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Article

Defining Quality Criteria for Nanoplastic Hazard Evaluation: The Case of Polystyrene Nanoplastics and Aquatic Invertebrate Daphnia spp.

by
Anita Jemec Kokalj
1,
Margit Heinlaan
2,
Sara Novak
1,
Damjana Drobne
1 and
Dana Kühnel
3,*
1
Biotechnical Faculty, University of Ljubljana, Večna pot 111, 1000 Ljubljana, Slovenia
2
National Institute of Chemical Physics and Biophysics, Akadeemia tee 23, 12618 Tallinn, Estonia
3
Helmholtz Centre for Environmental Research—UFZ, Permoserstr. 15, 03418 Leipzig, Germany
*
Author to whom correspondence should be addressed.
Nanomaterials 2023, 13(3), 536; https://doi.org/10.3390/nano13030536
Submission received: 5 January 2023 / Revised: 24 January 2023 / Accepted: 26 January 2023 / Published: 28 January 2023
(This article belongs to the Special Issue Ecotoxicology and Risk Assessment of Engineered Nanomaterials)
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Abstract

:
Polystyrene nanoparticles are the most investigated type of nanoplastics in environmental hazard studies. It remains unclear whether nanoplastic particles pose a hazard towards aquatic organisms. Thus, it was our aim to investigate whether the existing studies and data provided therein are reliable in terms of data completeness. We used the example of Daphnia spp. studies for the purpose of polystyrene nanoplastic (nanoPS) hazard evaluation. First, a set of quality criteria recently proposed for nanoplastic ecotoxicity studies was applied. These rather general criteria for all types of nanoplastics and different test organisms were then, in the second step, tailored and refined specifically for Daphnia spp. and nanoPS. Finally, a scoring system was established by setting mandatory (high importance) as well as desirable (medium importance) criteria and defining a threshold to pass the evaluation. Among the existing studies on nanoPS ecotoxicity for Daphnia spp. (n = 38), only 18% passed the evaluation for usability in hazard evaluation. The few studies that passed the evaluation did not allow for conclusions on the hazard potential of nanoPS because there was no consensus among the studies. The greatest challenge we identified is in data reporting, as only a few studies presented complete data for hazard evaluation.

1. Introduction

Global plastic pollution and the detection of micro- and nanoplastic particles in almost all environmental compartments and geographic regions has fuelled research into the possible effects of these particles on environmental organisms. One specific research question is whether nanoplastic particles exert specific and/or enhanced effects in organisms in comparison to microplastics [1,2,3,4]. However, studies have reported different conclusions regarding this question. For example, a very extensive meta-analysis of existing studies indicated that nanoplastics are less toxic than microplastic, but that nanoscale polystyrene particles (nanoPS) are more hazardous than other nanoplastic particles [5]. As reviewed by ref. [1], there are also indications of size-specific effects depending on the type of organism. However, it remains to be elucidated which size classes need specific attention for hazard evaluation, and whether additional particle characteristics play a role in hazardous effects. For example, it has been pointed out that current methodological approaches of nanoPS ecotoxicity testing have pitfalls, particularly regarding the particle additive consideration and other characteristics of the particles [2,6].
Hence, the overall goal of the current study was to assess the relevance (hereafter referred to as study quality, according to Armijo-Olivo, Stiles [7]) of existing aquatic ecotoxicity data on nanoPS for the purpose of hazard characterization. For this purpose, we conducted a case study on nanoPS toxicity towards the aquatic invertebrate, Daphnia spp. NanoPS was selected because it is the most used nanoplastic particle in laboratory ecotoxicity studies due to its commercial availability. Daphnia spp. are among the most used aquatic organisms in general and specifically used for the hazard characterization of nanoPS [1]. Hence, Daphnia spp. were selected for this study due to the availability of quite a number of studies. Because the process of quality evaluation of the studies was quite laborious, we focused on Daphnia spp.
Overall, 38 studies were assessed with regard to their quality (reporting completeness) for hazard evaluation based on a previously developed set of criteria [8] by 4 independent evaluators. We aimed to shortlist studies that adequately addressed the key characteristics of the test substance, organism, and system for (re)use of data in a scientific context (“fit-for-purpose” data). Our aim was to enhance the transparency in terms of research materials, study design, and methodology, thus supporting the reuse of data. Based on a comparison of the evaluation results and a discussion, the criteria were extensively refined and specified. Afterwards, the refined criteria were once again applied to all of the studies to evaluate their quality. To add to the criteria list provided by Jemec Kokalj, Hartmann [8], mandatory, desirable, and voluntary criteria were defined, and a scoring system was implemented to determine accepted and rejected publications. Finally, a hazard evaluation was performed based on the accepted publications. Special attention was paid to mechanistic endpoints, in particular, whether their inclusion would contribute to defining the specificity of nanoPS hazard.

2. Materials and Methods

2.1. Literature Collection

For the case study, research papers reporting the effects of nanoPS on daphnids were collected. The first publication list was extracted from a science communication article on the potential effects of nanoPS on humans and the environment (https://nanopartikel.info/en/knowledge/materials/polystyrene/, accessed on 1 November 2020). A more elaborated search was then performed via the Web of Science platform using keywords related to the chosen test organism (“daphnids,” “daphnia,” “daphnia magna,” “daphnia pulex”) AND/OR test material (“polystyrene,” “polystyrene nanoparticles,” “polystyrene nanoplastics”) AND/OR endpoint (“toxicity,” “effect,” “hazard”). The last search was performed in August 2022. Studies that focused on uptake and fate of nanoPS in daphnids were excluded from the list as the presence of particles inside the organism per se does not necessarily imply hazard. All plastic-related terms we used according to the definition given in ref. [8]. The final list of studies used in the analysis is provided in Supplementary information Table S1.

2.2. Data Extraction and Organisation in Matrix Table

Subsequently, all studies were categorized according to the number of parameters in the matrix table (Table S1). The respective parameters were selected based on our key questions and related to the experimental set-up as well as mechanistic information on toxicological mode-of-action.
In addition to the main text, annexes and supplementary information were also thoroughly screened since these commonly contain important experimental details (i.e., toxicity assays and related methods) as well as information on particle characteristics. This allowed for an evaluation of potential connections between particle characteristics and hazard. For example, the use of sodium azide (NaN3) as a preservative for nanoPS suspensions was addressed, because the literature data indicated this substance was hazardous towards aquatic organisms [5,6,9]. Studies that did not provide any information on the use of NaN3 were also labelled since the toxic effects reported in these studies could be attributed exclusively to nanoPS as other potentially toxic substances were not disclosed. In addition, information about surface charge/functionalization was listed since this parameter is also discussed as influencing the hazard of nanoPS [10]. Second, a subset of studies evaluating mechanistic endpoints was constructed (Table S1). By considering endpoints other than apical ones, for example oxidative stress parameters or differential gene expression, we aimed to assess the potential modes of action for nanoPS. An overview matrix table (Table S1) was constructed listing relevant information for all studies, such as NaN3 content of nanoPS suspensions, particle surface functionalization, and type of endpoint reported (apical/mechanistic).

2.3. Study Evaluation, Refinement of Quality Criteria, and Scoring System

This step aimed at evaluating all the retrieved studies with regard to their quality, based on criteria developed by Jemec Kokalj, Hartmann [8]. The basis for this parameter list specific for nano- and microplastic particles was two quality evaluation approaches developed for engineered nanomaterials [11,12]. The criteria consisted of a set of parameters that should be listed in ecotoxicity studies on nanoplastics to enable judgement about the reliability of the hazard data. The initial set of criteria [8] was the first general compilation intended for micro- and nanoplastics, while it did not consider specific questions related to a particular type of organism and plastic particle. Hence, for the purpose of this study, the initial set of criteria was further refined and specified for Daphnia spp. and nanoPS hazard. This was performed using an iterative process after the evaluation upon information analysis by 4 independent evaluators.
The criteria were sorted according to 5 different categories: (1) polymer particle characteristics, (2) organism characterization and testing parameters, (3) sample preparation (dispersion of as prepared or delivered nanoplastics in media used for biological experiments), (4) nanoplastic characteristics in exposure medium, and (5) documentation of study results (see Table 1). Further, a scoring system was developed, defining the number of mandatory, desirable (not mandatory but important), and voluntary (not important) criteria. Subsequently, each study was evaluated for whether the criteria were met, and the score was calculated based on the number of met criteria and their relative importance. In our further assessment, we only included studies with complete traceability of the reported results based on the information provided. For further analyses, only the studies that met all mandatory criteria were used.

3. Results and Discussion

3.1. Description of Studies Retrieved and Organization in a Matrix Table

In total, 38 scientific papers were retrieved for this analysis [6,9,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48]. As different experimental set-ups (e.g., different particle sizes, toxicity endpoints) were applied in the studies, each of these was considered an ecotoxicity data point. This resulted in 91 different ecotoxicity data points provided altogether by the reviewed 38 papers.
Regarding sample (nanoPS) preparation for toxicity testing, as indicated in the matrix table, 64% of studies did not specify whether NaN3 was present or removed, 18% studies published data despite this chemical being present, and 21% of studies applied steps to remove it (e.g., dialysis, ultracentrifugation). Two studies compared the results of nanoPS with and without NaN3 [6,9]. Regarding particle functionalization, 59% studies did not provide any data regarding this issue, 31% of studies reported amino (NH2) functionalization, 25% of studies reported -COOH group functionalization, and 13% reported the presence of fluorescent dyes. Reasons for the non-reporting of NaN3 use and surface functionalization seemed to be twofold; first there was a lack of awareness of the potential side-effects of NaN3 or the impact of surface functionalization, despite the bulk of data on the latter in nanomaterial hazard research [49]. Second, importantly, it is often not easy to obtain the relevant information from commercial suppliers of nanoPS particles. In addition, equipment and expertise to analyze these two characteristics might not be widely available.
Most of the studies used D. magna (79%), followed by D. pulex (18%), and D. galeata (3%). Different types of endpoints were assessed. Following the adverse outcome pathway concept, we classified the studies in two groups: apical endpoints (mortality/immobility, growth, feeding and egestion, swimming behavior, reproduction, embryonic development, body adsorption) and mechanistic endpoints (oxidative stress, detoxification, immune-related processes, neurotoxicity, energy metabolism, heart rate, changes in gut epithelium, moulting related processes) according to Jeong and Choi [50]. More studies assessed apical endpoints (58%; 53/91) than mechanistic endpoints (42%; 38/91) (Figure 1). Expectedly, the standard toxicity endpoint for Daphnia spp., mortality/immobility, was the most studied (23.7% of all endpoints) (Figure 1). Compared to other apical endpoints, 40% of studies investigated mortality/immobility, followed by reproduction (15%), swimming behavior (13%), and growth (11%) (Figure S1). Some of the toxicity endpoints, such as feeding rate, body adsorption, and swimming behavior, had specific relevance for particulate chemicals. For filter feeders such as Daphnia spp., physiological changes related to food intake are very relevant for nanoscale particles, as well as body adsorption e.g., ref. [18,51,52]. Additionally, physical interaction of particles is related to changes in moulting in daphnids or inflammatory changes in the gut epithelium upon ingestion [53]. Among the mechanistic endpoints, detoxification and oxidative stress were the most studied (each ~12% of all endpoints and ~30% of mechanistic endpoints). These endpoints are commonly used biomarkers for other substances as well [54]. Energy metabolism endpoints were the third most applied (18.4% of mechanistic endpoints) due to the presumed interference of particles with feeding and the energy budget [55] or lipid storage of animals [13].
In general, the endpoints used for hazard identification of nanoPS are also been commonly applied to other types of pollutants. For example, the commonly used positive control substance potassium dichromate (K2Cr2O7) due to its oxidative properties has also been used for evaluation of non-standardized toxicity endpoints such as swimming [56,57], feeding [58,59], heart rate [60], breathing [61], and neurotoxicity [62]. However, no data on body adsorption, growth, or embryonic development were available, as changes in these endpoints are probably not expected for this substance (Figure S2).

3.2. Refinement of Study Quality Criteria for Hazard Evaluation of NanoPS with Daphnia sp.

The criteria previously proposed for micro- and nanoplastics in general were taken as the basis for the evaluation of all nanoPS ecotoxicity studies with daphnids [8]. After an evaluation of the 38 studies by 4 independent experts, we came to the following conclusions:
(1)
The initial criteria needed additional explanation in order to avoid misinterpretation by the different evaluators. Detailed explanations for the existing criteria elaborated in this study are given in Table 1.
(2)
Some criteria needed nanoPS property-specific clarifications. NanoPS are usually sold as suspensions, hence some physico-properties cannot be provided (e.g., surface area) or are less commonly provided in general for nanoplastics (density, porosity, crystallography, radical production capacity, magnetic properties, surface charge, and concentration of particles prior/after the test). A common property for nanoPS commercial suspensions is the addition of NaN3, which acts as an antimicrobial additive. Specific attention was thus given to define criteria related to additives and impurities.
(3)
Authors commonly refer to standard guidelines being followed for daphnids, but this was often not true in all aspects. Specifically, when the authors referred to the use of a modified standard, the study should be thoroughly checked for all methodological details. For example, the type of medium was rarely reported, but a reference to the standard was made. However, the standard itself does not define which type of test medium should be used but provides several options. Hence, each study should specify the exact type of medium used. Additionally, the standards specifically define that some media parameters, such as pH and dissolved oxygen, should be measured, but this was rarely reported. When it comes to the use of a reference chemical in the tests, this criterion should be reported in studies regardless of the standard mentioned. This is because the standards do not define the reference chemical that should be tested in each experiment.
(4)
Searching for specific data in the manuscripts was not user friendly. We used the automatic built-in search tool in the pdf viewer whenever possible, but this was commonly not sufficient. The information in manuscripts was scattered and did not following a uniform nomenclature. Accordingly, this might have introduced a bias into the evaluation, as some criteria might have been met, but the information was not found by the evaluator. Additionally, data were available in the supplementary materials, which are not commonly the main focus of readers.
(5)
The authors did not commonly provide all data related to the nanoplastics tested or the test methodology but rather referred to previously published work. This led to additional work to search for previously published papers and these data might be missing for proper interpretation of the toxicity data provided. Hence, a minimum set of physico-chemical properties of nanoplastics and methodological details should be available in each study. Transparency, openness, and reproducibility of research are already recognized as vital features of scientific research and data reuse aligned with FAIR principles [63]. The minimum reporting information (MRI) as identified in our study covered fundamental aspects that should be included already in research planning and later in research reporting [64]. Only more complete and transparent reporting could facilitate data search and reuse as well as study quality evaluation [65]. We suggest an MRI, as presented in Supplementary information (Table S2).

3.3. Classification of Criteria Importance and Threshold Development

The overarching aim of the quality assessment of studies was to provide judgement whether a study passed the evaluation, i.e., whether the data were fit for the purpose of hazard evaluation. Such a scoring system was not yet in place in Jemec Kokalj, Hartmann [8]. Hence, in order to provide a system for acceptance or rejection of studies, criteria first needed to be ranked according to their importance. We relied on a ranking elaborated previously for nanomaterials in the scope of the GUIDEnano framework [11], the DaNa project [12], and our personal experience/judgement. All the criteria that were defined as mandatory (or red questions) by these existing frameworks were also considered mandatory in the current study (Table 1). Mandatory criteria were those that addressed the most important aspects of the development/reporting of the study and were considered as very relevant for the study to be considered of acceptable quality for hazard assessment [11]. The second category included criteria with “medium importance (desirable)” that would enhance the quality of the study and enable advanced interpretation of the toxicity data, but were not crucial for the study to be considered acceptable. The third category included the “not important (voluntary)” criteria, which referred to data that were important for other engineered nanomaterials but were not as relevant for nanoPS. Following this approach, we classified the studies meeting all the mandatory criteria as acceptable for hazard assessment and the rest as not acceptable.

3.4. Results of Study Quality Evaluation of NanoPS Toxicity Studies with Daphnia spp.

The results of the quality evaluation for each individual criterion are presented in Figure 2. Most of the studies passed the criteria related to test organism characterization and testing parameters, where at least 92% of all studies fulfilled the mandatory criteria. The criteria with the least records were related to natural particle control (0%) and verification of micro/nanoplastics background (0%). It has been suggested that natural particles could be tested to evaluate whether the effects of anthropogenic particles differ from natural ones [66]. Background contamination of laboratories is needed to rule out whether the effects are indeed on the account of the nanoplastics tested [67]. Compared to organism characterization and test parameters, much lower scores were found for physico-chemical properties of nanoPS. This result was in line with a previous review [2]. The data on impurities and additives, which are considered mandatory, are a bottleneck (only 36 % of studies reported impurities or additives, respectively). The rest of the mandatory criteria in this category were fulfilled by at least 82% studies. Very few studies provided information on sample preparation; surprisingly only 64% studies defined the type of test medium and 41% reported test medium conditions (pH, oxygen). Additionally, few studies reported nanoPS properties in the exposure medium, such as particle behavior and surface charge. These, however, are not considered mandatory but desirable criteria. With regard to statistics, 100% of the studies used relevant statistical approaches to evaluate the data, but very few studies defined the specific validation criteria and their acceptability for a certain study.
Taking into consideration the threshold that all mandatory criteria needed to be fulfilled in order for the study to pass the evaluation according to Fernández-Cruz, Hernández-Moreno [11,12], only 18% of studies (7 out of 38) passed. We considered this to be a very low score and hence only 7 studies were available for further analysis of nanoPS hazard in Daphnia spp.

3.5. Hazard Evaluation based on Accepted Studies

Hazard evaluation was performed based on the accepted publications, i.e., those having met all the mandatory criteria. Among the seven studies that were classified as high-quality studies according to our refined criteria (Table 1), six studies evaluated apical endpoints [6,25,28,32,38,48]. From these studies, five studies concluded that the tested concentrations of nanoPS induced acute toxicity to D. magna. However, Heinlaan, Kasemets [6] emphasized that nanoPS was acutely toxic to D. magna only in the presence of additives (undisclosed surfactants and biocidal NaN3) and no more toxicity was detected once the nanoPS suspension was dialyzed. The other five studies reported the use of stabilizers in particle synthesis, however, they either concluded that the concentrations of these chemicals were kept below the toxicity thresholds of D. magna [25], or that they were removed before the acute toxicity assays were performed [28,32,38,48].
Mechanistic endpoints were investigated in only one high-quality study [14]. An OECD-adopted acute toxicity test with prolonged exposure of 96 h was performed. NanoPS were tested in the presence and absence of humic acid. In this study, detoxification genes were used to characterize the toxic response of neonates to the nanoPS exposure. They concluded that nanoPS exposure induced immobility and resulted in upregulation of all genes studied, while humic acid was demonstrated to have a detoxifying effect against particulate toxicity.
All in all, there was no consensus on the hazard potential of nanoPS for Daphnia spp. even among the comparable high-quality studies.

4. Conclusions

In the absence of regulatory guidance for evaluation of the environmental hazard of polymers, data quality evaluation for the selection of fit-for-purpose data has been developed. As an example, a case study of polystyrene nanoplastics (nanoPS) hazard towards aquatic model organisms, Daphnia spp., was conducted.
Among the reviewed 38 studies on nanoPS ecotoxicity for Daphnia spp., only 18% of the studies passed the evaluation for usability in hazard evaluation. Due to the low number of studies containing fit-for-purpose data for hazard evaluation, we were unable to perform a hazard evaluation for nanoPS. As shown in the quite specific case of Daphnia spp. ecotoxicity, it became apparent that the reporting of key characteristics of the test substance, organism, and system is essential, and the criteria probably need refinement for other test systems and organisms.
According to the criteria defined and elaborated in this study, the majority of studies are not providing sufficient information on nanoPS characteristics (e.g., surface functionalization and additive content in suspension) and the test system to evaluate the hazard of nanoPS towards Daphnia spp.
Based on the 7 studies that passed our evaluation, no consensus was reached regarding nanoPS hazard. Specifically, mechanistic information was scarce that would have added to our knowledge of nanoPS mechanisms of action.
Overall, this study provides the following novelties:
  • Refining the existing criteria and providing an unambiguous description of each criterion for the selected case study served the purpose of re-evaluating data in scientific contexts (here: evaluation of polystyrene nanoplastic hazard);
  • A scoring system and threshold values for acceptance of studies/data for the purpose of hazard evaluation were developed;
  • A data reporting template for future studies, providing a list of criteria that authors can follow, was created that facilitated the retrieval of relevant information for data quality evaluation (Table S2).
  • No consensus on the hazard potential of nanoPS for Daphnia spp. was determined, even among the high-quality studies.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/nano13030536/s1, Figure S1: Share of ecotoxicity data points; Figure S2: Distribution of apical and mechanistic endpoints for potassium dichromate; Table S1: Matrix table; Table S2: Minimum reporting information.

Author Contributions

Conceptualization, A.J.K., D.D. and D.K.; Data curation, A.J.K.; Formal analysis, A.J.K., M.H., S.N. and D.K.; Funding acquisition, M.H., D.D. and D.K.; Methodology, A.J.K., M.H., S.N., D.D. and D.K.; Software, A.J.K.; Visualization, A.J.K. and S.N.; Writing—Original draft, D.K.; Writing—Review & editing, A.J.K., M.H., S.N., D.D. and D.K. All authors have read and agreed to the published version of the manuscript.

Funding

This work received funding from the European Union’s Horizon 2020 Research and Innovation programme, under Grant Agreement number 965367 (PlasticsFatE) and Grant Agreement Number 814530 (NANORIGO). D.K. received funding by the Federal Ministry of Education and Research (BMBF) (DaNa4.0 Information on the safety of new, innovative materials and nanomaterials, FKZ 03XP0282). Part of the research was funded by Slovenian Research Agency (J1-2482, P1-0184) and bilateral exchange agreement between ARRS and DAAD BI-DE/20-21-008. M.H. was funded by the Estonian Ministry of Education and Research (grant PRG1427).

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Share of ecotoxicity studies with the applied toxicity endpoints in nanoPS hazard studies using Daphnia spp. (n = 91, total number of ecotoxicity data points). Apical endpoints are shown as cold colors, mechanistic endpoints as warm colors.
Figure 1. Share of ecotoxicity studies with the applied toxicity endpoints in nanoPS hazard studies using Daphnia spp. (n = 91, total number of ecotoxicity data points). Apical endpoints are shown as cold colors, mechanistic endpoints as warm colors.
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Figure 2. Share of studies (%) fulfilling the refined criteria. Red asterisks (*) indicate the criteria considered as very important (mandatory) according to Table 1.
Figure 2. Share of studies (%) fulfilling the refined criteria. Red asterisks (*) indicate the criteria considered as very important (mandatory) according to Table 1.
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Table
Table 1. Data quality criteria and relevance for hazard evaluation of impact studies on polystyrene nanoplastics (nanoPS) using Daphnia spp. Criteria were designed based on Kokalj et al. (2021) where the rationale for the suitability for nanoplastics is provided. The relevance of criteria was classified as mandatory (very important), desirable (of medium importance), and voluntary (not important).
Table 1. Data quality criteria and relevance for hazard evaluation of impact studies on polystyrene nanoplastics (nanoPS) using Daphnia spp. Criteria were designed based on Kokalj et al. (2021) where the rationale for the suitability for nanoplastics is provided. The relevance of criteria was classified as mandatory (very important), desirable (of medium importance), and voluntary (not important).
No. CriteriaSpecific Guidance for Evaluator- Example for NanoPSRelevance
1. PrimaryProperties of Nanoplastics
1Polymer chemical composition (CAS No)This refers to the type of polymer, e.g., polystyrene (PS).Mandatory
2Origin of nanoplastics (commercial supply, laboratory production) including protocol of nanoplastic production and collection if relevantProtocol for field collection is not relevant for primary nanoPS.Mandatory
3Is the primary particle size stated?This refers to the diameter of particles. Mandatory
4Is the morphology (shape) stated or graphically presented?This criterion is also valid if a microscopy image is provided. Most nanoPS are spheres and hence commonly not explicitly mentioned in studies. Mandatory
5Is the specific surface area of nanoplastic powders (e.g., BET surface) given?This criterion is not valid for nanoPS in suspensions, because BET is measured on powders. Desirable
6Are surface charge and chemistry (any of the following: unlabeled, fluorescent label, functionalization, hydrophobic, hydrophilic, …)/coatings/modifications described?Special attention should be paid to information regarding labeling. If information on labeling is provided, this is considered as information on particle modification. This criterion is also valid if the study provides information that PS NPs are unlabelled.Mandatory
7Is the polymer density stated?This is not commonly reported for nanoPS.Voluntary
8Is porosity stated?This is not commonly reported for nanoPS.Voluntary
9Is the crystallography (crystalline or amorphous phase) and phase analysis (pure or mixed) stated?This is not commonly reported for nanoPS.Voluntary
10Is the concentration and/or identity of chemical impurities stated to the suspension? Or have any other steps (e.g., dialysis) been taken to remove impurities?Impurities are chemicals that are present in plastics and/or plastic suspension but were not added intentionally. They might originate from synthesis. If the authors report that no impurities were present, this is a valid information. This criterion is also fulfilled if any step to remove impurities (e.g., dialysis, ultracentrifugation) has been undertaken.Mandatory
11Is the concentration and/or identity of chemical additives stated for the suspension? Or have any other steps (e.g., dialysis; ultracentrifugation) been taken to remove additives?Additives are chemicals added to plastics and/or plastic suspension to improve their processability, properties, and performance. They are, thus, an essential part of the formulation. The most common additive of nanoPS suspension is sodium azide (NaN3). If the authors report that no additives were present, this criterion is also fulfilled.Mandatory
12Is the concentration and identity of other chemical pollutants stated?This refers to pollutants that might sorb to nanoPS during use and disposal and is relevant only for secondary nanoPS.Voluntary
13Other relevant information (i.e., radical production capacity, magnetic properties for composite nanoplastics particles with magnetic substances)This is not commonly reported for nanoPS.Voluntary
2. Organism Characterization and Testing Parameters
14Was the test species identified?Specific species of Daphnia are not required by the standard guidelines. Hence, different species can be used (D. magna, D. pulex, D. galeata, etc.) Mandatory
15Were any of the characteristics of the test organism (strain, sex, length, body weight, age, growth stage) stated?Any of these characteristics should be reported, preferably at least age. Mandatory
16Was the number of replicates per group given?If the test was done exactly according to the standard, it is assumed that this information was provided by referring to the standard.
If use of the Daphtox kit is referenced, it is assumed that this information is provided 		Mandatory
17Was the duration of the exposure stated (e.g., 48 or 96 h)? Mandatory
18Are the frequency and duration of the exposures, as well as the time-points of the observation explained?This applies to additional observations during the time of exposure. Desirable
19Was the type of exposure (e.g., static, flow through, diet) stated?This is usually not specifically defined, but if medium is not changed during the exposure, this refers to a static test. In chronic tests, the reference to the standard implies feeding the daphnids.Mandatory
20Are the number of concentrations and concentration range tested reported? Mandatory
21Were appropriate controls (e.g., solvent or negative control) used?Solvent control is usually not used with nanoPS, but negative control is.Mandatory
22Were parallel toxicity tests conducted with reference chemical or positive control (to ensure performance of the test system)?This criterion is fulfilled if the authors report on the use of reference chemical. Referring to standard guideline is not sufficient, because standards do not define that the reference chemical should be tested in each experiment.Desirable
23Was a reference (natural) particle used (to improve interpretation of environmental relevance)?This is not commonly reported for nanoPS.Desirable
24Was the biological endpoint of the toxicity study under evaluation stated and defined?Definition of endpoint is most important when mechanistic endpoints are studied.Mandatory
25Was the biological effect quantified (e.g., LC50, EC50, NOEC, LOEC, 25% effect, BCF, BAF) or statistical significance determined (for mechanistic endpoints)? Mandatory
26Was nano(micro) plastic background verified?This refers to background in the laboratory not in the organism. Desirable
3. Sample Preparation (Dispersion of as Prepared or Delivered Nanoplastic In Media Used for Biological Experiments)
27Was the type of test medium or vehicle used stated?Although the reference to the standard method is made, the type of test medium (M4, M7, Elendt, tap water) should be reported because the standard does not specifically define the type of test medium.Mandatory
28For ecotoxicity studies, were mandatory exposure medium conditions (at least pH and oxygen) measured? Or is this implied by mentioning the test method used, e.g., USEPA, OECD, ASTM?Standards specifically define that some properties, pH and dissolved oxygen, should be measured. This criterion is valid if the authors mention the use of the standard guideline. The test should be done exactly according to the standard. If the standard was modified, this criterion should be checked specifically in the paper.Mandatory
29For ecotoxicity studies, were any other exposure medium conditions measured?This includes hardness, conductivity, etc.Desirable
30Were the protocols of dispersion and characterization in the exposure medium identified? Or, were the protocols of preparation of exposure medium stated?This includes sonication, vortexing, mixing, etc.Mandatory
4. Nanoplastic Characteristics in Exposure Medium
31Is the particle behavior before or during the exposure described (i.e., agglomeration / aggregation / sedimentation / floating)?This includes any type of measurement to describe the behavior of particles in exposure medium, such as DLS, nanoparticle tracking analysis, visual observation of sedimentationDesirable
32Is the surface charge before or during the exposure described?This is not commonly reported for nanoPS.Voluntary
33Is the concentration before or during the exposure measured?This is not commonly reported for nanoPS.Voluntary
34Are the concentration and identity of leached chemicals from nanoplastics under experimental conditions stated?This is not commonly reported for nanoPS.Desirable
35Is the formation of the biocorona / biofouling described?This is not commonly reported for nanoPS.Voluntary
5. Study Results Documentation
36Was an appropriate statistical method or model used to determine toxicity?This refers to the method used to calculate effect values or statistical difference in comparison to control. The method should take into consideration the normality and homoscedascity of data.Mandatory
37Were the potential overload, attachment, and physical effects in organisms described?Usually the authors report the adsorption of particles or physical attachment.Desirable
38Were the test acceptability criteria stated (e.g., mortality in control must not exceed a certain percentage)? OR, were test acceptability criteria implied by mention of the test method used (e.g., USEPA, OECD, ASTM, etc)?Standards specifically define the validity criteria. This criterion is valid if the authors mention the use of the standard guideline. The test should be performed exactly according to the standard. If the standard was modified, this criterion should be checked specifically in the paper.Desirable
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Jemec Kokalj, A.; Heinlaan, M.; Novak, S.; Drobne, D.; Kühnel, D. Defining Quality Criteria for Nanoplastic Hazard Evaluation: The Case of Polystyrene Nanoplastics and Aquatic Invertebrate Daphnia spp. Nanomaterials 2023, 13, 536. https://doi.org/10.3390/nano13030536

AMA Style

Jemec Kokalj A, Heinlaan M, Novak S, Drobne D, Kühnel D. Defining Quality Criteria for Nanoplastic Hazard Evaluation: The Case of Polystyrene Nanoplastics and Aquatic Invertebrate Daphnia spp. Nanomaterials. 2023; 13(3):536. https://doi.org/10.3390/nano13030536

Chicago/Turabian Style

Jemec Kokalj, Anita, Margit Heinlaan, Sara Novak, Damjana Drobne, and Dana Kühnel. 2023. "Defining Quality Criteria for Nanoplastic Hazard Evaluation: The Case of Polystyrene Nanoplastics and Aquatic Invertebrate Daphnia spp." Nanomaterials 13, no. 3: 536. https://doi.org/10.3390/nano13030536

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