Introduction

Nanoparticles (NPs) have received great attention due to their unique properties and their extensive use in optical, physical, chemical, and biological processes, and also to their potential effects on the ecosystem and human health (Moore 2006; Wiesner et al. 2006). There are various studies that have been conducted on the widely used nanomaterials (NMs), such as fullerenes, TiO2, ZnO, CuO, and Ag (Matranga and Corsi 2012).

ZnO NPs are used in the production of pigments, semiconductors, and food additives (Manzo et al. 2013; Ma et al. 2013); in transparent UV protection films and chemical sensors; as preliminary materials for electronics (Meulenkamp 1998); and as UV-filters in sunscreens (Serpone et al. 2007; Manzo et al. 2013; Ma et al. 2013). Additionally, ZnO NPs have been shown to be effective in a range of environmental control technologies from remediation of environmental pollutants to medical disinfection (Hoffmann et al. 1995).

TiO2 NPs are used in photocatalytic water purification (Hagfeldt and Gratzel 1995; Wang et al. 2008), as UV-blocking agent in sunscreens (Popov et al. 2005), and possibly used in the new generation of solar cells (Usui et al. 2004).

Both ZnO and TiO2 NPs are widely used in sunscreens and cosmetics because of their photoactivity (Aitken et al. 2006). The international production of NPs for sunscreen products alone was approximately 1000 t during 2003–2004, consisting mainly of TiO2 and ZnO particles (Borm et al. 2006). The use of TiO2 and ZnO NPs in sunscreens means a major source of these materials in the marine environment (Baker et al. 2013). On average, 25 % of sunscreens will be washed off on immersion (Danovaro et al. 2008) and estimates indicate the potential for some 250 t of sunscreen-originated NPs to enter the marine environment each year (Wong et al. 2010).

Different research groups using different toxicity assays frequently reach diverse conclusions on the nanotoxicity and its underlying mechanism (Menard et al. 2011). Studies conducted on microalgae using ZnO NPs showed this metal oxide to cause growth inhibition of Pseudokirchneriella subcapitata (Franklin et al. 2007; Aruoja et al. 2009) and Dunaliella teriolecta (Manzo et al. 2013); decrease viable cells of Chlorella sp. (Chen et al. 2012); depress growth rate of four marine phytoplankton species including Skeletonema marinoi, Thalassiosira pseudonana, Dunaliella teriolecta, and Isochrysis galbana (Miller et al. 2010), inhibit photosynthetic activity of Anabaena flos-aquae cyanobacteria and to cause cell death in Euglena gracilis euglenoid microalgae (Brayner et al. 2010). Zhou et al. (2014) have reported that ZnO NPs were toxic to Chlorella vulgaris and the toxicity was dose-dependent. Although Zn (or ZnO) maybe nutritive at low concentrations, it becomes toxic when over accumulated (Franklin et al. 2007; Miao et al. 2010).

TiO2 NPs have shown similar effects on microalgae (e.g., Hund-Rinke and Simon 2006; Velzeboer et al. 2008; Cardinale et al. 2012). In contrast, TiO2 was found to have no effect on Pseudokirchneriella subcapitata photosynthetic activity after short-term exposure (4.5 h) at concentrations up to 100 mg L−1 (Velzeboer et al. 2008) and may even stimulate Pseudokirchneriella subcapitata growth rates at low concentrations (0–10 mg L−1) (Hartmann et al. 2010), facilitate electron transport in plants, and can stimulate photosynthesis (Hong et al. 2005; Lei et al. 2007). Furthermore, certain types of algae have been engineered to incorporate TiO2 into their tissues to enhance photo-efficiency (Jeffryes et al. 2008).

Hartmann et al. (2010) demonstrated the ecotoxicity of cadmium to algae in the presence and absence of 2 mg L−1 TiO2. They found that the presence of TiO2 in algal tests reduced the observed toxicity due to decreased bioavailability of cadmium resulting from sorption/complexation of Cd2+ ions to the TiO2 surface. Ma et al. (2013) have recommended in their review article that future investigations on NPs’ ecotoxicity should focus on the long-term effects of NPs at low-exposure levels (ZnO NPs was mentioned in particular).

Given these contrasting results of ZnO and TiO2 NPs, it is still not clear what the general impacts of these NPs on microalgae are. These studies have concentrated on the individual effect of these NMs. In this study, we went one step further and evaluated the cumulative effect of these NMs (ZnO and TiO2). The reason behind that is due to the fact that both ZnO and TiO2 NPs are commonly used in sunscreens, where the particles protect against cell damage by blocking UV light. TiO2 was found to be more effective in the UVB and ZnO in the UVA range, the combination of these particles assures a broad-band UV protection (Smijs and Pavel 2011). Moreover, the toxicity assay we performed lasted for 5 weeks after inoculating the microalgae with the NPs.

Aquatic systems are vulnerable to direct and indirect contamination by NMs; therefore, the potential toxicity of these NMs to aquatic biota should be evaluated (Gong et al. 2011). Coastal systems are particularly the eventual sink for NPs, discharged into the environment (Klaine et al. 2008). Marine algae are found in coastal ecosystems (Behrenfeld et al. 2006) and so, they are particularly vulnerable to contaminants associated with anthropogenic pollution (Manzo et al. 2013). Prokaryotic and eukaryotic microalgae are good food source for zooplankton and small marine organisms and play important roles as primary producers in the world oceans, and thus, the assessment of NPs effects on marine algae is an essential step to predict their potential impact on marine food webs and on the entire ecosystem they sustain (Manzo et al. 2013). Additionally, it was found that algae are more sensitive to NPs compared to other organisms like fish and invertebrates (Aruoja et al. 2009; Zhou et al. 2014). It is also worth mentioning that algae not only provide the basic nourishment for aquatic food web, but also contribute to the self-purification of polluted water (Ji et al. 2011).

With this background, this study was designed to assess the cumulative effect of ZnO and TiO2 NPs and each one individually at a particular concentration to microalgae. To the best of our knowledge, this is one of the first studies to test the cumulative effects of nanoparticles. Batch cultured Picochlorum sp. (Trebouxiophyceae, Chlorophyta) were employed as the testing organism to investigate their growth, morphological changes, and their chlorophyll a content under NPs exposure. This microalgae is chosen because it has been used in mariculture and there are many literature reports revealing its nutritional values. Picochlorum sp. belongs to the small unicellular algae (photoautotrophic picoplankton), which are important in many aquatic environments (Callieri 2008). They are major contributors to phytoplankton biomass and primary productivity in oligotrophic lakes and oceans (Li 1994), contributing 10 % of the global net primary production (Raven 1998), and more than 50 % of the primary production in oligotrophic waters (Agawin et al. 2000). Little is known about their diversity in the marine environment, in the open ocean in particular (Fuller et al. 2006). This could be due to difficulties in their identification by light microscopy. With the development of molecular approaches, the picophytoplankton diversity begun to be revealed (Moreira and Lopez-Garcia 2002).

The Muse™ Cell Analyzer was used in this study to give precise viable cell count after inoculating cells with NPs.

Materials and methods

ZnO and TiO2 NPs and structural characterization

ZnO and TiO2 NPs were purchased from Sigma-Aldrich. The particle size specified by the manufacturer is 50 and 21 nm for ZnO and TiO2, respectively. Stock suspensions of ZnO and TiO2 NPs were prepared in algal medium immediately before each experiment.

For the characterization of NPs, different techniques were used, including x-ray diffraction (XRD), scanning electron microscopy (SEM) equipped with electron dispersive spectrometer (EDS).

Phase purity was carried out by XRD measurements using high-resolution Rigaku Ultima IV diffractometer equipped with Cu-K radiation (λ = 1.5418 Å). Qualitative and quantitative phase analyses were performed using PDXL program. The refinements were carried out using rutile (a tetragonal structure having space group P42/mnm with lattice parameters a = 4.5937 Å and c = 2.9587 Å) and anatase (a tetragonal structure having space group I42/amd with lattice parameters a = 3.7842 Å and c = 9.5146 Å) phases for TiO2, and wurtzite structure for ZnO (a hexagonal structure having space group P63mc with lattice parameters a = 3.2427 Å and c = 5.1948 Å). During refinements, phase composition, lattice parameters, and microstructural parameters (crystallite size and microstrain) were refined.

Morphological observations and chemical analysis were carried using ZEISS EVO LS 10 scanning electron microscope (SEM) operating at an acceleration voltage of 20 kV equipped with x-ray energy-dispersive spectroscopy (EDX) Bruker 127 eV detector.

Biotoxicity assay

Picochlorum sp. culture

A pure culture of Picochlorum sp. was obtained from the National Mariculture Centre, Ministry of Municipalities and Urban Planning, Kingdom of Bahrain.

The microalga was grown in 2-L Erlenmeyer flasks containing 1 L of sterilized medium (prepared with filtered seawater) capped with loose cotton and placed in an illuminated incubator. The Walne’s medium was used and the initial pH value was 8. The culture was kept at 18 °C under continuous illumination of approximately 100 μmol m−2 s−1. The cell density of the culture was monitored microscopically every 24 h and the different growth phases were determined by counting with a hemocytometer and Olymous CX21 microscope. Morphological observations were conducted with a Zeiss HB100 microscope.

Algal growth assays

The Organization for Economic Cooperation and Development (OECD) 201 algal growth inhibition test guidelines were followed with some modification. The cultures were incubated with NPs at the lag phase and the viable cell counts and measurements of chlorophyll a concentrations were performed approximately every 2–4 days starting from the second day of inoculation of algae with NPs for 5 weeks.

ZnO and TiO2 NPs were used at 10 mg L−1 with three replicates for each test culture in addition to the control. The 10 mg L−1 concentration was chosen because Ji et al. (2011) found that the 6d EC30 of ZnO and TiO2 NPs of ca. 20 and 30 mg L−1, respectively. The first assay was run using ZnO NPs (10 mg L−1) only; the second with TiO2 (10 mg L−1) and the third with the addition of equal concentrations of ZnO and TiO2 (10 mg L−1 each). The cultures (control and treated algae) were incubated in an illuminated incubator under the above-mentioned conditions with continuous shaking (180 r/min). High shaking speed was selected to reduce aggregation and settlement of the NPs over the incubation period. Changes in viable cell concentration were recorded using MuseTM Cell analyzer (Millipore, USA). The Muse™ Cell analyzer uses patent-pending, miniaturized fluorescent detection and micro-capillary technology to deliver quantitative cell analysis of both suspension and adherent cells 2 to 60 μm in diameter.

Additionally, chlorophyll a concentrations were determined by collecting 30 mL of each treated algal culture. Chlorophyll a was extracted using 90 % acetone, followed by measurement by spectrophotometer (PerkinElmer UV spectrophotometer) following UNESCO protocol (Vohra 1966).

SEM observation of algal cells

Morphological observations of microalgae and chemical analysis were carried for few samples after incubation with the NPs. These analyses were performed using ZEISS EVO LS 10 scanning electron microscope (SEM) operating at an acceleration voltage of 20 kV equipped with x-ray energy-dispersive spectroscopy (EDX) Bruker 127 eV detector.

TEM observation of algal cells

Several algae samples from the different experiments were fixed using 4 % buffered formalin before transmission electron microscope (TEM) analysis. The ultrastructural changes of Picochlorum sp. induced by ZnO and TiO2 NPs were observed with high-angle annular dark-field scanning TEM (HAADF STEM) and energy-dispersive x-ray (EDX) analysis spectrometry (Bruker) using a Jeol 2100 electron microscope in order to map the elemental composition of the samples.

Statistical analysis

In the figures, values are drawn as mean ± standard deviation (SDEV) and were tested for statistical significance using one-way analysis of variance (ANOVA) followed by Tukey’s pairwise comparison using Minitab version 16. The level of significance was accepted at P < 0.05.

Results and discussion

Preliminary characterization

The preliminary characterizations of ZnO and TiO2 NPs were carried out by SEM, EDX, and XRD analyses before introducing them to the test organism. SEM images of ZnO and TiO2 NPs and EDX spectra are displayed in Fig. 1. Round-shaped NPs were observed in agglomerated conditions. SEM analysis confirmed the primary size given by the suppliers’ data. Figure 2 exhibits Rietveld refinements of XRD patterns of nanosized ZnO and TiO2. The estimated crystallite size of the 50-nm ZnO NPs was determined to be around 28 nm and that of the 21 nm of TiO2, containing two phases (85.4 wt% anatase and 14.6 wt% rutile), was found to be 19 nm for anatase and 20 nm for rutile, respectively. EDX spectrum of ZnO (Fig. 1a2) reveals Zn and O peaks with 51.94 and 48.06 at%, which is very close to the stoichiometric ZnO compound. Similarly, EDX spectrum of TiO2 (Fig. 1b2) reveals Ti and O peaks with 31.12 and 68.88 at%, which is very close to the stoichiometric TiO2 compound.

Fig. 1
figure 1

SEM and EDX showing the 50 nm ZnO NPs (a1, 2) and the 21 nm TiO2 NPs (b1, 2)

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Fig. 2
figure 2

Powder XRD patterns of the 50 nm ZnO (a) and 21 nm TiO2 NPs (b). A denotes anatase phase and R denotes rutile phase for TiO2

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Cumulative effect of ZnO and TiO2 NPs on algal growth

During the experiment, the initial pH was 8.0 and increased slightly to 8.19 ± 0.05 in the control and in the ZnO-treated culture, 8.2 in the culture inoculated with TiO2 and the highest pH was recorded in the ZnO/TiO2 treated culture (pH 8.22 ± 0.05) during the whole incubation period (35 days = 5 weeks).

The physicochemical properties may be different for the NPs from different sources, which may affect NPs interactions with organisms, though their effect is found to be largely related to their size or surface area (Nel et al. 2006). NPs may differ in their toxicological effects, depending on the particles’ chemical composition and size, test organisms, and test method (Ji et al. 2011). Additionally, NPs effects on algae in particular are dependent on particles type, dissolution, and the tendency to compete with other ionic nutrients for uptake (Baker et al. 2013).

For example, toxicity of TiO2 NPs to the green algae Desodesmus subspicatus has been found to be dependent on the NP-specific surface area. The smallest particles showed a clear concentration-effect relationship, whereas the larger ones caused less toxicity (Hund-Rinke and Simon 2006). Hartmann et al. (2010) observed a similar trend; the smaller TiO2 NPs gave rise to significant inhibition compared to the larger NPs.

As illustrated by the growth curves in Fig. 3, the control culture showed a normal growth curve and reached maximum concentration of viable cells during the exponential growth phase (6.8 × 104 cells mL−1). Cultures treated with the NPs showed a lower number of viable cells compared to the control during the first growth stages (Log-stationary phase). In contrast, during the decline phase, the number of viable cells was higher in the treated cultures with the highest concentration using ZnO and reached 26.7 × 104 cells mL−1, followed by TiO2-treated culture (16.9 × 104 cells mL−1) and ZnO/TiO2 (11.4 × 104 cells mL−1) and the lowest number was recorded in the control culture (4.5 × 104 cells mL−1). There was a significant difference between control and treated cultures during the exponential growth stage (P < 0.05). Additionally, there was a clear difference between ZnO-treated culture and the other cultures during the decline phase (P < 0.05).

Fig. 3
figure 3

Effects of ZnO and TiO2 NPs on the growth of Picochlorum sp. Values are reported as mean of three replicates ± standard deviation (concentration of viable cells × 104 mL−1)

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Figure 4 depicts the effect of the different NPs on the chlorophyll a concentration of Picochlorum sp. Generally, chlorophyll a curves showed similar tendency as growth curves. In the control culture, the highest concentration of chlorophyll a was achieved during the exponential growth phase (4.78 μg mL−1), which is in accordance with the viable cell concentration. In contrast, all treated cultures exhibited similar results as the control culture. The ZnO-treated culture displayed a high chlorophyll a concentration compared to the low cell count during the exponential phase (4.99 μg mL−1). TiO2- and ZnO/TiO2-treated cultures are still presenting high concentrations of chlorophyll a during the exponential phase (4.46 and 4.72 μg mL−1, respectively). During the decline phase, ZnO-treated culture exhibited the highest concentration of chlorophyll a (4.45 μg mL−1) followed by ZnO/TiO2- and TiO2-treated cultures (4.00 and 3.94 μg mL−1, respectively), and the lowest concentration was recorded in the control culture (3.09 μg mL−1). There was no clear difference between control and treated cultures during the different growth stages (P > 0.05). However, there was a significant difference between control and ZnO-treated cultures on the last day of the experiment (P < 0.05). At the early growth stages, ZnO and TiO2, either used individually or combined, were found to inhibit algal growth. This could be due to the shading effect imposed by NPs in the medium. This was confirmed by scanning electron microscopy that was used for morphological study of Picochlorum sp. incubated with the NPs (Fig. 5, S1, S2). The images obtained confirmed that the algal species were entrapped within agglomerated ZnO and TiO2 NPs (SEM of Picochlorum sp. incubated with both ZnO and TiO2 is shown in Fig. 5). This suggests that NPs aggregates may have reduced the light availability to the entrapped algal cells and consequently inhibited algal growth. As a result, the chlorophyll a content was also found to decrease. These results are in agreement with those reported by Sadiq et al. (2011) who tested the effect of <25 nm TiO2 NPs on two microalgae Scenedesnus sp. and Chlorella sp. In contrary, this study results are in contrast with results reported recently by Tong et al. (2015), where they tested the individual and combined toxicity of ZnO and TiO2 NPs using bacterial cells. They found that the toxicity of ZnO NPs dominated the toxicity of TiO2 and in some cases, reversed it. Though they found that the combined toxicity of nano-TiO2 and nano-ZnO is attenuated as compared to that of individual NPs.

Fig. 4
figure 4

Effects of ZnO and TiO2 NPs on chlorophyll a content of Picochlorum sp. Values are reported as mean of three replicates ± standard deviation (concentration of chlorophyll a μg mL−1)

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Fig. 5
figure 5

SEM image of Picochlorum sp. culture treated with ZnO and TiO2 NPs

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Aruoja et al. (2009) demonstrated that 50 nm ZnO NPs caused an inhibitory effect to Pseudokirchneriella subcapitata at a very low concentration (72 h EC50 = 0.04 mg L−1), which is in accordance with Franklin et al. (2007) findings (72 h EC50 for the same algal species to be 0.06 mg L−1). Both studies attributed the toxicity of ZnO NPs at this low concentration to be due to dissolved Zn (ions). In general, ZnO NPs were found to cause growth inhibition at low concentrations when tested using marine algal species (Miller et al. 2010; Miao et al. 2010; Wong et al. 2010; Peng et al. 2011; Miglietta et al. 2011; Manzo et al. 2013). The mechanism of toxicity of ZnO NPs to algae has been mostly attributed to particles dissolution (Ma et al. 2013).

TiO2 NPs caused an inhibitory effect to Pseudokirchneriella subcapitata as well (72 h EC50 = 5.8 mg L−1). TiO2 NPs were found to form aggregates and entrap algal cells and there are still some free cells (Aruoja et al. 2009).

In contrast, in this study, ZnO and TiO2 NPs enhanced algal growth and increased chlorophyll a concentration during the late growth stages. This could be due to the fact that microalgae can reverse the negative effect of NPs after long exposure to the NPs through settlement of the large aggregates.

TiO2 NPs also induced spinach seed germination and plant growth by regulating the germination of aged seeds and its vigor (Zheng et al. 2005). Furthermore, the presence of TiO2 NPs enhanced the dry weight, chlorophyll synthesis, and metabolisms in photosynthetic organisms. These positive effects are possibly due to the antimicrobial properties of NPs, which can increase strength and resistance of plants to stress. At the same time, NPs can sequester nutrients on their surfaces and thus serve as a nutrient stock to the organisms, especially those NPs having high specific surface area (Navarro et al. 2008).

Kulacki and Cardinale (2012) documented a range of negative and positive effects of TiO2 on algal growth using a range of freshwater algal taxa. They proposed three possible mechanisms that might explain the increase in algal biomass. First, TiO2 could reduce competition with bacteria for limiting nutrients. Second, TiO2 is photoactive in the presence of UV radiation, which leads to the generation of reactive oxygen species (ROS) (Hund-Rinke and Simon 2006). While ROS are normally thought of as having negative effects on algae and other organisms (Hund-Rinke and Simon 2006), these same oxygen radicals have the potential to breakdown natural organic matter and release nutrients that might stimulate the growth of algae and bacteria (Scully et al. 2003). In the third possible mechanism, algae were simply increasing cellular chlorophyll content. TiO2 was found to coat algal cells (Hartmann et al. 2010), which could lead to shading at the individual cell level. Algae have been shown to increase cellular chlorophyll in response to low light condition (Steele 1962; Jorgensen 1969). This is similar to our findings during the decline growth phase (Fig. 4).

The TiO2 NPs used in this study are composed of 85.4 % anatase and 14.6 % rutile. The anatase form of TiO2 has superior photocatalytic activity compared to the rutile crystal structure (Sayes et al. 2006). Sayes et al. (2006) found that the crystal structure of nano-TiO2 contributed to cytotoxicity, with anatase TiO2 showing more toxicity than rutile TiO2. The difference in crystalline structure may contribute to explain the differences in the observed toxicity. However, a correlation between crystalline structure and toxicity is not a straight forward behavior (Hartmann et al. 2010).

The pH of the treated cultures increased over the incubation time (highest pH was 8.22 for ZnO and TiO2 NP-treated cultures). Overall, the pH became more alkaline in this study. It is well known that the pH of the medium has the most significant influence on ZnO NPs dissolution (Miao et al. 2010). Ma et al. (2013) observed that more acidic conditions would favor ZnO dissolution. Moreover, the maximum Zn2+ concentration will increase by almost 100 times as pH decreased from 9 to 7. Additionally, the presence of anions (e.g., Cl−, SO4 2−) in artificial seawater can serve as Zn2+ binding ligands and thus accelerates ZnO dissolution.

HAADF STEM and EDX ultrastructural analyses

The mapping of the different samples showed precipitates or aggregates outside of the cells. No Zn nor Ti oxides could be detected inside the algae (Fig. 6, S3-S5).

Fig. 6
figure 6

HAADF imaging (a) and EDX mapping (b) of cultures treated with ZnO and TiO2 NPs

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Bioremediation of nanoparticles by algae

It is well known that NPs tend to aggregate in aquatic environments to form micrometer- sized particles and it is likely that this state of dispersion may reduce the influence of the particle size, shape, and surface properties on their ecotoxicity (Limbach et al. 2005; Handy et al. 2008).

As shown in Figs. 5 and 6, algal cells accelerated NPs aggregation and therefore reduced the negative effect of NPs to the free cells in solution, which is in accordance with the increase of the number of viable cells and chlorophyll a concentrations especially during the late growth stages (stationary and decline phases) in the treated cultures compared to the control. The phenomenon of aggregation has been reported elsewhere for the non-modified metal oxide NPs, for example, ZnO (Adams et al. 2006), TiO2 (Lovern and Kapler 2006), and CeO2 (Limbach et al. 2005), which is considered to be their inherent property (Gong et al. 2011). TiO2 particles were reported to have a strong tendency to agglomerate to larger particles size in dispersion. Therefore, the algal cells during the experiment have come across agglomerated NPs in the dispersion which is similar to Sadiq et al. (2011) findings.

In our study, the relatively high pH (varied between 8.19 and 8.22) of seawater recorded at the end of the experiment, also accelerated the aggregation of NPs. The ionic strength, the solution pH, and organic matter as well as the surface charge of NPs, all found to play key roles in the aggregation behavior of NPs (Gong et al. 2011). NPs are expected to be more soluble in freshwater with low pH and are more likely to aggregate in high ionic strength seawater (Quigg et al. 2013). Algae in this regard, can therefore promote sedimentation of NPs into the environment and are likely potential candidates for bioremediation and nanopollution (Gong et al. 2011).

NPs may serve as pollutant carriers. In this way, NPs may improve or decrease the bioavailability of other toxic substances to algae (Navarro et al. 2008; Hou et al. 2013). Yang et al. (2012a, b) investigated the interactions between standard pollutants (Cd2+), and TiO2 NPs, and the green alga Chlamydomonas reinhardtii (Chlorophyta). They observed that TiO2 NPs can alleviate the inhibitive effects of Cd2+ on the green alga. Cd2+ adsorption by TiO2 NPs decreased its ambient free ion concentration and its intracellular accumulation as well as its toxicity. Other researchers have also demonstrated that TiO2 NPs can remove heavy metals (Pb, Cd, Cu, Zn, Ni) from water (Debnath and Ghosh 2011; Engates and Shipley 2011). ZnO NPs are also effective in a range of environmental control technologies from remediation of environmental pollutants to medical disinfection (Hoffmann et al. 1995). Chen et al. (2012) found that the effects of ZnO NPs interacting with Chlorella sp. to be bilateral, i.e., the adsorption and aggregation of ZnO NPs compromised algal cell morphology, viability, and membrane integrity, resulting from zinc ion dissolution as well as possible mechanical cell damage induced by the NPs. On the contrary, algal cells displayed a remarkable capability of self protection by minimizing their surface area through aggregation mediated by the oppositely charged metal ions and suppressing zinc ion release from the NPs through exudation.

Figures 5 and 6 display the adsorption of NPs to algal cells, and that was also to be expected (Navarro et al. 2008). This will ultimately increase the cellular weight of algae and lead to their sedimentation. These processes of adsorption and sedimentation will help to remove pollutants from the medium.

Conclusion

To the best knowledge of the authors, this is one of the first systematic studies on cumulative effects of ZnO and TiO2 NPs on algal growth. It is very important to examine the effect of NPs using many different species of algae (marine and freshwater species) to gain an inclusive view of the effects of these NPs. The thrust of this study with Picochlorum sp. is to study the long-term chronic exposure to the metal oxides rather than of short-term acute exposure. We demonstrated that the toxicity and availability of nanoparticles to marine algae are reduced by their aggregation and sedimentation. During the early growth stages, NPs were found to have a negative effect on algal growth and chlorophyll a concentration. In contrast, the effect is reversed during the late growth stages. There was no significant difference between the effect of the NPs when they were used separately and when both ZnO and TiO2 were used together in the test (P > 0.05). Furthermore, marine algae are potential organisms for usage in nanopollution bioremediation in aquatic system because of their ability to adapt to the long exposure to NPs. The results of this study emphasize the importance of studying the possible interactions of different NPs in assessing their potential environmental risks.