1

Stable isotope labelling of metal/metal oxide nanomaterials for 1

environmental and biological tracing 2

Peng Zhang1,*, Superb Misra2, Zhiling Guo1, Mark Rehkämper3 & Eugenia Valsami-Jones1,* 3

1School of Geography, Earth and Environmental Sciences, University of Birmingham, Birmingham, UK. 4 2Materials Science and Engineering, Indian Institute of Technology Gandhinagar, Gujarat, India. 3Department of 5 Earth Science and Engineering, Imperial College London, London, UK. Correspondence should be addressed to P. 6 Z. (p.zhang.1@bham.ac.uk) and E.V.J (E.ValsamiJones@bham.ac.uk). 7 8

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ABSTRACT 14

Engineered nanomaterials are often compositionally indistinguishable from their natural counterparts 15

and thus their tracking in the environment or within biota requires the development of appropriate 16

labelling tools. Stable isotope labelling has become a well-established such tool, developed to assign 17

“ownership” or “source” to engineered nanomaterial enabling their tracing and quantification, especially 18

in complex environments. A particular methodological challenge for the stable isotope labelling is to 19

ensure the label is traceable in a range of environmental scenarios but without inducing modification of 20

the properties of the nanoamaterial and without loss of signal from the label, thus retaining realism and 21

relevance. This protocol describes the strategy for stable isotope labelling of several widely used metal 22

and metal oxide nanomaterials, namely ZnO, CuO, Ag, and TiO2, using isotopically enriched precursors, 23

namely 67Zn or 68Zn metal, 65CuCl2, 107Ag or 109Ag metal, and 47TiO2 powder. A complete synthesis 24

requires 1 to 8 days depending on the type of nanomaterial, the precursors used and the synthesis 25

methods adopted. The physicochemical properties of the labeled particles are determined by optical, 26

diffraction and spectroscopic techniques for quality control. The procedures for tracing the labels in 27

aquatic (snail and mussel) and terrestrial (earthworm) organisms and monitoring the environmental 28

transformation of labelled silver nanomaterials are also described. We anticipate this labelling strategy 29

can be adopted by industry to facilitate applications such as nanosafety assessments before 30

nanomaterials enter the market and environment as well as product authentication and tracking. 31

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INTRODUCTION 33

Why is labelling required for environmental and biological tracing? 34

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There has been a notable rise in the development and production of nanomaterials in recent years owing 35

to their broad range of applications, from antimicrobials and drug carriers to next generation computer 36

chips and advanced materials, to name but a few. Estimated global production for typical nanomaterials, 37

such as ZnO, Cu, Ag, TiO2, and Fe, were reported as 34,000, 200, 450, 88,000, and 42,000 tons per year, 38

respectively1. The release of engineered nanomaterials into landfills, soil, air, water, and other 39

environmental compartments is therefore inevitable. Substantial research funding and efforts have been 40

devoted to nanosafety studies in the past 15 years and the field has moved forward significantly. 41

However, until a satisfactory regulatory regime is established concerns will remain regarding their 42

potential impact on the environmental and human health2. 43

Evaluating biological responses to nanomaterials at environmentally relevant concentrations is a 44

crucial point in the field of nanosafety3. A particular challenge encountered in such studies is to 45

distinguish and detect the newly introduced nanomaterials in complex natural biological and 46

environmental matrices at low concentrations and against potentially high natural background values of 47

the same element, e.g., zinc, copper, and titanium. Traditional analytical approaches for measuring total 48

metal concentrations of the target elements, such as inductively coupled plasma mass spectrometry 49

(ICP-MS) techniques, have gone a long way in improving their sensitivity and accuracy in recent years, 50

but are challenged when low levels of nanomaterials need to be determined against high natural 51

background levels of the target element. In particular, it was recommended that nanomaterial exposures 52

should generate elevated elemental concentrations in the tested samples that exceed the background 53

levels by a factor of 10 or more4. This recommendation follows from the observation that elemental 54

concentrations can have uncertainties that exceed ± 10% of whilst the background levels of elements in 55

biological tissues and organisms can readily vary by more than 10% even when they grow under 56

essentially identical conditions5,6. Many published exposure scenarios have used elevated concentrations 57

4

of nanomaterials as a result of the analytical limitations, which, however, are generally not 58

environmentally realistic. For example, ZnO nanomaterial concentrations of 100 to 6400 mg kg-1 were 59

used in soil exposures of Eisenia fetida and Folsomia candida7,8; these levels are about 2 to 3 orders of 60

magnitude higher than the predicted environmental concentrations (PECs) of ZnO nanomaterials in soil9. 61

An important additional consideration is that nanomaterials are highly dynamic and prone to 62

transformation (physical, chemical, or biological) upon entering the environment or biological tissues10. 63

This applies even to materials that were previously considered “insoluble”, such as CeO2 nanomaterials, 64

which can release Ce3+ and transform into CePO4, Ce carboxylates etc11. Some other metal-based 65

nanomaterials (e.g., Ag, CuO/Cu2O, and ZnO nanomaterials) may, furthermore, dissolve quickly or 66

transform to structurally and/or chemically different phases. These processes further complicate 67

detection10. Introduction of a tracer (“label”) into the nanomaterial makes it possible to distinguish both 68

the original form and their transformation products from any natural background. 69

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Comparison of different labelling methods 71

A label may be an organic fluorescent dye, a foreign rare element of low natural abundance, or a less-72

abundant isotope (stable or radioactive) of the same constituent element(s) as in the nanomaterial. 73

Examples of fluorescent dyes include fluorescein isothiocyanate and rhodamine, which are used to trace 74

the uptake and distribution of nanomaterials (e.g., SiO2, graphene oxide) in cells and organisms12,13. 75

Labelling with a fluorescent dye, however, inevitably modifies the surface properties of the 76

nanomaterial and thus alters their environmental and biological behavior. Another commonly used 77

labelling technique is radioisotope labelling. For example, radioisotopes 141Ce, 59Fe, and 198Au are 78

frequently used for labelling of CeO2, Fe3O4, and Au nanomaterials and tracing in the environment and 79

organisms14-16. However, it is of more limited applicability due to the hazards involved in dealing with a 80

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radioactive substance. Moreover, the short half-life of many radioisotopes prevent their use in long term 81

environmental exposures of months to years. Labels in the form of fluorescent dyes and (if added as a 82

surface layer) radioisotopes can also detach from the core of the nanomaterial and thus may not 83

adequately replicate its real behaviour. For example, graphene oxide (GO) labelled with radioactive 125I 84

can gradually release the 125I in physiological fluids17. 85

Compared with the labelling methods above, stable isotope labelling of the nanomaterial itself (i.e. 86

where the nanomaterial is synthesized to have a unique isotopic composition throughout) is a safer as 87

well as more versatile and robust approach that has been well-established and has a range of essential 88

roles in many research fields such as earth18,19, environmental20, and life sciences21,22. Typical examples 89

in biological investigations are, for example, incorporation of different stable isotopes to support 90

accurate protein quantification in proteomics research and for the study of metabolic fluxes23,24. 91

Additionally, stable isotope labelling has potential for further development in the context of nanosafety 92

and environmental tracing of nanomaterials as well as new applications, such as e.g. quality control 93

(where the label could act as unique identifier) in products. 94

Different stable isotopes of the same element are fractionated during many natural processes such 95

as condensation, thermal diffusion, precipitation and biological reactions, and this leads to small mass-96

dependent variations in the isotopic abundances of the elements25. Tracing of the stable isotope labelled 97

nanomaterial takes place against this natural background and relies on the measurement of isotopic 98

changes in a system or compartment that results from the introduction of nanomaterials, prepared from a 99

highly enriched isotope of a constituent element 26. The isotopic changes that are induced by such 100

labelling are thereby orders of magnitude larger than the natural mass-dependent isotope fractionations 101

and can thus be readily detected even in complex natural samples. Once the nanomaterials are labelled, 102

the label can be detected at high selectivity in a large variety of bulk samples following dissolution using 103

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commonly available mass spectrometric techniques, such as quadrupole, sector-field or multiple-104

collector inductively coupled plasma mass spectrometry (ICP-MS), i.e. ICP-QMS, SF-ICP-MS, and 105

MC-ICP-MS. In situ detection of labelled products is also possible, for example using laser ablation 106

ICP-MS (LA-ICP-MS)27 or secondary ion mass spectrometry (SIMS) /nano-SIMS/time of flight SIMS 107

(TOF-SIMS) instrumentation28,29. 108

Another advantage of stable isotope labelling is its ability to monitor transformation of 109

nanomaterials (e.g., silver nanoparticles) in the environment. A critical question raised in toxicological 110

studies of metal-based nanomaterials is whether any observed toxic effects originate directly from the 111

particulates or the dissolved metal ions released from the nanomaterials.2 Addressing this question is 112

challenging due to the complex environmental and biological systems in which transformations take 113

place. In natural environments, silver nanoparticles can be oxidized to release Ag+ ions, which can then 114

be reduced again, as they move from one environmental compartment to another, to regenerate the silver 115

into a new material. These reactions can take place in limited space and time, so that the processes are 116

difficult to monitor. Using a double stable isotope labelling method, whereby enriched 107Ag 117

nanoparticles and 109AgNO3 were simultaneously employed, it was possible to monitor complex 118

transformation kinetics in aqueous media30, highlighting the capabilities of stable isotope labeling. 119

Although stable isotope labelling has many advantages compared with other labelling techniques, 120

it also has some limitations. Firstly, it can only be employed where more than one stable isotopes of the 121

element exist; notably gold has only one stable isotope and thus cannot be labelled using this technique. 122

Furthermore, the label can most practically be traced using ICP-MS bulk sample analysis, which is 123

destructive, requiring full digestion of the sample material using mineral acids. This in turn implies 124

sample availability in sufficient quantity and the adoption of appropriate health and safety measures and 125

precautions to prevent sample contamination. Additionally, compared with fluorescence labelling, the 126

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cost of stable isotopes is relatively high, and this should be considered in advance. Lastly, whilst in situ 127

imaging of stable isotopes is, in principle, possible by using, e.g. LA-ICP-MS and nano-/TOF-SIMS 128

techniques, such instrumentation is not widely available and the analyses require careful calibration 129

procedures. 130

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Stable isotope labelled metal/metal oxide nanomaterials 132

The authors, along with collaborators, have made major advances in the labelling approaches of 133

several particularly relevant nanomaterials, e.g., ZnO,4,31,32 CuO,33,34 and Ag35,36 nanomaterials. In 134

addition, TiO2 nanomaterials labelled with 47Ti and core/shell structured iron oxide@SiO2 labelled with 135

57Fe were also developed recently37,38. The labelling approaches were shown to be efficient and highly 136

sensitive for detecting the nanomaterials in diverse environmental matrices and biological tissues at low 137

concentrations, similar to the PECs of the nanomaterials. 138

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General concept of stable isotope labelling of nanomaterials 140

In general terms, the procedure of stable isotope labelling involves the synthesis of an nanomaterial 141

using a stable isotope enriched precursor as raw material (Figure 1). Isotopically enriched metal salts 142

may be directly used as precursors for nanomaterial synthesis. For example, isotopically enriched 143

65CuCl2 and 109AgNO3 were used directly as the precursors for synthesis of 65CuO nanomaterials and 144

109Ag nanomaterials 33,35. Enriched materials in the form of elemental metal or metal oxide are usually 145

transformed into soluble metal salts for further nanomaterial synthesis, typically by acid digestion31. 146

Direct milling of the metal/metal oxide powder could also, in principle, transform micron sized powders 147

or metal filings to labelled nanoparticulate material. Such milled particles are however, typically not of 148

uniform size and their size range cannot be controlled or modified precisely. Spark discharge reactions 149

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can alternatively be used to prepare isotopically labelled nanomaterials, but this requires specific 150

instrumentation which is not widely accessible39. In contrast, wet chemistry procedures can produce 151

nanomaterials with controllable size over a wide size range, and other desired properties (e.g. specific 152

structural form, complex composition). As such procedures are also accessible for most laboratories, 153

they are considered most suitable and common to produce stable isotope labeled nanomaterials. 154

The methods used for transforming precursors depend primarily on the procedures that are applied 155

for nanomaterial synthesis. For example, commercially available enriched Zn isotopes are usually in the 156

form of Zn metal or ZnO powder. To produce ZnCl2 or Zn(NO3)2 precursors, hydrochloric acid or nitric 157

acid digestion can be used. In our study, we refluxed the Zn metal powder in acetic acid to produce zinc 158

acetate as the precursor, which was optimal for the synthesis of ZnO nanomaterials by a simple 159

hydrolysis method40,41. 160

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Figure 1 here 162

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EXPERIMENTAL DESIGN 164

Precursor choice and preparation 165

Most metallic elements have more than one stable isotope, and nanomaterials produced from such 166

metals can hence be labelled with an enriched stable isotope. However, there are many considerations 167

for choosing the most suitable isotope for labelling (Figure 2). The primary factors to be considered are 168

possible spectral interferences during the mass spectrometric analyses, although such effects can be 169

reduced or avoided by using instrumentation such as collision/reaction cell ICP-MS42 or a chemical 170

separation of the element prior to the measurements, as is common for analyses by MC-ICP-MS5. For 171

example, 49Ti and 112Cd are unfavorable for labelling because their measurement is hindered by 172

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polyatomic interferences from 32S16O1H and 40Ca216O2 which have the same mass number as 49Ti and 173

112Cd. Elements such as Si, S and Ca are very common in biological and environmental samples, whilst 174

H, C, N, O, S and Cl are present in air or the reagents and acids that are used for sample preparation. 175

Argon-based interferences are also a major problem for the measurements, as polyatomic species are 176

formed with C, H, O, and N, e.g., 36Ar13C and 36Ar12C1H for 49Ti, and 40Ar216O2 for 112Cd. In these cases, 177

alternative isotopes, namely 46Ti or 47Ti and 114Cd or 116Cd, can be chosen for labelling. 178

The required enrichment level and quantity of the isotopes, as well as the cost of the enriched 179

material should be considered together at this stage. The enrichment levels of commercially available 180

isotopes range widely, from less than 30% (e.g., 29% for 116Cd) to more than 99% 181

(http://www.tracesciences.com/). To achieve high sensitivity, the use of highly enriched isotopes with a 182

relatively low natural abundance is always preferable. However, a more precise detection method (e.g, 183

MC-ICP-MS) can deliver similar tracing sensitivities even when an enriched isotope with high natural 184

abundance is used. For examples, MC-ICP-MS can deliver similar tracing sensitivity for more abundant 185

and hence cheaper 68Zn (~19% natural abundance) as less precise analyses by ICP-QMS can provide 186

using less abundant and hence more expensive 67Zn (~4% natural abundance). 187

The quantity of enriched material required depends on the experimental system to be studied as in 188

some cases, such as mesocosm exposures, large amounts of the labelled materials are needed. However, 189

the sensitivity of detection and the quantity used must be balanced against the cost of the isotopes, which 190

increases substantially for those of low natural abundance. For example, it was previously reported that 191

64Zn enriched to 99% and with natural abundance of 48% costs ~ $4.5 per milligram, while the price 192

jumps to ~$250 per milligram for 70Zn enriched to 95% and a natural abundance of 0.6%.4 193

The methods that are employed for the preparation of labeled nanomaterials should be chosen 194

carefully given the high cost of enriched isotopes and procedures with high yield and simple steps are 195

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much preferred. In addition, they are constrained by the precursor form required, which needs to be 196

available either from commercial sources or an “in-house” preparation. In most cases, metal/metal oxide 197

nanomaterials can be synthesized using simple metal salts as precursors, e.g., AgNO3 for synthesis of 198

Ag nanomaterials, CuCl2 for synthesis of CuO nanomaterials, and Zn acetate for ZnO nanomaterials. 199

These precursors can hence be readily prepared in the laboratory. In general, all factors mentioned above 200

should be considered and balanced to choose the isotopes that are most suitable for a given labelling and 201

tracing study. 202

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Figure 2 here 204

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Validation of synthesis method with no isotope labelling 206

Although there are many synthesis protocols available in the literatures for each type of nanomaterials, 207

many of these are not suitable for nanotoxicity studies. This reflects that such studies aim to evaluate 208

whether the metal core of nanomaterial is toxic or not, without considering the effects of any 209

contaminants or unwanted surface modifications. Many synthesis methods, however, involve toxic 210

chemicals, surfactants, and organic solvents, which may lead to false positive toxicity results for the 211

nanomaterials. A simple robust method that allows synthesis across the widest possible range of 212

parameters (e.g., size, shape, and surface charge) is preferable. For example, synthesizing nanomaterials 213

with different particle sizes using different methods may be associated with variable impurity contents 214

and structural changes, which may impact the results of the research in an unwanted manner. Methods 215

that involve multiple steps and organic reagents may also be not suitable if they are associated with low 216

synthesis yields. Finally, the synthesis should be robust and produce good yield in a reproducible 217

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manner. All these considerations can be readily verified in preliminary synthesis experiments that 218

employ natural (i.e., non-isotopically enriched) raw materials. 219

Taking ZnO nanomaterials as an example, there are two commonly used methods for the synthesis 220

of ZnO nanomaterials that employ a Zn acetate precursors, and these involve either hydrothermal 221

decomposition or forced hydrolysis. Preliminary work demonstrated that hydrothermal decomposition 222

only gave reaction yields of ~25% and the size of the ZnO nanomaterials was not uniform; as such, the 223

technique was deemed unsuitable for producing isotopically labelled nanomaterials. In comparison, 224

forced hydrolysis of the Zn acetate precursor in diethylene glycol (DEG) gives a yield of 75%, and the 225

size distribution of the ZnO nanomaterials is much more uniform. Furthermore, by manipulating the 226

synthesis conditions, specifically the precursor concentrations and reaction temperature, nanospheres 227

with different sizes can be obtained. The latter method is therefore preferred for producing isotopically 228

labelled ZnO nanomaterials31. 229

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Step 1: Synthesis of nanomaterials with enriched isotopes 231

Synthesis of ZnO nanomaterials labelled with enriched 67Zn or 68Zn (Step 1A) Zinc has five 232

stable isotopes, 64Zn, 66Zn, 67Zn, 68Zn, and 70Zn, with natural abundance of 49.17%, 27.73%, 4.04%, 233

18.45%, and 0.61%, respectively. Isotopically enriched material is available for all five Zn isotopes with 234

enrichment levels that can exceed 95%. Considering the cost and the sensitivity of detection, 67Zn and 235

68Zn are optimal for labelling ZnO. Commercially available isotopically enriched Zn is usually in the 236

form of Zn metal (sheets or filings) or micron sized ZnO powder. To synthesize ZnO nanomaterials, the 237

Zn metal needs to be transformed into a suitable precursor, whereby Zn acetate is the commonly used 238

precursor for synthesis of ZnO nanomaterials. Zn acetate is readily prepared from Zn metal by refluxing 239

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the latter (preferably in powder form) in concentrated acetic acid. After drying, the Zn acetate can be 240

employed for synthesis of ZnO nanomaterials by hydrolysis. 241

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Synthesis of CuO nanospheres and nanorods labelled with enriched 65Cu Cu has two stable 243

isotopes 63Cu and 65Cu with natural abundance of 69.17% and 30.83%, respectively. Enriched isotopes 244

of 63Cu and 65Cu are both commercially available with enrichments of up to 99%. Given the high 245

background concentrations of Cu in the environment and bio-organisms, enriched 65Cu (which has of 246

lower natural abundance) is recommended for tracing as this will provide better sensitivity. Since 247

65CuCl2 is available, this form can be directly used as precursor for synthesis of CuO nanomaterials by 248

wet chemistry. If the enriched Cu is purchased in the form of Cu metal or CuO powder, these materials 249

can be transformed into CuCl2 by digestion with hydrochloric acid. From CuCl2, CuO nanospheres and 250

nanorods can be synthesized by precipitation in alkaline solutions with or without the presence of glacial 251

acetic acid33. The yields of this method are 90% and 82% for nanospheres and nanorods, respectively, as 252

confirmed previously. 253

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Synthesis of Ag nanomaterials labelled with enriched 107Ag and 109Ag Since the two isotopes 107Ag 255

and 109Ag have similar natural abundances of about 50%, 107Ag or 109Ag are equally applicable for 256

labelling purposes. These two isotopes are usually available in the form of Ag metal powder. Most 257

commonly, Ag nanomaterials are synthesized from an AgNO3 precursor, which is readily produced by 258

digestion of the Ag metal powders in concentrated nitric. Following this, citrate-coated Ag 259

nanomaterials were synthesized by reaction of AgNO3 with sodium citrate and NaBH4. By changing the 260

synthesis conditions, e.g., different amounts of NaBH4, Ag nanomaterials with different sizes can be 261

obtained. The yield of the isotopically labelled Ag nanomaterials can reach up to 98%. 262

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Synthesis of TiO2 nanomaterials labelled with enriched 47Ti There are five stable isotopes of Ti, 264

namely 46Ti, 47Ti, 48Ti, 49Ti, and 50Ti. Except for 48Ti with the highest natural abundance of 73.72%, 46Ti, 265

47Ti, 49Ti, and 50Ti are all of low natural abundance (8.25%, 7.44%, 5.41%, and 5.18%, respectively). Of 266

these, 49Ti and 50Ti are not suitable as tracers due to serious polyatomic interference from 32S16O1H and 267

36Ar14N, respectively. Thus, only 46Ti and 47Ti can be used. Commercially enriched Ti isotopes are 268

usually in metal or metal oxide form, which need to be transformed into a suitable precursor (e.g., TiCl4) 269

that can be used for synthesis of TiO2 nanomaterials. It should be noted that dissolving TiO2 is 270

challenging due to its refractory nature. In particular, hydrochloric or nitric acid do dissolve TiO2 but a 271

fusion approach can be applied to facilitate dissolution of TiO243. The obtained precursor solution can 272

then be used for synthesis of TiO2 nanomaterials with an appropriate method, such as a microwave 273

assisted precipitation procedure. A published procedure that applies such an approach only achieves 274

yields of ~ 14% for labelled TiO2 however, which is not ideal and should to be improved in future 275

studies. 276

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Step 2: Quality control steps 278

The quality of the labelled nanoparticles can be established in a few key steps. Firstly, an appropriate 279

synthesis method should be chosen, to ensure that a good yield and suitable physicochemical properties 280

are obtained for the synthesis product. In particular, the synthesis procedure and product should be 281

tested initially using natural, non-isotopically enriched precursors. Such materials, however, will never 282

be exactly identical to the labelled precursors, such that caution should be exercised to minimize any 283

differences. Finally, a full physicochemical characterization of the labelled nanoparticles needs to be 284

performed and the results should be compared with data obtained for non-labelled nanomaterials. This 285

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comparison should yield no significant differences in the size, hydrodynamic diameter, morphology, 286

crystal structure, composition and surface chemistry, which are essential characteristics for reproducible 287

tracer studies. At the minimum, the synthesized nanomaterials hence need to be characterized by 288

transmission electron microscopy (TEM, Step 2A) or scanning electron microscopy (SEM) to confirm 289

size and shape, dynamic light scattering (DLS) or flow field-flow fractionation (FIFFF) to ascertain the 290

hydrodynamic diameter, X-ray powder diffraction (XRD) to determined crystallinity, and ICP-MS for 291

measurement of the chemical and isotopic composition. 292

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Step 3: Tracing of labelled nanomaterials in environmental samples 294

The techniques for tracing nanomaterials in aquatic organisms (snails and mussels) and terrestrial 295

organisms (earthworms) are described below, thus demonstrating the applicability of the stable isotope 296

labelling approach. In addition, a protocol for monitoring the transformation of Ag nanomaterials is also 297

provided. 298

Tracing labelled nanomaterials in aquatic and terrestrial organisms The exposure protocol should 299

be tailored to the organism, exposure method (dietborne vs waterborne) and environment (terrestrial vs 300

aquatic). Accordingly, the total number of individuals that are investigated in a single exposure can vary 301

from less than ten to several hundreds (REFS?). The minimum number of individuals that compromise a 302

single sample for analysis is thereby determined by the detection limit for the determination of the 303

labeled isotope. In many cases, for example when larger organisms such as earthworm and zebra 304

mussels are investigated, this can be a single organism. However, when smaller organisms (e.g., 305

Limnaea), low exposure levels or ICP-QMS detection without prior chemical separation are applied, it 306

may be necessary to pool several individuals to produce a single sample that contains a measurable 307

quantity of labelled nanoparticles. It is, furthermore, generally recommended that at least three replicate 308

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samples are produced for any single time point and experimental line. Conventional biological exposure 309

studies commonly employ more replicate samples but our experience suggests that this is typically not 310

required for stable isotope tracing of nanomaterials. This reflects, in particular, that the stable isotope 311

approach is not compromised by variable biological background levels of the target element. 312

The organisms need to be acclimatized at normal growth condition prior to exposure. For aquatic 313

species, the experiments are carried out in appropriate water tanks. It is essential to use plastic rather 314

than glass tanks, at least in the case of ZnO nanomaterial exposures because the latter have been shown 315

to release significant quantities of Zn44. The nanomaterial suspensions are dispersed by ultrasonication 316

before addition to the tank. During waterborne exposures, continuous stirring is required to reduce 317

aggregation and sedimentation of the nanomaterial , which can have a significant impact on 318

nanomaterial uptake by the organisms. To ensure the organisms are not affected by the stirring, they 319

should be kept suspended (e.g., on a sieve) above the stirring device. An identical number of individuals 320

that are not exposed to the nanomaterial but are otherwise treated in the same manner are used as a 321

control for the exposure. In some cases, an additional control line is required to check for any effects 322

from the nanomaterial coating or suspension matrix. For example, as labeled ZnO NPs are synthesized 323

by forced hydrolysis in DEG, past exposures of earthworms to 68ZnO NPs employed an additional 324

control line whereby DEG was added to the soils. 325

Earthworms of uniform size and weight are chosen for the terrestrial exposure and the organisms are 326

acclimatized to the system prior to the experiment. The earthworms have two pathways for Zn uptake: (i) 327

dermal uptake via direct contact with the soil and (ii) dietary uptake of soil. For dietary exposures, it is 328

important to allow the earthworms to void their guts for 2 h prior to the exposure. For dermal exposures, 329

earthworms are prevented from oral ingestion by sealing their mouth with medical hystacryl glue. The 330

earthworms are placed into soils spiked with the 68ZnO nanomaterial and sampled at suitable time points. 331

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The accumulation of Zn in the earthworms is then determined following the same procedure that is used 332

for snails and mussels described above. 333

After exposure, the soft tissues are dissected, dried, digested and analyzed by ICP-MS. To obtain 334

accurate results, complete digestion of the samples is critical to ensure full recovery of the element 335

constituents. This is particularly challenging for TiO2 nanomaterials due to their refractory nature, and a 336

fusion method is thus used to digest samples exposed to TiO2 nanomaterials. The element concentrations 337

in the solutions obtained after digestion can be determined by ICP-QMS or high precision MC-ICP-MS 338

by external calibration with a series of standard solutions made of the target element. All solutions are 339

also doped with an internal standard, to compensate for matrix suppression and signal drift during the 340

analyses. Publishd ICP-QMS studies thereby employed the elements Sc, Y and Ge as internal standards 341

for Zn and Cu, Ag and Ti measurements45, respectively, whilst Cu was used for the determination of Zn 342

by MC-ICP-MS32. 343

Since ICP-QMS provides sufficient sensitivity and accuracy for many stable isotope tracing studies 344

whilst use of MC-ICP-MS is associated with additional sample separation, the choice of analytical 345

method should be governed primarily by the experimental design (sampling frequency, nanomaterial 346

exposure concentrations, etc) and the data quality required. 347

348

Monitoring the transformation of labelled 107Ag nanomaterials and 109Ag+ ions The 349

effect of environmental factors such as light irradiation, natural organic matter, divalent cations, pH 350

value and temperature on the transformation of Ag nanomaterials are examined. A key step prior to the 351

ICP-MS measurments is the seperation of Ag nanomaterials and Ag+ ions after incubation, as the Ag+ 352

ions may otherwise adsorb on the Ag nanomaterial surfaces46, which will lead to erroneous results. To 353

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achieve this, the samples are treated with an EDTA solution for 10 minutes and ultrafiltrated prior to 354

measurement. 355

356

MATERIALS 357

BIOLOGICAL MATERIALS 358

In our study, we use snails and mussels as representatives of aquatic organisms, and earthworm as 359

representative of terrestrial organism. As suggested in Step 3, 10 of each organism is selected for each 360

treatment. Snails and mussels are kept in moderately hard synthetic water and fed with benthic diatom 361

Nitzschia paleawas and cynaobacteria (Synechocystis PCC6803), respectively31,37. Earthworm is raised 362

in moist soil and fed with horse manure. 363

364

REAGENTS 365

Synthesis of ZnO nanomaterials labelled with enriched 67Zn or 68Zn 366

• 67Zn or 68Zn metal (89% enrichment, Isoflex, Russia) 367

• Glacial acetic acid (CH3COOH; Sigma Aldrich, Cat. no. 1000631011) 368

! CAUTION A flammable and corrosive liquid, which can cause severe skin burns and eye 369

damage. Wear gloves, lab coat, face mask, and goggles and perform experiments in fume hood. 370

• Diethylene glycol (DEG; ReagentPlus 99%, Sigma Aldrich) 371

! CAUTION DEG may cause damage to organs (kidney) if swallowed. 372

• Ultrapure water (18.2 MΩ cm, e.g., Milli-Q) 373

374

Synthesis of CuO nanospheres and nanorods labelled with enriched 65Cu 375

• Copper chloride dihydrate (65CuCl2.2H2O; Trace Sciences, USA, 99% enrichment) 376

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• Glacial acetic acid (Sigma Aldrich, Cat. no. 1000631011) 377

• Sodium hydroxide pellets (NaOH; Sigma Aldrich, Cat. no. 795429) 378

! CAUTION CH3COOH and NaOH may cause severe skin burns and eye damage. Wear 379

protective gloves, protective clothing and eye protection. CH3COOH is flammable. Perform 380

experiments in fume hood. 381

• Ultrapure water 382

383

Synthesis of Ag nanomaterials labelled with enriched 109Ag or 107Ag 384

• Silver nitrate (109AgNO3 or 107AgNO3; Trace Sciences, USA, 99% enrichment) 385

! CAUTION: May be corrosive to metals and intensify fire. May cause severe skin burns and eye 386

damage. Wear gloves, lab coat and goggles, and handle in fume hood. Silver nitrate is toxic to 387

aquatic life and should not be disposed off in the sink. 388

• Trisodium citrate dihydrate (HOC(COONa)(CH2COONa)2.2H2O; Sigma Aldrich, Cat No.: 71320) 389

• Sodium borohydride (NaBH4; Sigma Aldrich, Cat No.: 71320) 390

! CAUTION When in contact with water, flammable gases may be released which can ignite 391

spontaneously. Sodium borohydride is hazardous and is toxic if swallowed. It can cause sever skin 392

burns and eye damage. Wear protective gloves, lab coat, and eye protection and perform 393

experiments in fume hood. 394

• Ultrapure water 395

396

Synthesis of TiO2 nanomaterials labelled with enriched 47Ti 397

• Ultrapure water 398

• Titanium dioxide (Micro-sized 47TiO2; Eurisotop, France, 95.7% enrichment) 399

19

• Hydrochloric acid (HCl; Sigma Aldrich, Cat. no. H1758) 400

• Ammonium hydrogen difluoride (NH5F2; Sigma Aldrich, Cat. no. 224820) 401

• Sodium hydroxide pellets (Sigma Aldrich, Cat. no. 795429) 402

! CAUTION HCl, NH5F2, and NaOH may cause severe skin burn and eye damage. Wear 403

protective gloves, lab coat, face mask, and eye protection. Handle in fume hood. NH5F2 is toxic if 404

swallowed. 405

406

Analytical procedure for tracing of labelled nanomaterials in environmental samples 407

• Hydrogen peroxide (H2O2; Sigma Aldrich, Cat no. 16911) 408

! CAUTION H2O2 cause serious eye damage. Wear protective gloves, face mask and eye 409

protection. Operate in fume hood. 410

• 2-(N-morpholino)ethanesulfonic acid (MES; Sigma Aldrich, Cat no. 163732) 411

• Boric acid (H3BO3; Sigma Aldrich, Cat no. B6768) 412

! CAUTION H3BO3 may damage fertility and the unborn child. Wear protective gloves, clothing, 413

face and eye protection. Operate in fume hood. 414

415

EQUIPMENT 416

• Ultrapure water system (Milli-Q, Merck Millipore) 417

• Electrothermal stirring heating mantle (Electrothermal, UK) 418

• Three neck flasks, condenser, conical flask, and beakers (Scientific Glass Laboratories ltd, UK) 419

• Teflon vessels and stainless steel autoclave (200 mL and 50 mL volume) 420

• Platinum crucible (Cole-Parmer, UK) 421

• Welding torch (Rothenberger, UK) 422

20

• Microwave digestion system (Synthos 3000, Anton Paar) 423

• Centrifuge (Thermo Scientific, Sorvall ST8) 424

• Ultracentrifuge (L8-80M Beckman Coulter, J6-MI) 425

• Amicon Ultra-15 Centrifugal Filter Units (Merck Millipore, Cat no. UFC900308) 426

• Dialysis membranes (Thermo Scientific, 3.5K MWCO, Cat no.: 68035) 427

• Dynamic Light Scattering (Malvern Zeta sizer Nano ZS, UK) 428

• Zeta cells (Malvern Panalytical, Cat no.: DTS1070) 429

• Polystyrene cuvettes (Malvern Panalytical, Cat No.: DTS0012,) 430

• TEM instrument (Hitachi 7100, Japan) 431

• TEM Grids (Cat No.: AGS160, Agar Scientific) 432

• XRD instrument (Bruker D8 Advantage) 433

• ICP-MS instrument (Perkin Elmer, Nexion 2000) 434

• MC-ICP-MS instrument (Nu Plasma HR) 435

• FIFFF instrument (Postnova Analytics, AF 2000) 436

• Solar simulator (Beifanglihui Co., SN-500) 437

REAGENT SETUP 438

Synthetic moderately hard (MOD) water Prepare synthetic moderately hard water according to the 439

standard guideline (US EPA, 2002) for nanomaterial exposure experiment47. Dissolve appropriate 440

amounts of MgSO4, NaHCO3, and KCl solids in ultrapure water and aerate overnight. Dissolve CaSO4 441

solids in ultrapure water separately and aerate overnight. Mix the above two solutions with a volume 442

ratio of 19:1 to achieve MOD water. The final concentrations of MgSO4, NaHCO3, KCl, and CaSO4 in 443

the MOD water are 96, 60, 4 and 60 mg/L, respectively. The MOD water is then used for rearing snails 444

21

or mussels and for the preparation of nanomaterial suspensions. It can be stored in sealed bottles at room 445

temperature (20 ~ 25 oC) for 1 month. 446

447

EQUIPMENT SETUP 448

Transmission electron microscope (TEM) The TEM imaging is performed on a Hitachi 7100 TEM 449

(Japan) operating at 100 kV. 450

X-ray diffraction (XRD) X-ray diffraction is performed using Bruker D8 Advantage with Cu−Kα 451

radiation. 452

Dynamic Light scattering (DLS) The colloidal stability of the nanomaterials is evaluated by 453

measuring the hydrodynamic size and zeta potential (Malvern Instruments, Nano ZS). Hydrodynamic 454

size is also evaluated by FIFFF. 455

Inductive coupled plasma optical mass spectroscopy (ICP-MS) The concentrations and isotope 456

compositions of the metals in samples are measured by ICP-QMS (Perkin Elmer, Nexion 2000) or MC-457

ICP-MS (Nu Plasam HR). 458

459

PROCEDURE 460

Synthesis of nanomaterials with enriched isotopes 461

1 | The protocol below contains the steps to produce five kinds of isotope labelled nanomaterials, i.e. 462

67ZnO nanospheres (8 nm) (Option A), 65CuO nanospheres (7 nm) (Option B), 65CuO nanorods (7 × 40 463

nm) (Option C), 107Ag nanospheres (26 nm) with citrate capping (Option D), and rice grain like TiO2 464

nanomaterials (10 nm)(Option E). 465

(A) Synthesis of 67ZnO nanomaterials ● TIMING 8 d 466

22

(i) Place a 100 mL three-necked round bottom flask into a heating mantle stirrer. Connect a Liebig 467

condenser to the central neck of the flask. Plug the second opening with silicone bung with 468

thermometer inserted. Place an oval shaped magnetic stirring bar in the flask. 469

(ii) Fixe the entire setup using clamps. Connect the water inlet and outlet tubing to the Liebig 470

condenser and the water outlet tubing to the outlet drain sink. Place the setup in a fume hood. 471

CRITICAL STEP The experimental setup must be fixed tightly. The water supply must be 472

switched on prior to heating the three-necked flask. The water flow must be checked thoroughly 473

to ensure that there is no water leaking from the tubing/glassware. 474

(iii) Weigh 500 mg of 67Zn metal powder and add into the flask through the second opening. 475

! CAUTION Zinc powder may catch fire spontaneously if exposed to air or release flammable 476

gases in contact with water. the powder is toxic if inhaled. Wear gloves, lab coat and face mask. 477

(iv) Add 50 mL of glacial acetic acid into the flask through the second opening. Plug the opening 478

with a silicone bung. 479

(v) Heat the flask to 90 °C with the heating mantle. Keep refluxing at 90 °C for 3 d to form a Zn 480

acetate precursor solution. 481

(vi) Turn down the temperature to 50 °C and keep the temperature for 2 d to obtain the dry Zn 482

acetate precursor. The yield is approximately 1 g. The precursor can be stored for at least 1 year 483

in a tightly sealed container at dry place. 484

? TROUBLESHOOTING 485

(vii) Dissolve 100 mg of the precursor in DEG at 60 °C with stirring for 70 h using the same 486

refluxing setup as above. 487

(viii) Heat the resulting mixture to 180 °C. Add 100 µL ultrapure water into the mixture and 488

keep the temperature at 180 °C for 1 h to force hydrolysis of the precursor. 489

23

! CAUTION High temperature. Ensure the tap water connected to the condenser is turned on 490

prior to the heating. 491

(ix) Terminate the reaction and cool the flask down to room temperature (25 °C). Collect the 492

precipitate and centrifuge the suspension at 15,000 g and 4 °C for 15 min. Discard the 493

supernatant and wash the precipitate four times with ultrapure water. 494

(x) Dry the precipitates in a vacuum oven at 50 °C overnight. 495

■ PAUSE POINT The obtained 67ZnO nanomaterial powders are very stable for at least 1 year 496

at room temperature if stored in a desiccator. 497

498

(B) Synthesis of 65CuO nanospheres ● TIMING 1 d 499

(i) Dissolve 0.512 g 65CuCl2.2H2O in 150 mL ultrapure water at room temperature to obtain a 500

0.02 M solution. Use a three-necked round bottom flask to prepare the solution. Wait till the 501

entire copper salt is dissolved in water, giving a light greenish colour. 502

CRITICAL STEP If CuCl2.2H2O sticks to the walls of the original container, to avoid 503

any loss of isotope, dissolve entire bottle of purchased 65CuCl2.2H2O (with given quantity) at 504

once. 505

■ PAUSE POINT The 65CuCl2.2H2O can be stored for at least 3 months in a tightly sealed 506

container at room temperature. 507

(ii) Add 500 µL of glacial acetic acid to the solution and manually shake to ensure it is mixed in 508

the flask. 509

(iii) Transfer the flask into a heating mantle stirrer and attach a Liebig condenser using clamps. 510

The experimental setup is identical to the setup used for the ZnO synthesis (Step 1A(i) and 511

(ii)). 512

24

(iv) Heat the solution to 100 °C under stirring. Keep a check on the inserted thermometer to 513

monitor the the rise in temperature inside the vessel. Once the temperature reaches about 95 514

°C, weigh 0.6 g of NaOH and keep near the set up. 515

! CAUTION NaOH pellets are hygroscopic and should not be left out for long. The pellets 516

must be solid and should not be moist before adding. Wear gloves, goggles and lab coat. 517

(v) At 100°C open the stopper in the top of the condenser, and drop in the NaOH pellets. The 518

solution turns black immediately upon addition of the NaOH as precipitation of 65CuO takes 519

place. 520

! CAUTION Opening of the condenser stopper must be done carefully and slowly. 521

CRITICAL STEP Ensure that all the NaOH pellets are all added at once. Failure to do 522

so will lead to greater inhomogeneity of the particle size and shape. 523

? TROUBLESHOOTING 524

(vi) Keep the temperature at 100 °C for 10 mins. Then switch off the heater and stirrer, and let the 525

suspension cool down naturally to room temperature. 526

? TROUBLESHOOTING 527

(vii) Dismantle the experimental set up and decant the entire suspension into four 50 mL 528

centrifuge tubes. Centrifuge the suspension at 15,000 g and 4 °C for 10 mins. Discard the 529

supernatant and wash the precipitates four times with ultrapure water. 530

(viii) Place the tubes in a heating oven (60 °C) overnight to produce 65CuO nanomaterials in 531

dry powder form. 532

n ■ PAUSE POINT 65CuO nanomaterials are stable in a desiccator filled with inert gas for at 533

least 6 months. 534

535

25

(C) Synthesis of 65CuO nanorods ● TIMING 1 d 536

(i) Prepare rod shaped 65CuO nanomaterials by following the same steps as those used for the 537

preparation of spherical shaped 65CuO nanomaterials, except for no addition of glacial acetic acid (Step 538

2B (ii)). 539

540

(D) Synthesis of 107Ag nanospheres ● TIMING 4 d 541

(i) Dissolve sodium citrate in 100 mL ultrapure water in a glass beaker to obtain a 0.3 mM solution. 542

Allow the solution to cool at 4 °C for 30 min. 543

(ii) Dissolve 107AgNO3 in 100 mL ultrapure water to obtain a 0.25 mM solution. Allow the solution 544

to cool at 4 °C in the dark for 30 min. 545

! CAUTION AgNO3 may cause severe skin burns and eye damage. Wear gloves, lab coat and 546

goggles. 547

CRITICAL STEP Light irradiation may cause reduction of Ag+ to Ag metal. The AgNO3 548

solution must be kept in the dark. 549

(iii) Mix sodium citrate and AgNO3 solutions in a conical flask and stir vigorously. 550

(iv) Dissolve NaBH4 in 10 mL ultrapure water to a concentration of 10 mM. 551

! CAUTION NaBH4 is toxic if swallowed. It may cause severe skin burns and eye damage. It 552

react with water and release extremely flammable H2. Wear gloves, lab coat, face mask and 553

goggle. Operate in fume hood. 554

CRITICAL STEP NaBH4 reacts with water. The NaBH4 solution must be prepared freshly 555

and used immediately. 556

(v) Add 6 mL NaBH4 solution to the mixture (step (iii)) and keep stirring for 10 min. Cover the 557

flask with aluminum foil. 558

26

(vi) Heat the solution slowly to boiling. Keep heating and stirring for 90 min. 559

(vii) Terminate the experiment and cool the solution overnight in the dark at room 560

temperature. 561

(viii) Remove non-reacted reagents by dialysis of the obtained Ag nanomaterial suspension in a 562

1 KDa molecular weight cut-off dialysis bag against 5 L of 0.15 mM sodium citrate solution for 563

72 h. 564

■ PAUSE POINT The concentrated 107Ag nanomaterials stock suspension can be kept in a 565

tightly closed container protected from light at 4 °C for several months. 566

? TROUBLESHOOTING 567

568

(E) Synthesis of 47TiO2 nanomaterials ● TIMING 7 d 569

(i) Digest 1 g of micrometer sized 47TiO2 powder in 200 mL concentrated HCl (37%) in a 500 mL 570

Teflon vessel with lid for 6 days. 571

! CAUTION Concentrated HCl is highly corrosive and volatile. Wear gloves, lab coat, face 572

mask and goggles during operation. Perform experiments in fume hood. Keep lid closed during 573

digestion. 574

(ii) After 6 days, add 11.4 g NH5F2 to the Teflon vessel at once and mix with Teflon stirring rod. 575

Close the lid, seal the vessel in a stainless steel autoclave, and heat at 200 oC in a microwave 576

digestion system for 2 h. The obtained precursor solution will be used for anatase crystallization. 577

! CAUTION NH5F2 is toxic and corrosive. Wear gloves, lab coats and goggles. As NH5F2 578

corrodes glass, avoid use of any glassware for the handling. As high temperatures and pressures 579

are produced during the experiment, the lid of Teflon vessel should be closed and the autoclave 580

must be sealed tightly before heating. 581

27

(iii) Add NaOH pellets to the precursor solution until mildly acidic conditions (pH = 6) are reached. 582

A white precipitate will immediately appear. 583

(iv) Centrifuge the precipitate at 12,000 g and 4 oC for 15 mins and wash the pellet with ultrapure 584

water. Repeat the washing three times. Resuspend the pellet in 100 mL ultrapure water and 585

adjust the pH to 6 with HCl. 586

! CAUTION NaOH and HCl are highly corrosive. Wear gloves, lab coats and goggles. 587

(v) Transfer the suspension into four Teflon vessels (maximum volume 50 mL) and screw the lids 588

tightly. Heat the vessels in a microwave system (Synthos 3000, Anton Paar) at 200 oC for 2 h. 589

! CAUTION High temperature and high pressure is produced, the lid of Teflon line should be 590

screwed tightly. 591

(vi) Switch off the microwave system and allow the vessels to cool down to room temperature. 592

Collect the products by centrifuging at 15,000 g for 15 min and washed four times with 593

ultrapure water. 594

(vii) Dry the product in an oven at 60 oC for 24 h. 595

■ PAUSE POINT The synthesized 47TiO2 nanomaterials are stable at least for 1 year at room 596

temperature if stored in a dry place. 597

598

Quality Control Steps ● TIMING 2 d 599

2 | Before using the labelled nanomaterials in environmental tracing studies, their quality should be 600

checked using a variety of characterization techniques, as feasible/necessary. It is anticipated that the 601

physicochemical properties of the labelled nanomaterials should not deviate from those of the non-602

labelled nanomaterials synthesized first, to ensure the methodology works without wasting costly 603

28

labelled material. The four characterisation techniques listed below provide a minimum level of quality 604

control. Fig. 3 shows typical results obtained using two of the techniques, TEM and DLS. 605

(A) TEM imaging ● TIMING 1 h 606

(i) Sonicate the nanomaterial suspensions for 15 min, and deposit 5 µL of nanomaterial suspension 607

on a carbon-coated copper TEM grid and leave for air-drying. 608

(ii) Image the particles; an appropriate imaging approach may be performed using a Hitachi 7100 609

TEM instrument operating at 100 kV. 610

(iii) Determine the average size of the nanomaterials by measuring 100 or more particles by ImageJ 611

software. 612

(B) XRD determination of crystal structure ● TIMING 1 h 613

(i) Perform XRD analyses of dry nanomaterial powders; this can be done, for example, using a Bruker 614

D8 Advantage XRD instrument with copper Kα radiation. Collect the data at angles (2 theta) from 0 to 615

90 degrees. 616

(ii) Compare the XRD peaks with the standard PDF (powder diffraction file) to assign the structure. 617

Make sure the crystal structure of the sample is identical to the structure of the non-labelled product. 618

(C) DLS analysis ● TIMING 1 h 619

(i) Prepare nanomaterial suspensions with appropriate concentrations, followed by sonication for 15 620

minutes before analysis. The optimal concentration for measuring the particle size of the nanomaterial 621

suspension mainly depends on particle size. It is recommended to make the measurements, at a 622

concentration of 0.5 g/L, if the anticipated particle size is smaller than 10 nm and 0.1 g/L if the 623

anticipated particle size is in the range of 10 to 100 nm 624

(https://www.malvernpanalytical.com/en/learn/knowledge-center/user-manuals/MAN0485EN). 625

29

(ii) Analyse the sample. A suitable instrument for such analysis is the Malvern Zetasizer. The 626

hydrodynamic size measured by DLS of the labelled nanoparticles should be identical with the non-627

labelled particles. 628

(D) Analysis by ICP-MS ● TIMING 6 – 7 d 629

(i) Weigh the dry nanomaterials and add 20 mg of the particles in 10 mL deionized water to prepare 630

stock suspensions of nanomaterials. 631

(ii) Digest the ZnO, CuO and Ag nanomaterials (100 µL, taken from the stock suspensions) in an HNO3 632

- H2O2 mixture with a volume ratio of 4:1 (total volume 10 mL) by heating either on a hot plate 633

(typically 3 hrs) or in microwave digestion system (typically 30 min). For the TiO2 nanomaterials, HCl 634

and NH5F2 are used successively for dissolution with the assistance of a microwave digestion system. 635

(iii) Dry the solutions down, suspend // dissolve the material in 2% HNO3 to a volume of 10 mL. Take 636

100 µL of the solution and dilute to 10 mL by 2% HNO3. 637

(iv) Determine the the trace concentration and isotope composition of the metal by ICP-MS. A suitable 638

instrument can be the PerkinElmer Nexion 2000 ICP-MS. 639

640

Tracing the labelled nanomaterials in environment 641

3 | Using the labelled nanomaterials in tracing experiments. Three examples are described here, 642

involving two aquatic (snail and mussel, (A)) and a terrestrial (earthworm, (B)) organisms, which are 643

tested for the traceability of the labels. The general procedure is to expose the snail, mussel or 644

earthworm to the nanomaterials and determine the accumulated metals in the soft tissues. In the final 645

example described (C), the tracing sensitivity of the labels is exemplified by monitoring the 646

transformation of Ag nanomaterials in aqueous systems. 647

648

30

(A) Tracing the labelled nanomaterials in aquatic organisms ● TIMING 2d - 4d 649

(i) Prepare MOD water for culturing of the snails or mussels according to the standard guideline 650

(US EPA 2002)47. 651

(ii) Prepare nanomaterial stock solutions depending on the final exposure concentrations. For 652

example, to expose the snail to 10 µg L-1 of nanomaterials in a 10 L tank, prepare 10 ml of a 100 653

mg L-1 stock solution. Sonicate the stock suspensions for 10 to 20 mins to disperse the 654

nanomaterials homogeneously. Add 1 mL of stock into the tank and mixed homogeneously with 655

a magnetic stirring bar. 656

? TROUBLESHOOTING 657

(iii) Place the snails or mussels with uniform size and weight on sieves and suspend them in tank 658

containing the nanomaterial suspension. Keep the suspension stirred at a speed of about ~ 600 659

rpm. 660

661 CRITICAL STEP The suspension should be stirred continuously to avoid sedimentation 661

of the nanomaterials and potential interference on their uptake by the organisms. Stirring speed 662

should not be too high to disturb the normal activity of snails or mussels, or too low to let the 663

nanomaterials sink to the bottom. 664

(iv) After completion of the exposure, collect the snails or mussels, and rinse their surfaces with 665

ultrapure water four times. Dissect the soft tissues for measurement of the metal concentrations. 666

(v) Dry the soft tissues in a freeze-drier for 24 h and grind the tissues into fine powders. 667

■ PAUSE POINT Samples are stable once dried. Avoid moisture. 668

! CAUTION Wear gloves and face mask to avoid inhalation of powder. 669

(vi) Weigh appropriate amounts of the dry tissues and digest using mineral acids. For samples 670

treated with ZnO, CuO and Ag nanomaterials, use an HNO3 – H2O2 mixture for digestion 671

31

following standard protocols. Transfer the solutions obtained after digestion into a 15 mL Falcon 672

tubes for analysis. For samples from exposures to TiO2 nanomaterials, place the samples in 673

platinum crucibles, ash at 700 oC in an oven and fuse the ashes with ammonium persulfate with a 674

welding torch for 10 min. Cool the crucible and repeatedly rinse the samples with 2% (v/v) 675

HNO3 to obtain the sample solution. The samples are then ready for analysis by ICP-MS. 676

! CAUTION High temperatures may cause severe skin burn. Wear arc welding gloves and 677

helmet during the welding. The flame of the welding torch may cause fire; work in a cleared-off 678

area away from inflammable substance. 679

CRITICAL STEP Samples need to be digested completely until a clear and transparent 680

solution is obtained. A fusion method using ammonium persulfate should be used for samples 681

containing TiO2 nanomaterials to ensure full recovery of Ti from the samples. 682

■ PAUSE POINT Sample solutions in Falcon tubes can be stored at 4 oC for up to 3 months 683

without impact on the metal concentrations. Longer-term storage of the solutions may be 684

associated with absorption of metals on the container walls. 685

(vii) Measure the metal concentrations of the solution by ICP-MS. If MC-ICP-MS is used to 686

determine the metal concentrations and isotope compositions, the target element needs to be 687

separated from the sample matrix by ion exchange chromatography using a suitable element-688

specific protocol41. 689

690

(B) Tracing the labelled nanomaterials in terrestrial organism ● TIMING 5d – 8d 691

(i) Dry the standard soil Lufa 2.2 soil at 60 oC in an oven overnight to remove soil fauna. 692

(ii) Weigh 0.6 kg dry soil and place into a plastic beaker. 693

32

(iii) Add the nanomaterial stock solution to the soil to obtain a concentration of 5 mg kg-1. 694

Thoroughly mix the soil and nanomaterials with a spatula or rod. Add 0.162 kg deionized water 695

to the soil to achieve a moisture content of 27%; this is equivalent to half of the maximum water 696

holding capacity (WHC) of the soil. 697

CRITICAL STEP The soil and nanomaterials need to be mixed thoroughly to achieve 698

a homogeneous distribution of the nanomaterials. This is critical for the subsequent 699

exposure experiment. 700

(iv) Allow the spiked soil to equilibrate for 2 days before the introduction of the earthworms. 701

(v) Collect earthworms (Lumbricus rubellus) from the field, keep them in moist soil and feed with 702

horse manure. 703

(vi) Choose earthworms of uniform size and weight and with developed clitellum. Transfer 704

earthworms to the clean Lufa 2.2 soli at 50% WHC. Allow acclimation of the earthworms at 15 705

oC for 2 days. 706

(vii) Wash the earthworms with deionized water and blot them dry on filter paper. Place the 707

earthworms on clean moist filter paper, allowing them to void the guts for 2 days. Change the 708

paper after 1 day to avoid coprophagy behavior. 709

CRITICAL STEP Voiding of gut is critical for dietary borne exposures. Two days is 710

sufficient for the earthworm species used in this study; for other species, the duration may 711

vary and should be examined. 712

(viii) For dermal-only exposures, seal the earthworm mouth using medical hystacryl glue after 713

removing mucus around the mouth region. 714

(ix) Add the earthworms to the nanomaterial-spiked soils, and collect samples at desired exposure 715

times. 716

33

(x) Digest and analyze the samples following the procedures described in 3A(iv) - (vii). 717

718

(C) Monitoring transformation of 107Ag nanomaterials and 109Ag+ ions in aquatic environments 719

● TIMING 4d 720

(i) Prepare 1 mg L-1 107Ag nanomaterials suspension by diluting the stock solution with deionized 721

water. Prepare 1 mg L-1 109Ag+ (AgNO3) solution in deionized water. Total volume depends on 722

experimental requirement. 723

! CAUTION Operate in the dark and avoid exposure to light. 724

(ii) Mix 20 mL of each solution in a 50 mL fluorinated ethylene propylene bottle. 725

(iii) To establish the effect of light radiation on the transformation, expose one set of bottles to light 726

in a solar simulator equipped with Xe lamps that produce an irradiation intensity of 550 W m-2 . 727

Another set of bottles is wrapped in two layers of aluminum foils and one layer of black plastic 728

bags as dark controls. 729

! CAUTION As Ag NPs and Ag+ are be sensitive to light irradiation, the bottles for the dark 730

control should be wrapped tightly. 731

(iv) To determine the effect of natural organic matter on the transformation, add Suwannee River 732

humic acid (SRHA) to the mixed solutions in Step (ii) to achieve a final concentration of 5 mg L-733

1. 734

(v) To investigate the effect of pH on the transformation in the presence of SRHA, maintain the pH 735

of the solutions in Step (iv) at 5.6, 7.4 and 8.5 using MES (10 mM, pH 5.6) and borate buffer 736

(1mM, pH 7.4 and 8.5) and adjust by addition of NaOH or HNO3. 737

34

(vi) To study the impact of cations, add stock solution of Ca(NO3)2 (40 g L-1) and Mg(NO3)2 (24 g L-738

1) to the solution in Step (ii) to obtain the final Ca2+ and Mg2+ concentrations of 40 mg L-1 and 24 739

mg L-1. 740

(vii) Take 5 mL samples from the bottles at each desired sampling time (up to 3 days). 741

! CAUTION Shake the bottles to homogenize the solutions prior to the sampling. 742

(viii) Mix the 5 mL samples with 2.5 mL EDTA solution (100 mM) for 10 min. 743

(ix) Separate Ag nanomaterials and Ag+ ions in the mixtures by ultrafiltration (30 kD MWCO). The 744

obtained filtrates are diluted in 2.5% HNO3 (v/v) for measurement by ICP-MS. 745

CRITICAL STEP EDTA effectively removes ions adsorbed on particle surfaces 746

without inducing Ag NM dissolution within 20 min, which is critical for the final measurements. 747

■ PAUSE POINT The solutions obtained in Step (ix) can be stored in Falcon tubes at 4 oC for 748

up to three month. Longer term storage may lead to loss of Ag due to the adsorption onto the 749

container walls. 750

Data reduction 751

4 | There are two approaches for quantifying the concentration of stable isotope labelled nanomaterials: 752

i) by measuring the concentration of the accumulated enriched isotope or ii) by measuring the changes 753

of a diagnostic isotope ratio. Using 67ZnO nanomaterials as an example, the concentration of the 754

accumulated enriched 67Zn in an exposed sample can be calculated from the total metal concentration 755

(p67 × [T67Zn]) minus the background concentration of 67Zn (p67 × [T66Zn]), i.e. the following equations: 756

Δ67Zn = p67 × ([T67Zn] – [T66Zn]) (1) 757

p67 indicates the relative abundance of 67Zn in calibration standards, [T67Zn] and [T66Zn] indicate the 758

total Zn concentrations inferred by the ICP-MS software. 759

p67 = Intensity [67Zn / (64Zn + 66Zn + 67Zn + 68Zn + 70Zn)] (2) 760

35

761

? TROUBLESHOOTING 762

Troubleshooting advice can be found in Table 1. 763

764

Table 1 here 765

766

● TIMING 767

Step 1A, Preparation of 67ZnO nanomaterials: 8 d 768

Step 1B, Preparation of 65CuO nanosphere: 1 d 769

Step 1C, Preparation of 65CuO nanorod: 1 d 770

Step 1D, Preparation of 109Ag nanomaterials: 4 d 771

Step 1E, Preparation of 47TiO2 nanomaterials: 7 d 772

Step 2A, TEM imaging: 1 h 773

Step 2B, XRD: 1 h 774

Step 2C, DLS analysis: 1 h 775

Step 2D, Analysis by ICP-MS: 6 – 7 d 776

Step 3A, Exposure of snails/mussels to nanomaterials and ICP-MS analysis: 2 – 4 d 777

Step 3B, Exposure of earthworms to nanomaterials and ICP-MS analysis: 5 – 8 d 778

Step 3C, Transformation of Ag nanomaterials and Ag+ ions in aquatic environment and ICP-MS 779

analysis: 4 d 780

781

ANTICIPATED RESULTS 782

Characterization of isotopically labelled nanomaterials 783

36

The isotope-labelled nanomaterials should have the same physicochemical properties (e.g., morphology, 784

size, surface charge, and crystal structure, etc.) as the non-labelled nanomaterials synthesized during 785

protocol development. This can be confirmed by quality control investigations of the nanomaterials 786

including using TEM/SEM, DLS, XRD, etc (Step 2). Figure. 3 shows images that characterize the size 787

and shape of labeled Ag and TiO2 nanomaterials and a comparison with the non-labelled counterparts. 788

The Ag NP1 and AgNP2 were synthesized using 10 mM and 1 mM solutions of NaBH4, respectively. 789

The hydrodynamic diameters of the Ag NP1 batch with or without 107Ag labelling were 26.8 ± 2.3 nm 790

and 24.7 ± 3.5 nm, respectively (Figure 3a), whilst the Ag NP2 batch had diameters of 17.4 ± 2.5 nm 791

(labeled) and 18.5 ± 3.3 nm (unlabeled) (Figure 3b). This demonstrated that the sizes of the Ag 792

nanomaterials synthesized from enriched 107Ag and natural Ag are identical. The TiO2 nanomaterials 793

that were prepared from enriched 47Ti and natural Ti show identical morphology (rice grain shaped) and 794

size (10.4 ± 3.3 nm and 12.9 ± 5.4 nm, respectively) (Figure 3c and 3d). A summary of the properties of 795

the stable isotope labelled nanomaterials is provided in Table 2. 796

797

Figure 3 here 798

Table 2 here 799

800

Enhanced tracing ability provided by stable isotope labelling 801

802 Figure 4 here 803

804 805

806

807

37

808

As an example of what results can be obtained in biological studies, snails (Lymnaea stagnalis) 809

and zebra mussels (Dreissena polymorpha) were exposed to a stable isotope labelled nanomaterial to 810

examine the tracing sensitivity of the labeling technique in an aquatic environment using the procedure 811

described in Step 3A. The average background concentrations of Zn and Cu in snails were 54 µg g-1 and 812

34 µg g-1, respectively, which is high relative to the predicted environmental concentrations (PECs) of 813

ZnO and CuO nanomaterials. For example, Gottschalk et al., modeled PECs for ZnO nanomaterials of 814

less than 0.5 µg L-1 for surface water, of 0.5 to 4 µg L-1 for waste water treatment plant effluents, of 10 815

to 16 µg g-1 for biosolids, and of 0.04 ~ 0.5 µg g-1 for sediment9. If stable isotope labeling is not used, Zn 816

in snails from ZnO nanomaterials is detectable only when the ZnO nanomaterial exposure 817

concentrations are higher than 5000 µg g-1 (Figure 4a), which does not constitute a realistic scenario. In 818

contrast, Zn additions of as little as 1 µg g-1 can be detected in snails when exposed to only 15 µg g-1 of 819

dietborne ZnO nanomaterials if an enriched label (67Zn) is used. This demonstrates that stable isotope 820

tracing provides a remarkably enhanced detection sensitivity. 821

For CuO nanomaterials, the newly accumulated Cu in snails exposed to CuO nanomaterials is not 822

detectable over a wide range of exposure concentrations (0.2 to 2000 µg L-1) unless the CuO 823

nanomaterials are prepared from enriched 65Cu (Figure 4b). 824

The predicted environmental concentrations of engineered Ag nanomaterials are 0.1 to 100 ng L-1 825

for surface water and 1 to 10 ng g-1 for sediments, and are thus similar to the respective natural 826

background concentrations of Ag 48. Stable isotope labelling is hence the only safe and reliable way for 827

tracing Ag nanomaterials at low environmentally realistic exposure concentration. Using 109Ag enriched 828

nanomaterials, even only 1 ng of newly accumulated Ag per gram of tissue can be detected (open 829

symbols, Figure 4c) in snails that were exposed to 6 ng L-1 Ag nanomaterials. However, if no tracer 830

38

used, quantifying the presence of the newly accumulated Ag requires exposure concentrations that are 831

two orders of magnitude higher. Similarly, for TiO2 nanomaterials, the newly accumulated Ti in mussels 832

(0.2 µg g-1) that were exposed to 47TiO2 nanomaterials at an environmental relevant concentration of 3.9 833

µg L-1 can be detected (Figure 4d). 834

The detection sensitivity can be further improved by tracing the change of a diagnostic isotope 835

ratios using a high precision MC-ICP-MS5. Taking 68Zn labelled ZnO nanomaterials as an example, the 836

68Zn/66Zn isotope ratio determined for an exposed sample is compared to the natural 68Zn/66Zn ratio 837

measured for a calibration standard (68Zn/66Znstd). The difference in isotope ratio can then be used to 838

calculate the proportion of enriched 68Zn present in the sample relative to the total and natural 839

background content of Zn. By measuring 68Zn/66Zn using MC-ICP-MS, 68Zn additions as low as 5 ng g-1 840

can be detected in natural samples with natural Zn backgrounds of 100 µg g-1. As such, the MC-ICP-MS 841

detection sensitivity is considerably better compared to that attainable by ICP-QMS, which can detect 842

68Zn additions that exceed ~120 ng g-1. However, the limitation of the MC-ICP-MS technique is that the 843

target elements need to be separated from the sample matrix before analysis. Such sample preparation is 844

laborious and requires special facilities, and this should be considered when the preferred instrumental 845

technique is chosen for an exposure experiment. 846

The performance of MC-ICP-MS is well demonstrated by a soil exposure of earthworms to labeled 847

68ZnO nanomaterials. After just 4 h of dermal exposure, an accumulation of 68Zn in the earthworms 848

equivalent to 0.03‰ of the total Zn content could be readily detected41. Using 68Zn isotope labelling, it 849

was also shown that the uptake kinetics of ZnO nanomaterial and ionic Zn by earthworm are similar 850

(Figure 4e and 4f), which probably reflects the rapid and complete dissolution of ZnO nanomaterials at 851

such low concentrations. However, different results were obtained in the study of Heggelund et al49, who 852

also exposed earthworm to ZnO nanomaterials and dissolved Zn but at much higher concentrations of up 853

39

to 2500 µg g-1. In particular, the latter study found lower toxicity when earthworms were exposed to 854

ZnO nanomaterials in comparison to those explosed to dissolved Zn, presumably due to limited ZnO 855

nanomaterials dissolution at the high exposure concentrations41. This demonstrated that stable isotope 856

labelling can provide unique mechanistic understanding on the uptake of nanomaterials at environmental 857

realistic exposure concentrations. 858

859

Transformation of Ag nanomaterials and Ag+ ion in aquatic environment 860

When Ag nanomaterials are released into the environment, they can be oxidized to release Ag+ 861

ions. Subsequently, the released Ag+ and any pre-existing Ag+ present can be reduced to produce a 862

mixed source regenerated Ag nanomaterials. Such reactions can be studied by stable isotope labeling, 863

using 107Ag nanomaterials and ionic109Ag+, thus each form of silver having its own distinct isotopic 864

composition. When 107Ag nanomaterials and 109Ag+ coexist, oxidation and dissolution of 107Ag 865

nanomaterials is the dominant reaction in a system of pure water (Figure 5a). However, in the presence 866

of dissolved organic matter (DOM), reduction of Ag+ and regeneration of Ag nanomaterials is the 867

dominant process (Figure 5b). Temperature, pH value and the presence of other divalent ions have a 868

significant impact on these transformation processes. For example, addition of Ca2+ and Mg2+ at 869

environmentally relevant concentrations had no strong effect on the reduction of 109Ag+ (Figure 5c). 870

However, it induced significant agglomeration of 107Ag nanomaterials, which considerably reduced the 871

effective surface area of the Ag nanomaterials and thus limited their subsequent dissolution in the dark 872

(Figure 5d). 873

These findings further highlight the power of the isotope labelling approach in tracing nanomaterials 874

in complex environment samples and systems. There is also significant potential for expanding the 875

double isotope labelling technique to other nanomaterials, in particular by labelling two different 876

40

elements. For example, this may be applicable to CdS quantum dots, an approach currently being 877

developed at the University of Birmingham. By labelling CdS with both 110Cd and 34S, it can be possible 878

to not only quantify the CdS present, but also detect environmental and biological transformation of CdS. 879

For example, to assess whether the labelled Cd and S remain together as the original particles or separate 880

and transform into distinct more stable forms with time. 881

Figure 5 here 882

Adaptation for industrial applications 883

Stable isotope labelling could find a number of industrial applications, whether to ensure safety or 884

authenticity of a product. In an industrial context, stable isotope labeling could be used to ensure the 885

authenticity of a product. More specifically, an isotopic label can link a material to its source following 886

accidental release, theft or any other scenario requiring unequivocal identification. In such cases, a 887

unique isotopic signature could represent an entire product line, a particular product or even a unique 888

batch. To save on the cost of the stable isotope label, the adaptation for industrial applications does not 889

need to employ full isotopic modification but could involve the synthesis procedures described above 890

(A-E) but with only a limited quantity of labeled material and the remaining synthesis precursor 891

unlabeled. This procedure would introduce an isotopic variation that is sufficient to differentiate a 892

labeled product from other similar materials (or batches) and background isotopic compositions, 893

whereby the proportion of enriched label present in the product is experimentally assessed prior to 894

release to ensure robust traceability. 895

896

ACKOWLEDGEMENTS 897

This work was supported by Marie Skłodowska-Curie Individual Fellowship (NanoLabels 750455 to 898

PZ; NanoBBB 798505 to ZG) under the European Union’s Horizon 2020 research program. Financial 899

41

support from MHRD-IMPRINT funds is appreciated. Partial funding from EU H2020 project ACEnano 900

(grant agreement no. 2016-720952) is also acknowledged. 901

902

AUTHOR CONTRIBUTIONS 903

P.Z., S.M., and Z.G. wrote the paper. E.V.J. and M.R. design part of the experiments and revised the 904

paper. S.M. performed part of the experiments. 905

COMPETING FINANCIAL INTERESTS 906

The authors declare no competing financial interests. 907

DATA AVAILABILITY 908

The data that support the plots within this paper are available from the corresponding author upon 909

reasonable request. 910

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1026

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1029

1030

1031

1032

47

TABLE 1 | Troubleshooting table. 1033

Step Problem Possible reason Solution 1A(vi) The yield of the precursor is very

low Zinc powder was not dissolved completely

Check the temperature, keep at 90 °C Extend the heating time

1B(v) No formation of black precipitate pH of the solution was not high enough

Add accurate amount of NaOH and add it all at once

1B(vi) Formation of light green precipitates

Copper chloride did not react completely

Keep the solution at 100 °C for a longer duration

1D(viii) The yield of Ag nanomaterials is too low

NaBH4 solution was not freshly prepared

Prepare fresh NaBH4 and use it immediately

3(ii) Nanomaterials not disperse homogeneously

The stock suspension was not sonicated sufficiently Stirring speed was too low

Ensure sufficient sonication of the stock Increase stirring speed up to 600 rpm

1034

1035

TABLE 2 | Summary of the properties of the stable isotope labelled nanomaterials synthesized by the 1036

protocols. 1037

67ZnO 65CuO 65CuO 107Ag 47TiO2 Size (TEM) 8 ± 1 nm 7 ± 1 nm 7 ± 1 nm

width 40 ± 10

nm length

28 ± 8 nm 10 ± 3 nm

Shape (TEM) Spherical Spherical Rod Spherical Rice grain-shaped

Crystalline phase (XRD)

Wurtize (ICDD 36-

1451)

Tenorite (ICDD 48-

1548)

Tenorite (ICDD 48-

1548)

FCC (ICDD 87-

0720)

Anatase (ICDD 21-

1272)

Hydrodynamic size (DLS)

38.6 ± 0.7 nm

82 ± 1 nm NA 26.8 ± 2.3 nm

98 ± 7 nm

1038

NA indicate the value is not available due to the shape of nanorod and its tendency to agglomerate. 1039

48

FIGURE LEGEND 1040

Figure 1. Scheme of stable isotope labelling of NMs. x indicates the atomic mass number of isotope M. 1041

99% indicates the enrichment level of the isotope. 1042

Figure 2. Considerations for the choice of suitable isotope and chemical form. 1043

Figure 3. Hydrodynamic diameters and TEM images. (a) Hydrodynamic diameter distributions of 107Ag 1044

NMs (synthesized in Step 1D) determined by FIFFF. Nat-Ag NP indicate the Ag NMs synthesized 1045

without labelling. (b) Hydrodynamic diameter distributions of 107Ag NMs synthesized with a different 1046

method (all steps are identical to Step 1D except for Step 1D (iv) where 1 mM of NaBH4 is used). (c) 1047

and (d) show the TiO2 NMs with and without 47Ti labelling, respectively. Adapted from previous 1048

publications35,37. 1049

Figure 4. Enhanced detection sensitivity for NMs provided by stable isotope labelling. (a) Zinc 1050

concentrations in snails exposed for 3-4 h to dietborne 67ZnO NMs. Triangles and circles represent two 1051

dietborne exposure modes, i.e. food (diatoms) labelled with 67Zn and diatoms mixed with 67ZnO. (b) 1052

Copper concentrations in snails exposed for 24 h to waterborne 65CuO NMs in synthetic MOD water. (c) 1053

Silver concentrations in snails exposed for 24 h to waterborne 109Ag NMs in synthetic MOD water. (d) 1054

47Ti concentrations in zebra mussel exposed for 1 h to waterborne 47TiO2 in the presence of 1 × 106 1055

c/mL of cyanobacteria as food source in synthetic MOD water. (e) and (f) show the 68Zn concentrations 1056

in unsealed and sealed earthworm, respectively, which were exposed to 68ZnO (red squares and line) and 1057

68ZnCl2 (blue circles and line). The solid red or blue lines across the exposure concentrations in a-d 1058

display the mean natural background concentrations of the metals measured in snails that were not 1059

exposed to any NMs. The shaded areas in a and b represent the error for the averaged concentrations, 1060

given as 1× (pink) and 3× (blue) the standard deviation (SD) of the mean (n = 140 in a; n=200 in b). The 1061

dotted lines in d show the SD of the mean (n=28). The open symbols in a and b represent the detectable 1062

49

67Zn and 65Cu (newly accumulated) after exposure to 67ZnO and 65CuO NMs; each concentration was 1063

derived from the total measured 67Zn and 65Cu concentrations minus background. The closed symbols in 1064

a and b represent the sum of the detectable 67Zn and 65Cu, and the background Cu concentrations. Error 1065

bars show SD, n = 10 samples per group. Adapted from previous publications.31,33,36,37 1066

Figure 5. Transformation of 107Ag NMs and 109AgNO3 in an aqueous system. (a) Change of the 109Ag 1067

concentrations in pure water. Error bars show SD of 4 replicates. (b) Fraction of dissolved 109Ag+ over 1068

time in the presence of DOM. (c) Fraction of dissolved 109Ag+ over time. (d) Release of 107Ag ions from 1069

the 107Ag NMs in dark and light conditions. Adapted from Yu et al.30 1070

1071

1072

1073

Precursors

Enriched isotopes

Metal salts

Elemental metal/metal oxides

NMsxM99%

NP-P180396B Valsami-Jones Fig 1

Stable isotope labeling of

nanomaterials

Polyatomic interference

Enrichment level

Price of isotope

Quantity required

Synthesis method

Detection method

NP-P180396B Valsami-Jones Fig 2

a

b

c

d

NP-P180396B Valsami-Jones Fig 3

TiO2 NP

47TiO2 NP

NP-P180396B Valsami-Jones Fig 4

a b

c d

e f

Ag in

flux

into

L. S

tagn

ails

(μg

g-1

day-

1 )

[47Ti

] in

mus

sels

(μg

g-1 )

Total Ag concentration (μg L-1) [47TiO2] NP exposure concentration (μg L-1)

[68Zn

] in

eart

hwor

m (μ

gg-

1 )

[68Zn

] in

eart

hwor

m (μ

gg-

1 )

Hours Hours

b

c d

a

NP-P180396B Valsami-Jones Fig 5