Telling the important stories of “no adverse effect” nanomaterials data

As editors we often state that we are open to receiving no-effect studies, but as authors we know that it can be very challenging to publish studies that do not provide exciting new insights into mechanisms of toxicity or explore new adverse outcomes or pathways. The occasion of publishing a well written and insightful no-effects paper by Dedman et al., (2021) which shows that environmentally relevant concentrations of titanium dioxide nanoparticles pose negligible risk to marine microbes,¹ presents the opportunity for us to reflect on what makes a great “no-effects” paper, and indeed to highlight the large range of such papers that are appearing in the literature, albeit often under different guises. For example, safer-by-design nanomaterials studies,² reduced impacts from environmentally aged nanomaterials,³ low-dose chronic exposure studies⁴ and many more, are actually no-effect studies.

The application of ‘omics’ technologies to nanomaterial toxicity and ecotoxicity assessment may provide novel insights beyond standard toxicology tests as to how we can differentiate between normal metabolic and repair functions versus those that lead to adverse effects. Key questions to explore include determining what these thresholds are and how we can determine them. Indeed, the distinction between no observed effect concentrations (NOEC) and no observed adverse effect concentrations (NOAEC), the statistically calculated highest exposure level at which there are no biologically significant increases in the frequency or severity of an adverse effect between the exposed population and its appropriate control, allows that some effects may be produced at this level, but that they are not above the threshold where one would consider adverse effects to occur. The concept of no observed transcriptomic adverse effect level (NOTEL) has been proposed as a means to explore the impacts of compounds beyond standard acute and chronic endpoints and to provide more insight regarding the mechanism of interaction for low concentrations and over time,⁵ and has also been applied to the assessment of nanomaterial toxicity.⁶ However, to date, the majority of nanomaterial toxicogenomics studies are restricted to just a single dose and timepoint, and represent only a few nanomaterial types. Few experiments incorporate multiple doses, multiple timepoints or both,⁷ such that the establishment of NOTELs is limited and insights into the no-adverse-effect thresholds are currently unavailable.

The impact of publication bias towards predominantly “effect” versus “no effect” papers has been well documented in the medical field, and it is widely agreed that the underreporting of negative results introduces bias into meta-analysis, which consequently misinforms researchers, doctors and policymakers.⁸ When no-effect data is unpublished and unavailable to the scientific community, others may repeat the same study, leading to wasted resources. That said, it is well-documented that it is easier to publish effects papers than no-effects papers; a meta-analysis of herbicide ecotoxicity papers found that studies that reported effects were published in journals with a greater mean impact factor than those that reported no effects (p < 0.05), and that papers reporting an effect had significantly more citations per year than those that did not (p < 0.05).⁹ There is thus a clear need to explore how to increase the information available regarding “no effect” studies to provide more balanced reviews, meta-analyses and predictive ecotoxicity models.

A key aspect of a good no-effect study is that the study is well framed from the outset with a clear study design and an interesting perspective; for example, Environmental Science: Nano reviewers and readers look for new mechanistic insights and studies that provide novel ways of examining the interactions of nanomaterials in the environment. For example, this could be research on environmentally-realistic low-dose and long-term exposure scenarios that ultimately demonstrate no acute impact, but investigate recovery pathways or organismal adaptation to exposure over time (see Fig. 1). Other areas of research where “no effects” are key include studies assessing a reduction of impacts following the intentional tailoring of nanomaterial properties – so-called safe by design approaches. Transformation in the environment can lead to “no effect” outcomes that are key to understanding the impact of nanomaterials in the environment and on organisms and may even be harnessed to target nanomaterials to specific environmental compartments or to facilitate interactions with specific organisms. Exploring the “why” of the no-effect is also essential, and we highlight some examples of such approaches here:

Fig. 1 Schematic illustration of the perturbation of a biological pathway by a nanomaterial or its environmental transformed form, and the potential consequences which include the induction of a repaid mechanism, induction of an adaptive stress response or an adverse outcome. Linking no adverse effect studies to the exploration of the biological pathways induced and the thresholds for recovery, adaptation or an adverse outcome will provide important insights to support the design of safer nanomaterials. Adapted from Firestone et al., 2010.¹⁸

- Environmentally realistic concentrations – as in the case of the study by Dedman et al., (2021) who used both research grade materials and particles extracted from real-world formulations, and who looked at both short-term effects and longer-term effects thus allowing the observation of the recovery and the overall determination of no adverse effects.

- Low uptake or limited bioavailability may dictate no effect for an otherwise toxic material. Exploring whether the no effect is due to a lack of interaction with or uptake by the test species, including ruling out any artefact of the study design (such as the nanomaterials settling out of the water column and thus being unavailable to organisms in the water column), is essential. For example, in the case of the no-effects of nanomaterials to the soil invertebrate Folsomia candida (Noordhoek et al., 2018) the absence of reproductive toxicity of the WCCo, CuO and Fe₂O₃ nanomaterials could partly be explained by low porewater concentrations, thus suggesting the low solubility or slow solubilisation of the WCCo, CuO and Fe₂O₃ nanomaterials tested, since soluble metal salts corresponding with these nanomaterials did affect F. candida survival and reproduction, at similar total concentrations.⁴

- Transformations of nanomaterial properties that alter bioavailability or toxicity – e.g., Ellis et al., (2020) compared the toxicity of pristine versus medium-aged silver and titanium dioxide nanomaterials in salt-only versus natural organic matter-containing medium and found that aging in either medium dramatically reduced chronic (reproductive) toxicity in Daphnia magna, including reduced effects in subsequent generations in both the continuously exposed and recovery generations.³ Understanding the interaction of nanomaterials with natural organic matter and/or suspended solids, as per Surette et al. (2021) which explored the impact of wastewater treatment plant constituents on nanomaterial fate and behaviour, is a critical step in gaining mechanistic insights into the reduction of nanomaterials’ toxicities, whether through reduced surface activity (passivation of the surface through eco-corona formation) or reduced bioavailability and uptake.¹⁹

- Adaptation or tolerance due to interaction with biomolecules in organisms – e.g., studies assessing the proteins secreted by organisms into their medium associated with normal organismal behaviour (particle-free controls) and processing of particles of low toxicity (environmentally aged) nanomaterials versus the pristine particles with higher toxicity effects, has shed new light on the potential of the secreted corona as a means to explore recovery pathways and/or adaption mechanisms¹⁰ and suggests that there is enormous scope for assessing recovery and exploring the aforementioned issue of no-effects versus no adverse effects. Indeed, given the high affinity of nanomaterials for proteins and other biomolecules, we can envisage studies whereby different nanomaterial compositions are added post-conditioning to the control organism medium to enrich specific proteins based on the affinity and binding strengths. Evidence of adaptation or tolerance can be explored using a combination of metabolomics, proteomics and transcriptomics, both directly on the organisms, on the nanomaterials recovered from or depurated by the organisms, or through analysis of the secreted biomolecules. Thus, there are vast new opportunities for screening beneficial material properties and designing greener products from the bottom up via the assessment of biocorona interactions.¹¹

- New methodologies to explore nanomaterial interactions or uptake, including novel luminescent particles used to assess ingestion and movement in or through the gut by C. elegans¹² or pH sensitive particles to assess changes in the gut pH of daphnids¹³ – such studies usually include some confirmatory studies to demonstrate the non- or low-toxicity of the particles, and as such provide important no-effect data that might be missed in standard literature searches.

- A safe by design approach to nanomaterial design to reduce the toxicity from a known physico-chemical parameter – many parameters of nanomaterials have been found to correlate with toxicity, including for example strained rings,¹⁴ nearly free silanols,¹⁵etc. For example, a recent paper showed that the oxidization rather than graphitization of nanodiamonds reduced growth inhibition, the severity of organelle damage and oxidative stress, and stimulated the expression of extracellular polymeric substances which further alleviated the adverse effects to Chlorella pyrenoidosa.² We note however that, as in the case of clinical trials to prove that a new medicine is as or more effective than the existing standard, careful thought needs to be given to the statistical power of the study and how to prove the null hypothesis, and the reporting of confidence intervals may be a useful approach.¹⁵ Of course, it should be noted that nanomaterials are complex entities and the specific design of the nanomaterial composition and properties may lead to unexpected effects, or changes in one property may affect others also, often with unexpected effects. Indeed, Buchman et al. (2019) found that, unexpectedly, increasing the nickel content in lithium intercalation compounds (complex metal oxide nanomaterials used in batteries) leads to increased particle stability and reduced dissolution, leading to quite different toxicities to bacteria (direct interaction) versus Daphnia (interaction with released ions).¹⁶ This also suggests scope for assessing the impacts of nanomaterials across species to understand their range of effects/no effects.

- Reduced bioavailability of co-contaminants – research is increasingly moving towards the applications of nanomaterials in agriculture, the remediation of soil and water, etc., and thus towards mixture toxicity where the interactions of co-pollutants with nanomaterials can enhance or reduce the co-pollutant toxicity depending on the binding affinities to nanomaterials, the duration of the complex etc. Thus, we can also envisage negative effects or no effects papers wherein the impact of a co-pollutant is reduced by the interaction with nanomaterials and the consequent reduced bioavailability. However, this too can be an effect in some cases e.g., where the impact of interacting with a nanomaterial is to reduce the bioavailability of essential nutrients from food, as reported by Li et al. (2021).¹⁷ We also call upon researchers publishing applications papers to include a small paragraph (even in the ESI) on the no adverse effects data they have to confirm the safe use of the specific application, as this will also help rebalance the overall picture.

We hope that we have inspired you to consider just some of the multiple ways to frame the story of your no-adverse-effect data, and look forward to redressing the balance of no effect data versus effect data, and driving the publication and citation of this data by our environmental nanomaterials research community, both on the health and safety side and on the applications side.

References

1. C. J. Dedman, A. M. King, J. A. Christie-Oleza and G. L. Davies, Environmentally relevant concentrations of titanium dioxide nanoparticles pose negligible risk to marine microbes, Environ. Sci.: Nano, 2021, 8.
2. C. Zhang, X. Huang, Y. Chu, N. Ren and S.-H. Ho, An overlooked effect induced by surface modification: different molecular response of Chlorella pyrenoidosa to graphitized and oxidized nanodiamonds, Environ. Sci.: Nano, 2020, 7(8), 2302–2312.
3. L.-J. A. Ellis, E. Valsami-Jones and I. Lynch, Exposure medium and particle ageing moderate the toxicological effects of nanomaterials to Daphnia magna over multiple generations: a case for standard test review?, Environ. Sci.: Nano, 2020, 7(4), 1136–1149.
4. J. W. Noordhoek, R. A. Verweij, C. A. M. van Gestel, N. M. van Straalen and D. Roelofs, No effect of selected engineered nanomaterials on reproduction and survival of the springtail Folsomia candida, Environ. Sci.: Nano, 2018, 5(2), 564–571.
5. E. K. Lobenhofer, X. Cui, L. Bennett, P. L. Cable, B. A. Merrick and G. A. Churchill, et al., Exploration of low-dose estrogen effects: identification of No Observed Transcriptional Effect Level (NOTEL), Toxicol. Pathol., 2004, 32(4), 482–492.
6. C. Pisani, J. C. Gaillard, V. Nouvel, M. Odorico, J. Armengaud and O. Prat, High-throughput, quantitative assessment of the effects of low-dose silica nanoparticles on lung cells: grasping complex toxicity with a great depth of field, BMC Genomics, 2015, 16(1), 315.
7. L. A. Saarimäki, A. Federico, I. Lynch, A. G. Papadiamantis, A. Tsoumanis and G. Melagraki, et al., Manually curated transcriptomics data collection for toxicogenomic assessment of engineered nanomaterials, Sci. Data, 2021, 8(1), 49.
8. A. Mlinarić, M. Horvat and V. Šupak Smolčić, Dealing with the positive publication bias: Why you should really publish your negative results, Biochem. Med., 2017, 27(3), 030201.
9. M. L. Hanson, L. E. Deeth and R. S. Prosser, Evidence of citation bias in the pesticide ecotoxicology literature, Ecotoxicology, 2018, 27(7), 1039–1045.
10. L.-J. A. Ellis and I. Lynch, Mechanistic insights into toxicity pathways induced by nanomaterials in Daphnia magna from analysis of the composition of the acquired protein corona, Environ. Sci.: Nano, 2020, 7(11), 3343–3359.
11. S. Lin, M. Mortimer, R. Chen, A. Kakinen, J. E. Riviere and T. P. Davis, et al., NanoEHS beyond toxicity – focusing on biocorona, Environ. Sci.: Nano, 2017, 4(7), 1433–1454.
12. Z. R. Jones, N. J. Niemuth, M. E. Robinson, O. A. Shenderova, R. D. Klaper and R. J. Hamers, Selective imaging of diamond nanoparticles within complex matrices using magnetically induced fluorescence contrast, Environ. Sci.: Nano, 2020, 7(2), 525–534.
13. A. Davis, F. Nasser, J. R. Lead and Z. Shi, Development and application of a ratiometric nanosensor for measuring pH inside the gastrointestinal tract of zooplankton, Environ. Sci.: Nano, 2020, 7(6), 1652–1660.
14. H. Zhang, D. R. Dunphy, X. Jiang, H. Meng, B. Sun and D. Tarn, et al., Processing pathway dependence of amorphous silica nanoparticle toxicity: colloidal vs pyrolytic, J. Am. Chem. Soc., 2012, 134(38), 15790–15804.
15. W. C. Blackwelder, "Proving the null hypothesis" in clinical trials, Controlled Clin. Trials, 1982, 3(4), 345–353.
16. J. T. Buchman, E. A. Bennett, C. Wang, A. Abbaspour Tamijani, J. W. Bennett and B. G. Hudson, et al., Nickel enrichment of next-generation NMC nanomaterials alters material stability, causing unexpected dissolution behavior and observed toxicity to S. oneidensis MR-1 and D. magna, Environ. Sci.: Nano, 2020, 7(2), 571–587.
17. C. Li, C. Ma, H. Shang, J. C. White, D. J. McClements and B. Xing, Food-grade titanium dioxide particles decrease the bioaccessibility of iron released from spinach leaves in simulated human gastrointestinal tract, Environ. Sci.: Nano, 2021 10.1039/D1EN00064K.
18. M. Firestone, R. Kavlock, H. Zenick and M. Kramer, The U.S. Environmental Protection Agency Strategic Plan for Evaluating the Toxicity of Chemicals. Journal of toxicology and environmental health Part B, Crit. Rev., 2010, 13, 139–162.
19. M. C. Surette, C. J. McColley and J. A. Nason, Comparing the fate of pristine and wastewater-aged gold nanoparticles in freshwater, Environ. Sci.: Nano, 2021, 8, 1109–1120.

---