Edible clay inclusion in the diet of oysters can reduce tissue residues of polychlorinated biphenyls

Abstract

Objective

Polychlorinated biphenyls (PCBs) are lipophilic and persistent environmental pollutants that are readily absorbed and accumulated in high concentrations in fatty tissues of humans and animals. Invertebrate animals, such as oysters, are vulnerable and sensitive to PCB contamination.

Methods

Previously, our in vitro isothermal studies have shown that acid processed montmorillonites (APM) can effectively bind PCBs and Aroclors. Therefore, in a novel application of this work, a dietary strategy for shellfish was developed using APM, and its parent clay to reduce exposures to PCBs in oysters. PCB residues in oysters with clay treatment at different dietary inclusion rates and durations were measured and compared to a washout treatment.

Results

The efficacy and safety of this strategy were supported by a significant reduction of PCB residues with the inclusion of a low level of APM (0.05%) during a 4-day treatment. Moreover, this sorbent strategy reduced PCB residues in oysters in a dose- and time-dependent manner.

Conclusions

Based on our results, it is possible that clay-based sorbents such as APM, can be included in the diet to significantly reduce exposures to PCBs.

Introduction

Polychlorinated biphenyls (PCBs) are chlorinated aromatic hydrocarbons that were discovered and initially characterized in 1881. The physical and chemical properties of PCBs, such as stability, resistance to degradation, oxidation and chemical agents, low vapor pressures, and fire resistance have resulted in their widespread use as electrical insulators and their persistence in the environment [1]. An important environmental concern about PCBs is their incorporation into the food chain. The problem can be magnified after flooding which can mobilize contaminated sediment. Benthic invertebrates feeding on the bottom consume PCBs and later pass them in the food chain to fish, humans and other creatures (Fig. 1). Importantly, PCB exposure from seafood accounts for over 90% of PCB exposure to humans [2]. There are several means by which PCB concentration can be mitigated, such as hydrolysis under extreme conditions and photodegradation. However, due to the overall stability of PCBs, environmental conditions are not likely to promote these chemical reactions.

Fig. 1.

PCBs and other hazardous chemicals can be mobilized in soil and sediment and accumulated through the food chain

Oysters are natural filter feeders that commonly grow in groups called an oyster reef (or bed) found in salt water or brackish water. They feed by pumping water through their gills, trapping particles of food as well as nutrients, and they are routinely exposed to clays and clay-containing sediments from the environment. Enhanced food uptake by oysters exposed to kaolinite clay has been reported in the literature [3]. Oyster culture is one of the most prominent forms of marine aquaculture in the USA, where approximately 14 thousand metric tons of oysters are harvested, and 2 billion oysters are consumed every year [4]. Also, algal feeds (containing microalgae concentrates) are used by shellfish hatcheries in more than 80 countries around the world. This makes them extremely vulnerable to water pollution, and exposure to PCBs from algae diets has been well documented [5–9]. Therefore, this pilot study was designed to characterize the ability of clays, when included in algae-based oyster diets, to reduce PCB uptake and residues.

Previously, we have reported that acid processed montmorillonite clay (APM) can effectively adsorb and detoxify coplanar and non-coplanar PCBs and Aroclors, based on in vitro and in silico studies [10]. This significant adsorption of toxins onto surfaces of APM was facilitated by its high surface area, high porosity, d₀₀₁ interlayer spacing and a polymorphic structure that provided a variety of active binding sites for PCBs. Importantly, APM was derived from a parent montmorillonite clay that has been shown to be safe for ingestion by both humans and animals [11]. The objective of this study was to determine if dietary sorbents such as APM can effectively reduce PCB uptake and residues in oysters. The effectiveness of various doses of sorbents and lengths of treatment were investigated and compared to a washout treatment.

Results and discussion

Total weight and shell length on Day 0 and Day 4, and soft tissue weight on Day 4 were measured and recorded. As shown in Table 1, there was no significant difference in the total weight and shell length change on Day 4 in all groups, including control and treatment with PCBs and/or clays (p > 0.5). This result is consistent with previous work, where 4 ppm PCB showed no observable adverse effects on oyster growth [12, 13], and suggests that the inclusion of APM and parent clay, up to 2% in the diet, should be safe for short-term consumption.

Table 1.

Effects of dietary addition of PCB and clays on oyster growth

	Total weight on day 0(g)	Total weight on day 4 (g)	Shell length on day 0 (mm)	Shell length on day 4 (mm)	Soft tissue weight on day 4 (g)
Basal diet	35.7 ± 4.56	36.1 ± 5.62	57.5 ± 1.25	57 ± 1	11.1 ± 1.76
2% montmorillonite	58.27 ± 2.28	58.43 ± 2.1	73.33 ± 1	73.67 ± 1.28	13.9 ± 0.95
2% APM	47.97 ± 1.98	48.47 ± 1.74	68.5 ± 0.65	69 ± 0.67	13.17 ± 0.74
4 ppm PCB	47.42 ± 4.68	48.05 ± 5.02	70.8 ± 1.75	71 ± 2	12.35 ± 1.43
4 ppm PCB + 2% montmorillonite	51.87 ± 4.89	52.17 ± 5.19	74.67 ± 0.38	74.2 ± 0.78	16.77 ± 1.81
4 ppm PCB + 2% APM	49.43 ± 3.83	50.27 ± 4.3	75.6 ± 1.22	75.33 ± 1.5	8.37 ± 1.64

Data are presented as mean ± SD. No significant differences were observed in total weight and shell length on day 4 compared to day 0

In this study, we determined that Crassostrea virginica oysters, at the chosen size, could easily consume 0.25 mL of diet per day. Also, we used PCB 153 in this study, since it has been shown to be the most significant PCB congener found in all oyster tissue compartments [5, 6]. As shown in Table 2, the PCB 153 uptake rate in oysters was linear with concentration in the diet as described by the equation y = 0.0497x + 0.1151 (r² = 0.86). This first-order kinetic model suggests that the short-term absorption of toxicant depends on exposure concentration, which agrees with the previous literature [12, 14]. The uptake of PCB 153 in oysters was the greatest (0.61 ± 0.05 μg) at the highest nominal exposure level in diets equal to 10 ppm (i.e., a total of 10 μg from 0.25 mL diet/oyster/day for 4 days). This indicated an uptake and accumulation rate (toxicant absorption efficiency) of approximately 6.1 ± 0.5%, which is consistent with a reported absorption range of 0.7–50% by C. virginica [15]. This study demonstrated that PCBs (that can be sorbed to components of the basal diet) are still bioavailable and that oysters can effectively assimilate PCBs via contaminated diets. Interestingly, there was a very low amount (0.022 ± 0.015 μg) of PCB 153 detected in the control oysters that were fed only basal diet. Since no PCB was detected in the algae-based diet prior to spiking, a possible source of the contamination could be from environment sources during their development. This trace level falls in the range of PCBs detected in oysters from lightly contaminated areas (i.e., Tampa bay, Florida with 63.2–112 ng/g dry weight) [16]. This data suggests that PCB existence in the environment is widespread and common and that large populations of oysters can be easily exposed to PCBs.

Table 2.

Change in PCB residues with increased dose (and after a 4-day washout)

Exposure (ppm)/treatment	0	2	4	Washout	8	10
PCB residues (ppm)	0.02 ± 0.015	0.24 ± 0.028	0.45 ± 0.079	0.36 ± 0.018*	0.49 ± 0.045	0.61 ± 0.05

^*p ≤ 0.05

Oysters are natural filters and are highly vulnerable to the accumulation of environmental contaminants [5]. Therefore, oysters may be used as sensitive indicators of PCB accumulation and reduction of residues and toxicity after treatment. The results of the washout treatment in Table 2 showed that PCB uptake was significantly reduced by 21 ± 4% to 0.36 ± 0.018 ppm after washing with clean sea water for 4 days (p ≤ 0.05). It is possible that the reduction rate can be increased with longer treatment duration. Although washout is an important step in the bioremediation process [17], it can be time-consuming and costly.

Previously, our laboratory has reported the high binding efficacy of APM for coplanar PCBs, non-coplanar PCBs and Aroclors based on in vitro and in silico studies [10, 18]. Importantly, parent montmorillonite [(Na,Ca)_0.3(Al,Mg)₂Si₄O₁₀(OH)₂·nH₂O] is the only clay that has been shown to be safe for animal and human consumption based on numerous animal studies and six human clinical interventions in Africa and the USA [11]. The treatment of clays, such as montmorillonites, with acid results in an increased surface area (approximately 1200 m²/g), mesopores with a diameter between 2 and 50 nm, low concentrations of trace metals including aluminum, calcium and sodium, and a low coefficient of linear expansibility in water. The pH of the final APM product is approximately 3, which is similar to acidic foods such as meat, cheese and chocolate. In fact, the inclusion of acidic food additives can serve as a common taste enhancer and increase palatability and consumption [19]. Also, acids, such as sulfuric acid, are permitted as food and feed additives to adjust pH [20, 21]. Thus, the inclusion of APM at a low concentration in the diet for short-term treatments should be safe.

Figure 2 showed that parent montmorillonite clay, at a 0.05% inclusion rate, reduced PCB exposure by 5 ± 1% (p ≤ 0.05); moreover, doses between 0.1 and 2% reduced PCB residues by 21 ± 7% to 27 ± 3% (p ≤ 0.05), which was similar to the reduction rate achieved by the washout treatment (21 ± 4%). This was in alignment with a previous study, where oysters co-exposed to sediment showed a slightly lower PCB accumulation than oysters exposed to PCBs alone [5]. The same experiment was repeated with co-exposure to sand (containing no clay) and results showed no significant difference of PCB uptake compared to the toxin control group (data not shown). This result suggested that the reduction of PCB residues resulted from adsorption onto clay surfaces, instead of competitive uptake of PCBs and clay particles from the diet. APM showed significantly more sorption of PCBs than parent clay. APM at an inclusion level as low as 0.05% reduced residues by 27 ± 9%, compared to 5 ± 1% by parent montmorillonite clay at the same rate (p ≤ 0.05). Additionally, APM at higher inclusion rates of 0.1%, 0.5%, and 2% significantly reduced PCB residues in a dose-dependent manner by 72 ± 9%, 90 ± 4% and 94 ± 2%, respectively (p ≤ 0.01). The possible mechanisms of enhanced sorption onto APM may result from its increased surface area, porosity and structural diversity that can lead to higher binding capacity and tighter binding.

Fig. 2.

PCB reduction percentage by clay treatment at inclusion rates ranging from 0.05 to 2% for 4 days (*p ≤ 0.05, **p ≤ 0.01)

The results from a time-course study in Fig. 3 showed that APM reduced PCB levels in a time-dependent manner and that the 4-day treatment consistently delivered the highest reduction percentage of PCB residues (p ≤ 0.01), compared to day 1 and day 2 treatments at the same APM inclusion rate. The 2-day treatment resulted in similar to slightly higher reduction compared to the 1-day treatment, suggesting that reduction is exponential with duration. Based on our results, low inclusion of APM (< 2%) requires a minimum 4-day administration to neutralize the majority of PCB contamination at 4 ppm. Additionally, the dose-dependent manner of APM was also shown for 1-day and 2-day treatment. Specifically, the lowest inclusion rate of APM at 0.05% slightly reduced PCB residues by 2 ± 2%, 11 ± 5% and 42 ± 8% (p ≤ 0.01), whereas the highest inclusion rate at 2% showed the highest reduction of 60 ± 3% (p ≤ 0.01), 55 ± 5% (p ≤ 0.01) and 88 ± 4% (p ≤ 0.01) for 1-day, 2-day and 4-day treatment, respectively. The dose-and time-dependent reduction of PCB residues by APM suggest that these materials can make a major impact on toxin contamination of oysters. This conclusion was supported by our in vitro isothermal results showing that APM had high binding capacity, affinity and enthalpy for PCBs and significantly protected a living organism (hydra vulgaris) against PCB and Aroclor toxicities [10]. Based on the results of the current study, it is possible that APM (and similar clays) could be administered through the diet and serve as a strategy to reduce PCBs in shellfish, fish and humans. The inclusion rate of APM and the length of treatment can be adjusted based on the contamination level.

Fig. 3.

PCB reduction percentage by APM treatment at inclusion rates ranging from 0.05 to 2% for 1 day, 2 days and 4 days (*p ≤ 0.05, **p ≤ 0.01)

Materials and methods

Oyster exposure to PCB‑contaminated diet

PCB-contaminated diet was prepared by mixing PCB 153 (purity > 99%, from Professor Stephen Safe at Texas A&M University, College Station, TX) with the basal diet commonly used for oyster culture (Shellfish Diet 1800®, Reed Mariculture Inc., CA) to yield an increasing concentration gradient of 0, 2, 4, 8 and 10 ppm (μg/mL) PCB. Concentrations were determined based on results from “range-finding” experiments in which no significant effects on oyster survival and microalgae viability were observed [22]. PCB-contaminated diet was gently stirred for 2 h at 24 °C on an electronic shaker under a chemical fume hood. The extent of mixing of PCBs with diet was found to be over 99.8%, and no PCB residues were detected in the basal diet.

Two-year-old C. virginica oysters of similar size (Table 1), that tested negative for virus and bacterial infections, were used in this study. Oysters were maintained in closed plastic containers to prevent evaporation. Each container included 5 L of sea water at 25 °C, pH 8.1 and 32‰ salinity, and each was aerated and thoroughly mixed using air stones to stimulate feeding and maintain sufficient dissolved oxygen. Each oyster was fed daily 0.25 mL of PCB-contaminated diets at 0–10 ppm for 4 days (n = 3 in each group).

Washout and clay treatment

To detect the effectiveness of a washout treatment suggested by EPA [23], following exposure to 4 ppm PCB-contaminated diet for 4 days, three oysters were extracted for PCB residues and another three were transferred to a new container in clean, aerated sea water and basal diet, and extracted at the end of 4 days.

Parent montmorillonite (TxESI, Inc., TX) [24, 25] and APM [26–28] at 0.05%, 0.1%, 0.5% and 2% w/v were individually administered in the PCB-contaminated diet at 4 ppm. Clay control groups contained 2% clays in the basal diet. The mixture of clay and diet was agitated for 2 h at 1000 rpm on an electronic shaker to ensure even distribution before administration to oysters. Each control and treatment group, containing three oysters, were maintained in separate plastic containers with 5 L of sea water and 0.25 mL of daily diet. In a time-course study, oysters were treated for 1, 2 or 4 days (in triplicate) to limit the effects of significant body weight change on the uptake of PCB153. At the end of the experiment, oysters were measured and extracted to determine PCB uptake and tissue residues.

PCB extraction and quantitation

Extraction of PCB residues was performed following previously described methods [29–31] with modification. Oyster soft tissues were removed from the shell with a sterile shucking knife and weighed. They were mixed with anhydrous sodium sulfate and homogenized using 150 Handheld Homogenizer (Fisher Scientific, MA) at 8000–10,000 rpm for 5 min until no visible chunks were observed [6]. The mixture was then extracted in a Soxhlet apparatus for 2 h at 80 °C with 100 mL petroleum ether. Extracts were concentrated to approximately 20 mL and transferred a Strata® NH2 solid-phase extraction column (55 μm, 70Å) (Phenomenex, CA). All extracts and 5 mL petroleum ether rinses of the extraction flask were passed through the column. The eluates were dried under a flow of nitrogen and dissolved with 1 mL of acetonitrile for analysis.

The determination of PCB 153 concentration was performed by a Waters Acquity® ultra performance LC/MS/MS equipped with triple quadrupole [32]. An Acquity® BEH C18 column (2.1 × 50 mm, 5 μm) was used and kept at 50 °C. A gradient elution using water with 0.1% formic acid (eluent A) and acetonitrile (eluent B) was carried out at a flow rate of 100 μL/min. The gradient program for elution was 0–3 min linear gradient from 92 to 100% B, 3–10 min 100% B and 10–11 min linear gradient from 100 to 92% B. Injection volume was 10 μL for each analysis. The ionization source was operated in negative-ion mode under the following conditions: discharge current 0 μA, vaporizer temperature 280 °C, spray voltage 3 kV, cone voltage 35 V, sheath gas pressure 20 psi, auxiliary gas 6 a.u., capillary temperature 120 °C and source temperature 300 °C. The monitored precursor and product ions were m/z 361 and 341.

All extracts were analyzed in batches that also included blank samples and standard calibration curves. Multi-level calibration curves for the quantification of PCB 153 were in a good correlation (r² > 0.99). The limit of detection (LOD) for PCB 153 was approximately 50 ppb with excellent reproducibility and sensitivity of the detection methods.

Statistical analysis

Data were analyzed using JMP Pro software 11.0.0 (SAS Institute Inc., NC). Data on shell length, total wet mass, wet flesh mass and PCB residues were expressed as mean ± SD. Comparison of means of PCBs between different groups was made using ANOVA with pairwise comparisons with a Tukey test. The level of significance was set at 5%.

Conclusions

PCBs are ubiquitous and persistent in the environment and the development of economically feasible and field-practical mitigation strategies are warranted to reduce PCB exposures. The results in an oyster model showed that (1) PCB uptake occurs in a dose-dependent manner by oysters fed PCB-contaminated diet; (2) PCB residues were reduced when APM, or montmorillonite clays were administered in the PCB-contaminated diet; (3) the percent of reduction was positively correlated with dose of sorbents and length of treatment; (4) reduction of PCBs was greater with APM versus parent clay versus washout. Based on these results, it is possible that edible clay inclusion in diets may significantly reduce PCB uptake in fish, other shellfish and humans.