Effects of suspended mineral coal dust on the energetic physiology of the Caribbean scallop Argopecten nucleus (Born, 1778)

Journal Pre-proof Effects of suspended mineral coal dust on the energetic physiology of the Caribbean scallop Argopecten nucleus (Born, 1778)

Z. Benitez-Polo, L.A. Velasco, m PII:

S0269-7491(19)35262-5

DOI:

https://doi.org/10.1016/j.envpol.2020.114000

Reference:

ENPO 114000

To appear in:

Environmental Pollution

Received Date:

13 September 2019

Accepted Date:

14 January 2020

Please cite this article as: Z. Benitez-Polo, L.A. Velasco, m, Effects of suspended mineral coal dust on the energetic physiology of the Caribbean scallop Argopecten nucleus (Born, 1778), Environmental Pollution (2020), https://doi.org/10.1016/j.envpol.2020.114000

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Effects of suspended mineral coal dust on the energetic physiology of the Caribbean scallop Argopecten nucleus (Born, 1778)

Z. Benitez-Polo* and L.A. Velasco

Laboratorio de Moluscos y Microalgas, Universidad del Magdalena, Carrera 32 No. 2208, Sector San Pedro Alejandrino, Santa Marta, Colombia Corresponding authors; Telephone and fax: 57 5 4217940 ext. 3011 *[email protected] and [email protected]

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Abstract The effects of increasing concentrations of suspended mineral coal dust on the energetic physiology of the Caribbean scallop Argopecten nucleus were studied, at a concentration range that is environmentally relevant and representative of areas proximate to coal loading and shipping ports. Adult hatchery-produced animals were exposed to different concentrations of coal dust, i.e. 0, 2, 9 and 40 mg L-1. At increasing concentrations of coal dust, the rates of filtration and pseudofeces production increased, while the rates of ingestion and absorption remained constant. The rates of oxygen consumption and ammonium excretion decreased, as well as the absorption efficiency and the scope for growth. Suspended coal dust particles, at concentrations higher than or equal to 2 mg L-1, were ingested preferentially over microalgae by A. nucleus, causing reductions in its absorption capability, metabolism and in the amount of energy for growth and reproduction, thus generating physiological stress.

Keywords: scope for growth (SFG), bioenergetics, absorption, metabolism, nanoparticles.

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Highlights 

First study about the effects of coal dust on traits related to energetic physiology of an animal.



Coal dust reduce the SFG of A. nucleus.



Coal dust is preferentially ingested over microalgae.

1. Introduction Mineral coal is the second most widely used fossil fuel in the world, and the first in terms of reserves for future use (Ahrens and Morrisey, 2005). The annual global production in the last 5 years has been around 7,702 Mt, with Australia, Indonesia, Russia and Colombia as the leading exporters, and China, India and Korea as the largest importers (IEA, 2018). The commercialization of this resource includes crushing, classification, storage and ultimately shipping of the material (UPME, 2012), which implies the risk of spills of coal dust in port areas by accidental and chronic causes. Accidental spills often occur by occasional sinking of ships and vessels, while chronic discharges are due to leaching of processed water used for coal refinement by flotation techniques, and water sprayed to suppress dust; cargo washing (i.e. the cleaning of ships’ holds and decks after offloading dry bulk cargoes by washing with water and discharging over the side); wind and water erosion of coastal stockpiles; and spills occurring during coal loading and unloading (Sydor and Stortz 1980; Ahrens and Morrisey, 2005; BCEMA, 2015). It is estimated that, during the last 30 years, nearly 114,000 t of fossil fuel have been spilled to the seafloor (CIIMAR, 2018), but the magnitude of chronic inputs remains unknown.

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Most of the mineral coal in accidental spills has a particle size larger than 2 mm, which is deposited at the seafloor in areas surrounding coal terminals and shipping vessels (Johnson and Bustin, 2006; Jaffrennou et al., 2007). Chronic spills, however, are composed mainly by coal dust or particulate mineral coal, which given its small size (< 53 µm), remains in suspension in the water column for a long time, with a wide lateral dispersion (i.e. 4 to 96 km from the spill point) due to the action of marine currents (Johnson and Bustin, 2006). Estimations of total suspended solids in coastal waters near some of the main coal marine ports of Australia, Indonesia, China and Colombia normally fluctuate between 10 and 511 mg L-1 (Goonetilleke et al., 2009; Garcés-Ordóñez et al., 2016, 2018; Song et al., 2017; Barakwan et al., 2019). Considering that the percentage of suspended coal in those zones vary between 18 and 42% (Franco-Herrera et al., 2011), the concentrations of suspended particulate mineral coal can oscillate between 1.8 and 215 mg L-1.

It has been demonstrated that coal dust has several negative effects on aquatic animals such as reduction of fertilization, larval settling, growth and/or survival of corals and fish (Pearce and McBride, 1977; Berry et al., 2016ab); obstruction and physical damage by coal particles on gills and digestive gland in bivalves (Henley et al., 2015), as well as mutagenic and carcinogenic alterations in fish (Campbell and Devlin, 1997). However, other studies show no evidence of detrimental effects caused by particulate coal on biological traits such as survival and oxygen consumption in crabs (Pearce and McBride, 1977; Hillaby, 1981), growth and survival in bivalves (Bender et al., 1987) and fish (Herbert and Richards, 1963; Pearce and McBride, 1977; Gerhart et al., 1981). Such discrepancies might be related to the

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wide range of coal dust concentrations used in these studies (i.e. 1 to 13,500 mg L-1), and differences in the tolerance and/or sensitivity to stress of the studied species.

Bivalve mollusks have been commonly used as sentinel organisms for biomonitoring programs of environmental water quality in marine ecosystems, given their particular characteristics such as reduced mobility, high tolerance to environmental changes, high sensitivity to stress, wide geographical distribution, they can be easily obtained and maintained in captivity, they are filter feeders and thus capable of absorbing nanoparticles (Baqueiro-Cárdenas et al., 2007; Koehler et al., 2008; Ward and Kach, 2009; Montes et al., 2012; Rocha et al., 2015).

One of the variables most commonly used as a bioindicator of stress and environmental quality in bivalves is the Scope for Growth (SFG), which represents the amount of energy that an organism has available for growth and reproduction, and is calculated based on the balance of energy inputs and outputs (Widdows and Donkin, 1991; Widdows et al., 1995). This physiological index is considered as one of the most sensitive to pollution-induced stress, and a very useful predictor of growth rate, as well as an instantaneous indicator of the condition and fitness of an organism (Grant and Cranford, 1991; Filgueira et al., 2011; Larsen et al., 2014).

Although the short-term response of energetic physiology variables in bivalves to increasing concentrations of suspended particulate matter have been well documented for

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natural seston (Barillé et al., 1997; Navarro and Widdows, 1997; Velasco and Navarro, 2005; Helson and Gardner, 2007), microalgae (Sejr et al., 2004; Velasco, 2006; Arrieche et al., 2011; Chávez-Villalva et al., 2013), sediment (Iglesias et al., 1996; Bacon et al., 1998; Velasco and Navarro, 2002; 2003; Kang et al., 2016; Navarro et al., 2016), microplastics (Rist et al., 2016; Xu et al., 2017¸ Gardon et al., 2018), graphite and aluminum (FosterSmith, 1975), volcanic ashes (Salas-Yanquin et al., 2018) and man-made nanoparticles (Liu et al., 2018), there are no reports of the effects of particulate mineral coal on such variables. According to these previous studies, the concentrations of particulate matter at which deficiencies in energy acquisition, availability and use for growth and reproduction begin to be evident, varies between 0.25 µg L-1 and 200 mg L-1, depending on the species and the type of particle they are exposed to. Particulate coal has different characteristics than particulate materials previously studied, including a greater content of organic compounds of high molecular weight and resistant to biodegradation such as kerogens (De Leeuw and Largeau, 1993; Ahrens and Morrisey, 2005), as well as components that can be highly toxic, such as polycyclic aromatic hydrocarbons (PAHs) and heavy metals (Ahrens and Morrisey, 2005), therefore it is possible that the negative effects of particulate coal occur at low concentrations.

Argopecten nucleus is a bivalve of the Caribbean, mostly found in sandy bottoms at depths of 5 to 50 m, including port coal zones such as the Guajira and Magdalena regions (Díaz and Puyana, 1994). This scallop species has a small size (50 mm length) and epibenthic habits, feeds by continuous filtration of seston and reaches an early sexual maturity at 3months-old and 20 to 25 mm length (Velasco, 2008). It exhibits simultaneous

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hermaphroditism and high fecundity (1.85 x 106 eggs animal-1 on average, Velasco et al., 2007). It displays the highest feeding rates and SFG values when it is maintained in laboratory conditions and fed with the microalgae Isochrysis galbana (Velasco, 2007) at concentrations of approximately 4.0 x 104 cells mL−1, 36 ppt and a temperature of 25°C (Velasco, 2006). The hatchery technology for seed production of this species has been recently developed (Velasco and Barros, 2007; 2008; 2009; Valderrama et al., 2016; Barros et al., 2018ab), which makes possible to obtain a significant number of individuals to be used in bioassays (Velasco and Barros, 2019; Rodríguez-Satizábal et al., 2015). With the exception of a study on bioaccumulation of coal particles in fish tissues (Franco-Herrera et al., 2011), there is a lack of knowledge about the effects of coal dust on the marine animals of the Caribbean Sea, which is required for developing effective policies for the control and mitigation of coal spills near port loading facilities. In order to be able to assess the impact of coal dust on the health of aquatic organisms, at a concentration range environmentally relevant and representative of areas influenced by coal loading and shipping ports, we studied the effects of increasing concentrations of coal dust on the energetic physiology of the Caribbean scallop Argopecten nucleus under laboratory conditions.

2. Materials and methods 2.1.

Obtaining and acclimation of experimental scallops

Adult individuals of A. nucleus were produced at the Laboratory of Mollusks and Microalgae, and cultured at the marine aquaculture lease of Universidad del Magdalena in Taganga Bay, Santa Marta, Colombia (Lat. 11º16’04′′ N, Long. 74°11’36′′ W), following the protocols of Velasco and Barros (2007; 2008; 2009), Velasco et al. (2007; 2009) and

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Barros et al. (2018ab). The seawater at the coastal area near the coal loading facilities in Santa Marta has temperatures between 23 and 33 ºC, salinity between 22 and 38, dissolved oxygen between 4.2 and 8.8 mg L-1, pH from 7.5 to 8.4, ammonium concentrations from 2 to 768 µg NH4-N L-1, concentration of suspended solids, i.e. seston, from 0.7 to 170.8 mg L-1, and a percentage of organic matter between 9.7 and 61% (Velasco et al., 2009; GarcésOrdóñez et al., 2016; Barros et al., 2018a; Garcés-Ordóñez, 2018). The percentage of suspended particulate coal was between 18 and 42% (Franco-Herrera et al., 2011), corresponding to estimated concentrations between 0 and 71 mg L-1.

A total of 50 adult individuals (mean ± SE shell length = 40.0 ± 0.4 mm, and mean ± SE soft tissues dry weight = 0.60 ± 0.02 mg) were transferred in wet conditions inside plastic coolers to the laboratory, where their shells were cleaned to remove epibionts and subsequently tagged. They were maintained by two weeks in rectangular tanks (250 L) with filtered (1 µm) and UV-treated seawater, with continuous aeration and controlled conditions of temperature (25 ± 1°C), salinity (36 ± 1), dissolved oxygen (6.02 ± 0.20 mg L-1), pH (8.1 ± 0.1) and ammonium concentrations (NH4-N) (4.48 ± 1.00 µg L-1). The individuals were drip-fed with the microalgae Isochrysis galbana at a rate of 2.3 x 108 cells h−1 animal−1, thus the concentration in the tank remained constant at 3.5 x 104 cells mL−1 (1.1 mg L-1). Biodeposits at the tanks’ bottom were removed daily, replacing 80% of the seawater volume.

2.2.

Experimental Design

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Once the scallops were acclimated to the laboratory conditions, three concentrations of coal dust were tested (2, 9 and 40 mg L-1), with an extra group as control with no coal. Each treatment was applied to 7 individuals randomly chosen among those perceived as more active (i.e. opening their valves and filtering food), which were individually placed in rectangular acrylic containers (0.8 L), following the design of Riisgård (1977), in order to minimize water recirculation and coal sedimentation. In addition, one container was maintained as control with a pair of empty valves, to correct the effect of retention and sedimentation of particles within the containers. The containers were kept with a continuous seawater flow (150 mL min-1) of similar quality and food concentration than the seawater used during acclimation., So, the total particulate matter (TPM) for the control was 1.1 mg L-1, and for the treatments were 3.1, 10.1 and 41.1 mg L-1, respectively. Each treatment was applied for 12 hours, including 10 h for acclimatization to experimental conditions and 2 h for feeding physiological estimations.

2.3.

Preparation and characterization of experimental seawater with microalgae and coal

The microalgae supplied as food derived from the collection of marine microalgae of Universidad del Magdalena (UMC-MA), which were cultured in batch systems using F/2 medium (Guillard, 1974). The particulate coal was obtained by crushing and sieving (40 µm) with filtered seawater (1µm) a sample of bituminous coal provided by a coal company located in the city of Santa Marta, Colombia. The coal stock solution was stored in cold conditions (-24 °C) until it was used in the experiments, to avoid microbial growth. The seawater containing microalgae and coal dust used in the experimental treatments was prepared by mixing specific volumes of microalgal culture and coal stock solution in a

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cylinder-conic tank (1,000 L) provided with strong bottom air injection, in order to minimize the agglomeration and/or sedimentation of particles. Microalgae and coal stock solutions were characterized by taking three samples previously homogenized (~100 mL).The number of microalgae cells or coal particles per mL was calculated under a light microscope by counting 3 subsamples (1 mL) using a Neubauer chamber. The diameter of algal cells and coal particles were measured with an ocular Vernier in 3 subsamples of 100 particles each, which were randomly sampled from stock solutions . The size distribution of particles smaller than 1 µm was analyzed using a DynaPro Plate Reader II (Wyatt Technology, Santa Barbara, CA, USA) equipped with a 380 nm laser. Briefly, 20 μL of each solution was loaded on a 384-well glass bottom plate (Cellvis, P384-1.5H-N) and data collected at 25 ºC. Data was analyzed using Dynamics software (Version 7.0.1, Wyatt Technology). The dry biomass was also estimated (mg mL-1), following the quantification methods for particulate matter by Strickland and Parsons (1972), using glass fiber filters Whatman GF/B with pore size of 1 µm. The organic content (mg mL-1) was determined by incinerating the filters with the samples in a muffle furnace (450°C x 3h) to deduct the weight of the ashes from the total biomass. During the exposure of scallops to experimental conditions, seawater samples from the control containers (with empty valves), were taken every 3 hours to estimate the seawater containing coal dust and microalgae in terms of total particulate matter (TPM: mg L-1), particulate organic matter (POM: mg L-1), particulate inorganic matter (PIM: mg L-1), coal content (number of particles mL-1) and microalgae content (number of cells mL-1), following the same methods previously described to determine total and organic dry biomass and number of particles.

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2.4.

Determination of physiological variables

The feeding physiological variables were estimated following the biodeposition method described by Iglesias et al. (1998) and later validated by Navarro and Velasco (2003), in conditions of high concentrations of particulate matter. Feces and pseudofeces (material agglomerated by mucus, that is filtered and expelled by the animal without digestion) produced by each animal during the last two hours of exposure to coal dust were quantitatively collected using Pasteur pipettes, and the total, organic and inorganic dry weight as well as the number of coal particles and microalgal cells were estimated with the same methods used to characterize the microalgae and coal stock solutions. Based on these data, the production rate of feces or organic ejection rate (OER: mg h-1) and inorganic ejection rate (IER: mg h-1), as well as the production rates of pseudofeces or organic rejection rate (ORR: mg h-1) and inorganic rejection rate (IRR: mg h-1) for each individual, were calculated. The clearance rates (CR) or quantity of water free of particles per unit of time and the filtration rate (FR) or number of particles removed from seawater per unit of time were calculated using the following equations: CR (L h-1 g-1) = IFR / PIM

(1)

FR (mg h-1 g-1) = IFR + OFR

(2)

IFR (mg h-1 g-1) = IRR + IER OFR (mg h-1 g-1) = IFR * (POM / PIM) where: IFR = inorganic filtration rate (mg h-1 g-1), OFR = organic filtration rate (mg h-1).

(3) (4)

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The ingestion rate (IR) or amount of particulate matter consumed per unit of time was calculated using the following equation: IR (mg h-1 g-1) = FR – RR

(5)

where: RR = IRR + ORR. The absorption efficiency (AE) and the absorption rate (AR) represent the capacity and velocity for transferring the ingested organic matter from the digestive tract to the body circulation, and they were calculated according to the Conover method (1966), following the equations of Iglesias et al. (1998): AE (%) = AR / OIR * 100

(6)

OIR (mg h-1 g-1) = OFR – ORR

(7)

AR (mg h-1 g-1) = OIR – OER

(8)

where: OIR = Organic ingestion rate. After the animals were exposed to coal dust, they were individually maintained for two hours in hermetic acrylic containers (0.8 L), previously rinsed with diluted hydrochloric acid and filled with seawater of equal quality to the seawater used for acclimation, but without food supply. Two control containers were set up, with no animals in them. At the end of the incubation, seawater samples from each container were collected using DBO bottles (300 mL) to determine the oxygen concentration according to the Winkler method modified by Carritt and Carpenter (Strickland and Parsons, 1972), and using micropipettes (5 mL) for estimating the ammonium concentration, based on the phenol-hypochlorite method described by Solorzano (1969). The oxygen consumption rate (OCR) and the ammonium excretion rate (UR) were estimated from the difference between the oxygen and ammonium concentrations in the experimental and the control containers (Widdows, 1985).

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Once the experiment was finished, the soft tissues of the scallops were dried at 70 ºC for 48 h, and then individually weighted to standardize the physiological rates to 1 gram of the animal dry weight and for the gonad maturity state, using the equation modified by Bayne et al. (1987): Yts = (1 g / We)b1*b2 * Ye

(9)

where Yts = physiological rate standardized to 1 gram of dry weight and maturity state 1, according to the scale of macroscopic maturity by Sastry (1963) Ye = non standardized rate, We = weight of the experimental animal, b1 = dependence of physiological rates on the animal size, b2 = dependence of physiological rates on the animal maturity state. The values used for “b” in ER were b1 = 0.98 and b2 = −0.67, in OCR: b1 = 1.52 and b2 = 0.32, and for UR: b1 = 1.16 and b2 = 0.11 (Velasco, 2006; 2007). These values were determined under experimental conditions similar to those applied in this study, but without coal supply.

Finally, the scope for growth (SFG) was estimated based on the equation for energetic balance by Widdows (1985), prior transformation of standardized physiological rates to energetic equivalents. SFG (J h-1 g-1) = A - (R + U)

(10)

where A = absorbed energy (J h-1) = AR (mg h-1g-1) * 11.40 (J mg-1) (Velasco, 2007); R = oxygen consumption (J h-1) = OCR (mL O2 h-1g-1) * 20.08 (J mL O2) (Gnaiger, 1983); U= ammonium excretion (J h-1) = UR (µg NH4-N h-1 g-1) * 24.8 (J mg NH4-N) (Elliot and Davinson, 1975).

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2.5.

Statistical Analysis

In order to determine the existence of significant differences for physiological rates between different coal dust concentrations, experimental data was analyzed by a One-Way Analysis of Variance, followed by the Bonferroni multiple comparison test, after confirmation of data normality and homoscedasticity. When required, data for some physiological variables was transformed. Thus, IR data was log transformed, while AE data was ranked, and AR and SFG data was applied the square root transformation. A Two-Way Analysis of Variance were used to determine the existence of differences in the organic and coal content of the suspended particulate matter in seawater and pseudofeces for the different treatments. Finally, in order to explore the correlation between physiological variables, the Pearson correlation test was applied. In all cases, an α value of 0.05 was used for significance. All statistical analysis were performed using the software Statgraphics Centurion XVII.

3.

Results

The size of cells of I. galbana varied between 2 and 6 µm, with predominance of sizes between 4 and 5 µm (Figure 1A). While the sizes of coal particles were in general smaller than 40 µm, a large fraction (81%) had a size smaller than 12 µm (Figure 1A). Within the group of coal particles smaller than 1 µm, the most frequent sizes were between 60 and 120 nm (Figure 1B). The organic content in cultures of I galbana was on average 78.2 ± 4.8%, while for the coal stock solution was 86.3 ± 0.1%.

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The clearance rate (CR) oscillated between 2.0 and 11.5 L h-1 g-1 (Figure 2A), being significantly lower at coal dust concentrations higher than or equal to 9 mg L-1, compared to the controls (gl = 3, F = 8.12, p = 0.0008). The filtration rate (FR) varied between 18.4 and 81.1 mg h-1g-1 (Figure 2B), while the pseudofeces production rate (RR) was between 0.0 and 60.7 mg h-1g-1 (Figure 2C). When the coal dust concentration increased, values of FR and RR significantly higher than those in controls were obtained (df = 3, p < 0.0001). The produced pseudofeces exhibited organic contents between 64 and 84%, and percentages of particulate coal between 82 and 93% (Figure3A), while the particles suspended in the water were composed of 82 to 85% (w/w) of organic material, and 89 to 98% of coal particles (Figure 3B). The organic content of pseudofeces were not significantly different than those found in particles suspended in the water column (df = 1, F = 5.10, p = 0.0347), except for the treatment of 2 mg L-1 of coal dust, where pseudofeces showed significantly lower values (Figure 3A). In contrast, significantly higher percentages of particulate coal suspended in the water were found, compared to pseudofeces (df = 1, F = 21.13, p = 0.0013), except for the treatment of 2 mg L-1 of coal dust, where values were not different (Figure 3B). The ingestion rate (IR) fluctuated between 18.4 and 28.8 mg h-1g1

(Figure 4A), without significant differences in the values corresponding to the different

coal dust concentrations and the controls (df = 3, F = 1.34, p = 0.2810). The absorption efficiency (AE) was between 49 and 88% (Figure 4B), being significantly lower than values corresponding to a coal dust concentration of 40 mg L-1 (df = 3, F = 8.86, p = 0.0003). The absorption rate (AR) was between 9.3 and 20.8 mg h-1g-1 (Figure 4C), with values statistically similar for the different coal dust concentrations tested (df = 3, F = 2.65, p = 0.0689). The existence of a significant positive correlation between FR and RR was

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confirmed (n = 31, r = 0.9395, p < 0.0001), as well as for AR, AE and IR (n = 31, r > 0.4955, p < 0.0046).

The oxygen consumption rate (OCR) fluctuated between 0.50 and 0.84 mL O2 h-1g-1 (Figure 5A), while the ammonium production rate (UR) was between 51.6 and 135.6 µg NH4-N h-1 g-1 (Figure 5B). Values significantly lower for OCR and UR were obtained in the treatment with the highest coal dust concentration tested, i.e 40 mg L-1 (df = 3, p < 0.0030). Significant correlations were found for OCR and UR with: FR and RR (n = 31, r < -0.6463, p < 0.0180) and AE (n = 31, r > 0.4649, p < 0.0084), while for the rest of the feeding rates, no significant correlations were found (n = 31, r < 0.4098, p > 0.1950). The scope for growth (SFG) fluctuated between -4.5 and 131.4 J h-1 g-1 (Figure 5C). SFG values decreased with increasing coal dust concentrations, and those corresponding to treatments with concentrations higher than or equal to 2 mg L-1 were significantly lower than the values for the control group with no coal (df = 3, F = 34.91, p < 0.0001). SFG exhibited significant positive correlations with AE, OCR and UR (n = 31, r > 0.4455, p < 0.0120), negative correlations with FR and RR (n = 31, r < -0.5305, p < 0.0021), and no correlation at all with IR and AR (n = 31, r < 0.2762, p < 0.5082).

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4. 4.1.

Discussion Feeding rates

The decrease of the clearance rate as well as the increase of the filtration rate in A. nucleus observed with increasing coal dust concentrations are similar to previous reports in bivalve species exposed to high concentrations of particulate matter in seawater (Iglesias et al., 1996; Barillé et al., 1997; Navarro and Widdows, 1997; Velasco and Navarro, 2002; 2005; Velasco, 2006). These responses have been explained by the reduction in the pump activity to avoid saturation by the larger number of particles retained by the gills when the animal pumps water in the same direction to its paleal cavity for respiration and feeding. Nevertheless, when bivalves are exposed to high concentrations of particulate matter, the filtration rate decrease, since the gills become saturated with particles, thus forcing the animal to reduce seawater pumping (Barillé et al., 1997; Navarro and Widdows, 1997; Velasco and Navarro, 2005). Such a trend has been reported for this same species exposed to the microalgae I. galbana at concentrations of 12 mg L-1 (Velasco, 2006), a response that was not observed in the present study at particle concentrations as high as 41.1 mg L-1. It is known that, for a given weight, microalgal particles represent a larger volume than rich mineral particles, such as sediments (Bayne et al., 1987). Therefore, it is possible that the gills of experimental scallops became saturated at lower concentrations if suspended particles are microalgae, in comparison to suspended particles of higher densities such as mineral coal. In addition, the efficiency of particle retention in scallop gills has been reported to be less than 100% for particles smaller than 5 µm, which is likely related to the lack of latero frontal cirri in their gill filaments (Cranford and Gordon, 1992; Ward and Shumway, 2004; Zhang et al., 2010). In the present study, around 22% of the coal dust

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particles used had a size inferior than or equal to 2 µm, and some of them were likely able to pass through the gills of A. nucleus, thus avoiding gill saturation at high coal dust concentrations.

The clearance rate values of A. nucleus (2-11 L h-1 g-1) were similar to those reported in this species and in other scallops (1-18 L h-1 g-1; Bricelj and Kuenstner, 1989; Cranford and Grant, 1990; Bacon et al., 1998; Velasco, 2007), but were relatively higher than those registered in clams (0.1-6 L h-1 g-1; Velasco and Navarro, 2002; 2005; Arambalza et al., 2014; Kang et al., 2016; Xu et al., 2017), oysters (0.1-7.5 L h-1 g-1; Kesarcodi-Watson et al., 2001; Pernet et al., 2008) and mussels (0.8- L h-1 g-1; Helson and Gardner, 2007; SalasYanquin et al., 2018). This response can be related to the opportunistic life strategy of A. nucleus that involves an early sexual maturity (3 months old) and short life (1-2 years), as well as a fast growth (Velasco, 2008) and high pumping activity (Velasco, 2006; 2007). Results suggest that A. nucleus is able to release coal particles until 11 L of seawater per hour, thus showing a high potential for depuration of coal dust polluted waters.

The increase of the pseudofeces production rate at higher coal dust concentrations is similar to previous reports for this species and other bivalves as response to rising concentrations of suspended particulate matter (Navarro and Widdows, 1997; Bacon et al., 1998; Velasco and Navarro, 2002; Velasco, 2006; Xu et al., 2017; Salas-Yanquin et al., 2018). These authors demonstrated that pseudofeces production is a mechanism that allows bivalves to regulate the excess of filtered particles and avoid the saturation of the digestive system,

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thus the production is enhanced when the abundance of particles in the environment is rising. The lower content of organic material and/or coal particles in pseudofeces produced by A. nucleus, in comparison to those measured in the particles suspended in the water, suggests that this species is able to perform a selection of particles prior to ingestion, based on the organic content or particle type, preferring to ingest coal particles of high organic content over the microalgae cells supplied. Pre-ingestive selection capacity has been reported before in other bivalve species exposed to natural seston or mixtures of microalgae and sediments, and it seems to be a strategy to improve the nutritional quality of the particles by preferentially ingesting microalgae and rejecting sediments and detritus particles of low organic content in the pseudofeces (Navarro and Widdows, 1997; Velasco and Navarro, 2002, 2005). Chemosensory cells in the mantle and labial palps of bivalves could detect the organic compounds of particles as has been previously suggested (Ward et al., 1992; Yahel et al., 2009). The present results suggest that, when coal dust is suspended in seawater, A. nucleus is incapable of improving the nutritional content of its feed through pre-ingestive selection, and instead, the quality of its feed can be further deteriorated, since animals might preferentially ingest coal particles over microalgal cells.

The similar values for the ingestion and absorption rates of A. nucleus exposed to different concentrations of coal dust are in agreement with previous reports for bivalves exposed to microplastic particles (Gardon et al., 2018) and to intermediate concentrations of suspended particles. Nevertheless, they differ from the low values observed for these rates in conditions of low and high particle concentrations in seawater (Navarro and Widdows, 1997; Bacon et al., 1998; Velasco and Navarro, 2002; 2005; Velasco, 2006). According to

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these studies, the increase in ingestion and absorption rates, in response to the availability of natural particles in seawater, represents a strategy to obtain nutrients in conditions of low food availability. Furthermore, the regulation of these rates, in response to higher particle concentrations, might allow the animals to compensate for the saturation of the digestive system through maximizing the residence time of food in the digestive tract, thus promoting a greater enzymatic activity and nutrients absorption. In the present study, a regulation of ingestion and absorption was confirmed for the lowest coal concentration (2 mg L-1), and it is unlikely that this regulation is related to saturation of the gills or the digestive tract, since the saturation was previously observed in individuals exposed to microalgae concentrations higher than or equal to 12 mg L-1 (Velasco, 2006). The nanoparticles suspended in the water can be ingested by bivalves, especially when they are agglomerated with organic matter, since then they can be slowly absorbed and digested by endocytosis in the digestive gland (Moore, 2006; Ward and Kach, 2009; Wegner et al., 2012). Therefore, it is possible that coal nanoparticles were ingested by A. nucleus mainly by forming agglomerations with microalgae, thus generating a regulatory effect on the ingestion and absorption rates even under conditions of low concentration of particles in the seawater.

The decline observed in the absorption efficiency of A. nucleus at a coal dust concentration of 40 mg L-1, in comparison to lower concentrations, is similar to other reports for bivalves exposed to high particle concentrations (Kesarcodi-Watson et al., 2001; Velasco and Navarro, 2003; 2005; Sejr et al., 2004; Arrieche et al., 2011; Chávez-Villalva et al., 2013; Gardon et al., 2018). Such a response has been explained by the occurrence of dilution of

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microalgal cells particles, and by the limited production of digestive enzymes. Although coal particles have a high organic content, they are apparently not considered a relevant source of biological energy, since they are composed by complex macromolecules which are resistant to biodegradation (De Leeuw and Largeau, 1993). Therefore, as noticed in the present study, the organic matter that was absorbed could have derived from the digestible or absorbable components of the suspended particles, present in both microalgae and coal nanoparticles. Considering that the cells of the digestive gland in bivalves can experience detrimental effects such as cytoplasm condensation, peeling, oxydative stress, lyposomal perturbation and necrosis, as a result of exposure to coal particles (Henley et al., 2015) and nanoparticles (Koehler et al., 2008; Canesi et al., 2012), it seems likely that a decrease in the absorption capacity of A. nucleus at the highest coal dust concentrations might be related also to the functional deterioration of the digestive gland caused by the greater availability of coal nanoparticles in seawater. 4.2.

Oxygen consumption and ammonium excretion rates

The reduction of oxygen consumption rates in A. nucleus, in response to increasing coal dust concentrations, contradicts the lack of effect of this material on the oxygen consumption of the crab Cancer magister (Hillaby, 1981), but agrees with the findings on bivalves exposed to different types of particles (Kesarcodi-Watson et al., 2001; Velasco and Navarro, 2003; Sejr et al., 2004; Velasco, 2006; Arrieche et al., 2011; Rist et al., 2016; Salas-Yanquin et al., 2018). In those studies, the observed reduction of metabolism was explained by the decrease in the energetic expenditure associated to the processes of filtration, pseudofeces production, ingestion and absorption. In the present study, no evidence of positive correlation for these variables was found, thus such a response could

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only be attributed to the obstruction of respiratory exchange or to damage on the gills tissue (Henley et al., 2015), especially due to the effect of nanoparticles that can go through gill epithelia and cause different biological responses in the cells (Koehler et al., 2008). On the other hand, a decrease in the oxygen consumption in bivalves has been reported at increasing concentrations of heavy metals in the water column, apparently via inactivation of important enzymes for oxidative phosphorylation processes in mitochondria (Anandraj et al., 2002; Lemus et al., 2012; Zhao et al., 2014; Polo-Osorio and Campos, 2016). Therefore, the reduction of oxygen consumption in A. nucleus related to increasing coal dust concentrations in seawater could be the result of an inhibition of respiratory and metabolic functions due to physical effects of particles, or maybe to chemical and biological effects of the heavy metals present in the coal dust nanoparticles.

The decrease of ammonium excretion rates of A. nucleus in response to increasing coal dust concentrations, does not agree with the trends observed in other bivalve species at low and moderate particle concentrations, but is comparable to results obtained in conditions of high coal dust concentrations (Kesarcodi-Watson et al., 2001; Velasco and Navarro, 2003; Sejr et al., 2004; Velasco, 2006). In those studies, a positive correlation between the ammonium excretion rate and the rates of absorption and ingestion was confirmed, suggesting that an increasing ammonium excretion rate might be a response to the availability of assimilated proteins that can be used for anabolism and catabolism. In the present study, the lack of association between these variables suggests that the reduction in the catabolism of assimilated proteins when coal dust concentration in seawater increases might represent a strategy for energetic saving under conditions of low availability of proteinaceous material

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in the food, or that the protein metabolism could have been affected by oxidative damage inflicted by the assimilated nanoparticles (Rocha et al., 2015; Liu et al., 2018). Thus, the influence of heavy metals in the coal nanoparticles can be neglected, since they commonly enhance the excretion rates in bivalves (Zhao et al., 2014; Boudjema et al., 2016; PoloOsorio and Campos, 2016).

4.1. Scope for Growth (SFG) The decrease of the SFG observed in A. nucleus at increasing coal dust concentrations, and the occurrence of negative values at the highest concentration tested, indicate a causal relationship between the concentration of coal dust and physiological stress in this species. Therefore, the animal is not able assign energy for growth and reproduction, decreasing also the energy allocated for basic vital functions. The reduction of SFG in bivalves exposed to high particle concentrations has been reported for microalgae (KesarcodiWatson et al., 2001; Sejr et al., 2004; Velasco, 2006; Arrieche et al., 2011), seston (Barillé et al., 1997), sediment (Velasco and Navarro, 2003; Kang et al., 2016), and microplastic (Gardon et al., 2018), as well as in the presence of aromatic hydrocarbons and heavy metals in the water column (Jeong and Cho, 2007; Zhao et al., 2014). In this study, such response can be related to the decrease in energy acquisition given the regulation of ingestion and absorption of nutrients, apparently exerted by the coal nanoparticles and the dilution of microalgae as a source for biological energy. When A. nucleus was exposed to the highest coal dust concentration, a considerable reduction was observed for the energy absorbed from food (44%), the aerobic metabolism (40%), the proteinaceous catabolism (62%) and the available energy for growth and reproduction (104%). Therefore, it is possible that

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bivalve populations living in areas under the influence of mineral coal loading and shipping ports, and hence exposed to coal dust concentrations higher than or equal to 2 mg L-1 for a period of time longer than or equal to 12 hours, might be experiencing debilitating conditions in their physiological state that can imply further ecological disadvantages due to an increased susceptibility to predation and pathogen agents, besides a low competitive performance. The coal concentration and exposure time at which negative effects were confirmed for the SFG of A. nucleus, were lower than those required to affect growth, fertilization and/or survival of corals, bivalves and fish (38 - 3,000 mg L-1; Herbert and Richards, 1963; Pearce and McBride, 1977; Bender et al., 1987; Campbell and Devlin, 1997; Berry et al., 2016ab). This suggest a higher sensitivity in bivalves in terms of SFG, which could be a useful stress bioindicator for coal-induced stress.

5. Conclusion Our study reveals that increasing concentrations of suspended coal dust in seawater caused a preferential ingestion of coal particles over microalgae cells in A. nucleus, as well as a reduction in

the clearance rate, digestive absorption, metabolism and the amount of

available energy for growth and reproduction, apparently due to the effects of nanoparticles absorption and enhanced microalgae dilution in the food. This suggests that coal dust at concentrations of 2 mg L-1 can impose a stress condition in experimental individuals, that might become critical at concentrations of 40 mg L-1. Further research is required to validate these results, in addition to analyze tissues and biological depositions of the exposed organisms to confirm the effects of coal nanoparticles.

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Founding support: Universidad del Magdalena Grant Res. 0388, 2017 and 0339, 2018.

Acknowledgements The authors would like to thank to Marisol Sepulveda of Purdue University for the coal nanoparticle analysis; to Maria Vicenta Valdivia for her help in the preparation of the english version of the manuscript and the staff of Laboratorio de Moluscos y Microalgas of Universidad del Magdalena for their help during the experiments, and to Asociación de Pescadores y Ostioneros de Taganga (ASPOTAG) for providing the experimental individuals.

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Figures legends

Figure 1. Frequency distribution of microparticles size in cultures of the microalgae Isochrysis galbana and in coal dust stock solution used in the experiments (A). Frequency distribution of nanoparticles size in coal dust stock solution used in the experiment (B).

Figure 2. Effect of the coal dust concentration on A. nucleus. A). Clearance rate (CR), B). Filtration Rate (FR) and C). Pseudofeces production rate (RR). Different letters indicate significant statistical differences (P < 0.05). Vertical bars represent the standard error.

Figure 3. Effect of the coal dust concentration on A. nucleus. A). Organic content and B). Coal content of seawater and rejected pseudofeces. Different letters indicate significant statistical differences (P < 0.05). Vertical bars represent the standard error.

Figure 4. Effect of the coal dust concentration on A. nucleus. A). Ingestion Rate (IR), B). Absorption Efficiency (AE) and C). Absorption Rate (AR). Different letters indicate significant statistical differences (P < 0.05). Vertical bars represent the standard error.

Figure 5. Effect of the coal dust concentration on A. nucleus. A). Oxygen Consumption Rate (OCR), B). Ammonium Production Rate (UR) and C). Scope for Growth (SFG). Different letters indicate significant statistical differences (P < 0.05). Vertical bars represent the standard error.

Particle frecuency (%)

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Coal

40 35 30 25 20 15 10 5 0 0

5

10

15

25

35

40

45

800

900 1000

20 15 10 5 0 0

100

200

300

400

500

600

Diameter (nm)

Figure 1.

30

Diameter (µm)

25 Particle frecuency (%)

20

Isochrysis galbana

700

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A. 14

ab

a

CR (L h-1g-1)

12 10 8

bc

6 4

c

2 0 0

2

9

40

B. 100

c

FR (mg h-1g-1)

80

b

60 40 20

ab a

0 0

C.

2

9

40

80

RR (mg h-1g-1)

d 60 40

c 20 0

b a 0

2

9

Coal dust concentration (mg Figure 2.

40

L-1)

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Organic content (%)

A.

Seawater

90

a

a

a

Pseudofeces a

a

80 b

70 60 50 2

9

Coal particle content (%)

B. 100 90

40 a

b c

bc

d

80 70 60 50 2

Figure 3.

9 40 Coal dust concentration (mg L-1)

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IR (mg h-1g-1)

A.

40 30 20 10 0

B. 100

0

2

a

a

9

40

a

AE (%)

80 b

60 40 20 0

C.

30

AR ( mg h-1g-1)

0

20

2

9

40

10

0 0

Figure 4.

2 9 Coal dust concentration (mg L-1)

40

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a ab

OCR (mL O2 h-1g-1)

A. 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0

b

0

UR (µg NH4 - N h-1g-1)

B.

ab

2

9

a

a

40

200 150

a

100 b

50 0 0

C. 160

2

9

40

a

SFG (J h-1g-1)

140 120 100

b

80 60 40

c

20

d

0 -20

Figure 5.

0

2 9 Coal dust concentration (mg L-1)

40

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Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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30-December -2019 Dr. C. Sonne Dr. E. Zeng Editors Dear Editor: The authors declare do not have any conflicts of interest related with this manuscript. The authors contributions to this study were: Z. Benitez-Polo performed the experiments, analyzed the data and wrote this manuscript. L.A. Velasco provided the original idea, advised the experiments and wrote this manuscript.

Best regards,

Zamir Benitez Polo, B.Sc.

Luz Adriana Velasco Cifuentes, Ph.D. Universidad del Magdalena

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Highlights 

First study about the effects of coal dust on traits related to energetic physiology of an animal.



Coal dust reduce the SFG of A. nucleus.



Coal dust is preferentially ingested over microalgae.