Transgenerational toxicity of nanopolystyrene particles in the range of μg L−1 in the nematode Caenorhabditis elegans

Li Zhaoa, Man Qua, Garry Wongb and Dayong Wang*a
aMedical School, Southeast University, Nanjing 210009, China. E-mail: dayongw@seu.edu.cn
bFaculty of Health Sciences, University of Macau, Macau, S.A.R., China

Received 3rd August 2017 , Accepted 24th October 2017

First published on 26th October 2017


The potential toxicity of nanoplastics to environmental organisms has gradually received great attention recently. We employed the in vivo assay system of Caenorhabditis elegans to investigate the possible transgenerational toxicity of nanopolystyrene particles and the underlying cellular mechanisms. After prolonged exposure, we observed the toxicity of nanopolystyrene particles at concentrations higher than 10 μg L−1. The transgenerational toxicity was further detected in nematodes exposed to nanopolystyrene particles at concentrations higher than 100 μg L−1. This observed transgenerational toxicity of nanopolystyrene particles might be mainly due to the translocation of nanopolystyrene particles into reproductive organs such as the gonad, which potentially in turn led to the transfer of nanopolystyrene particles to the next generation. Leachates from nanopolystyrene particles at concentrations in the range of μg L−1 did not contribute to the development of this transgenerational toxicity. Enhancement of intestinal permeability and extension of defecation cycle length provide the explanation for the observed accumulation and translocation of nanopolystyrene particles in reproductive organs. Therefore, our results demonstrate the potential transgenerational toxicity of nanopolystyrene particles in the range of μg L−1 in environmental organisms.



Environmental significance

It is now urgent to pay more attention to the toxic effects of nanoplastics on environmental organisms. Additionally, the knowledge on the transgenerational toxicity of nanoplastics is also very limited. In this study, after prolonged exposure, we detected the transgenerational toxicity of nanopolystyrene particles at concentrations higher than 100 μg L−1 in the in vivo assay system of C. elegans. This transgenerational toxicity was mainly due to the translocation of nanopolystyrene particles into the reproductive organ such as the gonad, which will in turn cause the transfer of nanopolystyrene particles to the next generation. Our results imply the potential development of transgenerational toxicity from nanopolystyrene particles in the range of μg L−1 in environmental organisms.

Introduction

It was estimated that the global plastic resin production could have reached 288 million metric tons in 2012.1 It was further calculated that 275 million metric tons of plastic waste was produced from 192 coastal countries in 2010, and approximately 4.8–12.7 million metric tons of plastic waste might be released into the ocean.2 Plastic debris as contaminants in the marine environment has been widely documented.3 In the environment, microplastic particles will be degraded gradually into nano-sized plastics, and the nanoplastics may be released directly into the environment.4 In recent years, the different aspects of toxicity of microplastics in different environmental organisms, such as zooplankton, bivalvia, polychaeta, echinoderms, crustaceans, fish, and seabirds, have been widely investigated.5–11 Considering the unique properties of nanoplastics, such as small size and large surface area, it is urgent to pay more attention to the toxic effects of nanoplastics on environmental organisms and to elucidate the underlying mechanisms for the observed toxicity of nanoplastics. Nevertheless, so far, the knowledge on the transgenerational toxicity of nanoplastics is still limited.

Caenorhabditis elegans has the typical properties of classic model animals, such as a small size, a short life cycle, a short lifespan, ease of cultivation and manipulation, and well-described genetic and molecular backgrounds.12,13 Meanwhile, it has been shown that C. elegans is sensitive to the toxicity of different environmental toxicants, including heavy metals, organic pollutants, fine particulate matter (PM2.5), and nanomaterials.14–21 Besides the endpoint of lethality, some sublethal endpoints including intestinal reactive oxygen species (ROS) production, locomotion behavior, and brood size were employed to assess the toxicity of certain environmental toxicants in nematodes.22–24 In nematodes, exposure to environmental toxicants could result in toxicity affecting the functions of both primary targeted organs (such as intestines) and secondary targeted organs (such as neurons and reproductive organs).25,26 Additionally, after the exposure, certain environmental toxicants, such as carbon-based nanomaterials, could be potentially translocated into the secondary targeted organs including reproductive organs through the intestinal barrier in nematodes.27–29 C. elegans is also a useful model for the study of transgenerational toxicity of certain environmental toxicants.30,31

In this study, we employed the in vivo assay system of C. elegans to investigate the possible transgenerational toxicity of nanoplastics. Moreover, we tried to determine the underlying cellular mechanisms for the observed transgenerational toxicity of nanoplastics. Our results highlight the potential transgenerational toxicity of nanoplastics in the range of μg L−1 to environmental organisms after long-term exposure.

Experimental

Physicochemical characterization of nanopolystyrene particles

The nanoplastics used were nano-sized (100 nm) polystyrene particles (Janus New-Materials Co., Nanjing, China). The commercial nanopolystyrene particles were 1% solid suspension. The nanopolystyrene particles used were fluorescently labeled with a fluorescent dye (Rhodamine B, Rho B) followed by carboxyl coating. The UV-vis absorption spectrum of nanopolystyrene particles labeled with Rho B shows an absorption band at ca. 530 nm (Fig. 1a). The fluorescence spectrum of nanopolystyrene particles labeled with Rho B indicates an absorption band at ca. 590 nm (Fig. 1b). The size distribution and zeta potential of nanopolystyrene particles labeled with Rho B were analyzed using Nano Zetasizer (Nano ZS90, Malvern Instrument, UK). Based on the analysis using Nano Zetasizer, the size of the nanopolystyrene particles labeled with Rho B was 108.2 ± 4.5 nm. The zeta potential of the nanopolystyrene particles labeled with Rho B was −9.698 ± 0.966 mV. In K medium, nanopolystyrene particles labeled with Rho B were stable for at least one week, and we did not observe obvious aggregation of nanopolystyrene particles in the K medium before the exposure (Fig. 1c). We transferred each of the exposed nematodes (24 h) into a fresh nanopolystyrene solution in the K medium. After 24 h or even 48 h exposure, we did not observe obvious aggregation of nanopolystyrene particles in the K medium (data not shown).
image file: c7en00707h-f1.tif
Fig. 1 Properties of nanopolystyrene particles labeled with Rho B. (a) UV-vis absorption spectrum of the examined nanopolystyrene particles. (b) Fluorescence spectrum of the examined nanopolystyrene particles. (c) Distribution of nanopolystyrene particles in K medium.

C. elegans strain and culture

The nematodes used in this study were wild-type N2, which were obtained from Caenorhabditis Genetics Center (funded by the NIH Office of Research Infrastructure Programs (P40 OD010440)). The nematodes were maintained on nematode growth medium (NGM) plates seeded with Escherichia coli OP50 at 20 °C.12 The age synchronous L1-larvae population was prepared as described previously.32 In order to prepare the L1-larvae population, adult hermaphrodite nematodes were lysed with a bleaching mixture (0.45 mol L−1 NaOH, 2% HOCl) in centrifuge tubes.

Exposure and toxicity assessment

Nanopolystyrene particles at the working concentrations (1, 10, 100, 1000, and 10[thin space (1/6-em)]000 μg L−1) were prepared by diluting the stock solution (1 mg mL−1) with the K medium just prior to the exposure. The nanopolystyrene solutions were sonicated for 30 min immediately prior to use in each experiment. Prolonged exposure to nanopolystyrene particles was performed from L1-larvae to adult day-1 (approximately 4.5 days) in the liquid solutions in 12-well sterile tissue culture plates at 20 °C in the presence of food (OP50). After the exposure, the examined nematodes were used for the toxicity assessment using the endpoints of intestinal ROS production, locomotion behavior, and brood size. To assess the possible effects of nanopolystyrene particles on the progeny of exposed nematodes, the eggs were transferred to a new NGM plate without the addition of nanopolystyrene particles.

We used the endpoint of intestinal ROS production to reflect the functional state of intestinal cells.33 The intestinal ROS production was analyzed as described previously.34,35 After the exposure, the nematodes were transferred into 1 μmol L−1 5′,6′-chloromethyl-2′,7′-dichlorodihydro-fluorescein diacetate (CM-H2DCFDA; Molecular Probes) solution for incubation for 3 h in the dark. After the labeling with CM-H2DCFDA, the examined nematodes were mounted on a 2% agar pad and observed at 488 nm excitation wavelength and with a 510 nm emission filter under a laser scanning confocal microscope (Leica, TCS SP2, Bensheim, Germany). The relative fluorescence intensity of ROS signals in the intestine was semi-quantified and expressed as relative fluorescence units (RFU). Forty nematodes were examined per treatment.

We used the endpoint of locomotion behavior to reflect the functional state of motor neurons.36 Head thrash and body bend were selected to evaluate the locomotion behavior and analyzed under a dissecting microscope as described previously.37,38 A head thrash is defined as a change in the direction of bending at the mid body. A body bend is defined as a change in the direction of the part of the nematode corresponding to the posterior bulb of the pharynx along the y axis, assuming that the nematode was traveling along the x axis. Fifty nematodes were examined per treatment.

We used the endpoint of brood size to reflect the reproductive capacity in nematodes.39 The brood size was analyzed as described previously.40 To analyze the brood size, the number of offspring at all stages beyond the egg was counted. Twenty nematodes were examined per treatment.

Nile red staining

Nile red staining was performed as described previously.41,42 Nile red (Molecular Probes, Eugene, OR) was dissolved in acetone to prepare a stock solution (0.5 mg mL−1) and stored at 4 °C. The stock solution was freshly diluted in 1× PBS buffer to obtain a working solution (1 mg mL−1) for the Nile red staining. Forty nematodes were examined per treatment.

Sudan black staining

Sudan black staining was performed as described previously.43 In organisms, Nile red can also be used to label fat storage.44 We assume that exposure to nanopolystyrene particles will not affect the Sudan black staining, if nanopolystyrene particles can only alter the intestinal permeability. The examined nematodes were washed in M9 buffer and fixed with 1% paraformaldehyde. The nematodes were subjected to 3 freeze–thaw cycles and dehydrated through an ethanol series. The nematodes were then stained overnight in a 50% saturated solution of Sudan black in 70% ethanol, rehydrated, and photographed. Forty nematodes were examined per treatment.

Defecation behavior analysis

The mean defecation cycle length was analyzed as described.29 The nematodes were examined for their fixed number of cycles, and a cycle period was defined as the interval between the initiations of two successive posterior body-wall muscle contraction steps. Forty nematodes were examined per treatment.

Statistical analysis

The data in this article were expressed as means ± standard deviation (SD). Statistical analysis was performed using SPSS 12.0 software (SPSS Inc., Chicago, USA). Differences between groups were determined using one-way analysis of variance (ANOVA), and a probability level of 0.05 was considered statistically significant. Graphs were generated using Microsoft Excel software (Microsoft Corp., Redmond, WA).

Results

Toxicity assessment of nanopolystyrene particles labeled with Rho B after prolonged exposure

To investigate the long-term effects of nanopolystyrene particles on nematodes, we performed prolonged exposure to nanopolystyrene particles labeled with Rho B from L1-larvae to adult day-1. We employed three endpoints (intestinal ROS production, locomotion behavior, and brood size) to assess the possible toxicity of nanopolystyrene particles to nematodes. After prolonged exposure, nanopolystyrene particles labeled with Rho B at the concentration of 1 μg L−1 did not induce significant induction of intestinal ROS production, a decrease in locomotion behavior, and reduction in brood size (Fig. 2). In contrast, after prolonged exposure, nanopolystyrene particles labeled with Rho B at concentrations higher than 10 μg L−1 resulted in significant induction of intestinal ROS production, a decrease in locomotion behavior, and reduction in brood size (Fig. 2).
image file: c7en00707h-f2.tif
Fig. 2 Toxicity assessment of nanopolystyrene particles labeled with Rho B after prolonged exposure. (a) Toxicity assessment of nanopolystyrene particles labeled with Rho B in inducing intestinal ROS production after prolonged exposure. (b) Toxicity assessment of nanopolystyrene particles labeled with Rho B in decreasing locomotion behavior after prolonged exposure. (c) Toxicity assessment of nanopolystyrene particles labeled with Rho B in reducing brood size after prolonged exposure. Prolonged exposure was performed from L1-larvae to adult day-1. Bars represent means ± SD. **P < 0.05 vs. control.

Transgenerational toxicity of nanopolystyrene particles labeled with Rho B after prolonged exposure

We next examined the possible development of transgenerational toxicity of nanopolystyrene particles in nematodes after prolonged exposure. In the F1 generation of nematodes exposed to nanopolystyrene particles labeled with Rho B (10 μg L−1), we did not observe significant induction of intestinal ROS production, a decrease in locomotion behavior, and reduction in brood size (Fig. 3). Nevertheless, in the F1 generation of nematodes exposed to nanopolystyrene particles labeled with Rho B at concentrations higher than 100 μg L−1, we could still detect significant induction of intestinal ROS production, a decrease in locomotion behavior, and reduction in brood size (Fig. 3).
image file: c7en00707h-f3.tif
Fig. 3 Transgenerational toxicity of nanopolystyrene particles labeled with Rho B after prolonged exposure. (a) Transgenerational toxicity of nanopolystyrene particles labeled with Rho B in inducing intestinal ROS production after prolonged exposure. (b) Transgenerational toxicity of nanopolystyrene particles labeled with Rho B in decreasing locomotion behavior after prolonged exposure. (c) Transgenerational toxicity of nanopolystyrene particles labeled with Rho B in reducing brood size after prolonged exposure. Prolonged exposure was performed from L1-larvae to adult day-1. Bars represent means ± SD. **P < 0.05 vs. control.

Toxicity assessment of leachate from nanopolystyrene particles labeled with Rho B after prolonged exposure

To determine whether the observed toxicity from nanopolystyrene particles labeled with Rho B is due to the effect of leachate, we prepared the leachate from nanopolystyrene particles by centrifuging (13[thin space (1/6-em)]000g for 20 min) nanopolystyrene particle solutions after the preparation of nanopolystyrene particle solutions for one week. After prolonged exposure, the leachates from nanopolystyrene particle solutions at concentrations of 10–1000 μg L−1 did not induce significant induction of intestinal ROS production, a decrease in locomotion behavior, and reduction in brood size (Fig. 4). In contrast, we only detected significant induction of intestinal ROS production, a decrease in locomotion behavior, and reduction in brood size in nematodes exposed to the leachate from 10[thin space (1/6-em)]000 μg L−1 of nanopolystyrene particles (Fig. 4).
image file: c7en00707h-f4.tif
Fig. 4 Toxicity assessment of the leachate from nanopolystyrene particles labeled with Rho B after prolonged exposure. (a) Toxicity assessment of the leachate from nanopolystyrene particles labeled with Rho B in inducing intestinal ROS production after prolonged exposure. (b) Toxicity assessment of the leachate from nanopolystyrene particles labeled with Rho B in decreasing locomotion behavior after prolonged exposure. (c) Toxicity assessment of the leachate from nanopolystyrene particles labeled with Rho B in reducing brood size after prolonged exposure. Prolonged exposure was performed from L1-larvae to adult day-1. Bars represent means ± SD. **P < 0.05 vs. control.

Distribution and translocation of nanopolystyrene particles labeled with Rho B in nematodes

We next examined the distribution and translocation of nanopolystyrene particles labeled with Rho B in nematodes. After prolonged exposure to nanopolystyrene particles in the range of μg L−1, we observed the accumulation of a large amount of nanopolystyrene particles in the intestine, especially in the middle and in the posterior regions of the intestine (Fig. 5). Meanwhile, a large amount of nanopolystyrene particles were observed in the tail (Fig. 5). Only a limited amount of nanopolystyrene particles were detected in the pharynx (Fig. 5). More importantly, we found an obvious accumulation of nanopolystyrene particles in both side arms of the gonads of nematodes (Fig. 5). In the progeny, we further detected the distribution of a certain amount of nanopolystyrene particles in the intestine (Fig. 5).
image file: c7en00707h-f5.tif
Fig. 5 Distribution and translocation of nanopolystyrene particles labeled with Rho B in nematodes. The pharynx (*) and the intestine (**) are indicated by asterisks. The single arrowhead indicates the gonad. The exposure concentration of nanopolystyrene particles was 10 μg L−1. Prolonged exposure was performed from L1-larvae to adult day-1.

Nanopolystyrene particles enhanced intestinal permeability after prolonged exposure

Induction of enhanced intestinal permeability is normally one of the important cellular contributors to toxicity induction of environmental toxicants in nematodes.15,18 We further used nano-sized (100 nm) polystyrene particles without fluorescence labeling (SmartyNano Technology Co., Suzhou, China) to investigate the effect of nanopolystyrene particles on intestinal permeability. The size of nanopolystyrene particles without fluorescence labeling was 104.7 ± 5.8 nm, and the zeta potential of nanopolystyrene particles without fluorescence labeling was −9.128 ± 0.482 mV. Similarly, prolonged exposure to nanopolystyrene particles without fluorescence labeling at concentrations higher than 10 μg L−1 induced significant intestinal ROS production, decreased locomotion behavior, and reduced brood size (data not shown). Additionally, transgenerational toxicity was detected in nematodes exposed to nanopolystyrene particles without fluorescence labeling at concentrations higher than 100 μg L−1 (data not shown). We used a lipophilic fluorescent dye, Nile red, to detect the possible effect of nanopolystyrene particle exposure on intestinal permeability. After prolonged exposure, nanopolystyrene particles at concentrations higher than 10 μg L−1 induced a significant increase in the relative fluorescence intensity of the Nile red signal in the intestine (Fig. 6a). Additionally, after prolonged exposure, nanopolystyrene particles at the concentration of 1 μg L−1 led to a moderate but significant increase in the relative fluorescence intensity of the Nile red signal in the intestine (Fig. 6a). Nevertheless, after prolonged exposure, we observed that nanopolystyrene particles at concentrations of 1–10[thin space (1/6-em)]000 μg L−1 did not obviously affect the fat storage labeled by Sudan black staining (Fig. 6b). Based on these data, we conclude the possibility that nanopolystyrene particles at concentrations higher than 1 μg L−1 may enhance the intestinal permeability in nematodes after prolonged exposure.
image file: c7en00707h-f6.tif
Fig. 6 Effect of nanopolystyrene particles on intestinal permeability after prolonged exposure. (a) Effect of nanopolystyrene particles on relative fluorescence intensities of Nile red signals after prolonged exposure. (b) Effect of nanopolystyrene particles on fat storage labeled by Sudan black staining after prolonged exposure. (c) Effect of nanopolystyrene particles on mean defecation cycle length after prolonged exposure. Prolonged exposure was performed from L1-larvae to adult day-1. Bars represent means ± SD. *P < 0.05 vs. control.

Nanopolystyrene particles prolonged the defecation cycle length after prolonged exposure

Besides the intestinal permeability, defecation behavior is also one important cellular contributor to toxicity induction of environmental toxicants in nematodes.15 Using mean defecation cycle length as the endpoint, we further examined the effect of nanopolystyrene particles without fluorescence labeling on mean defecation cycle length. After prolonged exposure, we did not observe an obvious alteration in mean defecation cycle length in nematodes exposed to nanopolystyrene particles at the concentration of 1 μg L−1 (Fig. 6c). In contrast, prolonged exposure to nanopolystyrene particles at concentrations of 10–10[thin space (1/6-em)]000 μg L−1 significantly prolonged the mean defecation cycle length (Fig. 6c).

Discussion

Previously, studies have examined the possible effects of short-term exposure to nanoplastics, such as nanopolystyrene, on environmental organisms.45,46 So far, the potential toxic effects of nanoplastic particles after long-term exposure on environmental organisms are still largely unclear. The potential toxic effects of microplastics or nanoplastics at environmentally relevant concentrations on environmental organisms have gradually received great attention.47 In the environment, the possible environmentally relevant concentrations of microplastics are in the range of ng L−1 or less than 1 μg L−1.48 However, so far, most of the used microplastic concentrations in experimental studies are far above the levels documented in the environment.49–55

In nematodes, prolonged exposure is helpful for detecting the long-term effects of certain environmental toxicants on animals.21,56 In this study, we observed that prolonged exposure to nanopolystyrene particles at concentrations higher than 10 μg L−1 induced significant intestinal ROS production, decreased locomotion behavior, and reduced brood size (Fig. 2). More importantly, we even detected an adverse effect on the function of the intestinal barrier in nematodes exposed to nanopolystyrene particles at the concentration of 1 μg L−1 (Fig. 6a). Therefore, our results imply that long-term exposure to nanopolystyrene particles at environmentally relevant concentrations (in the range of μg L−1) might potentially cause the toxicity on nematodes. With the copepod as the assay system, the chronic toxicity of nanopolystyrene (50 nm) in reducing the fecundity was observed in animals exposed to nanopolystyrene at concentrations higher than 100 μg L−1.57 Our results imply that C. elegans may be a sensitive in vivo assay system for the detection of toxicity from nanoplastic particles.

Using marine copepod as the assay system, it was reported that the transgenerational toxicity of 50 nm nanopolystyrene particles at concentrations higher than 1.25 mg L−1 could be observed.57 In this study, using C. elegans as the assay system, we detected the transgenerational toxicity of nanopolystyrene particles at concentrations higher than 100 μg L−1 (Fig. 3). Meanwhile, we found that only the toxicity of nanopolystyrene particles at the concentration of 10 μg L−1 was completely recovered in nematodes (Fig. 3). In nematodes, the transgenerational toxicity has also been detected after exposure to nanomaterials, such as Ag-nanoparticles, quantum dots, and graphene oxide,30,58,59 or heavy metals.60,61

For the observed transgenerational toxicity of nanopolystyrene particles from P0 generation to F1 generation, we tried to determine the possible underlying cellular mechanisms. Our results imply that the potential cellular mechanism for the observed transgenerational toxicity of nanopolystyrene particles may be largely due to the translocation of nanopolystyrene particles into the reproductive organs, such as the gonad, through the intestinal barrier. After prolonged exposure to nanopolystyrene particles in the range of μg L−1, we detected the accumulation of nanopolystyrene particles in both side arms of the gonads of nematodes (Fig. 5), which implies the potential transfer of nanopolystyrene particles to the next generation by entering the gonad. We further observed a certain amount of nanopolystyrene particles in the progeny of the exposed nematodes (Fig. 5). Nevertheless, we did not exclude the possibility that the observed transgenerational toxicity of nanopolystyrene particles may at least be partially due to the maternal effect.

Usually, it is considered that the leachate from microplastics or nanoplastics play an important role in inducing the toxicity in organisms.62,63 However, in this study, we did not detect the toxic effect of leachates from nanopolystyrene particles in the range of μg L−1 on nematodes using intestinal ROS production, locomotion behavior, and brood size as the endpoints (Fig. 4). We only found the toxicity in nematodes exposed to the leachate from nanopolystyrene particles at the concentration of 10[thin space (1/6-em)]000 μg L−1 (Fig. 4). Therefore, the induction of transgenerational toxicity of nanopolystyrene particles in the range of μg L−1 may not be due to the leachate from nanopolystyrene particles.

Several cellular mechanisms were further raised to explain the observed translocation of nanopolystyrene particles into the reproductive organs. One of the cellular mechanisms is that nanopolystyrene particles at concentrations higher than 10 μg L−1 significantly enhanced the intestinal permeability in nematodes after prolonged exposure (Fig. 6a), which in turn allowed the translocation of nanopolystyrene particles into the targeted organs through the intestinal barrier. The second cellular mechanism is that nanopolystyrene particles at concentrations higher than 10 μg L−1 could cause the significant induction of intestinal ROS production (Fig. 2a), which may contribute to the formation of damage on the intestinal barrier and the enhancement of intestinal permeability. Another cellular mechanism is that nanopolystyrene particles at concentrations higher than 10 μg L−1 could further prolong the mean defecation cycle length (Fig. 6c), which will lead to the severe accumulation of nanopolystyrene particles in the body of nematodes.

Conclusions

We employed the in vivo assay system of C. elegans to determine the possible transgenerational toxicity of nanopolystyrene particles and the underlying cellular mechanisms. After prolonged exposure, we detected the toxicity of nanopolystyrene particles at concentrations higher than 10 μg L−1. Moreover, we observed the transgenerational toxicity of nanopolystyrene particles at concentrations higher than 100 μg L−1. For the underlying cellular mechanism for this observed transgenerational toxicity, the development of transgenerational toxicity from nanopolystyrene particles might be mainly due to the translocation of nanopolystyrene particles into the reproductive organ such as the gonad, which might in turn lead to the transfer of nanopolystyrene particles to the next generation. The leachates from nanopolystyrene particles at concentrations in the range of μg L−1 did not contribute to the development of transgenerational toxicity. The enhancement of intestinal permeability and the prolonged defecation cycle length provide the important cellular basis for the accumulation and translocation of nanopolystyrene particles into the reproductive organs. Our results highlight the potential development of transgenerational toxicity from nanopolystyrene particles in the range of μg L−1 in environmental organisms.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This study was supported by University of Macau MYRG2017-00123-FHS.

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