1.4 ng/L in surface water and 20 μg/kg in soil treated with sludge [29].
So far, reviews on GFMs in the environment have focused on their source, fate and occurrence, environmental concentrations [27], properties [9] and characterisation by different analytical methods [30], their effects on organisms [2,4,31], and the applicability of existing standard ecotoxicity methods for their environmental risk assessment [3,32,33] as well as on their environmental applications and potentials in wastewater treatment [34,35].
The existing reviews have reported on environmental concentrations of GFMs and their ecotoxicological effects on aquatic organisms. Nevertheless, a comprehensive assessment of contemporary research trends about the suitability of the employed test systems in reflecting ecological complexity and, consequently, environmental relevance has not been conducted. As Table S1 illustrates, over the past decade, review articles published on similar topics have integrated and emphasized only the results of studies employing single-species tests. The assessment of multi-species test systems is virtually not demonstrated in these publications. Numerous studies have demonstrated that the transformation of GFMs can increase their toxicity through various mechanisms [36,37].
Thus, the aim of this paper was (i) to overview and understand current research trends in GFMs’ ecotoxicity assessment connected to their environmental risk characterisation and (ii) to further discuss the limitations and knowledge gaps about their environmental effects and the role of aquatic micro- and mesocosms in the life-cycle oriented environmental risk assessment of GFMs, (iii) as well as to identify promising areas for future research. The main novelty of the research is the focus on multi-species ecotoxicity tests, which is particularly important in terms of reliable impact assessment and environmental relevance. There is no example in the literature of such a complex discussion of micro- and mesocosm approaches in characterizing the ecotoxicity of GFMs.

2. Bibliometric Analysis

As a first step, a bibliometric analysis was carried out on 1 September 2023, using the Thompson Reuters database ISI (The Institute for Scientific Information) Web of Science in order to overview the temporal distribution of scientific research in connection with GFMs and related toxicity from 1990 to 2023. To this purpose, the combination of the keywords ‘graphene’ and ‘toxic’ was used in any title, abstract, or text words and a total number of 5729 candidate publications were identified.
As the prime objective of this review was to investigate current research trends in connection with the environmental and ecotoxic effects of GFMs, separate searches were conducted using the following keywords in combination with ‘graphene’—along with the ‘AND’ operation—‘communities’, ‘soil’, ‘water OR aquatic’, ‘wastewater’, ‘microcosm’, ‘mesocosm’.
As a second step, a deeper bibliometric analysis and a systematic and meticulous search of online databases including Web of Science (https://apps.webofknowledge.com/ (accessed on 14 September 2023)), Science Direct (https://www.sciencedirect.com/ (accessed on 14 September 2023)), PubMed (https://pubmed.ncbi.nlm.nih.gov/ (accessed on 14 September 2023)), Scopus (https://www.scopus.com/ (accessed on 14 September 2023)), complemented with Google Schoolar (https://scholar.google.com/ (accessed on 14 September 2023)) were conducted to retrieve relevant papers on the ecotoxicity of any forms of GFMs as recommended by Qualhato et al. [38]. The abstracts of all candidate articles were read until 1 September 2023. In addition, to have an extensive overview of the appropriate publications for the analyses, we also reviewed the reference section of each paper. Reviews were excluded after preliminary analysis. The abstracts were screened with the following exclusion criteria: articles that were not written in English, protocols, technical reports, and papers that did not fit the study aim. From all candidate papers, finally, 215 papers were retained and subjected to further analysis.
The applied test organisms in all relevant ecotoxicity studies were categorised as protozoa, algae, cnidaria, rotifera, planaria, nematoda, annelida, mollusca, crustacea, amphibia, fish and plants, while bacterial species except Aliivibrio fischeri were not included in the results reported here. Considering that an article could report the effects of GFMs on more than one test organism, the term ‘study’ was defined as a series of observations on a particular test organism, e.g., if one article reported only on a particular algal species it was considered to be one study, but if it reported an algal test organism, a crustacean, and a fish, it was considered to be three. This way, the number of studies presented in the results represents the number of interactions of the particular tested GFMs with all the applied test organisms in the particular paper, not the total number of publications.

3. Results and Discussion

3.1. Temporal Distribution of Studies

Results from our initial search showed that the number of scientific publications mentioning ‘graphene’ and ‘toxic’ has grown steadily since 2010 (Figure 1b). In 2022, there were 835 publications, roughly 2 times the amount from 5 years ago and roughly 20 times the amount from 10 years ago (Figure 1b). In the case of the strictly ecotoxicological effect-related papers, a phase of exploratory latency can be found between 2012 and 2014, followed by a phase of initial development of exploratory interest between 2015 and 2017 (Figure 1a). The trend of ecotoxicity-themed papers on GFMs’ effects is also worth highlighting, as their number seems to be stagnant from 2018 to date (typically 25–31 papers/year). In contrast to the prior five-year period marked by stagnation, the identification of 34 articles during the first nine months of 2023 signifies a modest uptick in the realm of GFMs’ ecotoxicity-themed studies for the entire calendar year of 2023 (Figure 1a).
From a biological or environmental impact assessment point of view, research is mainly focused on the investigation of GFMs on different microbial communities in the aquatic and soil ecosystems. Taking into consideration that the use of tests with increased environmental relevance in nano-ecotoxicology has been recommended to overcome the limitations of standardised protocols [32,39], terrestrial or aquatic microcosm and mesocosm studies are still under-represented in the risk analysis of engineered nanomaterials, specifically in the case of carbon-based nanomaterials, such as the representatives of the graphene family. The number of results for the search terms ‘graphene’ and ‘microcosm’ was found to be nine and even less (only two) in the case of ‘graphene’ and ‘mesocosm’ (Table 1).

3.2. Target Organism Groups Reported in Ecotoxicity Studies with GFMs

Test organisms applied in 215 papers on the ecotoxicological effects of GFMs were categorised into 12 distinctive groups of organisms. Literature analysis revealed that the three groups of organisms that are dominating ecotoxicological research on GFMs are fish (42%), algae (20%) and arthropoda (19%) predominantly applying small, planktonic crustaceans. Aquatic and terrestrial plant species represent 9% of all applied test organisms, while the remaining eight groups of organisms account for the remaining 10% (Figure 2).
To date, ecotoxicity studies with GFMs have been carried out predominantly using freshwater species (81%), while marine or terrestrial organisms have been used in 10% of all studies individually (Figure 3c). The majority (43%) of studies using vertebrate species as the target organism applied fish and, within the group of fish, test organism Danio rerio was the most investigated species as 83% of all studies with aquatic vertebrates were carried out with zebrafish (Figure 3a). Amongst invertebrate species, the toxic effects of GFMs were investigated with Daphnia magna in 33% of cases. Besides the planktonic crustacean, D. magna, the brine shrimp, Artemia salina, the mussel, Mytilus galloprovincialis, the earthworm, Eisenia fetida, and the house cricket, Acheta domesticus were applied in several cases (Figure 3b). Species used in only one or two cases accounted for 48% of all invertebrate studies. Focusing on the group of algae, five species were applied predominantly: Scenedesmus obliquus, Chlorella pyrenoidosa, Raphidocelis subcapitata, Chlorella vulgaris, Microcystis aeruginosa and Chlamydomonas reinhardtii. An amount of 80% of all algal species belonged to green algae, 14% belonged to cyanobacteria and 6% belonged to diatoms.

3.3. Ecotoxicological Effects of GFMs in Multispecies Test Systems

The scientific literature on the ecotoxicological effects of GFMs in multispecies test systems can be divided into two main domains: (i) studies conducted with bacterial communities and (ii) studies modelling complex ecosystems of test organisms with the hierarchical trophic organisation. These complex test systems may include bacterial species but principally do not rely on them exclusively.
This review was written with the ultimate aim to overview the state-of-the-art knowledge and latest findings on GFMs’ toxicity in complex ecosystem studies, namely micro- and mesocosm studies or any ecotoxicity studies modelling the trophic transfer of GFMs. Although the authors did not intend to give a detailed overview of the current scientific literature on the effects of GFMs on microbial communities, a brief summary was provided in order to give a more contrasting picture of the underrepresentation of complex ecosystem studies within the literature of multispecies test systems. As the current knowledge on the interactions of graphene-based materials with microbial communities has been summarised in the latest review of Braylé et al. [40], a brief summary of their findings and bibliometric analysis is given in Section 3.3.1.

3.3.1. Ecotoxicological Effects of GFMs on Bacterial Communities

The growing number of studies about the effects of GFMs on microbial communities in different environmental compartments indicates the emergence of this topic. Braylé et al. [40] also highlighted that the scientific literature on the interactions between GFMs and microbial communities is restricted to laboratory experiments mimicking environmental bioprocesses under controlled conditions. The absence of studies carried out in full-scale bioreactors or in natura systems was attributed to the lack of reliable GFMs quantification methods. It was also revealed that approximately half of the studies reported on GFMs effects in bioprocess test systems. The efficiency of wastewater treatment relies primarily on the functions of a diverse microbial community; however, WWTPs are ultimate repositories for GFMs [41]. Therefore, any potential toxic effects of GFMs need to be investigated essentially on wastewater microbial communities. Results confirmed the potential toxicity of high concentrations of GO and it was also revealed that the composition and dynamics of the microbial communities in the activated sludge changed under GFM exposure [42]. Furthermore, the impairment in microbial metabolic activity or shift in microbial diversity in WWTPs was reported by several articles [41,42,43,44].
Considering the different environmental compartments, studies on soil microbial communities were highly represented (27%). The effects of GFMs have been studied on bacterial communities from different aspects in recent years. Soil is a critical sink to GFMs after entering the environment as GFMs are able to migrate along the soil profile under the action of surface runoff and precipitation, which highly increases the possibility of their transformation in the terrestrial environment [45]. The physiology and structure of the microbial community are used as important indexes to characterise the adverse effects of toxic substances, as well as the influence of GFMs on soil properties [46]. Several authors investigated the effect of graphene on the bacterial community of soil ecosystems [47]; however, there is still an ongoing debate over whether and how graphene affects soil microbial community and enzyme activity [48]. It was reported that graphene may change the metabolism of soil microorganisms to a certain extent by adversely affecting the cell membrane integrity and by inhibiting crucial enzyme activities such as dehydrogenase, phosphatase, urease, and hydrogen peroxidase in a concentration-dependent manner [49]. However, other studies reported that the soil bacterial community was unaffected by graphene [50]. Positive effects of graphene on bacterial diversity indexes in Cd-contaminated Haplic Cambisols in Northeast China were reported as the species and abundance of bacteria varied with GO concentration, and GO significantly increased bacterial growth at 25 and 250 mg/L [51].
Microbial communities from surface water bodies (river, lake, estuary, and aquarium) were used for investigating the potential adverse effects of GFMs in small-scale batch incubation systems; however, the effects of GFMs on water bodies were slightly understudied [40].
In summary, most studies indicated the negative impacts of GFMs on the growth of bacterial communities, generally due to oxidative stress. In the presence of GFMs, several activities were found to be influenced, such as biochemical cycles and bioprocess yields due to the disruption of community structure. Results are heterogeneous, as environmental factors have a great influence on the fate and toxicity of GFMs. This way, studies carried out with different community compositions, in different environmental compartments, and also under different conditions are likely to produce contradictory results [40].

3.3.2. Ecotoxicological Effects of GFMs in Test Systems with Hierarchical Trophic Organisation

As standardized single-species-based assays fail to represent toxicological pathways implying interactions between organisms, the use of micro- and mesocosm test systems is essential in the investigation of the environmentally realistic effects of GFMs. In the current literature, the effects of GFMs in complex test systems of higher ecological relevance based on the approach of hierarchical trophic organisation of the applied organisms are understudied. After a thorough bibliometric survey, only nine articles were found that reported on the effects of GFMs in micro- or mesocosm test systems, or at least applied a trophic-transfer-mimicking exposure pathway. Four articles were considered to apply a real microcosm approach [52,53,54,55]. However, in one instance, authors indicated that a mesocosm experiment was carried out, but the test could rather be considered a microcosm experiment as the assembled test systems contained 1500 g of soil per pot and were incubated under laboratory conditions [55] and, e.g., were not assembled as an isolated part of the natural ecosystem or placed out to the natural environment. Four additional articles were discussed that applied any form of trophic chain exposure approach [33,56,57,58]. Freshwater ecosystems were mostly represented, while only one marine and one soil microcosm study was carried out. The presence of natural or artificial sediment phases was also scarce in these studies. Table 2 summarises the most important methodological parameters of scientific papers that reported on the effects of GFMs in trophic transfer studies or micro- or mesocosm experiments.
The number of studies investigating a particular type of GFM in test systems with the hierarchical trophic organisation was quite imbalanced toward GO in the current literature, as graphene multilayer nanoflakes, graphene, and rGO were investigated in one article, FLG was applied in two papers, while GO was tested in five scientific articles. The tested concentrations of GFMs are heterogenous in the different environmental compartments (Figure 4): the lowest concentration tested in freshwater or marine water phase was 1 ng/L, which is comparable to the predicted environmental concentration reported by Hong et al. [29] in surface waters. The lowest tested concentration in freshwater or marine sediment was 50 ng/L, while the lowest concentration tested in sediments was five orders of magnitude lower than the lowest concentration tested in soil. The concentration of GFMs in complex soil systems (10,000 mg/kg) was five orders of magnitude higher than the predicted environmental concentration reported in soil [29].

3.3.3. Micro- and Mesocosm Approaches for GFMs Ecotoxicity Characterisation

Evariste et al. [52] were the first who investigated the effect of GO in recirculated, large-volume microcosm test systems in environmentally relevant concentrations (0.05 and 0.1 mg/L) combining a water and a sediment compartment. As an uncontaminated test medium, commercial natural spring water was used. A reconstituted trophic chain was applied in order to model the interactions between pelagic and benthic microbial communities with aquatic organisms from higher trophic levels (decomposers, primary and secondary consumers). Redox potential, pH, dissolved oxygen, dissolved organic carbon, and nitrogen products (NO3+, NO2−, NH4+) were monitored in the test system. Test species were gradually introduced to the test system: 3 weeks after the development of the primary producers (bacterial community and the freshwater diatom, Nitzschia palea), primary and secondary consumers (Chironomus riparius midge, Pleurodeles waltii amphibian) were introduced to the test systems. Later, Xenopus laevis tadpoles were also added to the assembled microcosms. At the end of the experiment, the bacterial community was characterised by DNA isolation. In the case of C. riparius, mortality, growth, and teratogenicity were assessed, while for Pleurodeles larvae, mortality, growth, and genotoxic potential were determined. It was found that bacterial communities were affected by GO exposure; in addition, bacterial communities from the sediment were shown to be more impacted compared to those from the water compartment. Evidence was found for the genotoxic effect of GO in the Pleurodeles individuals. However, no toxicity was observed for chironomids, indirect effects were highlighted, resulting in changes in the decomposition of organic matter in the system.
In another study by Evariste et al. [53] the effect of GO and rGO was assessed toward a biofilm composed of the diatom N. palea associated with a bacterial consortium. GO and rGO were applied at 0.1, 1, and 10 mg/L concentrations in the short- (48 h) and in the long-term (144 h). Significant inhibition of bacterial growth by GO was reported from a 1 mg/L concentration level, as well as influence on the taxonomic composition of the diatom-associated bacterial consortium. Different effects of rGO were observed, as rGO exerted a weaker toxicity toward the bacterial consortium, whereas it influenced more strongly diatom physiology. Studying the interactions between the bacterial consortium and the diatom allowed authors to conclude, that diatoms benefited from diatom–bacteria interactions resulting in the potential to maintain or recover their carbon-related metabolic activities when exposed to GBMs.
The effect of multilayer graphene nanosheets (8–12 nm) was investigated in a microcosm experiment using the marine benthic worm, Hediste diversicolor [54]. The experiment was categorised as a microcosm experiment since natural sea sediment and seawater was applied to the experiments containing a natural phytoplankton community; however, the effect of graphene-nanosheets on the phytoplankton community was not investigated or discussed. Limited toxic effect of graphene was found. However, a significant elevation of catalase activity indicated the activation of defence mechanisms at the early stage of exposure, while cellular damage biomarkers (SOD, GST, GSH, MDA, and CBO) remained unaffected. Based on the acetylcholinesterase (AChE) activity neurotoxic effect was not expressed. Behavioural changes were reported as in the graphene-containing test systems, individuals were buried deeper in the sediment indicating an escape reaction and avoidance behaviour [54].
In one instance, the impact of graphene and graphene oxide was investigated in soil on the abundance and diversity of soil nematodes (29 genera) after growing a tall fescue plant (Festuca arundinacea) for 130 d using a laboratory pot experiment; however, data were not available on the effects concerning the soil microbial community [55]. Authors considered the test systems as mesocosms containing 1500 g topsoil and 50 g compost with 1 m/m% GFM contamination. Findings highlighted that the addition of GFMs had a negative influence on the composition and diversity of the nematode community, simplifying the community structure. Graphene or graphene oxide had no significant effects on the plant shoot biomass; however, both of them promoted the root growth of tall fescue.
An in situ macrocosm experiment was carried out in the eutrophic Lake Xingyun, southwestern China, evaluating the impacts of graphene on the photocatalysis of phytoplankton under environmental conditions. Significant changes in the community structure were found as Microcystis were significantly reduced, while the abundances of Anabaena and Aphanizomenon species greatly increased after graphene photocatalysis treatment. The abundances of Chlorophyta, Euglenophyta, Pyrrophyta, and Cryptophyta species significantly increased, whereas in the case of eutrophic diatom species decreased abundances were observed in the treated area [59].

3.3.4. Trophic Transfer Studies for GFMs Ecotoxicity Characterisation

The effect of 14C-labeled few-layer graphene (FLG) was determined modelling trophic transfer in the aquatic food chain by Dong et al. [56]. Direct uptake from FLG suspension was determined in the case of Escherichia coli, Tetrahymena thermophila, Daphnia magna, and Danio rerio. In trophic transfer experiments the transfer of FLG from bacteria to protozoa, from protozoa to Daphnia and from Daphnia to zebrafish was quantified along with the determination of biomagnification factors. Different concentration of FLG in the range of 1–1000 µg/L was tested depending on the experimental procedure of the different organisms. It was found that the test organisms had a high potential of accumulating graphene via direct uptake from the culture medium. It was also revealed that in the case of the food chain from E. coli to T. thermophila, there is a high potential for trophic transfer of FLG, while for the food chain from T. thermophila to D. magna and from D. magna to D. rerio, the likeliness is much lower. The main finding of the study was that in the case of T. thermophila, D. magna, and D. rerio, the burden measured for dietary uptake was higher than that via waterborne exposure in a similar nominal concentration, indicating that trophic transfer is a nonnegligible route for the bioaccumulation of graphene in organisms.
Su et al. [57] investigated the effect of algal food on the uptake and distribution of 14C-labeled few-layer graphene (~158 μg/L) in the freshwater snail, Cipangopaludina cathayensis. It was found, that in the presence of algae cells the accumulation of the few-layer graphene was significantly enhanced with a bioaccumulation factor of 2.7 (48 h exposure). The snail retained more than 90% of the accumulated few-layer graphene in the intestine; in addition, the accumulated graphene was able to pass through the intestinal wall and enter the intestinal epithelial cells. However, without the presence of algae cells, 1.3% of the few-layer graphene was transferred to hepatocytes; this phenomenon was not observed in the absence of the algae cells. The main finding of their studies is that algae cells may act as carriers enhancing the bioavailability of the few-layer graphene to the snails.
Malina et al. [58] studied the interaction of three differently oxidised GO with planktonic and benthic crustaceans. As the importance of the applied feeding strategy was observed, a pre-treatment with algae was introduced prior to the ecotoxicity tests with the aim to mimic environmentally more realistic conditions. As a result of the algal pretreatment of GO, a complete mitigation of acute toxicity of GOs to all organisms was observed. The eradication of oxidative stress caused by GOs was also discovered in the algae-pretreatment test systems, indicating that the pre-exposition of algal food is a crucial factor in GO’s overall environmental fate, hence the toxicity.
The effect of GO was tested on the green algae, Raphidocelis subcapitata at 1, 2, 4, 8, 16 and 32 mg/L exposure concentration for 96 h, applying two different algae media, the modified Keating algae culture medium (MA-MS) and the algae medium recommended by the OECD 201 test guideline [33]. Differences in the aggregation of GO were experienced in the different algae growth media. Hetero-aggregates were more prominent in the OECD medium than in the MA-MS medium. After 96 h, the growth rate of R. subcapitata was determined. In the OECD medium, IC50 (Inhibition Concentration) was determined to be 4.96 mg/L GO while in the MA-MS medium, it was 7.1 mg/L GO. In the second phase of experiments, Paratya australiensis shrimps were exposed to 2 or 8 mg/L GO for 14 days and no sign of stress, food avoidance or accumulation of GO in the gut due to the consumption of heteroaggregates was observed [33].
Although Lourerio et al. [60] investigated the effect of pegylated graphene oxide on the representatives of a trophic chain, Raphidocelis subcapitata, D. magna and D. rerio, real trophic transfer interactions were not assessed by this study. Considering the bioaccumulation of different representatives of the graphene family in a broader context, we found that the extent of bioaccumulation by different organisms and the effect of GFMs on one of the primary food sources of planktonic animals in the aquatic ecosystems, e.g., the entrapment of algal cells by GFMs [61], was reported by several authors [62,63,64,65,66,67,68].
All these results of ecotoxicity studies in test systems with hierarchical trophic organisation provided heterogeneous results on GFMs ecotoxicity, depending on the test species, environmental compartments and exposure conditions. The effects varied within a wide range, e.g., from complete mitigation of GO toxicity by algal pre-treatment [58] to enhanced bioaccumulation by algae acting as a carrier [57]. The authors addressed the challenges of characterising the tested GFM in complex environmental compartments which might become impossible under particular circumstances [40,54].

4. Current Status, Knowledge Gaps and Future Needs

The environmental risk assessment (ERA) of engineered nanomaterials has been mainly focused on the investigation of pristine forms [69]. Environmental research on aged nanomaterials is still very limited, especially in the case of ERA studies carried out in test systems of higher environmental relevance, such as microcosms and mesocosms [39]. Although, the use of micro- and mesocosms for studying the environmental fate and effects of engineered nanomaterials (ENMs), particularly in aquatic ecosystems is increasing [70], evaluating current scientific literature of GFMs, the lack of ecotoxicological studies of higher environmental relevance is striking even at first glance. However, these complex test systems would allow for the long-term characterisation of ecosystem responses to GFMs contamination and also could offer advantages over standard operating procedures as they allow for realistic contamination scenarios that incorporate natural complexity and synergies between contaminants and natural components.
Freixa et al. [71] published a comprehensive review article about the ecotoxicological effects of carbon-based nanomaterials in aquatic organisms based on approximately 100 articles from the past decade highlighting the following research needs: (1) using sublethal endpoints instead of the conventionally applied lethality, (2) investigation of the long term chronic effects instead of acute toxicity tests, (3) testing of environmentally relevant concentrations, (4) conduction of multispecies experiments assessing the exposure via the trophic chain. Although, there was a significant improvement in the first three research needs, the assessment of GFMs toxicity in the trophic chain is still in its infancy.
Various mesocosm designs consistently showed that ENMs entering the aquatic environment tend to be predominantly removed from the water column and accumulate in sediments in general [72]. Statistical analysis indicates that this accumulation is likely to occur on the long term, regardless of ENM physicochemical properties [39]. Consequently, this general scenario of potential risk is also relevant in the case of GFMs, especially for benthic species exposed to less reactive GFM nanoparticle species such as homo/hetero-aggregated and sulfidised forms but also with higher accumulation potential. On the other hand, the risk of planktonic species is associated with lower concentrations of potentially more reactive GFMs [73].
Future studies of hierarchical trophic organisation in complex test systems could address current knowledge gaps, by identifying the most environmentally relevant GFMs and determining realistic dosing strategies. However, some issues may discourage the scientific community from carrying out micro- and mesocosm studies with GFMs. Micro- and mesocosm studies could generate large and heterogeneous datasets that are difficult to reproduce.
The physicochemical characterization of GFMs in complex environmental compartments presents several challenges, including aggregation and sedimentation, surface modifications, matrix interferences, sample preparation, and the lack of standardised methods [74]. This issue has been raised related to microcosm studies, as no relevant quantification analysis could be performed [54]. Improving the understanding of GFM transformation during exposure is crucial, as during different experimental conditions (e.g., the media used for ecotoxicity studies) [75], the physical exclusion of algae from the water column by GO has proved to have implications on the food chain [33].
These difficulties of GFMs characterisation may discourage authors from using complex test systems. Researchers are hesitant to conduct tests on GFMs in complex systems due to challenges in accurately characterising their physicochemical properties. This is particularly true in systems involving soil phases or sediment phases in water [54]. This issue also has been previously observed in studies involving microbial communities [40].
Trophic transfer studies can be considered a promising approach in between single-species standard ecotoxicity test methods and complex microcosm test systems. Applying the trophic transfer approach, it has been proven that GO is not a hazardous material in complex aquatic environments because its acute toxicity can be successfully mitigated through interaction with algae even at very high concentrations (25 mg/L) [58]. Differences in the accumulation potential of GFMs were found in the case of crustacea and fish: D. magna had a much greater capability of accumulating graphene at similar concentration and exposure duration than D. rerio, which might be attributed to the difference in body size, organs complexity and feeding behaviours [56]. Despite the fact, that bioaccumulation or biomagnification studies seem to offer a better understanding of GFMs’ effects, they are typically performed with algae, small planktonic crustaceans or fish, therefore bioaccumulation studies on other groups of organisms (e.g., echinodermata, cnidaria and porifera) would be essential to better determine the relevance of studies on the frequently applied test organisms.
The fact, that marine and terrestrial ecotoxicity studies are underrepresented in GFMs toxicity studies has been commented on by several authors [73,76,77], as the lack of information on the toxic effect of GFMs in the marine or terrestrial environment is considered a main knowledge gap. Moreover, we know little about the transgenerational effects of GFMs [78,79]. Concerns have been also addressed about the uncertainty of the reliable life cycle assessment of GFMs that can be derived from current knowledge of their environmental impacts [80].
A total of 43 studies investigating the interactive effects of GFMs and other environmental contaminants have been identified to date, although five additional papers investigated the effect of humic acid on GFMs [81,82,83,84,85], and one study investigated the effect of alginate on graphene [57], these were not considered as co-contaminant studies. However, there is a growing number of studies on the combined effects of GFMs with other environmental contaminants, further investigation is needed to be able to draw reasoned conclusions on the synergistic or mitigation effects. Gaining insights into the interactions between GFMs and environmental pollutants is of outmost importance as GFMs exhibit high absorbent capabilities for removing heavy metals and organic pollutants from water matrices depending on particular colloidal properties [86,87,88,89,90].
With the growing development of novel magnetic nanocomposites and their expanding applications in modern life, it is imperative to heighten awareness of the potential environmental risks associated with their utilization. Consequently, the development of these nanomaterials should consistently incorporate an ecotoxicological assessment [91]. Latest findings indicated that magnetic graphenic nanocomposites exhibited noticeably lower cytotoxicity and ecotoxicity when compared to their corresponding control groups. This suggests the potential for developing environmentally safe nanocomposites that can be easily removed from the environment using magnetic fields and pose no acute toxicity to organisms like Artemia salina. [92]; however, given the contradictory findings in regard to their ecotoxic effects on D. magna [92,93,94,95], further research is needed to establish a clearer understanding of the impact of magnetic graphenic nanocomposites on the environment.

5. Conclusions and Future Perspectives

The relevant ecotoxicity characterisation connected to the environmental risk assessment of GFMs faces several critical knowledge gaps that need to be addressed to ensure their safe and sustainable use.
Standardized physicochemical methods for characterising and quantifying GFMs in complex environmental compartments and robust ecotoxicological data for a better understanding of their long-term fate and behaviour in environmentally relevant systems are urgently needed. In the reported ecotoxicity studies performed for the effect assessment of GFMs, less attention is paid to their physicochemical transformations during the testing period. The physicochemical characterisation of GFMs should be a critical element of all ecotoxicological investigations, at not only the beginning and end of the studies but also during testing, since the biological and biochemical processes taking place in living systems can generate changes in the structure and surface composition of GFMs that influence their manifested effects.
The identified knowledge gaps in the environmental risk assessment of GFMs have significant implications for the regulatory and scientific communities. The limited toxicological data on GFMs in complex test systems with greater environmental relevance hinder the accurate assessment of their potential ecological impacts. Additionally, the lack of understanding of the long-term environmental fate and behaviour of GFMs raises concerns about their potential accumulation and persistence in environmental systems, which may have far-reaching ecological consequences. The results of this study may contribute to the implementation of multi-species test systems with hierarchical trophic organisation into the environmentally realistic impact assessment of GFMs, as no such study has yet been published, which would highlight the lack of application of these systems in the current literature.
Based on the results of this review paper, the following issues are recommended to consider:
  • Performing more studies on GFMs effects at environmentally relevant concentrations;
  • Perform more field and laboratory studies with marine and terrestrial organisms;
  • Assess the ecotoxicity of GFMs in more environmentally relevant conditions, such as trophic transfer studies and multispecies exposures in micro- or mesocosms;
  • Gaining insights into the interactive effects between GFMs and environmental pollutants;
  • Investigate the stability of GFMs in aquatic environments as a function of concentration. Despite the widespread use of GFMs there is limited knowledge about their actual environmental concentrations. Therefore, it is imperative to develop appropriate methods and detection techniques to accurately determine the concentrations of GFMs in the environment;
  • The physicochemical characterisation of GFMs should be a critical element of all ecotoxicological investigations throughout the entire test period;
  • Encourage the publication of negative results.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/c9040090/s1. Table S1: Review articles published in the past 10 years within the scope of ecotoxicology or environmental application of GFMs. Refs. [96,97,98,99,100] are cited in Supplementary Materials file.

Author Contributions

Conceptualization, I.F.-K., K.L. and M.M.; methodology, I.F.-K. and M.M.; formal analysis, I.F.-K. and M.M.; investigation, I.F.-K. and M.M.; resources, I.F.-K. and M.M.; data curation, I.F.-K. and M.M.; writing—original draft preparation, I.F.-K. and M.M.; writing—review and editing, I.F.-K. and M.M.; visualization, I.F.-K.; supervision, M.M.; project administration, K.L. and M.M.; funding acquisition, K.L. and M.M. All authors have read and agreed to the published version of the manuscript.

Funding

The financial support for the research is greatly acknowledged to the Hungarian National Research, Development and Innovation Office Fund in the frame of the OTKA_K_128410, OTKA_K_143571 Research and TKP2021-EGA-02 Projects.

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author.

Acknowledgments

The authors are grateful to Emese Vaszita for her contribution to the language editing of the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Absolute and cumulative number of articles on the ecotoxicological effects of graphene-family materials (GFMs) (a) and number of results per year on entries ‘graphene’ AND ‘toxic’ (b). The search was conducted until 1 September 2023.
Figure 1. Absolute and cumulative number of articles on the ecotoxicological effects of graphene-family materials (GFMs) (a) and number of results per year on entries ‘graphene’ AND ‘toxic’ (b). The search was conducted until 1 September 2023.
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Figure 2. Percentage of studies investigating the toxic effects of GFMs categorized by the groups of the applied organisms. Literature search conducted between 1990 and 1 September 2023.
Figure 2. Percentage of studies investigating the toxic effects of GFMs categorized by the groups of the applied organisms. Literature search conducted between 1990 and 1 September 2023.
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Figure 3. Number of articles (%) by model systems used in ecotoxicological studies with graphene-family materials (GFMs). Number of studies per vertebrate species (a), invertebrate species (b), habitat (c), and algal species (d). Literature search conducted between 1990 and 1 September 2023.
Figure 3. Number of articles (%) by model systems used in ecotoxicological studies with graphene-family materials (GFMs). Number of studies per vertebrate species (a), invertebrate species (b), habitat (c), and algal species (d). Literature search conducted between 1990 and 1 September 2023.
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Figure 4. Tested GFMs log10 concentrations (left panel) and length of exposure (right panel) used in test systems with the hierarchical trophic organisation in different environmental compartments.
Figure 4. Tested GFMs log10 concentrations (left panel) and length of exposure (right panel) used in test systems with the hierarchical trophic organisation in different environmental compartments.
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Table 1. The number of entries using the following combinations of keywords in the ISI WoS database.
Table 1. The number of entries using the following combinations of keywords in the ISI WoS database.
Search KeywordsNumber of Results 1
‘graphene’ AND ‘toxic’5221
‘graphene’ AND ‘communities’1755
‘graphene’ AND ‘communities’ AND (‘water’ OR ‘aquatic’)453
‘graphene’ AND ‘communities’ AND ‘wastewater’124
‘graphene’ AND ‘communities’ AND ‘soil’110
‘graphene’ AND ‘microcosm’9
‘graphene’ AND ‘mesocosm’2
1 Literature search conducted between 1990 and 1 September 2023.
Table 2. Details of studies on the effects of GFMs in trophic transfer studies or micro- or mesocosm experiments.
Table 2. Details of studies on the effects of GFMs in trophic transfer studies or micro- or mesocosm experiments.
Ref.Test SystemTest OrganismTested Concentration [mg/L or mg/kg]Exposure PeriodEnvironmental CompartmentType of GFMApplied Ecotoxicity
Endpoint
[56]trophic transfer study (freshwater)Escherichia colibacterium0.05, 0.1, 0.25, 0.5, 12 hwater14C-labeled few-layer graphenecell density (OD600),
cell viability with MTT
Tetrahymena thermophilaprotozoon0.1, 0.2522 hwater14C-labeled few-layer graphenegrowth
Daphnia magnacrustacean0.005, 0.2520 hwater14C-labeled few-layer graphenegraphene uptake
Danio reriofish0.001, 0.054 weekswater14C-labeled few-layer graphenegraphene uptake
[57]trophic transfer study (freshwater)Scenedesmus obliquusgreen algae0.1, 124 hwater14C-labeled few-layer grapheneFLG bioaccumulation
Cipangopaludina cathayensismollusca 48 hwater14C-labeled few-layer grapheneFLG uptake
[52]microcosm
(freshwater)		Nitzschia paleadiatom0.05, 0.16 weekswatergraphene oxidegrowth, abundance
Chironomus ripariusinsect0.05 and 0.1 mg/L13 dayswatergraphene oxidemortality, growth and
teratogenicity
Pleurodeles waltiiamphibian0.05 and 0.1 mg/L10 dayswatergraphene oxidemortality, growth and
teratogenicity
Xenopus laevisamphibian--watergraphene oxideno endpoint (food for newt)
bacterial consortiumbacterium0.05 and 0.1 mg/L6 weekswater, sedimentgraphene oxidespecies distribution
[58]trophic transfer study (freshwater)Heterocypris incongruensostracoda0.39, 1.56, 6.25, 256 dayswatergraphene oxidemortality
Thamnocephalus platyuruscrustacean0.39, 1.56, 6.25, 2548 hwatergraphene oxidemortality
Daphnia magnacrustacean0.39, 1.56, 6.25, 2548 hwatergraphene oxidemortality, oxidative stress
[33]trophic transfer study (freshwater)Raphidocelis subcapitatagreen algae1, 2, 4, 8, 16, 3296 hwatergraphene oxidegrowth
Paratya australiensis)shrimp2, 814 dayswatergraphene oxidesurvival, molting, food intake
[53]microcosm
(freshwater)		Nitzschia paleadiatom0.1, 1, 1048 h, 144 hwatergraphene oxide and rGOviability, growth,
physiological effects
bacterial consortiumbacterium0.1, 1, 10 mg/L48 h, 144 hwatergraphene oxide and rGOsubstrate utilisation pattern, species distribution
[54]microcosm
(marine)		Hediste diversicolorannelid worm0.4, 4, 40, 40036 hsedimentgraphene multilayer nanoflakesoxidative stress, behavioural effects, neurotoxicity, cytotoxicity
Hediste diversicolorannelid worm4, 4024 dayssedimentgraphene multilayer nanoflakesoxidative stress, behavioural effects, neurotoxicity, cytotoxicity
phytoplankton communityphytoplankton4, 4024 dayswatergraphene multilayer nanoflakesbiodiversity indexes, abundance parameters
[59]macrocosm
(freshwater)		phytoplankton communityphytoplanktonnd5 monthswatera graphene photocatalysis netsabundance, species distribution
[55]mesocosm
(terrestrial)		nematodesnematode1 m/m%
(10,000 mg/kg)		130 dayssoilgraphene,
graphene oxide		biodiversity indexes,
abundance parameters
Festuca arundinaceaplant1 m/m%
(10,000 mg/kg)		130 dayssoilgraphene,
graphene oxide		dry biomass
microbial consortium 1 m/m%
(10,000 mg/kg)		130 dayssoilgraphene,
graphene oxide		no endpoint
a polypropylene net attached with graphene, nano-oxidized materials and quantum photosensitive materials.
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Fekete-Kertész, I.; László, K.; Molnár, M. Towards Understanding the Factors behind the Limited Integration of Multispecies Ecotoxicity Assessment in Environmental Risk Characterisation of Graphene-Family Materials—A Bibliometric Review. C 2023, 9, 90. https://doi.org/10.3390/c9040090

AMA Style

Fekete-Kertész I, László K, Molnár M. Towards Understanding the Factors behind the Limited Integration of Multispecies Ecotoxicity Assessment in Environmental Risk Characterisation of Graphene-Family Materials—A Bibliometric Review. C. 2023; 9(4):90. https://doi.org/10.3390/c9040090

Chicago/Turabian Style

Fekete-Kertész, Ildikó, Krisztina László, and Mónika Molnár. 2023. "Towards Understanding the Factors behind the Limited Integration of Multispecies Ecotoxicity Assessment in Environmental Risk Characterisation of Graphene-Family Materials—A Bibliometric Review" C 9, no. 4: 90. https://doi.org/10.3390/c9040090

APA Style

Fekete-Kertész, I., László, K., & Molnár, M. (2023). Towards Understanding the Factors behind the Limited Integration of Multispecies Ecotoxicity Assessment in Environmental Risk Characterisation of Graphene-Family Materials—A Bibliometric Review. C, 9(4), 90. https://doi.org/10.3390/c9040090

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