Green Algae as Carriers Enhance the Bioavailability of 14C-Labeled

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Green Algae as Carriers Enhance the Bioavailability of 14C-Labeled Few-Layer Graphene to Freshwater Snails Yu Su, Xin Tong, Chi Huang, Jiani Chen, Sijin Liu, Shixiang Gao, Liang Mao, and Baoshan Xing Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b05796 • Publication Date (Web): 28 Dec 2017 Downloaded from http://pubs.acs.org on December 29, 2017

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Environmental Science & Technology

TOC Art/ Abstract

ACS Paragon Plus Environment


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Green Algae as Carriers Enhance the Bioavailability of 14C-Labeled

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Few-Layer Graphene to Freshwater Snails

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Yu Su a, Xin Tong a, Chi Huang a, Jiani Chen b, Sijin Liu c, Shixiang Gao a,

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Liang Mao a, *, Baoshan Xing d a

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b

State Key Laboratory for Mineral Deposits Research, School of Earth Sciences and Engineering, Nanjing University, Nanjing 210093, China

8 c

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State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China

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State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing 210093, China

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d

Stockbridge School of Agriculture, University of Massachusetts, Amherst, MA 01003, USA.

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* Corresponding Author: Tel.: +86 25 89680393; Fax: +86 25 89680393; Email: [email protected].

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Abstract

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The waterborne exposure of graphene to ecological receptors has received much attention; however,

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little is known about the contribution of food to the bioaccumulation potential of graphene. We

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investigated the effect of algal food on the uptake and distribution of 14C-labeled few-layer graphene

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(FLG) in freshwater snails, a favorite food for Asian people. In a water-only system, FLG (~158

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µg/L) was ingested by and accumulated in the snails. Adding algae to the water significantly

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enhanced FLG accumulation in the snails, with a bioaccumulation factor of 2.7 (48 h exposure).

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Approximately 92.5% of the accumulated FLG was retained in the intestine; in particular, the

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accumulated FLG in the intestine was able to pass through the intestinal wall and enter the intestinal

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epithelial cells. Of them, 1.3% was subsequently transferred/internalized to the liver/hepatocytes, a

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process that was not observed in the absence of the algae. Characterizations data further suggested

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that both of the extra- and intracellular FLG in the algae (the algae-bound fraction was 30.2%)

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significantly contributed to the bioaccumulation. Our results provide the first evidence that algae as

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carriers enhanced FLG bioavailability to the snails, as well as the potential of FLG exposure to

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human beings through consuming the contaminated snails.

41 42 43 44 45 46 47 48 49 50 51 2



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Introduction

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Graphene-family nanomaterials (GFMs) uniquely combine various superior properties, making them

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attractive for multidisciplinary study and promising for numerous applications.1, 2 In the past few

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years, GFMs have moved from the research laboratory to the marketplace.3, 4 Rapid increases in

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industrial production (e.g., 400 tonnes/yr in China)4 and application will eventually lead to GFMs

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release into the environment. A few modeling studies suggest that the environmental concentrations

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of GFMs may be in the range of ppt to ppb, or even lower.5, 6 Under such low concentrations, we

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found that the carbon-14 labeled few-layer graphene (FLG) nanoparticles (NPs) were highly mobile

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in aquatic systems,7, 8 possibly having long-term and wide-ranging eco-toxicological impacts.9 For

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example, GFM exposure was reported to suppress the algal growth10 and inhibit the bacterial

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activity.11 However, the environmental risks of graphene largely depended on its physicochemical

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properties, such as lateral size and surface chemistry.12 Our previous works have shown that the

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suspended FLG NPs were readily taken up by aquatic organisms (e.g., water fleas,13 fishes,14 and

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sediment-dwelling oligochaetes15), this raises the importance of determining their potential for

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bioaccumulation (whether the FLG concentration in an organism exceeds that in its environmental

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matrix).16 Freshwater snails inhabiting lakes, ponds, and reservoirs may also ingest GFMs from

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water via filter feeding. Considering that several freshwater snail species are a favorite food for

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Asian people, it is very likely that GFMs will be transferred and pose exposure risks to human

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beings after the consumption of GFM-contaminated snails. However, this issue is often overlooked

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in previous GFM toxicity-related studies. To date, no study has quantitatively investigated the

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uptake, bioaccumulation, and distribution of GFMs in snails.

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In addition to waterborne exposure, dietary exposure may also act as a potential uptake route of

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GFMs in freshwater snails. As a primary producer, algae suspended in the water column provide a

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food base for most aquatic organisms, including freshwater snails. GFMs released in aquatic

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environments

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hetero-agglomeration.10 Freshwater snails take up GFMs along with the contaminated algal food will

will

inevitably

interact

with

ubiquitous

planktonic

algae

through

3


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be a pathway for GFMs to the animal. Therefore, understanding the relative importance of aqueous

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and dietary pathways is critical for better predicting the bioaccumulation potential of GFM in natural

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conditions. Exposure via algal food will affect NP availability to predators.17-19 For instance, Au NPs

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could be internalized to the epithelial cells of the digestive glands of freshwater clams Corbicula

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fluminea through dietary exposure;19 the freshwater snail Lymnaea stagnalis could efficiently

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assimilate Ag and CuO NPs from diatoms enriched in the NPs.17, 18 However, these studies did not

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clarify how NPs were associated with algal cells and hence affected their bioavailability to the

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predator. GFMs attached to the cell wall will interact with extracellular polymeric substances (EPS)

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excreted by algae,10 creating a biological surface coating on the particles, possibly enhancing cellular

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recognition and absorption in the predatory organisms.20 On the other hand, GFMs are able to cross

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the cell-wall barrier and be internalized to algal cells.21, 22 GFM associated with the prey (either

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sorbed to cell wall or internalized) will be available to ecological receptors at higher trophic levels.16,

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23

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important factors affecting the accumulation and distribution of GFM in aquatic organisms.14, 15, 24

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We hypothesized that dietary exposure may enhance GFM accumulation in freshwater snails when

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compared to exposure directly from the water, hence the potential of exposure to human beings.

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According to our recent work, the state of GFM agglomeration and surface chemistry are

In the present study, we focus on the interactions between

14

C-labeled FLG13 and the green

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algae Scenedesmus obliquus and their subsequent impacts on FLG accumulation and distribution in

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the freshwater snail Cipangopaludina cathayensis. S. obliquus is a model organism commonly used

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in NP eco-toxicity testing.25, 26 The edible C. cathayensis is a common gastropod in the family

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Viviparidae and has a wide distribution throughout central and southeastern China. The species feeds

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on epilithic and epiphytic algae and also consumes planktonic algae. Therefore, the objectives of this

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study were to (1) explore FLG uptake in the algae and how FLG associates with the algal cell, (2)

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quantify FLG concentration in the whole-body and main organs of the snail, and (3) calculate the

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FLG bioaccumulation factor (BAF) in the snails. To the best of our knowledge, this is the first study

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that quantified the GFM BAF in aquatic organisms. Our results will provide valuable insights into 4



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the roles of green algae on FLG transport and fate and may help address a key gap in understanding

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the ecological risk of FLG.

106 107

Materials and Methods

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Graphene Synthesis and Characterization. Synthesis and characterization of the 14C-labeled

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four-layer graphene are described elsewhere.13 The specific radioactivity of FLG was determined to

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be 16 ± 0.59 mCi/g (n = 3; uncertainties always indicate standard deviation values).24 From the

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Raman spectrum (Figure S1A), typical D-band (1343 cm-1), G-band (1579 cm-1), and 2D-band (2686

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cm-1) of graphene were observed in FLG.13 FLG surfaces consisted of 89% C, 6% O, 1.4% H, and

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3.6% N via X-ray photoelectron spectroscopy (Figure S1B).13 The detected oxygen-containing

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groups (e.g., C=O and C-OH) were introduced by the addition of 14C-phenol during FLG synthesis.24

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Using atomic force microscopy (Figure S1C,D), the lateral size and thickness of FLG were 60–590

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nm and 1.05–4.05 nm (n = 214), respectively.27 A stock suspension of 10 mg/L FLG was prepared

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through probe tip sonication as previously described.13,

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determined by mixing 1 mL of this suspension with 3 mL of a scintillation cocktail (Goldstar,

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Meridian) in triplicate and measuring radioactivity via liquid scintillation counting (LSC) (LS 6500,

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Beckman Coulter).

27

The concentration of FLG stock was

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Test Organisms. The freshwater algae S. obliquus (FACHB-14) were obtained from the

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Institute of Hydrobiology, Chinese Academy of Sciences (Wuhan, China). Batch cultures of S.

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obliquus were grown in sterilized SE medium (Bristol’s solution) at 25 ± 1 °C (12:12 h light/ dark

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photoperiod) with agitation three times a day.23 The algal cells were harvested during the exponential

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stage of their growth and centrifuged at 178 g for 10 min (4 °C), and the obtained pellet was

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re-suspended in artificial freshwater (AF water) (KCl, 1.2 mg/L; CaCl2·2H2O, 58.8 mg/L;

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MgSO4·2H2O, 24.7 mg/L; NaHCO3, 13.0 mg/L; ionic strength = 2 mM).13, 28 The concentrated algal

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suspension was centrifuged (16 g, 10 min, 4 °C) to remove the large agglomerates, and the

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supernatant was diluted with AF water and then used as the algal stock suspension. The optical 5


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density (OD) of the stock suspension was measured using an UV-vis spectrophotometer (Cary 50,

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Varian) at the maximum absorption wavelength of 685 nm (Figure S2A of the Supporting

132

Information, SI). The chlorophyll-a level of the stock suspension was measured using a modified

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method developed by Grace Analytical Lab.29 Detailed procedures are presented in the SI, and the

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calibration curve is shown in Figure S2B. The cell density of the stock suspension was determined

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using a Neubauer-improved counting chamber (Marienfeld, Germany). For cell enumeration, three

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replicate algal samples were counted by at least two counts per replicate.

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Freshwater snails C. cathayensis were collected from the rocky shores of Lake Yangshan,

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Nanjing (32°06′47.68″N 118°56′29.43″E) and acclimated in the laboratory in AF water at 25 ± 1 °C

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(12:12 h light/ dark photoperiod) for a week. The snails were fed with S. obliquus daily. One day

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prior to the uptake experiments, snails of restricted size range (average shell length of 2.3 ± 0.1 cm

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and wet weight of 2.8 ± 0.4 g (including the soft tissues and shell), n = 300) were cleaned carefully

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with a soft brush and then were transferred to clean AF water. Food was withheld during this period.

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Interactions between FLG and Algae. Two groups of settling experiments were performed

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following the modified method of Ma et al.30 One was to examine the independent settlings of

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individual FLG and algal suspensions, and the other was to investigate the co-settling of FLG-cell

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mixtures. Therefore, the discrepancy between co- and individual-settling curves was used to analyze

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the FLG-cell hetero-agglomeration. Briefly, FLG and the algae were suspended independently or

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together in 500 mL of AF water in glass containers (9.6 cm length × 8.4 cm width × 8.1 cm height).

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To evaluate the effect of cell density on FLG phase distribution, two algal suspensions with cell

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densities of 1.1 × 105 and 1.2 × 106 cells/mL were used. The cell density was commonly used for

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evaluating NP toxicity to algae.31, 32 The containers were left undisturbed in an incubator for up to 24

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h at 25 ± 1 °C (12:12 h light/ dark photoperiod). At designated time intervals, the individual FLG

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and algal suspensions or the FLG-cell mixtures (1 mL) were sampled and filtered through 3 µm

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polycarbonate membranes. Preliminary experiments testing FLG solid-liquid separation efficiency

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(or algae) by centrifugation and filtration (details in the SI) showed that this type of membrane 6



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ensured 100% retention of the algal cells and allowed the maximum (90.3%) free FLG passage

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(Table S1). The radioactivity of the filtrate (800 µL) was measured via LSC as described above

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(giving the free FLG concentration (C, µg/L)). The algal cells retained by the membranes were used

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to determine the chlorophyll-a levels as described above. The cell density (D, 106 cells/mL) was then

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calculated using the linear equations (Figure S2C, D) based on the OD685 value and chlorophyll-a

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level/cell counts. All settling experiments were conducted in triplicate to verify experimental

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reproducibility. The fraction of algae-bound FLG is defined as the difference between the C/C0 value

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measured in the absence and presence of the algae. The difference between the free FLG mass in the

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absence and presence of the algae together with the cell density was used to calculate the FLG mass

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bound to the algae (ng FLG per 106 cells).

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In another group of co-settling experiments, 1.2 × 106 cells/mL of the algal suspension was

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incubated for 24 h with 1 mg/L of FLG using the same procedure as described above. The

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FLG-contaminated algal cells were collected after filtration through 3 µm polycarbonate membranes

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and centrifugation at 178 g for 10 min (4 °C). Cellular ultrastructure was observed with a Zeiss

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Supra 55 field emission scanning electron microscopy (SEM) and an FEI Tecnai G2 F20 field

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emission transmission electron microscopy (TEM). The unexposed algal cells were used as controls.

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More information about the routine methods for cell sample preparation is presented in the SI. In

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addition, Raman spectra of the cell samples were acquired using a confocal Raman microscope

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(XploRA PLUS, Horiba Scientific) equipped with a laser excitation of 514 nm.14

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Uptake of Algae by the Snails. To investigate snail ingestion of algal food, five snails were

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transferred to 500 mL of algal suspension (1.1 × 105 and 1.2 × 106 cells/mL) in a glass container (9.6

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cm length × 8.4 cm width × 8.1 cm height) at 25 ± 1 °C (12:12 h light/dark photoperiod). At each

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time interval, three replicate containers were removed, the chlorophyll-a level was measured, and it

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was then converted to cell density using the same procedure as described above. To determine

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whether the FLG negatively affects the ability of the snail to ingest algae, another group of snails

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was exposed to algal suspensions spiked with FLG (158 ± 6 µg/L, n = 3) using the same procedure 7


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as described above. The tested concentration of FLG (158 µg/L) in this study was chosen based on

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previous modeling studies, which predicted that the concentrations of carbon-based nanomaterials in

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surface water were in the range of ppt to ppb.5, 6 The difference between the cell density measured at

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0 h and 24 h together with the number of the snails (i.e., 5) and the observation period (i.e., 24 h)

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were used to calculate ingestion rate of algae (number of cells consumed per individual snail and

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hour).33 In order to facilitate comparisons with feeding rates reported in the literature, the algae

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ingestion rate was transformed in unit of percentage of ingested food per g of snail wet weight per

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hour.

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Uptake of FLG by the Snails. Prior to the exposure experiment, FLG (158 ± 6 µg/L; n = 3)

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was mixed with the algal suspension (1.1 × 105 and 1.2 × 106 cells/mL) in a glass container (9.6 cm

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length × 8.4 cm width × 8.1 cm height), and then was left undisturbed for 24 h in an incubator at 25

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± 1 °C (12:12 h light/ dark photoperiod). After that, these suspensions were mixed again to allow the

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precipitated algal cells to re-suspend in water. The free FLG concentrations in the low- and

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high-density algal suspension were respectively determined to be 146 ± 2 and 110 ± 2 µg/L (n = 3)

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using the same procedures as described above. The FLG suspended independently in AF water (158

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± 6 µg/L; n = 3) was prepared as a control shortly before use. Five snails were exposed to the three

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FLG suspensions for up to 48 h (a long enough period for the snail finishing off the algal food) at 25

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± 1 °C (12:12 h light/ dark photoperiod). For all uptake experiments, the FLG suspensions were not

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renewed during the exposure period. The snails were not fed when they were exposed to FLG

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suspended in AF water alone. At each time point, three replicate containers were removed. The snails

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were rinsed with deionized (DI) water, anesthetized in an ice-water bath, and dissected. Main organs

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(i.e., gill, intestine, stomach, and liver) and remaining tissues of the snails from the same container

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were pooled, freeze-dried, and weighed. Then, these samples were oxidized in a Zinsser Analytic

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OX-500 biological oxidizer (BO), and the radioactivity was determined via LSC (detailed

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procedures are provided in the SI). The measured radioactivity was background-subtracted by that of

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the unexposed snails. Before the snails were removed, the concentration of free FLG was measured 8



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using the same procedure as described above. Control experiments (without the snails) were

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performed to determine the FLG losses caused by sedimentation. To evaluate the effect of algae on

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the FLG bioaccumulation, the BAF (L/g) was calculated as the ratio of the FLG concentration in the

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whole body (including FLG in the intestinal tract) or specific tissues of the snail (µg/g) and the

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waters (µg/L).16

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Quantification and Visualization of FLG in Intestinal Cells and Hepatocytes. Another

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group of snails was exposed to FLG alone and to FLG + algae (1.2 × 106 cells/mL) using the same

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procedure as described above. After exposure for 48 h, the intestines and livers of the snails were

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sampled. To remove feces in the gut lumen, 50 mL of DI water were injected quickly from one end

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of the intestine with a sterilized syringe. The eluted feces were dried and then the radioactivity was

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measured via BO and LSC. Thereafter, the clean intestinal tissues (or liver tissues) were minced into

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small pieces using a scalpel and mixed with 3 mL of phosphate buffered saline (PBS) buffer

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containing 5 mM EDTA.34 The mixture was pipetted up and down 50 times and filtered through a 45

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µm stainless steel mesh to retain tissue debris. Another 3 mL of PBS-EDTA solution was introduced

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to rinse the tissue debris. The isolated cells in the filtrate were collected through centrifugation at

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178 g for 5 min (4 °C). The supernatant was added to 10 mL of scintillation cocktail for radioactivity

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measurement. The intestinal epithelial cells (or hepatocytes) were observed using an inverted light

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microscope (Eclipse Ti, Nikon) at 400 × magnification. The tissue debris and isolated cells were

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freeze-dried and weighed; then, the radioactivity was measured. To further investigate the FLG

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cellular internalization, the cell samples were prepared and observed with TEM as described in the

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SI. The Raman spectra were also recorded using the same methods described above.

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Statistical Analysis. One-way ANOVA with Tukey’s multiple comparison tests were used to

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determine the significant differences in FLG concentrations in the algae or the snails among different

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treatment groups. Statistical difference was set at p < 0.05.

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Results and Discussion 9


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FLG Adherence to Algae. We first investigated the FLG-algae hetero-agglomeration by

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comparing FLG sedimentation in the absence and presence of the algae. From the individual settling

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profiles (Figure S3), neither the algal cells nor FLG was stable in AF water due to cell-cell or

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FLG-FLG agglomeration. The high-density algal suspension was less stable than the low-density

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one, approximately 0.7% (for 105 cells/mL) and 3.6% (for 106 cells/mL) of the total number of algal

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cells had settled out of water within 24 h. The impact of cell density on the stability of the algal

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suspension was consistent with the concentration-dependent agglomeration of colloids;7, 35, 36 an

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increase in density may result in more cell-cell collisions and subsequent attachment. In the absence

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of algae, the loss of free FLG caused by homo-agglomeration was measured to be 6% at 24 h (Figure

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1A). FLG presence did not have a notable impact on the stability of the algal suspension, as the D/D0

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values in the algal and FLG mixtures were not significantly (p > 0.05) different from that in the

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absence of FLG. However, the free FLG concentration was reduced by 2.3-fold (or 6.0-fold) after

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co-settling for 24 h with the low-density (or high-density) algal suspension when compared to in the

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absence of algae. This result is indicative of strong interactions between FLG and algal cells. By

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comparing the SEM images of the control (Figure 2A) and the FLG-contaminated (Figure 2B, C)

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algal cells, small and large FLG agglomerates were frequently observed adhering onto the cell walls.

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A similar result was reported by Zhao et al.10 on Chlorella pyrenoidosa upon being exposed to

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multi-layer graphene. According to the electrophoretic mobility measurements (provided in the SI),

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both the FLG ((-0.81 ± 0.038) ×10-8 m2/Vs) and the algae ((-1.31 ± 0.10) ×10-8 m2/Vs) were

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negatively charged in AF water. The EPS produced by S. obliquus mainly consisted of

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polysaccharides with a molecular weight of 127.9 kDa and total organic carbon of 27.8 mg /L at a

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cell density of 1.4 ×107 cells/mL.37, 38 The negative charges were acquired through deprotonation of

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the oxygen-containing groups on FLG7, 24 and the algal EPS. It is known that the EPS can strongly

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adsorb on NPs through a variety of mechanisms.39-41 We have demonstrated that the FLG had a high

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affinity for a model protein and polysaccharide in a previous work.8 The EPS covering on the cell

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surfaces may provide binding sites for the FLG and facilitate the FLG-cell hetero-agglomeration by 10



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overcoming electrostatic repulsive forces through electrostatic and hydrophobic interactions and

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through hydrogen bonding.10 At a given FLG concentration (~ 158 µg/L), an increase in the algal

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cell density (105 to 106 cells/mL) resulted in more binding sites being available for FLG, thus

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increasing the fraction of the algae-bound FLG from 7.6% to 30.2% after mixing for 24 h. However,

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FLG mass per unit cell was significantly (p < 0.05) decreased from (88.8 ± 1.2) ×10-6 ng to (34.1 ±

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3.0) ×10-6 ng at 24 h due to cell density dilution (Figure 1B). At a given algal cell density (106

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cells/mL), the number of binding sites for FLG was constant, FLG mass associated with individual

267

algal cell increased with the increasing initial FLG concentration (Figure 1C, D). These observations

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suggest that the algal cells possess great capacity to adsorb and accumulate FLG and consequently

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alter FLG phase distribution.

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Internalization of FLG to Algal Cells. Cellular ultrastructure was examined using TEM to

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investigate whether the FLG adhering on cell walls was able to enter the cell. Black particles were

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observed attaching onto cell wall and distributing in plasma membrane and protoplasm (Figure 2E).

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High-resolution TEM images (the enlarged parts from the points with yellow arrows in Figure

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2F–H) of these particles show visible lattice fringes with interplanar crystal spacing ranging from

275

0.32 to 0.33 nm, equal to the reported interlayer distance of graphite.42 Meanwhile, no lattice fringe

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was observed for the control cell (Figure 2D). Moreover, typical graphene D and G bands were

277

detected inside of the cells from Raman spectra shown in Figure 2I, further confirming the cellular

278

internalization of FLGs. Aligned with our results, recent studies also demonstrated that both pristine

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GO (lateral length, 0.5–5 µm) and the GO released from the embedded epoxy resin (lateral length,

280

70–130 nm) could enter Chlorella vulgaris cells.21, 22, 43 The cell wall is a key barrier for the entry of

281

large molecules to the algae.44 Because the lateral size (60–590 nm27) and hydrodynamic diameter

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(606 ± 29 nm in AF water) of FLG were larger than the diameter of pores embedded in the algal cell

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wall ( 0.05) different from those in the presence of 158 ± 6 µg/L of FLG (Figure 3B).

301

Hence, the co-existence of such low FLG doses did not affect the feeding behavior of the snail.

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Because significant cell wall attachment and cellular internalization of FLG occurred when the algae

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and FLG were mixed for 24 h, it is interesting to know whether the algae-bound fraction would

304

contribute significantly to the FLG influx within the snails.

305

High-Density Algae Enhanced the Bioaccumulation of FLG in the Snails. To investigate

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whether association with S. obliquus affected FLG uptake by C. cathayensis, the snails were exposed

307

to FLG alone or to the mixture of FLG with algae for 48 h. FLG whole-body levels in the snails are

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shown in Figure 4A. FLG was measurable in the snails exposed to FLG alone or to FLG + 105

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cells/mL for 12 h and was gradually reduced following two days of exposure. This observation

310

suggested that the snails eliminated the FLG more rapidly than they took up the FLG in the

311

treatments of FLG alone and FLG + 105 cells/mL. The impacts of algae on FLG phase distribution 12



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and hence the uptake and accumulation of FLG are discussed in later sections. Under these exposure

313

scenarios, the calculated BAF values in the whole body of snails were much less than 1, whereas the

314

BAF values in some intestinal samples were more than 1 (Table S2). These results indicated that the

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FLG bioaccumulation was not detectable on a whole-body level, which might be resulted from tissue

316

mass dilution. Conversely, the body burden of FLG showed an increasing trend over time when the

317

animals were exposed to FLG + 106 cells/mL. Moreover, the BAF value measured at 48 h (2.7 ± 1.8;

318

n = 5) was significantly (p < 0.05) increased (Table S2). During this period, the difference between

319

the body weight of the snails exposed to FLG alone and the combined treatments was less than 10%;

320

therefore, changes in the biomass could not account for the enhanced accumulation.

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We further investigated how and to what extent the interaction between FLG and algae affected

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the phase distribution of FLG and hence the FLG ingestion and bioaccumulation in the snails. Prior

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to the exposure (0 h), the pre-mixing process resulted in nearly 92.4% and 69.8% of the total added

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FLG freely suspended in 105 and 106 cells/mL of algae-amended water, respectively (Figure 3D).

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After exposure for 24 h, free FLG fractions in water were reduced rapidly to 36.4% (without algae),

326

34.0% (105 cells/mL), and 19.3% (106 cells/mL) (Figure 3C). Since the sedimentation contribution

327

to the free FLG loss was negligible, the reduction in the free FLG in water indicated the fractions of

328

free FLG uptake in the snails, which were calculated to be 63.6%, 58.4%, and 50.5% for the FLG

329

alone, the FLG + 105 cells/mL, and the FLG + 106 cells/mL, respectively. In addition to the free FLG,

330

7.6% and 30.2% of the total added FLG were respectively associated with the low- and high-density

331

algal suspensions at the start of the exposure period (Figure 1A). As mentioned previously, the snails

332

could take up approximately 42.8% (105 cells/mL) and 87.4% (106 cells/mL) of the total feeding

333

algae within 24 h (Figure 3B). There is no doubt that they would also take up the algae-bound FLG

334

with the ingestion of algal food. If the FLG particles were uniformly distributed among the algal

335

cells, the fraction of algae-bound FLG ingested by the snails at 24 h should be 3.2% (i.e., 7.6%

336

multiplied by 42.8%) and 26.4% (i.e., 30.2% multiplied by 87.4%) in the presence of low- and

337

high-density algae, respectively. In total, the snails took up more FLG (both free and algae-bound 13


Page 15 of 30


338

FLG) at 24 h from FLG + 106 cells/mL (76.9%) than from either FLG alone (63.6%) or FLG + 105

339

cells/mL (61.6%) (Figure 3D). Thus, we concluded that the presence of high-density algae

340

significantly altered the FLG phase distribution due to FLG-cell interactions; subsequently, rapid

341

ingestion of the algae-bound fraction contributed to FLG accumulation in the snails.

342

Effects of High-Density Algae on the FLG Distribution in the Snails. Distribution results

343

(Figure 4B, 4C, and S4) showed that in the absence of algae, 57.9% of the ingested FLG was

344

concentrated in the intestine (3–671 µg/g dw) at 12 h and then was completely depurated at 48 h,

345

which is consistent with changes in FLG whole-body levels in the snails (Figure 4A). Unlike the

346

intestines, the gills (0.01–6 µg/g dw), stomachs (0.04–4.4 µg/g dw), livers (0.09–0.3 µg/g dw), and

347

remaining tissues (0.04–4 µg/g dw) contained lower FLG concentrations after the two days of

348

exposure. In comparison with the FLG alone, the addition of 105 cells/mL of algae did not have a

349

significant effect on FLG distribution during the exposure period. However, the presence of 106

350

cells/mL of algae significantly (p < 0.05) increased the intestinal FLG mass from 36.4 ± 7.0 ng (FLG

351

alone) to 6752 ± 2331 ng at 48 h, which accounted for 92.5% of the total FLG mass in the snails.

352

The calculated BAF value in the intestines of the snails was 82 ± 54 (n = 5), indicating that FLG

353

bioaccumulation in the snails observed at 48 h in the presence of high-density algae primarily

354

resulted from the significant FLG accumulated in the intestine. Strikingly, when 106 cells/mL of

355

algae were added into the exposure media, the FLG mass in the liver was significantly (p < 0.05)

356

increased, accounting for 0.8% (0.05 ± 0.03 ng) and 1.3% (0.10 ± 0.08 ng) of the total FLG mass in

357

the snails at 24 h and 48 h, respectively. When the exposure period was extended to 120 h, the FLG

358

mass in liver (0.07 ± 0.03 ng) did not decrease significantly (p < 0.05). In the remaining tissues (gill,

359

0.3%; stomach, 3.6%; remaining tissue, 2.2%), the FLG mass sum merely accounted for ~6.1% of

360

the total FLG mass in the snails, and the accumulation of FLG in these tissues was scarcely observed

361

(BAF < 1) (Table S2 and S3).

362

We further explored the effect of high-density algae on the fate of FLG in the intestine. After

363

removal of feces from the gut lumen, only 0.05), FLG

410

transport from the intestinal epithelium to liver could occur with the extension of exposure time. In

411

the absence of the algae, most of the FLG ingested by the snails was in its free form, which may

412

agglomerate in the digestive tracts of the snail and have fewer interactions with the intestinal

413

epithelium from that bound to the algae.

414

Environmental Implications. In this study, the algae-bound FLG became more bioavailable to 16



Page 18 of 30

415

freshwater snails than the FLG freely suspended in water, indicating that interaction with algae may

416

represent a significant environmental process controlling the transport and fate of FLG in aquatic

417

systems. Our results for the dietary pathway of FLG to freshwater snails derived from a relatively

418

high algal cell density are probably not realistic for the natural environment. They do, however, help

419

to point out the importance of mode of exposure and clarify results of dietary exposure tests where

420

FLG-cell hetero-agglomeration will affect FLG phase distribution and where freshwater snails with a

421

strong filter feeding ability will take up and absorb the algae-bound FLG in tissues. Thus, algal-cell

422

density as an input parameter should be considered in the fate model for predicting the persistence

423

and exposure of FLG in aquatic ecosystems. More importantly, the propensity for algae-bound FLG

424

to accumulate in the intestines of the snail may increase the probability of transfer up the food chain,

425

thus creating exposure risk for upper-level predators, including human beings.

426 427

ASSOCIATED CONTENT

428

Supporting Information

429

Additional details of experimental methods and results as well as other supporting tables and

430

figures. This information is available free of charge via the Internet at http://pubs.acs.org/.

431

AUTHOR INFORMATION

432

Corresponding Author

433

Phone: +86-25 89680393; fax: 86-25 89680393; e-mail: [email protected].

434

Notes

435

The authors declare no competing financial interest.

436 437

ACKNOWLEDGMENTS

438

We acknowledge the financial support from the National Natural Science Foundation of China

439

(21377049, 21677074, and 21607072), a Foundation for the Author of National Excellent Doctoral 17


Page 19 of 30


440

Dissertation of PR China (201355), and the Fundamental Research Funds for the Central

441

Universities (021114380044, 021114380067, 021114380070).

442 443

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(55) Qu, G.; Liu, S.; Zhang, S.; Wang, L.; Wang, X.; Sun, B.; Yin, N.; Gao, X.; Xia, T.; Chen, J. J.;

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588 589 590 591 592 593 594 595 596 23


Page 25 of 30


597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612

FIGURE 1. Normalized concentrations (C/C0) of free FLG (not bound to algae) in AF water as a

613

function of time in the absence and presence of the algae S. obliquus (A, C). The results shown in (A)

614

were obtained via settling experiments, in which the initial concentration (C0) of FLG was 158 ± 6

615

µg/L (n = 3), and the initial algal cell density (D0) values were 1.1 × 105 and 1.2 × 106 cells/ mL. The

616

results shown in (C) were obtained via magnetic stirring experiments (the procedures are described

617

in the SI), in which the D0 value of the algal suspension was 1.2 × 106 cells/ mL, and the C0 values

618

of FLG were 103.0 ± 0.6 and 975 ± 12 µg/L (n = 3). The FLG masses bound to algal cells shown in

619

(B) and (D) were calculated based on the results shown in (A) and (C), respectively. Data points are

620

the mean and standard deviation values calculated from triplicate samples.

621 622 623 624 625 24



626

A

Control

B

Page 26 of 30

C

FLG

627 628 629 630 631 632

D

E

F

633 634 635 636

0.32 nm

637 638 639

G

H

I

640 0.32 nm 641 0.33 nm 642 643 644 645 646

FIGURE 2. Surface attachment and cellular internalization of FLG to the algae S. obliquus. The

647

algae (106 cells/mL) were exposed to 1 mg/L of FLG for 24 h. SEM images of control (A) and

648

FLG-exposed algal cells (B). The image in (C) is enlarged from (B). TEM images of sectioned

649

control (D) and FLG-exposed algal cells (E). High-resolution TEM images in (F–H) are taken from

650

the enlarged parts in (E), which show visibly ordered graphitic lattices with interplanar crystal

651

spacing ranging from 0.32 to 0.33 nm. Yellow arrows indicate FLG. Inside, the inside space of the

652

algal cell. Outside, the outside environment of the algal cell. (I) The Raman spectra of the sectioned

653

control (red line) and the FLG-exposed algal cells (blue line).

654 25


Page 27 of 30


A

105 algal cells/mL

106 algal cells/mL

without/with snails

without/with snails

655 656

FIGURE 3. (A) Time interval photos of green algae S. obliquus suspensions in the absence and

657

presence of the freshwater snails C. cathayensis. (B) Normalized cell density (D/D0) of the

658

suspended algae in AF water as affected by snail ingestion. (C) Normalized concentrations (C/C0) of

659

free FLG (not bound to algae) in AF water as affected by snail ingestion. The initial cell density (D0)

660

of the algal suspension was 1.1 × 105 and 1.2 × 106 cells/mL. The initial concentrations (C0) of free

661

FLG in the FLG alone, the FLG + 105 cells/mL, and the FLG + 106 cells/mL treatment groups were

662

158 ± 6, 146 ± 2, and 110 ± 2 µg/L (n = 3), respectively. Data points are the mean and standard

663

deviation values calculated from triplicate samples. (D) Changes in the fraction of free and

664

algae-bound FLG in water and the contributions of these FLG to the FLG uptake in the snails before

665

and after the FLG exposure.

666 667 668 669 26



Page 28 of 30

670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686

FIGURE 4. FLG concentrations in the whole-body (A), intestine (B), and liver (C) of C.

687

cathayensis. The snails were exposed to the FLG alone (without algae) or to the mixture of FLG and

688

algae (cell intensity was in the order of 105 or 106 cells/mL). The initial FLG concentration

689

suspended in water was 158 ± 6 µg/L (n = 3). Squares represent the means, lines indicate medians,

690

boxes represent 25th and 75th percentiles, error bars indicate minimum and maximum, and × marks

691

represent outliers. Asterisks indicate significant differences (*, p < 0.05; **, p < 0.01) between

692

treatment groups with or without algal addition. (D) Effects of exposure modes on FLG intestinal

693

distribution. Data points are the mean and standard deviation values calculated from three replicate

694

samples.

695 696 697 698 27


Page 29 of 30


699

A

D

B

E

700 701 702 703 704 705 706 707 708

0.33

709

0.34 0.33

710 711

0.33

0.34

712

0.34 713 714 715

C

F

716 717 718 719 720 721 722 723

FIGURE 5. Uptake and accumulation of FLG in intestinal epithelial cell and hepatocyte of the

724

snails exposed to the FLG mixture (158 ± 6 µg/L) and the algae (1.2 × 106 cells/mL) for 48 h. TEM

725

images of the intestinal epithelial cell (A) and hepatocyte (D). Inset: optical microscope images of

726

the cells. High-resolution TEM images (B) and (E) were taken from the enlarged parts (i.e., h1, h2,

727

and h3) of (A) and (D), respectively. The Raman spectra of the intestinal epithelial cell (C) and 28



Page 30 of 30

728

hepatocyte (F) (blue lines), control cells (red lines) were obtained from the snails without FLG

729

exposure.

29


Green Algae as Carriers Enhance the Bioavailability of 14C-Labeled

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