Introduction

The mass production, consumption, and inadequate treatment of waste water, have resulted in the contamination of waterbodies with plastics and microplastics (MPs). Microplastics are found as filaments and amorphous particles in freshwater and, plastispheres are becoming quite common in various oceans and seas around the world (Zhao et al., 2014; Mason et al., 2016; Amaral-Zettler et al., 2021). Microplastic density in different lakes is reported to range from 10–3 to 10 particles m−3 (Nava et al., 2023). The density of these particles in Pearl river, China is reported between 379 and 7924 particles m−3 (Lin et al., 2018). Freshwaters are also simultaneously exposed to complex mixtures of inorganic and organic pollutants (Altshuler et al., 2011; Guilhermino et al., 2021). Their mixtures and the continuous exposure could increase the adverse effects on aquatic microfauna (Wagner & Lambert, 2018; Guilhermino et al., 2021; Pestana et al., 2021).

The effects of the presence of multiple stressors, their interactions, and the acute and chronic exposure to aquatic organisms can be diverse, from mechanical damage by ingestion to intoxication associated with chemicals, with effects on demographic variables and species interactions or behaviour (Preston & Snell, 2001). Most studies on ecotoxicology with zooplankton have focused on testing the adverse effects on ecological variables such as survivorship, growth, and reproduction (Preston, 2002). Other characteristics, sensitive to the presence of contaminants, are feeding behaviour, escape mechanisms, swimming speed, and ecological interactions especially competition, and predation (Snell & Joaquim-Justo, 2007).

Aquatic communities usually are exposed to multiple stressors, and their interaction could have antagonist or synergistic effects (Sinclair & Arnott, 2015). The ingestion of MPs by aquatic organisms such as cladocerans (Nugnes et al., 2022), copepods (Setälä et al., 2018), and rotifers (Drago & Weithoff, 2021; Xue et al., 2021) has been demonstrated and attributed to their nonselective behaviour (Monakov, 2003; Sun et al., 2019). Early studies, however, indicated that rotifers can be selective in their prey preferences (Gilbert & Starkweather, 1977) and that large biotic and abiotic particles interfere in prey ingestion by metazoans (Moyo, 2022).

Rotifers are important in aquatic food webs as primary consumers linking lower and higher trophic levels (Wallace, 2002), with high sensitivity to environmental conditions and contamination (Wallace et al., 2006). They are sensitive bioindicators of water quality (Ejsmont-Karabin, 2012; Moreno-Gutiérrez et al., 2018) and are frequently used in toxicity tests due to their biological attributes (Sarma et al., 2006). There is also the potential for bioaccumulation or biotransference of microplastics to higher trophic levels if the size of the particles is very small (< 10 μm) and are actually consumed by the rotifers (Gambardella et al., 2018; Sun et al., 2019). Adverse conditions from multiple stressors can eventually modify ecological interactions, such as intra- and inter-specific competition (Gao et al., 2021).

Rotifers such as Brachionus havanaensis and Brachionus calyciflorus are common in freshwaters of pantropical regions (Koste, 1978). Many brachionids have similar food requirements (Dumont, 1977). However, there are few studies on the effects of microplastics on freshwater biota, and even less data available on the interactions between freshwater zooplankton, especially in the presence of multiple stressors. Species with low threshold food concentrations and high population growth rates often win in competition scenarios (Sarma et al., 1996). Several factors influence the population growth rates of rotifers; these include environmental variables such as temperature, food concentration and type, and rotifer characteristics such as their growth rates, capacity to withstand starvation and threshold food concentration (Kirk, 2002).

There are few studies that indicate the size of the microplastic particles in freshwaters. One such study by Eo et al. (2019) indicates that the size of most of the microplastics spheres in the Nakdong river in Korea ranges between 50 and 150 µm. Most studies indicate that the dominant microplastics in water and sediments are fibres and amorphous particles (Su et al., 2016; Nava et al., 2023). Brachionids are known to ingest particles in the 5–45 µm size range (Gilbert, 2022). Hence our choice for the selection of the microplastic particle size is within this range. In this work we evaluated the effect of 30 µm-sized beads of polystyrene microplastics at two concentrations (10 and 20 mg l−1) and at two temperatures (20 and 25 ºC) on population growth and competition of B. havanaensis and B. calyciflorus using 0.5 × 106 cells ml−1 of Chlorella vulgaris as food. We hypothesized that the higher MPs concentration would adversely affect the growth rate of the rotifers. Also, Brachionus calyciflorus, due to its higher growth rate, would be able to outcompete Brachionus havanaensis.

Material and methods

Culture of alga and rotifers

We obtained the rotifers Brachionus calyciflorus, Pallas, 1766 and Brachionus havanaensis, Rousselet, 1911 from a permanent waterbody, Lake Xochimilco (Mexico City, Mexico) and separately established a clonal population for each species under laboratory conditions. We used re-constituted moderately hardwater (here after called the EPA medium) for culturing rotifers. The EPA medium was prepared daily by dissolving 96 mg NaHCO3, 60 mg CaSO4, 60 mg MgSO4, and 4 mg KCl in 1 L of distilled water (Weber, 1993). The cultures of each species were maintained separately at 20 and 25 °C before using them for testing. The single-celled green alga Chlorella vulgaris which was used as food for rotifers, was batch-cultured on a defined medium (Bold’s basal) (Borowitzka & Borowitzka, 1988). Log phase alga was harvested, centrifuged and resuspended in a small volume of distilled water and stored at 4 °C until use. The algal density was quantified using a haemocytometer and required concentrations (0.5 × 106 or 1.0 × 106 cells.ml−1) were obtained using EPA medium for dilution for maintaining the cultures or the test individuals in the experiments (0.5 × 106 cells ml−1).

Microplastics

We used 30 µm diameter analytical grade polystyrene microplastic particles (SIGMA-ALDRICH laboratories). A stock solution of 80 mg l−1 was prepared in EPA medium. The stock solution was then sonicated at a frequency of 20 kHz at 10 watts for 3 min to separate the plastic beads.

Population growth

The experiments were set up in glass vials containing 20 ml of EPA medium with three concentrations; control group (0 mg l−1), 10 and 20 mg l−1 of polystyrene beads plus 0.5 × 106 cells ml−1 of C. vulgaris as food. We also set up negative controls with 20 mg l−1 of MPs but without algal food. Each treatment was set up with 4 replicates at two temperatures, 20 and 25 °C for Brachionus calyciflorus and Brachionus havanaensis cultured alone and together. Each test jar received 20 individuals of a mixed—age population, with juveniles (48–72 h old) in equal proportions of either rotifer species.

Following the start of population growth experiments, at every 24 h interval, the number of living individuals of either rotifer species was counted, individually, not by aliquots, and transferred to fresh medium with corresponding MPs and alga. The growth experiments were discontinuted after the populations in each test jar declined.

From the experiments we obtained the peak population density and day to achieve it at each treatment. The rate of population increase (r) was calculated based on a regression between the log natural-transformed population density vs time for each replicate (Princeé, 2016).

We statistically quantified the differences among the treatments for each variable using a two-way ANOVA, followed by Tukey post-hoc tests using SigmaPlot (ver. 11).

Results

Population growth of Brachionus calyciflorus grown at 20 °C and 25 °C in controls and in the presence of microplastics and in competition with B. havanaensis showed typical growth curves of a short initial phase, a long (ca. 7 days) exponential phase followed by a declinin the population (< a week) (Fig. 1). The presence of the competitor greatly reduced the population growth of B. calyciflorus. An adverse effect of the presence of microplastics was also evident.

Fig. 1
figure 1

Population growth curves (ind. ml−1) of Brachionus calyciflorus at 20 °C and 25 °C cultured alone (closed circles) and in the presence of Brachionus havanaensis as competitor (open circles) and with two concentrations (10 and 20 mg l−1) of microplastics (controls without microplastics)

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Brachionus calyciflorus had a population growth rate of 0.20 to 0.38 d−1 in the presence of the competitor and the microplastics. There was a significant impact of the presence of microplastics, the competitor, and the interaction of both of these factors on the population growth rate of B. calyciflorus at 20 °C (P < 0.01, 2-way ANOVA, Table 1). At 25 °C, there was a significant impact of the presence of the microplastics (P < 0.01, 2-way ANOVA, Table 1) but not on the presence of the competitor (P > 0.05, 2-way ANOVA, Table 1). There was a significant decrease (P < 0.05, Tukey posthoc test) in the population growth of B. calyciflorus in the presence of microplastics as compared to controls when cultured alone at both test temperatures (Fig. 2).

Table 1 Two-way ANOVA of the effects of microplastics concentration (MPs) and competition (with B. havanaensis) on B. calyciflorus for peak density (ind. ml−1), day of peak density and the rate of population increase (d−1) at 20° and 25 °C
Full size table
Fig. 2
figure 2

Population growth rate (d−1), peak population density (ind. ml−1), and day of peak population density of Brachionus calyciflorus alone (black bars) and with Brachionus havanaensis as competitor (gray bars) with different microplastic beads concentrations (10 and 20 mg l.−1) and controls without microplastics at 20 and 25 °C. Data bars carrying different alphabets represent significant differences among treatments (P < 0.05, two-way ANOVA and Tukey post-hoc tests)

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The peak population density of B. calyciflorus ranged between 5 and 9 ind. ml−1 at 20 °C and between 6 and 11 ind. ml−1 at 25 °C (Fig. 2). This variable was significantly lower, at both test temperatures, in the presence of microplastics and the competitor (P < 0.01, 2-way ANOVA, Table 1) with the exception of 20 mg L−1 microplastics at 20 °C where no significant difference was observed (P > 0.05, Tukey post-hoc test).

The day at which the peak population density (Fig. 2) was observed ranged between 7 and 11 at 20 °C and was not significantly different due to the presence of the microplastics or the competitor (P > 0.05, 2-way ANOVA, Table 1). At 25 °C, however, B. calyciflorus reached a peak population density significantly earlier in the presence of the competitor and the microplastics (P < 0.05, Tukey post-hoc test).

As in the case of B. calyciflorus, B. havanaensis showed growth curves with the three standard phases. In the presence of the competitor (B. calyciflorus), the population growth of B. havanaensis decreased in most treatments (Fig. 3).

Fig. 3
figure 3

Population growth curves (ind. ml−1) of Brachionus havanaensis at 20 °C and 25 °C cultured alone (closed circles) and in the presence of Brachionus calyciflorus as competitor (open circles) and with two concentrations (10 and 20 mg l.−1) of microplastics (controls without microplastics)

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The growth rate of B. havanaensis ranged between 0.28 and 0.43 d−1 at 20 °C and 0.35 and 0.49 d−1 at 25 °C (Fig. 4). There was a significant impact (P < 0.01) due to the presence of the competitor but not the microplastics (P > 0.05, 2-way ANOVA, Table 2) at 20 °C. On the other hand, there was a significant effect of the presence of the competitor and the microplastics (P < 0.05, 2-way ANOVA, Table 2) at 25 °C. The combined effects of the MPs and the competitor resulted in significantly lower growth rates (P < 0.05, Tukey post-hoc test) only at 20 °C.

Fig. 4
figure 4

Population growth rate (d−1), peak population density (ind. ml−1), and day of peak population density of Brachionus havanaensis alone (black bars) and with Brachionus calyciflorus as competitor (gray bars) with two concentrations (10 and 20 mg l.−1) of microplastics and controls without microplastics at 20 and 25 °C. Data bars carrying different alphabets represent significant differences among treatments (P < 0.05, two-way ANOVA and Tukey post-hoc tests)

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Table 2 Two-way ANOVA of the effects of microplastics concentration (MPs) and competition (with B. calyciflorus) on B. havanaensis for peak density (ind. ml−1), day of peak density and the rate of population increase (d−1) at 20° and 25 °C
Full size table

The peak population density of B. havanaensis at 20 °C and 25 °C ranged from 8 to 23 ind. ml−1, respectively (Fig. 4). There was a significant effect of the presence of the microplastics and the competitor on this variable at both the temperatures (P < 0.05, 2-way ANOVA, Table 2). The adverse impact of the presence of the competitor was more evident at 20 than at 25 °C (P < 0.05, Tukey post-hoc test).

The day at which B. havanaensis reached the peak population density ranged between 5 to 12 at the temperatures tested (Fig. 4). It was significantly different in relation to the presence of microplastics and the competitor at both test temperatures (P < 0.05, 2-way ANOVA, Table 2). The day at which the peak population density was observed was significantly later due to the presence of the competitor, especially at 25 °C (P < 0.05, Tukey post-hoc test).

Discussion

The size of the microplastic beads used in this study are within the size range that B. calyciflorus can ingest but larger than B. havanaensis can feed on (Monakov, 2003). In several tropical water bodies algal blooms are common. These are often dominated by prokaryotes and eukaryotes > 30 µm in size. Previous studies indicate that B. calyciflorus is also capable of ingesting cyanobacteria and algae of > 30 µm in size (Starkweather, 1981; Monakov, 2003; Soares et al., 2010). Here we observed that the adverse effect of competition was greater on B. calyciflorus while the adverse effect due to the presence of microplastics was greater on B. havanaensis.

Our study showed microplastics, larger than the preferred edible algal size, can affect rotifers without entering the zooplankton gut. We also found that the presence of microplastics resulted in different growth patterns, depending on the temperature, for either rotifer species. B. calyciflorus reached lower densities at 20 °C as compared to 25 °C. On the other hand, B. havanaensis was adversely affected due to microplastics at the higher test concentration only (20 mg l−1). These results probably reflect the effect of interference in grazing by the rotifers due to the presence of the microplastics. Zooplankton feeding is often inhibited due the presence of large sized phytoplankton in the medium. Gliwicz and Siedlar (1980) show that cladocerans reduce their carapace gape in the presence of large phytoplankton. The response of rotifers to the presence of large sized interfering particles is known to vary. Gilbert (1990) showed that the feeding of small rotifers such as Keratella and Synchaeta on Cryptomonas was unaffected due to the presence of the filamentous cyanobacterium Anabaena affinis. We too observed that the population growth rate of the small B. havanaensis was unaffected due to the presence of the microplastics. On the other hand, Rothhaupt (1990) has shown that rotifer feeding is inhibited in the presence of large (> 18 μm) particles. Drago and Weithoff (2021) also found that B. calyciflorus and B. fernandoi did not actually ingest microplastics > 5 μm, but the presence of these particles affected the growth rates of rotifers especially at lower food concentrations.

Our findings indicate that larger B. calyciflorus was more adversely affected than the smaller B. havanaensis. Field studies show that in the presence of large, interefering particles such as cyanobacterial colonies, B. havananensis is more abundant than B. calyciflorus (Gayosso-Morales et al., 2017). Microplastics in natural systems are rarely rounded particles, as tested here; they are more often fibrous or amorphous. It is therefore, quite likely that the presence of these particles will favour the presence of smaller rather than larger zooplankton.

In this study B. calyciflorus reached lower population densities than B. havanaensis. Previous studies also report that smaller bodied rotifers have lower threshold food concentrations and can reach higher population densities, as compared to larger taxa (Sarma et al., 2008; Gilbert, 2022). However, in competiton experiments, due to their capacity to better exploit the resources, in short term observations, larger species outcompete smaller ones. Here we observed that the percentage decline, with regard to temperature (20 and 25 °C), in the population of B. havanaensis was less (9 and 10%, respectively) than that of B. calyciflorus (37 and 25%, respectively) in the presence of the competitor. The increased susceptibility of B. calyciflorus to the presence of microplastics may also be due to the greater energy consumption of the species with higher swimming speeds as compared to B. havanaensis; further studies are needed to corroborate these hypotheses.

The adverse effects of lower concentration of the microplastics was more evident at 20 °C for both species. Our observations indicate that more than 90% of the microplastics sedimented within 8 h of setting up the experiment. Nevertheless, at the higher concentration of MPs, B. calyciflorus was not affected adversely, while B. havanaensis was. Our results show that the presence of microplastics may alter the outcome of competition among small and large zooplankton in natural communites, and thereby interfering with the size structure of planktonic communities.

The effect of environmental variables on the metabolic rates of rotifers are rarely studied (Miracle & Serra, 1989). However, inferences can be made from the available literature on the effect of biotic and abiotic factors on the demographic patterns of rotifers. In general, and especially for B. calyciflorus, several factors show a Q10 ≥ 2 which indicates an increased metabolic activity with a rise in temperature (Halsey et al., 2015). Here too, we observed an adverse effect due to the presence of the microplastics, competitor or both on the growth of either test species; the effect was greater on B. calyciflorus than on B. havanaensis. The time taken for the population to double the initial density was more in the case of B. calyciflorus than for B. havanaensis, especially in the presence of the competitor. Our personal observations also indicate that B. calyciflorus withstands starvation for 4 days at 20 °C and for 5 days at 25 °C; on the other hand, B. havanaensis can tolerate starvation for a longer period, 8 and 9 days, respective to the temperature. These factors explain the improved tolerance of B. havanaensis to the presence of the microplastics and the competitor. Thus, a synergistic effect of contaminants and interspecific interactions will result in changes in the community structure. Further studies on the effect of abiotic and biotic factors on the metabolic rates of rotifers will improve our understanding of these results.

Conclusions

Our study showed that microplastics caused adverse effects on the population growth and competitive outcome between two brachionid rotifers. Microplastics interacted with temperature and further caused adverse effects on the peak population density and rate of population increase of B. calyciflorus and B. havanaensis, separately and in mixed cultures. The adverse effects of microplastics were more severe for B. havanaensis than for B. calyciflorus, while competition had a greater adverse effect on B. calyciflorus. This study indicates that multiple stressors have different effects on species and will induce changes in zooplankton community structure in aquatic ecosystems.