Food-chain length determines the level of phenanthrene bioaccumulation in corals.

Food-chain length determines the level of phenanthrene bioaccumulation in corals.

Item Type Article

Authors Ashok, Ananya;Høj, Lone;Brinkman, Diane L;Negri, Andrew P;Agusti, Susana

Citation Ashok, A., Høj, L., Brinkman, D. L., Negri, A. P., & Agusti, S.

(2022). Food-chain length determines the level of phenanthrene bioaccumulation in corals. Environmental Pollution, 118789.

doi:10.1016/j.envpol.2022.118789 Eprint version Publisher's Version/PDF

DOI 10.1016/j.envpol.2022.118789

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Journal Environmental pollution (Barking, Essex : 1987)

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Environmental Pollution 297 (2022) 118789

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Food-chain length determines the level of phenanthrene bioaccumulation in corals

^☆

Ananya Ashok

^a,*

, Lone H ø j

^b

, Diane L. Brinkman

^b

, Andrew P. Negri

^b

, Susana Agusti

^a

aRed Sea Research Center (RSRC), King Abdullah University of Science and Technology (KAUST), Thuwal, Saudi Arabia

bAustralian Institute of Marine Science (AIMS), Townsville, Queensland, Australia

A R T I C L E I N F O Keywords:

PAHs Corals Phenanthrene Bioaccumulation Biomagnification

A B S T R A C T

Exposure from the dissolved-phase and through food-chains contributes to bioaccumulation of polycyclic aro- matic hydrocarbons (PAHs) in organisms such as fishes and copepods. However, very few studies have inves- tigated the accumulation of PAHs in corals. Information on dietary uptake contribution to PAHs accumulation in corals is especially limited. Here, we used Cavity-Ring-Down Spectroscopy (CRDS) to investigate the uptake rates and accumulation of a ¹³C-labeled PAH, phenanthrene, in Acropora millepora corals over 14 days. Our experiment involved three treatments representing exposure levels of increasing food-chain length. In Level W, corals were exposed to ¹³C-phenanthrene directly dissolved in seawater. In Level 1 representing herbivory, Dunaliella salina microalgal culture pre-exposed to ¹³C-phenanthrene for 48 h was added to the coral treatment jars. In Level 2 representing predation, corals were provided a diet of copepod (Parvocalanus crassirostris) nauplii fed on D. salina pre-exposed to ¹³C-phenanthrene. Bioconcentration factors (BCF) and bioaccumulation factors (BAF) were calculated as appropriate for all organisms, and biomagnification factors (BMF) were calculated for A. millepora.

We found that while phenanthrene uptake rates were not significantly different for the treatments, the accu- mulated concentration in corals was significantly higher in Level W (33.5 ±2.83 mg kg⁻¹) than in Level 1 (27.55

±2.77 mg kg⁻¹) and Level 2 (29.36 ±3.84 mg kg⁻¹). Coral log BAF values increased with food-chain length;

Level 2 log BAF (6.45) was higher than Level W log BCF (4.18) and Level 1 log BAF (4.5). Coral BMF was also higher for Level 2 than for Level 1. Exposure to dissolved or diet-bound phenanthrene had no significant effect on the coral symbionts’ photosynthetic efficiency (Fv/Fm) as monitored by pulse-amplitude-modulation (PAM) fluorometry, indicating the PAH can be accumulated without toxic effects to their Photosystem II. Our study highlights the critical role of dietary exposure for pollutant accumulation in corals.

1. Introduction

Polycyclic aromatic hydrocarbons (PAHs) are a large group of chemicals that are toxic to various marine organisms, bioaccumulative and persistent in the environment (French-McCay, 2002). Coral reefs, which support one-quarter of all marine biodiversity (Knowlton et al., 2010), are at risk of being exposed to PAHs pollution from direct oil spills near reefs (Keesing et al., 2018; Shigenaka, 2001) and from at- mospheric deposition, run-off and waste discharges (Burns and Brink- man, 2011; Kroon et al., 2020). Corals exposed to PAHs can experience several harmful effects including histopathological changes, reduced larval settlement and mortality at higher exposure concentrations (Nordborg et al., 2020; Turner and Renegar, 2017). Furthermore,

bioaccumulation (food-chain transfer) of such pollutants is a critical concern as it can lead to biomagnification (Arnot and Gobas, 2006).

Biomagnification occurs when the accumulated concentration of a pollutant in an organism exceeds that of its diet (Arnot and Gobas, 2006;

Borgå et al., 2004). Bioaccumulation can depend on several factors such as ecosystem type (Borgå et al., 2004), food chain structure (Kelly et al., 2007), organism physiology and environmental parameters such as temperature (Tao et al., 2018). For instance, Kelly et al. (2007) showed that the organic pollutant, ß-hexachlorohexane, biomagnified in a ma- rine mammalian food-chain while it did not in a piscivorous food-chain.

Field studies have shown that PAHs dilute with increasing food-chain steps in some ecosystems (Goutte et al., 2020; Wan et al., 2007) and potentially biomagnify in others (Froehner et al., 2011), with higher

☆This paper has been recommended for acceptance by Eddy Y. Zeng.

* Corresponding author.

E-mail address: ananya.ashok@kaust.edu.sa (A. Ashok).

Contents lists available at ScienceDirect

Environmental Pollution

journal homepage: www.elsevier.com/locate/envpol

https://doi.org/10.1016/j.envpol.2022.118789

Received 26 September 2021; Received in revised form 12 December 2021; Accepted 1 January 2022

Environmental Pollution 297 (2022) 118789

2 accumulation in apex marine predators (Cullen et al., 2019) and at higher trophic niches in lakes (Qadeer et al., 2019). As foundation species, corals play a crucial role in several reef food-chains. Different invertebrates and fish can feed directly on coral mucus, tissue or whole corals (Rice et al., 2019), and these organisms in turn become prey for higher organisms. By occupying multiple trophic niches, corals are vulnerable to PAHs accumulation through varied exposure routes such as direct bioconcentration from seawater across their large surface areas, from contact with sediments or from their diets which can range from suspended particulate material (Anthony, 2000) to herbivory (Conlan et al., 2019; Leal et al., 2014) to predation on zooplankton (Houlbr`eque et al., 2004; Houlbr`eque and Ferrier-Pag`es, 2009). Despite evidence that some pesticides, polychlorinated biphenyls and trace metals can accumulate and biomagnify in reef ecosystems (Dromard et al., 2018; Fey et al., 2019; Rumbold et al., 2018), accumulation of PAHs in corals has not been well studied.

Phenanthrene is a widespread three-ring PAH commonly studied as a marker for oil pollution. In laboratory studies, phenanthrene was readily accumulated from their diet by some aquatic organisms such as zebra fish (Danio rerio) and cichlids (Dimidiochromis kiwinge) (Xia et al., 2015).

Phenanthrene dietary accumulation is also reported for organisms such as copepods, Paracartia grani (Berrojalbiz et al., 2009), Pseudodiaptomus marinus (Arias et al., 2016), and the cladoceran Daphnia magna (Wang et al., 2021), which could also accumulate the PAH from seawater.

Bioaccumulation is identified as the dominant contributor to PAHs biomagnification in finfish (Jafarabadi et al., 2020; Wang et al., 2021, 2019; Xia et al., 2015). Existing studies determining the uptake of PAHs, including phenanthrene, by corals have only investigated bio- concentration (passive uptake from water) (Cook and Knap, 1983;

Kennedy et al., 1992; Knap et al., 1982; Solbakken et al., 1984), until recently when it was shown that corals fed phenanthrene-contaminated microalgae bioaccumulated the PAH to similar levels (Ashok et al., 2020). Therefore, knowledge on different pathways by which corals could accumulate PAHs and the impact of different exposure modes on coral physiology is limited. Since some corals commonly feed on co- pepods, which are known to accumulate PAHs both directly from seawater and by feeding (Berrojalbiz et al., 2009; Wang & Wang, 2006), we postulate that corals’ food chain could substantially contribute to the accumulation of PAHs, while direct absorption from seawater and feeding on other food items such as microalgae also remain highly relevant. Hence, here we exposed Acropora millepora, a common reef-building coral, to stable carbon isotope-labeled PAH, ¹³C-phenan- threne, via three exposure levels of increasing food-chain length: Level W (seawater - coral); Level 1 (seawater - microalga (Dunaliella salina) - coral) and Level 2 (seawater - microalga - copepod (Parvocalanus cras- sirostris) - coral), with the objectives of quantifying the accumulation of phenanthrene and comparing differences in uptake between the different exposure levels using the highly sensitive and robust tech- nique, CRDS. From the accumulated concentrations in coral tissue, algae and copepod biomass, we calculated BCF and BAF for these three or- ganisms at their respective exposure levels, and additionally BMFs were calculated for A. millepora. Further, we also examined the effects of phenanthrene accumulation through different exposure levels on coral photosystem health using PAM fluorometry.

2. Materials & methods

2.1. Culture and maintenance of experimental organisms

Cultures of the microalgae, Dunaliella salina, CS265 (Australian Na- tional Algae Supply Service, CSIRO Hobart, Australia) were grown at the National Sea Simulator (SeaSim) Algal Culture Facility in 0.2 μm filtered seawater (FSW) supplemented with f/2 culture medium. D. salina cul- ture conditions were maintained at 26 ^◦C, 35 ppt salinity, with a 16:8 h light:dark cycle with light intensity of 120 μmol photons m⁻² s⁻¹. A stock culture of the copepod, Parvocalanus crassirostris, obtained from

the Marine and Aquaculture Research Facility (James Cook University, Townsville) was scaled-up in large indoor tanks at the SeaSim Algal Culture Facility. Copepods were maintained on a mixed microalgal diet at 26 ^◦C, 35 ppt salinity and 12:12 h light:dark cycle with light intensity of <100 μmol photons m⁻² s⁻¹. Copepod nauplii (28–85 μm) were collected by size fractionation through filtration. Acropora millepora corals were collected from ~5 m depth at Davies Reef (18^◦50^′S, 147^◦37^′E) on the Great Barrier Reef in Australia, fragmented to ~2 cm length, healed and acclimatized at 26 ^◦C, 35 ppt salinity, with a 12:12 h light:dark cycle of 200 μmol photons m⁻² s⁻¹ for two weeks in the SeaSim facility’s flow-through systems at AIMS, Townsville.

2.2. Experimental design

We designed an experiment tracing the accumulation of ¹³C-phen- anthrene (¹³C14H10, 99 atom% purity, product number: 703125, CAS number: 1262770-68-2, Sigma-Aldrich Chemie GmbH) in A. millepora corals. A working solution of ¹³C-phenanthrene (2000 mg L⁻¹) was prepared in HPLC-grade methanol (Sigma-Aldrich) and sonicated to promote dissolution before use. The experiment consisted of treatments simulating three exposure levels of increasing coral food-chain length:

(a) In Level W, corals were exposed only to ¹³C-phenanthrene dissolved in seawater; (b) In Level 1 representing herbivory where some dissolved

13C-phenanthrene was not excluded, D. salina culture pre-exposed to

13C-phenanthrene in seawater for 48 h was added to the coral treatment jars and (c) In Level 2 representing predation, corals were given a diet of P. crassirostris nauplii fed on D. salina pre-exposed to ¹³C-phenanthrene.

In Level W corals could only bioconcentrate phenanthrene from seawater, while in Levels 1 and 2 they could bioaccumulate the PAH from their diet. Bioconcentration of ¹³C-phenanthrene at Level W was also quantified in D. salina. Likewise, Level 1 bioaccumulation was quantified in P. crassirostris nauplii from feeding on pre-exposed D. salina.

Coral fragments were maintained in glass jars containing 150 mL of FSW that was renewed daily. Each treatment had 12 replicate jars, each containing 3 coral fragments. Cultures of D. salina were pre-exposed to

13C-phenanthrene for 48 h, which was their average population doubling time, at the EC25 concentration of 182 μg L⁻¹ as described previously (Ashok et al., 2020). Exposure concentrations were selected to achieve a balance between toxicity, the potential decrease in con- centration from one food-chain step to the next, and to have sufficient signal detection in the CRDS. For Level W exposure, filtered seawater was spiked with ¹³C-phenanthrene at 182 μg L⁻¹, incubated for 48 h and added to the coral jars at 10% v/v. For Level 1 exposure, D. salina culture (15 mL) pre-exposed to ¹³C-phenanthrene was added to coral jars at 10%

(v/v). For Level 2 exposure, P. crassirostris nauplii were fed with 48 h¹³C-phenanthrene labeled D. salina culture for 5 h daily to avoid dep- uration of the pollutant via defecation, collected by filtration, resus- pended in FSW and provided to corals at a density of 3.6 ±0.6 nauplii mL⁻¹. Experiment controls consisted of corals maintained in plain FSW or fed unlabeled D. salina cells or fed unlabeled copepods for Level W, Level 1 and Level 2 exposure treatments respectively. The experiment was maintained for a total of 14 days, which is a time doubling from our previous shorter duration experiment (Ashok et al., 2020). Dissolved oxygen (mg L⁻¹) and water temperature (^◦C) were measured daily in 6 jars chosen at random using HQ30d equipped with an Intellical LDO101 oxygen probe (Hach, USA) and Horiba LAQUAact PC110.

2.3. ¹³C-phenanthrene accumulation

A. millepora fragments, D. salina and P. crassirostris nauplii were sampled at regular intervals for measurement of carbon isotopic composition and content using CRDS and calculation of ¹³C-phenan- threne accumulation. After determination of cell density by flow cytometry (BD Accuri™ C6 Plus bench-top flow cytometer with inbuilt BD Accuri™ C6 Plus software), D. salina cells were collected by filtering A. Ashok et al.

~100 mL of culture through glass fiber filters (GF/F, 25 mm diameter, Whatman™). The filters were dried at 60 ^◦C in a hot air oven, wrapped in aluminum foil and stored frozen at − 20 ^◦C until CRDS analysis.

Copepod nauplii were also collected on GF/F filters by filtering and preserved likewise for CRDS. Whole coral fragments were sampled on days 2, 4, and 7 with 6 replicates per day and thereafter on days 12 and 14 with 9 replicates per day, since, with greater exposure duration we expected larger variability in accumulated phenanthrene. The fragments were also stored wrapped in aluminum foil at − 20 ^◦C until CRDS anal- ysis. For details on sample processing and CRDS analytical conditions see Supporting Information (SI). Phenanthrene accumulation was calculated as described earlier in Ashok et al. (2020) (Details in SI).

Phenanthrene concentration in D. salina was converted to amount per cell (fg cell⁻¹) by dividing by respective abundance (counts mL⁻¹) and normalized to biomass dry-weight (mg kg⁻¹). Phenanthrene concen- tration in copepod nauplii was calculated as fg individual⁻¹ and con- verted to mg kg⁻¹. Dry weight of copepod nauplii was calculated from measured carbon content (mg C per filter) and nauplii concentration (Wiebe et al., 1975). Phenanthrene accumulated in coral tissue is also expressed as mg kg⁻¹, where dry weight was calculated as the mass difference of dried filters before and after filtration of air-brushed coral tissue slurry. BCF and BAF were calculated for corals in different treat- ments, and also for D. salina and P. crassirostris nauplii (Details in SI). In addition, BMF describing the increase in pollutant concentration from prey to predator was calculated for corals at exposure Levels 1 and 2 (Details in SI).

2.4. Dissolved ¹³C-phenanthrene measurement by GC-MS

Dissolved ¹³C-phenanthrene concentration in the experiment was measured using gas chromatography/mass spectrometry (GC/MS).

Filtered seawater (~100 mL) was collected from labeled D. salina culture at the end of 48 h exposure. Seawater samples from coral treatments (~100 mL) for T =0 h were collected from triplicate jars prepared with conditions such as addition of labeled seawater/algae/copepods iden- tical to the respective treatments except for addition of coral fragments.

Seawater samples (~100 mL) for T =24 h were collected directly from the coral treatment jars. Until analysis, all samples were stored in amber screw-capped bottles at 4 ^◦C after addition of 10% v/v dichloromethane (DCM). Surrogate standard (o-terphenyl) was added to each seawater sample and the samples were extracted three times with DCM. Com- bined extracts from each sample were dried with pre-combusted sodium sulfate (450 ^◦C, 4 h), filtered through DCM-extracted cotton wool and concentrated under nitrogen gas. Internal standards (biphenyl-d10, phenanthrene-d10 and perylene-d12) were added to the concentrated extracts prior to analysis using GC-MS in full scan (algal filtrates) and selective ion monitoring mode (seawater from coral jars). For more details see SI. Method blanks (FSW plus surrogate standard) were extracted and analyzed with each batch of samples.

2.5. Coral photophysiology measurements

Coral health on each sampling day was monitored by PAM fluor- ometry and with the coral health color chart as described in Siebeck et al. (2006). PAM fluorometry measured maximum quantum yield (Fv/Fm) of the coral symbiont’s Photosystem II as a measure of photo- synthetic efficiency (Schreiber, 2004). This technique measures emitted chlorophyll fluorescence under dark-adapted and under light-saturated conditions to estimate the levels of minimum (F0) fluorescence and maximum fluorescence (F_m) respectively with F_v/F_m =(F_m - F₀)/F_m. Briefly, at the start of the experiment and on each sampling day there- after, coral fragments were placed flat in a glass tray and dark-adapted for 15 min. The fragments were then transferred in their dishes to a Maxi-Imaging-PAM (I-PAM) (Walz, GmbH Germany) to obtain Fv/Fm as previously described (Bessell-Browne et al., 2017). Data files were processed using software, ImagingWinGigE V2.56p.

2.6. Data analysis

Data was analyzed using JMP Pro statistical software (v. 14.0). Plots were created using GraphPad Prism 9.1.0 (216). Values are reported as mean ±standard error (SE).

3. Results

3.1. Accumulation of ¹³C-phenanthrene in algae, copepods and corals D. salina pre-exposed to ¹³C-phenanthrene at Level W (directly from seawater, 48 h) showed stable carbon isotopic enrichment from − 18.26

±0.19 δ13C ‰ (N =3) to − 10.05 ±0.61 δ13C ‰, resulting in accu- mulation of 2.75 ±0.18 fg ¹³C-phenanthrene cell⁻¹ and 39.57 ±2.61 mg kg⁻¹ dry weight ¹³C-phenanthrene (N =30, averaged cell abundance 4.08 ×10⁵ ±0.27 ×10⁵ cells mL⁻¹). P. crassirostris nauplii pre-fed with

13C-phenanthrene-laden D. salina culture (5.24 ×10⁴ ±0.21 ×10⁴ cells mL⁻¹, ~5 h), representing Level 1 exposure, accumulated 1656 ±637 fg

13C-phenanthrene individual⁻¹ (N =6). The number of nauplii per filter averaged 2193 ±228 (mean ±SE). Based on an estimated dry weight of 0.24 ±0.01 μg individual⁻¹, the nauplii accumulated 8.04 ±2.60 mg kg⁻¹ phenanthrene in the biomass (N =6). Coral fragments used in the experiment had an average organic carbon content of 1.96 ±0.06 mg C fragment⁻¹ and an average biomass dry weight of 3.85 ± 0.13 mg fragment⁻¹ (N =113). Natural δ13C ‰ signal in corals was − 15.35 ± 0.39 (N =5). Exposure of corals to ¹³C-phenanthrene via all three Levels W, 1 and 2 resulted in enrichment of stable ¹³C isotope, confirming accumulation of phenanthrene in the fragments with time (Table 1).

Uptake rate of ¹³C-phenanthrene by corals was calculated as the slope of the regression of phenanthrene accumulated (mg kg⁻¹) versus time (d) (Fig. 1a, b, c). Uptake rates were calculated separately for an initial uptake period of 7 d and then for the total exposure period of 14 d (Table 1). One-way ANOVA showed no significant differences among the uptake rates at 7 d (p =0.65, F =0.43, DF =45) or 14 d (p =0.95, F

=0.041, DF =94) among the different levels (Fig. 1a, b, c). Uptake rates at 14 d were lower than 7 d for all treatments (Table 1). While the 14-day uptake rates of phenanthrene in Levels W and 1 were half the initial uptake rates, this only reduced by about 20% for Level 2 (Table 1).

However, accumulated ¹³C-phenanthrene concentrations (mg kg⁻¹) in Table 1

Measured δ13C (‰), carbon content (mg C fragment⁻¹) and calculated phen- anthrene accumulation (mg kg⁻¹) in Acropora millepora fragments over 14 days of ¹³C-phenanthrene exposure. All values are shown as mean ±SE.

Parameter Day Level W Level 1 Level 2

δ13C (‰) (mean ±SE) 2 −13.81 ±

0.49 −13.82 ±

0.22 −13.92 ± 0.16 4 −12.24 ±

0.19

−13.66 ± 0.34

−13.09 ± 0.12 7 −11.93 ±

0.31

−12.29 ± 0.16

−12.16 ± 0.19 12 −10.45 ±

0.36

−11.33 ± 0.18

−11.99 ± 0.23 14 −9.53 ±

0.31 −10.71 ±

0.32 −11.43 ±

0.24 Phenanthrene accumulated

(mg kg⁻¹) (mean ±SE) 2 13.06 ±

5.73 9.77 ±

1.23 8.41 ±

1.48

4 24.32 ±

3.17 11.03 ±

2.53 13.07 ±

3.59

7 31.28 ±

5.39 20.46 ±

1.04 20.28 ±

3.77 12 31.51 ±

4.63 23.53 ±

1.96 22.76 ±

1.42 14 33.52 ±

2.83 27.66 ±

2.77 29.36 ±

3.84 Uptake rate (mg kg⁻¹ d⁻¹) 7

days 3.54 ±

1.35 2.40 ±0.6 2.38 ± 0.85 14 days 1.41 ±

0.47 1.46 ±

0.24 1.54 ±

0.30

Environmental Pollution 297 (2022) 118789

4 corals tissues showed moderate differences among the treatments after 7 d (one-way ANOVA: p =0.0214; F =4.17; DF =48) and after 14 d exposure (one-way ANOVA: p =0.0253; F =3.8210; DF =97). Pair- wise comparison of mean accumulated phenanthrene concentration after 14 d using student’s t-test showed that the Level W had signifi- cantly higher phenanthrene concentrations than Levels 1 and 2, while the latter two were not significantly different in comparison to each other (Fig. 2).

3.2. Coral health

Fv/Fm, measured at the start of the experiment was 0.56 ±0.01 (N = 18). Overall mean Fv/Fm was not significantly different between controls and fragments exposed to ¹³C-phenanthrene at each time point (p = 0.2905; F-ratio =1.1234; DF =196). As exposure time increased Fv/Fm

tended to decrease in both controls and ¹³C-phenanthrene treatments (Fig. 3). Among treatments, while Fv/Fm was not affected in corals exposed at Level 2 after 14 d, those exposed at Level W had in com- parison reduced Fv/Fm (p =0.0054) (Fig. 3). Coral health color charts did not show apparent visual differences among treatments with time (S.

Fig. 1). Water quality parameters measured in randomly selected coral

treatment jars showed that dissolved oxygen generally decreased on average by 21.8% (n =11) at 24 h, and the seawater medium was renewed daily (S. Table 1). Water temperature remained stable throughout the experiment (S. Table 1).

3.3. Exposure concentrations of ¹³C-phenanthrene to corals

In Level W coral jars, the average seawater ¹³C-phenanthrene con- centration was 1.53 ±0.38 μg L⁻¹ (N =3) and 0.36 ±0.12 μg L⁻¹ (N = 3) at T =0 h and T =24 h, respectively. In Level 1, corals were fed D. salina pre-exposed for 48 h to phenanthrene at a density of 2.27 ×10⁴

±0.09 ×10⁴ cells mL⁻¹ and based on the accumulated concentration in the algae cells (2.75 ±0.18 fg cell⁻¹), this resulted in a dietary exposure concentration of 0.051 ±0.004 μg L⁻¹. Seawater filtrate from labeled D. salina culture had a residual ¹³C-phenanthrene concentration of 15.15 ±0.74 μg L⁻¹ (N =6) and after dilution in the coral treatment solution, this represented 1.30 ±0.26 μg L⁻¹ (N =3) and 0.36 ±0.16 μg L⁻¹ (N =3) at T =0 h and T =24 h, respectively. Most seawater ¹³C- phenanthrene concentration values were below the detection limit (<0.1 μg L⁻¹) in the Level 2 treatment. At Level 2, the dietary exposure concentration to corals from phenanthrene accumulated in copepods was estimated at 0.006 ±0.002 μg L⁻¹ (N =6). Measurements of ¹³C- phenanthrene in seawater using GC/MS at T =0 h and T =24 h are given in S. Table 2. From the mass difference of initial and final dissolved

13C-phenanthrene concentrations in Level W and Level 1 treatments corals were estimated to have absorbed approximately 76 and 72%, respectively, of the initial concentration.

3.4. BCF, BAF and BMF

Log BCF and log BAF values (L kg⁻¹) were calculated for corals and the dietary organisms, algae and copepod, at the Levels W, 1 and 2. BCF values represent accumulation from seawater only, while the BAF values represent accumulation from seawater + food. At all Levels, corals showed higher log BCF and log BAF values than D. salina (Level W log BCF =2.31 ±0.02 (N =30, calculated based on spiked ¹³C-phenan- threne concentration) and P. crassirostris nauplii (Level 1 log BAF =3.23

± 0.15 (Fig. 4). Despite higher overall accumulated phenanthrene concentration in corals at Level W, this pathway had a lower log BCF value compared to log BAF values for Levels 2 and 3 (Fig. 4). Log BAF increased with increase in food-chain length: corals at Level 2 had significantly higher log BAF (6.45) than Level W log BCF (4.188) or Level 1 log BAF (4.5) (p =<0.0001; F-ratio =477.3015; DF = 97) (Fig. 4). Coral Level W log BCF and Level 1 log BAF were also signifi- cantly different from one another as indicated by pair-wise Student’s t- test (p <0.0001). The BMF values calculated for corals showed that Level 1 BMF (0.6 ±0.04) was lower than Level 2 BMF (2.48 ±0.22), Fig. 1. Changes in phenanthrene concentration (mg kg⁻¹) in A. millepora coral tissues over 14 days’ time at the different exposure levels of (a) Level W (seawater - coral), (b) Level 1 (seawater – algae – coral) and (c) Level 2 (seawater – algae – copepod – coral).

Fig. 2. Accumulated ¹³C-phenanthrene concentration in corals after 14 d (mg kg⁻¹) from exposure to ¹³C-phenanthrene via Level W (seawater – coral), Level 1 (seawater – algae – coral) and Level 2 (seawater – algae – copepod – coral).

A. Ashok et al.

where a value exceeding 1 denoted the possibility of biomagnification with a longer food-chain.

4. Discussion

We found that A. millepora corals readily acquired the PAH, ¹³C- phenanthrene, at three exposure levels of increasing food-chain length with similar uptake rates. Higher tissue concentrations (mg kg⁻¹) was accumulated in Level W, where phenanthrene was directly dissolved in seawater, than in Level 2. Accumulation via exposure Levels 1 (seawater – algae – coral) and 2 (seawater – algae – copepod – coral) food-chains did not result in significantly different tissue phenanthrene concentra- tions in corals. We calculated BCF (L kg⁻¹) and BAF (L kg⁻¹) values for Level W, 1 and 2 organisms relative to the chemical concentration in their respective exposure sources. Coral accumulation at Level 2, where they were fed on copepods laden with phenanthrene-contaminated algae, resulted in higher log BAF than Level 1 log BAF or Level W log BCF. Coral log BAF exceeded its diet in both Level 1 (algae log BCF; 2.31) and Level 2 (copepod log BAF; 3.23) exposure. Further, we report higher BMF values in corals exposed at Level 2 (longer food-chain) than Level 1.

There are very few previous laboratory studies of PAHs accumulation in corals; however, field-studies reporting concentrations of PAHs in coral tissues offer useful comparative data on the dynamics of PAHs accumulation in corals. In natural environments, corals consistently show a high bioaccumulation potential by exhibiting tissue PAHs con- centrations greater than surrounding sediments, which are otherwise considered as final sinks for organic pollutants such as PAHs. For instance, in the Arabian Gulf, Jafarabadi et al. (2018) reported PAHs concentrations in coral tissue that were at least 15- and up to 36-fold higher than concentrations in nearby sediments. Likewise, Sabourin et al. (2012) reported PAHs concentrations in coral tissues from Gulf of Mexico to be about 19 times higher than ambient sediment concentra- tions (For further examples see S. Table 4). Low molecular weight PAHs (2–3 ring compounds) were predominant in biomass collected from field-exposed corals (Han et al., 2020; Jafarabadi et al., 2018; Ko et al., 2014) and phenanthrene is a dominant and frequently detected PAH (Jafarabadi et al., 2018; Xiang et al., 2018). Concentrations of up to 10 μg g⁻¹ of phenanthrene are reported in coral tissues from natural hab- itats (Sabourin et al., 2012). Although corals can acquire PAHs through multiple routes, reported biota-sediment concentration factors of PAHs are lower than coral BAFs in field studies indicating that these organisms may be primarily accumulating PAHs through their food-chains Fig. 3. Box plots with min-max whiskers of maximum photosynthetic efficiency

(Fv/Fm) of coral fragments in controls (no phenanthrene, light pink boxes) and treatments exposed to ¹³C-phenanthrene (dark pink boxes) at (a) Level W (seawater– coral), (b) Level 1 (seawater – algae – coral) and (c) Level 2 (seawater – algae – copepod – coral) at experimental timepoints of 2, 7 and 14 days. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4. ¹³C-phenanthrene log BCF for exposure at Level W (seawater – algae) or (seawater – coral) and log BAF for exposure at Level 1 (seawater – algae – copepod) or (seawater – algae - coral) and Level 2 (seawater – algae –copepod – coral).

Environmental Pollution 297 (2022) 118789

6 (Jafarabadi et al., 2018).

The few existing laboratory studies on PAHs accumulation in corals have examined uptake through bioconcentration (from dissolved phase only) (Kennedy et al., 1992; Solbakken et al., 1984). Lower uptake rates of phenanthrene in this study compared to those reported previously for the 6-ring PAH, benzo(a)pyrene, in corals Favia fragrum and Montastrea annularis (Kennedy et al., 1992), is possibly due to differences in chemical properties of the two PAHs, differences in coral species morphology and exposure times. A study investigating uptake of other pollutants such as lindane and phthalate esters in the coral, Diploria strigosa, also showed that coral uptake rates can vary for different chemicals (Solbakken et al., 1985). In other organisms such as zebra fish and cichlids, predation elevated PAHs uptake rates by up to ~40% (Xia et al., 2015).

The exposure concentrations of ¹³C-phenanthrene measured here are comparable to other experiments investigating bioconcentration and bioaccumulation of deuterated-PAHs in fish (Wang et al., 2021; Wang et al., 2019). While T =0 h concentrations in seawater were similar for Levels W and 1, this was mostly below detection level (<0.1 μg L⁻¹) for Level 2, indicating that the P. crassirostris nauplii fed on ¹³C-phenan- threne labeled D. salina did not excrete this pollutant into the seawater in substantial amounts over 24 h. Even longer retention times are re- ported for copepods. For instance, Agersted et al. (2018) reported that the Arctic copepod, Calanus hyperboreus did not eliminate accumulated phenanthrene for over three days. Similarly, pyrene, a four-ring PAH, was retained by copepods for over 77 days (Nørregaard et al., 2015).

Corals exposed at Level W accumulated significantly higher tissue concentration of phenanthrene after 14 d than Level 2 (p-value =0.012) and slightly more than Level 1 (p-value = 0.02), while accumulated concentrations of Levels 1 and 2 were not significantly different from each other. The higher accumulated concentration in coral tissue seen in Level W is likely due to direct partitioning of PAHs from the dissolved phase into the lipid-rich mucus layer overlying the large and intricate surface areas of corals. Acroporid corals can produce up to 4.8 L of mucus per square meter of reef per day (Wild et al., 2004) and studies by Han et al. (2020), Zhang et al. (2019), and more recently Jafarabadi et al.

(2021), have shown that coral mucus has high affinity for PAHs and other organic pollutants. The almost similar (p = 0.02) accumulated tissue concentrations seen in corals exposed at Levels W and 1 are also consistent with our previous study where A. millepora corals accumu- lated similar phenanthrene concentrations from dissolved-only and di- etary accumulation at the end of 6 days (Ashok et al., 2020), with likely contributions from the presence of dissolved phenanthrene transferred in addition to algae-bound phenanthrene in Level 1.

No significant differences in accumulated tissue phenanthrene con- centrations between corals exposed at Levels 1 and 2 demonstrated that A. millepora fed on either the microalgae D. salina or on the copepod P. crassirostris, depending on their availability. Coral species vary in their relative reliance on heterotrophic feeding (Conti-Jerpe et al., 2020) and in their preferred food items. While it is well documented that Acropora spp. can ingest zooplankton (Hoogenboom et al., 2015; Kuanui et al., 2016), few studies have specifically addressed herbivory in scleractinian corals. Recent studies to develop aquaculture feeds for Acroporid corals suggested however that herbivory may be an important feeding mode for Acroporids (Conlan et al., 2019). Accumulation from dietary uptake observed in this study is further in agreement with studies reporting field concentrations of PAHs in coral tissues consis- tently higher than sediments or seawater (Jafarabadi et al., 2018; Sab- ourin et al., 2012) (S. Table 4).

Toxicity resulting from dietary exposure to pollutants is rarely explored (Wang et al., 2017). Here we investigated the photosynthetic efficiency of the coral endosymbionts and found that exposure to phenanthrene did not significantly alter the Fv/Fm values measured in this experiment. Fv/Fm is a sensitive and non-intrusive parameter that detects changes indicative of the impairment of Photosystem II (Ralph et al., 2007). Toxicity from metals, herbicides and thermal stress has

been shown to reduce Fv/Fm in Symbiodinium ex hospite (Kuzminov et al., 2013; Mercurio et al., 2018; Van Dam et al., 2015) and in situ (Fisher et al., 2012). Toxicity studies on the effects of PAHs in corals have examined morphological, behavioral, histopathological changes, mor- tality and more recently, photosynthetic efficiency (Loya and Rinkevich, 1980; Renegar et al., 2017; Turner et al., 2021; Turner and Renegar, 2017). Renegar et al. (2016) examined the toxicity of high concentra- tions of a two-ring PAH, 1-methylnapthalene (640–25832 μg L⁻¹), to Porites muricata but found no significant differences among the treat- ments and controls at the end of exposure, as was also the case in our previous study (Ashok et al., 2020). Turner et al. (2021) also reported the relatively low sensitivity of Fv/Fm to phenanthrene compared to other hydrocarbons in five different coral species.

The coral log BAF values increased with food-chain length from Level 1 to Level 2 and was also higher than log BCF from exposure at Level W.

The log BAF values for phenanthrene in corals reported here are be- tween 2 and 4 magnitudes higher than those reported for nematodes (2.92) (Spann et al., 2015), freshwater isopods (2.8 ±0.5) (van Hattum and Cid Montanes, 1999), mussels (2.81) (Yakan et al., 2013), copepods such as Pseudodiaptomus marinus (3.80–3.86) (Arias et al., 2016) and Paracaria grani (2.2) (Berrojalbiz et al., 2009), and more similar to the log BCF of benzo(a)pyrene reported for copepods (4.95, Acartia eryth- raea) (Wang and Wang, 2006). The log BCF and BAF values of corals in this study are also higher than those reported for zebra fish (2.75–2.77) and cichlids (2.82) when considering the whole organism, while approaching closer to values reported for log BAFlipid reported for zebra fish (3.88 ±0.003) and cichlids (3.89 ±0.003) (Xia et al., 2015). BAF values measured here for P. crassirostris nauplii are comparable with those reported for other phenanthrene in other copepod species and lower than that reported for benzo(a)pyrene (S. Table 3). Measured coral log BCF and log BAF values here are also about 0.4–2 log magni- tudes higher than those reported earlier for A. millepora corals (Ashok et al., 2020), likely due to the longer experiment duration.

The higher BAF of phenanthrene seen here in corals with increasing food-chain length could be attributed to a complex-interaction of aspects such as food preference, greater retention of pollutants acquired through diet and elimination capacity of the organism. Organic pollutants such as γ–HCH and chlorobenzene that were not routinely considered bio- accumulative in laboratory studies based on dissolved-only uptake have BAFs up to ten-fold higher in field studies where dietary accumulation is bound to occur (Arnot and Gobas, 2006; Fisk et al., 2000; Kelly et al., 2007). Heterotrophic feeding can contribute up to 66% of all fixed carbon in corals and can meet 15–35% of their metabolic requirements (Houlbr`eque and Ferrier-Pag`es, 2009). Corals have demonstrated pref- erence for predation on zooplankton including copepods and can ingest up to two prey items per polyp per hour of ingestion (Ferrier-Pag`es et al., 2003; Kuanui et al., 2016; Sebens et al., 1996). In the case of finfish such as Danio rerio, dietary uptake contributed largely to body burden in laboratory food-chain experiments with the PAHs (phenanthrene and anthracene) body burden peaking after each feeding episode (Wang et al., 2021) whereas only water-borne uptake led to trophic dilution (Wang et al., 2021). It has also been shown that heterotrophic feeding can contribute to increased lipid content in corals of other species (Acropora cervicornis) (Towle et al., 2015) and this can also directly contribute to the accumulation of lipophilic pollutants such as PAHs when fed contaminated food. Furthermore, BMF values clearly demon- strate differences in bioaccumulation caused by different prey types (Borgå et al., 2004). Here, the coral BMF was higher in Level 2 than Level 1 suggesting biomagnification via a longer food-chain. However, BMF values can vary widely between organisms (Borgå et al., 2004), chemicals (Liu et al., 2020) and studies making intercomparisons with other food-webs complex. Further research is also needed to establish the role of corals’ physiological processes such as excretion or biotransformation on the retention of pollutants accumulated through different routes of exposure.

A. Ashok et al.

5. Conclusion

Corals have a unique physiology of occupying more than one trophic niche rendering them prone to acquire pollutants such as PAHs through multiple pathways. Here, we have shown that a¹³C-labeled PAH, phenanthrene, bioaccumulated substantially in reef-building corals of the species A. millepora exposed at different levels of increasing food- chain length such as direct uptake from seawater (Level W); uptake via feeding on contaminated microalgae (Level 1); and uptake via a more complex two-step food-chain where corals were fed copepods which were pre-fed phenanthrene labeled microalgae (Level 2). Our findings highlight corals’ capacity for accumulation of PAHs at similar rates at these different exposure levels. Of particular interest is the increased bioaccumulation factor at Level 2. When it comes to under- standing pollutant accumulation dynamics, it is necessary to consider how modes of exposure, trophic position of an organism and food-chain length can influence widespread environmental pollutants.

Credit author contribution statement

Ananya Ashok: Formal analysis, Investigation, Methodology, Vali- dation, Visualization, Writing – original draft, Writing – review &

editing. Lone Høj: Funding acquisition, Resources, Supervision, Vali- dation, Writing – review & editing. Diane L. Brinkman: Investigation, Resources, Writing – review & editing. Andrew P. Negri: Funding acquisition, Resources, Supervision, Validation, Writing – review &

editing. Susana Agusti: Conceptualization, Formal analysis, Method- ology, Funding acquisition, Resources, Supervision, Validation, Writing – original draft, Writing – review & editing.

Declaration of competing interest

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.

Acknowledgments

This research was funded by King Abdullah University of Science and Technology (KAUST) through baseline funding to S. Agustí and a center partnership project between the Red Sea Research Center (RSRC) and the Australian Institute of Marine Science (AIMS). We thank Florita Flores, Philip Mercurio, Tom Barker and Thomas Armstrong from AIMS and Malak Abdallah, Vijayalaxmi Dasari, Mongi Ennasri from KAUST for their valuable help and assistance with the experiments and samples analysis.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.

org/10.1016/j.envpol.2022.118789.

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