Investigating the role of ionoregulatory processes in the species- and

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Investigating the role of ionoregulatory processes in the species- and life-stage-specific differences in sensitivity of rainbow trout and white sturgeon to cadmium kamran shekh, Song Tang, Markus Hecker, and Som Niyogi Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b04828 • Publication Date (Web): 16 Oct 2018 Downloaded from http://pubs.acs.org on October 17, 2018

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Environmental Science & Technology

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Investigating the role of ionoregulatory processes in the species- and

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life-stage-specific differences in sensitivity of rainbow trout and

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white sturgeon to cadmium

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Kamran Shekh1*, Song Tang1,2†, Markus Hecker1, 2*, Som Niyogi1,3

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1

Toxicology Centre, University of Saskatchewan, Saskatoon, SK, S7N 5B3, Canada

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2

School of Environment and Sustainability, University of Saskatchewan, Saskatoon, SK, S7N

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5C8, Canada

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3

Department of Biology, University of Saskatchewan, Saskatoon, SK, S7N 5E2, Canada

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* Correspondence to: 44 Campus Drive, Toxicology Centre, University of Saskatchewan,

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Saskatoon, SK S7N 5B3, Canada. Tel: +1-306-966-5233; Fax: +1-306-966-4796; Email: Kamran

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Shekh ([email protected]) or Markus Hecker ([email protected]).

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†

Current affiliation: National Institute of Environmental Health, Chinese Center for Disease Control and Prevention, Beijing, 100021, China

ACS Paragon Plus Environment


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Abstract

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There are huge variations in life-stage- and species-specific sensitivities among the fishes to the

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exposure with metals; however, the physiological mechanisms underlying these differences are

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not well understood to date. This study revealed significant life-stage (larval, swim up and

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juvenile) and species-specific differences between two evolutionary distant species of fishes,

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rainbow trout (Oncorhynchus mykiss) and white sturgeon (Acipenser transmontanus) following

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acute exposures to Cd. Although the 96h LC50 of Cd was similar in both species at the larval stage,

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trout demonstrated an increased sensitivity to Cd at later life-stages as compared to sturgeon.

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Moreover, exposure to Cd disrupted calcium (Ca) uptake and whole body Ca levels in trout by a

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greater degree relative to that in sturgeon regardless of life-stage. Finally, white sturgeon

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demonstrated a lower affinity for Cd uptake relative to the more sensitive rainbow trout. This infers

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a differential nature of the interaction between Cd and Ca transport pathways in the two species

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and partially explains the differences in Cd sensitivity between rainbow trout and white sturgeon

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described previously. Overall, our results suggest that species- and life-stage-specific differences

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in sensitivity to waterborne Cd in fish are likely a function of the interplay between Cd uptake and

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Cd-induced disruption of Ca homeostasis.

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Introduction

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Fishes are the most diverse group of vertebrates. In North America alone, approximately 1,200

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freshwater fish species are currently known, which only represent 8.9% of the diversity of

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freshwater fishes on our planet.1 This diversity is reflected by huge differences in their sensitivity

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to environmental pollutants, including metals. The high diversity among fish species makes it

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logistically challenging to test every combination of toxicant and fish in order to estimate

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differences in sensitivity. Therefore, alternative approaches for more efficiently estimating

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differences in sensitivity among fishes to toxic chemicals are needed. Evaluating the physiological

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and biochemical traits in a species of unknown sensitivity to a chemical and then linking its

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phylogenetic relationship to other species of known sensitivity could be a useful predictive

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approach for sensitivity estimation.2 Hence, studies addressing the physiological and/or molecular

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basis of the species-specific differences in the sensitivity to contaminant exposure can be helpful

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in designing predictive models for evaluating differences in sensitivity to metals among fishes.

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However, such studies are scarce to date.

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Different species demonstrate significant life-stage-specific differences in the sensitivity

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to metals such as cadmium (Cd).3–6 Hence, the most sensitive life-stage in one species may not be

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the most sensitive life-stage in another species. These life-stage-specific differences in the

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sensitivity to metals complicate the interspecific comparison of toxicity data and pose a challenge

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for cross-species extrapolation of sensitivities to metals. For example, it has been suggested

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previously that juvenile white sturgeon are more sensitive to Cd than larval rainbow trout, whereas

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larval sturgeon are less sensitive to Cd than juvenile rainbow trout.3 Based on this and similar

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observations in other fish species 6, it appears that comparing the toxicity data of a life-stage in

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one fish species to a different life-stage of another fish species might result in misleading



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conclusions about the most sensitive species for any particular metal. It is generally believed that

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the earlier life-stages of fishes are more sensitive to the exposure with metals than juveniles or

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adults, which has been attributed to the higher surface area to volume ratio and underdeveloped

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detoxification mechanisms in larval fish.7–9 Although these principles explain the differences in

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sensitivity among many fish species, they do not seem to apply universally, as several previous

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studies reported that later early life-stages (late larvae/early juveniles) are more sensitive than

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embryonic or early larval life stages.3–5 The reasons for these conflicting data remain largely

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unexplained.

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Overall, there is a lack of comprehensive understanding of the mechanistic basis of the

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species and life-stage-specific differences in metal sensitivity in fishes. Understanding these

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differences in sensitivity and underlying mechanisms are of critical importance for making

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informed regulatory decisions and to enable the conduct of objective ecological risk assessments,

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since it helps in the identification and selection of the most sensitive life-stages and species for

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further testing.

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Cadmium (Cd) is a ubiquitous metal of environmental concern because of its presence at

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elevated concentrations in many ecosystems worldwide and its low threshold to cause adverse

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effects in aquatic organisms.10 The primary mechanism of its acute toxicity is the disruption of ion

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homeostasis, particularly Ca regulation.10,11 Several recent studies also proposed other

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mechanisms leading to toxicity of Cd such as oxidative stress, cell death and immunotoxicity.12–14

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Although information on the mechanisms of species- and life-stage-specific differences in the

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sensitivity of fishes to metals are scarce, some recent studies indicated that the molecular pathways

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driving metal and ion homeostasis and processes such as metal uptake, ionoregulatory disruption,


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metallothionein induction and oxidative stress response are likely among the most important

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contributing factors.5,11,15–18

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The aim of this study was to investigate the mechanistic basis of the species- and life-stage-

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specific differences in the sensitivity of two evolutionarily distinct fish species, rainbow trout

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(Oncorhynchus mykiss) and white sturgeon (Acipenser transmontanus), to Cd. These two species

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were selected because white sturgeon and rainbow trout have well-demonstrated significant

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species and life-stage-specific differences in the sensitivity to Cd.3–5 In addition, white sturgeon is

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an endangered species19 and information regarding its sensitivity to Cd in comparison to rainbow

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trout (a species most commonly used in environmental regulatory testing in North America) has

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important implications for the conservation of this species. We performed a comparative analysis

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of Cd uptake across different life-stages of rainbow trout and white sturgeon (larval, swim up,

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early juvenile) during acute exposure to waterborne Cd. Furthermore, the effect of different Cd

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concentrations on waterborne Ca uptake and whole-body Ca levels were compared across life-

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stages and species. Finally, life-stage- and species-specific responses of these physiological

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parameters were analyzed in relation to conventional acute toxicity endpoints for Cd (96h LC50).

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Materials and methods

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Experimental organisms, test chemicals and exposure media

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Embryos of white sturgeon and rainbow trout were obtained from the Nechako White Sturgeon

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Conservation Centre (BC, Canada) and Troutlodge (WA, USA), respectively, and were reared in

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the Aquatic Toxicology Research Facility at the University of Saskatchewan in a flow-through

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system maintained at approximately 13°C. Sturgeon were fed a diet of commercial frozen

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bloodworms (San Francisco Bay Brand, Newark, CA, USA) and rainbow trout were fed



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commercial trout food (Bio Vita Starter #0 Crumble, Bio-Oregon, British Columbia, Canada).

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Food was first introduced at 10 - 12 days post hatch (dph). Proper permits were obtained before

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research commenced (Fisheries and Oceans Canada SARA Permit XRSF 20 2013/SARA 305),

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and all procedures involving animals were approved by the University of Saskatchewan’s

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University Council on Animal Care and Supply (Protocol 20140079).

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Cadmium chloride hemi-pentahydrate (CAS 7790-78-5; purity 99.999%) was obtained

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from Sigma-Aldrich (St. Louis, MO, USA). All exposure waters were prepared by mixing reverse

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osmosis water and dechlorinated city of Saskatoon municipal tap water in the ratio of 3:1 in order

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to bring the hardness and alkalinity to a moderate level (~60 and 40 mg/L as CaCO3, respectively).

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Stock solution of Cd was prepared in this water and stored at 13°C for at least 24 hours before

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exposure.

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Acute Cd Toxicity Assessment

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Acute toxicity of Cd to early life stages of white sturgeon and rainbow trout was assessed by

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exposing larval (5dph), swim-up (15 dph), early juvenile (45 dph) and juvenile (75 dph) fish to

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increasing concentrations of Cd for 96h. Exposures were conducted in triplicate using high density

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polyethylene (HDPE) tanks of varying capacity based on fish size at different life-stages (5 or 25

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L) with 10 individuals per replicate tank (n = 3 with 10 fish per replicate). Fish were acclimated

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in normal exposure water (no added Cd) for 5 days prior to the initiation of Cd exposures for acute

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toxicity assays as well as for all other experimental exposures described below. No food was given

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during the exposure. Exposures were conducted at a temperature of 13±1°C under static renewal

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conditions and 16:8 h light/dark cycle. Half of the exposure media was replaced every day and fish

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were observed for mortality every 12 hours. Trout were exposed to 0, 0.625, 1.25, 2.5, 5, 10 and

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20 μg/L (0, 5.6, 11.1, 22.2, 44.5, 89, 177.9 nM) and sturgeon were exposed to 0, 5, 10, 20, 40, 80


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and 160 μg/L (0, 44.5, 89, 177.9, 355.8, 711.7, 1423.5 nM) of nominal Cd exposure concentrations

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(160 μg/L concentration was excluded in 75 dph). Water samples were collected at the beginning

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and the end of the exposure, filtered, acidified with nitric acid and stored at -20ºC until the analysis

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of Cd concentrations as described later.

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Effect of Cd on whole body Ca

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The effects of Cd on whole body Ca levels were estimated after 24 h exposure to Cd in the above

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listed life-stages of both fish species. For each of the life-stages, six fish (n=6) were exposed

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individually to 0, 1.25 and 5 μg/L (11.1 and 44.5 nM) of Cd concentrations in 500 mL HDPE cups.

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Half of the exposure media was replaced after 12 hours. The volume of exposure media in each

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cup was 250 mL (for 5 and 15 dph fish) or 500 mL (for 45 and 75 dph fish). Other experimental

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conditions such as water chemistry and temperature were the same as described for the acute

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toxicity assessment. At the termination of exposure, each fish was euthanised with Aquacalm

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(Metomidate hydrochloride) (Syndel Laboratories Ltd., BC, Canada), washed twice in deionised

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water, blotted dry, weighed and stored at -80°C until further analysis. For whole body Ca analysis,

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each fish was digested for 48 h in 5 volumes of 2N trace metal grade Nitric acid (EMD Millipore,

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Billerica, MA, USA) at 60 ºC. After digestion, samples were centrifuged at 15,000g and whole

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body Ca was measured in the supernatants using flame atomic absorption spectroscopy (AAnalyst

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800, Perkin Elmer, USA) after appropriate dilution in 1% nitric acid. One water sample was

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collected at random from one of the replicates in each exposure group at the end of the exposure,

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filtered through a 0.45 μm polycarbonate filter, acidified with trace metal grade nitric acid and

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stored at -20°C until the analysis of Cd concentrations, as described later.

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Whole body Cd uptake and the effect of Cd on whole body Ca uptake



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Short-term (3h) concentration-dependent whole body Cd uptake at the above described life-stages

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of each fish species was determined based on the methods described elsewhere.11 For each life-

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stage of either species, 42 HDPE cups of 120 or 500 mL capacity (7 sets of 6 cups, each set

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representing one Cd concentration tested at 6 replicates) were filled with 100 or 400 mL of the

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same water as described in acute toxicity assessment. Each set of cups was spiked with cadmium

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chloride hemi-pentahydrate solution to achieve approximate nominal Cd concentrations of 0.625,

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1.25, 2.5, 5.0, 10, 20, and 40 µg/L (5.6, 11.1, 22.2, 44.5, 89, 177.9, 355.8 nM) for rainbow trout.

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For white sturgeon, the nominal Cd exposure concentrations were 5, 10, 20, 40, 80 and 160 µg/L

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(89, 177.9, 355.8, 711.7, 1423.5 nM). Subsequently, each set of exposure cups was spiked with 3

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µCi/L

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were added to each cup (n = 6) and exposed to radiolabeled Cd for 3 h. Due to small size of 5 dph

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and 15 dph fish, 5-7 larvae per replicate were exposed together and pooled as individual replicate.

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At the end of exposure, fish were euthanised by an overdose of Aquacalm, rinsed separately

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in deionized water (Nanopure, Sybron Barnstead, Boston, USA) for 30 seconds, blotted dry and

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placed in polyethylene vials. Water samples (5 mL) were collected in duplicate at the beginning

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and end of the 3h exposure period and acidified with 50 µL concentrated trace metal grade nitric

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acid. Fish (whole body) and one set of water samples were analysed for radioactivity counts (CPM)

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using a Wallac Wizard 1480 automatic gamma counter (Perkin-Elmer, Shelton, Connecticut,

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USA). The other set of water was analysed for total Cd by graphite furnace atomic absorption

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spectroscopy as described later. The inward whole body influx (Jin) for Cd was calculated by an

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equation described previously (Eq. 1): 20

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Cd (as CdCl2, specific activity=3.45 mCi mg/L, Perkin-Elmer, USA). Individual fish

Jin = CPMwb / (SAw*t)


(1)

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Where CPMwb is the Cd109 radioactivity counts in whole body samples (CPM/g wet weight whole

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body), SAw is the mean specific activity of Cd109 in water (CPM L-1 water/total Cd in water) and t

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is the time of exposure.

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A similar set-up was used to analyse the life-stage- and species-specific differences in the effect

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of Cd on whole body Ca uptake. For each life-stage, three sets of cups were spiked with cadmium

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chloride to achieve nominal Cd concentrations of 0, 1.25, and 40 µg/L, and then spiked with 7

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µCi/L 45Ca (as CaCl2, specific activity=3.45 mCi mg/L, Perkin-Elmer, USA). Six cups were used

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for each Cd exposure concentration and individual fish were added to each cup (n = 6) and exposed

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for 3 h. After the exposure period, fish were euthanized, rinsed with deionized water and stored at

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-20ºC. Later, fish were digested in 5 times volume of 2N HNO3 at 600C for 48h and then

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centrifuged at 15,000g for 5 minutes. Supernatant was collected and diluted in the ratio of 1:5 with

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scintillation cocktail (Ultima Gold, PerkinElmer, ON, Canada) and counted for radioactivity using

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a Tricarb 2810TR beta scintillation counter (Perkin-Elmer, ON, Canada). One set of water samples

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was analysed for Ca45 radioactivity using aqueous scintillation fluor (500 µL water diluted 10x in

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scintillation fluor) (Optiphase, PerkinElmer, ON, Canada), and the other set of water samples was

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analyzed for total Ca content in water using flame atomic absorption spectroscopy. The inward

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whole body influx (Jin) for Ca was calculated as described above for whole body Cd influx (Eq. 1).

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Both, fish and water samples were kept overnight after adding scintillation fluid to reduce

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chemiluminescence. Quenching of Ca45 was taken into account by utilizing external standards ratio

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method, as described previously21,22. The quench curve was generated by adding known amounts

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of Ca45 to whole-body tissue samples in the same counting cocktail.

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Water Chemistry



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The same exposure media was used across all studies. Detailed water chemistry (Cl, DOC, Ca,

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Mg, K, Na, SO4) was analysed for water samples collected at the beginning and end of the project

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by the analytical laboratory of the Saskatchewan Research Council, Saskatoon, SK, Canada.

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Parameters such as hardness, alkalinity, pH, dissolved oxygen (DO), ammonia and temperature

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were measured at the beginning of the exposure and every 24 h thereafter. Hardness, ammonia and

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alkalinity were determined by using Nutrafin Test kits (Hagen, Canada) and pH, temperature and

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DO were measured by using YSI Quatro Multi-Parameter probe (Yellow Springs, OH, USA).

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Dissolved Cd concentrations were measured in the water samples collected as described in

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respective method sections using graphite furnace atomic absorbance spectroscopy (AAnalyst 800,

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Perkin Elmer, USA). The detection limit of the atomic absorption method was 0.005 µg/L. Quality

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control and quality assurance of Cd measurement was maintained by using appropriate method

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blanks and a certified Cd standard (Perkin Elmer, USA). In addition, a certified reference material

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(SLRS-6; National Research Council, Canada) was also analysed to validate the efficiency of the

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applied method, which yielded a 96% recovery rate of Cd.

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Statistical analysis

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The LC50s and concentration dependent Cd uptake parameters (affinity (Km) and maximum uptake

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rate (Jmax)) were calculated by using the two-parameter log-logistic function (LL.2) and Michaelis-

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Menten model (MM.2) functions, respectively, in the ‘drc’ package of R software version

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3.1.2.23,24 In cases where 50% mortality was not achieved at the greatest concentration tested, the

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LC50 was reported as being greater than the greatest concentration tested. Concentration dependent

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whole-body Cd uptake at different waterborne Cd concentration were calculated on the basis of

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freely available waterborne Cd (as Cd2+), which was estimated using Visual MINTEQ (version 3,

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Royal Institute of Technology, Stockholm, Sweden). Uptake parameters Km and Jmax among life-


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stages and species were analysed using 2-way ANOVA (life-stage x species). The effect of Cd on

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whole body Ca uptake rate and whole body Ca levels were analysed by using 3-way ANOVA,

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where the three independent variables were life-stage, species and exposure concentration, and the

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dependent variable was either whole body Ca level or whole body Ca uptake rate. All dependent

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variables were tested for normality and homogeneity of variance using the Shapiro-Wilk test and

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Bartlett’s test, respectively. Whenever assumptions for either normality or homogeneity of

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variance were not met, data was square-root transformed and tested. Whenever a statistical

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significance in ANOVA results was observed, a post-hoc Tukey’s multiple comparison test was

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applied. A p-value of 80 μg/L at 5 dph, 15 dph and 75 dph, respectively (Figure 1). White sturgeon demonstrated

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lesser sensitivity to Cd as compared to rainbow trout at all life-stages. The difference in sensitivity

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was greatest at 15 dph and 75 dph (35.5 and >27.3 times, respectively) as compared to 5 dph and

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45 dph (1.35 and 6.92 times, respectively) (Figure 1).

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The life-stage and species specific differences in the sensitivity of rainbow trout and white

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sturgeon to Cd observed in this study followed similar patterns noted in other studies.3–5 It has

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been suggested in these previous studies that sensitivity to metals such as Cd can change

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considerably in fishes during development over a very short duration (e.g. from 5 to 15 dph).

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Therefore, testing multiple early life-stages could be a better approach to estimate the true

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sensitivity of the early life-stage of a fish species. Together, these studies also suggested that in

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rainbow trout and white sturgeon, the earliest life-stage(s) are less sensitive than later life-stages,

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as opposed to observations in several other aquatic species with a variety of toxicants including

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metals.9 Moreover, in this study, rainbow trout were found to be several-fold more sensitive to Cd

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as compared to sturgeon during later life-stages. Comparative evaluation in previous studies also

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demonstrated that rainbow trout and several other salmonids are significantly more sensitive to

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waterborne Cd than other model fish species.25

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Species and Life-stage specific differences in the effects of Cd on whole body Ca

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The effect of Cd on whole body Ca levels across different life-stages of rainbow trout followed a

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similar pattern as observed with the acute toxicity of Cd, indicating a possible link between these

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two parameters. Rainbow trout exposed at 15 dph, 45 dph, and 75 dph to 1.25 and 5.0 μg/L Cd

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showed significant reduction in whole body Ca levels (44.3 – 61.3% reduction compared to

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controls) (Figure 2, Table S1). Although exposure to the same concentrations of Cd at 5 dph


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resulted in slight decrease in whole body Ca levels (1.5% and 9.5%), the effects were not

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statistically significant (Figure 2).

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Loss of Ca from the body (disruption of Ca homeostasis) is considered to be the major

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lethal mechanism of Cd toxicity in fish.10 While most studies investigating the effect of Cd on Ca

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levels in fishes have been conducted on plasma26, whole body Ca levels have also been

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successfully used for studying the toxic effects of metals such as Cd in organisms such as fish,

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midge larvae and sea urchin.27–29 The baseline (control) whole body calcium level in larval (5 dph)

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trout was lowest among all life stages and no significant effect of Cd on Ca levels occurred at this

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life-stage (9.58 µmol/g tissue, P = 0.783) (Figure 2). As the baseline Ca levels increased in later

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life-stages, the effect of Cd and its toxicity also became more prominent. A lower baseline of whole

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body Ca content in larval life-stages might indicate a lower requirement for Ca absorption from

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the external environment, which could result in a lesser whole body Ca uptake rate, and thus, lower

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ability for Cd to produce toxic effects assuming that Cd-induced loss of Ca is the main driver of

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Cd toxicity. The greater need for Ca during later life-stages of fish is expected due to skeletal

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growth. A number of studies with rainbow trout and other teleost species have also demonstrated

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a comparable trend in whole body baseline Ca concentrations with increasing age in growing

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fry.30,31 The greater need for Ca in later life-stages can potentially make them more susceptible to

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Cd toxicity. In white sturgeon, the baseline Ca levels increased gradually from 7.85 – 39.7 µmol/g

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tissue between 5 dph to 75 dph life-stage (Figure 2). However, in contrast to rainbow trout, Cd did

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not show any significant effects on whole body Ca levels regardless of the life-stages. Based on

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these results, it can be suggested that the whole body Ca levels are an indicator of life-stage-

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specific sensitivity to Cd in rainbow trout but not in white sturgeon.



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Across all life-stages, baseline whole body Ca levels were lower in white sturgeon as

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compared to rainbow trout (Figure 2). These findings are in agreement with previous observations

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of lower plasma and whole body Ca levels in sturgeon species relative to other fish species.32,33

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The lower Ca levels of sturgeons compared to other fish species has been suggested to be due to

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their cartilaginous skeleton.32 Higher baseline whole body Ca levels (reported here) and uptake

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rates (next section) in early life-stage rainbow trout suggest a higher demand of exogenous Ca and

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a resultant higher vulnerability to Cd in this species relative to early life-stages of white sturgeon.

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White sturgeon showed no significant reduction in whole body Ca levels in presence of Cd

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(1.25 and 5 µg/L), which might explain the lesser sensitivity of white sturgeon than rainbow trout.

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Considering the significantly lower toxicity (high LC50) of Cd in sturgeon than trout, 5 µg/L of Cd

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exposure concentration might not have been high enough to produce Ca disbalance in sturgeon.

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Nonetheless, significant decrease in Ca levels induced by the exposure of 5 µg/L Cd in the more

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sensitive species used in this study and the lack of similar effect in the less sensitive species at

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identical Cd exposure level further indicate the linkage between the disruption of Ca balance in

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the body and species-specific differences in Cd sensitivity. It is to be noted here that exposure to

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a higher Cd concentration of 40 µg/L resulted in a reduction of Ca uptake in white sturgeon (see

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below). Although we did not measure the effect of 40 µg/L Cd on the whole body Ca levels in

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white sturgeon, the decrease in the Ca uptake rate indicates that it might have resulted in the

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depletion of whole body Ca as well.

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Effect of Cd on whole body Ca uptake.

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Application of three-way ANOVA on the effect of Cd on whole body Ca uptake rate revealed a

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significant interaction between Cd exposure and species as well as Cd exposure and life-stage,


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which indicates that Cd exposure has species- and life-stage-specific effects on Ca uptake. Post-

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hoc analysis demonstrated that exposure to 1.25 µg/L Cd caused a significant reduction in Ca

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uptake at 15 and 45 dph rainbow trout but not at 5 dph (Figure 3). A higher Cd exposure

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concentration (40 µg/L) caused a significant reduction in Ca uptake in all life-stages of rainbow

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trout, however the magnitude of the effect was lower at 5dph (46.9% reduction) relative to that at

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15 and 45 dph (63.2 – 82.9% reduction) (Figure 3 and Table S2). Similarly, in white sturgeon,

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more sensitive life-stages, 5 dph and 45 dph, exhibited a higher magnitude of effect of Cd on Ca

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uptake (67.8% and 69.2% reduction, respectively) relative to that in the less sensitive life-stage,

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15 dph (no significant reduction) at 40 µg/L Cd exposure (Table S2). Between the species,

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exposure to the same Cd concentration (40 µg/L) led to a greater average reduction in Ca uptake

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in the more sensitive rainbow trout relative to white sturgeon at 15 and 45 dph (Figure 3 and Table

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S2). In other words, the effect of Cd on whole body Ca uptake was more prominent in those life-

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stages and species that were more sensitive to Cd which suggested a possible link between the

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species or life-stage-specific sensitivity to Cd and Cd-induced inhibition of Ca uptake.

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Overall, these observations indicate that quantitative differences in the capacity to affect

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waterborne Ca uptake might be a potential contributor in rendering species and life-stage specific

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differences in the sensitivity to waterborne Cd. Observations in this study are in agreement with

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some of the findings of previous studies. Studies with other fish species demonstrated that Ca

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uptake varies with species as well as life-stages.11,30,34 Baseline Ca uptake rate in 15 and 45 dph

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rainbow trout in our study was in a similar range as previously reported in juvenile rainbow trout.11

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For white sturgeon, the uptake rate of Ca has not been reported in any previous study. Moreover,

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it has also been reported previously that Cd elicits differential effects on Ca uptake at different

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life-stages in the same fish species.34 Similar to these previous findings, we also demonstrated here



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that the inhibitory effect of Cd on Ca uptake was variable between different species and among

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life-stages.

334

It is also notable that the effect of Cd on Ca uptake seemed to be dependent on baseline Ca

335

uptake rate. For example, 15 dph rainbow trout demonstrated a significantly higher baseline Ca

336

uptake rate than white sturgeon, and the effect of Cd on Ca uptake was more pronounced in trout

337

than sturgeon at this life-stage. At 5 dph, the baseline Ca uptake rate was not significantly different

338

between species and the Cd-induced reduction in Ca uptake was also comparatively similar in both

339

species (Table S2 and Figure 3). Hence, our data provided evidence of a relationship between the

340

baseline Ca uptake rate and the toxicity of Cd in both species. A similar relationship between Na

341

uptake rate and Ag toxicity has been suggested in juvenile rainbow trout, juvenile and adult

342

crayfish, and neonate and adult daphnids.35 Turnover of ions such as Ca is governed by their uptake

343

(influx) and efflux. Sensitivity to Cd is expected to be highly dependent on Ca turnover. Therefore,

344

species-specific differences in Ca efflux could be another important factor in determining the

345

differences in sensitivity to Cd. However, Ca efflux was not measured in this study. Further

346

research linking sensitivity to metals, baseline ion uptake and efflux rates and how these processes

347

are influenced by metals using a variety of species and life-stages are required to gain a more

348

comprehensive understanding of the differences in metal sensitivity in fish.

349

Species and Life-stage specific differences in Cd uptake

350

The short-term (3h) rate of Cd uptake in all life-stages exhibited saturation at higher range of Cd

351

exposure concentrations in both species (Figure 4). Michaelis-Menten analysis demonstrated no

352

significant difference in Km across different life-stages in both species (Table 1). The values of Km

353

ranged between 78.94 – 118.9 nM in rainbow trout and 197.3 – 282.8 nM in white sturgeon across

354

three life-stages. In contrast, both species demonstrated a trend of increased Jmax values from 5 dph


Page 16 of 28

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355

to 45 dph (Table 1). The Jmax value of waterborne Cd uptake in 5 dph rainbow trout was 0.04

356

(±0.0055) nmol/g wet wt./h, which increased to 0.12 (±0.0061) nmol/g wet wt./h in 45 dph rainbow

357

trout (p < 0.05). On the other hand, the Jmax value of 5 dph white sturgeon was 0.31 (±0.022)

358

nmol/g wet wt./h, which increased to 0.48 (±0.025) nmol/g wet wt./h in 45 dph white sturgeon (p

359

< 0.05).

360

The life-stage specific whole-body Km values of waterborne Cd uptake in rainbow trout

361

(78.94 – 118.7 nM) were comparable to the Km values (31.3 – 86.7 nM) previously reported in

362

juvenile rainbow trout in exposure water that contained similar Ca levels (100 - 500 µM) as the

363

water used in present study (300 µM).11 We used the negative logarithm of Km to obtain the affinity

364

constant of whole body Cd uptake (Log KCd-wb).36 This conversion was useful in directly

365

comparing the affinity of waterborne Cd uptake observed in this study with the waterborne Cd

366

uptake affinity reported previously in fish gills. The whole-body Km values across different life

367

stages of rainbow trout derived in the present study translate into log Kcd-wb values of 6.9-7.1. This

368

is comparable to the reported affinity constant of gill Cd uptake (log KCd-gill =7.1) in rainbow trout

369

exposed to Cd under similar water chemistry conditions as used in the present study.37 For white

370

sturgeon, log Kcd-wb values were in the range of 6.5 – 6.7 for all life stages.

371

The stable Km values across all life-stages in both species indicated that similar pathways

372

(e.g., transporters/channels) of Cd uptake are likely involved in waterborne Cd uptake at each life-

373

stage. A gradual increase in Jmax values from larval to later life-stages in both species could be

374

attributed to the gradual increase in the expression of epithelial transporters involved in Cd uptake

375

(e.g., epithelial calcium channel (ECaC), and plasma membrane Ca-ATPase) as suggested

376

previously.38 A comparison of species-specific waterborne Cd uptake parameters revealed that

377

generally white sturgeon had a lower affinity (higher Km) of Cd uptake than rainbow trout across



378

all corresponding life-stages. The lower affinity of Cd uptake in white sturgeon might also explain

379

its lower sensitivity to Cd compared to rainbow trout, at least in part. However, as discussed earlier,

380

the affinity of waterborne Cd (Km) did not change significantly across life-stages in both species,

381

indicating that the affinity of waterborne Cd uptake was not a contributing factor for life-stage

382

specific differences in Cd sensitivity in both species. The uptake sites for Cd that showed saturation

383

at higher Cd exposure concentrations are most likely represented by ECaCs and Ca-ATPases,

384

which are known to have a high affinity for Cd, and are thus characterized as the main sites of

385

toxic action for Cd in fish.38 An examination of life-stage and species specific differences in gene

386

and protein level response of ECaC and Ca-ATPase during waterborne Cd exposure may provide

387

further insights into the mechanisms underlying the differences in life-stage and species-specific

388

Cd sensitivity.

389

In conclusion, physiological processes related to acute metal sensitivity among fishes have

390

not been well characterized to date. In this study, significant species and life-stage specific

391

differences were found in Cd toxicity, an observation which was in agreement with previous

392

studies. The two evolutionary distant fish species, white sturgeon and rainbow trout, were shown

393

to present a high degree of variation in species and life-stage specific response to waterborne Cd

394

exposure: mortality, Cd uptake kinetics and Cd-induced disruption of Ca homeostasis. The 96h

395

LC50 of Cd was similar in both species at the larval stage; however, trout demonstrated a greater

396

sensitivity to Cd in later life-stages than sturgeon. We also observed that Cd disrupted Ca uptake

397

and whole-body Ca levels in trout by a greater degree relative to that in sturgeon, especially at

398

those life-stages where trout were significantly more sensitive than sturgeon. Finally, the

399

evaluation of waterborne Cd uptake across life-stages and species demonstrated that the less

400

sensitive species white sturgeon had a relatively lower affinity for Cd uptake compared to the more


Page 18 of 28

Page 19 of 28


401

sensitive species rainbow trout. Interspecific difference in Cd uptake affinity between sturgeon

402

and trout infers differential nature of interaction between waterborne Cd and Ca transport pathways

403

in the two species, which at least partly explain the differences in their Cd sensitivity. Overall, our

404

findings suggest that species and life-stage specific differences in Cd sensitivity could depend on

405

the interplay among Cd uptake and the pathways that regulate Ca uptake and Ca levels in the body.

406

Future studies with more species and metals are needed for generating additional data that can be

407

useful in further establishing the correlation between the physiological variables and acute metal

408

sensitivity, as characterized in this study. Well-defined mechanisms for species specific

409

differences in sensitivity to metals can be helpful in building phylogenetically based predictive

410

models for evaluating the sensitivity to metals among different fish species.

411

Acknowledgements

412

This work was supported by NSERC Discovery Grant to MH and SN, and through Canada

413

Research Chair Program to MH. KS was supported by Dean’s scholarship through the University

414

of Saskatchewan.

415

Supporting Information. Water quality parameters, measured concentrations, percent inhibition

416

of Ca uptake and whole body Ca

417

Reference

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537 538

Figure1: Acute toxicity (96 h LC50) of Cd to different life-stages of white sturgeon (blue) and rainbow trout (brown).

539

Error bars represent 95% confidence interval (n = 3 tanks with 10 fish per replicate). When an LC50 value could not

540

be calculated because mortality was less than 50% at the greatest concentration tested, it is represented as light

541

coloured bar.

542 543

544 545

Figure 2: Effect of 24 h exposure to waterborne Cd concentration on whole body Ca levels in rainbow trout and white

546

sturgeon. Data is presented as mean ± SEM values of whole body Ca (n = 6). Data was analysed using three-way

547

ANOVA followed by post-hoc Tukey test. Asterisks (*) demonstrate statistically significant differences in the

548

exposure group as compared to respective control group (p < 0.05). Significant interactions were recorded among all

549

life-stages, species and Cd exposure (p

Investigating the role of ionoregulatory processes in the species- and

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