1. Introduction
Benthic dinoflagellates have the capacity to produce a diverse suite of bioactive secondary metabolites that have been linked with seafood safety and human health concerns globally. One such group includes the neurotoxic ciguatoxins (CTXs) that have been linked to ciguatera poisoning and have been associated with some species and strains of epi-benthic dinoflagellates from the genus
Gambierdiscus and
Fukuyoa [
1,
2,
3,
4,
5]. Several studies have reported that the in situ dinoflagellate community assemblage can change with environmental factors (e.g., temperature) and that species and strains may have physiological niches [
6,
7,
8]. Likewise, long-term field studies have demonstrated that the CTX load of field-collected benthic microalgae is: (1) asynchronous with
Gambierdiscus abundance; (2) site specific; (3) seasonal; and (4) occurring in pulses in tropical reef ecosystems [
6,
9]. It has also been proposed that CTX environmental pulses are linked to the presence of highly toxic
Gambierdiscus strains rather than high overall algal biomass [
5,
6,
9,
10] potentially reducing the effectiveness of genus-level monitoring for these benthic HABs in terms of risk reduction. This presents some questions on the subsequent CTX load and CTX pulses that may occur in other demersal marine biota feeding on potentially toxicogenic epiphytic algae, aquatic invertebrates (e.g., amphipods), and other small grazers.
While still a working hypothesis in the field, the bioaccumulated CTX load in fish collected from ciguatera poisoning hotspots (and therefore ciguatera risk) is a function of the rate of toxin production by epi-benthic dinoflagellates (conceptual model in Lewis et al. [
11] and revisited in Lewis and Holmes [
12]). However, there remains a gap in understanding the timing between these processes. Field-based studies have provided evidence of a temporal lag of months to years occurring between the environmental triggers that increase toxin production, toxicity in upper-trophic-level fish, and increasing ciguatera cases [
13,
14]. Further, high fish toxicity (and resulting ciguatera prevalence) has been observed in high-site-fidelity fish when toxigenic dinoflagellate abundance and toxicity are low (or absent), suggesting a shift towards a non-toxic area [
15,
16], which leads to an assumption that bioaccumulated CTX is persistent in fish after the dinoflagellate source declines. However, field studies lack the ability to properly investigate ecologically relevant exposure routes (e.g., dietary, respiration, dermal) or the rates of uptake and depuration once a CTX source is removed, because total source removal can only be assumed.
Early studies attempted to investigate the retention of CTX-like toxicity (as a proxy for CTX, which was yet to be structurally elucidated) in a laboratory setting using fish that had naturally incurred toxin. For example, in Hawaii, Takata and colleagues (reported by [
17]) allowed wild-caught Lutjanids (
Lutjanus bohar,
L. gibbus) and a Serranid (
Variola louti) to depurate in aquariums at various intervals up to 14 months, while Banner et al. [
17] kept wild
L. bohar in holding ponds up to 30 months. In both studies, fish at the end remained toxic when fed to cats and mongoose. Davin et al. [
18] fed several species of marine and freshwater fish the ground flesh or extracts of barracuda (
Sphyraena) or whole
Gambierdiscus cells (both sourced near the Caribbean Antilles Islands), followed by depuration time up to 81 d. Many of the fish died or were intoxicated. Largemouth bass (
Micropterus salmoides) fed a high dose of cells recovered behaviorally but remained moderately toxic by intraperitoneal injection to mice after 81 days of clean feed, but fish fed lower doses were non-toxic after depuration. Recently, more laboratory studies have focused on CTX elimination using several exposure methods and in a variety of organisms. For instance, Ledreux et al. [
19] performed single-oral dose-recovery experiments with mullet (
Mugil cephalus) fed
G. polynesiensis cells and reported only 5% of the CTX activity occurred in tissues after 24 h when analyzed by the mouse neuroblastoma assay (N2a). Li et al. [
20] described multiple tissue kinetics of three Pacific CTX congeners, namely P-CTX-1 (CTX1B), P-CTX-2 (52-epi-54-deoxyCTX1B), and P-CTX-3 (54 deoxyCTX1B) extracted from eel (
Lycodontis javanicus). Toxins were added to a pelleted feed and given to juvenile orange-spotted grouper (
Epinephilus coioides) for 30 d and subsequently depurated for 30 d. The authors reported that CTX declined exponentially in depuration in some tissues (including muscle); however, CTX burden by the end of the study was not significantly different from the levels measured prior to depuration [
20]. In another recent report, Caribbean CTX (C-CTX-1) depuration was investigated in the muscle of the freshwater goldfish (
Carassius auratus) following a 43 d daily feeding of naturally C-CTX incurred amberjack (
Seriola sp.) flesh prepared in agarose [
21]. Estimated CTX toxicity (concentration) in muscle was reported to decline by approximately 86% out to 60 d post-exposure, but other tissues were not analyzed. Most recently, a depuration experiment using juvenile lionfish (
Pterois volitans) was reported after 30–41 d experimental feeding on the flesh of the naturally P-CTX-contaminated parrotfish (
Chlorurus microrhinos) [
22]. Extracts from pooled liver showed a gradual decline in CTX concentration in fish harvested during a 43 d depuration, where fish were switched to a non-toxic diet of farmed sea bream (
Sparus auratus). These two latter studies report depuration in single tissues (muscle and liver, respectively) and provide informative data on trends in bioaccumulation and depuration following oral exposure but were unable to capture cross-tissue distribution and did not examine fish growth that may contribute to the change in CTX tissue concentrations, but not body burden, as suggested by Holmes et al. [
23] in a recent field-based study and review. We propose that estimates of toxicokinetic rates that also incorporate growth dilution may be more valuable than CTX concentrations in tissues in studies aiming to predict or model CP risk in the food web across species.
In this study, we provide data on the kinetic rate of C-CTX-1 bioaccumulation and depuration in the ecologically relevant marine omnivore Lagodon rhomboides (pinfish). Replicate fish were either fed a C-CTX-1 (n = 40) formulated diet or matrix matched control (n = 37) for 20 d, and then placed into depuration for up to 99 d. CTX toxicity in the major tissue compartments of L. rhomboides were quantified and compared through time and across replicates compared to control fish using an in vitro mouse neuroblastoma (N2a-MTT) assay.
Sampling and CTX analysis of whole muscle, liver, and other pooled visceral contents (heart, spleen, gall bladder, intestine) throughout the time course revealed dynamic trends in tissue burden and kinetics. Further, modeling concentrations corrected for fish growth dilution revealed an increase in the estimated half-life of C-CTX-1 in L. rhomboides, highlighting growth as a major source of pseudo-elimination of accumulated CTX. This correction reveals that C-CTX (measured here as a CTX3C equivalents) can be retained for several months following removal from the toxin source and if not accounted for could result in error during CP risk assessment and analysis of food web transfer potential. This work should be considered when sampling mid-trophic-level fish with high site fidelity and in the development of temporal models of CTX cascades where the frequency of toxin pulses may be shorter than the CTX half-life in fish. These data have implications for CP risk analysis in fish and may help to explain high toxin loads in highly migratory species collected in non-endemic regions for ciguatera (e.g., barracuda, mackerel, amberjack).
4. Materials and Methods
4.1. Reagents and Chemicals
All solvents were HPLC grade obtained from Fisher Scientific (Waltham, MA, USA) and were acetone, MeOH, n-hexanes, CHCl3, and dimethyl sulfoxide (DMSO). Bond Elut silica solid-phase extraction (SPE) cartridges (100 mg and 500 mg) were from Agilent Technologies (Santa Clara, CA, USA). Ouabain octahydrate (O) and veratrine hydrochloride (V) used for in vitro assays were from Sigma (St. Louis, MO, USA). The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl- tetrazolium (MTT) was sourced from Alfa Aesar (Haverhill, MA, USA) and was prepared in sterile phosphate buffered saline (PBS) from Medicago (Quebec City, QC, Canada). Ciguatoxin 3C (CTX3C) was purchased from Wako Chemicals (Osaka, Japan) and a 50 ng mL−1 stock was prepared in LCMS grade MeOH and aliquoted in sealed amber vials maintained at −20 °C until use.
Adherent murine neuroblastoma cells (Neuro-2a; ATCC CCL-131) were originally purchased from the American Tissue Culture Collection (Manassas, VA, USA) and modified (i.e., OV desensitized) lines generated and maintained to ensure maximal and stable cell response to CTX in MTT-based assays prior to use (available on request). Powdered Roswell Park Memorial Institute (RPMI) 1640 medium (Millipore Sigma, Burlington, MA, USA) was prepared in 10 L batches with sterile ultrapure water (18 mΩ) and 1L aliquots filtered (polyethersulfone 0.2 µM Supor membrane; Pall Corp; Port Washington, NY, USA) into sterile bottles. Supplements included sterile L-glutamine (200 mM stock), sodium pyruvate (100 mM stock), and fetal bovine serum (all from Gibco; Grand Island, NY, USA). Trypsin-EDTA (0.025% stock) used in cell detachment and harvest was from Corning (Corning, NY, USA). Cell culture consumables including serological pipettes, tubes, flasks, and micro-well plates were from CellTreat (Shirley, MA, USA). Trypan blue (Fisher Scientific, Waltham, MA, USA) was prepared to 0.2% in sterile PBS (pH 7.4) and used in cell enumeration and viability assessment.
4.2. Controlled Exposure
4.2.1. Fish Collection and Acclimation
Wild pinfish, Lagodon rhomboides (Sparidae), were collected from Mississippi Sound (Dauphin Island, AL, USA) and Perdido Bay (Orange Beach, AL, USA) using hook-and-line with sabiki rigs. To reduce stress from handling, fish were directly transferred to an aerated, seawater-filled cooler using a specialized dehooking tool which does not require fish handling. Fish were transported in aerated, temperature and salinity-controlled tanks to the experimental wet-lab facility at the Dauphin Island Sea Lab (DISL) within one hour. Prior to transfer to a primary acclimation tank, a five-minute freshwater dip was performed to remove marine ectoparasites. This step was important to reduce the chance of disease in the closed system. The recirculating aquarium system was composed of a 450 L sump, 375 L head tank, and 120 L enclosure tank and filled with artificial seawater (Crystal Sea Marine mix; Mount Dora, FL, USA). To limit stress, environmental enrichment provided to each tank habitat included a two-inch bed of pool filter grade silica sand, 12 cm diameter × 12 cm length PVC tubes for structure, and artificial submerged aquatic vegetation. Fish were collectively monitored in the acclimation tanks in groups of 25 for three weeks for signs of parasitic diseases and behavioral abnormalities prior to transfer into individual tanks for experimental treatment. Two fish collections were performed to supply fish for the exposure study, once in January 2019 and again in May 2020. The water conditions across all systems were maintained on a 12 h light: 12 h dark cycle with weekly water changes (artificial seawater: 15–17 psu; 24 ± 2 °C; ammonia ≤ 0.1 ppm, nitrite ≤ 0.1 ppm, nitrate ≤ 40 ppm, pH = 8.1 ± 0.1).
4.2.2. Feed Formulation
Experimental (CTX feed) and control diets (no CTX) were created from a blend of fish meal, Mazuri Aquatic Gel Diet for Omnivorous Fish (sku: 1815252-409; Mazuri Exotic Animal Nutrition, St. Louis, MO, USA), and water. The fish meal consisted of white muscle tissue from either non-toxic great barracuda,
Sphyraena barracuda, collected from the northern Gulf of Mexico, Alabama (control) or
S. barracuda with naturally incurred C-CTX that were collected from the U.S. Virgin Islands during concurrent efforts. Fillets with skin, scales, and bones removed, were cut into small chunks and subsequently homogenized in an industrial food-grade stainless steel grinder (STX Turboforce 3000; Lincoln, NE, USA). From each fish, at least five replicate subsamples of minced
S. barracuda tissue were extracted and analyzed for the presence of CTX-like activity using N2a-MTT, and C-CTX-1 confirmed by liquid chromatography-mass spectrometry as previously reported [
50]. After verifying control and CTX fish, batches were pooled and mixed three times, then frozen in stainless steel trays and freeze dried. Dehydrated tissue (300 g) was powdered using a grain mill (HC-300; C-Goldenwall, Amazon, Seattle, WA, USA) at 28,000 rpm for 1 min and sieved to ≤500 µm to create a fine and uniform product. All fish powder was thoroughly mixed and kept in 500 g aliquots at −20 °C in airtight containers until use. Subsamples of the homogenized freeze-dried fish powder were taken for analysis by the N2a-MTT for final quantification of toxicity of the pelleted diet prior to further preparation. The control and CTX diets for
L. rhomboides were created by combining equal parts Mazuri gel powder and fish powder, then homogenized and combined with 60% water (by weight) that was warmed to 65 °C. The homogenized feed was transferred to a pastry piping bag with a round tip (Wilton size 10), then dispensed in even rows onto pre-weighed parchment paper. After fully dispensing the gel, wet weight was recorded, and the gel was cut into 5 mm pellets. The tray was then placed in a drying oven at 65 °C for approximately 1 h to remove approximately 2/3 water so that pellets retained a uniform shape. To maintain a consistent product across batches, the water content (as a percent of the final product) was calculated by subtracting the weight of solid ingredients in the mixture from the weight of the formulated food after drying, then dividing by the whole-food dry weight. Subsamples of pellets were weighed on an analytical balance to collect an average pellet weight.
4.2.3. Experimental Design
To accomplish optimal control and observation of fish, L. rhomboides were individually transferred to 12 L tanks attached to a closed recirculating aquarium system. Four recirculating aquarium systems with 10 to 15 tanks per system were used in this study. Fish were acclimated to the individual tanks (one fish per tank) for at least 2 weeks prior to the beginning of experimental treatment to ensure water quality parameters and fish health were stable. This acclimation period was in addition to the post-collection acclimation.
The experiments were designed to provide control over dietary intake of C-CTX-1 in individual fish so that the toxicokinetics of bioaccumulation and depuration could be analyzed across multiple tissues with sufficient replication for the subsequent statistical analyses. Tanks designated to receive control or CTX pellets were assigned a sampling day on the day prior to the start of experiments using a random number generator. Control fish received control pellets throughout trials while CTX fish received CTX pellets for up to 20 days (bioaccumulation phase) and then transitioned to the control feed for the depuration phase (see
Table 1 for sampling scheme). Both control and CTX pellets contained powderized
S. barracuda flesh (non-toxic or toxic, respectively) to match the nutritional intake across groups and maintain consistency (minus CTX) when fish were transitioned from the bioaccumulation to the depuration phase. Fish fed only the control pellets were designated for each sampling point to be used as matrix controls in assays, and to compare differences in behavior, and growth rate compared to the CTX treatment groups.
Data were collected from two feeding experiments (trial 1 and 2, hereafter) to allow adequate replication. The first experiment had maximum bioaccumulation and depuration phases of 20 days each with a total of 52 fish and was performed 5 March–13 April 2019. Fish were sampled as baseline controls prior to feeding on the first experimental day and on days 6, 10, and 20 of bioaccumulation and days 5, 10, and 20 of depuration. In the second experiment performed 30 July–25 November 2020, the bioaccumulation phase was replicated identical to trial 1, and depuration was extended out to a maximum of 99 days to increase the depuration course. Fish were collected on day 20 of the bioaccumulation phase to compare to trial 1 for reproducibility, and then on days 40, 70, and 99 of the depuration phase. Based on the lack of overt signs of intoxication during the first experiment and ample analytical controls, during the second experiment, remaining
L. rhomboides were allocated towards maintaining experimental replication in the CTX treatment group (CTX fish,
n = 4) and control replicates were reduced to
n = 2 per time point (except day 0,
n = 3) (
Table 1).
Daily feed requirements were calculated based on initial weights (g) of fish which were recorded using a water displacement method one or two days prior to the beginning of exposure trials. All fish were fed either control or CTX pellet food at the same time daily, normalized to body mass (approximately 1.8% of initial body weight day
−1). Fish were fed the same amount of food daily relative to the initial whole wet weight for the entire experiment. Individuals were closely observed to ensure all food was eaten during feeding times, and after acclimation
L. rhomboides were consistently consuming the pellets within seconds of entering the tank. Fish were also re-weighed at multiple points (at minimum initial and at time of sampling) throughout the study to track growth rate and to determine if somatic growth influenced C-CTX concentration as previously expected by others in P-CTX fish studies [
20,
33,
51].
4.2.4. Fish Sampling, Dissection, and Extraction
On a designated day and time of sampling, fish were fed and left undisturbed for 7 h. Fish were euthanized according to approved IACUC protocols by iced seawater immersion and mortality was confirmed by cessation for at least 10 min. A secondary spinal transection was performed to ensure mortality, and fish were then dissected. Dissected tissues were weighed whole including muscle, brain, liver, gonads (when present), and additional visceral organs combined (heart, spleen, pancreas, gall bladder, intestine). Swim bladder was discarded and not analyzed in this study. Stomach was removed from the viscera samples to limit residual CTX signal from undigested food in downstream toxicity assays; however, given reported gut clearance rates <24 h, the amount of residual CTX in the intestinal tract was expected to be below the limit of quantification (LOQ; described in
Section 4.3 Data Analysis) in the viscera on our assays.
Muscle subsamples (5–7 g) were extracted twice in acetone (2 mL g−1 tissue weight) by bead disruption (2.6 mm diameter; ceramic) using a Bead Ruptor 24 (Omni International; Kennesaw, GA) at a speed of 5 m/s for two cycles of 30 s duration each. Resultant homogenates were centrifuged at 2465× g for 5 min at room temperature (approximately 21 °C) between extractions to obtain the supernatant. Combined supernatants were placed at −20 °C for 18 h, then centrifuged (4 °C, 2465× g, 10 min), and supernatants dried under a gentle nitrogen stream (45 °C). Dried residues were reconstituted in 90% aq. MeOH (1 mL g−1 original weight) and partitioned twice using n-hexane (2 mL g−1 original weight) to remove non-polar lipids. The aq. MeOH phase was dried under nitrogen, and the resultant residue was partitioned with CHCl3: H2O (50:50, v:v). The CHCl3 layer containing CTX was collected, then water phase partitioned again with the same volume of CHCl3. Pooled CHCl3 fractions were dried, reconstituted in 500 µL CHCl3, and further cleaned by silica SPE. Powderized fish tissue (CTX and control) was extracted in a similar manner using a 5:1 solvent (mL) to tissue (g) weight ratio. Non-muscular tissues were extracted whole due to their small size and solvent ratios adjusted accordingly. Final tissue extracts were dissolved in 1 mL 100% MeOH and stored at −20 °C until analysis.
4.3. Toxin Analysis
4.3.1. Maintenance of Neuroblastoma Cells
Cells were maintained in vented 175 cm2 sterile culture flasks with RPMI medium supplemented with 5% heat-inactivated FBS, 1 mM sodium pyruvate, and 2 mM L-glutamine (complete media) and maintained in a humidified water jacketed incubator at 37 °C with 5% CO2: 95% atmospheric air. Cells were passaged every 48 h and maintained in exponential growth. During passaging and in preparation for seeding 96-well plates, cells were harvested with 0.025% trypsin-EDTA for <2 min, trypsin deactivated with 10% FBS-RPMI complete medium, centrifuged, supernatant discarded and cells washed two times in PBS. Duplicate aliquots of cells resuspended in 5% FBS-RPMI medium were enumerated on a hemocytometer to calculate growth rates, and passage % viability via trypan blue staining.
4.3.2. Neuroblastoma MTT Assay (N2a-MTT)
Sample extracts were tested for composite voltage-gated sodium channel (Na
V) response using a standardized in vitro mouse neuroblastoma assay (N2a-MTT) as previously described [
50] with toxin quantification of serially diluted samples evaluated and compared to a 9-point 2-fold serial dilution of CTX3C (initial dose equal to 20 pg well
−1 or 86.96 pg mL
−1). The European Food Safety Authority (EFSA) has developed guidelines outlining toxic equivalency factors for the various CTX-group toxins [
52]. CTX3C has been reported to be two-fold more toxic than C-CTX-1 based on intraperitoneal toxicity and was accepted here as the better certified reference standard compared to P-CTX1 which was reportedly ten-fold more toxic than C-CTX-1. The difference in CTX3C standard toxicity could cause lower estimates of C-CTX-1 content. For better understanding of toxin content, conversion from CTX3C eq. to C-CTX-1 can be done by multiplying CTX3C eq. concentrations by a factor of 2.
Due to the sample size of subsampled fish tissues, we were unable to perform additional LC-MS/MS analysis which requires much higher CTX concentrations compared to N2a-MTT. These analyses could have been accomplished by pooling tissues and/or extracts from replicate fish for each sampling point as described by others [
20] but this would have lost the experimental replication that we deemed critical to the validity of this study, so was delayed to a future study with larger fish specimens.
N2a cells from established OV adapted lines, were seeded into 96-well plates at a density of 3 × 105 cells per well, in complete RPMI media (200 µL). After 20 h, cells were dosed in triplicate with standards, controls, and sample extracts; all with and without OV. To prepare fish extracts for dosing, an extract aliquot dissolved in MeOH was transferred to a 1.5 mL microcentrifuge tube, dried under ultrapure N2(g), and redissolved in 5% FBS-RPMI complete media by vortex (30 s, room temperature). Cells in assay wells were carefully inspected by light microscopy prior to dosing and development. Positive controls (CTX-positive reference material), negative controls (containing PBS and medium), and assay controls with (sensitized) and without (non-sensitized) O/V (final well concentration: 0.22 mM ouabain/0.022 mM veratrine) were used to ensure quality assurance and control throughout the several hundred assays performed during this study. Sample wells were dosed with 10 µL of the fish extract solubilized in culture media or PBS (final well volume 230 µL). After a 20 h incubation, well contents were removed and MTT (1 mg/mL) diluted in 5% FBS-RPMI-1640 complete medium was added for 30 min. The resultant insoluble formazan product produced by mitochondrial activity of remaining live cells was solubilized in 100% DMSO (100 µL) with the colorimetric change measured within 5 min on a spectrophotometric microplate reader (µQuant; Biotek Instruments; Winooski, VT, USA) at 570 nm. Cells sensitized with O/V were used to assess CTX-like activity, while non-O/V-sensitized cells were used to monitor non-specific activity induced by sample extracts. When O/V-dependent toxicity was detected by at least a 20% difference between controls and wells dosed with fish extracts, a two-fold serial dilution of extract was prepared and assayed parallel to a CTX3C standard dilution series on the same day.
4.3.3. N2a-MTT Data Analysis
Raw data were analyzed using Microsoft Excel version 2013 (Microsoft Corporation, Redmond, WA, USA). Normalized data were analyzed with GraphPad Prism version 9.0.0 (GraphPad Software, San Diego, CA, USA).
Raw absorbance values for wells dosed with standards and fish extracts were normalized to the OV control wells (20 µL O/V + 10 µL PBS, or 30 µL PBS), to account for minor OV N2a mortality established during cell line adaptation as described by others [
53]. Triplicate absorbance responses within a single assay plate were deemed acceptable when the relative standard deviation was below 20%. To produce standard curves for quantification on the day of each assay, CTX3C standard doses (x-values) were logarithm transformed and fit by non-linear regression against the normalized response (y-values) in wells at each dose using a four-parameter logistic equation with variable slope (Y = Bottom + (Top/Bottom)/(1 + 10^((LogIC
50 − X) ∗ HillSlope). Toxicity of tissue extracts were estimated by interpolating the normalized responses (y-value) in wells onto the standard curve to estimate the dose (unknown x-value) in pg CTX3C equivalence. Values that fell between the effect concentration 20–80 (EC
20 to EC
80) which is the linear portion of the standard curve are acceptable, but we chose to use more strict parameters for quantification by accepting only values between EC
30 and EC
75 because full curves with a top and bottom plateau were not always possible based on the CTX concentrations in tissues. Interpolated results (pg CTX3C eq.) were divided by the tissue equivalence (mg TE) of the dose to calculate the concentration in ng CTX3C eq. g
−1 TE. The LOQ for CTX in each tissue type was determined by dividing the mean value of the EC
75 from the CTX3C dose–response curve and the maximum TE dosed in wells without a matrix induced effect defined as either growth enhancement >20% of the control wells or cell death associated with non-Na
v mechanisms (evaluated in sample wells without OV). All samples were analyzed in triplicate across 2–4 independent assays performed on separate days with a CTX3C standard curve prepared on the day of each assay.
4.4. Ciguatoxin Kinetics
4.4.1. Muscle, Liver, and Viscera Ciguatoxin Kinetics
Kinetics of CTX uptake and depuration in separate tissues were investigated through non-linear regression analysis of the experimentally determined CTX concentration in sample extracts against time for both phases using GraphPad Prism version 9.0.0 (model comparison function). When CTX was below detection levels due to a non-specific matrix effect, those replicates were excluded from analyses (see Results
Section 2.5). Normality of toxin data was evaluated using a Shapiro–Wilk test and models were fit using a least squares regression with no weighting and compared by an iterative process. Best fit models were compared based on the Akaike’s Information Criterion (AICc) which balances the goodness of fit using sum-of-squares and the simplicity (number of degrees of freedom) of the two models.
4.4.2. One-Compartment Model Kinetics
The combined CTX concentrations (Cfish) for the measured tissues were calculated for each fish by multiplying the quantified concentration for each tissue (muscle, liver, and viscera) by the whole tissue mass (conc. x mass = tissue burden), summing the tissue CTX burdens, and dividing by the total sum of the whole muscle, liver, and viscera mass. The Cfish was used to investigate a one-compartment model of CTX kinetics which assumes a homogenous concentration in the fish. While our analyses do not account for CTX in some compartments (kidney, stomach, and carcass), the included tissues (muscle, liver, viscera) contained the majority portion of CTX in the major tissue compartments in the fish. No CTX activity was detectable in brain or gonads, so these were excluded.
The uptake rate was calculated by linear regression of the measured
Cfish (ng g
−1) during the bioaccumulation phase against time (d = 0 to 20)
where the slope of regression is equal to the rate of increasing CTX concentration (
kuptake; ng g
−1 day
−1),
t is time (d), and
a is a constant which in this case is the y-intercept (ng g
−1).
The first-order depuration rate constant (
k2) was calculated using the methods outlined by Brooke and Crookes [
45]. A one-compartment model of exponential decay was fitted to the measured
Cfish during the depuration phase
where
Cfish(t) is the concentration (ng g
−1) measured at the time of sampling,
Cfish(i) is the initial concentration (ng g
−1) at the start of the depuration phase (day 20),
t is time (d), and
k2 is the overall depuration rate constant (day
−1). For curve fitting, the measured
Cfish in the depuration phase were natural Log transformed, i.e., Ln [
Cfish], to allow linear regression of Log-concentrations versus time in which the slope of regression is the
k2, and the coefficient of determination is used to confirm first-order kinetics. The
k2 calculated by these methods is the overall elimination rate constant including the sum of four first-order kinetic processes
where
kr,
km, and
ke (all in units day
−1) are the rate constant for elimination via respiration, metabolic transformation, and feces, respectively, and
kg is the rate constant for the change in concentration due to fish growth, a pseudo-elimination process. For an in-depth summary on each of the rate constants and application to overall
k2, see Gobas [
54] (pp. 4–9).
To correct the overall
k2 for growth of the fish,
kg was calculated using the growth rate data collected for each fish. Fish masses were applied to an exponential growth model
where
Wfish(t) is the wet weight (g) of the fish at any point,
Wfish(i) is the initial wet weight at the start of the experiment, and
kg is the growth rate constant (day
−1). To allow a linear fit, the inverse of measured weights was natural Log transformed, i.e., Ln (1/
Wfish), and plotted against time where the slope is equal to
kg (day
−1). The effect of growth, which results in diluted concentrations over time, was factored out of the depuration rate constant by subtracting the
kg from the overall
k2 to give the growth-corrected depuration rate constant (
k2 growth-corrected; day
−1)
and describes the rate constant for elimination processes that result in removal of CTX from the fish (
kr +
km +
ke) [
45].
4.4.3. Kinetic Modeling and Correction of Growth Dilution
The exponential growth Equation (4) was used to estimate the mass of each fish on day 20 (end of bioaccumulation phase) that entered the depuration phase using measured growth rate constants (kg) and solving for Wf(t). For trials 1 and 2, respectively, values of t were set to 20 and 22 days, because initial weights were collected on day 0 (trial 1) and two days prior to start (trial 2), while depuration was initiated on day 20 for both trials. The sum of muscle, liver, and viscera mass was estimated at day 20 using a linear correlation between whole fish mass (g) and the sum of tissue masses, i.e., Σ (whole muscle + liver + viscera; g), that were dissected from sampled fish for CTX analysis. The combined CTX burden in the whole muscle, liver, and viscera at day 20 was estimated from the linear relationship between the cumulative dose administered (total ng CTX3C eq. consumed = number of pellets fed daily × number of days in bioaccumulation phase × pellet CTX3C eq. conc.) and the combined CTX burden measured in fish that were sampled between 0 and 20 d. Estimates of total CTX burden and combined tissue mass at the end of the bioaccumulation phase (day 20) were divided for an estimate of an overall CTX concentration in the fish at the start of depuration (Cfish(i)).
A simulation model of our data was created using the estimated Cfish(i) to study the effect of fish growth on elimination of CTX. Final concentrations were simulated for each fish in the depuration phase at all time points using the one-phase exponential decay model (Equation (2)) where Cfish(i) is an estimate on day 20, time is delta time in depuration depending on when the fish was sampled, and overall k2 was measured from the linear plot depuration data. The resulting simulated concentrations were natural Log transformed (as previously described for the measured Cfish) and plotted against time (d) to compare the regression slope (k2) with the slope from the measured data. A correlation was then performed to check how well the simulated data fit the measured concentrations.
Simulated concentration data served as the baseline for growth-corrected models to compare the change in half-life (Ln [
2]/
k) under different conditions of
k2 growth-corrected. The growth-corrected models were produced by adjusting the depuration rate constant (
k2) in the simulation exponential decay model. Three growth-corrected models were produced using different values of
k2 growth-corrected that were calculated based on fish growth rates in the depuration phase, i.e., average growth rates at each sampled time point (Model 1), average growth rate based on ANOVA grouping (Model 2), and the individually measured growth rate (Model 3). Results of each model compared to the simulation are presented using the linear version of the exponential decay model where concentrations are natural Log transformed and plotted against time. The growth-corrected half-lives of the models were finally compared to the half-life calculated from growth correction by a simpler approach reported by others [
20,
55] that uses a growth correction factor, where measured concentrations in fish during the depuration phase are multiplied by (1 +
kg × time).
4.5. Statistical Analyses
Fish size (mass in g) at the initial time point across studies was analyzed using a non-parametric two-tailed Mann–Whitney U test since trial 1 weights were not normally distributed. Growth rate constants (kg) of control and treatment groups collected at the same time point were tested for normality using a Shapiro–Wilk test and compared for differences using a two-tailed t-test, respectively. Growth rate and toxicity data between control and exposure treatments were tested for normality and homoscedasticity using a Shapiro–Wilk test and Brown-Forsythe test, respectively, and residual plots were visually inspected as a parallel method prior to one-way ANOVA with Tukey’s multiple comparisons test. Growth trends were investigated by a linear regression plot of kg against time. A simple correlation was used to analyze the fit of the measured and model simulated Cfish(t) in the depuration phase prior to investigating growth correction of the model.