Percentages observed for the “immobilization” endpoint in young individuals, in relation to experimental exposure parameters.
The series of experiments presented in the paper served to clarify the effects of contemporary exposure to surfactant, microplastics (polyethylene and polyvinyl chloride), and nanoparticles (TiO2 and ZnO) on the model organism Daphnia magna. Exposure was evaluated with respect to the age of the organisms (“young”, 24 hours old, and “aged” 10 days old specimens), trophic status (feeding or fasting), and the simultaneous presence of a surfactant. All the above-mentioned substances are present in the wastewater coming from various environmental sources from cosmetic products. The experiments were conducted in compliance with the OECD 202:2004 guideline, which is also a reference for ecotoxicity tests required by REACH. The results showed that surfactants enhance effects of toxicity produced by the exposure to the microplastic + nanoparticle mixtures. The influence due to factors such as nutrition (effect in fasting >> feeding conditions) and the age of individuals (effects in older >> younger animals) is essential. Concerning young individuals, exposure to PE-TiO2 is the most significant in terms of effects produced: it is very significant, especially in the presence of surfactant (both under fasting and feeding conditions). On the contrary, exposure to the PE-Zn mixture shows the minor effects. The comparison with the literature, especially as regards the possibility of interpreting the toxicity trends for the various mixtures with respect to the individual elements that compose them, leads to hypothesize additive effects still to be investigated and confirms the greatest toxicity contribution of TiO2.
- metal-oxide nanoparticles
- fasting and feeding conditions
- municipal wastewater treatment plants
- toxicity tests
Municipal wastewater treatment plants (MWWTP) are renowned hot-spot sources of a wide variety of pollutants from human activities for the aquatic environments . Nutrients , surfactants , microplastics (MPs; ) and nanoparticles (NPs; ) are significant components of mixtures released by MWWTP, coming from different sources, not least that represented by cosmetic products.
NPs are chemicals, between 1 and 100 nm in size , both of natural (i.e., humic and fulvic acids, organic acids, fullerenes, and metals) and artificial origin (TiO2, ZnO) [6, 7]. Many products of common use (pharmaceutical and personal care products, plastic, rubber, paints, etc) are based on NPs and this contributes to their massive presence in the environment .
MPs can derive from industrial pellets used in the manufacture of plastic objects or from consumer products, such as cosmetics, abrasive products and objects containing microbeads and glitters (primary microplastics) or from the fragmentation of larger plastic objects (secondary microplastics) [3, 9, 10, 11]. They are pollutant of great environmental concern , affecting feeding habits, and reproductive success of many organisms . NPs and MPs enter the trophic chains when eaten by detritivores and filter feeders [14, 15, 16, 17].
Even if wastewater treatment processes retain a large fraction of plastic microparticles , in sewage the MPs not removed by plants reach the rivers and, ultimately, the sea . For what concerns NPs, discharges of nano-oxides occur due the sorptive removal of organic contaminants from wastewater  purification purposes, together with unintentional releases.
Both, MPs, and NPs are found in the mix of substances present in wastewater and water bodies, together with surfactants. Surfactants have direct toxicity on aquatic species  but can also vehicle other substances due to the formation of micelles  which can affect pollutant sorption/desorption from MPs surfaces . Surfactants could represent the way to increase the interaction among microplastics and animals and therefore lead to negative effects on exposed animals .
Toxic effects due to the exposure to complex mixtures differ from the exposure to single substances, even if compounds are at low levels . In this case, NPs and MPs toxicity could be affected both by the nutrient induced microalgal growth and by synergic/antagonistic interactive effects due to surfactants . The presence of metal-oxides NPs, MPs, and surfactants in effluents from MWWTP suggests deepening their ecotoxicity to assess the real effects on aquatic environments.
Despite the increasing interest, recent meta-data analysis underlined low standardization of in vitro tests  and the largest number of experiments performed on single kind MP/NP. In Europe, the placing on the market of new formulations implies the verification of their environmental compatibility in accordance with the current regulation (REACH).
This study aims to fill some important knowledge gaps on NPs and MPs ecotoxicity. It evaluates ecotoxicological responses of
2. Material and methods
2.1 Experimental design
The experiments were performed under the OECD 202:2004 guideline . Dispersions were made by suspension of tested particles in UNI EN ISO 6341:2012 standard freshwater. Experiments previously reported [28, 29] allowed to determine for each toxicant the dose permitting the survival of a significant fraction of the tested population until the end of the exposure time (96 h).
Figure 1 summarizes the experimental design. Mixtures of NPs (n-ZnO and n-TiO2) and MPs (PE and PVC) were used for the exposure experiments, adding/not adding a non-ionic surfactant (Triton X-100, CAS n. 9002-93-1; tested at 0.001% v/v according to ) to improve the dispersion of MPs and NPs in tested samples; results were compared to controls to test ecotoxicological effects of the NPs-MPs and NPs-MPs-surfactant mixtures. Furthermore, we also exposed to dispersions of microplastics + surfactant animals that at the beginning of the experiment had 10 days of life (called “aged”) to evaluate the effect of aging on the ecotoxicological responses. Animals were exposed under fasting conditions, selecting immobilization as endpoint and a contact time 24-48 h. Contextually to standard conditions required by OECD, animals were also exposed under feeding conditions and contact time was extended from 24 to 48 h to 96 h as suggested by the literature for tests performed on particulate toxicant  performing observations daily starting from T24 after the initial exposure. Experiments were made during an 8:16 dark/light exposure cycle .
ZnO and n-TiO2 were tested at 1.12 and 113.18 mg/L respectively; microplastic doses were 0.05 mg/L. The selection of microplastics to be tested was done according to Renzi et al. .
Dispersions of both single metal-oxides NPs, MPs and mixtures were characterized by microscopy coupled to Fourier Transformed-Infrared spectrometer (μFT-IR, model Nicolet i-10 MX equipped with ATR detector, Thermo®). The formation of clusters of nanoparticles in the mixtures was verified at the micrometric scale even in conditions with the addition of food. Survival rates % of exposed animals compared to negative controls were used as target endpoints.
2.2 Equipment and materials
Experimental condition for
2.3 Quality assurance and quality control
Ecotoxicological tests were performed following UNI EN ISO 17025 guidelines to ensure quality control of collected results. Laboratory performed experiments ensured to pass inter-calibration exercises performed on annual basis on
3. Results and discussion
The results obtained from the exposure experiments are summarized in Table 1 (young specimens) and Table 2 (aged specimens). In the following text, the term significant refers to statistically significant based on the results of the statistical analysis carried out on the results obtained from the exposure experiments considering immobilization as the endpoint.
|Feeding conditions (MPs 0.05 mg/L; n-TiO2 113.18 mg/L)||Feeding conditions (MPs 0.05 mg/L; n-ZnO 1.12 mg/L)|
|Fasting conditions (MPs 0.05 mg/L; n-TiO2 113.18 mg/L)||Fasting conditions (MPs 0.05 mg/L; n-ZnO 1.12 mg/L)|
|Feeding conditions (MPs 0.05 mg/L; n-TiO2 113.18 mg/L; Surfactant)||Feeding conditions (MPs 0.05 mg/L; n-ZnO 1.12 mg/L; Surfactant)|
|Fasting conditions (MPs 0.05 mg/L; n-TiO2 113.18 mg/L; Surfactant)||Fasting conditions (MPs 0.05 mg/L; n-ZnO 1.12 mg/L; Surfactant)|
As for both young and aged individuals, exposure to PE-TiO2 is the most significant in terms of effects: it is very significant, especially in the presence of triton (both fasting and feeding). On the contrary, exposure to the PE-Zn mixture shows the minor effects (Figure 2). This result, not linear if we consider that there is no clearly identifiable trend in the toxicity levels of the NPs/MPs, leads to think that more than the actual toxicity of the individual elements of the mixtures, there is a significant additivity effect. This estimation would also be legitimized by the comparison with previously obtained results: PVC, in presence of surfactant, resulted the most toxic among tested dispersions for both neonates and aged
On the other hand, in Renzi and Blašković , n-ZnO resulted less effective than n-TiO2 in leading to the target endpoints (immobilization and death) in exposed
3.1 Surfactant effect
The effect of surfactant (Triton X-100) was significant in all the experiments carried out on young organisms, compared to the control batches without the exposure to surfactant. For this reason, and to simplify the factors considered, making the observed effects more evident, it was decided to always add surfactant to the mixtures of contaminants to which adult organisms were exposed.
Figure 3 shows the contribution of surfactant presence/absence under different trophic conditions in young organisms exposed to NPs+MPs mixtures, in terms of percentages of mobile organisms at the target times. These effects could be because surfactants seem to improve the contact among microplastics and animals and therefore cause effects on exposed specimens . In a previous study , exposure to microplastics + surfactant showed the highest toxicity on
3.2 Feeding effect
The effect of feeding is almost always significant (Figure 3) on the toxicity of mixtures of contaminants on
In the case of adult individuals (Figure 2), the effect of feeding was significant in 5 out of 8 cases: in each experiment for PE+n-TiO2, partially following to exposure to Zn (with PE and PVC always at 96 hours) and with PVC+n-TiO2 at 24 hours.
It can be hypothesized that with the intake of food (phytoplankton) there is an increase in the metabolic rate, resulting in a better chance of detoxifying by
3.3 Aging effect
The results achieved provides evidence that the level of toxicity of NPs+MPs mixtures depends on the age of the animals, confirming what yet seen following tests with several kinds of MPs . The effect of aging (comparison between experiments carried out on young people and adults, Figure 2) was significant (affecting the possibility of remaining mobile or alive) in half of the experiments carried out. While results at 24, 48 and 72 hours appear controversial, the significance of a toxic effect clearly emerges at 96 hours, both for PE+n-ZnO and for PE+n-TiO2. Significant aging effect also for PE+n-TiO2 at 96 hours and PVC+n-TiO2 at 96 hours have been registered.
The results obtained in this study made it possible to identify the mixture PE+n-TiO2 as the most toxic following exposure of
Authors are grateful to BsRC research centre (Italy) that founded and completely supported this research. Funding: This work was supported by Bioscience Research Center [grant number RG2020009].
|MWWTP||municipal wastewater treatment plants|
|REACH||European Regulation on Registration, Evaluation, Authorisation and Restriction of Chemicals|
|OECD||organization for economic co-operation and development|
|μFT-IR||micro Fourier transform interferometer|
|ATR||attenuated total reflection|
|DOM||dissolved organic carbon|