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Inhibition effect of Na⁺ and Ca²⁺ on the bioaccumulation of perfluoroalkyl substances by Daphnia magna in the presence of protein

Corresponding Author

Xinghui Xia

School of Environment, Beijing Normal University, State Key Laboratory of Water Environment Simulation/Key Laboratory for Water and Sediment Sciences (Ministry of Education), Beijing, China

Address correspondence to [email protected].

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Andry Harinaina Rabearisoa,

Andry Harinaina Rabearisoa

School of Environment, Beijing Normal University, State Key Laboratory of Water Environment Simulation/Key Laboratory for Water and Sediment Sciences (Ministry of Education), Beijing, China

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Zhineng Dai,

Zhineng Dai

School of Environment, Beijing Normal University, State Key Laboratory of Water Environment Simulation/Key Laboratory for Water and Sediment Sciences (Ministry of Education), Beijing, China

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Xiaoman Jiang,

Xiaoman Jiang

School of Environment, Beijing Normal University, State Key Laboratory of Water Environment Simulation/Key Laboratory for Water and Sediment Sciences (Ministry of Education), Beijing, China

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Pujun Zhao,

Pujun Zhao

School of Environment, Beijing Normal University, State Key Laboratory of Water Environment Simulation/Key Laboratory for Water and Sediment Sciences (Ministry of Education), Beijing, China

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Haotian Wang,

Haotian Wang

School of Environment, Beijing Normal University, State Key Laboratory of Water Environment Simulation/Key Laboratory for Water and Sediment Sciences (Ministry of Education), Beijing, China

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Xinghui Xia,

Corresponding Author

Xinghui Xia

School of Environment, Beijing Normal University, State Key Laboratory of Water Environment Simulation/Key Laboratory for Water and Sediment Sciences (Ministry of Education), Beijing, China

Address correspondence to [email protected].

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Andry Harinaina Rabearisoa,

Andry Harinaina Rabearisoa

School of Environment, Beijing Normal University, State Key Laboratory of Water Environment Simulation/Key Laboratory for Water and Sediment Sciences (Ministry of Education), Beijing, China

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Zhineng Dai,

Zhineng Dai

School of Environment, Beijing Normal University, State Key Laboratory of Water Environment Simulation/Key Laboratory for Water and Sediment Sciences (Ministry of Education), Beijing, China

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Xiaoman Jiang,

Xiaoman Jiang

School of Environment, Beijing Normal University, State Key Laboratory of Water Environment Simulation/Key Laboratory for Water and Sediment Sciences (Ministry of Education), Beijing, China

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Pujun Zhao,

Pujun Zhao

School of Environment, Beijing Normal University, State Key Laboratory of Water Environment Simulation/Key Laboratory for Water and Sediment Sciences (Ministry of Education), Beijing, China

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Haotian Wang,

Haotian Wang

School of Environment, Beijing Normal University, State Key Laboratory of Water Environment Simulation/Key Laboratory for Water and Sediment Sciences (Ministry of Education), Beijing, China

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First published: 05 December 2014

https://doi.org/10.1002/etc.2823

Citations: 18

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Abstract

The authors investigated the individual effects of Ca²⁺ and Na⁺ on the bioaccumulation of 6 types of perfluoroalkyl substances (PFASs), including perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluoroundecanoic acid (PFUnA), and perfluorododecanoic acid (PFDoA), by Daphnia magna in water with 10 mg L⁻¹ bovine albumin or soy peptone. The bioaccumulation factors of PFASs by D. magna decreased linearly with the increase of Ca²⁺ and Na⁺ concentrations. The inhibition effect of Ca²⁺ was stronger than that of Na⁺, and the decreasing percentages of the body burden of PFASs in D. magna caused by the increment of 1 mmol L⁻¹ Ca²⁺ and 1 mmol L⁻¹ Na⁺ were 41% to approximately 48% and 2% to approximately 5%, respectively, in the presence of soy peptone. The partition coefficients (K_p) of PFASs between protein and water increased with rising Ca²⁺ and Na⁺ concentrations. The elevated K_p values led to the reduced concentrations of freely dissolved PFASs. This resulted in a decrease of PFAS bioaccumulation in D. magna, and the body burden of each PFAS was positively correlated with its freely dissolved concentration in water. The present study suggests that cations should be considered in the assessment of bioavailability and risk of PFASs in natural waters containing proteinaceous compounds. Environ Toxicol Chem 2014;9999:1–8. © 2014 SETAC

INTRODUCTION

Over the past 10 yr, perfluoroalkyl substances (PFASs) have increasingly attracted global attention because of their widespread application, environmental persistence, and potential toxicity to animals 1, 2. Some studies have shown that some PFASs such as perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) may induce potential carcinogenicity and reproductive toxicity on test organisms 3-5. The PFASs are globally distributed in the environment; their concentrations generally range from picograms to nanograms per liter for individual compounds in the aquatic environment, and they can reach micrograms per liter in some waters 6, 7. PFASs have a high bioaccumulation potential in organisms 8. For example, Lasier et al. 9, Rainie et al. 10, and Dai et al. 11 have demonstrated the bioaccumulation of PFASs in some aquatic organisms, including Lumbriculus variegatus, zebrafish (Danio rerio), rainbow trout (Oncorhynchus mykiss), and Daphnia magna.

Moreover, both laboratory studies and field monitoring have shown that PFASs tend to accumulate in protein-rich tissues and blood of exposed organisms 12-15. For instance, Jeon et al. 16 reported that perfluorinated compounds accumulated significantly in liver and serum of Sebastes schlegeli, a kind of black rockfish. Jones et al. 13 have demonstrated that PFOS was strongly bound with serum protein of fishes and birds. Butt et al. 17 also reported that PFASs accumulated in protein-rich tissues including blood, liver, and kidneys in whale and seabird species. Consequently, PFASs are considered as proteinophilic compounds 18, 19. Proteinaceous compounds are not only the main components of organisms including animals and plants but also are ubiquitous in aquatic systems, where they come from the wastewater of slaughterhouse, casein, fish, whey, and vegetable processing industries, with concentrations ranging from 1 mg L⁻¹ to 20 mg L⁻¹ 20. Feng et al. 21 reported that the protein concentration ranged from 1.8 mg L⁻¹ to 30.2 mg L⁻¹ in domestic wastewater of a typical region in the Zhejiang province of China. In our previous work 22, we provided evidence that proteins in aquatic environments also combine with PFASs and decrease their bioavailability, and a relatively higher concentration of proteins (>1 mg L⁻¹) will decrease the bioaccumulation of PFASs in D. magna.

In addition, some studies have reported that cations could combine with proteins, and the binding strength differs among different cations. For example, Kretsinger et al. 23 proposed that Ca²⁺ strongly binds to the protein containing a helix-loop-helix structural EF hand. Poonia and Bajaj 24 reported that Ca²⁺ has a higher affinity binding even in a 100-fold to 1000-fold excess than Mg²⁺ to protein. Speroni et al. 25 confirmed that Ca reinforces the protein structure by increasing the stability of existing interactions or promoting the formation of new ones, like hydrogen bonds, electrostatic interactions, or Ca bridges. Vrbka et al. 26 also studied the binding of a series of different proteins with ions and consistently found that sodium (Na⁺) binds at least twice as strongly to the protein surface as potassium (K⁺).

Ca²⁺ and Na⁺ are major cations in natural aquatic environments, with their concentrations ranging from several to thousands of milligrams per liter 27, 28. As mentioned, cations including Ca²⁺ and Na⁺ can affect the structure and conformation of proteins. Thus, we hypothesize that cations will affect the interaction of protein and PFASs and then exert influence on the PFAS bioavailability to, and bioaccumulation by, aquatic organisms. Moreover, the effects of cations will vary with their concentrations and types.

Therefore, the main objective of the present study was to investigate the individual effects of Ca²⁺ and Na⁺ on the bioaccumulation of 6 kinds of PFASs, including PFOS, PFOA, perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluoroundecanoic acid (PFUnA), and perfluorododecanoic acid (PFDoA), by D. magna in water with protein. Two types of model protein including soy peptone from plants and bovine albumin from animals were investigated, and the effects of cation concentrations were studied. Furthermore, the binding of PFASs to protein under different concentrations of Ca²⁺ and Na⁺ was examined, and the freely dissolved concentrations of PFASs were calculated to explore the effect mechanism of ions on the bioaccumulation of PFASs. The present study is the first to report the effect of ions on the bioaccumulation of PFASs by organisms in water containing protein compounds.

MATERIALS AND METHODS

Reagents

Perfluorooctane sulfonate (98%) was obtained from Tokyo Chemical Industries; PFOA (99.9%) and PFDA (99.9%) were obtained from Sigma-Aldrich; and PFNA (97%), PFUnA (95%), and PFDoA (95%) were obtained from Acros Organics. A stock solution containing these PFASs was prepared in a methanol to water solution (80:20, v/v) at a concentration of 200 mg L⁻¹ for each PFAS. Both [1,2,3,4–13C4] perfluorooctane sulfonate (MPFOS) and [1,2,3,4–13C4] perfluorooctanoic acid (MPFOA; purity > 99%) were purchased from Wellington Laboratories and used as recovery indicators. Chromatography-grade methanol was obtained from J.T. Baker. Methyl-tert-butyl ether (99.5%), tetrabutyl ammonium hydrogen sulfate, and ammonium acetate (98%) were obtained from Sigma-Aldrich; they were used to extract PFASs from D. magna. Sodium chloride (NaCl) and calcium chloride (CaCl₂) were obtained from Sinopharm Chemical Reagent. Soy peptone and bovine albumin were purchased from AMRESCO and Sigma-Aldrich, respectively. Sodium dihydrogen phosphate monohydrate (NaH₂PO₄ · H₂O) and anhydrous sodium hydrogen phosphate (Na₂HPO₄) were purchased from Fisher Chemical. Spectra6 dialysis bags of 7000 Da molecular weight were obtained from Sigma-Aldrich.

Cultivation of D. magna

The cultivation of D. magna was performed according to the chemical testing conditions described by Organisation for Economic Co-operation and Development 29. In brief, the cultivation of D. magna was conducted in artificial freshwater containing 0.294 g CaCl₂, 0.123 g MgSO₄, 0.064 g NaHCO₃, and 0.006 g KCl per liter deionized water (with Ca²⁺ = 2.64 mmol L⁻¹, Na⁺ = 0.76 mmol L⁻¹, and pH = 7.15). Daphnia magna were cultured under a 16:8-h light:dark photoperiod at 21 ± 0.5 °C and were fed a Scenedesmus subspicatus suspension twice per day. The detailed culture procedure can be found in our previous study 11.

Effect of Ca²⁺ and Na⁺ on PFAS bioaccumulation in the presence of protein

The bioaccumulation experiment procedure used in our previous study 22 was applied in the present study with some modification. In brief, each type of protein was prepared in artificial freshwater at a concentration of 10 mg L⁻¹. Each 500-mL polypropylene beaker received 200 mL protein solution and 1 mmol L⁻¹ to 10 mmol L⁻¹ Na⁺ (added as 120 g L⁻¹ NaCl solution) or 0.25 mmol L⁻¹ to 1 mmol L⁻¹ Ca²⁺ (added as 50 g L⁻¹ CaCl₂ solution; the experimental conditions are shown in Table 1). Next, 0.02 mL PFAS solution at a concentration of 50 μg mL⁻¹ was added to the beakers, obtaining a nominal concentration of 5 µg L⁻¹ for each PFAS. The beakers were shaken at 95 rpm at 21 °C in darkness for 72 h; a total of 10 D. magna (with the average wet wt of 2.07 ± 0.10 mg) were then added to each beaker and cultured for 72 h without food. After that, the 10 D. magna were collected, rinsed with artificial freshwater, dried by filter paper, and transferred to a 10-mL centrifuge tube. A wet weight of the D. magna was obtained, and the D. magna were then stored at –20 °C until extraction for the determination of the body burden of PFASs. A control experiment was conducted to study the PFAS bioaccumulation under different Ca²⁺ and Na⁺ concentrations without protein; a blank experiment was conducted lacking both proteins and PFASs. Each experiment set was carried out in triplicate. At the beginning and end of all tests, the hardness and pH of the experimental solutions were measured, and the results showed that their variations were 5% or less. After the bioaccumulation experiments, all of the daphnids survived. The PFAS bioaccumulation factors (BAF_ss; L kg⁻¹) based on the steady-state bioaccumulation were calculated according to Equation 1:

$urn:x-wiley:14381656:media:etc2823:etc2823-math-0001$ (1)

where C_B and C_w refer to the PFAS concentration in D. magna (µg kg⁻¹) and in the water phase (µg L⁻¹), respectively.

Table 1. Experimental conditions of perfluoroalkyl substances (PFAS) bioaccumulation and decreasing percentages of the body burden of PFASs in Daphnia magna caused by the increase of Ca²⁺ and Na⁺ concentrations compared with that of 0.76 mmol L⁻¹ Na⁺ and 2.64 mmol L⁻¹ Ca²⁺

Treatments	Na⁺ (mmol L^–1)	Ca²⁺ (mmol L^–1)	pH	Bovine albumin	Soy peptone
				Decreasing percentages of the body burden of PFASs
Control without protein	0.76a	2.64a	7.15a
Effect of Na⁺ in the presence of 10 mg L^–1 soy peptone or bovine albumin
Addition of 0 mmol L^–1 Na⁺	0.76	2.64	7.15
Addition of 1 mmol L^–1 Na⁺	1.76	2.64	7.19	3%–6%	2%–5%
Addition of 5 mmol L^–1 Na⁺	5.76	2.64	7.23	21%–33%	19%–29%
Addition of 10 mmol L^–1 Na⁺	10.76	2.64	7.28	39%–47%	32%–43%
Effect of Ca²⁺ in the presence of 10 mg L^–1 soy peptone or bovine albumin
Addition of 0 mmol L^–1 Ca²⁺	0.76	2.64	7.15
Addition of 0.25 mmol L^–1 Ca²⁺	0.76	2.89	7.17	9%–21%	6%–16%
Addition of 0.5 mmol L^–1 Ca²⁺	0.76	3.14	7.19	32%–43%	24%–34%
Addition of 1.0 mmol L^–1 Ca^2+c	0.76	3.64	7.24	46%–62%	41%–48%

a The composition of artificial freshwater for Daphnia magna cultivation.

Dialysis experiments

To study the association of PFASs with protein under the influence of Na⁺ and Ca²⁺, dialysis experiments were conducted with the method used in our previous study 22. In brief, 20 mL soy peptone or 20 mL bovine albumin stock solution at a concentration of 10 mg L⁻¹ was added to each dialysis bag. They were sealed and put in 500-mL polypropylene beakers with 200 mL deionized water, and they were then placed on a shaker at the speed of 90 rpm for 72 h in darkness to remove the components with molecular weights lower than 7000 Da. According to the preliminary experiments, no substantial bovine albumin and 4.3% of the soy peptone could pass through the dialysis bag. These results were considered to calculate the partition coefficients of PFASs between protein and water. The pretreated dialysis bags were put in beakers containing 200 mL artificial freshwater. Then 1 mmol L⁻¹ and 5 mmol L⁻¹ Na⁺ (as 120 g L⁻¹ NaCl solution) or 0 mmol L⁻¹, 0.25 mmol L⁻¹, and 1 mmol L⁻¹ Ca²⁺ (as 50 g L⁻¹ CaCl₂ solution) were added, respectively, to the solution. Specific amounts of PFAS solution were added to these dialysis bags, obtaining a nominal concentration of 5 µg L⁻¹ for each PFAS in the systems. The dialysis bags were resealed, and the beakers were again placed on a shaker at 120 rpm in darkness at 21 °C for 7 d. A preliminary experiment indicated that PFASs could pass through the dialysis bag freely, and a time of 72 h was long enough for PFASs to reach the equilibrium between the protein inside the dialysis bag and the water outside the dialysis bag. Each experiment consisted of 3 replicates. After equilibrium, water samples were collected from both the inside and outside of the dialysis bags for the determination of PFASs. The PFAS partition coefficients (K_p, L kg⁻¹) between protein and water were calculated using the following equation 22:

$urn:x-wiley:14381656:media:etc2823:etc2823-math-0002$ (2)

where C_free (μg L⁻¹) refers to the freely dissolved PFAS concentration in the system, which was equal to the PFAS concentration outside the dialysis bag; C_S (μg kg⁻¹) refers to the PFAS concentration associated with protein, which was calculated based on the difference in PFASs between the inside and outside of the dialysis bag.

Extraction and analysis of PFASs

An ion-pairing agent extraction technique was used to extract PFASs from water and D. magna samples, with some modification 30, 31. The extraction was conducted following the procedure descried in our previous study 11. In brief, each polypropylene centrifuge tube containing 10 D. magna or 4.5 mL water sample was spiked with 2 mL Na₂CO₃ (0.25 mol L⁻¹), 2 mL methyl-tert-butyl ether, 1 mL ion-pairing agent tetrabutyl ammonium hydrogen sulfate (0.5 mol L⁻¹, adjusted to pH 10), and 100 µL internal standards (10 ng MPFOA and MPFOS). The PFASs were analyzed with liquid chromatography–tandem mass spectrometry (Applied Biosystems API 3200 and Dionex Ultimate 3000) in an electrospray negative ionization mode. A 10-µL sample aliquot was injected into an Acclaim 120 C18 Column of 4.6 mm × 150 mm, and the mobile phase was 50 mmol L⁻¹ ammonium acetate and methanol at a 1 mL min⁻¹ flow rate. The detailed procedure can be found in our previous study 32.

Quality assurance and quality control

The procedure of quality assurance and quality control is described in the Supplemental Data. The limits of quantification (signal to noise [S/N] = 10) for the tested PFASs were between 0.01 µg L⁻¹ and 0.05 µg L⁻¹ with liquid chromatography–tandem mass spectrometry; and all procedural blank areas were lower than half limits of quantification. The repeatability of the standard curves with the correlation coefficients higher than 0.99 was confirmed before each determination. The recoveries of MPFOS, MPFOA, and the tested PFASs from the water samples and protein solutions were in the range of 86% to 108%. Recoveries of the mass spectrometric isotopes added to the D. magna samples ranged from 90% to 95%, and recoveries of the tested PFASs from the D. magna were in the range of 81% to 95% (Table S1). The recoveries of the indicators were used to correct all data of the tested PFASs. In the blank bioaccumulation tests, PFASs in the D. magna samples were below the detection limits. Furthermore, each PFAS accumulated in D. magna accounted for less than 2% of the whole amount added in the system; this indicated that PFAS bioaccumulation in D. magna did not exert significant influences on the PFAS concentrations and their equilibrium between protein and water in the system. For the dialysis bag experiments, PFASs could pass through the dialysis bag freely, with the PFAS concentration difference between the outside and inside of the dialysis bag being lower than 8% without protein in the system, and the mass balance results showed that the PFAS variations were lower than 10% in the system in the presence of protein.

Statistical analysis of data

All statistical analyses of the data were conducted using SPSS 18.0 for windows. The difference between each of the 2 compared groups was tested with one-factor analysis of variance (ANOVA). Duncan's multiple-range test was used to examine the differences among compared groups. Difference was considered statistically significant when the significance level was smaller than 0.05.

RESULTS AND DISCUSSION

Effect of ion concentrations on the PFAS bioaccumulation in D. magna

As shown in Figure 1, the body burden of PFASs in D. magna was ordered PFOA < PFNA < PFOS < PFDA < PFUnA < PFDoA for all of the treatments. The bioaccumulation of PFASs was positively correlated with the perfluoroalkyl chain length, and the body burden of PFOS was significantly higher than that of PFNA, with an equal perfluoroalkyl chain length. These findings were consistent with many reported results for Lumbriculus variegates, rainbow trout (Oncorhynchus mykiss), D. magna, and green mussels (Perna viridis) 9, 14, 20, 33. This indicated that the presence of protein and the concentrations of Na⁺ and Ca²⁺ did not exert influence on the order of 6 PFAS bioaccumulation in D. magna. In addition, as shown in Supplemental Data, Figure S1, the effect of Ca²⁺ and Na⁺ concentrations on the bioaccumulation of PFASs in D. magna was not significant in the absence of protein compounds. This suggests that although Ca²⁺ and Na⁺ are essential for D. magna, the variation of Ca²⁺ from 2.64 mmol L⁻¹ to 3.64 mmol L⁻¹ and the variation of Na⁺ from 0.76 mmol L⁻¹ to 5.76 mmol L⁻¹ will not exert significant influence on the bioaccumulation of PFASs in D. magna.

Details are in the caption following the image — **Figure 1**
Open in figure viewer PowerPoint

Effect of Na⁺ and Ca²⁺ concentrations on the bioaccumulation of perfluoroalkyl substances (PFAS) for 10 mg L⁻¹ soy peptone and 10 mg L⁻¹ bovine albumin by *Daphnia magna* (mean ± standard deviation, n = 3). Different letters (a, b, c, and d) mean significant (p < 0.05) differences among groups following analysis of variance and Duncan's multiple range tests. PFOA = perfluorooctanoic acid; PFNA = perfluorononanoic acid; PFOS = perfluorooctane sulfonate; PFDA = perfluorodecanoic acid; PFUnA = perfluoroundecanoic acid; PFDoA = perfluorododecanoic acid.

Compared with the body burden of PFASs in the absence of proteins, bovine albumin or soy peptone at a concentration of 10 mg L⁻¹ decreased the bioaccumulation of PFASs in D. magna under different Na⁺ and Ca²⁺ concentrations. For example, the presence of 10 mg L⁻¹ soy peptone reduced the body burden of PFASs by 7% to approximately 50% under different concentrations of Ca²⁺. In addition, the decrease of PFAS bioaccumulation caused by the bovine albumin was greater than that caused by soy peptone regardless of the Na⁺ and Ca²⁺concentrations (Supplemental Data, Figure S2), and this might be explained by the difference in characteristics of soy peptone and bovine albumin (Supplemental Data, Table S2).

In the presence of soy peptone or bovine albumin, the body burden and BAF_ss values of PFASs in D. magna decreased with increasing Na⁺ concentration (p < 0.05; Figure 1 and Supplemental Data, Table S3). For the systems with soy peptone, the body burden of PFASs was decreased by 2% to approximately 5% and 32% to approximately 43% when Na⁺ increased from 0.76 mmol L⁻¹ to 1.76 mmol L⁻¹, and 10.76 mmol L⁻¹, respectively (Table 1). For the systems with bovine albumin, the body burden of PFASs was decreased by 3% to approximately 6% and 39% to approximately 47% when Na⁺ increased from 0.76 mmol L⁻¹ to 1.76 mmol L⁻¹, and 10.76 mmol L⁻¹, respectively. Further analysis showed that the BAF_ss value of each PFAS in D. magna decreased linearly with the increase of Na⁺ concentration in the presence of soy peptone or bovine albumin (n = 4, r > 0.96, p < 0.05; Fig. 2).

Similar to Na⁺, the addition of Ca²⁺ to protein solutions also inhibited the PFAS bioaccumulation by D. magna. As shown in Figure 1, the body burden of all tested PFASs decreased gradually when Ca²⁺ concentration increased from 2.64 mmol L⁻¹ to 3.64 mmol L⁻¹. For example, when the Ca²⁺ concentration increased from 2.64 mmol L⁻¹ to 2.89 mmol L⁻¹, 3.14 mmol L⁻¹, and 3.64 mmol L⁻¹, the body burden of PFOS decreased from 1023 ± 53 ng g⁻¹ to 921 ± 41 ng g⁻¹, 780 ± 59 ng g⁻¹, and 545 ± 57 ng g⁻¹ for the systems in the presence of ng g⁻¹ soy peptone, and a similar result was obtained for the systems in the presence of bovine albumin (Fig. 1). For soy peptone, the PFAS body burden and BAFss values decreased by 41% to approximately 48% when the concentration of Ca²⁺ increased from 2.64 mmol L⁻¹ to 3.64 mmol L⁻¹. For bovine albumin, they decreased by 46% to approximately 62%. As shown in Figure 2, BAF_ss value of each PFAS in D. magna decreased linearly with the increase of Ca²⁺ concentration (n = 4, r > 0.96, p < 0.05 except for PFDoA).

Comparison of the effect of Ca²⁺ and Na⁺ on PFAS bioaccumulation in the presence of protein

According to the results shown in Figure 3, Ca²⁺ was more efficient at inhibiting the PFAS bioaccumulation in D. magna than Na⁺ with the presence of protein in water. For instance, when Ca²⁺ concentration was 2.64 mmol L⁻¹ and Na⁺ concentration increased from 0.76 mmol L⁻¹ to 1.76 mmol L⁻¹, the decreasing ratios of PFASs in D. magna caused by the increment of 1 mmol L⁻¹ Na⁺ were 2% to approximately 5% in the presence of 10 mg L⁻¹ soy peptone, and 3% to approximately 6% in the presence of 10 mg L⁻¹ bovine albumin. When Na⁺ concentration was 0.76 mmol L⁻¹ and Ca²⁺ concentration increased from 2.64 mmol L⁻¹ to 3.64 mmol L⁻¹, the decreasing ratios of PFASs in D. magna caused by the increment of 1 mmol L⁻¹ Ca²⁺ were 41% to approximately 48% and 46% to approximately 58% in the presence of soy peptone and bovine albumin, respectively. According to the results shown in Figure 2, the decreasing rates of BAF_ss value of each PFAS caused by the increase of Ca²⁺ were much higher than that caused by Na⁺. For example, the decrease in BAF_ss (L kg⁻¹) for PFASs was approximately 65 L kg⁻¹ to 175 L kg⁻¹and 5 L kg⁻¹ to 17 L kg⁻¹, caused by each increase in unit (mmol L⁻¹) [Ca²⁺] and [Na⁺], respectively, in the presence of bovine albumin, and that was 57 L kg⁻¹ to 185 L kg⁻¹ and 5 L kg⁻¹ to 16 L kg⁻¹, respectively, in the presence of soy peptone.

Influencing mechanism of ions on PFAS bioaccumulation in the presence of protein

Effects of ions on the partition of PFASs between protein and water

Except for PFOS, the partition coefficients (K_p) of PFASs between protein and water increased with perfluoroalkyl chain length for all of the treatments, and the K_p value between bovine albumin and water was significantly higher than that between soy peptone and water for each of the PFAS (Table 2). The partition coefficients increased with rising Na⁺ and Ca²⁺ concentrations (Table 2), and the effect of Ca²⁺ on the binding of PFASs and protein was much greater than that of Na⁺. According to the relationship between logK_p and log[Ca²⁺] as well as log[Na⁺] shown in Supplemental Data, Figure S3, the increase in logK_p for PFASs between soy peptone and water was approximately 2.4 mmol L⁻¹ to 5.4 mmol L⁻¹ and 0.2 mmol L⁻¹ to 0.7 mmol L⁻¹ for each increase in log unit [Ca²⁺] and [Na⁺], respectively, and that between bovine albumin and water was approximately 4.2 mmol L⁻¹ to 7.9 mmol L⁻¹ and 0.4 mmol L⁻¹ to 1.0 mmol L⁻¹, respectively. These results indicate that the binding of PFASs to proteins was dependent, not only on the concentration, but also on the type of cations.

Table 2. Partition coefficients (K_p, L kg⁻¹) of perfluoroalkyl substances (PFAS; represented as log value) between protein and water in the presence of different Ca²⁺ and Na⁺ concentrations

	PFOA	PFNA	PFOS	PFDA	PFUnA	PFDoA
Soy peptone
0.76 mmol L^–1 Na⁺, 2.64 mmol L^–1 Ca²⁺, pH = 7.15	3.83 ± 0.016	4.10 ± 0.058	4.51 ± 0.012	4.61 ± 0.001	5.05 ± 0.026	5.19 ± 0.101
0.76 mmol L^–1 Na⁺, 2.89 mmol L^–1 Ca²⁺, pH = 7.08	4.10 ± 0.010	4.30 ± 0.012	4.87 ± 0.052	5.03 ± 0.011	5.22 ± 0.020	5.69 ± 0.013
0.76 mmol L^–1 Na⁺, 3.64 mmol L^–1 Ca²⁺, pH = 7.11	4.21 ± 0.013	4.71 ± 0.014	4.93 ± 0.021	5.22 ± 0.011	5.75 ± 0.017	6.01 ± 0.019
1.76 mmol L^–1 Na⁺, 2.64 mmol L^–1 Ca²⁺, pH = 7.13	3.88 ± 0.006	4.14 ± 0.012	4.55 ± 0.026	4.65 ± 0.020	5.11 ± 0.037	5.22 ± 0.028
5.76 mmol L^–1 Na⁺, 2.64 mmol L^–1 Ca²⁺, pH = 7.17	4.18 ± 0.022	4.36 ± 0.033	4.69 ± 0.010	5.06 ± 0.021	5.30 ± 0.032	5.78 ± 0.018
Bovine albumin
0.76 mmol L^–1 Na⁺, 2.64 mmol L^–1 Ca²⁺, pH = 7.15	4.47 ± 0.059	4.65 ± 0.058	4.92 ± 0.072	5.17 ± 0.243	5.30 ± 0.097	5.60 ± 0.070
0.76 mmol L^–1 Na⁺, 2.89 mmol L^–1 Ca²⁺, pH = 7.08	4.94 ± 0.020	5.20 ± 0.021	5.37 ± 0.023	5.74 ± 0.019	6.10 ± 0.011	6.21 ± 0.026
0.76 mmol L^–1 Na⁺, 3.64 mmol L^–1 Ca²⁺, pH = 7.11	5.14 ± 0.035	5.34 ± 0.019	5.65 ± 0.014	6.04 ± 0.018	6.45 ± 0.016	6.78 ± 0.037
1.76 mmol L^–1 Na⁺, 2.64 mmol L^–1 Ca²⁺, pH = 7.13	4.84 ± 0.033	5.08 ± 0.045	5.22 ± 0.028	5.62 ± 0.019	5.73 ± 0.024	5.92 ± 0.023
5.76 mmol L^–1 Na⁺, 2.64 mmol L^–1 Ca²⁺, pH = 7.17	5.02 ± 0.035	5.18 ± 0.029	5.26 ± 0.018	5.79 ± 0.019	6.17 ± 0.016	6.31 ± 0.017

PFOA = perfluorooctanoic acid; PFNA = perfluorononanoic acid; PFDA = perfluorodecanoic acid; PFUnA = perfluoroundecanoic acid; PFDoA = perfluorododecanoic acid.

Because protein is one kind of dissolved organic matter (DOM), we compared the results obtained in the present study with others about DOM. A similar result was reported for the binding of anthracene, one kind of polycyclic aromatic hydrocarbon (PAH), to 25 mg L⁻¹ humic acid (pH = 7) that the partition coefficient increased with rising Na⁺ concentration 34. The increased partition coefficients of hydrophobic organic compounds (HOCs) between DOM and water with increasing ion strength have been found by other researchers. Gauthier et al. 35 found that the binding of pyrene, one kind of PAH, to humic acid increased with rising ionic strength when sodium chloride was used as the electrolyte. Jota and Hassett 36 reported an increasing binding of 2,2',5,5'-tetrachlorobiphenyl by humic acid when ionic strength was increased. Lee et al. 37 proposed a 3-stage variation model to describe DOM conformation change with increasing ionic strength, that is, change of DOM structural configuration, DOM aggregation, and salting-out effect. In addition, the salting-out effect of HOCs has been applied to explain the enhanced HOC sorption on humic substances after the addition of ions 38.

The salting-out effect also may occur for PFASs; a comparison of the solubility of PFOS in fresh water (680 mg L⁻¹) and ocean water (12.4 mg L⁻¹) confirms that the salting-out effect is significant for PFASs 39. Therefore, the increase of Ca²⁺ and Na⁺ concentrations could decrease the solubility of PFASs and elevate their binding to proteins. In addition, previous studies showed that the binding of cations to the negatively charged carboxylic groups of DOM could reduce the charge of DOM 40, 41. Because protein has a net negative charge in solution 42, the binding of cations to the negatively charged groups of protein, such as carboxylic groups, could also reduce the charge of protein. The PFASs also charge negatively in water solutions, and so the charge reduction of protein may result in the decrease of the repulsive force between PFASs and protein. Na⁺ and Ca²⁺ can also form a bridge between PFASs and protein; this leads to the enhancement of binding between PFASs and proteins. Therefore, the partition coefficients of PFASs between protein and water increased with rising cation concentrations. Tang et al. 43 also reported that the presence of Ca²⁺ increased the adsorption of PFASs to mineral by binding to the mineral surface and thereby increasing the electrostatic attraction between PFASs and mineral. Because Ca²⁺ is a divalent cation and Na⁺ is a monovalent cation, Ca²⁺ would exert a stronger salting-out effect and decrease the charge of protein more pronouncedly than Na⁺, resulting in greater effect on the binding of PFASs with protein. In addition, as mentioned before, for all of the treatments with different concentrations of Ca²⁺ and Na⁺, the partition coefficients of PFASs were positively correlated with their perfluoroalkyl chain length (Table 2). Therefore, both hydrophobic interactions and electrostatic attraction are important processes for the binding of PFASs to proteins.

Effects of ions on the freely dissolved PFAS concentrations

The freely dissolved concentration of each PFAS in the system was calculated using the following equations 22:

$urn:x-wiley:14381656:media:etc2823:etc2823-math-0003$ (3)

$urn:x-wiley:14381656:media:etc2823:etc2823-math-0004$ (4)

where C_free (µg L⁻¹) refers to the freely dissolved PFAS concentration; C_total (µg L⁻¹) refers to the total PFAS concentration in the system; C_p (kg L⁻¹) refers to the protein concentration in the system. K_p (L kg⁻¹) refers to the protein – water partition coefficient of individual PFAS with different Na⁺ and Ca²⁺ concentrations; here the K_p value with the initial PFAS concentration of 5 µg L⁻¹ was used because each PFAS was at a concentration of 5 µg L⁻¹ in the system in the bioaccumulation experiments. The results showed that the freely dissolved PFAS concentrations decreased with increasing Na⁺ and Ca²⁺ concentrations in the exposure system, and the decrease caused by Ca²⁺ was higher than that caused by Na⁺ (Supplemental Data, Table S4). Because only the freely dissolved PFASs are bioavailable to D. magna 20, the body burden of PFASs decreased with increasing Na⁺ and Ca²⁺ concentrations, and the decrease caused by Ca²⁺ was more pronounced than by Na⁺. In addition, the freely dissolved PFAS concentrations in the presence of bovine albumin were lower than that in the presence of soy peptone (Supplemental Data, Table S4), and this led to the lower body burden of PFASs in the system with bovine albumin. Further analysis showed that the body burden of each of the PFAS was positively correlated with its freely dissolved concentration in systems with different cation concentrations and different protein types (r > 0.64, n = 11; p < 0.05).

CONCLUSIONS

The presence of protein (10 mg L⁻¹ soy peptone or 10 mg L⁻¹ bovine albumin) inhibited the bioaccumulation of PFASs by D. magna, and both Ca²⁺ and Na⁺ could inhibit the bioaccumulation of PFASs further, with the body burden of PFASs decreasing linearly with the increase of Ca²⁺ and Na⁺ concentrations. The impact of Ca²⁺ on PFAS bioaccumulation by D. magna was greater than that of Na⁺ in the water system containing bovine albumin or soy peptone.

The partition coefficients (K_p) of PFASs between proteins and water increased with rising Ca²⁺ and Na⁺ concentrations in aqueous solution, and the effect of Ca²⁺ was stronger than that of Na⁺. The increase in logK_p for PFASs between soy peptone and water was approximately 2.4 mmol L⁻¹ to 5.4 mmol L⁻¹ and 0.2 mmol L⁻¹ to 0.7 mmol L⁻¹ for each increase in log unit [Ca²⁺] and [Na⁺], respectively. This suggests that both hydrophobic forces and electrostatic interactions play important roles in the association of PFASs with protein. The elevated partition coefficients of PFASs between protein and water caused by the increase of Ca²⁺ and Na⁺ concentrations led to the reduced concentrations of freely dissolved PFASs, resulting in the decreased body burden of PFASs in D. magna.

According to the present study, both Ca²⁺ and Na⁺ exerted significant influence on the PFAS bioavailability to and PFAS bioaccumulation by D. magna in the presence of proteins. Because protein compounds are ubiquitous in natural waters, and the variations of Ca²⁺, Na⁺, and other cation concentrations are significant, the present study suggests that both proteins and cations should be considered in the assessment of bioavailability and risk of PFASs in natural waters.

SUPPLEMENTAL DATA

Tables S1–S4.

Figures S1–S3 (225 KB).

Acknowledgment

The present study was supported by National Science Foundation for Distinguished Young Scholars, (No. 51325902), the Fund for Innovative Research Group of the National Natural Science Foundation of China (Grant No. 51421065), National Natural Science Foundation of China (No. 51279010), and Specialized Research Fund for the Doctoral Program of Higher Education (No. 20110003110030).

Supporting Information

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Citing Literature

Volume34, Issue2

February 2015

Pages 429-436

Inhibition effect of Na⁺ and Ca²⁺ on the bioaccumulation of perfluoroalkyl substances by Daphnia magna in the presence of protein

Abstract

INTRODUCTION