Toxicity effects of microplastics and nanoplastics with cadmium on the alga Microcystis aeruginosa

The extensive spread of microplastics (MPs) and nanoplastics (NPs) in the aquatic environment has attracted widespread attention. The toxicity of cadmium (Cd) combined with microplastics (MPs) and nanoplastics (NPs) toward freshwater algae Microcystis aeruginosa (M. aeruginosa) was investigated to evaluate the environmental behavior of the Cd complexation in fresh water. Cd alone has the highest toxicity to algae. Both MPs and NPs also have a negative effect on the growth of algae as individual components due to their adsorption of nutrients and disruption of the alga’s activity in a single MPs/NPs system. Compared with the single system, the toxicity of compound pollution including MPs + Cd and NPs + Cd becomes stronger, which presents a synergistic effect. In the presence of NPs, more extracellular polymeric substances (EPS) appeared, which helped to reduce the toxic effect on the algal cells. Moreover, MPs/NPs + Cd stimulate the production of microcystin-LR (MC-LR) under different treatments. Overall, the aquatic environmental assessment shows potentially elevated risks associated with combined MPs/NPs with Cd, which should be considered.


Introduction
With the sustained consumption of plastic products, the release of plastic waste into the environment has become an environmental hazard that increases with each passing decade. Jambeck et al. (2015) estimated that in 2010, 4.8 to 12.7 million metric tons of plasticwaste from 192 countries ended up in the Atlantic, Paci c, and Indian oceans as well as the Mediterranean and Black seas. Microplastics (MPs) are de ned as having a particle size smaller than 5 mm. This parameter was put forward for the rst time in a meeting hosted by NOAA (National Oceanic and Atmospheric Administration) (Hartmann et al. 2019).
Nanoplastics (NPs) are commonly de ned as having a particle diameter of less than 100 nm (Rist et al. 2017). They have been reported to have originated from the breaking down of plastic debris and polyethylene microbeads contained in commonly used chemical-based products (Peng et al. 2020), including personal care products (Hernandez et al. 2017). The abundance of MPs in surface water ranges from 379 to 7,924 particles/m 3 in industrial port waters ). Even higher amounts (102,550) of particles/m 3 have been found by Prata et al. (2019).
The persistence of MPs in fresh water highly affects the ecosystem with negative effects. Canniff (2018) revealed that MPs could serve as substrates for Raphidocelis subcapitata (a green algae) growth and provide energy for reproduction in the aquatic organism Daphnia magna (D. magna), a small planktonic crustacean. However, Wu et al. (2019) reported that MPs showed a negative effect on chlorophyll-a (Chla) contents and the photosynthetic activity of the freshwater algae Chlorella pyrenoidosa and Microcystis os-aquae, due to the accumulation of intracellular reactive oxygen species (ROS) and the interruption of photosynthetic electron transport. With the exposure of polyvinyl chloride (PVC) MPs, the highest inhibition of growth rate (I r ) of 45.8% was recorded by Ting et al. (2019) based on the marine dino agellate, Karenia mikimotoi, after 24 hours of contact time. This was due to physical blockage and aggregation.
During MP exposure in the environment, other contaminants containing organic matter and metal ions correlated with MPs, resulting in a complex effect on the aquatic ecosystem. Increased toxicity was reported by Sandra et al. (2018) based on the effects of combined MPs and chemical contaminants (PCB, BFRs, PFCs and methylmercury) on zebra sh. These effects on the zebra sh were revealed and compared with each contaminant's effect separately.  also found that the presence of MPs could increase cadmium (Cd) accumulation and cause oxidative damage in tissues, which eventually led to an enhanced toxicity to zebra sh. However, because of the strong adsorption capacity of glyphosate on amino-modi ed NPs, antagonistic effects appeared to inhibit the growth of the cyanobacterium, Microcystis aeruginosa (M. aeruginosa), a species of blue-green algae. The growth inhibition was attributed to the combined presence of NPs with pesticide ). Meanwhile, Ma et al. (2016) noticed that NPs showed more signi cant toxicity and physical damage to D.magna ( (1.5-5.0 mm) than MPs due to the higher adsorption of phenanthrene on the smaller size NPs.
Extracellular polymeric substance (EPS) consists of a complex mixture of carbohydrates, proteins, humic acid substances and deoxyribonucleic acid (DNA). EPS originates from microorganisms. After exposure to external stress, EPS produced by algae often easily interact with other contaminants and changes the ecological effects. Protein present in EPS can interact with NPs and change the surface charge and speci c surface area of NPs (Summers et al. 2018). Moreover, the protein corona presented on the altered surface of polystyrene (PS) NPs incubated with copper and EPS produced by the freshwater microalgae R.subcapitata, could eventually change the ecological effects in the combined system (Bellingeri et al. 2019).
For this study, the authors chose the heavy metal ion Cd as a typical metal ion found in fresh water. Cd is a non-essential metal and is presented as a highly toxic contaminant for biological functions in aquatic ecosystems (Samadani et al. 2018a). We investigated both PS MPs with an average size of 50 µm and PS NPs, which averaged 80 nm. Our research objectives were to compare the in uence of MPs and NPs on the growth of freshwater algae M. aeruginosa with and without Cd. M. aeruginosa is a species of freshwater cyanobacteria best known for its ecologically devastating algal blooms. The I r , Chl-a contents, and enzymatic activity were recorded to assess the toxic effects under the stress of PS and Cd in single and combined systems. EPS production by algae was analyzed under different treatments. Furthermore, the microcystin-LR (MC-LR) was also investigated to assess its ecological risk to fresh water. Results of this study will provide new insights for further investigating the combined effects of MPs and NPs with other pollutants in aquatic environments.

Experimental Methods
Algae and culture conditions M. aeruginosa was purchased from the Freshwater Algae Culture Collection at the Institute of Hydrobiology (FACHB) in Hubei, China. The algae were cultured in a BG-11 medium (Table S1) and maintained in a sterile growing environment in an oscillating light incubator at a room temperature of 25 ± 1 °C, with a pH value of 7.0 ± 0.1, and a rotating speed of 150 r/min. The light condition was adjusted to a light: dark cycle of 12: 12-h with the light intensity of 50 μmol photons/m 2 /s. In order to reduce the in uence of light intensity on the growth of algae, the asks were randomly arranged and changed every day.
Chemicals PS MPs were purchased from Guangdong Dongguan Xingwang Plastic Products Co., Ltd., China, with a particle size of about 50 μm. MPs need to be pretreated with 95 % ethyl alcohol and then with 5 % nitric acid to remove any residual organic or inorganic pollutants. After that, MPs were washed to attain a neutral state by ultrapure water and dried in air before usage. A certain number of MPs was weighted and dispersed into ultrapure water to prepare a concentration of 1,000 mg/L solution. Virgin PS NPs with a diameter of around of 80 nm were purchased from the BaseLine ChromTech Research Center in Tianjin, China, as a 2.5 % (w/v) suspension diffused in ethanol. The stock solution with a concentration of 1,000 mg/L was prepared by dilution with ultrapure water before the experiments.
The analytical grade cadmium chloride (CdCl 2 ) with 99 % purity was purchased from the Aladdin Bio-Chem Technology Co., Ltd. (Shanghai, China). A stock solution of 1000 mg/L of Cd was prepared in ultrapure water.

Algae toxicity test
Toxicity test of single pollutants on algae In order to evaluate single effects of MPs, NPs and Cd on M. aeruginosa, algae exposed with a different range of pollutants were investigated separately. A certain volume of Cd stock solution was transferred into a 250 mL conical ask containing 100 mL sterile medium until a variety of concentrations were obtained (i.e., 0.2, 0.5, 1.0, 1.5 and 2.0 mg/L). Due to the complex effect with Cd, ethylenediaminetetra acetic acid (EDTA) in the medium was removed to prevent the in uence of metal ions on the toxicity of algae. MPs and NPs with concentrations of 0.5, 1.0, 2.0, and 5.0 mg/L were also studied, respectively. Before the experiments, MP and NP solutions were dispersed well by ultrasound for 30 min to avoid agglomeration and sediment. The initial density of algae in the experiment was 4×10 6 cells/mL. Toxicity experiments were carried out for 96 h, and three parallel samples were set up for each sample under different conditions.

Effects of combined MPs/NPs and Cd on algae
The effect of the combined MPs/NPs and Cd on M. aeruginosa was also investigated and compared with the single system. The design of the toxicity experiments in the combined system is listed in Table 1. All the experiment conditions described in front part were maintained. The adsorption experiments of Cd on the MPs and NPs were also performed to assess the adsorbing behavior of metal ions on to plastics (details given in the SI). The density of M. aeruginosa algae was counted every 24 hours by an automatic cell counter (Countstar, Shanghai RuiYu Biotech Co., Ltd. China). One mL of algae suspension xed with 10 μL of Luger reagent was homogeneously mixed with an oscillator. 20 μL of the mixed sample was injected into a cell counting plate and automatically counted using CountStar Algae software. As described in the guideline 201 of the Organization for Economic Cooperation and Development (OECD), the I r was calculated as: where N t is the number of cells/mL at t (h), and N 0 is the control group number of cells/mL, ∆t is the time interval; μ 0 is the average speci c growth rate of the control group, and μ t is the average speci c growth rate of the algae at t (h).

Chl-a of algae
The Chl-a contents under the stress of Cd and PS in single and combined system was measured to quantify the variations in the phytoplankton biomass of algae. The suspension solution was extracted by acetone solution (details given in the SI). Chl-a (µg/L) was measured according to Eq. 3 as follows: where C a is the Chl-a contents inside the algae cell; V and V 1 represent the sample volume and acetonebased extract (mL) volume, respectively. OD is the absorbance at a certain wavelength, and σ is the optical path of the cuvette (cm).

Accumulation of cadmium ions in algal cells
After the exposure of Cd in single and combined systems, the metal ions absorbed in algae were analyzed. Algal samples were rst ltered through a 0.45 µm cellulose acetate membrane and then washed with 5 mL of 1 mM EDTA to remove adsorbed Cd on the surface of the cell. Then, lters containing algal cells were dried at 60 °C for 24 h. Next, the lter papers containing dried biomass were digested in 1 mL of 65 % HNO 3

Measurement of SOD activity and MDA content and ROS generation
The enzyme activities containing superoxide dismutase (SOD) and malondialdehyde (MDA) of algae under the stress of both Cd and PS in single and combined systems were assayed by using corresponding commercial kits (Jiancheng Bioengineering Institute, Nanjing, China). The algae cells were centrifuged for 15 min at 5,000 rpm to remove the suspension. Then, the cell was smashed by an ultrasonic cell disruptor to obtain homogenates. The absorbance was detected using the Synergy H1 multi-mode microplate reader (BioTek Instruments, Winooski, VT, USA). The SOD activity (U/mL) was monitored at 550 nm after the addition of the SOD assay kit. The MDA content (nmol/mL) was measured at 532 nm, using the MDA assay kit.

Statistical analysis
All the experiments were conducted in triplicate and the means ± standard was obtained. Statistical analysis was performed using Origin ® 9.0. We also found signi cant differences using the SPSS software statistical package version 22.0 (p < 0.05).

Results And Discussion
Effects

Effects of different concentrations of MPs/NPs on algal growth
A various concentration of MPs/NPs was studied to explore the effect of MPs and NPs on algae growth in both stand-alone MPs and NPs as well as the combined MP/NP system. After 96 h of exposure time, the I r gradually improved with the increasing concentration of polystyrene (PS) (Fig. 2a) in the single pollution system, due to the shielding effect and enhanced interaction between plastics and alga ). In the joint system of Cd and PS, even more inhibitory effects to algal growth became evident compared to PS alone. The results indicated that the addition of heavy metals (particularly Cd) may greatly increase their toxic effect on algae and play a key anti-algae role in the combined system. At the same time, it was also found that the I r of algal cells declined as the PS dose increased in the combined system. This may be related to the variances in the accumulation of Cd in cells along with the PS concentrations. Moreover, the I r of alga treated with NPs was slightly lower in the system where the alga was also treated with MPs, even though statistically, no signi cant difference was found in the performance of NPs compared with MPs.
To further understand the toxicity effect of MPs/NPs in the combined system, we also investigated the accumulation of Cd in algal cells under different concentrations of PS. After 96 h of exposure time, with the increasing concentrations of MPs/NPs, the accumulation of the Cd in cells decreased obviously (Fig.  2b). Meanwhile, in the presence of NPs, Cd concentration decreased more signi cantly than it did when in the presence of MPs. This could be explained by the higher adsorption capacities of metal ions onto NPs than MPs (Fig. S2). Although the accumulation of Cd inside the cells after the MPs and NPs treatments was distinct, the difference of the toxic effect on algae between them was not signi cant. This may be caused by the promote of higher number of EPS generated by algae under the stress of NPs, which could combine with more metal ion Cd in the solution (Shen et al. 2018). Meanwhile, the complex effect of the EPS with heavy metal ions could reduce the toxic effect of Cd in the combined system.

Effects of different concentrations of Cd on algal growth
With the increase of Cd concentration, the I r of alga increased continuously after 96 h of exposure (Fig.   3a). The inhibition effect on alga increased gradually with the highest inhibition rate reaching up to 92.

Effects of Cd and MPs/NPs on Chl-a with time
Chl-a is used as a standard index to re ect the growth and proliferation of algae as shown in Fig. 4. In the presence of Cd, the Chl-a content of algae was obviously reduced more than in the contact with MPs/NPs alone. The existence of Cd has a signi cant inhibitory effect on the Chl-a content of algae. The addition of Cd will reduce the biosynthesis and content of Chl-a by destroying the chloroplast structure, inhibiting the expression of photosynthesis-related genes, thereby thwarting the photosynthesis and growth of algae. The effect of NPs on the Chl-a content of algae was slightly greater than the effect of MPs, which is consistent with the results of Wu et al. (2019). This can be explained by the accumulation of intracellular ROS in the presence of NPs which can damage the cellular structure and hinder chlorophyll synthesis. The production of ROS can affect the function of chloroplasts, resulting in the disturbance of photosynthesis and metabolism ).
The combined system also had a high inhibition effect on the content of Chl-a which was lower than the inhibition effect of Cd alone but higher than that of PS alone. But the difference between MPs and NPs The toxicity of the combined system was lower than a single Cd system but higher than the MPs and NPs alone, which indicated that the presence of MPs and NPs could reduce the bioavailability of Cd in the composite system. The levels of antioxidant enzymes and growth inhibition rate were signi cantly increased in both Cd and combined treatment groups. The disruption of photosynthesis and the increase of the level of antioxidant enzyme may lead to membrane lipid peroxidation which cause the cell damage and growth inhibition. The addition of NPs in the joint system could decrease the activity of SOD and the content of MDA more obviously when compared to MPs, because NPs have a certain adsorption effect on Cd (Fu et al. 2019). It can also stimulate the algae to produce more EPS to complex heavy metals, thus reducing the toxic effect of Cd in the composite system (Mao et al. 2018).

ROS
Due to the detoxi cation mechanism of algae under the stress of pollutants, the algae may produce antioxidant enzymes to eliminate the damage caused by ROS (Wu et al. 2021). Both MPs, NPs and Cd could induce oxidative stress to cells which relate to the rise of ROS, leading to cell membrane damage and even death ultimately (Thiagarajan et al. 2019). The organism would develop an effective antioxidant defense system to maintain normal intracellular oxidative stress in response to excessive production of ROS. Therefore, the level of ROS was considered as a typical biomarker and has traditionally been used to evaluate the toxicity effect under stress (Miao et al. 2019).
As shown in Fig. 6, there was more ROS produced under the stress of Cd and MPs/NPs than the control group. The order of ROS percentages is listed as follows: Cd alone > MPs +Cd > NPs +Cd > NPs alone > MPs alone. The results con rmed that the more obvious toxicity was found in the presence of Cd compared to MPs/NPs alone, which is consistent with the SOD and MDA results. Thiagarajan et al. This study presents an analysis of both S-EPS and B-EPS contents of algae in different treatment groups.
As shown in Fig. 7(a), less effect on the polysaccharide content is noted at different treatment groups compared to the production of protein in S-EPS. The content of protein was signi cantly increased in S-EPS in the presence of Cd due to the self-protection mechanism of algae. Compared to MPs, NPs could accelerate the alga to produce more EPS due to the smaller particle size, easy agglomeration, enhanced interaction and higher toxicity (

Aggregation of the algae and MPs/NPs
As shown in Fig. 8(a), M. aeruginosa in the control group has integrity of cellular structure and easily aggregates. After contact with MPs, algae were adsorbing and surrounding the surface of MPs in order to obstruct the normal growth of algae (Fig. 8b) Fig. 8f. We also deduced that the presence of Cd not only promotes the algal cells but can also produce more EPS by self-protection, which also leads to the morphological damage of cells and the reduction of the algal cell density, resulting in the greater toxic effects (Wang et al. 2021).
In the composite system (Fig. 8d, e), more obvious EPS was generated by algae after the treatment of NPs and then MPs in the presence of Cd, which was in accordance with the former results of the EPS production. Meanwhile, the morphology of algae cells was damaged in the combined system shown in the SEM images, which revealed that the combined system of MPs/NPs with Cd had more of a negative effect on the algal cells than MPs or NPs alone. The existence of Cd with high toxicity in this system may contribution to this effect.

Extracellular and Intracellular MC-LR contents
M. aeruginosa is a representative species of cyanobacteria in freshwater, which is a main cause of the algal blooms. M. aeruginosa will release MCs that threaten the health of aquatic organisms. MC-LR, as one of the most typical types of MCs, contains both intracellular and extracellular MC-LR, which was considered in this research. The amount of extracellular MC-LR may be related to membrane lipid peroxidation and protein membrane synthesis ). Meanwhile, the dead algal cells also release intracellular MC-LR outside the cell, resulting in a higher extracellular MC-LR content.
As shown in Fig. 9, the maximum MC-LR both inside and outside the cells was produced under the stress of Cd, which was related to the stress reaction of cells. In addition, the osmosis of the metal ions through the membrane had the same effect on the algal metabolic activity that increased the permeability of the cell, thereby stimulating the release of the MC-LR (Xu et al. 2019). In comparison, NPs could promote more production and release of MC-LR than MPs due to the greater in uence on lipid peroxidation of a cell membrane, which in turn affects the uidity and permeability of membrane (Feng et al. 2020).
In the composite system, the generation of MC-LR was higher than in the single MPs/NPs system but lower than in the single Cd system. There was no signi cant difference between MPs and NPs in the combined system based on the comprehensive in uencing mechanism such as the membrane lipid peroxidation caused by cell membrane damage, the size of the algal density, cell lysis and oxidative stress ). In a word, the presence of MPs/NPs and Cd can induce MC-LR by M. aeruginosa, which gives rise to the cyanobacteria blooms and eutrophication of water especially in the existence with Cd.

Conclusions
Effect of MPs/NPs combined with Cd on M. aeruginosa was both investigated and compared with a single pollution system in this study. Both MPs and NPs do have a negative effect on the growth rate and antioxidant system of algae, while Cd has a signi cant toxic effect on algae by causing serious damage to the antioxidant system and destroying the integrity of algal cells. Compared to MPs, NPs have a greater toxic impact which has a major impact on the oxidative stress level of algae and produces more ROS in a single system. In the combined system, MPs/NPs could reduce the bioavailability of Cd to some extent, while the difference between them was not signi cant. This could be explained by the higher production of EPS under the stress of NPs as well as the aggregation abilities. Both MPs/NPs and Cd can stimulate the algae to produce MC-LR, while Cd as a stand-alone agent in icted the strongest oxidative damage to algal cells, thereby generating the most abundant MC-LR. In the combined system of MPs/NPs and Cd, the generation of MC-LR was higher than in the presence of MPs/NPs alone, which may give a potential risk to the water environment. The algal density of M. aeruginosa exposed to Cd (2 mg/L) alone, MPs (5 mg/L) alone, NPs (5 mg/L) alone, MPs (5 mg/L) + Cd (2 mg/L), and NPs (5 mg/L) + Cd (2 mg/L) with exposure time prolonged from 24 h to 96 h.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. supportingimformation.docx