**Abstract**

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.

**Keywords:** metal-oxide nanoparticles, surfactants, fasting and feeding conditions, microplastics, municipal wastewater treatment plants, toxicity tests

### **1. Introduction**

Municipal wastewater treatment plants (MWWTP) are renowned hot-spot sources of a wide variety of pollutants from human activities for the aquatic environments [1]. Nutrients [1], surfactants [2], microplastics (MPs; [3]) and nanoparticles

(NPs; [4]) 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 [5], 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 [8].

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, 9–11]. They are pollutant of great environmental concern [12], affecting feeding habits, and reproductive success of many organisms [13]. NPs and MPs enter the trophic chains when eaten by detritivores and filter feeders [14–17].

Even if wastewater treatment processes retain a large fraction of plastic microparticles [18], in sewage the MPs not removed by plants reach the rivers and, ultimately, the sea [19]. For what concerns NPs, discharges of nano-oxides occur due the sorptive removal of organic contaminants from wastewater [20] 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 [21] but can also vehicle other substances due to the formation of micelles [22] which can affect pollutant sorption/desorption from MPs surfaces [23]. Surfactants could represent the way to increase the interaction among microplastics and animals and therefore lead to negative effects on exposed animals [22].

Toxic effects due to the exposure to complex mixtures differ from the exposure to single substances, even if compounds are at low levels [24]. 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 [25]. 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 [25] 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).

*Daphnia magna* (Cladocera) is a crucial model freshwater species for ecotoxicological tests due to the well standardized procedural practice (i.e., OECD standards) for experimental exposure [26].

This study aims to fill some important knowledge gaps on NPs and MPs ecotoxicity. It evaluates ecotoxicological responses of *D. magna* exposed complex mixtures (NPs + MPs in presence/absence of surfactant). Effects attained under fasting conditions (OECD guidelines) are matched to those achieved following the exposure of animals under feeding conditions, supposed as natural and with animals of different ages.

#### **2. Material and methods**

#### **2.1 Experimental design**

The experiments were performed under the OECD 202:2004 guideline [27]. Dispersions were made by suspension of tested particles in UNI EN ISO 6341:2012 standard freshwater. Experiments previously reported [28, 29] allowed to

#### *Action of Surfactants in Driving Ecotoxicity of Microplastic-Nano Metal Oxides Mixtures… DOI: http://dx.doi.org/10.5772/intechopen.99487*

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 [22]) 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 [30] performing observations daily starting from T24 after the initial exposure. Experiments were made during an 8:16 dark/light exposure cycle [31].

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. [29].

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.

**Figure 1.** *Logic model of the exposure experiments on* D. magna*.*

### **2.2 Equipment and materials**

Experimental condition for *D. magna* (MicroBioTest Inc. ephippia) storage, hatches and preliminary treatments were the same described by Renzi and Blašković [28]. Collection of organisms was standardized at 90 h after the start of incubation. All reagents were purchased from Caelo or Sigma-Aldrich. Experimental conditions and sets were the same previously described by Renzi et al. [29] and Renzi and Blašković [28] to allow a comparison of reported data on toxicity of mixtures tested in this study with results obtained by previous research on single components of the tested mixtures. Also, experimental conditions are the same to allow a complete comparability of obtained results. Chemicals features and properties of nanoparticles, microplastic, and surfactant tested in this study are the same for chemicals reported by Renzi et al. [29] and Renzi and Blašković [28].

#### **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 *D. magna* immobilization. During experiments pH and DOM were measured to verify that they remained within the limits of acceptability defined by OECD (202:2004) [27]. Mortality of animals in negative controls shall be included within 0-10% not to invalidate tests. LC50 of chemical solution used as reference to test animals' responses was acceptable (0.6–2.1 mg/L). Test were performed in triplicates (n=3); results obtained by the exposure to samples were compared statistically with responses obtained by negative controls; significance (p<0.01) of observed differences among mean values was tested by T-test while differences among variances were explored by F-test (Prism® 4.0). Results reported in this study are mean values (standard deviation, SD), normalized concerning negative controls.
