Toxic Effects of Microplastics on Culture Scenedesmus quadricauda: Interactions between Microplastics and Algae

The number of microplastic particles in the environment is constantly increasing as a result of the decay of plastic waste, the incineration of which is associated with air emissions and the concentration of toxic combustion products in ash residues. Although numerous researchers have studied the effects of MPs on living organisms, only a small part of the published data is devoted to the study of the long-term toxic effects that MPs and combustion products of plastic have on phytoplankton organisms. The effect of different types of MPs and plastic incineration ash on the structural and functional growth parameters of a green microalga Scenedesmus quadricauda culture used as a test object was studied in a chronic experiment lasting 21 days. The development of the species was studied with the addition of five types of weathered MPs samples, obtained from macroplastics, collected in the supralittoral of the Barents Sea and one unweathered control sample at a concentration of 3 mg/L. In terms of changes in the number of Scenedesmus quadricauda cells, the following toxicity series was obtained in descending order: PU (polyurethane foam, weathered) > HDPE (food package, white, weathered) > HDPE (food package, red, weathered) > EPS (packaging material, weathered) > EPS (packaging material, unweathered) > PP (ship rope, weathered). In terms of the efficiency of photosynthesis (maximum quantum yield of PSII photochemistry (FV/FM)), polyurethane foam was found to be nontoxic, while other samples of MPs had a weak toxic effect. The effect of MPs on the culture caused a mosaic response, assessed by different parameters of the test object state: a strong inhibition of culture growth (with the addition of polyurethane foam) can be accompanied by a significant increase in thiobarbituric acid reactive substances (TBARS) in microalgal cells, while photosynthesis efficiency may not change. The toxicity of the residual ash obtained from the incineration of a mixture of weathered macroplastics was significantly higher than the toxicity of microplastics. Residual ash was studied at concentrations of 0.01, 0.1, 1, 10, 100, and 1000 mg/L and the toxicity was detected in terms of the change in the cell number only at a concentration of 1000 mg/L, at 0.01 mg/L in terms of the photosynthesis efficiency, and at 0.1 mg/L and above by the change in the amount of TBARS in microalgal cells.


INTRODUCTION
Plastic waste enters natural systems, where it undergoes mechanical fragmentation, chemical (oxidative and thermal) degradation and biodegradation, which contribute to the gradual transformation of bulk polymers into micro-and nanoparticles of plastic [1,2]. Micron and nanosized plastic particles quickly and widely spread in the marine environment, are easily transported to the oceans and to the seabed through food webs and aggregation processes, having an adverse effect on ecosystems [3]. In addition, microplastic particles (MPs) can be used as a vehicle for the transboundary transfer of microbial species [4], effec-tively adsorb persistent organic pollutants and inorganic contaminants due to their unique propertieshigh surface area and porosity [5], and ultimately have an adverse effect on all organisms of aquatic ecosystems as a result. However, little is known about the toxic effects of MPs on microalgae, which are among the most important primary producers of aquatic ecosystems.
MPs have minor effect on the growth of microalgae [6]. Micro-and, in particular, plastic nanoparticles can cause both growth inhibition [7,8] and its stimulation [9]. In most cases, it was not possible to find the values of the semieffective EC 50 concentration for

RESEARCH ARTICLE
MPs due to the high concentrations required for the induction of significant toxicity.
The presence of MPs leads to a decrease in the chlorophyll content [7,10] and photosynthetic activity in algae [10,11], regardless of growth retardation [7] and the shading effect [10], which is possibly associated with a decrease in the expression of photosynthetic genes [12], an increase in energy demand for mobility and disruption of gas exchange due to MP surface adsorption [13]. Moreover, MP can interfere with photosynthesis, disrupting the operation of the electron donor site and the reaction center of photosystem II (PSII), reducing the rate of electron transfer, which leads to the formation of reactive oxygen species and oxidative stress [11,13]. MPs can cause morphological changes in microalgae [11], internalize during cell division [14] or by capture by mixotrophic organisms [15]. It can also accumulate in exopolymer substances, reducing the availability of light, changing the bioavailability of carbon, and also increasing the frequency and strength of harmful algal blooms due to their ability to use MPs as a substrate for growth [9,11]. It is not yet clear from the literature data how the properties of MPs and adaptive responses of different types of microalgae affect the toxicity of different types of MPs.
The toxicity of MPs is influenced by a number of factors, including their concentration in the environment, type of polymer, size, presence of additives, chemical composition, and surface charge. As a rule, smaller and positively charged MPs are more toxic to microalgae [13,16]. The adsorption of pollutants into MPs can also enhance their effect [7].
Current levels of MPs in the environment do not significantly affect microalgae [17]. Most of the tested MP concentrations in experiments described in the literature are significantly higher than those found in the environment; however, many of these studies do not show significant effects of these polymers on algae.
Thus, there are very few and ambiguous data in the literature on the effect of MPs on microalgae, obtained mainly in short-term assays.
Industrial or spontaneous incineration of plastic waste is an alternative to landfilling, but it is associated with air emissions and the concentration of toxic combustion products in ash residues. Waste incineration is not a widespread practice in Russia; however, the ingress of plastic combustion products into the aquatic environment due to spontaneous disposal or accidental fires is highly probable in remote regions with limited possibilities for handling solid municipal waste.
The literature describes the formation of extremely toxic compounds in the composition of air emissions and ash residue formed during the incineration of plastic waste [18,19]; however, there are hardly any studies aimed at assessing the toxicity of plastic combustion products for aquatic organisms.
In this regard, the purpose of this work was to study the effect of different types of MP and its ash on the green microalga Scenedesmus quadricauda-in longterm experiments.
The development of this species was studied in normal conditions and when five MP samples-obtained from macrosamples taken in the supralittoral of the Barents Sea-and one intact sample that was not exposed to atmospheric influences were added to the medium at a concentration of 3 mg/L. Sampling site was the Barents Sea, supralittoral (upper boundary of winter storms), mainland, opposite the western end of the island Kildin. Sample date was August 30, 2020.
6. Intact EPS. Origin-packaging from household appliances for the laboratory; was not exposed to weather. Year of production: 2019. MP particle size: 130.2 ± 35.9 μm.
The selection of the concentration of 3 g/L of MP samples was determined by the results of a preliminary experiment, which showed the absence of their toxicity for the culture of S. quadricauda at 1 g/L.
Research methods. MP was obtained from macrosamples using an abrasive and a stainless-steel sieve with a mesh size of 300 μm, then plastic microparticles MOSCOW  were separated from the abrasive material residues by density separation in distilled water and dried. Particle sizes were determined on micrographs using the KOMPAS-3D v14 software (Ascon, Russia). The effect of ash obtained from the combustion of a mixture of MPs sampled from the supralittoral of the Barents Sea on S. quadricauda culture was evaluated at concentrations of 0.01, 0.1, 1, 10, 100, and 1000 mg/L. The bottom ash was obtained by burning a mixture of plastics in a Bullerjan-type furnace (Laotherm, Russia) at a temperature of approximately 400°C in proportions close to those observed on the coast of the Barents Sea. The composition of the mixture of plastics to obtain the bottom ash was as follows (mass fraction, %): 24.2% PP (industrial fishing net), 9.8% PP (food film), 6.1% EPS coarse (foam), 14.4% EPS dense (buoy), 2.4% PU (construction foam), 4.8% LDPE (low density polyethylene; high pressure polyethylene, LDPE; film, bags), 4.6% C/PAP (tetrapak food packaging), 8.4% HDPE (food bottles), 2.8% HDPE (film, bags), 22.5% HDPE (fragments of canisters, boxes and other containers). Experiments lasting 21 days were carried out in 100 mL conical flasks, to which 50 mL of medium in triplicate for each MP type, ash concentration, and control was added.
After adding MP and ash to the culture medium, EPS (intact), EPS (expanded polystyrene), PU (polyurethane foam), HDPE (white), and HDPE (red) particles floated on the surface and PP (polypropylene) and ash partially settled on the bottom of the flask. By the end of the experiment, all test materials partially settled except EPS (intact).
The main structural indicators for assessing the state of the population were changes in the number of cells (absolute and in comparison with the control) in the dynamics of its development. The number of cells was counted in a Goryaev chamber under a light microscope.
The functional state of algal cells was assessed by chlorophyll fluorescence measured using an AquaPen-C AP-C 100 fluorometer (Photon System Instruments, Czech Republic). Fluorescence was induced by red light with a wavelength of 630 nm for 2 s at a photon flux density of 3000 μmol photons m -2 s -1 . Before measurements, the samples were adapted to the dark for 10 min. The fluorescence intensity values at 50 μs (F 0 ) and 300 ms (F M ) were used to determine the maximum quantum yield of PSII photochemistry as [20].
The content of TBA-active products (products of the interaction of the end products of lipid peroxidation with 2-thiobarbituric acid) was determined according to Stewart and Bewley [21] with minor modifications. To 200 μL of the algal cell suspension, we added 1 mL of a solution of 0.25% thiobarbituric acid in 10% trichloroacetic acid. The obtained samples were incubated for 30 min at a temperature of 95°C in a water bath, after which the tubes were cooled for 5 min at a temperature of 0°C. The samples were then centrifuged for 15 min at 8000 rpm. Absorbance was measured at 532 nm and 600 nm. The concentration of TBA-active products was calculated taking into account the molar extinction coefficient of 156 mM -1 cm -1 .
The composition of emissions from the combustion of a mixture of various types of macroplastics in a furnace was determined by different methods: lead by the method of atomic emission spectroscopy with inductively coupled plasma (ICP-AES), CO and SO 2 using the OPTIMA 7 gas analyzer, hydrogen chloride (HCl) by the turbidimetric method, and benzo[a]pyrene by high performance liquid chromatography (HPLC).
The results were statistically processed using the Statistica Version 10 software (StatSoft Inc., United States). Data in the text and figures are presented as mean ± confidence interval (2ϭ). The normality of the distribution of parameter values was assessed using the Kolmogorov-Smirnov test. The assessment of the statistical significance of the differences between the control and experimental samples in experiments on algae was carried out using the Student's test; the differences were considered significant at p ≤ 0.05.

RESULTS AND DISCUSSION
In the first part of the work, the changes in various parameters the culture in the presence of MP were investigated. All types of MPs had a significant effect on the growth of the culture (Fig. 1a). At the same time, a significant inhibition of growth was noted for four types of MP: for EPS (Barents Sea) by 30% on day 3, PU (polyurethane foam) by 27-79% during the entire experiment, HDPE (white) by 41% on day 3, and HDPE (red) 37% on day 7. A significant stimulation was observed once for PP by 6% on day 21, for EPS (intact) by 29% on day 7, and for HDPE (white) by 23-27% on days 14 and 21. In the rest of the observation periods, the number of cells in the presence of six types of MP was at the control level.
The growth of the culture with the addition of rope MP particles was at the control level throughout the chronic experiment lasting 21 days. EPS (intact) caused a single, but rather significant and reliable growth stimulation (by 29%), which may indicate its weak toxicity.
Thus, in terms of inhibition of algae growth, PU was the most toxic, followed by HDPE (white), HDPE (red), and EPS (Barents Sea) in decreasing order of toxicity. PP (rope) was nontoxic.
The difference in toxicity between the EPS sample taken in the supralittoral of the Barents Sea and EPS (intact product) can be explained by the higher toxicity of the first sample due to its changes in natural sea conditions (oxidation, adsorption of substances). The long-term and significant (throughout the entire experiment) toxicity of PU for the test object under study is probably associated with the high toxicity of its organic components and their oxidation products.
Thus, according to the indicator of changes in the number of S. quadricauda cells for five MP samples taken in the supralittoral of the Barents Sea, and one intact sample of MP, the following toxicity series was obtained in decreasing order: PU > HDPE (white) > HDPE (red) > EPS > EPS (intact) > PP.
MP particles may contain toxic chemicals. According to the literature, the chemical hazard of plastic pollution can be associated with both components that make up synthetic materials [22] and toxic compounds present in the marine environment and adsorbed on the surface of plastic particles [23]. The first group may include monomers, processing aids, or by-products of production, while the second may include per-sistent organic pollutants or heavy metals. MP toxicity depends on the size of the particles, the stay period in water, the characteristics of the biofilm, and also on the chemical composition of the particles [24]. Thus, polyethylene, polypropylene, and polystyrene have fairly high sorption properties for a number of organic pollutants, while the monomers of polyethylene and polypropylene are quite inert with respect to biological objects [22], and the monomer of polystyrene, styrene, is acutely toxic. The hazard of polyurethane foam is associated, first of all, with the release into the environment of highly toxic hardeners, isocyanates, present in its composition. The toxic effect of the polyethylene samples we studied is possibly associated, among other things, with compounds sorbed from water since the plastic samples were collected in the water area with intensive navigation and the corresponding toxic load in the form of oil products, fuels and lubricants, etc.  Not only the structural indicators of the state of the laboratory microalgae population but also the functional indicators characterizing the physiological state of the test object under toxic exposure were used in this work in assessing the toxicity of the studied MP samples.
The maximum quantum yield of photochemistry of PSII (F V /F M ) of S. quadricauda upon addition of the studied MP samples to the culture changed insignificantly throughout the experiment: within the limits of up to 7% towards inhibition and up to 10% towards stimulation of growth. Nevertheless, these differences turned out to be significant and were of a long-term nature throughout the entire experiment, which indicates a weak toxic effect of most of the samples on the test culture. Polyurethane foam was an exception, in the presence of which the F V /F M values were at the control level (Fig. 1b).
Consequently, all MP samples, with the exception of polyurethane foam, had a weak toxic effect on the photochemical quantum yield-the value of photosynthesis efficiency (F V /F M ). And the polyurethane foam appeared to be nontoxic for this indicator.
Thus, the substances in the composition of the polyurethane foam cause prolonged inhibition of culture growth and affect the rate of cell division, but the value of the efficiency of cell photosynthesis remains within the normal range.
Oxidative stress was assessed by the content of TBA-active products in cells, which include a number of highly reactive compounds that act on all cell components, including DNA, and lead to disorganization of the cell membrane structure.
Observations of changes in the amount of TBAactive products in microalgae cells showed that it is the presence of polyurethane foam in the cultivation medium that leads to the formation of a very high level of these products from 158 to 567% in relation to the control during the entire experiment. In other cases, the increase was short-term (by 7 days) for EPS (Barents Sea) and HDPE (red) and the value of this indicator was 166 and 140%, respectively (Fig. 1b).
Consequently, the effect of MP on the culture of S. quadricauda is ambiguous and causes a mosaic response, assessed by different structural and functional indicators of the state of the test object. Namely, a strong suppression of culture growth can be accompanied by a significant increase in the content of TBAactive products in cells, while the value of the efficiency of photosynthesis does not change.
In the second part of the work, changes in the parameters of culture development under the influence of ash residue obtained from the combustion of a mixture of macroplastics in weight ratios close to those observed in the supralittoral of the Barents Sea were investigated. At all concentrations of the ash residue in a wide range of investigated concentrations from 0.01 to 1000 mg/L, no significant inhibition of the growth of the test culture was found (Fig. 2a). Significant stimulation by 16-19% was noted only at the highest concentration of 1000 mg/L on days 14 and 21. Such stimulation of culture growth may indicate the presence of components in the ash that affect the rate of cell division. Therefore, concentrations from 0.01 to 100 mg/L should be considered as nontoxic, and the concentration of 1000 mg/L of ash as slightly toxic.
The absence of a pronounced toxic effect of ash in the range of investigated concentrations on the studied culture in terms of the number of cells is, apparently, associated with a number of reasons. First, the plastic on the supralittoral is subject to degradation and oxidation. This can lead to the formation of oxidized products with a different toxicity than that of intact, original samples.
Firstly, the plastic on the supralittoral is subjected to degradation and oxidation. This can lead to the formation of oxidized products with a different toxicity than that of intact, original samples. In addition, toxic additives, such as bisphenol A and phthalates, are used in the production of plastics, which may have been released into the environment during the degradation of plastics before they are burned. Secondly, when plastic is burned, some other toxic components (for example, dioxins and furans) volatilize. Thirdly, the ratio of the mass fraction of different types of plastic for ash production shows a small percentage of toxic polyurethane foam (2.4%), and most of the plastic waste in the supralittoral of the Barents Sea are various containers and fishing nets.
The change in the maximum quantum yield of PSII photochemistry (F V /F M ) of S. quadricauda in the presence of different ash concentrations was insignificant (Fig. 2b). It should be noted that we observed the following: a significant decrease by 14% in this indicator at low ash concentrations of 0.01-0.1 mg/L, only single (on one of the observation dates) and weaker significant inhibition with medium concentrations (1 and 10 mg/L), and significant stimulation by 18% at high concentrations (100 and 1000 mg/L).
The effect we discovered in the literature is called "paradoxical," when low concentrations or levels of exposure to factors have a greater negative effect than higher ones, and can manifest itself under the influence of various factors (metals, antibiotics, ionizing radiation, pesticides, etc.) [25][26][27][28].
According to various authors, in the presence of low concentrations of a toxicant in cells, it is retained more and less is excreted from cells than at higher concentrations [28,29]. Since plastic in the environment adsorbs various pollutants and, in particular, toxic metals, residual amounts of them may be present in the ash, which then are accumulated in cells and, over time, can cause a toxic effect. Therefore, the suppression of the efficiency of photosynthesis with ash additions at a concentration of 0.01 and 0.1 mg/L can be explained by the cumulative effect of small doses of toxic ash components.
When various types of plastic are burned, dioxins, benzo[a]pyrene, and heavy metals are often found in ash [18,19]. We also detected many highly toxic volatile substances in the emissions from the combustion of a mixture of the studied types of plastics in the furnace, especially benzo[a]pyrene (0.74 ± 0.19 μg/m 3 ), CO (453 ± 32 ppm), SO 2 (77 ± 24 ppm), and HCl (4.39 ± 1.10 mg/m 3 ) as well as suspended solids, in particular, heavy metal-lead (0.091 ± 0.023 mg /m 3 ).
A significant increase in the amount of TBA-active products (by more than 20% compared to the control) in S. quadricauda cells in the presence of ash residue from a mixture of different types of plastic was detected once (only for one observation period) at ash concentrations of 0.1 mg/L (by 45% on day 21), 1 mg/L (by 21% on day 7), and 100 mg/L (by 37% on day 7) (Fig. 2b).
Thus, the toxicity of the ash residue was revealed in terms of changes in the number of cells only at the maximum tested concentration in the experiment (1000 mg/L), already at a minimum (0.01 mg/L) in terms of the maximum quantum yield of PSII photochemistry (F V /F M ), and at 0.1 mg/L and higher according to the change in the amount of TBA-active products in the cells.
Consequently, ash was much more toxic than the studied types of MPs since the toxic effect of ash in terms of the maximum quantum yield of PSII photochemistry (F V /F M ) was found already at 0.01 mg/L, at 1 g/L in terms of the number of cells, and the toxic effect was revealed at 3 g/L for MP by different indicators of the development of the test culture.
Thus, our studies have shown that different types of MPs at a concentration of 3 g/L have a negative effect on microalgae, inhibiting growth, altering the activity of photosynthesis, and causing oxidative stress in cells. The combination of different structural and functional indicators of the growth and state of the cell population of the microalgae S. quadricauda in a long-term experiment allow us to reveal the toxic effect of ash obtained by burning a mixture of plastics at lower toxic loads and to more adequately assess the toxic effect of the test sample.
Based on the analysis of literature data, it can be concluded that the current MP concentrations in the surrounding aquatic environment, which are several orders of magnitude lower than those studied by us, apparently do not have a toxic effect on microalgae. However, MP can disrupt the microalgae population by reducing the available nutrients, by suppressing their primary consumers, or by acting as a substrate. All of these changes depend on the specific properties of the MP, such as the type of polymer and size and surface charge, which are still poorly understood. Even small changes in the vital activity of microalgae populations as primary producers can lead to serious disruptions in the food chain and consequences for the functioning of aquatic ecosystems in general.
Modern experimental data on the toxicity of MPs do not give a unanimous opinion in assessing their toxicity. Therefore, further studies of the MP properties, their toxic effect on microalgae, the interaction of MPs with other substances in the environment, the establishment of the potential main mechanisms of their toxicity, and the identification of sensitive algal species and their adaptive reactions based on environmentally significant concentrations are necessary.

COMPLIANCE WITH ETHICAL STANDARDS
The authors declare that they do not have any conflict of interests.
This article does not contain any studies involving animals or human participants performed by any of the authors.