Article Cite This: Environ. Sci. Technol. XXXX, XXX, XXX−XXX
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Small-Sized Microplastics Negatively Affect Rotifers: Changes in the Key Life-History Traits and Rotifer−Phaeocystis Population Dynamics Yunfei Sun, Wenjie Xu, Qiujin Gu, Yitong Chen, Qiming Zhou, Lu Zhang, Lei Gu, Yuan Huang, Kai Lyu, and Zhou Yang*
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Jiangsu Key Laboratory for Biodiversity and Biotechnology, School of Biological Sciences, Nanjing Normal University, 1 Wenyuan Road, Nanjing 210023, China ABSTRACT: Most coastal waters are at risk from microplastics, which vary in concentration and size. Rotifers, as important primary consumers linking primary producers and higher trophic consumers, usually coexist with the harmful alga Phaeocystis and microplastics in coastal waters; this coexistence may interfere with rotifer life-history traits and ingestion of Phaeocystis. To evaluate the effects of microplastics on rotifers, we designed a series of experiments concerning rotifer Brachionus plicatilis life-history traits and rotifer−Phaeocystis (predator−prey) population dynamics under different concentrations and sizes of microplastics. The results showed that small-sized microplastics (0.07 μm) at high levels (≥5 μg mL−1) decreased rotifer survival and reproduction, prolonged the time to maturation, and reduced the body size at maturation, whereas large-sized microplastics (0.7 and 7 μm) had no effect on rotifer life-history traits. For rotifer− Phaeocystis population levels, small-sized microplastics (0.07 μm) significantly delayed the elimination of Phaeocystis by rotifers; this is the first study to test the effects of microplastics on predator−prey dynamics. The results of rotifer−Phaeocystis population dynamics are consistent with the changes in the life-history traits of rotifers and further confirm the negative effects of small-sized microplastics (0.07 μm) on rotifers. These findings help to reveal the effect of pollutants on predator−prey population dynamics.
1. INTRODUCTION In recent years, the marine alga Phaeocystis globosa has become a harmful bloom-forming alga with a worldwide distribution.1 It has a complex polymorphic life cycle, producing haploid cells and forming gelatinous colonies.2−4 P. globosa mainly exists in small colonies and as single cells in the preliminary phases and in the declining stage of blooms.2,5 P. globosa has been shown to cause negative effects on fish larvae and copepod production.5−7 Rapid outbreaks of algal biomasses and the production of P. globosa toxins have caused negative effects on marine coastal systems.8,9 To find effective methods to reduce the negative effects of harmful algae to marine ecosystems, chemical,10 physical,11 and biological methods12,13 have been extensively tested.14 The biological methods, especially using grazers from natural marine ecosystems, are considered as an optimal choice compared to the high cost and secondary pollution of physical and chemical measures. However, biological methods are affected by ambient environmental factors, including biotic factors and abiotic factors, such as climate, environmental pollution, and food changes.15−18 Many studies have indicated that heavy metals, temperatures, and food fluctuations not only affect zooplankton feeding on algae but also cause some damage to zooplankton.19,20 Previous studies have shown that rotifers can ingest the harmful alga P. globosa and grow a population without obvious negative effects,21 which emerges as © XXXX American Chemical Society
ecologically significant in controlling P. globosa in coastal ecosystems. Rotifers eliminating P. globosa are also affected by some environmental factors.15 At present, due to the impacts of anthropogenic activities, especially in the oceans, plastic pollution problems are continuously increasing and may become more and more serious in the future without effective controls.22 Each year, the manufacture, use, and degradation of plastic products make the plastic particles more difficult to collect and remove from the marine environment,23 which results in the widespread presence of plastic particles with different sizes and concentrations in the oceans.24 The sizes of plastic particles range from micro- to nanosize, and the particles smaller than 5 mm are commonly defined as microplastics.25 In natural surface waters, the concentration and distribution of microplastics has been increasing.26 The concentration of microplastics in the northeastern Pacific Ocean ranged from 8 to 9,200 particles m−3,27 whereas the concentration of nanoplastics in a few specific aquatic environments even can reach as high as 109 particles mL−1.28 Microplastics are easily swallowed by a variety of marine animals, ranging from small Received: Revised: Accepted: Published: A
May 15, 2019 July 2, 2019 July 3, 2019 July 3, 2019 DOI: 10.1021/acs.est.9b02893 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology
Figure 1. FTIR spectra showed that the microplastics (0.07, 0.7, and 7 μm) used in the experiments were polystyrene (A), and the TEM images showed the shape and size of the micro- and nanoplastics (B).
protozoans to large mammals29−33 and easily translocate through the food chain.34 Many studies have reported the harm of microplastics to various aquatic organisms.35,36 For example, microplastics can reduce the immune capacities of corals36 and slow down the development of sea urchins.35 Ingestion of large amounts of microplastics by aquatic organisms can reduce energy reserves and affect some physiological functions32,37 and, consequently, increase the mortality of Daphnia and copepods.37−40 Jeong et al. have also found that the relatively small-sized microplastics (0.05 μm) were more toxic to marine rotifers based on their decreased lifespan and fecundity.41 Microplastics are also carriers for some other pollutants (nanoparticles, heavy metals, and antibiotics), causing negative effects on marine species.42−44 However, a considerable number of studies have also found that microplastics are not harmful to aquatic animals.45,46 For example, when D. magna were exposed to microplastics at sizes of 73−75 μm, their survival and reproduction were not affected although the gut of D. magna was filled with microplastics;46 Beiras et al. tested several species of marine zooplankton using >1 μm microplastics and found that ingestion and contact with microplastics does not cause acute toxicological effects.45 As conflicting results exist, from this point of view, further research is needed to verify it. In coastal waters, rotifers, as important primary consumers linking primary producers and higher trophic consumers, usually coexist with the harmful alga P. globosa and with the emerging microplastic pollutants with different concentrations and sizes; this coexistence may interfere with rotifer life-history traits and grazing on P. globosa. To evaluate the effects of microplastics on rotifers for P. globosa control, in the present study we designed a series of experiments on the rotifer life-
history traits and rotifer−Phaeocystis (predator−prey) population dynamics, and we hypothesized that (1) Nanosized microplastics are more toxic to rotifers than are microsized microplastics; (2) Nanosized microplastics prolong the time to sexual maturity and decrease reproduction of rotifers; and (3) Nanosized microplastics affect predator−prey (rotifer−Phaeocystis) dynamics and delay the ability of rotifers to eliminate P. globosa populations. To test our hypotheses, we exposed rotifer Brachionus plicatilis to three levels of sizes (0.07, 0.7, and 7 μm) and five concentrations (0, 1, 5, 10, and 20 μg mL−1) of microplastics and recorded the key rotifer life-history traits and the rotifer−Phaeocystis population dynamics.
2. MATERIALS AND METHODS 2.1. Algae and Zooplankton. A stock culture of Phaeocystis globosa was axenically grown in an f/2 medium. The rotifer Brachionus plicatilis was cultured in aerated synthetic seawater (NaCl 24.54 g, KBr 0.1 g, KCl 0.7 g, H3BO3 0.003 g, Na2SO4 4.09 g, NaHCO3 0.185 g, NaF 0.003 g, CaCl2·2H2O 1.54 g, MgCl2·6H2O 11.10 g, and SrCl2·6H2O 0.017 g in every 1 L deionized water; salinity 33‰; pH = 8.3; DO > 6.0 mg L−1) and fed daily with P. globosa. Both the alga and rotifers were cultured at 25 °C with a light:dark cycle of 14 h:10 h at 20 μmol photons m−2 s−1 provided by fluorescent lamps. 2.2. Polystyrene Microplastics. The polystyrene microplastics with diameters of 0.07, 0.7, and 7 μm were purchased from BaseLine (Tianjin, China). As the microplastics can distribute in the deionized water relatively evenly in high concentration, a stock solution of polystyrene microplastics (500 μg mL−1) was prepared by distributing the microplastics in deionized water, and the specific testing solutions were B
DOI: 10.1021/acs.est.9b02893 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology
fixed with 75% ethanol. The body length (from the corona to the base of the tail) was measured under a light microscope using Imaging Software NIS-Elements BR (Nikon, Japan). 2.3.2. The Rotifer−Phaeocystis Population Dynamics Experiment. As we found that a 0.07 μm diameter of polystyrene microplastics had negative effects on rotifer lifehistory traits, we further conducted an experiment regarding the effects of microplastics of this size on rotifer ability to eliminate P. globosa. Therefore, in the population dynamics of the grazing experiments, we only used one diameter of microplastics (0.07 μm) with three levels (0, 1, 5 μg mL−1; i.e., 0, 2.04 × 109, and 1.02 × 1010 particles mL−1) to test the population dynamics of rotifers and P. globosa. We selected rotifer populations (1 ind mL−1) with different ages and transferred them to 100 mL of fresh f/2 medium in 150 mL glass flasks with an initial P. globosa abundance of 1 × 106 cells mL−1. All experiments were incubated at 25 °C and 20 μmol photons m−2 s−1 using a 14 h:10 h light:dark cycle. A total of 18 glass flasks were prepared, including three control groups (only algae were exposed to the three microplastics levels) and three treatment groups (both algae and rotifers were exposed to the three microplastics levels). The three controls and three treatments were each performed in triplicate. During the entire experimental duration, no fresh f/2 medium was added. All of the glass flasks were slightly shaken every 12 h to prevent the microplastics and algae from sinking to the bottom. The experiments continued until the algae were eliminated in all grazing treatments, and the rotifer populations decreased. In all treatments, 2 mL samples were collected daily and then separated for counting the abundance of the algae and rotifer. The rotifers in 2 mL samples were filtered on a mesh (50 μm size), rinsed with synthetic seawater, and preserved in 6-well culture plates, and then their abundances were counted under a stereoscopic microscope. The algae from the filtrates of the treatments and the controls were preserved in 2% Lugol’s solution, and the algal abundances were counted using a hemocytometer under a light microscope. We collected these data to calculate the ratio of P. globosa and rotifers (P/R), algal growth rates, and algal reduction ratios and to fit the rotifer and P. globosa population dynamics. 2.4. Statistical Analysis. In the rotifer−Phaeocystis population dynamics experiments, the population growth rates (δ) of P. globosa in the control groups were calculated as follows: δ (d−1) = (ln Nt1− ln Nt2)/(t1− t2), where Nt1 was the algal cell abundance at day t1, and Nt2 was the algal cell abundance at day t2. The ratio of P. globosa and rotifers on each day during the population dynamics of grazing experiment was calculated as P/R = Cpt/Crt, where Cpt and Crt represent P. globosa and rotifer population abundances at time t, respectively. These ratios vs time were plotted to reflect the change trends in the relative proportion of the two organisms during the grazing process under different concentrations of 0.07 μm microplastics. The population dynamics of both P. globosa and rotifers in the grazing experiments showed a “peak curve” trend, i.e., increasing first and then declining, thus a
obtained by diluting the stock solution of microplastics in autoclaved synthetic seawater (30 mL) daily. One mL of desired concentration of microplastics solution was sucked from the “30 mL” using a pipet and added to each well of the 24-well culture plates every day. To ensure the particles to be evenly distributed, the solution was also shaken and sonicated at 40 kHz for 2 min before being transferred to the wells.47 The constituency of the microplastics was confirmed using a Fourier Transform Infrared (FTIR) microscope (BRUKER, Vertex 70, Germany). Thirty-two sequential scans were made for each measurement at a spectral resolution of 4 cm−1 and wavenumbers from 4000 to 400 cm−1. The obtained FTIR spectra were analyzed and evaluated using the OMNIC spectral library. FTIR spectra analysis showed that the microplastics (0.07, 0.7, and 7 μm) used in the experiments were polystyrene (Figure 1A). The shape and size of microplastics were observed and measured under transmission electron microscopy (TEM) (Hitachi, H-7650, Japan), which confirmed that particles of microplastics used in the experiments are spherical and the sizes range from micro- to nanolevel (Figure 1B). 2.3. Experimental Protocol. 2.3.1. Life-History Experiment. The newly born rotifers less than 4 h were exposed to five concentrations (0, 1, 5, 10, and 20 μg mL−1) of polystyrene microplastics, which represented the present field extreme levels28 and the possible increased levels without effective control in the future. For each microplastics level, rotifers were also exposed to three different diameters ranging from those similar in size to the food, P. globosa, to the nano level, e.g., 7 μm, 0.7 μm, and 0.07 μm. Based on counting and calculation, the corresponding numbers of particles per mL for the five concentrations were as follows: (1) 7 μm: 0, 2.04 × 103, 1.02 × 104, 2.04 × 104, and 4.08 × 104 particles mL−1; (2) 0.7 μm: 0, 2.04 × 106, 1.02 × 107, 2.04 × 107, and 4.08 × 107 particles mL−1; and (3) 0.07 μm: 0, 2.04 × 109, 1.02 × 1010, 2.04 × 1010, and 4.08 × 1010 particles mL−1. There was a total of 13 combinations (4 microplastics concentrations × 3 sizes + 1 control), which were placed in 24-well culture plates. An individual rotifer was placed in each well with 1 mL of autoclaved synthetic seawater containing different levels of microplastics, and each treatment had 24 replications. All 24well culture plates were slightly shaken twice daily to maintain the microplastics and algae in suspension. During the life history experiment, the alga P. globosa (approximately 1 × 106 cells mL−1) was used as a diet, and the medium was renewed daily. To obtain accurate first reproduction times, the rotifer in each well was checked every 4 h under a stereomicroscope. After the first reproduction, the numbers of newborn offspring were counted and removed every 12 h until the initial maternal rotifers died. The experiment ended after all of the initial maternal rotifers died. In the life-history experiment, a set of life history parameters, e.g., the “time to first batch of eggs”, the “time to first brood”, the “body length after first brood”, the “total offspring per rotifer”, the “survival time” of the initial maternal rotifer, and the “number of the initial maternal rotifers producing offspring”, was determined according to the above record. To measure rotifer length after the first brood under different treatments, 24 replicates for each treatment (three different sizes of microplastics × five microplastics levels) were set up. Nine rotifers (after releasing the first brood of neonates) were randomly selected from each treatment and
t−t k 2
three-parameter Gaussian model C(t ) = Ke−0.5( b ) was used to fit the population dynamics of rotifers and P. globosa, where C(t) represents P. globosa or rotifer population abundance at time t, K represents the theoretical maximum population abundance, tk represents the time needed to reach the theoretical maximum population abundance, and b reflects the shape of the curve.48,49 From fitting the above population C
DOI: 10.1021/acs.est.9b02893 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology data by the model, we can obtain the maximum abundance and the time to maximum abundance, which can reflect the changes in rotifer−Phaeocystis population dynamics under different concentrations of microplastics. The reduction ratios (%) of P. globosa in the grazing treatments were calculated by comparison with the control group.15 A linear function was used to fit the data for each trait of the entire life history and, when the slope was significantly different from zero (α = 0.05), this indicated that the microplastics concentration had a significant effect on that trait. The interactive effect of microplastics sizes and concentrations on all of the life-history traits, except the “number of rotifers producing offspring”, were also assessed using two-way ANOVA (α = 0.05). One-way ANOVA (α = 0.05) was applied to evaluate the effects of microplastics concentrations on the growth rates of P. globosa, the maximum abundance of P. globosa and rotifers, and the time to maximum abundance. All data were expressed as the mean values ± SE, and all statistical analyses were performed using Sigmaplot 14.0.
3. RESULTS 3.1. Effects of Microplastics on Life History Traits. 3.1.1. Survival Time. In most treatments, rotifers survived well. Interestingly, the excretions of rotifer that ingested algae and microplastics showed regular strip shapes and were deposited at the bottom of the experimental vessel (Figure 2), whereas
Figure 3. Survival times of rotifers exposed to microplastics with different concentrations and sizes. Error bars indicate the standard error. Significant differences are indicated by different letters (P < 0.05).
3.1.2. Time to First Reproduction and the Body Length after the First Brood. The 0.07 μm microplastics with high levels (≥10 μg mL−1) had significant negative effects on the time to first reproduction, whereas the 0.7 and 7 μm microplastics did not (Figure 4), and there was a significant interaction between microplastics sizes and levels on this parameter (Table 1). For the 0.07 μm microplastics treatment, the time to first batch of eggs (Figure 4a) and brood (Figure 4d) increased significantly with increasing microplastic concentrations ranging from 5 to 20 μg mL−1, whereas there was no significant difference for the 0−5 μg mL −1 concentrations. The times for producing the first batch of eggs (97 h) and brood (176 h) in rotifers exposed to 20 μg mL−1 microplastics were 3.6 and 3.7 times longer than those exposed to 0 μg mL−1 microplastics (approximately 27 and 47 h). The fitted linear function also showed that the 0.07 μm microplastics had a significant effect on the time to first reproduction (Figure 4a, Table 2). In the 0.07 μm microplastics treatment, the body length after first brood decreased significantly with increasing microplastics concentrations, whereas the other two sizes of microplastics did not affect this parameter (Figure 5), and there was a significant interaction between microplastics sizes and concentrations on rotifer body length after the first brood (Table 1). The fitted linear function also showed that 0.07 μm microplastics had significant effects on the body length after the first brood (Figure 5, Table 2).
Figure 2. Fecal pellets excreted by the rotifers that ingested algae and microplastics.
no fecal pellets formed in the controls. Two-way ANOVA based on the survival data showed that 0.07-μm microplastics had a greater negative effect on survival time than the other two sizes of microplastics (Figure 3) and that there was a significant interaction between microplastics sizes and concentrations on rotifer survival time (Table 1). The survival times of the rotifers exposed to 10 and 20 μg mL−1 0.07-μm microplastics were significantly shorter than those cultured with 1 and 5 μg mL−1 0.07-μm microplastics (Figure 3a), whereas no significant difference in the survival times was detected among different concentration treatments of 0.7 and 7 μm microplastics (Figures 3b, 3c). The slope of the fitted linear function of the small-sized microplastics was significantly different from zero (α = 0.05, Table 2), indicating that the small-sized microplastics had a significant effect on the survival times, whereas the large-sized microplastics did not (Figure 3, Table 2). D
DOI: 10.1021/acs.est.9b02893 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology Table 1. Two-Way ANOVA on Survival Time, Time to First Reproduction, Body Length after First Brood, and Total Offspring of the Rotifer B. plicatilis Subjected to Various Microplastic Concentrations and Sizes trait survival time (Figure 3) microplastic size (A) microplastic concentration (B) AxB time to first batch of eggs (Figure 4) microplastic size (A) microplastic concentration (B) AxB time to first brood (Figure 4) microplastic size (A) microplastic concentration (B) AxB body length after first brood (Figure 5) microplastic size (A) microplastic concentration (B) AxB total offspring per rotifer (Figure 6) microplastic size (A) microplastic concentration (B) AxB
DF
SS
MS
F
Table 2. Equations and Associated Parameters for the Linear Response Fits to Data in Figures 3, 4, 5, and 6a life history trait 0.07-survival time (Figure 3a) 0.7-survival time (Figure 3b) 7-survival time (Figure 3c) 0.07-time to first batch of eggs (Figure 4a) 0.7-time to first batch of eggs (Figure 4b) 7-time to first batch of eggs (Figure 4c) 0.07-time to first brood (Figure 4d) 0.7-time to first brood (Figure 4e) 7-time to first brood (Figure 4f) 0.07-the body length after first brood (Figure 5a) 0.7-the body length after first brood (Figure 5b) 7-the body length after first brood (Figure 5c) 0.07-total offspring per rotifer (Figure 6a) 0.7-total offspring per rotifer (Figure 6b) 7-total offspring per rotifer (Figure 6c) 0.07-number of rotifers producing offspring (Figure 6d) 0.7-number of rotifers producing offspring (Figure 6e) 7-number of rotifers producing offspring (Figure 6f)
P
2 4
16251.370 132711.394
8125.685 33177.848
0.993 4.053
0.372 0.003
8
138065.479
17258.185
2.108
0.035
2 4
23907.856 22179.956
11953.928 5544.989
84.872 39.369
