Biological responses of Chironomus sancticaroli to exposure to naturally aged PP microplastics under realistic concentrations

Microplastic (MP) is yet another form of chronic anthropogenic contribution to the environment. MPs are plastic particles (<5 mm) that have been widely found in the most diverse natural environments, but their real impacts on ecosystems are still under investigation. Here, we studied the toxicity of naturally aged secondary polypropylene (PP) MPs after constant exposure to ultraviolet radiation (26 µm) to the third instar larvae of Chironomus sancticaroli, a dipteran species. The concentrations tested were 13.5; 67.5; and 135 items g−1 of dry sediment. C. sancticaroli organisms were investigated for fragment ingestion, mortality and changes in enzymatic biomarkers after 144 h of exposure. The organisms were able to ingest MPs from the first 48 h, and the amount of items internalized was dose-dependent and time-dependent. Overall, the results show that mortality was low, being significant at the lowest and highest concentrations (13.5 and 135 items g−1). Regarding changes in biochemical markers, after 144 h MDA and CAT activities were both significantly altered (increased and reduced, respectively), while SOD and GST levels were unchanged. In the present study, naturally aged polypropylene MPs induced biochemical toxicity in C. sancticaroli larvae, with toxicity being higher according to exposure time and particle concentration.


Introduction
Due to low cost and wide applicability, global plastics production has increased rapidly, reaching 367 million tonnes in 2020, not including fiber plastics (Plastics Europe 2021). Due to its intense use worldwide and lack of proper management systems, plastic has become a pollution problem of global proportions (Geyer et al. 2017;Horton and Dixon 2017). The investigation of microplastics (MPs) as contaminants of environmental matrices can be considered a relatively new area of study, although the presence of plastic particles in aquatic environments was initially reported in the early 1970s (Carpenter and Smith 1972). As a rule, MPs are particles up to 5 mm in diameter (Courtney et al. 2009), and can also be divided into small (<1 mm) and large (1 to 5 mm) MPs (Eriksen et al. 2014;Hanvey et al. 2017).
Although MP has been widely reported in aquatic ecosystems around the world, demonstrating its occurrence in water bodies (Cincinelli et al. 2017;Luo et al. 2019) and its toxic potential for certain groups of organisms, such as annelids, crustaceans and fish (Doyle et al. 2022;Qiao et al. 2022), there is a great lack of knowledge regarding other classes. So far, Diptera species have been superficially studied as having neglected toxic effects due to exposure to MPs. This group of organisms is considered extremely rich, comprising approximately 158 families and more than 159,000 recognized species (Ibáñez-Bernal et al. 2020), of which 46,000 are aquatic species. In addition, they are the only aquatic insects that colonize all continents, including Antarctica (Adler and Courtney 2019).
Chironomid larvae have at least one of their life cycle stages in aquatic environments and are considered models in toxicity tests due to their wide distribution in aquatic environments, ease of cultivation in the laboratory, short life cycle and their biological characteristics, which confer the ability to adapt to adverse environments (OECD/ OECD 2010; Rosa et al. 2014;Serra et al. 2017). The life cycle of Chironomus sancticaroli is characterized by four stages: egg, larva, pupa and adult (aerial). Of these, the first three are aquatic stages with benthic habits (Strixino and Strixino 1982). Due to their ecological role, Chironomidae larvae are an important biological representative of the benthic macrofauna and it is essential to understand their behavior and interaction with the various pollutants present in aquatic ecosystems. The species C. sancticaroli has been widely used in the literature as a bioindicator of environmental quality with several pollutants and chemical compounds (Pinto et al. 2021;Rebechi et al. 2021), including MPs (Palacio-Cortés et al. 2022). In the study carried out by Palacio-Cortés et al. (2022), C. sancticaroli larvae were able to ingest polyamide particles, however no mortality was observed after 96 h of exposure.
MPs are subject to several environmental factors that lead to chain breakage and plastic degradation. Among the environmental conditions that can act in this process, it is possible to mention temperature, weathering, intensity of ultraviolet radiation (UV), winds, physical friction, salinity and pH (Antunes et al. 2013, Liu et al. 2016, Wagner and Lambert 2017, Torres et al. 2020. Aged MPs present increased toxicity due to the release of monomers, additives and compounds generated by the reactions of these degradative processes (Hermabessiere et al. 2017, and the increase in the porosity of the particles, which will facilitate the sorption of chemical compounds present in the environment. In toxicity tests, the most studied particles refer to primary or artificially aged MPs, due to experimental practicality. However, even though the environmental distribution and toxicity of MPs have already been investigated, only limited information is available for the environmental transformation of this pollutant in the laboratory. Therefore, it is necessary to investigate the phototransformation of MPs under natural radiation to understand how the aging process can influence the potential risks of MPs to biota. Considering the scenario presented, the present study aimed to study the effects of exposure of C. sancticaroli larvae to naturally aged polypropylene MPs (PP; PP-MPs) through diet. In order to establish a pattern of response at the cellular and/or individual level, the organisms were evaluated for PP-MPs intake, changes in oxidative stress markers and mortality after 6 days of exposure.

Microplastic
The MP used in the tests was composed of polypropylene (PP) of bluish color, secondary, from the lid of a storage container that was exposed to ultraviolet radiation for an indefinite period, until the fragmentation of the material was visible. The plastic underwent high-energy milling and the fragments were passed through a system of metal sieves in a column coupled to a sieve (Godoy et al. 2019;Stock et al. 2019) with several mesh openings until it was obtained the size of 26 µm. To confirm the particle size, 100 particles were measured under a stereomicroscope (Zeiss Discovery V12). The particles were stained with the fluorescent dye Nile Red (99% pure, INLAB, Brazil), at a concentration of 300 mg L −1 (Prata et al. 2019). The stained particles were suspended in ultrapure water and the concentration was determined by manually counting 100 µL (n = 5) under a stereomicroscope (10 µL = 135 ± 12 items). The solution was kept at 4°C in the dark until used.
The shape of the particles was observed and characterized by a stereomicroscope (Zeiss Discovery V12) and by an inverted fluorescence microscope (Leica DMi8) and the chemical composition was performed by Fourier transform infrared spectroscopy (FTIR). The FTIR spectra were measured in a Bruker spectrometer, model Alpha, in the region of 400-4000 cm −1 , with a standard KBr beam splitter and a high sensitivity DLATGS detector. The spectra were recorded with the ATR (Attenuated Total Reflection): ATR Platinum module, equipped with a diamond crystal as a reflective element. The spectra were obtained with 128 accumulations and with a resolution of 2 cm −1 .

Cultivation of C. sancticaroli
The larvae of C. sancticaroli used were obtained from a continuous culture maintained in the laboratory (OECD 2011) by transferring newly laid eggs to glass vessels containing a thin layer of sediment and mineral water as a culture medium under constant aeration. The vessel containing the spawns were kept in an incubator at 25°C with a photoperiod of 12:12 h (light:dark) and monitored daily to obtain organisms 6 days old (third instar) after hatching for the experiment. The organisms were fed every 48 h with 1000 µL of a suspension of fish food (Tetramin) at a concentration of 5 g L −1 .
Exposure of organisms to PP microplastics C. sancticaroli larvae were exposed to 3 different concentrations of PP-MP, being 13.5; 67.5; and 135 items g −1 of dry sediment. The concentrations used were chosen based on monitoring studies carried out worldwide, which show that the average concentration of MPs in freshwater environments is 129 items g −1 (Yin and Zhao 2023). Thus, the results obtained in the present study can be considered environmentally relevant.
The experiment had a total duration of 144 h and was carried out in glass flasks. Each treatment consisted of six replicates and 10 g of calcined sediment, 200 mL of culture water and 1000 µL of a suspension used to feed the larvae in the culture (5 g of Tetramin L −1 ) were added to each replica. Food was offered every 48 h. The containers were kept at the same temperature (25 ± 1°C) and photoperiod (12:12, light:dark) conditions as the culture and under constant aeration (1 bubble.s −1 ). Each replica consisted of 10 organisms each.

Ingestion of PP microplastics by C. sancticaroli
Every 48 h, one larva was removed from each replicate (n = 6 per treatment) and fixed in 70% alcohol to assess PP-MPs ingestion. The presence of particles inside the digestive tract of the larvae was evaluated using inverted fluorescence microscopy (Leica DMi8).

Biomarkers of enzyme activity
At the end of the experiment, larvae of each replica were removed from the container and used for evaluation of potential changes in biochemical biomarkers (n = 9 for enzymatic reactions; n = 5 for malonaldehyde analysis). From these organisms, a homogenate (1:9, w/v) was prepared by steeping in cold potassium phosphate buffer (pH 7.4, 100 mM). The homogenates were centrifuged at 2000 × g for 20 min at 4°C, and the supernatants were collected to determine the biochemical biomarkers glutathione S-transferase (GST), catalase (CAT) and superoxide dismutase (SOD) and malonaldehyde (MDA) levels.
Protein contents were determined by the Bradford method using bovine serum albumin (BSA) as a standard (Bradford 1976). Biochemical biomarkers were determined three times using the same homogenate and the results used to calculate the specific activity of GST, CAT, SOD and levels of MDA.

Glutathione S-transferase (GST)
The activity of glutathione S-transferase (GST) was adapted from the method proposed by Habig et al. (1974). Assays were conducted in triplicate using 100 mM potassium phosphate buffer (pH 6.5), 1.0 mM EDTA, 9.5 mM reduced glutathione (GSH), 1.0 mM 1-chloro-2, 4-dinitrobenzene (CDNB) and 10.0 µL of homogenate. CDNB was used as a substrate for the conversion reaction of GSH into glutathione thiolate anion (GS−), through the GST enzyme. The formation of the S-(2,4-dinitrophenyl) glutathione conjugate was monitored to increase the absorbance at 340 nm for 5 min in the UV-VIS spectrometer. The molar extinction coefficient of CDNB was 9.6 mM −1 .cm −1 .

Catalase (CAT)
Catalase (CAT) activity was determined following the method described by Aebi (1984). Tests were conducted using 100 mM potassium phosphate buffer (7.0), 20.0 mM hydrogen peroxide (H 2 O 2 ) and 10.0 µL of homogenate. Activity was monitored by consumption of H 2 O 2 resulting in a decline in absorbance at 240 nm for 3 min in the UV-VIS spectrometer. The molar extinction coefficient for H 2 O 2 was 40.0 mM −1 .cm −1 . The enzymatic activity was expressed from the consumption of 1 mmol of H 2 O 2 min −1 mg of protein −1 .

Superoxide dismutase (SOD)
Superoxide dismutase (SOD) activity was analyzed by the reaction of pyrogallic acid with the sample, observed at 420 nm. In 2 mL microtubes, 1.3 mL of tris-EDTA buffer (5 mM, pH 8.0), 60 µL of the homogenate and 75 µL of the pyrogallol solution (15 mM) were added, and subsequently homogenized vigorously for 20 s. The assays were incubated for 30 min in the dark at 25°C. After incubation, the oxidation reaction was stopped with the addition of 65 µL of 1 N HCl. The same preparation was performed for the blank, using 60 µL of 100 mM potassium phosphate buffer pH 6.5. SOD activity was determined by the ability to inhibit the reduction of pyrogallol by superoxide radicals by 50% expressed as U/SOD.

Malonaldehyde (MDA)
Lipid peroxidation damage was evaluated through MDA levels, as described by Campos et al. (2014), with adaptations to better fit our samples. Assays were performed using 0.4% thiobarbituric acid (TBA), diluted in 100 mM potassium phosphate buffer (pH 2.5). In a 10 mL test tube, organisms of C. sancticaroli (n = 5) were added and macerated with 400 µL of mM potassium phosphate buffer (pH 7.4). Subsequently, 1 mL of 0.4% TBA was added, homogenized and incubated in a water bath at 95 ± 1°C for 45 min. After being cooled in an ice bath, the samples were centrifuged at 3.000 rpm for 5 min at 25°C and read at a wavelength of 532 nm. The blank solution was prepared from 500 µL of 100 mm potassium phosphate buffer pH 7.4 and 1 mL of 0.4% TBA. The same process was carried out for the standard control, adding 500 µL of 4.5 mM 1,1,3,3-tetraethoxypropane (TEP) and 1 mL of 0.4% TBA. Results were expressed as nmol.mL −1 of MDA.

Statistical analysis
Data were expressed as mean ± standard deviation (SD). All statistical analyzes were performed using Minitab v.14 software. Data normality was determined by Anderson-Darling. Significant differences between treatment and control groups were analyzed using Dunnett's test (p < 0.05).

Results and discussion
Characterization of PP-MPs PP-MPs that was fractionated from the lid of a naturally photoaged container to a size of 26 μm and fluorescence stained with Nile red was visualized under a fluorescence microscope (Fig. 1). The irregular morphology and expected size of the particles were confirmed. The measurement of 100 particles under a stereomicroscope showed that the maximum size of PP-MPs was 46.3 μm and the minimum size was 17.1 μm, while the mean diameter was 26 ± 5.2 μm.
FTIR analyzes showed that the MP samples were treated as PP particles (Fig. 2). After aging, the content of carbonyl groups is expected to increase compared to virgin materials, which characterizes oxidative degradation (Müller et al. 2018). This change in the polymer matrix can be observed by the presence of peaks 1714 cm −1 which correspond to carbonyl groups (C=O) (Romano et al. 2017).

Ingestion of microplastics
The number of PP-MPs ingested by larvae was higher according to the exposure time. Thus, larvae exposed to the lowest concentration (13.5 items g −1 ) ingested an average of 1.0 to 2.5 items of PP-MPs between 48 and 144 h. At concentrations of 65.2 and 135 items g −1 , the larvae ingested from 1.3 to 2.7 and from 2.8 to 4.0 PP-MPs between 48 and 144 h, respectively. The number of ingested particles was also proportional to the increase of PP-MPs concentration in the sediments in all treatments (Table 1).
Like other benthic organisms, Chironomus sp. are opportunistic omnivores that have a diet based on particulate organic matter (Armitage et al. 1995). In the environment, these organisms ingest a wide variety of food items without much selectivity (Cummins and Klug 1979). Therefore, eating habits make this genus susceptible to solid pollutants present in sediments, such as MPs. Several studies have already reported the ability to ingest MP particles by different species of Chironomus, both    (Scherer et al. 2017(Scherer et al. , 2019 and in environmental conditions (Nel et al. 2018). Images of C. sancticaroli larvae from the present study with particles in the gastrointestinal tract were recorded and can be seen in Fig. 3. Despite MPs ingestion being one of the prerequisites to induce toxicity in exposed organisms, the low volume of studies with this genus does not allow definitive outcomes to be inferred on the consequences of such interaction. Still, MPs ingestion by chironomids is thought to trigger gastrointestinal tract obstruction, which can lead to changes in food intake or nutrient absorption (Avio et al. 2015;Nel et al. 2018;Ziajahromi et al. 2018;Ziajahromi et al. 2018;Silva et al. 2019). In more extensively studied groups of organisms, such as Danio rerio, some studies have also reported that exposure to MPs can lead to neurotoxicity and behavioral changes (Chen et al. 2020;Wan et al. 2019). In an extensive study carried out with marine zooplankton, the authors reported two very relevant outputs for the study of MPs, which are: organisms ingest aged MPs in greater quantity than virgin particles and the ingestion rates are species-specific (Vroom et al. 2017). In this context, the comparison with biological responses after ingestion of other species can serve as a guide, but it is essential that more studies with C. sancticaroli are carried out to strengthen the understanding of the potential toxicity of MPs for this species.
The ecological relevance of studying aged particles is given by the implications of this process. As changes in the polymeric chemical structure occur, the behavior of MPs will tend to be more toxic (Hermabessiere et al. 2017. Changes in the chemical structure will promote not only the breaking of chains, but also the adsorption of several other compounds present in the environment, such as organic compounds (Bhagat et al. 2022;Yao et al. 2022). According to the results demonstrated by Luo et al. (2022), changes in the surface of PP after photoaging lead to a greater affinity of this polymer with organic matter. Thus, in the specific case of chironomids, a point that must be considered is that under environmental conditions, the intake may end up being increased as the particles are also aged, since the feeding of C. sancticaroli is not selective. If these particles are more easily ingested, this could also be a pathway for bioaccumulation and biomagnification of MPs and absorbed toxic organic compounds through the trophic chain. Thus, MPs phototransformation can influence the potential risks of MPs to biota through more than one toxicity pathway.

Mortality of C. sancticaroli after exposure period
Mortality data were calculated as cumulative mortality after 144 h of exposure and expressed as percentage ± SD. After 144 h, exposure to aged PP-MPs reduced the survival of organisms exposed to C1 and C3 concentrations (67.5 and 125 items g −1 of dry sediment, respectively) ( Table 2). This effect of decreasing longevity in animals stressed by MPs was amplified at the highest concentration (11.6 ± 1.3%).
Taking into account that mortality was about 3x higher at C3 concentration compared to the mortality presented by organisms treated with C1 concentration, which corresponds to 1/10 of the amount of MPs items, this suggests that the concentration interferes with the rate of survival, despite not following a linear curve. The concentration used in the C3 treatment is on the threshold of concentrations found under environmental conditions (He et al. 2020;Klein et al. 2015;Vermaire et al. 2017;Ding et al. 2019), indicating that the mortality of Chironomid larvae may be occurring in environments with these concentrations.
In a study performed with primary PP and a terrestrial invertebrate species, Metaphire guillelmi, no mortality was observed during the 14-day exposure time (Cheng et al. 2021). In nauplii and metanauplii of Artemia salina, a marine invertebrate, the LC50 was 40.947 μg/mL and 51.954 μg.mL −1 and the mortalities were directly proportional to the exposure concentration (Jeyavani et al. 2022). Thus, compared to what was observed in the results of the present work, aged PP-MPs may possibly cause greater mortality when interacting with different organisms and, like primary PP, this endpoint is dose-dependent. In a study with larval culture of acorn barnacle Amphibalanus amphitrite, and aged polystyrene MPs, mortality was observed to be time-dependent and size-dependent, with 3 µm particles being more toxic than 10 µm MPs (Nousheen et al. 2022). Thus, we can expect a greater reduction in survival when the MPs are smaller and the concentration and exposure time are greater.

Biomarkers of oxidative stress
To elucidate the potential effects of PP-MPs at the cellular level in C. sancticaroli larvae, changes in oxidative stress biomarkers (SOD, CAT, MDA and GST) were investigated. Overall, the investigated biomarkers had a distinct effect dependent on concentration and period of exposure to PP-MPs (Fig. 4). While SOD and GST showed no statistically significant changes (p > 0.05), CAT and MDA had their levels decreased and increased, respectively, in all treatments compared to the negative control, with the exception of MDA at low concentration, which showed no significant difference.
When the biota is exposed to different adverse conditions, such as the presence of pollutants, free radicals promote reactions with biological substrates, which can cause cellular damage and, consequently, trigger the malfunction of the metabolism as a whole (Samet and Wages 2018). The regulatory system against oxidative stress works to prevent and/or control cellular damage. Each marker plays a role within this system, CAT converts hydrogen peroxide into H 2 O and O 2 (Regoli and Giuliani 2014) and MDA is one of the by-products of lipid peroxidation (Esterbauer et al. 1991). Currently, the study of enzymatic alterations has increasingly been applied in research with MPs as indicators of cell damage (Han et al. 2022).
In the present study, CAT activity was significantly inhibited in larvae exposed to all concentrations tested (Fig. 4b). MDA levels were significantly increased in larvae exposed to 67.5 and 135 items g −1 of dry sediment (Fig. 4d). These results indicate that exposure to PP-MPs for 144 h caused a deregulation of antioxidant defenses. According to Paul-Pont et al. (2016), CAT may have its activity reduced in response to the ingestion of MP particles by invertebrates. Regarding the increase in MDA activity, the same pattern was observed in Corbicula fluminea, a freshwater bivalve, after exposure to polystyrene MPs (Fu et al. 2022). In this same study, SOD levels were also increased, which may have occurred due to the longer exposure time (between 7 and 42 days). Still on the results presented here, GST activity was not altered, unlike what was observed in previous studies with C. riparius larvae (Silva et al. 2021) exposed to polyethylene MPs (~40-48 µm) for 48 h and by other invertebrate species (Avio et al. 2015;Ribeiro et al. 2017). The concentrations used by these studies may also be a relevant factor, as the authors tested considerably higher concentrations, between 1.25 to 20 g of MPs kg −1 of sediment. In general, it is possible to notice a lack of consistency between different studies, which may be due to the specificity of each species, the types of MPs used and the exposure time. Conclusions C. sancticaroli organisms were able to ingest PP-MPs (26 µm). Mortality was only significant at the highest concentration tested (135 items g −1 of dry sediment). Effects at the cellular level were consistent for CAT and MDA and, the latter being altered at concentrations 67.5 and 135 items g −1 and CAT also at the lowest concentration, 13.5 items g −1 . It is still unclear whether aged PP-MPs can be a threat at the individual or community level, and more extensive and in-depth investigation is recommended with the C. sancticaroli species with longer exposure periods and with varying characteristic MPs. Chironomus sancticaroli are shown according to protein content. The asterisk symbol (*) represents a statistically significant difference (p < 0.05) in oxidative stress markers between the treatment and the negative control